Abstract
β-thalassemia is a common monogenic disease caused by mutations in the human β-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 β-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.
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Acknowledgements
Financial support from Academia Sinica and the National Science Council, Taiwan of the Republic of China (NSC 92-2113-M-001-071) is gratefully acknowledged. Hsin-Kai Liao and Yi-Ning Su contributed equally to this study.
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Liao, HK., Su, YN., Kao, HY. et al. Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations. J Hum Genet 50, 139–150 (2005). https://doi.org/10.1007/s10038-005-0234-z
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DOI: https://doi.org/10.1007/s10038-005-0234-z
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