Next-generation sequencing in molecular diagnosis: NUBPL mutations highlight the challenges of variant detection and interpretation. Hum Mutat

Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.
Human Mutation (Impact Factor: 5.14). 02/2012; 33(2):411-8. DOI: 10.1002/humu.21654
Source: PubMed


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.

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    • "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). "
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