Heritability of Retinal Vascular Morphology in a Danish Twin

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					Heritability of Retinal Vascular Morphology

       in a Danish Twin Population




                   A Ph.D. Thesis by
        Nina Charlotte Bille Brahe Taarnhøj, MD


              Department of Ophthalmology
                   Glostrup Hospital
                       Denmark
                         2008



                         -1-
The present Ph.D. thesis was handed in December 30th, 2007

The thesis was accepted by The Faculty of Health Science, University of Copenhagen for public
defence.

The thesis will be defended on March 11th, 2008 in Auditorium C, Glostrup Hospital, Denmark.

The supervisors are Michael Larsen, Professor, MD, Line Kessel, MD, Ph.D., Department of
Ophthalmology, Glostrup Hospital, Knut Borch-Johnsen, Professor, MD, Steno Diabetes Centre
and Thomas Martini Jørgensen, Risøe, Denmark.

The official opponents are: Fridbert Jonasson, Professor, MD, Reykjavik, Iceland, Peter Bjerre Toft,
Associate professor, MD, Department of Ophthalmology, Rigshospitalet, Denmark and Hans Ibsen,
Professor, MD, Medicinsk afdeling, Holbæk, Denmark.




Cover photo: Digital grey-scale fundus photograph of Nina Taarnhøj’s left eye.




Nina Taarnhøj
Printed in Denmark




                                               -2-
Preface
The present Ph.D. thesis was written during my position as a research fellow at the Department of
Ophthalmology, Herlev/Glostrup Hospital from 2004 to 2007. The principal purpose of the thesis
was to examine the genetic influence on retinal vascular morphology.

       I would like to express my sincere thanks to all who have helped and inspired me during my
work on this thesis. Thanks to Professor Henrik Lund-Andersen, MD, DMsc, for creating a very
encouraging and pleasant atmosphere in which research at a highly international level is made
possible. Thanks to Line Kessel, MD, Ph.D., Jesper Leth Hougaard MD, Ph.D., nurse Jette
Kvistgaard and photographer Hans Henrik Pedersen for conducting the twin study. Thanks to
Kirsten Ohm Kyvik, MD, PhD, University of Odense, scientific co-head and administrative leader
of the Danish Twin Registry and professor Thorkild I.A. Sørensen, MD, DMsc. and colleagues at
Institute of Preventive Medicine, Copenhagen University Hospital, Denmark, for making the twin
study possible and helping me with twin analysis. For her, statistical guidance and inspiring
discussions during this project I would like to warmly thank Birgit Sander, Ph.D. I am also very
grateful to Dorte Nellemann Thornit, MD, for being a fun and inspiring colleague and friend.

      To my primary supervisor Professor Michael Larsen, MD, DMsc, I want to express my
special gratitude for his interesting research ideas and experience which has provided me with a
great foundation for my research. Also special thanks to my co-supervisor Line Kessel, who has
been very engaged in my work and has been a really good support. Finally, I want to thank Inger
Christine Munch, MD, for great cooperation with the vessel analysis.

     I am very appreciative to Professor Bjørn Nicolaissen, who invited me to complete my
theoretical work at the Department of Ophthalmology, Ullevål University Hospital, Oslo, when I
moved to Norway in 2004.

     Finally, I would like to express my warmest thanks and deepest affection to my husband,
Frode and our son, Alexander for their patience, love and support.

     This Ph.D. project was made possible thanks to the financial support from The Danish
Medical Research Council, The Danish Eye Research Foundation, The Danish Eye Health Society,
Centre for Biomedical Optics and New Laser Systems Graduate School, The Danish Diabetes
Association, The Norwegian Association of the Blind and Partially Sighted, and The Research
Career Award Grant 8-2002-130 from The Juvenile Diabetes Research Foundation (Michael
Larsen).

                                                        Nina Taarnhøj, Copenhagen, March 2008




                                              -3-
The present thesis is based on the following papers:



Paper 1: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel,
Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen, Heritability of Cilioretinal
Arteries: A Twin Study. Invest Ophthalmol Vis Sci, 2005, Vol. 46, p. 3850-3854.



Paper 2: Nina C.B.B. Taarnhøj, Michael Larsen, Birgit Sander, Kirsten Kyvik, Line Kessel, Jesper
L. Hougaard, and Thorkild I.A. Sørensen, Heritability of Retinal Vessel Diameters and Blood
Pressure: A Twin Study. Invest Ophthalmol Vis Sci, 2006, Vol. 47, p. 3539-3544.



Paper 3: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel,
Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen. Straight Versus Tortuous Retinal
Arteries in Relation to Blood Pressure and Genetics. (Accepted for publication in British Journal of
Ophthalmology).

Paper 4: Nina C.B.B. Taarnhøj and Line Kessel. Review of Twin Studies in Ophthalmology. (In
revision).




                                                -4-
Contents:

1.     Introduction ............................................................................................................................. 7


2.     Background ............................................................................................................................. 8
     2.1.    The eye............................................................................................................................. 8
     2.2.    Anatomy and physiology of the retinal vascular system .................................................... 8

     2.3.    Retinal vessel abnormalities............................................................................................ 11
       2.3.1.       Hypertensive retinopathy ......................................................................................... 11
       2.3.2.       Branch retinal vein occlusion................................................................................... 13
       2.3.3.       Retinal vessel tortuosity........................................................................................... 13
     2.4.    Computer assisted methods for analysis of retinal vessel calibres.................................... 13


3.     Fundus photographic retinal vessel calibre studies ................................................................. 16
     3.1.    Introduction .................................................................................................................... 16
     3.2.    Associations of retinal arterial and venous calibres with eye disease ............................... 17
     3.3.    Associations between retinal arterial calibre and systemic disease................................... 18
     3.4.    Associations between retinal venous calibre and systemic disease................................... 23
     3.5.    Influence of environmental factors on retinal calibres ..................................................... 25
     3.6.    Discussion ...................................................................................................................... 26


4.     Clinical studies ...................................................................................................................... 28
     4.1.    Purpose........................................................................................................................... 28
     4.2.    Methods.......................................................................................................................... 28

       4.2.1.       Subjects ................................................................................................................... 28
       4.2.2.       Fundus photography and digital imaging ................................................................. 28
       4.2.3.       Computerized image measurement software ............................................................ 30
       4.2.4.       Visual grading of retinal vessel tortuosity and cilioretinal arteries............................ 33
       4.2.5.       Twin analysis........................................................................................................... 33




                                                                      -5-
     4.3.    Results............................................................................................................................ 35
       4.3.1.      Paper 1 (Heritability of cilioretinal arteries: A twin study) ....................................... 35
       4.3.2.       Paper 2 (Heritability of retinal vessel diameters and blood pressure: A twin study) .. 35
       4.3.3.      Paper 3 (Straight versus tortuous retinal arteries in relation to blood pressure and........
                   genetics) ................................................................................................................. 36
       4.3.4.      Paper 4 (Review of twin studies in ophthalmology) ................................................. 36
     4.4.    Discussion ...................................................................................................................... 37
     4.5.    Conclusions .................................................................................................................... 40


5.     Summary in English............................................................................................................... 41


6.     Summary in Danish ............................................................................................................... 42


Reference list ................................................................................................................................ 43


Papers 1 – 4 .................................................................................................................................. 51




                                                                      -6-
1. Introduction

The purpose of the present Ph.D. thesis was to examine the relative importance of genetic and
environmental factors on the morphology of retinal blood vessels in a population of young healthy
normotensive twins, the clinical perspective being to evaluate whether a single fundus photograph
may be of value in assessing an individual’s likelihood of having or developing cardiovascular
disease in the future. It is also potentially important to know if retinal vessel layout is influenced by
genetic factors when evaluating if there is a genetic susceptibility to how some diseases affect the
eye. The thesis is based on four papers; three which are covering heritability analysis of retinal
blood vessel calibres, retinal arterial tortuosity, and the presence of cilioretinal arteries and one
review which covers twin studies in ophthalmology. The first three studies derive from a clinical
cross-sectional twin study including 58 monozygotic (MZ) and 54 dizygotic (DZ) same-sex healthy
twin pairs, aged 20 to 46 years, recruited from the Danish Twin Register. The thesis covers aspects
of digital fundus imaging, retinal vessel diameter measurement techniques, twin analysis,
epidemiologic studies on retinal blood vessel diameters and their associations with ocular and
systemic diseases, and finally a summary and discussion of the papers.

   Clinical signs of systemic vascular diseases are reflected in the eye, and are easily observed with
ophthalmoscopy. The eye is like a window to the systemic microvasculature, and is the only place
in the body where it is possible to examine blood vessels of the central nervous system non-
invasively and non-destructively. Large epidemiologic studies have demonstrated associations
between retinal vessel abnormalities and glaucoma, cataract and age-related macular degeneration
and systemic diseases such as hypertension, cardiovascular disease, diabetes mellitus, and stroke.
These associations explain only a small part of the variation in retinal vessel morphology therefore
examining the genetic and environmental influence on retinal vessel layout may provide additional
insights into the aetiology and pathogenesis of these complex vascular diseases. The classic twin
model is a superb method for assessing the relative influence of genetic and environmental factors
on retinal vessel morphology. Monozygotic twins are 100% genetically identical for which reason
we assume that all observed phenotypic differences between the two twins in a pair must be
attributable to environmental factors, whereas dizygotic twins share only 50% of their genes and the
extent to which MZ twins are more alike than DZ twins is therefore assumed to reflect a genetic
influence on the phenotype in question.




                                                  -7-
2. Background
   2.1.    The eye
The eye, bulbus oculi, is a ball-shaped organ, consisting of clear refractive media surrounded by a
tough fibrous tissue, the sclera, lined inside by the choroid and retina (figure 1). Light passes
through the refractive media: cornea, anterior chamber, lens, and vitreous, to reach the
photoreceptors in the retina where it is transformed into neural impulses. The neural impulses are
transported from the retina through the optic nerve and optic radiation to the primary visual cortex
in the occipital pole of the brain, where they are perceived as images.1




Figure 1. Cross sectional schematic drawing of the human eye. From:
http://www.nei.nih.gov/photo/.



   2.2.    Anatomy and physiology of the retinal vascular system
The retina is supplied by the ophthalmic artery by two different systems: the choroidal circulation
that supplies the outer third of the retina and the branches from the central retinal vessels that
supply the inner two thirds of the retina. The blood-retinal barrier has tight junctions and is highly
selective. It serves to regulate the passage of pathogens and intravascular leukocytes, thus
protecting the eye from ‘surveillance’ by immune cells and infections.2, 3




                                                 -8-
Figure 2. A cross-section of the retina, showing the 10 layers of the retina and the blood supply,
Figure 2. A cross-section of the retina, showing the 10 layers of the retina and the blood supply,
with the retinal arteries and veins to the left and the choroid to the right. From a Ciba-Geigy
with the retinal arteries and veins to the left and the choroid to the right. From a Ciba-Geigy
magazine.
magazine.


The central retinal artery and vein
The central retinal artery and vein
The central retinal artery and vein are branches from the ophthalmic artery and vein. Approximately
The central retinal artery and vein are branches from the ophthalmic artery and vein. Approximately
one centimetre behind the eye, they pierce the optic nerve, and travel forward in the centre of the
one centimetre behind the eye, they pierce the optic nerve, and travel forward in the centre of the
optic nerve, to enter the eye through a gap in the lamina cribrosa. They both branch into one
optic nerve, to enter the eye through a gap in the lamina cribrosa. They both branch into one
superior and one inferior branch, which subdivide into nasal and temporal branches. The four major
superior and one inferior branch, which subdivide into nasal and temporal branches. The four major
arterial branches are functional end-arteries and they supply a quadrant of the retina each, with no
arterial branches are functional end-arteries and they supply a quadrant of the retina each, with no
overlaps. The large arterial and venous branches travel in the nerve fibre layer just beneath the inner
overlaps. The large arterial and venous branches travel in the nerve fibre layer just beneath the inner
limiting membrane. They branch into two different capillary networks, one plexus within the
limiting membrane. They branch into two different capillary networks, one plexus within the
ganglion cell layer and one plexus within the inner nuclear layer (figure 2). The fovea is a 500 m
ganglion cell layer and one plexus within the inner nuclear layer (figure 2). The fovea is a 500 !m
large capillary-free zone, allowing for light to pass through undisturbed; instead the fovea is
large capillary-free zone, allowing for light to pass through undisturbed; instead the fovea is
nourished from the choriocapillaris.2, 3 The retinal blood flow is autoregulated, and is believed to be
nourished from the choriocapillaris.2, 3 The retinal blood flow is autoregulated, and is believed to be
a combination of myogenic and metabolic mechanisms. Autoregulation of intraocular blood flow
a combination of myogenic and metabolic mechanisms. Autoregulation of intraocular blood flow
serves to ensure a constant level of blood flow, despite moderate variations in perfusion pressure.
serves to ensure a constant level of blood flow, despite moderate variations in perfusion pressure.
As a result of the autoregulation of retinal blood flow, the oxygen tension in the inner retina can be
As a result of the autoregulation of retinal blood flow, the oxygen tension in the inner retina can be
maintained at a normal level during variations in blood pressure or intra ocular pressure (IOP). But,
maintained at a normal level during variations in blood pressure or intra ocular pressure (IOP). But,


                                                 -9-
                                                 -9-
there is an upper limit for autoregulation, therefore a sufficient increase in arterial blood pressure
can overwhelm the autoregulation and result in increased retinal blood flow.3


The Choroid
The choroid is a highly pigmented, vascular, loose connective tissue that supplies the outer third of
the retina and also has a thermoregulatory role. It is supplied by the posterior ciliary arteries and is
situated between the sclera and the retina and consists of 5 layers: Bruch’s membrane, the
choriocapillaris with fenestrated capillaries, two vessel layers, and the suprachoroid.2 Choroidal
blood vessels do not respond to elevated IOP and significant reductions in choroidal blood flow
cause no measurable effect on choroidal blood flow, therefore they are believed to not have
autoregulation.3


Cilioretinal arteries
Occasionally the central retinal artery is assisted by one or more cilioretinal arteries, emerging from
the rim of the optic disc (figure 3). These arteries are derived directly from the circle of Zinn, which
is formed by small branches from the short posterior ciliary arteries, which also supply the choroid.
It is of clinical relevance that a temporal cilioretinal artery supplying the fovea may spare the fovea
in cases of central retinal artery occlusion.4




Figure 3. Fundus photograph with two temporal cilioretinal arteries (CRA) supplying the fovea.




                                                 - 10 -
    2.3.    Retinal vessel abnormalities

        2.3.1.      Hypertensive retinopathy
Hypertensive retinopathy represents the ocular end-organ damage secondary to arterial
hypertension. Pathological changes primarily affect the retina, but changes in the choroid and optic
nerve may also be involved. Essential arterial hypertension is a very common disease and is defined
as having mean systolic blood pressure       140mmHg and mean diastolic blood pressure      90mmHg.
Hypertension is associated with increased risk of stroke and cardiovascular disease. Hypertensive
retinopathy is usually asymptomatic and rarely results in loss of vision.

      The primary pathologic change in hypertensive retinopathy is a protective vasoconstriction,
seen clinically as focal arterial narrowing, generalized arterial narrowing and arterial straightening.
Physiologically, retinal arteries constrict in response to elevated blood pressure, as a result of
autoregulation. This is best seen in young people, because sclerosis in older patients´ arteries
prevents the same degree of narrowing.5 A further increase in blood pressure can break down the
autoregulation, and eventually result in arterial dilation and tortuosity.6, 7

      Long-standing hypertension can cause arteriosclerosis with thickening of the arterial walls
resulting in luminal narrowing. Arteriosclerotic vessel walls thicken because of intimal
hyalinization, medial hypertrophy, and endothelial hyperplasia.8 Arteriosclerotic changes manifest
as copper wiring and silver wiring of the retinal arteries. The age of the patient may complicate
interpretation of the fundus changes, because arterial sclerosis both occurs in hypertensive patients
and in the normal ageing population. Arteriovenous crossings in the retina share a common
adventitial sheath. When arteriosclerotic arteries cross over veins the vein distal to the constriction
can appear dilated, darker and more tortuous. Arteriovenous nicking (AV-nicking) is diagnosed
when the underlying vein becomes less apparent or maybe even disappears on either side of the
artery. AV-nicking is an unambiguous finding and is a characteristic of chronic hypertensive
retinopathy. Sustained hypertension leads to disruption of the blood-retinal barrier and vascular
leakage resulting in haemorrhages, retinal oedema, and formation of hard exudates. Occlusions in
the small end arterioles lead to retinal ischemia and is seen as cotton wool spots. In very severe
hypertension the optic nerve becomes oedematous, and the patient’s vision is threatened.5, 9

      A classification system for hypertensive retinopathy was introduced in 1939 by Keith, Barker,
and Wagener, based on the status of retinal arteries, and systemic arteries from pectoral muscle
biopsies (table 1).10 A classification that kept the vascular changes of hypertensive retinopathy, and


                                                   - 11 -
arterial sclerosis separate was introduced in 1952 by Scheie (table 2).5 The Keith-Barker-Wagener
classification is still used in the clinic today. In Western countries, hypertensive retinopathy group 1
and 2 are common, but patients that are treated with anti-hypertensive medicine seldom develop
retinopathy group 3 and 4 (table 1). Patients with hypertensive retinopathy group 3 and 4 are still
seen in the clinic on rare occasions.5, 9 Patients in group 4 are in a very critical state.

KEITH-BARKER-WAGENER CLASSIFICATION

Group 1       Mild to moderate narrowing or sclerosis of the arteries

Group 2       Moderate to marked generalized arterial narrowing, focal arterial narrowing, AV-
              nicking, and exaggeration of the light reflex

Group 3       As for Group 2, plus retinal oedema, cotton-wool spots, and retinal haemorrhages

Group 4       As for Group 3, plus papilledema

Table 1. The Keith-Barker-Wagener classification of hypertensive retinopathy.5

SCHEIE CLASSIFICATION

HYPERTENSION

Grade 1        Arterial narrowing

Grade 2        Grade 1, plus AV-nicking and focal arterial narrowing

Grade 3        Grade 2, plus retinal haemorrhages, hard exudates, and cotton-wool spots

Grade 4        Grade 3, plus papilledema

ARTERIAL SCLEROSIS

Grade 1        Barely detectable light reflex changes

Grade 2        Obvious increased light reflex changes

Grade 3        Copper-wire arteries

Grade 4        Silver-wire arteries

Table 2. The Scheie classification of hypertensive retinopathy.5


                                                   - 12 -
        2.3.2.      Branch retinal vein occlusion
Branch retinal vein occlusion (BRVO) is an important cause of unilateral quadrant vision loss. In
The Blue Mountains Eye Study, BRVO was associated with advancing age ( 70years), increasing
mean arterial blood pressure and arteriosclerotic retinal vessel signs (AV-nicking, focal arterial
narrowing, arterial wall opacification) and had a cumulative 10-year incidence of 1.2%.11 Branch
retinal vein occlusions are seen at arteriovenous crossings. When an arteriosclerotic artery
compresses a vein, the vein loses endothelial cells and thrombus formation leads to occlusion of the
vein, stagnation of blood flow and local retinal hypoxia in the area that is drained by the vein. The
clinical signs are leakage, haemorrhages, cotton wool spots, hard exudates, and oedema in the
affected zone. The vein dilates and becomes tortuous distal to the site of occlusion, because of
congestion and increased transmural pressure. The retinal artery supplying the affected area
becomes narrowed and sheathed. The patient complains of a sudden blurred vision or a visual field
defect.5, 9

        2.3.3.      Retinal vessel tortuosity
Studies on the variation in retinal vessel tortuosity and its association with various systemic
parameters in adults are limited. Some forms of arterial tortuosity are dominantly genetically
inherited and are caused by weakness in the vessel wall.12-15 Diabetic macular oedema16 and branch
retinal vein occlusion5 are associated with retinal venous tortuosity. Severe hypertension,6, 7, 17 and
retinopathy of prematurity (ROP) are associated with retinal arterial tortuosity.9,       18
                                                                                               Increased
transmural pressure results in dilation and elongation of arteries19 and tortuosity is observed when a
critical transmural pressure threshold of a passive tube is reached.20 This may also append to retinal
vessels when pathological stresses, such as severe hypertension and retinal hypoxia exceed the auto-
regulatory capacity. This was observed in retinal vessels of human newborns with acute perinatal
distress, where systemic metabolic distress (hypoxia and lactic acidosis) caused retinal arterial
dilation and tortuosity.21




    2.4.      Computer assisted methods for analysis of retinal vessel calibres
Historically, we can divide research on retinal vessel morphology into three eras: the visual
impression era, the quantitative era, and the digital imaging era. The first era of basic research used
visual impression and yielded limited data (figure 4). Retinal arterial narrowing in hypertensive




                                                - 13 -
retinopathy was first described by Gowers in 187622. Arteriovenous nicking as a sign of
hypertensive retinopathy was described by Marcus Gunn in 1898.23




Figure 4. Old fashioned funduscopy with a candle as light source.


      In the 1970s we moved into a quantitative era, thanks to the introduction of the wide-angle
fundus camera. Diameters were measured manually with a dial caliper based on visual identification
of the vessel outline on a magnified projected image on a white screen or by direct visual
measurement through a microscope with a screw-micrometer eye-piece.24,          25
                                                                                     In 1974 Parr and
Spears presented a more precise vessel measurement method. Negatives were mounted as slides
between two glass plates. The widths of the parent trunk, and of its two branches, were measured
with a measuring projector. They developed the Parr-Spears formula for the central retinal artery
equivalent by empirically measuring the relation between branches and their trunk vessels in
healthy eyes.26 In 1986, Olaf Brinchman-Hansen introduced a better technique where the fundus
photographs were measured with a computer controlled microphotometer.27 The above mentioned
methods were troublesome and time-consuming and had low intra- and inter-operator
reproducibility.
      A new era of digital imaging began in the 1990s. Like the rest of the imaging world today,
fundus photography also participated in the digital revolution. Advances in digital image analysis
have allowed objective, fast, and accurate semi-automatic quantitative measurements of retinal
vessel calibres.28 The advantages of these methods are the power to measure retinal vessel
diameters in eyes of large study populations and the ability to follow them over time. Another
advantage is the ability to use statistics in order to adjust for important systemic and environmental


                                                - 14 -
factors when analyzing the associations between retinal vessel calibre and ocular and systemic
disease. A computer assisted semi-automatic method has been used in recent large population
studies.28 Completely automatic fundus photographic vessel measurement programs that are quicker
and more accurate have recently been presented, but have not yet been tested in large population
studies.29-33

    The Zeiss Retinal Vessel Analyser is a commercially available system which comprises a
fundus camera, a video camera, a real time monitor and a computer with analyzing software for the
accurate measurement of real time retinal vessel diameters. Twenty five vessel diameter readings
can be obtained per second hence the vessel diameter can be measured as a function of time and can
therefore be used to assess the effect of different stimuli on vessel calibres. In contrast to other
vessel measurement software used in fundus photographs, the Retinal Vessel Analyser can only
measure the vessel diameter in relative units, ie the relative change in diameter over time.34-36 It is a
good tool in physiological studies of retinal vessel diameter changes.




                                                 - 15 -
3. Fundus photographic retinal vessel calibre studies
   3.1.    Introduction
Photographic assessment of retinal trunk vessel calibres has demonstrated significant statistical
effects of systemic variables, environmental conditions, and genetic factors. The aim of this chapter
is to give a short summary of the most important findings of the associations between retinal vessel
calibres and various systemic and environmental factors.

     Arteriovenous ratio (AVR) being a dimensionless figure does not vary with magnification. It
is also robust toward apparent vessel broadening in blurred images and can be made using
objective, automated image processing algorithms. Researchers used to interpret smaller AVR as an
indicator of arterial narrowing in patients with hypertension, assuming that veins were not
influenced by elevated blood pressure. Recent empirical studies of AVR and vessel diameters
however have shown that artery and vein diameters provide distinctly different correlations with
systemic diseases.

     The field of quantitative fundus photographic vessel studies is extensive. In terms of numbers
of participating subjects, most of the research has been made in six major studies, covering a total
of 34480 subjects, each of which has given rise to a multitude of publications:

a. The Atherosclerosis Risk in Communities Study (ARIC) is a prospective cohort study of
15792 persons aged 45 – 64 years, selected from 4 US communities, initiated in 1987 through 1989.
Fundus photos were taken during the third examination in 1993 through 1995. It investigated
cardiovascular disease and its risk factors among middle-aged people.

b. The Blue Mountains Eye Study (BMES) is an Australian population based cohort study of
common eye diseases that included 3355 participants at baseline in 1992-94, when mydriatic 30
degree fundus photos were taken. The study examined microvascular characteristics and their
association with ocular and systemic diseases.

c. The Beaver Dam Eye Study includes 4926 persons aged 43-84 years living in Beaver Dam,
Wisconsin. Baseline examinations were performed in 1988-1990. It investigated relationships
between microvascular characteristics and ocular and systemic disease.




                                                 - 16 -
d. The Cardiovascular Health Study (CHS) is a prospective cohort study initiated in 1989-1990,
in 4 communities in the USA. In 1997-98 fundus photos were taken of 2405 people, aged 69-97
years. It is a study of coronary heart disease and stroke among older people.

e. The Wisconsin Epidemiologic Study of Diabetic Retinopathy (XVIII and XIX ) included
996 persons diagnosed with diabetes before age 30 years who were taking insulin, and 225 controls
without diabetes, all from South Central Wisconsin. Baseline examinations were made in 1980-82.
The study examined diabetic retinopathy, retinal vein and artery diameters and other ocular
characteristics.

f. The Rotterdam Study is a population-based cohort study on chronic disease in the elderly,
including 7983 persons over age 55 years, from a district near the city of Rotterdam. 6780 persons
underwent eye examination at baseline in 1990-1993.

The following sections summarize the findings of these large-scale population studies.




    3.2.    Associations of retinal arterial and venous calibres with eye disease
End-stage glaucoma has long been described as being associated with narrow retinal vessels. It
remains unknown whether vessel narrowing precedes or follows retinal nerve fibre and ganglion
cell loss. In the BMES, eyes with glaucomatous damage had significantly narrower retinal arteries
(183±2.6 m) than eyes without glaucoma (194±0.4 m, p=0.0001) and also narrower arteries than
eyes with ocular hypertension (195±1.6 m, p=0.0002, after adjusting for age, gender, body mass
index (BMI), smoking, blood glucose level, mean arterial blood pressure (MAP), and refraction).
Eyes with open angle glaucoma were 2.7 times more likely to have generalized arterial narrowing
than eyes without glaucoma. A similar pattern was found for retinal vein diameters. There was also
a strong trend for narrower retinal arteries with increasing cup-to-disc ratios. Generalized retinal
arterial narrowing is an indicator of vascular abnormality, and thus appears to be associated with
optic nerve damage caused by open-angle glaucoma. It is not clear whether such retinal arterial
abnormality reflects an ischemic process leading to optic nerve damage, or whether it results from
an autoregulatory response to the loss of retinal neurons in glaucoma.37 A study of 64 patients with
primary open-angle glaucoma found a strong association between peripapillary arterial narrowing
and visual field defects in the corresponding hemifield. No statistical association was seen with
retinal vein narrowing or dilation.38



                                                - 17 -
      In the Beaver Dam Eye Study generalized arterial and venous narrowing were significantly
associated with the prevalence of nuclear cataract, defined as the odds ratio (OR) of the first versus
the fifth quintile of vessel calibres (OR 0.68; CI95 0.50, 0.93) and (OR 0.72; CI95 0.53, 0.98), for
arteries and veins, respectively. Smaller retinal artery diameter values at baseline were directly
correlated with the 10-year incidence of retinal pigment epithelium (RPE) depigmentation (OR
1.93; CI95 1.11, 3.34) and inversely correlated with the 10-year incidence of nuclear cataract (OR
0.71; CI95 0.50, 0.99, after controlling for age and gender). Larger retinal vein diameter values at
baseline were associated with the 10-year incidence of nuclear cataract (relative risk 0.60; CI95 0.42,
0.84).39 In contrast to the findings of the BMES37 arterial narrowing in the Beaver Dam Eye Study
was not associated with a large cup-to-disc ratio, high IOP, or glaucoma.39 The relation between
vessel diameters and nuclear cataract was unexpected and remains unexplained.

   Age-related macular degeneration and its precursor age-related maculopathy (ARM) are related
to arterial hypertension. Hence, it would appear likely that retinal arterial narrowing would be
associated with incident age-related maculopathy (ARM), but no such association was found in the
Beaver Dam Eye Study, only an association with incident RPE depigmentation was found.39 The
BMES Study also found no significant association between artery diameter and 5-year incident late
or early stage ARM, but its findings suggested that other structural changes such as AV-nicking and
focal arterial narrowing could either contribute to ARM progression or share common pathologies
with ARM.40




   3.3.    Associations between retinal arterial calibre and systemic disease
Association with age and gender
Generalized retinal arterial narrowing was associated with increasing age as has uniformly been
demonstrated in four different study populations. The arterial diameters decreased by 2.3 to 4.8 m
per decade,28, 41-43 the correlation persisting after adjustment for correlation between artery and vein
diameters.44 Data on the association with gender have been inconsistent. In the BMES41 and the
CHS,45 mean retinal arterial diameters were higher in women than in men across all age groups, but
this was not corroborated by the Beaver Dam Eye Study.42, 46




                                                 - 18 -
Association with current blood pressure
Recent data from eight large population studies showed that narrow retinal arteries were associated
with high blood pressure in adults28,   41-43, 47-51
                                                       and in children.52 The retinal artery diameter was
found to decrease by 2.8 m (p<0.0001) to 5.5 m (CI95 3.3, 7.6 m) for each 10mmHg increase in
MAP.41-43, 50 In the Rotterdam Study the association between current systolic blood pressure and
narrower arteries was strongest in the youngest age category (55 - 60 years), narrowing by 3.8 m
(CI95 2.8, 4.7 m) per standard deviation increase in systolic blood pressure, yet becoming non-
significant in persons above age 80 years. This may reflect that older people’s vessels are sclerotic
and have lost the ability to contract as a reaction to elevated blood pressure levels.49 People with the
highest quintile of current blood pressure were twice as likely to have generalized arterial narrowing
than those with the lowest quintile of blood pressure.48, 51 In children from Sydney and Singapore,
each 10 mmHg increase in systolic blood pressure was associated with narrowing of the arteries by
2.1 m (CI95 1.38, 2.79 m) and 1.43 m (CI95 0.27, 2.59 m), respectively.52



Association with past blood pressure
In the ARIC Study, blood pressure measured 3 to 6 years before fundus photography was
significantly associated with smaller artery diameters (as reflected by a lower AVR) in both men
and women, whether treated or untreated for hypertension. The association with previous blood
pressure was found after adjustment for current blood pressure, age, race, and smoking.47 In the
CHS, smaller arterial calibre was significantly associated with past systolic blood pressure
measured 8 years before fundus photography (OR 1.11, CI95 1.04, 1.27) per 10 mmHg increase in
blood pressure, after adjustment for current blood pressure and use of antihypertensive
medication.51 The findings were supported by the BMES, blood pressure measured 5 years prior to
fundus photography being independently associated smaller artery diameters.8 These findings
supported that generalized arterial narrowing represents cumulative effects from long-standing
arterial hypertension.



Association with future blood pressure

The ARIC study reported that normotensive individuals with the lowest quintile of AVR at baseline
were 60% more likely to develop hypertension than individuals with the highest quintile of AVR
within a 3-year period of observation.53 This was confirmed by prospective data from the BMES,


                                                   - 19 -
where narrow arteries were associated with increased risk of incident severe hypertension within a
5-year period (OR 2.6, CI95 1.7, 3.9, comparing the lowest and highest AVR quintiles).54 These
studies provided evidence that generalized arterial narrowing may precede the development of
severe hypertension.



Association with diabetes mellitus and diabetic retinopathy
In the ARIC study, lower AVR was associated with increased risk of diabetes mellitus, subjects in
the lowest AVR quartile being 71% more likely to develop diabetes than subjects in the highest
AVR quartile (OR, 1.71, CI95 1.13, 2.57), independently of known risk factors.55 However, it has
later been documented that the association with lower AVR was largely explained by venous
dilation in subjects with diabetes.56

      Larger arterial calibre was independently associated with the 4-year progression of
retinopathy. Smaller arterial calibre at baseline was associated with greater 4-year and 14-year
incidence of macular oedema. Arterial calibre was not associated with the incidence of proliferative
retinopathy. The findings suggested that retinal vascular calibre measurements in subjects without
retinopathy had little prognostic value for the development of retinopathy in persons with type 1
diabetes.57 In diabetic subjects, mean arterial calibre decreased by 3.23        m per increase in
retinopathy severity scale level and was smallest in eyes that had been treated with panretinal
photocoagulation for proliferative retinopathy. This was hypothesized to result from lower
volumetric flow secondary to lower metabolic needs and improved oxygenation after the
destruction of ischemic retinal tissue.58



Association with measures of atherosclerosis
In the ARIC Study generalised arterial narrowing, as reflected by lower mean AVR values, was
associated, after adjustment for past and present blood pressure, with carotid plaque59 and increased
carotid stiffness,60 but not with any other clinical indicator of atherosclerosis (cardiovascular
disease or stroke) or subclinical indicator of atherosclerosis (carotid or popliteal artery wall
thickness, popliteal plaque, or lower limb obstructive disease) or with plasma cholesterol. Lower
mean AVR was associated with higher plasma triglycerides, elevated white cells, higher fibrinogen
levels, reduced albumin, smoking, greater body mass index, and elevated blood pressure. Thus,
general retinal arterial narrowing shared many risk factors with atherosclerosis. Klein et al


                                               - 20 -
suggested that generalized arterial narrowing, defined by a lower mean AVR, occurred
independently of atherosclerosis and could therefore provide independent prediction for ischemic
diseases of the heart, brain and other organs.59 In the Rotterdam Study there was no association
between retinal arterial narrowing and any markers of atherosclerosis except increased carotid
intima-media thickness. Larger retinal venous diameters, on the other hand, were associated with
several markers of atherosclerosis after adjustment for blood pressure. The relationship between
AVR and atherosclerosis was mainly driven by larger vein diameters.49



Associations with stroke and sub-clinical cerebrovascular disease

There is evidence that small-vessel disease is related to both clinical and sub-clinical stroke but the
underlying mechanisms are not fully understood.61 Cerebral and retinal microcirculations share
similar anatomical, embryological, and physical characteristics.62 White matter lesions, which are
caused by small vessel abnormalities, were associated with increased risk of clinical stroke.63 In the
ARIC study, the relative risk of stroke increased with decreasing quintiles of AVR, with a relative
risk of 2.35 for the first versus the fifth quintile of AVR, independent of hypertension, in both men
and women.64 The Rotterdam study analyzed artery and vein diameters separately and found that
generalized arterial narrowing was not associated with an increased risk of stroke. The association
between smaller AVR and stroke was confirmed and the data revealed that venous dilation rather
than arterial narrowing explained this association.65, 66



Associations with coronary heart disease and cardiovascular mortality
In the ARIC Study, lower AVR was associated with increased risk of incident coronary heart
disease (CHD) and acute myocardial infarction in women, but not in men. Every 1 standard
deviation decrease in AVR was associated with a 39% increase in incident CHD of any type. This
association was independent of known risk factors such as mean arterial blood pressure, diabetes,
smoking, and plasma lipid levels and was seen in women with and without hypertension and/or
diabetes. Retinopathy, a marker of severe microvascular damage from hypertension, was associated
with an 83% increased risk of incident CHD in women, not in men. This study supported that
microvascular disease may play a more prominent role in the development of cardiovascular disease
in women than in men.67 The CHS reported that both smaller retinal arterial diameters (rate-ratio
2.0, CI95 1.1, 3.7, comparing largest and smallest arterial calibre quartiles) and larger vein diameters


                                                  - 21 -
(rate-ratio 3.0, CI95 1.6, 5.8, comparing largest with smallest vein calibre quartiles) were
independently associated with incident coronary heart disease in elderly men and women.68



Association with mortality
In the light of the many studies showing a relationship between retinal arterial narrowing and
various diseases and disease markers, an association would be expected between increased
mortality and retinal arterial narrowing. The ARIC Study found no association of smaller AVR with
either all-cause mortality, vascular-disease, or non-vascular-disease-related mortality.69 Pooled
analysis of data from the Beaver Dam and Blue Mountains Eye studies found that both smaller
arterial and larger venous diameters were associated with increased risk of CHD and stroke
mortality, independent of age, gender and vascular risk factors.70 Generalized arterial narrowing
was significantly associated with 10-year cardiovascular death in the Beaver Dam Study, with
multivariable adjusted odds ratio of 1.5 (CI95 1.1, 2.1), comparing presence versus absence of
arterial narrowing (defined as lowest quintile of AVR).71



Summary
In summary there is evidence suggesting that retinal arterial narrowing is associated with increasing
age, elevated blood pressure (past, current, and future), increasing degrees of macular diabetic
oedema, increasing severity of diabetic retinopathy, increased risk of coronary heart disease, and
increased mortality from CHD and stroke. Generalized arterial narrowing seems to be related to
chronically high blood pressure and may therefore reflect persistent structural damage from
hypertension, but there is also evidence that generalized arterial narrowing may precede the
development of hypertension. It remains to be shown whether increased blood pressure leads to
arterial contraction, as an autoregulatory response, which in turn leads to additional increases in
blood pressure, or vice versa. Lower mean AVR shared some risk factors with atherosclerosis, but it
was not directly associated with clinical and subclinical indicators of atherosclerosis. Low mean
AVR seemed to occur independently of atherosclerosis. Lower mean AVR was associated with
increased risk of diabetes mellitus and stroke, but it appeared that this association may be driven
mostly by venous dilation rather than arterial narrowing, reflecting the importance of analyzing the
effect of artery and vein diameters separately.




                                                  - 22 -
    3.4.     Associations between retinal venous calibre and systemic disease
Association with age

Generalized retinal venous narrowing was associated with increasing age in four different study
populations. Venous calibre decreased with 2.3 m (CI95 1.8, 2.9 m) to 4.5 m (CI95 0.56, 8.45 m)
per decade.28, 41-43



Association with current blood pressure
Retinal venous dilation was linearly and significantly associated with elevated blood pressure in
participants under the age of 80 years, when adjusting for arterial calibre. After age 80 the
association was insignificant, possibly due to the rigidity of the vessel walls or because of an effect
of survival.44



Association with future blood pressure
In the Rotterdam Study, smaller vein calibres were found to be significantly associated with 6-year
incidence of hypertension, but when adjusting for retinal arterial calibre, this association became
insignificant.72-74 In the BMES, retinal venous diameters were not associated with 5-year incident
hypertension in the first analysis,54 but when adjusting for retinal arterial calibres, incident
hypertension was independently associated with wider venous calibres (OR 1.28, CI95 1.09, 1.50).74
This finding was consistent with a study suggesting that prehypertensive damage to the
microcirculation leads to chronic retinal hypoperfusion, ischemia and venous dilation.75 The
correlation between retinal artery and vein diameters was 0.59 in The Rotterdam Study and the
BMES, and persons with narrower arteries were more likely to have narrower veins.72,           74
                                                                                                    This
confounding effect may explain the counterintuitive association between narrow veins and
hypertension in the Rotterdam Study.72



Association with diabetes mellitus and diabetic retinopathy
Wider retinal venous calibre has been reported to be independently associated with progression of
diabetic retinopathy, diabetic macular oedema, and incidence of proliferative diabetic retinopathy in
several studies.56-58, 76, 77




                                                - 23 -
Association with dyslipidemia and obesity
In three different study populations, dyslipidemia, such as high triglycerides and low HDL levels
were associated with venous dilation.76-78 The relationship between dyslipidemia and venous
dilation may hypothetically involve inflammatory factors and endothelial dysfunction, which also
play a role in the development of venous dilation in people with impaired glucose tolerance.59, 79
Increasing venous diameters were also related to obesity both in persons with diabetes and in the
general population during a 5-year period. Persons in the highest quintile of BMI were 70% more
likely than those in the lowest quintile to have generalized venous dilation (OR 1.7, CI95 1.3, 2.4).80



Association with direct measures of atherosclerosis
Larger venous diameter was associated with markers of atherosclerosis, inflammation and higher
cholesterol levels in the Rotterdam Eye Study.49 Retinal venous dilation was associated with
increased carotid intima-media thickness, a marker of early atherosclerosis, but the association was
largely explained by fasting insulin level in the Hoorn Study.81



Associations with stroke and sub-clinical cerebrovascular disease
Prospective data have demonstrated that larger retinal venous calibre was associated with an
increased risk of stroke, cerebral infarction, white matter lesions, and lacunar infarction.64-66 Each
standard deviation increase in venous diameter resulted in a 13% increased risk of stroke and a 15%
increased risk of cerebral infarction after adjustment for cardiovascular risk factors.65 Retinal
venous dilation was related to progression of cerebral small vessel disease, each standard deviation
increase in venous diameter resulting in a 1.8 times increased risk of marked periventricular white
matter lesion progression and in a 2.8 times increased risk of marked subcortical white matter lesion
progression.66 The CHS found that participants with larger retinal venous diameters were more
likely to develop incident stroke during a 5-year period (rate ratio, 2.2, CI95 1.1, 4.3, comparing
largest with smallest venous calibre quartile, after multivariable adjustment).




                                                 - 24 -
Associations with coronary heart disease
In the CHS, participants with higher retinal venous diameter had an increased 5-year risk of CHD,
11.7% (CI95 8.7, 15.8%) versus 8.1% (CI95 5.7, 11.6%, comparing largest with smallest venous
calibre quartile).68



Association with mortality
Larger venous calibres were significantly associated with CHD and stroke mortality, in a pooled
analysis of the Beaver Dam and the Blue Mountains Eye Studies.70



Summary
In summary, increasing age was associated with narrow retinal veins. Increased retinal vein
diameter was associated with elevated current and future blood pressure, increased risk of diabetes
mellitus, progression of diabetic retinopathy, dyslipidemia, obesity, markers of atherosclerosis and
inflammation, increased risk of stroke, white matter lesions, 5-year risk of coronary heart disease,
and CHD and stroke mortality. Venous dilation may be caused by the endothelial dysfunction
associated with microvascular disease processes in these systemic conditions. Venous dilation has
also been hypothesized to occur in response to lactic acidosis associated with retinal hypoxia in
eyes with severe diabetic retinopathy.58 These data indicated that retinal vein diameters may play
their own independent role in predicting cardiovascular diseases and risk of stroke. The mechanisms
underlying venous dilatation deserve more attention, as they may provide new clues into the
pathophysiology of small vessel disease in the heart and brain.




    3.5.    Influence of environmental factors on retinal calibres
Smoking was related to larger artery and vein diameters in three large population studies of vascular
abnormalities.49, 58, 68 Such a tendency was also found in the Danish Twin Study.43 The mechanism
may be that high carbon monoxide levels in smokers cause decreased oxygen delivery to the retina,
resulting in retinal hypoxia, which causes vessel dilation. There are no conclusive data on the
relation between retinal vessel calibres and alcohol consumption, oestrogen replacement therapy in
women, the use of ocular betablockers, or the use of systemic antihypertensive medications.




                                               - 25 -
   3.6.    Discussion
This review describes associations between quantitative retinal vascular calibre characteristics and
systemic and ocular diseases and disease markers. The studies substantiate the idea that fundus
photography may be used to advance our understanding of the pathogenesis of systemic and ocular
vascular disease.

      There is clear evidence of involvement of aging, hypertension, arteriosclerosis, inflammation,
endothelial dysfunction, smoking, and blood lipids in retinal vascular characteristics, to the extent
that retinal vessel abnormalities may precede arterial hypertension, stroke and coronary heart
disease.

      The clinical utility of using vascular calibres in cardiovascular risk prediction and in disease
management requires further evaluation. Previous studies have not established specific clinical
protocols or reference values graded by age, gender or blood pressure, and basic statistical measures
of applicability in individuals such as positive and negative predictive values for specific outcomes
have not been calculated. Because of various systemic, environmental and genetic effects it is
challenging to determine uniform reference values that can be applied across populations. It has
already been shown that separate reference values are needed for children,52 It remains to be
examined whether specific treatment can reduce retinal arterial narrowing and whether this will
result in reduced cardiovascular risk and mortality.

      More precise predictions for use in individuals may potentially be derived by applying
methods that enable absolute quantitative measurement of fundus dimensions. A system that could
measure absolute vessel diameters in the retina, while factoring in magnification would be a
potential advantage. This could be done by combining modern technologies, for example optical or
ultrasonographic measurements of corneal curvature and axial length together with fundus
photography to scale fundus images to true dimensions. Currently, retinal vessel measurements are
based on the assumption of a standard vertical disc diameter, such as the standard 1800 m used in
recent studies. Furthermore, electrocardiographic gating of fundus cameras could synchronize the
photograph to a standard phase of the cardiac cycle, thus reducing frame-to-frame variation.82, 83

   The results of the Danish Twin Study43 suggests that genetic factors determine much of the
variation in retinal vessel calibres. A genome-wide linkage study of the Beaver Dam population
found that retinal artery and vein diameters were linked to multiple gene loci in regions associated
with hypertension, endothelial dysfunction, and vasculogenesis.84 In conclusion, it is likely that


                                                - 26 -
genetic, systemic and environmental factors interact to contribute to variations in retinal artery and
vein calibres. Assessing these specific factors in new studies may allow for greater understanding,
prevention and treatment of complex vascular diseases in humans in the future.




                                                - 27 -
4. Clinical studies
   4.1.    Purpose
The purpose of the clinical studies that form the integral part of this thesis was to estimate the
relative influence of genetic and environmental factors on retinal artery and vein calibres, retinal
arterial tortuosity and the presence of cilioretinal arteries. This was done in a cross-sectional twin
study, using structural equation modelling.



   4.2.    Methods

       4.2.1.      Subjects
We examined 55 MZ and 54 DZ healthy same-sex, normotensive twin pairs aged 20 to 46 years,
recruited from the population-based Danish Twin Register.85

       4.2.2.      Fundus photography and digital imaging
We used a digital fundus camera (figure 5) to capture 50° digital grey-scale (red-free) fundus
photographs centred on the macula and optic disc and 50° colour fundus photographs on
transparency film. A green filter for red-free photographs was used to enhance the sharpness and
contrast of the blood vessels. The advantage of digital photography is immediate feedback control
of image focus and luminosity, enabling immediate adjustment of exposure settings. The clarity of
the fundus photograph is related to the focus of the camera, the clarity of the patient’s tear film,
anterior chamber, lens, and vitreous, since the bundle of illuminating and imaging rays pass twice
through these structures. Dry or swollen corneas, cataracts, and vitreous haemorrhages are examples
of conditions that can blur the image. Sharpness in a photograph is a visual phenomenon that is
difficult to quantify. An image is described as sharp when the borders defining the blood vessels are
distinct and clear. The sharpness of a final fundus photograph is also limited by the sharpness of the
specific camera’s optical system.86

     Digital technology has revolutionized the way in which we can acquire, store, and share
fundus images, and it gives us considerably many possibilities for quantitative assessment of retinal
pathological changes. The major advantage of digital imaging in ophthalmology is the ability to
conveniently enhance and adjust the photos in order to see small pathologic changes in the fundus.
The first step in digital imaging is digital image capture, which is the electronic recording of light
information, and its conversion to a set of numbers which are stored in an image file. Resolution


                                                - 28 -
refers to the number of specific points (pixels) of picture information contained in an image file.
High-resolution images contain a high number of pixels and can exhibit great image detail. 86




Figure 5.
Figure 5. A fundus camera is a specialized low-power microscope with an attached camera. The
camera illuminates the fundus of the eye. The light is projected through a set of filters and lenses
onto an annular mirror, creating a ring of illumination through the dilated pupil. The light reflects
off the retina, exits the pupil through the centre of the illumination ring, continues through the
central aperture of the annular mirror, and further through the fundus camera optics (lenses that can
compensate for refractive errors) to form an image at the film plane or the array sensor.86

      Magnification of the eye under fundus photography is very complex, and increases from the
centre and out towards the periphery because of the eyes ball shape. Magnification differs between
eyes due to differences in refraction, axial length, and distance and angle between the camera and

                                                - 29 -
the eye. Vessel diameter measurements are reported in absolute numbers. Setting a mean vertical
disc diameter to 1800 m, the measured distance in pixels in a digital image can be converted to m.
This method potentially introduces bias, since individuals have different size optic discs, refraction,
and axial length. To get around the magnification issue it would be necessary to go through a large
biometric analysis with measurements of cornea curvature, anterior chamber depth, axial length,
and refraction in each individual patient, which is far too comprehensive for large population
studies. Researchers within the field have accepted the magnification problem as bias, for which
there is no good solution at the moment.

        4.2.3.      Computerized image measurement software
A semi-automatic computer program was used to measure retinal vessel diameters on red-free
digital images in the present thesis. A blood vessel in a digital retinal photograph consists of a dark
(low intensity) blood column on a lighter (high intensity) background. Two edges line the blood
column. The vessel wall and the sleeve of plasma that surrounds the blood column are invisible.
The aim of edge detection is to mark the points in the digital image at which the luminous intensity
changes sharply. The vascular morphology measurement tool uses the active contour model, the so
called ‘snake’, which is initiated by the user, and subsequently delineates the blood column. It is
accurate, robust, fast, and easy to initiate.87

      The digital snake consists of a number of point coordinates. Each point is affected by internal
and external forces on the ‘snake’. The snake positions itself at a sub pixel level, meaning that the
snake points can take different positions inside an image pixel, and hence perform very precise
measurements. The snake is active, which means that the snake algorithm is iterative and has no
natural stopping criteria and it will continuously try to achieve an optimal position. This is done by
searching for features such as lines and edges with large differences in intensities of neighbouring
pixels in the image. The diameter of a vessel is measured as the mean of the width of each snake
joint, along the vessel that is marked by the grader (figure 6). The point distances between the snake
joints are 1 - 3 pixels.87




                                                  - 30 -
Figure 6. Vessel contour with smooth shape and evenly spaced measurement lines. The vessel
Figure 6. Vessel contour with smooth shape and evenly spaced measurement lines. The vessel
width (156!m) is the mean of the width of each snake joint.
width (156 m) is the mean of the width of each snake joint.

     Retinal vessel diameters were measured according to international standards (Vessel
     Retinal vessel diameters were measured according to international standards (Vessel
Measurement System, IVAN protocol, version 2, University of Wisconsin). Absolute distances
Measurement System, IVAN protocol, version 2, University of Wisconsin). Absolute distances
were calculated assuming a uniform vertical optic disc diameter of 1800 m. A standard grid
were calculated assuming a uniform vertical optic disc diameter of 1800 !m. A standard grid
containing three concentric circles were placed on the images. The grader identified arteries and
containing three concentric circles were placed on the images. The grader identified arteries and
veins, using red and blue lines respectively to delineate the vessels (figure 7). A segment of each
veins, using red and blue lines respectively to delineate the vessels (figure 7). A segment of each
vessel within Zone B most suitable for measurement was chosen based on image quality, contrast,
vessel within Zone B most suitable for measurement was chosen based on image quality, contrast,
straightness of the vessel, and distance from branching points and crossings. Whenever possible, it
straightness of the vessel, and distance from branching points and crossings. Whenever possible, it
was preferred to measure the whole length of the vessel within Zone B. The cursor was used to
was preferred to measure the whole length of the vessel within Zone B. The cursor was used to
mark the starting and endings points of the vessel segments.
mark the starting and endings points of the vessel segments.




                                               - 31 -
                                               - 31 -
Figure 7. Fundus image showing a standard grid with measured arteries and veins delineated in red
and blue, respectively. The inner circle demarcates an average optic disc, the middle circle
demarcates 0.5 disc diameters (DD) from the outer rim of the optic disc, and the outer circle
demarcates 1.0 DD from the outer rim of the optic disc. Zone B was defined as the region from 0.5
DD to 1.0 DD from the disc margin.

     At the end of the measurement session, the computer program located the six widest arteries
and the six widest veins, and included them in the calculation of the Central Retinal Artery
Equivalent (CRAE) and the Central Retinal Vein Equivalent (CRVE), using the formulas developed
by Knudtson et al.88 Equivalents for arteries and veins were found iteratively by pairing the largest
and smallest calibres, until only one calibre measure remained. The formulas used for pairing were:

CRAE:        !
             ˆ                    2
            W AA = 00.88 ( w12 + w2 w 2 )
            W = .88 (w12 + ) 2


CRVE:        ˆ
             ! 0.95 (w         2
            WV = 0.95 ( w12 + w2 )
               V               2



Where w1, w2, and W are, the widths of the narrower branch, the wider branch and the parent trunk,
       1   2
             88
respectively.88 AVR = CRAE/CRVE, a smaller AVR being a sign of smaller arterial diameter or
wider venous diameter.




                                               - 32 -
       4.2.4.      Visual grading of retinal vessel tortuosity and cilioretinal arteries
The presence of cilioretinal arteries and the degree of retinal vessel tortuosity were assessed by
visual grading of digital 50° red-free fovea- and disc centred fundus images on a 15 inch wide
computer screen, by two independent graders. Each grader examined all images twice at an interval
of two months and in random order, masked to the relatedness, zygosity and values of all
covariates. Discrepant classifications of cilioretinal arteries underwent independent arbitration by a
third investigator, who decided the classification, using colour transparencies when needed.
Discrepant classifications of vessel tortuosity were consensus-graded by the two graders in a joint
session.



       4.2.5.      Twin analysis
Humans have 23 pairs of chromosomes. A gene is a piece of deoxyribonucleic acid (DNA) that
codes for a certain phenotype and occupies a given locus on a chromosome. Alleles are sequences
of DNA that code for a gene. A combination of alleles makes an individual’s genotype and the
observed trait is called the phenotype.

      The classical twin model is based on the knowledge that MZ twins derive from a single
fertilized egg that divides and develops into two 100% genetically identical individuals, for which
reason all observed phenotypic differences between the two twins in a pair must be attributable to
environmental factors. DZ twins derive from two fertilized eggs and share 50% of their genes, just
like normal siblings. The extent to which MZ twins are more alike than DZ twins is therefore
assumed to reflect a genetic influence on the phenotype in question. It is necessary that zygosity is
diagnosed accurately by use of DNA markers.89

      Heritability is defined as the proportion of the total phenotypic variance that is attributable to
genetic variance. For continuous traits, such as blood pressure or vessel diameter, a rough estimate
of heritability (h2) can be made by doubling the difference between the intrapair correlation for MZ
and DZ twin pairs: h2 = 2(rMZ-rDZ). For example, if heritability of a trait is estimated to 80% in a
population, it means that most of the inter-individual variation is due to genetic differences among
them. Among different populations or at different times, the environmental and genetic influences
might differ, and therefore the heritability estimates can differ.

      For binary traits, typically a disease which may be diagnosed as present or absent, comparing
concordance rates for MZ and DZ twins can give information on whether the disease is influenced

                                                  - 33 -
by genes. When both members of a twin pair are affected by a disease it is called concordance, and
if the disease affects only one member of a twin pair it is called discordance. Pairwise concordance
rates can be calculated as number of concordant pairs divided by total number of concordant and
discordant pairs in the study population. If the pairwise concordance rate for MZ twins is
significantly higher than for DZ twins, for a given disease, it is influenced by genetic factors.

      When estimating heritability we often assume the genetic effects to be additive. Additive
genetic effects occur when alleles at a locus add up to affect a phenotype. However, sometimes the
effects of alleles in a locus are dominant (i.e. non-additive) because they interact, instead of adding
up. Another non-additive effect is epistasis which is the interaction of alleles at different loci.

     Recent advances in twin statistics have allowed estimation of heritability by means of
structural equation modelling by use of MX computer software.90, 91 Structural equation modelling
allows separation of the observed phenotypic variance into genetic and environmental components
while adjusting for covariates such as age, gender and systemic factors. The genetic contribution
can be divided into an additive (A) genetic variance component and a non-additive (D) genetic
variance component; dominance and epistasis. The environmental component can be subdivided
into a common (C) environmental variance component representing environmental factors affecting
both twins in a pair, a source of similarity and an unshared (E) environmental variance component
representing environmental factors not shared by twins, a source of dissimilarity, including random
factors and measurement errors.89 Heritability is defined as (A+D)/(A+D+C+E), in a given
population at a given time.92 The significance of variance components; A, D, C, E and various
covariates is assessed by removing each component sequentially from the full submodels, ACE or
ADE, and testing the deterioration in model fit after each component is dropped from the full
model. The criterion for best fitting model is based on Akaike’s information criterion (AIC) = - 2
times log likelihood + 2 * (number of free parameters in the model). The model with the lowest
AIC is used to determine the best fitting model. P-values below 0.05 are considered significant.




                                                  - 34 -
    4.3.    Results

        4.3.1.     Paper 1 (Heritability of cilioretinal arteries: A twin study)
The aim of the first study was to examine the relative influence of genetic and environmental
factors on the presence and location of cilioretinal arteries in the human retina. Cilioretinal arteries
were found to be present in 45.1% of participants and 28.8% of eyes. The majority of cilioretinal
arteries, 88.2%, were located temporally and 11.8% were located nasally. The pattern was divided
into seven different phenotypes: both eyes, any eye, one eye only, left eye only, left eye, right eye
only, and right eye. The heritability ranged from 55% (CI95 18, 81%) for the phenotype left eye to
84% (CI95 46, 98%) for the phenotype right eye only, the remaining phenotypes having heritability
estimates between 71% (CI95 0.40, 0.90%) and 74% (CI95 0.34, 0.94%), the average heritability of
all phenotypes being 71.4%. Unshared environmental factors accounted for the remaining variance.
There was no impact of gender, age, or mean arterial blood pressure on the presence of cilioretinal
arteries.



        4.3.2.    Paper 2 (Heritability of retinal vessel diameters and blood pressure: A
             twin study)
The second study was carried out to assess the relative influence of genetic and environmental
effects on retinal vessel diameters and arterial blood pressure in young normotensive twins without
cardiovascular disease. Mean vessel calibre values in the study population were 165.8 m (SD 14.9)
for arteries and 246.2 m (SD 17.7) for veins. Retinal vessel diameters were found to be governed
mainly by genetic factors, accounting for 70% (CI95 54, 80%) of the variance in arterial calibres and
83% (CI95 73, 89%) of the variance in venous calibres, after adjusting for age and arterial blood
pressure. The remainder variance was attributable to unshared environmental factors. For mean
arterial blood pressure the heritability was 61% (CI95 44, 73%), decreasing after correcting for
CRAE to 54% (CI95 0.34, 0.68%). An important finding was that retinal arterial diameter decreased
with 4.4 m per decade increase in age and by 5.5 m per 10mmHg increase in current arterial blood
pressure, and retinal vein diameter decreased by 4.5 m per decade increase in age. No significant
influence on retinal arterial or venous diameter was found for gender, body mass index, total
cholesterol, fasting blood glucose, or 2-h oral glucose tolerance test values, but there was a
tendency for smokers to have wider arteries and veins than non-smokers.




                                                 - 35 -
       4.3.3.    Paper 3 (Straight versus tortuous retinal arteries in relation to blood
            pressure and genetics)
The purpose of the third study was to examine the relative contributions of genetic and
environmental effects on the variation in retinal arterial tortuosity, and the association of this
characteristic with age, blood pressure, retinal vessel calibres, and other health indices. There was a
large inter-pair variation in arterial tortuosity and a three-level grading scale was developed for the
description of arterial tortuosity (straight, wavy and tortuous). Using structural equation modelling
the heritability of retinal arterial tortuosity was 82% (CI95 64, 92%), unshared environmental factors
accounting for the remaining 18% (CI95 8, 36%). Age, gender, current systolic blood pressure,
plasma lipid levels, smoking, and blood glucose levels were not associated with tortuosity. Only
weak effects were found of current mean arterial blood pressure and body mass index, increasing
values of both being significantly associated with decreasing tortuosity, but they were of null
clinical significance. The extensive inter-pair variation suggested that comparison against a baseline
fundus photograph is a prerequisite for evaluation of retinal arterial tortuosity as a biomarker of
cardiovascular health.



       4.3.4.      Paper 4 (Review of twin studies in ophthalmology)
The purpose of this review was to give an overview of recently published twin studies in
ophthalmology, as the last review was published in 1950. The studies performed after 1990 used
structural equation modelling to estimate heritability. In summary, anatomical structures such as
refractive error, axial eye length, lens thickness, corneal endothelial cell area, central corneal
thickness, central retinal thickness, eye colour, optic disc and cup diameter, retinal nerve fibre layer,
small hard drusen, and macular thickness in eyes without ocular disease had heritabilities between
70% and 99%. Whereas ocular diseases such as age-related macular degeneration, cataract and
glaucoma had heritability estimates of 13% to 71%, supporting the hypothesis that they are complex
multifactorial diseases that are also to a large extent influenced by age and systemic and
environmental factors. Examination of the relative contribution of genetic and environmental
factors on ocular morphology by use of twin studies can be of fundamental importance for the full
understanding of the aetiology of ocular diseases.




                                                 - 36 -
    4.4.    Discussion
The purpose of the clinical studies included in this thesis was to examine the relative influence of
genetic and environmental factors on retinal vessel morphology in young healthy normotensive
twins, the clinical perspective being to evaluate whether a single fundus photo may be of value in
assessing an individual’s likelihood of having or developing cardiovascular disease.

      The most important finding was that four different aspects of retinal vascular morphology,
namely the presence and location of cilioretinal arteries, retinal arterial calibre, retinal venous
calibre, and retinal arterial tortuosity were mainly determined by additive genetic factors, with
estimated heritabilities of 71.4%, 70%, 83% and 82%, respectively, unshared environmental factors
accounting for the rest of the variation. Narrow retinal arteries were significantly associated with
increasing age and mean arterial blood pressure. Narrow retinal veins were significantly associated
with increasing age. These results were in agreement with large population studies of older age
groups with more variation in blood pressure.28, 41, 42, 47 Decreasing arterial tortuosity was weakly
associated with increasing body mass index and increasing mean arterial blood pressure. As may
have been expected from the early layout of retinal vessels, the presence of cilioretinal arteries was
not associated with age, gender, or blood pressure.

      The heritability estimate of blood pressure decreased when adjusting for arterial diameter,
suggesting a genetic association between the two, as in agreement with the Beaver Dam genome-
wide linkage study on retinal vessel calibres, which found multiple gene loci in regions associated
with hypertension and vasculogenesis.84 The heritability results were also in agreement with the
Beaver Dam Eye Study, which demonstrated familial aggregation of retinal vessel calibres.93 The
Beaver Dam Eye Study and the present twin study provide the first evidence that retinal
microvascular status may have genetic determinants. The interdependence of arterial diameter and
arterial blood pressure is also evidence that they are strongly related, ie. retinal arterial narrowing
may not only be a response to elevated blood pressure but also a cause of increased arterial blood
pressure, as suggested in studies on the association between retinal arterial narrowing and incident
hypertension.53, 54

      We were the first to use structural equation modelling to estimate heritability of retinal vessel
characteristics while correcting for age, gender, blood pressure, body mass index, blood lipids,
blood glucose, and smoking. Another strength which is important to emphasize is that the subjects




                                                - 37 -
were young, healthy and without cardiovascular disease, thus when analyzing genetic and
environmental factors, aging and disease processes did not cause bias.

      It should be considered that the unshared environmental component included random factors
and measurement errors, therefore future methods with higher accuracy and less intergrader
variability may result in higher heritabilities for the phenotypes in question.

      The present studies were not designed to examine specific environmental factors. However,
smoking tended to be associated with wider retinal arteries and veins, as found in previous
studies.49, 58 The lack of statistical significance in the present study was possibly due to the young
age and the good health of the study population. A follow-up study of the twin population would be
of great interest to elucidate the effect of smoking on retinal vessel calibres.

      Correction for refractive error did not attenuate the heritability results in any of the three twin
studies. This may be because refractive error is highly heritable94-96 and since we tested the
difference between two twins in a pair with largely the same refraction, it was not possible to assess
the full influence of refraction.

      The power of a twin study is dependent on many factors: the effect being considered (e.g.
additive genetic influence or common environment), the size of the effect in the true world, the
sample size of the study, the probability level chosen, the type of measurement - ordinal or
continuous, and the proportion of MZ-to-DZ twins in the sample.97, 98 The present twin study was
limited by time and money, but power analysis beforehand suggested that inclusion of 114 twin
pairs was sufficient for the analysis of retinal vessel calibres and lens fluorescence, which were the
primary studies. Ordinal data (e.g. cilioretinal arteries) are much less informative than continuous
data (e.g. vessel diameters), therefore more twin pairs are needed in studies of ordinal data to
achieve the power equal to studies of continuous data.97 This was demonstrated by the larger
confidence intervals in the heritability of cilioretinal arteries compared to the heritability of vessel
diameters. Additional value may derive from increasing participant numbers in future twin studies.

      Another limitation of the present studies may be that the fundus images were not assessed by
electrocardiographically synchronized fundus photography. Diameter changes of 3.4% for retinal
arteries and 4.8% for retinal veins during the cardiac cycle have been observed in normal subjects.82
This variation factor may have resulted in under estimation of heritability of retinal vessel
diameters. However, Knudtson et al found that the variation in retinal vessel diameter was too wide



                                                  - 38 -
across images taken at the same point in the pulse cycle to detect differences across the pulse cycle.
The variation across images were suggested to be caused by short time variation in the patients
vessel calibres, inter and intragrader variability, different image quality, different positions of the
eye in front of the camera, and capture of images in different angles.99 Using
electrocardiographically synchronized fundus photography may eliminate one source of variation,
but improvement of the quality of fundus photography to reduce the variability across images is
also very important in future studies of retinal vessel calibres.

      In fundus images the vessel wall and the sleeve of plasma surrounding the column of blood
cells is transparent, therefore it was the column of formed blood cells rather than the actual internal
diameter of the vessel lumen that was measured in this thesis. In fluorescein angiography, the
fluorescein mixes with the plasma and the full lumen is visualized, but automatic measurements of
retinal vessel diameters in digitized red-free and fluorescein images were not significantly different
in a study describing a recently developed automatic vessel measurement method.32

      Retinal vascular changes, such as arterial narrowing and venous dilation, are associated with
diabetic retinopathy,57 arterial hypertension,41 coronary heart disease,67 stroke,65 and glaucoma.100
Increased retinal arterial tortuosity has been demonstrated to be associated with hypertension and
death from ischemic heart disease.101 Given these associations, understanding the genetic and
environmental determinants of retinal vessel layout may provide additional insights into the
pathogenesis of these complex diseases. Previous epidemiologic studies have focused mostly on the
association between retinal vessel characteristics and systemic risk factors. But, these associations
explain only a small part of the variation in retinal vasculature. In this thesis we have now
demonstrated that the remainder part of the variation in retinal vessel diameter and arterial
tortuosity may be largely due to additive genetic factors, accounting for 70-85% of the variation.

   Prospective cohort studies have demonstrated that narrow retinal arteries precedes the
development of hypertension.54,      102
                                           The present thesis suggests that the development of
hypertension may partly be caused by genetically determined arterial narrowing. Consequently, it
should be possible in principle to screen for retinal arterial narrowing early in life and thus to point
out individuals who are susceptible to develop hypertension and follow those closely, with the
clinical purpose of preventing development of hypertension. The potential utility of fundus
photography screening for cardiovascular risk remains to be examined in a prospective study.




                                                  - 39 -
    4.5.    Conclusions
In summary, the original studies included in this thesis have provided new knowledge of the
influence of genetic, environmental, and systemic factors on the retinal vessels in a population of
healthy young to middle-aged twins. This thesis demonstrated that genetic factors influence the
variation in retinal arterial tortuosity, retinal vessel calibre, and the presence of cilioretinal arteries.
Narrow retinal veins were associated with increasing age, and narrow retinal arteries were
associated with increasing age and mean arterial blood pressure. There was evidence of a weak
genetic association between retinal artery calibre and blood pressure, indicating that a genetic and
mechanistic link may be present between the two. The data from these studies suggest that a single
fundus photograph cannot yet be used in the evaluation of an individual patients’ risk of
cardiovascular disease because individual predictive values of risk estimates for cardiovascular
disease have yet to be defined. Longitudinal studies based on repeated fundus photographs over
many years, are likely to be more sensitive than cross sectional population studies in showing
effects of systemic health parameters on vascular morphology.




                                                   - 40 -
5. Summary in English

The clinical signs of many systemic vascular diseases, such as hypertension and diabetes mellitus,
are reflected in the fundus of the eye. Vascular abnormalities are easily observed by
ophthalmoscopy and the eye is like a window to the systemic vasculature. Large epidemiologic
studies have demonstrated the association between retinal vessel abnormalities and hypertension,
cardiovascular disease, diabetes mellitus, stroke and ocular diseases. These associations explain
only a small part of the variation in retinal blood vessels. Given, the association between retinal
vessel morphology and complex systemic diseases, understanding the genetic and environmental
determinants of vessel morphology may provide additional insights into the aetiology and
pathogenesis of these complex diseases. The classic twin study is superb at finding out if a trait is
genetically influenced – if it can demonstrate that monozygotic (MZ) twins are more similar for the
trait than dizygotic (DZ) twins, especially when using structural equation modelling. We wanted to
examine how much of the variation in retinal vessel morphology was explained by genetic and
environmental factors in a Danish twin population.

   The thesis is based on four papers, of which one is a review of twin studies in ophthalmology
and the other three cover heritability analyses of retinal blood vessel calibres, retinal arterial
tortuosity and the presence of cilioretinal arteries in digital red-free fundus images from 58 MZ and
54 DZ same-sex healthy normotensive twin pairs aged 20 to 46 years. This thesis demonstrated that
genetic factors influenced the variation in retinal arterial tortuosity, retinal vessel calibre, and the
presence of cilioretinal arteries. Straight arteries were associated with increasing body mass index
and mean arterial blood pressure. Narrow retinal veins were associated with increasing age, and
narrow retinal arteries were associated with increasing age and mean arterial blood pressure. There
was evidence of a weak genetic association between retinal artery calibre and blood pressure,
indicating that a genetic and mechanistic link may be present between the two. The data from these
studies suggest that a single fundus photograph cannot yet be used in the evaluation of an individual
patients’ risk of cardiovascular disease because individual predictive values of risk estimates for
cardiovascular disease have yet to be defined. Longitudinal studies based on repeated fundus
photographs over many years, are likely to be more sensitive than cross sectional population studies
in showing effects of systemic health parameters on vascular morphology.




                                                 - 41 -
6. Summary in Danish

I øjets nethinde kan man observere kliniske tegn på systemiske sygdomme, som for eksempel
forhøjet blodtryk og diabetes mellitus ved hjælp af oftalmoskop. Øjet kan betragtes som et vindue
ind til de systemiske kar, da mange abnormiteter i systemiske kar reflekteres i nethindens kar. Store
epidemiologiske studier har vist sammenhænge mellem nethinde arterie forsnævring/vene dilatation
og forhøjet blodtryk, hjertesygdom, diabetes mellitus og slagtilfælde. Sammenhæng mellem arterie
tortuositet i nethinden og forhøjet blodtryk og død pga. iskæmisk hjertesygdom er også blevet
påvist. Disse sammenhænge forklarer dog kun en brøkdel af variationen i nethindens kar. Vi
ønskede at finde ud af hvor meget af variationen som skyldtes genetiske og miljømæssige faktorer,
for at få en dybere forståelse af patogenesen til disse komplekse vaskulære sygdomme. Klassiske
tvillingstudier er fremragende til at finde ud af om en fænotype eller en sygdom er genetisk bestemt,
hvis de kan demonstrere at monozygote (MZ) tvillinger er signifikant mere ens end dizygote (DZ)
tvillinger, specielt ved brug af moderne tvillingstatistiske analysemetoder. Vi ønskede at undersøge
hvor stor del af variationen i tre forskellige typer af karmorfologi, som skyldtes genetiske og
miljømæssige faktorer i en dansk tvillingpopulation.

      Afhandlingen er baseret på fire artikler, hvoraf de tre af dem er arveligheds analyser af
henholdsvis kardiameter, arterie tortuositet og tilstedeværelse og placering af cilioretinale arterier.
Disse karakteristika er undersøgt på digitale fundus fotografier fra 58 MZ og 54 DZ raske,
normotensive tvillingepar af samme køn i alderen 20-46 år. Studierne viste at variationen i
kardiameter, arterie tortuositet og tilstedeværelse og placering af cilioretinale arterier var
hovedsagelig bestemt af genetiske faktorer i denne population. Desuden var mindre arterie diameter
associeret med stigende blodtryk og stigende alder og mindre vene diameter var associeret med
stigende alder. Nedsat tortuositet var associeret med øget middel arterie blodtryk og body mass
index.

      Det er ikke endnu muligt at forudsige noget om en individuel patients risiko for udvikling af
hjertekarsygdom baseret på karmål fra et enkelt fundus fotografi, fordi individuelle prædiktive
værdier og risk estimater for hjertekarsygdom mangler at blive defineret i større epidemiologiske
studier. Longitudinelle studier baseret på gentagne fundus fotografier over mange år, er muligvis
mere sensitive end tværsnits studier som dette, til at vise effekten af systemiske parametre på
nethindens kar morfologi.




                                                - 42 -
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                                              - 50 -
Papers 1 – 4


Paper 1: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel,
Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen, Heritability of Cilioretinal
Arteries: A Twin Study. Invest Ophthalmol Vis Sci, 2005, Vol. 46, p. 3850-3854.



Paper 2: Nina C.B.B. Taarnhøj, Michael Larsen, Birgit Sander, Kirsten Kyvik, Line Kessel, Jesper
L. Hougaard, and Thorkild I.A. Sørensen, Heritability of Retinal Vessel Diameters and Blood
Pressure: A Twin Study. Invest Ophthalmol Vis Sci, 2006, Vol. 47, p. 3539-3544.



Paper 3: Nina C.B.B. Taarnhøj, Inger C. Munch, Kirsten Kyvik, Birgit Sander, Line Kessel,
Thorkild I.A. Sørensen, Jesper L. Hougaard, and Michael Larsen. Straight Versus Tortuous Retinal
Arteries in Relation to Blood Pressure and Genetics. (Accepted for publication in British Journal of
Ophthalmology)




Paper 4: Nina C.B.B. Taarnhøj and Line Kessel. Twin Studies in Ophthalmology. (In review)




                                               - 51 -
Heritability of Cilioretinal Arteries: A Twin Study
Nina C. B. B. Taarnhøj,1 Inger C. Munch,1 Kirsten O. Kyvik,2 Birgit Sander,1 Line Kessel,1
Thorkild I. A. Sørensen,3 Jesper L. Hougaard,1 and Michael Larsen1

PURPOSE. To determine whether the presence of one or more                  tral retinal artery is assisted by one or more cilioretinal arteries.
cilioretinal arteries, a distinct element of the pattern of fundus         These arteries are derived directly from the circle of Zinn,
vessels, is genetically programmed, influenced by environmen-               which is formed by small branches from the short posterior
tal factors, or the result of random mechanisms of vascular                ciliary arteries, which also supply the choroid. It is of clinical
development.                                                               relevance that a temporal cilioretinal artery supplying the fovea
METHODS. The fundi of 112 pairs of healthy monozygotic and                 may spare the fovea in case of central retinal artery occlusion.1
dizygotic twins were examined using digital fundus photogra-               Furthermore, patients with advanced high-tension open-angle
phy and visual assessment of grayscale fundus photographs and              glaucoma have better preservation of the central visual field
color transparencies to detect the presence of cilioretinal ar-            and visual acuity in the presence of a temporal cilioretinal
teries.                                                                    artery, presumably because it is associated with a better blood
                                                                           supply to the prelaminar portion of the optic disc.2 In addition,
RESULTS. Cilioretinal arteries were present in 45.1% of partici-           the vascular layout of the retina may influence the pattern of
pants and 28.8% of eyes. The majority of cilioretinal arteries,            distribution of diabetic retinopathy and branch retinal vein
88.2%, were located temporally, and 11.8% were located na-                 occlusion.3
sally. Monozygotic twins had higher concordance rates for                      The prevalence of one or more cilioretinal arteries has
cilioretinal arteries than dizygotic twins. Tetrachoric correla-           previously been reported to be 49.5% of individuals and 32.1%
tions and Mantel-Haenszel odds ratios demonstrated statisti-               of eyes, based on a review of stereo fundus photographs and
cally significant evidence of a genetic effect underlying the               fluorescein angiograms from 1000 healthy persons.4 Using di-
presence of cilioretinal arteries (P     0.01). Statistical analysis       rect ophthalmoscopy, another study found a cilioretinal artery
supported the hypothesis that additive genetic factors influ-               prevalence of 26% of individuals in a population of 172 healthy
enced the presence of cilioretinal arteries with a heritability of         persons.5 In the present study, we examined fundus photo-
71.4%, the remaining variance being attributable to nonshared              graphs of 112 pairs of healthy twins (224 persons, 448 eyes) to
or random environmental factors.                                           assess the relative influence of genetic and environmental fac-
CONCLUSIONS. The presence or absence of one or more cilioreti-             tors on the presence of cilioretinal arteries.
nal arteries in healthy persons is markedly influenced by ge-
netic factors. (Invest Ophthalmol Vis Sci. 2005;46:3850 –3854)
DOI:10.1167/iovs.05-0177                                                   METHODS

S   ome vascular diseases in humans appear to be linked to the
    pattern of distribution of retinal blood vessels. Knowing
whether a variation in the distribution of retinal vessels is
                                                                           Subjects and Protocol
                                                                           We examined 59 monozygotic (MZ) and 55 dizygotic (DZ) same-sex
                                                                           twin pairs, aged 20 to 46 years. The persons were recruited from a
attributable to genetic or environmental factors may facilitate
                                                                           population-based register comprising twins born in Denmark between
elucidation of the pathogenesis of disease in the human retina.
                                                                           1870 and 1996 (The Danish Twin Registry, University of Southern
Consequently, retinal vascular patterns and the mechanisms
                                                                           Denmark, Odense, Denmark).6 Only subjects in self-assessed good
whereby they are laid out are of fundamental interest for
                                                                           health were invited to participate. Exclusion criteria for twin pairs
clinical ophthalmology. In primates, including humans, the
                                                                           were unclear refractive media, manifest eye disease, or fundus photo-
inner retina is supplied primarily or exclusively by the central
                                                                           graphs of unacceptable quality in either member of the pair. One MZ
retinal branch of the ophthalmic artery. Occasionally the cen-
                                                                           and one DZ twin pair did not want to have their fundus photographs
                                                                           taken. A total of 58 MZ and 54 DZ twin pairs were included in the
                                                                           present study, and all fundus photographs were of good quality. Zy-
     From the 1Department of Ophthalmology, Herlev Hospital, Uni-          gosity was determined by means of genetic markers, using nine mic-
versity of Copenhagen, Copenhagen, Denmark; the 2Danish Twin               rosatellite and restriction fragment length polymorphism markers. The
Registry, University of Southern Denmark, Odense, Denmark; and the         study was approved by the Medical Ethics Committee of Copenhagen
3
  Danish Epidemiology Science Center, Institute of Preventive Medi-        County and followed the tenets of the Helsinki Declaration, including
cine, Copenhagen University Hospital, Copenhagen, Denmark.                 informed consent. All persons underwent an ophthalmological exam-
     Supported by the Danish Eye Health Society; The Danish Associ-
                                                                           ination including refraction, visual acuity determination, slit-lamp
ation of the Blind; Center for Biomedical Optics and New Laser
Systems (BIOP) Graduate School; The Danish Research Agency; and            biomicroscopy, and fundus photography after pupil dilation (using
a Research Career Award (Grant 8 –2002-130) from the Juvenile Dia-         10% phenylephrine hydrochloride and 1% tropicamide) and a clinical
betes Research Foundation (ML).                                            examination including an oral glucose tolerance test and blood pres-
     Submitted for publication February 10, 2005; revised May 27,          sure measurement. Digital grayscale fundus photographs (20° and 50°,
2005; accepted August 19, 2005.                                            1024      1024 pixels) were recorded in red-free illumination (filter:
     Disclosure: N.C.B.B. Taarnhøj, None; I.C. Munch, None; K.O.           Wratten 54; Eastman Kodak, Inc., Rochester, NY) using a retinal cam-
Kyvik, None; B. Sander, None; L. Kessel, None; T.I.A. Sørensen,            era (model TRC-50X; Topcon Corp., Tokyo, Japan). The study design
None; J.L. Hougaard, None; M. Larsen, None                                 and photography protocol were as described by Kessel et al.7 and
     The publication costs of this article were defrayed in part by page
                                                                           Hougaard et al.,8 who conducted studies on the heritability of lens
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.     aging (autofluorescence) and retinal nerve fiber layer thickness in the
     Corresponding author: Nina C. B. B. Taarnhøj, Department of           present twin population.7,8
Ophthalmology, Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev,             Two independent observers evaluated the digital red-free grayscale
Denmark; ninat@dadlnet.dk.                                                 fundus photographs to assess the presence, number, and location of

                                                                            Investigative Ophthalmology & Visual Science, October 2005, Vol. 46, No. 10
3850                                                                        Copyright © Association for Research in Vision and Ophthalmology
IOVS, October 2005, Vol. 46, No. 10                                                                  Heritability of Cilioretinal Arteries           3851

                       TABLE 1. Clinical Characteristics and Cilioretinal Artery Phenotype of Study Participants

                                                                                      MZ Twins            DZ Twins
                               Characteristic or Phenotype                            (n 116)             (n 108)             P-Value

                       Pairs                                                               58                  54
                       Subjects                                                           116                 108
                       Eyes                                                               232                 216
                       Female/male subjects                                             62/54               64/44
                       Age* (years)                                                  34.9 7.5             35.0 7.2             0.93†
                       Arterial blood pressure* (mm Hg)                              85.3 10.0            86.3 8.6             0.42†
                       Cilioretinal artery phenotype
                         Any eye (both eyes one eye only)                                    52                  49            0.94‡
                         Both eyes                                                           15                  13            0.84‡
                         One eye only (left eye only right eye only)                         37                  36            0.82‡
                         Right eye only                                                      19                  21            0.55‡
                         Left eye only                                                       18                  15            0.73‡
                       Proportion of eyes with cilioretinal arteries, any
                            location                                                    67/232              62/216             0.97‡

                           * Values are means SD.
                           † Two-tailed t-test.
                           ‡ Hypothesis test of proportions.


cilioretinal arteries. A cilioretinal artery was defined as a retinal arterial           Tetrachoric correlations, as well as estimation of heritability and
branch noncontiguous with the central retinal artery, coursing through              best-fitting etiological model by means of structural equation mod-
a near-180° hook as it emerges from underneath the retinal pigment                  eling, were carried out using computer software (MX; Michael C.
epithelium at the rim of the optic disc. The location of a cilioretinal             Neale, Department of Psychiatry, Virginia Commonwealth Univer-
artery was classified as temporal or nasal with respect to the center of             sity; available at www.vcu.edu/mx/).13 These calculations are based
the optic disc. Discrepant classifications underwent independent arbi-               on a liability model, which assumes that the dichotomous distribu-
tration by a third investigator, who decided the classification, using               tion of cilioretinal arteries (present versus not present) reflects an
color transparencies when needed.                                                   underlying normally distributed liability of the population. When a
                                                                                    threshold value of the liability is exceeded, an individual is affected,
Statistics                                                                          otherwise not. The threshold reflects the prevalence of the trait.
The differences in sex, age, and blood pressure in persons with one                 These are standard assumptions in quantitative genetic analysis of
or more cilioretinal arteries compared to persons with no cilioreti-                categorical traits.14 The classical twin model is based on the assump-
nal arteries were assessed by 2 tests and two-sided t-tests. We                     tion that MZ twin pairs share all their genes, while DZ twin pairs,
performed a test of proportions to compare the phenotype propor-                    like other siblings, share on average 50% of their genes, and that
tions for MZ and DZ twin pairs. Test significance was evaluated at                   both MZ and DZ twin pairs share a common environment to the
the 5% level.                                                                       same extent. This means that a greater similarity among MZ than
    We compared MZ and DZ twin pairs by means of probandwise                        among DZ twin partners for a certain phenotype can be due only to
concordance rates, Mantel-Haenszel odds ratios, and tetrachoric corre-              genetic factors.15 Structural equation modeling quantifies sources of
lations, as described elsewhere.9 –11 Probandwise concordance rates                 individual variation by decomposing the observed phenotypic vari-
estimate the risk of having a certain phenotype, given that one’s twin              ance into genetic and environmental variance. The genetic contri-
partner has this phenotype. The odds ratio estimates the increased risk             bution can be further divided into an additive (A) genetic variance
of having a certain phenotype given that one’s twin partner has the                 component (representing the influence of alleles at multiple loci
phenotype, compared to the risk if one’s twin partner does not have                 acting in an additive manner) and a nonadditive (D) genetic vari-
the phenotype. The Mantel-Haenszel weighted odds ratio tests the                    ance component (representing intralocus interaction [dominance]
difference between the odds ratios for MZ pairs and DZ pairs, using                 and interlocus interaction [epistatis] of alleles). The environmental
computer software (Epi Info; available at http://www.cdc.gov/epi-                   component is subdivided into a common (C) environmental vari-
info).12 A statistically significant Mantel-Haenszel test provides evi-              ance component (representing environmental factors affecting both
dence for a genetic influence on the phenotype.                                      twins in a pair, a source of similarity) and a nonshared or random


TABLE 2. Probandwise Concordance Rates for the Presence and Location of Cilioretinal Arteries in Study Participants*

                                                             MZ Twins                                                      DZ Twins

                                      Probands in         Probands in           Concordance          Probands in       Probands in         Concordance
                                      Concordant          Discordant                Rate             Concordant        Discordant              Rate
Cilioretinal Artery Location             Pairs               Pairs              [% (95% CI)]            Pairs             Pairs            [% (95% CI)]

           Right eye                        22                  12              64.7 (46.0–83.4)          16                 18           47.1 (26.3–67.8)
           Left eye                         18                  15              54.5 (34.1–75.0)          10                 18           35.7 (13.0–58.5)
           Any eye                          38                  14              73.1 (59.5–86.7)          28                 21           57.1 (40.6–73.7)
           One eye                          20                  17              54.1 (34.7–73.5)          14                 22           38.9 (18.7–59.1)
           Both eyes                         8                   7              53.3 (22.8–83.9)           4                  9           30.8 ( 1.9–63.4)

     * A proband is the person in a twin pair that has one or more cilioretinal arterioles. In concordant pairs both twins are affected; in discordant
pairs only one twin is affected. Concordance rate is calculated as the number of probands in concordant pairs divided by the sum of probands in
concordant and discordant pairs.
3852     Taarnhøj et al.                                                                                                                                                                                                                IOVS, October 2005, Vol. 46, No. 10




                                                                                                                                                                                                                                                         both eyes, subject has one or more CRAs in each eye; left eye/right eye, subject has one or more CRAs in the left eye/right eye (includes subjects with CRAs in the other eye also); left eye only/right
                                                                                                                                                                                                                                                             * Any eye, subject has one or more cilioretinal arteries (CRAs) in one or both eyes (i.e., is affected by any kind of CRA); any location, subject has a CRA at any location (nasal, temporal, or both);

                                                                                                                                                                                                                                                         eye only, discordant pairs— one twin has CRAs in the left eye/right eye and the other twin does not have CRAs, concordant pairs— both twins have CRAs in the left eye/right eye, and neither has


                                                                                                                                                                                                                                                             ‡ Test of the difference between odds ratios for MZ and DZ twin pairs, taking into consideration the number of pairs in each group. Significant results indicate greater similarity among MZ twins
(E) environmental variance component (representing environmen-




                                                                                                                                                                                                                                      0.001)
                                                                                                                                                                                                                                      0.001)

                                                                                                                                                                                                                                      0.001)

                                                                                                                                                                                                                                      0.001)
                                                                                                                                                                                                                                       0.01)



                                                                                                                                                                                                                                      0.01)
                                                                                                                                                                                                                       Weighted OR‡




                                                                                                                                                                                                                                      0.01)
tal factors not shared by twins, a source of dissimilarity). The latter




                                                                                                                                                                                                                         Haenszel
                                                                                                                                                                                                                         Mantel-
component (E) also includes random factors and measurement
errors.16 Heritability is defined as the proportion of the total phe-




                                                                                                                                                                                                                                                             † Calculated as the cross product of concordant pairs * nonaffected pairs divided by the cross product of discordant pairs (twin A is proband) * discordant pairs (twin B is proband).
                                                                                                                                                                                                                                       4.55 (P



                                                                                                                                                                                                                                      9.67 (P
                                                                                                                                                                                                                                      8.37 (P
                                                                                                                                                                                                                                      5.17 (P

                                                                                                                                                                                                                                      5.09 (P
                                                                                                                                                                                                                                       3.3 (P
                                                                                                                                                                                                                                      8.71 (P
notypic variance attributable to genetic variance. The components
C and D cannot be estimated simultaneously in a twin study. We
fitted the ACE and ADE models and the submodels AE, CE, and E to
the data. The criteria for best-fitting model were based on Akaike’s
information criterion (AIC), goodness-of-fit 2 test, degrees of free-




                                                                                                                                                                                                                                      4.3 (0.41–41.6)
                                                                                                                                                                                                                                      2.9 (0.7–12.4)

                                                                                                                                                                                                                                      2.8 (0.7–11.1)

                                                                                                                                                                                                                                      3.4 (0.3–29.9)
                                                                                                                                                                                                                                      7.5 (0.6–91.0)
dom, and P-value. The model with the lowest negative AIC reflects




                                                                                                                                                                                                                                      2.4 (0.7–8.5)

                                                                                                                                                                                                                                      1.9 (0.4–8.7)
the best balance between goodness of fit and parsimony.




                                                                                                                                                                                                                                 DZ
    The phenotype any eye means that a person has one or more
cilioretinal arteries in one eye or in two (both) eyes, meaning that a




                                                                                                                                                                                                       [OR (95% CI)]
                                                                                                                                                                                                        Odds Ratio†
person is affected by any kind of cilioretinal artery. The phenotype
both eyes denotes a person with one or more cilioretinal arteries in
each eye. Any location denotes the presence of a cilioretinal artery at




                                                                                                                                                                                                                                      15.7 (2.0–148.4)




                                                                                                                                                                                                                                      32.7 (3.1–474.3)
                                                                                                                                                                                                                                      12.0 (1.3–124.2)
any location—nasal, temporal, or both.




                                                                                                                                                                                                                                      10.0 (2.2–48.4)
                                                                                                                                                                                                                                       9.7 (2.5–39.8)
                                                                                                                                                                                                                                      11.0 (2.5–52.4)
                                                                                                                                                                                                                                       5.5 (1.3–23.4)
    In this study, ascertainment is independent of affection status in




                                                                                                                                                                                                                                 MZ
that all twin pairs were enrolled in the study and then examined to
asses the prevalence of cilioretinal arteries. A proband is the affected
part in a twin pair. In discordant twin pairs there is one proband, and
in concordant twin pairs there are two probands.




                                                                                                                                                                                                                                      0.18–0.83)
                                                                                                                                                                                                                                      0.07–0.72)
                                                                                                                                                                                                                                      0.08–0.67)
                                                                                                                                                                                                                                      0.07–0.71)
                                                                                                                                                                                                                                      0.24–0.63)
                                                                                                                                                                                                                                      0.23–0.79)
                                                                                                                                                                                                                                      0.05–0.90)
RESULTS
                                                                           TABLE 3. Tetrachoric Correlations and Zygosity-Specific Mantel-Haenszel Odds Ratios for the Cilioretinal Artery Phenotypes




                                                                                                                                                                                                                                 DZ
The per-person prevalence of cilioretinal arteries, i.e., the

                                                                                                                                                                                                       Tetrachoric Correlation
presence of one or more cilioretinal arteries in either of a




                                                                                                                                                                                                                                      0.43 (
                                                                                                                                                                                                                                      0.38 (
                                                                                                                                                                                                                                      0.33 (
                                                                                                                                                                                                                                      0.37 (
                                                                                                                                                                                                                                      0.23 (
                                                                                                                                                                                                                                      0.37 (
                                                                                                                                                                                                                                      0.56 (
subject’s two eyes, was 45.1% (95% confidence interval [CI]:
38.6%–51.6%), 101 participants in a study population of 224.                                                                                                                                                [TC (95% CI)]
In MZ twins, the per-person prevalence of one or more
cilioretinal arteries was 44.8% (95% CI: 35.8%–53.9%), and
among DZ twins it was 45.4% (95% CI: 36.0%–54.8%). The
                                                                                                                                                                                                                                      0.74 (0.29–0.94)
                                                                                                                                                                                                                                      0.71 (0.36–0.90)
                                                                                                                                                                                                                                      0.72 (0.41–0.90)
                                                                                                                                                                                                                                      0.73 (0.39–0.91)
                                                                                                                                                                                                                                      0.57 (0.17–0.83)
                                                                                                                                                                                                                                      0.84 (0.44–0.98)
                                                                                                                                                                                                                                      0.68 (0.16–0.93)
per-eye prevalence of cilioretinal arteries, i.e., the presence
of one or more cilioretinal arteries in a given eye, was 28.8%
                                                                                                                                                                                                                                 MZ



(95% CI: 24.6%–33.0%; n        448) for the entire study popu-
lation, being 28.9% (95% CI: 23.0%–34.7%) in MZ twins and
28.7% (95% CI: 22.7%–34.7%) in DZ twins. Of the total




                                                                                                                                                                                                                                                         than DZ twins and therefore bolster evidence for genetic influence on the phenotype.
number of cilioretinal arteries, 88.2% (95% CI: 82.8%–93.7%)
were located temporally and 11.8% (95% CI: 6.3%–17.2%)
were located nasally. The per-person prevalence of cilioreti-
                                                                                                                                                                                                                                 DZ
                                                                                                                                                                                                       Nonaffected




                                                                                                                                                                                                                                      43
                                                                                                                                                                                                                                      30
                                                                                                                                                                                                                                      19
                                                                                                                                                                                                                                      28
                                                                                                                                                                                                                                      31
                                                                                                                                                                                                                                      42
                                                                                                                                                                                                                                      45


nal arteries was 49.0% (95% CI: 39.1%–58.9%) in men and
                                                                                                                                                                                                          Pairs




42.1% (95% CI: 33.4%–50.7%) in women. There was no
impact of sex ( 2, P 0.30), age (t-test, P 0.29), or mean
                                                                                                                                                                                                                                 MZ




arterial blood pressure (P         0.76) on the presence of
                                                                                                                                                                                                                                      47
                                                                                                                                                                                                                                      36
                                                                                                                                                                                                                                      25
                                                                                                                                                                                                                                      35
                                                                                                                                                                                                                                      34
                                                                                                                                                                                                                                      49
                                                                                                                                                                                                                                      48




cilioretinal arteries. Clinical characteristics and cilioretinal
phenotypes of the participants are described in Table 1;
there was no significant difference in the proportions (prev-
                                                                                                                                                                                                                                 DZ

                                                                                                                                                                                                                                       9
                                                                                                                                                                                                                                      17
                                                                                                                                                                                                                                      21
                                                                                                                                                                                                                                      18
                                                                                                                                                                                                                                      18
                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                       7
                                                                                                                                                                                                       Discordant




alence) of phenotypes between MZ and DZ twins.
    Probandwise concordance rates for cilioretinal artery phe-
                                                                                                                                                                                                          Pairs




notypes were consistently higher in MZ twins than in DZ twins
for all phenotypes, but the difference did not reach statistical
                                                                                                                                                                                                                                 MZ

                                                                                                                                                                                                                                       7
                                                                                                                                                                                                                                      12
                                                                                                                                                                                                                                      14
                                                                                                                                                                                                                                      12
                                                                                                                                                                                                                                      15
                                                                                                                                                                                                                                       5
                                                                                                                                                                                                                                       7




significance for any single phenotype (Table 2).
    Odds ratios were significantly higher for MZ twins than for
DZ twins for all phenotypes when using the Mantel-Haenszel
weighted odds ratio test for zygosity (Table 3). The odds ratio
                                                                                                                                                                                                                                 DZ

                                                                                                                                                                                                                                       2
                                                                                                                                                                                                                                       7
                                                                                                                                                                                                                                      14
                                                                                                                                                                                                                                       8
                                                                                                                                                                                                                                       5
                                                                                                                                                                                                                                       2
                                                                                                                                                                                                                                       2
                                                                                                                                                                                                       Concordant




results agreed with the tetrachoric correlations, where there
                                                                                                                                                                                                          Pairs




was a significantly higher correlation in MZ than in DZ twins
for all phenotypes (Table 3).
                                                                                                                                                                                                                                 MZ

                                                                                                                                                                                                                                       4
                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                      19
                                                                                                                                                                                                                                      11
                                                                                                                                                                                                                                       9
                                                                                                                                                                                                                                       4
                                                                                                                                                                                                                                       3




    Tetrachoric correlations and structural equation model-
                                                                                                                                                                                                                                                         CRAs in the other eye.




ing of the phenotypes both eyes (Table 4), one eye only, any
eye, right eye, right eye only, left eye, and left eye only
(Supplementary Tables, http://www.iovs.org/cgi/content/
                                                                                                                                                                                                                                      Right eye only
                                                                                                                                                                                                                       Artery, Any
                                                                                                                                                                                                                       Cilioretinal




                                                                                                                                                                                                                                      One eye only




full/46/10/3850/DC1) support that an AE model comprising
                                                                                                                                                                                                                                      Left eye only
                                                                                                                                                                                                                        Location*




additive genetic effects and random environmental effects
                                                                                                                                                                                                                                      Both eyes


                                                                                                                                                                                                                                      Right eye
                                                                                                                                                                                                                                      Any eye

                                                                                                                                                                                                                                      Left eye




was the best-fitting liability model and most aptly describes
all phenotypes. In both eyes, one eye only, any eye, left eye
only, and right eye, 71% to 74% of the phenotypic variance
IOVS, October 2005, Vol. 46, No. 10                                                         Heritability of Cilioretinal Arteries         3853

TABLE 4. Result of Structural Equation Modeling for Phenotype Both Eyes, Any Location
                          2
Model*       AIC                  df      P-Value             A*†                     D*†                    C*†                    E*†

 ACE          5.7      0.29       3         0.96        0.61 (0.00–0.94)        0.12 (0.00–0.82)               0             0.26 (0.06–0.70)
 ADE          5.7      0.33       3         0.96        0.74 (0.00–0.94)                0                      0             0.26 (0.06–0.66)
 AE           7.7      0.33       4         0.99        0.74 (0.34–0.94)                0                      0             0.26 (0.06–0.66)
 CE           6.8      1.24       4         0.88                0                       0              0.61 (0.24–0.84)      0.38 (0.16–0.75)
 E            1.08    11.1        5         0.05                0                       0                      0                1 (1.00–1.00)

    Boldface type indicates best fitting model.
    * Model components: A, additive genetic factors; D, nonadditive genetic factors; C, shared environment, E, nonshared environment.
    † Proportion of the total variation in prevalence of cilioretinal artery attributable to the model component [% (95% CI)].



was due to additive genetic factors. In right eye only, the             few or no twins.4 This could be because the twins were
heritability was 84%, and in left eye it was 55%. This means            healthy and perhaps had almost normal birth weights.
that factors identified as influencing the presence or absence               In a phylogenetic perspective there is a systematic variation
of cilioretinal arteries include additive genetic factors (her-         between species, meaning that the layout of the retinal vessels
itability) and nonshared or random environmental factors                must be governed by genetics. We found a consistent trend
unique to the individual. By convention, the latter includes            toward MZ twin pairs having higher probandwise concordance
random variation. We found sex to be insignificant when                  rates, odds ratios, and tetrachoric correlations. Insofar as vas-
included in the heritability analysis.                                  cular diseases of the posterior pole of the eye are influenced by
                                                                        the layout of the vascular bed, the implication is that there
                                                                        must be an element of congenital hereditary disposition to
DISCUSSION                                                              such disease. All confidence intervals for tetrachoric correla-
                                                                        tions were wide, suggesting that a larger study may have
The present study demonstrated that within a population of              detected an even stronger genetic effect in all phenotypes. A
224 twins, the presence or absence of one or more cilioretinal          twin study of the present size does not have enough power to
arteries was mainly influenced by genetic factors in all pheno-          detect a common environmental variance component (C) or a
types. The average heritability of all phenotypes was 71.4%.            nonadditive genetic variance component (D), which is indi-
The relative influence of specific genes versus environmental             cated also by the wide 95% CI of A and E.
or random processes was not assessed, because this requires                In summary, structural equation modeling demonstrated a
candidate genetic or environmental factors. For cilioretinal            marked additive genetic effect (heritability of 71.4%) and a
arteries, nongenetic effects may include the effects of non-            random environmental effect (28.6%) on the layout of the
shared intrauterine environment as well as random mecha-                arterial blood supply of the retina, as represented by the pres-
nisms of morphogenesis and measurement errors. The patterns             ence or absence of cilioretinal arteries in a population of
of distribution of retinal vessels are laid out completely at           healthy twins. Understanding the genetic and environmental
full-term birth.                                                        influence on the architecture of retinal vessels may facilitate
    Some support for a potential impact of preterm health on            elucidation of the pathogenesis of vascular disease in the hu-
retinal vessel layout has been found in the observation that            man retina.
preterm birth with a median birth weight of 1250 g is associ-
ated with a greater length index for arterioles and fewer num-
bers of branching points compared with those of children born           References
full term. These vascular abnormalities persist into adult-
hood.17 The development of retinal vessels is driven by relative           1. Brown GC, Shields JA. Cilioretinal arteries and retinal arterial
                                                                              occlusion. Arch Ophthalmol. 1979;97:84 –92.
anoxia of the immature avascular retina. The ontogeny of the
                                                                           2. Lee SS, Schwartz B. Role of the temporal cilioretinal artery in
intraocular blood supply involves programmed elimination of
                                                                              retaining central visual field in open-angle glaucoma. Ophthalmol-
vessels, most notably the hyaloid artery. During these pro-                   ogy. 1992;99:696 – 699.
cesses of vessel formation, reorganization, and disappearance,             3. Christoffersen N, Larsen M. Unilateral diabetic macular oedema
cilioretinal arteries may develop, hypothetically, as an enlarge-             secondary to central retinal vein congestion. Acta Ophthalmol
ment of the anastomoses of the posterior ciliary arteries with                Scand. 2004;82:591–595.
small branches from the hyaloid artery on the disc.18 This                 4. Justice J, Lehmann RP. Cilioretinal arteries. A study based on
theory was supported by a small study of 15 children with birth               review of stereo fundus photographs and fluorescein angiographic
weights       2500 g whose prevalence of cilioretinal arteries,               findings. Arch Ophthalmol. 1976;94:1355–1358.
53.3% ( 2 5,7; 0,01 P 0,05), was greater than that of 370                  5. Lorentzen SE. Incidence of cilioretinal arteries. Acta Ophthalmol
children with normal birth weights (27.3%).19 Despite the risk                (Copenh). 1970;48:518 –524.
of uneven intrauterine nutrition in MZ twins, because of their             6. Kyvik KO, Christensen K, Skytthe A, Harvald B, Holm NV. The
shared placenta and the risk of anastomoses and transfusion                   Danish Twin Register. Dan Med Bull. 1996;43:467– 470.
syndrome, this did not have any detectable influence on the                 7. Kessel L, Hougaard JL, Sander B, Kyvik KO, Sørensen TIA, Larsen
prevalence of cilioretinal arteries in this study, as seen by the             M. Lens ageing as an indicator of tissue damage associated with
equal prevalence of cilioretinal arteries in MZ twins and DZ                  smoking and non-enzymatic glycation—a twin study. Diabetolo-
                                                                              gia. 2002;45:1457–1462.
twins. We did not have information about birth weights in the
                                                                           8. Hougaard JL, Kessel L, Sørensen TIA, Kyvik KO, Larsen M. Evalu-
study population, but twins are normally born three to four                   ation of heredity as a determinant of retinal nerve fiber layer
weeks before term and are on average 1000 g lighter than                      thickness as measured by optical coherence tomography. Invest
singletons. This did not appear to have any detectable influ-                  Ophthalmol Vis Sci. 2003;44:3011–3016.
ence on the development of cilioretinal arteries in this study,            9. Hopper JL. Twin concordance. In: Armitage P, Colton T, eds.
since the prevalence of cilioretinal arteries was found to be                 Encyclopedia of Biostatistics. Vol. 6. London: Wiley, 1998:4626 –
comparable to that in a previous study of a population with                   4629.
3854    Taarnhøj et al.                                                                             IOVS, October 2005, Vol. 46, No. 10

10. Kyvik KO, Green A, Beck-Nielsen H. Concordance rates of insulin   14. Falconer DS, Mackay TFC. Introduction to Quantitative Genetics.
    dependent diabetes mellitus: a population based study of young        4th ed. London: Longman Group, 1996.
    Danish twins. BMJ. 1995;311:913–917.                              15. Day INM, Humphries SE. Genetics of Common Diseases. Oxford,
11. Ramakrishnan V, Goldberg J, Henderson WG, et al. Elementary           UK: BIOS Scientific Publishers, 1997: 19 –32.
    methods for the analysis of dichotomous outcomes in unselected    16. Plomin R, DeFries JC, McClearn GE, Rutter M. Behavioral Genet-
    samples of twins. Genet Epidemiol. 1992;9:273–287.                    ics. 3rd ed. New York: WH Freeman, 1997: 305–310.
12. Epi Info Version 3.3 [computer program]. Division of Public       17. Kistner A, Jacobson L, Jacobson SH, Svensson E, Hellstrom A. Low
    Health Surveillance and Informatics, Epidemiology Program             gestational age associated with abnormal retinal vascularization
    Office, U.S. Department of Health and Human Services.                  and increased blood pressure in adult women. Pediatr Res. 2002;
    2004. Available at: http://www.cdc.gov/epiinfo. Accessed June         51:675– 680.
    2005.                                                             18. Hayreh SS. The cilio-retinal arteries. Br J Ophthalmol. 1963;47:71– 89.
13. Neale MC, Boker SM, Xle G, Maes HH. MX Statistical Modeling.      19. Erkkila H, Laatikainen L. Characteristics of optic disc in healthy
    6th ed. Richmond, VA: 2002.                                           school children. Acta Ophthalmol (Copenh). 1979;57:914 –921.
Heritability of Retinal Vessel Diameters and Blood
Pressure: A Twin Study
Nina C. B. B. Taarnhøj,1 Michael Larsen,1 Birgit Sander,1 Kirsten O. Kyvik,2 Line Kessel,1
Jesper L. Hougaard,1 and Thorkild I. A. Sørensen3

PURPOSE. To assess the relative influence of genetic and envi-               arteries (general arterial narrowing and AV-nicking) often pre-
ronmental effects on retinal vessel diameters and blood pres-               cede the development and progression of severe hyperten-
sure in healthy adults, as well as the possible genetic connec-             sion,4,10,11 suggesting that these changes are an element in the
tion between these two characteristics.                                     pathogenesis of arterial hypertension rather than a conse-
METHODS. In 55 monozygotic and 50 dizygotic same-sex                        quence of it. Analysis of retinal vessel diameters in relatives has
healthy twin pairs, aged 20 to 46 years, interpolated diameter              shown higher correlations than in the background popula-
estimates for the central retinal artery (CRAE), the central                tion.12 In the present study, we have assessed the relative
retinal vein (CRVE), and the artery-to-vein diameter ratio (AVR)            influence of genetic and environmental effects on retinal vessel
were assessed by analysis of digital gray-scale fundus photo-               diameters and blood pressure in healthy young adult twins, as
graphs of right eyes.                                                       well as the influence of sex, age, blood pressure, body mass
                                                                            index, fasting blood glucose, glucose tolerance, and smoking.
RESULTS. The heritability was 70% (95% CI: 54%– 80%) for
CRAE, 83% (95% CI: 73%– 89%) for CRVE, and 61% (95% CI:
44%–73%) for mean arterial blood pressure (MABP). Retinal                   METHODS
artery diameter decreased with increasing age and increasing
                                                                            Subjects and Protocol
arterial blood pressure. Mean vessel diameters in the popula-
tion were 165.8       14.9 m for CRAE, 246.2          17.7 m for            This was a cross-sectional study of 59 monozygotic (MZ) and 55 dizygotic
CRVE, and 0.67       0.05 m for AVR. No significant influence                 (DZ) same-sex twin pairs, aged 20 to 46 years. The participants were
on artery or vein diameters was found for gender, smoking,                  recruited from a population-based register comprising twins born in Den-
body mass index (BMI), total cholesterol, fasting blood glu-                mark between 1870 and 1996 (The Danish Twin Registry, University of
cose, or 2-hour oral glucose tolerance test values.                         Southern Denmark, Odense, Denmark).13 All subjects in self-assessed
                                                                            good health were invited to participate. To avoid ascertainment bias, no
CONCLUSIONS. In healthy young adults with normal blood pres-
                                                                            post hoc exclusion of subjects found to have hypertension or diabetes was
sure and blood glucose, variations in retinal blood vessel diam-
                                                                            permitted. Exclusion was made of all twin pairs where one or both twins
eters and blood pressure were predominantly attributable to
                                                                            were found to have unclear refractive media, manifest eye disease, or
genetic effects. A genetic influence may have a role in individ-
                                                                            fundus photographs of unacceptable quality. Nine twin pairs were ex-
ual susceptibility to hypertension and other vascular diseases.
                                                                            cluded because of missing or ungradeable fundus photographs. The re-
The results suggest that retinal vessel diameters and the possi-
                                                                            maining 55 MZ and 50 DZ twin pairs were included in the study.
ble associated variations in risk of vascular disease are primarily
                                                                                Zygosity was determined by means of genetic markers using nine
genetic characteristics. (Invest Ophthalmol Vis Sci. 2006;47:
                                                                            microsatellite and restriction fragment length polymorphism (RFLP)
3539 –3544) DOI:10.1167/iovs.05-1372
                                                                            markers. The study was approved by the Medical Ethics Committee of
                                                                            Copenhagen County and followed the tenets of the Declaration of
H     aving narrow arteries is strongly associated with past and
      present hypertension, independent of other risk fac-
tors.1– 6 Epidemiologic studies support that genetic predispo-
                                                                            Helsinki, including informed consent.
                                                                                All persons underwent an ophthalmic examination, including re-
                                                                            fraction, visual acuity determination, slit lamp biomicroscopy, and
sition plays a role in the development of hypertension, but the
                                                                            fundus photography after pupil dilation using phenylephrine hydro-
underlying mechanisms are largely unknown.7–9 Recent stud-
                                                                            chloride 10% and tropicamide 1%. Blood pressure, body mass index
ies have shown that structural abnormalities in small retinal
                                                                            (BMI; defined as weight in kilograms divided by height in square
                                                                            meters), fasting blood glucose, total cholesterol, family disposition for
                                                                            diabetes mellitus, and smoking history were obtained before an oral
     From the 1Department of Ophthalmology, Herlev Hospital, Uni-           glucose tolerance test (OGTT) using 75 g glucose after a 12-hour
versity of Copenhagen, Copenhagen, Denmark; the 2Danish Twin                overnight fast, assessing whole capillary blood glucose at 0, 30, and
Registry, University of Southern Denmark, Odense, Denmark; and the          120 minutes after ingestion.
3
  Danish Epidemiology Science Center, Institute of Preventive Medi-             The mean arterial blood pressure (MABP) was calculated as 33% of
cine, Copenhagen University Hospital, Copenhagen, Denmark.
                                                                            the difference between the systolic and the diastolic blood pressure
     Supported by The Danish Eye Health Society, Center for Biomed-
ical Optics and New Laser Systems Graduate School, The Danish               plus the diastolic blood pressure. Hypertension was defined as systolic
Research Agency, and a Research Career Award Grant 8-2002-130 from          blood pressure 140 or diastolic blood pressure 90, and/or current
the Juvenile Diabetes Research Foundation (ML).                             use of antihypertensive medication. Diabetes was defined according to
     Submitted for publication October 19, 2005; revised March 4,           the current World Health Organization (WHO) criteria (i.e., fasting
2006; accepted May 24, 2006.                                                whole capillary blood glucose concentration 6.1 mmol L 1 or
     Disclosure: N.C.B.B. Taarnhøj, None; M. Larsen, None; B.               2-hour OGTT whole capillary blood glucose concentration 11.1
Sander, None; K.O. Kyvik, None; L. Kessel, None; J.L. Hougaard,             mmol L 1. Impaired glucose tolerance was defined as fasting whole
None; T.I.A. Sørensen, None                                                 capillary blood glucose concentration 6.1 mmol L 1 and 2-hour
     The publication costs of this article were defrayed in part by page
                                                                            OGTT whole capillary blood glucose concentration             7.8 mmol
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.      L 1.14 Smoking was categorized as being a current smoker or a current
     Corresponding author: Nina C. B. B. Taarnhøj, Department of            nonsmoker.
Ophthalmology, Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev,              Digital gray-scale fundus photographs (50°, 1024 1024 pixels) cen-
Denmark; ninat@dadlnet.dk.                                                  tered on the macula and the optic disc were recorded in red-free illumi-

Investigative Ophthalmology & Visual Science, August 2006, Vol. 47, No. 8
Copyright © Association for Research in Vision and Ophthalmology                                                                              3539
3540     Taarnhøj et al.                                                                                            IOVS, August 2006, Vol. 47, No. 8

TABLE 1. Clinical Characteristics and Retinal Vessel Calibers in Right Eyes of 105 Healthy Twin Pairs

                                                                 Monozygotic                                    Dizygotic

                                                          Male                 Female                  Male                  Female                  P‡

Subjects                                                  52                     58                    42                      58
Smoker/non-smoker subjects                               16/36                  25/33                 22/20                   25/33
Age* (ys)                                              36.1 (7.4)             34.3 (7.7)            35.9 (7.6)              35.1 (6.2)              0.55
Mean arterial blood pressure* (mm Hg)                  86.3 (8.0)             82.9 (9.0)            88.1 (9.0)              84.4 (7.7)              0.02
BMI* (kg/m2)                                           23.9 (2.6)             22.7 (2.5)            23.9 (3.0)              23.7 (3.7)              0.11
Total cholesterol*† (mmol/L)                            5.4 (1.1)              5.0 (0.9)             5.8 (1.3)               5.4 (0.8)              0.003
Fasting blood glucose*† (mmol/L)                        5.1 (0.5)              4.7 (0.4)             5.0 (0.4)               4.8 (0.4)              0.0001
2-h OGTT*† (mmol/L)                                     6.0 (1.6)              6.2 (0.9)             5.7 (1.4)               6.5 (1.2)              0.01
Artery diameter*§ ( m)                                165.1 (15.2)           165.5 (16.6)          168.0 (12.4)            165.2 (14.7)             0.78
Vein diameter*§ ( m)                                  246.1 (19.7)           247.1 (19.4)          246.4 (15.6)            245.1 (15.7)             0.95
AVR*                                                   0.67 (0.04)            0.67 (0.05)           0.68 (0.04)             0.67 (0.05)             0.61

    * Data are the mean SD.
    † Whole capillary blood.
    ‡ The variances are the same in the four groups, using the Bartlett test of variance homogeneity. One-way ANOVA testing the difference
between mean values in all four groups.
    § Central retinal artery equivalent diameter and central retinal vein equivalent diameter.


nation (filter: Wratten 54; Eastman Kodak, Inc., Rochester, NY) using a           where W represents the width of the trunk vessel, and w1 and w2 the
retinal camera (TRC-50X; Topcon Corp., Tokyo, Japan). The study design           width of each branch. The AVR was defined as CRAE/CRVE. Eyes were
and photography protocol has been described in previous publications on          considered ungradeable if one of the six largest arteries or veins could
lens fluorescence and retinal nerve fiber layer thickness.15,16                    not be measured or if the image was of poor quality (low contrast), as
    Vessel calibers were assessed using a custom-developed semiauto-             judged by the grader with reference to a standard image of least
mated computer algorithm (Visiopharm A/S, Hørsholm, Denmark). A                  acceptable quality and contrast. Absolute distances were found assum-
grid was placed on the 50° digital image, and only the diameters of              ing a uniform vertical optic nerve head diameter of 1800 m.
retinal vessels crossing the circular zone from 0.5 to 1.0 disc diameters
from the margin of the optic disc were analyzed. The grader chose the            Statistics
segment of each vessel within the circular zone that was deemed most
                                                                                 Retinal vessel calibers and clinical characteristics were normally dis-
suitable for measurement, based on image quality, contrast, straight-
                                                                                 tributed. Retinal vessel data were analyzed as continuous variables.
ness of the vessel, absence of branching and vessel crossings, and, if
                                                                                 Intragrader reproducibility was assessed using Pearson correlation and
possible, measured the whole length of the vessel within the circular
                                                                                 mean        SD of the differences of two independent gradings of 59
zone. When bifurcating or branching was found within the zone of
                                                                                 fundus photographs. Differences, between the four groups, (MZ male,
interest, the trunk was preferred to the branches, unless the trunk was
                                                                                 MZ female, DZ male, and DZ female pairs) in age, MABP, BMI, total
shorter than 80 m. The program identified the six largest arteries and
                                                                                 cholesterol, fasting blood glucose, 2-hour OGTT, and retinal vessel
the six largest veins and calculated the central retinal artery equivalent
                                                                                 parameters were compared by one-way ANOVA, assuming P 0.05 to
(CRAE), the central retinal vein equivalent (CRVE), and the artery-to-
                                                                                 be indicative of statistical significance. The difference in variance in the
vein ratio (AVR), according to the formulas described by Knudtson et
                                                                                 four groups was tested with the Bartlett’s test of variance homogene-
al.17 Equivalent diameters were found by pairing the largest- and the
                                                                                 ity. Pearson correlations were estimated between retinal vessel calibers
smallest-caliber vessels within a given class and proceeding to pair the
                                                                                 and relevant clinical characteristics for all the twins in the study.
resultant virtual vessels until only one remained, irrespective of the
                                                                                      Six twin pairs had a difference in refraction between twins A and B
actual branching pattern on the optic disc. The formulas used for
                                                                                 of more than 4 D and were tentatively excluded from the heritability
calculating trunk diameter from two branch diameters pairing were
                                                                                 analysis. Because this did not change the results, the full data without
                                                                                 statistical correction for refraction have been reported throughout.
                               ˆ
                Artery (CRAE): WA      0.88    w1 2    w2 2                           We compared vessel calibers and MABP between twin one and
                                                                                 twin two for the four groups: MZ male, MZ female, DZ male, and DZ
and                                                                              female pairs by means of intraclass (Pearson) correlations. Associations
                                                                                 of covariates with vessel calibers and blood pressure as well as estima-
                             ˆ
                Vein (CRVE): WV       0.95    w1 2    w2 2 ,                     tion of heritability by means of univariate structural equation modeling


TABLE 2. Correlations between Retinal Vessel Calibers and Clinical Characteristics in 105 Healthy Twin Pairs

Clinical Characteristics                                  CRAE*†                                    CRVE*†                                     MABP*

  MABP                                                0.42 (P    0.001)                         0.20 (P     0.01)
  Age                                                 0.27 (P    0.001)                         0.21 (P     0.01)                         0.22 (P   0.01)
  BMI                                                 0.12 (P    0.1)                           0.14 (P     0.05)                         0.40 (P   0.001)
  Total cholesterol                                   0.23 (P    0.001)                         0.15 (P     0.05)                         0.26 (P   0.001)
  Fasting blood glucose                               0.04 (P    0.2)                           0.11 (P     0.2)                          0.15 (P   0.05)
  2-h OGTT                                            0.07 (P    0.2)                           0.01 (P     0.2)                          0.04 (P   0.2)

     * Pearson correlation coefficient and probabilities of all the twins in the study (n 210). Similar patterns of correlations are obtained if the
analyses are limited to one of the twins (twin 1 or twin 2 chosen randomly) or only MZ or DZ twins.
     † Central retinal artery equivalent diameter and central retinal vein equivalent diameter.
IOVS, August 2006, Vol. 47, No. 8                            Heritability of Retinal Vessel Diameters and Blood Pressure                  3541

was made using MX computer software.18,19 Because of the limited         structural equation modeling. Structural equation modeling quantifies
number of twin pairs we only analyzed two or three covariates at a       sources of individual variation by decomposing the observed pheno-
time. The classic twin model is based on the assumption that MZ twins    typic variance into genetic and environmental variance. The genetic
have identical genotypes, for which reason all observed differences      contribution can be further divided into an additive (A) genetic vari-
between the two twins in a pair are attributable to environmental        ance component (representing the influence of alleles at multiple loci
factors. DZ twins, however, share on average 50% of their genes. The     acting in an additive manner) and a nonadditive (D) genetic variance
extent to which MZ twins are more alike than DZ twins is therefore       component (representing intralocus interaction [dominance] and interlo-
assumed to reflect a genetic influence on the phenotype in question.       cus interaction [epistasis] of alleles). The environmental component can
Heritability is defined as the proportion of the total phenotypic vari-   be subdivided into a common (C) environmental variance component
ance that is attributable to genetic variance (heritability   genetic    (representing environmental factors affecting both twins in a pair, a
variance/total phenotypic variance)20 and is calculated by means of      source of similarity) and an unshared/random (E) environmental variance




FIGURE 1. Scatterplot by zygosity
for CRAE diameter and CRVE diameter
in healthy twins. The intraclass corre-
lation data are shown in Table 3.
3542       Taarnhøj et al.                                                                                 IOVS, August 2006, Vol. 47, No. 8

                    TABLE 3. Intraclass Correlation Values by Gender and Zygosity for Vessel Diameters and Blood Pressure
                    in 105 Healthy Twin Pairs

                                                      Monozygotic                                 Dizygotic

                                               Male                Female                Male                     Female

                    Subjects                    52                   58                    42                      58
                    MABP*                0.59 (0.26, 0.79)    0.68 (0.41, 0.84)     0.47 (0.05, 0.75)      0.40 (0.04, 0.67)
                    CRAE diameter†       0.80 (0.60, 0.91)    0.80 (0.62, 0.90)     0.12 ( 0.33, 0.53)     0.50 (0.16, 0.73)
                    CRVE diameter‡       0.87 (0.72, 0.94)    0.88 (0.75, 0.94)     0.13 ( 0.53, 0.32)     0.36 ( 0.01, 0.64)

                         Pearson correlation coefficient (r) (95% CI) between twin 1 and twin 2. See Figure 1 for scatterplots
                    of the intraclass correlations for MZ and DZ twins.
                         * rmz 0.64 (CI95 0.45, 0.77) and rdz 0.45 (CI95 0.20, 0.65).
                         † rmz 0.80 (CI95 0.68, 0.88) and rdz 0.37 (CI95 0.11, 0.59).
                         ‡ rmz 0.87 (CI95 0.79, 0.92) and rdz 0.14 (CI95 0.14, 0.40).


component (representing environmental factors not shared by twins, a        diabetes before the study. All subjects were included in the anal-
source of dissimilarity, including random factors and measurement er-       ysis. Intragrader reproducibility, defined as the mean SD of the
rors).21 We fitted the ADE and ACE models and the submodels AE, CE, and      difference between two independent gradings from 59 eyes, was
E to the data. The criterion for best-fitting model was based on Akaike’s    0.32 1.89 m for CRAE and 0.02 1.71 m for CRVE, the
information criterion (AIC). The model with the lowest AIC reflects the      Pearson correlation being 0.991 for CRAE and 0.995 for CRVE.
best balance between goodness-of-fit and parsimony.19                            The distribution of CRAE, CRVE, and MABP demonstrated
                                                                            higher correlation in MZ twins than in DZ twins (Fig. 1), as did
RESULTS                                                                     the intraclass (Pearson) correlations (Table 3), similar patterns
                                                                            of intraclass correlations are obtained when analyzed accord-
In the study population of 210 healthy subjects aged 20 to 46               ing to gender (Table 3). The intraclass correlation being ap-
years, mean CRAE was 165.8         14.9 m (range, 125.9 –208.0),            proximately two times higher for MZ twins than for DZ twins
mean CRVE was 246.2 17.7 m (range, 198.4 –298.3 m), and                     indicated that an AE model was likely the best fit by structural
mean AVR was 0.67          0.05 m (range, 0.56 – 0.79). All vessel          equation modeling for both arteries and veins, which was
parameters and clinical characteristics demonstrated a normal               supported by low AIC values. This means that observed inter-
distribution. MZ male and female twin pairs and DZ male and twin            individual differences in vessel calibers were best explained by
pairs were of comparable age, MABP, BMI, and retinal vessel                 the effect of additive genetic factors (A) and unshared environ-
calibers (Table 1). There was no difference in variance in MABP             mental factors (E). For CRAE, after correcting for MABP, age,
and vessel calibers in the four groups, using the Bartlett’s test of        and gender, additive genetic factors (i.e., heritability) ex-
variance (Table 1). A tendency was noted for smokers to have                plained 70% (95% CI: 54%– 80%, Table 4) of the total pheno-
wider retinal arteries (167.8 vs. 164.4 m; P 0.10) and wider                typic variance in retinal artery diameter, unshared environmen-
retinal veins (248.6 vs. 244.4 m, P 0.08, t-test) than nonsmok-             tal factors accounting for the remaining 30% (95% CI: 20%–
ers. Blood pressure increased with age by 2.4 mm Hg per decade              46%). The results were independent of whether systolic or
(P 0.005). There was a significant correlation between CRAE                  diastolic blood pressure or MABP was used. Correction for
and MABP, age, and total cholesterol; between CRVE and MABP,                covariates while ignoring the effect of MABP resulted in in-
age, BMI, and total cholesterol; and between MABP and age, BMI,             creasing values of heritability after correction for smoking
total cholesterol, and fasting blood glucose (Table 2). None of the         (76%; Table 5) and 2-hour OGTT (77%; Table 5). Age and MABP
participants had arterial hypertension. Sixteen patients were               influenced the heritability results significantly and were both
found to have impaired glucose tolerance, and five patients were             inversely correlated with artery diameter, MABP by 5.45
found to have diabetes mellitus,14 each of them fulfilling only one            m/10 mm Hg (95% CI: 7.60 to 3.29) and age by 4.36
criterion for diabetes and none of them having had symptoms of                m/10 y (95% CI: 7.48 to 1.24). Gender, smoking, BMI,

TABLE 4. Model-Fitting Analyses of Retinal Vessel Diameters Corrected for MABP, Gender and Age in 55 Monozygotic and 50 Dizygotic Twin Pairs

                       Genetic Components                       Environmental Components                          Goodness-of-Fit Tests

                                                                                                            2
 Model                 A                      D                     C                    E                          df          P           AIC

Artery
  1. ADE       0.50 (0.00–0.80)       0.20 (0.00–0.79)                            0.30 (0.20–0.45)
  2. AE        0.70 (0.54–0.80)                                                   0.30 (0.20–0.46)        0.139     1         0.710         1.861
  3. E                                                                            1.00 (1.00–1.00)       42.664     2         0.000        38.664
  4. ACE        0.70 (0.28–0.80)                             0.00 (0.00–0.37)     0.30 (0.20–0.46)        0.139     0      incalculable     0.139
  5. CE                                                      0.53 (0.37–0.65)     0.47 (0.35–0.63)        9.751     1         0.002         7.751
Vein
  6. ADE       0.00 (0.00–0.83)       0.83 (0.00–0.88)                            0.17 (0.12–0.26)
  7. AE        0.83 (0.73–0.89)                                                   0.17 (0.11–0.27)        3.804     1         0.051         1.804
  8. E                                                                            1.00 (1.00–1.00)       72.507     2         0.000        68.507
  9. ACE        0.83 (0.63–0.89)                             0.00 (0.00–0.18)     0.17 (0.11–0.27)        3.804     0      incalculable     3.804
  10. CE                                                     0.56 (0.42–0.68)     0.44 (0.32–0.58)       33.269     1         0.000        31.269

     Boldface type indicates best fitting model. Data are the proportion of total variation attributable to model component (CI95 %). AIC, Akaike’s
information criterion.
IOVS, August 2006, Vol. 47, No. 8                             Heritability of Retinal Vessel Diameters and Blood Pressure                      3543

                   TABLE 5. Model-Fitting Analyses of Central Retinal Artery Diameter Corrected for Covariates in 55
                   Monozygotic and 50 Dizygotic Twin Pairs

                            Covariates                           A                         E                 AIC             P

                   Age and MABP                        0.70    (0.56–0.80)        0.30   (0.20–0.44)         1.946         0.816
                   Age, MABP, and gender               0.70    (0.54–0.80)        0.30   (0.20–0.46)         2.000         0.994
                   MABP, smoking, and age              0.70    (0.56–0.80)        0.30   (0.20–0.44)         2.000         0.995
                   MABP and gender                     0.71    (0.56–0.81)        0.29   (0.19–0.44)         1.967         0.856
                   MABP and smoking                    0.72    (0.57–0.81)        0.28   (0.19–0.43)         1.995         0.945
                   MABP, 2-h OGTT, and age             0.71    (0.56–0.80)        0.29   (0.20–0.44)         2.000         0.994
                   Age and smoking                     0.73    (0.60–0.82)        0.27   (0.18–0.40)         2.000
                   Age, smoking, and gender            0.73    (0.60–0.82)        0.27   (0.18–0.40)         2.000
                   Age and gender                      0.74    (0.62–0.83)        0.26   (0.17–0.38)         1.972         0.868
                   Age, gender, and cholesterol        0.74    (0.60–0.83)        0.26   (0.17–0.40)         2.000         0.997
                   Age and 2-h OGTT                    0.75    (0.62–0.83)        0.25   (0.17–0.38)         1.993         0.934
                   Age, 2-h OGTT, gender               0.75    (0.62–0.83)        0.25   (0.17–0.38)         2.000         0.998
                   Gender and smoking                  0.76    (0.64–0.84)        0.24   (0.16–0.36)         2.000
                   Gender and 2-h OGTT                 0.77    (0.65–0.84)        0.23   (0.16–0.35)         2.000

                        Data are the proportion of total variation attributable to model component (CI95 %). AIC Akaike’s
                   information criterion.


total cholesterol, fasting blood glucose, and 2-hour OGTT had                    We wanted to find out whether blood pressure and retinal
no significant effect on artery diameters or heritability.                    vessels share genes, but we found only minor interaction be-
   For CRVE, after correcting for MABP, age, and gender, additive            tween the two. The decrease in MABP heritability from 61% to
genetic factors were found to account for 83% (95% CI: 73%– 89%;             54% after adjustment for CRAE failed to reach statistical signif-
Table 4) of the phenotypic variance in retinal vein diameter and             icance. The most likely interpretation is that both genetic and
unshared environmental factors for the remaining 17% (95% CI:                nongenetic variance contribute and the former more than the
11%–27%). Vein diameters decreased with increasing age by                    latter, because if their interaction was mainly in their correla-
  4.50 m/10 years (95% CI: 8.45 to 0.56). Gender, MABP,                      tion in nongenetic variances, the heritability of CRAE would
diastolic blood pressure, systolic blood pressure, smoking, BMI,             not decrease by adjustment for MABP.
total cholesterol, fasting blood glucose, and 2-hour OGTT had no                 We found a nominal AVR heritability of 45%, but this per-
significant effect on vein diameters or heritability. For MABP, the           centage is likely to be lower than the true heritability, because
heritability was 61%. After adjustment for CRAE, the heritability            of the combination of environmental influences of CRAE and
decreased to 54%, and, after adjustment for CRVE, the heritability           CRVE, as CRAE (associated with blood pressure) and CRVE
decreased to 59% (Table 6).
                                                                             (associated with inflammatory markers and metabolic index)
                                                                             have quite different environmental associations.
DISCUSSION                                                                       The present study was designed to optimize fundus vessel
                                                                             imaging by applying direct digital imaging in red-free illumination
In the study population of 210 healthy, normotensive, adult twins,           rather than digitization of color diapositives. This procedure also
retinal vessel diameters were found to be governed mainly by
                                                                             allows immediate image quality control. The resolution of 1024
genetic factors, which accounted for 70% or more of the variance
                                                                             1024 pixels per 50° frame is lower than with the best currently
in artery diameters and 83% of the variance in vein diameters, the
remainder being attributable to unshared environmental factors.              available gray-scale sensors, but it is probably as good as that obtained
The heritability of vessel diameters was not affected by gender,             with three-chip color sensors of higher nominal resolution.
whereas correction for age and MABP was essential for the esti-                  Adjustment for refractive errors did not attenuate vessel diam-
mation of heritability. With adjustment for MABP, CRAE heritabil-            eter heritability in this study. Because refraction is highly herita-
ity decreased from 75% to 70% (Table 5), which may be because                ble22 and we are testing the difference between two twins in a
blood pressure shares a small proportion of heritability with                pair with largely the same refraction, we may not have been able
retinal vessel calibers. In contrast smoking, BMI, total cholesterol,        to assess the full influence of refraction. A previous study has
fasting blood glucose, 2-hour OGTT did not significantly influence             shown, however, that correction for refraction is unimportant in
artery and vein diameters in this study.                                     fundus vessel diameter studies.23
    Mean arterial blood pressure was also found to be governed                   The random acquisition of the fundus photographs in relation
mainly by genetic factors, with a heritability of 61%, when                  to the cardiac cycle in this study is likely to confer a higher level
controlling for gender and age. This result was in agreement                 of random variation than what can be achieved with electrocar-
with an earlier twin study finding 44% to 63% heritability of                 diograph-gated photography, but it is unlikely to offset the mean
ambulatory systolic and diastolic blood pressure.7                           results. Fundus vessel pulsation is detectable mainly in the largest


                   TABLE 6. Model-Fitting Analyses of Blood Pressure Corrected for Retinal Vessel Diameters, Gender, and
                   Age in 55 Monozygotic and 50 Dizygotic Twin Pairs

                   Phenotype                      Covariates                              A                            E

                      MABP                 Gender and age                        0.61 (0.44–0.73)              0.39 (0.27–0.56)
                      MABP                 Gender, age, and CRAE                 0.54 (0.34–0.68)              0.46 (0.32–0.66)
                      MABP                 Gender, age, and CRVE                 0.59 (0.42–0.72)              0.41 (0.28–0.58)

                        Data are the proportion of total variation attributable to model component (CI95 %).
3544     Taarnhøj et al.                                                                                   IOVS, August 2006, Vol. 47, No. 8

veins of the retina.24 In relation to heritability, added random           3. Wong TY, Klein R, Klein BE, Meuer SM, Hubbard LD. Retinal vessel
variation has the effect of decreasing the heritability.                      diameters and their associations with age and blood pressure.
    The vessel diameters found in this study were comparable to               Invest Ophthalmol Vis Sci. 2003;44:4644 – 4650.
those found in the Beaver Dam Eye Study,12 supporting that twins           4. Wong TY, Klein R, Sharrett AR, et al. Retinal arteriolar diameter
do not differ in retinal vessel calibers from singletons, as is also          and risk for hypertension. Ann Intern Med. 2004;140:248 –255.
true of blood pressure.7 The familial correlation of retinal vessel        5. Wang JJ, Mitchell P, Leung H, Rochtchina E, Wong TY, Klein R.
                                                                              Hypertensive retinal vessel wall signs in a general older
calibers was found to be 0.2 to 0.27 in the Beaver Dam Eye Study              population: the Blue Mountains Eye Study. Hypertension. 2003;
population.12 Genetic predisposition cannot be assessed in stud-              42:534 –541.
ies of familial correlations in natural families, because there is no      6. Wong TY, Hubbard LD, Klein R, et al. Retinal microvascular ab-
way to distinguish between genetic and shared familial environ-               normalities and blood pressure in older people: the Cardiovascular
mental influences on the trait. If there is no familial shared envi-           Health Study. Br J Ophthalmol. 2002;86:1007–1013.
ronment according to twin (and adoption) studies, then the fa-             7. Kupper N, Willemsen G, Riese H, Posthuma D, Boomsma DI, de
milial correlation may be multiplied by two to get the heritability           Geus EJ. Heritability of daytime ambulatory blood pressure in an
corresponding to that obtained in twin studies (i.e., 0.40 – 0.54),           extended twin design. Hypertension. 2005;45:80 – 85.
of which estimates the siblings usually has the higher one and the         8. McCaffery JM, Pogue-Geile MF, Debski TT, Manuck SB. Genetic
parent-offspring the lower one. Owing to the age differences and              and environmental causes of covariation among blood pressure,
other environmental differences, non-twin siblings are usually                body mass and serum lipids during young adulthood: a twin study.
                                                                              J Hypertens. 1999;17:1677–1685.
somewhat more different from DZ twins. Thus, a heritability of
                                                                           9. Poulsen P, Vaag A, Kyvik K, Beck-Nielsen H. Genetic versus envi-
0.7 in our study implies a correlation of 0.35 between the DZ twin            ronmental aetiology of the metabolic syndrome among male and
pairs, which is higher and not too far from that observed in most             female twins. Diabetologia. 2001;44:537–543.
non-twin siblings, which in the Beaver Dam Eye Study population           10. Smith W, Wang JJ, Wong TY, et al. Retinal arteriolar narrowing is
study was 0.20 to 0.23. The twin-based heritability can also be higher        associated with 5-year incident severe hypertension: the Blue
because of nonadditive genetic variance, which for the DZ twins               Mountains Eye Study. Hypertension. 2004;44:442– 447.
would mean that the genetic part of their between-twin correla-           11. Wong TY, Shankar A, Klein R, Klein BE, Hubbard LD. Prospective
tion is lower and the possible shared environmental contribution              cohort study of retinal vessel diameters and risk of hypertension.
to the correlation is higher. This can also fit with family studies,           BMJ. 2004;329:79.
where it seems likely that nontwin siblings share fewer environ-          12. Lee KE, Klein BE, Klein R, Knudtson MD. Familial aggregation of
ments than DZ twin pairs, and therefore would have a lower                    retinal vessel caliber in the beaver dam eye study. Invest Ophthal-
correlation than the DZ twin pairs. In addition, such nonadditive             mol Vis Sci. 2004;45:3929 –3933.
genetic variance can also explain the higher correlation between          13. Kyvik KO, Christensen K, Skytthe A, Harvald B, Holm NV. The
                                                                              Danish Twin Register. Dan Med Bull. 1996;43:467– 470.
nontwin siblings than between parent-offspring, as can a sibling-
                                                                          14. World Health Organization, Geneva. Definition, Diagnosis and Classi-
specific shared environment. Additional advantages of twin stud-               fication of Diabetes Mellitus and its Complications, report of a WHO
ies include the ability to assess the relative influence of genetics           Consultation. Geneva, Switzerland: WHO; 1999:1– 66.
and environment as well as lower susceptibility to cohort effects.        15. Kessel L, Hougaard JL, Sander B, Kyvik KO, Sørensen TIA, Larsen
    Narrow retinal arteries are independently associated with the             M. Lens ageing as an indicator of tissue damage associated with
presence of arterial hypertension and the risk of development of              smoking and non-enzymatic glycation: a twin study. Diabetologia.
hypertension in humans.4,10,11 Arteriolar narrowing has also been             2002;45:1457–1462.
found to precede the development of hypertension in rats.25 It            16. Hougaard JL, Kessel L, Sørensen TIA, Kyvik KO, Larsen M. Evalu-
may be reasonable to view retinal arterioles as effectors rather              ation of heredity as a determinant of retinal nerve fiber layer
than sensors in relation to arterial hypertension in the future. The          thickness as measured by optical coherence tomography. Invest
hypothetical inference may be that the antihypertensive medica-               Ophthalmol Vis Sci. 2003;44:3011–3016.
tions that most effectively reduce the morbidity and mortality            17. Knudtson MD, Lee KE, Hubbard LD, Wong TY, Klein R, Klein BE.
                                                                              Revised formulas for summarizing retinal vessel diameters. Curr
associated with arterial hypertension are the ones that most ef-              Eye Res. 2003;27:143–149.
fectively eliminate retinal arteriolar contraction, independent of        18. Neale MC. MX. Department of Psychiatry, Richmond, VA: Virginia
their effect on blood pressure. Arterioles also quite possibly act as         Commonwealth University; 2004.
sensors, as arterioles and blood pressure mutually affect each            19. Neale MC, Boker SM, Xie G, Maes HH. MX Manual, MX Statistical
other in a vicious cycle in hypertensive subjects.                            Modeling. 6 ed. Richmond, VA: Virginia Commonwealth University;
    Our study population was a healthy, normotensive young                    2003.
adult twin population, and the findings can be applied only to             20. Day INM, Humphries SE. Genetics of Common Diseases. Oxford,
normotensive subjects with a similar age range. In hyperten-                  UK: BIOS Scientific; 1997:19 –32.
sive populations, the mutual influence between blood pressure              21. Plomin R, DeFries JC, McClearn GE, Rutter M. Behav Genet. Third
and retinal vessel calibers is likely to be different from that of            ed. New York: WH Freeman; 1997:305–310.
normotensive subjects. In spite of this, our results may suggest          22. Lyhne N, Sjolie AK, Kyvik KO, Green A. The importance of genes
that retinal vessel calibers and the associated variations in risk            and environment for ocular refraction and its determiners: a pop-
of systemic disease may be a primary genetic characteristic                   ulation based study among 20 – 45 year old twins. Br J Ophthal-
                                                                              mol. 2001;85:1470 –1476.
rather than a reflection of variations in blood pressure.
                                                                          23. Wong TY, Wang JJ, Rochtchina E, Klein R, Mitchell P. Does
                                                                              refractive error influence the association of blood pressure and
References                                                                    retinal vessel diameters? The Blue Mountains Eye Study. Am J
                                                                              Ophthalmol. 2004;137:1050 –1055.
 1. Leung H, Wang JJ, Rochtchina E, et al. Relationships between age,     24. Knudtson MD, Klein BE, Klein R, et al. Variation associated with
    blood pressure, and retinal vessel diameters in an older popula-          measurement of retinal vessel diameters at different points in the
    tion. Invest Ophthalmol Vis Sci. 2003;44:2900 –2904.                      pulse cycle. Br J Ophthalmol. 2004;88:57– 61.
 2. Sharrett AR, Hubbard LD, Cooper LS, et al. Retinal arteriolar diam-   25. Norrelund H, Christensen KL, Samani NJ, Kimber P, Mulvany MJ,
    eters and elevated blood pressure: the Atherosclerosis Risk in            Korsgaard N. Early narrowed afferent arteriole is a contributor to the
    Communities Study. Am J Epidemiol. 1999;150:263–270.                      development of hypertension. Hypertension. 1994;24:301–308.
Straight versus tortuous retinal arteries in relation to blood pressure and genetics




Nina C.B.B. Taarnhøj1 MD, Inger C. Munch1 MD, Birgit Sander1 Ph.D., Line Kessel1 MD, Ph.D.,

Jesper Leth Hougaard1 MD, Ph.D., Kirsten Kyvik2 MD, Ph.D., Thorkild I. A. Sørensen3 Professor,

Dr Med Sci., Michael Larsen1,4 MD, Professor, Dr. Med.




1. Department of Ophthalmology, Glostrup Hospital, University of Copenhagen, Denmark

2. Danish Twin Registry, University of Southern Denmark, Odense, Denmark

3. Institute of Preventive Medicine, Copenhagen University Hospital, Denmark

4. Kennedy Centre, National Eye Clinic, Hellerup, Denmark




Corresponding author: Nina Taarnhøj, MD, Department of Ophthalmology, Glostrup Hospital,

Nordre Ringvej 57, DK-2600 Glostrup, Denmark. E-mail: ninat@dadlnet.dk


Telephone: +47 97070008, fax: +45 43234669




Key words: retinal arterial tortuosity, blood pressure, twin study, heritability




                                                   1
ABSTRACT

Background/aims: To assess the relative influence of genetic and environmental factors on retinal

arterial tortuosity and the association between tortuosity and various health indices in healthy young

to middle-aged persons.


Methods: This cross-sectional study included 57 monozygotic (MZ) and 52 dizygotic (DZ) same-

sex healthy twin pairs aged 20 to 46 years, who were characterized by determination of retinal

arterial tortuosity using a three-level scale (straight, wavy, tortuous), retinal vessel diameters,

arterial blood pressure and other health indices. Heritability was estimated using structural equation

modelling.


Results: Of 218 subjects, 79 (36.2%) had straight retinal arteries, 110 (50.5%) had wavy arteries,

and 29 (13.3%) had tortuous arteries. Heritability of tortuosity was 82% (CI95 64, 92%), with

unshared environmental factors accounting for the remaining 18% (CI95 8, 36%). Increasing values

of mean arterial blood pressure (MAP) and body mass index (BMI) were both associated with

decreasing levels of retinal arterial tortuosity.


Conclusion: The predominant determinant of arterial layout in the retina in this study population

was genetics, heritability accounting for 82% of the observed variation in tortuosity.




                                                    2
INTRODUCTION

Incidental observations of remarkable degrees of straightness or tortuosity of retinal blood vessels

may be evaluated as potential signs of morbidity, but there is little evidence to support the

diagnostic value of a single fundoscopic observation. Considerable variation in retinal vessel

tortuosity can be seen in young healthy subjects. Increased retinal arterial tortuosity has been

suggested to be associated with hypertension.1;2 A recent study demonstrated that less tortuous

retinal arteries were associated with increased risk of death from ischaemic heart disease,

independent of systolic blood pressure.3 Few studies have been performed on the normal variation

in retinal vessel tortuosity and its association with genetic and systemic factors. To evaluate

assessment of retinal vessel tortuosity as a means of monitoring systemic health, it is essential to

know what determines how retinal vessels are laid out early in life, before systemic risk factors and

disease have developed. The aim of the present study was to assess the relative contribution of

genetic and environmental factors on retinal vessel tortuosity, and the association between

tortuosity and various health indices in healthy adults.


MATERIALS AND METHODS


Participants


The study was a cross-sectional study of 57 monozygotic (MZ) and 52 dizygotic (DZ) same-sex

twin pairs, aged 20 to 46 years, all Caucasian and living in Denmark. The subjects were recruited

from the population-based Danish Twin Registry.4 The twins participated in a larger study of the

metabolic syndrome (The Geminakar Study).5 Exclusion criteria were pregnancy, breastfeeding,

known diabetes or cardiovascular disease and conditions preventing them from completing a

bicycle test. To avoid residual confounding of undiagnosed diabetes they underwent an oral glucose

tolerance test (OGTT). Sampling from this group was stratified according to age and gender, to get

                                                   3
an equal distribution of twin pairs along the age span in the two zygosity groups. DNA-based

microsatellite markers (AmpFISTR Profiler Plus Kit; PE Applied Biosystems, Perkin Elmer, Foster

City, CA, USA) were used to determine zygosity of the twins. Twin pairs where both twins lived on

the island of Sjaelland were invited to participate in a separate ophthalmic examination, for which

114 pairs of twins volunteered. Ophthalmic exclusion criteria included cataract and lens opacities

near the optical axis of the eye, and other manifest eye disease, such findings leading to the

exclusion of both twins in a pair. The study was approved by the Medical Ethics Committee of

Copenhagen County and followed the tenets of the Declaration of Helsinki, including informed

consent.


Clinical examination


Study examinations that have previously been described in detail 5;6 included oral glucose tolerance

testing, glucose and insulin levels, assessment of smoking habits, systolic and diastolic blood

pressure measurement, blood sampling, measurement of waist circumference, height and weight.

Body Mass Index (BMI) was calculated as weight (kg) divided by square of height (m). Systolic

and diastolic blood pressures were measured after 30 minutes rest, in a sitting position, using a

conventional mercury sphygmomanometer and hands-free stethoscope. Measurements were taken

three times and the mean was used for analysis. Mean arterial blood pressure (MAP) was calculated

as 33% of the difference between the systolic and the diastolic blood pressure, plus the diastolic

blood pressure. Hypertension was defined as systolic blood pressure      140 mmHg or diastolic blood

pressure   90 mmHg and /or current use of antihypertensive medicine. None of the participants had

arterial hypertension. Five participants in the eye study met one criterion for diabetes mellitus

according to current WHO7 criteria: four had fasting glucose     6.1mmol/L (whole capillary blood),

and one had 2-h glucose    11.1mmol/L. None of these subjects had diabetic retinopathy and all five

were included in the study. The eye examination included refraction, determination of visual acuity,
                                                   4
pupil dilation, slit-lamp biomicroscopy and fundus photography. Best corrected visual acuity was

determined using a Snellen decimal projection chart, and all eyes included had a visual acuity of at

least 0.9.


Fundus photography


Digital gray-scale fundus photographs (20° and 50°, TIFF 1024        1024 pixels), centred on the

macula and optic disc, after pupil dilation (with Phenylephrine Hydrochloride 10% and

Tropicamide 1%), were obtained using a fundus camera (model TRC-50X; Topcon Corp., Tokyo,

Japan) equipped with a digital back piece (MEGAPLUS model 1.4; Eastman Kodak, San Diego,

CA) and a PC-based image-management system (Ophthalmic Imaging Systems Inc., Sacramento,

CA). A green filter (filter: Wratten 54; Eastman Kodak, Inc., Rochester, NY) for “red-free”

photographs was used because it enhances the sharpness and contrast of blood vessels.


Fundus grading


Tortuosity was assessed by visual grading of one fovea-centred and one disk-centred fundus image

from each eye displayed on a computer display and viewed independently by two graders. The

graders were medical doctors with 3 years of experience in ophthalmology and especially trained in

fundus vessel grading. Fundus trunk vessel tortuosity was evaluated simultaneously for both of a

subject’s eyes, and reported as the average of the right and left eyes because the correspondence

between right and left eyes was high (Pearson correlation=0.85). A grading scale was developed for

the study based upon a preliminary inspection of the study photographs, which indicated that an

acceptable reproducibility could be achieved using a three-level scale for arterial tortuosity, whereas

the use of four or more levels led to inconsistent grading of a large proportion of cases. The

variation in venous tortuosity was found to be much smaller than that of the arteries and too small

to warrant an attempt at grading the entire study data set. The grading levels for retinal arterial

                                                    5
tortuosity were: category 1 (predominately straight vessels in all four quadrants), category 2 (mild

to moderate tortuosity, with one to two inflections of most arteries in all four quadrants), and

category 3 (prominent tortuosity with two or more inflections of most arteries in all four quadrants,

figure 1). Each grader examined all images twice at an interval of two months and in random order,

masked to the zygosity, relatedness and values of all covariates. The second grading was then used

together with the first to produce one final grading for each grader. The intra- and inter-grader

reproducibility was 0.79 (CI95 0.73, 0.86, n=218) and 0.68 (CI95 0.60, 0.77, weighted kappa,

n=218), respectively, demonstrating good agreement. The final gradings were found to disagree for

71 of 218 subjects. The fundus photographs from these 71 subjects were then consensus-graded by

the two graders in a joint session. The equation modelling yielded no significant difference in

heritability results for all five grading-sessions, heritabilities being between 84% and 87%, therefore

we chose to only report results from the final consensus grading, thinking that it was the best

grading, since two independent graders agreed.




                                                   6
Category 1




Category 2




Category 3




Figure 1 Categories 1, 2 and 3 for retinal arterial tortuosity in right and left eyes from three
different subjects of the study population.




                                                   7
Retinal vessel diameter measurement


Retinal vessel calibres were assessed using a custom-developed semi-automated computer

algorithm, described in detail elsewhere.8 The Central Retinal Artery Equivalent (CRAE) and the

Central Retinal Vein Equivalent (CRVE) were calculated using the formulae described by Knudtson

et al.9


Statistical analysis


The classical twin model is based on the assumption that MZ twins are 100% genetically identical,

for which reason all observed differences between the two twins in a pair are attributable to

environmental factors. DZ twins share on average 50% of their genes. The extent to which MZ

twins are more alike than DZ twins is therefore assumed to reflect a genetic influence on the

phenotype in question. Polychoric correlations, conditional probabilities, heritability estimates, and

regression coefficients for various covariates were estimated by use of structural equation modelling

using the MX computer software.10 The polychoric correlations were estimated because the

dependent variable, tortuosity, is categorical, and therefore we built our estimates on the assumption

that there is an underlying normally distributed continuous variable, which we cannot observe

directly. The polychoric correlations correspond to the intrapair Pearson correlations for continuous

traits. We assume that the categorical variable (tortuosity) changes value when the underlying

variable goes beyond the threshold values (there is two such thresholds, because there are three

tortuosity categories 0, 1 and 2). The model estimates the polychoric correlations for MZ and DZ

twins, respectively, for the underlying normally distributed variable. If the polychoric correlations

are significantly higher for MZ twins than for DZ twins, it indicates that genetics have an influence

on the phenotype in question.




                                                   8
              Heritability is the proportion of phenotypic variance that can be accounted for by

genetic differences among individuals for a particular trait in a particular population at a particular

time. The genetic contribution can be divided into an additive genetic variance component A,

representing the influence of alleles at multiple loci acting in an additive manner, and a non-additive

genetic variance component D, representing intra-gene interactions (dominance) and inter-gene

interactions (epistasis). The intra-pair variance due to the influence of the environment can be

subdivided into a common environmental variance component C, representing environmental

factors shared by twins during intra-uterine life and early childhood (a source of similarity), and an

unshared environmental variance component E, representing environmental factors within families

that are specific for each twin (hence, a source of dissimilarity which also includes random factors

and measurement error)11. Structural equation modelling was used to fit ACE and ADE models to

the observed data. The model with the lowest Akaike’s information criterion (AIC) was used to

determine the best-fitting model. AIC = - 2 times log likelihood + 2 * (number of free parameters in

the model). To adjust for the effects of age, gender and other covariates, allowance was made for a

linear effect on the thresholds in the model, resulting in additional regression coefficients to be

estimated. Due to lack of statistical power and for technical reasons it was decided to adjust for a

maximum of three covariates. Covariate selection was prioritized by first adjusting for covariates

with an established relation to vascular morphology, such as age, gender and blood pressure. BMI

was adjusted for because it was found to be significantly associated with tortuosity by use of

multiple regression analysis (SAS software, version 9.1.). Since MAP and BMI were correlated, we

estimated the heritability, adjusting for MAP and BMI separately. Reported p values for the

comparison of MZ and DZ twins were found to be robust with respect to deviations of data

distribution from normality and the presence of intra-pair correlation (STATA software, version 9).

The level of statistical significance was set at 0.05.


                                                    9
RESULTS


The study population of 57 MZ and 52 DZ same-sex twin pairs of both sexes aged 20-46 years,

demonstrated comparable distributions of age, gender, blood pressure, smoking habits, and other

study variables between the two subpopulations (table 1). Retinal arterial tortuosity grading

demonstrated that 79 subjects (36.2%) had predominantly straight arteries (category 1), 110

(50.5%) had wavy arteries (category 2), and 29 (13.3%) had tortuous arteries (category 3). For

concordant pairs (both twins in a pair having the same phenotype), the conditional probabilities

were higher for MZ than for DZ twins, whereas for discordant pairs (two twins in a pair having

different phenotypes), the conditional probabilities were smaller for MZ than for DZ twins,

indicating a genetic effect on this phenotype (table 2). For example, if twin 1 in a MZ pair has

tortuosity grade 1, the probability that twin 2 in the pair also has grade 1 is 0.72, whereas if twin 1

in a DZ twin pair has tortuosity grade 1 the probability that twin 2 also has tortuosity grad 1 is only

0.56 (table 2).


              Estimates for polychoric correlations were significantly higher for MZ twins, 0.81

(CI95 0.62, 0.92) compared to DZ twins, 0.40 (CI95 0.05, 0.67, p= 0.303, probability of MZ and DZ

twins being identical). It means that two twins in a MZ pair were more alike than two twins in a DZ

pair, demonstrating a genetic influence.


              Structural equation modelling estimated the heritability to be 82% (CI95 64, 92%),

unshared environmental factors accounting for the remaining 18% (CI95 8, 36%) of variation. Age

and gender were first included in the model but they were excluded without any loss of fit. When

including MAP as a covariate, the observed inter-individual differences in retinal arterial tortuosity

were best explained by the effect of additive genetic factors (A), common environmental factors (C)

and unshared environmental factors (E) (ACE model in bold type, table 3). The heritability estimate

                                                   10
for tortuosity was reduced to 68% (CI95 6, 92%), common environment contributing 12% (CI95 0,

64%) and unshared environment 20% (CI95 8, 40%), reflecting that arterial blood pressure may also

be partly heritable. MAP had an estimated regression coefficient of -0.02 (CI95 -0.03, -0.001, table

2). When including BMI in the ACE model, the heritability estimate was 79% (CI95 16, 92%),

common environment contributed 2% (CI95 0, 56%), and unshared environment 19% (8, 0.39%) of

variation. BMI had an estimated regression coefficient of -0.06 (CI95 -0.11, -0.02). The ADE model

was also tested, but the ACE model won on the lowest AIC value. Adjusting for other indices of

cardiovascular health: age, gender, systolic blood pressure, fasting blood glucose, total cholesterol,

LDL, HDL, triglycerides, smoking (pack years), retinal artery diameter, and retinal vein diameter

did not influence the heritability of retinal arterial tortuosity.




                                                     11
Table 1 Clinical characteristics of 218 healthy monozygotic and dizygotic twins


               Characteristic                MZ twins                  DZ twins         P-value *

Pairs                                        57                  52

Subjects                                     114                 104

Female subjects (%)                          54                  60                     0.67

Retinal arterial tortuosity

Category: 1/2/3 (%)                          32/52/16            41/49/10               0.17**

Smokers (%)                                  39                  47                     0.35

Pack years (smokers only)                    8.27 (6.04)         10.79 (7.96)           0.12

Age (years)                                  35.1 (7.5)          35.1 (7.1)             0.96

Diastolic blood pressure (mmHg)              70.3 (9.4)          70.7 (7.9)             0.78

Systolic blood pressure (mmHg)               115.3(12.7)         117.7(11.5)            0.24

Mean arterial blood pressure (mmHg)          85.3(10.0)          86.3(8.6)              0.50

Body mass index (kg/m2)                      23.5 (3.5)          23.9 (3.6)             0.50

Fasting blood glucose (mM)†                  4.88 (0.48)         4.88 (0.41)            0.99

Total cholesterol (mM)†                      5.24 (1.00)         5.54 (1.04)            0.08



Note: Values are given as mean (standard deviation), except pairs and subjects which are given in actual numbers.
Gender, smokers and tortuosity are given in percent.

* Robust t test (adjusted for clustering).

** Chi-Square test.

† Whole capillary blood samples




                                                           12
Table 2. Conditional Probabilities for Retinal Arterial Tortuosity in 57 Monozygotic and 52 Dizygotic Twin Pairs




      MZ twins                            Co-twin status of tortuosity category

                                    1                         2                       3

P(Twin status = 1| Co-
                             0.72 (0.53,0.86)         0.17 (0.09,0.29)         0.01 (0.00,0.11)
twin status)

P(Twin status = 2| Co-
                             0.28 (0.14,0.47)         0.71 (0.57,0.83)         0.37 (0.16,0.64)
twin status)

P(Twin status = 3| Co-
                            0.003 (0.00,0.06)         0.12 (0.05,0.23)         0.63 (0.35,0.83)
twin status)




       DZ twins                           Co-twin status of tortuosity category

                                    1                         2                       3

P(Twin status = 1| Co-
                             0.56 (0.37,0.72)         0.35 (0.22,0.51)         0.10 (0.01,0.38)
twin status)

P(Twin status = 2| Co-
                             0.42 (0.26,0.60)         0.51 (0.34,0.67)         0.70 (0.35,0.94)
twin status)

P(Twin status = 3| Co-
                            0.02 (0.002,0.10)         0.14 (0.06,0.26)         0.20 (0.01,0.58)
twin status)




Numbers are given in probability (P) and 95% confidence intervals.




                                                         13
        Table 3 Model-fitting analyses of retinal arterial tortuosity adjusted for gender, age and mean arterial blood pressure in
        57 monozygotic and 52 dizygotic twin pairs



                Genetic                      Environmental
                                                                                                 Covariates§
              Component                        Components


Model              A†                   C†                   E†                Gender                Age                 MAP


1.ACE             0.72                  0.08                0.20                 0.29                0.004               -0.02

               (0.08, 0.92)         (0.00,0.62)          (0.08,0.40)         (-0.09,0.68)        (-0.02,0.03)        (-0.03, 0.001)

2.ACE             0.68                  0.12                0.20                                     0.002               -0.02

               (0.06, 0.92)         (0.00, 0.64)         (0.08, 0.40)                            (-0.02, 0.03)       (-0.04, -0.001)

3.ACE*            0.68                  0.12                0.20                                                         -0.02

               (0.06, 0.92)         (0.00, 0.64)         (0.08, 0.40)                                                (-0.04, -0.001)

4.ACE             0.78                  0.04                0.18

               (0.16, 0.92)         (0.00, 0.57)         (0.08, 0.37)

 5.AE             0.82                   0                  0.18

               (0.64, 0.92)                              (0.08, 0.36)

 6.CE               0                   0.65                0.35

                                    (0.47, 0.78)         (0.22, 0.53)



        *Boldface type indicates best fitting model, based on Akaike’s Information Criterion, which was 393.5 in model 3 and
        between 395.2 and 401.9 in the other models.

        † Data are the proportion of total variation attributable to the model component (95% confidence interval). Model
        components are additive genetic factors A, shared environment C, unshared environment E.

        § Estimated regression coefficients (95% confidence interval).

        MAP mean arterial blood pressure.




                                                                    14
DISCUSSION


The results of the present study demonstrated that wide variations in retinal arterial tortuosity may

mostly be explained by the effect of genetic factors, which had a prominent impact in a study

population that was entirely free from arterial hypertension. Retinal arterial tortuosity demonstrated

a heritability of 82% without adjustment for covariates. Analysis of a wide range of systemic factors

revealed statistical effects only due to BMI and blood pressure. The regression coefficients for these

corresponding to a decrease in MAP of 0.02 mmHg for a one-step increase in retinal arterial

tortuosity and a decrease in BMI of 0.06 kg/m2 for a one-step increase in tortuosity; both effects

being of null clinical significance in this normotensive and healthy study population. Age, gender

and other cardiovascular risk factors were not found to be associated with retinal arterial tortuosity

in this study population. Our findings are in agreement with an earlier study that found no effect of

age on retinal arterial tortuosity in subjects without hypertension.12 When adjusting for MAP, the

heritability was reduced to 68%, suggesting some degree of shared genetic effects on arterial

tortuosity and arterial blood pressure, as was found for retinal vessel calibres and blood pressure.8

Although, this may just be a statistical consequence of the changes made in the basis for the

model’s fit to data after including blood pressure in the model, and should not be over interpreted.

The effect of common environment in this study was increased to 12% after adjusting for MAP,

which suggests that blood pressure may have an impact on the layout of retinal arteries early in life.

The effect of unshared environmental factors (18 - 20% in this study), may include those of non-

shared intrauterine environment, acute perinatal distress, random mechanisms of morphogenesis,

and errors of measurement. Other unshared environmental factors such as smoking and diet

(represented by blood lipids) did not have any influence on tortuosity in the present study.

Adjustment for refractive errors did not attenuate results in this study.



                                                   15
              The exact mechanisms that underlie the development and later modulation of the

vascular network are largely unknown. In response to increased transmural pressure, the canine

carotid artery has been shown to dilate and elongate.13 Qualitatively, this is what one would expect

of a passive elastic tube.14 Observation of such a passive behaviour may indicate that auto

regulatory capacity has been exhausted. The same behaviour can be observed in the retinal arteries

of newborns with acute perinatal distress, systemic hypoxia, and lactic acidosis.15 Preterm birth is

also associated with permanently increased retinal arterial and venous tortuosity and reduced

numbers of vascular branching points, independently of retinopathy of prematurity.16 Relief of acute

foetal distress is followed by decreasing retinal arterial tortuosity.17 Metabolic alterations and

consequent disease can also increase retinal vascular tortuosity, as seen in diabetic retinopathy.18


              The role of arterial hypertension in modulating retinal arterial dilation and tortuosity is

of particular interest. In monkeys and rats, high blood pressure has been shown to be associated

with increasing dilation and tortuosity.19;20 This finding has been replicated in humans in two cross-

sectional studies, one of which also found an effect of increased pulse pressure.1;2 In the present

cross-sectional study, which did not include subjects with arterial hypertension, we found the

opposite relation, namely decreasing retinal arterial tortuosity with increasing blood pressure.

Although, the change in blood pressure was so close to zero that it has null clinical significance in

the present study. A recent study found that ischemic heart disease (IHD) was associated with

reduced retinal arterial tortuosity, independent of arterial blood pressure.3 It is an interesting parallel

and supporting the finding that in the present study increasing BMI (a risk factor for IHD), was also

associated with decreasing retinal arterial tortuosity.


              It is important to emphasize that the subjects were young and healthy, because when

analyzing genetic and environmental factors, aging and disease processes should not cause any bias

in the results. It is hypothesized that most phenotypes vary in expression with age, and a trait may
                                                    16
be influenced by different genes at different ages, therefore using MZ and DZ twins instead of

families has the advantage of removing the age confounder from consideration.


              Since retinal vascular tortuosity is a continuum, semi-automatic digital image analysis

may have the potential to provide better measures of tortuosity and make it easier to compare results

with other studies, provided they use a standard method.3;21 But in the literature there is no such

standard method for measuring vessel tortuosity.21 Clinical assessment of retinal arterial tortuosity

based on a single examination appears to have little diagnostic value because we do not know if the

grade of tortuosity is a constantly changing parameter. Longitudinal studies based on repeated

fundus photographs over many years are likely to be more sensitive than cross sectional population

studies in showing effects of current ocular and systemic health parameters on vascular

morphology.


              In summary, the present study contributed further evidence of a pronounced genetic

effect on ocular morphology, as has been shown for retinal vessel diameters,8 cilioretinal arteries,22

small hard drusen,23;24 and retinal nerve fibre layer thickness.25 It also showed that there was a large

variation in tortuosity of retinal arteries, with 13% of the twins having tortuous arteries, although

they were normotensive, young, and healthy. The extensive inter-pair variation in vessel tortuosity

supports the hypothesis that comparison against a baseline fundus photograph is a prerequisite for

using evaluation of retinal arterial tortuosity to assess the impact of arterial blood pressure and other

health indices. A longitudinal follow-up study of this twin population, will allow us to determine

the influence of genetic factors and blood pressure on the age-related changes in retinal vascular

morphology.




                                                   17
                                        Reference List



1. de MARGERIE J, BOYD TA. A statistical investigation of the correlation of retinal arterial

   tortuosity with blood pressure and age. Trans.Can.Opthalmolog.Soc. 1961;24:6-17.


2. Wolffsohn JS, Napper GA, Ho SM et al. Improving the description of the retinal vasculature

   and patient history taking for monitoring systemic hypertension. Ophthalmic Physiol Opt.

   2001;21:441-9.


3. Witt N, Wong TY, Hughes AD et al. Abnormalities of retinal microvascular structure and risk

   of mortality from ischemic heart disease and stroke. Hypertension 2006;47:975-81.


4. Kyvik KO, Christensen K, Skytthe A et al. The Danish Twin Register. Dan.Med.Bull.

   1996;43:467-70.


5. Benyamin B, Sorensen TI, Schousboe K et al. Are there common genetic and environmental

   factors behind the endophenotypes associated with the metabolic syndrome? Diabetologia

   2007;50:1880-8.


6. Schousboe K, Visscher PM, Henriksen JE et al. Twin study of genetic and environmental

   influences on glucose tolerance and indices of insulin sensitivity and secretion. Diabetologia

   2003;46:1276-83.


7. World Health Organization, Geneva. Definition, Diagnosis and Classification of Diabetes

   Mellitus and its Complications, report of a WHO Consultation. 1-66. 1999. Geneva,

   Switzerland.

   Ref Type: Report



                                              18
 8. Taarnhoj NC, Larsen M, Sander B et al. Heritability of retinal vessel diameters and blood

     pressure: a twin study. Invest Ophthalmol.Vis.Sci. 2006;47:3539-44.


 9. Knudtson MD, Lee KE, Hubbard LD et al. Revised formulas for summarizing retinal vessel

     diameters. Curr.Eye Res. 2003;27:143-9.


10. Neale MC, Boker SM Xie G Maes HH. MX Manual, MX Statistical Modeling. 6. Richmond;

     2003. 2003.

     Ref Type: Computer Program


11. Plomin R, DeFries JC, McClearn GE et al. Behavioral Genetics. Fourth ed. New York, NY

     10010, USA: Worth Publishers and W.H. Freeman and Company, 2001.


12. Williams TD. Quantification of Arterial Tortuosity in Two Normotensive Age Groups.

     American Journal of Optometry & Physiological Optics 1982;59:675-9.


13. Van LP. Length-force and volume-pressure relationships of arteries. Biorheology

     1977;14:181-201.


14. Kylstra JA, Wierzbicki T, Wolbarsht ML et al. The relationship between retinal vessel

     tortuosity, diameter, and transmural pressure. Graefes Arch.Clin.Exp.Ophthalmol.

     1986;224:477-80.


15. Bracher D. Changes in peripapillary tortuosity of the central retinal arteries in newborns. A

     phenomenon whose underlying mechanisms need clarification. Graefes

     Arch.Clin.Exp.Ophthalmol. 1982;218:211-7.




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16. Hellstrom A, Hard AL, Chen Y et al. Ocular fundus morphology in preterm children.

     Influence of gestational age, birth size, perinatal morbidity, and postnatal growth. Invest

     Ophthalmol.Vis.Sci. 1997;38:1184-92.


17. Fahrni R, Thalmann J, Weber J et al. Central retinal arteries in the full-term newborn:

     decrease in width and tortuosity during uneventful adaptation. Helv.Paediatr.Acta

     1981;36:107-21.


18. Kristinsson JK, Gottfredsdottir MS, Stefansson E. Retinal vessel dilatation and elongation

     precedes diabetic macular oedema. Br.J.Ophthalmol. 1997;81:274-8.


19. Bhutto IA, Amemiya T. Vascular changes in retinas of spontaneously hypertensive rats

     demonstrated by corrosion casts. Ophthalmic Res. 1997;29:12-23.


20. Hayreh SS, Servais GE, Virdi PS. Retinal arterial changes in malignant arterial hypertension.

     Ophthalmologica 1989;198:178-96.


21. Hart WE, Goldbaum M, Cote B et al. Measurement and classification of retinal vascular

     tortuosity. Int.J.Med.Inform. 1999;53:239-52.


22. Taarnhoj NC, Munch IC, Kyvik KO et al. Heritability of cilioretinal arteries: a twin study.

     Invest Ophthalmol.Vis.Sci. 2005;46:3850-4.


23. Munch IC, Sander B, Kessel L et al. Heredity of small hard drusen in twins aged 20-46 years.

     Invest Ophthalmol.Vis.Sci. 2007;48:833-8.


24. Hammond CJ, Webster AR, Snieder H et al. Genetic influence on early age-related

     maculopathy: a twin study. Ophthalmology 2002;109:730-6.



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25. Hougaard JL, Kessel L, Sørensen TIA et al. Evaluation of Heredity as a Determinant of

    Retinal Nerve Fiber Layer Thickness as measured by Optical Coherence Tomography.

    Invest.Ophthalmol.Vis.Sci. 2003;44:3011-6.




                                              21
                          Review of Twin Studies in Ophthalmology


Authors:
Nina C.B.B.Taarnhøj and Line Kessel


Institution:
Department of Ophthalmology, Glostrup Hospital, University of Copenhagen, Denmark


Corresponding author:
Nina Taarnhøj, MD, Department of Ophthalmology, Glostrup Hospital
Nordre Ringvej 57, Dk-2600 Glostrup, Denmark
Phone: +47 97070008
Fax: +47 22119989
Email: ninat@dadlnet.dk


Key words:
Twin studies. Ophthalmology. Ocular diseases. Genetics.


Acknowledgements
This study was supported by: The Danish Medical Research Council; The Danish Eye Research
Foundation; The Danish Eye Health Society; Centre for Biomedical Optics and New Laser Systems
Graduate School; The Danish Diabetes Association; The Norwegian Association of the Blind and
Partially Sighted; Research Career Award Grant 8-2002-130 from The Juvenile Diabetes Research
Foundation (Michael Larsen).




                                               1
ABSTRACT

Twin studies provide a powerful tool to examine the relative influence of genetic and environmental
influence on human traits and diseases. Since the 1990’s, structural equation modelling has been
used by researchers in ophthalmology to estimate the heritability of ocular diseases. Twin studies
from the 1980’s to 2007 found that the traits with the highest heritability estimates (>90%) were:
refraction and it components, lens thickness, corneal endothelial cell area, central corneal thickness,
central retinal thickness and   20 small hard drusen in eyes without ocular disease. Eye colour,
optic disc and cup diameter, peripapillary atrophy, retinal nerve fibre layer, central retinal thickness,
macular thickness, macular pigment and retinal vascular morphology had heritability estimates of
70% to 90%. As expected, ocular diseases such as glaucoma, age-related macular degeneration
(AMD), and cataract had heritability estimates of 13% to 71%, supporting the theory that they are
complex multifactorial diseases that are also influenced by age, systemic and environmental factors.
Examination of the relative contribution of genetic and environmental factors on ocular morphology
can be of fundamental importance for the full understanding of the aetiology of ocular diseases.




                                                   2
INTRODUCTION

Examination of the relative contribution of genetic and environmental factors on ocular morphology
can be of fundamental importance for the full understanding of the aetiology of ocular diseases.
Twin studies provide a powerful method for disentangling complex genetic and environmental
interactions. The first classical twin study in ophthalmology was performed in 1922 by Walter
Jablonski who investigated intra-pair differences in total refraction and total astigmatism in
monozygotic (MZ) and dizygotic (DZ) twin pairs. He suggested that hereditary factors had a great
influence on the development of refraction.1 The last review on twin research in ophthalmology was
performed by Waardenburg in 1950. He concluded that refractive error, eye colour, corneal
astigmatism, corneal dystrophies, variations in optic disc size, cataract and many other ocular traits
were heritable.2 The twin studies at that time included limited numbers of twin pairs resulting in
low power, unequal numbers of MZ and DZ twin pairs, and the DNA (deoxyribonucleic acid)
technique was not developed so determination of twin zygosity was based on more insecure
methods. The aim of this review was to summarize findings from recent twin studies (1980-2007)
in ophthalmology.




TWIN METHODOLOGY

The classical twin model is based on the knowledge that MZ twins derive from a single fertilized
egg that divides and develops into two 100% genetically identical individuals, for which reason all
observed phenotypic differences between the two twins in a pair must be attributable to
environmental factors. DZ twins derive from two fertilized eggs and share 50% of their genes, just
like normal siblings. The extent to which MZ twins are more alike than DZ twins is therefore
assumed to reflect a genetic influence on the phenotype in question. It is necessary that zygosity is
diagnosed accurately by use of DNA markers.3

              Heritability is defined as the proportion of the total phenotypic variance that is
attributable to genetic variance for a given phenotype. For continuous traits, for example retinal
vessel diameters, a rough estimate of heritability (h2, additive genetic variance) can be made by
doubling the difference between the intrapair correlation (r) for MZ and DZ twin pairs: h2 = 2(rMZ-
rDZ). For dichotomous traits (for example having or not having a disease), concordance rates for MZ
and DZ twin pairs have to be compared. When both members of a twin pair are affected by a
                                                   3
disease it is called concordance, and if the disease affects only one member of a twin pair it is called
discordance. Pairwise concordance rates can be calculated as number of concordant pairs divided by
total number of concordant and discordant pairs in the study population. If the pairwise
concordance rate for MZ twins is significantly higher than for DZ twins for the disease in question,
it is assumed to be influenced by genetic factors.3

              When estimating heritability we often assume the genetic effects to be additive.
Additive genetic effects occur when alleles at a locus on a chromosome add up to constitute a
phenotype. However, sometimes the effects of alleles in a locus are dominant (i.e. non-additive)
because they interact instead of adding up. Another non-additive effect is epistasis which is
interaction of alleles at different loci. Among different populations or at different times, the
environmental and genetic influences might differ, and therefore the heritability estimates can
differ.3

              Recent advances in twin statistics have allowed analysis of covariance between MZ
and DZ twin pairs and estimation of heritability by means of structural equation modelling (model
fitting) using the MX computer software.4, 5 This method allows separation of the observed
phenotypic variance into genetic and environmental components while adjusting for covariates such
as age, gender and systemic factors. The genetic contribution can be divided into an additive (A)
genetic variance component and a non-additive (D) genetic variance component; dominance and
epistasis. The environmental component can be subdivided into a common (C) environmental
variance component, representing environmental factors affecting both twins in a pair, a source of
similarity and an unshared (E) environmental variance component representing environmental
factors not shared by twins, a source of dissimilarity, including random factors and measurement
errors.3 The heritability is defined as (A+D)/(A+D+C+E) in a given population at a given time.6




UTILITY AND LIMITATIONS OF TWIN STUDIES

The classic twin study design is the best existing method for estimating the relative influence of
environment and genes. Interpretation of heritability in twin studies is interesting in itself, but to be
useful, the results should be generalized to the general population. Although twins in general are
representative of the general population for a wide variety of phenotypes, with the exception of


                                                      4
birth weight,7 the problems which may affect the validity of the transformation of results from twin
studies to the general population should be discussed.

Can the assumptions for twin studies be met?
The first assumption that the effect of age and sex is the same across zygosity is always valid. The
equal environment assumption is based on the assumption that both MZ and DZ twin pairs share
intra-pair environment to the same extent and therefore any greater phenotypic similarity among
MZ twins compared to DZ twins must be due to genetic influence.8 The assumption is valid in
many circumstances; twins share the same womb, are exposed to the same environmental factors in
uterus (maternal smoking, drinking, drugs and infections), are born at the same time, are the same
age and are exposed to the same familial environment.3, 8 Due to common chorions and placentas
some MZ twins may suffer from complications related to inter-foetal vascular anastomoses leading
to unequal sharing of placenta and twin-transfusion syndrome causing large differences in birth
weight. Both MZ and DZ dichorionic twins can experience differences in nutrition supply and
differences in trans-placental transmitted teratogenes and infections.9 These effects can lead to
environmental differences among MZ and DZ twins and overestimation or underestimation of the
heritability, because it is difficult to distinguish between genetics and early environmental effects.
These prenatal adverse environmental effects may be important in the aetiology of common
diseases in adulthood. One approach to eliminate differences in prenatal environmental effects
would be to exclude all monochorionic MZ twin pairs and dichorionic MZ twin pairs with
significantly different birth weight. The value of gathering data on chorionicity is beyond doubt, but
usually unattainable. The data are not consistently entered in the patient’s records and twins are
normally recruited from registers with no available information on circumstances of birth.8

Are MZ twins really 100% genetically identical?
Another concern is that MZ twins are in theory genetically identical, but post-zygotic non-
disjunction, mutations and recombination, skewed X-chromosome inactivation in female MZ twins,
differences in tissue-specific methylation patterns and the timing of such events can lead to
differences in phenotype expression. These mechanisms might hold the key to why there is so much
we do not understand in disease aetiology.9
Are twins representative of the background population?
Finally, twins cannot always be compared to singletons. Some women frequently produce more
than one egg at each menstrual period and, therefore, are more likely to have DZ twins, this is

                                                   5
related to age, height and genetics.8 Artificial induction of ovulation and in vitro fertilization can
give rise to DZ twins. For these reasons, DZ twins may belong to a special group that does not
represent the background population.10
              Twins are on average born 3 weeks pre-term and are on average 1000 g lighter that
singletons and since being a twin is associated with caesarean sections, malformations, and other
gestation- and birth complications, some diseases may be more common in twins. However, studies
have shown evidence that mortality rates in twins and in the general population are similar after the
age of 6 and that twins can represent singletons in many complex traits and in cognitive
performance.8, 11-14 However, it is important to test if the disease or trait studied has the same
frequency in twins as in singletons and ensure that there is no association between disease and
zygosity.8

              The twin studies included in the present review were found on Pubmed by searching
on the following words: twin research AND eye, twins AND eye, twin studies AND eye, Hammond
C, Meyers SM and Teikari JM.




TWIN STUDIES IN OPHTHALMOLOGY

Refractive error and its components

Two different causes of myopia have been postulated: 1) environmental factors such as reading,
near-work and visual deprivation and 2) genetic factors. Several recent classical twin studies have
confirmed that refraction and its components are mainly genetically determined (table 1).15-25

              Both genetic factors and habits of reading were significantly associated with myopia
in Chinese school children.24 In 2001 Lyhne et al found that myopia was highly heritable and
significantly associated with longer education, an example of gene-environment interaction,
indicating that individuals that are genetically susceptible might develop myopia if exposed to
environmental factors such as near work (table 1).15

              The only twin study which did not report a high heritability of refractive errors was a
study by Angi et al. In children aged 3-7 years. They reported heritability estimates as low as 8%
and14% for right and left eye, respectively. However, the sample was very small, the children
included were very young which makes determination of refraction difficult, and one eye was found
                                                    6
to be myopic due to congenital cataract. Myopia was significantly related to increased axial length.
It was suggested that visual deprivation in early childhood, lead to postnatal eye growth, increased
axial length and myopia (table 1).26

              Large and high quality studies on refraction have been performed by Hammond22 and
Dirani.23 They used structural equation modelling on a large set of twins and were able to find
dominant genetic influences. Dominant genetic factors accounted for most of the variation in total-
and corneal astigmatism.22 Dirani estimated a high heritability of spherical equivalent, axial length,
anterior chamber depth, and corneal astigmatism of which involved both additive and dominant
genetic factors. Furthermore lower age and higher education levels were associated with more
negative refraction (table 1).23 There was no significant difference in refraction errors among
genders.

              The twin studies on refractive error and its determinants suggested that most of the
variance in refractive error was due to genetic factors but that individuals who are genetically
susceptible might develop myopia if exposed to environmental factors such as near-work or visual
deprivation, depending on the duration and age of onset. The influence of both additive and
dominant genetic influence suggested that multiple genes may be involved in the aetiology of
refractive error.

              In 1997 Løgstrup et al investigated the long term influence of insulin dependent
diabetes mellitus on lens thickness and refraction. They found a significant positive relation
between lens thickness and increasing duration of diabetes. The correlation coefficients were
similar in MZ and DZ twins, suggesting that the effect of the duration of diabetes on lens thickness
was independent of genetic factors.27 Increased lens thickness and decreasing anterior chamber
depth with increasing duration of diabetes were found to be responsible for the low degree myopic
refraction observed in diabetic patients.28




Cornea:

Central corneal thickness and endothelial cell area are highly heritable traits29, 30 but corneal
fluorescence which is related to non-enzymatic glycation of corneal proteins was mainly determined
by environmental factors, common environmental factors accounting for 56% and unshared

                                                    7
environmental factors for 19% of the variation (table 2).31 Smoking, impaired 2-hour oral glucose
tolerance and age were associated with corneal fluorescence and these factors accounted for 26%
(R2) of the variation in the Danish Twin Study.31




Lens:

Hammond et al investigated genetic and environmental factors in age-related nuclear and cortical
cataracts in the UK Adult Twin Study (table 3). The heritability was 48% for nuclear cataract and
53-58% for cortical cataract. Unshared environmental effects contributed 14% and age contributed
38% of the variance in nuclear cataract.32 Unshared environment contributed 26% and 37% and age
explained 16% and 11% of the variance in clinical and digital grading of cortical, respectively.33
These studies suggested that age was stronger associated with nuclear cataract than with cortical
cataract in women and that environmental factors contributed more to the development of cortical
cataract than to the development of nuclear cataract. Heritability was almost the same for cortical
and nuclear cataracts, although the genetic effect was additive in nuclear and mainly dominant in
cortical cataract.

              Kessel et al examined lens ageing in The Danish Twin Study by means of
autofluorescence. Increased autofluorescence is a sign of denaturation of the lens proteins, due to
aging and environmental factors. The heritability of lens autofluorescence was only 28% (additive),
shared environment accounting for 58% and unshared environment accounting for 14% of the
variation, after adjustment for age, smoking and 2-hour oral glucose tolerance (table 3). Age was
the most important factor, accounting for 53% (R2) of the variation. Tobacco smoking and glucose
metabolism were associated with increased lens autofluorescence, smoking accounting for 10% (R2)
of the variation (table 3).34




Age related macular degeneration

Age related Macular Degeneration (AMD) is a complex multifactorial disease and twin studies have
supplied evidence that there is an underlying genetic predisposition for AMD (table 4).35-37




                                                    8
                   In The U.K. Twin Study, Hammond et al analyzed the heritability of various
phenotypes associated with age-related maculopathy (ARM) in women and found that large
( 125 m) soft drusen were more heritable than small (>63 m) soft drusen. They also found that the
phenotype with the highest heritability was ‘ 20 small hard drusen’, with dominant genetic factors
accounting for 81% of the variation (table 4).35 Munch et al confirmed these results, estimating a
dominant heritability of 99% for the phenotype ‘ 20 small hard drusen’ in young and healthy twins
without AMD (table 4).38 These are important findings, which imply that for candidate gene studies
of early ARM, attention should be paid to phenotypes with large soft drusen or ‘ 20 small hard
drusen’.

                   The largest twin study on AMD at present included 840 male twins of which 509 had
maculopathy and 106 of these were diagnosed with advanced AMD. The heritability estimates for
AMD were 46% for the ordinal stage 5 grading (The Wisconsin age-related maculopathy grading
system), 67% for intermediate disease, and 71% for advanced disease (table 4).37




Pupil size:

Hammond et al investigated pupil size after dilation (with tropicamide 1% and phenylephrine 10%)
in the white female twins. Additive genetic factors contributed 78% of the variation, with
environmental factors and accounting for 22%. Iris colour and refractive error had little effect on
the pupil size (table 5).39




Iris structure and eye colour:

Larsson et al and Larsson and Pedersen investigated the human iris in a German twin study of 100
MZ, 99 DZ twin pairs and 99 age-matched randomly paired unrelated subjects. In this study the
heritability of nevi, crypts, contractional furrows and white dot rings were 58%, 66%, 78% and
78%, respectively, and that of eye colour was 85%, involving both additive and dominant factors
(table 5).40, 41

                   Bito et al investigated eye colour changes after age 6 years in a longitudinal study of
1513 twins and their parents. They found that most of the subjects achieved stable eye colour by age

                                                        9
6, but a subpopulation of 10-15% of subjects had changes in eye colour due to melanin content and
distribution and that these changes were influenced by genetic factors. There was evidence that eye
colour is predominantly determined by genetic factors and that it is not only a dominant trait, like
earlier hypothesised, but a complex trait with multiple genes contributing to the expression.42




Optic disc size and intraocular pressure

In 1992 Teikari and Airaksinen examined 10 MZ and 7 DZ twin pairs aged 34-35 years and
suggested that cup-to-disc ratio was mainly influenced by genetic factors. But, with the limited
number of twin pairs the statistical power was low, and heritability was not calculated.43

              In the Danish Twin Study, additive genetic factors (i.e. heritability) explained 77%,
70%, and 66% of the total phenotypic variance in vertical disc diameter, vertical cup diameter and
cup/disc ratio, respectively, after correction for age and gender (table 5).44




Glaucoma and intraocular pressure

The first population based twin study on open-angle glaucoma was performed by Teikari et al in
1987. The heritability of open-angle glaucoma was 13% (table 5).45 However, the diagnosis of
glaucoma was made by many different ophthalmologists who used different diagnostic criteria
which makes the results of the study hard to interpret. Gottfredsdottir et al found high concordance
rates for primary open-angle glaucoma and pseudoexfoliation syndrome in 50 MZ twins whereas
there was no concordance between twins and their spouses for these diseases. This indicated that
primary open-angle glaucoma and pseudoexfoliation syndrome may be influenced by genetic
factors.46

              In 2007, Pärssinen performed a twin study on intraocular pressure (IOP) in 94 MZ and
96 DZ female twin pairs aged 63-76 years without glaucoma diagnosis. The heritability of IOP was
64% , the rest of the variation in IOP being due to unshared environmental factors (table 5).47




                                                   10
Retinal thickness

Hougaard et al. found that peripapillary retinal nerve fibre layer thickness, measured by OCT, had a
heritability of 78% when adjusting for the effect of age and increasing to 85% when adjusting for
refraction.48 As a consequence of Hougaard’s study, Chamberlain et al. conducted a study of 109
pairs of healthy twins from the Australian Twin Registry. They found that macular thickness
measured by OCT had a heritability of 81%, 81% and 85% measured in the inner and outer macular
region and fovea, respectively, when adjusting for age, gender and axial length. They only tested
the ACE model, so it is not possible to say if dominant factors may have had an impact. They also
found that increased axial length and age were associated with decreased outer macular thickness
(table 5).49

               Healy et al. found that the heritability of peripapillary atrophy was 70%, adjusted for
the effect of refraction in the UK Adult Twin population. The heritability for spherical equivalent
was 88%. The prevalence of peripapillary atrophy increased with increasing myopia but
multivariate modelling found that only 3% of the genetic variance in peripapillary atrophy was due
to shared genetic factors with mean spherical equivalent (table 5).50

               Liew et al estimated the heritability of central retinal thickness (CRT) to 90% in a
population of 155 healthy female twin pairs. They also found that decreased CRT was associated
with myopia, but they did not adjust for refraction when analyzing the heritability of CRT, which
was a weakness of this study (table 5).51




Macular pigment

In 2005 Liew et al reported that macular pigment in 150 female twin pairs aged 18-50 y had an
estimated heritability of 67% measured with heterochromatic flicker photometry and 85% measured
with fundus autofluorescence, with unshared environmental effects accounting for the remaining
variance. There was no significant association between macular pigment and age in this relatively
young population (table 5).52




                                                   11
Retinal vessels:

The first twin study on retinal vascular patterns in retinal photographs was conducted by Huntzinger
and Christian in 1978. They examined retinal vascular length, vascular length between branch
points, number of retinal vessels crossing at the optic disc boundary, number of branch points as
given by retinal quadrant and by distance from the disc and vascular tortuosity in 37 MZ and 24 DZ
twin pairs. Vascular tortuosity was the only variable studied that revealed evidence of genetic
variation. The study was too small to gain enough statistical power and they did not estimate
heritability.53

                  Taarnhøj et al investigated retinal vessel morphology in the Danish Twin Study: The
heritability was 70% for retinal artery diameter and 83% for retinal vein diameter.54 Cilioretinal
arteries were present in 45.1% of participants and 28.8% of eyes. Additive genetic factors
influenced the presence of cilioretinal arteries with a mean heritability of 71.4%, the remaining
variance being attributable to unshared environmental factors.55 Tortuosity of retinal arteries
showed a heritability of 82% , unshared environmental factors accounting for the remaining 18%.56




                                                    12
DISCUSSION

In the present review, twin studies in ophthalmology from 1980 to 2007 showed that the traits with
the highest heritability estimates (>90%) were: refraction and it components, lens thickness, corneal
endothelial cell area, central corneal thickness, central retinal thickness and ‘ 20 small hard
drusen’. The estimated heritabilities of eye colour, optic disc and cup diameter, peripapillary
atrophy, retinal nerve fibre layer, central retinal thickness, macular thickness, macular pigment and
retinal vascular morphology were between 70% and 90%, which is considered high by twin
statisticians. Ocular diseases such as AMD, nuclear and cortical cataract had heritability estimates
of 45% to 71%. This was expected, because these are complex multifactorial diseases which are
apparently influenced by genes, but also to a large extent by age, systemic and environmental
factors. This was especially demonstrated in the analysis of corneal and lens autofluorescence
which were predominantly related to age, smoking and glucose metabolism.

              Results from twin studies are most often in agreement with genome wide linkage
studies, providing further evidence that they are reliable. Recently genome-wide linkage studies
have made it possible to scan the DNA, from venous blood samples of the study subjects, for linked
signals. Strong linkage to refractive error at four loci in the vicinity of the PAX6 gene on
chromosome 11 has recently been found. This suggest that PAX6 may play a role in the
development of myopia.57 Axial length, a major endophenotype for refractive error, was found to be
influenced by one or more genes on the long arm of chromosome 5.58

              Eye colour used to be considered a simple recessive Mendelian trait with brown eyes
being dominant over blue eyes. But more recently, by use of twin studies, it has been elucidated that
eye colour is actually a more complex trait with multiple genes contributing to the expression. The
physical basis of eye colour is the amount of melanin in the melanocytes in the iris. Brown eyes
have up to 70% higher concentrations of melanin than other eye colours. Genome-wide linkage
studies found that the region containing the gene OCA2 (oculo-cutaneous albinism type II) on
chromosome 15 explains most variation in eye colour.10, 59-61 Two studies also found significant
linkage on chromosome 11q in a region that contains the tyrosinase gene (TYR) which plays a role
in melanin formation in the eye.60




                                                  13
              Multiple linkage studies on AMD have not lead to a breakthrough, but at least 16
regions on ten different chromosomes have been found to be possible candidate genes for the
development of AMD.62

              The Beaver Dam Eye Study found that retinal arteriolar and venular calibres were
linked to multiple genetic loci. They did not find the exact genes that code for retinal blood vessel
calibre, but they were able to locate potential candidate genes in the linked regions that are
associated with endothelial dysfunction, angiogenesis and essential hypertension.63

              The revolution in molecular genetics has provided more effective tools for describing
the genome, but they do not have the strength to separate the genetic and environmental effects on
traits, like twin studies have. The findings from twin studies should encourage investigators to
pursue linkage studies to identify ocular disease-related genes. Longitudinal twin studies combined
with genome-wide linkage analysis on diabetic retinopathy, keratoconus, glaucoma, cataract, AMD
and other ocular diseases may provide additional insights into the pathogenesis of these
multifactorial complex diseases.




                                                  14
TABLE 1. Twin studies on refractive error and its determinants.

   Author            Year            MZ/DZ             Age         Phenotype         Heritability        Twin Study         Method
                                     (pairs)         (years)
     Hu16             1981            49/37           7-19           myopia               0.90            Shanghai,      h2 = 2(rMZ-rDZ)
                                                                                                           China
   Teikari17          1988          3676/8109         28-80          Myopia            0.82/1.00         The Finnish     h2 = 2(rMZ-rDZ)
                                                                                     (male/female)       Twin Cohort
   Teikari18          1989            42/30           30-31        Astigmatism            0.00                ”          h2 = 2(rMZ-rDZ)
   Teikari21          1990            80/111           60+          Hyperopia             0.75                ”          h2 = 2(rMZ-rDZ)
   Teikari19          1991            54/55           30-31          Myopia            0.74/0.61              ”          h2 = 2(rMZ-rDZ)
                                                                                     (male/female)
   Teikari20          1992            79/73           30-31          Myopia              0.91a                ”               SEM
    Angi26            1993            19/20            3-7          Refraction            0.14            Padua, Italy   h2 = 2(rMZ-rDZ)
   Valluri25          1999            20/19           12-73        Axial length           0.96            Washington     h2 = 2(rMZ-rDZ)
      “                 “               “               “           Spherical              1.0                “          h2 = 2(rMZ-rDZ)
                                                                    equivalent
   Lyhne15            2001            53/61           20-45         Refraction     0.94 (0.91, 0.96)a      Denmark           SEM
      “                 “               “               “          Axial length    0.94 (0.90, 0.96)a         “              SEM
      “                 “               “               “            Anterior      0.88(0.81, 0.92)d          “              SEM
                                                                  chamber depth
      “                “                “               “         Lens thickness   0.90 (0.85, 0.94)d          “             SEM
      “                “                “               “         Mean radius of   0.90 (0.84, 0.93)a          “             SEM
                                                                     corneal
                                                                    curvature
 Hammond22            2001           226/280          49-79          Myopia        0.90 (0.81, 0.95)a     UK Adult           SEM
                                                                                                         Twin Registry
      “                “                “               “           Hyperopia      0.89 (0.81, 0.94)a         “              SEM
      “                “                “               “           Spherical      0.86 (0.83, 0.89)a/        “              SEM
                                                                    equivalent     0.84 (0.81, 0.87)a
                                                                                       right/left
      “                “                “               “               Total      0.47 (0.37, 0.53)d/         “             SEM
                                                                   15
                                                                 astigmatism      0.49 (0.42, 0.55)d
                                                                                      right/left
      “                “                “              “           Corneal       0.61 (0.12, 0.71)d/           “              SEM
                                                                 astigmatism      0.42 (0.08, 0.66)d
                                                                                      right/left
   Dirani23          2006           345/267          18-88        Spherical      0.88 (0.86, 0.90) ad/    Australian          SEM
                                                                  equivalent     0.75 (0.72, 0.78)ad     Twin Registry,
                                                                                     male/female          Melbourne
      “                “                “              “         Axial length    0.94 (0.89, 0.99) ad/         “              SEM
                                                                                 0.92 (0.88, 0.96)ad
                                                                                     male/female
      “                “                “              “           Anterior      0.51 (0.47, 0.55) ad/         “              SEM
                                                                chamber depth    0.78 (0.72, 0.84)ad
                                                                                     male/female
      “                “                “              “           Corneal       0.50 (0.42, 0.58) ad/         “              SEM
                                                                 astigmatism     0.60 (0.53, 0.67)ad
                                                                                     male/female
    Yeh64            2007             33/10          18-67           Spherical           0.20             Taipei Twin     h2 = 2(rMZ-rDZ)
                                                                    equivalent                           Study, Taiwan
        “               “                “               “            Corneal            0.16                  “          h2 = 2(rMZ-rDZ)
                                                                     curvature
        “               “                “               “            Corneal            0.46                  “          h2 = 2(rMZ-rDZ)
                                                                  Astigmatism
        “               “                “               “           Spherical           0.56                  “          h2 = 2(rMZ-rDZ)
                                                                    aberration,
                                                                     Zernike 4
        “               “                “               “       RMS 4th order           0.32                  “          h2 = 2(rMZ-rDZ)
                                                                    abberation
        “               “                “               “            RMS of             0.44                  “          h2 = 2(rMZ-rDZ)
                                                                     spherical
                                                                    abberation
a                 d
  additive effect, dominant effect, SEM = structural equation modelling, RMS = root mean square
                                                                  16
TABLE 2. Twin studies on corneal characteristics.

    Author      Year    MZ/DZ            Age         Phenotype                Heritability         Twin Study            Method
                        (pairs)        (years)
Mäkitie30       1983     12/12          25-69        Endothelial       Intrapair correlation:   Finnish Twin Cohort   Fisher’s z-test
                                                       cell area        MZ/DZ:0.93/0.08                Study
     Toh29      2005    131/125         8-81           Central           0.95 (0.93, 0.96)a       U.K. Adult Twin         SEM
                                                       corneal                                        Registry,
                                                      thickness                                  Twin Eye Study in
                                                                                                   Tasmania, and
                                                                                                Brisbane Adolescent
                                                                                                    Twin Study
    Kessel31    2003     59/54         20-46            Corneal          0.25 (0.00, 0.67)a       The Danish Twin         SEM
                                                     fluorescence                                      Study

a
    additive effect, d dominant effect, SEM = structural equation modelling




                                                                       17
TABLE 3. Twin studies on lens characteristics.

      Author            Year            MZ/DZ            Age             Phenotype              Heritability   Twin Study      Method
                                        (pairs)        (years)
    Hammond32           2000            226/280         50-79       Age-related nuclear  0.48 (0.42, 0.54)a     UK Adult        SEM
                                                                          cataract                             Twin Registry
    Hammond33           2001            226/280         50-79        Cortical cataract                          UK Adult        SEM
                                                                      digital grading:   0.53 (0.45, 0.60)d    Twin Registry
                                                                     clinical grading:   0.58 (0.51, 0.64)ad
      Kessel34          2002             59/55          20-46      Lens autofluorescence 0.28 (0.08, 0.55)a     The Danish      SEM
                                                                                                                Twin Study
a
    additive genetic factors, d dominant genetic factors, SEM = structural equation modelling




                                                                       18
TABLE 4. Twin studies on age-related maculopathy

   Author           Year         MZ/DZ               Age          Phenotype        Heritability       Twin Study      Method
                                 (pairs)           (years)
 Hammond35          2002         226/280             (62)           Age-related  0.45 (0.35, 0.53)d     UK Adult       SEM
                                                                      macular                         Twin Registry
                                                                   degeneration
                                                                    Soft drusen  0.57 (0.50, 0.64)a         ”          SEM
                                                                        125 m
                                                                       20 hard   0.81 (0.77, 0.84)d         ”          SEM
                                                                       drusen
    Munch36          2007            58/52          20-46 (35)         20 hard    0.99 (0.82, 100)d    The Danish      SEM
                                                                       drusen                          Twin Study
   Seddon37          2005           210/181           (74.5)        Age-related  0.46 (0.12, 0.73)a    World War       SEM
                                                                      macular                         Two Veteran
                                                                   degeneration                       Twin Registry
                                                                       AMD,      0.71 (0.18, 0.88)a         ”          SEM
                                                                     advanced
                                                                    disease only
a                 d
  additive effect, dominant effect, SEM = structural equation modelling, AMD = Age-related macular degeneration




                                                                 19
TABLE 5. Twin studies on eye colour, optic disc size, intraocular pressure (IOP), glaucoma, retinal structures and retinal vessels.

   Author            Year          MZ/DZ               Age            Phenotype          Heritability       Twin Study          Method
                                   (pairs)           (years)
 Hammond39           2000          226/280            49-79         Pupil size after   0.78 (0.73, 0.82)a    UK Adult             SEM
                                                                        dilation                            Twin Registry
  Larsson40          2003          100/99              4-78           Iris colour      0.51 (0.29, 0.81)a     Germany             SEM
                                                                                       0.34 (0.03, 0.55)d
  Taarnhøj44         2007           59/55             20-46         Vertical disc      0.77 (0.65, 0.85)a    The Danish           SEM
                                                                      diameter                               Twin Study
       “               “              “                 “            Vertical cup      0.70 (0.55, 0.80)a        “                SEM
                                                                      diameter
      “                “              “                 “           Cup/disc ratio     0.66 (0.48, 0.77)a        “              SEM
   Teikari45         1987         4137/9162           30-90          Open-angle               0.13          The Finnish      Concordance*
                                                                      Glaucoma                              Twin Cohort
  Pärssinen47        2007           94/96             63-76              IOP           0.64 (0.53, 0.71)    The Finnish           SEM
                                                                                                            Twin Cohort
 Hougaard65          2003           25/25             20-45         Retinal nerve      0.82 (0.64, 1.00)a   The Danish       h2 = 2(rMZ-rDZ)
                                                                     fibre layer                            Twin Study
                                                                      thickness
Chamberlain49        2006           58/51          57.6/59.7           Macular         0.81 (0.73, 0.89)a    Australian           SEM
                                                 (mean MZ/DZ)         thickness                             Twin Registry
   Healy50           2007         209/263            49-79          Peripapillary      0.70 (0.54, 0.83)a    UK Adult             SEM
                                 Only female                           atrophy                              Twin Registry
    Liew51           2007          81/74              18-50         Central retinal    0.90 (0.85, 0.93)         “                SEM
                                                                      thickness
    Liew52           2005           76/74             18-50            Macular         0.67 – 0.85 (0.52,         “               SEM
                                                                       pigment               0.90)a
  Taarnhøj55         2005           58/54             20-46          Cilioretinal      0.74 (0.34, 0.94)a    The Danish           SEM
                                                                       arteries                              Twin Study

                                                                     20
  Taarnhøj54         2006          57/52             20-46         Retinal artery   0.70 (0.54, 0.80)a   “            SEM
                                                                       calibre
            54
   Taarnhøj          2006            57/52            20-46         Retinal vein    0.83 (0.73, 0.89)a   “            SEM
                                                                       calibre
            56
   Taarnhøj          2007            57/52            20-46        Retinal artery   0.82 (0.64, 0.92)    “            SEM
                                                                     tortuosity
a                 d
  additive effect, dominant effect, SEM = structural equation modelling
* Heritability = 2(%CMZ-%CDZ), where %CMZ=percentage of concordant pairs among MZ pairs and %CDZ= percentage of concordant pairs
among DZ pairs




                                                                   21
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                                               26

				
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