CFTR Haplotype Analysis Reveals Genetic Heterogeneity in the Etiology of Congenital Bilateral Aplasia of the Vas Deferens

Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.     

Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure

High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n = 60) and spermatogenic failure (n = 150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.     

Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens

Several recent studies have demonstrated the presence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in healthy males with infertility caused by congenital absence of the vas deferens (CBAVD), previously recognized as an idiopathic genetic condition distinct from CF. In order to document further the genetic commonality of these two disorders, we undertook a double screening of the entire coding and flanking sequences of the CFTR gene, by using single-strand conformational polymorphism analysis and denaturing gradient gel electrophoresis in 12 unrelated infertile men with abnormalities of the vas deferens and/or epididymis. This strategy allowed us to identify 11 DNA sequence alterations considered as CF-causing mutations and several variations. Despite this double analysis, only two patients out of eight with CBAVD could be demonstrated as compound heterozygotes for CF mutations.     

Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected

Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (E115). A second new mutation was found in exon 17b, viz., an AC substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (F508/L206W, F508/R74W+D1270N, Rl 17H/712-1GT), highlighting L206W, R74W/ D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%–10% necessary for normal protein function.     

Gène CFTR et infertilité masculine en 2001 Conseil génétique, diagnostic prénatal, diagnostic pré-implantatoire

As the vas deferens is also absent in the majority of CF (cystic fibrosis) males, it has been proposed that CBAVD (Congenital Bilateral Absence of Vas Deferens) males may present an incomplete or mild form of CF. Many studies using more extensive mutation analysis have confirmed the role of CFTR gene defects: 80% of CBAVD patients carry one or two mutations. Each patient with a diagnosis of CBAVD should also be examined for pulmonary and pancreatic signs, and sweat tests should be performed. In couples with CBAVD linked to CFTR mutations, the risk of having children with CF or infertility is increased if the female is also a carrier. The woman should be screened for the most frequent CFTR mutations according to her ethnic background. After screening for 80% of the mutations responsible for CF, the residual risk of being a carrier with negative screening is: Z=h(1?a)/(1?ah)=1/120 considering a carrier frequency of 1/25 in the general population. In the case of positive screening, antenatal diagnosis by chorionic villus sampling may be proposed. However, in some situations it is difficult to predict the phenotypic consequences for the child, particularly when a severe transmutation of a variable allele is identified. As these couples require medically-assisted reproduction techniques, pre-implantation genetic diagnosis appears to be more appropriate than antenatal diagnosis. Only embryos that inherit the non-mutated maternal CFTR allele are replaced in the uterus. Examination of childre born to couples with CBAVD is mandatory: immunoreactive trypsinogen assay at 3 days of age, sweat test at 3 months and clinical examination, especially looking for signs of CF. Identification of CFTR mutations in a CBAVD patient has important consequences for his family. Each sibling has a 50% risk of being a carrier and a 25% risk of inheriting the same genotype. The genetic counsellor must inform these siblings about the possible risk of having CF children if they carry CFTR mutations and if their partner is also a carrier.     

Les génotypes responsables de mucoviscidose ou d’absence bilatérale des canaux déférents ABCD

Over the last decade, the genetic basis for CBAVD has been identified by its association with CFTR gene mutations, and CBAVD is now generally considered to be a mild or incomplete form of CF. In this review, the author summarizes the main results of compilation of CFTR gene analysis conducted in French laboratories for 3,923 patients with CF and 800 males with CABVD. The degree of clinical expression can be affected by several variables, including the molecular mechanisms by which the various CFTR mutations impair or disrupt the function of the CFTR chloride channel. Phenotypic expression of CFTR mutational genotypes varies from severe, progressive pulmonary disease with pancreatic insufficiency (CF-PI), to mild pulmonary disease with pancreatic sufficiency (PS) or singleorgan forms of “CFTR-opathies”. In CF, a total of 310 different CFTR mutations accounting for 94% of 7,846 CF alleles have generated almost 500 different genotypes, comprising 2 severe mutations in 88% of cases (CF-PI), one severe mutation in trans to a mild mutation in 11% (CF-PS), and 2 mild mutations in 1% of identified genotypes. In CBAVD, 137 mutations scattered over the whole gene were identified in 60% of 1,600 CBAVD alleles during the study. Among the 150 characterized mutational CFTR genotypes, compound heterozygosity was the rule, and the most frequent CBAVD combinations were ΔF508/5T (35%), ΔF508/other mutation (30%, including ΔF508/R117H-7T: 5,6%), and 5T/other mutation (17%). No combination of two severe mutations was found in CBAVD (0%); by contrast with the CF population, 88% of genotypes identified in CBAVD comprised a severe mutation in trans to a mild mutation, and 12% consisted of 2 mild mutations. A total of 22 genotypes were shared by both CF and CBAVD. The role of the 5T allele as a splicing variant with variable, incomplete disease penetrance in CBAVD is reviewed. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and/or function. This study confirms the high molecular heterogeneity of CFTR mutations in CBAVD and emphasizes the importance of extensive CFTR analysis in these patients. Longterm follow-up studies of CBAVD patients are necessary in order to predict the phenotypic consequences of numerous CFTR mutational genotypes.     

Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? Analyses of the CFTR gene in 67 patients.

Congenital bilateral absence of the vas deferens (CBAVD) is an important cause of sterility in men. Although the genetic basis of this condition is still unclear, it has been shown recently that some of these patients carry mutations in their cystic fibrosis transmembrane conductance regulator (CFTR) genes. To extend this observation, we have analyzed the entire coding sequence of the CFTR gene in a cohort of 67 men with CBAVD, who are otherwise healthy. We have identified four novel missense mutations (A800G, G149R, R258G, and E193K). We have shown that 42% of subjects were carriers of one CFTR allele and that 24% are compound heterozygous for CFTR alleles. Thus, we have been unable to identify 76% of these patients as carrying two CFTR mutations. Furthermore, we have described the segregation of CFTR haplotypes in the family of one CBAVD male; in this family are two male siblings, with identical CFTR loci but displaying different phenotypes, one of them being fertile and the other sterile. The data presented in this family, indicating a discordance between the CBAVD phenotype and a marked carrier (delta F508) chromosome, support the involvement of another gene(s), in the etiology of CBAVD.     

Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype

To assess better the link between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis (CF), we compared sweat chloride values, analysis of the CFTR intron 8 poly(T) tract length and analysis of 10 exons in a population of 38 patients with CBAVD. The data indicate that this population can be divided into three groups of patients. In the first group of 15 patients with abnormal sweat chloride (> 60 mmol/l), the frequency of CF mutations is high. In the second group of 18 patients with equivocal sweat chloride (between 40 and 60 mmol/l), the frequency of the 5T variant is high; 6 patients have a F508 mutation and a 5T variant and 1 patient is homozygous for the 5T variant; a 5T variant has been detected in 3 other patients, and a F508 mutation in another patient. A third group of 5 patients is probably not related to CF: these patients have other congenital abnormalities of the urogenital tract, low chloride values (< 40 mmol/l) and apparently no abnormality of the CF gene.     

Implication of the Cystic Fibrosis Transmembrane Conductance Regulator Gene in Infertile Family Members of Indian CF Patients

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Among males with CF, 95% are infertile due to congenital absence of the vas deferens. We investigated the role of family history of infertility among CF subjects and characterized mutations in them. Among 50 CF subjects, four had a family history of infertility. A homozygous c.1521_1523delCTT mutation was detected in one, two had a compound heterozygous genotype (c.1521_1523delCTT/c.3717 + 10 kbC>T), and c.1521_1523delCTT mutation was identified on one allele of fourth CF subject. Genetic analysis of each infertile family members of CF subjects revealed the c.1521_1523delCTT mutation on one allele; however, no mutation could be identified on other allele. Haplotype analysis of the infertile family members showed that at least one of the alleles shared the same haplotype as that of the index case. It is suggested that the CFTR gene is implicated in the infertile members of the CF families. Failure to detect mutations on the other allele by SSCP analysis demands direct gene sequencing to detect mutations in the intronic or promoter region.     

Mukoviszidose – eine pleiotrope Ionenkanalerkrankung mit wesentlicher Lungenbeteiligung

The monogenic disease cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is inherited in an autosomal recessive fashion. Successful diagnosis of the disease is achieved by patient history, clinical assessment, genetic analysis of the CFTR gene and by in vivo measurement and ex vivo characterization of the basic defect in patient’s samples. Frequently, differential diagnosis of CFTR-related disorders such as congenital bilateral absence of the vas deferens (CBAVD), pancreatitis and bronchiectasis needs to be performed. Molecular therapeutics has been developed for some CFTR mutations and these will facilitate direct clinical use of the information provided by CFTR mutation analysis.     

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.     

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.     

Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels

The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.     

High allelic heterogeneity between Afro-Brazilians and Euro-Brazilians impacts cystic fibrosis genetic testing

Cystic fibrosis (CF) is an autosomal recessive disease caused by at least 1,000 different mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). To determine the frequency of 70 common worldwide CFTR mutations in 155 Euro-Brazilian CF patients and in 38 Afro-Brazilian CF patients, we used direct PCR amplification of DNA from a total of 386 chromosomes from CF patients born in three different states of Brazil. The results show that screening for seventy mutations accounts for 81% of the CF alleles in Euro-Brazilians, but only 21% in the Afro-Brazilian group. We found 21 different mutations in Euro-Brazilians and only 7 mutations in Afro-Brazilians. The frequency of mutations and the number of different mutations detected in Euro-Brazilians are different from Northern European and North American populations, but similar to Southern European populations; in Afro-Brazilians, the mix of CF-mutations is different from those reported in Afro-American CF patients. We also found significant differences in detection rates between Euro-Brazilian (75%) and Afro-Brazilian CF patients (21%) living in the same state, Minas Gerais. These results, therefore, have implications for the use of DNA-based tests for risk assessment in heterogeneous populations like the Brazilians. Further studies are needed to identify the remaining CF mutations in the different populations and regions of Brazil.     

Effect of genotype on selected clinical features of Polish cystic fibrosis adults

Cystic fibrosis (CF), the most common autosomal recessive disorder of Caucasians, is caused by the mutations in the gene encoding CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein. Until now, approximately 1000 mutations of the CFTR gene have been described. The genotype-phenotype relationships in CF are still not completely understood. This study was undertaken in an attempt to characterise the distribution of CFTR mutations and their effect on selected clinical parameters in a group of Polish CF adults. A total number of 38 adult CF patients (mean age 21.6 +/- 6.8); 18 females & 20 males were enrolled in the study. The CFTR gene identification was conducted with the use of PCR & InnoLipa-CF set. The assessed clinical parameters included: age at diagnosis, age, lung function test, X-ray scored in Brasfield score, weight & height. We found that: (1) the genotypes of the studied population were unevenly distributed (65.8%- genotype deltaF508/M), (2) a high percentage of 3849+10kbC-->T was noted, (3) patients homozygous for the deltaF508 mutation were diagnosed significantly earlier and had a lower body mass index, (4) no differences were observed in the patients' length of life or the progression of lung disease. Conclusions: 1. In comparison to other populations, Polish adult CF patients display a relatively higher frequency of mild mutations. 2. Late diagnosis of CF in the studied group may be partially caused by a high percentage of CFTR mutations connected with the mild course of the disease that are difficult to identify. 3. Cystic fibrosis should be more commonly taken into consideration in the differential diagnosis in adult patients with milder symptoms.     

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.     

Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies

Cystic fibrosis (CF) results from the absence or dysfunction of a single protein, the CF transmembrane conductance regulator (CFTR). CFTR plays a critical role in the regulation of ion transport in a number of exocrine epithelia. Improvement or restoration of CFTR function, where it is deficient, should improve the CF phenotype. There are >1000 reported disease-causing mutations of the CFTR gene. Recent investigations have afforded a better understanding of the mechanism of dysfunction of many of these mutant CFTRs, and have allowed them to be classified according to their mechanism of dysfunction. These data, as well as an enhanced understanding of the role of CFTR in regulating epithelial ion transport, have led to the development of therapeutic strategies based on pharmacologic enhancement or repair of mutant CFTR dysfunction. The strategy, termed 'protein repair therapy', is aimed at improving the regulation of epithelial ion transport by mutant CFTRs in a mutation-specific fashion. The grouping of CFTR gene mutations, according to mechanism of dysfunction, yields some guidance as to which pharmacologic repair agents may be useful for specific CFTR mutations. Recent data has suggested that combinations of pharmacologic repair agents may be necessary to obtain clinically meaningful CFTR repair. Nevertheless, such strategies to improve mutant CFTR function hold great promise for the development of novel therapies aimed at correcting the underlying pathophysiology of CF.     

Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele

Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.