囊性纤维化跨膜转导调节器在培养人子宫内膜上皮细胞的表达

目的　探讨雌、孕激素对体外培养人子宫内膜腺上皮细胞囊性纤维化跨膜转导调节器(CFTR)基因和蛋白质表达的影响。　方法　采用逆转录 聚合酶链反应(RT PCR)、原位杂交、免疫组织化学方法检测CFTR mRNA和蛋白质表达量的变化。　结果　RT PCR结果显示单纯雌二醇、雌二醇与孕酮合用、对照组均扩增出特异的 CFTR mRNA条带,孕酮组则未见到;原位杂交结果也证实雌二醇刺激CFTR mRNA的表达(P<0.05),孕酮则抑制CFTR mRNA的表达。免疫组织化学检测子宫内膜腺上皮细胞中CFTR蛋白质含量表明雌二醇显著刺激其生成。　结论　卵巢性激素雌二醇上调、孕酮下调体外培养人子宫内膜腺上皮细胞CFTR mRNA及蛋白质的表达,从而调节月经周期不同时相宫腔液微环境中水与电解质含量,适应不同的生殖阶段。     

中国先天性双侧输精管缺如患者CFTR基因全部外显子突变检测

目的:探讨我国先天性双侧输精管缺如患者CFTR基因检测的必要性。方法:采用PCR技术结合DNA直接测序的方法检测9例先天性双侧输精管缺如患者CFTR基因全部外显子的突变情况,并在NCBI和Cystic Fibrosis Mutation Database在线比对。结果:除非编码区突变和已经报道的SNP位点之外,9例先天性双侧输精管缺如患者中4例新发现4种不同于西方人已知突变类型的外显子区突变,均为杂合子错义突变。结论:中国先天性双侧输精管缺如患者CFTR基因外显子区存在不同于西方人的突变,有必要对中国先天性双侧输精管缺如患者进行CFTR基因突变检测。     

R560S: A class II CFTR mutation that is not rescued by current modulators

Background

New therapies modulating defective CFTR have started to hit the clinic and others are in trial or under development. The endeavour of drug discovery for CFTR protein rescue is however difficult one since over 2000 mutations have been reported. For most of these, especially the rare ones, the associated defects, the respective functional class and their responsiveness to available modulators are still unknown. Our aim here was to characterize the rare R560S mutation using patient-derived materials (rectal biopsies and intestinal organoids) from one CF individual homozygous for this mutation, in parallel with cellular models expressing R560S-CFTR and to assess the functional and biochemical responses to CFTR modulators.

Sweat chloride as a biomarker of CFTR activity: Proof of concept and ivacaftor clinical trial data

BackgroundWe examined data from a Phase 2 trial {NCT00457821} of ivacaftor, a CFTR potentiator, in cystic fibrosis (CF) patients with a G551D mutation to evaluate standardized approaches to sweat chloride measurement and to explore the use of sweat chloride and nasal potential difference (NPD) to estimate CFTR activity.MethodsSweat chloride and NPD were secondary endpoints in this placebo-controlled, multicenter trial. Standardization of sweat collection, processing, and analysis was employed for the first time. Sweat chloride and chloride ion transport (NPD) were integrated into a model of CFTR activity.ResultsWithin-patient sweat chloride determinations showed sufficient precision to detect differences between dose-groups and assess ivacaftor treatment effects. Analysis of changes in sweat chloride and NPD demonstrated that patients treated with ivacaftor achieved CFTR activity equivalent to approximately 35%–40% of normal.ConclusionsSweat chloride is useful in multicenter trials as a biomarker of CFTR activity and to test the effect of CFTR potentiators.     

How the sweat gland reveals levels of CFTR activity

CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·P_O·γ) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the simple and widely used sweat chloride test can be calibrated to provide more accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects (hereafter ‘CFTR activity’). Sweating stimulated by β-adrenergic agonists (β-sweat) is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity with carriers = 50% and CF = 0% of HC values set = 100%, but the β-sweat assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting mean sweat chloride values, taken from numerous studies and the CFTR2 database against mean β-sweat rates for CF, carriers and HC. The resulting inverse logarithmic relations indicate that sweat chloride values ≥60 mmol/L occur when CFTR activity is below 1.2% -10% of HC. These are lower than most previous estimates, which resulted from setting nasal potential difference (NPD) as linear rather than logarithmic measures of CFTR activity. Features of the sweat gland coil and duct are used to explain why readouts of CFTR activity are linear for β-sweat and logarithmic for sweat chloride. Sweat chloride values fall steeply for small increments of CFTR activity above zero—the most clinically relevant region. Thus, large health benefits can be achieved by restoring low levels of CFTR activity, especially if this is done before irreversible lung damage.Truncated Abstract: CFTR is an anion channel that causes cystic fibrosis (CF) when its activity, equal to channel number x open probability x conductance (n·P_O·γ) is absent or nearly so. CFTR modulators increase CFTR activity, but estimates of in vivo efficacy vary. This review shows how values from the sweat chloride test can be calibrated to provide accurate estimates of CFTR activity as a percent of the average for healthy control (HC) subjects. Sweating stimulated by β-adrenergic agonists is rate-limited by CFTR, producing a near linear, ratio scale of CFTR activity, but the assay is difficult to use. Here, sweat chloride is calibrated to CFTR activity by plotting it against mean β-sweat rates for different groups. The resulting logarithmic relations indicate that CF sweat chloride values occur when CFTR activity is below 1.2% -10% of HC, and that large health benefits can be achieved by restoring low levels of CFTR activity if this is done early.     

Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.     

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。     

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.     

Cardiovascular complications in cystic fibrosis: A review of the literature

Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to dysfunction of the CFTR protein. CFTR dysfunction leads to disease in the respiratory and gastrointestinal systems. Disorders of the cardiovascular system in individuals with CF are usually attributed to secondary effects from progressive lung disease. However, CFTR has been localized to vascular endothelium and smooth muscle, suggesting that CFTR dysfunction may directly impact cardiovascular function. As treatments for CF improve and life-expectancy increases, the risk of vascular disease may increase in prevalence related to primary and secondary CFTR dysfunction, chronic systemic inflammation, nutritional health and hyperglycemia in individuals with CF related diabetes. Here we review the available literature on CF and the cardiovascular system, examining the secondary effects and evidence for direct CFTR dysfunction in the heart, aorta, pulmonary vessels, and vasculature, as well as future directions and treatment options.     

CFTR stabilizes ENaC at the plasma membrane

CFTR was reported to regulate ENaC channel opening, decreasing ENaC activity in airways and increasing it in sweat ducts. We generated MDCK-I cell lines stably expressing tagged alphabetagammaENaC+CFTR or ENaC alone, and developed an assay to quantify cell-surface half-life of ENaC. Surprisingly, we found that co-expressed CFTR stabilizes ENaC at the plasma membrane, suggesting that CFTR regulates ENaC stability, not just opening.     

ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD :(1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or(2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or(3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.     

CFTR突变基因不影响先天性梗阻性无精子症患者ICSI治疗的成功率

为探讨囊性纤维化跨膜转运调节物（Cystic Fibrosis Transmembrane Conductance Regulator,CFTR)基因突变是否影响先天性梗阻性无精子症患者单精子卵浆内注射（Intracytoplasmic Sperm Injection,ICSI)治疗的成功率,本文对3例先天性梗阻性无精子症CFTR突变基因携带者和18例CFTR突变基因非携带者进行了ICSI的治疗。结果表明：先天性梗阻性无精子症CFTR突变基因携带者与CFTR突变基因非携带者ICSI治疗时受精率、卵裂率和妊娠率无显著性差异。结论：CFTR突变基因并不影响先天性梗阻性无精子症患者ICSI治疗的成功率,故这些患者在ICSI治疗前夫妻双方更有必要行CFTR突变基因的筛查。     

Defects in processing and trafficking of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is caused by inherited mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in epithelial tissues. Most mutations in CF patients result in rapid intracellular degradation of the CFTR protein. While this defect is thought to result from abnormal protein folding, it is unclear how mutant and wild-type (WT) proteins differ in structure, how the cell is able to distinguish these differences, and how the fate of the mutant protein is determined. By examining the initial steps of CFTR assembly into the endoplasmic reticulum (ER) membrane, it has recently been shown that CFTR utilizes two redundant translocation pathways to direct N-terminus folding events. Mutations that block one pathway therefore do not alter transmembrane topology, but rather appear to disrupt intracellular trafficking through perturbations in higher order tertiary structure. These studies suggest that cellular quality control machinery acts at least in part, by monitoring proper interactions between CFTR subdomains. The end result of this process is the conversion of misfolded CFTR into a membrane-bound, polyubiquitinated complex. This complex recruits cytosolic degradation machinery to the endoplasmic reticulum membrane where CFTR is degraded as it is extracted from the lipid bilayer. Understanding how cellular machinery mediates this process will be an important step in designing strategies to modify protein folding and degradation in CF and related ion channelopathies.     

Caution advised in the use of CFTR modulator treatment for individuals harboring specific CFTR variants

In December 2020, the U.S. Food and Drug Administration (FDA) expanded the list of CFTR variants approved for treatment with CFTR modulators drugs from 39 to 183. Clinicians should be aware that individuals harboring certain variants approved for treatment may not respond to or benefit from this therapy. After review, the expert panel leading the CFTR2 project identified four categories of variants that may not result in a clinical response to modulator treatment: 15 variants assigned as non CF-causing; 45 variants of unknown significance; six variants known or suspected to cause mis-splicing as their primary defect rather than an amino acid substitution; and eight variants known to occur together in cis with another deleterious variant not expected to lead to CFTR protein (nonsense or frameshift). The potential risks and benefits of CFTR modulator therapy should be considered carefully for individuals harboring these variants.     

Rescue of CFTR NBD2 mutants N1303K and S1235R is influenced by the functioning of the autophagosome

The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4?+?C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4?+?C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.     

Amplifiers co-translationally enhance CFTR biosynthesis via PCBP1-mediated regulation of CFTR mRNA

BackgroundCystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity.MethodsBiochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics.ResultsAmplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness.ConclusionsSmall molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.     

High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease.

We performed the complete screening of the CFTR gene in a group of 31 patients with COPD in order to investigate the impact of mutations and polymorphisms in the CFTR gene. The cumulative frequency of CFTR mutations (17.74%) was significantly higher than in our general population (P < 0.0001). The R75Q was significantly overrepresented in COPD patients (8.06%; P = 0.002). In all patients carrying the R75Q chronic bronchitis was a dominant symptom of COPD, and all were homozygous for the V470 allele. These findings suggest that R75Q mutation could be characteristic CFTR variant for COPD patients.     

Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.

BackgroundCystic fibrosis (CF) is the most common inherited disorder in Caucasian populations, with over 1400 mutations identified in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Mutations in the CFTR gene may be also causative for CBAVD (Congenital Bilateral Absence of the Vas Deferens). The type and distribution of mutations varies widely between different countries and/or ethnic groups, and is relatively unknown in Iran. We therefore performed a comprehensive analysis of the CFTR gene in Iranian CF patients.Methods69 Iranian CF patients, and 1 CBAVD patient, were analysed for mutations in the complete coding region, and its exon/intron junctions, of their CFTR genes, using different methods, such as ARMS (amplification refractory mutation system)-PCR, SSCP (single stranded conformation polymorphism) analysis, restriction enzyme digestion analysis, direct sequencing, and MLPA (Multiplex Ligation-mediated Probe Amplification).ResultsCFTR mutation analysis revealed the identification of 37 mutations in 69 Iranian CF patients. Overall, 81.9% (113/138) CFTR genes derived from Iranian CF patients could be characterized for a disease-causing mutation. The CBAVD patient was found to be homozygous for the p.W1145R mutation. The most common mutations were p.F508del (ΔF508) (18.1%), c.2183_2184delAAinsG (2183AA > G) (6.5%), p.S466X (5.8%), p.N1303K (4.3%), c.2789 + 5G > A (4.3%), p.G542X (3.6%), c.3120 + 1G > A (3.6%), p.R334W (2.9%) and c.3130delA (2.9%). These 9 types of mutant CFTR genes totaled for 52% of all CFTR genes derived from the 69 Iranian CF patients. Eight mutations, c.406-8T > C, p.A566D, c.2576delA, c.2752-1_2756delGGTGGCinsTTG, p.T1036I, p.W1145R, c.3850-24G > A, c.1342-?_1524 + ?del, were found for the first time in this study.ConclusionsWe identified 37 CFTR mutations in 69 well characterized Iranian CF patients, obtaining a CFTR mutation detection rate of 81.9%, the highest detection rate obtained in the Iranian population so far. These findings will assist in genetic counseling, prenatal diagnosis and future screening of CF in Iran.