Polymorphisms of Alcohol metabolizing enzymes in indigenous mexican population 2010

Vol. 34, No. 1
January 2010
Alcoholism: Clinical and Experimental Research
Polymorphisms of Alcohol Metabolizing Enzymes in
Indigenous Mexican Population: Unusual High Frequency
of CYP2E1*c2 Allele
Elizabeth Gordillo-Bastidas, Arturo Panduro, Daniela Gordillo-Bastidas, Eloy A. ZepedaCarrillo, Jesús J. Garcı́a-Bañuelos, José F. Muñoz-Valle, and Blanca E. Bastidas-Ramı́rez
Background: Alcohol abuse represents the major identified etiological factor of cirrhosis in
México. ADH1B, ALDH2, and CYP2E1 have been considered candidate genes in alcohol-related
diseases. Controversial results probably due to ethnic differences, among other factors, have been
reported. Mexican Mestizos (MES) derive from the combination of indigenous, Spaniard, and
African genes. Huichols (HUI) constitute an indigenous group from western Mexico with no
racial admixture. We determined ADH1B*2, ALDH2*2, and CYP2E1*c2 allele frequencies in
healthy HUI and MES from western Mexico. Lipid and hepatic profile were also carried out.
Methods: One hundred and one HUI and 331 MES subjects were studied. Genotype and allele
frequency were assessed through polymerase chain reaction–restriction fragment length polymorphism after DNA isolation from peripheral leukocytes. Commercial kits for lipid and hepatic
determinations were used.
Results: Polymorphic allele distribution in HUI was: 0% ADH1B*2, 0.5% ALDH2*2, 51.5%
CYP2E1*c2; in MES: 3.4% ADH1B*2, 0% ALDH2*2, 16.1% CYP2E1*c2. Frequency of
ADH1B*2 was statistically (p < 0.001) lower in HUI than MES. CYP2E1*c2 polymorphic allele
was significantly higher (p < 0.0001) in HUI than MES. Hepatic profile was normal in both
groups. HUI showed a better lipid profile than MES independently of genotype.
Conclusions: Huichols exhibited the highest CYP2E1*c2 allele frequency of the world documented up to this date; meanwhile, ADH1B*2 and ALDH2*2 were practically absent. This feature could be useful in the understanding of Mexican population gene composition, alcohol
metabolism, and alcoholic liver disease development. However, further association studies are
necessary. The heterogeneity of Mexican population was evidenced by the significantly different
distribution of CYP2E1*c2 allele observed among different regions of the country. Lipid and
hepatic values were not associated to genotype. This report constitutes the first study dealing with
gene polymorphisms of alcohol metabolizing enzymes conducted in HUI.
Key Words: Alcoholic Liver Disease, Alcoholism, Pharmacogenetics, Ethnicity, Racial
Admixture, Amerindian, Amplification Protocol.
A
LCOHOL ABUSE CONSTITUTES the major identified etiological factor of cirrhosis in México (MéndezSánchez et al., 2004). Alcohol dehydrogenase (ADH1B) and
aldehyde dehydrogenase (ALDH2) are the main enzymes
responsible for ethanol oxidation into acetaldehyde and ace-
From Instituto de Enfermedades Crónico-Degenerativas, Centro
Universitario de Ciencias de la Salud, Universidad de Guadalajara
(EGB, DGB, BEBR); Servicio de Biologı´a Molecular Hospital Civil
Fray Antonio Alcalde (AP); Instituto de Biologı´a Molecular en Medicina y Terapia Génica, Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara (JJGB); Instituto de Investigación en
Reumatologı´a y del Sistema Músculo Esquele´tico, Departamento de
Biologı´a Molecular y Genómica, Centro Universitario de Ciencias de
la Salud, Universidad de Guadalajara (JFMV); Universidad Autónoma de Nayarit, Hospital Civil Tepic Dr. Antonio González Guevara
(EAZC), Tepic, Nayarit, Me´xico.
Received for publication January 20, 2009; accepted September 2, 2009.
Reprint requests: Blanca E. Bastidas-Ramirez, Banderilleros # 8,
Col. Haciendas Tepeyac, C.P. 45053, Zapopan, Jal., Mexico; Fax:
(52)-(33)-1058-5200 ext. 3893; E-mail: [email protected]
Copyright 2009 by the Research Society on Alcoholism.
DOI: 10.1111/j.1530-0277.2009.01075.x
Alcohol Clin Exp Res, Vol 34, No 1, 2010: pp 1–8
tate, respectively. Cytochrome P4502E1 (CYP2E1) participates in alcohol oxidation to acetaldehyde as well, but in an
inducible manner, as it represents the major microsomal component of the ethanol oxidizing system (Zakhari, 2006).
CYP2E1 also catalyzes the activation of various hydrophobic
low molecular weight xenobiotics (acetone, benzene, phenol,
acetaminophen, inhalatory anesthetics) and food additives
(nitrosamines) (Xinxin and Kaminsky, 2003; Zhang et al.,
2006). Some genetic polymorphisms that influence the expression of these enzymes have been identified.
ADH1B polymorphism consisting in a point mutation in
exon 3, which results in the substitution of arginine by histidine in aminoacid 47 has been widely studied. The polymorphic allele ADH1B*2 originates a restriction site for MaeIII
and confers about 100-fold more catalytic activity to ADH1B
than the enzyme coded by the wild-type allele (Yoshida et al.,
1981).
A transition of G to A in exon 12 of the wild-type allele
ALDH2*1 at nucleotide 1510, giving rise to the change of glutamic acid by lysine at position 487, has been reported.
ALDH2*2 mutant allele is characterized by the loss of a
1
2
GORDILLO-BASTIDAS ET AL.
Ksp632I restriction site and a resulting enzyme with little or
no catalytic activity (Crabb et al., 1989; Hsu et al., 1985).
CYP2E1 polymorphism at the 5¢ flanking regulatory region
consisting in the substitution of C by T at position )1019, leads
to the lack of an RsaI restriction site. Compared with the most
common allele (c1), polymorphic allele (c2) is associated with a
10-fold higher transcriptional activity, elevated protein level,
and increased enzymatic activity (Hayashi et al., 1991; Tsutsumi et al., 1994; Ueno et al., 1996; Ueshima et al., 1996).
Adverse reactions due to acetaldehyde accumulation in
the oxidative pathway of ethanol constitute the phenotypical characteristic conferred by ADH1B*2, ALDH2*2, and
CYP2E1*c2 polymorphic alleles (Harada et al., 1982;
Higuchi, 1994). These gene polymorphisms, lipid alteration,
and oxidative stress represent some genetic and environmental
factors that have been associated to liver impairment (Bataller
and Brenner, 2005; Bataller et al., 2003; Hernández-Nazará
et al., 2008; LeCouteur et al., 2007; Matsuzawa et al., 2007).
On the other hand, ADH1B*2 and ALDH2*2 alleles have been
associated with a lesser consumption of alcohol and called ‘‘protector alleles’’ by some authors. However, controversial results
are reported (Bosron and Li, 1986; Vidal et al., 2004; Zintzaras
et al., 2006) probably because heterogeneity and racial admixture is playing an important role, among other factors.
Mexican population has been arisen by the admixture of
indigenous, Spaniard, and African genes (Jimenez-Sanchez
et al., 2008). Therefore, elucidation of the genomic structure
of ethnic groups constitute significant contributions to this
topic. Some reports exist in populations of Mexican origin
regarding these polymorphisms, but studies in indigenous
Mexican groups are very scarce, maybe because the access to
them is usually very difficult.
México is one of the 20 most populous countries accounting for 1.6% of the world population with approximately 103
million people (INEGI, 2006), including 62 different identified
ethnic groups comprising approximately 12 million people.
The heterogeneity of Mexican population may influence
genotype distribution from region to region. Huichols (HUI)
constitute an indigenous group of western central Mexico, living in the Sierra Madre Occidental in the states of Nayarit,
Jalisco, Zacatecas, and Durango (CDI, 2008).
The goal of this study was to assess genotype and allele frequency of ADH1B, ALDH2, and CYP2E1 polymorphisms in
healthy HUI and Mexican mestizos (MES) from western
Mexico and to compare them with other populations around
the world. Lipid profile and functional hepatic tests were also
performed in order to evaluate biochemical variations associated to polymorphisms. This is the first study conducted in
HUI analyzing polymorphisms of the enzymes involved in
ethanol metabolism.
SUBJECTS AND METHODS
Study Population
One hundred and one HUI and 331 MES clinically healthy representing the general population reporting a Mexican family history of
at least 3 generations, were included in this study. MES population
was constituted by health workers and students of western Mexican
origin from the Civil Hospital of Tepic, an urban population located
approximately 210 km apart from the HUI community. HUI were
recruited at a rural medical service after advertisement through their
own HUI governor. Indigenous ethnicity was self declared by the use
of traditional attire and HUI language, participation in ancestral religious ceremonies and living in a rural community of high degree of
poverty known as ‘‘Jesús Marı́a,’’ at the mountains in Sierra Madre
Occidental (INEGI, 2006).
Blood Samples
Peripheral blood samples were collected in plain tubes for biochemical determinations and EDTA containing tubes for DNA isolation and molecular analysis. Lipid and hepatic function profile were
performed in all the individuals included in our study. ADH1B polymorphism was assessed in 97 HUI and 218 MES, ALDH2 in 101
HUI and 227 MES, CYP2E1 in 99 HUI, and 239 MES.
DNA Isolation
Genomic DNA was isolated by modified Miller’s procedure
(Cuevas-Covarrubias et al., 2002; Miller et al., 1988). Briefly,
EDTA-whole blood was transferred to a 15-ml sterile polystyrene
conic tube and 3 ml of TTS (1 M baseTris pH 7.6 ⁄ 100 · Triton ⁄ 1 M
sucrose), were added. After mixing, the tube was centrifuged at
4500·g for 10 minutes at room temperature. Supernatant was discarded and the pellet resuspended in 1 ml TTS, mixed and transferred into an Eppendorf tube. After vortexing during 15 minutes,
the tube was centrifuged at 18,000·g for 2 minutes. Supernatant was
discarded. This procedure was carried out as many times as
necessary until a hemoglobin-free pellet was obtained. Afterwards,
pellet was resuspended in 570 ll of 5 mM NaCl, vortexed for 2 minutes, added 30 ll 10% SDS, and vortexed during 15 minutes. Then,
200 ll of saturated NaCl were added and the tube was vortexed for
15 minutes, before centrifuging at 17,500·g for 30 minutes at 4C.
Supernatant was poured into a sterile tube containing 2 ml of
absolute cold ethanol making DNA visible as white threads. Tubes
were kept at )20C to allow DNA precipitation. After 24 h, DNA
was collected in a 0.5-ml Eppendorf tube with a Pasteur pipette,
washed twice with 500 ll of cold 70% ethanol. DNA spectrophotometric quantification at 260 nm ⁄ 280 nm was conducted after eliminating remnant ethanol and resuspending DNA in 300 ll of
deionized water. DNA samples were maintained at )20C, until
molecular analysis was carried out.
Amplification and Digestion of DNA
Amplification of ADH1B, ALDH2, and CYP2E1 polymorphic
regions was performed by polymerase chain reaction (PCR) and ulterior restriction fragment length polymorphism analysis using the
following primers: ADH1B, sense 5¢-ATTTCAGGAATTTGGGTATGTT-3¢, antisense 5¢-GGCCTAAAATCACAGGAAGG-3¢ (Xu
et al., 1988); ALDH2, sense 5¢-CAAATTACAGGGTCAACTGCT3¢, antisense 5¢-CCACACTCACAGTTTTCTCTT-3¢ (Takeshita
et al., 1994); CYP2E1, sense 5¢-GTCCCTGCCACCTCACACT-3¢,
antisense 5¢-CCCTCTTCCACCTTCTATG-3¢ (Hayashi et al.,
1991).
After assaying various PCR amplification conditions to be able to
amplify the 3 polymorphic fragments in different tubes in the same
running of PCR, we selected the conditions described below. Final
volume of PCR reaction was 50 ll containing 1 lg of DNA, 0.3 lM
oligonucleotides, 2.5 mM MgCl2, 0.1 mM dNTP’s, 1 U Taq DNA
polymerase, and 5 ll of supplied buffer enzyme (10X). An initial
denaturation at 95C for 5 minutes, 30 cycles of PCR (95C 1 minute, 60C 30 seconds, 72C 30 seconds) and a final extension period
of 7 minutes at 72C were carried out in a DNA thermal cycler
HIGH FREQUENCY OF CYP2E1*c2 ALLELE IN HUICHOLS
3
(Applied Biosystems 2400, Foster City, CA). ADH1B amplicon size
was 161 bp, ALDH2 135 bp and CYP2E1 566 bp. Restriction
enzyme digestion was carried out in aliquots of the amplified DNA
products. ADH1B polymorphism was identified following enzymatic
digestion with 2 U of MaeIII for 3 hours at 55C; ALDH2 with 1 U
of Ksp632I for 2 hour at 37C; and CYP2E1 with 3 U of RsaI for
3 hours at 37C. PCR amplicons and restriction fragments were
electrophoresed and visualized on a 2% agarose gel stained with
ethidium bromide. Amplification and digestion were carried out by
triplicate in all cases.
ADH1B, ALDH2, and CYP2E1 Restriction Pattern and
Genotype Screening
Restriction fragments of ADH1B characterizing each genotype
were as follows: a nondigested fragment of 161 bp for *1 ⁄ *1 genotype; 161, 98, and 63 bp for *1 ⁄ *2 genotype; 98 and 63 bp for *2 ⁄ *2
genotype. For ALDH2, 112 and 23 bp corresponded to *1 ⁄ *1 genotype; 135, 112, and 23 bp to *1 ⁄ *2 genotype; and a nondigested fragment of 135 bp to *2 ⁄ *2 genotype. CYP2E1 exhibited 143 and
423 bp for the common genotype c1 ⁄ c1; 566, 423, and 143 bp for the
heterozygous genotype c1 ⁄ c2; and a nondigested fragment of 566 bp
for the polymorphic genotype c2 ⁄ c2.
Lipid and Hepatic Profile
Samples were taken in a fasting state of 12 hours. Serum determination of total cholesterol, cholesterol bound to high density
lipoproteins (HDL-c), cholesterol bound to low density lipoproteins
(LDL-c), cholesterol bound to very low density lipoproteins
(VLDL-c), triglycerides, alanine aminotransferase (ALT), aspartate
aminotransferase (AST), total bilirubin (TB), and albumin were performed using commercially available kits (Merck Co., Whitehouse
Station, NJ).
Fig. 1. Representative 2% agarose gel electrophoresis of restriction
fragments stained with ethidium bromide. Genotype of Arg47His ADH1B,
Glu487Lys ALDH2, and c1 ⁄ c2 CYP2E1 was determined after digestion with
MaeIII, Ksp632I, and RsaI, respectively. Lanes 1 and 2, homozygous; lanes
3 and 4, heterozygous; lanes 5 and 6, polymorphic.
Table 1. Genotype Distribution of ADH1B, ALDH2, and CYP2E1
Polymorphisms in Subjects From Western Mexico
Frequency
ADH1B
Statistical Analysis
Genotype and allele frequency differences between groups were
assessed through the Pearson chi-squared test or Fisher’s exact test
when n values lesser than 10. Quantitative parameters were compared
through ANOVA. Probability values less than 0.050 were considered
statistically significant.
Ethical Considerations
Written informed consent was obtained from all subjects and the
study protocol conformed to the ethical guidelines according to the
1975 Declaration of Helsinki.
RESULTS
We studied 101 healthy HUI, 65 women and 41 men, aged
20 to 78 years from the HUI community living in the Indigenous reservation ‘‘Jesús Marı́a,’’ located at the Nayar region
of Nayarit state at the Sierra Madre Occidental; and 331
healthy subjects, 193 women and 138 men, aged 31 to
75 years, from a MES population living in the city of Tepic,
Nayarit, in western Mexico.
The PCR amplification protocol for ADH1B, ALDH2, and
CYP2E1 utilized in this study provided an efficient reproducible time saving alternative that can be widely recommended,
since in a separate tube but in the same running of PCR the 3
polymorphic segments can be obtained. Restriction patterns
according to genotype were observed (Fig. 1).
Genotype
1⁄1
2⁄1
2⁄2
Allele
1
2
ALDH2
Genotype
1⁄1
2⁄1
2⁄2
Allele
1
2
CYP2E1
Genotype
c1 ⁄ c1
c1 ⁄ c2
c2 ⁄ c2
Allele
c1
c2
HUI % (n)
MES % (n)
p value
(n = 97)
(n = 218)
100.0 (97)
0.0 (0)
0.0 (0)
94.5 (206)
4.1 (9)
1.4 (3)
0.06
100.0 (194)
0.0 (0)
(n = 101)
96.6 (421)
3.4 (15)
(n = 227)
0.008
99.0 (100)
1.0 (1)
0.0 (0)
100.0 (227)
0.0 (0)
0.0 (0)
0.308
99.5 (201)
0.5 (1)
(n = 99)
100.0 (454)
0.0 (0)
(n = 239)
0.308
21.2 (21)
54.5 (54)
24.2 (24)
72.0 (172)
23.8 (57)
4.2 (10)
<0.001
48.5 (96)
51.5 (102)
83.9 (401)
16.1 (77)
<0.001
HUI, Huichols subjects; MES, Mestizos subjects.
Genotype distribution and allele frequency of ADH1B,
ALDH2, and CYP2E1 polymorphisms are shown in Table 1.
Similar distribution of ADH1B genotypes were observed both
in HUI and MES individuals, although HUI subjects
were monomorphic for ADH1B*1 ⁄ 1 genotype. Meanwhile,
4
GORDILLO-BASTIDAS ET AL.
frequency of the ADH1B*2 polymorphic allele in MES
(3.4%) attained statistical significance (p = 0.008) when compared with HUI (0.0%). Conversely, MES were monomorphic for ALDH2*1 ⁄ 1 genotype and polymorphic allele
frequency in HUI (0.5%) and MES (0.0%) did not show statistically significant difference. Regarding CYP2E1, significantly higher (p < 0.001) frequency of c2 allele was shown in
HUI (51.5%) in comparison with MES individuals (16.1%).
Statistically significant difference in genotype distribution
between HUI and MES (p < 0.001) was also observed.
Comparison of ADH1B*2 allele frequency found in HUI
and MES with frequency observed in other populations of the
world is shown in Table 2. ADH1B*2 frequency in HUI was
not statistically different from Eskimos (0.000) or Swedish
Lapps (0.005). Significantly lower (p < 0.050) frequency of
ADH1B*2 allele was observed in HUI (0.000) and MES (0.034)
when compared with Japanese (0.740 and 0.780) or Chinese
(0.690) populations, which exhibited the highest frequency
values. Slight, although statistically significant differences
(p < 0.050), were observed in MES and HUI when compared
with Caboclos, Germans, Mexican-Americans, and Otomies.
ALDH2*2 allele frequency observed in HUI and MES was
similar to other populations of the world, except Chinese and
Japanese (p < 0.050), who exhibited the highest frequencies
reported (0.263 and 0.267), as can be seen in Table 3. Caboclos, which constitute a specific type of Brazilian mestizos,
showed also significantly higher ALDH2*2 polymorphic allele
than the MES and HUI from our study.
CYP2E1*c2 allele frequency in HUI (0.515) was statistically higher (p < 0.050) than Chinese (0.300) and Japanese
(0.280) populations, and even higher than Otomies (0.300),
another Mexican population from central and north Mexico
(Table 4). CYP2E1*c2 allele frequency in MES (0.160) did
not statistically differ from Mexican-Americans (0.143 to
0.160), but they did (p < 0.05) when compared to Mexicans
from central and north Mexico (0.300) or Otomies (0.300).
Mean values of ALT, AST, TB, and albumin both in HUI
and MES were in the normal range showing no genotype association. Nevertheless, AST values 1.5 to 2.0-fold out of normal
range were observed in 3 individuals carrying the CYP2E1*c2
allele (data not shown). Regarding lipid profile, lower levels of
total cholesterol, LDL-c, VLDL-c, triglycerides, and higher
Table 2. Frequency of ADH1B Polymorphic Allele in Healthy Subjects
Population
n
Mexican Mestizos (western Mexico)
Huichol (indigenous Mexican group)
Caboclos (Brazilian Mestizos)
Germans
Chinese
Eskimos
Japanese (rural county)
Japanese
Mexican-Americans
Mexican Mestizos (nonspecified zone)
Otomies (indigenous group, Central Mexico)
Swedish Lapps
436
194
40
466
396
54
670
1,536
502
114
118
200
Wild-type allele
frequency (n)
0.977
1.000
0.900
0.959
0.310
1.000
0.260
0.220
0.947
0.947
0.932
0.995
(421)
(194)
(36)
(447)
(120)
(54)
(173)
(331)
(489)
(108)
(110)
(199)
Polymorphic allele
frequency (n)
0.034
0.000
0.100
0.041
0.690
0.000
0.740
0.780
0.053
0.053
0.068
0.005
(15)
(0)
(4)
(19)
(276)
(0)
(497)
(1,205)
(13)
(6)
(8)
(1)
p value
Reference
–
–b
<0.050<0.050
NS<0.050
<0.050<0.050
NSNS
<0.050<0.050
<0.050<0.050
NS<0.050
NS<0.050
NS<0.050
NSNS
This study
This study
Goedde et al., 1992
Goedde et al., 1992
Yang et al., 2007b
Goedde et al., 1992
Saito et al., 2003
Matsuo et al., 2006
Konishi et al., 2003
Goedde et al., 1992
Montaño Loza et al., 2006
Goedde et al., 1992
a
n, number of alleles; NS, nonstatistically significant.
a
Comparison versus Mexican Mestizos of our study.
b
Comparison versus Huichols of our study.
Table 3. Frequency of ALDH2 Polymorphic Allele in Healthy Subjects
Population
Mexican (western Mexico)
Huichols (indigenous group, Western Mexico)
Caboclos (Brazilian Mestizos)
Germans
Chinese
Eskimos
Japanese (rural county)
Japanese
Mexican-Americans
Mexican (nonspecified zone)
Otomies (indigenous group, Central Mexico)
Swedish Lapps
n
454
202
46
386
396
54
670
1,536
502
122
118
200
n, number of alleles; NS, nonstatistically significant.
a
Comparison versus Mexican Mestizos of our study.
b
Comparison versus Huichols of our study.
Wild-type allele
frequency (n)
1.000
0.995
0.826
1.000
0.737
1.000
0.733
0.703
0.994
1.000
1.000
1.000
(454)
(201)
(38)
(386)
(292)
(100)
(491)
(1,080)
(499)
(122)
(118)
(200)
Polymorphic allele
frequency (n)
0.000
0.050
0.170
0.000
0.263
0.000
0.267
0.297
0.006
0.000
0.000
0.000
(0)
(1)
(8)
(0)
(104)
(0)
(179)
(456)
(3)
(0)
(0)
(0)
p value
Reference
–
–b
<0.050<0.050
–NS
<0.050<0.050
NSNS
<0.050<0.050
<0.050<0.050
NSNS
NSNS
NSNS
NSNS
This study
This study
Goedde et al., 1992
Goedde et al., 1992
Yang et al., 2007b
Goedde et al., 1992
Saito et al., 2003
Matsuo et al., 2006
Konishi et al., 2003
Goedde et al., 1992
Montaño Loza et al., 2006
Goedde et al., 1992
a
HIGH FREQUENCY OF CYP2E1*c2 ALLELE IN HUICHOLS
5
Table 4. Frequency of CYP2E1 c2 Polymorphic Allele in Healthy Subjects
Population
n
Mexican (West Mexico)
Huichols (indigenous group, Western Mexico)
English
Chileans
Chinese
Japanese
Mapuches (indigenous group, Chile)
Mexican-Americans
Mexican-Americans
Mexican (Central and North Mexico)
Otomies (indigenous group, Central Mexico)
478
198
242
296
300
260
168
502
760
204
118
Wild-type allele
frequency (n)
0.839
0.485
0.983
0.840
0.697
0.720
0.750
0.857
0.840
0.700
0.700
(401)
(96)
(238)
(249)
(209)
(187)
(126)
(430)
(638)
(143)
(83)
Polymorphic allele
frequency (n)
0.161
0.515
0.017
0.160
0.303
0.280
0.250
0.143
0.160
0.300
0.300
(77)
(102)
(4)
(47)
(91)
(73)
(42)
(72)
(122)
(61)
(35)
p value
Reference
–
–b
<0.050<0.050
NS<0.050
<0.050<0.050
<0.050<0.050
<0.050<0.050
NS<0.050
NS<0.050
<0.050<0.050
<0.050<0.050
This study
This study
Grove et al., 1998
Quiñones et al., 2001
Tan et al., 2000
Iwahashi et al., 1998
Muñoz et al., 1998
Konishi et al., 2003
Wan et al., 1998
Mendoza-Cantú et al., 2004
Montaño Loza et al., 2006
a
n, number of alleles; NS, nonstatistically significant.
a
Comparison versus Mexican Mestizos of our study.
b
Comparison versus Huichols of our study.
Table 5. Lipid Profile in Huichol (HUI) and Mexican Mestizos (MES) From
Western Mexico
Variable (mg ⁄ dl)
HUI (n = 101)
MES (n = 331)
Total cholesterol
HDL-c
LDL-c
VLDL-c
Triglycerides
190.4
46.2
120.2
24.8
150.6
228.1
41.1
158.4
28.9
197.3
±
±
±
±
±
37.1*
10.6*
31.7*
10.6*
98.1*
±
±
±
±
±
49.2
10.8
46.4
15.9
123.6
Values represent the median ± standard deviation.
*p < 0.001.
levels of HDL-c in HUI than MES were observed; these differences were independent of genotype (Table 5).
DISCUSSION
This is the first study describing genotype and allele distribution of ADH1B, ALDH2, and CYP2E1 polymorphisms in
a HUI community of Mexico in comparison with MES and
other populations of the world.
Literature about the prevalence of ADH1B, ALDH2, and
CYP2E1 gene variations has been extensive due to the important participation of the enzymes encoded by these genes in
ethanol metabolism (Bosron et al., 1993; Tanaka et al., 1997).
Tables 2–4 compare our results with the corresponding frequencies reported in various populations around the world.
We observed that ADH1B*2 and ALDH2*2 alleles were relatively rare in HUI and MES subjects, as also stated before in
Otomies (Montaño Loza et al., 2006), Mexican-Americans
(Konishi et al., 2003; Wan et al., 1998; Yang et al., 2007b),
Germans, Eskimos, Swedish Lapps, and some Latin American ethnic groups (Goedde et al., 1992). In contrast, these
variant alleles are very common in Japanese and Chinese people (Eng et al., 2007; Matsuo et al., 2006; Saito et al., 2003;
Yang et al., 2007b). Thus, we could say that the presence of
these alleles is restricted almost only to East Asians.
A high frequency of the CYP2E1*c2 allele in HUI was
observed in our study. The prevalence reported by some
authors varies among populations: higher (0.190 to 0.300) in
Asians and lower (0.030 to 0.170) in Caucasians (Goedde
et al., 1992; Grove et al., 1998; Hamajima et al., 2002;
Iwahashi et al., 1998; Stephens et al., 1994; Tan et al., 2000).
A frequency of 0.300 and 0.250 of the c2 allele has been
revealed in Otomi and Mapuche subjects, respectively, attaining frequencies similar to Asians (Montaño Loza et al., 2006;
Muñoz et al., 1998). However, a frequency of 0.515 observed
in HUI in this study corresponds to the highest prevalence of
c2 allele reported up to this date. Meanwhile, MES individuals exhibited a frequency of 0.160, which is in the range
reported in Mexican-Americans (Konishi et al., 2003; Wan
et al., 1998; Yang et al., 2007a) and similar to Chileans
(Quiñones et al., 2001). Strikingly, Mexicans living in Mexico
city and Sabinas Coahuila (central and north Mexico, respectively) showed a high CYP2E1*c2 allele frequency of 0.300
(Mendoza-Cantú et al., 2004), which significantly differs from
the prevalence we are reporting in MES subjects from western
Mexico, yet both populations belong to the same country.
These results evidence Mexican population heterogeneity and
emphasize to be cautious when making general conclusions
about populations with an enriched ethnical background. It is
very important to stress that the virtual absence of the alcoholism ‘‘protecting’’ polymorphic alleles ADH1B*2 and
ALDH2*2 (Bosron and Li, 1986) observed, and the relatively
high frequency of the CYP2E1*c2 allele, may be contributing
to the public health problem of alcoholic cirrhosis in México
(Campollo et al., 1997; Méndez-Sánchez et al., 2004), where
the involvement of other polymorphisms, like Apo e2 allele,
has been recently described (Hernández-Nazará et al., 2008).
Also, it is necessary to determine the pathophysiological role
of this molecular feature in the heavy alcohol-drinking pattern reported in Mexican-Americans (Saunders et al., 1993).
Various CYP2E1 polymorphisms have been associated
with an increased expression of CYP2E1 or its catalytic activity; but contradictory results have been obtained in all cases
(Danko and Chaschin, 2005). CYP2E1*c2 is one of the most
studied polymorphisms due to its effect on transcription and
enzyme activity leading to CYP2E1 overexpression, free radical production and pathology association (Cichoz-Lach et al.,
6
2006; Gao et al., 2007; Hayashi et al., 1991; Lieber, 2004b;
Ueno et al., 1996; Ueshima et al., 1996; Watanabe et al.,
1994). The functional significance of this polymorphism may
be due to its localization in the element response to the
hepatic transcription factor HNF-1 (Hayashi et al., 1991).
CYP2E1*c2 is considered candidate gene for ALD development (Bataller et al., 2003). However, determination of genotype and allele distribution of other CYP2E1 variant forms in
HUI, may be of importance.
Individuals included in this study were clinically healthy
people representing the general population; notwithstanding,
liver function tests and lipid profile were determined to evaluate possible incipient alterations associated to genotype, as
these parameters could be indicative of liver dysfunction
(Hernández-Nazará et al., 2008). Mean hepatic function values were normal and not statistically different in HUI and
MES. Meaningless, but worth to mention is that 3 subjects
showing slightly higher than normal AST values owed the
CYP2E1*c2 allele. Although lack of significant genotype
association, HUI presented a better lipid profile than MES.
Recently, the absence of the Apo e2 allele and high prevalence
(28%) of the Apo e4 allele have been documented in HUI
(Aceves et al., 2006), which are related to hypertriglyceridemia and latest onset of liver cirrhosis, respectively (HernándezNazará et al., 2008). Besides, HUI are habituated to a low
fat, maize-rich diet, and high physical activity (Aceves
et al., 2006). These genetic and environmental factors may
explain the good lipid levels observed in HUI and may protect them of liver dysfunction in spite of the well-known
ancestral high consumption of alcohol among indigenous
groups as part of their way of life (Bruman, 2000) and the
unusual high frequency of the CYP2E1*c2 allele observed.
Nevertheless, well designed case–control studies to
elucidate the role of this polymorphic allele in pathologies
where oxidative stress constitute critical factors, like ALD
(Lu and Cederbaum, 2008), nonalcoholic steatohepatitis
(Lieber, 2004a), obesity and diabetes (Lieber, 2004b),
should be accomplished in HUI.
México is an American country that has been defined by
the National Commission for the Development of the Indigenous Peoples as a pluricultural nation. This category has been
assigned because of the great cultural wealth characterizing
México, since ethnic communities account for the 11 to 12%
of the total population (CDI, 2008).
Huichols constitutes one of the few indigenous groups still
remaining with its own ancestral cultural identity and considered to be of pure origin with no racial admixture living in
western central mountains of México, suggested to be of
Nahua origin (Krickeberg, 1961). Further evidence of the
peculiar genetic composition of HUI and the multiple ethnic
composition of the Mexican population consists in the significant different distribution of Apo e polymorphic alleles documented, where HUI highlight as the population presenting
the highest prevalence of Apo e allele (Aceves et al., 2006).
From the migrations point of view, and the supposed oriental origin of the America’s population, it is interesting to
GORDILLO-BASTIDAS ET AL.
notice that meanwhile Asians exhibit high frequency of the
CYP2E1*c2, ADH1B*2, and ALDH2*2 polymorphic alleles,
CYP2E1*c2 frequency was high in HUI but ADH1B*2 and
ALDH2*2 alleles were practically absent (Delanghe et al.,
2000; Horai et al., 1993). However, to have a better
understanding and to correctly interpret the meaning of
these findings, migration and historical analysis should be
conducted.
In conclusion, prevalence and haplotype association studies
to elucidate the role of CYP2E1 polymorphisms should be
addressed in different subgroups of the Mexican population.
From the pharmacogenetics and the pathophysiological perspective, HUI may represent a group of interest to investigate
not only the response to alcohol but also to other substances
metabolized by the CYP2E1 pathway, as well as for disease
susceptibility. In the past 500 years, we can assume that several HUI and other ethnic generations have been mixed with
MES contributing to the genetic MES composition. Studies
performed in ethnic groups represent a valuable source of
information to better understand the Mexican gene composition and to the implementation of health strategies to improve
life quality, since MES are considered the result of indigenous,
Spaniard, and African genes. Racial and lifestyle differences
may explain discrepancies in the establishment of the clinical
relevance of ADH1B, ALDH2, and CYP2E1 polymorphisms,
turning genotype and allele distribution studies a valuable scientific contribution.
ACKNOWLEDGMENTS
This study was supported by CONACYT grant SALUD2004-C01-104 and Universidad de Guadalajara REC ⁄ 787 ⁄
2005 to Blanca Estela Bastidas-Ramı́rez, PhD.
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