Rheumatoid arthritis in black South Africans: genomic susceptibility and methotrexate response

Abstract

Rheumatoid arthritis (RA) is a complex chronic inflammatory joint disease with no cure. The heritability of RA is estimated to be 60% and no large genome-wide association studies have been performed in African populations. RA is treated using methotrexate (MTX), an antifolate disease-modifying antirheumatic drug. Previous studies have shown that variations in MTX polyglutamation genes play a role in MTX response in RA patients. However, very little is known about the RA aetiopathogenesis and pharmacogenetics of MTX use in people of African ancestry. This study used a case-control genome-wide association study (GWAS) approach to assess genetic susceptibility to RA in 577 Black South Africans (BSA) representing seropositive RA cases and 1,612 population controls. The cases and controls were genotyped on the H3Africa consortium genotyping SNP array and imputed using the African Genome Resources reference panel. The association was tested using a logistic regression analysis assuming an additive model and adjusted for sex, age, smoking, and the first three principal components for possible population structure. This study confirmed the strong association between increased risk and genetic variants in the Human Leucocyte Antigen (HLA) region. Furthermore, this study identified four novel single nucleotide polymorphism (SNP) associations in the intron regions of the CPT1A gene, which has been shown to be differentially expressed in RA patients, compared to unaffected controls. GWAS results were followed by integration with functional annotation of variants obtained from data related to tissues and cells relevant to RA pathogenesis, revealing that the associated SNPs are likely involved in the regulation of the expression of CPT1A and other nearby genes.

In addition to the GWAS, this study investigated 247 BSA seropositive RA patients on MTX monotherapy to evaluate the association of 36 previously identified index SNPs spanning 19 genes, and an additional 7812 SNPs in the surrounding regions, with MTX response. Patients were treatment naïve at baseline and were categorised after 6 months of MTX treatment into two groups of either EULAR good/moderate responders (n=186) or poor responders (n=61). A logistic regression analysis with an additive model adjusting for sex, age, smoking and disease duration was used for association testing. The regional replication of previously reported MTX response loci identified a region on chromosome 9 with 14 significantly associated SNPs mapping to the intronic region of the CDK9 gene, of which four (rs1002095; rs2297214; rs3217740, rs3217751) are expression quantitative trait loci (eQTL) that potentially influence the expression of the folylpolyglutamate synthetase (FPGS) gene

v

involved in MTX polyglutamation and intracellular persistence of MTX polyglutamation. The regulation of the expression of the FPGS gene by eQTL variants mapping to the CDK9 gene may influence response to MTX therapy in BSA with seropositive RA.

Although the sample size in this study had limited power to test many of the previously identified RA genetic associations with confidence, our results indicate that the pharmacokinetics of MTX might have a different genetic underpinning in African populations and underlines the necessity of larger studies in populations from Africa. Our study is the first and largest GWAS of RA and pharmacogenetics of MTX in sub-Saharan Africa.