Based on the analysis of the most frequent mutations responsible for cystic fibrosis (CF), a higher than expected frequency of CF mutations was recently reported in men with infertility due to reduced sperm quality. To further document whether this condition is associated with severe or mild abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) functions, we carried out a complete scanning of CFTR sequences using a strategy that detects almost all 850 mutations and 150 polymorphisms reported to date in the CFTR gene. We have investigated a cohort of 56 patients with severe oligoasthenoteratozoospermia (OAT) and 50 controls from southern France for CFTR gene mutations and variations. The frequencies of CF-causing mutations and CFTR variations identified in this OAT sample did not differ significantly from the frequencies found in the normal population. However, we observed a 1.7-fold increase in the proportion of homozygotes for a specific CFTR haplotype (TG11-T7-G1540) in the OAT group (P = 0.025). Our results do not confirm a link between CF mutations and reduced sperm quality. Further studies are needed to substantiate the hypothesis that a combination of variants affecting expression and function of the CFTR protein is associated with male infertility.
Despite the exhaustive screening of F7 gene exons and exon-intron boundaries and promoter region, a significant proportion of mutated alleles remains unidentified in patients with coagulation factor VII deficiency. Here, we applied next-generation sequencing to 13 FVII-deficient patients displaying genotype-phenotype discrepancies upon conventional sequencing, and identified six rare intronic variants. Computational analysis predicted splicing effects for three of them, which would strengthen (c.571+78G>A; c.806-329G>A) or create (c.572-392C>G) intronic 5′ splice sites (5′ss). In F7 minigene assays, the c.806-329G>A was ineffective while the c.571+78G>A change led to usage of the +79 cryptic 5′ss with only trace levels of correct transcripts (3% of wild-type), in accordance with factor VII activity levels in homozygotes (1-3% of normal). The c.572-392C>G change led to pseudo-exonization and frame-shift, but also substantial levels of correct transcripts (approx. 70%). However, this variant was associated with the common F7 polymorphic haplotype, predicted to further decrease factor VII levels; this provided some kind of explanation for the 10% factor VII levels in the homozygous patient. Intriguingly, the effect of the c.571+78G>A and c.572-392C>G changes, and particularly of the former (the most severe and well-represented in our cohort), was counteracted by antisense U7snRNA variants targeting the intronic 5′ss, thus demonstrating their pathogenic role. In conclusion, the combination of next-generation sequencing of the entire F7 gene with the minigene expression studies elucidated the molecular bases of factor VII deficiency in 10 of 13 patients, thus improving diagnosis and genetic counseling. It also provided a potential therapeutic approach based on antisense molecules that has been successfully exploited in other disorders.
Editor—In the February 1998 issue of the Journal, Verlingue et al 1reported an absence of mutations in the promoter region of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. They analysed a region that spans over 3.9 kb of sequences upstream of the first CFTR exon, including the CFTR promoter, down to 1.3 kb within the first intron. These sequences, shown previously to contain potential regulatory elements,2-4 had been selected on the basis of conservation throughout evolution (phylogenetic footprints) from rodents to primates.2 3 Verlingue et al 1 analysed a cohort of 205 subjects including patients with classical cystic fibrosis (CF), disseminated bronchiectasis, or congenital bilateral absence of the vas deferens (CBAVD), carrying either one or no mutation after scanning all 27 CFTR exons by DGGE (denaturing gradient gel electrophoresis). They further screened 5.2 kb of targeted sequences spanning the CFTR promoter region, but were unable to detect any putative disease related mutation in their sample.
We report the first three nucleotide alterations in the CFTR minimal promoter, defined as a 250 bp fragment …
