Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/06/15/dmd.115.069153.DC1

1521-009X/44/9/1510–1515$25.00 http://dx.doi.org/10.1124/dmd.115.069153 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1510–1515, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Identification of Genetic Polymorphisms of CYP2W1 in the Three Main Chinese Ethnicities: Han, Tibetan, and Uighur

Yanwei Li,1 Xing Kang,1 Ge Yang, Penggao Dai, Chao Chen, and Huijuan Wang

National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University, Xi’an, China

Received December 21, 2015; accepted June 14, 2016

ABSTRACT CYP2W1 is an orphan member of the superfamily. data of nine exonic variants found in this study. CYP2W1*1A, *1B, *2, Recently, CYP2W1 has gained great research interest because of its *4,and*6 were detected as the main alleles/haplotypes. Moreover, unknown enzymatic function and tumor-specific expression property. one, three, and two ethnically specific haplotypes were observed in Downloaded from This study aims to investigate the genetic polymorphisms of the the Han, Tibetan, and Uighur samples, respectively. Then, the effects CYP2W1 in Chinese populations and explore the functions of the of four detected missense mutations (Ala181Thr, Gly376Ser, Val432Ile, detected variants. All of the nine exons and exon-intron junction and Pro488Leu) on the CYP2W1 function were predicted using regions of the CYP2W1 gene were sequenced in 150 Chinese three in silico tools: Polymorphism Phenotyping v2, Sorts Intolerant subjects, including 50 Han Chinese, 50 Tibetans, and 50 Uighurs. from Tolerant, and MutationTaster. The results showed that

A total of 26 genetic variants were identified in this study, and Gly376Ser and Pro488Leu may have deleterious effects. In summary, dmd.aspetjournals.org 19 polymorphisms were detected in each population. Frequency this study showed that the genetic pattern of CYP2W1 is interethni- comparison between populations showed that nine variants exhibited cally different among the three Chinese populations, and this finding significantly different allelic distributions. A total of 12 different can extend our understanding of population genetics of CYP2W1 in haplotypes were inferred from 150 samples by using the genotype the Chinese population. at ASPET Journals on September 24, 2021 Introduction Guengerich, 2012; Xiao and Guengerich, 2012). Thus, human CYP2W1 CYP2W1 is an extrahepatic cytochrome P450 (P450) , and is is a potential drug target for cancer therapy using antibodies and/or specific considered as an “orphan” P450 because of its unknown physiologic CYP2W1 activated prodrugs (Nishida et al., 2010; Travica et al., 2013). functions (Karlgren and Ingelman-Sundberg, 2007). Recent studies have Many of the P450 isoforms are highly polymorphic. The genetic reported that CYP2W1 is not expressed in adult human liver or in human polymorphisms in P450 can alter enzymatic activity and change the primary hepatocytes. Only low amounts of CYP2W1 mRNA were found metabolic rate of the drugs or procarcinogens catalyzed by the , in human adult extrahepatic and fetal tissues (Karlgren et al., 2006). resulting in varied drug responses or disease susceptibilities among However, CYP2W1 is highly expressed in several human tumor tissues, individuals (Wijnen et al., 2007). In a study that investigated the distribution especially colon tissues. Therefore, the expression of CYP2W1 seems of the genetic polymorphisms in a Japanese population, only six alleles tumor-specific (Karlgren et al., 2006; Choong et al., 2015). Further and five nonsynonymous single-nucleotide polymorphisms (nsSNPs) studies have demonstrated that the expression of CYP2W1 is regulated in CYP2W1 were reported (http://www.cypalleles.ki.se/cyp2w1.htm) by DNA methylation, and that the CYP2W1 gene is silenced by DNA (Hanzawa et al., 2008). Of the nsSNPs identified, Pro488Leu (CYP2W1*6) methylation after development and then reactivated in tumors by was found to be more common in the Japanese population than in hypomethylation (Karlgren et al., 2006; Gomez et al., 2007; Choong Caucasians (allele frequency: 0.368 vs. 0.06). In contrast, Ala181Thr et al., 2015). The in vitro expression of CYP2W1 in a heterologous (CYP2W1*2) is prevalent in Caucasians but rare in the Japanese (allele system revealed a broad range of catalytic activities toward several frequency: 0.21 vs. 0.014) (Hanzawa et al., 2008; Gervasini et al., 2010; chemicals, including arachidonic acid, benzphetamine, and several Stenstedt et al., 2013). Therefore, the distribution of CYP2W1 genetic procarcinogens (polycyclic aromatic hydrocarbon dihydrodiols, afla- polymorphisms is interethnically different. Still, further studies with toxin B1, and sterigmatocystin) (Wu et al., 2006; Yoshioka et al., 2006; different ethnic groups are needed for a more comprehensive understand- Eun et al., 2010; Nishida et al., 2010; Tan et al., 2011; Wang and ing of the distribution of CYP2W1 genetic polymorphisms in humans. Several studies have investigated the function of CYP2W1 genetic polymorphisms, and their conclusions are inconsistent with respect to the effect of CYP2W1 genetic variants on tumor susceptibility (Gervasini This work was supported by the National Natural Science Foundation of China [Grant J1210063] and the China Postdoctoral Science Foundation [Grant et al., 2010; Cardenas-Rodriguez et al., 2012; Stenstedt et al., 2013). One 2015M582695]. study showed that CYP2W1*2 is significantly associated with increased 1Y.L. and X.K. contributed equally to this work. colorectal cancer risk in a Caucasian population (Gervasini et al., 2010); dx.doi.org/10.1124/dmd.115.069153. however, another study debunked this relationship in another sample s This article has supplemental material available at dmd.aspetjournals.org. (Stenstedt et al., 2013). By using a human colon cancer cell expression

ABBREVIATIONS: LD, linkage disequilibrium; nsSNP, nonsynonymous SNP; P450, cytochrome P450; PolyPhen-2, Polymorphism Phenotyping v2; SIFT, Sorts Intolerant from Tolerant; SNP, single-nucleotide polymorphism; 39UTR, 39 untranslated region.

1510 Population Genetic Study of the CYP2W1 Gene 1511 system, the latter study also demonstrated that CYP2W1*2 and Results CYP2W1*6 do not alter the metabolic kinetics of CYP2W1 substrates CYP2W1 Genetic Polymorphisms and Distribution. A total of in vitro. Further studies are needed to characterize the function of 26 different genetic variations, including three novel ones in the CYP2W1 genetic polymorphisms and address their roles in disease CYP2W1 gene, were detected in 150 individuals (Table 1). The three susceptibility and drug metabolism. novel variants were all SNPs, located in the promoter (v01), intron7 (v21), The current study screened the genetic variants of CYP2W1 in three and 39UTR (v25), respectively. A total of nine variations were identified Chinese populations (Han, Tibetan, and Uighur) by sequencing the in the coding region, whereas the remaining variants were located in exonic sequences of the CYP2W1 gene in 150 samples. This process was the promoter (two variations), introns (13 variations), and 39UTR (two carried out to understand CYP2W1 genetic polymorphisms. The geno- variations). Four of the nine exonic SNPs were nsSNPs, located in type and haplotype distributions of CYP2W1 in Chinese populations exons 4, 7, and 9, giving rise to the amino acid exchanges Ala181Thr were investigated, and the effects of identified nsSNPs in these samples (v11), Gly376Ser (v20), Val432Ile (v23), and Pro488Leu (v24). One were predicted by using in silico tools. insertion variation (v09) in intron1 was also identified. The frequency of detected variations in specific populations was Materials and Methods calculated. First, 19 polymorphic variations were found in each Samples. Blood samples were collected from 150 unrelated healthy volunteers population (Table 2). They consist of 14 identical variations and five from three different nationalities: 50 Han Chinese from Shaanxi Province, ethnically specific variations. Second, statistical analysis showed that

50 Tibetans from Tibet, and 50 Uighurs from Xinjiang. All of the volunteers were nine variants (v3, v4, v05, v08, v9, v10, v13, v24, and v26) exhibited Downloaded from sophomores at Xizang Minzu University, aged about 19 years. The percentage of significantly different frequencies among three studied populations females in the Han, Tibetan, and Uighur samples is 52, 55, and 49%, respectively. (P , 0.05). Three variants (v04, v08, and v10) remained significantly This study was approved by the Ethics Committee of Northwest University, and different after Bonferroni correction. Third, of the four nsSNPs all participants signed an informed consent form before participating in the study. identified in this study, Gly376Ser (v20) was found to be heterozy- Genomic DNA Extraction. The genomic DNA samples were extracted from gous in one of the 50 Uighur subjects (Supplemental Table 2); m – 200 l of EDTA anticoagulated peripheral blood by using the QIAamp DNA Val432Ile (v23) presented with relatively low and comparable frequency blood mini kit (Qiagen, Shanghai, China). The extracted genomic DNA was dmd.aspetjournals.org (0.02–0.05, P = 1) among three populations; Ala181Thr (v11) exhibited dissolved in an elution buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) and stored at 280C. DNA concentration was measured by using a nanodrop significantly higher frequency in Uighurs (0.07) than in Hans (0.02) spectrophotometer (Thermo Fisher Scientific, Fitchburg, WI). (P = 0.03) and Tibetans (0.01) (P = 0.03); and Pro488Leu (v24) was CYP2W1 Genotyping. The 59 flanking region (21000 bp), all exons, their quite common in all studied populations, with significantly higher surrounding introns, and the 39 untranslated region (39UTR) of CYP2W1 were frequency in Tibetans (0.33) than in Uighurs (0.14) (P =0.002). sequenced to screen for CYP2W1 SNPs using the primers listed in Supplemental The allelic distributions of detected variations (Supplemental Table 2) Table 1. The location of these primers in the CYP2W1 sequence are shown in were also analyzed. The results showed that the allelic distribution of all Supplemental Fig. 1. The sequencing procedure was performed as previously variants was in a Hardy-Weinberg equilibrium (P . 0.05). Moreover, at ASPET Journals on September 24, 2021 described (Wang et al., 2011). nine variants (v3, v4, v05, v08, v9, v10, v13, v24, and v26) exhibited Data Analysis and Statistics. All polymorphisms were designated based on the nucleotide reference sequence (NC_000007.14) and the protein reference sequence (NP_060251.2). Allelic and genotypic frequencies were calculated TABLE 1 using the counting method [frequency of allele A or B = (AA or BB count + AB count /2)/(AA count + AB count + BB count)]. Pairwise linkage disequilibrium Genetic variations of CYP2W1 identified in this study (LD) across all SNPs and the Hardy-Weinberg equilibrium for each SNP were SNP rsID Location Nucleotide Changea Amino Acid Changeb assessed for each SNP using HAPLOVIEW version 4.2 (Broad Institute, Cambridge, MA). PHASE2.1 was used to reconstruct haplotypes and derive v01 Promotor 2717C.A haplotype frequencies (Stephens and Donnelly, 2001; Stephens and Scheet, v02 rs6768161064 Promotor 2668G.A . 2005). v03 rs2272375 Exon1 c.166 C T Leu56Leu v04 rs75358992 Intron1 IVS1+31 G.A Statistical analyses were conducted by using SPSS v.16.0 (IBM Corp., v05 rs3817576 Intron1 IVS1+170 A.G Armonk, NY). Fisher’s exact test was conducted to examine differences in the v06 rs79762569 Intron1 IVS1+247 A.G distributions of detected variant alleles and genotypes among the Han, Tibetan, v07 rs73259905 Intron1 IVS1+343 C.T and Uighur ethnic groups. Bonferroni correction was used for the adjustment of v08 rs3735689 Intron1 c.IVS2-26T.G multiple comparisons. A two-sided P value ,0.05 was considered statistically v09 rs201787342 Intron1 c.IVS2-25 ins C v10 rs3824081 Intron2 IVS3-5 T.C significant. . CYP2W1 v11 rs3735684 Exon4 c.541G A Ala181Thr Computational Prediction of the Functional Effect of nsSNPs. v12 rs201860979 Exon4 c.588G.A Val196Val In silico tools—Polymorphism Phenotyping v2 (PolyPhen-2) (Adzhubei et al., v13 rs9655345 Intron4 IVS5-186 A.G 2010). Mutation Tasten (Schwartz et al., 2014), and Sorts Intolerant from Tolerant v14 rs192750534 Intron4 IVS5-134 G.A (SIFT) (Kumar et al., 2009)—were used to predict the effects of identified CYP2W1 v15 rs772177532 Exon5 c.768 C.G Cys256Cys . nsSNPs on protein function. PolyPhen-2 predicts the possible impacts of amino v16 rs117425670 Intron5 IVS6-96 C T v17 rs113795030 Intron6 IVS6+17 T.C acid substitutions on the stability and function of human by using structural v18 rs61420495 Intron6 IVS6+18 G.A and comparative evolutionary considerations. A threshold of 0.15 was used, and a v19 rs377096714 Exon7 c.1035C.T Pro345Pro mutation is predicted to be “possibly damaging” if the prediction score is above v20 rs201612311 Exon7 c.1126G.A Gly376Ser 0.15 (Adzhubei et al., 2010). SIFT predicts whether an amino acid substitution v21 Intron7 IVS8-188T.C affects protein function. Such prediction is based on the degree of conservation of v22 rs370308301 Exon8 c.1236C.T Asp412Asp v23 rs78873069 Exon9 c.1294G.A Val432Ile amino acid residues in sequence alignments, which are derived from closely related . $ v24 rs3808348 Exon9 c.1463C T Pro488Leu sequences. The SNPs were classified as tolerated (cutoff value of 0.05) or v25 39UTR c.1886G.A damaging (cutoff value of #0.05) based on the prediction score (Kumar et al., v26 rs62433131 39UTR c.2208A.C 2009). MutationTaster is a Web-based application for the rapid evaluation of the disease-causing potential of DNA sequence alterations. Its analyses comprise rsID, reference SNP ID. aThe nucleotide change in the gene is given according to the CYP2W1 reference sequence evolutionary conservation, splice-site changes, the loss of protein features, and (NC_000007.14), where the A of the ATG translation initiation codon is denoted nucleotide +1. other changes that may affect the amount of mRNA (Schwarz et al., 2014). bThe accession number for the protein reference sequence is NP_060251.2. 1512 Li et al.

TABLE 2 Frequency of CYP2W1 variations and their comparison in different population samples

MAF Fisher’s Exact Test, P Bonferroni Correction, P Variants Minor Allele Han Tibetan Uighur Han versus Tibetan Tibetan versus Uighur Han versus Uighur Han versus Tibetan Tibetan versus Uighur Han versus Uighur v01 A 0 0 0.01 1 1 1 1 1 1 v02 A 0 0 0.01 1 1 1 1 1 1 v03 T 0.35 0.48 0.55 0.085 0.398 0.01a 1 1 0.26 v04 A 0.03 0.1 0.163 0.082 0.212 0.001a 1 1 0.026a v05 A 0.41 0.21 0.316 0.003a 0.107 0.186 0.078 1 1 v06 G 0.02 0.03 0 1 0.246 0.498 1 1 1 v07 C 0.02 0.03 0.082 1 0.132 0.057 1 1 1 v08 T 0.45 0.196 0.31 0.000a 0.097 0.058 0.005a 11 v09 Del C 0.41 0.213 0.31 0.003a 0.143 0.185 0.078 1 1 v10 T 0.41 0.19 0.33 0.001a 0.036a 0.305 0.026a 0.936 1 v11 A 0.02 0.01 0.07 1 0.065 0.17 1 1 1 v12 A 0.02 0.04 0 0.683 0.121 0.498 1 1 1 v13 A 0.41 0.21 0.27 0.003a 0.408 0.052 0.078 1 1 v14 A 0.02 0 0 0.498 1 0.498 1 1 1 v15 G 0 0.02 0 0.498 0.498 1 1 1 1 v16 T 0.276 0.3 0.26 0.755 0.637 0.873 1 1 1 Downloaded from v17 C 0.02 0.03 0 1 0.246 0.498 1 1 1 v18 A 0.02 0.03 0.02 1 1 1 1 1 v19 T 0 0 0.01 1 1 1 1 1 1 v20 A 0 0 0.01 1 1 1 1 1 1 v21 T 0.01 0 0 1 1 1 1 1 1 v22 T 0 0.01 0 1 1 1 1 1 1 v23 A 0.05 0.02 0.02 0.445 1 0.445 1 1 1 v24 T 0.25 0.33 0.14 0.275 0.002a 0.073 1 0.052 1 dmd.aspetjournals.org v25 A 0 0 0.01 1 1 1 1 1 1 v26 C 0.051 0.11 0.19 0.192 0.165 0.004a 1 1 0.104

MAF, minor allele frequency. aP , 0.05 significant differences in genotype frequency among the three studied respectively. Haplotypes 8–12 were defined by the Pro488Leu in exon 9. populations (P , 0.05). This is the characteristic variant of allele CYP2W1*6, with overall at ASPET Journals on September 24, 2021 Linkage Disequilibrium and Haplotype Analysis of CYP2W1. frequencies of 0.25, 0.33, and 0.14 in the Han, Tibetan, and Uighur Pairwise LD analysis was performed for the CYP2W1 gene using the samples, respectively. Of these, haplotypes 11 and 12, characterized by polymorphisms detected in this study. Variants with allelic frequen- the co-occurrence of Pro488Leu and another nsSNP (i.e,, Ala181Thr cies smaller than 0.01 were excluded from the pairwise LD analysis. and Pro488Leu, and Gly376Ser and Pro488Leu), were found to be Therefore, 19 variations remained in the following analysis. Big, heterozygous in one Tibetan and one Uighur subject, respectively. medium, and small linkage blocks across the locus were separately Functional Prediction of CYP2W1 nsSNPs. Possible effects of four observed in the Tibetan, Han, and Uighur populations, which contain detected nsSNPs on CYP2W1 protein function were predicted by 16, 11, and 7 polymorphisms, respectively (Fig. 1). Strong LDs among PolyPhen-2, MutationTaster, and SIFT (Table 4). All software programs v03, v05, v08, v09, v10, and v13 were observed in the three populations. predicted that Ala181Thr and Val432Ile were benign or probably A strong LD between v04 and v11 was observed in the Han and Uighur harmless variants. Gly376Ser was predicted by PolyPhen-2 and SIFT populations, but only a weak LD was found in the Tibetan population. as a harmful or damaging variant. In contrast, an opposite prediction was Complete LDs among v03, v04, and v26 were observed in the Uighur given by MutationTaster. Both MutationTaster and PolyPhen-2 in- population, but not in the Han and Tibetan populations. Moreover, a dicated that Pro488Leu may be harmless, although SIFT suggested it complete LD between v07 and v18 was observed in the Han and could cause a deleterious effect. Tibetan populations, but only a low degree of LD existed in the Uighur population. Then, based on the genotype data of the nine exonic SNPs detected in Discussion this study, the CYP2W1 haplotypes of three populations, respectively, CYP2W1 is an orphan P450 enzyme with a physiologic function that were constructed with the PHASE program. With 50 samples and nine remains unknown. However, its tumor-specific expression profile makes variants, the “best” PHASE reconstruction yielded seven (hap1–3, 6–9), it a potential anticancer drug target (Karlgren and Ingelman-Sundberg, eight (hap1–3, 5, 7, 8, 10, and 11), and seven (hap1, 2, 4, 6–8, and 12) 2007; Nishida et al., 2010; Travica et al., 2013). Previous studies have different haplotypes in the tested Han, Tibetan, and Uighur samples, shown that 30% of colon tumors presented high CYP2W1 expression respectively. All three populations shared the same haplotypes (hap1, 2, (Karlgren et al., 2006; Gomez et al., 2007), and such an expression 7, and 8) and possessed specific haplotypes as well. The most common difference could affect the metabolism of anticancer prodrugs. More- haplotypes in the three populations were haplotypes 1, 2, and 8, with over, P450 genetic polymorphism is an important factor that influences frequencies above 10%. As shown in Table 3, haplotypes 1, 2, 6, 7, and drug metabolism (Wijnen et al., 2007). Unlike other P450 members, the 8 were identical to the sequences of published alleles CYP2W1*1A, *1B, genetic polymorphism of CYP2W1 in humans has not been extensively *2, *4, and *6, respectively. Haplotypes 1–5, considered as a group of studied, especially in the Chinese population. Therefore, a comprehen- haplotypes, were characterized by not encoding amino acid changes, sive investigation of CYP2W1 genetic polymorphisms in the three main with total frequencies (i.e., the sum of the frequencies of haplotypes 1–5) Chinese populations is critical in gaining an extensive understanding of of 0.68, 0.66, and 0.77 in the Han, Tibetan, and Uighur samples, the CYP2W1 gene and function. Population Genetic Study of the CYP2W1 Gene 1513 Downloaded from dmd.aspetjournals.org

Fig. 1. Linkage disequilibrium of polymorphic sites in the CYP2W1 gene in the Han (A), Tibetan (B), and Uighur (C) populations. A standard color scheme is used to display LD. Bright red is for very strong LD (LOD = 2, D9 = 1), white is for no LD (LOD , 2, D9 , 1), andpinkred(LOD=2,D9 , 1) and blue indicate intermediate LD (LOD , 2, D9 = 1). LOD, logarithm (base 10) of odds. at ASPET Journals on September 24, 2021

The genetic variations in the CYP2W1 gene in the three main Five known alleles (CYP2W1*1A, *1B,*2, *4, and *6) were identified in ethnicities in China (Northern Han Chinese, Tibetan, and Uighur) were this study, whereas CYP2W1*3 and *5 were not found. The frequencies investigated using Sanger sequencing. The results showed that the of these alleles in the Chinese Han and Uighur populations obtained in distribution and profile of CYP2W1 genetic polymorphisms were this study were in agreement with those reported in a recent study interethnically different based on the comparison of frequencies in (Qi et al.,2015) (Supplemental Table 3). Moreover, CYP2W1*1A and *1B allele, genotype, and haplotype levels, as well as LD structure analysis. presented with comparable frequencies in the studied Han Chinese 1514 Li et al.

TABLE 3 Haplotypes of CYP2W1 constructed for the exonic SNPs detected in this study The reference haplotype was extracted from the sequence of CYP2W1*1A and is shown as hap1. For the detected haplotypes, identities with the reference sequence are denoted by blanks; capital letters indicate SNPs. Hyphens indicate the absence of a certain haplotype in the studied population. The CYP2W1 alleles are named according to the P450 allele nomenclature database (http://www. cypalleles.ki.se/cyp2w1.htm). —, the corresponding haplotype was absent in the population group.

No. of Carriers Frequency Variant ID v03 v11 v12 v15 v19 v20 v22 v23 v24 Allele Amino Acid Leu56Leu Ala181Thr Val196Val Cys256Cys Pro345Pro Gly376Ser Asp412Asp Val432Ile Pro488Leu Han Tibetan Uighur Han Tibetan Uighur hap1 C G G C C G C G C 36 17 29 0.36 0.17 0.29 *1A hap2 T 30 44 47 0.3 0.44 0.47 *1B hap3 T A 2 4 ― 0.02 0.04 0 hap4 T ―― 1 0 0 0.01 hap5 T ― 1 ― 0 0.01 0 hap6 T A 2 ― 7 0.02 0 0.07 *2 hap7 A 5 2 2 0.05 0.02 0.02 *4 hap8 T 24 30 13 0.24 0.3 0.13 *6 hap9 T T1――0.01 0 0 hap10 G T ― 2 ― 0 0.02 0 hap11 A T ― 1 ― 0 0.01 0 hap12 A T ―― 1 0 0 0.01 Downloaded from

population and the Japanese population, whereas CYP*2, *4,and*6 using different prediction tools. It was reported that the accuracy of demonstrated varied frequencies among these two populations (Hanzawa these three prediction methods typically reaches up to 85.7%, with a et al., 2008) (Supplemental Table 3). In addition, the comparison of the false-positive rate as high as 9–20% and a false-negative rate as high frequencies of four detected nsSNPs in the current study with the data as 14.2–31% (Ng and Henikoff, 2006) (http://homozygositymapper. dmd.aspetjournals.org obtained from the worldwide representative ethnic populations released org/MutationTaster/comparison.html). Therefore, the inconsistency by the 1000 Genome Project also demonstrated the diversified pattern of in the prediction results produced by different predictive tools and CYP2W1 allelic variations in different ethnic groups (Supplemental the disagreement between the prediction results and the previ- Table 3). ously reported experimental data could be explained. Hence, fur- Extensive studies have focused on the function of P450 nsSNPs ther in vitro function studies and metabolic phenotyping research because of their potential effects on drug metabolism, drug efficacy, are essential in addressing the functional role of CYP2W1 and its and disease susceptibility. However, few studies have investigated variants. at ASPET Journals on September 24, 2021 the function of CYP2W1 nsSNPs. A previous study has reported In conclusion, the present study investigated the genetic polymor- that Ala181Thr and Pro488Leu could not alter the expression level phisms of CYP2W1 in three main Chinese populations. The distribution of CYP2W1 protein in transfected SW480 cells and the metabolism of the identified variants and haplotype structures in these populations of the duocarmycin ICT2706 (a chloromethylindoline analogue of were analyzed, and results indicated the ethnic diversity of CYP2W1 duocarmycin), which could be converted into cytotoxic products genetic polymorphisms in different Chinese populations. Moreover, the capable of inducing rapid tumor cell death (Travica et al., 2013). possible effects of identified missense SNPs on protein function and Moreover, several studies have investigated the association of CYP2W1 protein structure were analyzed by in silico approaches. The predicted genetic polymorphisms with the susceptibility of individuals to effects suggested a possible altered CYP2W1 metabolic activity toward certain diseases; however, the conclusions were contradictory. One substrates. Such information would extend our understanding of study revealed that A181T could increase the risk of colorectal CYP2W1 genetic polymorphisms in humans. Further functional studies cancer (Gervasini et al., 2010), whereas another study debunked the are being carried out in the laboratory to further clarify the effect of associationofAla181ThrandPro488Leuwithcolorectalcancer genetic variants on CYP2W1 functions. (Stenstedt et al., 2013). In the present study, the possible impact of four detected nsSNPs on the CYP2W1 protein structure and function Authorship Contributions was predicted using three in silico tools: PolyPhen-2, SIFT, and Participated in research design: Chen, Wang. MutationTaster. The prediction results of Ala181Thr and Val432Ile Conducted experiments: Li, Kang, Yang. were uniform and indicated they might be benign variants, whereas Performed data analysis: Li, Kang, Wang. the results of Gly376Ser and Pro488Leu were highly inconsistent Wrote or contributed to the writing of the manuscript: Dai, Chen, Wang.

TABLE 4 Predicted effect of four nsSNPs

Parameters Gly376Ser Ala181Thr Val432Ile Pro488Leu PolyPhen-2 Score 0.936 0.011 0.004 0.042 Function Possibly damaging Benign Benign Benign MutationTaster Function Probably harmless Probably harmless Probably harmless Probably harmless SIFT Score 0.05 0.07 1 0 Function Damaging Tolerated Tolerated Damaging Population Genetic Study of the CYP2W1 Gene 1515

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