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Clinical Chemistry 50: 73-79, 2004; 10.1373/clinchem.2003.023077
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(Clinical Chemistry. 2004;50:73-79.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Barbara Foglieni1, Laura Cremonesi1, Maurizio Travi2, Anna Ravani3, Antonino Giambona4, Maria Cristina Rosatelli5, Chiara Perra6, Paolo Fortina7 and Maurizio Ferrari1,8,a

1 Unit of Genomics for Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Raffaele, Milan, Italy.
2 Dipartimento di Medicina di Laboratorio, Laboratorio di Genetica Medica, Istituti Clinici di Perfezionamento, Milan, Italy.
3 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Università di Ferrara, Ferrara, Italy.
4 Divisione di Ematologia II, Azienda Ospedaliera Vincenzo Cervello, Palermo, Italy.
5 Laboratorio Genetica Molecolare, Dipartimento di Scienze Biomediche e Biotecnologie, Università degli Studi di Cagliari, Cagliari, Italy.
6 Ospedale Regionale Microcitemico, Cagliari, Italy.
7 Department of Medicine, Center of Translational Medicine, Thomas Jefferson University, Jefferson Medical College, Philadelphia, PA.
8 Diagnostica e Ricerca San Raffaele S.p.A., Milan, Italy.

aAddress correspondence to this author at: Unit of Genomics for the Diagnosis of Human Pathologies, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale San Raffaele, Via Olgettina 60, 20132 Milan, Italy. Fax 39-02-26432640; e-mail ferrari.maurizio{at}hsr.it.

Background: ß-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 ß-thalassemia alleles in the Mediterranean area.

Methods: 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.

Results: 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.

Conclusions: 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 ß-thalassemia in other regions of the world.




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