title: Validation of dHPLC for molecular diagnosis of beta-thalassemia in Southern Italy.
abstract: Beta-thalassemia, the most common hereditary anemia in the Mediterranean area, results from over 200 causative mutations in the beta-globin locus. The aim of this study was to validate a denaturing high-performance liquid chromatography (dHPLC)-based assay for postnatal and prenatal molecular diagnosis of beta-thalassemia in Southern Italy. Sixty beta-thalassemic patients, affected either by thalassemia intermedia or thalassemia major, were analyzed in a blind study. We also carried out prenatal molecular diagnosis in 12 couples at-risk for having affected offspring. Chorionic villi samples were subjected to dHPLC analysis upon molecular characterization of the parental beta-globin alleles. Direct sequence analysis was used to validate each result, showing an accuracy rate of 100% for dHPLC. Overall, our protocol was able to identify the responsible mutations in all 96 analyzed subjects (including 12 prenatals in at-risk pregnancies), detecting the eight most common mutations in Southern Italy. Three rare mutations (one of which, reported here for the first time) that standard mutation detection methods failed to reveal, were also identified. dHPLC assay proved to be a reliable, rapid, and sensitive method for detecting both common and rare mutations within the beta-globin gene. Because of this property our protocol has the potential to be implemented for mutational screening in different areas of high prevalence for beta-thalassemia.
authors.affiliation: CSS-Mendel Institute, Viale Regine Margherita 261, 00198 Rome, Italy. a.colosimo@css-mendel.it
authors.forename: Alessia
authors.initials: A
authors.lastname: Colosimo
authors.forename: Valentina
authors.initials: V
authors.lastname: Guida
authors.forename: Anna
authors.initials: A
authors.lastname: Scolari
authors.forename: Alessandro
authors.initials: A
authors.lastname: De Luca
authors.forename: Giandomenico
authors.initials: G
authors.lastname: Palka
authors.forename: Luciana
authors.initials: L
authors.lastname: Rigoli
authors.forename: Anna
authors.initials: A
authors.lastname: Meo
authors.forename: Damiano Carmelo
authors.initials: DC
authors.lastname: Salpietro
authors.forename: Bruno
authors.initials: B
authors.lastname: Dallapiccola
mesh_terms: D015193:Chorionic Villi Sampling; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005315:Fetal Diseases / Q000175:diagnosis* / Q000235:genetics*; D005914:Globins / Q000235:genetics; D006801:Humans; D007558:Italy; D008297:Male; D009154:Mutation; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D016037:Single-Blind Method; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
chemical_list: D005914:Globins