P. Capasso

A novel donor splice site characterized by CFTR mRNA analysis induces a new pseudo-exon in CF patients.

BACKGROUND The CFTR gene is tightly regulated and differentially expressed in many mucosal epithelial cell types. There is evidence of an increasing number of genomic variations in the intronic regions influencing mRNA splicing, and also the level of normal CFTR transcript. METHODS In the present study, we investigate the molecular defect by RT-PCR analyzing the mRNA of 25 cystic fibrosis (CF) patients in whom only one or no CF allele had been identified after DNA analysis (of all the exons of the CFTR gene). RESULTS mRNA analysis led to the detection of a cryptic exon in two patients: the new exon is a 104 bp insertion between exons 10 and 11 and is caused by a new point mutation c.1584+18672 bp A>G (http://www.hgvs.org/mutnomen/) discovered in intron 10; moreover, they showed the absence of exon 9 skipping. CONCLUSIONS Our results confirm the utility of RNA analysis in discovering new mutations and in investigating their effect on normal splicing processes.

Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis ☆

OBJECTIVE The sweat test remains the gold standard for the diagnosis of Cystic Fibrosis (CF) even despite the availability of molecular analysis of Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR). We investigated the relationship between CFTR mutation analysis and sweat chloride concentration in a cohort of subjects with borderline sweat test values, in order to identify misdiagnosis of CF. DESIGN AND METHODS In the period between March 2006 and February 2008 we performed 773 sweat tests in individuals referred for suspect CF. Ninety-one subjects had chloride values in the border-line range. Clinicians required CFTR gene complete scanning on 66 of them. RESULTS The mean value of sweat chloride in the DNA negative subjects was lower than in those with at least one CFTR mutation. Our data indicate that 39 mEq/l is the best sensitivity trade off for the sweat test with respect to genotype. CONCLUSIONS To optimise diagnostic accuracy of reference intervals, it may be useful to modify from 30 to 39 mEq/l the threshold for sweat chloride electrolytes.

Cystic Fibrosis Newborn Screening: Distribution of Blood Immunoreactive Trypsinogen Concentrations in Hypertrypsinemic Neonates

The IRT screening test for the use in diagnosing newborns with CF has a high sensitivity but is not very specific resulting in a large number of screened positive infants found to have a normal sweat test. The aim of this study was to analyze the differences in b-IRT levels among different groups of newborns positive to NBS.Population data included all b-IRT positive (>99th centile) neonates born in Lombardia from 2000 to 2007. The hypertrypsinemic newborns were divided into four groups, according to CF status (noncarrier, carrier, CFTR-RD, CF).Among a total of 717,172 newborns screened within the study period, 7,354 newborns were found positive to NBS and were included in the study. An overall statistically significant difference in b-IRT levels was found among the four groups (p < 0.001), while b-IRT values did not differ between noncarriers and carriers. b-IRT levels had a low predictive accuracy in correctly identifying the four different groups (c-index: 0.60), but the accuracy was high in discriminating between classic CF and carrier or noncarrier status in neonates positive to NBS. The IRT level on the initial blood specimen obtained at birth differs based on the CF genotype, although a wide range of individual variation may occur.

A wide methodological approach to identify a large duplication in CFTR gene in a CF patient uncharacterised by sequencing analysis

BACKGROUND PCR-based diagnostic procedures are not able to characterise 6% of CF alleles. Recently, the application of array-CGH and of CFTR mRNA analysis has allowed the identification of new copy number mutations and splicing defects, that account for 2% and 13% of CF alleles, respectively, in the Italian population. METHODS Here, we report the characterisation of a large duplication in CFTR gene through different methods: MLPA assay, RT-PCR and high-resolution array-CGH. RESULTS We identified a large duplication, involving exons 6b-16, in a patient heterozygous for F508del mutation. This duplication produces an abnormal transcript with an out of frame addition of 2244 nucleotides and leads to the insertion of 8 amino-acid residues in the protein, followed by a stop codon. CONCLUSIONS We propose a wide methodological approach based on MLPA assay, RT-PCR and high-resolution array-CGH to routinely analyse CF patients uncharacterised for one or both CFTR alleles.