CFTR gene mutations in sarcoidosis

Sarcoidosis is a complex disease of multiorgan granulomatous inflammation. Genetic susceptibility is involved in the pathogenesis of the disorder. Two successive studies from Italy have shown a high frequency of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suffering from sarcoidosis. We have genotyped a panel of 63 families with two or more affected siblings for the CFTR gene mutation R75Q, which was found to be present in three of 26 cases of the Italian study. Although R75Q was present in seven families, it was neither associated with the sarcoidosis phenotype in the German population (P=0.5), nor was it linked to sarcoidosis (P=0.54). In addition, a screening for 34 functional CFTR mutations was performed in a subset of 54 patients from 25 families. These patients were known to be concordant for at least one parental copy of the CFTR gene. With the exception of the mayor CF mutation deltaF508, which was present in three patients and absent in one patient from two families, we did not find any other CF mutation in these 54 patients. Our results do not support the hypothesis that CFTR mutations have a major influence on the pathogenesis of sarcoidosis.     

Identification and characterization of CFTR gene mutations in Indian CF patients

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study was performed on Indian CF patients (n = 50) to investigate the spectrum of mutations in the CFTR gene and their association with intragenic and extragenic marker haplotypes. We report identification of 14 previously known and eight novel mutations, namely 3986-3987delC, 876-6del4, 1792InsA, L69H, S158N, Q493L, I530L and E1329Q. The frequency of delta F508 was found to be 27%. Absolute linkage between delta F508 and the KM.19-GATT-TUB9-M470V-T854T haplotype (2-2-1-1-1) predicts a relatively recent appearance of delta F508 in Indian CF patients. Low frequency of delta F508 mutation and detection of eight novel and thirteen rare mutations reflect a heterogeneous spectrum of mutations in Indian CF patients. Failure to detect mutations in 34% of alleles indicates the possible presence of gross deletions involving one or more exons or may indicate the location of the molecular defects in either the noncoding parts of the gene or in the promoter region, which warrants analysis of those regions.     

Identification of 5 novel mutations in the AGXT gene

In order to identify additional genotypes in primary hyperoxaluria type 1, we sequenced the AGXT genes of 9 patients. We report 5 new mutations. Three are splice-site mutations situated at the end of intron 4 and 8 (647-1G>A, 969-1G>C, 969-3C>G), one is a missense mutation in exon 5 (D183N), and one is a short duplication in exon 2 (349ins7). Their consequence is always a lack of enzymatic activity of the Alanine-Glyoxylate Aminotransferase (AGT); for 4 of them, we were able to deduce that they were associated to the absence of AGT protein. These mutations are rare, as they have been found on one allele in our study (except 969-3C>G present in 2 unrelated families), and have not been previously reported.     

Identification of seven novel mutations in the GAN gene

Giant axonal neuropathy (GAN) is a severe early onset neurodegenerative disorder affecting both the peripheral nerves and the central nervous system. The diagnosis is based on the presence of characteristic giant axons on nerve biopsy. In GAN, the integrity of the intermediate filament network is altered. Indeed, abnormal accumulation of the intermediate filaments has been reported in different cell types, including in the swollen axons, which are filled with neurofilaments. We identified the defective protein, gigaxonin, of unknown function, and reported fourteen distinct mutations in twelve families of various origins. Two additional mutations have been recently reported. In the present study, we analysed the GAN gene in 6 families, and identified seven novel mutations: three nonsense and two missense mutations and two deletions. In addition, the molecular result for an already reported family was re-evaluated. In this family, the R269Q "polymorphism" is in fact the pathogenic mutation.     

Identification of 12 novel mutations in the SLC3A1 gene in Swedish cystinuria patients

Cystinuria is an autosomal recessive disorder that affects luminal transport of cystine and dibasic amino acids in the kidneys and the small intestine. Three subtypes of cystinuria can be defined biochemically, and the classical form (type I) has been associated with mutations in the amino acid transporter gene SLC3A1. The mutations detected in SLC3A1 tend to be population specific and have not been previously investigated in Sweden. We have screened the entire coding sequence and the intron/exon boundaries of the SLC3A1 gene in 53 cystinuria patients by means of single strand conformation polymorphism (SSCP) and DNA sequencing. We identified 12 novel mutations (a 2 bp deletion, one splice site mutation, and 10 missense mutations) and detected another three mutations that were previously reported. Five polymorphisms were also identified, four of which were formerly described. The most frequent mutation in this study was the previously reported M467T and it was also detected in the normal population with an allelic frequency of 0.5%. Thirty‐seven patients were homozygous for mutations in the SLC3A1 gene and another seven were heterozygous which implies that other genes may be involved in cystinuria. Future investigation of the non‐type I cystinuria gene SLC7A9 may complement our results but recent studies also suggest the presence of other potential disease genes. Hum Mutat 18:516–525, 2001. © 2001 Wiley‐Liss, Inc.     

Identification of rare and novel mutations in the CFTR genes of CF patients in Southern England

Cystic fibrosis patients referred to two genetics centres in southern England and not found to carry common CF-associated mutations in one or both of their CFTR genes have been subjected to an extensive mutation search. The whole of the coding region of the CFTR gene, all intron–exon boundaries and 5′ and 3′ untranslated regions have been examined by a combination of single stranded conformational polymorphism analysis and chemical mismatch detection; 48 chromosomes with rare mutations have been identified, including 7 novel mutations, 182delT in exon 1, G27X in exon 2, Q151X in exon 4, Q220X in exon 6a, Q525X in exon 10, 3041delG in exon 16, and 4271delC in exon 23. © 1994 Wiley-Liss, Inc.     

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected—K166E, L568X, and 3121-2 A→G (in homozygosity)—accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations. Hum Mutat 9:136–147, l997. © 1997 Wiley-Liss, Inc.     

Screening practices for mutations in the CFTR gene ABCC7

Cystic fibrosis transmembrane conductance regulator (CFTR) gene studies are now one of the most frequent activities in clinical molecular genetics laboratories. The number of requests is growing, owing to the increasingly wide range of recognized CFTR gene diseases (cystic fibrosis, congenital bilateral absence of the vas deferens, disseminated bronchiectasis, allergic bronchopulmonary aspergillosis and chronic pancreatitis), and the availability of efficient molecular tools for detecting mutations. A growing number of tests capable of simultaneously detecting several frequent CF mutations are being developed, and commercial kits are now available. The most recent kits detect nearly 90% of defective alleles in Caucasians, a rate high enough for carrier screening and for the majority of diagnostic requests. However, because of the wide variety of molecular defects documented in the CFTR gene, only a limited number of laboratories have mastered the entire panoply of necessary techniques, while other laboratories have to refer certain cases to specialized centers with complementary and/or scanning tools at their disposal. A good knowledge of CFTR diseases and their molecular mechanisms, together with expertise in the various techniques, is crucial for interpreting the results. Diagnostic strategies must take into account the indication, the patient's ethnic origin, and the time available in the framework of genetic counseling. This review presents the methods most frequently used for detecting CFTR gene mutations, and discusses the strategies most suited to the different clinical settings.     

经典型苯丙酮尿症苯丙氨酸羟化酶基因的新突变鉴定

目的研究经典型苯丙酮尿症(phenylketonuria, PKU)基因突变.方法应用聚合酶链反应,单链构象多态分析和DNA直接测序等技术,对内蒙古地区32个PKU家系苯丙氨酸羟化酶(phenylalanine hydroxylase, PAH)基因第3～12外显子进行了鉴定分析. 结果检出14种PAH基因点突变R243Q (12/64)、Y356X(6/64)、Y204C(5/64)、R261Q(2/64)、Y161S(2/64)、R252Q(1/64)、R111X(2/64)、D282G(1/64)、S303P(1/64)、G239D(1/64)、R413P(1/64)、IVS7nt+2(2/64)、IVS4nt+3(1/64)、IVS9nt+34(2/64),经检索国际PAH基因突变数据统计库(截至到2004年7月),确认IVS4nt+3(G>C)、IVS9nt+34(G>A)为国际首次发现的新突变,S303P(T>C) 、D282G(A>G)为国内首次报道的新突变.结论内蒙古人群苯丙氨酸羟化酶基因存在突变的多样性,R243Q、Y356X、Y204C是PAH基因的突变热点.     

Identification of novel mutations in the PCCB gene in European propionic acidemia patients

Propionyl‐CoA carboxylase (PCC) is a biotin‐dependent enzyme located in the mitochondrial matrix. Mutations in the PCCA and PCCB genes, which encode the a and b subunits of this heteropolymer, result in propionic acidemia (PA). We report the molecular analysis of b‐deficient patients from Spain and Austria. Subjects were screened for defects affecting the PCCB gene by direct sequencing from genomic PCR products, restriction digests and mRNA analysis by RT‐PCR. Study by western blot of the presence of immunoreactive b‐PCC protein was also performed. A total of four novel sequence variations were found including the point mutations V205D, and M442T, and the frameshift mutation 790‐791insG. Additionaly, a new point change, L17M, was identified on the same allele as 790‐791insG. The missense changes described above were not found in at least 40 control chromosomes analyzed. The Austrian patients were homozygous for V205D. One of the Spanish subjects was heterozygous for M442T and the known mutation c1170insT. The other Spanish patient carried L17M+790‐791insG on one allele, and the described mutation E168K on the other mutant chromosome. The mutations V205D and M442T were confirmed at RNA level and also we have detected the presence of immunoreactive b‐PCC protein translated from these mutant alleles. The patient having L17M+790‐791insG and E168K also presented immunoreactive b‐PCC protein. However, no cDNA product was obtained from the chromosome carrying L17M+790‐791insG. We propose that 790‐791insG, which causes a frameshift and a premature stop codon, is responsible for this finding. In any case, the translation from this mutant cDNA would produce a severily truncated peptide and, in consequence, a non‐functional protein. Expression analysis of all these changes will help us to clarify their structural/functional consequences. Hum Mutat 14:89–90, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of a novel mutation of CFTR gene in a Korean patient with cystic fibrosis

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in Caucasians, but rare in Asians. The mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene are responsible for CF. To date, less than 5 cases of CF have been reported and a few of them diagnosed based on the genotype of the CFTR gene in Korea. We encountered a 4-month-old Korean infant with CF and the diagnosis was confirmed by CFTR gene mutation analysis. The patient underwent surgical operation, due to meconium ileus at birth. He suffered by recurrent respiratory infections, failure to thrive, fatty liver with hepatomegaly, and cholestasis. The mutations of the CFTR gene were identified in the patient and his parents. The patient was a compound heterozygote with a nonsense mutation of c.263T>G, resulting in an amino acid change of p.Leu88X in exon 3. It was previously described in a Korean patient with CF. The other is a novel mutation; c.2089-2090insA mutation (p.Arg697LysfsX33) in exon 13. The mutation c.263T>G was inherited from his father, and the c.2089-2090insA mutation from his mother. Respiratory infection was recovered by supportive care, and cholestasis was improved slowly with sufficient feeding and supplementation of pancreatic exocrine enzymes. He is 19- month old now and shows catch-up growth. We report a novel CFTR mutation in a Korean infant with CF.     

Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome

The SLC12A3 gene encodes the thiazide-sensitive Na-Cl co-transporter (NCCT) expressed in the apical membrane of the distal convoluted tubule of the kidney. Inactivating mutations of this gene are responsible for Gitelman syndrome (GS), a disorder inherited as an autosomal recessive trait. We searched for SLC12A3 gene mutations in 21 Italian patients with the clinical and biochemical features of GS (hypokalemia, hypomagnesemia, metabolic alkalosis, hypocalciuria, and the absence of nephrocalcinosis). All coding regions with their intron-exon boundaries were analyzed using PCR and SSCP techniques followed by sequencing analysis. We identified 21 different mutations evenly distributed throughout the gene without any mutation hot-spot. Fifteen are novel variants, including 12 missense mutations, one deletion, one deletion-insertion and one splice site mutation: R158Q, T163M, W172R, G316V, G374V, G463E, A464T, S615W, V677M, R852S, R958G, C985Y, 2114-2120delACCAAGT, 2144-2158delGCCTTCTACTCGGATinsTG, and 531-2A>G.     

Molecular screening of the CFTR gene in men with anomalies of the vas deferens: identification of three novel mutations

Many studies have shown that congenital absence of the vas deferens (CAVD) is a genital cystic fibrosis transmembrane conductance regulator (CFTR)-mediated phenotype, with a broad spectrum of abnormalities causing male infertility. The genotype of these patients includes mutations in the CFTR gene, e.g. DeltaDeltaF508, R117H and the T5 allele; all of which are commonly found in CAVD. In this study we have screened the entirety of CFTR gene in 47 males with anomalies of the vas deferens: 37 cases of congenital bilateral absence of the vas deferens, three cases of congenital unilateral absence of the vas deferens and seven cases of obstructive azoospermia with hypoplastic vas deferens. Among the 94 chromosomes studied, 65 mutations, of which three are novel (2789+2insA, L1227S, 4428insGA), were identified. The majority of patients (63.8%) had two detectable CFTR gene mutations. Furthermore, high frequencies of the DeltaDeltaF508 mutation (44.7%), the T5 allele (36.2%) and R117H mutation (19.1%) were observed.     

Identification of novel mutations in the RB1 gene in Mexican patients with retinoblastoma

Retinoblastoma (RB) is a childhood tumor of the eye with an average incidence of one case in every 15,000-20,000 live births and occurs in sporadic or hereditary form. This cancer results from loss or inactivation of the RB1 gene located at 13q14.1. This gene encodes for a 110 Kd nuclear phosphoprotein (pRB) that plays a major role in cell proliferation control. Different types of mutations in the RB1 gene have been reported, but point mutations are the most common. There are no molecular studies on RB1 gene mutation in Mexican patients. In this study, 19 patients with bilateral or unilateral RB were analyzed. Genetic and cytogenetic studies were carried out. Detection of RB1 gene mutations was done using single-strand conformational polymorphism (SSCP). Five conformational polymorphisms were identified in different exons. In all cases, SSCP sequence showed new non-described mutations that produced a frameshift on the open reading frame. The identification of mutations in the RB1 gene contributes to basic knowledge of this neoplasia and permits the possibility to offer adequate genetic counseling to relatives at risk.     

两个斑驳病家系KIT基因突变研究

目的 对2个斑驳病家系进行致病基因突变分析,为患者及其家系成员提供遗传咨询和生育指导.方法 分别采集家系1两例患者(先证者及其父亲)、家系2先证者及3名表型正常家系成员的外周血,提取外周血DNA和RNA.应用PCR、逆转录PCR及测序等技术,从基因组水平和表达水平对此两家系先证者和患者进行KIT基因诊断,并初步探讨检测到的突变对KIT基因功能的影响.结果 家系l中两例患者KIT基因均存在IVS12+ 2_+7delinsACATCTTTA的杂合突变,该突变在cDNA水平导致KIT基因c.1765-1779del突变,在氨基酸水平导致p.Gly592Ala/del:E12突变,使得KIT基因剪切位点发生改变,即其中一条cDNA第12外显子被跨越、未转录.家系2中先证者KIT基因存在c.2401A＞C突变,3位表型正常的家系成员未见该突变.结论 确诊了两个斑驳病家系的致病原因.家系1患者KIT基因均存在IVS12+ 2_+ 7delinsACATCTTTA的杂合突变,该突变为人类基因突变数据库未记载的、新的剪切突变;家系2先证者KIT基因存在c.2401A＞C突变,结合3位表型正常的家系成员KIT基因未见c.2401A＞C突变,推测该突变为先证者患斑驳病的致病突变可能性大.为此两家系进行遗传咨询和产前诊断提供了理论依据.