Ethnicity influences the distribution of cytochrome P450 3a4 gene polymorphism.

CYP3A4, a member of CYP3A family, exhibits an interindividual variation in the level of gene expression and enzyme production. It is hypothesized that such variations in the level of gene expression are caused by polymorphisms within the regulatory element of the gene. The variant most studied is at position -290 and involves an A to G transition. We have studied DNA samples from 206 African-American and 108 Caucasian patients and controls. DNA was tested in a PCR assay using sequence specific nucleotide primers and Taq polymerase. The amplification product was analyzed by 2% agarose gel electrophoresis. Genotypes were detected based on the presence or absence of the amplification product. CYP3A4 G genotype was present with a higher frequency in African-American individuals as compared with Caucasians (83% VS 3%, p < 0.0001, Relative Risk = 3.9). The homozygous AA allele was predominantly present in Caucasians (97%) but only 17% in African-Americans (p < 0.0001, Relative Risk = 2.5). In contrast, the homozygous GG allele was only detected in African-American group (14.6%). Consequently, the frequency distribution of heterozygous AG alleles were higher in African-Americans (68.4%) as compared with Caucasians (3%) (p < 0.0001; Relative Risk = 2.56). The frequency distribution of the AG heterozygous genotype was significantly lower in African-American CTx patients (30.4%) as compared with African-American RTx patients (68%) or African-American unrelated controls (82%) (p < 0.001, Relative Risk = 0.3; p < 0.0001, Relative Risk = 0.2 respectively). Interestingly, 52% of African-American patients, who had undergone cardiac transplantation (CTx) carried the GG allele as compared with 14% of the patients with renal transplantation (RTx) (p < 0.0002, Relative Risk = 4.3), and 3% of unrelated controls (p < 0.0001, Relative Risk = 6.0). Suggesting that possession of the CYP 3A4-G variant might influence the underlying clinical characteristics that have exposed these patients to an end stage heart or kidney failure. In summary, CYP3A4 genotype demonstrated remarkable interindividual variations between African-American and Caucasian populations, and a gene dose effect was present in African-American patients. Such polymorphism in CYP3A4 gene might play a role in disease susceptibility independent of the clinical course associated with variation in drug metabolic rates and immunosuppressant clearance in transplantation.

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Cytochrome P-450 (CYP-450) is a heme-containing enzyme and the isoform CYP3A, accounts for about 60% of the total CYP-450 family. The members of CYP 3A are mainly found in the liver, intestine and peripheral blood cells (Shimada et al., 1994; Cholerton et al., 1992). The CYP 3A plays an important role in bioactivation of environmental carcinogens, the metabolism of a variety of drugs and anticancer agents (Shimada et al., 1994; Shinderman et al., 2003; Koch et al., 2002). Four different CYP 3A isoforms: CYP 3A4, CYP 3A5, CYP 3A7, and CYP 3A43 have been identified (Gellner et al., 2001), which are located in tandem on chromosome 7. CYP 3A4 is the major enzyme, which metabolizes immunosuppressive drugs, such as Cyclosporine, Rifampin, and Tacrolimus (Neylan, 1998; Chenhsu et al., 2000; Pichard et al., 1992). In addition, many antibiotics and calcium channel blockers are metabolized by CYP3A4 (Watkins, 1992; Bertz and Granneman, 1997). The catabolic activity of the CYP 3A4 may vary up to 90-fold in different individuals (Kuehl, 2001). Because of individual's variability in drug uptake and clearance, this might have a great impact on the outcome of allograft survival after transplantation.

CYP 3A4 exhibits an interindividual variation in the level of gene expression (Wandel et al., 2000; Amirimani et al., 2003). It is hypothesized that such variations in the level of expression are caused by polymorphism within the regulatory region of the gene (Amirimani et al., 2003). The majority of variations are single nucleotide polymorphisms (SNPs) and might affect the level of enzyme production. However, the functional significance of such polymorphism is unknown (Amirimani et al., 2003; Rebbeck et al., 1998; Galant et al., 2001).

A transition of (A [right arrow] G) at position -290 from transcription site of CYP 3A4 gene, caused by a point mutation in the 5'-flanking region of the gene, has been reported (Rebbeck et al., 1998; Sata et al., 2000). It is speculated that the mutated genotype (CYP 3A4-G) might have a clinically significant role in pharmacogenetics of drug metabolisms because of its effect on the level of CYP 3A4 expression and activity (Amirimani et al., 2003; Ball et al., 1990). There have been reports demonstrating an association between CYP3A4 genotypes and disease conditions, including tumors of higher clinical stages and grades in prostate cancer (Rebbeck et al., 1998; Paris et al., 1999; Kittles et al., 2002), secondary leukemia (Felix et al., 1998), and breast carcinoma (Galant et al., 2001). However, some studies disagree with the association of this variant and the risk of breast cancer (Sprudle et al., 2002). Such discrepancies suggest that there might be population differences in the frequency distribution of the genotypes causing the clinical phenotypes. Indeed, the frequency of CYP 3A4-G variant varies substantially in different populations. It has been reported at a low frequency in Caucasians (2-9%), high in African-Americans (53%) and absent in Chinese (0%) (Wandel et al., 2000; Ball et al., 1990; Walker et al., 1998). In addition, a CYP 3A4-G variant has been found more frequently in Caucasians who had low levels of CYP 3A4 protein production as compared with Caucasians with higher levels of CYP 3A4 protein (Lamba et al., 2002). This indicates the significance of the CYP 3A4-G in regulation of the gene transcript, and furthermore, it might suggest variation in the level of expression in the population. Given the importance of functional activity of the CYP 3A4 in drug metabolism and clearance, the association with various disease states, particularly, differences in various population, assessment of the genotype variation in the population might have important consequences for patients such as allograft recipients who are subjected to multiple drug therapy. Our initial study demonstrated that African-Americans in Mississippi, predominantly carry the CYP3A4-G allele. Since the majority of transplant patients at the study center were African-Americans, this study was designed to investigate the frequency distribution of CYP 3A4 genotype variations in African-American individuals as compared with Caucasian patients and controls. The knowledge of the frequency distribution in the population will have a major impact in establishing the clinical relevance of CYP3A4 genotyping in disease susceptibility and for optimization of drug dose and therapy.

MATERIALS AND METHODS

Blood samples. Two hundred six African-American and 108 Caucasian individuals were studied. Of these, 65% were patients, who either had undergone cardiac or renal transplantation. One hundred eight unrelated controls from the same geographic area were studied. Informed consent was obtained as part of a protocol approved by the University of Mississippi Medical Center Review Board for obtaining blood samples from the participants.

Genomic DNA. DNA was isolated from whole blood using a modification of Blin and Staford method (Blin and Stafford, 1976), followed by phenol extraction and precipitation with 3 M sodium acetate and ethanol. DNA was stored in TE Buffer (10 mM Tris/2 mM EDTA; pH 8.0) at 4 [degrees]C until analysis of CYP 3A4 genotypes by polymerase chain reaction (PCR) method.

Polymorphisms. CYP3A4 genotypes were determined by amplification of a 186-bp CYP 3A4-G or a 187-bp -A DNA fragment. The primers were specific either for G or A polymorphism. A third set of primers were used to amplify a 440-bp generic fragment flanking 3' from the polymorphic site as a positive control, to confirm the integrity of the amplification pattern. The primer sequence and the location relevant to the CYP 3A4 gene are given in Table 1.

PCR. One hundred ng genomic DNA was amplified at initial cycle of 94 [degrees]C for 2 minutes followed by 25 cycles of 94 [degrees]C for 30 sec and 72 [degrees]C for 60 sec and an additional cycle of 72 [degrees]C for 2 minutes. The amplified DNA fragments were visualized by 2% agarose gel based on the presence or absence of the target DNA fragment (Figure 1).

Statistical analysis. The Frequency of the genotypes was compared using contingency 2 X 2 table of Fisher's exact test. A p values less than 0.05 was considered statistically significant. An INSTAT3 program was used to analyze the relation of CYP 3A4 genotypes based on a relative risk (RR) value obtained between patients and controls. The RR was calculated to categorize the study subjects into two groups of positive or negative for the possession of the CYP 3A4 genotypes.

[FIGURE 1 OMITTED]

RESULTS

The study group comprised 206 transplant patients of which 90 had undergone cardiac transplantation (CTx) and 116 had undergone renal transplantation (RTx). In addition, a total of 108 controls, 67 African-American and 41 Caucasians were included in this study. The control subjects had no history of disease and were from the same geographic region as the patients.

The frequency distribution of the CYP 3A4-A and -G genotypes in African-American and Caucasian groups is given in Table 2. The frequency was analyzed based on gender and ethnicity. The CYP 3A4-G variant was present in 3% of Caucasian individuals, whom were all male, but among African-American groups, it was present in both male and female (79.4% and 87% respectively) and the difference was statistically significant (Table 2). The CYP 3A4-A genotype was present in a higher frequency in Caucasians as compared with African-American (p < 0.0001, Relative Risk = 0.6) individuals. Overall, 90% of the study population carried the CYP 3A4-A genotype. In contrast, the CYP 3A4-G genotype was detected in only 3 out of 108 (2.8%) Caucasians as compared with 171 (83%) of African-Americans (p < 0.0001, Relative Risk = 3.9).

The CYP 3A4, AA homozygous allele was found in 35 (17%) African-American individuals as compared with 105 (97%) Caucasians (p < 0.0001; Relative Risk = 2.5). Whereas the homozygous GG allele was detected in 30 (14.6%) African-American group, but it was absent in Caucasians.

The frequency distribution of the CYP 3A4 was analyzed in patients and controls. We observed significant differences in CYP 3A4 genotype distribution among the two groups of patients with CTx or RTx. The CYP 3A4-A genotype was detected only in 11 (48%) of the African-American patients with CTx as compared with 100 (86%) of the African-American patients with RTx (p < 0.0002, Relative Risk = 0.3), and 65 (95%) of controls (p < 0.0002; Relative Risk = 0.2), shown in Table 3. On the other hand, the CYP 3A4-A genotype was present in 100% of both the Caucasian patients and controls. The frequency distribution of homozygous AA allele was 100% in Caucasian patients with CTx, and 93% of the Caucasian controls, but it was only 17.4% in African-American patients with CTx, 18% with RTx and 15% in African-American controls. As shown in Table 3, the data demonstrated a significant frequency difference across the ethnic population.

There was no CYP 3A4-G genotype, found in Caucasian CTx patients. However, 12 (52%) of the African-American patients with CTx, carried a homozygous GG allele as compared with 16 (14%) of the patients with RTx, and 2 (3%) of the controls (p < 0.0002, Relative Risk = 4.3; p < 0.0001, Relative Risk = 6.0 respectively). The frequency distribution of the AG heterozygous allele was lower in African-American CTx patients (30.4%) as compared with African-American RTx patients (68%) or African-American controls (82%), (p < 0.002, Relative Risk = 0.3; p < 0.0001, Relative Risk = 0.2 respectively). Only, 3 out of 41 (7.3%) Caucasian controls were heterozygous for the AG allele. A comparison of gender difference between African-American RTx and CTx and the presence of CYP 3A4 variants are shown in Figure 2. The frequency distribution of the CYP 3A4 homozygous G and A were inversely present in female and male patients with CTx or RTx. The GG alleles were a 1.5-fold increased in males with CTx as compared with females with CTx, but, it was a 1.5-fold decreased in male patients with RTx as compared with females. In contrast, the homozygous AA genotypes were 1.2-fold decreased in male patients with CTx as compared with females, but it was 2-fold increased in males with RTx. The differences were statistically significant where a comparison was made between the same gender and disease. As shown in Figure 2, the CYP 3A-GG was present in 40% of female with CTx as compared with 17.3% with RTx (p < 0.0005; Relative Risk = 1.7). Similarly, it was found in 61.5% of males with CTx as compared with 11% of males with RTx (p < 0.0002; Relative Risk = 6.6). While these variations must have some associations with clinical characteristics of disease, the small number of subjects and disease variations in these patients has limited the stratification of the genotypes and the underlying clinical characteristics in this study.

[FIGURE 2 OMITTED]

DISCUSSION

CYP3A4 genotype demonstrated remarkable interindividual variation between the African-American and Caucasian population as assessed by direct analysis of blood nucleated cells using single nucleotide polymorphism (SNP) detection. CYP 3A4 plays a major biological role in the metabolism of numerous compounds, including, steroid hormones, glucocorticoids, and carcinogens (Shinderman et al., 2003; Pichard et al., 1992; Zhao et al., 2002; Kobayashi et al., 2000). In addition, many antibiotics and immunosuppressive agents such as cyclosporine, and tacrolimus are metabolized by CYP 3A4 (Shimada et al., 1994; Neylan, 1998; Chenhsu et al., 2000; Hesselink et al., 2003). The study of CYP 3A4 genotype variation has at least a two-fold importance. First, the ethnic distribution of the CYP 3A4-G variant, 83% in African-American and 3% in Caucasian, may confer genetic susceptibility to diseases. Second, the CYP3A4 genotype variation might account for the differences in drug clearance between different individuals, which might affect the outcome of therapeutic management and intervention. In addition, the difference observed between African-American CTx and RTx patients in this study might have an association with clinical background of the individuals who experienced an end stage heart or kidney failure and whom were predisposed to the cardiac or kidney transplantation. Thus, this study was designed to analyze the frequency distribution of the genotypes in the population, and to investigate whether clinically different populations of patients, such as cardiac and kidney transplant recipients, might genetically have a common background for CYP 3A4 genotypes.

To our surprise, the frequency distribution of CYP3A4-G variant was 1.5-fold higher in the African-American population in our study than previously reported by others (Paris et al., 1999; Walker et al., 1998; Tayeb et al., 2000). This inconsistency between the observations might be due to a higher percentage of population admixtures present in other studies, given that the CYP 3A4-G variant is less common in Caucasians and a recent report (Zeigler-Johnson et al., 2002) have shown a high frequency distribution of the CYP 3A4-G variant among the African population. As shown in Table 3, number of subjects with the CYP 3A4-G genotype was almost rare in the Caucasian group as compared with African-American [3(3%) vs 171(83%), p < 0.0001, Relative Risk = 3.9]. Previous studies have shown an array of frequencies in different ethnic populations. The G variant of CYP 3A4 was nonexistent in Chinese and Asians, 6.5% in Caucasians, 8.9% in Saudis, 11% in Latinos, and 79.2% in Africans (Walker et al., 1998; Tayeb et al., 2000; Zeigler-Johnson et al., 2002). Including this study, the frequencies significantly vary (53%-83%) between African-Americans from different geographic regions (Rebbeck et al., 1998; Paris et al., 1999; Kittles et al., 2002; Walker et al., 1998). The variations in frequency distribution of the genotypes become significantly important where the prevalence of the disease varies considerably between populations. Such associations for CYP 3A4-G variant have been observed between African-American and Caucasian men with prostate cancer (Paris et al., 1999; Walker et al., 1998).

The CYP 3A4-A was a dominant genotype in Caucasian patients with CTx (100%) but not in African-American patients with CTx (48%), p < 0.0002, Relative Risk = 0.6 (Table 3). The CYP 3A4-A genotype difference between CTx patients was independent of the ethnicity, since the CYP3A4-A genotype was present with high frequency in African-American, both RTx (86%) and control (97%) groups. While the variable frequency of the CYP 3A4 homozygous GG in African-American CTx vs RTx patients requires further investigation, in general, this data have demonstrated a significant variation in the frequency distribution of the CYP 3A4 genotypes between African-American and Caucasian population, and in particular, between the African-Americans from other regions of the United States. The substantial variation in frequency distribution of homozygous CYP 3A4-G genotype in African-American patient population demonstrated here was intriguing with regards to the difference between CTx and RTx patients (Figure 2). In terms of clinical characteristics, they both suffer from a multiple complex diseases including vasculopathy, hypertension and autoimmunity, which these diseases are associated with a complex interaction between environmental and genetic factors. Thus, a proportional variation in disease association with CYP 3A4 genotypes as well as proportional differences in prevalence of the disease and gender effect might contribute to the outcome as seen in CTx and RTx patients in this study.

Two important issues relevant to the frequency distribution of homozygous GG allele between African-American CTx and RTx patients might be raised: (1) Clinical characteristics that exposed these patients to transplantation, (2) Prevalence of cancer such as prostate, breast and secondary leukemia among these patients. At present the underlying disease characteristics for these patients are under investigation. We know that cardiovascular disease (CVD) was in large, a major cause of the heart failure in these patients. Thus, considering the complexity of the nature of CVD, a small population size such as this has offered a limited sufficient interpretation. Our results, however, suggests that variation in frequency distribution of CYP 3A4 might have a great impact in stratification of clinical manifestation of disease between CTx and RTx. The relationship between functional expression of the CYP 3A4 genotype and the clinical response to a drug clearance may considerably be depended to the status of a disease. Such observations are complicated by several factors, including involvement of CYP 3A4 in gastrointestinal and hepatic activity of Cyclosporine metabolism and other drugs (Lindholm et al., 1988; Lown et al., 1994; Schuetz et al., 2000; Combalbert et al., 1989), resulting in a variable absorption in a variety of clinical settings. Studies to observe precise quantification levels of CYP 3A4 gene expression and protein production are in progress. Such studies require a sufficient follow-up and patient management and at present it is not the focus of this study.

Table 1. Sequence-Specific CYP 3A4 Amplification Primers.

Genotype                 5'-3' sequence             Location    Fragment
                                                                size
                                                                (bp)

(A) forward  ATG-AGG-ACA-GCC-ATA-GAG-ACA-AGG-GCA-A  -789, -810  187
    reverse  GAA-TCA-CAC-ACA-CAC-CAC-TCA-CTG-ACC-   -971, -946
             TC
(G) forward  TGA-GGA-CAG-CCA-TAG-AGA-CAA-GGG-TAG    -790, -813  186
(C) forward  AAC-AGG-CGT-GGA-AAC-CCA-AT             -535, -554  440

The sequence for reverse amplification primer was same for both alleles
and the control. A: forward primer for amplification of allele A; G:
forward primer for amplification of allele G and C: forward primer for
amplification of positive control.

Table 2. Frequency distribution of CYP 3A4 genotypes in African-
Americans and Caucasians.

                       AFAM*
Genotype    Male       Female     Total
            n = 107    n = 99     n = 206
            + (%)      + (%)      + (%)

A           92 (86)    84 (85)    176 (85.4) (a)
G           85 (79.4)  86 (87)    171 (83) (b)
AA          22 (20.6)  13 (13)     35 (17) (c)
AG          70 (65.4)  71 (72)    141 (68.4) (d)
GG          15 (14)    15 (15.2)   30 (14.6) (e)

                       CAU*
Genotype    Male       Female      Total
            n = 79     n = 29      n = 108
            + (%)      + (%)       + (%)

A           79 (100)   29 (100)    108 (100) (a)
G            3 (3.8)    0 (0)        3 (3) (b)
AA          76 (96)    29 (100)    105 (97) (c)
AG           3 (4)      0 (0)        3 (3) (d)
GG           0 (0)      0 (0)        0 (0) (e)

* AFAM stands for African-American and CAU stands for Caucasian
a: p < 0.0001, Relative Risk = 0.6
b: p < 0.0001, Relative Risk = 3.9
c: p < 0.0001, Relative Risk = 2.5
d: p < 0.0001, Relative Risk = 2.56
e: p < 0.0001, Relative Risk = NA

Table 3. CYP 3A4 genotype distribution in patients and controls in
African-American and Caucasian populations.

                                 AFAM*
Genotype    CTx            RTx           Control       PValue
            n = 23         n = 116       n = 67
            + (%)          + (%)         + (5)

A           11 (48) (a)    100 (86)      65 (95)        a
G           19 (83)         95 (82)      57 (85)       NS
AA           4 (17.4)       21 (18)      10 (15)       NS
AG           7 (30.4) (b)   79 (68)      55 (82) (c)   b, c
GG          12 (52) (d,e)   16 (14) (f)   2 (3)        d, e, f

                         CAU*
Genotype     CTx         Control         PValue
             n = 67      n = 41
             + (%)       + (%)

A            67 (100)    41 (100)        NS
G             0 (0)       3 (7.3)        NS
AA           67 (100)    38 (93)         NS
AG            0 (0)       3 (7.3)        NS
GG            0 (0)       0 (0)          NS

* AFAM stands for African-American and CAU stands for Caucasian
a: AFAM CTx was compared with AFAM controls, p < 0.0002; Relative Risk =
0.2
b: AFAM CTx was compared with AFAM RTx, p < 0.002; Relative Risk = 0.3
c: AFAM CTx was compared with AFAM controls, p < 0.0001; Relative Risk =
0.2
d: AFAM CTx was compared with AFAM RTx, p < 0.0002; Relative Risk = 4.3
e: AFAM CTx was compared with AFAM controls, p < 0.0001; Relative Risk =
6.0
f: AFAM RTx was compared with AFAM controls, p < 0.02; Relative Risk =
1.46

ACKNOWLEDGMENT

Many thanks to the patients and the clinical staff at the UMMC, for their support and cooperation. DTA acknowledges D. Olga McDaniel, Ph.D. (Mentor) for her laboratory support and contribution to the final development of the manuscript; Robin Rockhold, Ph.D., Director of the Base-Pair Program at UMMC, and Ms Cindy Cook, Coordinator of the Base-Pair Program at Murray High School, for their support. This study was supported by funds from Biomedical Research Grant #BR57125 and the Base-Pair Program which is sponsored by the Howard Hughes Medical Institute and the University of Mississippi Medical Center.

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David T. Arrington, Jr. (1,2), Brenda D. Mangilog (2), Lee Y. Tee (2), Sebron Harrison (2), Xinchun Zhou (2), and D. Olga McDaniel (2,3,4)

(1) Murrah High School (Base Pair Program), Jackson, MS 39202 and Departments of (2) Surgery and (3) Neurology, University of Mississippi Medical Center, Jackson, MS 39216

(4) Author for correspondence: Telephone: [phone], FAX: [phone], Email: [e-mail]