Marie-Claude Pasquet

Influence of the Duplication of CFTR Exon 9 and Its Flanking Sequences on Diagnosis of Cystic Fibrosis Mutations

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact, present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions.

CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes

Genotype–phenotype correlations in cystic fibrosis (CF) may be difficult to establish because of phenotype variability, which is associated with certain CF transmembrane conductance regulator (CFTR) gene mutations and the existence of complex alleles. To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a collaborative genotype–phenotype correlation study, collected epidemiological data, and investigated structure–function relationships for single and natural complex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg;Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys]. Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Sixty‐four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Functional studies performed on all single and natural complex mutants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splicing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associated with CFTR‐related disorders. The present study emphasizes the importance of comprehensive genotype–phenotype and functional studies in elucidating the impact of mutations on clinical phenotype. Hum Mutat 33:1557–1565, 2012.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene mutations in North Egyptian population: implications for the genetic diagnosis in Egypt

Cystic fibrosis (CF) occurrence in Arab populations is not common and still remains underidentified. Furthermore, the lack of disease awareness and diagnosis facilities have mislead the identification of cystic fibrosis for decades. The knowledge about cystic fibrosis (CF) in Egypt is very limited, and a few reports have drawn attention to the existence of CF or CFTR-related disorders (CFTR-RDs) in the Egyptian population. Therefore a comprehensive genetic analysis of the CFTR gene was realized in patients of North Egypt. DNA samples of 56 Egyptian patients were screened for the CFTR gene mutations. The 27 exons and their flanking regions of the CFTR gene were amplified by PCR, using the published primer pairs, and were studied by automated direct DNA sequencing to detect disease-causing mutations. Moreover, large duplication/deletion was analysed by MLPA technique. CFTR screening revealed the identification of thirteen mutations including four novel ones: c.92G>A (p.Arg31His), c.2782G>C (p.Ala928Pro), c.3718-24G>A, c.4207A>G (p.Arg1403Gly) and nine previously reported mutations: c.454A>T (p.Met152Leu), c.902A>G (p.Tyr301Cys), c.1418delG, c.2620-15C>G, c.2997_3000delAATT, c.3154T>G (p.Phe1052Val), c.3872A>G (p.Gln1291Arg), c.3877G>A (p.Val1293Ile), c.4242+10T>C. Furthermore, eight polymorphisms were found: c.743+40A>G, c.869+11C>T, c.1408A>G, c.1584G>A, c.2562T>G, c.3870A>G, c.4272C>T, c.4389G>A. These mutations and polymorphisms were not previously described in the Egyptian population except for the c.1408A>G polymorphism. Here we demonstrate the importance of the newly discovered mutations in Egyptian patients and the presence of CF, whereas the p.Phe508del mutation is not detected. The identification of CFTR mutations will become increasingly important in undocumented populations. The current findings will help us expand the mutational spectrum of CF and establish the first panel of the CFTR gene mutations in the Egyptian population and design an appropriate strategy for future genetic diagnosis of CF.

N1303K (c.3909C>G) mutation and splicing: implication of its c.[744-33GATT(6); 869+11C>T] complex allele in CFTR exon 7 aberrant splicing.

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasians. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys, old nomenclature: N1303K) is one of the most common worldwide mutations. This mutation has been found at high frequencies in the Mediterranean countries with the highest frequency in the Lebanese population. Therefore, on the genetic level, we conducted a complete CFTR gene screening on c.3909C>G Lebanese patients. The complex allele c.[744-33GATT(6); 869+11C>T] was always associated with the c.3909C>G mutation in cis in the Lebanese population. In cellulo splicing studies, realized by hybrid minigene constructs, revealed no impact of the c.3909C>G mutation on the splicing process, whereas the associated complex allele induces minor exon skipping.

Complexity of phenotypes induced by p.Asn1303Lys-CFTR correlates with difficulty to rescue and activate this protein

Cystic Fibrosis is the most common recessive autosomal rare disease found in Caucasian. It is caused by mutations on the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) that encodes for a protein located on the apical membrane of epithelial cells. c.3909C>G (p.Asn1303Lys) is one of the most common worldwide mutations located in nucleotide binding domain 2. The effect of the p.Asn1303Lys mutation on misprocessing was studied by immunofluorescence and western blotting analysis in presence and absence of treatment. To evaluate the functionality of potentially rescued p.Asn1303Lys-CFTR, we assessed the channel activity by radioactive iodide efflux. No recovery of the activity was observed in transfected cultured cells treated with VX-809. Thus, our results suggest that multiple drugs may be needed for the treatment of c.3909C>G patients in order to correct and activate p.Asn1303Lys-CFTR as it shows folding and functional defects.

WS21.5 Evolution of CFTR exon 10 and duplications in primates

Objectives: Previous studies conducted on the human CFTR gene have reported that exon 10 and its flanking introns are present in multiple copies in the human genome. The c.1392+6insC and c.1392+12G>A variations are found in all duplicated sequences, but are not authentic mutations in CFTR. To understand the recent evolution of this part of the genome, the comparison of this CFTR region in humans and in 8 different primates had been performed. Methods: DNA extraction, sequencing and BLAST analyses were performed on the studied species. Results and Conclusion: A homologous sequence of the CFTR gene was obtained in the 9 species. We observed also that the two mutations are present in the CFTR gene except for Gorilla and Human but they only reappeared in the duplicated human sequences, suggesting that these duplications are only in human. To explain these observations, we suggest three hypotheses which permitted to better understand the molecular evolution of the CFTR gene in primates: duplications and elimination of mutations on CFTR gene, or horizontal transfer and duplications, or founder effect. Furthermore, whatever was the origin of the duplicated sequences in Human, their presence in all individuals could be explained by a selective advantage not yet defined. These results underline the importance of studying the duplicated sequence of the human CFTR gene that can appear in different chromosomes or in the gene itself as example several duplications of a part of intron 24 are detected in this intron. This study gives novel insights in understanding CFTR gene evolution in primates and uses, for the first time, the CFTR gene as a significant tool in the evolutionary medicine.