Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

doi:10.1016/S0888-7543(05)80376-3

Genomics

Volume 18, Issue 3, December 1993, Pages 693-697

https://doi.org/10.1016/S0888-7543(05)80376-3 Get rights and content

Abstract

We have previously shown that about 85% of the mutationsin 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay (7). In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region and their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in our population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available.

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Cited by (69)

Analysis of the CFTR gene in Iranian cystic fibrosis patients: Identification of eight novel mutations
2008, Journal of Cystic Fibrosis
Citation Excerpt :
If a mutation was identified, so that a mutation on both CFTR genes of a patient was characterized, the remainder exons were not analysed further, except for some missense mutations in order to detect or exclude the presence of other mutations in cis. Amplification of all 27 CFTR exons, including flanking intronic regions, was performed using CFTR gene specific primers, which are commonly used in many studies for CFTR analysis [21,22]. The PCR products were purified using small purification columns.
Cystic 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.
69 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).
CFTR 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.
We 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.
High incidence of the CFTR mutations 3272-26A → G and L927P in Belgian cystic fibrosis patients, and identification of three new CFTR mutations (186-2A → G, E588V, and 1671insTATCA)
2007, Journal of Cystic Fibrosis
We have analyzed 143 unrelated Belgian patients with a positive diagnosis of cystic fibrosis (CF) for mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. An initial screening for 29 CFTR mutations led to mutation identification in 89.9% of the tested chromosomes. Subsequently an extensive analysis of the CFTR gene was performed by denaturating gradient gel electrophoresis (DGGE) in those patients with at least one unknown mutation after preliminary screening. In addition to 10 previously reported mutations we identified 2 new mutations 186-2A → G and E588V. A third new mutation 1671insTATCA was identified during routine screening for ΔF508. Two mutations were detected with a higher frequency than expected: 3272-26A → G, which is the second most common mutation after ΔF508 in our CF population with a frequency of 3.8%, and L927P (2.4%). The clinical data is presented for the mutations 186-2A → G, E588V, 3272-26A→G and L927P. The mutation data are useful for the Belgian population to supplement the initial screening set of mutations.
Mutational spectrum of cystic fibrosis patients from Córdoba province and its zone of influence: Implications of molecular diagnosis in Argentina
2006, Molecular Genetics and Metabolism
Cystic Fibrosis (CF) is an autosomal recessive disorder affecting 1/2000–4000 newborns in Caucasian populations. This lethal disease mainly affects respiratory and digestive organs as well as fertility in man. So far, the CF prevalence and mutational spectrum have showed specificity among populations and regions, making it necessary to establish them in each one. In this study, we present the spectrum and frequency of CFTR gene mutations in CF patients from Córdoba (a province with 3.1 millions inhabitants in the middle of Argentina) and its zone of influence, to offer an accurate genetic testing. The study includes 78 families in which 98 patients fulfilled clinical criteria to CF diagnosis. The strategy for the molecular diagnosis comprised analysis of 21 common mutations, microsatellite haplotypes and the complete CFTR gene analysis using scanning techniques followed by sequencing of the abnormal migration patterns. Our first step led us to the identification of 10 mutations that represented 76% of alleles. Another four mutations (p.R1066C, c.1811 + 1.6kbA > G, c.711 + 1G > T, and p.G85E) were found based on the microsatellite haplotype–mutation association. Finally, 14 mutations were characterized after the CFTR gene scanning, three of them are not previously described (p.G27R, c.622-2A > G, and p.W277R). In summary, we have identified 27 mutations accounting for 94.23% of CF alleles. This characteristic mutational spectrum highlights the 14 most frequent mutations (>1%) in the Córdoba region.
High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease
2004, Journal of Cystic Fibrosis
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.
Cystic fibrosis transmembrane conductance regulator (CFTR) gene defects in patients with primary sclerosing cholangitis
2002, Journal of Hepatology
Background/Aims: Because biliary tract lesions that resemble those of primary sclerosing cholangitis (PSC) may occur in cystic fibrosis (CF), we examined the prevalence and influence of CF transmembrane conductance regulator (CFTR) gene mutations in PSC patients.
Methods: Genomic DNA was analyzed in 29 consecutive PSC patients and in 115 healthy control individuals. A scanning method followed by direct DNA sequencing was used to scan the CFTR coding regions.
Results: Four patients (13.8%) were heterozygous for a CFTR mutation, including a new putative severe CF-causing mutation (N782K), and three mild defects (L997F, D1270N, and S1235R). The comparison of PSC patients with healthy controls showed no significant difference in the frequency of CFTR mutations (P=0.415). In addition, two patients (6.9%) were heterozygous for the IVS8-5T allele, which is not significantly different from the 5–6%-prevalence in the general population. Unusual clinical features including a severe outcome in childhood, with a lethal outcome at age 22, and biliary aspergillosis were recorded in patients with a CFTR mutation.
Conclusions: The proportion of CF carriers is not significantly higher in PSC patients than in the general population. The possibility that CFTR mutations may contribute to a severe clinical course in PSC patients is worth further examining.
G970R-CFTR Mutation (c.2908G>C) Results Predominantly in a Splicing Defect
2021, Clinical and Translational Science

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Short communicationDetection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

Abstract

Mol. Cell. Probes

Cell

J. Biol. Chem.

Genomics

Trends Genet.

Genomics

Genomics

Screening for five mutations detects 97% of cystic fibrosis (CF) chromosomes and predicts a carrier frequency of 1:29 in the Jewish Ashkenazipopulation

Am. J. Hum. Genet.

Demonstration that CFTR is a chloride channel by alteration of its anion selectivity

Science

Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchialepithelium

EMBO J.

Extensive postranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts ofthe cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis

J. Clin. Invest.

Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA

Nature Genet.

Short communication
Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene