Our results indicate that the newly developed rapid pyrosequencing method for analyzing the c.855C > T, c.441C > T, c.923A > G, and c.472G > A SNPs is a feasible and accurate technique. The allele frequencies obtained by this method in 122 Korean subjects suggested that the results were generally most similar to those reported in the Japanese population [23]. To our knowledge, this is the first study to analyze non-synonymous FMO3 SNPs using a pyrosequencing method.
Various methods have been suggested for the analysis of the targeted SNPs. For example, FMO3-related SNPs were detected by PCR-restriction fragment length polymorphism [24], real-time PCR [25], or direct sequencing methods [26]. The automated sequencing method was first introduced in the 1970s by Frederick Sanger [27]. The principle of this method is the use of dideoxynucleotide triphosphates as a DNA sequence termination technique. Our pyrosequencing method for analyzing FMO3 SNPs was designed based on the solution-based pyrosequencing method suggested by Ronaghi et al. in 1998, which is a simple method suitable for automation using apyrase, DNA polymerase, and luciferase that eventually detects light emission by pyrophosphate production during DNA synthesis [28]. The major advantages of this method are its simplicity, feasibility, sensitivity, and specificity compared to conventional sequencing systems [29]. Therefore, we assumed that our developed method was suitable for our study aims to precisely, rapidly, and cost-effectively measure the occurrence of targeted SNPs in a relatively large sample size.
SNPs are the most frequently occurring sequence variations in the human genome and often vary among different ethnic groups. Among the currently reported non-synonymous FMO3 SNPs, we chose to analyze the c.855C > T, c.441C > T, c.923A > G, and c.472G > A SNPs, which are relatively common and known to be functionally effective in East Asian populations as described previously [9, 23]. The allele frequencies observed in this study were comparable to those reported in the Japanese population, while only c.923A > G and c.472G > A were similar to those found in the Chinese population. Among the analyzed FMO3 SNPs, c.855C > T was the most common in the Korean population, and the result was similar to that reported in a smaller Korean population (n = 41, MAF = 0.329) [9].
Several previous studies have investigated functional SNPs in FMO3, although detailed pharmacokinetic studies based on FMO3 genetic polymorphisms are scarce. For example, c.441C > T and c.855C > T are associated with fast tacrolimus elimination in Chinese patients [30]. Furthermore, c.855C > T and c.472G > A have been shown to affect the pharmacokinetics of sulindac in women who underwent preterm labor [9]. c.923A > G has been associated with reduced nicotine dependence in European Americans [31]. Considering these ethnic and inter-individual differences in SNPs and their suspected clinical roles, personalized dosing, pharmacokinetics, and pharmacodynamics studies for drugs based on FMO3 SNPs may present a novel research direction.
In conclusion, the designed pyrosequencing method was successfully applied to identify the c.855C > T, c.441C > T, c.923A > G, and c.472G > A SNPs. In Korean subjects, c.855C > T (rs909530) was most frequently found among the four non-synonymous FMO3 SNPs. Significant differences were observed when the frequencies of these alleles were compared to those of other ethnic groups; however, they were most similar to those reported in the Japanese population.