Myasthenia gravis (MG) is an antibody-mediated autoimmune disorder of the neuromuscular junction characterized by fatigable muscle weakness. Genetic susceptibility contributes importantly to MG risk, with many GWAS-identified variants located in noncoding regions. Noncoding risk variants often act through cis-regulatory elements such as enhancers and can alter expression of downstream targets, including long noncoding RNAs (lncRNAs). Among lncRNAs in the major histocompatibility complex (MHC) region, HCG27 and HCG9 have been implicated in immune regulation in other autoimmune diseases, but their roles and the impact of lncRNA-related SNPs in MG subtypes have not been systematically defined.
The authors aimed to identify MG-associated lncRNA regulatory SNPs, determine whether any act as functional enhancer variants that regulate lncRNA expression, and assess potential downstream effects of altered lncRNA levels on immune cell behavior relevant to MG pathogenesis.
An age-, sex-, and ethnicity-matched case–control study was conducted. The genotyping cohort comprised 161 patients with MG and 161 healthy controls recruited at the Second Affiliated Hospital of Harbin Medical University between June 2024 and February 2025. Among the MG patients, 131 were acetylcholine receptor antibody (AChR-Ab) positive; antibody status for 30 patients was unknown. Healthy controls had no personal or family history of autoimmune or neuromuscular disorders. Standard clinical criteria, electrophysiology, and pharmacological testing were used to diagnose MG. Exclusion criteria included severe cardiopulmonary disease, severe infections, pregnancy, most malignancies other than thymoma, and other autoimmune diseases.
Regulatory SNPs were screened using MG GWAS datasets, and selected candidate lncRNA-related SNPs were genotyped in the cohort using SNaPshot minisequencing. To prioritize SNPs associated with disease, the investigators applied two machine learning approaches: LASSO regression and random forest. These methods were used to identify SNPs that contributed most strongly to classification between cases and controls or between clinical subtypes.
The study identified significant candidate SNPs on chromosomes 1, 6, 11, 17, and 18. In the full case–control comparison, no overall genotype or allele frequency differences reached significance. However, subtype-specific analyses revealed associations: HCG27 rs9263872 (TT), rs9263875 (GG), and rs28362336 (GG) were associated with generalized myasthenia gravis (gMG), while the HCG9 rs3823381 T allele was associated with ocular MG (oMG). The rs9263875 G allele specifically associated with gMG. Machine learning analysis identified rs28362336 as a risk factor for MG.
The manuscript reports these subtype-specific associations rather than an overall case–control effect, indicating genetic heterogeneity between oMG and gMG.
Bioinformatic annotation of the genomic region harboring rs28362336 indicated chromatin features consistent with enhancer activity. ChIP-PCR experiments demonstrated enrichment of enhancer-associated histone marks H3K4me1 and H3K27ac, as well as RNA polymerase II (Pol II), at the rs28362336 locus upstream of HCG27. These data support the concept that rs28362336 resides in an active cis-regulatory element capable of influencing transcription of nearby genes or lncRNAs.
The authors also performed eQTL analysis; combined multiomic evidence and ChIP validation were used to support a model in which the SNP modulates transcription factor binding and local chromatin state to affect HCG27 expression. Specific transcription factor binding changes at the allele level were a focus of the analysis, and ChIP-PCR provided experimental support for regulatory activity at the locus.
Quantitative real-time PCR (qRT-PCR) showed that HCG27 expression was increased in peripheral samples from MG patients compared with controls. The increase was particularly notable in gMG patients carrying the rs28362336 GG genotype, consistent with an allelic effect on lncRNA expression. These findings link the genotype at rs28362336 to altered HCG27 levels in patient samples, supporting the SNP’s functional relevance.
Fluorescence in situ hybridization (FISH) localized HCG27 within CD4+ T cells. Functional perturbation experiments evaluated the effects of HCG27 modulation in these cells: overexpression of HCG27 promoted CD4+ T-cell proliferation and inhibited apoptosis, while knockdown experiments yielded reciprocal effects. Western blot analysis and CCK-8 proliferation assays were used to quantify these cellular responses. These results indicate that elevated HCG27 can drive T-cell survival and expansion, cellular processes that could plausibly influence autoimmune responses in MG.
This integrative study combines GWAS-guided SNP selection, targeted genotyping, machine learning prioritization, epigenetic validation, expression analysis, and cell-based functional assays to investigate lncRNA-related regulatory variants in MG. The key finding is that rs28362336, located upstream of HCG27, behaves as an enhancer-associated SNP with allele-dependent effects on HCG27 expression and associates with risk of generalized myasthenia gravis in subtype analyses. Increased HCG27 expression in patients and its capacity to promote CD4+ T-cell proliferation and inhibit apoptosis support a potential mechanistic link between the SNP, lncRNA dysregulation, and immune-mediated pathology.
These results underscore the importance of interrogating noncoding regulatory variation and lncRNA biology to explain genetic susceptibility and clinical heterogeneity in MG. The authors propose that HCG27 and its regulatory SNPs may serve as molecular markers for subtype stratification and as potential targets for mechanistic study.
The reported associations were subtype-specific rather than observed across the full case–control cohort; the manuscript includes statistical analyses and machine learning outputs to support findings but full effect sizes and all p-values are provided in the original article. The study cohort comprised 161 patients and 161 controls recruited at a single center; additional replication in independent and larger cohorts would be necessary to confirm generalizability. The manuscript provides multiomic and experimental data supporting enhancer activity at rs28362336 and a functional role for HCG27 in CD4+ T cells, but further work is required to define precise transcription factor interactions and downstream molecular pathways mediating the lncRNA’s effects.
Overall, the study identifies rs28362336 as a regulatory SNP associated with gMG and links this variant to increased HCG27 expression and pro-proliferative effects in CD4+ T cells, implicating an enhancer-SNP–lncRNA axis in MG pathogenesis.