Large genomic rearrangements in the CFTR gene contribute to CBAVD

Abstract

Background: 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). 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.

Methods: We 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.

Results: We 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.

Conclusion: Identification 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.

Figure 1

A) Confirmation of complex deletion c.54-5811_c.164+2186del8108ins182 [CFTRdele2] in CBAVD. Rearrangement c.54-5811_c.164+2186del8108ins182 (upper panel) consists of a gross deletion of 8108 bp spanning exon 2 (dotted area) and an insertion of 182 bp (grey area) at the deletion junction between the nucleotide 54-5811 of intron 1 (IVS1 nt 18294) and the nucleotide 164+2186 of intron 2 (IVS2 nt 2186) [according to the recommended international nomenclature with the A of the ATG start codon numbered as +1]. The 182-bp insertion is part of intron 3 between IVS3 nucleotides 6780 and 6961 but in inverted orientation (arrow). This complex in/del is also termed c.186-5811_c.296+2186del8108+ins182 with the A of the ATG translation start codon numbered as +133 in accordance with the GenBank reference sequence for the CFTR gene on chromosome 7 (NM_000492.2) and the CF mutation database [4]. It was recently reported as IVS1-5811_IVS2+2186del8108ins182 in one patient with cystic fibrosis [27]. A specifically-designed junction fragment amplification test (lower panel) confirmed the presence of the heterozygous deletion, indicated by specific PCR products on 2% agarose gels (left) by comparison with the non deleted allele. The deletion breakpoint junctions (indicated by vertical bars) and inserted sequences were determined by direct sequencing (right). B) Confirmation of complex deletion c.3964-3890_c.4443+3143del9454ins5 [CFTRdele22_24 or CFTRdele25_27] in CBAVD. Rearrangement c.3964-3890_c.4443+3143del9454ins5 (upper panel) consists of a gross deletion of 9454 bp (dotted area) encompassing exons 25 to 27 [22 to 24], the stop codon and the poly(A) signal. A small insertion of 5 bases (TAACT) was observed at the junction but is too small to allow any determination of its origin (arrow). The same rearrangement has recently been described in one patient with cystic fibrosis as c.4096-3890_c.4575+3143del9454ins5 with the A of the ATG translation start codon numbered as +133 in accordance with the GenBank reference sequence for the CFTR gene on chromosome 7 (NM_000492.2) or as IVS21-3890Stop+3143del9454insTAACT [15]. A specifically-designed junction fragment amplification test (lower panel) confirmed the presence of the heterozygous deletion, indicated by specific PCR products on 2% agarose gels (left) by comparison with the non deleted allele. The deletion breakpoint junctions (indicated by vertical bars) and inserted sequences were determined by direct sequencing (right).

Figure 2

Exon phasing of the CFTR gene (Universal Mutation Database [25]). The CFTR gene comprises 27 exons which were previously numbered 1–24 [23] with subdivisions A and B for exons 6, 14 and 17, recognized as distinct units after the initial publication of the gene (GenBank NM_000492.2). Here we presents the numerotation of CFTR exons as described in UCSC (University of California Santa Cruz) Human Genome databases and updated as March 2006 Assembly [24]. Each exon is presented as a blue box with the international numbering within the box, and the familiar numbering above it. Each extremity of the box represents the specific phasing of the exon. Left end of exons: 1) vertical extremity: the exon begins by the first nucleotide of a codon; 2) curve: the exon begins by the second nucleotide of a codon; 3) arrow: the exon begins by the third nuceotide of a codon. Right end of exons: 1) vertical extremity: the exon ends by the last nucleotide of a codon; 2) light blue curve: the exon ends by the first nucleotide of a codon; 3) arrow: the exon ends by the second nucleotide of a codon. Large-scale deletions can introduce a translational frameshift (and lead to a premature termination codon) when two exons are joined end to end (for example, exons 1 and 4). When two exons in the same phase are joined, it is assumed that no frameshift occurs and that a shorter protein with an internal deletion may be produced.