A novel nonsense variant in SUPT20H gene associated with Rheumatoid Arthritis identified by Whole Exome Sequencing of multiplex families

Abstract

The triggering and development of Rheumatoid Arthritis (RA) is conditioned by environmental and genetic factors. Despite the identification of more than one hundred genetic variants associated with the disease, not all the cases can be explained. Here, we performed Whole Exome Sequencing in 9 multiplex families (N = 30) to identify rare variants susceptible to play a role in the disease pathogenesis. We pre-selected 77 genes which carried rare variants with a complete segregation with RA in the studied families. Follow-up linkage and association analyses with pVAAST highlighted significant RA association of 43 genes (p-value < 0.05 after 106 permutations) and pinpointed their most likely causal variant. We re-sequenced the 10 most significant likely causal variants (p-value ≤ 3.78*10-3 after 106 permutations) in the extended pedigrees and 9 additional multiplex families (N = 110). Only one SNV in SUPT20H: c.73A>T (p.Lys25*), presented a complete segregation with RA in an extended pedigree with early-onset cases. In summary, we identified in this study a new variant associated with RA in SUPT20H gene. This gene belongs to several biological pathways like macro-autophagy and monocyte/macrophage differentiation, which contribute to RA pathogenesis. In addition, these results showed that analyzing rare variants using a family-based approach is a strategy that allows to identify RA risk loci, even with a small dataset.

Fig 1. Description of the study design.

This schema of the study presents the 3 main steps of the analysis and, the resulting number of variants selected through each sub-analysis. (A) The selection of candidate variants was performed on the discovery set and resulted in the identification of 77 candidate SNVs (B) The association analysis was applied on the discovery set and 98 controls extracted from the 1000 genomes project. The 43 genes significantly associated with RA were re-tested after removing the candidate variant identified in step A and, the candidate variant was tested alone (C) The SNVs with the strongest RA association were re-sequenced in the extended families of the discovery, plus 9 new multiplex families.

Fig 2. Principal component analysis (PCA) results of samples processed for pVAAST analysis.

The PCA is based on the 30 samples from the discovery set and 98 CEU controls extracted from the 1000 genomes databases. This plot represents the 2 first principal components which respectively account for 2.12% and 1.87% of the genetic variability. The color code represents the population source: each family sequenced by WES has its own color described in the legend, the CEU controls are in grey. The shape of each dot represents the RA status of the represented individual.

Fig 3. Gene SUPT20H and its product: p38IP.

Abbreviations: NLS: Nuclear Localization signal, PEST: Proline (P) Glutamic acid (E) Serine (S) Threonine. (A) Gene model of SUPT20H. The red arrow indicates the exon including the nonsense variant identified by WES. The exons color code refers to the protein domain it encodes for. (B) On top, p38IP protein model of 733 amino-acid and on the bottom, the positions of p38IP interactors binding sites.

Fig 4. Representation of the pedigree 3 and the variants identified in his members.

All the individuals represented here were part of the validation set but only the ones marked with a red arrow were part of the discovery set. Each of them is represented by a circle (if female) or a square (if male). Their filling color corresponds to their status: black if affected by RA or white if unaffected by the disease. The presence of a variant is represented by its HGVS id. The absence of this id indicates a homozygote genotype for the reference allele.