A large-scale study of the random variability of a coding sequence: a study on the CFTR gene

Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (ngapproximate to100-150 genes). In the present investigation, a large random European population sample (average ngapproximate to1500) was studied for a single gene, the CFTR ( Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q>0.005), much lower than that of the synonymous ( S) substitutions, but they showed a similar rate of subpolymorphic (q<0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic

Associations with 5 CFTR Mutations in »Grande Brière«, an Isolate Located in Southern Brittany

The variability at three microsatellites in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) locus has been studied for frequent mutations encountered in an isolated population of »Grande Brière«, a small region located in Southern Brittany. Fluorescent multiplex PCR of these microsatellites were assayed in 16 Cystic Fibrosis (CF) families carrying 5 different mutations. The four most frequent haplotypes on df508 chromosomes were the same as those found in Northern France and Europe but the distribution of these haplotypes provides new enlightenment on the population origin of this insular community

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Mitochondrial DNA A3243G mutation involved in familial diabetes, chronic intestinal pseudo-obstruction and recurrent pancreatitis

AimsTo report on a family with five members who carry the A3243G mutation in mitochondrial tRNA for leucine 1 (MTTL1) and present with diabetes, chronic intestinal pseudo-obstruction (CIPO) and recurrent pancreatitis, and to screen for this mutation in a cohort of 36 unrelated patients with recurrent pancreatitis. Methods The mutation was quantified in several tissue samples from patients. Respiratory chain activity was studied in muscle biopsies and fibroblast cultures. In addition, the thymidine phosphorylase gene (TP) involved in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and three genes involved in chronic pancreatitis – PRSS1, SPINK1 and CFTR – were sequenced in affected patients. Finally, the MTTL1 gene was examined in 36 unrelated patients who had recurrent pancreatitis, but no mutations in the PRSS1 and SPINK1 genes. Results Heteroplasmy for the mtDNA A3243G mutation was found in all tissue samples from these patients, but no mutations were found in the genes coding for thymidine phosphorylase, PRSS1, SPINK1 and CFTR. Also, none of the 36 unrelated patients with recurrent pancreatitis were carrying any MTTL1 mutations. Conclusion The mtDNA A3243G mutation associated with the gastrointestinal manifestations observed in the affected family should be regarded as a possible cause of CIPO and unexplained recurrent pancreatitis. However, the mutation is probably only weakly involved in cases of isolated recurrent pancreatitis

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

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations

An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

© 2017 The Author(s). Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range