The consanguineous pedigree with Mendelian form of RA.

<p>The consanguineous pedigree consists of 49 individuals from 4 generations. The pedigree included 8 individuals affected with RA (colored in black) and 1 ACPA-positive unaffected subject subject (colored in gray). Four RA cases for whom whole-exome sequencing was conducted were indicated with asterisks. Genotypes of the identified <i>PLB1</i> p.G755R mutation was indicated by the combination of “+” (mutated allele) and “-” (reference allele).</p

Results of the GWAS meta-analysis of European RA case-control cohorts in the <i>PLB1</i> locus.

a<p>Based on NCBI Build 37/hg19.</p>b<p>Conditioned with rs116018341.</p>c<p>AA risk haplotype was not observed in the imputation reference panel.</p><p>RA; rheumatoid arthritis, ACPA; anti-citrullinated protein antibodies.</p

IBD mapping of the pedigree with RA.

<p>(A) We investigated the novel non-parametric linkage analysis method which enabled the IBD mapping for the disease with any types of inheritance modes. Affected cases should carry one or two copy of the mutation which resides on a single ancestral haplotype in IBD, thus, SNPs adjacent to the causal mutation lose homozygous genotypes for at least one of the alleles. Our method searched the regional IBD stretches where SNP genotypes of the affected cases followed this rule, and then imputed presence or absence of the ancestral haplotype in the other unaffected subjects separately. (B) Results of the IBD mapping in the consanguineous pedigree with RA. Mapped IBD stretches are indicated as the bands colored in red. As the pedigree members used for the IBD mapping increased (left panel; 5 RA cases and 1 ACPA-positive unaffected subject, right panel; all available subjects), IBD stretches narrowed down (see detailed process in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087645#pone.0087645.s003" target="_blank">Table S2</a>). Candidate causal SNVs and Indels obtained after whole-genome exome sequencing were indicated as the triangles colored in blue and orange, respectively. The variants included and not included in the IBD stretches of each stages are indicated with filled and non-filled colors. Finally, only one SNV at 2p23 was included in the defined IBD stretch (right panel).</p

Association of the <i>PLB1</i> locus in RA GWAS meta-analysis.

<p>(A) Coding regions of <i>PLB1</i> and p.G755R mutation identified in the consanguineous RA pedigree. <i>PLB1</i> consists of 58 exons (NM_153021), and p.G755R (c.2263G>C) mutation was located at exon 33 (the black triangle). (B) Regional association of <i>PLB1</i> in RA GWAS meta-analysis including 8,875 RA cases and 29,367 controls from the European populations. Upper panel showed the results of nominal association, and the lower panel showed the results of conditional analysis with rs116018341, the top SNP in the nominal associations. The red diamond-shaped dots represent P-values of the SNPs in the GWAS meta-analysis, and the intensity of the red color in the dots represents the <i>r²</i> value with the most significantly associated SNP. Stepwise logistic regression analysis demonstrated multiple independent signals driven by non-coding variants. (C) H3K4me3 peak of T_reg primary cells in the <i>PLB1</i> locus. Non-coding RA risk SNP of rs116018341 overlapped with one of the H3K4me3 peaks as the SNP located in the most vicinity of the peak summit (a vertical dashed red line).</p

Results of rare variant tests for <i>PLB1</i> coding variants in the European RA case-control cohort.

a<p>Low-frequency rare coding variant (MAF≤0.01) obtained from deep sequencing of 1,088 RA cases and 1,088 controls were selected.</p><p>RA; rheumatoid arthritis, ACPA; anti-citrullinated protein antibodies.</p><p>BURDEN; burden test, VT; variable threshold test, FRQWGT; frequency-weighted test, CALPHA; C-alpha test, SKAT; sequence kernel association test.</p

Results of the validation assay for candidate variants derived from exome sequencing.

a<p>Genes of which variants were shared among 5 RA cases and 1 ACPA+ unaffected subject are indicated.</p>b<p>Based on NCBI Build 37/hg19.</p>c<p>Mid-P value of Fisher's exact test for RA cases and unaffected subjects are indicated.</p><p>RA; rheumatoid arthritis, ACPA; anti-citrullinated protein antibodies.</p

Description of the study design.

<p>Our study consists of analysis on three sources of data: (1) rare risk variant detection in the consanguineous pedigree with Mendelian form of RA (A–C), (2) regional association analysis using RA GWAS meta-analysis of European populations (D), and (3) target deep exon sequencing of the European RA case-control cohort (E). (A) We conducted IBD mapping of the pedigree using genome-wide SNP genotype data. (B) Whole-exome sequencing was performed for the 4 affected RA cases of the pedigree. (C) By integrating the results of IBD mapping and whole-exome sequencing, and subsequently conducting the validation assay, we identified a non-synonymous mutation of <i>PLB1</i> associated with RA segregation. (D) We evaluated the regional association of the <i>PLB1</i> locus using RA GWAS meta-analysis including 8,875 RA cases and 29,367 controls. (E) Deep exon sequencing of <i>PLB1</i> and gene-based rare variant test was conducted for 1,088 RA cases and 1,088 controls.</p

<i>TYK2</i> protein-coding variants identified by exon-sequencing of RA cases and controls.

<p>Using dense genotyping, we demonstrate that three <i>TYK2</i> protein-coding variants predicted to be damaging, P1104A, A928V, and I684S, protect against RA (highlighted in red). By exon-sequencing in 1,118 RA cases and 1,118 controls, we identified 23 additional missense variants predicted to be damaging (PolyPhen-2 and SIFT), with no strong evidence of association to RA in gene-based association tests. The <i>TYK2</i> coding exons, the protein domains, and the minor allele count (MAC) of the rare variants (MAC<5) in cases and controls are shown.</p

Results from stepwise conditional analysis of the <i>TYK2</i> locus.

<p>We fine-mapped the <i>TYK2</i> locus using Immunochip data available for 7,222 ACPA+ RA cases and 15,870 controls (MAF>0). (A) In the meta-analysis, the best signal of association was at the <i>TYK2</i> missense variant P1104A (rs34536443).(B) Conditional on P1104A, the best signal of association was at the <i>TYK2</i> missense variant A928V (rs35018800). (C) Conditional on P1104A and A928V variants, the best signal of association is at the <i>TYK2</i> missense variant I684S (rs12720356). (D) Conditional on the 3 RA-protective variants in <i>TYK2</i>, we observed no additional signal of association at the locus (best signal is rs3176768, P = 0.01). P-values from meta-analyses of logistic regressions results from 6 Immunochip collections are shown. The three <i>TYK2</i> missense variants predicted to be damaging and independently associated with RA risk are highlighted in green.</p