Article

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.

0 Bookmarks
 · 
102 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Thalassemia is one of the most common autosomal single-gene disorder worldwide. The highest prevalence of the disease is in the "thalassemia belt" which includes the Mediterranean region, parts of the Middle East, the Indian subcontinent, the southern parts of the Far East, Pakistan and South-East Asia. This study aimed to detect the common molecular abnormalities of the beta thalassemia syndrome in Pakistan. The study was conducted at the Institute of Hematology, Baqai Medical University, Karachi, Pakistan from August 2004 to November 2007. Blood samples of patients with beta thalassemia major (n = 400) were collected from hospital transfusion centres and diagnostic laboratories in different districts of Karachi representing five major ethnic groups including Punjabi, Pathan, Sindhi, Baluchi and Urdu speaking. All the samples were analysed for five common mutations by using the polymerase chain reaction technique ARMS (amplification of refractory mutation system). The data revealed five common mutations including IVS 1-5(G→C), Fr 41/42(-CTTT), Fr 8/9 (+G), IVS 1-1 and Del 619. These accounted for 90% of the total beta thalassemia genes in Pakistan. The IVS 1-5(G→C) was found to be the most common beta thalassemia gene in the Pakistani population with a frequency of 44.4% present in all major ethnic groups. The results of this study will be helpful in the establishment of a large scale prenatal diagnosis programme in Pakistan.
    Sultan Qaboos University medical journal 12/2009; 9(3):305-10.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Craniosynostosis, the premature fusion of 1 or more sutures of the skull, is a common congenital defect, with a prevalence of 1 in 2500 live births. Untreated progressive craniosynostosis leads to inhibition of brain growth and increased intracranial and intraorbital pressure. The heterogeneity of clinical phenotypes and the overlap of the various associated syndromes render the correct diagnosis of the different craniosynostoses particularly difficult. To identify 10 common mutations in the genes for fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3), we developed a microelectronic microchip assay that exploited the PCR multiplexing format and coupled it with serial addressing and probe hybridization on the same pad. For the molecular characterization of patients who tested negative in the microchip screening, we also developed conditions for denaturing HPLC (DHPLC) analysis of the most mutated regions of FGFR2 and FGFR3 and the entire coding region of the TWIST1 gene. In our cohort of 159 patients with various craniosynostosis syndromes, mutations were found in 100% of patients with Apert syndrome, 83.3% with Pfeiffer syndrome, 72.7% with Crouzon syndrome, 50.0% with Saethre-Chotzen syndrome, 27.7% with plagiocephaly, 31.8% with brachicephaly, 20% of complex cases, and 6.9% of mixed cases. No mutations were found in syndromic cases. The combined microchip-DHPLC strategy allows rapid and specific molecular diagnosis of craniosynostosis and is an effective tool for the medical and surgical management of these common congenital anomalies in a newborn or an infant with a developmental defect of the cranial vault.
    Clinical Chemistry 11/2007; 53(10):1767-74. · 7.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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. · 2.37 Impact Factor

Full-text

View
0 Downloads
Available from