Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations

Abstract

b-thalassemia is a common monogenic disease caused by mutations in the human b-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 b-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.

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.…”

Gene expression and promoter polymorphisms of DNA methyltransferase 3B in nasopharyngeal carcinomas in Taiwanese people: A case-control study

“…The DNMT3B -283T/C, -579G/T, C46359T polymorphisms were determined by a MALDI-TOF based mini-sequencing genotyping method as previously described (24,25). The primer sets used for amplification of each SNP region were as described previously (26).…”

“…There was no statistically significant difference in age and gender between NPC cases and controls. The genotypes of the three DNMT3B SNPs were determined by means of a MALDI-TOF-based mini-sequencing method (24,25), the primer sets used for generating three separate DNA fragments covering each of the three DNMT3B SNPs, the mini-sequencing primer, and the molecular weight of mini-sequencing products are as we described previously (26). The PCR products were randomly selected to determine their DNA sequences by auto-sequencing analysis.…”

Molecular Diagnostics in Transfusion Medicine: MALDI-TOF MS for Blood Bankers- The Alternative Approach for Genotyping

“…Because of the minimal DNA amount requirements and short amplicon length, even formalin-fixed and paraffin-embedded tissue or ancient DNA samples can be processed. Successful applications of this system in the fields of forensics, oncology, pharmacogenetics, and haematology, including β-thalassemia genotyping have been previously reported [38,[45][46][47][48]. Additionally, MALDI-TOF MS has been successfully employed in genotyping foetal, paternally inherited SNPs [49].…”