Correlation between CYP2D6*10 Gene Mutation, and Structure and Function of its Encoding Protein

Purpose: To investigate the gene polymorphism of CYP2D6*10 (C188T) in the Hui people and study its correlation between CYP2D6*10 gene mutation and structure and function of its encoding protein. Methods: 150 unrelated Hui ethnic group volunteers participated in this study. A total of 500 µL heparin-treated blood from each volunteer was extracted with the TIANGEN DNA Mini Kit. Allele specific amplification PCR and Gene sequencing were used to detect the CYP2D6 alleles *10. Bioinformatics and computer modeling methods were used to predict the spatial structure and function of the protein encoded by the wild type gene and mutant gene. Results: The mutation frequency of C188T allele (T) of CYP2D6*10 in Ningxia Hui people was 47.5 %, compared with Turkish (14.5 %), Ethiopia (8.6 %), Spanish (1.9 %), and they were significantly different, (p < 0.01;) The result from ProtParam shows that mutant protein was more unstable than the wild-type protein. The isoelectric point, molecular weight and hydrophilicity were similar in terms of mutant protein and wild-type protein. Analysis of the gene sequence of CYP2D6*10 using DNAStar/Protein software indicates that the mutant protein had one more Gamier-Robson Turn while MotifScan analysis showed that the wild-type protein had 2 P450 enzyme activation sites and that there was none in the mutant protein. Analysis using SignalP demonstrated that the wild-type protein had signal peptide while the mutant protein had none. Analysis using TMHMM Server showed that both of them had a transmembrane region. The foregoing differences between the mutant protein and the wild-type protein could influence the activity of CYP2D6. Conclusion: Gene mutation can change the spatial structure and function of CYP2D6. This change may be the main reason for the decreased activity of the enzyme.


INTRODUCTION
CYP2D6 is responsible for the metabolism of many endogenous as well as exogenous compounds [1]. It metabolizes more than 25% of all therapeutic drugs [2]. The metabolism of these substrates is highly dependent on the amount of active CYP2D6 enzymes expressed in the liver. At present, more than 150 relevant alleles of CYP2D6 gene have been identified [3], with varying frequencies between ethnic groups [4].There is a large inter-individual variation in the enzyme activity of CYP2D6, resulting in enzymes with high, low, or no activity [5,6].
There is a close relationship between CYP2D6 genotype and phenotype [7][8][9]. The CYP2D6*10 allele. which results in a P34S substitution (C188T) change the protein's primary structure encoded by CYP2D6, thereby influencing the protein's advanced structure and the active CYP2D6 enzyme. Genetic diagnosis can be used to predict an individual's phenotype reliably and may facilitate improved drug efficacy and diminished risk for adverse drug reactions [10,11].
Ningxia Hui nationalities are a special population in China. There is no report on mutation frequency of C188T allele (T) of CYP2D6*10 in Ningxia Hui nationalities. Therefore, in this study, 150 healthy people were selected to detect the frequency of C188T allele of CYP2D6 as well as analyze the differences in spatial structure and function between the wild type protein and the mutant protein using bioinformatics methods. This will provide the theoretical basis for applying gene polymorphism to guide clinically personalized medicine.

EXPERIMENTAL Subjects
One hundred and fifty (150) unrelated Hui ethnic group volunteers participated in this study after giving written informed consent. They were healthy volunteers recruited during their routine check-up in Yinchuan, China. This study was approved by Ethics Committee of Ningxia Autonomous Region people's Hospital (approval ref. no. 20110920) [12]. A total of 500 µL heparintreated blood from each volunteer was extracted with the TIANGEN DNA Mini Kit.

Comparison of protein structure and function
Using bioinformatics analysis software was used to predict and analyze the similarities and differences of the mutant and wild-type protein structure and function encoded by the CYP2D6*10 gene.

Statistical analysis
Allele frequency was determined via direct counting and the standard goodness-of-fit test was used to test the Hardy-Weinberg equilibrium.
The distribution of genotypes in the study was obtained by Chi-square test. All statistical analyses were performed using SPSS software, and significance was defined as p < 0.05.

The product of PCR
The mutation frequency of allelic genes of CYP2D6*10 in the region of Ningxia Hui nationality was 47.5 % (Fig 1), compared to Turkish (14.5 %), Ethiopia (8.6 %), and Spanish (1.9 %) [14]; this is significantly different, (p < 0.01), as shown in Table 2. Analysis of the gene sequence of CYP2D6*10 using DNAStar/Protein software indicates that CYP2D6*10 (T instead of C) produced the amino acid "S" instead of "P". When the differences  between wild-type protein and mutant protein of CYP2D6*10 are compared using ProtParam (ExPASy), it is evident that the instability index of mutant protein is higher than wild-type protein, although both were above the threshould 40 and were neutral in solution.

Prediction of the protein's second structure and activation sites
Analysis of the gene sequence of the amino acids encoded by the PCR product made by DNAStar/Protean software indicate that the mutant protein had one more Gamier-Robson Turn in the 33rd site, as shown in Figures 2 and   3. MotifScan analysis shows that the wild-type protein possessed 2 P450 enzyme activation sites while the mutant protein had none.

Post-translational modification and structural features of sequence
Analysis with SignalP demonstrated that the wildtype protein had signal peptide while mutant protein did not have. The cleavage site was between the 30th and 31st (between RWA and AR). However, anaysis with TMHMM Server v. 2.0 shows that both of them had a transmembrane region.

DISCUSSION
Bioinformatics is a art and science which use calculations in biological research areas such as genomics, transcriptomics, proteomics, genetics and evolution; in other words, bioinformatics acquire and interpret biological data using computing and analytical tools.
Cytochrome P4502D6 (CYP2D6) is the most polymorphic P450 oxidative metabolism enzyme system. Several studies have reported the association between the lipid-lowering efficacy of simvastatin and genetic polymorphisms of CYP2D6. Nordin found that a significant correlation between LDL-C reduction and reduced expression of CYP2D6 [15]. Mulder found that the cholesterol-lowering effect of simvastatin was influenced by CYP2D6 polymorphism [16]. However, Prueksaritanont concluded that the efficacy of simvastatin is not influenced by CYP2D6 polymorphism [17].
The analytic result of online protein assayer has shown that the mutant protein is more instable than wild-type protein and the instability of the mutant protein is related to the reduction of enzyme activity. Analysis of the gene sequence of CYP2D6*10 made by DNAStar/Protein software indicated that the mutant protein has one more Gamier-Robson Turn in the 33rd site. The result also indicate that the mutation site of CYP2D6*10 (C188T) in the spatial structure may affect the spatial arrangement of other amino acids, which will, in turn, alter the structure and function of CYP2D6*10 and lead to the decline of enzyme activity. It also revealed that the wildtype protein possesses 2 P450 enzyme activation sites while the mutant protein had none, based MotifScan analysis. It has also been shown that the wild-type protein belongs to the family of P450 protein enzyme, which might become potential target drugs whereas the mutant protein having lost this functional site may result in the decline of its activity.
As part of amino acid sequence in one end of protein, signal peptide could be used for guiding the target protein to precisely locate in the cell by getting through the membrane of the cellorganelle. C-score shows that the mutant protein has no signal peptide whereas the wild-type protein does. Internal signal peptide sequence indicates that the wild-type protein is a secreted protein while the mutant protein having lost this function may exert an influence on its enzyme activity. However, analysis of TMHMM Server showed that both of them had a transmembrane region.

CONCLUSION
Our results suggest that the CYP2D6 gene polymorphism determines its enzyme activity and might influence an individual's behavior. In future, clinicians could choose rational drugs and apply the most suitable dosage to reduce and avoid the occurrence of adverse reactions so as to achieve an ideal "personalized treatment".