Assessment of Target Enrichment Platforms Using Massively Parallel Sequencing for the Mutation Detection for Congenital Muscular Dystrophy

Abstract

Sequencing individual genes by Sanger sequencing is a time-consuming and costly approach to resolve clinically heterogeneous genetic disorders. Panel testing offers the ability to efficiently and cost-effectively screen all of the genes for a particular genetic disorder. We assessed the analytical sensitivity and specificity of two different enrichment technologies, solution-based hybridization and microdroplet-based PCR target enrichment, in conjunction with next-generation sequencing (NGS), to identify mutations in 321 exons representing 12 different genes involved with congenital muscular dystrophies. Congenital muscular dystrophies present diagnostic challenges due to phenotypic variability, lack of standard access to and inherent difficulties with muscle immunohistochemical stains, and a general lack of clinician awareness. NGS results were analyzed across several parameters, including sequencing metrics and genotype concordance with Sanger sequencing. Genotyping data showed that both enrichment technologies produced suitable calls for use in clinical laboratories. However, microdroplet-based PCR target enrichment is more appropriate for a clinical laboratory, due to excellent sequence specificity and uniformity, reproducibility, high coverage of the target exons, and the ability to distinguish the active gene versus known pseudogenes. Regardless of the method, exons with highly repetitive and high GC regions are not well enriched and require Sanger sequencing for completeness. Our study demonstrates the successful application of targeted sequencing in conjunction with NGS to screen for mutations in hundreds of exons in a genetically heterogeneous human disorder.

Utility of next generation sequencing in genetic diagnosis of early onset neuromuscular disorders

“…Our study confirms that mutations in the genes for the extracellular matrix protein collagen VI α are a prevalent cause of CMD, with a spectrum of presentations ranging from BM to UCMD 8 14 21. Case 52, with merosin positive CMD and suspected limb girdle muscular dystrophy (LGMD), actually had a de novo variant of COL6A3 predicted to abolish a donor splice site function.…”

“…We also utilised the NGS for the diagnosis of mitochondrial disorders 11–13. Both targeted and whole exome NGS sequencing approaches have been tested for the molecular diagnosis of CMD and congenital myopathies in a small subset of patients 14 15…”

The Use of Autozygosity Mapping and Next-Generation Sequencing in Understanding Anterior Segment Defects Caused by an Abnormal Development of the Lens

“…Since data interpretation can be arduous, it is currently practical to target sequencing at specific regions of the genome -for example, all protein-coding regions (approx. 2% of the entire genome), as these contain the vast majority of variants underlying Mendelian disease [31,[34][35][36][37] , or a group of genes associated with a genetically heterogeneous condition [38][39][40][41][42][43][44] . The latter technique has proven extremely useful when applied to heterogeneous diseases of the eye [42,[45][46][47][48][49] .…”

Next-generation sequencing in childhood disorders

“…A recently published study used targeted NGS to sequence 34 autosomal recessive deafness genes, achieving a genetic diagnosis in 9/24 patients 39. Other examples include retinitis pigmentosa,24 38 40–42 Usher syndrome,43 44 inherited arrhythmias,45 congenital muscular dystrophy,46 mitochondrial diseases47 48 and ataxia 49…”