Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness

Abstract

Purpose: Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology.

Methods: Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions.

Results: We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation.

Conclusion: Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.

Keywords: genetic diagnosis; limb-girdle weakness; neuromuscular disease; next-generation sequencing; targeted exome analysis.

Fig. 1. Summary of the solved rate and sequential analysis in the MYO-SEQ cohort.

Suspected pathogenic variants were detected in a total of 520 patients (52%). Initially, 468 (47%) patients were solved through a screening of the MYO-SEQ gene list of 169 neuromuscular disorder (NMD) genes; of these, 450 (45%) had pathogenic variants in one of 72 genes and 18 (2%) had pathogenic variants in two genes. Pathogenic variants in eight of these genes alone accounted for half of the solved patients (26%); the most common disease detected in our cohort was limb-girdle muscular dystrophy (LGMD) R1 calpain3-related (CAPN3). Variants in 64 genes accounted for the remaining patients with one causal gene. The analysis strategy was extended by a further 260 genes, solving an additional 2% of the cohort. Copy-number variations (CNVs) were detected in 26 (3%) patients, SMN1 deletions in five (0·5%) patients and transcriptional perturbations in one patient. n = 1001. GS genome sequencing.

Fig. 2. Solved rate by country of origin.

The MYO-SEQ solved rate (in dark gray) was higher in countries such as Egypt and Turkey where the infrastructure for genetic testing for prescreening is not as widely available, and lower in Western European countries where genetic prescreening of common limb-girdle muscular dystrophy (LGMD) genes is routinely performed. Calculated for referring centers submitting more than 20 samples.

Fig. 3. Breakdown of the suspected pathogenic variants and genotypes in the MYO-SEQ cohort.

(a) Zygosity of the solved patients’ variants (n = 506; 489 patients, 20 with an additional gene to report). (b) Type of variants suspected to be pathogenic. Initiation and stop loss occurred twice each (n = 865). (c) The 865 occurrences were accounted for by 520 distinct variants, of which 119 were reported as pathogenic in ClinVar and 401 were novel in their association to disease at the time of the analysis. (d) Of the 119 distinct variants reported in ClinVar, 70 were detected in only one individual (unique) while 49 were detected in multiple patients. Of the 401 distinct variants that were novel in their association to disease, 285 were detected in only individual cases, while 116 were detected in multiple families.