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Genetic Analysis of
Multiple       Sclerosis


   in   EuropeanS

    Jesus College, Cambridge

        3 – 4 April 2000
                                                                                     2




SESSION 1


The analysis of complex traits
Chairman : Kees Lucas




Alastair Compston opened the meeting saying how pleased he was at the
enthusiastic response to GAMES. He acknowledged the UK and Northern Ireland
Multiple Sclerosis Societies, the Wellcome Trust, the Dutch Multiple Sclerosis
Society and PE Biosystems for supporting the meeting, and thanked the Cambridge
team, especially Katrina Dedman and Julia Gray for organising the event.
GAMES follows a tradition which began in 1984 with groundbreaking epidemiological
studies. Following the 1989 meeting in Cambridge, DNA collection started in earnest.
Initial efforts concentrated on biological and immunological candidate genes for
which markers were available, but it soon became evident that an alternative
approach systematically screening the whole genome would be needed. Four
linkage based whole genome screens have been completed. No regions of linkage
with genome-wide significance have yet been identified, although several regions of
                           1 2 3 25
interest have been found          . Heterogeneity and the presence of epistatic effects
were illustrated by segregating the results according to      HLA haplotype4. It has
become clear that the genes conferring susceptibility to MS individually have only a
modest effect. A change of strategy from linkage to family based association studies
on a large scale now seems necessary.
The GAMES experiment should accelerate and could solve the problem of the
search for MS susceptibility genes. Our hope is that in five years the current map will
have been refined, some peaks will have disappeared from the list of potential
candidates for the location of susceptibility genes, whilst others will undoubtedly
have become more significant. Some of these regions may be ubiquitous and some
domestic to certain populations. Hopefully someone will have spotted a relevant
gene
                                                                                    3


John Todd described his experience in the genetic dissection of another complex
disease, IDDM. One of the main difficulties is to conduct a study with sufficient
power. The main requirements for such studies are that large sample sizes and high
throughput automated technology 5 are needed 6. Nominal p-values of at least 5x10-8
are required before claiming positive results (genomewide significance)7. Further
correction for multiple testing involved in genome screens is needed, especially
when analyses are stratified.
For genes with modest effects (s <2) the number of samples required to achieve
genome-wide significance can realistically only be achieved if either case-control or
family based association studies, rather than sib pair based linkage studies are
performed. The power of case control studies can be increased by using more
controls than cases (given that controls are generally easier to collect). These
controls could be used for studies of several diseases. Background population allele
frequencies can be estimated by looking at non-transmitted alleles from parents of
simplex cases. An experiment comparing populations from the United Kingdom,
Sardinia, Finland and the United States has demonstrated that linkage disequilibrium
(LD) appears to be similar in each population, and rather more extensive than
previously suggested. Microsatellites up to 1 centimorgan apart are often found to be
in LD.
Professor Todd argued that the identification of susceptibility genes would ultimately
require “direct” association mapping, i.e. the testing of all known Single Nucleotide
Polymorphisms (SNPs), which would necessarily include the causative ones, in very
large case control populations. He described developments, which could potentially
                                    8 9                                        10
make such an approach realistic        , including the role of DNA pooling       . He
suggested that autoimmune loci may be shared between diseases and summarised
the evidence for chromosome 18 and 2 (CTLA4).




David Clayton discussed problems associated with the statistical analysis of
               7
complex traits . The power to detect the relevant genes is limited by their modest
genetic effects, as reflected by the small recurrence risks which they determine. An
alternative to typing all available polymorphisms is to rely on linkage disequilibrium
and type a smaller numbers of markers (“indirect association studies”).
Demonstration of population association requires markers to be very close to the
functional gene (or the marker actually to be the functional gene). In terms of power,
case control studies are the method of choice when testing for association, although
                                                                                        4


the selection of controls is crucial since unobserved population stratification may
create bias. However, this problem only has a substantial effect in extreme
situations, when it should be easy to spot. Family based association studies (i.e.
TDT) protect from effects of stratification, but are less powerful because of the intra-
                                11
familial genetic similarities    . The effects of multiple testing have to be taken into
account and require more rigorous significance levels to be employed. A linear
correlation exists between log (significance) and required sample size. Thus,
increasing significance levels to the high levels suggested by Risch should not be
prohibitive. Linkage analysis does not depend on the vagaries of population history
and, for many of the genes involved in complex traits, it may well be possible to
demonstrate linkage with genome-wide significance if efforts are made to collect
sufficient families. In short, it would be premature to abandon linkage and efforts with
this method should continue.


Arne Svejgaard described the relation of HLA and multiple sclerosis. The HLA
complex was sequenced in 1996. It extends over 3.6 Mbp (3.6 cM) on the short arm
of chromosome 6. It has 128 functional genes, 40% of which have immune function.
The associations between multiple sclerosis and HLA A3, DR2 and B7 were
                                     12
demonstrated in the 1970's            .   Spurkland et al. demonstrated in 1997 that
susceptibility is conferred both by HLA DRB1*1501 and HLA DQB1*060213. The
association with TNF results from linkage disequilibrium. Madsen et al. showed
recently in an animal model that HLA-DR2 can mediate both induced and
spontaneous disease resembling MS, by presenting an MBP self-peptide to T cells14.
Narcolepsy, probably caused by alterations in the Orexin 2 receptor, is highly
associated with the same HLA haplotype as multiple sclerosis15.


General problems arising in the study of candidate genes were discussed by Jan
Hillert. The strategy for suggesting the right candidate is to choose genes with
specific biologic function and those mapping to an area of interest from previous
genome screens or implicated in animal models16. Replicating associations is made
difficult because of the genetic and allelic heterogeneity and the epistatic effects.
Relevant polymorphisms may be old and linkage disequilibrium therefore only
extending over relatively short distances. Positive findings should be confirmed with
linkage and association in at least two separate populations. Candidate genes of
interest in multiple sclerosis include CTLA417 18, which is important in T cell activation
(negative signal). Apo E alleles (which are associated with sporadic and familial
                                                                                     5

                                                               19
Alzheimer's Disease) may affect clinical course and severity     and the Interleukin 1
          20
complex        which may have a combined effect with HLA-DR on susceptibility to
relapsing multiple sclerosis and disease progression. Future candidate gene studies
should benefit from information emerging through sequencing the human and
mouse/rat genomes.


Bertrand Fontaine stressed that linkage and association based studies can be
employed in the search for candidate genes, which may not only be chosen to look
for disease susceptibility but also for treatment response or clinical course.
Difficulties in candidate gene approach include the high number of potential
candidates, and the possible absence of intragenic polymorphic markers within the
region of interest; for example, the oligodendrocyte growth factor complex offers very
promising candidate genes, but consists of more than 50 different factors, which
have effects on oligodendrocyte migration, differentiation, regeneration, survival or
proliferation, making it is difficult to select the most promising candidate. There may
be a role for one of the oligodendrocyte growth factors (TGFb3) in interaction with
HLA421. Future studies need to employ large sample sizes and consider clinical or
para-clinical subgroups as well as stratification for HLA.




The Cambridge group then talked about their vision of the GAMES experiment.
Alastair Compston showed a map illustrating where the various groups expressing
interest are based and emphasised that attending the meeting does not imply
signing up to a commitment. Some groups have already formed consortia (i.e. the
Nordic group), whilst others work independently. Several groups have significant
experience in the genetics of multiple sclerosis.
GAMES is a multi-centre linkage disequilibrium screen of the whole genome in
several European populations. 6000 microsatellite markers have already been
purchased and are available for those investigators wishing to play GAMES. The
sequence will be a screen of the genome using 6000 markers in DNA pooled from at
least 200 cases and 200 controls, followed by a second screen using family based
samples (a minimum of 200 trio families pooled into two samples). This means, that
four pools will be screened by each GAMES player (requiring a total of 24,000
genotypes). In addition to these association studies, efforts to collect multiplex
families will continue, and some linkage genome screens will also be performed
                                                                                      6


within GAMES. There are likely to be several types of GAMES players. Type 1
players will use markers provided by Cambridge, but will collect and screen samples
locally; those will be groups who already have access to fluorescence based
sequencing machines using ABIs 373, 377, 310 and 3700. Type 2 players will send a
member of the group to Cambridge to genotype and analyse samples which have
been collected and pooled locally. Type 3 players will collect and pool samples
locally and send these to Cambridge for screening by a member of the Cambridge
team. Type 4 players will collect their own samples but will then be visited by a
researcher from Cambridge to help with pooling and DNA extraction before the
samples are sent to Cambridge for genotyping and analysis.
GAMES will not inhibit local efforts and will support domestic projects, but players
must commit themselves to a spirit of collaboration and, after initial publication, make
their results available to the GAMES consortium.


Stephen Sawcer reviewed the theoretical background leading to the GAMES
experiment and discussed some practical issues. Since this provides the basis upon
which GAMES is designed and is therefore of special interest to all participants, his
talk is summarised separately and in more detail in Appendix 1.




Session 2


Multiple Sclerosis in Europe
Chairman: Ian McDonald




Annette Oturai represented the Nordic group consisting of seven centres operating
in four countries. Activity of the centres is co-ordinated by twice yearly meetings. A
central database and DNA bank are located in Copenhagen. The group has received
EU grants since the 1980s for the identification of candidate genes/loci . The
collection of 200 sib-pairs is almost completed, and a linkage screen is currently
                                                                                       7


ongoing. The collection of 1000 trios has just started, and at least 100 cases and
controls have been collected by each centre. Possible susceptibility loci identified in
                                                                      22
Denmark include two on chromosome 5 (D5S1953, D5S667)                      and one on
chromosome 17 (D17S787). A strong association of multiple sclerosis with HLA DR
2 is confirmed by the Danish group, demonstrating an odds ratio of 3.7.
Anne Spurkland reported on the progress of sample collection in Norway, where
cases and controls have already been collected for work on HLA23, immunoglobulin
regions and T-cell activation genes. A locus of interest is the SHC2D2A gene, which
lies close to the CD1 region; its product is expressed on activated T-cells. It is
implicated in EAE and some alleles are associated with multiple sclerosis even
though there is no linkage to flanking markers.
Jan Hillert explained that over 2,300 MS patients have already been collected in
Sweden, of which 1,300 are included in a registry. No effect of HLA on disease
severity has been demonstrated and a current project compares features of the 96
most benign cases with the 96 most severe ones.
Special features of the Finnish population were discussed by Pentti Tienari. Familial
clustering and high prevalence rates (i.e. 200/100,000 in Seinajoki) in West Finland
make the population particularly interesting for genetic analysis24. A genome screen
in multiple sclerosis demonstrated regions of interest on chromosomes 5, 6,17
        25
and18    . To date, 1527 families have been identified and 150 trios and 27 multiplex
families are collected.




Jeff Gulcher represented DeCode, the major centre for genetic studies in Iceland.
The Icelandic population offers unique opportunities since it consists of large
extended pedigrees for which about 36,000 ancestors are known. Databases of
family trees extending back more than five hundred years frequently reveal
relationships between individuals, most of whom are unaware of their extended
family. A genealogical approach, based on the fact that haplotypes may be identical
for these distantly related affected individuals, is currently being employed for the
                                        26
analysis of several complex diseases      . Definitions of phenotypes are broad, and
phenotype transmission is used to decide which of the extended families will be
included in the analysis. Using this approach it has been possible to identify at least
one locus with genome-wide significance in stroke patients (ischaemic and
haemorrhagic). Psoriasis is another example where a locus with a Lod score of 1.5
before and over 5 after HLA segregation could be identified in 300 related patients.
                                                                                     8


Iceland has currently 330 living Multiple Sclerosis patients, and blood has been
collected from 270. A genome-wide scan involving two thirds of the patients has not
identified any regions of unequivocal linkage.




Marcin Mycko explained that the prevalence of multiple sclerosis in Poland is
90/100,000, with about 14,000 individuals currently affected. The Polish population is
genetically homogeneous and there are few minorities. Blood has already been
collected from 700 affected individuals, including 50 mutiplex families. About 90
cases have been used for previous candidate gene studies, looking at the HLA
region and 2 polymorphisms of ICAM-127. Future studies looking at other candidate
genes are underway.




The group from Holland was represented by speakers from two centres. Chris
Polman from Amsterdam reported that previous attempts to dissect the genetic base
of multiple sclerosis have mainly taken the candidate gene approach, looking for
cytokine polymorphisms which are associated with clinical course or disability. The
                                                                        20
interleukin 1 receptor agonist is associated with disease progression     ; interleukin
     28
10        is associated with TNF production. Gerhard te Meerman from Groningen
emphasised that the dissection of complex diseases is compromised by weak effects
attributable to the involved genes. He pointed out that accurate determination of
allele frequencies is crucial for showing association and that power is limited by
features such as allele frequency mismatching. He felt that before 'blowing the start
whistle' for GAMES, two or three times as many markers were needed, the artefacts
inherent to estimating allele frequencies using pooled DNA had to be resolved, and
the possibility of null alleles limiting the usefulness of the markers in different
populations checked by direct sequencing of each marker in each population. He
proposed examining specific genetic regions in detail rather than performing a whole
genome screen. Mapping specific genes in the HLA region, for example, and
identifying the disease polymorphism might prove more useful than conducting a
potentially under-powered experiment and this should be postponed until appropriate
technology is available.
                                                                                      9


Herwig Carton reviewed the epidemiologic features of multiple sclerosis in Belgium
29
 . There are 10,000 affected individuals. The relative risk for family members is 12
                                                               30
for first degree relatives and 3 for second degree relatives     . Sample collection is
currently in progress from 675 affected patients and 2400 relatives. Several multiplex
families are included in this number. The genetic heterogeneity of multiple sclerosis
is reflected by the development of a multiple sclerosis like illness in patients with
Leber’s optic atrophy. The LHON registry in Belgium comprises 103 patients of
which 5 suffer from Harding’s disease, in which the clinical phenotype is identical to
classical multiple sclerosis with the exception that optic nerve involvement is more
severe 31.




Professor Jörg Epplen talked about work being performed by the Bochum group
                                                                                      32
from Germany. Projects are being conducted on several autoimmune diseases
(multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease are vasculitis) in
which the same control groups can be used. In Wegener’s Granulomatosis, only
5/10 microsatellite or SNP markers from the proteinase 3 gene region, which is
already implicated, show association with the disease. The HLA association in
multiple sclerosis illustrates that normal alleles in specific combinations can be
                                         33
important for conveying susceptibility    . An association study of the NFB cascade
is being performed in over 400 probands. SNPs may show limited polymorphisms.
The p50 and p65 gene in Caucasians, for example, has only two SNP
polymorphisms associated with the gene. Frauke Zipp from Berlin explained the
present lack of interaction between potential GAMES players in Germany. About 100
cases and controls have so far been collected in Berlin, but access to the Serono
database may provide an additional 700 cases. Although there is higher TNF,
lymphotoxin and IFN production in cases, TNF productions by T cell clones does
not correlate with TNF alleles, HLA might influence proinflammatory cytokine
production, but so far no correlation has been demonstrated in animal models.
                                                                                     34
Current work in Berlin looks at candidate genes from the apoptosis context             ,
especially those involved in T-cell regulation.


Northern Ireland has contributed significantly to the establishment of genetic
epidemiologic studies, participating in the UK sib-pair study and urban-rural
difference in risk. Stanley Hawkins explained that there is little migration in Northern
Island; in the North East, 71% of probands were born in the region and 91% in
                                                                                      10


Northern Ireland. Primary progressive multiple sclerosis accounts for 9– 25% of
patients, and has distinct features; less female preponderance, later onset and less
MRI lesion-load. There is lack of agreement on what constitutes PPMS; in Ireland
this means progression of disability from onset, allowing for episodes of deterioration
during inter-current illness. Long-term follow-up studies of patients considered to
have benign MS (i.e. EDSS <3 10 years after diagnosis) show that most have
progressed significantly 20 years after diagnosis. No HLA association with clinical
                                                   35 36
subgroups or disability has been demonstrated          . Sample collection in Northern
Ireland has already been performed in 174 RR/SP patients, 113 PP patients and 220
controls.




Graeme Stewart reviewed the epidemiology of multiple sclerosis in Australia, which
has a landmass comparable to the United States excluding Alaska, but only about 20
million inhabitants. The first British settlement was established 1788 and the
Federation formed in 1901. Even though the population is multicultural, most
inhabitants originate from the United Kingdom or Ireland. About 80% live in inner
city areas. Despite a common genetic background, the prevalence of multiple
sclerosis shows a striking variation with latitude, ranging from 75/100,000 in the
south to 12/100,000 in the sub-tropical areas. No association between risk of
developing multiple sclerosis and country of origin has been demonstrated. Blood
samples from 200 cases, 200 controls and 206 trio families have been collected;
87% are of Northern European origin and only 2% from outside Europe. An
Australian longitudinal database will be established from April 2000 to contain
information on about 2000 patients. A workshop planned for June 2000 will further
define plans for collecting prospective data and correlating genotypes with clinical
aspects. The Tasmanian population, which is ideal for genealogical studies, offers a
special opportunity in Australia. Several extended pedigrees have been identified
and a genome screen with 300 markers is underway.




Michel Clanet spoke for the French group. A national collaboration has existed in
France since 1994 with clinicians at 3 centres and a laboratory in Paris. The group is
mainly funded by the Multiple Sclerosis Society and so far has collected 261 simplex
families and 97 multiplex families. All patients fulfil the Poser or Goodkin criteria and
                                                                                  11


have been personally examined by a member of the group who records EDSS,
clinical course, duration and progression.
Francoise Clerget-Darpoux debated the most promising strategy for GAMES,
acknowledging that power calculations have to assume many unknown factors; the
size of the genetic effects sought, the extent of interaction and the presence of
heterogeneity are all unsolved; the optimum number of markers is unclear;
increasing the number will inflate the number of false positives. Questions about
what happens at the end of the "GAME" need to be addressed: who "owns"
interesting results when they become available; when and by whom will the meta-
analysis be performed; and who plays in what order? Any search for genes is
associated with risk, and the GAMES experiment is no exception, but the prospects
of success make that risk worthwhile.




SESSION 3


Multiple Sclerosis in Southern Europe
Chairman : Kees Lucas




Luca Massacesi spoke for the group from Northern Italy. A focused linkage based
genome screen looking at 14 promising areas in 41 Italian and 28 Sardinian
multiplex families was published in 199937. Analysis was separate for the continental
and Sardinian data set. Even though, the linkage scores obtained were low, some
markers (at 2p11, 3q21.1, 7p15.2 and 22q13.1) stood out as promising. Simon
Broadley then performed a linkage-based screen of the whole genome in 40
multiplex families using 322 markers. Regions of interest could be identified on
chromosomes 5, 10 and 16, but no area of genome-wide significance was
demonstrated. There was no peak in the HLA region, illustrating the low power of
linkage studies with relatively small sample sizes. Hits on chromosome 2 and 22
overlap with the UK screen, and others on chromosomes 8 and 10 have been shown
to be important in diabetes. A peak on chromosome 7 overlaps with an autoimmunity
cluster described by Becker.     Luigi Grimaldi described candidate gene studies
performed in Milan. Interleukin 1 receptor agonist is associated with occurrence of
                                                                                            12

                                                                         38
 disease and clinical course variability in 339 Italian MS patients . PECAM-1 gene
 39                             40
  and APOE polymorphisms             fail to show association with the risk of developing MS
 or clinical course. Two groups in the Milan area are currently recruiting patients for
 GAMES. So far blood samples from 325 sporadic cases, 110 trios and 155 multiplex
 families have been collected.




Maria Trojano explained that the prevalence of Multiple Sclerosis Southern Italy is
50/100.000 and currently about 2000 patients, including 300 from multiplex families
attend regular clinics. Multiple Sclerosis candidate genes41, already examined, include
various cytokines, metalloproteinase and mitochondrial mutations. A corporation of six
centres in South Italy aims to register all familial neurological diseases, define
predictive laboratory and diagnostic tests and analyse genetic factors. The first phase
of the plan includes a network creation where each centre will be connected to an
electronic database. Patients will be recruited if they fulfil Poser criteria for definite or
probable MS. Patients will be segregated in clinical subtypes during the second phase
and clinical events/disease progression recorded 42. The aim is to collect blood from
2000 cases, 1000 parents and 500 controls and perform TDT and case/control
studies on 7 microsatellite markers and 200 SNP’s in addition to looking for
mitochondrial mutations. Maria Liguori reported that 1200, including 87 multiplex
families patients, are currently being followed at the University Hospital of Bari,. For
the GAMES experiment, blood has already been collected from 125 MS sporadic
cases, 112 parents, 28 sib-pairs and 150 controls.




 Francesca Coraddu discussed epidemiologic features of Sardinia, where
 prevalence (150 /100,000) and incidence ( 80/ year) of multiple sclerosis are much
 higher than in surrounding Mediterranean countries, suggesting that isolated
 population growth has concentrated susceptibility factors.The special genetic basis
 of susceptibility is reflected by the established association with DR3 (DRB1*0301,
 DQA1*0501, DQB1*0301) and DR4 (DRB1*0405, DQA1*0501, DQB1*0302)
 haplotypes, which are distinct from those seen in Europeans of Caucasian origin 43.
                                                                                      13


A linkage genome screen in 56 multiplex Sardinian families, including 46 sibling
pairs, 3
sibling trios and 7 parent child pairs, screened with 327 microsatellite
markers,demonstrated no region of genome-wide
significance. However, regions of interest were identified on Chr 1q,Chr 10q and Chr
11p where the MLS reaches 1.8. The "Regional Multiple Sclerosis Centre" in Cagliari
has established a DNA bank for Southern Sardinia. At present 551 trios families, 62
multiplex families and 190 sporadic cases have been collected .




Giovannie Savettieri represented Sicily, where epidemiological surveys have been
conducted in 5 centres including follow-up studies in 3. Enna was found to have
prevalence rates of 53/100 000 in 1975 compared to 113.3/100.000 in 1995;
                                         44                                           45
prevalence was 43/100 000 in 1981             and 72.4/100 000 in 1998 in Monreale     ,
32/100.000 in 1975 and 51/100 000 in 198146 in Agrigento and 50/100.00 in
Caltanisetta City and Baghara in 1980 and 1994 respectively. No genetic studies
have yet been been performed in Sicily. The gradient in prevalence which contrasts
with Malta (4/100.000) is unexplained.




Xavier Montalban from Barcelona indicated that prevalence rates of 53-65/100 000
have been established Spain in the last 10 years. A collaboration of 3 neurological
centres which includes 25 Neurologists and 3 DNA Labs will be coordinated from
Barcelona (UNIC). The group in Barcelona, Madrid and Valencia. Funding is available
from the Spanish Multiple Sclerosis Society. Pablo Villoslada reported that blood for
GAMES has already been collected from 265 Simplex families, 25 Multiplex families,
200 cases and 200 controls. Special features of Spain which may be interesting for
genetic research are the Canary and Baleares Islands and several partially genetic
isolated areas( the Basque area in the North and Galicia in the Northwest).


        .
                                                                                      14


Berta Silva reviewed the epidemiology of multiple sclerosis in Portugal. There is a
national register of patients aiming to study the epidemiology of Multiple Sclerosis.
Prevalence was found to be 30/100.000 in 1996 and 49/100.000 in 1998.                  In
preparation for GAMES, blood samples from about 100 cases have already been
collected. Further collection of cases, controls and trio families is planned and will be
facilitated by recruiting probands from the National Register.


.




Pimpi Reyes-Yanez discussed epidemiologic features of the Canary Islands. The
population is Caucasian with genetic influences from Spaniards, Romans, Vikings,
Africans and others. The prevalence of Multiple Sclerosis was 6.8/100 000 in Las
Palmas in 1983 and of 18.3/100 000 in Lanzarote in 1987. The difference is probably
methodological but both regions show lower prevalence compared to mainland Spain
                                                                            47
(50/100 000). Susceptibility is associated with DR15 and also DR4            . The DR4
haplotype is different from unique Sardinian DR4 , but the same as the HLA DR4
associations seen in Jordan, Malaga or Turkey. This association may represent
genetic input from the Eastern Mediterranean.




                                                             48
John Milonas explained that studies in Northern Greece            between 1970 and 1984
demonstrated an incidence of 1.8/100 000/yr and prevalence of 29/100 000. Further
studies are needed, because the figures are probably higher, reflecting better
knowledge of the disease and better means of investigation. About 680 patients with
clinically definite MS (9% of which have a benign course) characterised in
Thessaloniki between 1990 and 2000 could potentially be recruited for GAMES.
Collaborations exist with centres in Crete, Athens and Padras who are also recruiting
patients, and blood collection is due to start next month.




Mefkure Eraksoy reported that the earliest publications on Multiple Sclerosis in
Turkey date from 1934 when Uzman suggested that the disease only occurs in
                                                                                       15


those who visit Europe. Further studies in 1949, 1954 and 1998 established that
clincal presentation and prognostic factors are similar to those seen in other
                      49
European countries      . The Turkish MS Study Group was established in 1993.
Currently 1075 patients with clinically definite, laboratory supported clinically definite
or laboratory supported probable MS are being followed in Istanbul. Patients are
seen mainly in two large hospitals, but also by 249 neurologists practising in the city.
At the University Hospital in Istanbul, 5% of neurological patients have MS, and
about 20-25% are newly diagnosed per year. Female/Male ratio is 2:1 and 3.2% are
multiplex families. Special epidemiological features of Turkey includes the high rate
of consanguineous marriages (1:5).




The first epidemiology studies of MS in Cyprus were undertaken in the early 1990’s.
Koulis Kyriallis reported that similar prevalence figures were found in the Greek
                            50
and Turkish communities      . Middleton and Dean found prevalence rates of 45/100
000 (similar to Sicily) in 3 rural parts of Cyprus in 1991. More recent numbers from
1999 show a prevalence 67/100 000 for clinically definite MS in the Greek population
of the island, with 15% constituting familial cases. The increased incidence and
prevalence figures in the last 30 years are probably due partly to increased use of
MRI imaging technology. However, preliminary data suggest that incidence
continues to rise so that prevalence rates may come close to 100/100 000, as in
northern European countries. Demographic features of MS patients seem to be
similar to other European countries. Blood has already been taken and DNA
extracted from 296 patients, 290 parents and 200 controls.




Multiple sclerosis seems to be rare in Arabs and the earliest reports date back to
1958 when studies were carried out in Iraq. Amir Al-Din explained that more recent
                            51
studies studies in Kuwait        suggest that prevalence rates for Palaestinians (23/
100.000) may be higher compared to other Arabs (13/100.000), particularly those
from the Gulf     (7.5/100.000). These figures are not influenced by migration of
Palaestinians during the wars of 1948 and 1957, and are similar in Jordan
(prevalence of Palaestinians 33/100.000 compared to 16/100.000 in Jordanians)52.
Studies on HLA susceptibility factors in Palaestinians53 show association with DR2,
DR3, and DQw1. DR1 and DR5 may have protective effects, as has already been
suggested in Kuwait where all patients were negative for DR1. The lack of a DR2
                                                                                    16


association in Kuwaiti Arabs is probably due to high prevalence of this allele in
controls. Multiple sclerosis in Jordan has different demographic features compared
to European countries. The country has a much younger population with a 60%
increase in < 14 year olds, creating a pyramidal age-stratification curve. There are
no other differences regarding prognosis, imaging and phenotype. The King Hussein
Medical Centre is the main neurological centre in Amman (Jordan) with a register of
800 patients. Information on some pedigrees still needs documentation but blood
collection, DNA extraction and pooling could be done locally.




SESSION 4


A consensus on how to play GAMES
Chairman : Professor I. McDonald




Mefkure Eraksoy talked about her experiences whilst organising the Turkish
GAMES group. She visited Cambridge for several weeks, to obtain more information
about GAMES and find out how the work is organised. Whilst still in Cambridge, she
formed of the Turkish Multiple Sclerosis Study Group (TMSGSG) which involves 34
neurological centres, at least one of which is located in each region of Turkey. The
majority involves University Hospitals. Funding for the experiment and ethical
approval are currently being applied for. Blood has already been collected from 200
controls, 180 cases and 180 trio families. Information about specific family structures
is being recorded and this is especially important in view of the high rate of
                                                                                       17


consanguinous marriage in Turkey. Professor Eraksoy said, that she encountered no
specific problems when recruiting centres for the experiment.




Mara Giordano discussed practical issues of pooling DNA samples. As a proof of
principle, she described an experiment pooling DNA from 400 individuals. DNA was
quantitated by Pico Green Fluorescent Assay and diluted to a final concentration of
20ng/ul. The pools contained 200 ul (4ng) of each DNA and 5 ul (100 ng) of the pool
was used for each PCR. Pools of different sizes were created after dividing the 400
individuals into 5 subgroups. In the first stage experiment, each subgroup pooled
together was analysed; in the second stage, 4 pools each containing 2 subgroups; in
the third stage 3 pools each containing 3 subgroups; and finally one pool of all the
individuals together. When comparing results with those achieved by individual
genotyping, it could be demonstrated that reliability increased with pool size, due to a
reduction of experimental and sampling errors. In the discussion that followed,
Professor Lucas suggested that the Cambridge group should provide a detailed
protocol describing methods to be used for pooling DNA. Stephen Sawcer pointed
out that it would not be possible (or appropriate) to be prescriptive since facilities
available to each group vary. He pointed out that several methods for constructing
pools have been used and are published (that suggested in appendix 4 is based on
the existing literature).. Each group should test their own pool using a positive control
such as the TNFa microsatellite which is known to be in LD with HLA or an SNP,
previously typed individually, should be typed in the samples pool. Although checking
the validity of pools is appealing, it is not clear how validity checking might be
performed and the tolerances which would be considered appropriate are undefined,
as are actions to take in the event of failure to meet these tolerances.


Clinical classification, definition of cases and the possible inclusion of subgroups is
another crucial issue of practical importance for GAMES. Michel Clanet stressed
the importance of strict inclusion criteria, especially in view of the fact that up to 10%
rate of mis-diagnosis in families. Cases classified as definite multiple sclerosis
should fulfil the following 6 criteria:
-   at least one objective abnormality on clinical examination,
-   predominant involvement of at least one CNS long tract
-   two or more episodes or stepwise progression with abnormal CSF
-   two or more CNS areas invoved ( including paraclinical evidence)
                                                                                     18


-   age between 10 and 60 years
-   alternative diagnosis excluded by appropriate tests
Only probands with definite or probable MS (5 out of the 6 criteria) should be
included. Most groups have used the Poser54 or the Goodkin55 criteria for previous
studies. The need for longitudinal evaluation of cases, i.e. use of progression indices
measuring EDSS per disease duration, should be addressed. The question of
stratification on clinical course arises, particularly when separating primary
progressive cases from other forms of the disease. Chris Polman drew attention to
the fact that any sub grouping invariably leads to loss of power. However if the
primary progressive group is to be separated, strict criteria must be employed. In the
ensuing discussion, it became clear that most GAMES players would be in favour of
separating primary progressive cases. Ian McDonald mentioned that new criteria for
the diagnosis of primary PPMS are due to be published in the Annals of Neurology
this year. Inclusion in the GAMES PPMS subgroup would require patients to be
definite PPMS (includes abnormal CSF) or probable PPMS (may allow for normal or
not available CSF result) according to these criteria. Benign cases should be
included in the relapsing remitting/secondary progressive group.




Oluf Andersen addressed ethical considerations. There is general agreement about
informed consent and ethics local committee approval as the basic requirement for
any study involving patients. The exact guidelines to be followed vary between local
authorities. The Swedish Research Council requires consent for storage of DNA and
informed consent for each new purpose         (a general acceptance for unspecified
causes is not acceptable). Exceptions are studies which closely resemble the original
aim. Information must be given on methods of storage and storage time, and donors
must be informed that storage can be terminated at their request. International
collaborations require that only coded material is distributed. Jeff Gulcher
emphazised the special ethical considerations in a small country where researchers
may know participating patients56. To ensure collaboration by hospitals and general
practitioners, a strict system of data protection is required. In Iceland, a third party
system exists where neither the participating doctors nor researchers have access to
the key necessary to decode patient numbers. Researchers requiring clinical
information must contact the third party who makes the information available to
them57. Bar codes rather than numbers on blood samples provide an additional level
                                                                                     19


of security. In the course of discussion, it was decided that the third party system
would be good for the GAMES experiment. Ian McDonald agreed that the
International Federation of Multiple Sclerosis Societies might be a suitable institution
and offered to discuss this internally.




Alastair Compston initiated the final discussion of the first GAMES meeting by
reviewing several issues that had been raised.       Whilst the experiment may lack
power, there are a lot of unknown factors necessary to make this judgement and
GAMES is the most powerful experiment that can currently be performed. On the
question of using either SNP based genome screens or a dense microsatellite map,
the latter is currently available and GAMES already uses 85 - 90% of informative
microsatellites. GAMES provides a unique opportunitiy to look at areas of the
genome which have never been systematically examined in several countries.
Taking a straw poll of all those present at the meeting it appeared that the vast
majority wanted to participate. The approximate number of type 1, type 2, type 3 and
type 4 players was 8,12,6 and 2 respectively.
One of the practicalities for GAMES is resources. Substantial funding for the 6000
microsatellite markers, the technical equipment and the screening of the UK
population has already been provided in Cambridge by the Wellcome Trust.
Application for an additional set of 400 markers for linkage studies in the
Scandinavian, Turkish and Australian population has been made to the MS Societies
of Great Britain and Northern Ireland. Application to the European Community for
funding a Marie Curie host institution are currently ongoing. If successful this would
provide a permanent salary for type 2 players to cover their stay in Cambridge. If a
type 2 player comes with funding, the Mary Curie salary could be distributed to other
GAMES players. Costs not covered by this scheme would need to be applied for
locally by the participating groups, such as those needed for sample collection, DNA
extraction and pooling. Type 1 players would need to cover the running costs of their
lab and genotyping, although markers will be provided by Cambridge, at the cost of
plates onto which they are aliquoted. The issue of how individual groups should best
apply for funding was raised. The option of applying individually carries the
disadvantage that several groups might compete for the same grants. The
alternative is to apply as a consortium. Michel Clanet was in favour of the latter and
offered to co-ordinate such an initiative.
                                                                                       20


International collaborations are prone to cause conflicts of interest and it is important
that the conditions of GAMES are transparent to all players. All groups are free to
publish their own results and      follow up interesting findings resulting from their
screen. Results obtained by each group will be made available to the rest of the
team after a suitable interval and included in the meta - analysis. Evolving data will
be published on a GAMES website. It still needs to be decided if all players or only
those who have made already made their data available would have access. It is not
required that other GAMES players are co-authors in interim publications (although
the Wellcome trust must be acknowledged). The meta-analysis will be published by
the GAMES group, not individual authors. A schedule for type 2 players will have to
be worked out and some groups may want to change to type 1 or type 3 in order to
move faster. One rate limiting step for the experiment is access for type 2 players
wishing to visit Cambridge. Possible solutions include recruiting a second centre.
Iceland offered this facility. A second alternative is to purchase a second ABI 3700. .
Stephen Sawcer pointed out some practical issues raised in the meeting, including
the theoretical possibility of multiplexing, the arrangement of plates to markers with
similar PCR conditions, and the allocation of chromosome X markers on separate
blocks.


Ian McDonald closed the first GAMES meeting by wishing everybody good luck and
demonstrating true sportsmanship by being the first GAMES player to be seen
wearing the official T-shirt.




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