Next-generation sequencing still needs our generation's clinicians

In this issue of Neurology Genetics, Tian et al report on a robust and comprehensive NGS-based panel testing for 236 neuromuscular-related genes. They report that this targeted capture approach resulted in an excellent yield, identifying deleterious mutations in 29 of 35 families (83%) studied in their cohort of patients with clinically heterogeneous neuromuscular diseases, including congenital myopathies, limb-girdle muscular dystrophies (LGMDs), Charcot-Marie-Tooth (CMT) disease, metabolic myopathies, myotonic syndromes, and ion channel diseases. They considered the diagnosis to be definitive in 21 families (60%) and likely in an additional 8 families (23%). The authors correctly point out that the strength of their NGS and capture approach is the excellent coverage (with a mean depth of approximately 1,000-fold) and the considerable number of genes targeted in parallel...

It is essential to have a solid understanding of the extended phenotype (which may include histology, physiology, or imaging) before ordering genetic testing not only to ensure that the appropriate panel is ordered but especially to recognize those conditions for which NGS sequencing is unable to reliably identify the pathogenic genetic cause. In fact, this is the case for the most common genetic neuromuscular disorders, including trinucleotide repeat expansions (myotonic dystrophy, Friedreich ataxia), deletions, duplications, or, in particular, “copy number neutral” inversions of multiple exons—as NGS is now getting better at detecting larger deletions or duplications (Duchenne/Becker muscular dystrophy)—deletions situated in highly similar duplicated genomic regions (spinal muscular atrophy), contractions or duplications of genomic regions (facioscapulohumeral muscular dystrophy and CMT type 1A), abnormal methylation (Prader-Willi syndrome, an important differential diagnosis in the hypotonic infant), and chromosomal disorders (molecular karyotype by array comparative genomic hybridization may be better here). For this reason alone, deep phenotyping and a hierarchical differential diagnosis are imperative when selecting the genetic testing methodology appropriate for an individual patient.

NGS, like every form of genetic testing, not only requires such careful “pre-testing” phenotyping but also requires an equally careful “post-testing” analysis. The post-testing challenges for the clinician arise from interpreting the results reported by the genetic laboratory. Using this approach of “pre-test” deep phenotyping, NGS, and “post-test” clinical plausibility checking, the potential outcomes of NGS for the clinician include (1) genetic diagnosis confirmed, as suspected; (2) genetic diagnosis confirmed, but not as suspected; (3) incomplete genetic diagnosis in a highly suspected gene, such as a missing second pathogenic allele in a recessive disease; (4) genetic variants of unknown significance (VUS) in more than 1 gene, which are possibly causative; and (5) no genetic diagnosis achieved. For the purpose of this discussion, we will not consider the equally vexing problem of the identification of unexpected secondary findings on WES or WGS that are unrelated to the patient's underlying clinical diagnosis but may have significant implications for future medical management...

When considering scenario 3 (potentially “missing” mutation), it is helpful to realize that in contrast to WES, NGS-based panels typically cover all genes included in the panel at superior depth. Deletion/duplication testing (to assess for copy number variations) can supplement NGS, thus decreasing the risk of “missed” mutations. Coverage for WES may be more incomplete, with only 80%–85% of the exome captured and at a lower depth...

One of the main hurdles in broad panel-based testing, especially in WES and WGS, is scenario 4, the interpretation of a large number of sequence VUS, which can be incredibly time consuming and may result in incorrectly assigned pathogenicity or missed causative variants. It is the laboratory's responsibility to help evaluate any variants found with regard to their predicted pathogenicity (based on allele frequency, in silico analysis tools, data in published reports and databases, and tools for predicting the functional effects of amino acid substitutions such as PolyPhen and SIFT, as detailed nicely in Tian et al.). Given the number of sequence variants reported by WES, it is essential for the clinician to have strategies in place for sorting through the variants reported by the genetic laboratory as potentially deleterious. This list will be narrowed by the laboratory to initially include only those with disease association; however, the likely still considerable number of variants can be more successfully analyzed with a hypothesis-driven condensed candidate gene list in mind, based on a hierarchical clinical diagnosis of a well-phenotyped patient.

A. Reghan Foley, MD, Sandra Donkervoort, MS, CGC and Carsten G. Bönnemann, MD.  Next-generation sequencing still needs our generation's clinicians  Neurol Genet August 2015  Published online August 13, 2015.