Expanding genotype/phenotype of neuromuscular diseases by comprehensive target capture/NGS

Abstract

Objective: To establish and evaluate the effectiveness of a comprehensive next-generation sequencing (NGS) approach to simultaneously analyze all genes known to be responsible for the most clinically and genetically heterogeneous neuromuscular diseases (NMDs) involving spinal motoneurons, neuromuscular junctions, nerves, and muscles.

Methods: All coding exons and at least 20 bp of flanking intronic sequences of 236 genes causing NMDs were enriched by using SeqCap EZ solution-based capture and enrichment method followed by massively parallel sequencing on Illumina HiSeq2000.

Results: The target gene capture/deep sequencing provides an average coverage of ∼1,000× per nucleotide. Thirty-five unrelated NMD families (38 patients) with clinical and/or muscle pathologic diagnoses but without identified causative genetic defects were analyzed. Deleterious mutations were found in 29 families (83%). Definitive causative mutations were identified in 21 families (60%) and likely diagnoses were established in 8 families (23%). Six families were left without diagnosis due to uncertainty in phenotype/genotype correlation and/or unidentified causative genes. Using this comprehensive panel, we not only identified mutations in expected genes but also expanded phenotype/genotype among different subcategories of NMDs.

Conclusions: Target gene capture/deep sequencing approach can greatly improve the genetic diagnosis of NMDs. This study demonstrated the power of NGS in confirming and expanding clinical phenotypes/genotypes of the extremely heterogeneous NMDs. Confirmed molecular diagnoses of NMDs can assist in genetic counseling and carrier detection as well as guide therapeutic options for treatable disorders.

Figure 1. Coverage depth of 4,815 coding exons of the neuromuscular disease panel

Figure 2. Muscle pathology of patients 2, 26, 11, 9, 32, and 48

(A) Markedly increased lipid droplets in both number and size were observed in patient 2 with homozygous SLC25A20 mutations (oil red O). (B) Forty percent myofibers with centralized nuclei were found in patient 26 with compound heterozygous RYR1 mutations (hematoxylin and eosin). (C) Multiminicore pattern was observed in patient 11 with compound heterozygous SEPN1 mutations (nicotinamide adenine dinucleotide tetrazolium reductase). (D) Prominent nemaline rods were seen in patient 9 with heterozygous ACTA1 mutations (modified Gomori trichrome). (E) Nonspecific myopathic change except for internalized nuclei and mild fiber size variation was shown in patient 32 with compound heterozygous TCAP mutations (hematoxylin and eosin). (F) Similar to patient 9, typical intracytoplasmic nemaline rods were present in patient 48 with compound heterozygous NEB mutations (modified Gomori trichrome).

Figure 3. Confirmation of single exon deletion and point mutation in RYR1 detected by next-generation sequencing

(A) Sanger sequence for heterozygous c.9658A>G (p.T3220A). (B) Exon 39 heterozygous deletion was detected by copy number variation analysis. (C) Designed primers for exon 39 deletion. Forward primer: F, reverse primer: R, the total length between the primers is 4.3 kb. (D) DNA gel for exon 39 deletion; patients 25 and 26 have extra small fragments on the gel (∼2.7 kb).