Beta-thalassemia microelectronic chip: a fast and accurate method for mutation detection.

Unit of Genomics for Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Raffaele, Milan, Italy.
Clinical Chemistry (Impact Factor: 7.15). 02/2004; 50(1):73-9. DOI: 10.1373/clinchem.2003.023077
Source: PubMed

ABSTRACT beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area.
We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes.
Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip.
The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.

  • Source
    Journal of Biosensors & Bioelectronics. 01/2012;
  • [Show abstract] [Hide abstract]
    ABSTRACT: β-thalassemia is one of the most common genetic disorders worldwide. Concerted efforts are being made to prevent the disease, as the medical and economic burden of thalassemia represents a major public health problem. The molecular diagnosis of the β-globin mutations that cause the disease currently involves a combination of classic methodologies. A microarray-based assay for parallel one-shot detection of mutations has been developed, but the assay remains too expensive for routine application. We developed a cost-effective plastic fiber-based DNA chip for the fast and reliable detection of 25 types of β-thalassemia mutations. Assay conditions were established and genotyping was successfully performed on a genomic sample from a β-thalassemia patient. Our data show that this β-thalassemia genotyping chip is an advantageous platform for mass genotyping because of its low cost, rapid results, and reliability.
    International journal of hematology 07/2012; 96(3):301-7. · 1.17 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: β-Thalassemias and abnormal hemoglobin variants are among the most common hereditary abnormalities in humans. Molecular characterization of the causative genetic variants is an essential part of the diagnostic process. In geographic areas with high hemoglobinopathy prevalence, such as the Mediterranean region, a limited number of genetic variants are responsible for the majority of hemoglobinopathy cases. Developing reliable, rapid and cost-effective mutation-specific molecular diagnostic assays targeting particular populations greatly facilitates routine hemoglobinopathy investigations. We developed a one-tube single-nucleotide primer extension assay for the detection of eight common Mediterranean β-thalassemia mutations: Codon 5 (-CT); CCT(Pro)->C-, Codon 6 (-A); GAG(Glu)->G-G, Codon 8 (-AA); AAG(Lys)->-G, IVS-I-1 (G->A), IVS-I-6 (T->C), IVS-I-110 (G->A), Codon 39 (C->T), and IVS-II-745 (C->G), as well as the hemoglobin S variant beta 6(A3) Glu>Val. We validated the new assay using previously genotyped samples obtaining 100% agreement between independent genotyping methods. Our approach, applicable in a range of Mediterranean countries, offers a combination of high accuracy and rapidity exploiting standard techniques and widely available equipment. It can be further adapted to particular populations by including/excluding assayed mutations. We facilitate future modifications by providing detailed information on assay design.
    PLoS ONE 01/2012; 7(10):e48167. · 3.53 Impact Factor


Available from