Distribution of CFTR mutations in cystic fibrosis patients of Tunisian origin: identification of two novel mutations.

Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by mutations in the CF transmembrane conductance regulator gene (CFTR). More than 500 molecular defects have been reported to date. The distribution of these mutations is both heterogeneous and population related. In Mediterranean populations, 20-30% of CF alleles remain unidentified. We have studied a sample of 39 CF patients of Tunisian origin and have used a GC clamp DGGE assay to scan the CFTR gene. Two novel mutations have been found, but we have been unsuccessful in finding any mutation in 40% of these alleles. These results suggest that, in this Mediterranean population, additional mutations may lie elsewhere in the promoter region or in introns not yet analyzed.

Strategy linking several analytical methods of neonatal screening for sickle cell disease.

BACKGROUND: The French national programme for the neonatal screening of sickle cell disease (SCD) was set up in 1995. This screening is targeted at newborn infants at risk. Over 5 years, 115,480 newborn infants were tested from 80 maternity departments from the northern part of the Paris area. 250 Patients with SCD were identified--that is, one in 462 newborn infants tested. Carriers for a haemoglobin (Hb) variant are frequent (5.34%). Some uncommon Hb variants were also identified, which gave rise to pitfalls to the testing when associated with HbS: HbKorle-Bu, HbHope, HbBougardirey-Mali, and HbLadésirade (4% of SS-like profiles). OBJECTIVE: An effective screening strategy was developed to avoid these false positive and false negative responses. METHODS: Isoelectric focusing (IEF), the method of primary screening, is rapid and inexpensive. Cation exchange high performance liquid chromatography (CE-HPLC), which is automated, fast, and quantitative was selected as a secondary method. RESULTS: IEF diagnosed normal profiles in 89% of the tested samples from newborn infants. CE-HPLC identified most of the common Hb variants by their retention time and the measure of HbA/HbS ratio, important for the differential diagnosis between an asymptomatic HbS carrier and an HbS/beta+thal compound heterozygote. Furthermore, the high sensitivity of the CE-HPLC detected as little as 0.5% of a Hb variant. This avoided false negatives in samples from premature or transfused newborn infants. All samples with SS-like profiles were confirmed with a second CE-HPLC with another programme. A combination of these three methods confirmed the status of 99.7% of the samples from the tested newborn infants. Some cases required a reverse phase-HPLC method (for gamma-globin or alpha-globin chain variants). Finally, some exceptional samples required confirmation by testing DNA extracted with Güthrie paper for a precise diagnosis. CONCLUSIONS: This effective strategy combining several methods dramatically reduces the risk of errors. Many families are thus spared unnecessary worrying recalls. The only unavoidable cause of false positives remains the HbS/hereditary fetal Hb (HPFH).