Frequencies of CYP3A5*1/*3 Variantsin a Moroccan Population and Effect on Tacrolimus

Daily Dose Requirements in Renal Transplant Patients

Mustapha Elmachad,1,2,* Driss Elkabbaj,3,* Fatiha Elkerch,2 Fatima Zahra Laarabi,2

Amina Barkat,4 Zouhir Oualim,3 and Abdelaziz Sefiani1,2

The cytochromes P450 are a superfamily of oxidative enzymes, which are implicated in the metabolism of a largenumber of endogenous substances as well as exogenous chemicals. The cytochrome P450 (CYP3A5) appears toplay an important role in drug metabolism activity. The most frequent mutation in the CYP3A5 gene, affectingits activity, consists of a G6986A transition within intron 3. In this study, we determined the allelic frequency ofCYP3A5*3 in a Moroccan population, consisting of 108 individuals including 10 renal transplant patients. About8.33% (9/108) of the subjects were homozygous wild-type (CYP3A5*1/*1), 37.04% (40/108) were heterozygous(CYP3A5*1/*3), and 54.63% (59/108) were homozygous (CYP3A5*3/*3). Therefore, CYP3A5*3 variant was themost frequent allele detected at 73.15%. In the second part of this work, we assessed the influence of the CYP3A5polymorphism on tacrolimus doses required for 10 renal transplant patients who are receiving tacrolimus asimmunosuppressive therapy. Our results showed that, during the first 3 months after kidney transplantation,the tacrolimus daily requirements for heterozygous patients (CYP3A5*3/*1) were higher compared with ho-mozygous patients (CYP3A5*3/*3) (0.133 – 0.026 vs. 0.21 – 0.037 mg/kg/day). After the third month the differ-ence was also observed, whereby the mean of tacrolimus daily requirements for patients with CYP3A5*3/*3and CYP3A5*1/*3 was 0.053 – 0.013 and 0.08 – 0.014 mg/kg/day, respectively. This first study in Morocco pro-vides genetic data related to the frequency of genetic polymorphisms of CYP3A5 and opens the perspective todevelop other pharmacogenetic studies.

Introduction

The cytochromes P450 are a superfamily of oxidativeenzymes, which are implicated in the metabolism of a

large number of endogenous substances as well as exogenouschemicals (Thompson et al., 2004). The cytochromes P450s areinvolved in the oxidation of *40%–60% of commonly useddrugs. CYP3A4 and CYP3A5 are two major isoforms ofCYP3A subfamily expressed in adult human tissues. CYP3A4is found in the liver and small intestine, whereas CYP3A5 ispolymorphically expressed in the liver, small intestine, kid-ney, and other organs (Murray et al., 1999; Paine et al., 2006).These isoforms represent the predominant functional forms ofhuman CYP3A (Lin et al., 2002).

Many studies have been focused on the identification ofsequence variation affecting enzyme activity in the CYP3A5gene. The most frequent mutation in this gene consists of a

G6986A transition within intron 3 (CYP3A5*3). This mutationcreates an alternative splice site resulting in a frame shift andtruncation of the protein (Kuehl et al., 2001; Lamba et al., 2002).Individuals who are carrying two copies of a CYP3A5*3 alleleshow a markedly reduced level of CYP3A5 protein expressionand activity (Kuehl et al., 2001).

Many reports suggested that the CYP3A5*3 mutantallele may play an important role in interindividual dif-ferences of response to drug therapy (Ingelman-Sundberget al., 1999; Tsuchiya et al., 2004). The association betweentacrolimus, an immunosuppressive agent for preventingor treating graft rejection in organ transplantation, andCYP3A5 polymorphism was largely investigated in severalstudies. It has been demonstrated that renal transplantpatients who are CY3A5 expressors (CYP3A5*1) required ahigher dose of tacrolimus to achieve therapeutic troughconcentration than nonexpressors (CYP3A5*3) (Thervet

1Centre de Genomique Humaine, Faculte de Medecine et de Pharmacie, Universite Mohammed V Souissi, Rabat, Morocco.2Departement de Genetique Medicale, Institut National d’Hygiene, Rabat, Morocco.3Service de Nephrologie Dialyse et Transplantation Renale, Hopital Militaire d’instruction Mohammed V, Rabat, Morocco.4Centre National de Reference en Neonatologie et en Nutrition, Rabat, Morocco.*These authors contributed in the same way to the realization of this work.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 16, Number 6, 2012ª Mary Ann Liebert, Inc.Pp. 644–647DOI: 10.1089/gtmb.2011.0240

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et al., 2003; Haufroid et al., 2004; Macphee et al., 2005;Quteineh et al., 2008).

In this study, we aimed to determine the allelic frequency ofCYP3A5*3 in the Moroccan population. Moreover, we as-sessed the impact of this polymorphism on the daily doserequired for 10 renal transplant patients undergoing treat-ment with tacrolimus.

Materials and Methods

Subjects

In this study we have genotyped a group of 108 individu-als, including 98 healthy newborn infants and 10 renaltransplant patients (7 men and 3 women). Newborn infantsare from different regions of Morocco and the Moroccan ori-gin of their parents and grandparents was confirmed. All re-nal transplant patients received tacrolimus as their mainimmunosuppressant; the characteristics of patients are shownin Table 2. Informed consent for DNA analysis was obtainedfrom patients and parents of newborns.

DNA samples and polymerase chainreaction conditions

Genomic DNA was extracted from peripheral blood sam-ples, using a salt extraction method. The concentration ofgenomic DNA was determined by measuring the absorbanceby NanoDrop ND-2000 (Nanodrop Technologies). DNA wasstored at - 20�C before use. One hundred nanograms of ex-tracted DNA was amplified in a final volume of 50 mL. Thepolymerase chain reaction (PCR) mixture contained 10mL of5 · buffer (2 mM MgCl2), 0.1 mM each of the deoxynucleotidetriphosphates, 1.5 U of Go Taq (Promega), and 0.6 mM of eachprimer.

The forward primer was 5¢aaacatataaaacattatggagagtgg3¢and reverse primer was 5¢cacacccaggaagccagact3¢. The PCRconditions were 1 min denaturation at 95�C; 35 cycles of 15 s at95�C, 15 s at 59�C, and 30 s at 72�C; and a final extension stepat 72�C for 7 min. Two microliters of purified product wasamplified using a Big Dye kit (Applied Biosystems) as indi-cated in the manufacturer’s protocol in a final volume of13 mL. The PCR consisted of an initial denaturation step at96�C for 10 s, 25 cycles at 96�C for 10 s, 50�C for 5 s, and 60�Cfor 4 min. The final product was sequenced using an Abi-Prism 310 sequencer (Applied Biosystems) (Fig. 1).

Results

To determine the frequency of CYP3A5 polymorphism inthe Moroccan population we genotyped 98 healthy newborninfants in addition to 10 renal transplant patients: 8.33% (9/108) subjects were homozygous wild-type (CYP3A5*1/*1),37.04% (40/108) were heterozygous (CYP3A5*1/*3), and54.63% (59/108) were homozygous (CYP3A5*3/*3) (Table 1).Therefore, CYP3A5*3 variant was the most frequent alleledetected at 73.15% (Table 1).

In this study we have also explored the effect of theCYP3A5 genotype on the daily dose required for 10 renaltransplant patients. The characteristics and genotypes ofsubjects participating in this study are shown in Table 2.Among 10 renal transplant patients genotyped, 6 subjectswere homozygous for the CYP3A5*3 allele, while 4 subjectswere heterozygous. During the first 3 months after kidneytransplantation, the mean of required doses of tacrolimus forpatients carrying homozygous genotype was 0.133 – 0.026 mg/kg/day, while for patients with a heterozygous genotypethe mean was 0.21 – 0.037 mg/kg/day. After the third month,the mean of tacrolimus daily requirements for patientswith CYP3A5*3/*3 and CYP3A5*1/*3 was 0.053 – 0.013 and0.08 – 0.014 mg/kg/day, respectively.

In the study group, no acute rejection episodes wereobserved. However we reported, in patients with the twodifferent genotypes, some known adverse events due to ta-crolimus use (diabetes de novo and neuropathy) (Table 2).

FIG. 1. Direct sequencing results for the G6986A mutation in CYP3A5 gene: (A) profile of homozygous CYP3A5*3/*3genotype; (B) profile of homozygous CYP3A5*1/*3 genotype; (C) profile of homozygous CYP3A5*1/*1 genotype.

Table 1. CYP3A5 Allele Frequencies in Morocco

and Other Population

CYP3A5

Population n *1 *3 Reference

Moroccans 108 0.268 0.732 This studyChinese 34 0.245 0.755 Balram et al. (2003)Japanese 200 0.233 0.767 Fukuen et al. (2002)Caucasians 428 0.061 0.939 Dally et al. (2004)Spaniard 204 0.086 0.914 Sinues et al. (2007)Afro-Americans 34 0.721 0.279 Floyd et al. (2003)Ghanaian 200 0.85 0.15 Kudzi et al. (2010)

n, number of individuals.

CYP3A5*1/*3 VARIANTS IN MOROCCAN POPULATION 645

Discussion

The family of cytochrome P450 is one of the major targetsfor pharmacogenetic studies. This family metabolizes morethan 50% of drugs prescribed. It has been shown that certainmutations affecting genes coding for enzymes of this familyhave an influence on hepatic metabolism of many drugs in thebody (Cholerton et al., 1992; Nebert and Russell, 2002). Anexample that illustrates this relationship is the effect of theCYP3A5 gene on metabolism of tacrolimus, an immunosup-pressant used for prevention and treatment of transplant re-jection. Many studies have explored this relationship anddemonstrated that there is an effect of the CYP3A5 genotypeon daily doses required for renal transplant patients. Indeed,patients with the genotype CYP3A5*1/*1 have an increasedintestinal and liver metabolism, which will lead to a rapidelimination of tacrolimus from the body, and the daily doserequired to achieve adequate trough levels will be higher incontrast to patients carrying the CYP3A5*3/*3 genotype(Thervet et al., 2003; Haufroid et al., 2004; Macphee et al., 2005;Quteineh et al., 2008).

In our study, we analyzed the distribution of CYP3A5 allelefrequency in the Moroccan population. Then we comparedthese frequencies with those of other populations reported inthe literature. In the Moroccan population the CYP3A5*3allele was more frequent with a 73.2%, while the frequency ofthe CYP3A5*1 allele was 26.8%.

Previous studies published in other populations showedthat the CYP3A5*3 allele was the most prevalent in Caucasianpopulations with a frequency exceeding 90%, whereas inAfrican and African-American populations the CYP3A5*1allele was the most frequent (Floyd et al., 2003; Dally et al.,2004; Kudzi et al., 2010). Thompson et al. (2004) showed thatthe frequency of the CYP3A5*3 allele shows extreme varia-tions across human populations and is significantly correlatedwith distance from the equator. The results obtained in ourstudy are concordant with these observations. Indeed, wenote that the CYP3A5*3 allele in the Moroccan population hasa frequency intermediate between those of European popu-lations and African populations.

Several studies that evaluated the effect of CYP3A5 geneticpolymorphisms on tacrolimus dose requirement demon-strated that there is a significant difference between renaltransplant patients heterozygous or homozygous forCYP3A5*1 allele compared with CYP3A5*3 homozygotes(Thervet et al., 2003; Quteineh et al., 2008). Our results showedthat during the first 3 months of transplantation, individualscarrying CYP3A5*3/*3 need lower doses to achieve the bloodtacrolimus targeted trough level, than those carryingCYP3A5*1/*3. The same results were observed after the thirdmonth. Nevertheless, the limited number of patients does notallow statistically significant conclusions.

The possible presence of Moroccan patients homozygousfor CYP3A5*1/*1 (8.33%) should be considered by physiciansduring treatment after transplantation to achieve the desiredimmunosuppression, as this genotype is associated with a needfor significantly higher doses of tacrolimus and risk of devel-oping acute graft rejection episodes (Quteineh et al., 2008).

Conclusion

In conclusion, this is the first study performed in a popu-lation from Morocco, in which a distribution of the CYP3A5

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646 ELMACHAD ET AL.

variants was investigated in a group of newborn infants. Ourresults showed that frequency of the CYP3A5*1 variant seemsto be higher as compared with the Caucasians and lower thanis found in black Africans. Furthermore, comparison of dosesrequired for 10 renal transplant patients showed a differencebetween patients homozygous for CYP3A5*3 and those whowere heterozygous; this difference needs to be explored byincreasing the number of cases, even if many previous studiesshowed a statistically significant difference only betweenCY3A5*1/*1 and CY3A5*3/*3 genotypes.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Mustapha Elmachad, Ph.D.

Departement de Genetique MedicaleInstitut National d’Hygiene

27, Avenue Ibn BatoutaB.P. 11400 Rabat

Morocco

E-mail: elmachad@hotmail.fr

CYP3A5*1/*3 VARIANTS IN MOROCCAN POPULATION 647