HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit an electronic Letter to the Editor about this paper Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Guida, V. Articles by Dallapiccola, B. Search for Related Content PubMed PubMed Citation Articles by Guida, V. Articles by Dallapiccola, B. Related Collections Molecular Diagnostics and Genetics

Denaturing HPLC-Based Assay for Molecular Screening of Nondeletional Mutations Causing -Thalassemias

¹ Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS)-Casa Sollievo Sofferenza (CSS), San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy;² Department of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy;³ Department of Biomedical Sciences, University "G. D’Annunzio", Chieti, Italy;⁴ Centro Microcitemia Associazione Nazionale Microcitemia Italiana-ONLUS, Rome, Italy;⁵ Human Genetic Laboratory, Galliera Hospital, Genoa, Italy;⁶ Laboratorio di Patologia Genetica IRCCS Oasi Maria Santissima, Troina, Italy;

^aaddress correspondence to this author at: CSS-Mendel Institute, Viale Regina Margherita 261, 00198 Rome, Italy; fax 39-06-44160548, e-mail v.guida@css-mendel.it

The first 20% of the full text of this article appears below.

-Thalassemias (OMIM 141850 and 141800; GenBank accession no. NT037887) are recessively inherited hemoglobin disorders caused by loss of function of either one of the two duplicated -globin genes (1 and 2), both located on chromosome 16p13.3 (1)(2). More than 95% of -thalassemia phenotypes result from meiotic unequal recombinational events between the highly homologous 1- and 2-globin loci, which lead mainly to large genomic deletions (3–100 kb), which remove one to four -globin genes, and rarely to -gene triplication or quadruplication. Although less frequent, at least 48 different nondeletional mutations (including point mutations and deletions/insertions of a few nucleotides), mostly located in the 2-globin gene, have also been reported as causative mutations of ⁺-thalassemia (3)(4). At present, molecular identification of this type of nucleotide mutation is carried out by specific PCR amplification of the 2 or 1 gene, followed by methods that have only a limited rate of detection (60–80%) and are technically demanding, such as single-strand conformation polymorphism (SSCP) analysis and denaturing gradient gel electrophoresis (5)(6), or are costly, cumbersome, and time-consuming, such as direct sequencing and reverse dot-blot analysis (7)(8).

We have evaluated the performance of a relatively simple and semiautomated technique, denaturing HPLC (DHPLC), that separates heteroduplex and homoduplex molecules on a stationary phase under partially denaturing conditions (9)(10). We tested a Transgenomic^TM Wave DHPLC-based protocol for the molecular identification of -globin gene nondeletional mutations in 50 wild-type individuals and 50 heterozygous carriers of Italian origin whose genes had previously been molecularly defined by restriction endonuclease digestion of PCR fragments and/or reverse dot-blot analysis.

Blood samples were collected from heterozygous individuals and healthy controls at the Centro Studi Microcitemie (Rome), the . . . [Full Text of this Article]