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 ␤ (HBB) gene cause ␤-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 ␤-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. (J Mol

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

“…25 Multiplex minisequencing has also been developed in combination with subsequent detection using a variety of platforms including capillary electrophoresis, 26 denaturing high-performance liquid chromatography, 27,28 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 29,30 However, these high-end techniques are best suited for use in a well-equipped central laboratory because of their expense and the high level of expertise required.…”

Fetal RHD genotype detection from circulating cell‐free fetal DNA in maternal plasma in non‐sensitized RhD negative women

“…To examine the performance of fetal RHD genotyping as a routine test in all RhD negative women, high‐throughput methods with robotic isolation of fetal DNA have been described that offer a cost‐effective alternative to routine administration of anti‐D (van der Schoot et al , 2006; Finning et al , 2008; Legler et al , 2009). Another technique for analysis of ccff DNA is MALDI‐TOF MS which combines flexibility, accuracy, automated analysis and high‐throughput data generation (Ding and Cantor 2003; Jurinke et al , 2004; Li et al , 2006; van der Schoot et al , 2008; Ding 2008; Oeth et al , 2009; Thongnoppakhun et al , 2009; Farkas et al , 2010; Tynan et al , 2010). In a companion publication we have also showed that fetal sexing can reliably be performed using the MALDI‐TOF MS technology (Akolekar et al , 2010).…”

Rational Design of New ThiazoloThiazole Dyes as Input Energy Units in Molecular Dyads

“…Mass spectrometry was used to provide a platform amenable to interrogation of > 90 DNA polymorphisms to identify candidates that are paternally inherited so as to differentiate maternal from fetal DNA. Mass spectrometry is the preferred platform for assays of density of many tens to hundreds of targets due to not only accuracy but also flexibility of panel design upstream of detection (Thongnoppakhun et al , 2009; Farkas et al , 2010). In the laboratory‐developed test, male results are reported directly.…”