Arylamine N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms' frequencies in Nigerian populations

Abstract

The study was carried out to investigate the distribution of arylamine N-acetyltransferase 2 (NAT2) allele frequencies associated with slow acetylation in healthy individuals from the three major Nigeria ethnic groups comprising of Hausa, Ibo and Yoruba. The single nucleotide polymorphisms (SNPs) in the NAT2 gene from three hundred unrelated subjects comprising, Hausa (N=98), Ibo (N=101) and Yoruba (N=101) who consented to the study were genotyped by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) techniques (481 C>T, 590 G>A) and DNA sequencing (191 G>A, 857 G>A). The allele frequencies of the investigated SNPs indicates that NAT2*4, wild-type (34%; 95% confidence interval (CI): 22-38%) is the most prevalent allele in Hausa, NAT2*6, G>A (29%; 95% CI: 22-37%) is the most common in Ibo while NAT2*5, 481 C>T (33%; 95% CI: 21-37%) is the most recorded in Yoruba populations. The most prevalent alleles in the three populations are the wild type, NAT2*4 and the defective alleles, NAT2*5 and NAT2*6. The frequencies of NAT*7, 857 G>A and NAT2 *14, 191 G>A are relatively low in these populations except for the 11% recorded for NAT2*14 in the Ibo population. Overall, the NAT2 defective alleles (NAT2*5, *6, *7 and *14) were found to be 66%, 72% and 71% in the Hausa, Ibo and Yoruba populations, respectively. The single nucleotide polymorphism frequencies of NAT2 gene showed a higher prevalence of the slow acetylator alleles in this study, which was in agreement with previous findings in some African populations.

Full text

African Journal of Pharmacy and Pharmacology Research Vol. 1(1) pp. 001-006, March 2011
Available online http://www.interesjournals.org/AJPPR
Copyright © 2011 International Research Journals
Full Length Research Paper
Arylamine N-acetyltransferase 2 (NAT2) single
nucleotide polymorphisms’ frequencies in Nigerian
populations
*Benjamin U. Ebeshi1, 2, Oluseye O. Bolaji1 and Collen M. Masimirembwa3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria
2Department of Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island,
Bayelsa State, Nigeria
3African Institute of Biomedical Science and Technology (AiBST), Harare, Zimbabwe
Accepted 28 February, 2011
The study was carried out to investigate the distribution of arylamine N-acetyltransferase 2 (NAT2)
allele frequencies associated with slow acetylation in healthy individuals from the three major Nigeria
ethnic groups comprising of Hausa, Ibo and Yoruba. The single nucleotide polymorphisms (SNPs) in
the NAT2 gene from three hundred unrelated subjects comprising, Hausa (N=98), Ibo (N=101) and
Yoruba (N=101) who consented to the study were genotyped by polymerase chain reaction/restriction
fragment length polymorphism (PCR/RFLP) techniques (481 C>T, 590 G>A) and DNA sequencing (191
G>A, 857 G>A). The allele frequencies of the investigated SNPs indicates that NAT2*4, wild-type (34%;
95% confidence interval (CI): 22-38%) is the most prevalent allele in Hausa, NAT2*6, G>A (29%; 95% CI:
22-37%) is the most common in Ibo while NAT2*5, 481 C>T (33%; 95% CI: 21-37%) is the most recorded
in Yoruba populations. The most prevalent alleles in the three populations are the wild type, NAT2*4
and the defective alleles, NAT2*5 and NAT2*6. The frequencies of NAT*7, 857 G>A and NAT2 *14, 191
G>A are relatively low in these populations except for the 11% recorded for NAT2*14 in the Ibo
population. Overall, the NAT2 defective alleles (NAT2*5, *6, *7 and *14) were found to be 66%, 72% and
71% in the Hausa, Ibo and Yoruba populations, respectively. The single nucleotide polymorphism
frequencies of NAT2 gene showed a higher prevalence of the slow acetylator alleles in this study, which
was in agreement with previous findings in some African populations.
Key words: Pharmacogenetics; N-acetyltransferase 2; Genotyping, Nigerian populations
INTRODUCTION
N-acetylation polymorphism is one of the earliest
discovered pharmacogenetic polymorphisms of the
phase II drug metabolism affecting acetylation of selected
substrates such as, sulfamethazine, dapsone, prizidilol,
isoniazid, hydralazine, food derived heterocyclic amines
and carcinogens such as 2-naphthylamine, benzidine and
2-aminoflourane (Dietz et al., 2000; Svensson and Hein,
2005). The polymorphism on the N-acetyltranferase 2
(NAT2) gene cluster arises from point mutations, which
can be divided into two broad groups: those mutations
*Corresponding author E-Mail: ben.beshi@gmail.com ; Tel.
+234-8059817538
which do not cause changes in the coded amino acids
(silent mutations) and those which result in amino acid
changes. Some of the silent mutations include the
following nucleotide changes: 111C>T, 282C>T, 481C>T
and 759C>T. The silent mutations might not be important
in pharmacogenetics but may be very important in
explaining evolutionary relationship of species or
geographically separated populations (Hein et al.,
2000a).
The point mutations, individually or in combination, give
rise to the different NAT2 allelic variants. Literature
reports show more than 13 point mutations in the NAT2
gene, giving rise to at least 53 allelic variants (Hein et al.,
2008). At least 9 of the 13 mutations of the NAT2 gene:
190C>T, 191G>A, 341T>C, 434A>C, 499G>A, 590G>A,

002 Afr. J. Pharm. Pharmacol. Res.
803A>G, 845A>C and 857G>A, result in substitution of
one amino acid with another while the others are silent
mutations-(Hein-et-al.,-2008;http://www.
louisville.edu/medschool/pharmacology/NAT.html). The
most important mutations in terms of NAT2 activity are
those occurring at positions 191, 341, 590 and 857,
which either alone or in combination with other mutations
result in NAT2 enzyme variants with altered activity,
stability or specificity (Hein et al., 2000b; Shishikura et al.,
2000; Hein et al., 2008). Different combinations of the
point mutations, give rise to different allelic variants e.g.
the two mutations, 282C>T and 191G>A are designated
as NAT2*14D while 282 C>T and 590G>A are
designated as NAT2*6A. Usually, when naming NAT2
alleles, the “most functionally significant” nucleotide
substitution is considered. Thus, NAT2 alleles possessing
191G>A are assigned to the NAT2*14 cluster, alleles
containing 341T>C are assigned to the NAT2*5 cluster,
alleles having 590G>A are assigned to the NAT2*6
cluster; the alleles with 857G>A are assigned to the
NAT2*7 cluster (Shishikura et al., 2000). Generally, while
NAT2*14 have been observed mainly among black
populations (Lin et al., 1993), the frequency of NAT2*7
are highest among the Asians compared to other ethnic
groups (Butcher et al., 2002; Hein et al., 2006). Frazier et
al. (2001) reported that individuals heterozygous for
NAT2*7 have been shown to have a higher risk of
developing colorectal cancer On the basis of genotypes,
individuals carrying at least one wild type allele are
classified as fast acetylators while those carrying two
mutant alleles are regarded as slow acetylators.
Reports in literature indicate a strong association
between acetylator phenotype and various drug-induced
disorders. Since heterocyclic amines cannot be N-
acetylated but can be activated by O-acetylation following
N-hydroxylation, humans with rapid acetylation
phenotypes may be more prone to carcinogenic influence
of heterocyclic amines (Deitz et al., 2000). Human
prostate epithelial cells express NATs and they are
capable of activating heterocyclic amines (Hein et al.,
2006). Also, NAT2 genotypes have been associated with
prostate cancer and in patients who have bladder cancer
(Rovito et al., 2005). In spite of the great ethnic variability
associated with NAT2 as well as its importance in
aromatic amines and carcinogens metabolism, its
polymorphisms have rarely been studied in Nigerian
populations, which informed this present study. The
objective of the present study therefore, was to perform a
genetic analysis of NAT2 gene using PCR/RFLP and
sequencing techniques in order to determine the
frequency of the null phenotypes in the Hausa, Ibo and
Yoruba ethnic populations of Nigeria.
MATERIALS AND METHODS
Subjects
Three hundred healthy, unrelated subjects consisting of 215 males
and 85 females, aged 18 to 45 years, who met the study inclusion
criteria, were randomly selected, from the three major Nigerian
ethnic groups of Hausa, Northern region (N=98), Ibo, Eastern
region (N=101) and Yoruba, Western region (N=101) of Nigeria.
Details of the study procedures were explained to the potential
subjects after which, they were given an opportunity to make an
independent decision to participate in the study. Eligible subjects
were enrolled after signing the c onsent form and were classified as
belonging to a particular ethnic group based on family history up to
two previous generations. The ethics committee of the Obafemi
Awolowo University Teaching Hospital, Ile-Ife, approved the study.
Genomic DNA preparation
Qualified personnel withdrew 5 ml of blood sample fr om each
participant using a syringe by veno-puncture into labelled EDTA
tubes. After collection, the blood s amples were fr ozen at –20oC until
further analysis. The DNA was prepared using the QIAamp DNA
Blood Mini kits (Qiagen, Belgium) in accordance with the
manufacturer’s protocols. The quality and the quantity of DNA was
determined using UV spectrophotometer (Shimadzu, Japan). DNA
samples were then stored at 4 oC prior to genotyping analysis and
aliquot of the sample was stored at –20 oC for long-term use.
Genotyping
Polymorphism on the NAT2 gene was investigated by checking for
mutations at positions 191, 341, 481, 590 and 857. The change 481
C>T and 590 G>A was diagnosed by PCR/RFLP due to loss of
enzyme restriction sites on NAT2 gene in accordance to the
methods of Abe et al. (1993) and Delomene et al. (1996).
The general PCR conditions were: an initial denaturation step for 4
minutes at 94oC, followed by 35 cycles of denaturation at 94 oC for
30-60 sec, a specific primer annealing temperature from 50-59 oC
for 30-60 sec, a specific extension temperature of 72 oC for 0.5 to 1
minute (details in Table 1). The last cycle was followed by a 7-
minute extension step at 72 oC. The PCR products were visualized
on 2% agarose gel electrophoresis by loading of the amplified
product on the gel while 10 µ L of the diluted DNA marker was
loaded to the gel for analysis. The gel was ran at 100 V, allowing
migration of 2.5-3 cm. The result of the gel was viewed using a Gel
Photo system GFS1000 (Fran Techtum Lab, Sweden).
DNA Sequencing
PCR products of 20 DNA samples from each of the three ethnic
groups were sequenced for 857 G>A, NAT2*7 and 191 G>A,
NAT2*14. The sequencing of PCR product was performed using an
ABI Prism® 3730 DNA analyzer with DNA sequencing analysis
softwareTM, version 3.6.1 (Applied Biosystems, Brussels, Belgium).
The s equencing reaction mixture consisted of a total volume of 12
µL, which is made up of 5 µL of purified PCR products added to a
strip tube containing 2 µL of Big DyeTM terminator version 3.0, 1 µL
of 5 x sequencing buffer, 1 µL of sequencing primer (2 µM) and 3
µL of double distilled water. The sequencing cycles consisted of
initial denaturation of DNA by incubating the reaction mixture at 96
oC f or 1 min, f ollowed by 25 cycles of denaturation at 96 oC for 10
sec, primer annealing at 50 oC for 5 sec and primer extension at 60
oC for 4 min.

Ebeshi et al. 003
Table 1. Primers sequence and PCR conditions for NAT2 genotyping and sequencing
Gene (allele) Primers sequence dbSNP PCR/Sequencing conditions Restriction
enzymes
Agarose gel
fragment pattern
NAT2*5 F 5’-TGACGGCAGGAATTACATTGT-3’
R 5’CCTTGTTTTATTTGGGAACACA-3’ rs1801280 35 cycles of 94
o
C at 30 sec, 55
o
C
at 30 sec, 72 oC at 60 sec Asp718 419 +139 bp (wt)
559 bp (mt)
NAT2*6 F 5’-TGA CGG CAG GAATTACATTGT-3’
R 5’CCTTGTTTTATTTGGGAACACA-3’ rs1799930 35 cycles of 94
o
C at 30 sec, 55
o
C
at 30 sec, 72 oC at 60 sec TaqI 19+142+169+227bp (wt)
396 bp (mt)
NAT2*7 F 5’-TGA CGG CAG GAATTACATTGT-3’
R 5’CCTTGTTTTATTTGGGAACACA-3’ rs1799931 25 cycles of 96
o
C at 10 sec, 50
o
C
at 5 sec, 60 oC at 4 min. BamHI 515+43 bp (wt)
559 bp (mt)
NAT2*14 F 5’-GAAGCATATTTTGAAAGAATTGG-3’
R 5’-GCATTTTAAGGATGGCCT-3’ rs1801279 25 cycles of 96
o
C at 10 sec, 50
o
C
at 5 sec, 60 oC at 4 min. NA NA
NA: not applicable
Identification of SNPs was carried out using the novoSNP
v2.1.9 software package (Weckx et al., 2005). Reference
sequence was NC_000008.9 for NAT2. The identified
SNPs were c ompared with the NCBI Single Nucleotide
Polymorphism database (dbSNP)
(http://www.ncbi.nlm.nih.gov/SNP). As SNPs can cause the
introduction of pre-microRNA (miRNA) sites, this was
included as part of the annotation in the novoSNP analysis
procedure. Frequencies of SNPs were calculated using
Genepop (Raymond and Rousset, 1995).
Statistical analysis
Allele and genotype frequencies were obtained by direct
counting and were tested for Hardy-Weinberg equilibrium
based on the chi-square (χ2) test of observed versus
predicted using the Graphpad Instat statistical software,
rejecting the null hypothesis if p<0.05.
RESULTS
The PCR product of 559bp fragment of the NAT2
gene after visualization on agarose gel
electrophoresis is shown in Figure 1. For single
nucleotide polymorphisms on the NAT2 gene to
be detected using RFLP after co-digestion of the
PCR product of 559bp fragment with the three
restriction enzymes namely, ASP718 for NAT2*5,
Taq1 for NAT2*6 and BamH1 for NAT2*7 based
on discriminatory pattern of wild-type sequence
are shown in Figures 2 and 3. The presence of a
228bp fragment is indicative of 481 C>T, NAT2*5
(loss of ASP718 restriction site). The appearance
of a 279 bp fragment indicates the presence of
590 G>A, NAT2*6 (loss of Taq 1 restriction site)
and the presence of a 142bp fragment identifies
857 G>A, NAT2*7 allele (loss of Bam H1
restriction site).
The allele frequencies of the NAT2
polymorphisms in the Hausa, Ibo and Yoruba
populations with varied acetylation capacity are
summarized in Table 2. The NAT2*4, wild-type
allele is differentiated by the absence of all
dysfunctional alleles is most prevalent in Hausa
(34%) compared to the Ibo and Yoruba with no
significant difference (P>0.05) in these
populations. Of the alleles associated with slow
acetylation, the most prevalent were NAT2*5
(33%; 95% CI: 21-37%) and NAT2*6 (33%; 95%
CI: 22-37%) in Yoruba and Hausa, respectively,
with no significant differences (P>0.05) in the
three populations. On the other hand, the NAT2*7
allele was the least prevalent in the studied
populations, however, the NAT2*14 allele
frequency showed a significant difference
(P<0.05) in the three populations studied being
highest in Ibo with frequency of 11% compared to
the 3% and 8% recorded in Hausa and Yoruba,
respectively. The distribution of the alleles
(P<0.05) was in Hardy-Weinberg equilibrium.
DISCUSSION
The five nucleotide transitions 191 G>A, 341
T>C, 481 C>T, 590 G>A, and 857 G>A that are
responsible for the NAT2 alleles were genotyped
in the three major Nigerian ethnic groups. A slow
genotype could be defined by a possible
combination of any of the five single nucleotide
polymorphisms (SNPs). The allele frequencies of
the investigated SNPs indicates that NAT2*4,
wild-type (34%) and NAT2*6, G>A (33%) were the
most prevalent alleles in Hausa while NAT2*5,
481 C>T (33%) was the most prevalent in Yoruba.
The most prevalent alleles in the three
populations were the wild type, NAT2*4 and the
defective alleles, NAT*5 and NAT*6. The
frequencies of NAT*7, 857 G>A and NAT2*14,

004 Afr. J. Pharm. Pharmacol. Res.
500bp
559bp
NGH1
NGH2
NGH3
NGH4
MW
MW
Figure 1. ypical results of Amplified 559bp fragment of NAT2 on a 2% agarose gel electrophoresis
for Subjects (NGH1, NGH2, NGH3 and NGH4)
MW: DNA marker
UC
BamH1
ASP718
Taq1
MW
MW
559 bp
419 bp
396 bp
147 bp
227 bp
559 bp
500 bp
Figure 2. Typical results of Digestion of 559bp fragment with restriction enzymes for various NAT2 alleles,
BamH1 (NAT2*7), ASP718 (NAT2*5) and Taq1 (NAT2*6) showing heterozygous mutation for NAT2*5 and NAT2*6
alleles and homozygous mutation for NAT2*7.
UC: undigested control, MW: DNA marker
NGH13
MW
MW
Undigested
contol
BamH1
ASP718
Taq1
500 bp
559 bp
169 bp
227 bp
Figure 3. Results of Digestion of 559bp fragment with restriction enzymes for various NAT2 alleles, BamH1
(NAT2*7), ASP718 (NAT2*5) and Taq1 (NAT2*6) showing homozygous mutation for NAT2*5 and NAT2*7 and
homozygous wild-type for NAT2*6 alleles

Ebeshi et al. 005
Table 2. Frequencies of N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms (SNPs) in Hausa, Ibo and
Yoruba populations of Nigeria
NAT2 Alleles Effect
Hausa
N/total (%)
Ibo
N/total (%)
Yo
ruba
N/total (%)
Combined
N/total (%)
NAT2*4
(wild-type) Normal 68/196 (34) 56/202 (28) 58/202 (29) 182/600 (30)
NAT2*5
(C > T) I114T 54/196 (27) 56/202 (28) 66/202 (33) 176/600 (29)
NAT2*6
(G > A) R197Q 66/196 (33) 58/202 (29) 54/202 (27) 178/600 (30)
NAT2*7
(G > A) G286E 2/40 (3) 2/40 (4) 2/40 (3) 6/120 (3)
NAT2*14
(G > A) R64Q 2/40 (3) 5/40 (11) 4/40 (8) 11/120 (7)
Table 3. NAT2 allele frequencies in the Nigerian populations in this study compared to the other populations in literature
Ethnic groups
NAT2
Allele frequencies (%)
NAT2*5 NAT2*6 NAT2*7 NAT2*14
Tanzanian 34 21 3 13
Shona 31 21 6 14
Venda 39 22 5 11
Kikuyu 58 24 - -
Luo 34 22 3 14
Maasai 42 27 4 9
San 20 8 - -
Orientals 5 25 13 0
Chinese 6 31 16 0
Japanese 2 19 10 0
Koreans 3 19 11 0
Caucasian 49 27 2 0
Swedes 51 28 2 0
Germans 46 27 4 0
Americans 45 28 2 0
African Americans 30 22 2 9
*Hausa 27 33 3 3
*Igbo 28 29 4 11
*Yoruba 33 27 3 8
*The ethnic groups in asterisk are results from this study, the non-asterisk are literature data compilations
191 G>A are relatively low in these populations except for
the 11% recorded for NAT2*14 in the Ibo population. The
NAT2 slow alleles were found among the Hausa, Ibo and
Yoruba at the frequencies of 66%, 72% and 71%,
respectively. Although these frequencies may not be
absolute in terms of NAT2 slow genotype as some of the
slow acetylation alleles especially, NAT2*5 and NAT2*6
occurs not mutually exclusive with the NAT2*4 that
accounts for fast acetylation phenotype. The frequencies
of the slow alleles in the three major Nigerian ethnic
groups are higher than frequencies reported in other
African populations such as, Tanzanians (49%), South
African Vendas (38%) and Zimbabweans (52%)
(Dandara et al., 2003) but comparable to the frequency of
56% reported in the Gabonese, a fellow West African
ethnic population. The overall prevalence of NAT2*5
(29%) in the Nigerian populations, was lower than the
value of 40% reported for Caucasians (Lin et al., 1994).
The frequency of the NAT2 slow alleles in the Nigerian
populations is higher to the frequency of 35-60% reported
in the Caucasians (Leff et al., 1999) and also following
literature data compilations as shown in Table 3. The
high frequencies of the NAT2 slow acetylation alleles in
this study is further justified by the reported incidence of
41% slow acetylator phenotype observed in the Nigerian
population (Eze and Obidoa, 1978).
It is a usual phenomenon that humans are constantly,
exposed to aromatic and heterocyclic amine carcinogens
through cigarette smoke (Rovito et al., 2005) and
consumption of well done meat (Hein et al., 2006).
Cigarette smoking and consumption of well done meat
have been shown to be risk factors for breast cancer in

006 Afr. J. Pharm. Pharmacol. Res.
some human epidemiological studies (Hein et al., 2000a).
Individuals who are slow acetylators are at higher risk of
drug side effects, such as isoniazid-induced peripheral
neuropathy, drug-induced lupus erythematosus and
sulphonamides-induced hypersensitivity among HIV
patients being treated of opportunistic infections
(Svensson and Hein, 2005; Hein et al., 2006). Slow
acetylators have also been associated with increased risk
of occupationally induced bladder cancer, and colon
cancer induced by smoking and mutagens that occur in
cooked meat (Hein et al., 2000b) and of developing
Parkinson’s disease (Bandmann et al, 1997). Peripheral
neuropathy triggered by isoniazid-induced over dosage
may be a major adverse effect in populations with high
frequencies of slow acetylators (Bell et al., 1993). The
findings of high frequency of the NAT2 variants, which
contribute to slow acetylator phenotypes in this study,
may predispose the Nigerian populations to adverse drug
effects when treated with isoniazid. Thus, the application
of NAT2 genotyping may be important in optimising the
treatment of the resurgent cases of tuberculosis in
HIV/AIDS patients in Nigeria in particular and indeed
Africa in general.
ACKNOWLEDGEMENTS
The authors acknowledge the cooperation of Staff of the
African Institute of Biomedical Science and Technology
(AiBST) Harare, Zimbabwe and also thank Alice Matimba
of the Department of Molecular Genetics, AiBST for her
contribution in the genetic data analysis.
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