Rapid Pyrosequencing Method for FMO3 Non-Synonymous Genetic Variant Evaluation in A Korean Population

Background: The aim of this study was to develop a feasible pyrosequencing method to detect non-synonymous single nucleotide polymorphisms (SNPs) of the avin-containing monooxygenase 3 (FMO3) gene and compare the ethnic differences in the frequencies of these alleles. Methods and Results: This pyrosequencing method was used to identify four non-synonymous FMO3 SNPs, including c.855C>T (rs909530), c.441C>T (rs1800822), c.923A>G (rs2266782), and c.472G>A (rs2266782). The allele frequencies of these SNPs in 122 unrelated Korean subjects were analyzed, and were as follows: 44.7% for c.855C>T, 23.4% for c.441C>T, 23.0% for c.923A>G, and 27.1% for c.472G>A. Linkage disequilibrium (LD) analysis showed that c.923A>G and c.472G>A were in strong LD (D′ = 0.8289, r 2 = 0.5332). Conclusions: The designed pyrosequencing method was successfully applied to identify the c.855C>T, c.441C>T, c.923A>G, and c.472G>A SNPs. The frequencies were similar to those reported previously in a Japanese population. However, in general, large differences between ethnicities were found.

These mutations have shown frequent functional inter-individual and inter-ethnic variability, and their signi cance is associated with the pharmacokinetics of certain chemicals (e.g., sulindac and ranitidine) [9]. Related disease manifestations have been described previously [13,14]. One of the most commonly studied topics is the impact of FMO3 gene mutations in trimethylaminuria (also known as sh odor syndrome) patients [12,15]. The FMO3 enzyme is known to increase plasma trimethylamine N-oxide (TMAO) levels by converting trimethylamine (TMA) derived from the gut microbiome [16]. Therefore, the single nucleotide polymorphisms (SNPs) responsible for FMO3 loss-of-function may result in increased plasma TMA levels [10]. Moreover, increased levels of TMAO are associated with greater risk of cardiovascular disorders because they affect atherosclerosis pathogenesis [17]. Recently, it was reported that a mutation in FMO3 was responsible for sulindac pharmacokinetics in Korean and Chinese populations [9,18]. Because the pharmacogenetics of the FMO3 gene play a crucial role in its substrate disposition, it is necessary to develop a feasible method to detect SNPs and validate the analysis for future research. Therefore, this study aimed to develop a rapid, feasible pyrosequencing method to detect nonsynonymous FMO3 SNPs c.855C > T (rs909530), c.441C > T (rs1800822), c.923A > G (rs2266780), and c.472G > A (rs2266782), all of which have been reported to be functional and commonly present in the Korean population [9,18], and compare allele frequencies with those reported in other ethnic groups.

Subjects and methods
Genomic DNA samples were obtained from 122 unrelated Korean subjects who provided written informed consent. The protocol for the assay was approved by the institutional review board of Anam Hospital, Korea University Medical Center (Seoul, Korea).
Polymerase chain reaction (PCR) conditions and FMO3 genotyping using the pyrosequencing method Genomic DNA was isolated from peripheral blood leukocytes as previously described [19,20]. We developed a pyrosequencing method to identify the functional SNPs of the FMO3 gene: c.855C > T (rs909530), c.441C > T (rs1800822), c.923A > G (rs2266780), and c.472G > A (rs2266782). The primers used in the PCR analysis for FMO3 genotyping and pyrosequencing are listed in Table 1. PCR reactions were performed to amplify sequences to identify each FMO3 SNP using newly developed primer sets after biotin was attached to the 5' end of each forward (or reverse) primer using the PSQ Assay Design software (Biotage AB, Uppsala, Sweden).
125 60 132 60 primer incorporated into each well. For strand separation, all liquids were removed using a vacuum prep workstation (Pyrosequencing AB, Uppsala, Sweden). The beads captured on probes were incubated in 70% ethanol, and the solution was ushed through the lters for 5 s. The beads were then treated with a denaturing solution (0.2 M NaOH) that was ushed through the lters for 5 s. A wash buffer (10 mM Trisacetate, pH 7.6) was used to rinse the beads for 5 s. All liquid was completely drained from the probes, and the beads were released into a PSQ 96 Plate Low (Pyrosequencing AB) containing the sequencing primer. The PSQ 96 Plate Low was heated at 85°C for 2 min, and the reactions were allowed to cool to room temperature. The resulting mixture was analyzed using a PSQ 96MA Pyrosequencer (Pyrosequencing AB). The pyrosequencing accuracy was validated by direct DNA sequencing of randomly selected samples using the same genomic DNA.

Statistical analysis
Genetic equilibrium and linkage disequilibrium (LD) were tested according to the Hardy-Weinberg equation using SNPAnalyzer version 9.0 (DYNACOM Co., Ltd., Yokohama, Japan). The chi-square test was used to assess the consistency of the pyrosequencing method. p < 0.05 (two-tailed) was considered to be statistically signi cant. D′ and r 2 are standard measurements for LD [21]. D′ values were calculated as D/D max , where D is the coe cient of LD ranging from − 0.25 to 0.25. Generally, the standardized value D′ is preferred because D is often affected by allele frequencies [22].

Results
Each SNP for c.855C > T, c.441C > T, c.923A > G, and c.472G > A was identi ed and compared to the predesigned pyrosequencing histogram (Fig. 1). Representative peaks for each SNP analysis are shown in Fig. 2. The sequenced data obtained from the pyrosequencing method were randomly selected and validated by direct DNA sequencing, which showed 100% concordance, thereby indicating 100% speci city and sensitivity.
The observed allele frequencies for FMO3 genetic analysis in the Korean population (n = 122) using our pyrosequencing method were as follows: 44.7% for c.855C > T, 23  The expected and observed frequencies were compared using the Hardy-Weinberg equation.
The analyzed allele frequencies were compared to those investigated in other ethnicities and those reported in the HapMap database ( Table 3). The data were very limited, especially in European and African populations; however, the trend of frequencies in FMO3 polymorphisms in our study was most similar to those published previously in the Japanese population. Only the frequencies of c.923A > G and c.472G > A appeared to be similar in the Chinese population. The occurrence of the c.923A > G polymorphism showed some similarity to the minor allele frequency (MAF) of the HapMap data on Utah residents with Northern and Western European ancestry from the CEPH collection reported by the National Center for Biotechnology Information SNP database (https://www.ncbi.nlm.nih.gov/snp), while others showed substantial differences.

Discussion
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 rst study to analyze nonsynonymous 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 rst 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 speci city 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. Signi cant 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. Declarations