title: Simple, efficient, and cost-effective multiplex genotyping with matrix assisted laser desorption/ionization time-of-flight mass spectrometry of hemoglobin beta gene mutations.
abstract: A number of common mutations in the hemoglobin beta (HBB) gene cause beta-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 beta-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.
authors.affiliation: Departments of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand .
authors.forename: Wanna
authors.initials: W
authors.lastname: Thongnoppakhun
authors.forename: Surasak
authors.initials: S
authors.lastname: Jiemsup
authors.forename: Suganya
authors.initials: S
authors.lastname: Yongkiettrakul
authors.forename: Chompunut
authors.initials: C
authors.lastname: Kanjanakorn
authors.forename: Chanin
authors.initials: C
authors.lastname: Limwongse
authors.forename: Prapon
authors.initials: P
authors.lastname: Wilairat
authors.forename: Anusorn
authors.initials: A
authors.lastname: Vanasant
authors.forename: Nanyawan
authors.initials: N
authors.lastname: Rungroj
authors.forename: Pa-Thai
authors.initials: PT
authors.lastname: Yenchitsomanus
mesh_terms: D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D005821:Genetic Techniques / Q000191:economics; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000175:diagnosis / Q000235:genetics
chemical_list: D015415:Biomarkers; D055544:beta-Globins