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             APRIL 1
Instructions                                                  Standard Investigator Grant
To apply for a research grant, please complete the             This grant is awarded a maximum of $50,000
application form, attach any additional required               per year up to two years’ duration, the maximum
materials and e-mail to                   total award being $100,000. These grant
(Applications submitted by fax will NOT be                     applications may be in any area of spinal
accepted.) This application form may also be found             deformity research, although an additional
on the SRS Web site at If you have                funding source for grants focusing on the
any questions, please call 414-289-9107 or e-mail at           etiology of scoliosis is available in conjunction Application deadlines is April 1.                with the Cotrel Foundation. These grant
                                                               application require evidence that the
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                                                               complete the proposed research. As such,
 Applicants do not need to be members of the                  preliminary data is generally required. The
  SRS.                                                         application is limited to 15 pages.
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                                                               funding and/or time necessary to complete the
  developed to support research for independent
                                                               project is required.
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  this issue and avoid funding to these projects.            Selection Process
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 Small Exploratory Grant                                    following criteria:
  This grant is designed specifically for new
  investigators who have a preliminary concept               Significance: Does it address the significance of the
  they would like to develop into a research                 topic, either health burden or biological importance?
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Statement of Conditions                                         the Chairman and to the Society by both the
It is understood that any award approved and funded             responsible Institutional Financial Officer and
by the Scoliosis Research Society will be made on               the Primary Investigator no later than four (4)
the following conditions:                                       months after the termination of the grant.
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  the same person or project is received from other           returned to the Society.
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  furnished to the Chairman of the Research Grant
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  Committee by September 1 until the project is
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              Application for Research Funding “Standard Grant”
                                        Application Deadline: April 1
                          Applications must be submitted via e-mail to

Title of Project: Understanding Scoliosis: Addressing the need for a large cell repository and clinical database

Principal Investigator:
Name:             International Consortium for Vertebral Anomalies and Scoliosis (ICVAS)
Address:          The Stowers Institute for Medical Research, 1000 E. 50th St.
City, State, Zip/Postal Code:       Kansas City, MO, 64110
Country:          USA
Phone Number: (816) 926-4462

Project Period: 06/2008 to 06/2010

Total Amount Requested: $           $50, 000 per year for 2 years                              (Max: $50,000 per
year for 2 years)

Institutional Approval:    YES

Was Institutional Research Committee approval obtained? YES

Fiscal Officer:
Name: Holly Welsh
Title:   Treasurer
Address: ICVAS at the Stowers Institute, 1000 E. 50th St.
City, State, Zip/Postal Code: Kansas City, MO 64110
Country: USA
Phone Number: (816) 926-4462

Proposal Abstract: (Purpose and Description)

The International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) is a non-profit
organization whose mission is to better understand the etiology of scoliosis and vertebral anomalies
in humans. ICVAS attempts to achieve this mission by collecting and storing cell lines and clinical
data from patients with vertebral anomalies and scoliosis. This collection of samples and data will
prove to be a valuable resource to scientists, clinicians and SRS members. The potential value of
this project is infinite due to the fact that:
    1. Patients with congenital scoliosis are rare, therefore, limiting the size and power of many
    2. A large database collecting clinical data and tracking clinical outcomes does not exist
    3. Any member of ICVAS can obtain data or specimens, promoting work in this field
    4. By creating cell lines, there is a continuous source of DNA, allowing work to continue in the
    5. The project will ultimately expand to include idiopathic scoliosis, therefore, further fulfilling
         the mission of SRS
As outlined in the grant, the founding members of ICVAS support a candidate gene approach for
better understanding the genetics of scoliosis. This support is based on the success of pilot studies
in their individual labs and studies in the literature. However, their potential projects utilizing the
samples and data from ICVAS are only an example of the tremendous work that could be done by
making the most of this valuable resource.

Project Budget Summary:
Note: The preferred grant duration is two years.
 Period                          1st year               2nd year     3rd year               Total
 Personnel                   $10,000               $10,000                           $20,000

 Materials                   $40,000               $40,000                           $80,000
 Total Requested             $100,000 Total over two years

Supplemental Information:

Is work currently in progress in this research?

Have you applied elsewhere for support of this project?

If requesting funding more than $25,000 a year, are you able to secure additional moneys if the SRS only
approves partial funding?

Assurance Statement: (Grant will not be considered if box is not checked)
X The application summarized on these pages and further documented in the succeeding pages is submitted to
the Scoliosis Research Society for consideration.

Name: Olivier Pourquié (in the name of ICVAS)                                    Date: 03/25/08
                                  Principal Investigator

Name: Holly Welsh                                                                Date: 03/25/08
                                  Fiscal Officer

Detailed Application:

A.       Responsible Individuals:
Principal Investigator:     Olivier Pourquié (in the name of ICVAS)
Degrees:            Ph.D.
Present Position:           Investigator
Department:         Pourquié Laboratory
Institution:                Stowers Institute for Medical Institute
Address:            1000 E. 50th St.
City, State, Zip/Postal Code:          Kansas City, MO, 64110
Country:            USA

Collaborating Investigator:            Benjamin Alman (in the name of ICVAS)
Degrees:            M.D.
Present Position:           Head, Division of Orthopedic Surgery, Sr. Scientist, Program in Developmental
& Stem Cell Biology, Hospital for Sick Children
Department:         Orthopedics
Institution:        Hospital for Sick Children, University of Toronto
Address:            555 University Avenue

City, State, Zip/Postal Code:          Toronto, Ontario
Country:                    Canada

Institution where work will be done: Coriell Institute and various institutions that house ICVAS members
Address: ________________
City, State, Zip/Postal Code:____________________

B.        Proposed Budget (1st year)
                      Personnel                       % Effort              Salary            Fringe
 Database support                                                     $10,000

 Total Personnel Cost:                            $

                 Materials and Supplies                            Purpose                      Cost
                  (itemize major items only)
                                                      lymphoblastoid cell line creation
     Coriell Medical Institute                                                            $40,000
                                                      and storage

           Equipment (itemize major items only)                    Purpose                      Cost

Total cost for first year: $50,000

C.        Proposed Budget (2nd year)
                      Personnel                       % Effort              Salary            Fringe
 Database support                                                     $10,000

 Total Personnel Cost:                            $

                 Materials and Supplies                            Purpose                      Cost
                  (itemize major items only)
                                                      lymphoblastoid cell line creation
     Coriell Medical Institute                                                            $40,000
                                                      and storage

           Equipment (itemize major items only)                    Purpose                      Cost

Total cost for second year: $50,000

D.    Facilities Available: Describe the general facilities available for your research, including laboratory and
      hospital space and major items of permanent equipment:

After comparing other cell culturing services, ICVAS intends to use the Coriell Cell Repository to
create and store the lymphoblastoid cell lines and to distribute DNA from these cell lines to
interested ICVAS members. Staffed by experts in microbiology, molecular biology, cytogenetics,
cell biology, the cell repository at Coriell plays a vital role in modern biomedical research. The
repository houses many cell culture collections including those from the NIH, the National Institute
on Aging, the Juvenile Diabetes Foundation International, and the American Diabetes Association.
(See appendix for the quote and a description of these services.) The Stowers Institute for Medical
Research will house the ICVAS database. Specifically, this database has been paritally created in
the laboratory of Dr. Olivier Pourquie and housed on one of the institute’s servers. Coordination of
the proposed project will be ensured by Holly Welsh, a certified genetic counselor based in the
Pourquie laboratory. Given the collaborative nature of ICVAS, the screening for candidate gene
mutations will take place in participating members’ laboratories around the world, with their own
funding. Currently, the members of ICVAS include the Board of Directors (Benjamin Alman,
Olivier Pourquie and Philip Giampietro) and the Executive Council (Sally Dunwoodie, Peter
Turnpenny, Kenro Kusumi, Amaka Offiah, Alberto Santiago-Cornier, and Valerie Cormier-Daire).
There are also general members, whose numbers will undoubtedly increase to include additional
researchers and clinicians. Please see attached letters of support.

E.    Biographical Sketches: For Principal Investigator and any Collaborating Investigators, including date of
      birth, education (BA, BS, MS, PhD, MD) postgraduate training (internship, residency, fellowship), hospital
      and academic appointments, thesis titles and supervisors, and any relevant publications. Attach copies –
      five (5) pages maximum per investigator.

F.    Research Plan: This section should detail the specifics of the grant proposal. Use separate plain white 8.5
      x 11 inch paper for this section of the request. The research plan should answer at a minimum the following
      questions: Attach copies – 10 pages maximum.

             1.    Why is the work important?
             2.    What do you plan to do?
             3.    What has already been done?
             4.    How long will it take you to do the work?

It should be further organized as follows:
     I.       Specific Aims: A concise statement of what the research is intended to accomplish and/or why
              hypothesis is to be tested.
     II.      Significance: Briefly describe the background of the proposal, relating it to existing knowledge and
              published work. Identify the gaps in existing knowledge which the research is intended to fill and
              state concisely the importance of the project.
     III.     Materials and Methods: Detail the experimental design and procedures to be used to accomplish the
              Specific Aims of the Project. Describe a tentative sequence or timetable of investigation and the
              means by which the data will be analyzed and interpreted.
     IV.      Supporting Data: Briefly describe any previous work done on this project. Cite results obtained by
              yourself and others on this or closely related work (cite not more than five (5) personal publications).
              Those investigators with less than two (2) personal publications related to the proposed project
              should submit a letter of recommendation from a recognized investigator in the field.

                             Understanding Scoliosis:
        Addressing the need for a large cell repository and clinical database
I. Specific Aims

Congenital scoliosis is a rare deformity of the spine, occurring in 1 to 2 out of every 10,000 births,
negatively impacting health, with progressive deformity resulting in cardiopulmonary compromise
and/or neurological deficits. In addition, congenital scoliosis is often associated with anomalies of
other organ systems, most commonly the renal, neural and cardiac systems. Clinical management of
these patients is problematic primarily because it is difficult to predict the long-term effects of
anomalies associated with other organ systems. Most forms of congenital scoliosis are thought to be
sporadic in nature, but in fact, limited information is available on familial incidence.

Because congenital vertebral malformations are relatively uncommon and under studied, it is a
daunting task for any single group to accumulate the necessary clinical material to make progress in
understanding the underlying etiology. Therefore, we have established the International
Consortium for Vertebral Anomalies and Scoliosis (ICVAS) to bring together scientists who are
interested in the patterning of the vertebral precursors in the embryo and clinicians who are
involved in the treatment of patients with pathologies in which the process is disrupted. The mission
of ICVAS is to better understand the etiology of scoliosis and vertebral anomalies in humans. The
consortium attempts to achieve this mission by collecting and organizing DNA samples and clinical
data from a wide array of patients with vertebral anomalies in a repository. This collection will
ultimately prove to be a valuable resource for basic scientists, clinicians and families interested in or
affected by scoliosis.

Aim 1. To develop a repository of lymphoblastoid cell lines from patients with vertebral
Clinicians (geneticists and orthopedic surgeons) who are ICVAS members will obtain blood
specimens from patients with vertebral anomalies. These blood samples will be sent to Coriell
Institute for Medical Research where lymphoblastoid cell lines will be created. Members of ICVAS
can then receive DNA from these cell lines for molecular testing. Anonymous clinical information
from these patients will be entered into the ICVAS database hosted at the Stowers Institute for
Medical Research and made available to ICVAS members. (see letters of support)

Aim 2. To create a database of clinical phenotypes from patients with vertebral anomalies
An ICVAS database has been partially created to store and to analyze clinical data from patients
with vertebral anomalies. This database will interface with the Manteia database that was developed
in the Pourquié laboratory at the Stowers Institute, in which all phenotypic and expression data of
the segmentation process in vertebrate model organisms (fish, chick and mouse) will be stored. In
addition, the database will be able to track clinical progression and potentially provide insight into
the natural history of vertebral anomalies.

Aim 3. To screen for mutations in candidate genes in congenital scoliosis
Using the ICVAS database, stratification and clustering of the human vertebral phenotypes will be
used in combination with phenotypic comparisons with mouse segmentation mutations to predict
candidate genes or pathways that are more likely to be affected in patients with scoliosis. These
candidate genes will subsequently be sequenced by ICVAS scientific members using their own
independent funding in their individual laboratories. (see letters of support)

One of the overall goals of this project is to better understand the underlying causes of congenital
scoliosis in order to provide better stratification, diagnosis and treatment of this problematic
disorder. The outcomes from the ICVAS repository and the ICVAS database are limitless. In the
short term the project will provide a better correlation between genotype and phenotype and
potentially insight into clinical progression. In turn, this could lead to prenatal diagnosis options
and potential drug interventions targeting the signaling pathways involved in somitogenesis. The
collection of lymphoblastoid cell lines and the ICVAS database will undoubtedly enrich future
research and could potentially increase the rate at which the etiology of scoliosis is understood.

II. Significance

                                 Overview of Congenital Scoliosis

Congenital forms of scoliosis involve structural malformations of the spine including segmental
malformations (hemivertebrae or wedge vertebrae) and vertebral fusion and bars, suggestive of
defects in early developmental patterning. 1, 2 Conditions that fall into this category include:
generalized vertebral malsegmentation (e.g., spondylocostal dysostosis), regionalized conditions
(e.g., Klippel-Feil syndrome) or conditions involving only one or two vertebrae (e.g., Alagille
syndrome). 3 Congenital scoliosis can also be associated with anomalies of other systems, most
frequently involving renal, cardiac or neural abnormalities. 4 Clinical management of patients with
a congenital spinal deformity is problematic not only due to the inherent vertebral malformation, but
also due to the many additional anomalies that can be associated. An improved knowledge base of
the disease mechanism will enhance the ability to predict the course of the disease in patients,
particularly in children. This will substantially improve patient outcomes by allowing earlier
surgical intervention, lowering the associated complication rates and allowing improved overall
correction of the curvature.

Although most cases of congenital scoliosis were previously thought to be sporadic, recent evidence
points to a considerable genetic component. In a study of 400 patients with vertebral anomalies, a
familial occurrence rate of 20% was reported. 5 Interestingly, in this study idiopathic scoliosis was
also found to be associated with familial congenital scoliosis, suggesting that the distinction
between the two may not be absolute. This high familial occurrence rate was confirmed in a
separate study population by Dr. Kenro Kusumi. 6 Additionally, in these and other studies, it is
clear that a significant subset of patients (25%) with congenital scoliosis exhibit other associated
anomalies that commonly include the cardiac and renal systems, as well as spinal cord defects. 7
Although there are a few families within congenital scoliosis patient cohorts that could be utilized to
define genetic linkage, the variability of clinical phenotypes and associated anomalies indicate that
currently, it is difficult to define groups of families that are likely to have the same underlying
molecular defect. A major goal of this study will be to develop a database of clinical phenotypes in
this disorder, to aid in defining the underlying syndromes and to prioritize families for further
genetic analysis.

There is some confusion regarding the classification of congenital vertebral malformations in
different subgroups. Thus far, three important subtypes have been characterized, and the associated
mutations have been identified. Remarkably, all the mutated genes are associated with Notch
signaling, a well-known pathway involved in the control of vertebral segmentation during
embryogenesis. Homozygosity linkage mapping in consanguineous Arab-Israeli and Pakistani
pedigrees with a particular form of spondylocostal dysostosis (SCD1) led to the discovery of
multiple mutations in the Notch ligand DLL3. 8 Mutated DLL3 in humans results in abnormal

vertebral segmentation throughout the entire spine, with all vertebrae losing their normal form and
regular three-dimensional shape. A milder form of spondylocostal dysostosis (SCD2) has been
associated with a 4 bp duplication in the MESP2 gene. 9 Recently, a mutation in another Notch
pathway member, LFNG, was found in a family with spondylocostal dysostosis (SCDO3). 10.

                                      Molecular Embryology

The vertebral column is formed from a subset of cells, the sclerotome, that is derived from the
segmental somites. Somites begin to form soon after gastrulation and arise in a progressive manner,
as paired epithelial structures bud off from the unsegmented presomitic mesoderm (PSM) on either
side of the developing spinal cord. As they mature, somites become subdivided into three regions:
the sclerotome, myotome and dermatome. The sclerotome goes on to form the ossified structures of
the vertebrae and the ribs, while the dermatome forms the dermis of the back, and the myotome
forms the axial skeletal musculature as well as some other non-axial muscles. As the somites form,
they gain anterior-posterior positional identity that is later translated into the different shape and
form of the vertebrae from the cervical through the thoracic to the lumbar. The ordered segmental
structure of the somites provides the scaffold that determines the later segmental migration of the
motor neurons exiting the spinal cord and the intersomitic blood vessels and thus, is key to the
normal integrated development of the body axis. The striking segmented pattern of the human spine
is established during embryogenesis when somites, the precursor of the vertebrae, are rhythmically
added to the forming posterior part of the embryo. The periodicity of somite distribution and
production has led to postulate the existence of an oscillator or clock acting in the cells of the PSM.
The oscillator is thought to set the pace of the segmentation process by generating a periodic signal
subsequently translated into the periodic array of somite boundaries. The first evidence of such a
molecular oscillator, termed the segmentation clock, came from the observation of periodic
expression of c-Hairy1, a basic helix-loop-helix (bHLH) transcription factor in PSM cells in the
chick embryo. 11 A growing number of genes (“cyclic genes”) that exhibit a seemingly dynamic
expression pattern in the PSM has now been characterized in fish, frog, birds and mammals,
suggesting that the segmentation clock has been conserved in vertebrates, including humans. An
important output of the oscillator is the periodic, coordinated and mutually exclusive activation of
the Notch/FGF and Wnt signaling pathways in the PSM, which likely plays a critical role in the
initial definition of the segmental domain. Thus far, the three mutations associated with human
congenital scoliosis have been found in genes associated with the segmentation clock mechanism.

Data from mouse mutants have already proved to be highly predictive in identifying candidate
genes for congenital scoliosis. For instance, identification of a mutation in the mouse Notch ligand
Dll3 as the cause of a severe spine segmentation defect in the mouse Pudgy mutant led to the
prediction that the human DLL3 homologous gene was located in the 19q13.1 region. 12, 13 This is
syntenic with the mouse chromosome 7 interval containing the Dll3 gene; thus, Dll3 was considered
a candidate gene potentially involved in spondylocostal dysostosis in consanguineous families,
demonstrating linkage to 19q13.1. 8, 14-16 This was consistent with previous observations supporting
the role of the Notch signaling pathway in mouse and chick in the segmentation of the embryonic
somites from which the vertebrae develop. 17 Recently, another gene involved in mouse somite
patterning, MESP2, has also been shown to be mutated in another form of spondylocostal
dysostosis. 9

Classical linkage analysis for vertebral anomalies is limited due to the paucity of genetic
information available on these patients. Therefore, we believe that the study of mutant mice
showing spine segmentation defects will provide candidate genes for a number of human congenital

anomalies. Thus, cross-comparison of mouse phenotypes of mutations in genes involved in somite
formation and patterning with phenotypes of human congenital scoliosis patients will aid in defining
additional candidate genes for the human scoliosis syndromes.

Whereas only genes associated with the Notch pathway have been implicated in congenital scoliosis
thus far, Notch is not the only pathway involved in the control of the segmentation of vertebral
precursors. Recently, it has been shown that the Wnt, FGF and retinoic acid (RA) signaling
pathways also play important roles in determining the regular patterning of the developing somites.
The Axin2 gene, which is a negative regulator of Wnt signaling, also shows oscillating expression in
the PSM, out of phase with the Notch components. Mutation of Wnt3a inhibits oscillatory gene
expression and leads to severe truncation of the axis, suggesting that Wnt signaling is required
upstream of the Notch pathway to establish the somite clock. Microarray studies in the mouse have
identified a group of cyclic genes belonging to the Wnt pathway. Mutation of several of these Wnt-
associated genes, such as the secreted Wnt inhibitor Dkk1 or the transcription factor SP5 in mouse,
leads to spine segmentation defects that are usually different from those associated with the Notch
pathway. Because these defects are often less severe than those seen with the Notch pathway
mutants, they are good candidates to account for milder forms of congenital scoliosis. Recent
overexpression and loss-of-expression studies suggest that FGF and RA act in opposing gradients in
the PSM to establish the actual position where the somite boundary will form. In addition, RA is
required to ensure coordination of patterning on the left and right sides; blocking RA action leads to
desynchronization of the clock on either side of the midline. 18 This finding has potential
implications for scoliosis syndromes which are often biased in a left-right direction. Therefore, all
of these genes should be considered as potential candidate genes and could potentially be associated
with human vertebral anomalies.


The International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) was founded in 2006
by scientists and physicians interested in elucidating the etiology of scoliosis. The mission of
ICVAS is to better understand the etiology of scoliosis and vertebral anomalies in humans. The
consortium attempts to achieve this mission by collecting and organizing DNA samples and clinical
data from a wide array of patients with vertebral anomalies. This collection will ultimately prove to
be a valuable resource for basic scientists, clinicians and families interested in or affected by
congenital scoliosis.

ICVAS was incorporated in the state of Delaware in 2007 and was granted non-profit status
(501(c)3) by the federal government the same year. The Consortium consists of two governing
bodies. The first governing body is the Board of Directors, comprised of a Chairperson (Dr.
Benjamin Alman), a President (Dr. Olivier Pourquié), a Secretary (Dr. Phillip Giampietro) and a
Treasurer (Holly Welsh, M.S.). The Board is elected to two-year terms by the second governing
body, the Executive Council. The Executive Council is currently comprised of the founding
members of the Consortium: Dr. Benjamin Alman, Dr. Olivier Pourquié, Dr. Philip Giampietro, Dr.
Sally Dunwoodie, Dr. Peter Turnpenny, Dr. Kenro Kusumi, Dr. Amaka Offiah and Dr. Valerie
Cormier-Daire. In addition, an Advisory Board has been created which offers counsel to the Board
and the Executive Council. The Advisory Board consists of individuals who have made major
contributions to the field of vertebral malformations. A study coordinator/certified genetic
counselor, Holly Welsh, directs the basic functions of the consortium at the Stowers Institute for
Medical Research in Kansas City, Missouri.

The general members of ICVAS consist of researchers and clinicians from around the world. In
order to become a member of ICVAS, an individual must agree to specific requirements as
delineated in a membership document. First, an individual must agree to contribute samples or
novel research ideas. Second, he/she must agree to acknowledge ICVAS in any publications
generated from work using ICVAS samples or data. Third, he/she must agree to work within the
ethical framework set forth by their institutional Internal Review Board (IRB).

Finally, ICVAS has created a novel approach to categorizing vertebral anomalies on X-ray. This
novel classification system was recently published in Developmental Dynamics and will be
submitted to additional publications in the near future 19(see appendix). The need for this system is
evident in the fact that the current nomenclature is not standardized and fails to address the diversity
of radiologic phenotypes. The goal of the ICVAS classification system is to create a less confusing,
more clinically applicable system that can be readily transferred between humans and animal
models. This classification system will be used when describing radiologic findings in the ICVAS

Of course, one major hurdle in establishing such a consortium is funding. During its initial phase of
development (December 2004- November 2006), the candidate gene approach was validated (see
supporting data below), the seeds were planted for the consortium’s development, and the Stowers
Institute provided generous financial support. The cost to fund the developments of this initial
phase was $34,000. During the second phase of ICVAS development (December 2006- January
2008) the consortium was incorporated, legal documents finalized, and liability insurance secured
costing approximately $180,000. Again, the Stowers Institute for Medical Research funded these
endeavors. During this phase of development the University of Toronto also agreed to contribute
$25,000 to seed the ICVAS cell repository. During these initial phases of development, ICVAS
established a solid framework for success, propelling the consortium to publicize its mission (see, attract members and seek funding from additional sources.

III. Materials and Methods

Aim 1. To develop a repository of lymphoblastoid cell lines from patients with vertebral
A key component of ICVAS is the collection of samples and data under an informed consent that
allows clinical, laboratory and DNA data to be shared with all investigators. ICVAS will provide an
infrastructure for resource utilization that will enable research by providing centralized facilities for
access to human samples and data. As part of this protocol, blood will be collected and
lymphoblastoid cell lines established to provide a renewable source of DNA in order to facilitate
future studies on scoliosis. The use of cell lines is of vital importance due to the scarcity of such
patients. The ICVAS protocol related to the storage and retrieval of the specimens is in the process
of being submitted to Western IRB. Currently, only patients with congenital vertebral anomalies
and infantile scoliosis are eligible to contribute samples and data. However, as ICVAS develops
and funding is secured, eligibility will be expanded to include patients with idiopathic scoliosis.

Patients with vertebral anomalies will be recruited during routine orthopedic clinic visits. Upon
agreement to participate in an ICVAS study, patients will sign a consent form (approved by the
institution’s IRB). Blood will be drawn and labeled with an ICVAS identification number. Samples
will then be sent via overnight courier to Coriell Institute (see Appendix) where leukocyte cultures
will be established and cultures cryopreserved in liquid nitrogen for future use. When patients agree
to take part in an ICVAS study, they will be asked a series of questions about their medical history.

This clinical data will then be submitted, without identifiers, to the ICVAS database. Patients who
agree to enroll in an ICVAS study will not incur any charges related to the study, nor will they be
compensated for their involvement. ICVAS will be the administrator of the repository of
lymphoblastoid cell lines and also the administrator of the usage agreements with recipient

A timetable for the creation of a cell repository from patients with vertebral anomalies is, in part,
dependent upon the rate at which samples are collected. A collaborative relationship has been
established between Dr. Nigel Price at Children’s Mercy Hospital in Kansas City, Missouri and Dr.
Olivier Pourquié (see letter of support). Assuming that funding is available, a collection of samples
from Children’s Mercy Hospital could be secured as early as June of 2008. It is anticipated that 30
samples could be collected from this center in 2008. In addition, it is anticipated that an additional
30 samples could be collected in 2009. Dr. Philip Giampietro, one of the founding members of
ICVAS, has consents and samples from 90 patients with vertebral anomalies. Assuming that
funding is granted, he is willing to contact some of these patients to obtain consent for samples to be
sent to the ICVAS repository. These samples are the property of Marshfiled Clinic. This could
generate approximately 30 samples (see letter of support). ICVAS also has support from other
orthopedic surgeons such as Dr. Lee Segal at Phoenix Children’s Hospital (see letter of support).
Therefore, we believe that we can recruit several orthopedic surgeons from major medical centers to
agree to become members of ICVAS and contribute samples. These future members will generate
approximately 150 samples in 2008-2009. See Table 1 below.

                    Table 1: Timetable for the Creation of
                              Lymphobalstoid Cell Lines
                    Source of Patient  Potential Number of
                    Material           Samples
                    Children’s Mercy   60 samples by Dec 2009
                    Marshfield Clinic  30 samples by Dec 2009
                    Phoenix Children’s 30 samples by Dec 2009
                    Future ICVAS       150 samples
                    Total number of    270 samples
                    samples in ICVAS
                    repository by Dec

Aim 2. To create a database of clinical phenotypes from patients with vertebral anomalies
Dr. Olivier Tassy, a bioinformatics specialist, has created the ICVAS database in the laboratory of
Dr. Olivier Pourquié which is housed on a VMWARE server at the Stowers Institute. This server is
a dual Intel Xeon 5160 64 bits 3Ghz dual core processor with four gigabytes of RAM memory and
40 gigabytes hard drive. The database runs on LINUX Red Hat 3.4 and was created using the
PostgreSQL database management system (DBMS). Members of ICVAS are able to access the
database through, a domain which has already been purchased by the Consortium.
The database offers members several levels of security, requiring passwords to access the server and
to enter data, as well as securing each member’s individual data from being accessed by other

Members of ICVAS will use the database to enter anonymous clinical data gathered from patients
with vertebral anomalies who have consented to contribute a sample to the ICVAS cell repository.
This clinical data will then be stored in the database under each patient’s ICVAS identifier, along
with a link to the sample at the ICVAS repository at Coriell Institute. Through the ICVAS database,
members will be able to obtain the necessary paperwork from Coriell to order samples. In addition,
the database can store and analyze the clinical information from patients with vertebral anomalies
and house copies of pertinent de-identified records and x-rays. Therefore, members interested in a
patient with a specific vertebral anomaly or a specific pattern of anomalies will be able to select
patients, comparing data or ordering samples. As mentioned previously, the ICVAS database will
have the capability to interface with the Manteia database.

Manteia is a database created to gather a wide variety of information concerning the segmentation
process in vertebrate model organisms (fish, chick and mouse). This data includes genomic
expression and phenotypic descriptions collected either from public databases or generated in Dr.
Olivier Pourquié’s laboratory. Mouse phenotypes are described using the mammalian phenotype
ontology created by Mouse Genome Informatics (MGI) at the Jackson Laboratory in Bar Harbor,
Maine. This ontology provides a formatted set of terms to describe a multitude of phenotypic
features in mice. Manteia will be reorganized and extended to take into account the ICVAS
nomenclature (see appendix) for spinal defects and other human phenotypic findings, making it
possible to use the same data model to describe both human and animal developmental defects. This
will allow researchers to compare the phenotypic findings between human subjects and the
experimental data available for model organisms such as mice. Based on the candidate genes that
have been identified in mice, researchers could then extrapolate the target genes to determine if
there is a phenotypic effect in humans. These candidate genes could then be tested in model
organisms, while human blood samples from the repository could be used to confirm these

In addition to improving the existing database, an additional arm of the database will be created that
allows for ICVAS members to track associations between mutations and clinical course.
Deciphering such a molecular classification of vertebral anomalies in humans would be invaluable
for clinicians, as it would help them predict the evolution of the disease and potentially improve
decision making with respect to the need for surgery. Tracking the clinical outcomes of spinal
defects will also provide great insight into the natural history of such differences. As with the
ICVAS cell repository, the database will ultimately expand to include patients with idiopathic

Aim 3. To screen for mutations in candidate genes in congenital scoliosis
Select samples from the ICVAS repository will be screened for mutations in candidate genes. This
selection will be based on the knowledge of mutations that cause congenital scoliosis in mice and of
genes dysregulating the somite oscillatory clock. Members of ICVAS will prioritize the candidate
genes tested by comparing phenotypic information from humans and mice to select genes for
analysis. If a mutation is identified, phenotypically similar patients would be tested, determining
which clinical characteristics are associated with the genotype. Ultimately, focused studies will be
undertaken testing a larger patient cohort to determine if the genotype can indeed be used to predict
the phenotypic characteristics.

Candidate genes will be selected from genes identified in a screen of somite cycling genes and from
known genes identified in mice that develop congenital scoliosis. The list of current candidate genes
are listed in Table 2. This list is not meant to be inclusive, but includes genes for which mouse data

are available and suggestive of possible involvement in somite patterning. However, the
combination of a more detailed phenotypic classification in humans and additional genetic
screening in mice will influence the choice of candidate genes. Additionally, it is expected that the
group will move rapidly to high throughput sequencing strategies such as 454 or Solexa, which will
permit the simultaneous sequencing of a large number of candidate genes and of their associated
genomic regions.

                  Table 2: Genes Implicated in the Oscillator
                           Clock During Somite Formation
                  Gene               Chromosome Location
                  DLL3               19q13
                  MESP2              15q26.1
                  LFNG               7p22
                  HES7               17p13.2
                  NOTCH1             9q34.3
                  HES1               3q28-q29
                  HEY2               6q21
                  AXIN2              17q24
                  NKD 2              5p15.3
                  NKD1               16q12
                  LRP6               12p13.3-p11.2
                  DKK1               10q11.2
                  CYP26              10q23-q24
                  RALDH2             15q22

Genotype results could then be correlated with detailed information in the clinical database and
trends in clinical findings (e.g., associations with anomalies in other organ systems or with certain
morphologies of spinal malformations). Based on this analysis, ICVAS members could provide
clear information about the association of specific clinical characteristics with specific mutations.

IV. Supporting Data

   Pertinent publications
   1. Palmeirim I., Henrique D., Ish-Horowicz D., and Pourquié O. (1997). Avian hairy gene
       expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis.
       Cell 91:639-48.
   2. Pourquié O. (2003). The segmentation clock: converting embryonic time into spatial pattern,
       Science 301(5631):328-30.
   3. Dubrulle J. and Pourquié O. (2004). fgf8 mRNA decay establishes a gradient that couples
       axial elongation to patterning in the vertebrate embryo. Nature 427(6973):419-22.
   4. Vermot J. and Pourquié O. (2005). Retinoic acid coordinates somitogenesis and left-right
       patterning in vertebrate embryos. Nature 435:215-20.
   5. Dequeant ML, Glynn E, Gaudenz K, Wahl M, Chen J, Mushegian A, Pourquie O. (2006) A
       complex oscillating network of signaling genes underlines the mouse segmentation clock.
       Science Dec 8;314(5808):1595-8. Epub 2006Nov9.

   1. Purkiss SB, Driscoll B, Cole WG, Alman B. Idiopathic scoliosis in families of children with
       congenital scoliosis. Clin Orthop Relat Res 2002 Aug;(401):27-31.

   2. Alman BA, Raza SN, Biggar WD. Steroid treatment and the development of scoliosis in males
      with duchenne muscular dystrophy.J Bone Joint Surg Am 2004 Mar;86-A(3):519-24.
   3. Alman BA. A classification for genetic disorders of interest to orthopaedists. Clin Orthop
      Relat Res 2002 Aug;(401):17-26.
   4. Hopyan S, Gokgoz N, Poon R, Gensure RC, Yu C, Cole WG, Bell RS, Juppner H, Andrulis
      IL, Wunder JS, Alman BA. A mutant PTH/PTHrP type I receptor in enchondromatosis. Nat
      Genet 2002 Mar;30(3):306-10.
   5. Li CF, Wei RY, Baliko F, Bapat B, Alman BA. An association between the 4G polymorphism
      in the PAI-1 promoter and the development of aggressive fibromatosis (desmoid tumor) in
      familial adenomatous polyposis patients. Fam Cancer 2006 Dec 8.

The segmentation clock was initially identified in the Pourquié laboratory, which has been
subsequently focusing on the elucidation of the molecular mechanisms underlying this oscillator.
The Pourquié laboratory has further identified several of the cyclic genes (including Lfng mutated in
SCD3 in humans) in chick and mouse embryos and characterized the role of FGF signaling in the
control of somite boundary formation. Furthermore, they have demonstrated the role of RA in the
control of somite symmetry. Recently, they undertook a microarray screen to identify cyclic genes
in the mouse which led to the identification of novel genes and pathways associated with the
segmentation process. Some of these genes show vertebral anomalies when mutated in the mouse,
thus constituting interesting candidate genes for sequencing in human patients.

The candidate gene approach proposed in this application was validated in a pilot screen carried out
by the Pourquié laboratory in collaboration with the Dr. John Emans from the Department of
Orthopedic Surgery at Boston Children’s Hospital. Dr. Pourquié selected a list of six genes based on
their previous association with human congenital scoliosis and on their mouse mutant phenotypes.
The coding sequence of the six genes was sequenced at the Stowers Institute in a cohort of 30
patients with various degrees of vertebral anomalies. A novel homozygous mutation was identified
in a 12-year-old female, of Puerto Rican descent, with a severe form of spondylothoracic dysplasia
(STD). This patient had a single-base pair substitution mutation (307G>T) in Mesp2. The mutation
results in the replacement of a glutamic acid codon (GAG) at position 103 with a stop codon (TAG)
and the creation of a novel SpeI restriction enzyme site

Spondylothoracic dysplasia (MIM 277300) has been characterized as an autosomal recessive
disorder with high prevalence in the Puerto Rican population which currently comprises 49% of the
STD cases reported in the medical literature.20 Patients with STD have a short stature due to a short
rigid neck and thorax, and a “crab like” chest resulting from the fusion of the ribs at the costo-
vertebral junctions 20. Because of the similarity of the phenotype observed in the Puerto Rican
patient from Boston with the classical STD patients from Puerto Rico, a collaboration was
established with Dr Alberto Santiago-Cornier, who has worked extensively with these patients. The
Mesp2 gene was sequenced in a set of eight families. All of the affected offspring were found to
exhibit the same recessive mutation as that identified in the patient from Boston, arguing in favor of
a founder effect. Therefore, this approach led to the identification of the molecular nature of a
classic form of STD in the Puerto Rican population, thus validating a candidate gene approach. The
results of this pilot project are under revision for publication in the American Journal of Human
Genetics (February 2008)


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