Profiling of pathogenic variants in Japanese patients with sarcoglycanopathy

Abstract

Background: Sarcoglycanopathies (SGPs) are limb-girdle muscular dystrophies (LGMDs) that can be classified into four types, LGMDR3, LGMDR4, LGMDR5, and LGMDR6, caused by mutations in the genes, SGCA, SGCB, SGCG, and SGCD, respectively. SGPs are relatively rare in Japan. This study aims to profile the genetic variants that cause SGPs in Japanese patients.

Methods: Clinical course and pathological findings were retrospectively reviewed in Japanese patients with SGP. Genetic analyses were performed using a combination of targeted resequencing with a hereditary muscle disease panel, whole genome sequencing, multiplex ligation-dependent probe amplification, and long-read sequencing. The structures of transcripts with aberrant splicing were also determined by RT-PCR, RNA-seq, and in silico prediction.

Results: We identified biallelic variants in SGC genes in 53 families, including three families with LGMDR6, which had not been identified in Japan so far. SGCA was the most common causative gene, accounting for 56% of cases, followed by SGCG, SGCB, and SGCD, at 17%, 21%, and 6%, respectively. Missense variants in SGCA were very frequent at 78.3%, while they were relatively rare in SGCB, SGCG, and SGCD at 11.1%, 18.2%, and 16.6%, respectively. We also analyzed the haplotypes of alleles carrying three variants found in multiple cases: c.229C > T in SGCA, c.325C > T in SGCB, and exon 6 deletion in SGCG; two distinct haplotypes were found for c.229C > T in SGCA, while each of the latter two variants was on single haplotypes.

Conclusions: We present genetic profiles of Japanese patients with SGPs. Haplotype analysis indicated common ancestors of frequent variants. Our findings will support genetic diagnosis and gene therapy.

Keywords: Genetic Profile; Haplotyping; Japan; Large deletion; Limb-Girdle muscular dystrophies; RNA-Seq; Sarcoglycanopathies; Skeletal muscles, in silico prediction; Variants.

Fig. 1

STRAD flow diagram showing the genetic analysis carried out. We started with sequencing analyses, such as Sanger sequencing, panel analysis, whole-exome sequencing, or whole-genome sequencing. Aberrant splicing was detected using RNA-seq or RT-PCR in patients with exon–intron boundary or intronic variants. MLPA was applied to patients who were not diagnosed by sequencing analysis, and the breakpoints of a large SGCG deletion were identified using long-read sequencing

Fig. 2

Locations of amino-acid changes caused by identified genetic variants in predicted protein structures. Predicted three-dimensional structures of α-sarcoglycan (Q16586 (SGCA_HUMAN)), β-sarcoglycan (Q16585 (SGCB_HUMAN)), γ-sarcoglycan (Q13326 (SGCG_HUMAN)), and δ-sarcoglycan (Q92629 (SGCD_HUMAN)), obtained from Alphafold Protein Structure Database (https://alphafold.ebi.ac.uk/). Red circles indicate the positions of missense variants. Red squares indicate in-frame deletions or insertions. Gray circles denote the positions of truncation of normal sequences in truncation variants. Yellow and white arrowheads show the 4 amino-acid deletion (13–16) caused by c.38-2A > C in SGCA and p.K32E encoded by c.94A > G in SGCD, respectively

Fig. 3

Frequencies of variants and variant types in patients with sarcoglycanopathies. The frequency of (a) causative genes in Japanese patients with sarcoglycanopathies, and (b)–(e) variants in each causative gene: b SGCA, c SGCB, d SGCG, and e SGCD. Gray sections in pie charts indicate variants detected one family. f Effects of gene variants on protein products

Fig. 4

Genome structures in patients with deletion of SGCG exon 6, SGCG exon 1 to 6 and the whole SGCG gene. a SGCG exon 6 deletion. The breakpoints were the same in all patients. b SGCG exon 1 to 6 deletion. c Whole gene deletion of SGCG; similar sequences (identity: 94%) were present in the 5' and 3' breakpoints. Repeat sequence schema were retrieved from Ensembl (https://asia.ensembl.org/index.html). SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; LTR, long terminal repeat

Fig. 5

Splicing abnormalities and MaxEnt scores generated by analysis of intronic variants. a F1 SGCA intron 1: c.37 + 6 T > C. Sashimi plots of RNA-seq data using control and patient muscles are shown. b–e RT-PCR products obtained from control and patient muscle samples separated on agarose gels and schema showing aberrant splicing events with MaxEnt scores. White squares are normal-sized exons and gray squares are altered-sized exons. In the right schema, genetic variants are shown in red and new splicing sites are shown in light blue. * indicate the non-specific products which were not related to SGC genes. b F2 SGCA intron 1: c.38-2A > C. c F3 SGCA intron 2/exon 3: c.158-2_167del. d F25 SGCA intron 5: c.584 + 1G > A and F26 SGCA intron 5: c.584 + 5G > T. e F37 SGCB intron 5: c.753 + 5G > A. f F53 SGCD intron 6: c.502 + 24695G > T. RT-PCR and Sashimi plots of RNA-seq data are shown; sequences 5' (upstream) of the variants included a possible branch point site, a polypyrimidine tract, and a splice acceptor site. ctrl, control sample; Ref, reference sequence

Fig. 6

Haplotype analyses of alleles containing common single nucleotide polymorphism (SNP) variants. a Patients with SGCA c.229C > T. b Patients with SGCB c.325C > T. c Patients with SGCG exon 6 deletion. Bases in blue font indicate minor SNP alleles and bases in red font indicate variants. Orange shaded cells indicate homozygosity for minor SNP alleles. Yellow shaded cells indicate heterozygosity for minor SNP alleles. Blue shaded cells indicate homozygosity for major SNP alleles