Genetic landscape and novel disease mechanisms from a large LGMD cohort of 4656 patients

Abstract

Objective: Limb-girdle muscular dystrophies (LGMDs), one of the most heterogeneous neuromuscular disorders (NMDs), involves predominantly proximal-muscle weakness with >30 genes associated with different subtypes. The clinical-genetic overlap among subtypes and with other NMDs complicate disease-subtype identification lengthening diagnostic process, increases overall costs hindering treatment/clinical-trial recruitment. Currently seven LGMD clinical trials are active but still no gene-therapy-related treatment is available. Till-date no nation-wide large-scale LGMD sequencing program was performed. Our objectives were to understand LGMD genetic basis, different subtypes' relative prevalence across US and investigate underlying disease mechanisms.

Methods: A total of 4656 patients with clinically suspected-LGMD across US were recruited to conduct next-generation sequencing (NGS)-based gene-panel testing during June-2015 to June-2017 in CLIA-CAP-certified Emory-Genetics-Laboratory. Thirty-five LGMD-subtypes-associated or LGMD-like other NMD-associated genes were investigated. Main outcomes were diagnostic yield, gene-variant spectrum, and LGMD subtypes' prevalence in a large US LGMD-suspected population.

Results: Molecular diagnosis was established in 27% (1259 cases; 95% CI, 26-29%) of the patients with major contributing genes to LGMD phenotypes being: CAPN3(17%), DYSF(16%), FKRP(9%) and ANO5(7%). We observed an increased prevalence of genetically confirmed late-onset Pompe disease, DNAJB6-associated LGMD subtype1E and CAPN3-associated autosomal-dominant LGMDs. Interestingly, we identified a high prevalence of patients with pathogenic variants in more than one LGMD gene suggesting possible synergistic heterozygosity/digenic/multigenic contribution to disease presentation/progression that needs consideration as a part of diagnostic modality.

Interpretation: Overall, this study has improved our understanding of the relative prevalence of different LGMD subtypes, their respective genetic etiology, and the changing paradigm of their inheritance modes and novel mechanisms that will allow for improved timely treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials.

Figure 1

Major contributing LGMD genes. (A) Molecular diagnosis has been established in 27% of the patients. A majority of these patients had a pathogenic variant in one of the following genes CAPN3 17%(175/1003), DYSF 16%(167/1003), FKRP 9%(87/1003), and ANO5 7%(72/1003) indicating that these genes are likely the major contributors to LGMD phenotype. (B) Number of unique pathogenic variants identified. Numbers of identified pathogenic variants were compared among the major contributing LGMD genes to understand the allelic heterogeneity of these genes. DYSF,CAPN3, and COL6A1 were identified with the most pathogenic variants including 133, 95 and 40, respectively, in each gene, indicating more allelic heterogeneity in these genes.

Figure 2

Types of variants identified in the tested LGMD patients. Variants were classified according to standards and guidelines of the American College of Medical Genetics and Genomics. Around 23% of the identified variants are pathogenic. Around 72% of the variants are interpreted as variants of uncertain significance (VUS) because majority of LGMD subtypes are poorly studied and currently limited knowledge available.

Figure 3

Homozygosity identified in major LGMD genes. Percentage of homozygous pathogenic variants identified in major LGMD genes.

Figure 4

Multigenic inheritance in LGMD. Pathogenic variants identified in more than one LGMD genes in two patients with unusual disease presentation and progression indicating complex inheritance patterns of LGMD. (A) Patient with homozygous variants in both ANO5 and SGCA genes. NGS reads indicated the identification of homozygous missense pathogenic variants c.2272C>T (p.R758C) and c.850C>T (R284C) in ANO5 and SGCA genes, respectively. (B) Rapid disease progression was observed in a 16‐year‐old male (arrow) with two pathogenic variants in ANO5 gene and one pathogenic variant in COL6A2 gene indicating multiple gene contributions for an unusual presentation. His mother, a 40‐year‐old female with one pathogenic variant each in ANO5 and COL6A2 shows unspecified myopathy with elevated creatine phosphokinase (CPK).

Figure 5

Segregation analysis of a deep intronic DYSF variant. Variant c.4886 + 1249G>T in DYSF gene was identified in a large family with LGMD2B. A deep intronic variant in DYSF gene alters mRNA splicing and ultimately results in inframe insertion of a new pseudoexon in dysferlin. Percentages indicate levels of expression of dysferlin protein in peripheral blood mononuclear cells (PBMCs) compared to the control.