Analysis of the CFTR gene in the Spanish population: SSCP-screening for 60 known mutations and identification of four new mutations (Q30X,A120T, 1812-1 G→A,and 3667de14)

In order to determine the spectrum of CF mutations in the Spanish population, we have analysed 40 unrelated Spanish CF patients, with at least one chromosome negative for mutations ΔF508, G542X, and N1303K. Exons l–7,10–14a,15,16,17b,18–21 of the CFTR gene were studied by Single Strand Conformation Polymorphism (SSCP) analysis, using 60 known CF mutations as controls. SSCP screening allowed us to detect 28 different mutations in 52 CF chromosomes, and to identify four new mutations (Q30X in exon 2, A120T in exon 4, 1812-lG→A in intron 11 and 3667del4 in exon 19). Further analysis of the four new mutations in a total of 950 Spanish CF chromosomes showed a final frequency of 0.4%, 0.1%, 0.1%, and 0.1% for 1812-1G→A, Q30X, A120T, and 3667del4, respectively. No mutations were detected in exons 1, 3, 14a, 16, and 18. We have also detected 10 intragenic polymorphisms and DNA sequence variants and have analysed their frequencies in our population. The total of 28 mutations identified in the 80 CF chromosomes highlight the molecular heterogeneity of CF in the Spanish population. © 1994 Wiley-Liss, Inc.     

Imaging CFTR in its native environment

Application of atomic force microscopy (AFM) on isolated plasma membranes is a valuable method to study membrane proteins down to single-molecule level in their native environment. The cystic fibrosis transmembrane conductance regulator (CFTR), a protein of the adenosine triphosphate-binding cassette transporter superfamily, is known to play a crucial role in maintaining the salt and water balance on the epithelium and to influence processes such as cell volume regulation. A mutation in the gene encoding for CFTR results in cystic fibrosis (CF), a very common lethal genetic disease. Identification of CFTR within the cell membrane at the single-molecule level makes it feasible to visualize the distribution and organization of CFTR proteins within the cell membrane of healthy individuals and CF patients. We were able to show that human red blood cells have a CFTR distribution comparable to that of epithelial cells and that the number of CFTR in cells derived from CF patients is strongly reduced. Studies on CFTR-expressing oocytes disclose CFTR dynamics upon CFTR activation. We observed that cyclic adenosine monophosphate induces an insertion of CFTR in the plasma membrane and the formation of heteromeric CFTR-containing structures with yet unknown stoichiometry. The structure of CFTR was identified by high-resolution scans of immunogold-labeled CFTR, revealing that CFTR forms a tail-to-tail dimer with a central pore. In conclusion, these studies show that AFM experiments on isolated plasma membranes allow not only quantification and localization of membrane proteins but also provide insight in their dynamics at a single-molecule level.     

PKU in Minas Gerais State,Brazil: Mutation Analysis

This work was undertaken in order to ascertain the PKU mutational spectrum in Minas Gerais, Brazil, the relative frequency of the mutations in the State and the origin of these mutations by haplotype determination. Minas Gerais is a trihybrid population formed by miscegenation from Europeans, Africans and Amerindians. All 13 exons of the PAH gene from 78 PKU patients were analyzed, including splicing sites and the promoter region. We identified 30 different mutations and 98% of the PAH alleles were established. A new mutation (Q267X) was identified as well. The most common mutations found were V388M (21.2), R261Q (16.0%), IVS10‐11G>A (15.3%), I65T (5.8%), IVS2+5G>C (5.8%), R252W (5.1%), IVS2+5G>A (4.5%), P281L (3.8%) and L348V (3.2%). These nine mutations correspond to 80% of the PKU alleles in the state. Haplotypes were determined to characterize the origin of the PAH alleles. The majority of the mutations found, with respective haplotypes, are frequent in the Iberian Peninsula. However, there were some mutations that are rare in Europe and four previously unreported mutation‐haplotype associations. I65T and Q267X were found in association with haplotype 38 and may be African in origin or the result of miscegenation in the Brazilian population.     

A novel double deletion underscores the importance of characterizing end points of the CFTR large rearrangements

Large genomic rearrangements in patients with cystic fibrosis (CF) account for up to 16–24% of CF alleles negative for point mutations in European populations. Herein, we identified a new large rearrangement removing exon 19 in a young CF patient, who hitherto harbored only the F508del mutation. By using LightCycler technology, we successfully and rapidly delineated the deletion end points by determining the relative copy number of a set CFTR sequence from introns 18 to 19. Fine mapping of the sequences bordering its break points was achieved using direct sequencing. We reported the first complex CFTR rearrangement containing two successive deletion events putatively linked. We evidenced the presence of short direct repeats in the vicinity of the deletions suggesting a possible replication slippage model. In this report, we also discussed the putative molecular mechanism and consequences of this complex gene rearrangement, unprecedented in CF. This complex deletion illustrates the importance of delineating the genomic rearrangement to improve our knowledge of the CFTR mutational spectrum and to better understand the molecular mechanism controlling the CFTR expression.     

Single-stranded conformation polymorphism analysis of the CFTR gene in slovenian cystic fibrosis patients: Detection of mutations and sequence variations

Cystic fibrosis (CF) mutations have been identified in Slovenian CF patients using single-stranded conformation polymorphism (SSCP) analysis. The entire coding region and all of the splice junction sites were screened in 24 patients. By varying the electrophoretic conditions and composition of the gel, 16 different nucleotide changes have been observed in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Three newly described mutations and four previously reported mutations were found. In addition two new polymorphisms have been identified. Of 35 non-ΔF508 chromosomes examined, mutations were detected on 25.7%, raising the proportion of Slovenian CF alleles characterized to 67.5%. Because of the high sensitivity of the SSCP technique most of the remaining uncharacterized CF mutations probably lie in large introns, promoter sequences, or putative regulatory regions not yet analyzed. © 1993 Wiley-Liss, Inc.     

Screening for cystic fibrosis mutations in southern France: identification of a frameshift mutation and two missense variations.

In the search for mutations in the cystic fibrosis gene in patients from the Mediterranean area, we have analysed exons 4, 9, 10, 19, and 21 by the single-strand conformation polymorphism (SSCP) technique in 50 patients with at least one non-delta F508 chromosome. Ten samples demonstrated a shifted band, four in exon 19 and six in exon 21. Sequencing of the PCR fragments has led to the identification of three new sequence alterations, two in exon 19 (3737 delA and I1234V), and one in exon 21 (N1303H). We also analysed the frequency of two known intronic polymorphisms in front of exon 19 (C to A change at nucleotide 3601-65) and exon 21 (G to A change at position 4006-200).     

Notable contribution of large CFTR gene rearrangements to the diagnosis of cystic fibrosis in fetuses with bowel anomalies

Grade III fetal bowel hyperechogenicity and/or loop dilatation observed at the second trimester of pregnancy can be due to several disease conditions, including cystic fibrosis (CF). Screening for frequent CF mutations is performed as a first step and, in certain situations, such as when a frequent CF mutation is found in the fetus, the increased risk of CF justifies an in-depth study of the second allele. To determine the contribution of large CFTR gene rearrangements in such cases, detected using a semiquantitative fluorescent multiplex PCR (QFM-PCR) assay, we collated data on 669 referrals related to suspicion of CF in fetuses from 1998 to 2009. Deletions were found in 5/70 cases in which QFM-PCR was applied, dele19, dele22_23, dele2_6b, dele14b_15 and dele6a_6b, of which the last three remain undescribed. In 3/5 cases, hyperechogenicity was associated with dilatation and/or gallbladder anomalies. Of the total cases of CF recognized in the subgroup of first-hand referrals, deletions represent 16.7% of CF alleles. Our study thus strengthens the need to consider large CFTR gene rearrangements in the diagnosis strategy of fetal bowel anomalies, in particular in the presence of multiple anomalies.     

Molecular study of the tumour suppressor gene PTEN in gastric adenocarcinoma in Brazil

Abstract Among all tumours diagnosed worldwide, gastric adenocarcinoma is the second most frequent type of malignancy. In Brazil, it is estimated to be the fifth most frequent type of neoplasia. According to the classification of Laurén, these tumours are divided into well differentiated and ill differentiated gastric adenocarcinomas. There are studies suggesting that the first type develops through remodulation of genes involved in the suppressor pathway and the second through remodulation of genes belonging to the mutational pathway. The gene PTEN is located in region 10q23 and is altered in several human tumours. In gastric cancer, this gene is thought to take part in the suppressor pathway. In our study, DNA was obtained from 48 gastric adenocarcinoma samples, amplified, screened for all exons of the PTEN gene by PCR-SSCP and then confirmed by sequencing. There was only one sample that presented an alteration and that was a transversion. Our results corroborate the hypothesis that somatic alterations in the PTEN gene are rare events in gastric cancer.     

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 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A

To provide a National database, 1,410 unrelated hemophilia A (HA) patients were investigated using screening methods denaturing high-performance liquid chromatography (DHPLC), conformational-sensitive gel electrophoresis (CSGE)] and/or direct sequencing. F8 gene mutations were identified in 877 (81%), 146 (82%), and 133 (89%) families with severe, moderate, or mild HA, respectively. Among the 382 different mutations detected, 217 (57%) have not previously been reported in the F8 Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database. Mutations leading to a null allele accounted for 82, 15%, and less than 1% of severe, moderate, or mild HA, respectively. A missense mutation was identified in 16%, 68%, and 81% of severe, moderate, or mild HA, respectively. They included 105 missense mutations (48%), 41 small deletions (19%), 25 splice site mutations (12%), 24 nonsense mutations (11%), 18 insertions (8%), three large deletions (1%), and one deletion plus insertion. Unreported mutations were distributed throughout the F8 gene, as they affected all F8 exons but exon 20. We report a wide spectrum of mutations collected in a large National database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counseling and medical care of HA families in Italy.     

Molecular characterization of the <Emphasis Type="Italic">env</Emphasis> gene from Brazilian field isolates of Bovine Leukemia Virus

Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions. In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found in the aminoacid sequences of the gp51 protein were localized in the G and H epitopes. Using the SIFT software, it was predicted that they should not alter the protein functions. Phylogenetic analyses showed that partial or complete env gene sequences grouped in three or four phylogenetic clusters, respectively. The sequences from the Brazilian isolates had similar mutation rates as compared to samples from other countries, and belonged to at least two phylogenetic clusters.     

Genetic characterization of Rabies virus isolated from cattle between 1997 and 2002 in an epizootic area in the state of São Paulo, Brazil

The biogeographical history of rabies can be reconstructed using molecular data. This work describes the genetic characterization of the Rabies virus variant that circulates in the Desmodus rotundus (vampire bat) population in an epizootic area and is transmitted to herbivorous livestock. The N and G genes of this virus were sequenced, and the phylogenetic trees generated were topologically concordant. Three genetic clusters were identified in the epizootic area and were designated RD1, RD2 and RD3. The results show that the origins of the epizootics in areas RD1 and RD2 were different and that the epizootic in area RD3 was the result of expansion of that in area RD2. The two genes analyzed are conserved, and their identities, which are greater than 98%, were maintained over time and space. The genetic sequences in this study were compared with others retrieved from GenBank, and the high identity of the N and G genes was also shown to be maintained over time and space. The results suggest that the D. rotundus lineages of the Rabies virus from the Atlantic coast of South America are highly conserved.     

Molecular study of the tumour suppressor gene PTEN in gastric adenocarcinoma in Brazil

Abstract   Among all tumours diagnosed worldwide, gastric adenocarcinoma is the second most frequent type of malignancy. In Brazil, it is estimated to be the fifth most frequent type of neoplasia. According to the classification of Laurén, these tumours are divided into well differentiated and ill differentiated gastric adenocarcinomas. There are studies suggesting that the first type develops through remodulation of genes involved in the suppressor pathway and the second through remodulation of genes belonging to the mutational pathway. The gene PTEN is located in region 10q23 and is altered in several human tumours. In gastric cancer, this gene is thought to take part in the suppressor pathway. In our study, DNA was obtained from 48 gastric adenocarcinoma samples, amplified, screened for all exons of the PTEN gene by PCR-SSCP and then confirmed by sequencing. There was only one sample that presented an alteration and that was a transversion. Our results corroborate the hypothesis that somatic alterations in the PTEN gene are rare events in gastric cancer.     

A novel mutation of RPGR gene in an X-linked Chinese family with retinitis pigmentosa

PurposeTo localize and identify the gene and mutations causing an X-linked Chinese family with retinitis pigmentosa.MethodsAn XLRP Chinese family was ascertained and patients underwent ophthalmological examinations. Blood samples were collected and genomic DNA was extracted. Linkage scan was performed on genomic DNA from affected and unaffected family members using microsatellite markers flanking 17 known autosomal dominant loci and markers covering the entire X chromosome. Mutation screening of RPGR gene was carried out by direct DNA sequence analysis.ResultsA genome wide scan yielded a lod score of 2.7 at θ = 0 with DXS1068 and 3.29 at θ = 0 with DXS993. This region harbors the RPGR gene. Direct DNA sequence analysis reveals one base pair deletion, gORF15 + 556delA, in all affected individuals. The deletion results in the frameshift change of RPGR gene and produces a truncated protein.ConclusionsWe identified a novel mutation, gORF15 + 556delA (p.Lys184fs), in a Han Chinese family with retinitis pigmentosa. This mutation expands the mutation spectrum of RPGR and helps to study molecular pathogenesis of RP further.     

Anti-Pseudomonas aeruginosa IgG subclass titers in patients with cystic fibrosis: Correlations with pulmonary function,neutrophil chemotaxis,and phagocytosis

To explore possible mechanisms for the association between elevated immunoglobulin levels and lower pulmonary function in cystic fibrosis patients, we measured serum IgG subclass levels and anti-P. aeruginosa IgG subclass titers and correlated levels with neutrophil phagocytosis and chemotaxis. Serum was obtained from 13 cystic fibrosis patients colonized with the same serotype ofP. aeruginosa, 12 noncolonized patients, and 12 normal volunteers. All anti-P. aeruginosa IgG subclass titers were elevated in serum from colonized patients. IgG₃ level and anti-P. aeruginosa IgG₃ titer were inversely correlated with pulmonary function. Phagocytosis ofP. aeruginosa by neutrophils correlated with serum IgG₃ level and was increased by opsonization with serum from colonized patients. Chemotactic index was increased in serum from colonized patients and inversely correlated with pulmonary function chest roentgenogram score. Chemotactic index directly correlated with anti-P. aeruginosa IgG₃ titer and serum IgG₃. These data demonstrate that cystic fibrosis patients with increased IgG₃ levels are in poorer clinical condition and that their serum enhances neutrophil function. Such patients may have increased pulmonary inflammation with subsequent lung damage.