Published online before print May 21, 2009

This Article Full Text Full Text (PDF) All Versions of this Article: jmoldx.2009.080151v1 11/4/334    most recent Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thongnoppakhun, W. Articles by Yenchitsomanus, P.-t. Search for Related Content PubMed PubMed Citation Articles by Thongnoppakhun, W. Articles by Yenchitsomanus, P.-t.

Journal of Molecular Diagnostics 2009, Vol. 11, No. 4
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080151

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations

Wanna Thongnoppakhun^*, Surasak Jiemsup, Suganya Yongkiettrakul, Chompunut Kanjanakorn^*, Chanin Limwongse^*, Prapon Wilairat, Anusorn Vanasant, Nanyawan Rungroj^* and Pa-thai Yenchitsomanus^*

From the Departments of Research and Development, * and Medicine, Faculty of Medicine Siriraj Hospital, and the Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok; and the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathumthani, Thailand

A number of common mutations in the hemoglobin β (HBB) gene cause β-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous β-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.