الفهرس 
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Abstract
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Summary and Conclusions
Epilepsies are among the most common neurological disorders worldwide and can have a major impact on a child’s development. When seizures are poorly controlled, they may be disabling and interfere with the child’s ability to learn. Diagnosis of epilepsy is essentially clinical, based on a clear history of epileptic seizures and confirmed by available medical history, seizure description, neurological examination, biochemical tests and electroencephalography (EEG).
Valproic acid (VPA) is widely used for treating children with epilepsy. Although VPA is affordable and effective in the control of multiple seizure types, large interindividual variability exists in its pharmacokinetics and pharmacodynamics. Such variability may reflect functional consequences of genetic polymorphisms in genes encoding drug metabolizing enzymes giving rise to variability in the metabolism and clinical response to VPA.
The hepatic biotransformation of VPA involves three major metabolic ways, including uridine diphosphate glucuronosyltransferases (UDPGT) mediated pathway which accounts for up to 50% of administered dose. UGT isozymes were found to be highly polymorphic, and some of the polymorphisms can lead to both transcriptional and functional changes of these enzymes. Some studies have identified several polymorphisms in the UGT1A6 gene, including 552A>C and 541A>G, and were found to be associated with the variability in glucuronidation of their substrates in the in vitro models. Further, genotype-phenotype association studies were conducted both in adult and pediatric patients, investigating the plausible influence of polymorphic genetic variants on VPA pharmacokinetics. However, results obtained were controversial, heterogeneous populations were included in these studies; moreover, no studies were validated in Egyptians or Caucasian ethnicity.
The present study aimed at evaluating comprehensively the effect of UGT1A6 polymorphism at 552A>C and 541A>G nucleotide sites on the clinical response to valproic acid in Egyptian children with epilepsy, regarding VPA serum concentration (Css), consumed dosage, seizure control, and adverse drug reactions (ADRs).
The study was a prospective, observational one conducted in 50 pediatric patients aging 2-18 years. The cases were recruited from Pediatric Neurology Clinic, Children’s Hospital, Ain Shams University, during the period from April 2010 till September 2011. All patients underwent thorough history taking, physical and neurological examination added to
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EEG study. Laboratory investigations included complete blood count, liver enzymes assay (including ALT) as well as renal function test (whenever indicated), done as baseline and repeated every 6 months during the course of therapy. All patients were diagnosed as idiopathic epilepsy and seizure classification was considered according to the guidelines of the International League Against Epilepsy (1989).
Patients who completed the study (48 patients) were divided into 2 groups comprising established group (n=30) and new onset epilepsy group (n=18) with an average duration of therapy of 3.52 ± 2.72, and 0.83 ± 0.36 years respectively. For each patient the following information was recorded: gender, weight (kg), age, consanguinity, seizure classification, triggering factors, VPA maintenance dose (mg/kg/day). Valproic acid was administered as monotherapy in the dosing range of 7.0 -39.6 mg/kg/day (mean 20.6 ±7.14 mg/kg/day). The maximum maintenance dose received averaged 22.2 ± 10 mg/kg/day (range, 7.7‒39.6 mg/kg/day).
For all patients, steady state trough level samples for VPA assay were taken after at least 1 month when a stable dose was given and after ensuring patient’s compliance. Venous blood samples (5 ml) were collected immediately before the morning dose and separated into two tubes, one of which was immediately stored at −70°C until used for DNA isolation and the other was centrifuged immediately to obtain serum then stored at − 20°C until used for drug analysis. Valproic acid serum levels were measured by homogenous enzyme immunoassay technique (Cobas®c 311, Roche diagnostics, USA).
Genomic DNA was extracted from peripheral blood lymphocytes using the blood DNA extraction kit according to the manufacturer’s recommendations (Qiagen, USA). The presence of UGT1A6 (541A>G and 552A>C) variants was identified by PCR restriction fragment length polymorphism (RFLP) using restriction enzymes NsiI for the 541A>G substitution and Fnu4HI for the 552A>C substitution. Digested samples were run on a 2% agarose gel and stained with ethidium bromide.
All patients were clinically monitored for seizure severity using Chalfont seizure severity scale and for seizure frequency. Besides, a checklist was used for recording ADRs.
The average age of seizure onset was 5.8 ± 4.1 years (range, 0.4‒16 years). Thirty five patients (72.9%) had generalized seizures (with generalized tonic clonic seizures amounting to 58.3%), compared to 13(27.1%) having focal seizures.
The genotypic distributions were all consistent with Hardy–Weinberg equilibrium proportions and genotype frequencies showed similar distribution at both UGT1A6 541 A>G and 552A>C polymorphic loci. They were 0.521, 0.333, 0.146 for wild-type, heterozygous
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variant and homozygous variant genotypes, respectively. Average Css was 59.96 ± 27.18 μg/ml (range, 20‒124 μg/ml).
For 552A>C polymorphism, the homozygous and heterozygous polymorphic variant group (AC & CC) had significantly lower CDRs of VPA than wild-genotype. It was also demonstrated that variant allele carriers (G and C allele) at both polymorphic sites had significantly lower CDRs than wild-type allele carriers. It may be inferred that patients with variant genotypes of 541A>G and 552A>C need lower doses of VPA to achieve the same target therapeutic levels than patients with wild-types, although, no significant association was detected in this study between the maximum consumed VPA dosage and different genotypic groups. Increasing the number of patients may have better demonstrated the functional consequences of genetic polymorphism contribution to the variability in dosage requirement which is recommend to be done in future studies.
In this work, it was also found that patients who have homozygous variant genotypes (GG) of 541A>G polymorphism, had significantly more cognitive side effects than wild-type (AA), while homozygous variant genotype carriers (CC) of 552A>C were less prone to fatigue as an ADR to VPA treatment than the wild-type. Although the current study failed to detect a significant association between seizure control as assessed by seizure frequency through the criteria set by the study and UGT1A6 541A>G and 552A>C genotypes, wild type allele carriers of 552A>C showed a significantly higher score on the Chalfont seizure severity scale than variant genotype carriers.
Conclusion
from the results of this work, it can be concluded that:
1. UGT1A6 polymorphisms at nucleotide positions 541A>G and 552A>C may be associated with increased VPA metabolism in Egyptian epileptic children.
2. Homozygous genetic variants were found to have significantly lower CDRs versus wild genotypes in both UGT1A6 541A>G and 552A>C polymorphisms.
3. A significant association exists between certain ADRs, namely, fatigue and cognitive adverse effects and certain genotypes of both UGT1A6 552A>C and 541 A>G polymorphisms.
4. A significant association exists between seizures severity score on Chalfont scale and certain genotypes of 552A>C polymorphism.
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Recommendations:
1. Genetic association studies of larger sample sizes are needed as well as independent replication of results, warranting collaboration between multiple centers which facilitates epilepsy pharmacogenetic studies.
2. Investigate the association of the combined effects of two genotypes, involving other polymorphic genes of drug metabolizing isozymes e.g CYP4B, CYP2C9, CYP2A6, UGT2B7, UGT1A3 and pharmacokinetic and/ pharmacodynamic aspects of VPA as the impact of single genetic variant is likely to be small due to the multifactorial influences on drug response.
3. As drug therapy experts, clinical pharmacists should actively engage in clinical pharamcogenetics service both in clinical practice environments as well as in conducting research.
4. Multiple-occasion drug analysis is recommended to avoid any possible daily fluctuations of drug level.
5. More accurate methods for DNA genotyping than RFLP method.