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

Large genomic rearrangements in the CFTR gene contribute to CBAVD

<p>Abstract</p> <p>Background</p> <p>By performing extensive scanning of whole coding and flanking sequences of the <it>CFTR (Cystic Fibrosis Transmembrane Conductance Regulator</it>) 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 <it>CFTR </it>locus in the 32 CBAVD patients with only one or no mutation.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or <it>CFTRdele2</it>], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or <it>CFTRdele 22_24</it>], in two males carrying a typical CBAVD mutation on the other parental <it>CFTR </it>allele. We present the first bioinformatic tool for exon phasing of the <it>CFTR </it>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.</p> <p>Conclusion</p> <p>Identification of large rearrangements further expands the <it>CFTR </it>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.</p

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

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

International audienceIn 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

Variants in CFTR untranslated regions are associated with Congenital Bilateral Absence of the Vas Deferens

International audienceBackground: CBAVD (Congenital Bilateral Absence of the Vas Deferens), a frequent cause of obstructive azoospermia, is generated by mutations in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) 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. In this report, we explored 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. We also investigated whether regulatory elements located within the intronic tetrarepeat might influence the CFTR expression. We evidenced 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 the most susceptible to a reduction in the normal CFTR transcripts levels, and as two mild mutations are sufficient to induce CBAVD phenotype, our findings raise the possibility that these uncommon variants may be a novel cause of CBAVD

Molecular epidemiology of DFNB1 deafness in France

Abstract 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.</p

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