|Year : 2020 | Volume
| Issue : 2 | Page : 92-97
CYP2C9 polymorphisms are associated with phenytoin toxicity in South-Indian epileptic patients
Rakesh Reddy Reddigari1, SV Naveen Prasad1, Vengamma Bhuma1, P VGK Sarma2, Appa Rao Anumolu1
1 Department of Neurology, SVIMS, Tirupati, Andhra Pradesh, India
2 Department of Biotechnology, SVIMS, Tirupati, Andhra Pradesh, India
|Date of Submission||09-May-2020|
|Date of Acceptance||30-May-2020|
|Date of Web Publication||18-Jul-2020|
Dr. S V Naveen Prasad
Department of Neurology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh - 517 507
Source of Support: None, Conflict of Interest: None
Background and Aims: The contribution of CYP2C9 in phenytoin metabolism is generally modest, but it increases with increasing serum phenytoin concentration. In the current study, we sought to study the association of CYP2C9*2 and CYP2C9*3 polymorphism with clinical and biochemical phenytoin toxicity in epileptic patients presenting to a tertiary care hospital in South-India.
Methods: In total, 50 patients on phenytoin therapy and clinically diagnosed with phenytoin toxicity in neurology outpatient department/casualty/intensive care unit and medicine wards were considered as cases. A total of 50 patients on phenytoin therapy without any evidence of toxicity were considered as controls. CYP2C9*2 (exon-3) and CYP2C9*3 (exon-7) gene polymorphisms were studied using the allele specific PCR method.
Results: Out of 100 patients, CYP2C9*2 polymorphism was seen in 8 (8%) patients in which 6 (12%) were cases and 2 (4%) were controls. CYP2C9*3 polymorphism was seen in 23 (23%) patients out of which 17 (34%) were cases and 6 (12%) were controls. Mean serum phenytoin level in cases was 24.23 ± 1.3 μg/ml, whereas in controls, it was 17.10 ± 0.6 μg/ml and the difference was statistically significant with P value <0.0001. Mean serum phenytoin level among patients with CYP2C9 polymorphism was 25.21 ± 4.72 μg/ml, whereas among patients without polymorphism, it was 17.51 ± 3.51 μg/ml (p < 0.0001).
Conclusion: Our findings conclude that the presence of CYP2C9*2 and CYP2C9*3 polymorphisms are associated with increased serum phenytoin levels and increased risk of clinical toxicity with phenytoin.
Keywords: CYP2C9, epilepsy, phenytoin toxicity, polymorphism, South-India
|How to cite this article:|
Reddigari RR, Naveen Prasad S V, Bhuma V, Sarma P V, Anumolu AR. CYP2C9 polymorphisms are associated with phenytoin toxicity in South-Indian epileptic patients. J NTR Univ Health Sci 2020;9:92-7
|How to cite this URL:|
Reddigari RR, Naveen Prasad S V, Bhuma V, Sarma P V, Anumolu AR. CYP2C9 polymorphisms are associated with phenytoin toxicity in South-Indian epileptic patients. J NTR Univ Health Sci [serial online] 2020 [cited 2020 Dec 5];9:92-7. Available from: https://www.jdrntruhs.org/text.asp?2020/9/2/92/289896
| Introduction|| |
Epilepsy is one of the most common neurological disorders worldwide and is the second most common and frequently encountered neurological condition. At the global level, it is estimated that nearly 70 million people suffer from epilepsy, in which nearly 12 million are expected to reside in India.,
Phenytoin is an anticonvulsant medication and is probably the most widely used antiepileptic drug., It is highly effective in generalized tonic clonic seizures and focal seizures. Phenytoin binds and inhibits voltage-dependent sodium channels, which are found on both neuronal and cardiac tissue. The increased membrane threshold for depolarization after sodium channel blockade lowers the susceptibility of neuronal tissue to epileptogenic stimuli.
Phenytoin metabolism varies depending upon its serum concentration. Hydroxylation by the cytochrome P450 (CYP) system is the critical step. Phenytoin metabolism follows first-order kinetics in the therapeutic range and mild overdoses. At higher concentrations, the cytochrome P450 enzyme system becomes saturated, leading to zero-order kinetics in which a fixed amount of phenytoin is eliminated over a given period. This results in a prolonged half-life (24 to 230 h in overdose) and contributes to increased serum concentrations.,
CYP2C9 is an important cytochrome P450 (CYP) enzyme with a major role in the oxidation of both xenobiotics and endogenous compounds. The contribution of CYP2C9 in phenytoin metabolism is generally modest, but it increases with increasing serum phenytoin concentration and in situ ations where the contribution of CYP2C9 is reduced due to the expression of less functional genetic variants or to the presence of metabolic inhibitors.
The CYP2C9 gene is highly polymorphic, and 60 different allelic variants have been described. The wild-type enzyme (CYP2C9*1) is characterized by the presence of isoleucine at position 359 and arginine at position 144. Previous studies have shown the association of CYP2C9*2 and CYP2C9*3 alleles with increased toxicity in patients on phenytoin therapy., In the current study, we sought to study the association of CYP2C9*2 and CYP2C9*3 polymorphism with clinical and biochemical phenytoin toxicity in epileptic patients presenting to a tertiary care hospital in South-India.
| Materials and Methods|| |
This prospective, case-control study was conducted in the Department of Neurology, Sri Venkateswara Institute of Medical Sciences between May 2017 and December 2018. This study was approved by the Institutional Ethics Committee of SVIMS, Tirupati (IEC No. 644; dated 13/06/2017). A written informed consent was obtained from all of the study participants.
A total of 50 patients on phenytoin therapy and clinically diagnosed with phenytoin toxicity in neurology outpatient department/casualty/intensive care unit and medicine wards were considered as cases. In all, 50 patients on phenytoin therapy without any evidence of toxicity were considered as controls.
- Patients on phenytoin therapy (4–8 mg/kg body weight) and diagnosed to have clinical features of phenytoin toxicity were considered as cases.
- Patients on phenytoin treatment for at least 2 months and not showing any signs of phenytoin toxicity were considered as controls.
- Patients willing to participate in the study.
- Patients with abnormal liver and renal function tests.
- Patients with pre-existing neurological disorder known to cause ataxia, nystagmus.
- Patients not willing to participate in the study.
Study procedures and assessments
A detailed history, which consists of presenting complaints, history of present illness, personal history, drug history, was recorded. All the participants were subjected to a detailed physical examination, neurological examination, and examination of other systems.
Serum phenytoin levels, serum electrolytes, renal function test, and liver function test were performed in all the cases. Serum phenytoin level was measured using the homogenous enzyme immunoassay method.
Procedure for Genetic analysis of CYP2C9*2 (exon 3) and CYP2C9*3 (exon 7) genes
Genomic DNA isolation
Genomic DNA was isolated from EDTA-treated blood samples of patients on the phenytoin treatment using QIAamp DNA Mini spin-column [Qiagen] DNA extraction kit. Extracted DNA samples were analyzed on 1% agarose gel electrophoresis.,,
The CYP2C9*2 (exon 3) and CYP2C9*3 (exon 7) gene sequences were retrieved from NCBI (ID: NM_000771.3). Allele specific oligonucleotide primers for the CYP2C9*2 (exon 3) and CYP2C9*3 (exon 7) genes were designed using Pimer-3 (v. 0.4.0) online tool and validated manually. The designed primers were synthesized from Sigma-Aldrich. The below mentioned allele specific CYP2C9*2 (exon 3; 258 bp) and CYP2C9*3 (exon 7; 125bp) primers were used for PCR amplification.
CYP2C9*2 (exon 3) allele specific primers
Forward primer: 5'-GCATTGAGGACTGTGTTC AAGAG-3' &
Reverse primer: 5'-AGTAATCAATGATAGGAG AAAAAT-3'
CYP2C9*3 (exon 7) allele specific primers
Forward primer: 5'-AGGAAGAGATTGAACGTG TGA-3' &
Reverse primer: 5'-GCTGGTGGGGAGAAGGTCA AGGTA-3'
The PCR reaction mixture consisted of 100 pmoles of each primer, 100 μmol of dNTPs mix, 10 mM Tris-HCl (pH: 8.8), 1.5 mM MgCl2, 1U of Hot start Taq DNA polymerase (Merck Biosciences Pvt Ltd), and 0.5 μg of DNA in a final volume of 50 μl. Amplification parameters included an initial denaturation step for 5 min at 94° C; 35 Cycles at 94°C for 40 s of denaturation, at 56°C for 40 s of annealing, and at 72°C for 60 s of amplification which was followed by a final extension step at 72°C for 5 min in a Master cycler gradient Thermo cycler (Eppendorf, Hamburg, Germany). The amplified PCR products were analyzed on 1.5% agarose gel electrophoresis. Samples having CYP2C9*2 and CYP2C9*3 polymorphisms showed fluorescent bands on 1.5% agarose gel as the amplification of the genes was done, while samples without these polymorphisms showed no such bands due to inability of amplification of the genes.
Data was captured on predesigned Microsoft excel spread sheets. All the categorical variables were expressed as frequencies with percentages. All the continuous variables were expressed as mean with standard deviation, and the differences between groups were tested with independent Student's t-test. The relationship between cytochrome P450 polymorphisms and phenytoin toxicity was assessed by Fischer's exact test as appropriate. All the statistical analysis was performed using SPSS v. 20.0 (IBM SPSS, Somers, NY, USA). A P value ≤0.05 was considered as statistically significant.
| Results|| |
Current study has enrolled 50 patients who were on phenytoin therapy and diagnosed to have phenytoin toxicity clinically as cases and 50 patients who were on phenytoin therapy without any evidence of phenytoin toxicity as controls. Demographic details are summarized in [Table 1]. The mean age of the study patients is 40.42 ± 13.32 years. Mean age of the cases and controls was 41.32 ± 13.77 and 39.52 ± 12.93 respectively (p = NS). Majority of both cases and controls were in third and fourth decade. Male: female ratio was 28:22 in cases and 29:21 in controls.
Electrophoretogram of isolated genomic DNA is shown in [Figure 1]a. PCR amplification of CYP2C9*2 (exon 3) and CYP2C9*3 (exon 7) genes revealed that 8 patients showed CYP2C9*2 mutation (C430T) resulting in Arg144Cys amino acid variation [Figure 1]b, while 23 patients showed CYP2C9*3 mutation (A1075C) resulting in Ile359Leu amino acid substitution [Figure 1]c.
|Figure 1: Electrophoretograms. (1a) 1% agarose gel electrophoretogram showing lane p1–p17 genomic DNA isolated from patients on phenytoin therapy. (1b) PCR amplification of CYP2C9*2 (exon 3) gene having CYP2C9*2 polymorphism. (1c) PCR amplification of CYP2C9*3 (exon 7) gene having CYP2C9*3 polymorphism|
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Comparison of CYP2C9*2 and CYP2C9*3 polymorphism in cases and controls
Out of 100 patients, CYP2C9*2 polymorphism was seen in 8 (8%) patients out of which 6 (12%) were cases and 2 (4%) were controls. CYP2C9*3 polymorphism was seen in 23 (23%) patients out of which 17 (34%) were cases and 6 (12%) were controls [Table 2].
|Table 2: Number of Patients with CYP2C9*2 and CYP2C9*3 Polymorphism in Cases and Controls|
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Adverse event profile of cases
Gingival hyperplasia was the most common side effect among cases, which was seen in 28 patients followed by ataxia seen in 21 patients, nystagmus in 14 patients, dizziness in 11 patients, megaloblastic anemia in 4 patients, dermatological side effect in 4 cases, and hirsutism in 3 patients. [Figure 2], [Figure 3], [Figure 4] depict adverse event profile in all cases, in CYP2C9*2 and CYP2C9*3 cases, respectively.
Comparison of serum phenytoin level between cases and controls
Mean serum phenytoin level in cases was 24.23 ± 1.3 μg/ml, whereas in controls it was 17.10 ± 0.6 μg/ml, and the difference was statistically significant with P value < 0.0001 [Table 3].
|Table 3: Comparison of Serum Phenytoin Level Between Cases and Controls.|
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We found statistically insignificant difference between patients with CYP2C9*2, CYP2C9*3 polymorphisms and patients without polymorphism in terms of mean age, age at onset of seizures, duration of seizures, duration of phenytoin use, and mean dose of phenytoin given.
Comparison of serum phenytoin level between patients with and without CYP2C9*2 and CYP2C9*3 polymorphism
Mean serum phenytoin level among patients with CYP2C9*2 and CYP2C9*3 polymorphisms was 25.21 ± 4.72 μg/ml, whereas among patients without polymorphism, it was 17.51 ± 3.51 μg/ml. Statistically significant difference (P < 0.0001) was seen with the serum phenytoin level between patients with and without CYP2C9*2 and CYP2C9*3 polymorphisms [Table 4].
|Table 4: Comparison of Serum Phenytoin Level Between Patients with and Without CYP2C9*2 and CYP2C9*3 Polymorphism.|
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| Discussion|| |
In this study, 50 epileptic patients who had clinical symptoms/signs of phenytoin toxicity were considered as cases and 50 epileptic patients without phenytoin toxicity were considered as controls. Mean age of the cases was 41.32 ± 13.77 and controls was 39.52 ± 12.93. Majority of both cases and controls were in third and fourth decade. Among 50 cases, 27 patients were on monotherapy with phenytoin, 12 were on dual antiepileptic drugs, and 11 patients were on ≥3 antiepileptic drugs. Among 50 controls, 37 patients were on monotherapy with phenytoin, 8 patients were on dual antiepileptic drugs, and 5 patients were on ≥3 antiepileptic drugs. Mean serum phenytoin level among cases was 24.23 ± 1.3 μg/ml, whereas among controls, it was 17.10 ± 0.6 μg/ml with P < 0.0001. CYP2C9*2 polymorphism was seen in 8 patients (8%), which include 6 cases (12%) and 2 controls (4%); P = 0.14. CYP2C9*3 polymorphism was seen in 23 patients (23%) out of which 17 were cases (34%) and 6 were controls (12%); P = 0.009. Mean serum phenytoin level among patients with CYP2C9*2 and CYP2C9*3 polymorphism was 25.21 ± 4.72 μg/ml, whereas among patients without polymorphism, it was 17.51 ± 3.51 μg/ml (P < 0.0001).
Phenytoin toxicity often occurs in patients who have predisposing factors for toxicity such as malnourishment, chronic renal failure, hepatic dysfunction and inhibition of phenytoin metabolism by other drugs. However, even in the absence of these predisposing factors, patients can develop phenytoin toxicity which leads to the speculation of genetic defects. Several pharmacogenetic studies have shown that genetic defects in the drug-metabolizing enzymes encoded by cytochrome P450 2C9 and 2C19 (CYP2C9 and CYP2C19, respectively). Phenytoin is predominantly metabolized by the polymorphic hepatic CYP2C9 which accounts for 90% of its metabolism. CYP2C9*2 and CYP2C9*3 are the two most common allelic variants that result in a significant reduction in the metabolism of various CYP2C9 substrates.
Our study had higher allelic frequency of CYP2C9*2 and CYP2C9*3 polymorphism in patients with epilepsy when compared to other studies.,,,, When patients with phenytoin toxicity (cases) are considered, Kesavanet al. had 7.8% of cases with CYP2C9*2 polymorphism while it is 12% in our study and frequency of CYP2C9*3 polymorphism was 23.3% in Kesavanet al. while it is 34% in our study. In few studies, the frequency of CYP2C9*2 polymorphism was not observed even in a single case,,, whereas it was observed in 9% and 4.5% in the studies done by Twardowschy et al. and Kesavan et al. The frequency of CYP2C9*3 polymorphism was higher (23%) in our study when compared with previous other studies that ranges from 7% to 13.6%.,,,,
Our study findings are correlating with the studies done by Kesavan et al. (22.29 ± 2.62 vs 9.21 ± 2.41) and Ozkaynakci et al. (27.95 ± 1.85 vs 14.25 ± 2.34) in which serum phenytoin levels were higher in patients with CYP2C9*2 and CYP2C9*3 polymorphisms when compared to patients without CYP2C9 polymorphisms.
Previous studies and case reports by Lee et al. and Kesavan et al. showed that cutaneous adverse reactions are more with CYP2C9*3 polymorphism. In our study, cutaneous adverse reactions are seen in 4 patients, and all these patients had CYP2C9*3 polymorphism. Ataxia and nystagmus are the most common clinical findings in patients with CYP2C9*2 and CYP2C9*3 polymorphisms similar to that of findings seen in study by Kesavan et al.
Present study has showed a significant association of CYP2C9*2 and CYP2C9*3 polymorphisms with phenytoin toxicity and the elevated serum phenytoin level. This is due to poor metabolism of phenytoin in these patients causing elevated serum phenytoin levels leading to toxicity. Similar results were reported by Kesavan et al. and Lee et al.
Though about 60 polymorphic alleles of CYP2C9 gene were reported, we have studied only 2 prevalent polymorphisms of CYP2C9 gene. Homozygous or heterozygous state of CYP2C9*2 and CYP2C9*3 has not been determined in this study. This study is single centric, and sample size is also relatively small. Hence, further multicenter studies with larger sample size are needed to validate/generalize our findings.
| Conclusions|| |
Our study findings conclude that the presence of CYP2C9*2 and CYP2C9*3 polymorphisms are associated with increased serum phenytoin levels and increased risk of clinical toxicity with phenytoin.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This work was supported by Sri Balaji Arogyavaraprasadini Scheme of SVIMS University, Tirupati [Grant No: SBAVP-RG/MD/46].
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ngugi AK, Bottomley C, Kleinschmidt I, Sander JW, Newton CR. Estimation of the burden of active and life-time epilepsy: A meta-analytic approach. Epilepsia 2010;51:883-90.
Sander JW. The epidemiology of epilepsy revisited. Curr Opin Neurol 2003;16:165-70.
Chaudhry AS, Urban TJ, Lamba JK, Birnbaum AK, Remmel RP, Subramanian M, et al
. CYP2C9*1B promoter polymorphisms, in linkage with CYP2C19*2, affect phenytoin autoinduction of clearance and maintenance dose. J Pharmacol Exp There 2010;332:599-611.
Fields MC, Labovitz DL, French JA. Hospital-onset seizures: An inpatient study. JAMA Neurol 2013;70:360-4.
Glauser T, Ben-Menachem E, Bourgeois B, Cnaan A, Guerreiro C, Kälviäinen R, et al
. Updated ILAE evidence review of antiepileptic drug efficacy and effectiveness as initial monotherapy for epileptic seizures and syndromes. Epilepsia 2013;54:551-63.
Giancarlo GM, Venkatakrishnan K, Granda BW, von Moltke LL, Greenblatt DJ. Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro
: Inhibition by sulfaphenazole, omeprazole, and ticlopidine. Eur J Clin Pharmacol 2001;57:31-6.
Holcomb R, Lynn R, Harvey B Jr, Sweetman BJ, Gerber N. Intoxication with 5,5-diphenylhydantoin (Dilantin): Clinical features, blood levels, urinary metabolites, and metabolic changes in a child. J Pediatr 1972;80:627-32.
Anderson GD. Pharmacogenetics and enzyme induction/inhibition properties of antiepileptic drugs. Neurology 2004;63:S3-8.
Ragueneau-Majlessi IBM, Levy RH. Phenytoin and other hydantoins. Interactions with other drugs. In: Levy RH, Mattson RH, Meldrum BS, Perucca E, editors. Antiepileptic Drugs. 5th
ed. Philadelphia: Lippincott Williams & Wilkins; 2002. p. 581-90.
Romkes M, Faletto MB, Blaisdell JA, Raucy JL, Goldstein JA. Cloning and expression of complementary DNAs for multiple members of the human cytochrome P450IIC subfamily. Biochemistry 1991;30:3247-55.
Hennessy S, Leonard CE, Freeman CP, Metlay JP, Chu X, Strom BL, et al
. CYP2C9, CYP2C19, and ABCB1 genotype and hospitalization for phenytoin toxicity. J Clin Pharmacol 2009;49:1483-7.
Kesavan R, Narayan SK, Adithan C. Influence of CYP2C9 and CYP2C19 genetic polymorphisms on phenytoin-induced neurological toxicity in Indian epileptic patients. Eur J Clin Pharmacol 2010;66:689-96.
Sambrook J, Russel WD. Molecular Cloning-A Laboratory Manual. 3rd
ed.. New York: Cold Spring Harbor Laboratory Press; 2001.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.
Latheef K, Rajasekhar D, Vanajakshamma V, Aparna BR, Chaudhury A, Sarma PVGK. Association of MTHFR, IL-6 and ICAM-1 gene polymorphisms with Coronary artery disease in South-Indian ethnic subset: A case-control study. J Cardiovas Dis Res 2018;9:115-22.
Hashimoto Y, Otsuki Y, Odani A, Takano M, Hattori H, Furusho K, et al
. Effect of CYP2C polymorphisms on the pharmacokinetics of phenytoin in Japanese patients with epilepsy. Biol Pharm Bull 1996;19:1103-5.
Odani A, Hashimoto Y, Otsuki Y, Uwai Y, Hattori H, Furusho K, et al
. Genetic polymorphism of the CYP2C subfamily and its effect on the pharmacokinetics of phenytoin in Japanese patients with epilepsy. Clin Pharmacol Ther 1997;62:287-92.
Soga Y, Nishimura F, Ohtsuka Y, Araki H, Iwamoto Y, Naruishi H, et al
. CYP2C polymorphisms, phenytoin metabolism and gingival overgrowth in epileptic subjects. Life Sci 2004;74:827-34.
Twardowschy CA, Werneck LC, Scola RH, De Paola L, Silvado CE. CYP2C9 polymorphism in patients with epilepsy: Genotypic frequency analyzes and phenytoin adverse reactions correlation. Arq Neuropsiquiatr 2011;69:153-8.
Ozkaynakci A, Gulcebi MI, Ergeç D, Ulucan K, Uzan M, Ozkara C, et al
. The effect of polymorphic metabolism enzymes on serum phenytoin level. Neurol Sci 2015;36:397-401.
Lee AY, Kim MJ, Chey WY, Choi J, Kim BG. Genetic polymorphism of cytochrome P450 2C9 in diphenylhydantoin-induced cutaneous adverse drug reactions. Eur J Clin Pharmacol 2004;60:155-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]