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Project protocol
Genetics of multiple sclerosis on the Faroe Islands
Ph.D Student Stefanie Binzer
Main supervisor:
Egon Stenager lektor, klinikchef, Sydjysk
Skleroseklinik (Sønderborg, Vejle, Esbjerg),
Neurologisk afd, Sygehus Sønderjylland, DK-6400
Sønderborg og Institut for Regional Forskning,
Syddansk Universitet.
Associate supervisors:
Jan Hillert, Professor of Neurology, Department of
Clinical Neuroscience, Karolinska Institute.
Kirsten Ohm Kyvik, MD, Ph.d, MPM, Head of Institute,
Associate Professor, Institute of Regional Health
Services Research
Introduction
Multiple sclerosis (MS) is a chronic inflammatory neurological disease
which occurs in young and middle-aged people1. MS is one of the commonest
causes of neurological handicap in young people in the Nordic countries2. The
aetiology is unknown but both genetic and environmental factors seem to be
implicated and autoimmune factors contribute greatly to the pathophysiology
of the disease3.
No one knows how long MS has existed, but there are records dating
back from the 13th century describing physical features which would now be
described as MS4. During the 1830ese Robert Carswell discovered strange
damage to the spinal cord and in 1870 Jean Martin Charcot established the
connection between the symptoms of MS and nerve damage. His postmortem brain studies lead him to discover scars and “plaques” in the brain. In
1884 Pierre Marie described his theory that MS was triggered by an infection.
During the following years several theories about the cause of MS were put
forward, most believing it to be caused by viruses or toxins. In 1916 detailed
microscopic analysis of post-mortem brains showed that there was damage to
myelin sheets. During the 1960ese the first medication to improve attacks was
used, as ACTH resulted in quicker recovery from attacks. In the 1980ese MRI
revolutionised the concept of MS as it showed that it is a constant ongoing
disease even though symptomatic relapses only occur sporadically. 5 During
the 1990ese the importance of genes became apparent as large studies of
twins reared apart demonstrated that genes play a role in the development of
MS.6 Beta-interferons were approved for the first time in patients in 1993 and
this was the first drug to reduce the frequency of attack.7 Since then much
focus has been on trying to understand the cause of MS and develop drugs
which will halt the illness. With the advent of the human genome project,
genes have become the crux of much MS research.8
Genes are important contributors as there is an increased relative risk
of 15-20 among siblings and concordance rate of 30% between identical
twins.9 To date, the genes that are widely accepted to influence susceptibility
to MS are the Human Leukocyte Antigen (HLA) complex gene cluster on
6p21.3,10 (easily identified both by candidate gene associations and wholegenome linkage), the interleukin 7 receptor alpha chain gene (IL7RA) and the
interleukin 2 receptor alpha chain gene (IL2RA).11 Several other genes are
believed to contribute to the susceptibility of MS either by independent action
or through interaction with others resulting in a highly complex
pathophysiology.9 Lately, the list of MS genes has become longer and to date
approximately 20 non-HLA genes have achieved genome-wide significance
and are thus widely recognized.12
However in the past year it has become increasingly clear that there
are limitations to the gene discoveries in MS as well as in other complex
diseases. It has been recognized that genes discovered in large case-control
studies adhere to the common-disease/common variant principle and thus are
very weakly acting, even together. This may prompt complementary strategies
for gene search in MS, for instance by focusing on isolated populations where
shared chromosomal segments may be identified with a higher efficiency.
Certainly, isolated populations seem to demonstrate good potential for
searching for variants contributing to complex traits.13
Background
MS on the Faroe Islands has been an interesting subject for the past
60 years since John Kurtzke formulated his hypothesis about the
development of MS on the Faroe Islands. Kurtzke’s hypothesis is that during
the Second World War the British troops lived on the Faroe Islands and they
brought with them a supposed virus called primary MS affection
(PMSA).14,15,16,17,18 The Faroes population apparently got exposed to this virus
which then resulted in four consecutive MS epidemics.19
The Faroe Islands is a relatively isolated group of islands and its
population therefore constitutes one of the few still existing isolated population
groups. This relative isolation has probably resulted in a genetic bottle-neck
phenomenon and caused the Faroese to share long runs of homozygosity.
This makes the Faroese a very interesting population to study complex
genetic disease in since a more homogenous genetic make-up probably gives
better opportunities to find new genes which are possibly associated with MS.
Indeed some studies have used more genetically homogeneous and
geographically isolated populations in the search for susceptibility genes in
complex disorders.
In an isolated population in the western part of Finland (Ostrobothnia)
Tienari et al found a high incidence and prevalence of MS. In the group of MS
patients all originating from Ostrobothnia they identified specific haplotypes of
the myelin basic protein (MBP) gene, which suggests a founder effect. 20 The
importance of a founder effect has also been demonstrated by Binzer et al
which found a high prevalence of multiple sclerosis (253/100,000) in an
isolated community in northern Sweden. (Överkalix). Genealogical
investigations showed that out of the 33 patients identified, 22 belonged to a
single pedigree.21 Studies have also been conducted in the relatively isolated
population of Sardinia, where Marrosu et al found a higher than average
prevalence of MS in a small village in Sardinia. Geneological data showed
that all 11 MS patients from that village descended from 3 pairs of ancestors,
and the study concluded that “MS familial aggregation in Sardinians is
influenced by genetic factors and that founder effect and the isolation of
Sardinia can be considered causes of the enrichment of "etiologic" MS
genes”.22 High incidences of MS have also been found in genetic isolates in
Värmland, Sweden23,24 and in Holland.25 Recent studies by Imrell et al have
also examined related MS cases with reduced heterogeneity and found five
chromosomal regions of interest,26 showing that there is basis for studying MS
in isolated populations.
During our preliminary research we identified a family on the Faroe
Islands where 14 members had MS. Seven of them had the primary
progressive type of MS (PPMS), which is a greater percentage than expected.
We HLA-typed 6 of the still living family members and it appear as though a
certain HLA-haplotype is seen in those with PPMS. The genetic results and
the high incidence in this family question Kurtzke’s theory and point at the
importance of genes in the development of MS.
As this initial pilot study only had few participants no firm conclusions
can be made and therefore we would like to include all MS patients on the
Faroe Islands in our future studies in order to further examine these
preliminary interesting findings.
With this study we hope to be able to contribute to a greater
understanding of the importance of genetic and environmental factors in MS
and expand our knowledge about which genes are associated with an
increased susceptibility of developing MS.
Purpose
In our study we would like to examine the following parameters:
1) Characterization of prevalent MS patients in the Faroe Islands
genealogically and clinically and collect physical material (blood samples for
DNA and serum)
2) Characterize MS patients for exposure to known MS environmental factors
by questionnaire such as smoking habits, sun exposure and childhood and
adolescent diseases as well as by EBV status
3) Genotype MS patients and controls for HLA-DR and HLA-A
4) Genotype MS patients and controls with a microarray-based technique for
genome-wide SNP analysis for shared chromosomal segments
5) Examine the epidemiology of MS on The Faroe Islands as a follow-up of
Kurtzke’s epidemiology theory
Hypothesis
Hypothesis: An isolated genetically homogenous population as in the
Faroe Islands offers a unique opportunity to efficiently identify genes of
importance for the development of MS; and MS on the Faroe Island cannot be
explained by Kurtzke’s proposed theory.
Materials and methods
1) 35 people on the Faroe Islands + 60 controls
2) 10 people from the Faroe Islands with MS living in Denmark + controls
From both groups we will:
a. Collect blood samples
b. Perform a clinical examination
c. Obtain genealogical data
On the Faroe Islands we have identified approximately 35 patients with
MS. Our inclusion criterion was that our participants had been diagnosed with
MS according to the MacDonald Criteria. Our exclusion criterion was people
under the age of 18 years. We will collect 30 ml venous blood in
EDTA/heparin vacutainers. We will also collect blood from 60 healthy controls
not directly related to the MS patients. The controls will be gender and age
matched. The blood will be stored in a biobank at the genetic resource centre
on the Faroe Islands. 10mls of blood will be kept for possible future analyses.
DNA will be extracted from the remaining blood and sent to the Karolinska
Institute (KI) in Stockholm, Sweden for further genetic analyses. The same will
happen to the blood samples collected from the 10 Faroese with MS currently
living in Denmark and belonging to “Sydjysk skleroseklinik (Esbjerg, Vejle og
Sønderborg)”. However, the remaining 10 mls of blood from these 10 patients
will be saved for possible future further analysis at the OPEN biobank in
Odense, Denmark.
Genealogy from the Faroese family database (Ættarbandsskráin) will
be obtained in collaboration with Ílegusavnið (Genetics Resource
Centre)(GRC) on the Faroe Islands. GRC will send notification to Dátueftirlitið
(Data Protection Authority of the Faroes) about the project.
Stefanie Binzer will travel to the Faroe Islands and obtain relevant
information from the patients’ medical records and perform neurological
examinations on the patients.
DNA samples will be typed for HLA-DR and HLA-A genes by a
standardized PCR-SSP method with 2-digit resolution. Samples will be
genome-widely investigated for a large number of markers with a microarray-
based technique, most likely on the Illumina platform, exact choice of assay
will be decided according to availability since techniques are developing
rapidly.
Statistical analyses
We will perform descriptive statistics, i.e. frequency analyses, normality
check and cross tabulations. We will also execute parametric and nonparametric comparisons between MS patients and controls. Recurrence risk
in different groups of relatives will be estimated as the prevalence in different
groups, taking proband status into account.
HLA genotypes will be analyzed by regression analysis.
Joint carriage of one or several known susceptibility genes will be used to
assess genetic load in patients compared with controls adapted from de
Jager, 2009.27
Analyses of shared chromosomal segments in recessive and dominant
models will be assessed using PLINK according to the technique suggested
by Imrell, hopefully with a new software being developed in the lab.
Project organisation
1) Stefanie Binzer: Ph.D student. Will be responsible for gathering the
clinical data from the patients. Will furthermore HLA type the DNA
samples and interpret the data and be the main author of the planned
articles.
2) Egon Stenager, klinikchef, Sydjysk Skleroseklinik (Sønderborg, Vejle,
Esbjerg): Main supervisor and responsible for the clinical aspect of the
project.
3) Kirsten Ohm Kyvik, Institute of Regional Health Services Research:
Associate supervisor and responsible for the genetic epidemiological
aspects of the project.
4) Jan Hillert, Department of Clinical Neuroscience, Karolinska Institute:
Associate supervisor and responsible for the genetic aspects of the
project
5) Sigurd Vang, Genetic Resource Centre on the Faroe Islands:
responsible for the genealogical data.
6) Bjarke Rogvi-Hansen, Queen Alexandrines Hospital/Landssjukrahusid,
Faroe Islands & Department of Neurology, Rigshospitalet: responsible
for the clinical aspects on the Faroe Islands.
7) Michael Binzer, Sydjysk Skleroseklinik (Sønderborg, Vejle, Esbjerg):
Will participate in the clinical aspect of the project which involves the
MS patients living in Denmark. He has furthermore previously worked
with MS in isolated populations.
8) Kerstin Imrell, Department of Neuroscience, Karolinska Institute: Will
assist with the genetic aspects of this study as she has extensive
knowledge of statistical analysis in genetics of isolated populations.
Budget
Salary:
1) Ph.d. salary app. 480.000kr + 200.000kr salary to “laborant”.
Expenses:
1) Ph.d.-school 35.000kr/year, which among other things will pay for
courses.
2) Computer with statistic software and Office + printer app. 10.000kr
3) Telephone bill: approximately 1000kr
4) Biobank (OPEN)……
5) HLA typing reagents approximately 20.000kr.
6) Microarray-based GWAS analysis app. 120.000kr for cases and app.
4000kr for every control – amount pending the existence of such data
in the Faroe population
7) Conference participation and presentation of results app. 12.-15.000kr
for European and 40.000kr for international
8) Preliminary total amount: 991.000kr (+240.000kr if GWAS analysis
done one all 60 controls)
Timeline
This timeline is pending Stefanie Binzer being able to get the jobs
described below. The timeline may have to be changed if jobs are not
attainable.
1) May 2010: Cand Med.
2) September 2010-August 2011: “klinisk basisuddannelse” at
Sydvestjysk sygehus.
3) October 2010: 2 weeks on the Faroe Islands for data collection
4) September 2011: One month on the Faroe Islands for data collection
5) October 2011-December 2011: “Neurologisk intro-stilling” which I plan
to apply for at “Neurologisk afdeling, Sygehus Sønderjylland”.
6) 6 months (January 2012-July 2012): Karolinska Institute, genetic
analyses and interpretations.
7) July 2012-March 2013: “Neurologisk intro-stilling”, I plan to apply for
extension of that position.
8) April 2013: Hope to qualify in order to obtain a “Neurologisk blokstilling” at “Neurologisk afdeling, Sygehus Sønderjylland”.
9) May 2013-August 2015: completion of Ph.D
10) Total of 36 months Ph.D: 2 weeks (sep 2010) + 1month (Sep 2011) +
6 months (Jan-July 2012) + 28 months and 2 weeks (May 2013-August
2015)
Publications
This project will end with a Ph.D dissertation which will consist of three or
four articles:
1) The first article is already published under the title: ”Multiple Sclerosis
in a family on the Faroe Islands”. Acta Neurol Scand. 2010;121(1):169.
2) The second article will include the genealogy and clinical picture of MS
on the Faroe Islands. This will also include exposure to known MS
environmental factors which will have been obtained through
questionnaires as well as HLA-A and HLA-DR genotypes.
3) The third article will focus on the genetic aspects. Patients and controls
will be genotyped with a microarray-based technique for genome-wide
SNP analysis for shared chromosomal segments.
4) A possible 4th article might examine the epidemiology of MS as a
follow-up to Kurtzke’s theory.
The preliminary list of authors include: Stefanie Binzer, Michael Binzer,
Kerstin Imrell, Jan Hillert, Kirsten Kyvik, Sigurd Vang, Bjarke Rogvi-Hansen
and Egon Stenager.
Ethical considerations
This project focuses on a limited number of individuals, actually all
patients with MS in a small community. Thus, although all published data will
be anonymised, there is a chance that individuals may be recognised by
others.
Although MS genes discovered so far are typically very weakly acting
and carry little useful information regarding the risk of carriers, it is principally
possible, and in this case even hoped, that there is a chance of detecting
genetic variants carrying a greater risk and thus a higher level of informativity
regarding MS risk for carriers. These factors need to be made clear in the
patient information while applying for ethical permission.
Literature list
1) Hauser, S. L. & Oksenberg, J. R. The neurobiology of multiple
sclerosis: genes, inflammation, and neurodegeneration. Neuron 52,
61–76 (2006).
2) Tienari PJ, Sumelahti M, Rantamäki T et al. Multiple sclerosis in
western Finland: evidence for a founder effect. Clinical Neurology and
Neurosurgery. 2004;106(3):175-179
3) Compston A, Coles A. Multiple sclerosis. Lancet 2002;359:1221–31.
4) http://www.historyofms.org/
5) Maravilla KR, Weinreb JC, Suss R et al. Magnetic resonance
demonstration of multiple sclerosis of plaques in the cervical cord. AJR
Am J Roentgenol. 1985;144(2):381-385
6) Ebers GC, Yee IM, Sadovnick AD et al. Conjugal multiple sclerosis:
population-based prevalence and recurrence risk in offspring.
Canadian Collaborative Study group. Ann Neurol. 2000;48(6);927-931
7) Knobler RL, Greenstein JI, Johnson KP et al. Systemic recombinant
human interferon-beta treatment of relapsing-remitting multiple
sclerosis: pilot study analysis and six-year follow-up. J Interferon Res.
1993;13(5):333-340
8) International Multiple Sclerosis Genetics Consortium. Risk alleles for
multiple sclerosis identified by a genomewide study. N. Engl. J. Med.
2007;357:851–862
9) Oksenberg JR, Baranzini SE; Sawcer S et al. The genetics of multiple
sclerosis: SNPs to pathways to pathogenesis. 2008;9:516-526
10) Hauser, S. L. & Oksenberg, J. R. The neurobiology of multiple
sclerosis: genes, inflammation, and neurodegeneration. Neuron.
2006;52:61–76
11) Lundmark F, Duvefelt K, Lacobaeus E et al. Variation in interlukin 7
receptor alpha chain (IL7R) influences risk of multiple sclerosis.
2007;39(9):1108-1119
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come. Am J Hum Genet. 2010;86(4):621-625
13) Jakkula E, Leppä V, Sulonen AM. Genome-wide association study in a
high-risk isolate for multiple sclerosis reveals associated variants in
STAT3 gene. Am J Hum Genet. 2010;86(2):285-291
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cause. Journal of Neurovirol 2000;6(2):134-140
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17) Kurtzke J F. Epidemiologic Evidence for Multiple Sclerosis as an
Infection. Clinical Microbiology Reviews. 1993;6(4):382-427
18) Kurtzke JF, Heltberg A. Multiple sclerosis in the Faroe Islands: an
epitome. The Journal of clinical epidemiology. 2001;54:1-22
19) Kurtzke JF, Hyllested K. Multiple sclerosis: an epidemic disease in the
Faeroes. Trans Am Neurol Assoc. 1975;100: 213-5.
20) Tienari PJ, Sumelathi ML, Rantamäki T et al. Multiple sclerosis in
western FInland : evidence for a founder effect. Clinic Neurol and
Neurosurg. 2004;106:175-179
21) Binzer M, Forsgren L, Holmgren G et al. Familial clustering of multiple
sclerosis in a northern Swedish rural district. J Neurol Neurosurg
Psychiatry. 1994;57:497-499
22) Marrosu M, Lai M, Cocco E, Loi V, Spinicci G, Pischedda M, et al.
Genetic factors and the founder effect explain familial MS in Sardinia.
Neurology 2002;58:283–8.
23) Callander M, Landtblom AM. A cluster of multiple sclerosis cases in
Lysvik in the Swedish county of Värmland. Acta Neurol Scand.
2004;110(1):14-22
24) Boström I, Callander M, Kurtzke JF et al. High prevalence of multiple
sclerosis in the Swedish county of Värmland. Mult Scler.
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25) Hoppenbrouwers IA, Cortes LM, Aulchenko YS et al. Familial
clustering of multiple sclerosis in a Dutch genetic isolate.
26) Imrell K. Conquering Complexity: Successful strategies for finding
disease genes in multiple sclerosis. Thesis. Department of clinical
Neurosience. Karolinska Institute. 2009.
27) De Jager PL, Chibnik LB, Cui J et al. Integration of genetic risk factors
into a clinical algorithm for multiple sclerosis susceptibility: a weighter
genetic risk score. Lancet Neurol. 2009;8(12):1111-1119