Distribution of allelic and genotypic frequencies of NAT2 and CYP2E1 variants in Moroccan population.

BACKGROUND
Several pathogenesis and genetic factors influence predisposition to antituberculosis drug-induced hepatotoxicity (ATDH) especially for isoniazid (INH). However, the major susceptibility genes for ATDH are N-acetyltransferase 2 (NAT2) and cytochrome P450 2E1 (CYP2E1). NAT2 gene determines the individual's acetylator status (fast, intermediate or slow) to metabolize drugs and xenobiotics, while CYP2E1 c1/c1 genotype carriers had an increased risk of ATDH. Polymorphisms of the NAT2 and CYP2E1 genes vary remarkably among the populations of different ethnic origins. The aim of this study was to determine, for the first time, the frequency of slow acetylators in Moroccan population by genotyping of NAT2 gene variants and determining the genotype c1/c1 for CYP2E1 gene, in order to predict adverse effects of Tuberculosis treatment, particularly hepatotoxicity.


RESULTS
The frequencies of specific NAT2 alleles were 53%, 25%, 2% and 4% for NAT2*5, NAT2*6, NAT2*7 and NAT2*14 respectively among 163 Moroccan studied group. Genotyping of CYP2E1 gene, by real-time polymerase chain reaction using TaqMan probes, revealed frequencies of 98.5% for c1/c1 and 1.5% for c1/c2 among 130 Moroccan studied group.


CONCLUSION
The most prevalent genotypes of NAT2 gene in Moroccans are those which encode slow acetylation phenotype (72.39%), leading to a high risk of ATDH. Most Moroccans are homozygous for c1 allele of CYP2E1 gene which aggravates hepatotoxicity in slow acetylators. This genetic background should be taken into account in determining the minimum dose of INH needed to treat Moroccan TB patients, in order to decrease adverse effects.


Background
Pharmacogenetics refers to genetic differences in metabolic pathways which can affect individual responses to drugs, both in terms of therapeutic effect as well as adverse effects. Pharmacogenetics is generally regarded as the study or clinical testing of genetic variation that gives rise to differing responses to drugs. Its purpose is to optimize the therapeutic decisions based on the genome of the individual and the target molecule. Medicines are developed and used together with pharmacodiagnosis tools to achieve desired drug efficacy and safety.
Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis. TB remains to date one of the major public health problems in the world, with 8.6 million of incident cases, 12.0 million prevalent cases and 1.3 million deaths in 2012 [1]. In Morocco, 27,429 new cases of TB were reported in 2012, an incidence of 83 new cases per 100,000 inhabitants, according to the epidemiological services of the Moroccan Ministry of Health (Unpublished data).
The main drugs to treat TB are isoniazid (INH), rifampicin (RMP) and pyrazinamide (PZA), ethambutol (EMB) and/or streptomycin used in combination for 6 months or more [2]. Tuberculosis treatment cause adverse drug reactions (ADRs), including hepatitis, gastrointestinal intolerance, kidney failure, cutaneous and hematological reactions, which can lead to therapy discontinuation or more serious morbidity and mortality [3]. Among firstline anti-TB drugs, INH is the most effective but also the one that can easily cause hepatotoxicity.
The pharmacogenetic interest of these data lies in adjusting the dose of isoniazid based on genotype and phenotype found in the patient in order to prevent hepatotoxicity [21].
Polymorphisms of the NAT2 and CYP2E1 genes vary remarkably among the populations of different ethnic origins. Acetylators phenotypes in Moroccan population were previously reported to have a higher frequency of phenotypic slow acetylators [25].
The aim of this study was to determine the distribution of allelic and genotypic frequencies of NAT2 and CYP2E1 variants in Moroccan controls in order to estimate the prevalence of slow acetylators among Moroccans, who are facing the risk to develop hepatotoxicity after TB treatment.

Results
In this study we identified twelve different genotypes for NAT2 gene. The most of them encode for the slow acetylator phenotype. Genotypes of the four variants of NAT2 gene and their corresponding phenotypic profiles are presented in Table 1 Table 2.
The allelic and genotypic distribution of the rs2031920 variant of CYP2E1 in the studied group is reported in Table 3. The c2/c2 genotype was not found in the studied group. It seems that the majority of Moroccans are carriers of c1/c1 genotype with a frequency of 98.5% [95% CI 96-100]. CYP2E1 genotypic frequencies of Moroccans compared with different populations of the world are shown in Table 4.

Discussion
This results show that slow acetylators are the most frequent in our Moroccan population, and the NAT2*5 allele is the most represented. Comparing the allele frequencies of the four NAT2 variants reported in Table 2, the distribution pattern of NAT2*14 in Tunisian and NAT2*6, NAT2*7 in Caucasians populations did not vary significantly from our population (p > 0.05). On the other side, the allelic distribution in our population is different from other populations as Tunisians for the three alleles NAT2*5, NAT2*6 and NAT2*7 and from Caucasians for two alleles NAT2*5 and NAT2*14, this difference is statistically significant (p < 0.05) [26,32]. This difference with Tunisian neighbors could be explained by the origins of the Moroccan and Tunisian populations. Since about 8000 years ago native Berbers have been the major population group in all the North African regions, but through the centuries, Berbers have mixed differently with many other ethnic groups, Phoenicians, Carthaginians, Romans, Vandals, Byzantines, and Arabs whereas Ottoman rule reached Tunisia only [41,42]. Other explanation of the difference between our population and Tunisians could be a selection bias, and the limited size of their group controls.
Studies have shown variation in the distribution of NAT2 alleles among different populations, where four major groups could be distinguished according to the frequency of NAT2*5 and NAT2*6 alleles and according to the presence of NAT2*7 and NAT2*14 alleles. NAT2*5 allele is the most common among Caucasian, Egyptian and Omanian populations as in our population [26,29,31], while Asians such as South Korean and Japanese populations, have less NAT2*5 and more NAT2*7 [26,27,33]. NAT2*6 variant is at the second position in our population similarly to Caucasians [26]. NAT2*14 allele, at the third position in our population, is rare in Caucasians and absent in Omanian and Southern Korea populations [26,27,31].
Junichi Azuma et al. 2012 [21], proposed in their recent study about NAT2 genotypes and impact on doses of INH to increase the recommended dose of 5 mg/kg by the World Health Organization (WHO) to 7.5 mg/kg in rapid acetylators, maintain it in intermediate acetylators, and reduce it to 2.5 mg/kg in slow acetylators. As Moroccans are mainly slow acetylators, we propose according to our results to reduce the dose of INH in TB patients carrying slow acetylators genotypes, in order to prevent hepatotoxicity and to decrease the cost of managing adverse events.
For the polymorphism rs2031920 of the CYP2E1 gene, our studied group consisted of 130 controls, originated from different regions of Morocco. From our results, it appears that there is a high frequency of c1/c1 genotype in Moroccan population (Table 3), which aggravates hepatotoxicity in slow acetylators patients under TB treatment. In Table 4, we compared our results with  those of other reported populations. The frequencies of c1/c1, c1/c2 and c2/c2 genotypes in Moroccans were close to those found in Caucasians [35,37,38].

Conclusion
In conclusion, this preliminary study shows that more than 70% of Moroccan subjects are carriers of NAT2*5, NAT2*6, NAT2*7 and NAT2*14 genotypes compatible with a slow acetylators status, and therefore, they are sensitive to lower doses of TB treatment. We should take into account this high prevalence of slow acetylators in order to decrease adverse effects, especially knowing that a vast majority of Moroccans are also homozygous for the c1 allele of CYP2E1 gene, which aggravates hepatotoxicity.

Studied population
Blood samples were collected from umbilical cords of 163 unrelated newborns. They originated from different regions of Morocco and the Moroccan origin of their parents and grandparents was confirmed. Informed consent for DNA analysis was obtained from the parents. Ethics approval was obtained from the local committee of National institute of Hygiene in Rabat for this study.

Genotyping protocol
Genomic DNA was extracted from three mL of blood using the salting-out method [43]. The quality and quantity of the DNA were controlled by A260/A280 using a Nanodrop spectrophotometer ( Genotyping of SNPs of both genes was performed with an allele-specific probe of SNP using allele-specific realtime polymerase chain reaction (StepOne Real-Time PCR System; Applied Biosystems7500, Foster City, CA, USA) using TaqMan (Applied Biosystems, Warrington, UK) probes. This method combines PCR and mutation detection in a single step. A hybridization probe is cleaved by the 5' nuclease activity of Taq DNA polymerase only if the specific sequence is successfully amplified. Two TaqMan probes are used, one for each allele. TaqMan probes consist of a 18-22 bp oligonucleotide probe which is labeled with a reporter fluorophore at the 5' end and a quencher fluorophore at the 3' end. Allelic discrimination was obtained by the post-PCR read of fluorescence intensity.
For the four polymorphisms of NAT2 gene, the primers sequences were from Drug metabolism genotyping assay of the manufacturer's (Applied Biosystems) which reference is (p / n 4362038).
Genotype frequencies in our population were calculated in accordance with the Hardy-Weinberg equilibrium. Intervals confidence 95% were calculated for phenotypic genotypic and allelic frequencies.

Statistical analysis
Statistical analysis was performed using the SPSS (Statistical Package for the Social Sciences) version 17.0 for Table 4 Genotypic frequencies of rs2031920 polymorphism of CYP2E1 gene in different populations of the world Turkish [34] Germanians [35] Taiwanese [9] Serbians [36] French [37] English [38] Brazilians [28] Chinese [26] Indians [39] Spanish [40] Moroccans (This study) windows. The chi-square test was used to determine whether there is a significant difference between the expected frequencies and the observed frequencies relative to NAT2 gene distribution in the studied group versus other populations. Statistical significance was assumed at the p < 0.05.