Figure 1.
Description of the study design.
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 PLB1 associated with RA segregation. (D) We evaluated the regional association of the PLB1 locus using RA GWAS meta-analysis including 8,875 RA cases and 29,367 controls. (E) Deep exon sequencing of PLB1 and gene-based rare variant test was conducted for 1,088 RA cases and 1,088 controls.
Figure 2.
The consanguineous pedigree with Mendelian form of RA.
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 PLB1 p.G755R mutation was indicated by the combination of “+” (mutated allele) and “-” (reference allele).
Figure 3.
IBD mapping of the pedigree with RA.
(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 Table S2). 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).
Table 1.
Results of the validation assay for candidate variants derived from exome sequencing.
Figure 4.
Association of the PLB1 locus in RA GWAS meta-analysis.
(A) Coding regions of PLB1 and p.G755R mutation identified in the consanguineous RA pedigree. PLB1 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 PLB1 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 r2 value with the most significantly associated SNP. Stepwise logistic regression analysis demonstrated multiple independent signals driven by non-coding variants. (C) H3K4me3 peak of Treg primary cells in the PLB1 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).
Table 2.
Results of the GWAS meta-analysis of European RA case-control cohorts in the PLB1 locus.
Table 3.
Results of rare variant tests for PLB1 coding variants in the European RA case-control cohort.