Genome sequencing with comprehensive variant calling identifies structural variants and repeat expansions in a large fraction of individuals with ataxia and/or neuromuscular disorders

Abstract

Introduction: Neuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals.

Methods: In this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added.

Results: In the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes.

Discussion: Our results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders.

Keywords: ataxia; genome sequencing; neuromuscular disorders; repeat expansion; single nucleotide variant; structural variant.

Figure 1

Clinical workflow and bioinformatic pipeline. Patients of different ages were referred for clinical genome sequencing due to neuromuscular disorder phenotypes. The size of the circle behind the phenotype corresponds to the size of the subgroup. Genome sequencing was analyzed using an in-house analysis pipeline including base calling (1), alignment to reference (2), and variant calling and prioritizing (3). Finally, called variants were filtered in silico with the NMD gene panel (4) and classified according to the ACMG/AMP criteria (5). Clinically relevant variants (selected class 3, class 4, and class 5) were reported to the referring doctor.

Figure 2

Description of the cohort of NMD patients. (A) Bar chart showing the distribution of patient ages. (B) Bar chart showing the distribution of phenotype subgroups as well as the relation between prenatal cases, pediatric patients, and adult patients in each phenotype group. Patients 0–19 years of age (yellow), ≥20–90 years of age (green), and prenatal cases (blue).

Figure 3

Four individuals with SVs detected in NMD genes. (A) Homozygous tandem duplication of CRRPA in an individual with muscular dystrophy, CK 90, and mild ID. Duplication is shown in copy number variant (CNV) plot from chromosomal microarray with a 1M oligonucleotide slide. (B) Homozygous deletion of exon 7–11 in DYSF in an individual with limb girdle muscular dystrophy and disease onset in their twenties. The deletion is shown in the integrative genomics viewer (IGV) where there are no reads (gray arrows) mapping to the deleted region. (C) De novo deletion of exon 2–3 in INF2 in an individual with the onset of sensory motor neuropathy in early childhood and renal failure before the age of 20 years. The heterozygous deletion is shown in IGV displaying the two breakpoints with a sudden drop and a rise in coverage (arrows), respectively. (D) Whole-gene deletion of TTBK2 in an individual with the onset of ataxia around 40 years of age and sensory-motor neuropathy at nearly 50 years of age. Deletion is shown in CNV plot from chromosomal microarray.

Figure 4

Diagnostic yield. (A) Pie chart showing the percentage of all patients with pathogenic or likely pathogenic findings in NMD genes (dark blue), with a bar chart showing the distribution of SNVs/INDELs (green), SVs (yellow), and STRs (pink). For visualization, patients with VUS in NMD genes (purple) and pathogenic findings in non-NMD genes (light blue). (B) Pie chart showing the percentage of pediatric patients with pathogenic or likely pathogenic findings in NMD genes (dark blue), with a bar chart showing the distribution of SNVs/INDELs (green), SVs (yellow), and STRs (pink). For visualization, patients with VUS in NMD genes (purple) and pathogenic findings in non-NMD genes (light blue). (C) Pie chart showing the percentage of adult patients with pathogenic or likely pathogenic findings in NMD genes (dark blue), with a bar chart showing the distribution of SNVs/INDELs (green), SVs (yellow), and STRs (pink). For visualization, patients with VUS in NMD genes (purple) and pathogenic findings in non-NMD genes (light blue).

Figure 5

Genetic findings across phenotype subgroups. Bar chart showing the percentage of patients with pathogenic or likely pathogenic SNVs/INDELs (green), SVs (yellow), and STRs (pink). For visualization, patients with pathogenic findings in non-NMD genes (blue) and VUS in NMD genes (dark gray). Line chart showing the diagnostic yield in each phenotypic subgroup.