1. A MHC-chromosome 2 interaction in JRA affected sibpairs.
M.L. Sudman1, L. Guyer2, M. Ryan2, M.A. Keddache1, W.M. Brown3, C.D. Langefeld3, S.S. Rich3,
C. Valis3, W.C. Nichols1, M.B. Moroldo2, S.D. Thompson2, D.N. Glass2.
1) Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 2) Division of Rheumatology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 3) Wake Forest University School of Medicine, Winston-Salem, NC.
Abstract
Juvenile Rheumatoid Arthritis (JRA) is a complex genetic disorder and is the most common chronic rheumatic inflammatory condition in childhood, with an estimated 50,000 children diagnosed in the U.S. The disease is phenotypically and genetically heterogeneous and distinct from adult onset RA. A genome scan (386 microsatellite markers) was done on 121 families containing 247 affected children (NIAMS supported JRA Affected Sibpair [ASP] Registry). In addition, individuals were typed for HLA-DR. Pedigree disequilibrium tests at the DRB1 locus revealed excess transmission of the DR8 haplotype to offspring with pauciarticular JRA (haplotype-specific P = 2.0 X 10-6). Other haplotypes, including HLA-DR11, 13 and 4, are well documented to be associated with susceptibility (or resistance) to one or more subtypes of JRA. To evaluate potential interactions between HLA-DR and other genomic regions, nonparametric linkage analysis was performed on 33 pedigrees with offspring concordant for the presence of at least one DR8 allele. The strongest evidence for linkage in these pedigrees was near D2S168 at 35 cM (2p25, LOD=6.00). Fine mapping with 13 additional microsatellites produced a LOD=6.09 and reduced the LOD-1 support interval to 7 cM ( cM). Suggestive evidence for linkage in these 33 pedigrees was observed at D14S292 at cM (14q32, LOD=1.84). Repeating these analyses on the pedigrees with at least one offspring having HLA-DR4 yielded a LOD=2.48 at 3p27, and on pedigrees concordant for the presence of either DR8, DR11 or DR13 yielded a LOD=2.34 at 17q25. Linkage disequilibrium analyses based on a dense SNP map of the 2p25 region are underway.
Introduction
(Methods cont.)
Genotyping
A genome scan was conducted using 386 microsatellite polymorphisms from Applied Biosystem’s MD10 Linkage Mapping Set. Amplification was done using standard 10μl PCR reactions, and products were pooled according to panels and processed on an ABI3700 Fragment Analysis System. Genotypes were generated from these ABI3700 sample files using Applied Biosystem’s GeneMapper 2.0 software.
Low resolution HLA-DR allele typing was performed using GenoVision’s allele-specific PCR.
Analysis
PDTPHASE was used to test for association between markers and disease phenotypes.
Nonparametric multipoint linkage analysis using the NPL(pairs) statistic was performed using GeneHunter-Plus. The one parameter exponential allele sharing model (asm) was used to compute likelihood ratio z-scores, nominal LOD scores and the single parameter, δ, that is a reflection of deviation from null allele-sharing.
(Results cont.)
Association Between Family DR8 Status & JRA Clinical Classifications
(Fisher's Exact Test testing for differences in the
clinical characteristics amongst the three groups)
Juvenile Rheumatoid Arthritis, the most common chronic rheumatic inflammatory condition of children, is estimated to affect more than 50,000 children in the U.S. The heterogeneity of its clinical phenotypes makes diagnoses difficult, and evidence has shown JRA to be distinct from adult onset Rheumatoid Arthritis.
The American College of Rheumatology (ACR) has defined classifications of JRA based on systemic involvement (fevers, rash, and involvement of other organ systems besides the musculoskeletal system) and mode of disease onset (≤4 joints, ≥5 joints). The disease onset type is defined by the clinical phenotype during the first 6 months of disease; patients with ≤4 affected joints during the first 6 months of disease are designated as having pauciarticular onset disease, and patients with ≥5 affected joints are designated as having polyarticular onset disease. The disease course type is determined by the number of affected joints after the first 6 months of disease; patients with ≤4 affected joints after 6 months of disease are classified as having a pauciarticular course, and those with ≥5 affected joints after 6 months of disease are classified as having a polyarticular course. Patients are further classified according to age at onset; those for whom disease developed before their sixth birthday are classified as having early-onset JRA, and those for whom disease developed on or after their sixth birthday are classified as having late-onset JRA.
There is a sibling risk ratio for a full sib of a child with JRA (estimated to be 15), which is consistent with other autoimmune disorders, such as Type I Diabetes Mellitus and Lupus.
Also consistent with other autoimmune disorders, the HLA region has been shown to have a major genetic effect in some JRA subtypes. Family-based studies have shown that the HLA class II allele DR8 has a higher than expected frequency of transmission (haplotype-specific P = 2.0 X 10-6), and that there is excess allele sharing of DR8 and DR11.
A recent genome-wide scan was conducted on our cohort of 128 affected sibpairs. Results from this GWS support the hypothesis that multiple genes, including at least 1 in the HLA region, influence susceptibility to JRA. The strongest evidence of linkage to JRA was observed near D6S276/HLA-DRB1 (6p22-21, LOD=2.26), while linkage analyses performed on JRA subtypes (stratified by onset age, onset, and course type) show considerable evidence of linkage at 7q11 in early polyarticular subjects, and at 19p13 in pauciarticular subjects.
Conditional linkage analyses on stratified families based on HLA typing, specifically the DRB1 locus, show strong evidence of linkage on chromosome 2 in DR8 sibpairs. To further investigate this interaction, we stratified our families based on concordance/discordance for DR8 and repeated the analyses on these subgroups.
Subtype
Concordant DR8-
(0 affected sibs with DRB1*8 allele)
Discordant DR8
(1 affected sib with DRB1*8 allele)
Concordant DR8
(2 affected sibs with DRB1*8 allele)
p-value
Observed
Expected
Polyarticular
Onset 17
13.0 1
1.6 2
5.4
0.1282
Pauciarticular 35
38.4 3
4.8 21
15.8
0.0767
Early Onset 26
29.9
3.4 19
12.3
0.0084
Late Onset 18
16.9 4
2.1 7
0.1506
Early
Course 5
4.6
0.6
1.9
1.0000 15
18.9
2.4 13
7.8
0.0435
Late
3.2
0.4837
0.2205
24.1
3.0 16
9.9
0.0299 32
23.4
2.9
9.7
0.0010
Results
Early Onset subtype is associated with concordant DR8 sibs
Early Pauciarticular is associated with concordant DR8 sibs
Pauciarticular Course is associated with concordant DR8 sibs
DR8 is protective against Polyarticular Course
Summary
 There is excess transmission of the HLA class II allele DR8 to offspring with pauciarticular JRA (haplotype-specific P = 2.0 x 10-6).
A LOD = 6.09 at 2p25 (LOD-1 interval cM) is observed in 33 pedigrees with offspring concordant for the presence of a DR8 allele.
Suggestive evidence for linkage is observed at 14q32 (LOD = 1.84) in these 33 pedigrees.
A LOD of 2.48 at 3p27 is observed in pedigrees with at least one offspring having HLA-DR4.
A LOD = 2.34 at 17q25 is observed in pedigrees with offspring concordant for either DR8, DR11, or DR13.
Preliminary TDT have been run with our microsatellite data, and we are investigating suggestive haplotype blocks in this region.
 NPL analysis in 80 families with 0 affected siblings having DR8:
LOD = 0.13
LOD-1 interval cM
 NPL analysis in 10 families with only one affected sibling having DR8:
LOD = 1.03
LOD-1 interval cM
 NPL analysis in 43 families with at least one affected sibling having DR8:
LOD = 3.05
LOD-1 interval cM
 NPL analysis in 33 families with an affected concordant full sib pair for DR8:
LOD = 6.09
LOD-1 interval cM
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 Wilcoxon Signed Test testing for difference in the NPL scores of the 3 groups indicates a distinct difference amongst the groups (0 DR8 sibs, exactly 1 DR8 sib, concordant DR8 sibs):
p-value <0.001
Methods
Subject Recruitment
Subjects were recruited by The JRA Affected Sibpairs Registry from across U.S.A. and Canada (supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases). At the time of analysis, the registry included 512 individuals from 121 pedigrees, yielding 128 JRA affected sibpairs (ASPs). Blood was collected from patients with JRA, their unaffected siblings, and their parents (grandparents were included where available). This study was approved by the Institutional Review Board of the Cincinnati Children’s Hospital Medical Center, and appropriate consent was obtained from all subjects and family members. DNA was isolated from mono-nuclear cells using standard salting-out procedures.
What’s next
We are currently investigating a 20 Mb region on chromosome 2 that encompasses the 7 cM region at 2p25 by using a SNP map composed of 768 SNPs, ~26 kb spacing. No candidate genes have yet been identified.