The NQO1 allelic frequency in hindu population of central India varies from that of other Asian populations.

CONTEXT: THE ENZYMES ENCODED BY THE POLYMORPHIC GENES NAD (P) H: quinone oxidoreductase 1 (NQO1) play an important role in the activation and inactivation of xenobiotics. This enzyme has been associated with xenobiotic related diseases, such as cancer, therapeutic failure and abnormal effects of drugs. AIM: The aim of the present study was to determine the allelic and genotypic frequencies of NQO Hinf I polymorphisms in a Hindu population of Central India. SETTINGS AND
DESIGN: Polymorphisms of NQO1 were determined in 311 unrelated Hindu individuals.
MATERIALS AND METHODS: Polymerase chain reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) analysis in peripheral blood DNA for NQO1 Hinf I polymorphism was used in 311 unrelated Hindu individuals. STATISTICAL ANALYSIS: Allele frequencies were calculated by direct counting. Hardy Weinberg Equilibrium was evaluated using a Chi-square goodness of fit test.
RESULTS: The observed allelic frequency was 81% for C (wild) and 19% for T (mutant) in the total sample.
CONCLUSIONS: The allelic frequency of "C" was higher than in other Asians (57%), but similar to Caucasians (81%). The genotype distributions for Hinf I polymorphisms were in Hardy-Weinberg equilibrium.

Introduction

Phase I enzyme DT-diphorase [NAD (P) H: quinone oxidoreductase 1 (NQO1)] converts toxic benzoquinone into hydroquinone in an obligate two-electron reduction reaction and is a key enzyme in polycyclic hydrocarbon metabolism in humans.[1] This reaction competes with one-electron reduction reactions by cytochrome P-450, producing the semiquinones, which generate free radicals and reactive oxygen species via redox cycling. The NQO1 is capable of maintaining these quinones in their reduced form, and therefore, detoxifies them.[23]

Two polymorphic variants of NQO1 have been identified: a C-to-T change at nucleotide 609 yields a serine substitution and a T-to-C change at nucleotide 464 results in tryptophan replacement of arginine. The C 609 T effectively inactivates the enzyme due to decreased catalytic activity and stability of NQO1 protein.[45] So, it is supposed that NQO1 has a crucial role in cancer susceptibility. Among Northern Europeans and Caucasian Americans, the gene frequency is about 79% for the wild-type allele and 21% for the mutated allele.[67] The frequency of the mutated allele is known to be slightly higher among African Americans and considerably higher among Hispanics and Asians.[8] NQO1 enzyme activity is found to be normal in individuals with wild-type alleles. It is variably reduced in individuals who are heterozygotes for the polymorphism.[9] The NQO1 protein and its activity are absent in those who are homozygous for the point mutation.[10] It is reported that inhibition of NQO1 activity by dicoumarol induces degradation of p53. This indicates that NQO1 plays a role in p53 stabilization, which has a crucial role in cell cycle regulation.[11] In various studies, NQO1 null alleles have shown to be associated with benzene related leukemia,[12] cancer of lung,[13] and colon.[14] A study suggested that NQO1 can modulate the susceptibility for breast cancer[15] Chang Gun Cho and coworkers concluded that NQO1 139Arg alleles were associated with tobacco dose-dependent increase in risk of head and neck squamous cell carcinoma (HNSCC), and NQO1 genotype polymorphisms may play an important role in the development of smoking-related HNSCC.[16] However, Hongwei Chen and coworkers have found no relation between NQO1 null allele and lung cancer.[17] But this does not render the significance of NQO1. It is also a highly inducible enzyme. Synthetic antioxidants such as butyrate hydroxyanisole and extracts of cruciferous vegetables, including broccoli, have been shown to be potent inducers of NQO1. This inducibility has led to the suggestion that NQO1 can play an important role in cancer chemoprevention and therapy.

Materials and Methods

Blood was drawn from the 311 normal, healthy, unrelated Hindu individuals [Table 1]. All the subjects were the regular residents of the Vindhyan region, the heart land of India. This name originated from Vindhya Range, which is a range of older rounded mountains and hills in the west-central Indian subcontinent, which separates the Indian subcontinent into northern India (the Indo-Gangetic plain) and southern India. The study was approved by the ethical council of APS University, Rewa. Blood samples were drawn by intravenous injection and 3–5 ml of blood was collected in ethylenediamine tetraaceticacid (EDTA) containing bowls and was stored at −20°C until use. Genomic DNA was extracted from whole blood by a slight modification of salting out procedure described by Miller and coworkers.[18] A C-to-T change at nucleotide 609 in NQO1 genes yields a serine substitution, which creates a Hinf I cleavage site. Primer was designed to amplify the 172 bp sequence to study this polymorphism. The oligonucleotide sequences (primers) used were those described by Jianhui Zhang et al.[19] For each DNA sample, 25 μl of polymerase chain reaction (PCR) mixture was prepared containing 5 μl template DNA (final concentration 100–200 ng/μl), 2.5 μl of 10× Taq polymerase buffer (10 mM Tris HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.005% Tween-20, 0.005% NP-40; final concentration 1×; Genetix Biotech Asia Pvt. Ltd., New Delhi, India), 0.5 μl of 10 mM dNTPs (Banglore Genei, Bangalore, India), 0.5 μl of 25 pmol/μl of forward and reverse primers specific for NQO1 genes, 0.5 μl of 5 U/μl of Taq DNA polymerase (final concentration 1 U; Genetix Biotech Asia Pvt. Ltd., India) and sterile water to make up the volume of reaction mixture to 25 μl. Thermal profile used for the amplification of desired segment of gene was as follows: initial denaturation at 95°C for 5 min and 30 cycles of denaturation at 94°C for 45 sec, annealing at 62.9°C for 45 sec and extension at 72°C for 1 min, followed by final extension at 72°C for 07 min. PCR products were separated on 2% agarose gel (2% w/v, Sigma, Signa Adrich, Bangalore, India) using a 100-bp molecular weight (MW) marker to confirm the PCR product size of 172 bp.

Table 1

Clinical features of study population

Clinical features	Males	Females
Total no.	136	175
Sex ratio (%)	43.73	56.27
Age	33.43 ±13.96
Mean ± SD	32.00
Age range	11–73

For restriction digestion of the C-to-T substitution at nucleotide 609 non-coding region, which creates an Hinf I restriction enzyme cleavage site, the reaction mixture included 0.2 μl of 10,000 U/ml Hinf I restriction enzyme (final concentration 2.5 U), 2.5 μl of 10× GENAI buffer c (final concentration 1×; 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM Di This Thretal (DTT), pH 7.9), 10.0 l of PCR product and 10 μl of sterile water. Reaction was incubated for 24 hours at 37°C for complete digestion. Ten microliters of digested PCR product was loaded on 2% agarose gel. Electrophoresis was done at 80 V in 1× Tris-borate EDTA buffer (89 mM Tris pH 7.6, 89 mM boric acid, 2 mM EDTA pH 8.0). A 100-bp gene DNA ladder (Genetix, India) was run concurrently as a molecular weight marker. The gel was than stained with ethidium bromide (10 mg/ml). The products were visualized using an ultraviolet transilluminator. The gel picture was captured using a digital camera and gel documentation software (Vilber Lourmate, Cedex I, France).The NQO1 wild-type allele shows a 172-bp PCR product resistant to enzyme digestion, whereas the null allele shows a 131-bp and 41-bp band in 2% agarose gel.

Results

In our investigation, the polymorphism of NQO1 genes was studied by PCR-RFLP. The C-to-T substitution at nucleotide 609 creates the restriction site for Hinf I. The PCR products of wild-type allele (CC) were 172 bp long and were undigestable by Hinf I at standard condition. Heterozygous condition (CT) appeared as 172, 131 and 41 bp long DNA fragments were produced after digestion with Hinf I. The null alleles (TT) were detected as the 131 and 41 bp long fragments were produced after digestion with Hinf I at standard condition. The genotype frequency of CC, TT and CT genotypes scored 70.59, 5.15 and 24.24%, respectively, in males. In females, is the percentages were CC = 66.86%, TT = 9.14% and CT = 24% [Table 2 Figure 1] The association of genotype frequency distribution in both the groups (Hindu males and Hindu females) was studied and it was observed that there were no statically significant differences as the χ2 and P values were 0.3768 and 0.5393, respectively. The distribution of C and T alleles in both the groups was C = 71.32% and T = 28.68% in male Hindus and C = 67.43% and T = 32.57% in females [Table 2 Figure 2]. The association between allelic distribution in both the groups was studied by Chi2(with Yates correction) test and values obtained were χ2 = 1.810 and P = 0.4046. All the values were nonsignificant, which showed that the frequency distribution of C and T alleles in both the groups was statically not Significant [Table 2].

Table 2

Distribution of NQO1 genotypes and allele frequencies and their association between study groups

Category	No.	Wild allele C	Mutant allele T	CC	TT	CT
Male	136	No. (%) 97 (71.32)	No. (%) 39 (28.68)	No. (%) 96 (70.59)	No. (%) 07 (5.15)	No. (%) 33 (24.26)
Female	175	118 (67.43)	57 (32.57)	117 (66.86)	16 (9.14)	42 (24)
Chi square		1.810 NS			0.3768 NS
P value (with Yates correction)		0.4046 NS			0.5393 NS

Figure 1

NQO1 Genotype distribution among Hindu Male and Female Population (In %)

Figure 2

NQO1 Allelic distribution in Hindu Male and Female Population (In %)

Discussion

NQO1 is a key enzyme in carcinogen metabolism and its allelic frequency varies by ethnicity. The variable frequency of NQO1 makes some populations more susceptible for getting cancerous disease due to the inability to detoxify the carcinogen by null allele. This fact led us to study the frequency of NQO1 in a population of Vindhyan region (Central India). Our study pointed at some important features of Hindu population, which were unknown until this study. Our results showed that the frequency of NQO1 in Hindu population was different from other Asian populations and slightly similar to Caucasian population.

The NQO1 Hinf I genotype frequency of our population was not statically different with that reported in Maharashtrian Indians where CC = 52.3%, CT = 42.48% and TT = 5.18%.[20] But an increase in heterozygosity was observed in Maharashtrian Indians than in our population. It could be due to random mixing of various ethnic groups in Maharashtrian population in the modern age. But increase in heterozygosity surprised us because the marriage between the close communities is common among the Maharashtrians.

Our results showed that the frequencies of different polymorphs of NQO1 genes are statically distinct in our population than in Caucasian and other Asian populations, although the differences are much evident between our population and other Asian populations [Table 3 Figure 3]. The frequencies observed in our population also did not match with the Caucasian, Chinese and Koreans, where the frequencies reported were CC = 79%, CT = 16% and TT = 05% in Caucasian,[22] CC = 34%, CT = 49.7% and TT = 16.3% in Chinese,[23] and CC = 94.5%, CT = 5.2% and TT = 0.3% in Koreans.[24] This showed that these populations are distinct from our study population. The Swedish population showed a slight difference with our population, where the frequencies reported were CC = 69.4%, CT = 28.9% and TT = 1.7%.[25] But the distribution of TT (mutant) allele was less in Swedish than in our population. The genotype frequencies of our population also matched with the population of different Iranian ethnic groups[26] on comparison, where the recorded frequencies were CC = 59.5%, CT = 31% and TT = 9.5% in Fars, CC = 64%, CT = 24% and TT = 12% in Mazzandarani and CC = 69%, CT = 28.6%, TT = 2.4% in Turks. But an increase in TT (mutant) genotype was observed in our population and in two Iranian ethnic groups (Fars and Mazzandarani) than in Turks and other world populations. The results are in agreement with the phenomena of common ancestry of Indian and Iranian population.

Table 3

NQO1 allelic frequency in Caucasian, Asian and Hindu male, female population (%)

Population	C	T	References
Hindu males	71.32	28.68	Present study
Hindu females	67.43	32.57	Present study
Caucasian	81.1	18.9	Kiyohara et al.[21]
Asian	57	43

Figure 3

NQO1 allelic distribution in Caucasian, Asian and Hindu Male, Female populations (In %)

The allelic distributions are found to be in accordance with other findings in Caucasian population (C = 81.1% and T = 18.9%),[27] as there were no significant differences observed. Significant differences were seen when our findings were compared with that of others in various Asian populations, where the frequency of C allele was reported to be 57% and that of the T allele was 43%, as calculated in a previous finding by Kiyohara et al.[21] This difference was also evident when our results were compared with the individual findings of Choi et al.[28] in Korean and Fowke et al.[29] in Chinese populations, where the frequencies were recorded as C = 58.5% and T = 41.5% in Korean and frequency of C allele was 59.3% and T allele was 40.7% in Chinese. These results did not surprise us because in calculation of NQO1 Hinf I allele, Kiyohara et al. did not include the frequencies of Indian population and their findings were solely based on the results from Korean, Chinese and Japanese populations, which are totally distinct from our population and belong to different races. This finding suggests that the Indian population cannot be correlated with the rest of the Asian population.