Novel Multiple Sclerosis Predisposing Genetic Variants

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Suvi P. Kallio
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
Prevalence and familial occurrence of MS are exceptionally high in a Finnish
population subisolate, Southern Ostrobothnia, presumably due to enrichment
of predisposing genetic variants within this region. Previous linkage scan on
MS pedigrees from Southern Ostrobothnia detected three main MS loci on
chromosomes 5p, 6p (HLA) and 17q. In this thesis work an effort was made
to localize MS predisposing alleles of the linked loci outside the HLA region by
studying familial MS cases from the Southern Ostrobothnia isolate.
This thesis provides an example of how extended families from special
populations can be utilized in fine-mapping of the linked loci. A first relatively
rare MS variant was here identified utilizing the strength of a Finnish population
subisolate. The identified haplotype, flanking
the complement component 7 (C7) gene, seems to have a fairly large effect on
genetic susceptibility of MS, potentially by regulating activity of the complement
system, which has previously been suggested to have an important role in
pathogenesis of MS.
.!7BC5<2"HIDMKI!
ISBN 978-952-245-097-5
National Institute
for Health and Welfare
P.B. 30 (Mannerheimintie 166)
FI - 00271 Helsinki, FINLAND
Telefon: +358 20 610 6000
www.thl.fi
16
Novel Multiple Sclerosis
Predisposing Genetic Variants
Outside the HLA Region
Novel Multiple Sclerosis Predisposing Genetic Variants Outside the HLA Region
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous
system. Both environmental factors and several predisposing genes are required
to generate MS. Despite intensive research these risk factors are still largely
unknown, pathogenesis of MS demyelination is poorly understood, and no
curative treatment exists.
RESEARCH
Suvi P. Kallio
RESEARCH
Suvi P. Kallio
16
2009
16
Suvi P. Kallio
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
Academic dissertation
To be presented with the permission of the Medical Faculty,
University of Helsinki, for public examination in the Lecture Hall 2,
Biomedicum Helsinki, on June 26th, at 12 noon.
National Public Health Institute,
Helsinki, Finland
and
National Institute for Health and Welfare,
Helsinki, Finland
and
Department of Medical Genetics,
University of Helsinki, Finland
RESEARCH 16
Helsinki 2009
© Suvi P. Kallio and National Institute for Health and Welfare
Cover graphic: A heat map illustrating prevalence of MS in Scandivavia. The more
red is the area, the higher is the prevalence. Obtained from tutorial ”Minulla on
MS” with a permission of the copyright holder Dr. Juhani Ruutiainen, The Finnish
MS Society. Original data from Kurtzke 1974.
Layout: Christine Strid
ISBN 978-952-245-097-5 (printed)
ISSN 1798-0054 (printed)
ISBN 978-952-245-098-2 (pdf)
ISSN 1798-0062 (pdf)
Helsinki University Print
Helsinki, Finland 2009
Supervised by
Academy Professor Leena Peltonen-Palotie
Finnish Institute for Molecular Medicine and
University of Helsinki
Department of Medical Genetics
Helsinki, Finland and
The Sanger Institute
Cambridge, UK
Adjunct Professor Janna Saarela
Finnish Institute for Molecular Medicine and
University of Helsinki
Department of Medical Genetics
Helsinki, Finland
Reviewed by
Professor Anne Kallioniemi
University of Tampere
Institute of Medical Technology
Tampere, Finland
Professor emeritus Arne Svejgaard
University Hospital of Copenhagen
Department of Clinical Immunology
Copenhagen, Denmark
Opponent
Professor Leif Groop
University of Lund
Department of Endocrinology
Malmö, Sweden
To my family
Abstract
Suvi P. Kallio. Novel Multiple Sclerosis Predisposing Genetic Variants Outside the
HLA Region. National Institute for Health and Welfare (THL), Research 16/2009.
139 Pages. Helsinki 2009. ISBN 978-952-245-097-5
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous
system (CNS) affecting approximately 0.1 % of Europeans. Similar to what occurs
in numerous other complex diseases, both environmental factors and several
predisposing genes are required to generate MS. However, despite long-standing
and intensive research, these risk factors are largely unknown and the pathogenesis
of MS demyelination is still poorly understood. Hence, no accurate diagnostic tools
or curative treatment exists.
Both prevalence (0.2 %) and familial occurrence of MS are exceptionally
high in a Finnish population subisolate, Southern Ostrobothnia, presumably due
to enrichment of predisposing genetic variants within this geographical region.
Previous linkage scan on large MS pedigrees from Southern Ostrobothnia detected
three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in
several other populations have also provided independent evidence for the location
of MS susceptibility genes in these regions, and further, these loci are syntenic to the
experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents,
supporting their role in predisposition to autoimmune demyelination.
In this thesis work an effort was made to localize MS predisposing alleles of
the linked loci outside the HLA region and to better understand the molecular
mechanisms of MS. Taking into account that MS most probably is not a unitary
disorder, but instead may represent an overlapping spectrum of related disorders,
we have minimized the genetic and environmental heterogeneity by studying
familial MS cases from the Southern Ostrobothnia isolate.
A scan of the 17q locus provided evidence for association with variants of the
protein kinase C alpha (PRKCA) gene (p = 0.0001). Modest evidence for association
with PRKCA was observed also in MS families from Canada.
Analysis of the 5p locus revealed one region, flanking the complement
component 7 (C7) and hypothetical protein LOC133558 (FLJ40243) genes. The
identified relatively rare haplotype seems to have a fairly large effect on genetic
susceptibility of MS (frequency 12 % in MS cases and 4 % in controls, p = 0.000003,
OR = 2.73). Evidence for association with alleles of the region and MS was also seen
in more heterogeneous populations. Convincingly, plasma C7 protein levels and
total complement activity correlated with the risk haplotype identified.
The fairly strong association with the haplotype flanking C7 stimulated us to
study other complement cascade genes in MS. Previous publications have provided
functional evidence for involvement of C3 in autoimmune demyelination. However,
Research 16
THL 2009
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
7
the data of this work suggests that variation in the complement component coding
genes outside 5p is not associated with genetic susceptibility of MS, at least in
Finland.
Finally we used a candidate gene approach to identify potential MS loci. Lossof-function mutations of the DAP12 and TREM2 genes cause a recessively inherited
CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located
in MS regions on 6p and 19q, and we tested them as potential candidate genes
in the Finnish MS sample. No evidence for association with MS was observed,
and the Finnish PLOSL mutation was not over-represented among Finnish MS
cases compared to controls (carrier frequency 5/1,000). Thus, the highly conserved
DAP12 and TREM2 genes unlikely have a role in genetic susceptibility of MS.
This thesis contributes to the existing studies of complex disease genetics by
providing an example of how extended families from special populations can be
utilized in fine-mapping of the linked loci. A first relatively rare MS variant was
here identified utilizing the strength of a Finnish population subisolate. This variant
seems to have an effect on activity of the complement system, which has previously
been suggested to have an important role in pathogenesis of MS. Thus, according
to these results the role of the complement system in MS should be explored more
indepth.
Keywords: multiple sclerosis, MS, complex disease, association analysis, linkage,
complement cascade
8
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
Research 16
THL 2009
Abstract in Finnish
Suvi P. Kallio. Novel Multiple Sclerosis Predisposing Genetic Variants Outside
the HLA Region [Multippeliskleroosille altistavien geenivarianttien paikantaminen HLA-alueen ulkopuolelta]. Terveyden ja hyvinvoinnin laitos (THL), Tutkimus
16/2009. 139 sivua. Helsinki 2009. ISBN 978-952-245-097-5
Multippeliskleroosi (MS-tauti) on krooninen tulehduksellinen keskushermostotauti, johon sairastuu noin 0.1 % eurooppalaisista. Kuten monien muidenkin monitekijäisten sairauksien kohdalla, sekä ympäristötekijät että useat alttiusgeenit
yhdessä johtavat MS-taudin puhkeamiseen. Pitkäaikaisesta ja intensiivisestä tutkimuksesta huolimatta suurin osa näistä MS-taudin riskitekijöistä on kuitenkin vielä tunnistamatta ja MS-taudissa tapahtuvan myeliinituhon syntymekanismit ovat
huonosti ymmärrettyjä. Näin ollen MS-taudille ei ole olemassa täsmällistä diagnostiikkaa tai parantavaa hoitoa.
Sekä MS-taudin esiintyvyys (0.2 %) että suvuittainen esiintyminen ovat poikkeuksellisen korkeat erään Suomen väestöisolaatin alueella, Etelä-Pohjanmaalla.
Tämä johtuu luultavasti perinnöllisten MS-taudin alttiustekijöiden rikastumisesta
tälle maantieteelliselle alueelle. Suomalaisessa kytkentätutkimuksessa on aiemmin
Etelä-Pohjanmaan MS-sukuja tutkimalla tunnistettu kolme pääasiallista MS-taudin geenipaikkaa kromosomeissa 5p, 6p (HLA) ja 17q. Myös muissa väestöissä tehdyt kytkentäanalyysit tukevat löydöstä. Lisäksi nämä geenipaikat vastaavat jyrsijöiden kokeellisen autoimmuunienkefalomyeliitin (EAE) alttiusalueita, vahvistaen
näiden kromosomien merkitystä autoimmuuni-demyelinaation kehittymisessä.
Tässä väitöskirjatutkimuksessa on pyritty paikantamaan MS-taudille altistavia geenivariantteja HLA-alueen ulkopuolisilta kytkentäalueilta ja siten ymmärtämään paremmin MS-taudin syntymekanismeja. Koska MS-tauti ei luultavasti ole
vain yksi sairaus vaan ennemminkin kirjo samankaltaisia sairauksia, pyrimme minimoimaan perinnöllisten alttiustekijöiden ja ympäristötekijöiden vaihtelun tutkimalla MS-sukuja Etelä-Pohjanmaan isolaattialueelta.
Kromosomin 17q alttiusalueen analyysissä havaittiin proteiinikinaasi C alfa
-geenin (PRKCA) varianttien liittyvän MS-alttiuteen (p = 0.0001). Myös kanadalaisia MS-perheitä tutkimalla todettiin assosiaatio PRKCA-geeniin.
Kromosomin 5p alttiusalueen analyysissä havaittiin yhdellä suhteellisen harvinaisella haplotyypillä olevan suurehko vaikutus MS-alttiuteen (yleisyys 12 %
MS-potilailla ja 4 % kontrolleilla, p = 0.000003, OR = 2.73). Tämä haplotyyppi
sivuaa komplementti komponentti 7 (C7) ja hypoteettinen proteiini LOC133558
(FLJ40243) geenejä. Alueen alleelien havaittiin liittyvän MS-alttiuteen myös heterogeenisemmissa väestöissä. Lisäksi plasman C7-proteiinitasojen sekä komplementtisysteemin aktiivisuuden havaittiin korreloivan tunnistetun riskihaplotyppin
kantajuuteen.
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Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
9
Suhteellisen vahva assosiaatio C7-geeniin osuvaan haplotyyppiin kannusti
tutkimaan myös muiden komplementtikomponentteja koodaavien geenien osuutta MS-taudissa. Lukuisissa aiemmissa tutkimuksissa on havaittu yhteys C3-proteii­
nitasojen ja immunologisen myeliinituhon välillä. Tässä tutkimuksessa kromosomin 5p ulkopuolisten kompementtigeenien ei kuitenkaan voitu todeta liittyvän
MS-alttiuteen suomalaisilla.
Väitöskirjan viimeisessä osatyössä tutkittiin MS-taudin ehdokasgeenejä.
DAP12 ja TREM2 geenien toimimattomuuteen johtavat mutaatiot aiheuttavat peittyvästi periytyvän keskushermoston valkean aineen sairauden, PLOSL:n. Mielenkiintoista on, että kyseiset geenit sijaitsevat kromosomien 6p ja 19q alueilla, joiden
on havaittu kytkeytyvän myös MS-tautiin. Osatyössä selvitettiinkin, altistavatko
DAP12 ja TREM2 myös MS-taudille. Näiden geenien ei todettu assosioituvan MSalttiuteen, eikä suomalaisen PLOSL-mutaation havaittu rikastuneen Suomen MSpotilaille verrattuna verrokkeihin (kantajuus 5/1000).
Tämä väitöskirjatyö tukee monitekijäisten tautien geneettistä tutkimusta kuvaamalla, miten erityisväestöjen sukuaineistoja voidaan hyödyntää alttiusvarianttien paikantamiseksi kytkentäalueilta. Tutkimuksessa tunnistettiin suomalaista väestöisolaattia aineistona hyödyntäen ensimmäinen suhteellisen harvinainen
MS-taudille altistava geenivariantti. Tämä variantti vaikuttaisi säätelevän komplementtiaktiivisuutta, jolla on jo aiemmin todettu olevan tärkeä rooli MS-taudin patogeneesissa. Havaitut tulokset kannustavatkin tutkimaan tarkemmin komplementtisysteemin roolia MS-taudin kehittymisessä.
Avainsanat: multippeliskleroosi, MS-tauti, monitekijäinen sairaus, assosiaatioanalyysi, kytkentä, komplementtikaskadi
10
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
Research 16
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Contents
Abstract
Abstract in Finnish
Abbreviations................................................................................................................. 13
List of original publications......................................................................................... 15
1
Introduction. ................................................................................................... 17
2 Review of the literature. ........................................................................... 18
2.1 Multiple sclerosis.......................................................................................... 18
2.1.1 Epidemiology and diagnosis............................................................ 18
2.1.2 Pathological hallmarks and theories of pathogenesis................... 22
2.2 Genetics of comples diseases....................................................................... 25
2.2.1 The human genome........................................................................... 25
2.2.2 Sequence variation............................................................................ 26
2.2.3 Strategies to identify genes underlying complex traits................. 28
2.2.4 The Finnish population and its subisolates.................................... 31
2.3 Genetics of multiple sclerosis...................................................................... 34
2.3.1 Genome-wide linkage screens of MS.............................................. 34
2.3.2 MS candidate gene studies............................................................... 37
2.3.3 The first genome-wide association scan of MS.............................. 40
2.3.4 Other strategies to map MS predisposing genetic variants.......... 42
3
Aims of the study............................................................................................ 44
4 Materials and methods................................................................................ 45
4.1 Study sample.................................................................................................. 45
4.1.1 Finnish MS sample............................................................................ 45
4.1.2 Selection of cases and controls for the Finnish GWA study........ 46
4.1.3 Study samples from more heterogeneous populations................. 48
4.2 Laboratory methods and statistical analyses............................................. 49
5 Results and discussion. ................................................................................ 52
5.1 Fine-mapping of the linked loci on chromosomes 17 and 5 (I, II)........ 52
5.1.1 MS locus on chromosome 17q (I)................................................... 52
5.1.2 MS locus on chromosome 5p (II)................................................... 58
5.2 A follow-up study: variation in other complement cascade
genes in MS (III)........................................................................................... 66
5.3 Candidate genes for immune-mediated demyelination on
MS linked loci (IV)....................................................................................... 68
6
Concluding remarks...................................................................................... 72
7
Acknowledgements........................................................................................ 74
References....................................................................................................................... 76
Original publications
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Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
11
Abbreviations
2H2D2A
AITD
BBB
C3
C5
C7
C9
CD-CV
CEU
CNS
CNV
CSF
CTLA-4
DNA
EAE
EBV
FLJ40243
FYB
GWA
HGP
HHRR
HHV-6
HLA
IBD
IBS
ICAM2
IRF5
IL2RA
IL7R
LD
LOD
MAC
MAF
MBP
MHC
MHC2TA
MRI
MS
NK
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SH2 domain protein 2A
Autoimmune thyroid disease
Blood-brain barrier
Complement component 3
Complement component 5
Complement component 7
Complement component 9
Common disease-common variant
Caucasians of European origin
Central nervous system
Copy number variation
Cerebrospinal fluid
Cytotoxic T lymphocyte antigen 4
Deoxyribonucleic acid
Experimental autoimmune encephalomyelitis
Epstein-Barr virus
Hypothetical protein LOC133558
Fyn binding protein
Genome-wide association
Human genome project
Haplotype-based haplotype relative risk
Human herpesvirus-6
Human leukocyte antigen
Identical by descent
Identical by state
Intercellular adhesion molecule 2
Interferon regulatory factor 5
Interleukin 2 receptor, alpha chain
Interleukin 7 receptor
Linkage disequilibrium
Logarithm of odds
Membrane attack complex
Minor allele frequency
Myelin basic protein
Major histocompatibility complex
MHC class II transactivator
Magnetic resonance imaging
Multiple sclerosis
Natural killer cell
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
13
OR
PBMC
PCR
PECAM1
PLOSL
PRKCA
PP
PTPN22
RA
RNA
RR
SLE
SNP
SO
SP
T1D
TCC
TDT
TREM2
TYROBP
14
Odds ratio
Peripheral blood mononuclear cell
Polymerase chain reaction
Platelet/endothelial cell adhesion molecule 1
Polycystic lipomembranous osteodysplasia with
sclerosing leucoencephalopathy
Protein kinase C alpha
Primary progressive
Protein tyrosine phosphatase, non-receptor type
Rheumatoid arthritis
Ribonucleic acid
Relapsing remitting
Systemic lupus erythematosus
Single nucleotide polymorphism
Southern Ostrobothnia
Secondary progressive
Type 1 diabetes
Terminal complement complex
Transmission disequilibrium test
Triggering receptor expressed on myeloid cells 2
TYRO protein tyrosine kinase binding protein
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
Research 16
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List of original publications
This thesis is based on the following original articles referred to in the text by their
Roman numerals:
I
Saarela J, Kallio SP, Chen D, Montpetit A, Jokiaho A, Choi E, Asselta R,
Bronnikov D, Lincoln MR, Sadovnick AD, Tienari PJ, Koivisto K, Palotie
A, Ebers GC, Hudson TJ, Peltonen L (2006) PRKCA and multiple sclerosis:
association in two independent populations. PLoS Genet 2(3):e42.
II Kallio SP, Jakkula E, Purcell S, Suvela M, Koivisto K, Tienari PJ, Elovaara I,
Pirttilä T, Reunanen M, Bronnikov D, Viander M, Meri S, Hillert J, Lundmark
F, Harbo HF, Lorentzen ÅR, De Jager PL, Daly MJ, Hafler DA, Palotie A,
Peltonen L and Saarela J (2009) Use of a genetic isolate to identify rare disease
variants: C7 on 5p associated with MS. Hum Mol Genet 18(9):1670-1683.
III Leppä V, Kallio SP, Kemppinen A, Koivisto K, Tienari PJ, Elovaara I, Pirttilä
T, Reunanen M, Meri S, Palotie A, Peltonen L and Saarela J. Variation
in complement cascade genes in MS patients and population controls.
Submitted.
IV Sulonen A-M*, Kallio SP*, Ellonen P, Suvela M, Elovaara I, Koivisto K, Pirttilä
T, Reunanen M, Tienari PJ, Palotie A, Peltonen L and Saarela J (2009) No
evidence for shared etiology in two demyelinative disorders, MS and PLOSL.
J Neuroimmunol 206(1-2):86-90.
* These authors contributed equally to the study.
These articles are reproduced with the kind permission of their copyright holders.
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Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
15
1
Introduction
Multiple sclerosis (MS), affecting approximately two million people worldwide,
is the most common cause of neurological disability in young adults in the
developed world (Oksenberg and Barcellos 2005). MS is a clinically heterogeneous
demyelinating disease of the human central nervous system (CNS) with a putative
autoimmune pathogenesis and complex inheritance. However, despite longstanding and intensive research, both genetic and environmental risk factors are
still largely unknown. Thus, pathogenesis of MS is poorly understood and no
curative treatment exists.
Genetics provides tools to dissect the molecular backround of MS, of which the
target tissue is challenging to study using conventional cell biological approaches.
During this thesis study, the emphasis in genetic mapping of complex diseases has
largely shifted from linkage studies in families to genome-wide association (GWA)
studies in unrelated cases and controls. However, it also has become obvious that
these large GWA studies primarily expose common variants contributing to disease
pathogenesis with modest effects, and alternative strategies are needed to identify
relatively rare, more penetrant alleles, which most probably give rise to a familial
concentration of cases.
Previous linkage scan on large Finnish MS pedigrees detected three main
MS loci on chromosomes 5p, 6p (HLA) and 17q, and linkage studies in several
other populations have provided independent evidence for the location of MS
susceptibility loci in these regions. In this thesis study current knowledge of human
genetics as well as strengths of Finnish population history have been utilized to
localize MS predisposing genetic variants of the linked loci outside the HLA region
and, further, to better understand the molecular mechanisms of MS susceptibility.
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Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
17
2 Review of the literature
2
Review of the literature
2.1 Multiple sclerosis
2.1.1 Epidemiology and diagnosis
The onset of MS happens typically in early adulthood. Despite the fact that most
patients die due to unrelated reasons, MS causes significant neurological disability
and no curative treatment is available. Based on twin and population studies MS
has a complex inheritance: both environmental factors and several predisposing
genes are required to generate the disease. The sibling relative risk (λs) of 20-40,
and higher concordance rate of ~20% for monozygotic twins compared to ~5%
for dizygotic twins demonstrate a moderate genetic component for MS (Sadovnick
1993, Dyment et al. 2004). The role of childhood environmental factors on MS
susceptibility is supported by migration studies: if migration occurs during
childhood, the migrant acquires the new region’s susceptibility to MS, whereas
migration occuring later in life has little effect on MS susceptibility (Gale and
Martyn 1995). Notably, dizygotic twins of MS patients seem to have a higher risk
to get the disease than siblings of patients, even though there is no difference in
genetic sharing (Ebers 2008) (Table 1). This may reflect the importance of the
environmental factors of prenatal period and early childhood on MS susceptibility.
On the other hand, adoption studies have provided convincing evidence that the
aggregation of MS within families is largely explained by shared genes rather than
a shared environment (Ebers et al. 1995) (Table 1).
Table 1. Population-based prevalence rates of MS in relatives of patients.
Genetic sharing
Relationship
0%
General population (European origin)
1/1000
0%
Adoptive sibling
1/1000
12.5%
Cousin
7/1000
25%
Half sibling
20/1000
50%
Child
30/1000
50%
Parent
30/1000
50%
Full sibling
30/1000
50%
Dizygotic twin
100%
Monozygotic twin
1
18
Prevalence1
40–50/1000
200–300/1000
Estimates from Ebers 2008 and Dyment et al. 2004.
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
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2 Review of the literature
Similar to most diseases with a putative autoimmune etiology, MS affects
twice as many females as it does males. The prevalence of MS is highest (1/1,000) in
populations of Northern European descent living in temperate climate (Compston
1997), especially in the coastlines of Scandinavia, Iceland, the British Isles and in
countries settled by their inhabitants. Thus, it has been proposed that the Vikings
may have disseminated the risk alleles of MS, and the alleles may have entered
Finland via them along the rivers in the Southwestern Finland (Tienari 2004).
Notably, even though the prevalence of MS is quite uniform in Europe, both its
incidence and prevalence are two times higher in Southern Ostrobothnia in Western
Finland (Sumelahti et al. 2000, Sumelahti et al. 2001, Wikström and Palo 1975),
most probably due to enrichment of risk alleles within this population subisolate
(Figure 1). Further, there are also more familial MS cases in this isolated region
than elsewhere in Europe (Wikström 1975b). MS is extremely rare in certain ethnic
groups, including sub-Saharan Africans and Maori of New Zealand (Pugliatti et al.
2002). The uneven geographical distribution of MS is currently considered to be due
to both regional variation in frequency of genetic risk factors and unubiquitously
distributed environmental risk factors (Ebers 2008).
Figure 1. Special features of MS in distinct parts of Finland.
The prevalence of MS is two times higher in Southern Ostrobothnia than in other parts of
Finland, and there are also more familial MS cases (large MS pedigrees) in this isolated
region.The Southern Ostrobothnia subisolate region is marked with grey color. Values
are obtained from Sumelahti et al. 2000, Sumelahti et al. 2001, Sumelahti et al. 2003,
Wikström and Palo 1975. F/M = female to male ratio. RR = relapsing remitting MS.
Due to largely unknown pathogenesis, no specific diagnostic test for MS
exists. The diagnosis is mainly based on symptoms and clinical findings, supported
by laboratory and radiologic data. MS is a spectrum of various neurological
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19
2 Review of the literature
symptoms and findings, which are caused by inflammatory demyelinating lesions,
axonal damage and inflammatory burst. Charasteristic to MS is that lesions, which
develop in the brain, the optical nerve and the spinal cord, affect different sites,
separated in time. In consequence, the symptoms of the disease are protean. The
most typical symptoms are unilateral painful loss of vision (optical neuritis), diplopia,
problems with senses of feeling and spastic pareses of the limbs, ataxia and vertigo,
tremor, dysarthria, bladder dysfunction, constipation, erectile impotence and fatigue
(Compston and Coles 2002). Revised McDonald’s diagnostic criteria (McDonald
et al. 2001, Polman et al. 2005), representing an update for previously used Poser’s
diagnostic criteria (Poser et al. 1983), are currently used for MS diagnosis (Table 2).
TABLE 2. McDonald’s diagnostic criteria for MS
RR
PP
Clinical Presentation
Additional Evidence Needed for Diagnosis
≥ 2 attacks and
≥ 2 objective clinical lesions
-
1 attack and
≥ 2 objective clinical lesions
Dissemination in time
(MRI)
≥ 2 attacks and
1 objective clinical lesion
Dissemination in space
(MRI / CSF+ and ≥ 2 MRI lesions)
1 attack and
1 objective clinical lesion
Dissemination in time and space
(see abowe)
Insidious neurological
progression suggestive
of MS (≥ 1 year)
Two of the following:
* brain MRI +
* spinal cord MRI +
* CSF +
RR = relapsing remitting; PP = primary progressive; MRI = magnetic resonance imaging;
CSF = cerebrospinal fluid
Most MS patients (80-85%) experience a relapsing-remitting (RR) disease
characterized by attacks followed by periods with no new signs of disease activity
(Compston and Coles 2002, Inglese 2006) (Figure 2). However, the majority of
patients with this clinical subtype will evolve into a secondary progressive (SP)
MS within three decades, leading to chronic increases of symptoms and disability.
Approximately 10-20% of MS patients experience a primary progressive (PP) form
of the disease, characterized by chronic increase of symptoms and disability straight
from the onset of the disease (Compston and Coles 2002, Inglese 2006) (Figure
2). A very rare clinical subtype is a malignant MS, leading to severe neurological
symptoms or death within a short period after the disease onset.
20
Novel Multiple Sclerosis Predisposing
Genetic Variants Outside the HLA Region
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2 Review of the literature
FIGURE 2. Clinical subtypes and progression of MS.
A relapsing-remitting (RR) MS is characterized by attacks followed by periods with no
new signs of disease activity. The majority of patients with this clinical subtype will evolve
into a secondary progressive (SP) MS, leading to chronic increase of disability. A primary
progressive (PP) MS is characterized by chronic increase of symptoms and disability
straight from the onset of the disease. Modified from Oksenberg and Barcellos 2005 by
permission from Macmillan Publishers Ltd: [Genes and Immunity], copyright (2005).
MS subtypes are important for both prognosis and therapeutic decisions, since
current MS therapies are immunomodulatory, partially protecting against relapses,
but being ineffective against progressive symptoms (Inglese 2006). However, in a
recent study it was shown, that the clinical course of MS is typically similar between
affected siblings but not between affected parents and their children (Hensiek
et al. 2007) (Figure 3). Thus, familial factors influence the clinical course of MS,
but different clinical subtypes of MS can exist in one pedigree. No effect of family
concordance for disease severity was observed (Figure 3). The authors concluded
that different clinical subtypes of MS most probably represent a continuous
spectrum of inflammation and neurodegeneration, balance of which is determined
by both genetic and environmental risk factors; Individuals with a low threshold
for neurodegeneration would more probably manifest disease progression, whereas
individuals with a lower risk of this component but more inflammatory activity
would tend to the relapsing remitting phenotype. Further, they suggest that the
analysis of genetic studies should be stratified according to clinical course rather
than disease severity (Hensiek et al. 2007). However, taking into account that
different clinical subtypes of MS exist in one pedigree and most probably represent
a continuous spectrum of inflammation and neurodegeneration, stratification of
the study sample according to the clinical course in order to identify genetic risk
factors for MS may be unnecessary and even lead to loss of statistical power.
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FIGURE 3. Intrafamilial correlation for various clinical features in multiplex MS families
(n = 1,083). Affected parent-child pairs and affected sibling pairs also analysed separately.
Modified from Hensiek et al. 2007.
The clinical course of MS is typically similar between affected siblings but not between
affected parents and their children. Thus, familial factors influence the clinical course of
MS, but different clinical subtypes of MS can exist in one pedigree. No effect of family
concordance for disease severity was observed. The authors suggested that the analysis of
genetic studies should be stratified according to clinical course rather than disease severity.
Concordance for age of onset was observed for all family members studied. *p<0.05,
**p<0.001. Reproduced with a permission of the copyright holder.
2.1.2 Pathological hallmarks and theories of pathogenesis
The pathological hallmarks of MS are inflammatory demyelinating lesions (plaques)
within the CNS white matter. Pathogenesis leading to development of these lesions
is currently unknown, but several theories exist.
According to data obtained from experimental autoimmune encephalomyelitis
(EAE), an animal model of MS caused by immunizing rodents with myelin
peptides, autoreactive peripherally activated CD4+ T lymphocytes enter the CNS by
penetrating the blood-brain barrier (BBB), recognize myelin as foreign and attack
it, and simultaneusly activate the complement cascade, stimulate other immune
cells like macrophages/microglia and B lymphocytes, and trigger expression of
cytokines and antibodies (Gold et al. 2006). In the CNS, more epitopes of destructed
myelin are represented to CD4+ T cells in the context of human leukocyte antigen
(HLA) class II molecules. This process called epitope spreading further accelerates
the inflammatory reaction and leads to a vicious cycle of myelin destruction and
CNS inflammation. However, active MS lesions have been shown to be colonised
also by CD8+ T lymphocytes, their pathogenetic relevance still being unclear
(Babbe et al. 2000).
There is still a need for many explanations for tissue pathogenesis. The key
questions are: why do the T lymphocytes get activated against myelin and how
do they reach the CNS via BBB, which is normally unpermeable to lymphocytes.
The most apparent explanation is that the trigger to both autoreactivity and
decreased BBB integrity is an infection, which must be fairly ubiquitous within the
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population and infect people before early adulthood. According to the molecular
mimicry hypothesis, lymphocytes have a sensitization to myelin proteins because
of homologous sequences found on antigenic viral proteins (Libbey et al. 2007).
One of the candidate viruses is Epstein-Barr virus (EBV), a human herpes virus
infecting 80-90% of the general population (Ascherio and Munch 2000, Ebers 2008).
A systematic review of eight case-control studies comparing EBV seropositivity
in MS cases and unaffected controls found increased odds of MS among EBV
seropositive individuals (Ascherio and Munch 2000). Further, in a recent study,
evidence for EBV infection in over half of brain-infiltrating B cells and plasma cells
was observed in the post-mortem brain tissue of MS patients, potentially indicating
an important role for EBV reactivation in MS pathogenesis (Serafini et al. 2007).
However, it remains unclear whether homing of EBV infected B cells into the CNS
is a primary event in MS or just a consequence of an unknown disease process.
Another hypothesis called the neural hypothesis suggests that a latent viral
infection within the CNS leads to chronic infection of neurons, which in turn
causes release of tissue antigens, increases permeability of the BBB and activates
autoreactivity against myelin (Prat and Antel 2005). Among the candidate
pathogens are herpes viruses and especially the human herpesvirus-6 (HHV-6),
which is the causative agent in a common febrile rash (exanthema subitum) of
children (Christensen 2007). Interestingly, the cellular receptor for HHV-6 is a
complement regulatory protein MCP (Santoro et al. 1999). It was recently shown
that ~10% of the CSF samples of MS patients are positive for HHV-6 and the total
prevalence of human herpesviruses in the CSF of patients is around 15% compared
to 2.3% for controls (Alvarez-Lafuente et al. 2008). However, it is worth noting that
a reactivation of herpes viruses, including EBV and HHV-6, is typical for immunemediated diseases and is possibly just a consequence of rather than a reason for
MS.
Despite numerous attempts, no causal pathogen has been unequivocally
linked to MS and the role of infectious diseases on MS predisposition still remains
unproven. After all, even if a particular virus is not involved in MS pathogenesis,
virus infections generally can predispose to MS by increasing production of
cytokines, activating the complement system, altering the permeability of the BBB
and boosting the autoreactive response against the CNS. On the other hand, MS
patients may have defects in the BBB itself, facilitating penetration of lymphocytes
into the CNS, and failure of regulatory T lymphocytes, which normally keep
autoreactive T cells in control, might lead to proliferation and activation of
autoreactive T lymphocytes and enhance the chronic CNS inflammation (Zozulya
and Wiendl 2008).
Can something be learned about the MS etiology by studying the pathology
of demyelinating lesions, the hallmarks of MS? Recently, four different
immunopathological patterns of MS were characterized based on the composition
of early active plaques in a large set of biopsies and necropsy samples (Luchinnetti et
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al. 2000). Based on this study, demyelination can be induced by T cells, macrophages
and their toxic products (pattern I), by antibodies and the complement system
(pattern II), by distal oligodendrogliopathy and apoptosis (pattern III) or by primary
degeneration of oligodendrocytes in periplaque white matter (pattern IV) (Figure
4). Interestingly, these pathological patterns differ between patients but remain
similar through the disease course, suggesting that MS is more heterogeneous
disorder than expected and that most probably genetic heterogeneity also exists
(Luchinnetti et al. 2000). The clinical significance of the pathological patterns was
verified in a recent publication, in which pathological patterns of 19 MS patients
treated with plasma exchange for an attack of fulminant demyelination were
retrospectively studied (Keegan et al. 2005). All patients with pattern II (n=10),
but none with other patterns, achieved functional neurological improvement after
the plasma exchange, which depletes antibodies and complement. Notably, no
clear correlation between the immunopathological patterns of demyelination and
the classical clinical subtypes of MS (RR, PP) have been observed (Pittock et al.
2005). It is still unknown whether there is concordance in the immunopathological
patterns of MS between members of the same pedigree.
FIGURE 4. Pathological mechamisms behind the four distinct demyelination patterns of MS.
Four different immunopathological patterns of MS according to Luchinnetti et al. 2000.
Demyelination is induced by T cells (T), macrophages (M) and their toxic products (pattern
I), by antibodies (Y) and complement (C) (pattern II), by distal oligodendrogliopathy and
apoptosis (dashed line) (pattern III) or by primary degeneration of oligodendrocytes in
periplaque white matter (pattern IV).
Pattern II, characterized by immunoglobulins and complement activation, is
the most common pathological subtype of MS, accounting for over 50% of patients
(Luchinnetti et al. 2000). The complement system is a biochemical cascade of the
innate immune system that helps to clear pathogens and cellular debris from an
organism. Activation of C3 by the classical, lectin or alternative pathway leads to
activation of the terminal components (C5-C9), which then form the TCC (C5b-9)
(Figure 5). The TCC makes a transmembrane channel (membrane attack complex,
MAC) in the cell membrane of the target cell, resulting in osmotic lysis of the target.
Complement system has been shown to have an important role in pathogenesis of
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many immunological diseases like hereditary angioedema, membranoproliferative
glomerulonephritis, hemolytic uremic syndrome and SLE (Meri 2007), and based
on several publications it may play an important role also in the etiology of MS
(see Results and Discussion). Interestingly, oligodendrocytes, myelin forming
cells of the CNS, are especially sensitive to complement mediated injury due to
relative deficiency of regulatory proteins, which normally protect host cells from
complement-mediated lysis (Scolding et al. 1998).
FIGURE 5. Complement cascade.
Activation of C3 by the classical, lectin or alternative pathway leads to activation of the
terminal components (C5-C9), which then form a transmembrane channel (C5b-9 =
membrane attack complex, MAC) in the cell membrane of the target cell, resulting in
osmotic lysis of the target. Host cells are protected from complement-mediated lysis by
regulatory proteins (marked as grey thunderbolts).
2.2 Genetics of complex diseases
2.2.1 The human genome
James Watson and Francis Crick discovered the structure of deoxoribonucleic acid
(DNA) already 55 years ago (Watson and Crick 1953) (Figure 6), but the number
of human genes and their exact order in the map of human chromosomes was not
known until the beginning of this century, when the human genome was sequenced
by the Human Genome Project (HGP) (Lander et al. 2001). The project revealed the
3.2 billion base pairs long human genome to contain about 20,000-25,000 protein
coding genes (HGP website, http://www.ornl.gov/sci/techresources/Human_
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Genome/home.shtm). This is much less than expected. Indeed, less than 2% of
the human genome encodes proteins, while the rest of the sequence is composed
of introns, promoters, other regulatory regions, non-translated RNA and so called
“junk DNA”, for which the function is still largely unknown. It has become clear,
that the human genome is much more than the sum of its genes.
2.2.2 Sequence variation
Humans are genetically approximately 99.5% the same (Levy et al. 2007). The
rest 0.5% makes individuals genetically different and mainly explains why one
individual is more susceptible to a certain hereditary disease than another. This
genetic difference is caused by sequence variation (Table 3).
Variation in the human genome arises from mutations. Mutations occurring in
the germline are further transmitted to offspring. The variants can be silent, modify
protein products of genes or alter gene expression (Table 3). Single-nucleotide
polymorphisms (SNPs) constitute the great majority of sequence variations: at least
6.6 million polymorphic SNPs are known to exist in the human genome (dbSNP
build 129, validated SNPs, http://www.ncbi.nlm.nih.gov/projects/SNP/). Since
our knowledge of the human genomic sequence is currently based on only a few
sequenced individuals, the magnitude of genetic variation is still largely unknown.
To identify majority of interindividual sequence variation and to better understand
the role of this variation in human diseases, a large sequencing effort called The
1,000 Genomes Project was launched year 2008 (http://www.1000genomes.org).
TABLE 3. The main types of sequence variation in the human genome.
26
Variation
Description
Potential role in diseases
Single nucleotide
polymorphism (SNP)
A single nucleotide of DNA
sequence differs compared to
the reference sequence.
Altered protein product or
gene expression.
Microsatellite,
minisatellite
Tandem arrays of repeat units.
Altered protein product or
gene expression.
Structural variation,
copy number variation
(CNV)
Insertions, deletions,
translocations, inversions,
(segmental) duplications. Gains
or losses of DNA segments
compared to the reference
sequence.
Altered protein product,
gene dosage or gene
expression.
Rearrangement of the
genome (non-allelic
homologous
recombination)
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FIGURE 6. The Human Genome.
The 3.2 billion base pairs long human genome contains about 20,000-25,000 protein
coding genes and numerous sequence variations, including SNPs, which appear roughly
1/1000 nucleotide sites. DNA is packed into 23 pairs of chromosomes preserved in the
nucleus of a cell. Modified from National Human Genome Research Institute (http://
www.genome.gov).
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2.2.3 Strategies to identify genes underlying complex traits
To identify genetic variants predisposing to hereditary diseases, sequence variation
is studied in affected cases, their family members and healthy controls. When the
molecular mechanism leading to a disease is known, particular candidate genes
can be selected based on their biological function. However, for many complex
diseases, including MS, the etiology is largely unknown and hypothesis-free
mapping methods might be more productive. One strategy to identify susceptibility
genes of common diseases is the linkage approach, which aims to identify loci that
co-segregate with the disease within multiplex families (multiple affected individuals
in one pedigree) or affected sib pairs (Botstein and Risch 2003). After a genomewide linkage scan, typically using a set of less than 1,000 polymorphic markers, has
identified candidate loci, fine-mapping of these regions is usually needed to identify
the causative variants. The subsequent association analysis aims to identify a marker
allele, which co-segregates with the disease across individuals or families (Botstein
and Risch 2003). Further, findings should usually be repeatable in independent
study samples, and functional studies are needed to confirm biological significance
of the finding. Notably, positional cloning, although being successful in gene
identification of Mendelian diseases, have had substantially more limited success
in complex diseases, which are influenced by multiple genes with modest effects,
gene-gene interactions as well as allelic and locus heterogeneity (Hirschhorn and
Daly 2005). However, a few success stories in complex polygenic disease genetics
have already been reported, including the discovery of the NOD2/CARD15 gene
as an inflammatory bowel disease susceptibility gene. Linkage to chromosome 16
was identified already in 1996 (Hugot et al. 1996), and fine mapping of this locus
finally led to the identification of NOD2/CARD15 (Hugot et al. 2001), rare variants
of which are associated with inflammatory bowel disease.
Recently, progress in high-throughput genotyping technologies and a better
understanding of the human genome have enabled genome-wide association (GWA)
scans, which have become popular and have largely replaced the conventional
whole-genome linkage studies. In this new, more sensitive mapping method the
genome is covered with 300,000-1,000,000 SNPs, each of which is then tested for
association, typically in large study samples of several thousands or even tens of
thousands of individuals (McCarthy et al. 2008). However, like other mapping
methods, the GWA approach also has its problems. Perhaps most importantly,
the huge number of tests performed unavoidably leads to false positive results,
highlighting the importance of replication of the findings (Pearson and Manolio
2008).
Every variation in the genome still can not be studied before sequencing
technologies get more cost-efficient. Instead, information produced by the
HapMap project is utilized to “tag” the common variation of the human genome
(The International HapMap Consortium 2003). The fundamental idea behind
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tagging is that when a new mutation arises in the genomic sequence, where specific
SNP allele already exists, the combination of SNP alleles, a haplotype, is further
passed down to descendents as a unit unless a rare recombination event breaks the
link. Due to this non-random association of alleles at closely linked chromosomal
loci, linkage disequilibrium (LD), so called haplotype-tagging SNPs provide
information not only about themselves but also about several other SNPs located
nearby (Hirschhorn and Daly 2005). Hence, even when the actual causative variant
is not tested, the genetic variants in proximity and in LD will be co-inherited
more often in the same haplotype with the disease variant than expected under
independent assortment and will show association with the trait. The amount of
LD between two variants varies, to some extent, between different populations due
to genetic drift, natural selection, mutations, recombinations, ancestral population
demographics and mating patterns (Varilo and Peltonen 2004). Due to population
history of Finland, the genome of Finns exhibits an increase in LD compared to
mainland Europe and Africa, thus making gene mapping especially advantageous
in Finland (Jakkula et al. 2008).
It is worth noting that the HapMap project focuses only on common variation
(minor allele frequency is at least 1-5% on the population level). For any disease
allele frequency, the power of an association study is greatest when the marker and
disease allele frequencies match. Thus the GWA panels based on HapMap data
are not optimized to detect genomic variants or rare genetic variants, which may
also have a significant role on susceptibility of many common complex diseases
(Hirschhorn and Daly 2005). According to the common disease-common variant
(CD-CV) theory, common complex traits are mainly caused by genetic variants,
which have a relatively high frequency and are found in all human populations
(Reich and Lander 2001). However, these kinds of variants must have only small
effects on the disease phenotype, since common variants with high penetrance
would already have been detected in genome scans (McCarthy et al. 2008) (Figure
7). Common variants of complex diseases can be detected by studying large
international study samples. For example a variant of the interleukin 7 receptor
(IL7R) gene has a frequency of approximately 70% in the general population and it
is further slightly enriched among MS patients from several populations (Lundmark
et al. 2007, Gregory et al. 2007). An alternative opinion also exist, postulating that
common diseases are caused by relatively rare mutations, each having a moderate
or high effect on disease phenotype (Bodmer and Bonilla 2008). Further, there
may be different mutations in different populations and the mutations may be even
family specific. This seems to hold true, for example, for autism (Abrahams and
Geschwind 2008). Notably, the HapMap tagging SNPs, detecting mainly common
haplotypes, may not capture the relatively rare disease alleles very well, making
mapping of these variants challenging. Only studies in special populations with
unusual histories or exceptional pedigrees at high risk might provide sufficient
number of cases to explore the association (Weiss and Terwilliger 2000) (Figure 7).
It has also been suggested that, since environmental factors have so important role in
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FIGURE 7. Identification of low and high frequency variants of complex diseases.
Current GWA studies are designed to identify common small effect variants of common
diseases, and the rare variation has remained largely uncharacterized, mainly due
to challenges in identifying such variants using the current methods. Modified from
McCarthy et al. 2008. Adapted by permission from Macmillan Publishers Ltd: [Nature
reviews genetics], copyright (2008).
the development of complex diseases, individual genetic variants must be rare and
have a small effect on disease trait (Weiss and Terwilliger 2000), making detection
of this kind of variants extremely challenging. For most complex diseases the truth
probably lies between these extremities, and both rare and common variants,
together with environmental factors, have a role in disease predisposition.
The current GWA studies aim to identify common variants of complex
diseases, and the rare variation has remained largely uncharacterized, mainly due to
challenges in identifying such variants with current platforms favouring common
variants (Hirschhorn and Daly 2005, McCarthy et al. 2008, Frazer et al. 2009). Is
there any point to try to map the rare variation when common variants more likely
have an effect on disease phenotype globally? Firstly, the relatively rare variants with
at least moderate penetrance most probably give rise to a familial concentration of
cases and therefore explain the linkage identified in large pedigrees and provide
opportunities for family specific diagnostic testing (Frazer et al. 2009). Secondly,
common variants are common also among healthy population and typically increase
the probability to get the disease only marginally. Thus, these variants do not
necessarily much improve either diagnostics or understanding of the pathogenesis
of the disease. For example, studies of several immunological diseases have revealed
associations with interleukin genes (a catalog of published GWA studies at http://
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www.genome.gov/gwastudies), as expected. To better understand the molecular
mechanisms behind the diseases of unknown pathogenesis, relatively rare variants
with higher impact on disease phenotype should also be looked for, since they
provide information of defective metabolic pathways (Frazer et al. 2009).
2.2.4 The Finnish population and its subisolates
The human species originated in Africa around 150,000 years ago, of which the
first waves of migration occurred approximately 100,000 years ago (Cavalli-Sforza
2007). After that several waves of migration have occurred. Finland was inhabited
mainly from two immigration waves, occurring about 4,000 years ago from East
and 2,000 years ago from South (Kittles et al. 1998). In the 16th century the internal
migrations within Finland created regional subisolates, which were established
typically by only a few founders (Figure 8A) (Varilo and Peltonen 2004). Since then,
multiple bottlenecks like famines, wars and infectious diseases have temporarily
reduced the population size, causing loss of genetic variation, and the subsequent
rapid population expansion characterized by relative imbreeding and isolation has
remarkably reduced allelic diversity (Varilo and Peltonen 2004) (Figure 8B).
FIGURE 8. Charasteristics of the population history of Finland.
A. First only the coastal region of Finland was inhabited (early settlement). An internal
migration movement originated mainly from south Savo in the 16th century, resulting
in genetically isolated subpopulations established by only few founders and isolated by
distance (late settlement). The Vikings might also have disseminated their genome into the
Southwestern Finland. The map is modified from Peltonen et al. 1999. Reproduced with
a permission of the copyright holder. B. Multiple bottlenecks, temporarily reducing the
population size and causing loss of genetic variation, and the subsequent rapid population
expansion have remarkably reduced allelic diversity.
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Due to this unusual population history of Finland, the Finnish genome,
especially in the young subisolates, shows a decrease of genetic diversity and
an increase in LD compared to other parts of Europe and especially to Africa
(Varilo and Peltonen 2004). This has been a key to success in positional cloning of
monogenic diseases in Finland and can further be beneficial in the identification
of genetic variants of complex diseases, as the common HapMap markers of the
GWA panels capture more variation through haplotype blocks in Finland than in
more heterogeneous populations (Service 2006, Jakkula et al. 2008). On the other
hand, high LD can complicate identification of actual causative variants. Hence, in
some cases fine-mapping might be more meaningful in more outbred populations
(Varilo and Peltonen 2004).
FIGURE 9. The population substructures within Finland.
Pairwise IBS sharing data of samples from ten distinct early- and late-settlement
subpopulations is visualized with multidimensional scaling. The coloured dots indicate
samples from the corresponding coloured areas of the map. Modified from Jakkula et al.
2008. Reproduced with a permission of the copyright holder.
The population history of Finland has led to uneven geographical distribution
of disease alleles. Thus, the prevalence of several traits varies significantly between
different subisolates, and typically birthplaces of the patients’ grandparents
represent regional clustering (Norio 2003). Similar population substructure can
still be detected at a very high resolution by studying the “genetic fingerprint” of
Finnish individuals (Jakkula et al. 2008, Salmela et al. 2008) (Figure 9). Sample
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sizes of the population subisolates are often too small to detect common disease
alleles with modest effects, but the subisolate populations can be especially valuable
for identification of rare, high-impact variants of the isolate-enriched diseases. The
majority of affecteds are identical by descent (IBD), meaning that chromosomes
descending from a common ancestral chromosome carry the same disease allele
in similar haplotypes, making the genetic backround of complex diseases resemble
that of monogenic disease (Varilo and Peltonen 2004) (Figure 10). However, use of
isolates in gene mapping can expose to false positive associations due to population
stratification (cases and controls originate from genetically distinct population
subsets having distinct allele frequencies due to population history) (Hirschhorn
and Daly 2005), and attention should be paid to selection of study sample, even
when studying a seemingly homogeneous population like Finns (Jakkula et al.
2008). Another way of avoiding stratification is to use family-based study samples.
FIGURE 10. The use of LD and the founder effect in identification of the disease alleles.
Chromosomes descending from a common ancestral chromosome carry the same disease
allele (arrow) in similar haplotypic backround. However, recombinations have limited the
shared haplotype (white). Modified from Varilo and Peltonen 2004. Reproduced with a
permission of the copyright holder.
Incidence, prevalence and familial occurrence of MS are exceptionally high
in Southern Ostrobothnia (SO) (Sumelahti et al. 2000, Sumelahti et al. 2001,
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Wikström and Palo 1975), which is approximately 2,000 years old subisolate in
Western Finland (Figure 1). Notably, SO is a relatively old subisolate, and the extent
of LD and the length of homozygous segments are not as substantial as in younger
subisolates of Finland (Jakkula et al. 2008).
2.3 Genetics of multiple sclerosis
2.3.1 Genome-wide linkage screens of MS
Linkage approach has been successful in gene identification of Mendelian diseases,
but it has had substantially more limited success in genetic mapping of complex
diseases (Hirschorn and Daly 2005). In MS, several genome-wide linkage scans,
usually performed using sparse marker maps and small study samples, have resulted
in identification of numerous potential disease loci. However, only a handful of
them have been repeatable. The first genome-wide scans were published in 1996–
1997 and studied affected sibling pairs and MS families from UK, US (Americans
of European descent), Canada and Finland (Sawcer et al. 1996, Haines et al. 2006,
Ebers et al. 1996, Kuokkanen et al. 1996 and 1997). Evidence for a shared MS locus
was observed in all four studies for 6p21 (MHC region). Other potential regions of
consensus were 2q24-33, 3q21-24, 5p14-tel, 5q13-23, 7q21-22, 10q21-22, 17q2224, 18p11 and 19q13 (bolded in table 4).
To increase power to detect linkage, the data of the first four genome scans and
five additional genome-wide screens was combined in a meta-analysis, resulting in
719 MS families from US, Australia, Canada, Finland, Italy, Scandinavia, Sardinia,
Turkey and UK (GAMES 2003). The HLA locus provided strongest evidence for
linkage and the loci on chromosomes 17q21 and 22q13 were the next strongest
findings. Further, the International Multiple Sclerosis Genetics Consortium
(IMSGC) performed a high-density linkage screen utilizing 4,500 SNPs in 730
multiplex MS families of Northern European descent (IMSGC 2005). Again, the
peak logarithm of odds (LOD) score 11.7 was found in the HLA locus, and no other
locus reached genome-wide significance. Promisingly, the second most significant
LOD score was again detected on chromosome 17q.
The HLA gene cluster on chromosome 6p21 (Figure 11) is no doubt the
strongest susceptibility locus for MS. As a matter of fact, immunologist discovered
HLA-DR2 as a risk factor for MS already before molecular geneticists using
serological methods (Jersild et al. 1973). HLA molecules are heterodimeric cell
surface glycoproteins presenting antigens to T lymphocytes. Association with
HLA locus and MS has been observed in most populations studied and with
different clinical subtypes of MS (McDonnell 1999). The association signal
primarily arises from the HLA-DRB1*1501-DQB1*0602 haplotype (recognized by
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TABLE 4. Suggestive linkage regions according to the first MS genome scans
Chromosome
Region
1
p35C, p21U
2
p23A, p21C, q24-33F,C
3
p25C, p14C, q21-24A,C,F, q26C
4
p16C, q26-28C, q31-35A
5
p14-12(-tel)F,C, q11-13U, q13-23A,C
6
p21F,A,C,U, q14C
7
p21C, p15U, p14C, q11A, q21-22A,C, q32-35A
9
p24-22A, q34A
10
q21-22A,F, q26C
11
p15A, q22C
12
p13U, q23-24A,
13
q33-34A
14
q32C
15
q21C
16
p13A, q12C
17
q22-24F,U
18
p11F,C,A, q21C
19
q13F,A,C
22
q12-13U
X
p21C, p11C, p22C, q26C
U = UK (Sawcer et al. 1996); A = American (Haines et al. 2006); C = Canadian (Ebers et al.
1996); F = Finnish (Kuokkanen et al. 1996 and 1997).
FIGURE 11. The MHC region on chromosome 6p21-23.
MCH class I and II code for HLA molecules involved in antigen presentation, whereas
class III includes genes for complement proteins (C2 and C4), tumor necrosis factor (TNF)
and cytochrome P450 (CYP21). Modified from Oksenberg and Barcellos 2005. Adapted
by permission from Macmillan Publishers Ltd: [Genes and Immunity], copyright (2005).
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the HLA-DR2 serotype) and especially from the HLA-DRB1 gene located on the
major histocompatibility complex (MHC) class II segment (Lincoln et al. 2005).
However, identification of the actual MS predisposing variant within the HLA
locus has been extremely complex due to extensive polymorphism and LD across
the region. The allele frequency of the MS risk haplotype is approximately 35%
in Finnish MS patients and 15% in unaffected population controls (unpublished
result). Notably, this HLA-DRB1*1501-DQB1*0602 haplotype has a relatively
low frequency in Africa (frequency of 4% in African Americans, 6% in African
American MS patients; Oksenberg et al. 2004), suggesting that positive selection
of the haplotype has occurred in Europeans, potentially due to some infectious
pathogen specific to Europe (Oksenberg and Barcellos 2005). The low frequency
of the HLA-DRB1*1501-DQB1*0602 haplotype may mostly explain the low MS
prevalence in Africa. Even though African Americans are known to carry other MS
risk haplotypes (for example HLA-DRB1*0301-DQB1*0200), the effect size of these
haplotypes seems to be substantially smaller than that of the HLADRB1*1501DQB1*0602 haplotype in Caucasians (Oksenberg et al. 2004).
Roles for HLA class I loci in MS have also been suggested in several studies
(Fogdell-Hahn et al. 2000, Harbo et al. 2004, Yeo et al. 2007, Brynedal et al. 2007).
However, many of the reports suggesting association between HLA class I and MS
are likely to be secondary to LD with class II loci. This was supported by the recent
study, in which class I and II interactions were analysed in 1,260 individuals from
almost 300 MS families (Chao et al. 2007). Overtransmission of the HLA-A and
HLA-B alleles could be detected only in HLA-DRB1*15 positive but not in HLADRB1*15 negative MS families.
The HLA locus has been estimated to explain 14-50% of the genetic
susceptibility of MS (Hafler et al. 2005), and the population attributable risk of MS
has been estimated to be 48% for HLA-DRB1*1501 positive individuals (Svejgaard
2008). Hence, a substantial fraction of the genetic component still remains to
be explained. In addition to HLA, whole genome scans and other more targeted
linkage approaches in several populations have provided evidence for the location
of susceptibility genes on chromosomes 5p and 17q (Ebers et al. 1996, Oturai et al.
1999, Eraksoy et al. 2003, Sawcer et al. 1996, Larsen et al. 2000, Dyment et al. 2001,
IMSGC 2005) (Figure 12). Similarly, the Finnish genome-wide linkage analysis in
multicase MS families from Southern Otsrobothnia, together with other mapping
methods, has revealed HLA and two wide regions on chromosomes 5p15-q11 and
17q22-q24 as the main MS susceptibility loci (Kuokkanen et al. 1996, Kuokkanen
et al. 1997) (Figure 12). Interestingly, these regions are syntenic to the EAE
susceptibility loci of rodents, supporting their role in predisposition to autoimmune
demyelination (Butterfield et al. 1998, Sundvall et al. 1995, Jagodic et al. 2001). In
addition, some evidence for linkage and association to the myelin basic protein
(MBP) locus on 18q and a region on 19q has also been observed in Finnish MS
families (Tienari et al. 1992, Reunanen et al. 2002).
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Importantly, only loci with moderate to high effect on disease outcome can be
detected using linkage approach. If the CD-CV hypothesis holds true, very many
these kinds of predisposing variants do not exist for MS. Secondly, linkage analyses
utilizing exceptional MS families as a study sample can detect only variants with at
least moderate penetrance, and this kind of variants must be relatively rare (Figure
7). As mentioned above, the most effective mapping of relatively rare variants is
carried out in populations with unusual histories, like Southern Ostrobothnia in
Finland. By contrast, common, low effect variants of MS might be more meaningful
to map using the sensitive GWA method and large study samples.
FIGURE 12. The main MS susceptibility loci in Finnish pedigrees.
Evidence for linkage to these regions has been observed also in other populations, and the
loci are syntenic to EAE susceptibility loci of rodents.
2.3.2 MS candidate gene studies
Numerous candidate genes for MS (genes coding for proteins with meaningful
biological function regarding MS) have been studied in several populations, but
the findings have mainly been unconsistent between different sample sets. Notably,
most of these studies have been performed using small or modest sized study
samples and sparse marker maps, potentially leading to either false positive or false
negative results. Variants outside the linked loci tend to have small effect sizes, and
the smaller the effect is, the larger should the sample size be to detect association.
Further, the pathogenesis of MS is largely unknown and thus, selection of particular
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candidate genes based on their biological function can be misleading. Some of the
numerous MS candidate studies are described below.
There is commonly an overlap in families with different autoimmune diseases
like rheumatoid arthritis (RA), autoimmune thyroid disease (AITD) and type 1
diabetes (T1D) (Maier and Hafler 2008). Similarly, a number of genetic loci show
association with several autoimmune diseases, suggesting that these immunemediated diseases may, at least partially, share a common molecular backround.
Increased prevalence rates of various autoimmune diseases, including AITD and
T1D, have been reported also among relatives of MS probands (Barcellos et al.
2006, Midgard et al. 1996, Broadley et al. 2000), and MS seems to occur more
frequently, for example, in families with systemic lupus erythematosus (SLE) than
in the general population (Corporaal et al. 2002). However, based on a recent study,
no excess of common autoimmune diseases could be identified in MS patients or
their families when the data was adjusted for sex (Ramagopalan 2007 A). Neither
does MS explicitly fit into the “genetic cluster of autoimmunity”. For example, the
cytotoxic T lymphocyte antigen 4 (CTLA-4) gene, involved in the regulation of T
cell proliferation, show association with T1D and AITD (Kavvoura and Ioannidis
2005, Kavvoura et al. 2007), but discrepant results have been reported in MS.
Nominal evidence for association has been observed in two Scandinavian studies
(Harbo et al. 1999, Ligers et al. 1999) whereas no linkage or association to the
region have been detected in several other studies (Dyment et al. 2002, Lorentzen
et al. 2005, Bonetti et al. 2004, Greve et al. 2008, Wray et al. 2008). Similarly, no
association between the protein tyrosine phosphatase (PTPN22) gene, a regulator
of T cell receptor signalling associated with SLE, RA and T1D (Vang et al. 2007),
and MS has been observed (Begovich et al. 2005). IL2RA, being strongly associated
both with MS and T1D, as well as the STAT3 gene, associated with MS, T1D
and Crohn’s disease, are obviously exceptions (see 2.3.3 and 5.1.1). Moreover,
some evidence for association with MS have been observed with the interferon
regulatory factor 5 (IRF5) gene (Kristjansdottir et al. 2008), which is associated also
with SLE, RA and inflammatory bowel disease (Dideberg et al. 2007, Sigurdsson
et al. 2007, Demirci et al. 2007), and the MHC class II transactivator (MHC2TA)
gene, which is associated also with RA (Swanberg et al. 2005). However, replication
of these findings is still needed to validate the role of IRF5 and MHC2TA in genetic
susceptibility of MS.
Most of the MS candidate genes studied possess immunological functions.
One of them is IL7R, which codes for a receptor of regulator of lymphopoiesis, IL7.
A Swedish research group selected 66 candidate genes based on their immunological
functions and/or location in linked regions, IL7R located on the linked region of
chromosome 5p being one of them (Zhang et al. 2005). In a small Swedish sample
set of 670 cases and 670 controls nominal evidence for association with IL7R was
observed (p=0.004). The IL7R association was further validated in two simultaneous
publications studying rather large study samples from Scandinavia, US (European
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descent), UK and Belgium (Lundmark et al. 2007, Gregory et al. 2007). Moreover,
the nonsynonymous SNP rs6897932 (T244I) in the alternatively spliced exon 6
of IL7R was reported to most likely be the causative variant. The MS-associated
variation T244I is located in a transmembrane domain of the IL7R protein. The
associated allele C of SNP rs6897932 is the major allele (allele frequency ~70%) and
has only a modest effect on MS susceptibility (OR 1.2-1.3) (Lundmark et al. 2007,
Gregory et al. 2007). Thus, it is very unlikely that the risk variant of IL7R alone
explains the linkage observed to chromosome 5p in MS families. Some functional
data for the MS-associated variant of IL7R already exists. The SNP rs6897932 has
been shown to influence the amount of soluble (non-functional) and membranebound (functional) isoforms of the IL7R protein by putatively disrupting an exonic
splicing silencer, individuals carrying the C allele having higher levels of circulating
soluble receptor (Gregory et al. 2007). Further, levels of IL7R and IL7 transcripts
have been reported to be higher in the CSF of MS patients than in that of unaffected
controls (Lundmark et al. 2007). On the other hand, this is not necessarily due to the
genetic variant of IL7R, but can just be a consequence of the active inflammatory
process of MS CNS.
Numerous other immunological candidate genes have also been studied as
candidate genes for MS, including complement components 6 and 7, complementlike perforin and regulators of T lymphocytes, to mention but a few. In a small
study by Chataway et al., only three SNPs of C7 and one SNPs of C6 were genotyped
(Chataway 1999). Suggestive evidence for linkage and association with C7 and MS
was observed, but correction for multiple testing diluted the signals and the authors
concluded that C6 and C7 do not confer susceptibility to MS. However, the study
was underpowered.
Perforin is involved in CD8+ T cell and natural killer (NK) cell mediated
cytotoxity. After perforin and granzymes are released from these cells on a target
cell upon its recognition, perforin forms MAC resembling pores on the target
cell membrane allowing entry of apoptosis triggering granzymes into the target.
Homozygous mutations of its gene PRF1 cause a rare immune deficiency syndrome
due to decreased capacity of the immune system to clear viral infections, and it
has been suggested that some heterozygous variations may also favor development
of several autoimmune diseases (Cappellano et al. 2008). Hence, PRF1 has been
studied also as a candidate gene for MS. Recently the entire coding region of
PRF1 was sequenced in 190 MS patients and 268 controls, and frequency of the
exonic variations of the perforin gene was observed to be higher in patients than
in controls (17% vs 9%) (Cappellano et al. 2008). The finding was replicated in a
larger independent study sample. The authors suggested that the variations of PRF1
may be important for MS development by altering perforin activity and thus by
delaying virus clearance, potentially favoring development of molecular mimicry
(Cappellano et al. 2008).
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In a Norwegian study sample nominal evidence for association with the SH2
domain protein 2A (2H2D2A) gene and MS has been observed (Dai et al. 2001,
Lorentzen et al. 2008). 2H2D2A is a good candidate gene for MS since it encodes a
T cell specific adaptor protein, which is important for normal differentiation and
activation of T cells. However, replication of the association in other populations
has been problematic (Lorentzen et al. 2008).
Myelin basic protein (MBP) is a key player in myelin maintenance and repair
and is a potential target for immune-mediated demyelination, the MBP gene being
thus one of the MS candidates. Further, the golli form of MBP has been shown
to negatively regulate signal transduction in T lymphocytes (Feng et al. 2004).
Evidence for linkage and association between MS and MBP has been observed in
MS samples from Southern Ostrobothnia in Finland (Tienari 1992, Tienari 1998,
Pihlaja et al. 2003). However, the findings mainly have not been confirmed in other
populations studied (Pihlaja et al. 2003).
2.3.3 The first genome-wide association scan of MS
It has been suggested that association studies in complex diseases should involve
at least 2,000 cases and 2,000 controls to achieve significance level where p-values
<5x10-7 would more commonly be true positives than false positives (Wellcome
Trust Case Control Consortium (WTCCC) 2007). Even larger, international study
samples are needed to identify common MS variants with very small effects. On
the other hand, allelic and locus heterogeneity most probably exists in clinically
heterogeneous MS, and large study samples combining cases from several
populations can be disadvantageous in identification of relatively rare susceptibility
variants, which may be even population specific (Bodmer and Bonilla 2008).
Recently, the first MS GWA study was published, involving 931 trio families
and 2,431 controls from UK and US (IMSGC 2007). A total of 70 SNPs were
selected for validation based on association signal (p<0.0001 for families (Figure
13), p<0.001 for the case-control set). Further, SNPs showing only very modest
association with MS (p<0.01) but located in proximity to autoimmune loci were
also selected for validation, resulting in 40 SNPs. Thus, the study design was
actually partially candidate gene-based. Despite the fairly small study sample of
the first stage of the study, validation of the findings was performed using a largish
study sample of 2,322 MS cases, 5,418 unaffected controls and 1,540 trio families.
Three non-HLA markers in two genes exceeded the p-value threshold suggested
by the Wellcome Trust (WTCCC 2007): rs6897932 in the IL7R gene (p=2.94x10-7)
and rs12722489 and rs2104286 in the IL2RA gene (p=2.96x10-8 and p=2.16x10-7,
respectively). However, it is worth noting that IL7R showed only trivial level of
significance in the first phase of the study and was actually selected for the study
based on its immunological function and the previous publications (Zhang et al.
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2005, Lundmark et al. 2007, Gregory et al. 2007). Weak evidence for association
in the replication sample was observed also with some of the SNPs selected for
validation based on association observed in the first phase (SNPs located on the
following genes: RPL5, CD58, FAM69A, ANKRD15 and CBLB) (IMSGC 2007), but
none of these SNPs exceeded the p-value threshold suggested by the Wellcome
Trust (WTCCC 2007).
FIGURE 13. Association results of the first stage of the MS GWA scan.
P values (shown as -log10 values) for results of transmission disequilibrium testing (TDT)
in 931 MS trio families are plotted across the genome. SNP with p<0.0001 (dashed line) in
this analysis were selected for validation. Modified from IMSGC 2007 with a permission
of the copyright holder.
IL2RA was selected as a candidate gene for MS due to its association with
another immune-mediated disease, type 1 diabetes (WTCCC 2007). The IL2RA
gene, located on chromosome 10p15, codes for interleukin 2 receptor alpha chain.
IL2R in turn mediates the action of the T-cell growth factor IL2. Originally, two
SNPs (rs12722489, rs2104286), being in LD (r2=0.5) with each other, showed
association with MS (IMSGC 2007). Later, SNP rs12722489 was showed to provide
the primary association (IMSGC 2008). Notably, even the modest LD between the
SNPs rs12722489 and rs2104286 was enough to reveal the primary association of
the common risk allele when the study sample was large enough. Like the risk
allele of IL7R, the common susceptibility variant of IL2RA has only a modest
effect on MS susceptibility, with an OR of ~1.2 (IMSGC 2007 and 2008). The risk
variants of IL7R and IL2RA have further been genotyped in a large set of over
20,000 individuals from Australia, Belgium, Denmark, Finland, France, Germany,
Ireland, Italy, the Netherlands, Norway, Sardinia, Spain, Sweden and UK (IMSGC
2008) and in 600 multiplex MS families from Canada (Ramagopalan et al. 2007b)
to refine understanding of the findings. Association was replicated in all but three
populations. In the large combined study sample impressing p-values of even 1x1023
were obtained, even though the variants are common and have only modest effect
on disease outcome. Associations could not be replicated in three populations,
namely Australia and Ireland for IL7R and Holland for IL2RA Notably, the small
case-control sample from Holland was significantly underpowered (power to detect
an OR of 1.2 was only <10%).
All the 17 SNPs showing even weak evidence for association in the first MS
GWA scan were later genotyped in a large set of 1,134 MS cases and 1,265 controls
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from Australia (Rubio et al. 2008). Again, no association could be detected with
IL7R and MS, even though the statistical power was estimated to be almost 90%;
the risk allele of IL7R was actually less frequent in Australian MS cases than in
controls. In this study associations with KIAA0350, IL2RA, RPL5 and CD58 could
be replicated.
At least the risk allele of IL7R seems to be independent of the HLA
(Lundmark et al. 2007). Interestingly, on contrary to the prevalence of MS, the risk
alleles of both IL7R and IL2RA are more common among non-white populations
than in populations of European origin (IMSGC 2008). These variants explain very
small proportion of the genetic risk of MS, which has been estimated to be only
0.2% (IMSGC 2007). However, it is still not clear whether the variants identified are
the actual causative ones, and fine-mapping and functional studies are required to
fully understand the role of IL7R and IL2RA in pathogenesis of MS.
Whereas this first GWA study of MS highlighted the power of collaboration in
identification of genetic risk variants, it also made clear that identification of other
MS risk genes with even smaller effects of disease outcome can only be revealed by
studying much larger samples. Hence, the extended consortium, supported by the
Wellcome Trust, has started even larger MS GWA scan (http://www.neurodiscovery.
harvard.edu/research_initiatives/imsgc.html). In total this study will examine
approximately 20,000 patients and 20,000 non-MS controls. Hopefully this large
international collaborative project, of which massive data will be analysed during
the next couple of years, will reveal novel MS genes and pathways and results in
better understanding of MS pathogenesis.
2.3.4 Other strategies to map MS predisposing genetic variants
One method of association analysis is admixture mapping, which can be used
when two populations have different prevalence of a disease and there exists a third
population admixed of these first two populations (Zhu et al. 2008). Prevalence
of MS is extremely low in Africans and much higher in European-Americans,
whereas the prevalence in African-Americans is in between, suggesting that MS
predisposing genetic variants most probably exist in the European genome but not
in the African genome. In an admixture study for MS, genomes of African-American
MS-patients were analysed to find regions that have an increased proportion of
European ancestry due to potential risk alleles of European origin (Reich et al.
2005). Strongest evidence for association was found on the centromeric region of
chromosome 1. Later, modest association with the CD58 gene of the admixture
locus was detected in the MS GWA scan (IMSGC 2007). The CD58 gene encodes
the CD58 antigen which, together with its counterreceptor CD2, optimizes immune
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recognition and, on the other hand, promotes differentiation of regulatory T cells
(Arthur et al. 2008), thus being a good candidate for MS.
Another way to try to dissect the molecular backround of MS is to compare
transcriptional profiles of MS patients and unaffected controls using microarray
technology. In MS lesions, overexpression of inflammation-related genes and
underexpression of myelin component coding genes have been observed, reflecting
an important role for the immune system in MS and suggesting that ineffective
remyelination may predispose to chronic demyelination and neuronal damage
(Lock et al. 2002).
Finally, it is worth noting that the role of structural variation and epigenetics
in MS still remains mainly uncharacterized. For example, rare and even family
specific high penetrance CNVs (both de novo and inherited variation) seem to have
an important role in genetic susceptibility to autism (Abrahams and Geschwind
2008). The role of this kind of more complex variation should also be dissected in
more detail in future studies of MS.
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Aims of the study
The aim of this thesis was to better understand the genetic architecture of MS,
pathogenesis of which is largely unknown. The following specific aims were
addressed:
1. To identify MS predisposing risk alleles within the two wide MS linkage loci
on chromosomes 17q and 5p utilizing the strength of the Finnish population
history (I, II).
2. To study relevant biological pathways for MS based on the findings (III).
3. To test if allelic variation of the DAP12 and TREM2 genes, mutations of which
cause a recessively inherited white matter disease PLOSL, would have an
impact on another immune-mediated demyelinating disease, MS (IV).
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4.1 Study sample
4.1.1 Finnish MS sample
The Finnish MS samples have been collected from the hospital districts of Helsinki,
Kuopio, Tampere, Oulu and Seinäjoki (Southern Ostrobothnia). The diagnosis
of MS has strictly followed Poser’s diagnostic criteria (Poser et al. 1983). All
individuals have given their informed consent and the study has been approved by
the Ethics Committee for Ophthalmology, Otorhinolaryngology, Neurology and
Neurosurgery in the Hospital District of Helsinki and Uusimaa (Decision 46/2002,
Dnro 192/E9/02). Unfortunately, only little clinical data for the MS patients was
available during this thesis study.
The Finnish study sample is described in Figure 14. The family-based study
material from Southern Ostrobothnia MS high-risk region consists of 22 Finnish
multiplex MS families with two to six affected cases per pedigree, and ~140 MS
patients with their parents and/or unaffected siblings. The multiplex families have
previously been utilized in the Finnish genome-wide linkage scan (Kuokkanen et
al. 1997). For case-control analyses, ~390 regional population controls have also
been collected from Southern Ostrobothnia. In addition, ~730 unrelated MS cases
and ~960 population controls have been collected from other parts of Finland.
At the time of the first publication (I), no population control samples were
available and only MS families were studied. Case-control samples have since been
utilized to increase statistical power (II-IV). However, to verify the findings and
to avoid false positive association signals induced by population stratification, the
families have been studied as well (II). The number of Southern Ostrobothnian MS
families has slightly diminished during this thesis study due to revised genealogical
data.
A small proportion of the Finnish genotyping sample was utilized for further
studies. The genes of interest were sequenced in approximately ten Southern
Ostrobothnian MS cases and population controls (I, II), and expression of the
genes were tested in lymphocyte and mononuclear cell samples of ~10-20 cases and
controls from Southern Ostrobothnia (I, II). Further, serum and plasma samples,
as well as some clinical data, were collected for 20 Southern ostrobothnian MS
cases (II). Finally, 174 Finnish MS cases and 172 population contols, of which half
originated from Southern Ostrobothnia, were utilized in the CNV analysis (IV).
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FIGURE 14. Study samples used in this thesis.
The rounded number of MS families, MS cases and population controls from different
countries used in original publications (I-IV).
4.1.2 Selection of cases and controls for the Finnish GWA study
We utilized the data of the Finnish GWA study (Jakkula et al., manuscript in
preparation) to screen the chromosome 5p linked region (II). Specifically, 72 MS
cases, having either one parent born in the high-risk region of Southern Ostrobothnia
and a family history of MS (n=8) or both parents born in Southern Ostrobothnia
(n=64), have been genotyped for the GWA. Of these 72 MS cases, 41 belonged to
either one (n=14) or both (n=27) of the two large interconnected mega-pedigrees,
which we were able to construct via genealogical studies (Figure 15). However, no
1st degree relatives were included. 68 identity-by-state (IBS) matched population
controls from Finnish genome-wide studies (from a total of 227 control individuals
with GWA data) were used as the control set. We used genome-wide SNP data
and IBS, identity-by-descent (IBD) and multidimensional scaling analyses to select
these controls so that their genetic background would be similar with the cases,
as parental birthplace information was not available for all the controls (Figure
16). Fourteen of the controls were known to have both parents born in Southern
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Ostrobothnia (Figure 16, SOB ctrls) while 13 of the controls were known to live in
Southern Ostrobothnia (Figure 16, SOB living ctrls) and 41 controls were part of
the Health 2000 project (Figure 16, H2000 selected ctrls).
The genomic inflation factor (a comparison of unassociated genetic markers
with those of control subjects for potential differences in allele frequency related
to imperfect matching between case subjects and control subjects) was 1.0758 for
our GWA data set, which suggests that cases and controls are well-matched (no
difference over the majority of markers tested) and thus, there is no large-scale
population stratification within our final study set. Importantly, no 1st degree
relative pairs were found in any pair combination (case-case, case-control, controlcontrol) according to the IBD sharing estimates.
FIGURE 15. Finnish mega-pedigrees.
Majority of the MS cases of the Finnish GWA study belonged to either one or both of the
two large interconnected mega-pedigrees constructed via genealogical studies.
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FIGURE 16. Selection of the controls for the Finnish GWA study.
Multidimensional scaling of pairwise IBS sharing data.
4.1.3 Study samples from more heterogeneous populations
In this thesis study a Finnish population subisolate has been utilized in finemapping. To study the role of the findings in more heterogeneous populations,
MS families, cases and controls have been obtained from the collaborators of the
Canadian Collaborative Project on the Genetic Susceptibility to MS (Figure 14,
Canada), the Nordic MS Genetics Network (Figure 14, Sweden and Norway) and
the Partners Multiple Sclerosis Center in Boston, Massachusetts (Figure 14, US).
The Canadian cohort consisted simplex, extended, affected sibling pair and affected
parent-child pair families. All the samples used were of Northern European descent,
and the diagnosis of MS has strictly followed Poser’s or McDonald’s diagnostic
criteria (Poser et al. 1983, Polman et al. 2005). All individuals have given their
informed consent and the study has been approved by the ethics committees of the
institutions involved.
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4.2 Laboratory methods and statistical analyses
The methods used in this study are described in Table 5. Most of the SNPs have
been genotyped using the Illumina 317K and the Illumina Golden gate assays and
the Sequenom’s MassArray system (Sequenom, San Diego, CA, USA), either with
the hME or the iPLEX reaction. The Sequenom method utilizes chip arrays and
matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry (Gabriel and Ziaugra 2004, Gabriel et al. 2009). Genotype counts
between MS cases and unaffected controls were compared using Pearson’s chisquare statistics (II-IV). The standard measure of effect in the case-control study
is the odds ratio (OR), defined as the odds of exposure among cases divided by
the odds of exposure among controls. Transmission disequilibrium test (TDT)
(Spielman et al. 1993), haplotype-based haplotype relative risk (HHRR) (Terwilliger
and Ott 1992) analysis as well as the Gamete Competition option of the Mendel 7.0.0
program (Lange et al. 2001) were used to monitor for association in MS families
(I, II). In TDT analysis the transmission of alleles from heterozygous parents to
affected children is compared to the expected 1:1 ratio. In HHRR, the two parental
alleles, which have not been transmitted to the affected child, are combined to
form the marker genotype of the “control individual”, overcoming the stratification
problem of the case-control design (Terwilliger and Ott 1992). TDT utilizes only
genotype data of full trios. This is worth noting since only ~30% of the Finnish and
Canadian MS families studied had both parents available. HHRR uses genotype
data of cases also when data for both parents is not available. Further, HHRR is
able to make use of families in which both parents are not heterozygotes for a given
marker (Terwilliger and Ott 1992). The TDT-based Gamete Competition analysis
utilizes the genotype data of the whole pedigree by treating transmission to normal
children as complementary to transmission to affected children and is better
adapted to missing data than the classical TDT test (Lange et al. 2001). Notably,
if more than one affected child per family is used, all the family-based methods
can confound linkage and association. Statistical significance of an association
analysis is usually defined with p-value, which is the probability of obtaining by
chance a result at least as extreme as that observed, even when the null hypothesis
(no association) is true and no real difference exists. The smaller the p-value, the
more strongly the test rejects the null hypothesis and the more unlikely the result
is explained by chance alone. A p-value of 0.05 (corresponding to a 5% probability)
or less is commonly used to reject the null hypothesis.
Illumina HumanHap300 SNP chip (Illumina, San Diego, CA, USA) was used
to genotype samples of the Finnish GWA study. High quality SNPs (n=3,981)
mapping to 11.1-56.0 Mb of chromosome 5 were used in the sliding window 5 SNP
haplotype analyses (II), which were performed using the PLINK program (Purcell
et al. 2007).
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This study utilized two methods for multiple testing correction. In SNPSpD
method (Nyholt 2004) the effective number of variants was estimated and the
significance threshold was adjusted according to estimated number of independent
SNPs (I). In permutation method (Chuchill and Doerge 1994) a false positive rate
was obtained by generating several data sets by breaking the link between genotype
and phenotype data (II).
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TABLE 5. Methods used in this thesis study.
Laboratory method
Original publication
DNA and RNA extraction, quality controls
Polymerase chain reaction (PCR)
Agarose gel electrophoresis
Genotyping
Allele-specific primer extension on microarray
Pyrosequencing
Sequenom MassArray
Illumina BeadArray
Illumina HumanHap300 SNP chip
ABI Prism DNA Sequencer
Quantitative real time (RT) PCR using TaqMan
Sequencing using ABI Prism DNA Sequencer
Complement level quantification
Radial immunodiffusion (Mancini technique)
Enzyme-linked immunosorbent assay (ELISA)
I-IV
I-IV
I-IV
Statistical method or analysis program
Original publication
SNP and primer selection
dbSNP database
HapMap tagger
Primer3
Sequenom AssayDesigner
Sequenom MassArray Typer
Linkage and association analyses
PLINK
Pedcheck
Chi-square test
Merlin
MLINK
TDT, Analyze package
HHRR, Analyze package
Mendel Gamete Competition
Haploview
Phase
Genepop
GeneMapper
Correction for multiple testing
Permutation using Haploview
SNPSpD
ABI Prism 7900HT Sequence Detection System
Sequencher
Mann-Withney U test
Copy number variation analysis using SeLoFit
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I
I
I-IV
I
II
IV
I, II, III
I, II
II
II
I-IV
II, III
I, II
I-IV
I-IV
II, III
I, II
I-IV
I
IV
I
I
II
I-IV
I, II
I
IV
I, II
I
I, II
I, II
II
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5
Results and discussion
5.1 Fine-mapping of the linked loci on chromosomes 17 and 5 (I, II)
In addition to HLA, whole genome linkage scans in several populations, including
Finns, have provided evidence for the location of MS susceptibility genes on
chromosomes 5p and 17q (Figure 12). Further, these regions are syntenic to the
EAE susceptibility loci of rodents, supporting their role on predisposition of
autoimmune demyelination. An effort has previously been made to narrow these
wide linked loci by haplotype analysis in Finnish MS families (Saarela et al. 2002,
Riise-Stensland et al. 2005). The 17q ~20Mb linked locus has been restricted to a
~3.4Mb region (Saarela et al. 2002), which was further fine-mapped in this thesis
study (Figure 17A). For chromosome 5p no gene in the restricted region was
found to explain the linkage observed (unpublished), thus, in this thesis study the
complete linked region was futher scanned through to pinpoint a MS predisposing
gene (Figure 18).
Notably, most probably MS is not a unitary disorder, but may represent an
overlapping spectrum of related disorders (Lucchinetti 2000, Barcellos et al. 2002).
To minimize the genetic and environmental heterogeneity, MS patients and families
from the homogeneous Southern Ostrobothnian MS high-risk isolate have been
utilized as a study sample in these fine-mapping efforts (Figure 14).
5.1.1 MS locus on chromosome 17q (I)
Fine-mapping of the chromosome 17q linked locus was started at the time when
no extensive marker maps, HapMap or high-throughput genotyping techniques
existed. In the first stage of the study, the previously restricted 3.4Mb region was
flanked and covered with a sparse marker map of 67 unevenly distributed SNPs
(Figure 17A), which were genotyped in a small set of 63 Finnish MS families. Nine
of the SNPs studied showed some evidence for association (p<0.05) in TDT or
HHRR tests, four of them mapping to the protein kinase C alpha (PRKCA) gene
(Figure 17A). The second stage of the study focused on PRKCA and the 1Mb flanking
sequence. Over 200 SNPs were selected from the dbSNP database to somewhat
evenly cover the target region (Figure 17A). These markers were genotyped in
the whole set of Finnish nuclear MS families from Southern Ostrobothnia and
in a large set of Canadian MS families. The strongest p-valuewise evidence for
association was observed with SNP rs887797 (p=0.0001), which is located in
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intron 3 of the PRKCA gene (Figure 17A). This association observed seemed to
be independent of the HLA. Although the same SNP of PRKCA failed to reveal
association in Canadian families, two SNPs next to it provided suggestive evidence
for association in this more heterogeneous study sample (Figure 17A). However,
some evidence for association with MS was observed also with SNPs mapping to
other genes within the critical region (Figure 17A), and not all of these SNPs were
in high LD with PRKCA.
Further, all the exons, the promoter as well as parts of the intron 3 of the
PRKCA gene were sequenced in ten MS patients and eight unaffected population
controls, but no causative variant was identified. To dissect the allelic backround
in more detail, haplotypes over the critical region were constructed. A haplotype,
flanking introns 3 and 8 of PRKCA, was observed to be over-represented in Finnish
MS cases compared to their healthy family members (OR 1.34, 95th CI 1.07-1.68),
whereas another haplotype of the same region showed association in Canadian
MS pedigrees (OR 1.64, 95th CI 1.39-1.94). In a small sample of MS cases and their
unaffected family members (n=20) a slight correlation, although not statistically
significant, with PRKCA expression in CD4- blood mononuclear cells and the
putative risk allele was observed, PRKCA expression being lower in individuals
with two copies of the risk allele compared to carriers of only one copy of the allele
(Figure 17B).
The study represented a large-scale fine-mapping effort of that time. When this
fine-mapping study was started only few SNPs were known to exist in chromosome
17q according to public databases. No tagging SNP information was available even
when the second stage of the study was started, and not all the common variation
was captured. Further, at the time there was a debate in science community on how
to construct and analyse haplotypes. Thus, the study really emphasizes the rapid
progress in the field of human genetics.
The first stage of the study was conducted in a small set of MS families, of
which only a fraction could be utilized in the TDT-based analyses. However,
association with a SNP in the PRKCA gene was observed, and this association was
validated in a larger set of Finnish MS families. Thus, SNPs within PRKCA seem to
be in LD with some yet unidentified MS variant, at least in the Finnish population
subisolate. At the time we published our results, modest evidence for association
with PRKCA (p=0.001) was observed also in UK population (Barton et al. 2004).
Specifically, in that study 35 SNPs of PRKCA were genotyped in a small sample of
184 MS cases and 340 controls from UK. The associated variants were located in
the 5’ and 3’ ends of the large 0.5Mb PRKCA gene, and no evidence for association
was detected with SNPs of the intron 3. Notably, the 5’ end association was later on
suggested to be a false positive finding: no evidence for association with the same
variants was observed in 947 MS families from UK (Ban et al. 2005).
PRKCA (located at 17q24.2) encodes a protein kinace C type alpha, which
is a calcium-activated, phospholipid-dependent serine- and threonine-specific
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enzyme. When activated by diacylglycerol, PKC phosphorylates a range of cellular
proteins. PRKCA is fairly ubiquitously expressed.
Interestingly, it has been shown that PKC alpha plays a critical role in signal
transduction pathway via which the cytokine CCL2 induces permeability of
the blood-brain barrier (BBB) (Stamatovic et al. 2006), making PRKCA a good
functional candidate for MS predisposition. However, based on our data MS
cases carrying two PRKCA risk alleles seem to express less PKC alpha compared
to individuals with one copy of the risk allele. Obviously, this is in discordance
with the hypothesis that BBB of MS patients is anomalously permeable, facilitating
penetration of lymphocytes into the CNS.
Like potential causative variants of other linked regions, the putative MS variant
of the 17q locus is most probably relatively rare (Figure 7). Thus, it is not totally
surprising that no very strong evidence for association with any of the SNPs within
the 17q locus was observed in the first international MS GWA, which has been
optimized to detect common variation (IMSGC 2007). By contrast, such relatively
rare variant most probably should have been revealed by the Finnish GWA study
(Jakkula et al., manuscript in preparation). However, none of the variants within
the restricted 3.4Mb region provided strong evidence for association in the Finnish
scan. Allele frequency of the most promising SNP of the PRKCA gene (rs887797)
remained to be different between the MS cases and the unaffected population
controls studied (MS 0.80 versus controls 0.75, p>0.05), but this variant unlikely
explains the linkage observed to 17q.
The wide linked region of chromosome 17q has previously been restricted by
selecting a region that was shared by all affecteds from each of the 20 multiplex
families studied (Saarela et al. 2002). In fact, the affecteds shared the whole linked
region in all but three families. Thus the MS variant may be located also outside
the 3.4Mb region, which was shared by all affected individuals of each family.
Hence, like in question of chromosome 5p linked locus, we later decided to scan
through the complete region under the original wide 17q linkage peak utilizing the
Finnish GWA data. Indeed, variants of the STAT3 (signal transducer and activator
of transcription) gene, located outside the restricted 3.4Mb region, provided
strong evidence for association with MS (p~5x10-5) (Jakkula et al., manuscript in
preparation). The finding was further replicated in case-control samples from six
populations of Northern European origin (a combined CMH analysis p=2.65 x 10-10)
(Jakkula et al., manuscript in preparation). SNPs within STAT3 have provided
nominal evidence for association also in the international MS GWA scan (p=0.002
in the first phase, p=0.03 in the validation phase), and these SNPs were observed to
be in LD with the associated variants of the Finnish scan (r2>0.7 according to the
HapMap CEU data). Interestingly, STAT3 has been reported to show association
also with Crohn’s disease, ulcerative colitis and T1D (WTCCC 2007, Barrett et al.
2008, Franke et al. 2008, Fung et al. 2008), the risk allele of MS being protective
for Crohn’s disease and ulcerative colitis. The protein encoded by STAT3 acts as a
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transcription activator when activated in response to certain cytokines and growth
factors (including IL5, IL6, interferons, HGF, LIF and BMP2). Interestingly, in
a recent study it was shown that mice with targeted deletion of STAT3 in CD4+
T-cells do not develop EAE (Liu et al. 2008). The authors hypothesize that STAT3
may have a critical role in shaping T–cell repertoire: activation of STAT3 seems to
be required for generation of Th17 lineage and restriction of the Th1 lineage. The
role of STAT3 in MS predisposition will certainly be tested in future studies.
Other biologically highly relevant candidate genes within the 17q linked region
are ICAM2 and PECAM1 (both located at 17q23.3), which code for intracellular
adhesion molecules involved in, for example, transendothelial migration of
lymphocytes. The Illumina SNP chip used in the Finnish GWA scan includes
only five SNPs in PECAM1 and no SNPs in ICAM2. None of the studied SNPs
provided evidence for association with MS in the Finnish GWA scan (Jakkula et
al., manuscript in preparation). Neither have previous candidate gene based studies
revealed evidence for association between PECAM1 and MS (Nelissen et al. 2000,
Nelissen et al. 2002, Sciacca et al. 2000), but notably, all of these studies analysed
only one microsatellite polymorphism in a fairly small study sample. Thus, the role
of these genes in MS predisposition can not be definitely excluded.
Interestingly, the restricted 3.4Mb region on 17q24 is flanked by palindromic
segments and highly homologous duplicated sequences. These can predispose to
large chromosomal rearrangements by nonallelic homologous recombination (Chen
et al. 2004). Further, this region is inverted in the chimp and human with respect
to the order in the mouse genome (Chen et al. 2004) (Figure 17A). Considering
the complex structure of this chromosomal region, the SNPs showing association
with MS could be in LD with a yet unidentified CNV or structural variant,
affecting potentially even several genes within the critical region. Unfortunately,
this could not be tested in Finnish MS, since the marker map of the GWA panel
was relatively sparse at the duplicated regions, which could potentially predispose
to the rearrangements.
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FIGURE 17. Fine-mapping of the MS locus on chromosome 17q.
A. The linked locus has previously been restricted to a 3.4Mb region, which is flanked
by highly duplicated sequences (vertical lines) and which is inverted in the chimp and
human compared to the mouse genome. In the first stage of this study, 67 SNPs over the
critical region were genotyped in 63 Finnish MS families. Associated SNPs are marked
as black triangles. In the second stage, over 200 SNPs of PRKCA and the flanking 1Mb
were genotyped in two MS samples from Finland and Canada. Strongest evidence for
association was observed with SNP rs887797. B. An allelic variant of PRKCA was
observed to be over-represented in Finnish MS cases compared to their healthy family
members, and a correlation with this risk allele (one allele n=7; two alleles n=9) and
PRKCA expression in CD4- mononuclear cells was observed.
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FIGURE 18. Fine-mapping of the MS locus on chromosome 5p.
A. The linked region was screened with a five SNP haplotype association analysis from the
Illumina HumanHap 300 chip genotypes. The –log(p-values) for the haplotypes are shown
as blue dots. The analysis revealed one region with omnibus p-values 10-4 (C7-FLJ40243).
B. The haplotype showing strongest evidence for association was extended with PLINK to
both orientations until reaching sites with increased number of degrees of freedom (DF) in
recombination spots. C. The identified 59kb risk region, extending from 3’ end of C7 to 3’
end of FLJ40243, can be divided into a low LD region and a more clear block structure in
heterogeneous populations according to the HapMap CEU data.
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5.1.2 MS locus on chromosome 5p (II)
The first MS GWA scan (IMSGC 2007), together with other studies (Zhang et al.
2005, Lundmark et al. 2007, Gregory et al. 2007), revealed IL7R associated with
increased risk of MS. Notably, IL7R is located in chromosome 5p MS linked region
(Figure 12). However, the associated allele C of the likely causative variant rs6897932
of IL7R is very common also among healthy population, and it has been estimated
to explain only a tiny fraction of the variance in the risk of development of MS. As
speculated above, common variants with low penetrance, like IL7R, unlikely give
rise to a familial concentration of cases. Thus, it is likely that other MS predisposing
variant(s) also exists in the 5p linked region.
We first studied whether the variants of IL7R contribute to MS susceptibility
in Finland. We genotyped SNP rs6897932 as well as three additional SNPs from
previous association studies in the whole Finnish study sample of over 900 MS
cases, of which 200 originated from Southern Ostrobothnia isolate, and 1,300
population controls (Figure 14). Only modest association was observed (rs6897932
p=0.002, OR 1.24, 95th CI 1.09-1.41), the odds ratio corresponding to that observed
in other studies.
To study whether other loci on 5p could be identified as susceptibility loci for
MS in Finland, we scanned through the complete ~45 Mb linked region utilizing
the SNP data of the Finnish GWA study (Figure 18A). Specifically, the Finnish GWA
has been aimed to enrich relatively rare, penetrant variants, which most probably
give rise to a familial concentration of MS cases. The 72 MS patients studied had
parents born in the Southern Ostrobothnia MS high-risk region, characterized
with isolation and a founder effect, and majority of the patients were noticed to
belong to either one or both of the two large interconnected mega-pedigrees, which
we were able to construct via genealogical studies (Figure 15). Even though most
of the MS patients were distantly related, none of them were first-degree relatives
with each other. We hypothesized that the relatively short history, with common
ancestors only 14-16 generations ago, might expose shared haplotypes between the
distantly related MS cases. To avoid large-scale population stratification, we used
68 IBS-matched Finns as controls.
The haplotype analysis over the 5p linked locus revealed one region, located
over 5 Mb centromeric from IL7R (Figure 18A). This haplotype, covering the 3’
ends of the C7 (complement component 7) and FLJ40243 (hypothetical protein
LOC133558) genes, had a frequency of 0.18 in MS cases compared to 0.04 in controls
in the GWA sample (p=0.0001). Due to the small sample size of the GWA scan, we
could not correct the result for multiple testing. Thus, to validate the finding, we
genotyped the haplotype in an independent set of 125 Southern Ostrobothnian
MS cases and over 350 population controls from the same geographical region
and were able to replicate the association (p=0.0004). To estimate the effect size
of the identified 59kb risk haplotype, the two study sets from the isolate were
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combined. Frequency of the C7-FLJ40243 risk allele was 0.12 among MS cases and
0.04 among population controls, resulting to a p-value of 3x10-6 (pperm=5x10-5) and
a fairly high odds ratio of 2.73 (95th CI 1.67-4.47). Convincingly, also familybased association between the critical region and MS was observed (p=0.006),
suggesting that the association observed is not just a false positive finding caused
by population stratification. Due to a low frequency of the C7-FLJ40243 risk allele
and the relatively small sample size we could not test whether the association was
independent of the HLA and the PRKCA.
We sequenced the coding regions of the C7 and FLJ40243 genes in 8 MS cases
and 8 controls from the isolate. Four of the MS cases were known to carry two
copies, four MS cases one copy, and eight controls no copies of the C7-FLJ40243
risk haplotype. We identified altogether nine SNPs. Four nonsynonymous SNPs
were located within C7, one of them being a novel variant. Two nonsynonymous
and three synonymous SNPs were located within FLJ40243. However, none of
the variants was in tight LD with the risk allele, thus, these SNPs are not likely
candidates for the causative variant.
In addition, the C7 promoter and most of the non-coding sequence covering
the 3’ end of C7 were sequenced through. The sequenced region reached from
the beginning of the intron 12 of C7 to a recombination hotspot between the C7
and FLJ40243 genes. Several polymorphisms were found, but again, no definite
causative variant was identified (unpublished result). As evolution conserves
function, we carefully looked at the conserved motives within the sequenced
region. One such sequence motive was noticed to be located in the middle of the
C7 risk haplotype in intron 14 (~41,003,460–41,003,640 Mb according to UCSC,
hg18 assembly, Mar2006). The function of this motive is unknown. Interestingly,
the same conserved sequence is found in several locations of the human genome.
However, there was no variation in the sequence of the conserved motive between
the individuals studied.
There is a potential micro-RNA binding site within the 3’ end of C7, allele A of
the SNP rs1061429 (A/C) enabling binding of miR-591. However, the major allele C
was present in the identified risk haplotype as well as in most of the other haplotypes.
Instead, a SNP rs3805226 could be a good candidate since it is in complete LD
with the risk haplotype of the isolate and is located in a conserved element within
intron 15 of C7. According to the transcription binding site predicting softwares
SNP rs3805226 could potentially alter binding of a brain specific transcription
factor Brn2. However, to verify the functionality of the SNP, changes in DNA
binding capability should be tested for example using the electrophoretic mobility
shift assay (EMSA). Interestingly, of the vertebrates with conservation information
available for this SNP rs3805226 in the USCS Genome Browser (http://genome.
ucsc.edu/), almost all carry an allele A (Opossum, Elephant, Armadillo, Dog, Cow,
Horse, Rabbit, Mouse, Rhesus, Macaque, Chimp), exept Tenrec (C), whereas the
genomes of Cat and Rat contain the rare allele G. However, eventhough the isolate
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enriched haplotype contains the rare allele G, it is not present in risk haplotypes
of the more heterogeneous populations. Thus, this SNP is not a likely candidate
for the causative variant, at least globally. Sequencing of the rest of the non-coding
regions and the intergenic sequence is still warranted.
FIGURE 19. Frequency of the identified C7-FLJ40243 risk haplotype in various populations
according to the Human Diversity Panel data.
The haplotype was observed to be relatively rare globally, having the highest frequency in
Eastern populations. It has further been enriched in MS pedigrees of Southern Ostrobothnia,
having a frequency of 12% among the MS cases of this population subisolate.
The identified C7-FLJ40243 risk haplotype seems to have a fairly large effect
on genetic susceptibility of MS, at least in the Finnish MS isolate where it has got
enriched due to the founder effect and isolation. We monitored the frequency of
this haplotype in various populations utilizing the Human Diversity Panel SNP
data (http://www.cephb.fr/en/hgdp/diversity.php/). Interestingly, the haplotype
was observed to be relatively rare globally, found at the ~4% of alleles in the general
European population, being almost absent in the Africans, Southern Americans
and Oceanians, and having the highest frequency of ~6% in Eastern populations
(Figure 19).
The advantages of the Finnish population isolates are that most of the affected
individuals typically share the same major risk allele and that the relatively rare
variants can be exposed by the common HapMap markers due to the wide LD
intervals. However, the relatively rare variants identified using an isolated population
are much more challenging to detect in more heterogeneous populations with
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distinct LD patterns. As expected, the LD pattern of the 59kb risk region identified
here was noticed to differ between the isolate and the general European population:
based on the GWA data, the haplotype seems to cover only one haplotype block in
the isolate whereas the critical region can be divided into a low LD region and a
more clear block structure according to the HapMap CEU data (Figure 18C).
We studied the identified C7-FLJ40243 risk region also in case-control
samples from more heterogeneous populations, namely Finland outside the MS
high-risk region, Sweden, Norway and US (Figure 14). The risk allele of the isolate
had a comparable frequency in control samples of the isolate (0.04) and the other
populations of Northern European origin (0.03-0.06) but it was not over-represented
among the MS cases from these more heterogeneous populations. However, the
Finnish MS cases were observed to carry another risk allele (freq MS 0.20, controls
0.16, p=0.003), which was also marginally over-represented in the MS cases from
Sweden and US (Sweden: MS 0.18, Ctrl 0.15; US: MS 0.17, Ctrl 0.14), and a third
allele was over-represented among the Norwegian cases (MS 0.05, Ctrl 0.03).
Use of population isolates is a double-edged sword. Our strategy was to study
familial MS cases having both parents born in Southern Ostrobotnia, exposing
the most extreme load of similar genetic background. As expected, the LD pattern
of the identified risk region differed between the isolate and other populations,
thus, it was not totally surprising that unequivocal association could not be
detected. There seems to be more allelic heterogeneity within the region in more
heterogeneous populations, and the potential risk variant may be carried in diverse
allelic backrounds. Only hard functional evidence or identification of the actual
causative variant may enable uniform replication of the finding. Moreover, it is
also possible that, like in many monogeneous diseases, there are various causative
mutations within the same gene in different populations and in that case replication
of the original allelic association in a more heterogeneous population might not be
even feasible.
Notably, it is likely that MS is a more heterogeneous disorder that expected, and
most probably genetic heterogeneity also exists. The MS cases from the Southern
Ostrobothnia isolate presumably carry the same predisposing genes and have
been exposed to same environmental factors, thus they are likely to experience the
same immunopathologicals pattern of MS. Taking into account our results, it is
tempting to speculate that majority of the MS cases of the Finnish MS subisolate
experience the most common pathological subtype of MS, pattern II, characterized
by immunoglobulins and complement activation and that the C7-FLJ40243 risk
allele identified predisposes especially to this type of disease. Hence, sorting the MS
cases of the more heterogeneous populations according to the immunopathological
pattern type could potentially reduce the genetic heterogeneity and unmask the
association. Further, taking into account the frequencies of the identified MS risk
haplotype in different populations, it possibly has drifted into Europe from East,
and it could be worth the effort to study the C7-FLJ40243 in Asian MS.
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The C7 gene is an excellent candidate for MS and there already exists strong
evidence for involvement of the complement system in MS and EAE pathology
(see discussion of study III). The seventh component of the complement system
is a component of the terminal complement complex (TCC, C5b-9) which, when
assembled on a cell membrane, forms the cytolytic MAC complex. Interestingly, C7
is a critical limiting factor of complement activation: only when the local expression
of C7 is sufficient, C7 binds to preformed C5b6 and the resulting C5b-C7 complex
is able to insert into the phospholipid membrane to start the formation of the MAC
(Thompson and Lachmann 1970). C7 has been reported to be synthesized at least
by endothelial cells, polymorphonuclear cells, macrophages, platelets, fibroblasts,
synovial tissue and even in the CNS by astrocytes and oligodendrocytes (Gasque et
al. 1995, Hogasen et al. 1995, Langeggen et al. 2000, Hosokawa et al. 2003, Morgan
and Gasque 1997). However, we observed no expression of C7 in peripheral blood
mononuclear cells (PBMCs), of which we had RNA available.
To study whether the identified haplotype has an effect on C7 protein levels,
we collected plasma samples of 20 MS patients and 32 unaffected controls. Eleven
of the cases and thirteen of the controls were known to carry the MS risk allele.
Eventhough most of the C7 levels were within the reference range, a correlation
between the protein level and the risk allele carriership was observed, carriers of
the risk allele having slightly more circulating C7 protein in plasma compared
to non-carriers. Convincingly, this correlation was seen both in MS cases and in
unaffected controls (Figure 20A).
We hypothesized that the complement system of MS cases could overall
be more active than that of unaffected controls and that the observed increase
in C7 levels could potentially affect the three complement activation pathways:
classical (CP), alternative (AP) and lectin (MBL). Therefore, we studied the total
complement activity in same individuals by measuring the number of TCC formed
as a consequence of activation of each pathway. As expected, the complement
system was significantly more active in MS cases than in controls (Figure 20B).
Importantly, the cascade was most active in MS cases carrying the identified
C7FLJ40243 risk allele, suggesting that this allele further boosts the complement
system when it gets activated, which happens for example in chronic inflammatory
diseases, including MS.
In the future, it would be interesting to study protein levels of the three TCC
components located on 5p, namely C7, C6 and C9, also in CSF samples of MS cases
and unaffected controls. It would also be interesting to test whether the myelin
forming oligodendrocytes are injured more easily when being in contact with the
serum of the carriers of the identified C7-FLJ40243 risk allele. The hypothesis would
be that the carriers of this risk allele have more active complement system compared
to noncarriers and that excess of functional TCCs following the complement
activation would lead to more efficient destruction of the oligodendrocytes, which
are fairly defenceless against the complement mediated lysis (Scolding et al. 1998).
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Notably, the potential causative variant of the C7-FLJ40243 region is still
unidentified. Both C7 and FLJ40243 are present at the corresponding location
of the mouse genome 15 (Mouse Genome Informatics database). The function
of the other gene of the risk haplotype region, FLJ40243, is still unresolved but it
is known to encode for a protein. Based on the human GNF Expression Atlas 2
Data (http://genome.ucsc.edu/) FLJ40243 is expressed at extremely low levels fairly
ubiquitously. To test whether FLJ40243 also is a good biological candidate for MS,
we monitored its expression in several human tissues. In concordance with the
GNF Expression Atlas data, FLJ40243 was observed to be expressed at extremely
low levels at least in spleen, lymph node, fetal liver and fetal skeletal muscle. No
expression was observed in PBMCs, of which we had RNA available, thus, we could
not test whether the identified C7-FLJ40243 risk allele has an effect on FLJ40243
expression in MS.
The C7-FLJ40243 region and IL7R are 5 Mb apart from each other and there is
no LD between these two genes. To test whether these two variants are independent
risk factors for MS susceptibility, we calculated how large proportion of all the
studied Finnish and Swedish individuals carrying a certain number of the IL7R
and C7-FLJ40243 risk alleles were affected. Figure 21 shows how these two MS risk
factors contribute to the probability of developing the disease in an additive way:
individuals carrying the C7-FLJ40243 risk allele are in higher risk to get the disease
compared to non-carriers, and the risk is even higher when an additional IL7R
risk allele is present (Figure 21A). However, at least C7 and IL7R are expressed in
different tissues and cell types, unlikely acting in same cellular pathways in MS.
Interestingly, in addition to the identified C7-FLJ40243 region, two other
haplotypes within the MS linked locus also provided a p-value <0.001 in the original
scan: an intergenic region in 5p15.2 and the FYB region in 5p13.1 at 39.2 Mb
(Figure 18a). FYB encodes a FYN-binding protein isoform 2, which acts as an
adapter protein of the FYN and LCP2 signaling cascades in T cells and modulates
the expression of interleukin-2 (IL-2). Interestingly, Fyb-deficient T lymphocytes
of mice are defective in adhering to mouse Icam1, to human ICAM2 (the gene is
located on chromosome 17q MS linked region), and to other substrates mediated
by integrins (Griffiths et al. 2001). Thus, FYB is also a good candidate gene for
MS.
Besides IL7R, C7 and FYB, the chromosome 5p MS linked locus encompasses
also several other immunological genes, which potentially might also have a role
in MS susceptibility. Importantly, this thesis study demonstrates the complexity of
the genome regions initially identified as potential loci for common diseases and
suggests that several independent genetic risk factors may exist in a single locus
showing evidence for linkage in many populations.
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FIGURE 20. C7 protein levels and the complement activity in 20 Finnish MS cases and 32
unaffected controls.
Carriers of the risk allele are indicated with black dots and the non-carriers with open
dots. A. Plasma complement component 7 protein levels (C7) were noticed to be higher in
carriers of the risk haplotype (+) compared to non-carriers (-). Reference range: 80-120%.
B. Amount of serum terminal complement complexes (TCC), formed as a consequence
of the activation of classical (CP) and alternative (AP) pathways, was observed to be
significantly higher in MS patients compared to controls. Interestingly, the complement
system was most active in MS cases carrying the identified C7-FLJ40243 risk allele. No
statistically significant difference was observed in the lectin pathway (MBL). Dashed lines
indicate the mean values of carriers and noncarriers of the risk haplotype among MS
cases. Reference values: CP>60%, AP>40%, MBL>10%.
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FIGURE 21. Co-effect of the C7-FLJ40243 and IL7R risk alleles on MS predisposition.
Y-axis refers to the proportion of the MS cases among all the individuals of the study sets
carrying a certain number of the IL7R (0, 1 or 2 copies) and C7-FLJ40243 (0 or 1 copies)
risk alleles. The absolute number of the MS cases in each risk group is shown within the
bars in parenthesis. Groups with two copies of the C7-FLJ40243 risk alleles were excluded
from the analysis due to the small number of individuals (2 C7-FLJ40243 risk alleles and
0 IL7R risk alleles: Southern Ostrobothnia MS n=0; other parts of Finland and Sweden
MS n=4). A. Southern Ostrobothnia MS high-risk region. B. Two study sets from more
heterogeneous population, Finland outside the high-risk region and Sweden, combined.
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5.2 A follow-up study: variation in other complement cascade genes in MS (III)
After we had observed association with a haplotype flanking the C7 gene, we
wanted to study both genetic and genomic variation of the other complement
cascade genes in MS. Interestingly both functional studies in humans as well as
genetic studies in EAE rodents have previously provided evidence for involvement
of C3 and TCCs in autoimmune demyelination. In a recently published study, CSF
samples of patients with MS and patients with other neurological diseases were
compared in order to identify genes that are differentially expressed between the
groups and potentially indicate relevant biological pathways for MS. Two important
factors of complement-mediated inflammation were identified: clusterin, a
regulator of complement activity, and C3 (Stoop et al. 2008). Further, depositions
of complement components have been detected in active MS lesions (Barnett and
Prineas 2004, Compston et al. 1989), and increased levels of TCC in the CSF of
MS patients have been shown to correlate with neurological disability (Sellebjerg
et al. 1998). Moreover, C6-deficient rats (unable to form the TCC) as well as C3deficient mice have attenuated EAE, the animal model of MS, and show little to no
demyelination compared to wild-type littermates (Nataf 2000, Mead 2002, Szalai
2007). In contrast, deletion of C5 has no significant effect on the course of EAE
(Weerth 2003).
In addition to C7, the third and the fifth components of the cascade are
critical limiting factors of the complement activation (Figure 5). Thus, we first used
HapMap tagger to capture most of the common variation in the C3 and C5 genes
and genotyped the tagging SNPs in the whole Finnish case-control sample (Figure
14). The SNPs did not show any evidence for association in the well-matched casecontrol sample from the Southern Ostrobothnia MS high-risk isolate. Suggestive
evidence for association was observed with three SNPs of the C3 gene (p=0.003)
when the whole Finnish sample set was analysed, but this association was then
noticed to be due to population stratification, thus being a false positive finding
and highlighting importance of properly matched controls, even in the relatively
non-admixed population like Finns.
Next, we monitored for potential MS associated variation in other complement
cascade related genes utilizing the SNP data of the two MS GWA studies (IMSGC
2007; Jakkula et al., manuscript in preparation). SNPs showing nominal evidence
for association in these studies (at least two SNPs within the gene or the surrounding
10kb region having >5% difference in case-control allele frequencies in the Finnish
GWA or p<0.03 in the international GWA) were selected for validation. The 23
potentially interesting SNPs were then genotyped in the whole Finnish case-control
sample. However, none of these SNPs provided significant evidence for association
in this expanded Finnish sample.
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Interindividual variation in the copy-number of the fourth component of the
complement cascade is known to exist, the number of total C4 genes (C4A and
C4B) varying between two and six and four copies being the most common count
(Yang 2007). We hypothesized that high copy-numbers of C4 might predispose
to MS by potentially leading to higher complement activity. Thus, we examined
the CNV for total C4 in 174 MS cases and 172 population contols. The variation
showed a pattern close to normal distribution both in the cases and in the controls,
the majority having four copies of C4, as expected (Figure 22). Case-control copynumber frequencies observed in this study were comparable to known copy number
frequencies of healthy European Americans (Yang 2007). Importantly, no evidence
for association with C4 CNV and MS was observed (p>0.05).
FIGURE 22. C4 copy number variation.
Frequency of different copy numbers (2-6) in Finnish MS cases (black columns; n=174)
and population controls (grey columns; n=172).
The data of this work suggests that variation in the complement component
coding genes outside 5p is not associated with genetic susceptibility of MS, at
least in Finland. However, since previous publications have provided functional
evidence for involvement of both TCCs and C3 in autoimmune demyelination, it
would still be interesting to study also C3 protein levels in CSF samples of Finnish
MS cases and unaffected controls.
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5.3 Candidate genes for immune-mediated demyelination on MS linked loci (IV)
Polycystic lipomembranous osteodysplasia with sclerosing leucoencephalopathy
(PLOSL), also known as Nasu-Hakola disease, is a recessively inherited rare
disease of the bone and the white matter of the brain. The estimated population
prevalence of PLOSL is 1-2x10-6 (Hakola 1990). The first symptoms of the disease
are typically pain and fractures in wrists and ankles at early adulthood, followed
by neuropsychiatric symptoms, dementia and premature death. Notably, the most
prominent feature of PLOSL is myelin loss in the CNS (Paloneva et al. 2001).
PLOSL is caused by mutations either in the DAP12 (TYROBP, TYRO protein
tyrosine kinase binding protein) or in the TREM2 (triggering receptor expressed on
myeloid cells 2) gene (Paloneva et al. 2000, Paloneva et al. 2002). All Finnish patients
carry a homozygous 5.3 kb PLOSLFin-deletion of DAP12, whereas inactivating point
mutations of TREM2 have been found in PLOSL patients of other populations
(Paloneva et al. 2002). Together DAP12 and TREM2 form a signalling receptor
complex (Figure 23), which is expressed in various cell types of the myeloid lineage
and has quite recently been discovered to be an important regulator of the innate
immune system (Klesney-Tait et al. 2006). However, the ligands for TREM2 as well
as the downstream effects of the DAP12-TREM2 mediated signal transduction are
still largely unrecognized.
The fairly small DAP12 (3.9kb) and TREM2 (4.7kb) genes are located on
chromosomes 19q13.12 and 6p21.1, respectively (Figure 24). Interestingly, linkage
to these loci has been reported also in families affected by another, more common
immune-mediated demyelinating disease, MS (Ebers et al. 1996, Haines et al. 1996,
Sawcer et al. 1996, Kuokkanen et al. 1997, Reunanen et al. 2002). Even though no
MS cases are known to exist in the Finnish PLOSL pedigrees, the number of Finnish
PLOSL families is too small to make any final conclusions. Thus, we wanted to test
if allelic variation in DAP12 or TREM2 predisposes also MS.
Since homozygous PLOSLFin-deletion of DAP12 results in severe white matter
changes and premature death in Finnish PLOSL, we hypothesized that the same
mutation as heterozygous form could lead to a milder, relapsing phenotype like
MS. According to the prevalence of PLOSL, carrier frequency of this 5.3 kb deletion
has previously been estimated to be 2.4/1000 in Finland (Hakola 1990), but since
high throughput genotyping of the deletion has been difficult, its prevalence in
the healthy Finnish population has never been established. Neither has carrier
frequency of this mutation been previously checked in MS.
To test our hypothesis, we genotyped the DAP12 deletion in 744 unrelated
Finnish MS cases (randomly selected from the 900 Finnish MS cases to equally
represent our full study set) and in the whole set of 1,350 Finnish controls (Figure
14) using an in-house developed high throughput method. Two carriers of the
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FIGURE 23. The DAP12-TREM2 receptor complex.
The DAP12 and TREM2 proteins form a signalling receptor complex, which is expressed
in various cell types of the myeloid lineage.
deletion were identified among the MS cases, corresponding to carrier frequency
of 2.7/1,000. Importantly, the clinical picture of these MS patients did not differ
from that of the non-carriers. DNA was available for the first degree relatives of
one of the carriers, and they were also observed to be carriers. Of the studied MS
cases 138 originated from the Southern Ostrobothnia MS high-risk isolate. None of
these Otrobothnian MS cases carried the mutation. Six carriers of the deletion were
identified among the controls, corresponding to carrier frequency of 4.9/1,000.
Thus, the previous estimation of the PLOSLFin carrier frequency in Finland was
now shown to be slightly underestimated. Importantly, the DAP12 deletion was not
observed to be over-represented among the MS cases.
We further made an effort to study the role of allelic variation of the highly
conserved DAP12 and TREM2 in MS by linkage and association analyses. All
putative SNPs (n=24) mapping to DAP12 and TREM2 were initially selected from
public databases. However, only one of these SNPs was found to be polymorphic
(minor allele frequency (MAF) ≥ 0.05 in Europeans) and was selected for
genotyping. To find more polymorphisms, we re-sequenced parts of DAP12. We
found four novel non-coding SNPs, but again, only one of these was observed
to have a MAF ≥ 0.05 in Finns, and this SNP was included in the genotyping
panel. Further, 15 polymorphic SNPs flanking the TREM2 gene were selected for
genotyping to capture possible variation in this highly conserved locus. The final
set of 17 SNPs was genotyped in the whole Finnish case-control sample (Figure
14). No evidence for association was observed with any of the SNPs. Due to a low
number of polymorphic SNPs in TREM2 and DAP12, two STS markers nearby
these genes were also genotyped in Finnish multiplex MS families (Figure 14), but
no evidence for linkage was observed.
Both microglia, the resident immune cells of the CNS, and oligodendrocytes,
myelin forming cells of the CNS, express DAP12 and TREM2 (Kaifu et al. 2003,
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Roumier et al. 2004, Kiialainen et al. 2005, Takahashi et al. 2007). However, no
expression of TREM2 and only modest expression of DAP12 was detected in
PBMCs, of which we had RNA available. Further, there was no difference in
expression levels of DAP12 between the 15 MS patients and 6 unaffected controls
studied (unpublished data).
As has been demonstrated previously, the low number of variants in DAP12
and TREM2 most probably indicates the crucial role of this receptor complex in
immune response modulation. Fenoglio et al. analysed the known polymorphisms
of the TREM2 coding regions in 100 patients with Alzheimer’s disease, 56 patients
with frontotemporal lobe degeneration, 78 patients with MS and 140 population
controls (Fenoglio et al. 2007). None of the SNPs were polymorphic in this Italian
study sample. Further, they sequenced the coding regions of TREM2 in Alzheimer’s
patients and healthy controls but no new mutations were found.
FIGURE 24. The DAP12 and TREM2 genes and their chromosomal locations.
The only SNP with MAF>5% in the general European population is marked with an
arrow.
The lack of polymorphisms in DAP12 and TREM2 is intriguing but makes these
genes very difficult to study. The Affymetrix 550K SNP panel used in the first MS
GWA study included only one SNP in TREM2 (MAF 0.02 in Finland) and no SNPs
in DAP12. Likewise, the Illumina HumanHap300 SNP panel used in the Finnish
GWA study included only one SNP in DAP12 (genotyped also in this study) and
no SNPs in TREM2. Neither do the latest GWA panels cover these genes properly.
Thus, the future GWA studies most probably do not bring more enlightenment for
potential involvement of genetic variation of DAP12 and TREM2 in MS.
None of the SNPs in the TREM2 coding regions was polymorphic in Italian
MS patients (Fenoglio et al. 2007). Furthermore, we have sequenced 80% of the
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DAP12 gene in Finnish MS patients and found no new mutations. We also studied
the non-coding polymorphisms of DAP12 and TREM2 and found no evidence for
association with MS. However, we did not study the rarest variants (MAF<5% in
general European population) in MS, and to finish up the study, those variants
should also be tested. Another way to try to dissect the role of the DAP12TREM2 receptor complex in MS is more extensive deep sequencing, which could
potentially reveal some rare sporadic mutations. However, these mutations most
probably would not explain the linkage observed to the DAP12 and TREM2 loci.
Further, the future genome wide association studies hopefully will bring some
enlightenment for potential involvement of copy number variations in these genes
in MS pathogenesis.
The pathogenesis of PLOSL, like that of MS, is mostly unknown. MS and
PLOSL share some common features. However, both the clinical picture and
the pathology of these two diseases are also dissimilar in many respects. Firstly,
PLOSL patients, lacking functional DAP12 or TREM2, suffer from a dramatic and
progressive loss of CNS white matter in the deep frontal and temporal white matter
(Paloneva et al. 2001), whereas in MS demyelination occurs both in the brain and
in the spinal cord, usually in the relapsing-remitting way. Secondly, colonization of
activated T lymphocytes is characteristic to MS CNS but not to PLOSL. Moreover,
DAP12 and TREM2 are expressed also in osteoclasts, and thus, PLOSL patients
with inactivating point mutations of the receptor complex develop osteoporosis,
but such bone abnormalities have not been observed to be over-represented among
MS patients.
We have confirmed that the Finnish PLOSL mutation is not enriched among
Finnish MS patients. To conclude, the DAP12-TREM2 receptor complex unlikely
has any role in genetic susceptibility to MS in Finland, and the strong linkage of 6p
is most probably explained by the HLA region, whereas the linkage of chromosome
19 is most probably due to variation in some other locus than DAP12. On the other
hand, 19q linkage has not been unequivocally replicated in multiple populations
and it may also exemplify a false positive finding.
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6 Concluding remarks
Despite long-standing and intensive research the etiology and pathogenesis of
MS are still poorly understood and few predisposing genetic variants have been
identified. In this thesis study an effort was made to better understrand the
molecular backround of MS taking advantage of genetics. Traditionally, linkage
approach has been used to map the susceptibility loci of genetic diseases. However,
the importance of linkage studies in complex diseases has been under debate
during this thesis study, and the emphasis in genetic mapping has largely shifted
from genomewide linkage studies in families to genome-wide association studies
in unrelated cases and controls. These kinds of large analyses in mixed populations
are optimized to detect common variants of complex diseases, but will not bring
out the relatively rare, penetrant variants, which give rise to a familial concentration
of cases.
Here an effort was made to identify MS predisposing genetic variants within
the most promising non-HLA loci showing evidence for linkage to MS. Taking into
account that MS most probably is not a unitary disorder, but instead may represent
an overlapping spectrum of related disorders, we have minimized the genetic and
environmental heterogeneity by studying familial MS cases from the Southern
Ostrobothnian MS high-risk isolate. Presumably, most of these MS patients share
the same risk alleles, which can be exposed by the common HapMap markers due
to the wide LD intervals. In reference to the aims listed for this thesis, the following
findings were presented:
1. In the scan of the 5p linked locus, strongest evidence for association was
detected with a haplotype flanking the complement component 7 (C7) gene.
The identified haplotype is relatively rare, has become enriched in Finland and
especially in Southern Ostrobothnian MS pedigrees and seems to have a fairly
large effect on genetic susceptibility of MS. Interestingly, there are already
multiple lines of evidence to suggest the involvement of the complement
system in MS. Plasma C7 levels and complement activity were here observed
to correlate with the risk haplotype identified, the complement system being
most active in MS cases carrying the risk allele. The identified risk variant
may predispose especially to the most common pathological subtype of MS,
pattern II.
The scan of the 17q linked locus showed evidence for association with
variants of the protein kinase C alpha (PRKCA) gene. Thus, these variants are
likely to be in LD with the putative causative variant of the locus, at least in the
Finnish isolate. Another variants of PRKCA provided nominal evidence for
association with MS also in Canadian MS families. We would conclude that
the MS risk locus on 17q is more complex than previously assumed and might
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contain multiple genes, different genes potentially playing role in different
families.
2. The strong association with the C7 region stimulated us to study other
complement cascade genes in the Finnish case-control sample. No evidence
for association could be observed with the complement component coding
genes outside 5p and MS.
3. The highly conserved DAP12 and TREM2 genes, located on the MS
linked regions of chromosomes 6p and 19q, unlikely have a role in genetic
susceptibility of MS. Most importantly, the Finnish PLOSL-mutation is not
over-represented among the Finnish MS cases.
This thesis work provides an example of how extended families from special
populations can be utilized in fine-mapping of the linked loci, even in this new
era of complex genetics. The study also suggests that the genome regions initially
identified as potential loci for common diseases most probably are more complex
than assumed; It appears that there exists at least two independent risk variants
within the chromosome 5p MS locus, and the same may hold true with other
loci showing evidence for linkage with MS in several populations. Moreover, the
commonly accepted conseption that the same allele of the same genetic variant
should be repeatable globally to verify the significance of the finding is here
challenged. Such may be feasible in question of the common genetic variants, but
a new mindset is needed to define less common and more penetrant variants of
complex diseases. Finally, the study highlights the rapid process of both knowledge
and the technologies of Human genetics during the past few years.
In future, classification of MS patients according to the immunopathological
patterns of demyelination could be relevant, since distinct patterns might have
different molecular backround which, in turn, might require different therapeutic
strategies. Such classification could also enable more straightforward replication
of the findings by reducing the genetic heterogeneity of mixed populations and
thereby unmasking the association.
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7
Acknowledgements
This study was carried out at the Department of Molecular Medicine, National
Public Health Institute, and the Department of Chronic Disease Prevention,
National Institute for Health and Welfare, Helsinki Finland during the years 20022009. The former and present Director General of the Institutes, Jussi Huttunen
and Pekka Puska, as well as the Head of the Department of Molecular Medicine,
Anu Jalanko, are acknowledged for providing excellent research facilities.
Professor Leif Groop is acknowledged for accepting the role as an Opponent
in my thesis defense. Professor emeritus Arne Svejgaard and Professor Anne
Kallioniemi are warmly thanked for thorough revision of this thesis. Their
constructive comments led to some very essential improvements. I would also like
to thank Päivi Saavalainen and the members of my thesis committee, Aarno Palotie,
Pentti Tienari, Per-Henrik Groop and Petteri Arstila, for both critical comments
and support.
I wish to thank all the participating MS patients and their families. Without
their voluntary assistance none of these conceptions could have been made.
Financial support for this thesis was provided by the National Institutes of
Health (NIH) (grant RO1 NS 43559), the Center of Excellence for Disease Genetics
of the Academy of Finland, the Paulo Foundation, the Sigrid Juselius Foundation, the
Finnish Cultural Foundation, the Biocentrum Helsinki Foundation, the Research
Foundation of the Helsinki University Central Hospital, the Neuropromise EU
project (grant LSHM-CT-2005-018637), the Multiple Sclerosis Foundation of
US, the Canadian Institutes of Health Research (CIHR), the European Molecular
Biology Organization (EMBO), Genome Canada and Genome Quebec, the US
National Center for Research Resources (grant U54 RR020278), the SGENE EU
project (grant LSHM-CT-2006-037761), Simons Foundation (R01MH7142501A1) and a Harry Weaver Neuroscience Scholar Award from US National Multiple
Sclerosis Society, which all are gratefully acknowledged.
I wish to express my deepest gratitude to my supervisors,
Professor Leena Peltonen-Palotie and Adjunct Professor Janna Saarela. I have had
a priviledge to be supervised by two brilliant female scientists who have managed
to get me to do my best. In the enthusiastic and inspiring atmosphere created by
them I have had a unique chance to grow both as a scientist and as a person.
My sincere thanks go to our collaborators, Pentti Tienari, Keijo Koivisto,
Irina Elovaara, Tuula Pirttilä, Mauri Reunanen, the Nordic MS genetics Network,
the collaborators from US and Canada, Joe Terwilliger, Mark J Daly, Markku
Viander and Seppo Meri, for providing their study samples and expertise for these
projects.
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I am also deeply indebted for the senior researchers of our laboratory, Marjo
Kestilä, Samuli Ripatti, Kaisa Silander, Ismo Ulmanen, Teppo Varilo, Markus
Perola, Anu Loukola, who have helped me in many ways during these years.
Special thanks also go to the wonderful secretaries Sari Kivikko, Tuija Svanbäck
and Mika Kivimäki, the DNA extraction team of Minttu Jussila and Outi Törnwall
as well as the sequencing team of Pekka Ellonen. Anne Nyberg, Sisko Lietola, Anne
Vikman, Elli Kempas, Siv Knaappila, Lea Puhakka and especially Minna Suvela
are acknowledged for skilful technical assistance. Jari Raikko, Juha Saharinen, Juha
Knuuttila, Juri Ahokas and Hannu Turunen, thank you for the IT-support and all
the help. Päivi Hauhia and Christine Strid are thanked for the assistance in the
publication process.
“Missukat” Anu, Ansku, Virpi, Eveliina, your company has made this work
joyful – we have had great time, great discussions (and great coffee too). Many
thanks also to all who have helped me with my project and/or made the lab such
a nice place to work at: Roxy, Denis, Anna, Kati, PP, Annu, Jussi, Emma, Marika,
Olli, Nora, Karola, Sampo, Joni, Mervi, Kirsi, Annika, Jarkko, Johannes, Pia, Tintti,
Juho, Annina, Tero, Markus, and anyone I failed to mention here. Mari, Tämer,
Ulla and Petteri from ”family Kankkunen”, Anne and Mari from ”Pachamamas”,
Salla, Laura, Varpu, Henri, Liisa, Markku, Petra, Hanna-Mari and all friends from
the Medical School, my dearest friends, thank you for lending me an ear and
maintaining the balance between work and relationships.
Last but not least, I warmly thank my family. Father, you have been my role
model; I admire your determination and resilience. Antti, thank you for your
unconditional love, faith and support.
Without you all I could not fulfill my dreams today.
Helsinki, May 13th, 2009
Suvi Kallio
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