Introduction
Statins are inhibitors of Hydroxymethylglutaryl-CoA (HMG-CoA) reductase and statin therapy is used for prevention of atherosclerosis [1]. People who take statins respond differently to these drugs. Genetic differences are one of the factors in these different responses [2]. Given that the serum level of Statins increases in patients with poor metabolizers and results in side effects that occur more rapidly, it is important to diagnose poor metabolizers [3, 4]. One of the most significant enzymes involved in drug metabolism is the cytochrome P450 2D6 (CYP2D6) [6]. This enzyme is coded by the CYP2D6 gene, which is one of the most polymorphic genes in the cytochromes family [12]. The CYP2D6*4 polymorphism is the most frequent non-functional allele in the Middle East [14]. In this study, we aim to assess the association between CYP2D6*4 and response to atorvastatin among patients with high low-density lipoprotein (LDL) in northern Iran.
Methods
A total of 180 patients (127 females and 53 males) with high LDL level were recruited in this study. All patients were treated with 10, 20 or 40 mg atorvastatin (Sobhan Darou, Tehran, Iran). The level of LDL was measured before and after atorvastatin therapy. 
DNA was extracted from the whole blood sample using the DNP kit (CinnaGen, Tehran, Iran). Genotyping of CYP2D6*4 allele was performed using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. Specific primers were designed for both wild type (G) and mutant (A) sequences using the online NCBI primer blast and Primer3Plus tools. The PCR was performed in 20 μl volume, containing 10 μl master mix, 2 μl DNA, various volume of forward and reverse primers, and distilled water. The PCR included 0.5 μl of each forward and reverse primers to amplify wild-type (WT) sequence (forward: 5’-CCGCATCTCCCACCCCCAG-3’, reverse: 5’-AGACTCCTCGGTCTCTCGCT-3’) and 1 μl for each primer to amplify mutant sequence (forward: 5’-TTGGAGTGGGTGGTGGATGG-3’, reverse: 5’-GGGGCGAAAGGGGCGTCT-3’) (Table 1).


The PCR started with 5 minutes of initial denaturation at 95ºC, followed by three-step cycles: incubation for 30 seconds at 95ºC, 30 seconds at annealing temperature (68.5ºC for WT sequence and 71.5 for mutant sequence), and 30 seconds at 72ºC. The PCR was finalized by incubation at 72ºC for 7 minutes. The three-step cycle was repeated 28 times to amplify the WT sequence and 27 times to amplify the mutant sequence (Figure 1).  The results were validated by the Sanger sequencing method in BIONEER company (Daejeon, South Korea) (Figure 2). Statistical analysis was performed in SPSS v.16 software. The amount of reduced LDL in patients with different genotypes (WT/WT and CYP2D6*4/WT) based on the used doses of atorvastatin was compared by Kruskal-Wallis test. The Wilcoxon signed-rank test was used to compare the amount of reduced LDL with baseline LDL level and secondary LDL level (after treatment).
Results
In the patients, the percentage of the WT allele G was 93% (n=335) and the percentage of CYP2D6*4 allele A was 7% (n=25). The *4/*4 (AA) genotype was not observed in any patients. The majority of patients (n=155, 86.1%) were normal homozygous (WT/WT) with GG genotype. The percentage of WT/*4 (GA) genotype was 13.9% (n=25). In all three dosage groups, the number of CYP2D6*4 allele was lower than that of WT allele. The frequency of CYP2D6*4 and WT alleles was almost the same among the three dosage groups. 
The amount of LDL reduction was not significantly different between patients with WT/WT and CYP2D6*4/WT genotypes in any dosage groups (P>0.05) (Table 2). 


There was no significant association between different genotypes and LDL parameters (baseline LDL level, secondary LDL level, and LDL reduction rate) (P>0.05), although the amount of reduced LDL was significant in individuals with two different genotypes using different dosages of atorvastatin (10, 20, and 40 mg) compared to baseline and secondary LDL levels.
Discussion
There is no association between CYP2D6*4 and response to atorvastatin. Low allelic frequency might be the reason for non-significance of this association. Considering the low frequency of CYP2D6*4 variant and *4/*4 genotype, CYP2D6*4 variant probably is not related to poor metabolizers, especially poor metabolizers of atorvastatin in northern Iran. Therefore, to distinguish poor metabolizers in this region, further studies on other genes are recommended.

Ethical Considerations
Compliance with ethical guidelines
Informed written consent was obtained from all participants. This study was approved by the Research Ethics Committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1396.340).

Funding
The data presented in this research is a part of the general Ph.D. thesis of Mrs. Razia Asadullahpour, and it was done with the support of the Research and Technology Vice-Chancellor of Gilan University of Medical Sciences.

Authors' contributions
Conceptualization: Mehdi Evazalipour; Supervision: mehdi evazalipour and fardin mirbolouk methodology: Mehdi Evazalipour, Omid Goodarzvand, Sara Dabirian, and Mohammad Sadegh Alipour; Software: Mehdi Evazalipour, Omid Goodarzvand and Mohammad Sadegh Alipour; Data curation: Mehdi Evazalipour, Raziyeh Asadollahpour, Mohammad Sadegh Alipour, and Ehsan Zamani; Editing & review: Mehdi Evazalipour, Anvarsadat Kianmehr, and Ehsan Zamani; Investigation: Raziyeh Asadollahpour, Faeze khaghani, Fardin Mirbolouk, Omid Goodarzvand, Anvarsadat Kianmehr, Sara Dabirian, and Ehsan Zamani; Writing original draft: and Visualization: Faeze khaghani; Writing: Anvarsadat Kianmehr. 

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors are supported by the Research Council of the University of Medical Sciences Guilan appreciates and thanks.

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