Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation associated with a congenital bilateral absence of vas deferens

Abstract:   Cystic fibrosis transmembrane conductance regulator ( CFTR ) gene mutations associated with cystic fibrosis have been reported to be rare in Japanese patients with congenital bilateral absence of vas deferens (CBAVD).
A 28-year-old Japanese male was referred for infertility. Vas deferens and epididymis were not palpable bilaterally. Semen analyses showed azoospermia with volumes below 2.0 ml. Serum follicle-stimulating hormone value was slightly elevated. Seminal fructose concentration was also very low. Scrotal ultrasonography showed absence of the bodies and tails of the right and left epididymides. Imaging studies showed cystic dysplasia of the right seminal vesicle and agenesis of the left seminal vesicle. A CFTR gene mutation of I556V was found.
Recent studies show that prevalence of CFTR gene mutation in Japanese CBAVD patients may be approximately equal to that of the Caucasian population. Genetic counselling may be recommended for any couple attempting assisted reproduction technology when the man has CBAVD.     

聚合酶链反应单链构象多态银染技术检测国人先天性双侧输精管缺如患者囊性纤维化跨膜转运调节物基因突变

为了探讨囊性纤维化跨膜转运调节物 (CFTR)基因在中国人先天性双侧输精管缺如 (CBAVD)患者中的突变频率及热点 ,应用聚合酶链反应 -单链构象多态 (PCR-SSCP)、银染技术及 PCR产物直接序列分析的方法检测了 32例 CBAVD患者 CFTR基因第 2、3、4、5、6 a、8、10、11、12、13、15A、17b、19A、2 0、2 1、2 3外显子区域上的突变情况。结果 :2例存在 PCR- SSCP电泳迁移的改变 ,经测序确认了 CFTR基因突变的性质。结论 :PCR- SSCP银染技术检出中国人 CBAVD患者 CFTR基因突变为 :ΔF50 8,2 2 5del C。所以此方法作为检测 CFTR基因突变是可行的     

先天性双侧输精管缺如患者囊性纤维化跨膜转运调节物基因⊿F508突变的研究

目的研究男性先天性双侧输精管缺如(CBAVD)患者其囊性纤维化跨膜转运调节物(CFTR)基因第10号外显子区域上△F508的突变情况。方法应用聚合酶链反应(PCR)及PCR产物直接测序的方法检测了32例CBAVD患者和32例健康生育男性CFTR基因第10号外显子区域上△F508的突变情况。结果两组检查均没有发现△F508突变。结论△F508突变是国外白种人CBAVD患者最常见的一种基因突变,而对中国人来说检测该突变意义可能不大。     

先天性双侧输精管缺如患者囊性纤维化跨膜传导调节因子基因的检测

目的 探讨先天性双侧输精管缺如(CBAVD)患者与囊性纤维化跨膜传导调节因子(CFTR)基因突变的关系. 方法 收集2007年5月至2009年5月85例CBAVD患者.CBAVD诊断依据:无精子;性激素4项正常;双侧输精管未触及;双睾丸体积正常,附睾饱满淤积.另设健康已生育男性85例作为对照.抽取外周血,应用聚合酶链反应-单链构象多态及PCR产物直接序列测定法检测患者及对照组CFTR基因第10,11外显子,比较两组的突变情况. 结果 CBAVD组85例,CFTR基因突变10例,占11.8％,分别是I556V突变4例,M469V突变2例,E527N、△F508、L558S、S485C各1例.对照组85例均未见突变.两组间比较差异有统计学意义(x2=8.606,P=0.003).结论 CBAVD主要由CFTR基因突变引起,CFTR基因突变的位点与频率与西方白种人有所不同.     

Congenital bilateral absence of the vas deferens and cystic fibrosis

Summary CF and CBAVD are really just ends of a clinical spectrum. The type and nature of the mutations in the CF gene probably determine the phenotypic expression of the patient. Perhaps all patients homozygous for F508, for example, will have severe pulmonary and pancreatic disease as well as absent vasa, whereas those with other combinations, such as F508/D1270N, will be unaffected in terms of pulmonary and pancreatic function but will have absent vasa. Besides contributing to a better understanding of the nature of CBAVD, this linkage of CF and CBAVD most importantly mandates genetic screening and counseling for appropriate family members and even the patient's spouse. In addition, a broader understanding of CF is now at hand, as this brings a whole new cohort of patients under the CF umbrella. Many of these will have at least one, if not two, rare or novel CF gene mutations. Once all of these mutations have been detected and defined, our knowledge of the CF gene, its mutations, and their implications will be dramatically expanded.     

CFTR gene mutations in Japanese individuals with congenital bilateral absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is a monosymptomatic disease confined to the male reproductive system with similarity to the phenotype of cystic fibrosis (CF), and mutations in the CFTR gene are highly prevalent in Caucasian CBAVD patients. While CF is very rare in Japan, CBAVD is not. Our previous study demonstrated high prevalence of the 5T allele in the CFTR gene in Japanese CBAVD patients. We analyzed whole exons of the CFTR gene in 19 CBAVD patients and 53 normal individuals using polymerase chain reaction amplification-single strand conformation polymorphism analysis and direct sequencing. Three missense mutations (W216X, G1349S, Q1352H) were found in seven CFTR alleles, and the 5T allele was positive in 11 of 38 CFTR patient alleles. Consequently, 47% of CFTR chromosomes in the patients were affected, and 11 individuals (58%) had at least one mutated CFTR allele. In contrast, three of 53 normal individuals (5.7%) had a missense mutation in one of the CFTR genes, but no 5T allele was detected (both P<0.0001). Mutations of the CFTR gene are closely associated with Japanese patients with CBAVD.     

The cystic fibrosis transmembrane regulator (CFTR) in the kidney

The cystic fibrosis transmembrane regulator (CFTR) is a Cl - channel. Mutations of this transporter lead to a defect of chloride secretion by epithelial cells causing the Cystic Fibrosis disease (CF). In spite of the high expression of CFTR in the kidney, patients with CF do not show major renal dysfunction, but it is known that both the urinary excretion of drugs and the renal capacity to concentrate and dilute urine is deficient. CFTR mRNA is expressed in all nephron segments and its protein is involved with chloride secretion in the distal tubule, and the principal cells of the cortical (CCD) and medullary (IMCD) collecting ducts. Several studies have demonstrated that CFTR does not only transport Cl - but also secretes ATP and, thus, controls other conductances such as Na+ (ENaC) and K+ (ROMK2) channels, especially in CCD. In the polycystic kidney the secretion of chloride through CFTR contributes to the cyst enlargement. This review is focused on the role of CFTR in the kidney and the implications of extracellular volume regulators, such as hormones, on its function and expression.     

Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols

Congenital bilateral absence of vas deferens （CBAVD） is a manifestation of the mildest form of cystic fibrosis （CF） and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator （CFTR） gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach： （i） first, a reverse dot-blot analysis was performed to detect mutations with regional impact; （ii） next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and （iii） finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.     

先天性双侧输精管缺如患者睾丸组织中CFTR蛋白的表达及意义

目的通过观察中国CBAVD患者CFTR蛋白在睾丸组织中的表达水平,探讨其与睾丸生精功能之间的关系。方法免疫组化检测66例中国先天性双侧输精管缺如患者睾丸组织CFTR蛋白表达水平并对睾丸生精功能进行评分,探讨先天性双侧输精管缺如患者CFTR蛋白的表达情况以及其与睾丸生精功能的关系。结果免疫组化结果显示CFTR蛋白可表达于睾丸内支持细胞及生精上皮细胞,以细胞膜和细胞浆为主;CFTR蛋白在CBAVD患者睾丸生精上皮及支持细胞的表达以阳性和弱阳性为主,比例分别为50%(33/66)和37.87%(25/66),部分患者可见阴性表达,比例为4.54%(3/66);睾丸生精Johnsen评分7~10分的比例分别为4.45%(3/66)、40.9%(27/66)、42.42%(28/66)和12.23%(8/66)。部分患者存在生精功能障碍。CFTR蛋白在睾丸组织中的表达与生精功能强正相关性,相关系数为0.785(P<0.01)。结论BAVD患者睾丸内的CFTR蛋白表达以阳性和弱阳性为主;部分CBAVD患者存在生精功能障碍,CFTR蛋白在睾丸组织中的表达水平与生精功能存在强正相关性。     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis

BackgroundThe pancreas is one of the primary organs affected by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. While exocrine pancreatic insufficiency is a well-recognized complication of cystic fibrosis (CF), symptomatic pancreatitis is often under-recognized.ResultsThe aim of this review is to provide a general overview of CFTR mutation-associated pancreatitis, which affects patients with pancreatic sufficient CF, CFTR-related pancreatitis, and idiopathic pancreatitis. The current hypothesis regarding the role of CFTR dysfunction in the pathogenesis of pancreatitis, and concepts on genotype–phenotype correlations between CFTR and symptomatic pancreatitis will be reviewed.Symptomatic pancreatitis occurs in 20% of pancreatic sufficient CF patients. In order to evaluate genotype–phenotype correlations, the Pancreatic Insufficiency Prevalence (PIP) score was developed and validated to determine severity in a large number of CFTR mutations. Specific CFTR genotypes are significantly associated with pancreatitis. Patients who carry genotypes with mild phenotypic effects have a greater risk of developing pancreatitis than patients carrying genotypes with moderate–severe phenotypic consequences at any given time.ConclusionsThe genotype–phenotype correlation in pancreatitis is unique compared to other organ manifestations but still consistent with the complex monogenic nature of CF. Paradoxically, genotypes associated with otherwise mild phenotypic effects have a greater risk for causing pancreatitis; compared with genotypes associated with moderate to severe disease phenotypes. Greater understanding into the underlying mechanisms of disease is much needed. The emergence of CFTR-assist therapies may potentially play a future role in the treatment of CFTR-mutation associated pancreatitis.     

Assisted reproduction technology for patients with congenital bilateral absence of vas deferens

PURPOSE: We investigate the frequency of cystic fibrosis transmembrane conductance regulator gene mutations in Japanese patients with congenital bilateral absence of the vas deferens, and assess treatment outcomes of assisted reproduction interventions. MATERIALS AND METHODS: In 10 Japanese patients with bilateral congenital absence of the vas deferens genetic analysis was performed for known frequent mutations of the cystic fibrosis transmembrane conductance regulator gene using polymerase chain reaction amplification followed by dot-blot hybridization with the allele-specific oligonucleotide probes and direct sequencing. Intracytoplasmic sperm injection using spermatozoa retrieved from the testes was performed in 7 of the couples. RESULTS: No known mutations of the gene were detected in the patients. However, analysis of the polythymidine tract polymorphism in intron 8 revealed 30% allele frequency of 5T. Pregnancy was achieved in 7 cycles of intracytoplasmic sperm injection using spermatozoa retrieved from the testes. CONCLUSION: The 5T variant in intron 8 polythymidine tract was identified with high allelic frequency in Japanese patients with congenital bilateral absence of the vas deferens, suggesting that the disease in Japan is also partially caused by this particular mutation of the cystic fibrosis transmembrane conductance regulator gene. Modern assisted reproduction technology offers an important option for patients with congenital bilateral absence of the vas deferens.     

Molecular screening of CFTR gene in Egyptian patients with congenital bilateral absence of the vas deferens: a preliminary study

In the current study, we enrolled 14 Egyptian infertile males with isolated congenital bilateral absence of the vas deferens (CBAVD). Screening for the most commonly reported 36 CFTR mutations, and the intron 8 (T)n splice variant was performed by multiplex PCR followed by reversed hybridisation. Samples with the 5T variant were picked for DNA sequencing of intron 8/exon 9 region to identify the number of adjacent TG repeats. The p.Phe508del and the p.Ser1251Asn mutations were detected in heterozygous state in three patients (10.7% of alleles) and in one patient (3.6% of alleles), respectively, while the 5T variant was detected in five patients (28.6% of alleles). Among those five patients, four had TG12 repeats and one had TG13 repeats confirming the pathogenic penetrance of all 5T alleles in Egyptian CBAVD patients. The allelic frequencies of the mutations p.Phe508del, p.Ser1251Asn and the 5T variant in 60 Egyptian cystic fibrosis patients were 24.2%, 3.3% and 2.5% respectively. The mutation p.Ser1251Asn was detected for the first time in isolated CBAVD patient in our study. Due to the high prevalence of p.Phe508del mutation and 5T variant in Egyptian CBAVD patients, we recommend their screening initially, ideally followed by full CFTR gene sequencing in unidentified patients.     

The CFTR polymorphisms poly-T,TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.     

Sperm retrieval and intracytoplasmic sperm injection outcomes in men with cystic fibrosis disease versus congenital bilateral absence of the vas deferens

Recent data suggest that cystic fibrosis transmembrane conductance regulator(CFTR)gene alterations negatively impact male fertility beyond obstruction.We sought to compare gene alterations,sperm retrieval rates,and intracytoplasmic sperm injection(ICSI)outcomes among men with cystic fibrosis(CF)disease and congenital bilateral absence of the vas deferens(CBAVD)only.We retrospectively evaluated all men who underwent surgical sperm retrieval at two academic,high-volume andrology centers from 2010 to 2018.Only men with documented CFTR alterations and obstructive azoospermia from either CBAVD or CF were included.Differences between groups for CFTR abnormality,sperm retrieval,and ICSI outcomes were statistically analyzed.Overall,39 patients were included with 10 in the CF and 29 in the CBAVD groups.Surgical sperm retrieval rates were significantly lower in the CF group for sperm concentration(14.8×10⁶ml^-1vs 61.4×10⁶ml^?1,P=0.02)and total motile sperm count(2.9 million vs 11.4 million,P=0.01).This difference was only predicted by homozygous delta F508 CFTR mutations(P<0.05).The CF group also demonstrated a significantly higher rate of rescue testicular sperm extraction(70.0%vs 27.6%,P<0.03)and lower fertilization rate with ICSI(32.5%vs 68.9%,P<0.01).In conclusion,those with CF demonstrated lower sperm quality,greater difficulty with sperm retrieval,and worse ICSI outcomes compared with CBAVD-only patients.Homozygous delta F508 CFTR mutations appear to significantly impair spermatogenesis and sperm function.     

先天性单侧输精管缺如合并无精子症CFTR基因突变检测

目的:探讨先天性单侧输精管缺如(CUAVD)合并无精子症患者囊性纤维化跨膜转导(CFTR)基因全外显子检测的结果与意义。方法:抽取CUAVD合并无精子症6例患者外周血行CFTR全外显子突变及多态性检测,测序结果与UCSC Genome Browser on Human Dec.2013 Assembly进行在线比对及分析。结果:6例CUAVD合并无精子症患者中,1例第6号外显子中可检测到1个已知错义突变c.592GC,2例患者第10号外显子前非编码区域发现c.1210-12T[5]剪切突变,且该2例患者合并第11号外显子上V470单倍体。结论:CUAVD合并无精子症患者CFTR全外显子基因突变有一定的检出率,有必要对这部分患者进行CFTR基因突变检测。