1. ¹Institute of Chemistry, Academia Sinica, Taipei, 11529, Taiwan, Republic of China
2. ²Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan, Republic of China
3. ³Department of Chemical Engineering, National Taipei University of Technology, Taipei, Taiwan, Republic of China
4. ⁴Department of Chemistry, National Changhua University of Education, Changhua, Taiwan, Republic of China

Correspondence: Yu-Ju Chen, Institute of Chemistry, Academia Sinica, Taipei, 11529, Taiwan, Republic of China. Fax: +886-2-27831237. E-mail: yjchen@chem.sinica.edu.tw

Received 10 November 2004; Accepted 17 January 2005; Published online 11 March 2005.

Abstract

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