Abstract
BackgroundBy performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole CFTR locus in the 32 CBAVD patients with only one or no mutation.MethodsWe developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing.ResultsWe detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or CFTRdele2], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or CFTRdele 22_24], in two males carrying a typical CBAVD mutation on the other parental CFTR allele. We present the first bioinformatic tool for exon phasing of the CFTR gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame.ConclusionIdentification of large rearrangements further expands the CFTR mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.
Highlights
By performing extensive scanning of whole coding and flanking sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD)
In the current study we present the result of our investigations concerning the involvement of large rearrangements in the 32 remaining samples with one or no CFTR small mutation
DNA sequencing showed that deletions breakpoint junctions appeared to be the same as those recently described in two patients with cystic fibrosis as IVS1-5811_IVS2+2186del8108ins182 [27] or
Summary
By performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). In about 85% of cases, CBAVD is recognized as an autosomal recessive disorder (MIM≠277180) associated with mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR, symbolized ABCC7) [1,2,3]. Routine testing for the most prevalent mutations in classical CF misses most CFTR gene alterations in the CBAVD phenotype, which can be detected only by scanning the 27 CFTR coding and flanking sequences [8,9]. Despite exhaustive analysis of the CFTR gene, a proportion of mutations still remains unidentified in CBAVD, ranging from 15 to 40% depending both on the technologies used and the ethnicity of patients
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