Validation of dHPLC for Molecular Diagnosis of β-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.

Analysis of fetal blood using capillary electrophoresis system: a simple method for prenatal diagnosis of severe thalassemia diseases

“…The use of CVS is preferable as it allows the possibility to obtain results at an earlier stage of pregnancy. The PCR assay is highly sensitive, could be used with small amount of fetal DNA sample and involved technical developments and improvements (22–24). However, misdiagnosis using PCR alone can occur either with maternal contamination or allelic dropout.…”

A Novel Frameshift Mutation (+A) at Codon 18 of the β-Globin Gene Associated with High Persistence of Fetal Hemoglobin Phenotype and δβ-Thalassemia

“…In contrast to α-thalassemia, where deletions in the α-globin gene cluster account for the disease, the molecular defects causing β-thalassemia are usually point mutations involving only 1 or few nucleotide(s) [4]. For instance, β⁰39-thalassemia is caused by a stop codon mutation that leads to premature termination of β-globin chain synthesis [5]; the β⁰IVSI-1 mutation suppresses correct maturation of the β-globin RNA precursor, while in thalassemia with the β + IVSI-110 mutation normal and abnormal spliced β-globin RNA precursors coexist [6]. …”

Rapid genotyping of known mutations and polymorphisms in β-globin gene based on the DHPLC profile patterns of homoduplexes and heteroduplexes

“…DHPLC has been extensively used for mutational screening of β-globin alleles and other disease-causing alleles and polymorphisms by heteroduplex analysis using partially-denaturing conditions [12][13][14][15]. One advantage of the present method is the use of a proven mutation detection technology typically used for screening of unknown mutations for genotyping of known mutations.…”

“…Denaturing high-performance liquid chromatography (DHPLC) has been shown to be an automated, highly sensitive, and reliable genotyping platform for detecting point mutations, particularly used for screening of unknown mutations, based on the separation of heteroduplexes from homoduplexes by ion pair reverse-phase HPLC under partially-denaturing conditions [12][13][14][15]. In this study, we developed an assay to effectively screen for the known mutations of β-thalassemia in the Chinese population by DHPLC.…”