Despite the extensive efforts over the past years, a significant percentage of CF alleles still bear undetected CFTR mutations. This percentage is variable in different ethnic groups and also depends on the methodologies used to search CFTR mutations. The incidence of CF alleles bearing unknown CFTR mutations in Italy ranges between 3 to 20%, as reported by the website of the Italian CF laboratories (spazioinwind.libero.it/laboratoricf). This percentage is about 10%, 20%, and 12%, for Veneto, Campania-Basilicata, and Lazio regions respectively, from which the CF patients for the present research will be recruited. The number of alleles bearing unknown CFTR mutations further increases if CF patients bearing “atypical” phenotypes are considered.
Conventional PCR-based methodologies commonly used, can detect single point mutations and little deletions/insertions involving few nucleotides. In fact, the more than 1000 CFTR mutations identified so far (Cystic Fibrosis Mutation Database), are mainly single nucleotide substitutions, microdeletions and microinsertions. By contrast, only a few large deletions have been found so far in the CFTR gene. For this reason, it has been suggested that genomic rearrangements could account for a significant percentage of unidentified disease alleles. These mutations, if present in heterozygosis, elude current analytical techniques, including direct sequencing, and denaturing high performance liquid chromatography (DHPLC).
The most frequent large deletion occurring in CFTR gene was described by Dork et al. (2000). Indeed, this 21 kb deletion removes exons 2 and 3 and is the second most common mutant CFTR allele observed in Central and Eastern Europe; the haplotype analysis suggested a common origin for this allele (Dork et al., 2000).
To date, few large germline rearrangements of the CFTR gene have been identified fortuitously either by noting a uniparental inheritance pattern, or through failure to amplify target sequences if such mutations are present in homozygosis. In other words, none of these rearrangements was found by applying a systematic screening procedure. Southern blotting, the classical technique for detecting large genomic rearrangements, is rather laborious and therefore rate-limiting for large-scale analysis. This led to the an underestimation of gross gene mutations in CF, also limiting the study of the mechanisms underlying large genomic rearrangements involving the CFTR gene.
To fill this gap in the knowledge of the molecular genetics of CF, the group by prof. Ferec (University of Brest, France) performed a systematic screening of the CFTR gene large rearrangements using the recently introduced technique of quantitative multiplex PCR amplification of short fluorescent fragments (QMPSF; Charbonnier et al., 2000).
QMPSF is based on the simultaneous amplification of multiple short target sequences followed by the rapid and reliable quantification of the fluorescence of each amplicon. This technique has been successfully used to screen the mismatch repair (Charbonnier et al., 2000, 2002), C1 inhibitor (SERPING1), (Duponchel et al., 2001), and BRCA1 genes (Casilli et al., 2002). The combination of QMPSF with quantitative DHPLC and sequencing was used to identify and characteryze genomic rearrangements within the CFTR gene, allowing to resolve a significant fraction of previously uncharacterized CFTR alleles.
The aims of this project are: i) to scan CFTR gene rearrangements with the novel QMPSF technique in a large population of patients bearing CF or CF atypical/related forms carrying one or both CFTR mutations undetected by current scanning procedures and coming from different Italian regions; ii) to study the breakpoints of the CFTR rearrangements possibly found to make ligth on the mechanisms underlying such type of mutations, iii) to analyse the frequencies of the identified CFTR gene rearrangements in large populations of CF patients and of normal control individuals.
The result of this study could significantly expand the known spectrum of genomic variants in the CFTR gene, improving the diagnostic possibility for the Italian population. Moreover these results could add to our knowledge the mechanisms underlying genomic rearrangements in inherited disease.