CYP3A4*1G gene Polymorphism on Javanese People

Abtract Most of drugs are metabolized by cytochrome P 450 (CYP) enzyme. Cytochrome P450 3A4 is the cytochrome that is involved in metabolizing more than 60% of all medicine used in human. The variation of this CYP3A4 gene will affect the catalytic activity of this enzyme. Recently, CYP3A4*1G in intron 10 was found in Chinese and Japanese population. There is a substitution of G to A at position 82266 in intron 10. The purpose of this research was to investigate the frequency of allele and genotype CYP3A4*1G. Samples were taken from bloods of the subjects of the research. The examination of CYP3A4*1G was conducted by RTLP-PCR method.As the results of this research, the frequency of CYP3A4*1G in Javanese people is CYP3A4*1/*1 0.25, CYP3A4*1/*1G 0.55 and CYP3A4*1G/*1G 0.20. Frequency of allele G: 0.53, allele A: 0.47. The Fisher’s exacttest shows that the allele and genotype frequencyis p. 1.000. The allele and genotype frequency of Javanese people isstill in Hardy-Weinberg equilibrium.


Introduction
The response to drugs delivery is influenced by many factors.These factors included drug's factors and host factors.Host factors that affect clinical response are age, nutritional status, and physiologic organs including genetics factors.Genetic variation that currently focused on attention is genetic polymorphism in individual that leads to differences in treatment response.Polymorphism in individual can influence the pharmacokinetic profile of drugs, including absorption, distribution, metabolism and elimination.This happens because these polymorphism can cause level of drugs does not achieve the expected therapeutic levels (sub therapeutics), or even cause toxic therapeutics.Sub therapeutic levels can cause the treatment failure and toxic therapeutics level can lead to excessive drugs effect (Alvirevi et al, 2006) CYP3A4 enzyme is present in liver and gastrointestinal tract (Tomaszewski et al., 2008;Hsieh, et al., 2001) and inrenal dan prostatparencim (Tomaszewski et al., 2008).Human liver caontains the most abundant isoenzyme of CYP450 (Gao et al., 2008).It has 18 isoform numbers with a molecular weight of 57,1kD (Tomaszewski et al., 2008).CYP3A4 is involved in the metabolism of more than 50% of drugs in human (Van Schaik et al., 2000 ;Wang et al., 2005), among of these are alprazolam, amiodaron, amlodipin, amitriptilin, atorvastatin, deksametason, dektrometorpan, diazepam, digoksin, diltiasem, ketokonazole, ondansentron, terfenadin, progesterone, nateglinid and others (Tomaszewski et al., 2008).

Sutrisna et al.
Polymorphism can be found on the enzyme of drugs metabolism gene, drugs traport gene, and drugs target gene.Polymorphism that is most widely mapping is on drug metabolism gene.Most of drugs are metabolized by cytochrome P450 (CYP).
From the various CYP that has been mapped, CYP3A4 metabolizes more than 60% of medicines given to human.The research on the Malaysia's population showed that there were 2,1% CYP3A4*18 (Ruzilawati et al., 2007).Mapping of the CYP3A4*1G polymorphism has not been done in Indonesia and Malaysia.
The purpose of this research is to investigate the allele and genotype frequency of CYP3A4*1G on Javanese people.

Subjects
The Javanese people patients that took cure in Balai Kesehatan Paru Masyarakat (BKPM) Klaten and Balai Kesehatan Paru Masyarakat (BKPM) Yogyakarta.Inclusion criteria were age>14 years, Javanese ethnic (three generation and over) while exclusion criteria was family relationships (father, mother, grandmother, grandfather, children and grandchildren).

DNA isolation
DNA isolation was performed by using Wizard®genomic DNA purification kit Promega, which steps were done as refered to manufacture recommendation.Briefly, 100 µL of cell lysis solution was added in 300 µL of buffy coat samples were incubated 10 min.On room temperature, mix solutions were centrifuged 13.000 for 1 min.After supernatant was discarded, 300 uL of solution was added on the residue.The mixture was vortexed 10-15 sec.The result of the mixture was added by 1,5 mL of RNA solution, then was vortexed 10-15 sec.These mixtures were incubated on 37 0 C then stored in room temperature and added by 100 µL of protein presipitation solution.Mixture then was vortexed on 10-15 sec and centrifuged 13.000 for 3 min on 37 0 C. Three hundreds µL of supernatant was taken and put into 1,5 mL tube then added by 300 µL isopropanol.The solution was mixed by inversion until the white treads-like strands of DNA form a visible mass, then centrifuged on 13.000 for 1 min at 37 0 C. The supernatant was discarded and the residue was added by 300 uL of 70% ethanol.It was mixed a few time and centrifuged on 13.000 for 1 min on 37 0 C. The ethanol was removed after inversion.The residue was added by DNA rehydration solution (100 µL for 300 µL sample volume) and incubated at 65 0 C for 60 min.Periodically, the solution is mixed by gently tapping the tube.The samples were stored at 4 0 C.

Polymerase chain reaction CYP3A4*1G
A total of 12,5mL master mix , 6,5 mL dH2O, forward 5'-CAC CCT GAT GTC CAG CAG AAA CT-3' dan 2µL reverse 5'-AAT AGA AAG CAG ATG AAC CAG AGCC-3' and 2µL DNA (25µL total) were mixed and run by PCR.Amplification conditions as follows: 94 0 C for 7 min, the 35 cycles by 30 secs on 94 0 C, 1 min on 62 0 C, dan 1 minon 72 0 C with 5 min final extensionat 72 0 C.After PCR amplification, 8 µL of PCR products of 287 bp was digested by 10 U RsaI for 12 h at 37 0 C. The digested PCR products were analyzed by electrophoresis on 2% agarose gel and detected by ethidium bromide.The bands of DNA fragments were visualized by UV light (Gao et al., 2008)

Results
A total 60 subjects whom fulfilled the inclusion and exclusion criteria were used as test subjects.
The sample material was derived from blood buffy coat.The DNA purity was measured from ratio of absorbance at A260/280.The results of these samples were 1,7-2.DNA concentration was 50µg/ mL with dilution.DNA which had isolated then amplified by RFLP-PCR.Results can be seen in Figure 1.
The digested RFLP-PCR-products that analyzed by 2% agarose gel electrophoresis is shown on Figure2.
The genotype and allele frequency can be seen in Table 1 and 2. Table 2 showed that the sum of frequency of (G)+frequency (A)=1, and nG 2 + 2nGnA + nA 2 =1.The frequency of genotype and allele was not deviated from   Hardy-Weinberg equilibrium on p. 1,000.The comparison of this study with research in Chinese population can be seen in Table 3.

Discussion
The result of this study showed that the frequency of polymorphism of CYP3A4*1/*1 was 0,25, CYP3A4*1/*1G was 0,55 and CYP3A4*1G/*1G was 0,20.Frequency of allele G was 0,53, while the frequency of allele A was 0,47.By comparing to two studies on Chinese populations, it showed that the frequency of variant type/mutant type CYP3A4*1G/*1G of Javanese population is much higher than Chinese population.The result of the genetics findings is diferent from anthropological studies.Anthropological studies states that the island of Java had inhabited by Homo sapiens since 40,000 years ago.The earliest inhabitants were austromelanesid ethnic that since 10.000 years ago were mongolidizied.This process became more intensive during last 1000 years.The Javanese were formed since 2168 years ago (Glinka, 2006), so there is a relationship between Mongolian and Javanese ethnics.The frequency of mutant/ variant type in this research is relatively large (55% in heterozygotes and 20 % homozygous of CYP3A4*1G/*1G).
Based on this result, therapy of drugs which are substrates of CYP3A4 must be carried carefully.Several drugs had been known as inhibitors and inductors.The drugs known as inductors CYP3A4 such as antiepileptic drugs (classphenobarbital, phenytoin, carbamazepine, felbamate, lamotrigine, oxcarbazepine, primidone, rufinamid & topiramate), cyclophospamid, dexamethason, erythromisin, griseofulvin, l a n s o p r a z o l e , n e v i r a p i n , o m e p r a z o l , pioglitasone, prednisone, rifampin,troglitason.The drugs known as inhibitor of CYP3A4 are chlorampenicol, cimetidin, cyprofloxasin, flukonazole, itrakonazole, nevirapin, norfloxasin, varikonazole, mibefradile, estradioleand (Tomaszewski et al., 2008).Therapy of drugs which are substrates of CYP3A4 together with its inhibitor may increase drugs plasma level.This can cause adverse effect.Conversely, the therapy of drugs that are substrates of CYP3A4 together with its inductor may decrease drugs plasma level and can cause failure of the treatment.

Table 2 .
Frequency of genotype& allele on subject test

Table 1 .
Characteristic of subject test based on sex