Variants in CFTR untranslated regions are associated with congenital bilateral absence of the vas deferens.

BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD), a frequent cause of obstructive azoospermia, is generated by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite extensive testing for point mutations and large rearrangements, a small proportion of alleles still remains unidentified in CBAVD patients. METHODS AND RESULTS: Mutation scanning analysis of microsatellite variability in the CFTR gene identified two undescribed 4 bp sequence repeats (TAAA)(6) and (TAAA)(8) in intron 9 in two CBAVD patients heterozygote for either the -33G→A promoter transition or the classical [TG12T5] CBAVD mutation. This study explores the putative impact of this promoter variant by using a combination of web based prediction tools, reporter gene assays, and DNA/proteins interaction analyses. Results of transiently transfected vas deferens cells with either the -33G wild-type or the -33A variant CFTR directed luciferase reporter gene confirmed that the -33A variant, which alters the FOXI1 (Forkhead box I1) binding, significantly decreases the CFTR promoter activity. It was also investigated whether regulatory elements located within the intronic tetrarepeat might influence the CFTR expression. There was evidence that both the (TAAA)(6) and the (TAAA)(8) alleles modulate the CFTR transcription and the binding affinity for FOX transcription factors, involved in the chromatin architecture. CONCLUSIONS: As the vas deferens seems to be one of the tissues most susceptible to a reduction in the normal CFTR transcripts levels, and as two mild mutations are sufficient to induce CBAVD phenotype, these findings raise the possibility that these uncommon variants may be a novel cause of CBAVD.

WGA allows the molecular characterization of a novel large CFTR rearrangement in a black South African cystic fibrosis patient.

By performing extensive scanning of whole coding and flanking sequences of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, we had previously identified the CF-causing mutations in black South African patients of different ethnic groups suspected with the disease. Of ten samples analyzed, there were six remaining that had either one (n = 2) or two (n = 4) unidentified CFTR alleles that have now been tested for large rearrangements using a semiquantitative fluorescent PCR assay. A novel deletion encompassing CFTR exon 2 was detected in one patient who was heterozygous for the mutation 3120+1G>A. The Caucasian deletion involving the same exon [c.54-5811_c.164+2186del8108ins182] was ruled out. The DNA had been stored for more than 12 years and only minute quantities remained. We thus used a whole-genome amplification method based on multiple displacement amplification to generate sufficient amounts of DNA to characterize the intronic breakpoints and identify the deletion at the genomic level. Mapping and sequencing the breakpoint junctions revealed a novel large deletion [c.54-1161_c.164+1603del2875]. We have designed a simple test to specifically detect the presence or absence of this large rearrangement. This study reports the first large CFTR rearrangement in a black South African CF patient, further defining the molecular spectrum of CF that will be useful for improving genetic testing and counseling in this region.

Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders.

Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis.

Large genomic rearrangements in the CFTR gene contribute to CBAVD.

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.

Are p.I148T, p.R74W and p.D1270N cystic fibrosis causing mutations?

BACKGROUND: To contribute further to the classification of three CFTR amino acid changes (p.I148T, p.R74W and p.D1270N) either as CF or CBAVD-causing mutations or as neutral variations. METHODS: The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms. RESULTS: Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone. CONCLUSION: These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified.

Molecular epidemiology of DFNB1 deafness in France.

BACKGROUND: Mutations in the GJB2 gene have been established as a major cause of inherited non syndromic deafness in different populations. A high number of sequence variations have been described in the GJB2 gene and the associated pathogenic effects are not always clearly established. The prevalence of a number of mutations is known to be population specific, and therefore population specific testing should be a prerequisite step when molecular diagnosis is offered. Moreover, population studies are needed to determine the contribution of GJB2 variants to deafness. We present our findings from the molecular diagnostic screening of the GJB2 and GJB6 genes over a three year period, together with a population-based study of GJB2 variants. METHODS AND RESULTS: Molecular studies were performed using denaturing High Performance Liquid Chromatograghy (DHPLC) and sequencing of the GJB2 gene. Over the last 3 years we have studied 159 families presenting sensorineural hearing loss, including 84 with non syndromic, stable, bilateral deafness. Thirty families were genotyped with causative mutations. In parallel, we have performed a molecular epidemiology study on more than 3000 dried blood spots and established the frequency of the GJB2 variants in our population. Finally, we have compared the prevalence of the variants in the hearing impaired population with the general population. CONCLUSION: Although a high heterogeneity of sequence variation was observed in patients and controls, the 35delG mutation remains the most common pathogenic mutation in our population. Genetic counseling is dependent on the knowledge of the pathogenicity of the mutations and remains difficult in a number of cases. By comparing the sequence variations observed in hearing impaired patients with those sequence variants observed in general population, from the same ethnic background, we show that the M34T, V37I and R127H variants can not be responsible for profound or severe deafness.

High heterogeneity of CFTR mutations and unexpected low incidence of cystic fibrosis in the Mediterranean France.

In this report, we present updated spectrum and frequency of mutations of the CFTR gene that are responsible for cystic fibrosis (CF) in Languedoc-Roussillon (L-R), the southwestern part of France. A total of 75 different mutations were identified by DGGE in 215 families, accounting for 97.6% of CF genes and generating 88 different mutational genotypes. The frequency of p.F508del was 60.23% in L-R versus 67.18% in the whole country and only five other mutations (p.G542X, p.N1303K, p.R334W, c.1717-1G>A, c.711+1G>T) had a frequency higher than 1%. The mutations were scattered over 20 exons or their border. This sample representing only 5.7% of French CF patients contributed to 24% of CFTR mutations reported in France. This is one of the highest molecular allelic heterogeneity reported so far in CF. We also present the result of a neonatal screening program based on a two-tiered approach "IRT/20 mutations/IRT" analysis on blood spots, implemented in France with the aim to improve survival and quality of life of patients diagnosed before clinical onset. This 18-month pilot project showed an unexpected low incidence of CF (1/8885) in South of France, with only six CF children detected among 43,489 neonates born in L-R, and 13 among 125,339 neonates born in Provence-Alpes-Côte-d'Azur (PACA).

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene.

In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.

Cystic fibrosis transmembrane conductance regulator mutation spectrum in patients with cystic fibrosis in Tunisia.

AIM: To determine the frequency and types of mutations causing cystic fibrosis (CF) in Tunisia. METHODS: We analyzed the complete coding region and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 68 unrelated patients suffering from the classical form of the disease. RESULTS: Twelve different CFTR mutations accounted for 90% (123/136) of CF alleles, including F508del (47.06%), E1104X (16.18%), N1303K (6.62%), 711 + 1T > G (5.88%), W1282X (4.41%), G542X (3.67%), R1158X (1.47%), 4016insT (0.74%), and R785X (0.74%). Three novel mutations were detected in this study: I1203V (1.47%), 1811 + 5A > G (0.74%), and 4268 + 2T > G (1.47%). Fifty patients (74%) were homozygous, among which 28 (41.17%) for F508del and 10 (14.7%) for E1104X. CONCLUSIONS: Ninety-seven percent of patients were found with at least one CFTR mutation. This study contributes to a better knowledge on CF-causing mutations in different regions in Tunisia and demonstrates that a complete scanning of CFTR sequences is necessary to implement efficient programs for CF genetic screening and counseling in this part of North Africa.