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Analysis of CYP2J2 polymorphisms in the Chinese Uyghur population

Background: Genetic characteristics of CYP2J2 in different populations may be helpful to explore inter-ethnic variability in drug response and disease susceptibility. There is no information about the genetic profile of CYP2J2 in the Chinese Uygur population. Methods: We used PCR and direct sequencing to investigate the whole CYP2J2 in 100 unrelated healthy individuals of Chinese Uygur populations. Chi-square test was used to compare genotyping data of CYP2J2 in the Chinese Uygur population with other races. The SIFT and PolyPhen-2 online tools were used to predict the protein function of the novel non-synonymous mutations in CYP2J2. The CADD software was used to predict pathogenicity for mutations. Results: We detected twenty-eight polymorphisms in the CYP2J2, including five new mutations, three alleles (*1, *7 and *8), and three genotypes (*1/*1, *1/*7 and *1/*8) of CYP2J2 in Chinese Uyghur population. The allele frequencies of CYP2E1*1, *7 and *8 were 96%, 3.45%, and 0.5%, respectively. Interethnic comparison found that *1 was significantly higher than Taiwanese and African-Americans; and*7 was relatively lower when compared with Taiwanese and African-Americans (p < 0.05). Furthermore, the protein prediction results revealed that the five non-synonymous mutations could influence the protein structure and function. Conclusion: The observations of this study give rise to useful information on CYP2J2 polymorphisms in Chinese Uygur individuals. We hope the results will indicate important clinical implications for the use of medications metabolized by CYP2J2.


Introduction
The cytochrome P450 (CYP) enzymes play central roles in catalyzes oxidative reactions and bioactivation, accounting for almost 75% of the total drug metabolism (X. Liu et al., 2013;Shah & Breslin, 2010). Nowadays Cytochromes P450 have been the focus of study by toxicologists and pharmacologists. Currently fifty-seven CYP genes involving three families (CYP1, CYP2 and CYP3) have been detected to contribute to the oxidative metabolism of various compounds (Arici & Özhan, 2017). The cytochrome P450, family 2, subfamily J, polypeptide 2 (CYP2J2), an important member of CYP superfamily, which is encoded by CYP2J2 gene, is highly expressed in cardiovascular system (Delozier et al., 2007). The CYP2J2 has prominent role in cardiac protection because of its ability to catalyze arachidonic acid to epoxyeicosatrienoic acids that possess potent anti-inflammatory, vasodilatory, and fibrinolytic properties (Spiecker et al., 2004). Variations in the coding regions of CYP2J2 gene may lead to changes in CYP2J2 expression and/or enzymatic activity, and result in altered CYP2J2-dependent metabolism of AA, and ultimately lead to abnormal heart function. For example, CYP2J2 SNP (single nucleotide polymorphism) such as G-76T, has been repeatedly considered an increased factor for coronary artery disease (S. Wu, Moomaw, Tomer, Falck, & Zeldin, 1996 ). Therefore, we systematically screened the whole CYP2J2 gene from 100 unrelated healthy individuals of Chinese Uygur populations to explore their polymorphisms and compared their frequencies with previous observations of other ethnic groups. These genetic factors can be used to predict the drug metabolizing potential of patients before starting therapy. We hope the present work will contribute to prediction for potential risks of drug toxicity and individualized therapy based on CYP2J2 genetic polymorphisms.

Study populations
A total of 100 unrelated healthy volunteers (aged 19-52 years) included 50 males and 50 females were randomly recruited in our study. The inclusion and exclusion criteria were as follows: the subjects selected were judged to be of good health by medical examination; all of these participants were Chinese Uygur populations; their ancestors had lived in the Xinjiang Autonomous Region for at least three generations; volunteers who had any type of medical disease, being pregnant or lactating, drug or alcohol addiction, and organ transplant were excluded from the study.

Ethics approval and consent to participate
The study protocol and consent form were reviewed and approved by Ethics Committee of the Xizang Minzu University and conducted in compliance with the ethical principles for medical research involving human subjects of the Helsinki Declaration. All participants provided written informed consent prior to study enrollment.

DNA extraction
Peripheral venous blood sample (5 mL) was taken into vacutainer tubes containing EDTA (Ethylene diamine tetra-acetic acid) from each subject on an empty stomach during their routine health examination. Then, the samples were stored at −20•C until use.
Genomic DNA was extracted from the blood samples using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag. Co. Ltd., Xi'an, China) following the manufacturer's standard procedures. DNA concentration and purity were evaluated using a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).

SNP Genotyping
Amplification primers for the promoter region, the 3'-UTR region, all exons and intronexon boundaries of CYP2J2 gene were designed by online software Primer 3 Input (version 4.1.0) and synthesized by the Sangon Biotech (Shanghai, China). Primers for PCR (polymerase chain reaction) amplification and sequence are shown in Table 1. Each PCR was performed in 10ul volume system including 5μl HotStar Taq Master Mix (Qiagen, Germany), 0.5μl each primer (0.25uM), 3μl RNase-free water and 1ul template genomic DNA (20ng/ul). PCR product that have single band with prospective size in agarose gel was regarded as qualified. Subsequently, PCR products were purified using TIANgel Midi Purification Kit (TIANGEN, Beijing, China) and sequenced using ABI BigDye Terminator Cycle Sequencing Kit (version 3.1, Applied Biosystems, Thermo Fisher Scientific, Inc., USA) on an ABI Prism3100 sequencer (Applied Biosystems, Thermo Fisher Scientific, Inc., USA).

Statistical analysis
The Microsoft Excel (Redmond, WA, USA) and SPSS 20.0 statistical packages (SPSS, Chicago, IL, USA) were used to perform statistical calculations. The initial analysis of the sequences including base calling, fragment assembly, and detection of SNPs, insertions, and deletions were performed by Sequencher 4.10.1 software (http://www.genecodes.com/). We used Blast online software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to screen the actual genetic differences. The CYP2J2 variants were identified and named based on the wildtype CYP2J2 nucleotide sequence NG_007931.1 in NCBI (national center for biotechnology information) database and CYP allele nomenclature (http://www.cypalleles.ki.se/). Statistical differences in the distributions of allele frequency between the Chinese Uygur and other ethnic populations were evaluated using Chi-square test. P value < 0.05 was considered statistically significant, and all statistical tests were two sided. We used χ 2 test to examine HWE (hardy-weinberg equilibrium) for each genetic variant. Haploview software version 4.2 was used for analyses of linkage disequilibrium and haplotype construction.
Each variant was given a score based on the impact of its mutation on protein function.

Genetic variants
A total of twenty-eight different SNPs in CYP2J2 were determined by direct sequence in the present study Chinese Uygur population. The identified SNPs, position, region, nucleotide change, allele, amino-acid effect, flanking sequence and the corresponding frequencies are listed in Table 2. Rs1155002 (18644 G>A) locating in intron 5 was found to have the highest frequency of 48%, followed by rs1570693 (15285 A>C) in intron 4 with the frequency of 34%. We found five novel polymorphisms, -273 G>C, 10664 G>A, 15023 A>G, 15062 A>G and 32811 C>T, which have not previously been reported in either the NCBI database or the Human Cytochrome P450 Allele Nomenclature Committee tables.
Among of them, 15023 A>G and 15062 A>G within coding region exon 4, caused the amino acid alterations. Four SNPs were found to cause synonymous mutations and five resulted in non-synonymous mutations. The mutation -76 G>T locating in CYP2J2 promotor with a frequency of 7%, was reported to lose the binding site of the Sp1 transcription factor and led to a decreased enzyme activity referring to published studies (Spiecker et al., 2004).

Interethnic comparison
In order to understand the interethnic variabilities of CYP2J2 genetic profiles, we further compared CYP2J2 distribution patterns between Chinese Uygur population and previously published other major races including East Asians, Caucasians and Africans (Table 4). The allele frequency of CYP2J2*1 in the Chinese Uygur population was significantly higher than Taiwanese and African-Americans (p < 0.05). Furthermore, we found that the allele frequency of CYP2J2*7 in the Chinese Uygur population was relatively lower when compared with Taiwanese (12%) and African-Americans (11.27% and 13.7%). The allele frequency of CYP2J2*7 in Mongolians (3.39%) and Tatars (3.65%) were both similar to our results. However, the allele frequency of CYP2J2*8 is currently not reported in other races.

Linkage disequilibrium analysis
We used Haploview to evaluate LD between SNPs with a minor allele frequency higher than 5%. The D' values on the square is a measure of the LD extent for each pair of SNPs ( Figure 1). Two LD blocks in the CYP2J2 gene were determined. Strong LD was observed between each pair of rs4388726 (33266 T>A), rs2280273 (33440 A>G) and rs2271798 (19114 T>C) in block 1 (Figure 1). The three SNPs rs3820538 (10522 C>T), rs11572245 (10982 G>C) and rs1570693 (15285 A>C) were also found strong LD in block 2.

Predicted protein function for non-synonymous SNPs
We found five non-synonymous SNPs in the present study (15023 A>G, 15052 C>T, 15062 A>G, 18892 G>A, and 21748 G>A). They caused the amino acid substitutions (Asn190Ser, Arg200Cys, Tyr203Cys, Gly312Arg and Ala355Thr respectively). The protein prediction results of the five nonsynonymous variant from the SIFT analysis indicated that all the missense mutations were predicted to be intolerant with scores of 0.00-0.05 (Table   5). The CADD analysis also indicated that all the missense mutations were predicted to be pathogenicity with scores of 4.21, 5.26, 5.93, 2.52 and 13.29, respectively (Table 5). The result performed by PolyPhen-2 analysis showed that 15023 A>G, 15052 C>T, 15062 A>G, and 18892 G>A seemed to be probably damaging (Figure 2). HumVar values of 15052C>T and18892 G>A were 1.000, while 15023 A>G and 18892 G>A were close to 1.000.
However, 21748 G>A was classified to benign mutation with a PolyPhen-2 value of 0.015.

Discussion
In the present study, we sequenced the whole CYP2J2 gene in 100 Chinese Uyghur populations. The results identified CYP2J2 genetic variants including five novel polymorphisms, three alleles (*1, *7 and *8), and three genotypes *1/*1, *1/*7 and *1/*8) of CYP2J2 in Chinese Uyghur population. We also compared the allele frequencies of CYP2J2*1, CYP2J2*7 and CYP2J2*8 with previous observations of other ethnic populations, and found that CYP2J2 *1 was significantly higher than Taiwanese and African-Americans; CYP2J2*7 was relatively lower when compared with Taiwanese and African-Americans (p < 0.05). Furthermore, the protein prediction results revealed that the variants (15023 A>G, 15052 C>T, 15062 A>G, 18892 G>A, and 21748 G>A) could influence the protein structure and function.
P450 pharmacogenetics has been one of the important areas of pharmacogenetic variability. Genetic trait of cytochrome P450 genes subfamily offers the opportunity to identify sources of inter-individual variability in drug disposition and response (Fohner et al., 2013). On the other hand, functional polymorphisms in P450 genes can affect the enzyme activity and relate to human disease. Hundreds of CYP2J2 polymorphisms have been identified currently. CYP2J2*7 resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and decreased CYP2J2 promoter activity (Spiecker et al., 2004). Besides coronary artery disease, it has been implicated in other cardiovascular disease. Ping-Yen Liu et al demonstrated that CYP2J2*7 T allele was synergistically associated with the risk of premature MI (myocardial infarction), particularly in smokers.

The present result was quite different from our previous analysis in the Chinese Wa and
Zhuang population (Zhang, Chen, et al.; Zhang, Cheng, et al.). A total of fourteen CYP2J2 genetic varieties were identified in the Chinese Wa population, twelve of which were also found in the Chinese Uighur population. Only one allele CYP2J2 *1 was identified in the Chinese Wa population and no common synonymous and nonsynonymous mutations were found. We identified seventeen SNPs in the Chinese Zhuang population, including two common synonymous mutations associated with the Chinese Uighur population. We found that the most common polymorphism in the Chinese Uygur population, rs1155002, is also the highest among the Chinese Wa and Zhuang population. Its frequency in Chinese Uygur population (48%) and Zhuang (34%) population were much less than that in the Chinese Wa population (72%). This common intron SNP is an important SNP that has been recognized as a risk factor for hypertension (S. N. Wu et al., 2007). CYP2J2*7 is an allele that has been receiving attention and has appeared in many races. Wang et al. first analyzed the CYP2J2*7 single nucleotide polymorphism in the Chinese population (Wang et al., 2006). They indicated that the allele frequency of CYP2J2*7 was 2.6% in Chinese Han, 17% in Africa, 5.49% in White, 13% in Asian and 4.23% in Korean population. The allele frequency was comparable to that of Korean, but significantly lower than those of African and White. Here we expanded the groups and further expound that CYP2J2*7 variant represents a relatively rare polymorphism in most East Asians. But in Taiwanese, the allele frequency of CYP2J2*7 was distinctly higher and comparable to African-Americans. This indicates that although in the same continent, there are still differences among diverse ethnic groups. Possible explanations for these differences include: genetic background, cultural variants and other factors, such as living environment and lifestyles.
Five non-synonymous mutations Asn190Ser, Arg200Cys, Tyr203Cys, Gly312Arg and Ala355Thr were observed in the Chinese Uygur population. Asn190Ser and Tyr203Cys were never identified before. Gly312Arg was predicted as probably damaging and affecting protein function. Sang et al. reported that Gly312Arg variant was co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and its catalytic activities were quantified.
Compared with the wild-type CYP2J2, recombinant CYP2J2 Gly312Arg variant showed almost complete loss of enzymatic activity (Lee et al., 2005a).
In addition to Ala355Thr, protein functional analysis of the other four mutations showed consistency. However, polyPhen-2 protein function predicted results showed that Ala355Thr was benign, whereas SIFT predicted it was damaging. We deem that the inconsistency is reasonable because algorithms of different bioinformation tools are based on different training data. PolyPhen-2 indicates benign variants occurring at residues that were polymorphic across multiple species. SIFT assess the pathogenicity of a missense variant based on the sequence homology and a conservation value (Lai et al., 2015;J. Wu, Li, & Jiang, 2014). As to chemical properties, the replacement from alanine to threonine was non-conservative. According to related analysis, the sensitivity of SIFT and PolyPhen was both reasonably high (69% and 68%) but their specificity was low (13% and 16%) (Flanagan, Patch, & Ellard, 2010). Therefore, we think predictions should be interpreted with caution before reporting novel missense changes and further experimental evidence should be sought.
In this study, some limitations should be taken into consideration. First, the sample size included 100 individuals is relatively small, therefore, it is needed to continue collecting samples to confirm our findings in the future study. Second, other genes, which are equally important for CYP450 enzyme activity, such as CYP2S1 and CYP2R1, should be studied in the further research. Finally, the function analyses of mutations in the CYP2J2 gene were not performed. Therefore, further studies are warranted to investigate the function of mutation site.

Conclusions
In conclusion, we detected twenty-eight polymorphisms in the CYP2J2, including five new mutations, three alleles (*1, *7 and *8), and three genotypes (*1/*1, *1/*7 and *1/*8) of CYP2J2 in the Chinese Uyghur population. Five non-synonymous mutations among them could influence the protein structure and function. The allele the allele frequency of *1 and *7 were significantly differences compared with other races. Overall, our results provide a basic profile of CYP2J2 in the Chinese Uygur population. We hope the data may be helpful to plan candidate gene-trait association studies and population-specific research on pharmacogenetics.