Raeesa Gupte, PhD
Conventional diagnosis of neurological disorders involves a battery of neuropsychological and biochemical tests followed by techniques such as electroencephalography, electromyography, and non-invasive CT, MRI, or PET imaging. Many of these tests and techniques are cumbersome, highly variable, time consuming, and expensive, and despite extensive testing, the genetic etiology of complex neurological disorders often remains undiagnosed.
The advent of next-generation sequencing (NGS) technology has led to exponential growth of information about the genetic underpinnings of neurological disorders. As opposed to conventional Sanger sequencing that sequences a single DNA fragment at a time, NGS allows multiple parallel sequencing of millions of DNA fragments simultaneously. The high throughput and accuracy of NGS makes it suitable for use as a clinical diagnostic for neurological diseases with complex phenotypes attributed to multiple genes.
Accurate diagnosis with targeted panel sequencing
In targeted sequencing, specific genes or regions known to be associated with a disease or phenotype are sequenced. It requires target enrichment wherein disease-focused genes are captured or amplified, followed by massive parallel sequencing. Multigene panels are most suitable for disorders that have a characteristic phenotype associated with a couple hundred gene variants with a high potential to cause disease. Targeted NGS panels for neuromuscular disorders, Charcot-Marie-Tooth disease, ataxia, epilepsy, myopathies, and neuropathies are available commercially and custom-designed for use in clinical research.
NGS panels may include 50 to 300 genes with 30x to 400x coverage depth. Coverage depth is an important consideration in the clinical setting since high coverage depth indicates more accurate variant identification and greater confidence in the sequencing approach. Targeted panels allow clinical laboratories to test multiple genetic markers with short turnaround times while using small amounts of patient samples. By limiting screening to known causal genes, targeted sequencing panels keep costs low, simplify clinical reports by decreasing the number of variants of unknown significance, and avoid ethical dilemmas related to accidental discoveries not related to the disease category.
Currently, targeted multigene panels are the most common NGS approaches used in clinical laboratories. However, focusing on a limited number of genes is both a strength and a drawback of this approach. Based on the disorder and the number of genes screened in the panel, the diagnostic yield of targeted sequencing panels varies widely. For example, the diagnostic yield for epilepsy gene panels ranged from 10 percent to 50 percent, and for neuromuscular disorders ranged from 4 percent to 33 percent. Therefore, sequence variations in known genes explain only a fraction of the genetic causes underlying neurological disorders while overlooking the contribution of novel genes. Furthermore, inclusion of additional genetic markers in a previously validated panel may alter its overall performance. That makes redesign and revalidation significant challenges when novel variants are discovered and existing panels need to be upgraded.
Whole exome sequencing for highly heterogeneous diseases
The exome encompasses the protein-coding regions of the genome. Defects in these protein-encoding regions are the genetic cause of a majority of neurological disorders. Whole exome sequencing (WES) covers approximately 1.5 percent of the human genome, totaling about 50 million base pairs. WES is a promising approach in cases where targeted multigene panels have failed to identify a disease-causing variant. Additionally, it can be used to identify the genetic cause of diseases with high clinical heterogeneity including intellectual disability, autism spectrum disorders, idiopathic neuropathies, and multiple congenital abnormalities. It has been used to identify novel rare variants involved in the pathogenesis of Parkinson’s disease, multiple sclerosis, epilepsy, and early onset Alzheimer’s disease.
Despite the name, state of the art WES techniques cannot sequence the entire exome. Due to variability in sample enrichment during exon capture, the average coverage depth of WES is about 20x. However, only 85 to 90 percent of all coding exons get the 20x depth required for clinical confidence. For instance, a study on idiopathic peripheral neuropathy using WES reported that 7 percent of exons of known neuropathy genes had less than 10x coverage and 2 percent were not covered at all. Another drawback to WES is that it requires considerable infrastructure for bioinformatic analysis and data storage, leading to higher costs. Since bioinformatic workflows and criteria for identifying novel pathogenic variants are not standardized across laboratories, the same NGS raw data may identify different variants. Lastly, WES may identify unexpected sequence variations not related to the disease under investigation, raising ethical issues about risk communication.
Whole genome sequencing identifies coding and non-coding variants
Although sequencing of protein-coding exons has improved the diagnostic yield of neurological disorders with Mendelian inheritance, other complex disorders remain undiagnosed. This is because a majority of genetic mutations occur in intronic non-protein coding regions of the genome. Such mutations may influence regulatory elements and gene promoters involved in disease susceptibility. Whole genome sequencing (WGS) can be used to identify both coding and non-coding variants that cause or increase the risk of neuropsychiatric disorders. Recently, the largest known WGS study of patients with schizophrenia identified ultra-rare variants in a genome structure that controls interactions between genes and regulatory elements.
Due to its prohibitive cost, WGS is not commonly used as a clinical diagnostic. As NGS continues to become faster and cheaper, WGS may be used to predict disease risk. However, limitations of WES, including massive bioinformatic processing power, high number of variants of unknown significance, and ethical concerns for reporting incidental disease risk are also shared by WGS.
Transforming diagnosis and treatment of neurological disorders
Identifying the genetic cause of a disease averts the use of improper therapies and additional invasive or expensive diagnostic testing. Whole-exome and whole-genome sequencing are used in clinical research to identify novel disease-causing variants in protein-coding and non-coding DNA. The diagnostic potential of these techniques is limited by high costs and data processing power.
Commercial and custom-designed multigene sequencing panels are increasingly used by clinical diagnostic laboratories. Used along with traditional diagnostic techniques, targeted NGS panels can help improve the accuracy of neurological diagnoses and ensure therapeutic success. When used early in the diagnostic process, NGS panels can keep costs low by preventing the use of excessive diagnostic tests. Thus, rapid advances in NGS and bioinformatics have the potential to transform the diagnosis and treatment of neurological disorders.