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Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy
Accepted Manuscript Title: Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy Authors: Miguel A. L´opez-Garc´ıa, Iris A. Feria-Romero, H´ector Serrano, Dar´ıo Rayo-Mares, Pietro Fagiolino, Marta V´azquez, Consuelo Escamilla-N´un˜ ez, Israel Grijalva, David Escalante-Santiago, Sandra Orozco-Suarez PII: DOI: Reference:
S1734-1140(17)30028-2 http://dx.doi.org/doi:10.1016/j.pharep.2017.01.007 PHAREP 621
To appear in: Received date: Revised date: Accepted date:
26-7-2016 10-1-2017 16-1-2017
Please cite this article as: Miguel A.L´opez-Garc´ıa, Iris A.Feria-Romero, H´ector Serrano, Dar´ıo Rayo-Mares, Pietro Fagiolino, Marta V´azquez, Consuelo Escamilla-N´un˜ ez, Israel Grijalva, David Escalante-Santiago, Sandra Orozco-Suarez, Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy, http://dx.doi.org/10.1016/j.pharep.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy
Miguel A López-García1-2, Iris A Feria-Romero2, Héctor Serrano1, Darío Rayo-Mares3, Pietro Fagiolino4, Marta Vázquez4, Consuelo Escamilla-Núñez 5, Israel Grijalva2, David EscalanteSantiago2, Sandra Orozco-Suarez2*.
1
Programa de Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Unidad-Iztapalapa, Ciudad de México, México 2
Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México 3
Servicio de Neurología, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México 4
Departamento de Ciencias Farmacéuticas de la Facultad de Química. Universidad de la República. Montevideo, Uruguay. 5
Instituto Nacional de Salud Pública, Cuernavaca, México
*Address for correspondence: Dra. Sandra Orozco Suárez. 1Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social. Av. Cuauhtémoc 330, Col. Doctores, 06720, Ciudad de México. México (52) (55) 5578-0240; *[email protected]
Acknowledgements
The Institute of Science and Technology of GDF PIFUTP/08-127 and National Council of Science and Technology (CONACYT) 248513 number grants supported this project.
ABSTRACT. Background: Identified the polymorphisms of CYP2D6, CYP2C9, CYP2C19 and CYP3A4, within a rigorously selected population of pediatric patients with drug-resistant epilepsy. Method: The genomic DNA of 23 drug-resistant epilepsy patients and 7 patients with good responses were analyzed. Ten exons in these four genes were genotyped, and the drug concentrations in saliva and plasma were determined. Results: The relevant SNPs with pharmacogenomics relations were CYP2D6*2 (rs16947) decreased your activity and CYP2D6*4 (rs1065852), CYP2C19*2 (rs4244285) and CYP3A4*1B (rs2740574) by association with poor metabolizer. The strongest risk factors were found in the AA genotype and allele of SNP rs3892097 from the CYP2D6 gene, followed by the alleles A and T of SNPs rs2740574 and rs2687116, respectively from CYP3A4. The most important concomitance was between homozygous genotype AA of rs3892097 and genotype AA of rs2740574 with 78.3% in drug-resistant epilepsy patients as compared to 14.3% in control patients. Conclusion: The results demonstrated the important role of the CYP 3A4*1B allelic variant as risk factor for developing drug resistance and CYP2D6, CYP2C19 SNPs and haplotypes may affect the response to antiepileptic drugs. Keywords. Refractory epilepsy, CYPs, SNPs, anticonvulsant drugs.
1. Introduction About 35% of patients with epilepsy are refractory to treatment despite several polytherapy regimens [1, 2, 3]. Clinically, drug resistance is associated with the time of onset (before the first year), type (usually febrile seizures), the high frequency of seizures prior to drug administration and the presence of structural lesions. Pharmacokinetic theory proposes that the overexpression of transporter proteins in the blood brain barrier and the expression of certain allelic variants of metabolizing enzymes (CYP450) modify the concentrations of AEDs that enter the brain [4]. The CYP450 enzymes (CYPs) are accountable for the metabolism of approximately 90% of all clinically prescribed drugs; the first three CYP families are part of oxidative enzymes (traditionally called metabolic enzymes phase I). CYP34A4 is the most important hepatic CYP, and represents more than a third of hepatic CYPs. Others, CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, are conclusively important for the metabolism of drugs; these first families of CYPs include genes that are highly polymorphic, meaning there are frequent genetic variations that affect their function [4,5,6,7]. CYP2D6 is highly polymorphic, with over 100 known allelic variants [8]. Some polymorphisms lead to a complete loss of CYP2D6 function, while others reduce its activity. These polymorphisms seem to cause large inter-individual and ethnic differences in CYP2D6 activity in vivo. CYP2C9 and CYP2C19 are responsible for the catalysis of the oxidation and metabolic clearance of up to 20 % of clinically important anticonvulsant drugs, as phenytoin [9,10]. CYP2C9*2 and CYP2C9*3 are recognized as the main CYP2C9 variants [11] and have reduced catalytic activity compared with the wild type (CYP2C9*1)[12)]. CYP2C19 acts on 5–10 % of drugs in current clinical use, including antidepressants and barbiturates [13].
At least 28 variant alleles for CYP2C19 have been identified, the most
extensively described of which are CYP2C19*2 and CYP2C19. Both CYP2C19*2, which causes a 40-nucleotide deletion and a frame shift, and CYP2C19*3, which leads to a premature stop codon, result in the production of a truncated protein without enzymatic activity [14]. More than 30 SNPs have been reported in CYP3A4; the most well characterized are CYP3A4*1B and CYP3A4*22 due to their effects on the functional activities of the encoded enzymes. The variability of CYP3A5 protein expression is attributed to three alleles (CYP3A5*3, CYP3A5*6 and CYP3A5*7), all of which are associated with the reduced/abolished expression of CYP3A5. The distribution of CYP3A alleles varies across ethnic groups. For instance, 8.8% and 8.0% of Mexican subjects from Mestizo and Tepehuano, respectively, carried the CYP3A4 * 1B allele [15].
Variations in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes could influence interindividual variations in AED metabolism that may be responsible for the drug-responsive or drugresistant phenotype. These genetic variants have been correlated with at least three classes of phenotypes based on the extent of drug metabolism: fast (FM), extensive (EM), and poor (PM) metabolizers; these phenotype classes result in low, normal and high blood levels of the parent drugs, respectively. Whether the polymorphisms of these genes are associated with AED resistance is still not clear; for this reason, this study identified SNPs of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 that were associated with the metabolism of antiepileptic drugs (AEDs).. The aim of this study was to perform a non-inferential exploratory study to identify reported nucleotide changes in a rigorously selected pediatric patients with similar clinical drug-like and seizures characteristics with AED-resistant epilepsy (ADR) and patients with good response to AEDs (CTR)
2. Methods 2.1. Patients and sample collection An observational study with 23 drug-resistant (cases) and 7 seizure-controlled pediatric epileptic patients (controls) was performed, while avoiding inbreeding between the patient’s biological parents. The inclusion criteria for AED-resistant epileptic patients were: (1) patients had to have demonstrable epileptic focus through EEG and without radiological focal structural lesions, (2) classified as resistant to pharmacological treatment, (3) treated with two or more drugs (Table 1) at appropriate doses, (4) serum levels within therapeutic range for at least six months of continuous treatment and under the supervision a neurologist pediatrician, (5) a frequency of 3 seizures per month, (6) 1 to 16 years of age, and (9) either gender. The asymptomatic control patients had to be seizure-free for at least 6 months before the study. 2.2. Amplification and sequencing Genomic DNA was extracted from leukocytes from patient blood samples using a commercial kit (Genomic DNA Purification kit, Thermo Scientific®, Fremont, California, USA) according to the supplier’s recommendations.
The single nucleotide polymorphisms (SNPs) from exons 1 (rs1065852), 3 (rs1058164), 5 (rs35742686), and 6 (rs16947) were located in Gen Bank accession NG_008376.3, corresponding to the CYP2D6 gene; exons 3 (rs1799853) and 7 (rs1057910) were located in Gen Bank accession NG_008385.1, corresponding to the CYP2C9 gene; exons 4 (rs4986893) and 5 (rs4244285) were located in Gen Bank accession NG_008384.2, corresponding to the CYP2C19 gene; and, 5’ UTR region (rs2740574) and exon 6 (rs55901263 and rs113667357) was located in Gen Bank accession NG_008421.1, corresponding to the CYP3A4 gene. These polymorphisms were selected based on the frequency reported in Hispanic and MestizeMexican populations (Pub Med Gene bank). Each amplification reaction was performed with 5μl (5 ng/ μl) of genomic DNA in a 50 μl total reaction volume containing 5 μl of 10x reaction buffer, MgCl2 (concentration depending on the primer set), 1 μl of 10 mM dNTPs (Thermo Scientific®, Fremont California USA), 1 μl of each 10 mM flanking primer (IDT, San Diego, California, USA), and, 1 μl of Taq polymerase (Thermo Scientific®, Fremont, California, USA). PCR was performed in a DNA engine system® thermocycler (Bio-Rad®, Hercules, California, USA) with a cycle program of 94°C for 5 min, 37 cycles of 94°C for 60 s, annealing temperature (T° h) for 60 s, 72°C for 35 s, and one extension cycle of 10 min at 72°C. The primers, MgCI 2 concentration, and T°h for each amplification reaction are listed in Table 2. The amplification products were purified with the Gene JET Gel Extraction kit (Thermo Scientific®, Fremont, California, USA) and 100 ng of amplicon was sequenced in a 5 μl reaction using a BigDye v 3.1 sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s recommendations. Sequencing was performed in a DNA engine system® thermocycler (BioRad®, Hercules, California, USA) with a cycle program at 94°C for 1 min, 30 cycles of 94°C for 10 s, 50°C for 5 s, and 60°C for 4 min After a purification with a DyeEx 2.0 Spin Kit (Qiagen, Duesseldorf, Germany), the fragments were then analyzed in an Automated Sequencing ABI Prism 310 Gene Analyzer (Applied Biosystems, Foster City California USA). 2.3. Drug monitoring . Salivary Sampling Saliva and blood samples were simultaneously taken from the patient after fasting and oral hygiene. Two sequentially-collected salivary fractions, S1 (1 mL) and S2 (1 mL), were obtained prior to morning dose (predose). Saliva was stimulated with citric acid, and collected in recipients with sodium bicarbonate when CBZ was intended to be quantified. Non buffered collection was
needed for PHT determination. VPA saliva concentrations were determined by high performance liquid chromatography (HPLC) as described previously [16].
Plasma samples were used for valproic acid (VPA) and carbamazepine (CBZ) detection by immunofluorescence with a polarized light system (FPIA, Dade Behring, USA) according to the supplier’s recommendations 2.4. Statistical Analysis. Clinical data from patients was analyzed with a nonparametric and an unpaired test (p <0.05) with the Graph Pad Prism software version 4.00 (Graph Pad Software Inc ®, USA). Allelic and genotype ratios were analyzed by odd ratio crude (ORs) using software available at http://ihg.gsf.de/cgi-bin/hw/hwa1.pl
3. Results 3.1. Polymorphisms in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes within a Mexican population. Chromatograms from amplicons were analyzed in this study to identify expected polymorphisms and to determine the relative frequencies of alleles and genotypes (Table 3). Polymorphisms studied, which had been reported in Hispanic population, however not all were detected, such as; rs35742686 in CYP2D6, rs1799853 and rs1057910 in CYP2C9, rs4986893 in CYP2C19, rs55901263 or rs113667357 in CYP3A4. On the other hand we identified the new polymorphisms rs1058164 in CYP2D6, rs9332120 in CYP2C9 and rs2687116 in CYP3A4 by sequencing the entire fragment, including exons and parts of the introns. The relative frequencies of the studied alleles were compared with their frequencies in Hispanic, Mexican-Mestize and Caucasian relevant alleles as reference populations. The alleles T and C corresponding to SNPs rs1065852 in exon 1 of CYP2D6 and rs9332120 in an intron region of CYP2C9 occurred with a higher frequency in CTR patients (0.429 and 0.214, respectively) compared with ADR patients (0.196 and 0.109, respectively) in our study population or with minor allele frequencies (0.238 and 0.144, respectively). The most relevant results in allele analysis were in allele A corresponding to SNP rs3892097 in the intron region between exons 3
and 4 of the CYP2D6 gene appeared in a higher frequency in ADR patients (0.934) compared with CTR patients (0.643) and the MAF (0.093) value. The alleles A and A corresponding to the SNPs rs28371725 in the intron region between exons 6 and 7 of the CYP2D6 gene and rs4244285 in exon 5 of the CYP2C19 gene were present only in ADR patients (0.065 and 0.109, respectively), but only rs28371725 was higher than its MAF value (0.064). The alleles A and T corresponding to SNPs rs2740574 in the 5’UTR region and rs2687116 in the intron region between exons 6 and 7 of the CYP3A4 gene occurred with higher identical frequencies in ADR patients (0.957) compared with CTR patients (0.714). Moreover, the highest allelic frequency in both cases occurred as a polymorphic change (A in rs2740574 and T in rs2687116) and not the ancestral allele (G) (Table 3). 3.2. Alleles to the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 Additionally, five alleles from The Human Cytochrome P450 (CYP) Allele Nomenclature Database correlated with different SNPs found in our population (Table 4 and 5). The families of alleles *2, *4 and *14 were identified in the CYP2D6 gene. In CTR patients, the genotype *1/*1 or the wild type, as well as *1/*4 and *2/*14, had the same frequency (0.142), while the genotypes *4/*14 and *1/*2 had a frequency of 0.285. In the ADR patients, genotypes *1/*1, *1/*4 and *4/*14 showed the same frequency (0.086), genotypes *2/*2, *2/*14 and *14/*14 exhibited the same frequency (0.043), and genotype *1/*2 had the highest frequency at 0.521. The wild type or CYP2D6*1 alleles were reported to have normal enzyme activity, while *2, *4 and *14 reduced the enzymatic activity. Table 5 describes the genotype enzyme activity based on the alleles. The rs4244285 SNP in the CYP2C19 gene corresponded to the family of allele *2. CTR patients showed only genotype *1/*1 or the wild type. ADR patients presented the wild type genotype at a frequency of 0.739 and genotype *1/*2 with a frequency of 0.217. Allele *2 presented no enzyme activity, while reduced activity was reported with genotype *1/*2. Finally, the SNP rs2740574 in the CYP3A4 gene corresponded to allele CYP3A4*1B. In CTR patients, the genotype *1A/*1B had a frequency of 0.428 and *1B/*1B had a frequency of 0.571. In ADR patients, the genotype *1A/*1B appeared with a frequency of 0.086 and *1B/*1B with a frequency of 0.913.
4. Discussion.
In
this
study,
pediatric
patients
with
epilepsy
underwent
a
regimen
of
long-term
pharmacotherapy. One factor related to the lack of seizure control is the inadequate monitoring of plasma AED concentrations Fagiolino et al., [24] proposed the quantification of AED levels in saliva using tandem drug monitoring. Monitoring drug concentrations in saliva provides advantages over monitoring drug concentrations in plasma because there is a significant relationship between saliva/plasma drug levels and the expected capillary/venous drug levels. We observed that salivary drug concentrations in most patients were at sub-therapeutic levels (Table 1). However, most drug levels in the plasma of CTR patients were at therapeutic levels. Individual genetic variations were directly related to AED concentrations because not all patients had the same capacity for AED absorption, distribution and metabolism. Different polymorphisms affect drug metabolism at different stages, which in turn affects bioavailability. Therefore, equal doses of a drug produce different concentrations in plasma and saliva, which provide access to the central nervous system [24]. 4.1. Polymorphisms in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes The cytochrome gene P450 2D6 (CYP2D6) is one of the most extensively studied genes with pharmacogenetic relevance due to its involvement in the metabolism of commonly-used drugs, most of which have narrow therapeutic indices [25, 26, 27]. The SNP rs1058164, which was identified in our study population, has not been reported previously in the Mexican-Mestizo population [17,18]. However has been reported in other populations [28].This SNP (rs1058164,) was found in a higher proportion in its heterozygous form (GC) in the ADR group compared with the CTR group. The closest reference was the Caucasian population, which had a frequency of 1.000 (PubMed GenBank). The SNP rs3892097 occurred at a higher frequency in the drugresistant group compared with the controls and reference populations. The SNP rs1065852 was reported previously as PM in the mutated homozygous form. ADR in our patients occurred with a frequency that coincided with the frequency reported in the Mexican-Mestizo population [17]. CYP2D6 variants were primarily associated with PM and the alteration of metabolite concentrations in the bloodstream. However, the SNP rs16947 was associated with PM due to a mutation that decreased its enzyme activity [5, 20, 29, 30, 31]. This variant has not been reported previously in the Mexican-Mestizo population, and then has been reported in multiple other populations, including the Hispanic population. Patients in this study exhibited a higher frequency of the heterozygous variant (CT) in the ADR group; indeed, the mutated homozygous
variant TT frequencies were more than twice those of the Hispanic and Caucasian populations [17, 28]. The CYP2C19 gene is expressed primarily in the liver, where it represents 20% of the liver metabolizing enzymes and metabolize a variety of anticonvulsants drugs [32]. Patients in our study who had the rs9332120 SNP were associated with the PM phenotype. The heterozygous form of this SNP appeared at a lower frequency than the wild type variant TT, with different values for the CTR and ADR groups. The differences in the frequencies of the TC values were greater in the ADR group compared with the CTR group. Importantly, there are no reports of this SNP in the Mexican-Mestizo population [17]. SNPs from the CYP2C19 gene have been widely studied and phenotypically identified as PM [33]. These SNPs are extensively involved in the metabolism of anticonvulsants, such as benzodiazepines and phenytoin. The SNP rs424285 induces a defect in the splicing region that results in the disruption of replication [29]. The SNP rs424285 occurred most frequently in our study population as the wild type in homozygous GG form; this frequency was higher in the CTR group. The ADR was closer to the Caucasian population with a heterozygous GA form [24]. The CYP3A4 enzyme showed high inter-individual variability that was influenced by environmental factors (e.g., alcohol, smoking and drug use); these factors contributed to 20% of its variability. Variants rs4244285 and rs2687116 of the homozygous or heterozygous genotypes occurred in both patient groups in our study population. However, the SNP rs2740574 in the 5'UTR region was a pathogenic; the AA genotype in particular exhibited a higher frequency in the ADR group compared with the control group. Among the pharmacogenetic markers in CYP3A4, the polymorphism SNP rs2740574 increased gene expression by changing the transcription factor binding affinity [33]. SNP rs2740574 plays an important role in enzyme activity [34, 35]. notably, Sosa et al [17] reported that the *1B variant (also known as SNP rs2740574) exhibited low frequencies for Mestizos. This variant occurs more frequently in European populations, including Greeks with a frequency of 0.940, followed by the English (0.940), Dutch (0.901), French (0.820), Portuguese (0.707) and Spanish (0.05)[7, 13]. Clinical observations suggest that certain combinations of AEDs may be associated with beneficial effects and adverse pharmacodynamic interactions [36]. Antiepileptic therapy in medical practice is based on the type of epilepsy and sometimes requires the prescription of relatively contraindicated drugs (e.g., valproic acid and carbamazepine or valproic acid and
phenytoin). Phenytoin, valproic acid, and tiagabine are highly bound to serum proteins, and displacement from protein binding sites may occur, especially the displacement of phenytoin by valproic acid; however, these interactions do not result in changes in clinical effects, but may be of importance for the interpretation of drug level monitoring data [37, 38]. The total concentration of the affected drug is decreased, but the concentration of the unbound, which is pharmacologically active, is unchanged. The concomitant concentration of CBZ and phenytoin in our study population demonstrated sub-therapeutic levels with VPA. Notably, this observation coincided with the concomitant presence of the homozygous genotype of SNP rs16947, which is a polymorphism associated with FM. Additionally, the presence of the heterozygous genotype of CYP2C19 SNP rs4244285, which directly affects the metabolism of VPA, was also identified [39]. Our analysis of this polymorphism demonstrated that 87% of patients had the drug-resistant homozygous genotype, compared with 4% of controlled patients. The presence of this polymorphism and sub-therapeutic concentrations of VPA in the saliva were used to identify drug-resistant patients (Table 1). However, we must not rule out that this drug interaction might exist in these patients With this study we found in population of pediatric patients with epilepsy the SNPs rs1058164, rs16947, and rs28371725 of CYP2D6, rs9332120 of CYP2C9, rs4244285 of CYP2C19 and rs2687116 of CYP3A4 which have not been previously presented in Mexican-Mestizo studies of the general population, which suggests the Hispanic heritage of our population. As expected for rs1058164, all polymorphisms were found in the reference Hispanic population. Only the rs28371725 and rs4244285 SNPs appeared in the ADR patients. 4.2. Alleles analysis Haplotype study (Table 4, 5) revealed that the CYP2D6*2 (A), CYP3A4*1B (T) and CYP2C19*2 (A) haplotype was associated with AED resistance. It has been shown that patients with variant alleles of the CYP3A49 and CYP2C19 genotypes are at increased risk for drug resistance, although the overall contribution of CYP2C19 towards the metabolism of some AEDs was less important than other CYPs [40, 41]. Drug resistance is a complex phenotype resulting from the contribution of numerous genes. In addition to drug metabolizing enzymes, the expression of multidrug transporters also influences phenytoin and carbamazepine disposition and may account for inter-individual pharmacokinetic variability [16, 42]. The CYP2C19*2 and CYP2C19*3 variant alleles are the most characterized alleles of the CYP2C19 gene [43, 44].
CYP2C19*2 is the most common allele among Caucasians; the frequency of 20.83% found in our DR group was close to the values reported in studies of different Caucasian populations [45, 46], Sachsen et al. [47] detected similar allele frequencies of the CYP2C19*2 and *3 variants in a Turkish population (12 and 0.4 %, respectively). The most common polymorphic CYP2D6 variants in other populations are the variants *3 and *4, which result in decreased enzyme activity and lead to poor metabolizer phenotypes [48]. In our study, we found in CYP2D6*4 a low frequency of 21% in CTR group and 9% in ADR group. These results were similar to the results of Aynacioglu et al [48] who evaluated 404 individuals (CYP2D6*4 allele frequency was 11%), and Aydin-Sayitoglu et al [49] who evaluated 140 individuals (*4 allele the frequency was14%). Additionally CYP2D6*14 allele frequency was of 21% in CTR group and 11% in ADR group. 4.3. Methodological considerations These results highlight the importance of conducting an analysis from two perspectives (both SNPs and alleles) because heterozygous genetic variants are present in a large number of patients. Additionally, in this study we employed a strategy to rigorously select pediatric patients with AED-resistant epilepsy and patients with good seizure control to decrease the potential influence of confounding variables associated with the disease and to develop a non-inferential exploratory search based on known polymorphisms and unreported new characteristics of the Mexican population. Our epileptic population had not been studied, which allowed us to conduct association studies of the new SNPs and AEDs used to treat different forms of epilepsy. We selected exons that contained previously reported polymorphisms in Mexican and Hispanic populations to avoid sequencing entire genes and limit the focus to the sample size and variability in treatments. However, it is difficult to perform these analyses in patients with drugresistant epilepsy who have received the same pharmacological treatment. Therefore, the main goal was to standardize the clinical variables. 5. Conclusions
This approach was designed to find an association between CYP450 polymorphisms and drugresistant epilepsy in Mexican epileptic children. The results confirm the importance of the CYP3A4*1B variant in conferring a multiple drug-resistant phenotype against AEDs. Large-scale efforts are needed to unravel the genetic determinants of the AED response. These results may
aid clinicians’ decisions on appropriate treatment and provide an explanation for the failure of some antiepileptic drugs (i.e., valproic acid) to give the expected response. Because variability in the pattern of genetic variation between populations translates into differences in drug responses, understanding CYP variability will improve rational drug use and has public health significance. However, given the small sample size, further research is needed to determine the prevalence of these polymorphisms in larger pharmacoresistant epilepsy patient populations to validate the new polymorphisms found and those previously reported.
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Figure captions Table 1. Mean and standard deviation of drug concentrations of patients with AED-resistant epilepsy and patients with good response to AEDs Table 2. Primers used on CYP2D6, CYP2C9, CYP2C19 and CYP3A4 fragment amplification. The primer design was performed using the web page http://www.yeastgenome.org/cgi-bin/webprimer, T°h was selected by a temperature gradient, and the MgCl2 concentration was initially with 2.0 mM. PCR was performed gradually until a single sharp band was obtained. Table 3. Genotype and allelic frequencies, relatives of SNPs, MAF value, amino acid changes and odds ratio found in CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes. The relative frequencies are presented for the eight SNPs reported from CYP2D6, 2C9, C19 and 3A4 database genes (Pub Med Gene Bank), 14 controlled and 46 drug-resistant patients (2n or Chrom. Sample), were obtained by direct sequencing. The position indicates the nucleotide change in CYP2D6, 2C9, C19 and 3A4 genes which were obtained from RefSeqGene NG_008376.3, NG_008385.1, 008384.2 and NG_008421.1 respectively. An amino acid change was obtained from SNP page and its location from www.uniprot.org web site. The odds ratio (OR) represents the allele risk or mutated using the online software from http://ihg.gsf.de/cgibin/hw/hwa1.pl. ADR: drug-resistant. CTR: controlled. MAF: Minor Allele Frequency, values updated in December 2014. Table 4. Clinical data of patients with AEDs-resistant epilepsy (ADR) and patients with good response to AEDs (CTR) and relative frequencies from most relevant Alleles. TLE: Temporal lobe epilepsy, FRT: Frontal, R: Right, L: Left, PCS: Partial complex seizures, GEN: Generalized seizures, CTS: Clonic-tonic seizures: ABS: Absent seizures, ATS: Atonic seizures. VPA: Valproic acid, CBP: Carbamazepine, LEV: Leviracetam, TOP: Topiramato, LMGT: Lamotrigine, PHT: Phenytoin, CLB: Clobazan, PRIM: Primidone
Table 5. The relative frequencies are presented for the ten genotypes reported from CYP2D6, C19 and 3A4 database (Human Cytochrome P450 Allele Nomenclature), 7 controlled and 23 drug-resistant patients, were obtained by direct sequencing. ADR: drug-resistant. CTR: controlled
Tables Table 1. Drug Valproic acid Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/l Carbamazepine Dose (SD), /24h Concentration saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Lamotrigine Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Phenytoin Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Levetiracetam Dose (SD), μmol/24h Concentration/saliva (SD), μmol/l Concentration/plasma (SD), μmol/l
CTR n Mean (SD)
ADR n Mean (SD)
5 791.66 (62.08) 5 2.3 (1.3) 3 82.1 (32.5 )
14 711.80(295.97) 13 0.69(0.069) 11 43.88 (15.97)
1 254.23 1 1.0 0 -
2 2 1
2 39.06(0.0) 1 0.351 0 1 587.2 -
354.33(55.51) 0.27(0.19) 2.32 -
4 2 3
49.20(37.30) 0.357(0.03) 9.85(14.88)
5 4
764.7(335.29) 1(1) -
Table 2 Primer
MgCl2 (mM)
T°h
Size (pb)
Gen Exon
Fw
1
5’-TTTATAAGGGAAGGGTCACGC-3’
5’-TTTCACCCACCATCCATGTT-3’
1.5
56
375
3
5’-AAGGTGGATGCACAAAGAGTG-3’
5’-AAGAGACCGTTGGGGCGAA-3’
1.5
57
264
5
5’-ACTTGTCCAGGTGAACGCAGA-3’
5’-ATTCCTCCTGGGACGCTCAA-3’
1.25
59
294
6
5’-AAAAGGTTGGACCAGTGCATC-3’
5’-TGTTGGAGGAGGTCAGGCTTA-3’
1.25
60
416
3
5’-TAGGTGTGCATGTGCCTGTTT-3’
5’-CCCCTGAAATGTTTCCAAGA-3’
1.5
57
490
7
5’-TGGCAGTTACACATTTGTGCA-3’
5’-TAGCCCCAAACTGGAAACAA-3’
1.5
58
500
CYP2C1 9
4
5’-TGTGTTGATTTTATGCATGCC-3’
5’-CTTTTCCAGATATTCACCCCA-3’
1.5
57
460
5
5’-TTACAACCAGAGCTTGGCAT-3’
5’-CCTTGACCTGTTAAACATCCG-3’
1.5
58
350
CYP3A4
5’ UTR
5’-AAGGGATGACATGCAGAGGC-3’
5’-TCACAAACCCTGTCATCAT-3’
1.5
63
804
6
5’-TAGGGCCAGCTGCATCACT-3’
5’-TCACAAACCCTGTCATCAT-3’
1.5
61
570
CYP2D6
CYP2C9
Rv
Table 3
PROTEIN ASSOCIATION Gene
SNP
Sample
Allele Frequency
Genotype Frequency
MAF
aa, change
Localization
ODDS RATIO Allele
Genotype Heterozogous Homozygou s
CYP2D6 CTR
C=0.571 T=0.429
C/C=0.428 C/T=0.286 T/T=0.286
rs1065852
T=0.238/1192 ADR
C=0.804 T=0.196
C/C=0.740 C/T=0.130 T/T=0.130
CTR
G=0.429 C=0.571
G/C=0.858 C/C=0.142
rs1058164
C=0.401/2008 136; G>C; Val>Val ADR
G=0.500 C=0.500
G/G=0.217 G/C=0.566 C/C=0.217
CTR
G=0.357 A=0.643
G/A=0.714 A/A=0.286
ADR
G=0.065 A=0.934
G/A=0.130 A/A=0.870
CTR
G=0.571 A=0.429
G/G=0.286 G/A=0.572 A/A=0.142
rs3892097
rs16947
A=0.093/466
ADR
G=0.565 A=0.435
G/G=0.261 G/A=0.609 A/A=0.130
CTR
G=1.000
G/G=1.000
ADR
G=0.935 A=0.065
G/G=0.870 G/A=0.130
CTR
T=0.786 C=0.214
T/T=0.572 T/C=0.428
rs9332120 ADR
T=0.891 C=0.109
T/T=0.783 T/C=0.217
CTR
G=1.000
G/G=1.000
PPGP region, NH2terminal
[C]<->[T] 0.324
[CC]<->[CT] 0.265
Alfa-Helix chain
[G]<->[C] 0.750
[GG]<->[GC] [GG]<->[C/C] 0.189 0.333
[G]<->[A] 7.963
[GG]<->[GA] 0.636
[GG]<->[AA] 8.200
[G]<->[A] 1.026
[GG]<->[GA] 1.167
[GG]<->[AA] 1.000
Intron between 3 and 4 exon
A=0.359/1799 296; G>A; Cys>Arg
rs28371725
CYP2C9
34; C>T; Ser>Pro
Alfa-Helix chain
[CC]<->[TT] 0.265
A=0.064/318
Intron between 6 and 7 exon
[G]<->[A] 2.333
[GG]<->[GA] 2.561
[GG]<->[AA] 0.366
C=0.144/722
Intron between 2 and 3 exon
[T]<->[C] 0.447
[TT]<->[TC] 0.243
[TT]<->[CC] 0.370
[G]<->[A] 3.843
[GG]<->[GA] 4.459
[GG]<->[AA] 0.405
CYP2C19
rs4244285
A=0.221/1109 227; G>A; Pro>Pro ADR
G=0.891 A=0.109
Alfa-Helix chain
G/G=0.783 G/A=0.217
CYP3A4 CTR
A=0.714 G=0.286
A/A=0.429 A/G=0.571
rs2740574 ADR
A=0.957 G=0.043
A/A=0.913 A/G=0.087
CTR
T=0.714 G=0.286
T/T=0.429 T/G=0.571
T=0.957 G=0.043
T/T=0.913 T/G=0.087
rs2687116 ADR
G=0.231/1156
5’ UTR region
[G]<->[A] 8.000
[GG]<->[GA] 0.556
[GG]<->[AA] 6.200
G=0.220/1103
Intron between 6 and 7 exon
[G]<->[T] 8.000
[GG]<->[GT] 0.556
[GG]<->[TT] 6.200
Table 4
Case
Age
Sex
P001 P002
15 8
M F
Main Focus TLE TLE-R
P003 P004
10 14
F M
TLE TLE
P005 P006
6 11
M F
TLE TLE
P007 P008 P009 P010 P011 P012
13 11 14 11 8 15
M M F M M F
TLE F- TEMP F- TEMP TLE TLE TLE
P013 P014 P015 P016 P017
3 12 13 9 11
F F F F M
FRT-R TLE-R TLE-R TLE-L TEM-B
P018 P019 P020
11 13 1
F F F
FRT-L FRT-R FRT-L
P021 P022 P023 C001 C002 C003 C004 C005 C006 C007
3 14 10 1 9 7 12 15 12 15
M M F M F F M F M M
TLE-R FRT- B TLE-L FRT-R TLE-R TLE-R TLE-R FRT-R TLE TLE
Seizures Type PCS PCS with GEN PCS CTS with GEN GEN CTS with GEN PCS PCS PCS PCS PCS, GEN CTS with GEN PCS,ABS PCS CTS,ABS AS, GEN PCS with GEN TCS T-GEN PCS with GEN PCS PCS TCS PCS PCS PCS ATS PCS PCS PCS
Drugs
CYP 3A4
ALLELE CYP2D6 CYP2C19
VP,CLO,LV
*1B *1B
*1/*2 *1
*1B *1B
*1B *1A/*1B
*4/*14 *1/*2
*1B *1A/*1B
*1B *1B
*1/*2 *14/*14
*1B *1B
*1B *1B *1B *1B *1A/*1B *1B
*1/*4 *1 *1/*2 *1/*2 *2/*14 *1/*2
*1B *1B *1A/*1B *1B *1B *1A/*1B
*1B *1B *1B *1B *1B
*1/*4 *1/*2 *1 *1 *4/*14
*1B *1B *1B *1B *1A/*1B
*1B *1B *1B
*1/*2 *1/*2 *1/*2
*1B *1B *1A/*1B
*1B *1B *1B *1A/*1B *1A/*1B *1B *1B *1A/*1B *1B *IB
*1/*2 *2 *1/*2 *4/*14 *1/*4 *4/*14 *1/*2 *2/*14 *1/*2 *1
*1B *1B *1B *1A/*1B *1B *1B *1B *1B *1B *1B
TOP CBZ, TOP CBZ VP, TOP CBZ, TOP LEV LEV, PHT CBZ, TOP VPA,CLB VPA,LEV,PRM LEV VPA, PTH, CLB LEV,VPA,CLON OX,LEV OX,TOP LEV TOP VPA, TOP VPA,TOP,OX VPA,CBZ VPA,TOP VPA,OX PHT VPA,LMT VPA TOP,OX TOP,LEV VPA,CBZ LEV
Table 5 Genotype, SNP, genotype frequency and enzyme activities in CYP2D6, CYP2C19 and CYP3A4 genes. Genotype Gene Genotype SNP Sample Activity Frequency CTR 0.142 *1/*1 Wild-Type Normal ADR 0.086 CTR 0.285 Wild-Type *1/*2 Decreased rs16947 ADR 0.521
CYP2D6
CTR
0.142
ADR CTR
0.086 0.285
ADR
0.043
rs16947 rs1065852
CTR
0.142
ADR
0.043
*4/*14
rs16947 rs1065852
CTR
0.285
ADR
0.086
*14/*14
rs16947 rs1065852
CTR
0.000
ADR
0.043
*1/*1
Wild-Type
CTR
0.739
Normal
*1/*2
Wild-Type rs4244285
ADR
0.217
Decreased
*1A/*1B
Wild-Type rs2740574
CTR
0.428
*1B/*1B
rs2740574
ADR CTR ADR
0.086 0.571 0.913
*1/*4
Wild-Type rs1065852
*2/*2
rs16947
*2/*14
CYP2C19
CYP3A4
Decreased Decreased Decreased Decreased Decreased
Decreased Decreased
S1734-1140(17)30028-2 http://dx.doi.org/doi:10.1016/j.pharep.2017.01.007 PHAREP 621
To appear in: Received date: Revised date: Accepted date:
26-7-2016 10-1-2017 16-1-2017
Please cite this article as: Miguel A.L´opez-Garc´ıa, Iris A.Feria-Romero, H´ector Serrano, Dar´ıo Rayo-Mares, Pietro Fagiolino, Marta V´azquez, Consuelo Escamilla-N´un˜ ez, Israel Grijalva, David Escalante-Santiago, Sandra Orozco-Suarez, Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy, http://dx.doi.org/10.1016/j.pharep.2017.01.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy
Miguel A López-García1-2, Iris A Feria-Romero2, Héctor Serrano1, Darío Rayo-Mares3, Pietro Fagiolino4, Marta Vázquez4, Consuelo Escamilla-Núñez 5, Israel Grijalva2, David EscalanteSantiago2, Sandra Orozco-Suarez2*.
1
Programa de Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Unidad-Iztapalapa, Ciudad de México, México 2
Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México 3
Servicio de Neurología, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Ciudad de México, México 4
Departamento de Ciencias Farmacéuticas de la Facultad de Química. Universidad de la República. Montevideo, Uruguay. 5
Instituto Nacional de Salud Pública, Cuernavaca, México
*Address for correspondence: Dra. Sandra Orozco Suárez. 1Unidad de Investigación Médica en Enfermedades Neurológicas, Hospital de Especialidades Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social. Av. Cuauhtémoc 330, Col. Doctores, 06720, Ciudad de México. México (52) (55) 5578-0240; *[email protected]
Acknowledgements
The Institute of Science and Technology of GDF PIFUTP/08-127 and National Council of Science and Technology (CONACYT) 248513 number grants supported this project.
ABSTRACT. Background: Identified the polymorphisms of CYP2D6, CYP2C9, CYP2C19 and CYP3A4, within a rigorously selected population of pediatric patients with drug-resistant epilepsy. Method: The genomic DNA of 23 drug-resistant epilepsy patients and 7 patients with good responses were analyzed. Ten exons in these four genes were genotyped, and the drug concentrations in saliva and plasma were determined. Results: The relevant SNPs with pharmacogenomics relations were CYP2D6*2 (rs16947) decreased your activity and CYP2D6*4 (rs1065852), CYP2C19*2 (rs4244285) and CYP3A4*1B (rs2740574) by association with poor metabolizer. The strongest risk factors were found in the AA genotype and allele of SNP rs3892097 from the CYP2D6 gene, followed by the alleles A and T of SNPs rs2740574 and rs2687116, respectively from CYP3A4. The most important concomitance was between homozygous genotype AA of rs3892097 and genotype AA of rs2740574 with 78.3% in drug-resistant epilepsy patients as compared to 14.3% in control patients. Conclusion: The results demonstrated the important role of the CYP 3A4*1B allelic variant as risk factor for developing drug resistance and CYP2D6, CYP2C19 SNPs and haplotypes may affect the response to antiepileptic drugs. Keywords. Refractory epilepsy, CYPs, SNPs, anticonvulsant drugs.
1. Introduction About 35% of patients with epilepsy are refractory to treatment despite several polytherapy regimens [1, 2, 3]. Clinically, drug resistance is associated with the time of onset (before the first year), type (usually febrile seizures), the high frequency of seizures prior to drug administration and the presence of structural lesions. Pharmacokinetic theory proposes that the overexpression of transporter proteins in the blood brain barrier and the expression of certain allelic variants of metabolizing enzymes (CYP450) modify the concentrations of AEDs that enter the brain [4]. The CYP450 enzymes (CYPs) are accountable for the metabolism of approximately 90% of all clinically prescribed drugs; the first three CYP families are part of oxidative enzymes (traditionally called metabolic enzymes phase I). CYP34A4 is the most important hepatic CYP, and represents more than a third of hepatic CYPs. Others, CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, are conclusively important for the metabolism of drugs; these first families of CYPs include genes that are highly polymorphic, meaning there are frequent genetic variations that affect their function [4,5,6,7]. CYP2D6 is highly polymorphic, with over 100 known allelic variants [8]. Some polymorphisms lead to a complete loss of CYP2D6 function, while others reduce its activity. These polymorphisms seem to cause large inter-individual and ethnic differences in CYP2D6 activity in vivo. CYP2C9 and CYP2C19 are responsible for the catalysis of the oxidation and metabolic clearance of up to 20 % of clinically important anticonvulsant drugs, as phenytoin [9,10]. CYP2C9*2 and CYP2C9*3 are recognized as the main CYP2C9 variants [11] and have reduced catalytic activity compared with the wild type (CYP2C9*1)[12)]. CYP2C19 acts on 5–10 % of drugs in current clinical use, including antidepressants and barbiturates [13].
At least 28 variant alleles for CYP2C19 have been identified, the most
extensively described of which are CYP2C19*2 and CYP2C19. Both CYP2C19*2, which causes a 40-nucleotide deletion and a frame shift, and CYP2C19*3, which leads to a premature stop codon, result in the production of a truncated protein without enzymatic activity [14]. More than 30 SNPs have been reported in CYP3A4; the most well characterized are CYP3A4*1B and CYP3A4*22 due to their effects on the functional activities of the encoded enzymes. The variability of CYP3A5 protein expression is attributed to three alleles (CYP3A5*3, CYP3A5*6 and CYP3A5*7), all of which are associated with the reduced/abolished expression of CYP3A5. The distribution of CYP3A alleles varies across ethnic groups. For instance, 8.8% and 8.0% of Mexican subjects from Mestizo and Tepehuano, respectively, carried the CYP3A4 * 1B allele [15].
Variations in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes could influence interindividual variations in AED metabolism that may be responsible for the drug-responsive or drugresistant phenotype. These genetic variants have been correlated with at least three classes of phenotypes based on the extent of drug metabolism: fast (FM), extensive (EM), and poor (PM) metabolizers; these phenotype classes result in low, normal and high blood levels of the parent drugs, respectively. Whether the polymorphisms of these genes are associated with AED resistance is still not clear; for this reason, this study identified SNPs of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 that were associated with the metabolism of antiepileptic drugs (AEDs).. The aim of this study was to perform a non-inferential exploratory study to identify reported nucleotide changes in a rigorously selected pediatric patients with similar clinical drug-like and seizures characteristics with AED-resistant epilepsy (ADR) and patients with good response to AEDs (CTR)
2. Methods 2.1. Patients and sample collection An observational study with 23 drug-resistant (cases) and 7 seizure-controlled pediatric epileptic patients (controls) was performed, while avoiding inbreeding between the patient’s biological parents. The inclusion criteria for AED-resistant epileptic patients were: (1) patients had to have demonstrable epileptic focus through EEG and without radiological focal structural lesions, (2) classified as resistant to pharmacological treatment, (3) treated with two or more drugs (Table 1) at appropriate doses, (4) serum levels within therapeutic range for at least six months of continuous treatment and under the supervision a neurologist pediatrician, (5) a frequency of 3 seizures per month, (6) 1 to 16 years of age, and (9) either gender. The asymptomatic control patients had to be seizure-free for at least 6 months before the study. 2.2. Amplification and sequencing Genomic DNA was extracted from leukocytes from patient blood samples using a commercial kit (Genomic DNA Purification kit, Thermo Scientific®, Fremont, California, USA) according to the supplier’s recommendations.
The single nucleotide polymorphisms (SNPs) from exons 1 (rs1065852), 3 (rs1058164), 5 (rs35742686), and 6 (rs16947) were located in Gen Bank accession NG_008376.3, corresponding to the CYP2D6 gene; exons 3 (rs1799853) and 7 (rs1057910) were located in Gen Bank accession NG_008385.1, corresponding to the CYP2C9 gene; exons 4 (rs4986893) and 5 (rs4244285) were located in Gen Bank accession NG_008384.2, corresponding to the CYP2C19 gene; and, 5’ UTR region (rs2740574) and exon 6 (rs55901263 and rs113667357) was located in Gen Bank accession NG_008421.1, corresponding to the CYP3A4 gene. These polymorphisms were selected based on the frequency reported in Hispanic and MestizeMexican populations (Pub Med Gene bank). Each amplification reaction was performed with 5μl (5 ng/ μl) of genomic DNA in a 50 μl total reaction volume containing 5 μl of 10x reaction buffer, MgCl2 (concentration depending on the primer set), 1 μl of 10 mM dNTPs (Thermo Scientific®, Fremont California USA), 1 μl of each 10 mM flanking primer (IDT, San Diego, California, USA), and, 1 μl of Taq polymerase (Thermo Scientific®, Fremont, California, USA). PCR was performed in a DNA engine system® thermocycler (Bio-Rad®, Hercules, California, USA) with a cycle program of 94°C for 5 min, 37 cycles of 94°C for 60 s, annealing temperature (T° h) for 60 s, 72°C for 35 s, and one extension cycle of 10 min at 72°C. The primers, MgCI 2 concentration, and T°h for each amplification reaction are listed in Table 2. The amplification products were purified with the Gene JET Gel Extraction kit (Thermo Scientific®, Fremont, California, USA) and 100 ng of amplicon was sequenced in a 5 μl reaction using a BigDye v 3.1 sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer’s recommendations. Sequencing was performed in a DNA engine system® thermocycler (BioRad®, Hercules, California, USA) with a cycle program at 94°C for 1 min, 30 cycles of 94°C for 10 s, 50°C for 5 s, and 60°C for 4 min After a purification with a DyeEx 2.0 Spin Kit (Qiagen, Duesseldorf, Germany), the fragments were then analyzed in an Automated Sequencing ABI Prism 310 Gene Analyzer (Applied Biosystems, Foster City California USA). 2.3. Drug monitoring . Salivary Sampling Saliva and blood samples were simultaneously taken from the patient after fasting and oral hygiene. Two sequentially-collected salivary fractions, S1 (1 mL) and S2 (1 mL), were obtained prior to morning dose (predose). Saliva was stimulated with citric acid, and collected in recipients with sodium bicarbonate when CBZ was intended to be quantified. Non buffered collection was
needed for PHT determination. VPA saliva concentrations were determined by high performance liquid chromatography (HPLC) as described previously [16].
Plasma samples were used for valproic acid (VPA) and carbamazepine (CBZ) detection by immunofluorescence with a polarized light system (FPIA, Dade Behring, USA) according to the supplier’s recommendations 2.4. Statistical Analysis. Clinical data from patients was analyzed with a nonparametric and an unpaired test (p <0.05) with the Graph Pad Prism software version 4.00 (Graph Pad Software Inc ®, USA). Allelic and genotype ratios were analyzed by odd ratio crude (ORs) using software available at http://ihg.gsf.de/cgi-bin/hw/hwa1.pl
3. Results 3.1. Polymorphisms in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes within a Mexican population. Chromatograms from amplicons were analyzed in this study to identify expected polymorphisms and to determine the relative frequencies of alleles and genotypes (Table 3). Polymorphisms studied, which had been reported in Hispanic population, however not all were detected, such as; rs35742686 in CYP2D6, rs1799853 and rs1057910 in CYP2C9, rs4986893 in CYP2C19, rs55901263 or rs113667357 in CYP3A4. On the other hand we identified the new polymorphisms rs1058164 in CYP2D6, rs9332120 in CYP2C9 and rs2687116 in CYP3A4 by sequencing the entire fragment, including exons and parts of the introns. The relative frequencies of the studied alleles were compared with their frequencies in Hispanic, Mexican-Mestize and Caucasian relevant alleles as reference populations. The alleles T and C corresponding to SNPs rs1065852 in exon 1 of CYP2D6 and rs9332120 in an intron region of CYP2C9 occurred with a higher frequency in CTR patients (0.429 and 0.214, respectively) compared with ADR patients (0.196 and 0.109, respectively) in our study population or with minor allele frequencies (0.238 and 0.144, respectively). The most relevant results in allele analysis were in allele A corresponding to SNP rs3892097 in the intron region between exons 3
and 4 of the CYP2D6 gene appeared in a higher frequency in ADR patients (0.934) compared with CTR patients (0.643) and the MAF (0.093) value. The alleles A and A corresponding to the SNPs rs28371725 in the intron region between exons 6 and 7 of the CYP2D6 gene and rs4244285 in exon 5 of the CYP2C19 gene were present only in ADR patients (0.065 and 0.109, respectively), but only rs28371725 was higher than its MAF value (0.064). The alleles A and T corresponding to SNPs rs2740574 in the 5’UTR region and rs2687116 in the intron region between exons 6 and 7 of the CYP3A4 gene occurred with higher identical frequencies in ADR patients (0.957) compared with CTR patients (0.714). Moreover, the highest allelic frequency in both cases occurred as a polymorphic change (A in rs2740574 and T in rs2687116) and not the ancestral allele (G) (Table 3). 3.2. Alleles to the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 Additionally, five alleles from The Human Cytochrome P450 (CYP) Allele Nomenclature Database correlated with different SNPs found in our population (Table 4 and 5). The families of alleles *2, *4 and *14 were identified in the CYP2D6 gene. In CTR patients, the genotype *1/*1 or the wild type, as well as *1/*4 and *2/*14, had the same frequency (0.142), while the genotypes *4/*14 and *1/*2 had a frequency of 0.285. In the ADR patients, genotypes *1/*1, *1/*4 and *4/*14 showed the same frequency (0.086), genotypes *2/*2, *2/*14 and *14/*14 exhibited the same frequency (0.043), and genotype *1/*2 had the highest frequency at 0.521. The wild type or CYP2D6*1 alleles were reported to have normal enzyme activity, while *2, *4 and *14 reduced the enzymatic activity. Table 5 describes the genotype enzyme activity based on the alleles. The rs4244285 SNP in the CYP2C19 gene corresponded to the family of allele *2. CTR patients showed only genotype *1/*1 or the wild type. ADR patients presented the wild type genotype at a frequency of 0.739 and genotype *1/*2 with a frequency of 0.217. Allele *2 presented no enzyme activity, while reduced activity was reported with genotype *1/*2. Finally, the SNP rs2740574 in the CYP3A4 gene corresponded to allele CYP3A4*1B. In CTR patients, the genotype *1A/*1B had a frequency of 0.428 and *1B/*1B had a frequency of 0.571. In ADR patients, the genotype *1A/*1B appeared with a frequency of 0.086 and *1B/*1B with a frequency of 0.913.
4. Discussion.
In
this
study,
pediatric
patients
with
epilepsy
underwent
a
regimen
of
long-term
pharmacotherapy. One factor related to the lack of seizure control is the inadequate monitoring of plasma AED concentrations Fagiolino et al., [24] proposed the quantification of AED levels in saliva using tandem drug monitoring. Monitoring drug concentrations in saliva provides advantages over monitoring drug concentrations in plasma because there is a significant relationship between saliva/plasma drug levels and the expected capillary/venous drug levels. We observed that salivary drug concentrations in most patients were at sub-therapeutic levels (Table 1). However, most drug levels in the plasma of CTR patients were at therapeutic levels. Individual genetic variations were directly related to AED concentrations because not all patients had the same capacity for AED absorption, distribution and metabolism. Different polymorphisms affect drug metabolism at different stages, which in turn affects bioavailability. Therefore, equal doses of a drug produce different concentrations in plasma and saliva, which provide access to the central nervous system [24]. 4.1. Polymorphisms in the CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes The cytochrome gene P450 2D6 (CYP2D6) is one of the most extensively studied genes with pharmacogenetic relevance due to its involvement in the metabolism of commonly-used drugs, most of which have narrow therapeutic indices [25, 26, 27]. The SNP rs1058164, which was identified in our study population, has not been reported previously in the Mexican-Mestizo population [17,18]. However has been reported in other populations [28].This SNP (rs1058164,) was found in a higher proportion in its heterozygous form (GC) in the ADR group compared with the CTR group. The closest reference was the Caucasian population, which had a frequency of 1.000 (PubMed GenBank). The SNP rs3892097 occurred at a higher frequency in the drugresistant group compared with the controls and reference populations. The SNP rs1065852 was reported previously as PM in the mutated homozygous form. ADR in our patients occurred with a frequency that coincided with the frequency reported in the Mexican-Mestizo population [17]. CYP2D6 variants were primarily associated with PM and the alteration of metabolite concentrations in the bloodstream. However, the SNP rs16947 was associated with PM due to a mutation that decreased its enzyme activity [5, 20, 29, 30, 31]. This variant has not been reported previously in the Mexican-Mestizo population, and then has been reported in multiple other populations, including the Hispanic population. Patients in this study exhibited a higher frequency of the heterozygous variant (CT) in the ADR group; indeed, the mutated homozygous
variant TT frequencies were more than twice those of the Hispanic and Caucasian populations [17, 28]. The CYP2C19 gene is expressed primarily in the liver, where it represents 20% of the liver metabolizing enzymes and metabolize a variety of anticonvulsants drugs [32]. Patients in our study who had the rs9332120 SNP were associated with the PM phenotype. The heterozygous form of this SNP appeared at a lower frequency than the wild type variant TT, with different values for the CTR and ADR groups. The differences in the frequencies of the TC values were greater in the ADR group compared with the CTR group. Importantly, there are no reports of this SNP in the Mexican-Mestizo population [17]. SNPs from the CYP2C19 gene have been widely studied and phenotypically identified as PM [33]. These SNPs are extensively involved in the metabolism of anticonvulsants, such as benzodiazepines and phenytoin. The SNP rs424285 induces a defect in the splicing region that results in the disruption of replication [29]. The SNP rs424285 occurred most frequently in our study population as the wild type in homozygous GG form; this frequency was higher in the CTR group. The ADR was closer to the Caucasian population with a heterozygous GA form [24]. The CYP3A4 enzyme showed high inter-individual variability that was influenced by environmental factors (e.g., alcohol, smoking and drug use); these factors contributed to 20% of its variability. Variants rs4244285 and rs2687116 of the homozygous or heterozygous genotypes occurred in both patient groups in our study population. However, the SNP rs2740574 in the 5'UTR region was a pathogenic; the AA genotype in particular exhibited a higher frequency in the ADR group compared with the control group. Among the pharmacogenetic markers in CYP3A4, the polymorphism SNP rs2740574 increased gene expression by changing the transcription factor binding affinity [33]. SNP rs2740574 plays an important role in enzyme activity [34, 35]. notably, Sosa et al [17] reported that the *1B variant (also known as SNP rs2740574) exhibited low frequencies for Mestizos. This variant occurs more frequently in European populations, including Greeks with a frequency of 0.940, followed by the English (0.940), Dutch (0.901), French (0.820), Portuguese (0.707) and Spanish (0.05)[7, 13]. Clinical observations suggest that certain combinations of AEDs may be associated with beneficial effects and adverse pharmacodynamic interactions [36]. Antiepileptic therapy in medical practice is based on the type of epilepsy and sometimes requires the prescription of relatively contraindicated drugs (e.g., valproic acid and carbamazepine or valproic acid and
phenytoin). Phenytoin, valproic acid, and tiagabine are highly bound to serum proteins, and displacement from protein binding sites may occur, especially the displacement of phenytoin by valproic acid; however, these interactions do not result in changes in clinical effects, but may be of importance for the interpretation of drug level monitoring data [37, 38]. The total concentration of the affected drug is decreased, but the concentration of the unbound, which is pharmacologically active, is unchanged. The concomitant concentration of CBZ and phenytoin in our study population demonstrated sub-therapeutic levels with VPA. Notably, this observation coincided with the concomitant presence of the homozygous genotype of SNP rs16947, which is a polymorphism associated with FM. Additionally, the presence of the heterozygous genotype of CYP2C19 SNP rs4244285, which directly affects the metabolism of VPA, was also identified [39]. Our analysis of this polymorphism demonstrated that 87% of patients had the drug-resistant homozygous genotype, compared with 4% of controlled patients. The presence of this polymorphism and sub-therapeutic concentrations of VPA in the saliva were used to identify drug-resistant patients (Table 1). However, we must not rule out that this drug interaction might exist in these patients With this study we found in population of pediatric patients with epilepsy the SNPs rs1058164, rs16947, and rs28371725 of CYP2D6, rs9332120 of CYP2C9, rs4244285 of CYP2C19 and rs2687116 of CYP3A4 which have not been previously presented in Mexican-Mestizo studies of the general population, which suggests the Hispanic heritage of our population. As expected for rs1058164, all polymorphisms were found in the reference Hispanic population. Only the rs28371725 and rs4244285 SNPs appeared in the ADR patients. 4.2. Alleles analysis Haplotype study (Table 4, 5) revealed that the CYP2D6*2 (A), CYP3A4*1B (T) and CYP2C19*2 (A) haplotype was associated with AED resistance. It has been shown that patients with variant alleles of the CYP3A49 and CYP2C19 genotypes are at increased risk for drug resistance, although the overall contribution of CYP2C19 towards the metabolism of some AEDs was less important than other CYPs [40, 41]. Drug resistance is a complex phenotype resulting from the contribution of numerous genes. In addition to drug metabolizing enzymes, the expression of multidrug transporters also influences phenytoin and carbamazepine disposition and may account for inter-individual pharmacokinetic variability [16, 42]. The CYP2C19*2 and CYP2C19*3 variant alleles are the most characterized alleles of the CYP2C19 gene [43, 44].
CYP2C19*2 is the most common allele among Caucasians; the frequency of 20.83% found in our DR group was close to the values reported in studies of different Caucasian populations [45, 46], Sachsen et al. [47] detected similar allele frequencies of the CYP2C19*2 and *3 variants in a Turkish population (12 and 0.4 %, respectively). The most common polymorphic CYP2D6 variants in other populations are the variants *3 and *4, which result in decreased enzyme activity and lead to poor metabolizer phenotypes [48]. In our study, we found in CYP2D6*4 a low frequency of 21% in CTR group and 9% in ADR group. These results were similar to the results of Aynacioglu et al [48] who evaluated 404 individuals (CYP2D6*4 allele frequency was 11%), and Aydin-Sayitoglu et al [49] who evaluated 140 individuals (*4 allele the frequency was14%). Additionally CYP2D6*14 allele frequency was of 21% in CTR group and 11% in ADR group. 4.3. Methodological considerations These results highlight the importance of conducting an analysis from two perspectives (both SNPs and alleles) because heterozygous genetic variants are present in a large number of patients. Additionally, in this study we employed a strategy to rigorously select pediatric patients with AED-resistant epilepsy and patients with good seizure control to decrease the potential influence of confounding variables associated with the disease and to develop a non-inferential exploratory search based on known polymorphisms and unreported new characteristics of the Mexican population. Our epileptic population had not been studied, which allowed us to conduct association studies of the new SNPs and AEDs used to treat different forms of epilepsy. We selected exons that contained previously reported polymorphisms in Mexican and Hispanic populations to avoid sequencing entire genes and limit the focus to the sample size and variability in treatments. However, it is difficult to perform these analyses in patients with drugresistant epilepsy who have received the same pharmacological treatment. Therefore, the main goal was to standardize the clinical variables. 5. Conclusions
This approach was designed to find an association between CYP450 polymorphisms and drugresistant epilepsy in Mexican epileptic children. The results confirm the importance of the CYP3A4*1B variant in conferring a multiple drug-resistant phenotype against AEDs. Large-scale efforts are needed to unravel the genetic determinants of the AED response. These results may
aid clinicians’ decisions on appropriate treatment and provide an explanation for the failure of some antiepileptic drugs (i.e., valproic acid) to give the expected response. Because variability in the pattern of genetic variation between populations translates into differences in drug responses, understanding CYP variability will improve rational drug use and has public health significance. However, given the small sample size, further research is needed to determine the prevalence of these polymorphisms in larger pharmacoresistant epilepsy patient populations to validate the new polymorphisms found and those previously reported.
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Figure captions Table 1. Mean and standard deviation of drug concentrations of patients with AED-resistant epilepsy and patients with good response to AEDs Table 2. Primers used on CYP2D6, CYP2C9, CYP2C19 and CYP3A4 fragment amplification. The primer design was performed using the web page http://www.yeastgenome.org/cgi-bin/webprimer, T°h was selected by a temperature gradient, and the MgCl2 concentration was initially with 2.0 mM. PCR was performed gradually until a single sharp band was obtained. Table 3. Genotype and allelic frequencies, relatives of SNPs, MAF value, amino acid changes and odds ratio found in CYP2D6, CYP2C9, CYP2C19 and CYP3A4 genes. The relative frequencies are presented for the eight SNPs reported from CYP2D6, 2C9, C19 and 3A4 database genes (Pub Med Gene Bank), 14 controlled and 46 drug-resistant patients (2n or Chrom. Sample), were obtained by direct sequencing. The position indicates the nucleotide change in CYP2D6, 2C9, C19 and 3A4 genes which were obtained from RefSeqGene NG_008376.3, NG_008385.1, 008384.2 and NG_008421.1 respectively. An amino acid change was obtained from SNP page and its location from www.uniprot.org web site. The odds ratio (OR) represents the allele risk or mutated using the online software from http://ihg.gsf.de/cgibin/hw/hwa1.pl. ADR: drug-resistant. CTR: controlled. MAF: Minor Allele Frequency, values updated in December 2014. Table 4. Clinical data of patients with AEDs-resistant epilepsy (ADR) and patients with good response to AEDs (CTR) and relative frequencies from most relevant Alleles. TLE: Temporal lobe epilepsy, FRT: Frontal, R: Right, L: Left, PCS: Partial complex seizures, GEN: Generalized seizures, CTS: Clonic-tonic seizures: ABS: Absent seizures, ATS: Atonic seizures. VPA: Valproic acid, CBP: Carbamazepine, LEV: Leviracetam, TOP: Topiramato, LMGT: Lamotrigine, PHT: Phenytoin, CLB: Clobazan, PRIM: Primidone
Table 5. The relative frequencies are presented for the ten genotypes reported from CYP2D6, C19 and 3A4 database (Human Cytochrome P450 Allele Nomenclature), 7 controlled and 23 drug-resistant patients, were obtained by direct sequencing. ADR: drug-resistant. CTR: controlled
Tables Table 1. Drug Valproic acid Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/l Carbamazepine Dose (SD), /24h Concentration saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Lamotrigine Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Phenytoin Dose (SD), μmol/24h Concentration/saliva (SD), μmol/L Concentration/plasma (SD), μmol/L Levetiracetam Dose (SD), μmol/24h Concentration/saliva (SD), μmol/l Concentration/plasma (SD), μmol/l
CTR n Mean (SD)
ADR n Mean (SD)
5 791.66 (62.08) 5 2.3 (1.3) 3 82.1 (32.5 )
14 711.80(295.97) 13 0.69(0.069) 11 43.88 (15.97)
1 254.23 1 1.0 0 -
2 2 1
2 39.06(0.0) 1 0.351 0 1 587.2 -
354.33(55.51) 0.27(0.19) 2.32 -
4 2 3
49.20(37.30) 0.357(0.03) 9.85(14.88)
5 4
764.7(335.29) 1(1) -
Table 2 Primer
MgCl2 (mM)
T°h
Size (pb)
Gen Exon
Fw
1
5’-TTTATAAGGGAAGGGTCACGC-3’
5’-TTTCACCCACCATCCATGTT-3’
1.5
56
375
3
5’-AAGGTGGATGCACAAAGAGTG-3’
5’-AAGAGACCGTTGGGGCGAA-3’
1.5
57
264
5
5’-ACTTGTCCAGGTGAACGCAGA-3’
5’-ATTCCTCCTGGGACGCTCAA-3’
1.25
59
294
6
5’-AAAAGGTTGGACCAGTGCATC-3’
5’-TGTTGGAGGAGGTCAGGCTTA-3’
1.25
60
416
3
5’-TAGGTGTGCATGTGCCTGTTT-3’
5’-CCCCTGAAATGTTTCCAAGA-3’
1.5
57
490
7
5’-TGGCAGTTACACATTTGTGCA-3’
5’-TAGCCCCAAACTGGAAACAA-3’
1.5
58
500
CYP2C1 9
4
5’-TGTGTTGATTTTATGCATGCC-3’
5’-CTTTTCCAGATATTCACCCCA-3’
1.5
57
460
5
5’-TTACAACCAGAGCTTGGCAT-3’
5’-CCTTGACCTGTTAAACATCCG-3’
1.5
58
350
CYP3A4
5’ UTR
5’-AAGGGATGACATGCAGAGGC-3’
5’-TCACAAACCCTGTCATCAT-3’
1.5
63
804
6
5’-TAGGGCCAGCTGCATCACT-3’
5’-TCACAAACCCTGTCATCAT-3’
1.5
61
570
CYP2D6
CYP2C9
Rv
Table 3
PROTEIN ASSOCIATION Gene
SNP
Sample
Allele Frequency
Genotype Frequency
MAF
aa, change
Localization
ODDS RATIO Allele
Genotype Heterozogous Homozygou s
CYP2D6 CTR
C=0.571 T=0.429
C/C=0.428 C/T=0.286 T/T=0.286
rs1065852
T=0.238/1192 ADR
C=0.804 T=0.196
C/C=0.740 C/T=0.130 T/T=0.130
CTR
G=0.429 C=0.571
G/C=0.858 C/C=0.142
rs1058164
C=0.401/2008 136; G>C; Val>Val ADR
G=0.500 C=0.500
G/G=0.217 G/C=0.566 C/C=0.217
CTR
G=0.357 A=0.643
G/A=0.714 A/A=0.286
ADR
G=0.065 A=0.934
G/A=0.130 A/A=0.870
CTR
G=0.571 A=0.429
G/G=0.286 G/A=0.572 A/A=0.142
rs3892097
rs16947
A=0.093/466
ADR
G=0.565 A=0.435
G/G=0.261 G/A=0.609 A/A=0.130
CTR
G=1.000
G/G=1.000
ADR
G=0.935 A=0.065
G/G=0.870 G/A=0.130
CTR
T=0.786 C=0.214
T/T=0.572 T/C=0.428
rs9332120 ADR
T=0.891 C=0.109
T/T=0.783 T/C=0.217
CTR
G=1.000
G/G=1.000
PPGP region, NH2terminal
[C]<->[T] 0.324
[CC]<->[CT] 0.265
Alfa-Helix chain
[G]<->[C] 0.750
[GG]<->[GC] [GG]<->[C/C] 0.189 0.333
[G]<->[A] 7.963
[GG]<->[GA] 0.636
[GG]<->[AA] 8.200
[G]<->[A] 1.026
[GG]<->[GA] 1.167
[GG]<->[AA] 1.000
Intron between 3 and 4 exon
A=0.359/1799 296; G>A; Cys>Arg
rs28371725
CYP2C9
34; C>T; Ser>Pro
Alfa-Helix chain
[CC]<->[TT] 0.265
A=0.064/318
Intron between 6 and 7 exon
[G]<->[A] 2.333
[GG]<->[GA] 2.561
[GG]<->[AA] 0.366
C=0.144/722
Intron between 2 and 3 exon
[T]<->[C] 0.447
[TT]<->[TC] 0.243
[TT]<->[CC] 0.370
[G]<->[A] 3.843
[GG]<->[GA] 4.459
[GG]<->[AA] 0.405
CYP2C19
rs4244285
A=0.221/1109 227; G>A; Pro>Pro ADR
G=0.891 A=0.109
Alfa-Helix chain
G/G=0.783 G/A=0.217
CYP3A4 CTR
A=0.714 G=0.286
A/A=0.429 A/G=0.571
rs2740574 ADR
A=0.957 G=0.043
A/A=0.913 A/G=0.087
CTR
T=0.714 G=0.286
T/T=0.429 T/G=0.571
T=0.957 G=0.043
T/T=0.913 T/G=0.087
rs2687116 ADR
G=0.231/1156
5’ UTR region
[G]<->[A] 8.000
[GG]<->[GA] 0.556
[GG]<->[AA] 6.200
G=0.220/1103
Intron between 6 and 7 exon
[G]<->[T] 8.000
[GG]<->[GT] 0.556
[GG]<->[TT] 6.200
Table 4
Case
Age
Sex
P001 P002
15 8
M F
Main Focus TLE TLE-R
P003 P004
10 14
F M
TLE TLE
P005 P006
6 11
M F
TLE TLE
P007 P008 P009 P010 P011 P012
13 11 14 11 8 15
M M F M M F
TLE F- TEMP F- TEMP TLE TLE TLE
P013 P014 P015 P016 P017
3 12 13 9 11
F F F F M
FRT-R TLE-R TLE-R TLE-L TEM-B
P018 P019 P020
11 13 1
F F F
FRT-L FRT-R FRT-L
P021 P022 P023 C001 C002 C003 C004 C005 C006 C007
3 14 10 1 9 7 12 15 12 15
M M F M F F M F M M
TLE-R FRT- B TLE-L FRT-R TLE-R TLE-R TLE-R FRT-R TLE TLE
Seizures Type PCS PCS with GEN PCS CTS with GEN GEN CTS with GEN PCS PCS PCS PCS PCS, GEN CTS with GEN PCS,ABS PCS CTS,ABS AS, GEN PCS with GEN TCS T-GEN PCS with GEN PCS PCS TCS PCS PCS PCS ATS PCS PCS PCS
Drugs
CYP 3A4
ALLELE CYP2D6 CYP2C19
VP,CLO,LV
*1B *1B
*1/*2 *1
*1B *1B
*1B *1A/*1B
*4/*14 *1/*2
*1B *1A/*1B
*1B *1B
*1/*2 *14/*14
*1B *1B
*1B *1B *1B *1B *1A/*1B *1B
*1/*4 *1 *1/*2 *1/*2 *2/*14 *1/*2
*1B *1B *1A/*1B *1B *1B *1A/*1B
*1B *1B *1B *1B *1B
*1/*4 *1/*2 *1 *1 *4/*14
*1B *1B *1B *1B *1A/*1B
*1B *1B *1B
*1/*2 *1/*2 *1/*2
*1B *1B *1A/*1B
*1B *1B *1B *1A/*1B *1A/*1B *1B *1B *1A/*1B *1B *IB
*1/*2 *2 *1/*2 *4/*14 *1/*4 *4/*14 *1/*2 *2/*14 *1/*2 *1
*1B *1B *1B *1A/*1B *1B *1B *1B *1B *1B *1B
TOP CBZ, TOP CBZ VP, TOP CBZ, TOP LEV LEV, PHT CBZ, TOP VPA,CLB VPA,LEV,PRM LEV VPA, PTH, CLB LEV,VPA,CLON OX,LEV OX,TOP LEV TOP VPA, TOP VPA,TOP,OX VPA,CBZ VPA,TOP VPA,OX PHT VPA,LMT VPA TOP,OX TOP,LEV VPA,CBZ LEV
Table 5 Genotype, SNP, genotype frequency and enzyme activities in CYP2D6, CYP2C19 and CYP3A4 genes. Genotype Gene Genotype SNP Sample Activity Frequency CTR 0.142 *1/*1 Wild-Type Normal ADR 0.086 CTR 0.285 Wild-Type *1/*2 Decreased rs16947 ADR 0.521
CYP2D6
CTR
0.142
ADR CTR
0.086 0.285
ADR
0.043
rs16947 rs1065852
CTR
0.142
ADR
0.043
*4/*14
rs16947 rs1065852
CTR
0.285
ADR
0.086
*14/*14
rs16947 rs1065852
CTR
0.000
ADR
0.043
*1/*1
Wild-Type
CTR
0.739
Normal
*1/*2
Wild-Type rs4244285
ADR
0.217
Decreased
*1A/*1B
Wild-Type rs2740574
CTR
0.428
*1B/*1B
rs2740574
ADR CTR ADR
0.086 0.571 0.913
*1/*4
Wild-Type rs1065852
*2/*2
rs16947
*2/*14
CYP2C19
CYP3A4
Decreased Decreased Decreased Decreased Decreased
Decreased Decreased