Complete ascertainment of intragenic copy number mutations (CNMs) in the CFTR gene and its implications for CNM formation at other autosomal loci

Abstract

Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2-3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript been routinely underascertained. Finally, our analysis of ±20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements.

Genes, mutations, and human inherited disease at the dawn of the age of personalized genomics

“…Conrad et al [2010] further noted that these estimates did not allow for purifying selection, and so they probably represent ''a lower bound on the true rate.'' There is also an ascertainment bias to contend with, duplications being significantly harder to identify than deletions [Quemener et al, 2010].…”

“…For most inherited disorders, the mutation detection rate is already fairly high (490%), although this success rate is often achieved by combining different mutation detection methodologies, for example, to screen for exon deletions and copy number variants as well as more subtle lesions [Quemener et al, 2010]. At least some of the ''missing lesions'' may nevertheless be found by screening extragenic functional elements.…”

Discovery of variants unmasked by hemizygous deletions

“…42,44 Intragenic deletions, encompassing one to several exons, account for 2-5% of the spectrum of deleterious mutations in Mendelian disorders. [7][8][9][10][11][12][13][14][15] In case an inherited or de novo intragenic deletion is combined with a deletion of the entire gene transmitted by a healthy parent no intact alleles of a particular gene will be found in the patient, which amounts to a recessive mode of gene action. In agreement with this inference is the finding that numerous patients within the MR/MCA spectrum carry novel or rare CNVs at two or even more loci.…”

“…5,6 The recent flurry of publications regarding losses of a single or several exons in selected genes by using targeted arrays underscore that current routine diagnostic methods may miss potentially pathogenic copy number variations (CNVs). [7][8][9][10][11][12][13][14][15] In this study, we investigated whether unmasking of recessive alleles may contribute to the phenotype of patients with idiopathic MR/ MCA by analyzing all coding sequences in hemizygous deletion regions of patients who inherited a novel deletion from a healthy parent. To do so, we performed multiplexed genomic enrichment and next-generation sequencing of the entire coding sequence of all genes in the deletion regions of 20 patients.…”

Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells

“…triplex, quadruplex, Z-DNA, cruciforms, slipped structures) at the breakpoint junctions of chromosomal alterations (gross deletions and duplications) associated with human genetic disease, including cystic fibrosis, mental retardation, and multiple congenital anomalies (1). These observations have served to extend the generality of previous work that aimed to elucidate the molecular mechanisms underlying recurrent translocations (2-6) and the genetic instability observed in many model systems (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), both of which were suggestive of a direct mutagenic role for non-B DNA.…”