Genetic polymorphism of CYP2D6 gene among Egyptian
Ahmed A.A. Alia,
, Nahla M. Wassimb
, Moataz M. Dowaidarb
, Ahmed E. Yaseenb
Department of Zoology, College of Science, Sohag University, Egypt
Department of Zoology, College of Science (Suez), Suez Canal University, Egypt Received 13 October 2012; revised 11 December 2012; accepted 19 December 2012 Available online 13 March 2013
KEYWORDS CYP2D6; Polymorphism; Hypertension
Abstract Background: Hypertension is a cardiovascular disease that is affected by environmental, demographic and genetic factors.
Objective: This study aims to determine the frequency of the CYP2D6*1, *3, *4 and *5 variants among hypertensive cases and cases with obesity and cases with cardiac complications.
Subjects and methods: DNA was isolated from peripheral blood samples that were collected from 123 hypertensive cases and from 429 healthy non-related controls by using the Magna pure system. Genomic DNA was used to determine the frequency of CYP2D6*1, CYP2D6*3, CYP2D6*4 and CYP2D6*5 allelic variants by the application of the light cycler polymerase chain reaction (Real-time PCR) technique.
Results: Comparing cases of hypertension and controls as regard to the genotypic allelic variants of CYP2D6 gene, hypertensive cases showed a signiﬁcantly higher wild genotype 1/1 compared to controls (85.4% vs. 74.8%, p = 0.01) with a lower frequency of mutant genotype 4/4 (1.6% vs. 8.6%, p = 0.008) This phenomenon was manifested among cases of subgroups with obesity that had signiﬁcantly lower mutant homozygous forms than obese controls (2.3% vs. 9.5%, p 0.04) and cases with cardiac complications (88.2% vs. 74.8%, p = 0.01).
Conclusion: CYP2D6polymorphism is positively associated with hypertensive cardiac complica-tions as well as hypertensive obese cases.
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Cardiovascular diseases are complex disorders that are af-fected by environmental, demographic and genetic factors. It is evaluated that the genetic element associated with blood pressure variation ranges from 30% to 50% (Tanira, 2005). Evidence from animal models and human studies suggests that cytochrome P450 plays a mechanistic role in the development of hypertension. However, the human cytochrome P450 (CYP) – containing more than 59 functional genes and 58 pseudo-genes – are playing an important or dominant role in the
* Corresponding author.
E-mail addresses: AAh592003@yahoo.com,email@example.com. sa(A.A.A. Ali).
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metabolism of some endogenous compounds, therapeutic drugs, and other xenobiotics (Zhou et al., 2009; Bradford, 2002). One of the most important polymorphic P450 genes is CYP2D6, the debrisoquine hydroxylase. (Bertilsson, 1995; Sistonen et al., 2007). Although this CYP2D6 makes up only 1.5% of the total cytochrome P450 isoforms, it metabolizes up to one-quarter of all prescribed drugs such as antidepres-sant, antipsychotic, antiarrhythmic, opiates and antihyperten-sive drugs (Murphy et al., 2001).
The CYP2D6 gene is localized on chromosome 22q13 (Heim and Meyer, 1990). It is highly polymorphic variant, more than 120 alleles had been. These alleles are resulting from point mutations, rearrangements, additions, deletions and duplications (Ma et al., 2002; Evans & Relling, 2004). Among the common allelic variants, individuals with two functional alleles (wild-type allele, CYP2D6*1), and with one functional allele are considered extensive metabolizers (EMs) (Gardiner &Zanger et al., 2001; Gardiner and Begg, 2006). In addition, individuals with whole gene deletion (null allele) are considered poor metabolizers (PM) e.g. CYP2D6*5. There are also indi-viduals with inactivating alleles produced from single nucleo-tide polymorphisms alone or in combination, deletion or insertion of a single or multiple base(s), e.g. CYP2D6*3 and CYP2D6*4 are considered PM (Linder et al., 1997; Sachse et al., 1997; Bradford, 2002; Kirchheiner et al., 2001; Zanger et al., 2004; Guzey and Spigset, 2004). Published studies have determined that the frequency of the alternative CYP2D6 phe-notypes varies signiﬁcantly among different racial/ethnic groups. Genetic variation of drug metabolizing enzymes also has been shown as one of the major causes of the inter-individ-ual variability to drug response. Therefore the impact of CYP2D6polymorphism on the clearance of or response to a series of cardiovascular drugs was addressed (Zhou, 2009).
Although the frequencies of CYP2D6 mutant alleles have been studied extensively in different populations, limited infor-mation is available for those of the Middle East populations. The objective of the current study was to determine the fre-quencies of the CYP2D6*1, *3, *4 and *5 variants among Egyptian hypertensive cases and with complicated subgroups comparing with healthy individuals.
Subjects and methods
As a pilot study ahead of a future wide scale genetic analysis of hypertensive cases, a convenient sample of 123 cases (83 males and 40 females) with an age mean ± SD of 50.93 ± 15.43 was selected for the study. Of these cases, 22.8.3% had a positive family history of hypertension, 28.5% had a positive parental consanguinity, 10.6% were smokers, 71.5% were obese and 39% had type 2 diabetes. For comparison, 429 normal healthy unrelated subjects (227 males and 202 females) with an age mean and SD of 47.65 ± 11.15 years were taken from the same locality as controls.
Cases were furthermore stratiﬁed according to the associa-tion of other complicaassocia-tions into 4 subgroups including: Gp I: Hypertension with obesity 88 (71.5%), Gp II: Hypertension with diabetes 48 (39%), Gp III: Hypertension with cardiac complication 76 (61.8%) and Gp IV: Hypertension with renal complication 22 (17.9%).
Obesity was diagnosed on the basis of the deﬁnitions estab-lished by the World Health Organization (WHO) in 1997 and published in 2000, that deﬁnes the body mass index (BMI) of
obesity as being 30 or greater (WHO, 2012). On the other hand, type 2 diabetes was diagnosed on the basis of blood su-gar during fasting time (12–14 h), being higher than 126 mg/dL at least for two times; or with symptoms of hyperglycemia plus a random plasma glucose being higher than 200 mg/dl (11.1 mmol/l) (American Diabetes Association, 2009). The other cases with cardiac and renal complications were diag-nosed by renal and cardiology departments.
DNA extraction and ampliﬁcation
Genomic DNA was isolated from collected peripheral blood samples using MagNA Pure LC instrument (Roche Molecular Biochemicals, Mannheim, Germany), according to the manu-facturer’s standard. Oligonucleotide primers and ﬂuores-cence-labeled hybridization probes were designed for ampliﬁcation and sequence-speciﬁc detection of corresponding polymorphism (TIB MolBiol; Berlin, Germany). During PCR the amplicon was detected using two speciﬁc hybridization probes, one labeled with ﬂuorescein and one with LightCycler Red 640 (LCRed640). The absence of a CYP allele introduces a destabilizing mismatch, which results in a decreased melting temperature. For mutation detection with the LightCycler, a 20 ll reaction was performed. The reaction mixture contained: 1 · LightCycler DNA Master Hybridization Mix (Roche), 2.0 mM MgCl2, 5 pmol of each primer, 4 pmol Sensor probe, 8 pmol Anchor probe, and 200 ng genomic DNA. PCR condi-tions were: 120 s at 95C for DNA denaturation, 40 cycles (0 s at 95C (denaturation), 10 s at 55 C (annealing), 40 s at 72 C (extension)). Next a melting curve analysis was performed by heating at 95C for 60 s, followed by cooling at 40 C for 120 s and gradual heating (0.1C/s) up to 80 C. After the melting curve analysis a ﬁnal cooling was performed at 40C for 30 s. To analyze the melting curves the corresponding melt-ing peaks were calculated by plottmelt-ing the ﬁrst negative deriva-tive of the ﬂuorescence with respect to the temperature ( dF/ dT vs. T)Fig. 1.
Probes used in the RT PCR ‘‘variable base is highlighted in yellow if the probe is wild type speciﬁc or green for the poly-morphism speciﬁc probes’’:
Statistical analysis was done using the SPSS software program version 17. Allelic and genotypic frequencies were compared and statistically analyzed using the Fisher’s exact test and Odds ratio (OR) with 95% conﬁdence intervals (95% CI). Conformity of genotype distributions with Hardy–Weinberg (HW) equilibrium was evaluated by Chi-square analysis. For all tests, a p-value < 0.05 was considered to be statistically signiﬁcant.
Comparing cases of hypertension and controls as regards the genotypic allelic variants of CYP2D6 gene showed a signiﬁcantly
higher wild genotype 1/1 among hypertensive cases compared to controls (85.4% vs. 74.8%, OR 1.96, 95% Cl = 1.1–3.4, p= 0.01) with a lower frequency of mutant genotypes (1.6% vs. 8.6%, OR 0.2, 95% Cl = 0.04–0.7, p = 0.008) (Table 1). This phenomenon was manifested among cases of subgroups with obesity that had signiﬁcantly lower mutant homozygous forms than obese controls (2.3% vs. 9.5%, OR 0.2, 95% Cl = 0.05– 0.95, p = 0.04) (Table 2) and cases with cardiac complications (1.3% vs. 8.6%, OR 0.1, 95% Cl = 0.02–1.05, p = 0.03) (Table 3). Other subgroups were neglected because their genotyping was not conﬁrmed. Regarding the allelic frequency, hypertensive cases had also a signiﬁcant higher frequency of the wild type allele 1 (91.9% vs. 82.6%, OR 2.3, 95% Cl = 1.4–3.8, p = 0.001) with a signiﬁcantly lower frequency of the mutant alleles compared to controls (8.1% vs. 16.9% OR 0.4, 95% CI = 0.3–0.7, p = 0.001) (Table 1).
Based on the polymorphism detection, the present study iden-tiﬁed the prevalent wild genotype (CYP2D6 1/1) and the most common inactive genotypes (mutant or poor metabolic) partic-ularly CYP2D6 4/4 in hypertensive cases and healthy controls.
As we did not get any previous studies associating hyper-tension with genotypic variants of CYP2D6, we have this study to reach a conclusion in this regard.
As regards the genotypic variants of CYP2D6 gene, the present study showed a signiﬁcantly higher wild genotype 1/1 among hypertensive cases compared to controls with a lower frequency of mutant genotypes (poor metabolic). This phenomenon was manifested among cases of subgroups with obesity that had signiﬁcantly lower mutant homozygous forms than obese controls and cases with cardiac complications. Hypertensive cases with diabetes and cases with renal complication were neglected because their genotyping was not conﬁrmed. Whereas comparing cases of hypertension and controls as regards the allelic variants of CYP2D6 gene showed hypertensive cases exhibited a signiﬁcantly higher fre-quency for allele 1 than controls.
Actually the impact of CYP2D6 polymorphisms on the clin-ical outcome of hypertensive drugs had been extensively pub-lished in several literatures (Arnett et al., 2006; Lefebvre et al., 2007; Zateyshchikov et al., 2007; Bijl et al., 2009; Goryachkina et al., 2008; Rau et al., 2009). Depending on which alleles are present in an individual, a wide range of metabolic activities was observed among subjects: poor (PM), intermediate (IM), extensive (EM) and ultrarapid (UM) (Zanger et al., 2004;
Table 1 Frequency of genotypic and allelic variants of CYP2D6 gene among hypertensive cases compared to controls.
1/1 1/M M/M# Allele 1 Allele M n(%) n(%) n(%) n(100%) n(%) n(%) Controls n = 429 321 (74.8) 71 (16.6) 37 (8.6) 858 713(83.1) 145 (61.9) Cases n = 123 105 (85.4) 16 (13) 2 (1.6) 246 226 (91.9) 20 (8.1) Dominant 1/1 vs. 1/M + M/M# Additive 1/M vs. 1/1 + M/M Recessive M/M vs. 1/1 + 1/M Allele 1 Allele M p 0.01* 0.4 0.008* 0.00097* 0.00097* OR (95% CI) 1.96 (1.1–3.4) 0.2 (0.4–1.4) 0.2 (0.04–0.7) 2.3 (1.4–3.8) 0.4 (0.3–0.7) #
M = mutant alleles of CYP2D6 i.e. alleles 3, 4 and 5.
* psigniﬁcant < 0.05.
Table 2 Frequency of genotypic and allelic variants of CYP2D6 gene among obese hypertensive cases compared to controls.
1/1 1/M M/M# Allele 1 Allele M n(%) n(%) n(%) n(100%) n(%) n(%) Obese controls n = 318 237 (74.5) 51 (16.0) 30 (9.5) (636) 525(82.5) 111(17.5) Obese Complication n = 88 74 (84.1) 12 (13.6) 2 (2.3) 176 160(90.9) 16(9.1) Dominant 1/1 vs. 1/M + M/M# Additive 1/M vs. 1/1 + M/M Recessive M/M vs. 1/1 + 1/M Allele 1 vs. Allele M Allele M vs. Allele 1 p 0.07 0.6 0.04* 0.007* 0.007* OR (95% CI) 1.8 (0.97–3.4) 0.8 (0.4–1.6) 0.2 (0.05–0.95) 2.1 (1.2–3.7) 0.5 (0.3–0.8)
# M = mutant allels of CYP2D6 i.e. alleles 3, 4 and 5. *
Kirchheiner et al., 2004; Sistonen et al., 2007; Spina and de Leon, 2007). PM alleles (e.g.*3,*4,*4· n,*5,*6,*7,*8,*11 or *45) cause absent enzymatic activity and, possibly, an increased risk of adverse drug reactions even with a routine therapy with CYP2D6substrates (Marez et al., 1997; Sachse et al., 1998). In addition, many of the studies have shown that some ethnic groups have speciﬁc CYP2D6 phenotypes such as CYP2D6*10, found in Asians and CYP2D6*17, found in Africans (Bertilsson, 1995; Ma et al., 2002; Evans & Relling, 2004).
On the other hand, the frequency of the phenotype of poor metabolizers differs among ethnic groups. Less than 1% of Asians, 2–5% of African–Americans, and 6–10% of Cauca-sians are poor metabolizers of CYP2D6 (Kalow et al., 1980). Whereas, the frequencies of CYP2D6*4, allele are described among different ethnic populations: 20.7% in Caucasians (Sachse et al., 1997), 7.8% among African Americans (Wan et al., 2001), 17.84% in Greeks (Arvanitidis et al., 2007), 18.4% in Dutch (Tamminga et al., 2001), 18.2% in Russians (Gaikovitch et al., 2003), 20.7% in Germans (Sachse et al., 1997), 15.3% in Italians (Scordo etal., 2004), 33.4% in Faroese population (Halling et al., 2005), 13.8% in Spanish (Menoyo et al., 2006), 14% in Croatians (Bozina et al., 2003), 3.5% in the Saudi population (McLellan et al., 1997) and 2% in the UAE population (Qumsieh et al., 2011).
CYP2D6polymorphism is positively associated with hyperten-sive cardiac complications and hypertenhyperten-sive obese cases. Actu-ally some caution-in the interpretation of the data-is warranted because of the relatively small number of studied subjects, especially in subgroup analysis. In this respect, we recommend a wider scale analysis recruiting a large sample of hypertensive cases and also involving a wide genome association study to conﬁrm these results and search for other important interactive genomic markers.
Authors are grateful to Prof. Dr Ahmad Settin, College of Medicine, Mansura University for helping in collection and diagnosis of cases.
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1/1 1/M M/M# Allele 1 Allele M n(%) n(%) n(%) n(100%) n(%) n(%) Controls n = 429 321 (74.8) 71 (16.6) 37 (8.6) 858 713 (83.1) 145(61.9) Cardiac Complication n = 76 67 (88.2) 8 (10.5) 1 (1.3) 152 142(93.4) 10(6.6) Dominant 1/1 vs. 1/M + M/M# Additive 1/M vs. 1/1 + M/M Recessive M/M vs. 1/1 + 1/M Allele 1 vs. Allele M Allele M vs. Allele 1 p 0.01* 0.2 0.03* 0.002* 0.002* OR (95% CI) 2.5 (1.2–5.2) 0.6 (0.3–1.3) 0.1 (0.02–1.05) 2.9 (1.5–5.6) 0.3 (0.2–0.7) #
M = mutant allels of CYP2D6 i.e. alleles 3, 4 and 5.
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