The increase in genome scanning data, derived from clinical genetics practice, is producing a wealth of information on human sequence variability. The critical issue is to identify if a given nucleotide change results in a benign polymorphism or a disease-causing mutation. We have focused on one specific gene expression step, pre-mRNA processing, where we can functionally define the effect of nucleotide changes and in turn the patient's mutation can shed light on the basic pre mRNA splicing mechanisms. Our results show that several nucleotide changes in CFTR exon 12 induce a variable extent of exon skipping that leads to reduced levels of normal transcripts. This is the case in both natural mutations D565G and G576A (the latter having previously considered a neutral polymorphism) and several site-directed silent substitutions. We demonstrate here that this phenomenon is due to the interference with a new regulatory element that we have named composite exonic regulatory element of splicing (CERES). The effect of single nucleotide substitutions at CERES cannot be predicted by neither SR matrices nor enhancer identification. The recognition and characterization of splicing abnormalities, caused by exon sequence variations at CERES elements, may represent a frequent disease-causing mechanism that also relates to the phenotypic variability. Our results indicate that even the most benign looking polymorphism in an exon cannot be ignored as it may affect the splicing process. Hence, appropriate functional splicing assays should be included in genotype screenings to distinguish between polymorphisms and pathogenic mutations.

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

Aspergillus fumigatus is commonly found in cystic fibrosis (CF) airways. Our aim was to assess the relationship between A. fumigatus chronic colonization and lung function in CF patients. A case-control study of CF patients born from 1989 to 2002 was performed. Medical records were reviewed from the time of initial diagnosis until December 2013. Chronic colonization was defined as two or more positive sputum cultures in a given year. Each patient chronically colonized with A. fumigatus was matched with three control patients (never colonized by A. fumigatus) for age, sex, and year of birth (±3 years). A number of parameters were recorded and analyzed prospectively. The primary outcome measure was the difference in forced expiratory volume in 1 s (FEV1) in percent predicted between groups. Linear mixed models were used for longitudinal analyses to evaluate the relationship between A. fumigatus chronic colonization and lung function during a 7-year period and study the lung function 4 years before the time of enrollment (t0). Twenty patients had chronic colonization and were matched with 60 controls. A significant difference in lung function was detected throughout the 7-year period after adjustment for confounders (est = 8.66, p = 0.020). Four years before t0, FEV1 baseline was the only factor associated with the course of lung function (est = 0.64, p< 0.001) and was significantly different between groups (p = 0.001). In conclusion, a decreased FEV1 baseline appears to be a risk factor for chronic colonization by A. fumigatus, which, in turn, may cause a faster deterioration of lung function.

Our data indicate that vitamin K supplementation may have a beneficial role in bone health in CF children.