Next-generation sequencing in neuromuscular diseases

Abstract

Purpose of review: Neuromuscular diseases are clinically and genetically heterogeneous and probably contain the greatest proportion of causative Mendelian defects than any other group of conditions. These disorders affect muscle and/or nerves with neonatal, childhood or adulthood onset, with significant disability and early mortality. Along with heterogeneity, unidentified and often very large genes require complementary and comprehensive methods in routine molecular diagnosis. Inevitably, this leads to increased diagnostic delays and challenges in the interpretation of genetic variants.

Recent findings: The application of next-generation sequencing, as a research and diagnostic strategy, has made significant progress into solving many of these problems. The analysis of these data is by no means simple, and the clinical input is essential to interpret results.

Summary: In this review, we describe using examples the recent advances in the genetic diagnosis of neuromuscular disorders, in research and clinical practice and the latest developments that are underway in next-generation sequencing. We also discuss the latest collaborative initiatives such as the Genomics England (Department of Health, UK) genome sequencing project that combine rare disease clinical phenotyping with genomics, with the aim of defining the vast majority of rare disease genes in patients as well as modifying risks and pharmacogenomics factors.

Figure 1A. A timeline depicting the key events in the history of genomics

Genetic research and the involvement of new technologies have played a major role in the increase of the amount of DNA sequence generated per person per year at a greater cost efficiency. From the discovery of DNA in 1870, to the introduction of chain terminators to DNA sequencing and separation of DNA tracts by polyacrylamide gel electrophoresis (PAGE) through to the implementation of whole exome (WES) and whole genome sequencing (WGS) in diagnostic and clinical research. Without the integration of the DNA sequencing chemistry and DNA cloning, the sequencing of whole genomes would not be possible today. The innovation of laser detection systems and the progress in computational hardware and analytical software have greatly accelerated the speed of DNA sequencing, as well as its cost and labour needs.

Figure 1B

Schematic representation comparing the ease of analysis compared to the amount of genomic data generated per run.

Figure 2. Filtering and functional analysis technique for whole exome sequencing

Data analysis of exomes involves the alignment of sequences, removing duplicates and annotating the data sequence and variants. When investigating disease-causing mutations, common polymorphisms and non-coding amino acid changes are removed to leave a list of unique or very rare (<0.01% of the population) variants that are either heterozygous or homozygous.

Figure 3. An example of exome sequencing filtering strategy in a recessive family

The analysis of multiple family members is often very important in proving a genetic variant is pathogenic. A. Exome filtering of variants. B. Family tree and C. Exome Viewer visualising the mutation.

Figure 4. A deletion in exon 7 of the (Periaxin) PRX gene, investigated by a gene panel and visualized with an interactive software

In the top panel, the patient has part of the gene deleted (indicated by a circle) which is determined by the greatly reduced coverage depth of that region (red). The bottom panel shows a healthy control patient which has a uniform coverage depth throughout exon 7 of PRX gene.

Figure 5

A timeline of discovery of genes involved in Charcot-Marie-Tooth disease. In the early 2000’s linkage analysis and Sanger sequencing is widespread but requires larger families. After 2009/2010 the advent of next generation sequencing has allowed new disease genes to be identified in smaller and rarer families. X axis = number of genes, Y axis = year.

Figure 6. Genomics England and Genomics England Clinical Interpretation Partnership (GeCIPs) flow diagram

The major objectives of the Neurology and Neurodegeneration research plan are to advance and optimise whole genome sequencing validation, interpretation and interrogation. This will significantly accelerate our understanding of disease pathophysiology and key mechanistic pathways enabling the future development of new therapies. Specific aims include:

1. Development of data capture and similarity scoring algorithms to delineate rare and severe neurological and neurodegenerative disorders into homogeneous phenotypic groups.

2. Identification of novel disease genes, genetic risks and modifying factors to enable comprehensive clinical diagnostic testing, prediction of disease onset and severity.

3. Gene discovery will lead to the identification of novel pathways that will be investigated and modeled to advance our understanding of disease pathophysiology and mechanisms.

4. Training to develop the next generation of NHS technologists, scientists and clinicians in genomic medicine to sustain a thriving effective team for the future.

5. Future collaboration with industry to utilise pathway discovery and identify novel series of medicines, vaccines, and pharmacogenomics to deliver precision patient treatments based on genomics.