Objective
Neuromuscular diseases (NMDs) are a group of >200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, ...resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests.
Methods
To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)‐based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD‐associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES).
Results
We found that NMD comprehensive panel testing has a 3‐fold greater diagnostic yield (46%) than single gene testing (15–19%). Sanger fill‐in of low‐coverage exons, copy number variation analysis, and thorough in‐house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES.
Interpretation
Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first‐tier approach. Ann Neurol 2015;77:206–214
Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale ...sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.
Gene sequencing panels are a powerful diagnostic tool for many clinical presentations associated with genetic disorders. Advances in DNA sequencing technology have made gene panels more economical, ...flexible, and efficient. Because the genes included on gene panels vary widely between laboratories in gene content (e.g., number, reason for inclusion, evidence level for gene–disease association) and technical completeness (e.g., depth of coverage), standards that address technical and clinical aspects of gene panels are needed. This document serves as a technical standard for laboratories designing, offering, and reporting gene panel testing. Although these principles can apply to multiple indications for genetic testing, the primary focus is on diagnostic gene panels (as opposed to carrier screening or predictive testing) with emphasis on technical considerations for the specific genes being tested. This technical standard specifically addresses the impact of gene panel content on clinical sensitivity, specificity, and validity—in the context of gene evidence for contribution to and strength of evidence for gene–disease association—as well as technical considerations such as sequencing limitations, presence of pseudogenes/gene families, mosaicism, transcript choice, detection of copy-number variants, reporting, and disclosure of assay limitations.
About half of people with trisomy 21 have a congenital heart defect (CHD), whereas the remainder have a structurally normal heart, demonstrating that trisomy 21 is a significant risk factor but is ...not causal for abnormal heart development. Atrioventricular septal defects (AVSD) are the most commonly occurring heart defects in Down syndrome (DS), and ∼65% of all AVSD is associated with DS. We used a candidate-gene approach among individuals with DS and complete AVSD (cases = 141) and DS with no CHD (controls = 141) to determine whether rare genetic variants in genes involved in atrioventricular valvuloseptal morphogenesis contribute to AVSD in this sensitized population. We found a significant excess (p < 0.0001) of variants predicted to be deleterious in cases compared to controls. At the most stringent level of filtering, we found potentially damaging variants in nearly 20% of cases but fewer than 3% of controls. The variants with the highest probability of being damaging in cases only were found in six genes: COL6A1, COL6A2, CRELD1, FBLN2, FRZB, and GATA5. Several of the case-specific variants were recurrent in unrelated individuals, occurring in 10% of cases studied. No variants with an equal probability of being damaging were found in controls, demonstrating a highly specific association with AVSD. Of note, all of these genes are in the VEGF-A pathway, even though the candidate genes analyzed in this study represented numerous biochemical and developmental pathways, suggesting that rare variants in the VEGF-A pathway might contribute to the genetic underpinnings of AVSD in humans.
Accurate interpretation of DNA sequence variation is a prerequisite for implementing personalized medicine. Discrepancies in interpretation between testing laboratories impede the effective use of ...genetic test results in clinical medicine. To better understand the underpinnings of these discrepancies, we quantified differences in variant classification internally over time and those between our diagnostic laboratory and other laboratories and resources. We assessed the factors that contribute to these discrepancies and those that facilitate their resolution. Our process resolved 72% of nearly 300 discrepancies between pairs of laboratories to within a one-step classification difference and identified key sources of data that facilitate changes in variant interpretation. The identification and harmonization of variant discrepancies will maximize the clinical use of genetic information; these processes will be fostered by the accumulation of additional population data as well as the sharing of data between diagnostic laboratories.
ABSTRACT
Revolutionary changes in sequencing technology and the desire to develop therapeutics for rare diseases have led to the generation of an enormous amount of genomic data in the last 5 years. ...Large‐scale sequencing done in both research and diagnostic laboratories has linked many new genes to rare diseases, but has also generated a number of variants that we cannot interpret today. It is clear that we remain a long way from a complete understanding of the genomic variation in the human genome and its association with human health and disease. Recent studies identified susceptibility markers to infectious diseases and also the contribution of rare variants to complex diseases in different populations. The sequencing revolution has also led to the creation of a large number of databases that act as “keepers” of data, and in many cases give an interpretation of the effect of the variant. This interpretation is based on reports in the literature, prediction models, and in some cases is accompanied by functional evidence. As we move toward the practice of genomic medicine, and consider its place in “personalized medicine,” it is time to ask ourselves how we can aggregate this wealth of data into a single database for multiple users with different goals.
The ability of a single technology, next-generation sequencing, to provide both sequence and copy number variant (CNV) results has driven the merger of clinical cytogenetics and molecular genetics. ...Consequently, the distinction between the definition of a sequence variant and a CNV is blurry. As the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for interpretation of sequence variants address CNV classification only sparingly, this study focused on adapting ACMG/AMP criteria for single-gene CNV interpretation.
CNV-specific modifications of the 2015 ACMG/AMP criteria were developed and their utility was independently tested by three diagnostic laboratories. Each laboratory team interpreted the same 12 single-gene CNVs using three systems: (1) without ACMG/AMP guidance, (2) with ACMG/AMP criteria, and (3) with new modifications. A replication study of 12 different CNVs validated the modified criteria.
The adapted criteria system presented here showed improved concordance and usability for single-gene CNVs compared with using the ACMG/AMP interpretation guidelines focused on sequence variants.
These single-gene CNV criteria modifications could be used as a supplement to the ACMG/AMP guidelines for sequence variants, allowing for a streamlined workflow and a step toward a uniform classification system for both sequence and copy number alterations.
Clinical genetics laboratories have recently adopted guidelines for the interpretation of sequence variants set by the American College of Medical Genetics (ACMG) and Association for Molecular ...Pathology (AMP). The use of in silico algorithms to predict whether amino acid substitutions result in human disease is inconsistent across clinical laboratories. The clinical genetics community must carefully consider how in silico predictions can be incorporated into variant interpretation in clinical practice.Please see related Research article: https://doi.org/10.1186/s13059-017-1353-5.
In the last decade, there has been a flood of new technology in the sequencing arena. The onset of next-generation sequencing (NGS) technology has resulted in the vast increase in genetic diagnostic ...testing available to the ordering physician. Whole exome sequencing (WES) has become available as a diagnostic test performed in certified clinical laboratories. This has led to increased presence in the diagnostic marketplace, increased consumer awareness, and the question has been raised by various stakeholders to whether there is sufficient stringent regulation of WES and other NGS-based tests. We discuss the various WES services currently available in the marketplace, current regulation of WES as a laboratory developed test, the proposed FDA involvement in its oversight as well as the response of various laboratory groups that provide these diagnostic services. Overall, a rigorous process oversight and assessment of inter-lab reproducibility is strongly warranted for WES as it is used as a diagnostic test, but regulation should be mindful of the excessive administrative burden on academic and smaller diagnostic laboratories.
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