Abstract
CYP2W1 is an orphan member of the cytochrome P450 superfamily. Recently, CYP2W1 has gained great research interest because of its unknown enzymatic function and tumor-specific expression property. This study aims to investigate the genetic polymorphisms of the CYP2W1 gene in Chinese populations and explore the functions of the detected variants. All of the nine exons and exon-intron junction regions of the CYP2W1 gene were sequenced in 150 Chinese subjects, including 50 Han Chinese, 50 Tibetans, and 50 Uighurs. A total of 26 genetic variants were identified in this study, and 19 polymorphisms were detected in each population. Frequency comparison between populations showed that nine variants exhibited significantly different allelic distributions. A total of 12 different haplotypes were inferred from 150 samples by using the genotype data of nine exonic variants found in this study. CYP2W1*1A, *1B, *2, *4, and *6 were detected as the main alleles/haplotypes. Moreover, one, three, and two ethnically specific haplotypes were observed in the Han, Tibetan, and Uighur samples, respectively. Then, the effects of four detected missense mutations (Ala181Thr, Gly376Ser, Val432Ile, and Pro488Leu) on the CYP2W1 protein function were predicted using three in silico tools: Polymorphism Phenotyping v2, Sorts Intolerant from Tolerant, and MutationTaster. The results showed that Gly376Ser and Pro488Leu may have deleterious effects. In summary, this study showed that the genetic pattern of CYP2W1 is interethnically different among the three Chinese populations, and this finding can extend our understanding of population genetics of CYP2W1 in the Chinese population.
Introduction
CYP2W1 is an extrahepatic cytochrome P450 (P450) enzyme, and is considered as an “orphan” P450 because of its unknown physiologic functions (Karlgren and Ingelman-Sundberg, 2007). Recent studies have reported that CYP2W1 is not expressed in adult human liver or in human primary hepatocytes. Only low amounts of CYP2W1 mRNA were found in human adult extrahepatic and fetal tissues (Karlgren et al., 2006). However, CYP2W1 is highly expressed in several human tumor tissues, especially colon tissues. Therefore, the expression of CYP2W1 seems tumor-specific (Karlgren et al., 2006; Choong et al., 2015). Further studies have demonstrated that the expression of CYP2W1 is regulated by DNA methylation, and that the CYP2W1 gene is silenced by DNA methylation after development and then reactivated in tumors by hypomethylation (Karlgren et al., 2006; Gomez et al., 2007; Choong et al., 2015). The in vitro expression of CYP2W1 in a heterologous system revealed a broad range of catalytic activities toward several chemicals, including arachidonic acid, benzphetamine, and several procarcinogens (polycyclic aromatic hydrocarbon dihydrodiols, aflatoxin B1, and sterigmatocystin) (Wu et al., 2006; Yoshioka et al., 2006; Eun et al., 2010; Nishida et al., 2010; Tan et al., 2011; Wang and Guengerich, 2012; Xiao and Guengerich, 2012). Thus, human CYP2W1 is a potential drug target for cancer therapy using antibodies and/or specific CYP2W1 activated prodrugs (Nishida et al., 2010; Travica et al., 2013).
Many of the P450 isoforms are highly polymorphic. The genetic polymorphisms in P450 genes can alter enzymatic activity and change the metabolic rate of the drugs or procarcinogens catalyzed by the enzymes, resulting in varied drug responses or disease susceptibilities among individuals (Wijnen et al., 2007). In a study that investigated the distribution of the genetic polymorphisms in a Japanese population, only six alleles and five nonsynonymous single-nucleotide polymorphisms (nsSNPs) in CYP2W1 were reported (http://www.cypalleles.ki.se/cyp2w1.htm) (Hanzawa et al., 2008). Of the nsSNPs identified, Pro488Leu (CYP2W1*6) was found to be more common in the Japanese population than in Caucasians (allele frequency: 0.368 vs. 0.06). In contrast, Ala181Thr (CYP2W1*2) is prevalent in Caucasians but rare in the Japanese (allele frequency: 0.21 vs. 0.014) (Hanzawa et al., 2008; Gervasini et al., 2010; Stenstedt et al., 2013). Therefore, the distribution of CYP2W1 genetic polymorphisms is interethnically different. Still, further studies with different ethnic groups are needed for a more comprehensive understanding 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 et al., 2010; Cardenas-Rodriguez et al., 2012; Stenstedt et al., 2013). One study showed that CYP2W1*2 is significantly associated with increased colorectal cancer risk in a Caucasian population (Gervasini et al., 2010); however, another study debunked this relationship in another sample (Stenstedt et al., 2013). By using a human colon cancer cell expression system, the latter study also demonstrated that CYP2W1*2 and CYP2W1*6 do not alter the metabolic kinetics of CYP2W1 substrates in vitro. Further studies are needed to characterize the function of CYP2W1 genetic polymorphisms and address their roles in disease susceptibility and drug metabolism.
The current study screened the genetic variants of CYP2W1 in three Chinese populations (Han, Tibetan, and Uighur) by sequencing the exonic sequences of the CYP2W1 gene in 150 samples. This process was carried out to understand CYP2W1 genetic polymorphisms. The genotype and haplotype distributions of CYP2W1 in Chinese populations were investigated, and the effects of identified nsSNPs in these samples were predicted by using in silico tools.
Materials and Methods
Samples.
Blood samples were collected from 150 unrelated healthy volunteers from three different nationalities: 50 Han Chinese from Shaanxi Province, 50 Tibetans from Tibet, and 50 Uighurs from Xinjiang. All of the volunteers were sophomores at Xizang Minzu University, aged about 19 years. The percentage of females in the Han, Tibetan, and Uighur samples is 52, 55, and 49%, respectively. This study was approved by the Ethics Committee of Northwest University, and all participants signed an informed consent form before participating in the study.
Genomic DNA Extraction.
The genomic DNA samples were extracted from 200 µl of EDTA–anticoagulated peripheral blood by using the QIAamp DNA blood mini kit (Qiagen, Shanghai, China). The extracted genomic DNA was dissolved in an elution buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) and stored at −80°C. DNA concentration was measured by using a nanodrop spectrophotometer (Thermo Fisher Scientific, Fitchburg, WI).
CYP2W1 Genotyping.
The 5′ flanking region (−1000 bp), all exons, their surrounding introns, and the 3′ untranslated region (3′UTR) of CYP2W1 were sequenced to screen for CYP2W1 SNPs using the primers listed in Supplemental Table 1. The location of these primers in the CYP2W1 sequence are shown in Supplemental Fig. 1. The sequencing procedure was performed as previously described (Wang et al., 2011).
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 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 (LD) across all SNPs and the Hardy-Weinberg equilibrium for each SNP were assessed for each SNP using HAPLOVIEW version 4.2 (Broad Institute, Cambridge, MA). PHASE2.1 was used to reconstruct haplotypes and derive haplotype frequencies (Stephens and Donnelly, 2001; Stephens and Scheet, 2005).
Statistical analyses were conducted by using SPSS v.16.0 (IBM Corp., Armonk, NY). Fisher’s exact test was conducted to examine differences in the distributions of detected variant alleles and genotypes among the Han, Tibetan, and Uighur ethnic groups. Bonferroni correction was used for the adjustment of multiple comparisons. A two-sided P value <0.05 was considered statistically significant.
Computational Prediction of the Functional Effect of CYP2W1 nsSNPs.
In silico tools—Polymorphism Phenotyping v2 (PolyPhen-2) (Adzhubei et al., 2010). Mutation Tasten (Schwartz et al., 2014), and Sorts Intolerant from Tolerant (SIFT) (Kumar et al., 2009)—were used to predict the effects of identified CYP2W1 nsSNPs on protein function. PolyPhen-2 predicts the possible impacts of amino acid substitutions on the stability and function of human proteins by using structural and comparative evolutionary considerations. A threshold of 0.15 was used, and a mutation is predicted to be “possibly damaging” if the prediction score is above 0.15 (Adzhubei et al., 2010). SIFT predicts whether an amino acid substitution affects protein function. Such prediction is based on the degree of conservation of amino acid residues in sequence alignments, which are derived from closely related sequences. The SNPs were classified as tolerated (cutoff value of ≥0.05) or damaging (cutoff value of ≤0.05) based on the prediction score (Kumar et al., 2009). MutationTaster is a Web-based application for the rapid evaluation of the disease-causing potential of DNA sequence alterations. Its analyses comprise evolutionary conservation, splice-site changes, the loss of protein features, and other changes that may affect the amount of mRNA (Schwarz et al., 2014).
Results
CYP2W1 Genetic Polymorphisms and Distribution.
A total of 26 different genetic variations, including three novel ones in the CYP2W1 gene, were detected in 150 individuals (Table 1). The three novel variants were all SNPs, located in the promoter (v01), intron7 (v21), and 3′UTR (v25), respectively. A total of nine variations were identified in the coding region, whereas the remaining variants were located in the promoter (two variations), introns (13 variations), and 3′UTR (two variations). Four of the nine exonic SNPs were nsSNPs, located in exons 4, 7, and 9, giving rise to the amino acid exchanges Ala181Thr (v11), Gly376Ser (v20), Val432Ile (v23), and Pro488Leu (v24). One insertion variation (v09) in intron1 was also identified.
Genetic variations of CYP2W1 identified in this study
The frequency of detected variations in specific populations was calculated. First, 19 polymorphic variations were found in each population (Table 2). They consist of 14 identical variations and five ethnically specific variations. Second, statistical analysis showed that nine variants (v3, v4, v05, v08, v9, v10, v13, v24, and v26) exhibited significantly different frequencies among three studied populations (P < 0.05). Three variants (v04, v08, and v10) remained significantly different after Bonferroni correction. Third, of the four nsSNPs identified in this study, Gly376Ser (v20) was found to be heterozygous in one of the 50 Uighur subjects (Supplemental Table 2); Val432Ile (v23) presented with relatively low and comparable frequency (0.02–0.05, P = 1) among three populations; Ala181Thr (v11) exhibited significantly higher frequency in Uighurs (0.07) than in Hans (0.02) (P = 0.03) and Tibetans (0.01) (P = 0.03); and Pro488Leu (v24) was quite common in all studied populations, with significantly higher frequency in Tibetans (0.33) than in Uighurs (0.14) (P = 0.002).
Frequency of CYP2W1 variations and their comparison in different population samples
The allelic distributions of detected variations (Supplemental Table 2) were also analyzed. The results showed that the allelic distribution of all variants was in a Hardy-Weinberg equilibrium (P > 0.05). Moreover, nine variants (v3, v4, v05, v08, v9, v10, v13, v24, and v26) exhibited significant differences in genotype frequency among the three studied populations (P < 0.05).
Linkage Disequilibrium and Haplotype Analysis of CYP2W1.
Pairwise LD analysis was performed for the CYP2W1 gene using the polymorphisms detected in this study. Variants with allelic frequencies smaller than 0.01 were excluded from the pairwise LD analysis. Therefore, 19 variations remained in the following analysis. Big, medium, and small linkage blocks across the locus were separately observed in the Tibetan, Han, and Uighur populations, which contain 16, 11, and 7 polymorphisms, respectively (Fig. 1). Strong LDs among v03, v05, v08, v09, v10, and v13 were observed in the three populations. A strong LD between v04 and v11 was observed in the Han and Uighur populations, but only a weak LD was found in the Tibetan population. Complete LDs among v03, v04, and v26 were observed in the Uighur population, but not in the Han and Tibetan populations. Moreover, a complete LD between v07 and v18 was observed in the Han and Tibetan populations, but only a low degree of LD existed in the Uighur population.
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, D′ = 1), white is for no LD (LOD < 2, D′ < 1), and pink red (LOD = 2, D′ < 1) and blue indicate intermediate LD (LOD < 2, D′ = 1). LOD, logarithm (base 10) of odds.
Then, based on the genotype data of the nine exonic SNPs detected in this study, the CYP2W1 haplotypes of three populations, respectively, were constructed with the PHASE program. With 50 samples and nine variants, the “best” PHASE reconstruction yielded seven (hap1–3, 6–9), eight (hap1–3, 5, 7, 8, 10, and 11), and seven (hap1, 2, 4, 6–8, and 12) different haplotypes in the tested Han, Tibetan, and Uighur samples, respectively. All three populations shared the same haplotypes (hap1, 2, 7, and 8) and possessed specific haplotypes as well. The most common haplotypes in the three populations were haplotypes 1, 2, and 8, with frequencies above 10%. As shown in Table 3, haplotypes 1, 2, 6, 7, and 8 were identical to the sequences of published alleles CYP2W1*1A, *1B, *2, *4, and *6, respectively. Haplotypes 1–5, considered as a group of haplotypes, were characterized by not encoding amino acid changes, with total frequencies (i.e., the sum of the frequencies of haplotypes 1–5) of 0.68, 0.66, and 0.77 in the Han, Tibetan, and Uighur samples, respectively. Haplotypes 8–12 were defined by the Pro488Leu in exon 9. This is the characteristic variant of allele CYP2W1*6, with overall frequencies of 0.25, 0.33, and 0.14 in the Han, Tibetan, and Uighur samples, respectively. Of these, haplotypes 11 and 12, characterized by the co-occurrence of Pro488Leu and another nsSNP (i.e,, Ala181Thr and Pro488Leu, and Gly376Ser and Pro488Leu), were found to be heterozygous in one Tibetan and one Uighur subject, respectively.
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.
Predicted effect of four nsSNPs
Functional Prediction of CYP2W1 nsSNPs.
Possible effects of four detected nsSNPs on CYP2W1 protein function were predicted by PolyPhen-2, MutationTaster, and SIFT (Table 4). All software programs predicted that Ala181Thr and Val432Ile were benign or probably harmless variants. Gly376Ser was predicted by PolyPhen-2 and SIFT as a harmful or damaging variant. In contrast, an opposite prediction was given by MutationTaster. Both MutationTaster and PolyPhen-2 indicated that Pro488Leu may be harmless, although SIFT suggested it could cause a deleterious effect.
Discussion
CYP2W1 is an orphan P450 enzyme with a physiologic function that remains unknown. However, its tumor-specific expression profile makes it a potential anticancer drug target (Karlgren and Ingelman-Sundberg, 2007; Nishida et al., 2010; Travica et al., 2013). Previous studies have shown that 30% of colon tumors presented high CYP2W1 expression (Karlgren et al., 2006; Gomez et al., 2007), and such an expression difference could affect the metabolism of anticancer prodrugs. Moreover, P450 genetic polymorphism is an important factor that influences drug metabolism (Wijnen et al., 2007). Unlike other P450 members, the genetic polymorphism of CYP2W1 in humans has not been extensively studied, especially in the Chinese population. Therefore, a comprehensive investigation of CYP2W1 genetic polymorphisms in the three main Chinese populations is critical in gaining an extensive understanding of the CYP2W1 gene and function.
The genetic variations in the CYP2W1 gene in the three main ethnicities in China (Northern Han Chinese, Tibetan, and Uighur) were investigated using Sanger sequencing. The results showed that the distribution and profile of CYP2W1 genetic polymorphisms were interethnically different based on the comparison of frequencies in allele, genotype, and haplotype levels, as well as LD structure analysis. Five known alleles (CYP2W1*1A, *1B,*2, *4, and *6) were identified in this study, whereas CYP2W1*3 and *5 were not found. The frequencies of these alleles in the Chinese Han and Uighur populations obtained in this study were in agreement with those reported in a recent study (Qi et al.,2015) (Supplemental Table 3). Moreover, CYP2W1*1A and *1B presented with comparable frequencies in the studied Han Chinese population and the Japanese population, whereas CYP*2, *4, and *6 demonstrated varied frequencies among these two populations (Hanzawa et al., 2008) (Supplemental Table 3). In addition, the comparison of the frequencies of four detected nsSNPs in the current study with the data obtained from the worldwide representative ethnic populations released by the + Genome Project also demonstrated the diversified pattern of CYP2W1 allelic variations in different ethnic groups (Supplemental Table 3).
Extensive studies have focused on the function of P450 nsSNPs because of their potential effects on drug metabolism, drug efficacy, and disease susceptibility. However, few studies have investigated the function of CYP2W1 nsSNPs. A previous study has reported that Ala181Thr and Pro488Leu could not alter the expression level of CYP2W1 protein in transfected SW480 cells and the metabolism of the duocarmycin ICT2706 (a chloromethylindoline analogue of duocarmycin), which could be converted into cytotoxic products capable of inducing rapid tumor cell death (Travica et al., 2013). Moreover, several studies have investigated the association of CYP2W1 genetic polymorphisms with the susceptibility of individuals to certain diseases; however, the conclusions were contradictory. One study revealed that A181T could increase the risk of colorectal cancer (Gervasini et al., 2010), whereas another study debunked the association of Ala181Thr and Pro488Leu with colorectal cancer (Stenstedt et al., 2013). In the present study, the possible impact of four detected nsSNPs on the CYP2W1 protein structure and function was predicted using three in silico tools: PolyPhen-2, SIFT, and MutationTaster. The prediction results of Ala181Thr and Val432Ile were uniform and indicated they might be benign variants, whereas the results of Gly376Ser and Pro488Leu were highly inconsistent using different prediction tools. It was reported that the accuracy of these three prediction methods typically reaches up to 85.7%, with a false-positive rate as high as 9–20% and a false-negative rate as high as 14.2–31% (Ng and Henikoff, 2006) (http://homozygositymapper.org/MutationTaster/comparison.html). Therefore, the inconsistency in the prediction results produced by different predictive tools and the disagreement between the prediction results and the previously reported experimental data could be explained. Hence, further in vitro function studies and metabolic phenotyping research are essential in addressing the functional role of CYP2W1 and its variants.
In conclusion, the present study investigated the genetic polymorphisms of CYP2W1 in three main Chinese populations. The distribution of the identified variants and haplotype structures in these populations were analyzed, and results indicated the ethnic diversity of CYP2W1 genetic polymorphisms in different Chinese populations. Moreover, the possible effects of identified missense SNPs on protein function and protein structure were analyzed by in silico approaches. The predicted effects suggested a possible altered CYP2W1 metabolic activity toward substrates. Such information would extend our understanding of CYP2W1 genetic polymorphisms in humans. Further functional studies are being carried out in the laboratory to further clarify the effect of genetic variants on CYP2W1 functions.
Authorship Contributions
Participated in research design: Chen, Wang.
Conducted experiments: Li, Kang, Yang.
Performed data analysis: Li, Kang, Wang.
Wrote or contributed to the writing of the manuscript: Dai, Chen, Wang.
Footnotes
- Received December 21, 2015.
- Accepted June 14, 2016.
↵1 Y.L. and X.K. contributed equally to this work.
This work was supported by the National Natural Science Foundation of China [Grant J1210063] and the China Postdoctoral Science Foundation [Grant 2015M582695].
This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- LD
- linkage disequilibrium
- nsSNP
- nonsynonymous SNP
- P450
- cytochrome P450
- PolyPhen-2
- Polymorphism Phenotyping v2
- SIFT
- Sorts Intolerant from Tolerant
- SNP
- single-nucleotide polymorphism
- 3′UTR
- 3′ untranslated region
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
References
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Population Genetic Study of the CYP2W1 Gene
