Núria Morral

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences.

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.

CA/GT microsatellite alleles within the cystic fibrosis transmembrane conductance regulator (CFTR) gene are not generated by unequal crossingover.

The gene responsible for cystic fibrosis (CF) has recently been identified, and a three-nucleotide deletion (delta F508 mutation) that results in the loss of a phenylalanine residue in the first putative ATP-binding domain of the predicted protein (CF transmembrane conductance regulator, CFTR) has been found to be the major CF mutation. Although several other mutations have been identified in the CFTR gene, most of them are very rare, making their application to genetic diagnosis difficult. While characterizing the genomic region encompassing the CF locus, we have identified three CA/GT blocks that flank exon 9 of the CF gene. One of the CA/GT blocks exhibits a highly informative variable number of dinucleotide repeats (VNDR) polymorphism. This intragenic VNDR microsatellite should, by itself, provide full information for genetic analysis in approximately 80% of CF families and will help elucidate the associations between DNA polymorphism haplotypes and specific gene mutations. Haplotype analyses of CF chromosomes with and without the delta F508 mutation suggest that the different alleles are generated by slipped-strand mispairing within the dinucleotide repeat during DNA replication, rather than by unequal crossingover within a recombination hot spot.

Multiplex PCR amplification of three microsatellites within the CFTR gene

Multiplex PCR amplification has been developed for three highly polymorphic microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) located in intronic regions of the CFTR (cystic fibrosis (CF) transmembrane conductance regulator) gene. The triplex PCR reaction required different concentrations of each pair of primers and labeling of primers in the same reaction. Total informativity is obtained in 90.25% of couples requiring analysis of polymorphisms, and when triplex microsatellite analysis is combined with analysis for the six most common CF mutations in the Spanish population, informativity reaches more than 99%.

Analysis of the CFTR gene confirms the high genetic heterogeneity of the Spanish population: 43 mutations account for only 78% of CF chromosomes

We have analysed 972 unrelated Spanish cystic fibrosis patients for 70 known mutations. Analysis was performed on exons 1, 2, 3, 4, 5, 6a, 6b, 7, 10, 11, 12, 13, 14a, 14b, 15, 16, 17b, 18, 19, 20 and 21 of the cystic fibrosis transmembrane regulator gene using single strand conformation polymorphism analysis and denaturing gradient gel electrophoresis. The major mutation ΔF508 accounts for 50.6% of CF chromosomes, whereas another 42 mutations account for 27.6% of CF chromosomes, with 21.8% of Spanish CF chromosomes remaining uncharacterised. At present, we have identified 36 mutations that have frequency of less than 1% and that are spread over 15 different exons. This indicates that, in the Spanish population, with the exception of ΔF508 (50.6%) and G542X (8%), the mutations are not concentrated in a few exons of the gene nor are there any predominating mutations. This high degree of genetic heterogeneity is mainly a result of the different ethnic groups that have populated Spain and of the maintenance of separated population sets (Basques, Arab-Andalusian, Mediterranean, Canarian and Gallician). The high proportion of CF chromosomes still unidentified (21.8%) together with association analysis with intragenic markers suggest that at least 100 different mutations causing CF are present in our population.

Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers

We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, ΔF508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272‐26A → G, R1162X, and 3849 + 10kbC → T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

Identical intragenic microsatellite haplotype found in cystic fibrosis chromosomes bearing mutation G551D in Irish, English, Scottish, Breton and Czech patients.

Mutation G551D of exon 11 of the cystic fibrosis transmembrane conductance regulator gene is one of the most common mutations in patients of European origin. In order to test the hypothesis that the mutation is identical by descent in these patients, we have studied haplotypes for the three intragenic microsatellite markers IVS8CA, IVS17bTA and IVS17bCA from 92 patients bearing this mutation, who had been referred to laboratories in Ireland, Scotland, England, France (Brittany) and the Czech Republic. In all cases we found that only haplotype 16-7-17 is associated with mutation G551D. Our results support the hypothesis of identity by descent of all cystic fibrosis chromosomes bearing mutation G551D in these patient populations, and suggest that given the combined mutation rate of the microsatellite markers, there is a low probability (p < 0.05) that the haplotype where mutation G551D first occurred remained unaltered for more than 170 generations.

Cystic fibrosis in a low-incidence population: two major mutations in Finland

The incidence of cystic fibrosis (CF) in Finland, 1∶25 000 newborn, is one of the lowest in Caucasian populations. The ΔF508 mutation accounts for 18/40 (45%) of CF chromosomes in Finland. Other mutations were therefore sought among the remaining 55%. Twelve out of 40 chromosomes (30%) were found to carry 394delTT, whereas G542X and 3732delA were each detected in one chromosome. Eight mutations remained unidentified using a testing panel for 26 mutations. Mutation 394delTT was associated exclusively with haplotype 23-36-13. Five unknown mutations were associated with different haplotypes for microsatellite markers, whereas three shared the same haplotype. Most ΔF508 mutations and all unidentified mutations originated from regions of old and dense settlement in the coastal regions, whereas 394delTT was geographically clustered and enriched in a rural location, consistent with a local founder effect. The remote location of Finland and her population history give a plausible explanation for the rarity of CF in Finland.

Cystic fibrosis in Spain: high frequency of mutation G542X in the Mediterranean coastal area

We have determined the frequency of deletion ΔF508 and mutation G542X, a nonsense mutation in exon 11 of the cystic fibrosis (CF) gene, in a sample of 400 Spanish CF families. Mutation G542X represents 8% of the total number of CF mutations in Spain, making it the second most common mutation after the ΔF508 deletion, which accounts for 48% of CF chromosomes. G542X has a higher frequency in the Mediterranean coastal area (14%) and in the Canary Islands (25%). About 70% of G542X chromosomes are from Andalucia, Múrcia, Valencia, Catalunya and the Canary Islands. The ΔF508 deletion has its highest frequency in the Basque Country (83%). Mutation G542X is associated with the same rare haplotype that is found in association with the ΔF508 mutation. The haplotype homogeneity found for G542X, even when intragenic microsatellites (IVS8CA, IVS17BTA and IVS17BCA) are considered, allows us to postulate that this mutation arose from a single mutational event. The geographic distribution of mutations ΔF508 and G542X suggests that ΔF508 was present in the Iberian Peninsula before the Indo-European invasions, and that G542X was introduced into Spain, via the Mediterranean Sea, probably by the Phoenicians, between 2500 and 3000 years ago.

COMPLETE DETECTION OF MUTATIONS IN CYSTIC FIBROSIS PATIENTS OF NATIVE AMERICAN ORIGIN

An increased incidence of cystic fibrosis (CF) has been reported in some populations of Native Americans of the Southwest such as the Pueblo, which is a genetic isolate. As the most common mutation found in Caucasians (ΔF508) was absent and only one chromosome carried the G542X mutation, we decided to analyze the entire coding sequence of the CFTR gene in eight Pueblo CF patients. We have identified four different mutations: G542X, R1162X, 3849+10kbC→T, and D648V that account for these 16 haplotypes. The R1162X was found on 11 chromosomes. Using intragenic microsatellites, we have compared the haplotypes of those chromosomes to those of Italian origin where the R1162X mutation was initially reported. These haplotypes turned out to be identical, suggesting a common origin and an admixture with Italian or Spanish settlers, followed by typical founder effect. In contrast the 3849+10kbC→T mutation, which was found on three chromosomes, is associated with different haplotypes than those on chromosomes carrying the same mutation in Caucasians. A novel mutation, D648V, observed on one chromosome has not been found outside the Pueblo population.

A tetranucleotide repeat polymorphism in the cystic fibrosis gene

SummaryWe describe a tetranucleotide (GATT) repeat polymorphism in the cystic fibrosis transmembrane conductance regulator gene.