Integration of Sequence Data from a Consanguineous Family with Genetic Data from an Outbred Population Identifies PLB1 as a Candidate Rheumatoid Arthritis Risk Gene

Integrating genetic data from families with highly penetrant forms of disease together with genetic data from outbred populations represents a promising strategy to uncover the complete frequency spectrum of risk alleles for complex traits such as rheumatoid arthritis (RA). Here, we demonstrate that rare, low-frequency and common alleles at one gene locus, phospholipase B1 (PLB1), might contribute to risk of RA in a 4-generation consanguineous pedigree (Middle Eastern ancestry) and also in unrelated individuals from the general population (European ancestry). Through identity-by-descent (IBD) mapping and whole-exome sequencing, we identified a non-synonymous c.2263G>C (p.G755R) mutation at the PLB1 gene on 2q23, which significantly co-segregated with RA in family members with a dominant mode of inheritance (P = 0.009). We further evaluated PLB1 variants and risk of RA using a GWAS meta-analysis of 8,875 RA cases and 29,367 controls of European ancestry. We identified significant contributions of two independent non-coding variants near PLB1 with risk of RA (rs116018341 [MAF = 0.042] and rs116541814 [MAF = 0.021], combined P = 3.2×10−6). Finally, we performed deep exon sequencing of PLB1 in 1,088 RA cases and 1,088 controls (European ancestry), and identified suggestive dispersion of rare protein-coding variant frequencies between cases and controls (P = 0.049 for C-alpha test and P = 0.055 for SKAT). Together, these data suggest that PLB1 is a candidate risk gene for RA. Future studies to characterize the full spectrum of genetic risk in the PLB1 genetic locus are warranted

Integration of sequence data from a consanguineous family with genetic data from an outbred population identifies PLB1 as a candidate rheumatoid arthritis risk gene

Integrating genetic data from families with highly penetrant forms of disease together with genetic data from outbred populations represents a promising strategy to uncover the complete frequency spectrum of risk alleles for complex traits such as rheumatoid arthritis (RA). Here, we demonstrate that rare, low-frequency and common alleles at one gene locus, phospholipase B1 (PLB1), might contribute to risk of RA in a 4-generation consanguineous pedigree (Middle Eastern ancestry) and also in unrelated individuals from the general population (European ancestry). Through identity-by-descent (IBD) mapping and whole-exome sequencing, we identified a non-synonymous c.2263G>C (p.G755R) mutation at the PLB1 gene on 2q23, which significantly co-segregated with RA in family members with a dominant mode of inheritance (P = 0.009). We further evaluated PLB1 variants and risk of RA using a GWAS meta-analysis of 8,875 RA cases and 29,367 controls of European ancestry. We identified significant contributions of two independent non-coding variants near PLB1 with risk of RA (rs116018341 [MAF = 0.042] and rs116541814 [MAF = 0.021], combined P = 3.2×10-6). Finally, we performed deep exon sequencing of PLB1 in 1,088 RA cases and 1,088 controls (European ancestry), and identified suggestive dispersion of rare protein-coding variant frequencies between cases and controls (P = 0.049 for C-alpha test and P = 0.055 for SKAT). Together, these data suggest that PLB1 is a candidate risk gene for RA. Future studies to characterize the full spectrum of genetic risk in the PLB1 genetic locus are warranted. © 2014 Plenge et al

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

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

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

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

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

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