Case: 32 year old man presented with progressive muscle weakness and loss of ambulation that began in adolescence. The patient is of nonconsanguineous Nepalese descent and has no family history of LGMD. On exam the patient had significant proximal weakness in the arms and legs and a CK of approximately 2,000. Whole-exome sequencing (WES) revealed a homozygous VUS lying in a 5’ donor splice site of SGCA intron 1 (c.37+6T>C). SGCA codes for alpha-sarcoglycan, a structural protein found in the sarcolemma. Recessive pathogenic variants in SGCA have been attributed to clinical presentation of LGMD2D.
Methods and Results: To analyze the transcriptomic consequence of this SGCA variant, we conducted total RNA-seq with skeletal muscle tissue biopsied from the tibialis anterior. Alternative splicing analysis conducted with the R package FRASER revealed 17 differentially spliced (z-score |2|) instances of SGCA. Visualization of SGCA read alignment in Integrative Genomics Viewer (IGV) suggested a retained intron event correlated with the VUS (gnomAD MAF = 7.13e -6). The patient was shown to have roughly 25-30% SGCA and alpha-sarcoglycan expression, revealed from TaqMan qPCR and western blotting, respectively.
We performed a minigene assay to provide functional evidence supporting aberrant splicing. The VUS disrupted the canonical splice site, resulting in a ~50 bp intron inclusion event stemming from use of a 5’ cryptic donor site. This event causes a frameshift culminating in a premature stop codon (PTC) in exon 2. We were unable to amplify SGCA exon 1-exon 2 with patient-derived RNA, indicating potential nonsense-mediated degradation (NMD) of the transcript.
Conclusions: RNA-seq was successfully able to provide evidence of pathogenicity associated with a VUS by annotating a variant’s impact on muscle transcriptomics. We present a novel LGMD2D splice junction variant that likely results in NMD.