G542X Mutations Research Articles

The Most Common Mutations in the CFTR Gene in the Population of Bosnia and Herzegovina

Aims: Cystic fibrosis is an autosomal recessive multisystem disease caused by a mutation of the CFTR gene. To date, more than 1900 mutations of this gene are known. Studies have shown that the most common mutation is delF508. In Bosnia and Herzegovina, the prevalence of individual mutations in the general population has not been thoroughly studied, so this study aimed to determine the prevalence of the mutation concerning the countries of the region and the rest of the world.
 Study Design: Retrospective study.
 Place and Duration of Study: Thirty-nine subjects with suspected Cystic fibrosis were referred to the Center for Genetics of the Medical Faculty in Sarajevo between 2018-2020.
 Methodology: 29 common CFTR gene mutations were analysed with the ELUCIGENE CF29 v2 kit (Elucigene Diagnostics, UK) using four multiplex PCR.
 Results: The most common mutation in our study was the F508 deletion, present in 14 subjects (73.68%). R347P and G542X mutations were confirmed in two subjects in the heterozygous state in combination with delF508 (M) 5.26% of each of these mutations. 621+1G>T was found in a homozygous state in one subject, while in another, it was in a heterozygous state in combination with delF508(M) mutation, 10.52%. Mutation 2184 delA was found in one subject in the homozygous state with a total frequency of 5.26%.
 Conclusion: Subjects with cystic fibrosis in Bosnia and Herzegovina are most often carriers of the delF508 mutation. Considering the existence of many mutations and that it is difficult to test them all, targeting the most common mutations in a clinical environment might help in approving therapy, and increasing patients’ quality of life.

A CASE OF AUTOSOMAL-DOMINANT HYPER-IGE SYNDROME MASQUERADING AS CYSTIC FIBROSIS

A CASE OF AUTOSOMAL-DOMINANT HYPER-IGE SYNDROME MASQUERADING AS CYSTIC FIBROSIS

Sheep models of F508del and G542Xcystic fibrosismutations show cellular responses to human therapeutics.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del / F508del and CFTRG542X / G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR −/− sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del / F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X / G542X lambs showed modest restoration of CFTR function following inhibition of nonsense‐mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life‐limiting disorder.

92 Correction of the CFTR G542X mutation using CRISPR/Cas9 genome editing in ovine-bovine interspecies embryos

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have recently generated 3 CF sheep models: a CFTR−/− model (Fan et al. 2018 LCI Insight 3:e123529; https://doi.org/10.1172/jci.insight.123529) and 2 additional models where we introduced human G542X and F508del mutations into the sheep genome (unpublished). Correction of CFTR mutations in zygotes with gene-editing techniques could be a permanent solution to cure this disease. To assess the efficiency of mutation correction invitro by CRISPR/Cas9, we utilised embryos generated by ovine-bovine interspecies SCNT (iSCNT) due to limited access to sheep oocytes. First, we evaluated the developmental capacity of reconstructed iSCNT embryos, in which nucleus donors were derived from ovine fibroblasts and recipient cytoplasm from enucleated bovine oocytes. These iSCNT embryos were able to develop to 16- to 32-cell stage (3/30, 10.0%), which allowed the genotyping of each embryo using PCR-restriction fragment length polymorphism assays and Sanger sequencing. Then, specific single-guide RNAs (sgRNAs) and 101-bp single-stranded oligodeoxynucleotides (ssODNs) were designed and synthesised to correct the G542X mutation in the sheep CFTR gene. We optimized the concentrations of Cas9:sgRNA ribonucleoproteins (RNPs) for 1-cell stage embryonic injection. Mutation analysis of embryos was conducted at 3 days post injection. Genotyping results showed that we achieved high efficiencies (95.7–100%) of mutations (indels) at targeting loci after injection of different concentrations of Cas9:sgRNA RNPs (0.02 µg:0.6 pmol/µL to 1.4 µg:40 pmol/µL). Furthermore, when an RNP (1.4 µg:40 pmol/µL) was co-injected with a ssODN (80 pmol/µL), both targeting the G542X mutation, the mutation was successfully corrected in the genome of iSCNT embryos generated using G542X fibroblasts as nucleus donors at an efficiency of 5.7% (3/53) via homology-directed repair mechanism. During the invitro culture of iSCNT embryos, we did not observe significant difference (P>0.05, unpaired t-test) in cleavage rates between embryos with or without injection (85.5% vs. 89.0%). Off-target analysis of those mutated and G542X-corrected embryos is in progress. Our strategy overcomes the limitation of oocyte source and provides an opportunity to mimic the editing of any other gene in embryos of different species.

95 Invitro correction of F508del and G542X mutations in sheep fibroblasts of cystic fibrosis models

Cystic fibrosis (CF) is a human genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Among the ∼2000 known CF mutations, the F508del mutation is found in 84% and G542X in 4.6% of the CF patients in the United States. The F508del mutation occurs in exon 11 and is characterised by deletion of the “CTT” nucleotides, resulting in deletion on the phenylalanine residue at the position 508 of CFTR. This causes misfolding of the CFTR protein, which is further degraded by proteases. The G542X mutation is a nonsense mutation found in exon 12 and associated with nonsense-mediated decay of the mutant transcript causing the absence of protein production. Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. We hypothesised that gene editing may be an effective tool to correct these mutations and permanently cure this genetic disease. Thus, in this study, we evaluated the efficiency of CRISPR/Cas9-meditated gene knock-in to correct the F508del and G542X mutations in sheep fibroblasts invitro. We designed single guide (sg)RNAs using the Benchling software (https://benchling.com/academic) and approximately 100bp of single-stranded oligodeoxynucleotides (ssODNs) targeting the mutation sites at exon 11 and 12 to introduce either “CTT” or change the “T” to “G” nucleotide in genome of F508del or G542X CF sheep cells, respectively. Each of Cas9/sgRNA ribonucleoproteins was transfected into sheep fibroblast cells along with ssODNs using the Lonza-4D-NucleofectorTM (Lonza) system for homology-directed repair. The transfected cells were subsequently cultured in Dulbecco’s modified Eagle medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. DNA was extracted 48h post-transfection to validate mutation efficiency. PCR products of the exons 11 and 12 were ligated into T-vector, and bacterial colonies were selected based on blue/white screening. In total, we isolated 32 single cell bacterial colonies for each mutant. Sequencing results indicate that “CTT” was introduced in 4/26 (15.3%) plasmid colonies, and “T to G” replaced in 13/31 (41.9%) colonies. Therefore, our results indicate that the F508del and G542X mutations can be effectively corrected in CF sheep fibroblasts invitro using a CRISPR/Cas9 approach.

Efficient Gene Editing at Major CFTR Mutation Loci

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

7 Frequency of five CFTR gene mutations in miscegenated cystic fibrosis population of the Brazilian Northeast

Introduction There are more than 1,900 known mutations in the CFTR gene. In the state of Bahia, in the northeastern area of Brazil, there are high rates of miscegenation, principally white/black miscegenation. The knowledge about genetics of cystic fibrosis (CF) in this population is scarce. Objective To determine the prevalence of five CFTR gene mutations in cystic fibrosis patients being assisted at Professor Edgard Santos Teaching Hospital in Salvador, Brazil. Methods It was a cross-sectional study involving 47 patients of 0–20 years of age with cystic fibrosis. The molecular analysis for DF508, G542X, G551D, R553X and 3120+1G→A mutations was performed by standard methods. Results The median age of the patients was 5.6 years at the time of diagnosis. The allele frequency of DF508 and G542X mutations was 23.9% (21/88) and 2.7 (2/74), respectively. So far the mutations G551D, R553X and 3120+1G→A have been studied in 21 patients. Three patients had the splicing mutation 3120+1G→A and the other mutations were not observed. Conclusion The diagnosis of the disease was late. The frequency of the DF508 mutation was lower than that one found in Caucasian populations, but the frequency of G542X mutation was similar to the one observed in this population. The 3120+1G→A mutation was found in three patients.

Frequency of CFTR, SPINK1, and Cathepsin B Gene Mutation in North Indian Population: Connections between Genetics and Clinical Data

Objectives. Genetic mutations and polymorphisms have been correlated with chronic pancreatitis (CP). This study aims to investigate the association of genetic variants of cystic fibrosis transmembrane conductance regulator (CFTR) and serine protease inhibitor Kazal type 1 (SPINK-1) genes and Cathepsin B gene polymorphisms with CP and to associate genetic backgrounds with clinical phenotypes. Methods. 150 CP patients and 150 normal controls were enrolled consecutively. We analyzed SPINK-1 N34S and IVS3+2T>C gene mutations by PCR-restriction-fragment length polymorphism (RFLP). The identification of DF508, G551D, G542X, R117H, and W1282X mutations was carried out by ARMS-PCR. S549N mutation, IVS8 polyTn polymorphism, and Cathepsin B Lec26Val were analysed by PCR-RFLP, nested PCR, and PCR-RFLP plus sequencing, respectively. Results. We found a significant association of SPINK1 (N34S) gene polymorphism. IVS1−37T>C polymorphism shows linkage with 101A>G. 300 chromosomes belonging to the CFTR subgroup exhibited minor allele frequency of 0.04, 0.03, 0.03, 0.013, 0.006, and 0.02 for DF508, G452X, G551D, S549N, R117H, and IVS8 T5, respectively. Except for R117H and IVS8 T5 polymorphisms, all other mutations showed significant variation. Conclusion. Analysis of potential susceptibility variants is needed to support nature of the genes and environment in pancreatitis. This data may help establish genetic screening and prenatal setup for Indian population.

CFTR mutation analysis and haplotype associations in CF patients

Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator ( CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/ trans phase of the mutations without need for parent studies.

The prevalence of common CFTR mutations in Iranian infertile men with non-CAVD obstructive azoospermia by using ARMS PCR techniques

To evaluate five common cystic fibrosis trans-membrane conductance regulator (CFTR) mutations (ΔF508, G542X, R117H, W1282X and N1303K) in the Iranian infertile men with noncongenital absence of vas deferens (CAVD) obstructive azoospermia. The common CFTR gene mutations were tested on blood samples from 53 infertile men with non-CAVD obstructive azoospermia and 50 normal men as control individuals. Genomic DNA is extracted from the whole blood and the common CFTR mutations have been detected by the amplification refractory mutation system (ARMS) techniques. The common CFTR mutations were found positive in 5/53)9.43%(for ΔF508 and 4/53)7.55%(for G542X mutation of all patients tested. Also, no CFTR mutations were detected in the normal men. The common CFTR mutations were detected in 9/53(17%) infertile men with non-CAVD obstructive azoospermia. Pre-treatment CFTR mutation analysis remains critical to distinguish cystic fibrosis (CF) genotypes for men with non CAVD obstructive azoospermia.

Protein–energy malnutrition as the first manifestation of cystic fibrosis in infancy

The protein-energy malnutrition (PEM) that is characterized by hypoproteinemia, edema, and anemia has been reported in 5-13% of infants with cystic fibrosis (CF). Due to the surprising higher incidence of PEM as the first presenting manifestation of CF in Macedonia, the aim of the present study was to evaluate the possible risk factors in its development. Clinical and laboratory profiles (hemoglobin, red blood cell count, total serum protein, serum albumin and liver enzyme levels) and genotype data were analyzed in 115 newly diagnosed infants with CF, during the period 1990-2006. PEM manifested in 39 CF infants (33%), usually within the first 5 months of life and in breast-fed infants. Mean hemoglobin, red blood cell count, total serum protein and serum albumin values in the PEM subgroup were, respectively, 76.0 g/L, 2.4 x 10(12)/L, 38.0 g/L and 16.6 g/L. Clinically significant liver involvement was found in 22 patients (56.4%) with PEM. Concerning the molecular basis of CF in these patients, PEM was always associated with triangle upF508, G542X, N1303K and other severe mutations. PEM is a common manifestation of CF in infancy. Early infant age, breast-feeding, impaired liver function and the presence of severe cystic fibrosis transmembrane conductance regulator mutations are predisposing factors for the development of PEM.

XV-2c/KM19 haplotypes analysis of cystic fibrosis patients from western Mexico

The analysis of polymorphic markers within or closely linked to the cystic fibrosis transmembrane regulator (CFTR) gene is useful as a molecular tool for carrier detection of known and unknown mutations. To establish the association between mutations in the CFTR gene in western Mexican cystic fibrosis (CF) patients, the distribution of XV2c/KM19 haplotypes was analyzed by PCR and restriction enzyme digestion in 384 chromosomes from 74 CF patients, their unaffected parents, and normal subjects. The haplotype analysis revealed that haplotype B was present in 71.9% of CF chromosomes compared to 0% of non-CF chromosomes. The F508del and G542X mutations were strongly associated with haplotype B (96.7% and 100% of chromosomes, respectively). The haplotype distribution of the CF chromosomes carrying other CFTR mutations had a more heterogeneous background. Our results show that haplotype B is associated with CFTR mutations. Therefore, haplotype analysis is a suitable alternate strategy for screening CF patients with a heterogeneous clinical picture from populations with a high molecular heterogeneity where carrier detection programs are not available. In addition, it may be a helpful diagnostic tool for genetic counseling and carrier detection in the relatives of CF patients and in couples who are planning to have children.

Design, Development, Validation, and Use of Synthetic Nucleic Acid Controls for Diagnostic Purposes and Application to Cystic Fibrosis Testing

We have designed, tested, and validated synthetic DNA molecules that may be used as reference standard controls in the simultaneous detection of mutations in one or more genes. These controls consist of a mixture of oligonucleotides (100 to 120 bases long) each designed for the detection of one or more disease-causing mutation(s), depending on the proximity of the mutations to one another. Each control molecule is identical to 80 to 100 bases that span the targeted mutations. In addition, each oligonucleotide is tagged at the 5' and 3' ends with distinct nucleic acid sequences that allow for the design of complementary primers for polymerase chain reaction amplification. We designed the tags to amplify control molecules comprising 32 CFTR mutations, including the American College of Medical Genetics minimum carrier screening panel of 23, with one pair of primers in a single tube. We tested the performance of these controls on many platforms including the Applied Biosystems/Celera oligonucleotide ligation assay and the Tm Bioscience Tag-It platforms. All 32 mutations were detected consistently. This simple methodology allows for maximum flexibility and rapid implementation. It has not escaped our notice that the design of these molecules makes possible the production of similar controls for virtually any mutation or sequence of interest.

Genetic examination of children suffering from cystic fibrosis.

CFTR protein (cystic fibrosis trans membrane conductance regulator) is expressed in multiple epithelial tissues, including upper and lower respiratory tracts, pancreas, sweat glands and gastrointestinal tract. More than 800 mutations and 100 polymorphic variants of DNA sequences were identified in patients with CF (Cystic fibrosis) and CFTR-diseases. In this study, genetic CFTR analysis of the children suffering from chronic lung disease (cystic fibrosis) is presented. They are treated and regularly controlled at the Pediatric hospital Sarajevo. CFTR analysis was done in 9 cases, 4 boys (44.4%) and 5 girls (55.55%). There are 3 children (33.3%) in the age group 1 to 3 years, 1 child (11.1%) in the age group 3 to 6 years, 3 children (33.3%) in the age group 6 to 9 years and 2 children (22.2%) in the age group 9 to 12 years. Genetic analysis was conducted at the Medical center for molecular biology, School of Medicine, Ljubljana. PCR method with PAGE and direct sequestration on ABI PRISM 31 was applied. The majority of children (7 children, i.e. 77.77%) had CFTR mutation Delta F 508 whilst one child had G542X mutation and one child R 1174 mutation. The purpose of this study is to emphasize the need for CFTR gene identification in the institutes of our country.

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation (deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

First Study of CF Mutations in the CFTR Gene of Iranian Patients: Detection of F508, G542X, W1282X, A120T, R117H, and R347H Mutations

Thirty-seven unrelated Iranian CF families were screened for the presence of seven common mutations (DeltaF508, G542X, W1282X, G551D, N1303K, 1717-1G-->A, and 621-1G-->T) using ARMS PCR and exons 4 and 7 of the CFTR gene by SSCP method. This study resulted in the identification of 26.8 per cent of all CF alleles: DeltaF508 (16.2 per cent), W1282X (4 per cent), G542X (2.7 per cent), R117H (1.3 per cent), R347H (1.3 per cent), and A120T (1.3 per cent) mutations were detected. To the best of our knowledge, it is the first report of an Asian subject carrying the A120T mutation. Our findings suggest heterogeneity in the Iranian population, stressing the need to draw attention to sequence analysis in order to find population-specific mutations.

CFTR gene: molecular analysis in patients from South Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator ( CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), ΔF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventy-seven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a non-radioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The ΔF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.

Cystic fibrosis mutations in Costa Rica.

Using polymerase chain reaction amplification of DNA in dried blood spots and a nonisotopic reverse dot blot hybridization method, we performed molecular genetic analysis for 6 and for 16 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 24 unrelated Costa Rican individuals with cystic fibrosis (CF). While many countries and ethnic groups have been surveyed for CF mutations since the cloning of CFTR, Costa Rica has not heretofore been studied. Moreover, Costa Rica represents an especially intriguing population because of its mixed European-African-Amerindian origins and the existence of a detailed historical record of the founding Spanish families. Thus, such a study may reveal not only the population frequencies of various mutant alleles in this country, but also something about their geographic migrations and ethnic founder effects. The most common CF mutation in Caucasians, deltaF508, was found in only 11 (23%) of the CF chromosomes studied, while the G542X mutation, relatively rare in the general population but more common in southern Europe, was observed in 12 (25%). None of the other mutations tested was found in any of the subjects. We failed to detect the second mutant allele in 17 subjects and could not detect either allele in 4 subjects. The high prevalence of the G542X mutation in our cohort, which exceeds that of both the general Caucasian population and the American Hispanic population, reflects the strong genetic influence of the original Spanish founding families of Costa Rica. These results highlight important differences in Costa Rican CF genotypes as compared both to other North American and European populations and to American Hispanics, raising important implications about isolated founder effects and strategies for population screening in that country.

Mutations and polymorphisms in CFTR genes in infertile men with oligospermia or azoospermia

Impaired infertility of the male partner is causative or contributOry to in up to one half of all couples unable to conceive spontaneously. A considerable number of genes are now known that have an essential function in human reproduction and which, when deleted or mutated, can cause pathologic changes in the male reproductive system. Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of obstructive azoospermia in otherwise healthy men. It is also present in 95% of men with an autosomal recessive systematic disease--cystic fibrosis. However, clinically affected CF patients present a spectrum of genital phenotypes ranging from normal fertility to severely impaired spermatogenesis and CBAVD. Cystic fibrosis and most cases of CBAVD are caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) gene. The aim of this study was to test the possible involvement of the CFTR gene in the aetiology of male infertility other than CBAVD. Twenty one infertile men with oligo or azoospermia were analysed for the presence of mutations and polymorphisms in the CFTR gene. Patients were divided in two groups according to the spermatogram: 1) patients with obstructive azoospermia (V < 2 mL, pH < 7.2, low level of a-glucosidase and fructose and absence of spermatozoa; 2) patients with impaired spermatogenesis or sperm maturation. We performed direct detection for the following mutations: delta F508 and delta 1507 (heteroduplex analysis), 621 + 1 G-->T, and N1303K (PSM--PCRmediated site-specific mutagenesis), A455E, 1717-1 G-->A, S549N, R560T, W1282X, R334W, R347P, R117H, 3849 + 10 kb C-->T and Tn, F508C, 1507V, 1506V polymorphisms (reverse dot blot method). G542X, R553X and GSS1D mutations were tested by SSCP (Single Strand Conformation Polymorphism). We also performed indirect detection of mutations and polymorphisms in 3, 5, 6a, 8, 9, 11, 12, 14a, 14b, 15, 17b, 18, 20, 21 and 23 exons by DGGE (Denaturant Gradient Gel Electrophoresis). Differences between frequencies were tested by chi-square statistic, p values of less than 0.05 were considered statistically significant. Among 42 chromosomes from infertile men with oligo or azoospermia we detected 7 mutations in CFTR gene (16.7%), which was significantly (p = 0.0319) more frequent than in general population (2%). Frequency of 5T allele in analysed group was high (11.9%) compared to general population (5%), but not statistically significant (0.0938). The most common mutation in the group of 10 men with obstructive azoospermia was delta F508. It was detected on one chromosome in five patients. In three of these patients with 4F508 mutation on the other chromosome we found 5T allele on polymorphic Tn locus. In one patient, heterozygous for delta F508 mutation, 711 + 3 A-->G mutation on the other chromosome was detected. In the group of 11 infertile men with impaired spermatogenesis or sperm maturation we detected one mutation--delta F508. Two patients from this group had 5T variant on one chromosome. We analysed 21 infertile men with oligo or azoospermia not caused by endocrine or inflammatory character, or chromosome mutations. Within this group frequency of CFTR mutations was increased compared to general population (p = 0.0319), suggesting that CFTR gene may be involved in the aetiology of infertility in men with oligo or azOospermia. In the group of patients with obstructive azoospermia 50% had at least one mutation, but only 10% had mutations in both chromosomes. One of the possible explanations would be that mutations are in the promoter region, introns or exons that were not included in analyses. The second explanation could be that some cases of obstructive azoospermia are only partially (or not) related to CFTR gene. In the group of patients with impaired spermatogenesis or sperm maturation, the frequencies of CFTR mutations and 5T allele were also increased compared to general population, but lower than in the group with obstructive azoospermia. This fact could mean that the influence of some other genes is higher in this condition than in the case of obstructive azoospermia. We concluded that CFTR gene plays a role in the aetiology of obstructive azoospermia and that is also could be involved in some cases of impaired spermatogenesis and sperm maturation. Due to the high incidence of CFTR mutations in patients with obstructive azoospermia we suggest screening of CFTR mutations before assisted reproduction.

XV-2c/KM-19 haplotype analysis of cystic fibrosis mutations in Mexican patients.

We analyzed 97 unrelated Mexican cystic fibrosis (CF) patients and their first-degree relatives to study the association of XV2C/TaqI/KM19/PstI haplotypes with CF mutations in this population. Haplotype phases could be established in 148 CF and 110 normal chromosomes, and haplotype distributions of normal and CF chromosomes differed significantly (P < 0.001). DeltaF508 and G542X mutations accounted for 56% of CF chromosomes and were found to be associated with haplotype B in 97.2% and 72.7% of chromosomes, respectively. The haplotype distribution of CF chromosomes carrying other rare and unknown mutations was similar to that of normal chromosomes (P > 0.05), haplotypes A and C being the most frequent. This is in accordance with the extensive heterogeneity and the spectrum of mutations reported in Mexican CF patients. We also report the haplotype distribution of all informative chromosomes bearing rare mutations; some were found to be associated with previously reported haplotypes, whereas others were found on different haplotypes. Recombination or recurrence of mutations may explain these different associations, although other intragenic markers must be used to better understand the origin and dispersion of CF mutations in our country. XK haplotype analysis allowed carrier detection among sibs in 24.3% of families, showing that this method may be useful for carrier detection in populations with high allelic heterogeneity.