Next-generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an individual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild-type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch-site mutation in addition to the missense mutation. We demonstrate that the branch-site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus-specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow-up to confirm mutation pathogenicity.
"RNA studies may be used to further validate and confirm the consequences of a splice site variant. Examples of other pathogenic non-coding or splice site variants include a GATA2 intron 5 self-regulating binding site mutation in MonoMAC syndrome (Hsu et al. 2013); a TERT promoter mutation in melanoma (Horn et al. 2013); a variant in the 3'UTR of HDAC6 in dominant X-linked chondrodysplasia, affecting miRNA-mediated posttranscriptional regulation (Simon et al. 2010); and a pathogenic branch-site mutation in NUBPL in a patient with mitochondrial complex I deficiency (Tucker et al. 2012). "
[Show abstract][Hide abstract] ABSTRACT: Massively parallel sequencing (MPS) has become a powerful tool for the clinical management of patients with applications in diagnosis, guidance of treatment, prediction of drug response, and carrier screening. A considerable challenge for the clinical implementation of these technologies is the management of the vast amount of sequence data generated, in particular the annotation and clinical interpretation of genomic variants. Here, we describe annotation steps that can be automated and common strategies employed for variant prioritization. The definition of best practice standards for variant annotation and prioritization is still ongoing; at present, there is limited consensus regarding an optimal clinical sequencing pipeline. We provide considerations to help define these. For the first time, clinical genetics and genomics is not limited by our ability to sequence, but our ability to clinically interpret and use genomic information in health management. We argue that the development of standardised variant annotation and interpretation approaches and software tools implementing these warrants further support. As we gain a better understanding of the significance of genomic variation through research, patients will be able to benefit from the full scope that these technologies offer. This article is protected by copyright. All rights reserved.
Human Mutation 04/2014; 35(4). DOI:10.1002/humu.22525 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing has become a powerful tool for testing genetically and clinically heterogeneous conditions such as mitochondrial disorders. A recent study published in Science Translational Medicine underscores the considerable clinical benefits of targeted next-generation sequencing for the diagnosis of mitochondrial disorders. The findings also suggest that the genetic heterogeneity that can result in mitochondrial disease appears much broader than previously thought.
Genome Medicine 03/2012; 4(3):22. DOI:10.1186/gm321 · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The current diagnostic approach for mitochondrial disorders requires invasive procedures such as muscle biopsy and multiple biochemical testing but the results are often inconclusive. Clinical sequencing tests are available only for a limited number of genes. Recently, massively parallel sequencing has become a powerful tool for testing genetically heterogeneous conditions such as mitochondrial disorders.
Methods: Targeted next-generation sequencing was performed on 26 patients with known or suspected mitochondrial disorders using in-solution capture for the exons of 908 known and candidate nuclear genes and an Illumina genome analyzer.
Results: None of the 18 patients with various abnormal respiratory chain complex (RCC) activities had molecular defects in either subunits or assembly factors of mitochondrial RCC enzymes except a reference control sample with known mutations in SURF1. Instead, several variants in known pathogenic genes including CPT2, POLG, PDSS1, UBE3A, SDHD, and a few potentially pathogenic variants in candidate genes such as MTO1 or SCL7A13 were identified.
Conclusions: Sequencing only nuclear genes for RCC subunits and assembly factors may not provide the diagnostic answers for suspected patients with mitochondrial disorders. The present findings indicate that the diagnostic spectrum of mitochondrial disorders is much broader than previously thought, which could potentially lead to misdiagnosis and/or inappropriate treatment. Overall analytic sensitivity and precision appear acceptable for clinical testing. Despite the limitations in finding mutations in all patients, the present findings underscore the considerable clinical benefits of targeted next-generation sequencing and serve as a prototype for extending the clinical evaluation in this clinically heterogeneous patient group.
Pediatrics International 04/2012; 54(5):585-601. DOI:10.1111/j.1442-200X.2012.03644.x · 0.73 Impact Factor
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