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Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to myasthenia gravis.

ABSTRACT: The muscle acetylcholine receptor is the major target of the autoimmune response in generalized myasthenia gravis. To investigate the role of the gene encoding the alpha subunit of the receptor (CHRNA), two stable polymorphic d[(GT).(CA)]dinucleotide repeats, designated HB and BB, were characterized within the first intron of CHRNA. The HB*14 allele conferred a relative risk for myasthenia gravis of 2.5 in 81 unrelated patients compared with 100 control subjects. Very significantly, family analysis based on haplotype segregation data indicated that parental haplotypes associated with HB*14 always segregated to the child with myasthenia gravis (P < 0.0002 for the comparison with the transmission of haplotypes not bearing HB*14), whereas their transmission to unaffected siblings was equilibrated. Myasthenia gravis patients also showed a high frequency of microsatellite variants unseen in controls. These findings implicate the CHRNA in susceptibility to myasthenia gravis.

SUBMITTER: Garchon HJ

PROVIDER: S-EPMC43849 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to myasthenia gravis.

Garchon H J HJ Djabiri F F Viard J P JP Gajdos P P Bach J F JF

Proceedings of the National Academy of Sciences of the United States of America 19940501 11

The muscle acetylcholine receptor is the major target of the autoimmune response in generalized myasthenia gravis. To investigate the role of the gene encoding the alpha subunit of the receptor (CHRNA), two stable polymorphic d[(GT).(CA)]dinucleotide repeats, designated HB and BB, were characterized within the first intron of CHRNA. The HB*14 allele conferred a relative risk for myasthenia gravis of 2.5 in 81 unrelated patients compared with 100 control subjects. Very significantly, family analy ...[more]

PMID: 7910962

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Project description:Background and objectivesAutoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation.MethodsAn HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry.ResultsSerum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset.DiscussionA novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.

Project description:Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients' thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

Dataset Information

Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to myasthenia gravis.

Publications

Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to myasthenia gravis.

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