Consortium of Multiple Sclerosis Centers Annual Symposium

Multiple Sclerosis Genetics A Tale of Two Molecules

Stephen L. Hauser, M.D.University of California, San Francisco

June 1, 2007

Sir Augustus d’Este (1794-1848), from the collection of the Victoria and Albert Museum, London.

Multiple Sclerosis

λλ

Chronic, multifocal CNS disorder

λλ

Immune-mediated myelin destruction

λ

Secondary neurodegeneration

λ

Prevalence has increased

λ

Gender dimorphism

λ

Influence of environment

λ

Racial and familial clustering

λ

Empirically based therapies

Multiple Sclerosis Impediments to Development of Therapies,

Preventions, and Cures

1. Underlying disease heterogeneity

2. Inadequate animal models

3. Uncertain relationship between inflammatory and degenerative stages

4. Limited repertoire of useful biomarkers

5. Very limited knowledge of presymptomatic events and triggers

It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts. Sherlock Holmes, A Scandal in Bohemia

It is a capital mistake to theorize before one has data. Insensibly one begins to twist facts to suit theories, instead of theories to suit facts.

Sherlock Holmes, A Scandal in Bohemia

Recurrence Rate Estimates for MS

Relationship

Monozygotic twin

Sibling, two affected parentsSibling, one affected parent

Dizygotic twinSiblingParent

Child

Half siblingAunt or uncle

Nephew or niece

Cousin

AdopteeGeneral population

0 5 10 15 20 25 30 35 40

0%

25%

50%

100%

12.5%

Genetic Identity

Age-adjusted lifetime risk

λs =1

λs =190

λs =6.5

λs =20

λs =150

Modified from: Ebers G. et al. Lancet Neurol. 3:104, 2004

Familial Aggregation of MS

1-866-MSGENES

0.0%

1.0%

2.0%

3.0%

4.0%

5.0%

6.0%

7.0%

8.0%

9.0%

10.0%

Alopecia areata Autoimmune hepatitisCrohn's DiseaseUlcerative colitisMeniere's disease MCTDPernicious anemiaPsoriasisRaynaud's phenomenonRheumatoid arthritisSystemic lupus erythematosusGraves' disease/hyperthyroidismHashimoto's thyroiditis/hypothyroiUveitis/iritisAsthmaType 2 DiabetesHeart AttackEmphysema

Autoimmune Disorders in High Risk for MS Families

Barcellos L. et al: Lancet Neurol 5:924, 2006

Disease genes

Genes with rare alleles with strong penetrance and effects (i.e. deletions, expansions, etc.)

Susceptibility genes

Genes with common or rare alleles with weak, but cumulative effects and penetrance and effects (i.e. polymorphisms)

Monogenic disorderMutation

Gene A

Complex disorderGene variations

Gene C

Gene BGene A

Gene D

Mendelian inheritance patterndominant or recessive Complex inheritance pattern

Genetic risk in the population

Impact of mutations/variations in a single gene on disease phenotype

Environment

Post-genomic modifications

Genetic risk in different families

30% AB C

D

Family 2

30%

Family 1

100%

Family 1 Family 2 Family 3

100%

Family 3

Modified from Peltonen & McKusic. Science 291:1224, 2001

Population Sample After Many Generations

Founding Mutation

gene gene

exon

rSNP iSNP cSNP sSNP

amino acidsubstitution

no amino acidsubstitution

Possible phenotype change

(regulatory SNP) (intron SNP)A/A

G/G

A/G

Single Nucleotide Polymorphisms (SNPs)

Haplotype Mapping

• Long-range correlations among SNPS in the human genome organized as haplotype blocks

• The quantum element of genetic variation on a population-wide scale is not the individual base pair or SNP, but rather 5-100 kb blocks of sequence unbroken by recombination in modern human evolution

This suggests that it may be possible to use only a small fraction of the total genetic variation to serve as an adequate test of the remainder

Populations of Mixed Ancestry: African-Americans

• Typical MS does not occur in black Africans • MS occurs in AA but, compared to WA, is less

common, more severe and less responsive to treatment• African chromosomes have a longer population history

(shorter haplotypes) and greater genetic diversity• These features make possible trans-racial comparisons

to define causal genetic variants • Admixture mapping can identify regions of excess

ancestry in the ethnic group at higher risk for a disease

The History of Human Migration

Northern European chromosome

African chromosome

Detecting Admixture Association

Patterson et al. Am J Hum Genet 74: 979, 2004

Putative Disease Locus

100%

50%

0%

20cM 40cM 60cM 80cM 100cM 120cM 140cM

Position on chromosome 19 (centimorgans)

Perc

ent E

urop

ean

Ance

stry

African American Controls

African Americans with MS

International MS Genetics Consortium

LINKAGE ASSOCIATION ADMIXTURE HLA

VanderbiltDuke UCSFMS Genetics Group

Broad

HarvardU. Cambridge

MontrealMinnesotaNS/LIJ Karolinska

Imperial CollegeIMAGEN

Chr1

0.0

1.0

2.0

3.0

Chr2

0.0

1.0

2.0

3.0

Chr3

0.0

1.0

2.0

3.0

Chr4

0.0

1.0

2.0

3.0

Chr5

0.0

1.0

2.0

3.0

Chr7

0.0

1.0

2.0

3.0

Chr8

0.0

1.0

2.0

3.0

Chr9

0.0

1.0

2.0

3.0

Chr10

0.0

1.0

2.0

3.0

Chr11

0.0

1.0

2.0

3.0

Chr12

0.0

1.0

2.0

3.0

Chr13

0.0

1.0

2.0

3.0

Chr15

0.0

1.0

2.0

3.0

Chr16

0.0

1.0

2.0

3.0

Chr17

0.0

1.0

2.0

3.0

Chr18

0.0

1.0

2.0

3.0

Info

Chr19

0.0

1.0

2.0

3.0

Chr20

0.0

1.0

2.0

3.0

Chr21

0.0

1.0

2.0

3.0

Chr22

0.0

1.0

2.0

3.0

ChrX

0.0

1.0

2.0

3.0

Chr14

0.0

1.0

2.0

3.0

Chr6

0.0

2.0

4.0

6.0

8.0

10.0

12.0

cM

Info

International Multiple Sclerosis Genetics Consortium, Am J Hum Genet 77.454-467, 2005

High Density Linkage Screen: 1000 families

HLA

-3-2-10123456789

1-5

1-10

5

1-20

5

2-21

2-12

1

2-22

1

3-61

3-16

1

4-40

4-14

0

5-38

5-13

8

6-30

6-13

0

7-41

7-14

1

8-60

8-16

0

9-88

10-2

2

10-1

22

11-5

6

11-1

56

12-9

9

13-3

3

14-9

14-1

09

15-1

10

16-8

7

17-6

2

18-4

1

19-3

5

20-4

1

21-4

5

X-1

4

X-1

14

Genetic position in centimorgans (Chromosome-Position)

LOD

sco

re(lo

g ba

se 1

0 of

like

lihoo

d)

Chromosome 1 peak

Published scan: 605 cases, 1043 controls

Whole Genome Admixture Scan: African-Americans

-3-2-10123456789

1-5

1-10

5

1-20

5

2-21

2-12

1

2-22

1

3-61

3-16

1

4-40

4-14

0

5-38

5-13

8

6-30

6-13

0

7-41

7-14

1

8-60

8-16

0

9-88

10-2

2

10-1

22

11-5

6

11-1

56

12-9

9

13-3

3

14-9

14-1

09

15-1

10

16-8

7

17-6

2

18-4

1

19-3

5

20-4

1

21-4

5

X-1

4

X-1

14

Genetic position in centimorgans (Chromosome-Position)

LOD

sco

re(lo

g ba

se 1

0 of

like

lihoo

d)

Chromosome 1 peak

Published scan: 605 cases, 1043 controlsCurrent scan: 882 cases, 1056 controls

Whole-Genome Association: 1000 Trios

HLA

HLA

CD25

CD25DBC1

RPL5

ALK

Whole-Genome Replication: 3000 Cases/Controls

0

10

-log1

0(p)

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300Position

High Density HLA Genotyping: 1000 Triosq p

Class II Class IClass III

0

1

2

3

-log1

0(p)

0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300Position

DRB1*1501 Excluded; (324 Trios)

HLA-CMICA LOC729792

-log1

0(p)

3

10

-log1

0(p)

q p

Class II Class IClass IIID

PB

2D

PB

1D

PA

1

DQ

A

DM

AD

MB

TAP

1

TAP

2D

OB

DR

B1

DR

B5

DR

A

DQ

B1

DQ

A1

LMP

2

LMP

7

Tapa

sin

LTB

TNFa

LTA

C4A

C4B

C2

BF HS

PA

1BH

SP

A1A

HS

PA

1L

HLA

-B

HLA

-C

MIC

-A

MIC

-B

HLA

-E

HLA

-AH

LA-G

HLA

-F

Chromosome 6

MO

G

BTN

PR

L

q p

Class II Class IClass IIID

PB

2D

PB

1D

PA

1

DQ

A

DM

AD

MB

TAP

1

TAP

2D

OB

DR

B1

DR

B5

DR

A

DQ

B1

DQ

A1

LMP

2

LMP

7

Tapa

sin

LTB

TNFa

LTA

C4A

C4B

C2

BF HS

PA

1BH

SP

A1A

HS

PA

1L

HLA

-B

HLA

-C

MIC

-A

MIC

-B

HLA

-E

HLA

-AH

LA-G

HLA

-F

1990s

MO

G

BTN

PR

L

q p

Class II Class IClass IIID

PB

2D

PB

1D

PA

1

DQ

A

DM

AD

MB

TAP

1

TAP

2D

OB

DR

B1

DR

B5

DR

A

DQ

B1

DQ

A1

LMP

2

LMP

7

Tapa

sin

LTB

TNFa

LTA

C4A

C4B

C2

BF HS

PA

1BH

SP

A1A

HS

PA

1L

HLA

-B

HLA

-C

MIC

-A

MIC

-B

HLA

-E

HLA

-AH

LA-G

HLA

-F

2004: African-American genomes

MO

G

BTN

PR

L

q p

Class II Class IClass IIID

PB

2D

PB

1D

PA

1

DQ

A

DM

AD

MB

TAP

1

TAP

2D

OB

DR

B1

DR

B5

DR

A

DQ

B1

DQ

A1

LMP

2

LMP

7

Tapa

sin

LTB

TNFa

LTA

C4A

C4B

C2

BF HS

PA

1BH

SP

A1A

HS

PA

1L

HLA

-B

HLA

-C

MIC

-A

MIC

-B

HLA

-E

HLA

-AH

LA-G

HLA

-F

2007: African-American genomes

MO

G

BTN

PR

L

modifier

q p

Class II Class IClass IIID

PB

2D

PB

1D

PA

1

DQ

A

DM

AD

MB

TAP

1

TAP

2D

OB

DR

B1

DR

B5

DR

A

DQ

B1

DQ

A1

LMP

2

LMP

7

Tapa

sin

LTB

TNFa

LTA

C4A

C4B

C2

BF HS

PA

1BH

SP

A1A

HS

PA

1L

HLA

-B

HLA

-C

MIC

-A

MIC

-B

HLA

-E

HLA

-AH

LA-G

HLA

-F

2007: High resolution mapping

MO

G

BTN

PR

L

protective

modifier

Alaβ71

The Major Genetic Effect on MS Superimposed HLA-DRβ1*1501 and DRβ1*1503 allelic

structures

Allelic and Functional Association of the Interleukin 7 Receptor α

Chain Gene with MS

F3F3

CK

COOHCOOH

α-chain (CD127) IL2R γc -chain (CD132)

Location5p13 humans15 4.6 cM mouse

DistributionThymocytesT, B cellsMonocytesEndothelial cells

SignalingPI3KStat5

JAK1 JAK3

Genomic Conversion Approach to Identify Candidate Genes in MS

Gene Location Number of SNPs

MMP19 12q14 4

CCL2 17q12 5

IL7Rα 5p13.2 (6) 14

SNPselector (Xu et al Bioinformatics 21:4181, 2005) was used to identify SNPs within exons, untranslated and conserved regions within the gene

r 2=0.97

r 2=0.95PDT analysis of IL7R SNPs

760 MS families (197 multi-case families, 1055 MS patients)

SNP Position

(bp)

Minor/majorallele

MAF Gene region

Type Aminoacid

change

OverallPDT p-value

rs13188960* 35889076 T/G 0.24 upstream 0.0023rs7718919 35891753 T/G 0.13 upstream 0.8707rs1389832* 35894478 T/C 0.34 intron 1 0.0804rs1494558* 35896825 A/G 0.34 exon 2 Non-synon. I66T 0.0249rs11567705 35896909 G/C 0.24 intron 2 0.0018rs969128 35896916 G/A 0.13 intron 2 0.9343rs1494555* 35906947 C/T 0.33 exon 4 Non-synon. V138I 0.0327rs7737000 35907030 T/C 0.13 exon 4 Synonymous H165H 0.6801rs1494554* 35909629 C/A 0.29 intron 5 0.7175rs6897932* 35910332 T/C 0.24 exon 6 Non-synon. T244I 0.0006rs987107* 35910984 T/C 0.29 intron 6 0.7856rs987106* 35911350 T/A 0.47 intron 6 0.0134rs3194051* 35912031 G/A 0.29 exon8 Non-synon. I356V 0.7856rs1494571 35915844 C/G 0.28 downstream 0.9276

P-values meeting Bonferroni-corrected significance threshold and respective risk allele are shown in

bold italic. Minor allele frequency (MAF) estimated from genotyped founders. SNPs genotyped by the

HapMap project (http://www.hapmap.org) are denoted with an *.

Replication of the SNP rs6897932 Risk Effect in Independent Datasets

Dataset N P valueUS (familial) 760 (1055 patients) 0.0006

US (c/c) 438/479 0.05

US AA (c/c) 700/300 NS

UK (fam) 1338 trios 0.03

UK-Belgium (c/c) 1077/2725 0.0006

Combined (fam) 2098 0.0001

Combined (White c/c) 1515/3204 0.000005

IL7Ra Alternative Transcripts

Transcripts that include exon 6 membrane bound

Transcripts that exclude exon 6 soluble receptor

rs6897932 T244I locates in exon 6, which codes a trans-membrane domain

The MS associated C allele resulted in a 2 fold increase in skipping exon 6 (more [short product] soluble receptor)

CT T C T C

U DExon 6

DU

B

(326bp)

(232bp)

HeLa cells DT3 cells AT3 cells

0

20

40

60

80

100

T T TC C C

HeLa DT3 AT3

C.

C alleleT allele-RT

20

40

60

80

100

C alleleT allele

Inc

Sk

D

E.

T7 XbaI XhoI

(94bp)(614bp) (573bp)

SP6

pl-11

Pl-11-IL7R382=T

Pl-11-IL7R172=C

or

A

IL7Ra and Homeostatic Control of T-cell Function

IL7Rα

expression by mature T cells is dramatically influenced by extrinsic factors, including cytokines and antigens.

The more receptor a cell expresses, the more signal it receives for survival and proliferation

The more receptor a cell expresses, the more IL7 it consumes, thereby depriving neighboring cells of their survival signals

EBV

MBP

Crossreactive TCR

DRADRB1

DRB5

MBP TCR

CD4+ Treg cell

B7

CD4+

Th1 cell

CTLA4

NK cell

KIR

CD8+

T cell

HLA-C

DRB1*1501 DRB5*0101

HLA-C

APC

Immune Interactions: The Genetic Evidence

IL7RaIL7Ra

IL7Ra

Current Directions

• Most of the MS genome remains unidentified– Collection of larger datasets in progress

• New approaches– Epigenetics– Copy number variation, differential allelic expression – Whole genome sequencing– Biological heterogeneity

• Genotype-phenotype• Pharmacogenomics

• Identify earliest triggering events in individuals at risk for MS

datadatadata

Patient enrollment

Clinical, epidemiologic MRI Genomics Expression

data

Data Integration

The Genetic Multiple Sclerosis Associations Project

HLA DRB1*1501 Influences MRI Lesion Load in Early MS

1.97*1.79*

1.44

Q2/Q1 Q3/Q1 Q4/Q1

DRB1*1501-DRB1*1501+

T2-Lesion Volumes Stratified by Quartiles

Odd

s R

atio

Individuals at Risk for MS The IRIS Study

• A longitudinal study of ~8000 unaffected first degree relatives of new onset MS cases collected across North America, including 900 discordant twins

• Longitudinal surveillance: Genetic markers, Viral responses (e.g. EBNA1); DRB1*1501 restricted T-cell responses to MBP89-96; autoantibodies; neuroimaging

• Goal to identify “preclinical autoimmunity” state prior to MS onset and factors associated with transition to MS

• Primary prevention with vitamin D supplementation?

The UCSF MS Group