Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort

Abstract

Original research article INTRODUCTIONSignificant advances in copy-number detection have broadened the mutation spectrum for many clinical genetic disorders. 1,2 Intragenic deletion mutations are of considerable frequency in many disease genes, such as PAX6, CDKL5, and STXPB1. Recurrent rearrangements between segmentally duplicated sequences are also associated with a number of syndromic disorders. 3 For these known disorders, targeted gene testing by multiplex ligation-dependent amplification or exon-focused arrays has been useful. With the increasing uptake of exome sequencing into the clinical diagnostic approach, the need for testing previously uncharacterized genes for pathogenic copynumber variation (CNV) is a significant consideration, not only to detect aberrations in genes that may cause disease when haplo-insufficient but also in genes associated with recessive disorders for which the mutation has been identified in only one of the alleles by exome sequencing. 4 Whereas exome sequencing is still gaining popularity as a powerful clinical tool, whole-genome chromosomal microarray analysis (CMA) has become an indispensable screening method that is now routinely used as a first-tier test for children with intellectual disability, developmental delay, or congenital anomalies. 5 In less than 10 years, the CMA designs have evolved from low-resolution arrays containing large bacterial artificial chromosome clones or <100,000 oligonucleotide probes to high-resolution versions with more than 1 million probes. 6 As a result, several groups have identified single-gene pathogenic aberrations that boost the analytical sensitivity of CMA. 7 However, although some of these more recent arrays have higher density at disease genes, they do not all cover every exon in those genes and are therefore not capable of detecting some pathogenic intragenic mutations. Separately, data from exon-focused arrays have shown that up to 40% of intragenic mutations can involve just one or two exons within a gene, and therefore it is essential to cover all exons within targeted genes. 1 Copy-number detection in clinical genetic testing eventually will occur entirely through examination of next-generation data, whereas array comparative genomic hybridization (aCGH) and other assays will serve as complementary and confirmatory methods. 8 To complement whole-exome sequencing (WES) or whole-genome sequencing data in a meaningful way, an array with coverage of virtually all exons is essential. Until the time that WES/whole-genome sequencing can be used routinely and reliably for copy-number detection, a whole-exome array can be used as the ultimate whole-genome CMA platform. Purpose: Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its ut...

Clinical application of whole-exome sequencing across clinical indications

“…Copy-number variants (CNVs) were also called from the WES data using a relative coverage method we have described previously. 17 All potentially diagnostic UPD and CNV results were confirmed before reporting by an appropriate orthogonal measure, such as chromosome microarray, exon aCGH, multiple ligation-dependent probe amplification, Sanger sequencing, or qPCR.…”

Mutations in DDX3X Are a Common Cause of Unexplained Intellectual Disability with Gender-Specific Effects on Wnt Signaling

“…Exome sequencing and data analysis were performed essentially as previously described, 10 and sequencing was performed in the probands and their unaffected parents (trio approach). 11 To replicate these findings, we examined a second cohort of 957 individuals (543 males, 414 females) with intellectual disability or developmental delay from GeneDx (sequencing methods as previously published 12 ) and a third cohort of 4,295 individuals with developmental disorders (2,409 males, 1,886 females) from the Deciphering Developmental Disorders (DDD) study (UK). 13 In most individuals in these cohorts, a SNP array or array CGH had been performed as well and, based on the clinical findings, in several individuals additional metabolic testing, Fragile X testing, or targeted Sanger analysis of different genes associated with ID was completed previously.…”

“…Our data also suggest that the genetic architecture of DDX3X-mediated pathology in males is different. All 7,9,23,24,26), a wide nasal bridge and/or bulbous nasal tip (e.g., individuals 11,13,15,16), narrow alae nasi and/or anteverted nostrils (e.g., individuals 2, 8,9,12,14,18,24,27,32,35), and hypertelorism (e.g., individuals 5,7,8,20,27). Informed consent was obtained for all 30 individuals shown.…”

Next generation sequencing‐based copy number analysis reveals low prevalence of deletions and duplications in 46 genes associated with genetic cardiomyopathies

“…2013; Retterer et al. 2014). Emerging data show that overall, intragenic deletions or duplications constitute an important portion of the so‐called missing variation.…”