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Genes and Immunity (2002) 3, 464–469
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A genome screen for linkage in Australian sibling-pairs
with multiple sclerosis
M Ban1,2, GJ Stewart2, BH Bennetts3, R Heard2, R Simmons4, M Maranian1, A Compston1 and
SJ Sawcer1
1
Neurology Unit, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK; 2Institute for Immunology and Allergy Research,
Westmead Millennium Institute, Westmead Hospital, University of Sydney, NSW, Australia; 3Department of Paediatrics and Child
Health, Children’s Hospital at Westmead, University of Sydney, NSW, Australia; 4Australian National Register of Multiple Sclerosis
Families, Canberra Hospital, ACT, Australia
The role of genetic factors in determining susceptibility to multiple sclerosis is well established but, despite the global
distribution of the disease, systematic efforts to locate susceptibility genes have concentrated exclusively on populations from
the Northern Hemisphere. We performed a genome wide screen of linkage in the Australian population using a panel of 397
microsatellite markers in 54 affected sibling-pairs. Multipoint linkage analysis revealed four regions of suggestive linkage (on
chromosomes 2p13, 4q26-28, 6q26 and Xp11) and 18 additional regions of potential linkage (at 1q43-44, 3q13-24, 4q24, 4q3134, 5q11-13, 6q27, 7q33-35, 8p23-21, 9q21, 13q31-32, 16p13, 16p11, 16q23-24, 17p13, 18p11, 20p12-11, Xp21-11 and Xq2328). Our results contribute to the available data adding new provisional regions of linkage as well as increasing support for
areas previously implicated in genetic susceptibility to multiple sclerosis.
Genes and Immunity (2002) 3, 464–469. doi:10.1038/sj.gene.6363910
Keywords: multiple sclerosis; linkage genome screen; genetic susceptibility
Introduction
Multiple sclerosis is a demyelinating disorder of the
central nervous system with a complex aetiology in
which unknown environmental factors are thought to
trigger disease in genetically susceptible individuals.1
Epidemiological analysis of twins,2,3 half-sibs4 and
adoptees5 confirm the involvement of genetic factors
while the role of environmental agents is demonstrated
by the results of migration studies.6 In Australia,
studies of the prevalence of multiple sclerosis have
been particularly influential showing a latitudinal
relationship similar to that seen in the Northern
Hemisphere with lower rates in northern regions closer
to the equator (11/100 000) than in the south (74/
100 000).7,8 However, overall, these prevalence rates are
lower than those seen in Northern Europe, even though
the majority of the Australian population originates from
Northern Europe, suggesting that, despite expressing the
same susceptibility genes, Australians may be at relatively reduced risk of multiple sclerosis through differential exposure to the causative environmental risk
factors.
To date seven whole genome screens for linkage have
been completed in multiple sclerosis, five in Northern
European9–13 and two in Southern European populaCorrespondence: GJ Stewart, Associate Professor, Institute for Immunology and Allergy Research, Westmead Millennium Institute, Westmead
Campus, University of Sydney, Westmead, NSW 2145, Australia.
E-mail [email protected]
tions.14,15 None of these screens identified statistically
unequivocal evidence for linkage but nearly all found
more regions of potential linkage than would be
expected to have occurred by chance alone. Several
regions show some degree of consistency between
screens, including the major histocompatibility complex
(MHC) on chromosome 6p21.
A systematic search for linkage in multiple sclerosis
has not previously been performed in a population from
the Southern Hemisphere. Here, we report a genome
screen for linkage in 54 Australian affected sibling-pairs
with multiple sclerosis using a panel of 397 microsatellite
markers.
Results
Results of the multipoint non-parametric linkage
analysis are shown graphically in Figure 1. A
Maximum lod score (MLS) value of greater than 1.8,
the threshold of suggestive linkage, was seen on
chromosomes 2p13, 4q26-28, 6q26 and Xp11. Another
18 regionsFon chromosomes 1q43-44, 3q13-24, 4q24,
4q31-34, 5q11-13, 6q27, 7q33-35, 8p23-21, 9q21, 13q31-32,
16p13, 16p11, 16q23-24, 17p13, 18p11, 20p12-11, Xp21-11
and Xq23-28Fhad MLS values X0.7, indicating potential linkage. The mean genetic information extracted
from the 54 families across the genome was 61%, ranging
from 16% on chromosome 19pter to 86% on chromosome
4 in a region covered by particularly informative
markers.
Australian MS linkage screen
M Ban et al
465
Figure 1 MLS calculated by MAPMAKER/SIBS at each point
along the genome is shown for each chromosome. The x-axis is
proportional to the length of the corresponding chromosome and
the map position of the markers is indicated by the tick marks. In
each case the y-axis is scaled from 0 to 2.5.
Discussion
We have completed a genome wide screen for linkage in
54 Australian affected sibling-pairs with multiple sclerosis using 397 markers and identified four regions of
suggestive linkage and 18 additional regions of potential
linkage. Under the null hypothesis of no susceptibility
for multiple sclerosis, a marker map of this density
would only be expected to generate a single region with
an MLSX1.8 and approximately 10 regions with MLS
values X0.7.16,17 As the number of observed peaks
greatly exceeds that expected by chance at both MLSX0.7
and X1.8, it is likely that at least some of these regions
harbour susceptibility genes. This interpretation is
further supported by the fact that two of the four regions
providing suggestive linkage have previously featured
in multiple sclerosis genome screens: a regions close to
chromosome 6q26 was observed in the UK,9 American,10
Nordic13 and Italian14 genome screens, whereas the
Figure 1 (continued)
suggestive linkage on chromosome Xp11 was previously
reported in the UK screen.9
Several of our regions of potential linkage also
coincide with those suggested in previous multiple
sclerosis genome screens. A region identified in the
present study on chromosome 1q43-44 was also seen in
the Italian screen,14 and linkage at 4q31, 5q31, 7q33-34
and 18p11 was additionally observed in the American
screen.10 Similarly, our potential linkage on chromosome
16p matches that observed in the Nordic13 and American
screen,10 as well as in a meta-analysis of the original
American, Canadian and British linkage screens.18 Since
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Australian MS linkage screen
M Ban et al
466
Figure 1
(continued)
16p was not identified in the two screens in Southern
European populations,14,15 this observation emphasises
that alternate combinations of genes may determine susceptibility to multiple sclerosis in different populations
(allelic and locus heterogeneity). Despite the wellestablished association of the MHC region with susceptibility to multiple sclerosis in several populations
including Australia,19,20 we only found modest evidence
for linkage in this region. While this result is intriguing
Genes and Immunity
Figure 1 (continued)
as greater than 90% of our subjects were of Northern
European origin, it reflects the limited statistical power
of the linkage approach in small samples of affected
family members.
Australian MS linkage screen
M Ban et al
Figure 1
(continued)
Although replication of linkage is the essence of
narrowing the search for chromosomal regions of
interest, failure to implicate any one chromosomal region
across several screens does not necessarily undermine
confidence that a susceptibility gene is genuinely
encoded at that site. Variation in the evidence for linkage
observed between studies can occur for a number of
reasons. For example, the genes conferring susceptibility
may differ between populations (genetic heterogeneity).
More importantly, the limited statistical power of
individual linkage screens means that random variation
is expected to play a large part in the final result. Since
the power is influenced not only by the number of
families studied but also by the frequency of susceptibility alleles in the population screened, variation in the
extent of evidence for linkage is to be expected.21
The two novel regions of suggestive linkage observed
on chromosome 2p13 and 4q26-28 in our screen harbour
a number of interesting immunological candidates. For
example, IL-2 and IL-21 which are both involved in T and
B cell proliferation map to chromosome 4q26-27, while
the cluster of genes for the immunoglobulin kappa light
chain and CD8 are encoded at chromosome 2p12. These
candidates are worthy of further study although previous study of IL-2 in the UK yielded negative results.22
A number of investigators have suggested that some
genetic factors influence susceptibility to a general
autoimmune disease diathesis against which background
additional genes determine which autoimmune condition develops. Studies showing increased prevalence
of autoimmunity in first-degree relatives of multiple
sclerosis patients23 and a higher than expected concordance of linkage results across genome screens from
different autoimmune diseases24 support this hypothesis.
A number of regions identified in our study overlap with
those found in other autoimmune disease linkage
screens. Chromosome 1q43-44 has been implicated in a
genome screen of rheumatoid arthritis25 as well as in
three independent studies of systemic lupus erythema-
tosus.26–28 Chromosome 16q24 also showed evidence of
linkage in inflammatory bowel disease29 as well as two
independent screens of rheumatoid arthritis,25,30 and the
region on 4q24 showed the strongest evidence of linkage
outside the HLA region in a genome screen of rheumatoid arthritis.25 In addition, the region of suggestive
linkage on chromosome Xp11 overlaps with a region
linked to insulin-dependent diabetes mellitus.31,32 Evidence also exists for an autoimmune susceptibility gene
on chromosome 4q26-28. An obvious positional candidate within this area is the IL-2 gene, which has been
proposed as a possible autoimmunity gene on the basis
of linkage to insulin-dependent diabetes mellitus in
the NOD mouse and experimental autoimmune encephalomyelitis (the mouse model for human multiple
sclerosis).33,34
Our study is consistent with the hypothesis that
susceptibility to multiple sclerosis is determined by the
action of many genes, each exerting only modest effects
individually. Four regions of suggestive linkage (2p13,
4q26-28, 6q26 and Xp11), two of which (2p13 and 4q2628) have not previously been reported, are identified.
These provisional results require further exploration
through the use of a positional candidate approach and
fine mapping of the regions in order to refine characterisation of genetic predisposition to multiple sclerosis in
Australian and other Northern European populations.
467
Materials and methods
Families
The index cases and their affected siblings were recruited
by the Australian National Register of Multiple Sclerosis
Families, Canberra Hospital (Canberra) and the Institute
for Immunology and Allergy Research, Westmead
Hospital (Sydney). All patients gave informed written
consent for genetic analysis and satisfy criteria for
clinically definite (66%), clinically probable (9%), laboratory-supported definite (19%) or laboratory-supported
probable (6%) multiple sclerosis.35 Families with affected
half-siblings or sib-ships with more than two affected
were not included. Although DNA was available from
parents and/or unaffected siblings in 60% of families,
these additional samples were not genotyped for reasons
of genotyping efficiency. Others have shown using these
markers that typing parents and unaffected family
members results in only a modest increase in the amount
of information extraction.13,36
Markers
A total of 397 dinucleotide microsatellite markers from
the Applied Biosystems Medium Density Linkage Mapping Set (LMS-MD10) were used. The markers were
observed to have a mean heterozygosity of 78% and a
mean PIC (polymorphism information content) of 75%.
Polymerase chain reaction (PCR) amplification
of markers
DNA was extracted from whole blood using a rapid
salting out method.37 PCR amplification of markers was
carried out using True Allelet Premix (Applied Biosystems) with the manufacturer’s recommended conditions
in a final volume of 15 ml on either a GRI Tetrad or a 9700
thermal cycler (Applied Biosystems). PCR products were
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Australian MS linkage screen
M Ban et al
468
pooled in predetermined panels prior to electrophoresis
on a 3700 Genetic Analyser (Applied Biosystems). Semiautomated genotyping was performed using the GENESCAN/GENOTYPER software (Applied Biosystems).
Statistical methods
As the mode of inheritance is unknown in multiple
sclerosis, a non-parametric multipoint linkage analysis
was performed using the MAPMAKER/SIBS program
(version 2.0).38 This calculates the MLS at each point on a
chromosome, using all the available genotypes, and
computes the proportion of information extracted by the
markers typed. Areas of potential linkage were identified
as positive MLS values taking X0.7 as the nominal 5%
significance threshold and X1.8 as suggestive of linkage.
Marker allele frequencies were estimated using the
SPLINK program (version 1.07).36 As no parents were
genotyped in this study, the data were tested with
the SIBERROR program (version 1.0)39 prior to linkage
analysis in order to exclude half or unrelated siblings.
Acknowledgements
We thank S Adams, V Perich, N Hartley, M Bugeja,
S Surkus and S Teutsch for the collection and preparation
of the DNA samples, J Gray for valuable technical
support and the Neurologists of Australia for verifying
the diagnosis of the patients. This work was supported
by the Multiple Sclerosis Society of Great Britain and
Northern Ireland (Grant No 659/01), the Wellcome Trust
(Grant No 057097/Z/99Z), and the National Health and
Medical Research Council of Australia (Grant No 981580
and 153990).
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