Estimation of Lewis Blood Group Status by Fluorescence Melting Curve Analysis in Simultaneous Genotyping of c.385A>T and Fusion Gene in and c.59T>G and c.314C>T in .

Lewis blood group status is determined by two fucosyltransferase activities: those of -encoded fucosyltransferase (Se enzyme) and -encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in and a fusion gene between and its pseudogene are the cause of most Se enzyme-deficient alleles ( and ), and c.59T>G and c.314C>T in are tag SNPs for almost all nonfunctional alleles (, , , and ). In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and using a pair of primers that collectively amplify , se, and . Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and assay system by adding primers and probes to detect c.59T>G and c.314C>T in . We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose and genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, /, 385A/T, 385A/, and 385T/. In addition, the triplex FMCA successfully identified both and genotypes, although the resolutions of the analysis of c.385A>T and were somewhat reduced compared to that of the analysis of alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.