Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as "splicing mutations," but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002-1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5'- and 3'-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002-1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75.

Prenatal Diagnosis and Screening of the Haemoglobinopathies.

The most important aspects of carrier detection procedures, genetic counselling, population screening and fetal diagnosis of the thalassaemias and sickle cell anaemia are reviewed. Carrier detection can be made retrospectively, i.e. following the birth of an affected child, or prospectively. Most carrier detection and genetic counselling in population at risk for alpha-thalassaemia an sickle cell anaemia is retrospective. However, some prospective carrier screening programmes for sickle cel anaemia are ongoing in Cuba and Guadeloupe and very limited screening for alpha-thalassaemia is in progress in some South East Asian populations. As regards beta-thalassaemia, several programmes, based on carrier screening and counselling of couples at marriage, preconception, or early pregnancy, have been operating with several populations at risk in the Mediterranean. These programmes have been very effective, as is proved by the fact that the target population has improved its knowledge of thalassaemia and its prevention, and by the marked decline that has been observed in the incidence of thalassaemia major. Carrier detection is carried out by haematological methods, followed by mutation detection by DNA analysis. Prenatal diagnosis is accomplished by mutation analysis on PCR-amplified DNA from chorionic villi. Future prospects include automation of the process of mutation detection, simplification of preconception and preimplantation diagnosis, and fetal diagnosis by analysis of fetal cells in the maternal circulation.