Next generation sequencing in a large cohort of patients presenting with neuromuscular disease before or at birth

Abstract

Background: Fetal akinesia/hypokinesia, arthrogryposis and severe congenital myopathies are heterogeneous conditions usually presenting before or at birth. Although numerous causative genes have been identified for each of these disease groups, in many cases a specific genetic diagnosis remains elusive. Due to the emergence of next generation sequencing, virtually the entire coding region of an individual's DNA can now be analysed through "whole" exome sequencing, enabling almost all known and novel disease genes to be investigated for disorders such as these.

Methods: Genomic DNA samples from 45 patients with fetal akinesia/hypokinesia, arthrogryposis or severe congenital myopathies from 38 unrelated families were subjected to next generation sequencing. Clinical features and diagnoses for each patient were supplied by referring clinicians. Genomic DNA was used for either whole exome sequencing or a custom-designed neuromuscular sub-exomic supercapture array containing 277 genes responsible for various neuromuscular diseases. Candidate disease-causing variants were investigated and confirmed using Sanger sequencing. Some of the cases within this cohort study have been published previously as separate studies.

Results: A conclusive genetic diagnosis was achieved for 18 of the 38 families. Within this cohort, mutations were found in eight previously known neuromuscular disease genes (CHRND, CHNRG, ECEL1, GBE1, MTM1, MYH3, NEB and RYR1) and four novel neuromuscular disease genes were identified and have been published as separate reports (GPR126, KLHL40, KLHL41 and SPEG). In addition, novel mutations were identified in CHRND, KLHL40, NEB and RYR1. Autosomal dominant, autosomal recessive, X-linked, and de novo modes of inheritance were observed.

Conclusions: By using next generation sequencing on a cohort of 38 unrelated families with fetal akinesia/hypokinesia, arthrogryposis, or severe congenital myopathy we therefore obtained a genetic diagnosis for 47% of families. This study highlights the power and capacity of next generation sequencing (i) to determine the aetiology of genetically heterogeneous neuromuscular diseases, (ii) to identify novel disease genes in small pedigrees or isolated cases and (iii) to refine the interplay between genetic diagnosis and clinical evaluation and management.

Fig. 1

Pedigrees for families in which mutations were identified from next generation sequencing of a proband. Pedigrees and segregation of the mutation/s identified within each family is shown for pedigrees not previously described elsewhere. Probands denoted by arrowheads. (a) Family 16 and (b) Family 20 with homozygous KLHL40 mutations; (c) Family 14: X-linked MTM1 mutation; (d) Family 6, (e) Family 8 and (f) Family 13 with compound heterozygous mutations of RYR1; (g) Family 9: homozygous NEB mutation; (h) Family 10: compound heterozygous mutation of CHRND; i Family 15: homozygous mutation of CHRNG; (j) Family 1: de novo mutation of MYH3; (k) Family 11: dominantly-inherited mutation of MYH3. Pedigrees for Family 2²³, 3-4²⁴, 5²², 7⁵⁷, 12²⁰ and 38³⁰ are published previously

Fig. 2

Evolutionary conservations of substituted residues in three families harbouring novel missense substitutions. Evolutionary conservation of the substituted amino acid in KLHL40 in Family 20 (a), RYR1 in Family 6 (b) and CHRND in Family 10 (c)

Fig. 3

Histology of muscle biopsies from four families with mutations identified in the proband. Family 16 (a-c): h&e indicating variation in myofibre diameter (a) and Gomori trichrome staining showing dark purple regions suggesting nemaline bodies (arrows) (b). Electron micrograph, arrows indicate miliary nemaline bodies (c). (d) H&E stain of muscle from the proband in Family 14, indicating variation in myofibre size, central and internal nuclei. (e) Staining for NADH-TR in muscle from the proband in Family 14 with arrows indicating reduced central staining indicative of minicores. (f) H&E staining of muscle from the proband in Family 13 showing muscle tissue embedded in fibro-adipose tissue, with severe myopathic, non-specific changes. (g) H&E staining of muscle from the proband in Family 8, demonstrating a severe non-specific picture

Fig. 4

Expression of wild-type (αβδε) and mutant (αβδC257Rε) acetylcholine receptors (AChR) in HEK 293 cells. AChR expression was determined through the binding of ¹²⁵I α-Bungarotoxin (¹²⁵I α-BuTx) to AChR on the cell surface (n = 6). Note: numbering of the mutation includes the pre-peptide sequence