Molecular analysis of the cystic fibrosis gene reveals a high frequency of the intron 8 splice variant 5T in Egyptian males with congenital bilateral absence of the vas deferens

It has previously been shown that defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are largely responsible for the condition of congenital bilateral absence of the vas deferens (CBAVD), without associated renal abnormalities, in Caucasian populations. To assess the involvement of the CFTR in CBAVD in a population with presumed low cystic fibrosis (CF) frequency, we have analysed 20 CBAVD males from Egypt for the presence of 12 common Caucasian CFTR mutations and the intron 8 5T splice variant, IVS-5T, known to be a major cause of CBAVD in Caucasian patients. In 16 of the males without associated renal abnormalities only one deltaF508 carrier was identified, but an exceptionally high frequency of the IVS-5T variant was found (14 of 32 alleles or 43.7%), confirming that this variant is involved in many cases of CBAVD, even in populations where CF is rare. CFTR mutations or the IVS-5T variant were found neither in the remaining four patients with associated renal abnormalities nor in the spouses of the 20 CBAVD patients. However, one patient was homozygous for a leucine to proline substitution at amino acid position 541 (L541P) of the CFTR. It is as yet not clear whether this change is involved in CBAVD in this male.     

The complex relationships between cystic fibrosis and congenital bilateral absence of the vas deferens: clinical, electrophysiological and genetic data

Congenital bilateral absence of the vas deferens (CBAVD) is found in 1-2% of infertile males and in most male cystic fibrosis (CF) patients. CF and some of the CBAVD cases were found to share the same genetic background. In this study, 21 males with CBAVD had extensive physical and laboratory testing for symptoms of CF. Possible defective cellular chloride transport was measured by interstitial current measurement of rectal suction biopsies. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation analysis was performed for 10 common CFTR mutations. CF-related symptoms were found in six men. On laboratory testing slightly abnormal liver and pancreatic function was found in seven patients. The sweat test was found to be abnormal in four patients; interstitial current measurement showed defective chloride excretion in 11 patients. CFTR gene mutations were found in 66% of the patients: eight were compound heterozygotes; in six, only one common mutation could be detected. The 5T allele in one copy of intron 8 was found in four men. CBAVD appears to be a heterogeneous clinical and genetic condition. A CFTR gene mutation was found in both copies of the allele or interstitial current measurement showed defective chloride excretion in 14/21 cases. Genetic counselling is clearly indicated for couples seeking pregnancy through epididymal or testicular sperm aspiration and intracytoplasmic sperm injection.     

Molecular analysis of the IVS8-T splice variant 5T and M470V exon 10 missense polymorphism in Iranian males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is responsible for 2-6% of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. To investigate CBAVD at the molecular level in Iran, we have characterized the mutations in the CFTR gene in 106 patients with this condition. None had clinical manifestations of cystic fibrosis (CF). We also analysed a DNA variant (the 5T allele) in a noncoding region of CFTR, which causes reduced levels of the normal CFTR protein and M470V exon 10 missense polymorphism. Five of the 106 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Eighty-five patients had a mutation in at least one copy of CFTR, and of these patients, 46 had one 5T allele (in 11 cases, two alleles and in 35 cases, just one allele of 5T was detected). In 21 patients, no CFTR and 5T mutations were found (19.81%). 5T/M470 genotype was found in 19 patients, 5T/V470 was found in 3 and 5T with heterozygote form of M470V was found in 24 CBAVD patients. In CBAVD patients, 28 F508del carriers were identified. Most of our patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a CF mutation in the other copy is the most common cause of CBAVD in Iran. The 5T allele mutation has a wide range of clinical presentations and revealed a high frequency, occurring in patients with CBAVD or moderate forms of CF and infertile men.     

Cystic fibrosis mutation screening in CBAVD patients and men with obstructive azoospermia or severe oligozoospermia

Congenital bilateral absence of the vas deferens (CBAVD) found in otherwise healthy infertile males, is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles, and is considered a genital form of cystic fibrosis (CF). The CF gene may also be involved in the aetiology of male infertility in cases other than CBAVD. The present study was undertaken to test the involvement of CFTR gene mutations in 14 CBAVD males and additionally in cases of male infertility caused by obstructive azoospermia (n = 10) and severe oligozoospermia (n = 3). The entire coding region of the CFTR gene was analysed using denaturing gradient gel electrophoresis (DGGE). The three allele (5T, 7T, 9T) polymorphic tract of thymidines in intron 8 (IVS8-polyT) of which the 5T allele acts as a mild mutation, causing reduced levels of normal CFTR mRNA due to deletion of exon 9, was also analysed. Of the 14 CBAVD cases, four (28.6%) were found to have mutations in both copies of the CFTR gene, six (42.8%) had one CFTR mutation, and in the remaining four (28.6%) no CFTR mutations were found. Of the 10 cases with obstructive azoospermia, three (30%) had one CFTR mutation and in the remaining seven (70%) no mutations were found. None of the three severe oligozoospermia cases carried a CFTR mutation. The frequency of the IVS8(5T) allele was 14.3% (4/28) for the CBAVD cases and 5% (1/20) for the obstructive azoospermia cases, none of the severe oligozoospermia males carried the IVS8-5(5T) allele. The data indicate that while there is a strong association between male infertility caused by CBAVD and mutations in the CFTR gene, cases of obstructive azoospermia without CBAVD also seem to be associated with CFTR gene mutations.      

CFTR (TG)m(T)n polymorphism in patients with CBAVD in a population expressing low incidence of cystic fibrosis

As it is well established that an association exists between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis gene mutations, we investigated CFTR(TG)m(T)n polymorphism within a Taiwanese population that exhibits a very low incidence of CF. Sixty-three patients with CBAVD and 86 age-matched normal control subjects were evaluated. Temporal temperature gradient gel electrophoresis was used for CFTR mutational analysis. No major CFTR mutation was found in the patient series. A single prominent CFTR mutation, IVS8-5T, was present; however, (50.8% of 63 cases and 33.3% of 126 alleles), and exhibited a high prevalence of 12 or 13 TG repeats (93.8% of 32 cases and 95.2% of 42 alleles with IVS8-5T). Although these results are similar to those of Japanese CBAVD patients, they are higher than the common frequency (about 21%) found among Caucasian CBAVD patients. The very high percentage (42.9%) of patients with no CFTR mutations is also an ethnic characteristic. We concluded that CBAVD patients from Taiwan, who express a very low incidence of CF, were less affected by CFTR mutations, with the exception of IVS8-5T linked to either 12 or 13 TG repeats, which does exhibit a high prevalence among CBAVD patients tested.     

Mutation spectrum of the CFTR gene in Taiwanese patients with congenital bilateral absence of the vas deferens

BACKGROUND: Clinically affected cystic fibrosis (CF) patients present a spectrum of genital phenotypes ranging from normal fertility to moderately impaired spermatogenesis and congenital bilateral absence of vas deferens (CBAVD). Little is known about the CF incidence in the Taiwanese population. It has been shown that the CBAVD in men without clinical evidence of CF is associated with a high incidence of mutated CFTR (cystic fibrosis transmembrane conductance regulator) alleles. In order to understand the involvement of the CFTR gene in the aetiology of Asian/Taiwanese male infertility, we screened the entirety of the CFTR gene in 36 infertile males with CBAVD. METHODS: Temporal temperature gradient gel electrophoresis (TTGE) followed by direct DNA sequencing was used. RESULTS: Five mutations, p.V201M, p.N287K, c.-8G > C (125G > C), p.M469I and p.S895N, were found in five of the patients. p.N287K occurred in the first transmembrane-spanning domain, p.M469I in the first ATP-binding domain and p.S895N in the second transmembrane-spanning domain, were novel. In addition, seven homozygous and seven heterozygous 5T alleles in the intron 8 poly(T) tract were found. The overall frequency of CFTR mutant alleles in Taiwanese CBAVD males was 26 out of 72 = 36%. This finding was lower than the published frequency of CFTR mutations in other ethnic CBAVD patients (ranging from 50 to 74%). The frequency of p.M470V in Taiwanese CBAVD patients is not significantly different from that in the general population (P = 0.12). CONCLUSIONS: The results of this study add to the short list of Taiwanese/Asian CFTR mutations. Unlike Caucasian patients, the CFTR mutations cannot account for the majority of Taiwanese CBAVD. This is consistent with the low incidence of CF in the Asian/Taiwanese population. Furthermore, the mutation spectrum of CFTR in CBAVD patients does not overlap with the Caucasian CFTR mutation spectrum.     

Two novel missense and one novel nonsense CFTR mutations in Iranian males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia. Nearly 75% of men with CBAVD have at least one detectable common cystic fibrosis (CF) transmembrane conductance regulator (CFTR) mutation. To study the involvement of CFTR mutations in the Iranian population with presumed low CF frequency, we analysed 112 Iranian CBAVD males. Three Iranian CBAVD males with no clinical CF phenotype indicated by a normal karyotype, normal pancreatic function and sweat chloride concentration and no Y chromosome microdeletions were studied for CFTR mutations, IVS8-5T mutations and M470V exon 10 missense polymorphism. The entire coding sequence of each gene was analysed using a combination of the denaturing gradient-gel electrophoresis or by single-strand conformation analysis and direct DNA sequencing. Also, 52 fertile males were tested as controls to rule out polymorphism. This approach allowed us to detect one novel nonsense mutation (K536X) in the nucleotide-binding domain 1 (NBD1) region and two novel missense mutations (Y122H and T338A) in the M2 and M6 regions of CFTR gene in our studied population, which were not reported previously. Also, the conservation of changed nucleotide and amino acid in mutated regions was analysed by aligning with nine different species. K536X nonsense mutation (transversion) was found in the first NBD (NBF1), which plays an important regulatory role in CFTR function. It was, therefore, considered as a severe allele responsible for elevated sweat chloride levels and obstructive azoospermia. Because Y122H and T338A mutations were compound heterozygote with the IVS8-5T, it is difficult to judge the severity of these mutations and their role in the CBAVD phenotype.     

Histological and genetic analysis and risk assessment for chromosomal aberration after ICSI for patients presenting with CBAVD

Intracytoplasmic sperm injection (ICSI) has opened a new field in the treatment of male infertility, leading to a debate concerning its genetic safety. In this study we present an analysis of 11 patients presenting congenital bilateral absence of the vas deferens (CBAVD). In all 11 cases, genetic counselling, histological analysis of testicular biopsies, cystic fibrosis transmembrane conductance regulator (CFTR) mutation screenings of both partners and spermatozoa three-colour fluorescent in-situ hybridization (FISH) analysis were performed. A total of 31 CFTR mutations were screened and mutations were found in eight out of 11 cases, with DeltaF508 being the most common mutation found. Histological analyses showed that seven out of 11 patients had normal tubule/membrane/interstitium (TMI) and Johnsen scores, while the remaining four patients had mild impairment of testicular parenchyma. The average aneuploidy rate was 6.8 +/- 3.9% compared with two control subjects with 4.4 and 5.4% aneuploidy rates respectively, using FISH analysis. After ICSI, the fertilization and pregnancy rates were 66.2 and 22.7% respectively. Thus, in our case of CBAVD, the risk of chromosomal aberration following ICSI, in the absence of a CFTR mutation in the male patient and/or in his partner, was not higher than in normal fertile men. Furthermore, the pregnancy success rate following ICSI of these CBAVD patients was comparable to the general ICSI population, even when histological analysis showed limited spermatogenesis.     

Nasal epithelial ion transport and genetic analysis of infertile men with congenital bilateral absence of the vas deferens

It has been suggested that congenital bilateral absence of thevas deferens (CBAVD), an important cause of male infertility,is a variant of cystic fibrosis (CF). This study describes adefect in chloride conductance across the nasal epithelium ofsubjects with CBAVD which is dissimllar to that found in patientswith CF. It also demonstrates normal sodium transport acrossthe nasal epithelium in these men, in contrast to patients withCF who exhibit increased sodium absorption. The increased frequencyof CFTR mutations in these men implicates the CFTR gene in thepathogenesis of this disorder. Genetic analysis of men withCBAVD who were heterozygous for a known CFTR mutation failedto identify a second mutation within any of the exons or intronsof the CFTR gene. These results demonstrate that most men presentingwith CBAVD are not compound heterozygotes for mutations withinthe CFTR gene and can be distinguished from individuals withatypical or asymptomatic CF on the basis of the bioelectricproperties of their nasal epithelium. We postulate that mutationsin the promoter region or at other regulatory sites of the CFTRgene may be reponsibie for the CBAVD phenotype in a proportionof cases.     

Characterization of cystic fibrosis conductance transmembrane regulator gene mutations and IVS8 poly(T) variants in Portuguese patients with congenital absence of the vas deferens

BACKGROUND: Cystic fibrosis conductance transmembrane regulator (CFTR) gene mutations and IVS8 poly(T) variants in Portuguese patients with bilateral (CBAVD) and unilateral (CUAVD) congenital absence of the vas deferens remain to be evaluated. METHODS: Patient screening was carried out by PCR, denaturing gradient gel electrophoresis and DNA sequencing. RESULTS: CFTR mutations were found in 18 out of 31 (58.1%) CBAVD and in three of four (75%) CUAVD patients. The most frequent mutations were F508del and R334W in CBAVD and G542X in CUAVD, with the allelic frequencies of R334W (6.5%) and G542X (25%) being particular to the Portuguese population. The 5T allelic frequency was 3.5% in the fertile male population, 25% in CUAVD and 27.4% in CBAVD patients. The combined frequency of mutations (CFTR+5T) was increased in CBAVD to 22 out of 31 (71%). The frequency of CFTR mutations was compared with that of patients with secondary obstructive azoospermia (OAZ; one out of 16, 6.3%) and non-obstructive azoospermia (NOAZ; two out of 22, 9.1%) with conserved spermatogenesis, which were similar to the general population. However, whereas the 5T allelic frequency in OAZ was similar to that of the general population (3.1%), it was increased in NOAZ cases (14.3%). CONCLUSIONS: Data confirm that CFTR+5T mutations represent the most common genetic abnormality in CAVD, and suggest that cases of NOAZ may be associated with the 5T allele.     

Mutations of the CFTR gene in Turkish patients with congenital bilateral absence of the vas deferens

BACKGROUND: Mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) can cause congenital bilateral absence of the vas deferens (CBAVD) as a primarily genital form of cystic fibrosis. The spectrum and frequency of CFTR mutations in Turkish males with CBAVD is largely unknown. METHODS: We investigated 51 Turkish males who had been diagnosed with CBAVD at the Hacettepe University, Ankara, for the presence of CFTR gene mutations by direct sequencing of the coding region and exon/intron boundaries. RESULTS: We identified 27 different mutations on 72.5% of the investigated alleles. Two-thirds of the patients harboured CFTR gene mutations on both chromosomes. Two predominant mutations, IVS8-5T and D1152H, accounted for more than one-third of the alleles. Five mutations are described for the first time. With one exception, all identified patients harboured at least one mutation of the missense or splicing type. Presently available mutation panels would have uncovered only 7-12% of CFTR alleles in this population cohort. CONCLUSIONS: Although cystic fibrosis is relatively rare in Turkey, CFTR mutations are responsible for the majority of CBAVD in Turkish males. Because of a specific mutation profile, a population-specific panel should be recommended for targeted populations such as CBAVD in Turkey or elsewhere.     

Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutatations. Mutations in brief no. 138. Online

Congential bilateral aplasia of vas deferens (CBAVD), a form of male sterility, has been suggested to represent a "genital" form of cystic fibrosis (CF), as mutations in the CFTR gene have been identified in most patients with this condition. Interestingly, the 5T allele in intron 8 appeared to be the most frequent mutation associated with CBAVD. However, the molecular basis of CBAVD is not completely understood. We have analysed the complete coding and flanking CFTR sequences by PCR-DGGE in 64 men with CBAVD from southern France with the aim to list any sequence alteration. Fourty-two of the 64 patients (65.6%) had mutations on both copies of the CFTR gene, including one patient with two mutations in the same copy (DF508 + A1067T). The 5T allele was present in 21/64 cases (33%). Six of the 28 different mutations identified in this study had never been described previously, and appeared to be specific to CBAVD (P111L, M244K, A1364V, G544V, 2896insAG,-33G->A).     

MALT1基因2种转录本在正常人和B细胞白血病患者中的表达特点

 目的：比较正常人和B细胞白血病患者外周血中黏膜相关淋巴样组织淋巴瘤转位基因1（MALT1）2种异构体的分布和表达差异。方法：根据MALT1基因的结构特点,设计2对引物,通过建立2种转录本的逆转录聚合酶链式反应（RT-PCR）方法分析8例B细胞急性淋巴细胞白血病(B-ALL)、8例B细胞慢性淋巴细胞白血病（B-CLL）和8例正常人的外周血单个核细胞(PBMC)中MALT1的表达情况。实时定量PCR分析各组样本的MALT1 mRNA表达水平。结果：所有样本中均能得到所预期的PCR产物,其中第1对引物可以扩增出2条片段,分别为MALT1转录本1（包含第5、6、7、8、9外显子）和MALT1转录本2（包含第5、6、8、9外显子）,第2对引物只能扩增出1条包含转录本1的片段,所有PCR产物均通过核苷酸序列分析证实。MALT1 2种转录本均在外周血中有表达,并且MALT1转录本2表达水平高于转录本1。通过灰度分析发现,在B-ALL中,MALT1转录本1的灰度与正常人相比显著增高（P＜0.05）,而MALT1转录本2显著降低（P＜0.05）;在B-CLL中,MALT1 2种转录本的灰度与正常人相比无显著差异（P＞0.05）。此外,B-ALL中,MALT1 mRNA表达水平（中位数：1.253）低于正常人（中位数：1.976）（P=0.05）;而B-CLL中,MALT1 mRNA的表达水平与正常人相比无显著差异（P＞0.05）。结论：MALT1 2种转录本均表达于正常和B细胞白血病样本中,但B-ALL中两者的表达水平有所差异,同时,B-ALL的MALT1 mRNA表达水平也有所降低。B-ALL中MALT1基因表达的异常可能影响MALT1信号通路而与疾病的发生发展相关。     

Cystic fibrosis gene mutations and infertile men with primary testicular failure

It has been proposed that the gene responsible for cystic fibrosis, called the cystic fibrosis transmembrane conductance regulator (CFTR) gene, may play an important role in the process of spermatogenesis. A group of azoospermic men with primary testicular failure underwent CFTR mutation analysis, including assessment of the intron 8 polythymidine tract (IVS8-T tract). An association was not found between CFTR mutations or the 5T variant of the IVS8-T tract and the primary testicular failure phenotype. This finding suggests that CFTR does not play a significant role in the aetiopathogenesis of primary spermatogenic dysfunction. Therefore, the abnormal testicular histological findings in some post-pubertal men with cystic fibrosis may be a result of nutritional deficiency or testicular obstruction rather than a primary defect in spermatogenesis. In addition, the decreased sperm count in oligozoospermic men with CFTR mutations may be secondary to partial reproductive tract obstruction and not abnormal spermatogenesis. Lastly, routine screening of men with primary testicular failure for CFTR gene mutations is not warranted.     

Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutations

Congenital bilateral aplasia of vas deferens (CBAVD), a form of male sterility, has been suggested to represent a "genital" form of cystic fibrosis (CF), as mutations in the CFTR gene have been identified in most patients with this condition. Interestingly, the 5T allele in intron 8 appeared to be the most frequent mutation associated with CBAVD. However, the molecular basis of CBAVD is not completely understood. We have analysed the complete coding and flanking CFTR sequences by PCR-DGGE in 64 men with CBAVD from southern France with the aim to list any sequence alteration. Fourty-two of the 64 patients (65.6%) had mutations on both copies of the CFTR gene, including one patient with two mutations in the same copy (DF508 + A1067T). The 5T allele was present in 21/64 cases (33%). Six of the 28 different mutations identified in this study had never been described previously, and appeared to be specific to CBAVD (P111L, M244K, A1364V, G544V, 2896insAG, -33G→A). Hum Mutat 11:480, 1998. © 1998 Wiley-Liss, Inc.