11/6/2011

1

Large-scale, high-density genotyping performed by the

Sjögren’s Genetics Network using the ImmunoChip

identifies PRKRA as a novel Sjögren's syndrome

susceptibility locus and confirms associations with IRF5,

BLK, and MHC

Christopher J. Lessard, Indra Adrianto, John A. Ice, Jennifer Kelly, Roland Jonsson, Gabor G.

Illei, Maureen Rischmueller, Gunnel Nordmark, Xavier Mariette, Corinne Miceli-Richard, Marie

Wahren Herlenius, Torsten Witte, Michael Brennan, Roald Omdal, Patrick M. Gaffney, James

A. Lessard, Wan-Fai Ng, Nelson Rhodus, Barbara Segal, R. Hal Scofield, Judith A. James,

Juan-Manuel Anaya, John B. Harley, Courtney G. Montgomery, and Kathy L. Moser

American College of Rheumatology Annual Meeting

Chicago, IL

November 6, 2011

Disclosures

• NONE

Complex Etiology of SS: Genetics + Environment

• Potential environmental factors:

- Viral infection:

• EBV (Epstein Barr virus)

• CMV (cytomegalovirus)

• HCV (Hepatitis C virus)

• HHP-6 (Human Herpes virus 6)

- UV light

• The genetics of SS are largely unexplored:

- Twin concordance estimates unavailable

- Genome-wide linkage studies in families not performed

- ~30 Candidate genes evaluated to date, ~ ½ show positive association

• Relatively small (typically < 500 cases), case-control association studies

• Most consistent associations found with HLA molecules involved in antigen recognition

Revised criteria for diagnosis of Sjögren’s syndrome; Ann. Rheum. Dis. 61 (6):554-8.

Anti-Ro or Anti-La

Dry Eyes Patient report

Dry Mouth/PG swelling Patient report

Biopsy:

Nat Clin Pract Rheumatol 2: 262–269

Focal lymphocytic sialoadenitis Focal score >1

Schirmer’s Test or

Lissamine Green

Staining

Whole unstimulated salivary flow <1.5 ml in 15 min

www.intelligentdental.com www.salujaeyecare.com

www.salujaeyecare.com Nat Clin Pract Rheumatol 2: 262–269

gulfmd.com

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Classification Criteria

Objective

• Comprehensively evaluate loci previously

reported in related inflammatory conditions.

Methods-Genotyping

• ImmunoChip:

• An Illumina iSelect custom array (~196,000

markers) designed by a consortium of

multiple inflammatory phenotypes

• SGENE cohort

• Replication:

• Illumina OMNI1-Quad array

• OMRF SS cohort

11/6/2011

2

Methods-Quality Control

• Standard Sample and SNP QC:

- Clean cluster plots, Hardy-Weinberg proportions,

missingness, minor allele frequency >1%, etc.

- Assessment of population stratification using

EIGENSTRAT

• ImmunoChip (IC): ~120,000 SNPs tested in 1203 SS

cases (from SGENE) and 2023 healthy controls

• Replication: ~4000 overlapping SNPs between

OMNI1-Quad and IC tested in 424 SS cases (from

OMRF and UKPSSR) and 2120 healthy controls

Methods-Statistics

• Logistic regression implemented in PLINK

• Adjusted for first 4 PCs and sex

• Correction for multiple testing

• Bonferroni correction threshold: p = 4.2 x 10-7

• Meta-analysis between ImmunoChip and replication

• Weighted Z-scores using METAL

Summary of Results

• 1466 SNPs with p < 4.2 x 10-7

• Peak association observed was at HLA-DRB1

• p = 4.15 x 10-42; OR = 2.76 (95%CI = 2.39-3.20)

MHC

Replication within the MHC

• Peak association after Replication:

• HLA-DRA p = 4.71 x 10-69

26 28 30 32 34

0

10

20

30

40

50

60

70

26 28 30 32 34

0

10

20

30

40

50

60

70

Mb

-lo

g10(P

)

0

20

40

60

80

100

Reco

mb

. R

ate

(cM

/Mb

)

HLA Class I HLA Class III HLA Class II

HLA-BHLA-C HLA-DR HLA-DQGWAS

Meta-Analysis

Recomb. Rate

31.6 31.8 32.0 32.2 32.4

55

60

65

70

HLA Class III/Mb

BAT1 BAT2 MSH5

C2

STK19

TNXB C6orf10

Summary Outside MHC

• IRF5-TNPO3, BLK, and STAT4 with p < 4.2 x 10-7

• IL12A & CXCR5 with p < 4 x 10-4

HLA-DRB1 (P=4.15x10-42)

IL12A

IRF5-TNPO3

BLK

CXCR5

STAT4

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Biological Function • Interferon regulatory factor 5 (IRF5) – 7q32

- Transcription factor involved in type I IFN responses

- Controls inflammatory and immune responses

- Critical in the production of pro-inflammatory

cytokines (TNF-α, IL12, and IL6)

• B lymphoid tyrosine kinase (BLK) – 8p23-p22

- Src family of tyrosine kinases

- Expression restricted to B cells (1st in pro-B cell and

down-regulated in plasma B cells)

- Transduces signal downstream of the B cell receptor

Biological Function • Signal transducer and activator of transcription 4 (STAT4) – 2q32

- Member of the STAT family of transcription factors (IFN-γ, etc.)

induced by IL12, IL23, and type I IFNs

- Regulates the differentiation of T helper cells

• Interleukin-12A (IL12A) – 3q25

- Encodes the p35 subunit of IL12, secreted by monocytes and

dendritic cells, stimulates the production of IFN-γ

- Responses to IL12 are mediated through STAT4 and

NOS2A/NOS2

• Chemokine (C-X-C motif) receptor 5 (CXCR5) – 11q23

- Receptor for B-lymphocyte chemoattractant (BLC)

- Involved in the migration of B cells into B cell follicles of spleen

and Peyer’s patches

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PRKRA • Reported association in abstract with PRKRA at 2q32

• ImmunoChip: p = 6.44 x 10-24, OR = 0.47

• Omni1-Quad: p = 1.04 x 10-6, OR = 0.43

• Meta-analysis: p = 1.3x10-27

From Chida et al. 2001, Immunogenetics 52(3-4) 186-194.

Summary and Future Directions

• Identified and replicated associations within the

previously implicated loci MHC, BLK, IRF5, and

STAT4

• Identified and replicated novel associations at

IL12A and CXCR5

• PRKRA association likely due to pseudogene

located adjacent to peak MHC effect within HLA-

DRB1

• Continue replication of regions not in common

with OMNI1-Quad

• Identify functional/causal variants and

pathophysiological relevance to SS

Acknowledgements

Kathy Moser

Courtney Montgomery

R. Hal Scofield

Judith James

Patrick Gaffney

Jennifer Kelly

John Ice

Indra Adrianto

Erin Rothrock

Judy Harris

He Li

Cherilyn Pritchett

James Robertson

Kiely Grundahl

Laura Battiest

Kyla Crouch

Mehdi Radfar

Justin Rodgers

Adam Adler

Stuart Glenn

Kimberly Hefner

Eliza Chakravarty

Lida Radfar

Donald Stone

Rhea Siatkowski

David Lewis

Glen Houston

OUHSC

Roland Jonsson Tom Gordon

Gabor Illei

Maureen Rischmueller

Gunnel Nordmark

Corrine Miceli-Richard

Marie Wahren-Herlenius Michael Brennan

Roald Omdal

Fai Ng

Juan-Manuel Anaya

Torsten Witte

Xavier Mariette

James Lessard Ben Rybicki for ACCESS

Ken Kaufman

John Harley

James Chodosh

SGENE Collaborators

University of MN Barbara Segal

Nelson Rhodus

Pamala Hughes

Raj Gopalakrishnan

Michael Rohrer

NIH/NIAD

NIH/NIAMS

NIH/NIDCR

OMRF

Sjögren’s Syndrome Foundation

Phileona Foundation

Arthritis Foundation

American College of Rheumatology

The Barrett Predoctoral Scholarship

Fund-OMRF

Funding Agencies

SGENE: The Sjogren’s Genetics Network

Founded in 2009, coordinating center = Moser lab, OMRF

International group interested in contributing DNA samples and clinical data for

genetic studies

Currently:

15 contributing sites (plus subsites)

>2000 SS cases (AECG criteria), 2000 controls

Developing consistent clinical dataset

Goal: Continue to expand sites

Overall: 15,000+ cases

Contact Info:

Moserk@omrf.org

Lessardc@omrf.org