β-Thalassemia Microelectronic Chip: A Fast and Accurate Method for Mutation Detection

Department of Medicine , Thomas Jefferson University, Filadelfia, Pennsylvania, United States
Clinical Chemistry (Impact Factor: 7.91). 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.

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Available from: Anna Ravani, Jan 15, 2015
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    • "Nowadays a number of systems for molecular detection of the β-thalassemia mutations is commercially available, which are not completely automated and quite expensive. Among them, the oligonucleotide microchip based-assays have been proposed many times for the large-scale detection of mutations in genetic diseases, including β-thalassaemia.15 Given the alternative features of high throughput and automation, the DNA chip has the potential to become a valuable method in future applications of mutation detection in medicine. "
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    ABSTRACT: Prenatal diagnosis of β-thalassemia was accomplished for the first time in the 1970s by globin chain synthesis analysis on fetal blood obtained by placental aspiration at 18-22 weeks gestation. Since then, the molecular definition of the β-globin gene pathology, the development of procedures of DNA analysis, and the introduction of chorionic villous sampling have dramatically improved prenatal diagnosis of this disease and of related disorders. Much information is now available about the molecular mechanisms of the diseases and the molecular testing is widespread. As prenatal diagnosis has to provide an accurate, safe and early result, an efficient screening of the population and a rapid molecular characterization of the couple at risk, are necessary prerequisites. In the last decades earlier and less invasive approaches for prenatal diagnosis were developed. A overview of the most promising procedure will be done. Moreover, in order to reduce the choice of interrupting the pregnancy in case of affected fetus, Preimplantation or Preconceptional Genetic Diagnosis (PGD) has been setting up for several diseases including thalassemias.
    Mediterranean Journal of Hematology and Infectious Diseases 11/2009; 1(1):e2009011. DOI:10.4084/MJHID.2009.011
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    • "Several assays have been developed to genotype common b-thalassemia mutations, including amplification refractory mutation analysis (Fortina et al. 1992), DNA-probe assays (Ugozzoli et al. 1998), fluorescent multiplex PCR (Sherlock et al. 1998), real-time PCR (Vrettou et al. 2004), and microelectronic chip-based assays (Foglieni et al. 2004). Each of these methods has advantages and limitations, and a highly accurate, highthroughput method to simultaneously genotype common HBB alleles has yet to be developed and widely implemented. "
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    ABSTRACT: Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by "mass tuning" the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common beta-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.
    Journal of Human Genetics 02/2005; 50(3):139-50. DOI:10.1007/s10038-005-0234-z · 2.46 Impact Factor
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    ABSTRACT: Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real-time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male-bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long-term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild-type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild-type allele signals.
    Annals of the New York Academy of Sciences 07/2004; 1022(1):105-12. DOI:10.1196/annals.1318.017 · 4.38 Impact Factor
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