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Editor's note
Open Access | 
10.1172/JCI171497
Removing uncertainty from variants of unknown significance
Elizabeth M. McNally
Find articles by McNally, E. in: PubMed | Google Scholar
Published June 15, 2023 - More info
J Clin Invest. 2023;133(12):e171497. https://doi.org/10.1172/JCI171497.
© 2023 variants et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
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Abstract
Genetic testing is essential for patients with a suspected hereditary myopathy. More than 50% of patients clinically diagnosed with a myopathy carry a variant of unknown significance in a myopathy gene, often leaving them without a genetic diagnosis. Limb-girdle muscular dystrophy (LGMD) type R4/2E is caused by mutations in β-sarcoglycan (SGCB). Together, β-, α-, γ-, and δ-sarcoglycan form a 4-protein transmembrane complex (SGC) that localizes to the sarcolemma. Biallelic loss-of-function mutations in any subunit can lead to LGMD. To provide functional evidence for the pathogenicity of missense variants, we performed deep mutational scanning of SGCB and assessed SGC cell surface localization for all 6,340 possible amino acid changes. Variant functional scores were bimodally distributed and perfectly predicted pathogenicity of known variants. Variants with less severe functional scores more often appeared in patients with slower disease progression, implying a relationship between variant function and disease severity. Amino acid positions intolerant to variation mapped to points of predicted SGC interactions, validated in silico structural models, and enabled accurate prediction of pathogenic variants in other SGC genes. These results will be useful for clinical interpretation of SGCB variants and improving diagnosis of LGMD; we hope they enable wider use of potentially life-saving gene therapy.
Authors
Chengcheng Li, Jackson Wilborn, Sara Pittman, Jil Daw, Jorge Alonso-Pérez, Jordi Díaz-Manera, Conrad C. Weihl, Gabe Haller
Interpretation of genetic testing results is complicated by the high degree of genetic variation present in every individual genome. Most inherited single-gene disorders are caused by rare gene variants, but it is often not possible to distinguish between rare pathogenic variants and rare benign variants using current classification systems. These classification systems rely heavily on comparing disease prevalence with the population frequency of genetic variants, both of which may be inadequately reported. This ambiguity can lead to genetic tests yielding results called variants of unknown significance (VUSs), and uncertain results can be frustrating for both health care participants and providers. Reliable functional assays of all variants in a gene and/or protein provide additional critical information to improve genetic interpretation.
Over 50% of patients with myopathy are found to have at least one VUS in a myopathy gene, complicating genetic diagnosis (1). A subtype of limb-girdle muscular dystrophy (LGMD) is caused by recessive mutations in the SGCB gene. SGCB, which encodes β-sarcoglycan, together with α-, γ-, and δ-sarcoglycan, forms a four-protein transmembrane complex (SGC) that stabilizes the muscle plasma membrane, and mutations in any of the sarcoglycan subunits can lead to LGMD (2, 3). Data that indicate native protein expression can aid in the genetic diagnosis for these muscle disorders, but gathering protein localization information requires an invasive muscle biopsy.
In this issue of the JCI, Li and colleagues assessed the impact of missense variants by performing deep mutational scanning of the SGCB gene and evaluating the cell-surface localization of the sarcoglycan complex for all 6,340 possible amino acid changes (4). The functional assay scored missense variants based on cell surface production of the sarcoglycan complex. For known pathological variants, the degree of cell surface production correlated with the clinical outcome of loss of ambulation. Variants with less severe functional scores often correlated with slower disease progression, demonstrating a link between variant function and disease severity. The authors used AlphaFold2 to identify domains in this single-pass type 2 transmembrane protein enriched for pathological variants. These findings can aid in the clinical interpretation of SGCB variants and enhance LGMD diagnosis, and, with clinical trials ongoing for β-sarcoglycan gene replacement therapy, accurate diagnosis is an essential step.
The predicted structural information in Li et al. not only identified regions in which pathogenic variants clustered, but described AlphaFold2 modeling that incorporated multiple sarcoglycan subunits alongside β-sarcoglycan that also helped identify interface residues and predicted pathogenicity of γ-sarcoglycan and δ-sarcoglycan residues (4). This work provides a clinically useful assay to interpret genetic variation in the sarcoglycan transmembrane complex, and this method can be extended to genes encoding other transmembrane proteins and cell-surface complexes (4).
Currently, clinical diagnosis of sarcoglycan-deficient LGMD is challenging owing to the overlap in phenotype between different sarcoglycanopathies and the phenotypic heterogeneity of other genetically defined LGMDs. Obtaining a genetic diagnosis can help determine genotype-phenotype correlations and provide useful interpretation for patients and families.
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ISSN: 0021-9738 (print), 1558-8238 (online)