OBJECTIVE: To identify potential causative markers involved in the development of early-onset myasthenia gravis (EOMG) in the MHC and non-MHC regions that may interact with the HLA-B*08:01 allele.METHODS: We analyzed 583 MG patients and identified 5 patients homozygous for the disease-associated ancestral haplotype 8.1 (HLA-A*01:01, B*08:01, DRB1*03:01, DQB1*02:01). We also analyzed more than 9000 controls and selected 24 for further investigation. We subsequently conducted a fine mapping analysis through high-throughput sequencing of the MHC region (from upstream of the GPá5 gene to downstream of the ZBTB9 gene). For the interaction analysis we analyzed a total of 150,090 SNPs equally distributed throughout the genome in the individuals that were homozygous for the main susceptibility HLA allele HLA-B*08:01 and investigated the expression of the genes located close to the observed susceptibility variants.RESULTS: The overall coverage of the 4.79 Mb MHC region ranged between 96.57% and 97.41%. We identified 705 new variants in the MHC region (673 SNPs and 32 InDels). However, no significant differences were found between patients and controls within the MHC region of the ancestral 8.1 haplotype. As the susceptibility gene is considered to be located close to the HLA-B locus, complete sequencing of the surrounding 200 kb was carried out in the 5 patients and 24 controls. No significant differences where observed, suggesting that the HLA-B molecule itself is the susceptibility factor for EOMG. We also observed two new susceptibility loci specific for MG HLA*08:01 patients (P < 3.33 ? 10-7). These loci map to an intronic OVCH1 variant (rs10492374; P = 1.90 ? 10-8) and a 5' downstream CNPY2 variant (rs10783780; P = 3.33 ? 10-7) on chromosome 12. Individuals heterozygous for GA*rs10492374 showed an increased expression of the OVCH1 gene. The rs10783780 genotypes were not associated with CNPY2 mRNA levels, but the MG HLA*08:01 patients present a lower expression of this gene than the healthy controls.CONCLUSION: Our results showed that when we control for the influence of the ancestral haplotype 8.1, no polymorphism was demonstrated to be associated with EOMG development within the MHC region suggesting that the HLA-B*08:01 allele is the unique genetic factor within the HLA region responsible for EOMG development in patients who carry the ancestral haplotype 8.1. Our study also identified two novel polymorphisms as risk factors for MG HLA-B*08:01 positive patients which regulate the expression of the OVCH1 and CNYP2 genes.
Adaptor Proteins, Signal Transducing Adolescent