Comprehensive functional characterization of SGCB coding variants predicts pathogenicity in limb-girdle muscular dystrophy type R4/2E
Chengcheng Li, Jackson Wilborn, Sara Pittman, Jil Daw, Jorge Alonso-Pérez, Jordi Díaz-Manera, Conrad C. Weihl, Gabe Haller
Chengcheng Li, Jackson Wilborn, Sara Pittman, Jil Daw, Jorge Alonso-Pérez, Jordi Díaz-Manera, Conrad C. Weihl, Gabe Haller
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Research Article Genetics Muscle biology

Comprehensive functional characterization of SGCB coding variants predicts pathogenicity in limb-girdle muscular dystrophy type R4/2E

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

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Figure 1

Overview of SGCB functional screen.

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Overview of SGCB functional screen. Steps for generating and testing SGC...
Steps for generating and testing SGCB variant function in ADG-HEK human cells (stably expressing SGCA, SGCD, and SGCG). (i) Generation of mutation libraries by cloning synthesized pools of mutant oligos into a WT backbone. (ii) Creation and transduction of lentiviral libraries derived from the mutant plasmid libraries. (iii) Staining and imaging of transduced cells for YFP and HA antibody staining. Cells with pathogenic variants (G167S) fail to effectively transport intracellular SGCB (green) to the cell surface (red), while WT demonstrates robust total protein and cell surface expression of SGCB. (iv) Transduced cells sorted using FACS for HA staining into 4 bins. (v) Sequencing of cells with each bin of HA staining. (vi) Calculation of functional scores from mutation prevalence in each bin of HA staining. The resulting functional score is negative for deleterious variants or positive for functionally neutral variants.