Mol Biol Rep DOI 10.1007/s11033-014-3341-0
PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations Elena Larrieta-Carrasco • Victor Acun˜a-Alonzo • Rafael Vela´zquez-Cruz • Rodrigo Barquera-Lozano • Paola Leo´n-Mimila • Hugo Villamil-Ramı´rez • Marta Menjivar • Sandra Romero-Hidalgo • Nahu´m Me´ndez-Sa´nchez • Vanessa Ca´rdenas • Manuel Ban˜uelos-Moreno • Yvonne N. Flores • Manuel Quiterio • Jorge Salmero´n • Fausto Sa´nchez-Mun˜oz • Teresa Villarreal-Molina Carlos A. Aguilar-Salinas • Samuel Canizales-Quinteros
•
Received: 25 September 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with Elena Larrieta-Carrasco and Victor Acun˜a-Alonzo have contributed equally to this study.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3341-0) contains supplementary material, which is available to authorized users. E. Larrieta-Carrasco Departamento de Gastroenterologı´a, Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n (INCMNSZ), Mexico, Mexico V. Acun˜a-Alonzo P. Leo´n-Mimila H. Villamil-Ramı´rez M. Menjivar S. Canizales-Quinteros (&) Unidad de Geno´mica de Poblaciones Aplicada a la Salud, Facultad de Quı´mica, Universidad Nacional Auto´noma de Me´xico (UNAM)-Instituto Nacional de Medicina Geno´mica (INMEGEN), Mexico, Mexico e-mail: [email protected] V. Acun˜a-Alonzo R. Barquera-Lozano Laboratorio de Gene´tica Molecular, Escuela Nacional de Antropologı´a e Historia (ENAH), Mexico, Mexico
serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels C40 U/L and 564 controls with ALT \40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7 %, and varied according to obesity status: 14.4 % in normal weight, 19.9 % in overweight and 24.5 % in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91–5.20; P = 7.1 9 10-6) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95 % CI 1.50–3.33; P = 8.1 9 10-5). This is S. Romero-Hidalgo Laboratorio de Geno´mica Computacional, INMEGEN, Mexico, Mexico N. Me´ndez-Sa´nchez Unidad de Investigacio´n del Hı´gado, Fundacio´n Me´dica Sur, Mexico, Mexico M. Ban˜uelos-Moreno Y. N. Flores M. Quiterio J. Salmero´n Unidad de Investigacio´n Epidemiolo´gica y en Servicios de Salud, Instituto Mexicano del Seguro Social, Cuernavaca, Morelos, Mexico Y. N. Flores UCLA Department of Health Policy and Management, Fielding School of Public Health, Johnson Comprehensive Cancer Center, Los Angeles, CA, USA
R. Vela´zquez-Cruz V. Ca´rdenas ´ seo, INMEGEN, Laboratorio de Geno´mica del Metabolismo O Mexico, Mexico
123
Mol Biol Rep
the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations. Keywords I148M/PNPLA3 Alanine transaminase Native Mexican Mexican Mestizos Abbreviations PNPLA3 Patatin-like phospholipase domain-contain 3 NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis ALT Alanine transaminase AST Aspartate transaminases IMSS Mexican Institute of Social Security BMI Body mass index HOMA-IR Homeostasis model assessment for insulin resistance HDL-C High-density lipoprotein cholesterol OR Odds ratio
(PNPLA3) gene, as the strongest genetic factor associated with NAFLD and elevated ALT and aspartate transaminase (AST) levels in a large multiethnic population [14]. Interestingly, the most striking effects of the PNPLA3 I148M variant were observed in Hispanics who had the highest 148M risk allele frequency [14]. In addition, Native American ancestry was recently associated with higher ALT levels in a Mexican–American cohort [15]. Because the Mexican population resulted from the admixture of mainly European (Spaniard) and Native American populations, it is likely that the elevated 148M allele frequency resulted from the contribution of the Native American gene pool. The aim of the present study was to assess the frequency of the 148M risk allele and whether it is associated with serum ALT levels in Native Mexican and Mexican Mestizo individuals.
Materials and methods Study population Native American populations
Introduction Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) [1]. Elevated levels of alanine transaminase (ALT) have been associated with NAFLD and may be considered as a sensitive indicator of liver-cell injury [2]. Several studies have suggested that both liver fat accumulation and ALT levels vary among different populations, with Hispanics having the highest prevalence of NAFLD and elevated ALT levels [3–9]. According to heritability estimations, 20–60 % of ALT level variability can be attributed to genetic factors [10–13]. A genome-wide association study identified the nonsynonymous I148M variant (rs738409) in the patatin-like phospholipase domain-containing 3
J. Salmero´n Centro de Investigacio´n en Salud Poblacional, Instituto Nacional de Salud Pu´blica, Cuernavaca, Mexico F. Sa´nchez-Mun˜oz Departamento de Inmunologı´a, Instituto Nacional de Cardiologı´a ‘‘Ignacio Cha´vez’’ (INCICh), Mexico, Mexico T. Villarreal-Molina Laboratorio de Enfermedades Cardiovasculares, INMEGEN, Mexico, Mexico C. A. Aguilar-Salinas Departamento de Endocrinologı´a y Metabolismo, INCMNSZ, Mexico, Mexico
123
We evaluated a total of 919 unrelated Native American adults aged 18–91 years, including 350 Nahuas from Central Mexico, 179 Totonacs from Eastern Mexico, 86 Seris from Northern Mexico, 120 Coras from Western Mexico, and 184 Zapotecs from Southwestern Mexico. Only subjects born in their communities who speak their native language were included. The study was approved by the Ethics Committees of all participating Institutions, local authorities gave their approval to participate, and a translator was used as needed. Metabolic measurements were only available in the Nahua and Totonac subjects. Mexican Mestizo population A total of 705 Mexican Mestizo individuals working at the Mexican Institute of Social Security (IMSS) of the state of Morelos were recruited for a case–control study. This study was performed comparing individuals with elevated and non-elevated ALT levels, defined as ALT measurements C40 U/L (141 cases) or \40 U/L (564 controls) in two or more measurements over a 6 month period. The study protocol was approved by the IMSS Ethics Committee and all participants provided written informed consent prior to their inclusion. Anthropometric and biochemical measurements Weight and height were measured and all individuals. Waist circumference was measured in 367 indigenous
Mol Biol Rep
subjects and in all Mexican Mestizo individuals. BMI was calculated as weight in kilograms/height in meters squared. Participants were grouped according to BMI as normal weight (BMI B 24.9 kg/m2), overweight (25 B BMI B 29.9 kg/m2), or obese (BMI C 30 kg/m2). Biochemical parameters were measured in all participants in a fasting state as previously described [16]. The homeostasis model assessment for insulin resistance (HOMA-IR) was used to estimate insulin sensitivity in Indigenous Mexican population [17]. Serum levels of ALT and AST were determined only in Mexican Mestizo, Nahua and Totonac individuals using commercially available standardized methods (Beckman Coulter, Fullerton, CA, USA). I148M/PNPLA3 genotyping All participants were genotyped for rs738409 I148M variant using TaqMan assays (ABI Prism 7900HT Sequence Detection System; Applied Biosystems, Foster City, CA, USA). No discordant genotypes were observed in 39 duplicate samples. Deviation from Hardy–Weinberg equilibrium was not observed for this polymorphism. Statistical analysis Student’s t test was used to compare clinical and biochemical characteristics of normal weight to overweight and obese individuals. Differences among genotypes for categorical variables were evaluated using a chi square test. Associations of I148M genotype with quantitative variables were analyzed by linear regression analysis adjusting for age, gender, BMI and ethnicity when appropriate. A multiple linear regression model was used to assess the interaction between waist circumference and the I148M polymorphism on ALT levels, adjusting for the variables mentioned above (SPSS V15, statistical package; Chicago, IL, USA).
Results Association of PNPLA3 I148M with ALT and AST levels in Native Americans PNPLA3 148M risk allele frequencies were similar among the five Native groups (mean 0.73), ranging from 0.62 in Seris to 0.85 in Coras (Table 1). Anthropometric and biochemical measurements were available only in the Nahua and Totonac populations (Table 2). Although the frequency of elevated ALT levels was higher in Nahuas (20.9 %) than in Totonac individuals (15.5 %), the difference was not statistically significant (P = 0.135). The
Table 1 PNPLA3 I148M genotypes and allele distribution by native Mexican population Population
N
Genotypes N (%) I148I
I148M
M148M
M-allele frequency
Seri
86
9 (10.5)
47 (54.7)
30 (34.9)
0.62
Cora
120
4 (3.3)
28 (23.3)
88 (73.3)
0.85
Nahua Totonac
350 179
31 (8.9) 13 (7.3)
151 (43.1) 63 (35.2)
168 (48.0) 103 (57.5)
0.70 0.75
Zapotec
184
11 (6.0)
71 (38.6)
102 (55.4)
0.75
overall frequency of elevated ALT levels was 18.7 %, and varied according to obesity status: 14.4 % in normal weight, 19.9 % in overweight and 24.5 % in obese individuals. Moreover, the frequency of elevated ALT levels was higher in male individuals, although gender differences were statistically significant only for comparisons in the entire sample and in overweight individuals (P = 0.001). Because ALT and AST levels showed no significant differences between I148I homozygous and I148M heterozygous individuals in the entire sample (P [ 0.21 and P [ 0.43, respectively), associations were tested under a recessive model (I148I and I148M vs. M148M). Mean ALT and AST levels were 26.4 and 19.1 % higher respectively in M148M homozygous as compared to I148I/ I148M individuals (P = 5.3 9 10-6 and 2.0 9 10-6 respectively, after adjustment for age, gender, BMI and ethnic group). On stratification according to gender, the association of the M148M genotype with ALT levels was statistically significant only in females, in the entire sample (P = 6.6 9 10-6), as well as in the overweight and obesity groups (P = 3.8 9 10-5 and 0.014, respectively). Very similar results were observed on comparing elevated vs non-elevated ALT levels (Table 3). The M148M genotype was not significantly associated with any other metabolic parameters in Native Mexican individuals (Supplementary Table 1). Moreover, no significant interactions between BMI or waist circumference and I148M polymorphism were observed for ALT levels (P = 0.549 and P = 0.256, respectively). Association of PNPLA3 I148M with elevated ALT and AST levels in Mexican Mestizos PNPLA3 148M risk allele frequency in Mexican Mestizos was 0.58. The M148M genotype was significantly associated with elevated ALT levels in the entire simple (OR = 2.24, 95 % CI 1.50–3.33; P = 8.1 9 10-5 adjusted for gender, age and BMI). On stratification according to gender, the association of the M148M genotype with elevated ALT levels remained significant only in women
123
Mol Biol Rep Table 2 Anthropometric and biochemical parameters of Nahua and Totonac populations according to BMI All
Normal weight
Overweight
Obese
N (%)
529
195 (36.9)
236 (44.6)
98 (18.5)
Male (%)
144 (27.2)
69 (35.4)
56 (23.7)*
19 (19.4)*
47.6 ± 15.1
50.3 ± 18.1
45.8 ± 13.5*
46.5 ± 11.3*
BMI (kg/m )
26.5 ± 4.3
22.3 ± 2.0
27.2 ± 1.4*
33.0 ± 2.7*
Waist circumference (cm)
89.4 ± 13.6
81.6 ± 12.8
90.3 ± 8.8*
102.5 ± 13.2*
HOMA-IR
2.4 (1.5–4.2)
1.6 (0.9–2.3)
2.9 (2.1–4.6)*
4.9 (3.3–7.9)*
Age (years) 2
Total cholesterol (mg/dL)
183.5 ± 40.4
179.2 ± 41.2
185.5 ± 40.5
187.3 ± 38.2
Triglycerides (mg/dL)
162.0 (111.0–229.0)
130.0 (95.0–189.0)
177.0 (124.0–235.0)*
189.0 (139.5–279.5)*
HDL-C (mg/dL)
40.5 ± 12.6
44.8 ± 15.5
38.4 ± 10.2*
37.0 ± 8.3*
ALT (U/L)
26.0 (21.0–36.0)
24.0 (20.0–31.0)
27.0 (21.0–37.0)*
30.5 (23.0–39.5)*
Male Female
30.5 (23.0–41.0) 25.0 (20.0–33.0)
25.0 (21.5–33.5) 24.0 (19.8–30.0)
37.0 (28.0–49.8) 25.0 (20.0–34.0)
33.0 (30.0–44.0) 29.0 (23.0–38.0)
P
1.3 9 10-4
0.357
2.7 9 10-5
0.217
31.0 (26.0–38.0)
31.0 (27.0–37.0)
31.0 (25.0–38.0)
31.0 (26.0–40.0)
Male
33.0 (29.0–43.8)
33.0 (29.0–44.0)
36.0 (31.0–43.0)
31.0 (24.0–44.0)
Female
29.0 (25.0–37.0)
29.5 (26.0–35.3)
29.0 (25.0–36.0)
30.0 (26.0–40.0)
P
5.8 9 10-5
0.006
0.002
0.527
AST (U/L)
ALT C40 U/L (%) Male
99 (18.7)
28 (14.4)
47 (19.9)
24 (24.5)*
42 (29.2)
13 (18.8)
22 (39.3)
7 (36.8)
Female
57 (14.8)
15 (11.9)
25 (13.9)
17 (21.5)
P
0.001
0.214
0.001
0.224
Data are means ± standard deviations, medians (interquartil range) or N (%). HOMA-IR and triglycerides were log-transformed before analysis BMI body mass index, HOMA-IR homeostasis model of assessment insulin resistance index, HDL-C high density lipoprotein cholesterol, ALT alanine transaminase, AST aspartate transaminase * P \ 0.05 compared to normal weight and P \ 0.05 compared to overweight
(OR = 3.21, 95 % CI 1.98–5.21; P = 2.5 9 10-6) (Table 4). As in the indigenous population, the effect of the interaction between BMI or waist circumference and the I148M polymorphism on ALT levels was not significant (P = 0.579 and P = 0.747, respectively).
Discussion Although the Mexican indigenous population includes over 10 million individuals [18], there are very few studies analyzing genetic associations and the impact of metabolic disease in these populations [19, 20]. It has been suggested that Native American ancestry is associated with higher ALT levels in Mexican–Americans [15]. However, there is only one previous study evaluating the ALT levels in a Mexican indigenous population (Tepehuanos) [21]. In the present study, the frequency of ALT levels C40 U/L in Nahuas and Totonacs was 18.7 %, with median values (26 U/L) similar to those reported in Tepehuanos (30.5 U/L). In contrast, the prevalence of elevated ALT levels was higher than in white and black populations of the Third
123
National Health and Nutrition Examination Survey (7.1 and 8.1 %, respectively) [7]. Elevated ALT levels were more frequent in overweight and obese than in normal weight subjects, in accordance with previous reports [22, 23]. To the best of our knowledge, this is the first study to estimate the frequency of elevated ALT levels in over 500 Mexican indigenous individuals from central Mexico. The association of I148M PNPLA3 with elevated ALT levels was highly significant in both Mexican Indigenous and Mestizo populations. Although this association has been previously reported in Mexican–American adults [24, 25], this is the first study showing evidence of an association of this polymorphism with chronically elevated ALT levels in Mexican Mestizo individuals. Interestingly, although the percentage of males with elevated ALT levels was higher in those bearing M148M genotype, the association was significant only in Native Mexican and Mestizo women. The lack of significance in males may be partially due to the reduced sample size and/or to the lower percentage of obese males compared to females; however it is clear that the effect size and level of significance of these associations was much higher in women. This is consistent
Mol Biol Rep Table 3 ALT levels of Nahua and Totonac populations according to PNPLA3 I148M genotypes stratified by BMI
I148I
I148M
M148M
P*
All N = 529 ALT (U/L)
44 (8.3) 23.0 (21.0–30.8)
214 (40.5)
271 (51.2)
25.0 (20.0–32.3)
28.0 (21.0–40.0)
5.3 9 10-6
Male
25.0 (22.0–33.8)
30.0 (23.0–42.0)
34.0 (25.0–44.0)
0.065
Female
23.0 (19.0–29.0)
24.0 (19.0–30.0)
27.0 (20.0–38.8)
6.6 9 10-6
2 (2.0)
27 (27.3)
70 (70.7)
7.1 9 10-6
Male
2 (4.8)
18 (42.9)
22 (52.4)
0.270
Female
0 (0.0)
9 (15.8)
48 (84.2)
3.9 9 10-6
N = 195
22 (11.3)
71 (36.4)
102 (52.3)
ALT (U/L)
ALT C40 U/L (%)
Normal weight 23 (20–29)
24 (20–29)
25 (20–34)
Male
23 (21–25)
25 (21–37)
27 (22–37)
0.163 0.314
Female
26 (21–29)
23 (20–28)
24 (19–34)
0.353
0 (0.0)
8 (28.6)
20 (71.4)
0.019
0 (0.0) 0 (0.0)
6 (46.2) 2 (13.3)
7 (53.8) 13 (86.7)
0.542 0.020
N = 236
15 (6.4)
99 (41.9)
122 (51.7)
ALT (U/L)
23 (19–30)
24 (19–34)
ALT C40 U/L (%) Male Female Overweight
30 (24–39)
35 (24–43)
39 (34–59)
0.105
Female
22 (19–26)
23 (18–30)
27 (21–38)
3.8 9 10-5
0 (0.0)
11 (23.4)
36 (76.6)
3.4 9 10-4
Male
0 (0.0)
7 (31.8)
15 (68.2)
0.109
Female
0 (0.0)
4 (16.0)
21 (84)
0.001
7 (7.1)
44 (44.9)
47 (48.0)
Obese
ALT alanine transaminase
N = 98
* P values calculated by linear regression adjusted for age, sex, BMI and ethnicity
ALT (U/L)
P value calculated by multiple logistic regression analysis adjusted for age, sex, BMI and ethnicity as covariates when was appropriate
5.1 9 10-5
Male ALT C40 U/L (%) Data are medians (interquartil range) or N (%)
30 (24–43)
32 (22–40)
28 (23–36)
33 (22–43)
Male
40 (32–62)
33 (26–51)
30 (25–36)
0.585
Female
23 (20–33)
26 (23–31)
34 (22–44)
0.014
ALT C40 U/L (%) Male Female
0.048
2 (8.3)
8 (33.3)
14 (58.3)
0.039
2 (28.6) 0 (0.0)
5 (71.4) 3 (17.6)
0 (0.0) 14 (82.4)
0.252 0.014
with previous findings suggesting sex differences in the genetic association of PNPLA3 polymorphisms and liver enzymes [25, 26]. Even though the magnitude of the association of PNPLA3 with ALT levels and risk of liver damage has been reported to be modulated by abdominal fat in children and adults [27, 28], interactions between waist circumference and I148M polymorphism for ALT levels were not significant in Mestizos or indigenous individuals. Several studies have shown that this polymorphism is associated with NAFLD in Hispanics [29–31]. Although elevated ALT levels have been correlated with the presence of hepatic fat on imaging in adults [32], a limitation of the present study was the lack of imaging techniques to diagnose fatty liver. Another limitation to be considered is that the analysis in Mexican Mestizos was not tested for
admixture, however the association of this polymorphism with elevated ALT levels is consistent with previous observations in Mexican–Americans [24]. In addition, other factors affecting ALT levels such as alcohol consumption and liver infection were not assessed. In this regard, although Mexico is known to be a low endemic area for hepatitis B and C infection, a high frequency of occult hepatitis B has been reported in Indigenous Mexicans [33]. However, it is noteworthy that the PNPLA3 polymorphism is also associated with liver injury in individuals with hepatitis infection and alcoholic fatty liver disease [34, 35]. In summary, the Mexican indigenous population has the highest frequency of the 148M/PNPLA3 risk allele (0.73), compared to African American (0.18), Caucasians (0.26–0.32), Taiwanese (0.39), or other Hispanic
123
Mol Biol Rep Table 4 Association of PNPLA3 M148M genotype with elevated ALT levels in Mexican Mestizos I148I
I148M
M148M
P
All (N = 705) Cases ALT C40 U/L
21 (14.9)
51 (36.2)
69 (48.9)
111 (19.7)
281 (49.8)
172 (30.5)
Cases ALT C40 U/L
8 (15.4)
23 (44.2)
21 (40.4)
Controls ALT \40 U/L Female
17 (13.6)
58 (46.4)
50 (40.0)
Cases ALT C40 U/L
13 (14.6)
28 (31.5)
48 (53.9)
Controls ALT \40 U/L
94 (21.4)
223 (50.8)
122 (27.8)
Controls ALT \40 U/L
8.1 9 10-5
Male 0.965
2.5 9 10-6
Data are N (%). P value calculated by multiple logistic regression analysis adjusted for age, sex and BMI when was appropriate ALT alanine transaminase
populations (0.48–0.61) [27, 29, 30, 35–38]. This suggests that the high frequency of this allele in Hispanics, including Mexican Mestizos, is due to their indigenous component. The M148M genotype may be considered as an important risk factor for liver damage in Mexican Indigenous and Mestizo populations. Acknowledgments This research was supported by grant SALUD2009-01-113861 from the Consejo Nacional de Ciencia y Tecnologı´a (CONACyT). The study of the IMSS cohort is supported in part by grants from IMSS (FIS/IMSS/PROT/G12/1117) and CONACyT (26267M, SALUD-2008-01-87783 and SALUD-2011-C01-161930). PLM is in the Ciencias Bioquı´micas PhD program from Universidad Nacional Auto´noma de Me´xico (UNAM) and HVR is in the Ciencias Biolo´gicas y de la Salud PhD program from Universidad Auto´noma Metropolitana (UAM), and were supported by the PhD fellowships number 244112 and 234714 from CONACyT, Me´xico.
References 1. Angulo P (2002) Nonalcoholic fatty liver disease. N Engl J Med 346:1221–1231 2. Pratt DS, Kaplan MM (2000) Evaluation of abnormal liverenzyme results in asymptomatic patients. N Engl J Med 342:1266–1271 3. Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH (2004) Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 40:1387–1395 4. Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, Terrault NA (2005) Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 41:372–379
123
5. Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, Landt CL, Harrison SA (2011) Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 140:124–131 6. Fraser A, Longnecker MP, Lawlor DA (2007) Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999–2004. Gastroenterology 133:1814–1820 7. Clark JM, Brancati FL, Diehl AM (2003) The prevalence and etiology of elevated aminotransferase levels in the United States. Am J Gastroenterol 98:960–967 8. Ioannou GN, Boyko EJ, Lee SP (2006) The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999–2002. Am J Gastroenterol 101:76–82 9. Flores Y, Yee HF, Leng M, Bastani R, Escarce J, Salmero´n J, Morales L (2008) Risk Factors for Chronic Liver Disease in Blacks, Mexican Americans, and Whites in the United States: results From NHANES IV, 1999–2004. Am J Gastroenterol 103:2231–2238 10. Brouwers MC, Cantor RM, Kono N, Yoon JL, van Kallen CJ, Bilderbeek-Beckers MA, van Greevenbroek MM, Lusis AJ, de Bruin TW (2006) Heritability and genetic loci of fatty liver in familial combined hiperlipidemia. J Lipid Res 47:2799–2807 11. Makkonen J, Pietila¨inen KH, Rissanen A, Kaprio J, Yki-Ja¨rvinen H (2009) Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: a study in monozygotic and dizygotic twins. J Hepatol 50:1035–1042 12. Day CP (2010) Genetic and environmental susceptibility to nonalcoholic fatty liver disease. Dig Dis 28:255–260 13. Alisi A, Cianfarani S, Manco M, Agostoni C, Nobili V (2012) Non-alcoholic fatty liver disease and metabolic syndrome in adolescents: pathogenetic role of genetic background and intrauterine environment. Ann Med 44:29–40 14. Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC, Hobbs HH (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 40:1461–1465 15. Qu HQ, Li Q, Lu Y, Fisher-Hoch SP, McCormick JB (2012) Translational genomic medicine: common metabolic traits and ancestral components of Mexican Americans. J Med Genet 49:544–545 16. Villarreal-Molina MT, Aguilar-Salinas CA, Rodrı´guez-Cruz M, Rian˜o D, Villalobos-Comparan M, Coral-Vazquez R, Menjivar M, Yescas-Gomez P, Ko¨nigsoerg-Fainstein M, Romero-Hidalgo S, Tusie-Luna MT, Canizales-Quinteros S (2007) The ATP-binding cassette transporter A1 R230C variant affects HDL cholesterol levels and BMI in the Mexican population: association with obesity and obesity-related comorbidities. Diabetes 56:1881–1887 17. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412–419 18. Ferna´ndez Ham P, Tuira´n Gutie´rrez A, Ordorica Mellado M, Salas y Villago´mez G, Camarena Co´rdova RM, Serrano Carreto E (2006) Comisio´n Nacional para el Desarrollo de los Pueblos Indı´genas (CDI)-Programa de las Naciones Unidas para el Desarrollo (PNUD). Informe sobre Desarrollo Humano de los Pueblos Indı´genas de Me´xico 2006. http://www.cdi.gob.mx/idh/ informe_desarrollo_humano_pueblos_indigenas_mexico_2006.pdf. Accessed Sept 2013 19. Aguilar CA, Talavera G, Ordovas JM, Barriguete JA, Guille´n LE, Leco ME, Pedro-Botet J, Gonzalez-Barranco J, Go´mez-Pe´rez FJ, Rull JA (1999) The apolipoprotein E4 allele is not associated with an abnormal lipid profile in a Native American population following its traditional lifestyle. Atherosclerosis 142:409–414
Mol Biol Rep 20. Acun˜a-Alonzo V, Flores-Dorantes T, Kruit JK, Villarreal-Molina T, Arellano-Campos O, Hu¨nemeier T, Moreno-Estrada A, OrtizLo´pez MG, Villamil-Ramı´rez H, Leo´n-Mimila P, VillalobosComparan M, Jacobo-Albavera L, Ramı´rez-Jime´nez S, Sikora M, Zhang LH, Pape TD, Granados-Silvestre MA, Montufar-Robles I, Tito-Alvarez AM, Zurita-Salinas C, Bustos-Arriaga J, CedilloBarro´n L, Go´mez-Trejo C, Barquera-Lozano R, Vieira-Filho JP, Granados J, Romero-Hidalgo S, Huertas-Va´zquez A, Gonza´lezMartı´n A, Gorostiza A, Bonatto SL, Rodrı´guez-Cruz M, Wang L, Tusie´-Luna T, Aguilar-Salinas CA, Lisker R, Moises RS, Menjivar M, Salzano FM, Knowler WC, Bortolini MC, Hayden MR, Baier LJ, Canizales-Quinteros S (2010) A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans. Hum Mol Genet 19:2877–2885 21. Lares-Asseff I, Lujı´n-Garcı´a A, Sosa-Macı´as M, Lazalde-Ramos B, Loera-Castan˜eda V, Galaviz-Herna´ndez C, Villanueva-Fierro I (2012) Biochemical differences in ethnic groups in Durango, Mexico. Ethn Dis 22:102–105 22. Luyckx FH, Desaive C, Thiry A, Dewe´ W, Scheen AJ, Gielen JE, Lefe`bvre PJ (1998) Liver abnormalities in severely obese subjects: effect of drastic weight loss after gastroplasty. Int J Obes Relat Metab Disord 22:222–226 23. Golik A, Rubio A, Weintraub M, Byrne L (1991) Elevated serum liver enzymes in obesity: a dilemma during clinical trials. Int J Obes 15:797–801 24. Qu HQ, Li Q, Grove ML, Lu Y, Pan JJ, Rentfro AR, Bickel PE, Fallon MB, Hanis CL, Boerwinkle E, McCormick JB, FisherHoch SP (2012) Population-based risk factors for elevated alanine aminotransferase in a South Texas Mexican–American population. Arch Med Res 43:482–488 25. Li Q, Qu HQ, Rentfro AR, Grove ML, Mirza S, Lu Y, Hanis CL, Fallon MB, Boerwinkle E, Fisher-Hoch SP, McCormick JB (2012) PNPLA3 polymorphisms and liver aminotransferase levels in a Mexican American population. Clin Invest Med 35:E237– E245 26. Sookoian S, Pirola CJ (2011) Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 53:1883–1894 27. Giudice EM, Grandone A, Cirillo G, Santoro N, Amato A, Brienza C, Savarese P, Marzuillo P, Perrone L (2011) The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat. PLoS One 6:e27933 28. Graff M, North KE, Franceschini N, Reiner AP, Feitosa M, Carr JJ, Gordon-Larsen P, Wojczynski MK, Borecki IB (2013) PNPLA3 gene-by-visceral adipose tissue volume interaction and
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
the pathogenesis of fatty liver disease: the NHLBI family heart study. Int J Obes 37:432–438 Romeo S, Sentinelli F, Cambuli VM, Incani M, Congiu T, Matta V, Pilia S, Huang-Doran I, Cossu E, Loche S, Baroni MG (2010) The 148M allele of the PNPLA3 gene is associated with indices of liver damage early in life. J Hepatol 53:335–338 Goran MI, Walker R, Le KA, Mahurkar S, Vikman S, Davis JN, Spruijt-Metz D, Weigensberg MJ, Allayee H (2010) Effects of PNPLA3 on liver fat and metabolic profile in Hispanic children and adolescents. Diabetes 59:3127–3130 Palmer ND, Musani SK, Yerges-Armstrong LM, Feitosa MF, Bielak LF, Hernaez R, Kahali B, Carr JJ, Harris TB, Jhun MA, Kardia SL, Langefeld CD, Mosley TH Jr, Norris JM, Smith AV, Taylor HA, Wagenknecht LE, Liu J, Borecki IB, Peyser PA, Speliotes EK (2013) Characterization of European ancestry nonalcoholic fatty liver disease-associated variants in individuals of African and hispanic descent. Hepatology 58:966–975 Chang Y, Ryu S, Sung E, Jang Y (2007) Higher concentrations of alanine aminotransferase within the reference interval predict nonalcoholic fatty liver disease. Clin Chem 53:686–692 Roman S, Tanaka Y, Khan A, Kurbanov F, Kato H, Mizokami M, Panduro A (2010) Occult hepatitis B in the genotype H-infected Nahuas and Huichol native Mexican population. J Med Virol 82:1527–1536 Tian C, Stokowski RP, Kershenobich D, Ballinger DG, Hinds DA (2010) Variant in PNPLA3 is associated with alcoholic liver disease. Nat Genet 42:21–23 Valenti L, Colombo M, Fargion S (2011) Modulation of the effect of PNPLA3 I148M mutation on steatosis and liver damage by alcohol intake in patients with chronic hepatitis C. J Hepatol 55:1470–1471 Santoro N, Kursawe R, D’Adamo E, Dykas DJ, Zhang CK, Bale AE, Calı´ AM, Narayan D, Shaw MM, Pierpont B, Savoye M, Lartaud D, Eldrich S, Cushman SW, Zhao H, Shulman GI, Caprio S (2010) A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology 52:1281–1290 Lin YC, Chang PF, Hu FC, Yang WS, Chang MH, Ni YH (2011) A common variant in the PNPLA3 gene is a risk factor for nonalcoholic fatty liver disease in obese Taiwanese children. J Pediatr 158:740–744 Larrieta-Carrasco E, Leo´n-Mimila P, Villarreal-Molina T, Villamil-Ramı´rez H, Romero-Hidalgo S, Jacobo-Albavera L, Gutie´rrez-Vidal R, Lo´pez-Contreras BE, Guillen-Pineda LE, Sa´nchez-Mun˜oz F, Bojalil R, Mejı´a Domı´nguez AM, Me´ndezSa´nchez N, Domı´nguez-Lo´pez A, Aguilar-Salinas CA, CanizalesQuinteros S (2013) Association of the I148M/PNPLA3 variant with elevated alanine transaminase levels in normal-weight and overweight/obese Mexican children. Gene 520:185–188
123
PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations Elena Larrieta-Carrasco • Victor Acun˜a-Alonzo • Rafael Vela´zquez-Cruz • Rodrigo Barquera-Lozano • Paola Leo´n-Mimila • Hugo Villamil-Ramı´rez • Marta Menjivar • Sandra Romero-Hidalgo • Nahu´m Me´ndez-Sa´nchez • Vanessa Ca´rdenas • Manuel Ban˜uelos-Moreno • Yvonne N. Flores • Manuel Quiterio • Jorge Salmero´n • Fausto Sa´nchez-Mun˜oz • Teresa Villarreal-Molina Carlos A. Aguilar-Salinas • Samuel Canizales-Quinteros
•
Received: 25 September 2013 / Accepted: 21 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with Elena Larrieta-Carrasco and Victor Acun˜a-Alonzo have contributed equally to this study.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3341-0) contains supplementary material, which is available to authorized users. E. Larrieta-Carrasco Departamento de Gastroenterologı´a, Instituto Nacional de Ciencias Me´dicas y Nutricio´n Salvador Zubira´n (INCMNSZ), Mexico, Mexico V. Acun˜a-Alonzo P. Leo´n-Mimila H. Villamil-Ramı´rez M. Menjivar S. Canizales-Quinteros (&) Unidad de Geno´mica de Poblaciones Aplicada a la Salud, Facultad de Quı´mica, Universidad Nacional Auto´noma de Me´xico (UNAM)-Instituto Nacional de Medicina Geno´mica (INMEGEN), Mexico, Mexico e-mail: [email protected] V. Acun˜a-Alonzo R. Barquera-Lozano Laboratorio de Gene´tica Molecular, Escuela Nacional de Antropologı´a e Historia (ENAH), Mexico, Mexico
serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels C40 U/L and 564 controls with ALT \40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7 %, and varied according to obesity status: 14.4 % in normal weight, 19.9 % in overweight and 24.5 % in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91–5.20; P = 7.1 9 10-6) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95 % CI 1.50–3.33; P = 8.1 9 10-5). This is S. Romero-Hidalgo Laboratorio de Geno´mica Computacional, INMEGEN, Mexico, Mexico N. Me´ndez-Sa´nchez Unidad de Investigacio´n del Hı´gado, Fundacio´n Me´dica Sur, Mexico, Mexico M. Ban˜uelos-Moreno Y. N. Flores M. Quiterio J. Salmero´n Unidad de Investigacio´n Epidemiolo´gica y en Servicios de Salud, Instituto Mexicano del Seguro Social, Cuernavaca, Morelos, Mexico Y. N. Flores UCLA Department of Health Policy and Management, Fielding School of Public Health, Johnson Comprehensive Cancer Center, Los Angeles, CA, USA
R. Vela´zquez-Cruz V. Ca´rdenas ´ seo, INMEGEN, Laboratorio de Geno´mica del Metabolismo O Mexico, Mexico
123
Mol Biol Rep
the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations. Keywords I148M/PNPLA3 Alanine transaminase Native Mexican Mexican Mestizos Abbreviations PNPLA3 Patatin-like phospholipase domain-contain 3 NAFLD Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis ALT Alanine transaminase AST Aspartate transaminases IMSS Mexican Institute of Social Security BMI Body mass index HOMA-IR Homeostasis model assessment for insulin resistance HDL-C High-density lipoprotein cholesterol OR Odds ratio
(PNPLA3) gene, as the strongest genetic factor associated with NAFLD and elevated ALT and aspartate transaminase (AST) levels in a large multiethnic population [14]. Interestingly, the most striking effects of the PNPLA3 I148M variant were observed in Hispanics who had the highest 148M risk allele frequency [14]. In addition, Native American ancestry was recently associated with higher ALT levels in a Mexican–American cohort [15]. Because the Mexican population resulted from the admixture of mainly European (Spaniard) and Native American populations, it is likely that the elevated 148M allele frequency resulted from the contribution of the Native American gene pool. The aim of the present study was to assess the frequency of the 148M risk allele and whether it is associated with serum ALT levels in Native Mexican and Mexican Mestizo individuals.
Materials and methods Study population Native American populations
Introduction Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) [1]. Elevated levels of alanine transaminase (ALT) have been associated with NAFLD and may be considered as a sensitive indicator of liver-cell injury [2]. Several studies have suggested that both liver fat accumulation and ALT levels vary among different populations, with Hispanics having the highest prevalence of NAFLD and elevated ALT levels [3–9]. According to heritability estimations, 20–60 % of ALT level variability can be attributed to genetic factors [10–13]. A genome-wide association study identified the nonsynonymous I148M variant (rs738409) in the patatin-like phospholipase domain-containing 3
J. Salmero´n Centro de Investigacio´n en Salud Poblacional, Instituto Nacional de Salud Pu´blica, Cuernavaca, Mexico F. Sa´nchez-Mun˜oz Departamento de Inmunologı´a, Instituto Nacional de Cardiologı´a ‘‘Ignacio Cha´vez’’ (INCICh), Mexico, Mexico T. Villarreal-Molina Laboratorio de Enfermedades Cardiovasculares, INMEGEN, Mexico, Mexico C. A. Aguilar-Salinas Departamento de Endocrinologı´a y Metabolismo, INCMNSZ, Mexico, Mexico
123
We evaluated a total of 919 unrelated Native American adults aged 18–91 years, including 350 Nahuas from Central Mexico, 179 Totonacs from Eastern Mexico, 86 Seris from Northern Mexico, 120 Coras from Western Mexico, and 184 Zapotecs from Southwestern Mexico. Only subjects born in their communities who speak their native language were included. The study was approved by the Ethics Committees of all participating Institutions, local authorities gave their approval to participate, and a translator was used as needed. Metabolic measurements were only available in the Nahua and Totonac subjects. Mexican Mestizo population A total of 705 Mexican Mestizo individuals working at the Mexican Institute of Social Security (IMSS) of the state of Morelos were recruited for a case–control study. This study was performed comparing individuals with elevated and non-elevated ALT levels, defined as ALT measurements C40 U/L (141 cases) or \40 U/L (564 controls) in two or more measurements over a 6 month period. The study protocol was approved by the IMSS Ethics Committee and all participants provided written informed consent prior to their inclusion. Anthropometric and biochemical measurements Weight and height were measured and all individuals. Waist circumference was measured in 367 indigenous
Mol Biol Rep
subjects and in all Mexican Mestizo individuals. BMI was calculated as weight in kilograms/height in meters squared. Participants were grouped according to BMI as normal weight (BMI B 24.9 kg/m2), overweight (25 B BMI B 29.9 kg/m2), or obese (BMI C 30 kg/m2). Biochemical parameters were measured in all participants in a fasting state as previously described [16]. The homeostasis model assessment for insulin resistance (HOMA-IR) was used to estimate insulin sensitivity in Indigenous Mexican population [17]. Serum levels of ALT and AST were determined only in Mexican Mestizo, Nahua and Totonac individuals using commercially available standardized methods (Beckman Coulter, Fullerton, CA, USA). I148M/PNPLA3 genotyping All participants were genotyped for rs738409 I148M variant using TaqMan assays (ABI Prism 7900HT Sequence Detection System; Applied Biosystems, Foster City, CA, USA). No discordant genotypes were observed in 39 duplicate samples. Deviation from Hardy–Weinberg equilibrium was not observed for this polymorphism. Statistical analysis Student’s t test was used to compare clinical and biochemical characteristics of normal weight to overweight and obese individuals. Differences among genotypes for categorical variables were evaluated using a chi square test. Associations of I148M genotype with quantitative variables were analyzed by linear regression analysis adjusting for age, gender, BMI and ethnicity when appropriate. A multiple linear regression model was used to assess the interaction between waist circumference and the I148M polymorphism on ALT levels, adjusting for the variables mentioned above (SPSS V15, statistical package; Chicago, IL, USA).
Results Association of PNPLA3 I148M with ALT and AST levels in Native Americans PNPLA3 148M risk allele frequencies were similar among the five Native groups (mean 0.73), ranging from 0.62 in Seris to 0.85 in Coras (Table 1). Anthropometric and biochemical measurements were available only in the Nahua and Totonac populations (Table 2). Although the frequency of elevated ALT levels was higher in Nahuas (20.9 %) than in Totonac individuals (15.5 %), the difference was not statistically significant (P = 0.135). The
Table 1 PNPLA3 I148M genotypes and allele distribution by native Mexican population Population
N
Genotypes N (%) I148I
I148M
M148M
M-allele frequency
Seri
86
9 (10.5)
47 (54.7)
30 (34.9)
0.62
Cora
120
4 (3.3)
28 (23.3)
88 (73.3)
0.85
Nahua Totonac
350 179
31 (8.9) 13 (7.3)
151 (43.1) 63 (35.2)
168 (48.0) 103 (57.5)
0.70 0.75
Zapotec
184
11 (6.0)
71 (38.6)
102 (55.4)
0.75
overall frequency of elevated ALT levels was 18.7 %, and varied according to obesity status: 14.4 % in normal weight, 19.9 % in overweight and 24.5 % in obese individuals. Moreover, the frequency of elevated ALT levels was higher in male individuals, although gender differences were statistically significant only for comparisons in the entire sample and in overweight individuals (P = 0.001). Because ALT and AST levels showed no significant differences between I148I homozygous and I148M heterozygous individuals in the entire sample (P [ 0.21 and P [ 0.43, respectively), associations were tested under a recessive model (I148I and I148M vs. M148M). Mean ALT and AST levels were 26.4 and 19.1 % higher respectively in M148M homozygous as compared to I148I/ I148M individuals (P = 5.3 9 10-6 and 2.0 9 10-6 respectively, after adjustment for age, gender, BMI and ethnic group). On stratification according to gender, the association of the M148M genotype with ALT levels was statistically significant only in females, in the entire sample (P = 6.6 9 10-6), as well as in the overweight and obesity groups (P = 3.8 9 10-5 and 0.014, respectively). Very similar results were observed on comparing elevated vs non-elevated ALT levels (Table 3). The M148M genotype was not significantly associated with any other metabolic parameters in Native Mexican individuals (Supplementary Table 1). Moreover, no significant interactions between BMI or waist circumference and I148M polymorphism were observed for ALT levels (P = 0.549 and P = 0.256, respectively). Association of PNPLA3 I148M with elevated ALT and AST levels in Mexican Mestizos PNPLA3 148M risk allele frequency in Mexican Mestizos was 0.58. The M148M genotype was significantly associated with elevated ALT levels in the entire simple (OR = 2.24, 95 % CI 1.50–3.33; P = 8.1 9 10-5 adjusted for gender, age and BMI). On stratification according to gender, the association of the M148M genotype with elevated ALT levels remained significant only in women
123
Mol Biol Rep Table 2 Anthropometric and biochemical parameters of Nahua and Totonac populations according to BMI All
Normal weight
Overweight
Obese
N (%)
529
195 (36.9)
236 (44.6)
98 (18.5)
Male (%)
144 (27.2)
69 (35.4)
56 (23.7)*
19 (19.4)*
47.6 ± 15.1
50.3 ± 18.1
45.8 ± 13.5*
46.5 ± 11.3*
BMI (kg/m )
26.5 ± 4.3
22.3 ± 2.0
27.2 ± 1.4*
33.0 ± 2.7*
Waist circumference (cm)
89.4 ± 13.6
81.6 ± 12.8
90.3 ± 8.8*
102.5 ± 13.2*
HOMA-IR
2.4 (1.5–4.2)
1.6 (0.9–2.3)
2.9 (2.1–4.6)*
4.9 (3.3–7.9)*
Age (years) 2
Total cholesterol (mg/dL)
183.5 ± 40.4
179.2 ± 41.2
185.5 ± 40.5
187.3 ± 38.2
Triglycerides (mg/dL)
162.0 (111.0–229.0)
130.0 (95.0–189.0)
177.0 (124.0–235.0)*
189.0 (139.5–279.5)*
HDL-C (mg/dL)
40.5 ± 12.6
44.8 ± 15.5
38.4 ± 10.2*
37.0 ± 8.3*
ALT (U/L)
26.0 (21.0–36.0)
24.0 (20.0–31.0)
27.0 (21.0–37.0)*
30.5 (23.0–39.5)*
Male Female
30.5 (23.0–41.0) 25.0 (20.0–33.0)
25.0 (21.5–33.5) 24.0 (19.8–30.0)
37.0 (28.0–49.8) 25.0 (20.0–34.0)
33.0 (30.0–44.0) 29.0 (23.0–38.0)
P
1.3 9 10-4
0.357
2.7 9 10-5
0.217
31.0 (26.0–38.0)
31.0 (27.0–37.0)
31.0 (25.0–38.0)
31.0 (26.0–40.0)
Male
33.0 (29.0–43.8)
33.0 (29.0–44.0)
36.0 (31.0–43.0)
31.0 (24.0–44.0)
Female
29.0 (25.0–37.0)
29.5 (26.0–35.3)
29.0 (25.0–36.0)
30.0 (26.0–40.0)
P
5.8 9 10-5
0.006
0.002
0.527
AST (U/L)
ALT C40 U/L (%) Male
99 (18.7)
28 (14.4)
47 (19.9)
24 (24.5)*
42 (29.2)
13 (18.8)
22 (39.3)
7 (36.8)
Female
57 (14.8)
15 (11.9)
25 (13.9)
17 (21.5)
P
0.001
0.214
0.001
0.224
Data are means ± standard deviations, medians (interquartil range) or N (%). HOMA-IR and triglycerides were log-transformed before analysis BMI body mass index, HOMA-IR homeostasis model of assessment insulin resistance index, HDL-C high density lipoprotein cholesterol, ALT alanine transaminase, AST aspartate transaminase * P \ 0.05 compared to normal weight and P \ 0.05 compared to overweight
(OR = 3.21, 95 % CI 1.98–5.21; P = 2.5 9 10-6) (Table 4). As in the indigenous population, the effect of the interaction between BMI or waist circumference and the I148M polymorphism on ALT levels was not significant (P = 0.579 and P = 0.747, respectively).
Discussion Although the Mexican indigenous population includes over 10 million individuals [18], there are very few studies analyzing genetic associations and the impact of metabolic disease in these populations [19, 20]. It has been suggested that Native American ancestry is associated with higher ALT levels in Mexican–Americans [15]. However, there is only one previous study evaluating the ALT levels in a Mexican indigenous population (Tepehuanos) [21]. In the present study, the frequency of ALT levels C40 U/L in Nahuas and Totonacs was 18.7 %, with median values (26 U/L) similar to those reported in Tepehuanos (30.5 U/L). In contrast, the prevalence of elevated ALT levels was higher than in white and black populations of the Third
123
National Health and Nutrition Examination Survey (7.1 and 8.1 %, respectively) [7]. Elevated ALT levels were more frequent in overweight and obese than in normal weight subjects, in accordance with previous reports [22, 23]. To the best of our knowledge, this is the first study to estimate the frequency of elevated ALT levels in over 500 Mexican indigenous individuals from central Mexico. The association of I148M PNPLA3 with elevated ALT levels was highly significant in both Mexican Indigenous and Mestizo populations. Although this association has been previously reported in Mexican–American adults [24, 25], this is the first study showing evidence of an association of this polymorphism with chronically elevated ALT levels in Mexican Mestizo individuals. Interestingly, although the percentage of males with elevated ALT levels was higher in those bearing M148M genotype, the association was significant only in Native Mexican and Mestizo women. The lack of significance in males may be partially due to the reduced sample size and/or to the lower percentage of obese males compared to females; however it is clear that the effect size and level of significance of these associations was much higher in women. This is consistent
Mol Biol Rep Table 3 ALT levels of Nahua and Totonac populations according to PNPLA3 I148M genotypes stratified by BMI
I148I
I148M
M148M
P*
All N = 529 ALT (U/L)
44 (8.3) 23.0 (21.0–30.8)
214 (40.5)
271 (51.2)
25.0 (20.0–32.3)
28.0 (21.0–40.0)
5.3 9 10-6
Male
25.0 (22.0–33.8)
30.0 (23.0–42.0)
34.0 (25.0–44.0)
0.065
Female
23.0 (19.0–29.0)
24.0 (19.0–30.0)
27.0 (20.0–38.8)
6.6 9 10-6
2 (2.0)
27 (27.3)
70 (70.7)
7.1 9 10-6
Male
2 (4.8)
18 (42.9)
22 (52.4)
0.270
Female
0 (0.0)
9 (15.8)
48 (84.2)
3.9 9 10-6
N = 195
22 (11.3)
71 (36.4)
102 (52.3)
ALT (U/L)
ALT C40 U/L (%)
Normal weight 23 (20–29)
24 (20–29)
25 (20–34)
Male
23 (21–25)
25 (21–37)
27 (22–37)
0.163 0.314
Female
26 (21–29)
23 (20–28)
24 (19–34)
0.353
0 (0.0)
8 (28.6)
20 (71.4)
0.019
0 (0.0) 0 (0.0)
6 (46.2) 2 (13.3)
7 (53.8) 13 (86.7)
0.542 0.020
N = 236
15 (6.4)
99 (41.9)
122 (51.7)
ALT (U/L)
23 (19–30)
24 (19–34)
ALT C40 U/L (%) Male Female Overweight
30 (24–39)
35 (24–43)
39 (34–59)
0.105
Female
22 (19–26)
23 (18–30)
27 (21–38)
3.8 9 10-5
0 (0.0)
11 (23.4)
36 (76.6)
3.4 9 10-4
Male
0 (0.0)
7 (31.8)
15 (68.2)
0.109
Female
0 (0.0)
4 (16.0)
21 (84)
0.001
7 (7.1)
44 (44.9)
47 (48.0)
Obese
ALT alanine transaminase
N = 98
* P values calculated by linear regression adjusted for age, sex, BMI and ethnicity
ALT (U/L)
P value calculated by multiple logistic regression analysis adjusted for age, sex, BMI and ethnicity as covariates when was appropriate
5.1 9 10-5
Male ALT C40 U/L (%) Data are medians (interquartil range) or N (%)
30 (24–43)
32 (22–40)
28 (23–36)
33 (22–43)
Male
40 (32–62)
33 (26–51)
30 (25–36)
0.585
Female
23 (20–33)
26 (23–31)
34 (22–44)
0.014
ALT C40 U/L (%) Male Female
0.048
2 (8.3)
8 (33.3)
14 (58.3)
0.039
2 (28.6) 0 (0.0)
5 (71.4) 3 (17.6)
0 (0.0) 14 (82.4)
0.252 0.014
with previous findings suggesting sex differences in the genetic association of PNPLA3 polymorphisms and liver enzymes [25, 26]. Even though the magnitude of the association of PNPLA3 with ALT levels and risk of liver damage has been reported to be modulated by abdominal fat in children and adults [27, 28], interactions between waist circumference and I148M polymorphism for ALT levels were not significant in Mestizos or indigenous individuals. Several studies have shown that this polymorphism is associated with NAFLD in Hispanics [29–31]. Although elevated ALT levels have been correlated with the presence of hepatic fat on imaging in adults [32], a limitation of the present study was the lack of imaging techniques to diagnose fatty liver. Another limitation to be considered is that the analysis in Mexican Mestizos was not tested for
admixture, however the association of this polymorphism with elevated ALT levels is consistent with previous observations in Mexican–Americans [24]. In addition, other factors affecting ALT levels such as alcohol consumption and liver infection were not assessed. In this regard, although Mexico is known to be a low endemic area for hepatitis B and C infection, a high frequency of occult hepatitis B has been reported in Indigenous Mexicans [33]. However, it is noteworthy that the PNPLA3 polymorphism is also associated with liver injury in individuals with hepatitis infection and alcoholic fatty liver disease [34, 35]. In summary, the Mexican indigenous population has the highest frequency of the 148M/PNPLA3 risk allele (0.73), compared to African American (0.18), Caucasians (0.26–0.32), Taiwanese (0.39), or other Hispanic
123
Mol Biol Rep Table 4 Association of PNPLA3 M148M genotype with elevated ALT levels in Mexican Mestizos I148I
I148M
M148M
P
All (N = 705) Cases ALT C40 U/L
21 (14.9)
51 (36.2)
69 (48.9)
111 (19.7)
281 (49.8)
172 (30.5)
Cases ALT C40 U/L
8 (15.4)
23 (44.2)
21 (40.4)
Controls ALT \40 U/L Female
17 (13.6)
58 (46.4)
50 (40.0)
Cases ALT C40 U/L
13 (14.6)
28 (31.5)
48 (53.9)
Controls ALT \40 U/L
94 (21.4)
223 (50.8)
122 (27.8)
Controls ALT \40 U/L
8.1 9 10-5
Male 0.965
2.5 9 10-6
Data are N (%). P value calculated by multiple logistic regression analysis adjusted for age, sex and BMI when was appropriate ALT alanine transaminase
populations (0.48–0.61) [27, 29, 30, 35–38]. This suggests that the high frequency of this allele in Hispanics, including Mexican Mestizos, is due to their indigenous component. The M148M genotype may be considered as an important risk factor for liver damage in Mexican Indigenous and Mestizo populations. Acknowledgments This research was supported by grant SALUD2009-01-113861 from the Consejo Nacional de Ciencia y Tecnologı´a (CONACyT). The study of the IMSS cohort is supported in part by grants from IMSS (FIS/IMSS/PROT/G12/1117) and CONACyT (26267M, SALUD-2008-01-87783 and SALUD-2011-C01-161930). PLM is in the Ciencias Bioquı´micas PhD program from Universidad Nacional Auto´noma de Me´xico (UNAM) and HVR is in the Ciencias Biolo´gicas y de la Salud PhD program from Universidad Auto´noma Metropolitana (UAM), and were supported by the PhD fellowships number 244112 and 234714 from CONACyT, Me´xico.
References 1. Angulo P (2002) Nonalcoholic fatty liver disease. N Engl J Med 346:1221–1231 2. Pratt DS, Kaplan MM (2000) Evaluation of abnormal liverenzyme results in asymptomatic patients. N Engl J Med 342:1266–1271 3. Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH (2004) Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 40:1387–1395 4. Weston SR, Leyden W, Murphy R, Bass NM, Bell BP, Manos MM, Terrault NA (2005) Racial and ethnic distribution of nonalcoholic fatty liver in persons with newly diagnosed chronic liver disease. Hepatology 41:372–379
123
5. Williams CD, Stengel J, Asike MI, Torres DM, Shaw J, Contreras M, Landt CL, Harrison SA (2011) Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 140:124–131 6. Fraser A, Longnecker MP, Lawlor DA (2007) Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999–2004. Gastroenterology 133:1814–1820 7. Clark JM, Brancati FL, Diehl AM (2003) The prevalence and etiology of elevated aminotransferase levels in the United States. Am J Gastroenterol 98:960–967 8. Ioannou GN, Boyko EJ, Lee SP (2006) The prevalence and predictors of elevated serum aminotransferase activity in the United States in 1999–2002. Am J Gastroenterol 101:76–82 9. Flores Y, Yee HF, Leng M, Bastani R, Escarce J, Salmero´n J, Morales L (2008) Risk Factors for Chronic Liver Disease in Blacks, Mexican Americans, and Whites in the United States: results From NHANES IV, 1999–2004. Am J Gastroenterol 103:2231–2238 10. Brouwers MC, Cantor RM, Kono N, Yoon JL, van Kallen CJ, Bilderbeek-Beckers MA, van Greevenbroek MM, Lusis AJ, de Bruin TW (2006) Heritability and genetic loci of fatty liver in familial combined hiperlipidemia. J Lipid Res 47:2799–2807 11. Makkonen J, Pietila¨inen KH, Rissanen A, Kaprio J, Yki-Ja¨rvinen H (2009) Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: a study in monozygotic and dizygotic twins. J Hepatol 50:1035–1042 12. Day CP (2010) Genetic and environmental susceptibility to nonalcoholic fatty liver disease. Dig Dis 28:255–260 13. Alisi A, Cianfarani S, Manco M, Agostoni C, Nobili V (2012) Non-alcoholic fatty liver disease and metabolic syndrome in adolescents: pathogenetic role of genetic background and intrauterine environment. Ann Med 44:29–40 14. Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC, Hobbs HH (2008) Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 40:1461–1465 15. Qu HQ, Li Q, Lu Y, Fisher-Hoch SP, McCormick JB (2012) Translational genomic medicine: common metabolic traits and ancestral components of Mexican Americans. J Med Genet 49:544–545 16. Villarreal-Molina MT, Aguilar-Salinas CA, Rodrı´guez-Cruz M, Rian˜o D, Villalobos-Comparan M, Coral-Vazquez R, Menjivar M, Yescas-Gomez P, Ko¨nigsoerg-Fainstein M, Romero-Hidalgo S, Tusie-Luna MT, Canizales-Quinteros S (2007) The ATP-binding cassette transporter A1 R230C variant affects HDL cholesterol levels and BMI in the Mexican population: association with obesity and obesity-related comorbidities. Diabetes 56:1881–1887 17. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412–419 18. Ferna´ndez Ham P, Tuira´n Gutie´rrez A, Ordorica Mellado M, Salas y Villago´mez G, Camarena Co´rdova RM, Serrano Carreto E (2006) Comisio´n Nacional para el Desarrollo de los Pueblos Indı´genas (CDI)-Programa de las Naciones Unidas para el Desarrollo (PNUD). Informe sobre Desarrollo Humano de los Pueblos Indı´genas de Me´xico 2006. http://www.cdi.gob.mx/idh/ informe_desarrollo_humano_pueblos_indigenas_mexico_2006.pdf. Accessed Sept 2013 19. Aguilar CA, Talavera G, Ordovas JM, Barriguete JA, Guille´n LE, Leco ME, Pedro-Botet J, Gonzalez-Barranco J, Go´mez-Pe´rez FJ, Rull JA (1999) The apolipoprotein E4 allele is not associated with an abnormal lipid profile in a Native American population following its traditional lifestyle. Atherosclerosis 142:409–414
Mol Biol Rep 20. Acun˜a-Alonzo V, Flores-Dorantes T, Kruit JK, Villarreal-Molina T, Arellano-Campos O, Hu¨nemeier T, Moreno-Estrada A, OrtizLo´pez MG, Villamil-Ramı´rez H, Leo´n-Mimila P, VillalobosComparan M, Jacobo-Albavera L, Ramı´rez-Jime´nez S, Sikora M, Zhang LH, Pape TD, Granados-Silvestre MA, Montufar-Robles I, Tito-Alvarez AM, Zurita-Salinas C, Bustos-Arriaga J, CedilloBarro´n L, Go´mez-Trejo C, Barquera-Lozano R, Vieira-Filho JP, Granados J, Romero-Hidalgo S, Huertas-Va´zquez A, Gonza´lezMartı´n A, Gorostiza A, Bonatto SL, Rodrı´guez-Cruz M, Wang L, Tusie´-Luna T, Aguilar-Salinas CA, Lisker R, Moises RS, Menjivar M, Salzano FM, Knowler WC, Bortolini MC, Hayden MR, Baier LJ, Canizales-Quinteros S (2010) A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans. Hum Mol Genet 19:2877–2885 21. Lares-Asseff I, Lujı´n-Garcı´a A, Sosa-Macı´as M, Lazalde-Ramos B, Loera-Castan˜eda V, Galaviz-Herna´ndez C, Villanueva-Fierro I (2012) Biochemical differences in ethnic groups in Durango, Mexico. Ethn Dis 22:102–105 22. Luyckx FH, Desaive C, Thiry A, Dewe´ W, Scheen AJ, Gielen JE, Lefe`bvre PJ (1998) Liver abnormalities in severely obese subjects: effect of drastic weight loss after gastroplasty. Int J Obes Relat Metab Disord 22:222–226 23. Golik A, Rubio A, Weintraub M, Byrne L (1991) Elevated serum liver enzymes in obesity: a dilemma during clinical trials. Int J Obes 15:797–801 24. Qu HQ, Li Q, Grove ML, Lu Y, Pan JJ, Rentfro AR, Bickel PE, Fallon MB, Hanis CL, Boerwinkle E, McCormick JB, FisherHoch SP (2012) Population-based risk factors for elevated alanine aminotransferase in a South Texas Mexican–American population. Arch Med Res 43:482–488 25. Li Q, Qu HQ, Rentfro AR, Grove ML, Mirza S, Lu Y, Hanis CL, Fallon MB, Boerwinkle E, Fisher-Hoch SP, McCormick JB (2012) PNPLA3 polymorphisms and liver aminotransferase levels in a Mexican American population. Clin Invest Med 35:E237– E245 26. Sookoian S, Pirola CJ (2011) Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 53:1883–1894 27. Giudice EM, Grandone A, Cirillo G, Santoro N, Amato A, Brienza C, Savarese P, Marzuillo P, Perrone L (2011) The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat. PLoS One 6:e27933 28. Graff M, North KE, Franceschini N, Reiner AP, Feitosa M, Carr JJ, Gordon-Larsen P, Wojczynski MK, Borecki IB (2013) PNPLA3 gene-by-visceral adipose tissue volume interaction and
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
the pathogenesis of fatty liver disease: the NHLBI family heart study. Int J Obes 37:432–438 Romeo S, Sentinelli F, Cambuli VM, Incani M, Congiu T, Matta V, Pilia S, Huang-Doran I, Cossu E, Loche S, Baroni MG (2010) The 148M allele of the PNPLA3 gene is associated with indices of liver damage early in life. J Hepatol 53:335–338 Goran MI, Walker R, Le KA, Mahurkar S, Vikman S, Davis JN, Spruijt-Metz D, Weigensberg MJ, Allayee H (2010) Effects of PNPLA3 on liver fat and metabolic profile in Hispanic children and adolescents. Diabetes 59:3127–3130 Palmer ND, Musani SK, Yerges-Armstrong LM, Feitosa MF, Bielak LF, Hernaez R, Kahali B, Carr JJ, Harris TB, Jhun MA, Kardia SL, Langefeld CD, Mosley TH Jr, Norris JM, Smith AV, Taylor HA, Wagenknecht LE, Liu J, Borecki IB, Peyser PA, Speliotes EK (2013) Characterization of European ancestry nonalcoholic fatty liver disease-associated variants in individuals of African and hispanic descent. Hepatology 58:966–975 Chang Y, Ryu S, Sung E, Jang Y (2007) Higher concentrations of alanine aminotransferase within the reference interval predict nonalcoholic fatty liver disease. Clin Chem 53:686–692 Roman S, Tanaka Y, Khan A, Kurbanov F, Kato H, Mizokami M, Panduro A (2010) Occult hepatitis B in the genotype H-infected Nahuas and Huichol native Mexican population. J Med Virol 82:1527–1536 Tian C, Stokowski RP, Kershenobich D, Ballinger DG, Hinds DA (2010) Variant in PNPLA3 is associated with alcoholic liver disease. Nat Genet 42:21–23 Valenti L, Colombo M, Fargion S (2011) Modulation of the effect of PNPLA3 I148M mutation on steatosis and liver damage by alcohol intake in patients with chronic hepatitis C. J Hepatol 55:1470–1471 Santoro N, Kursawe R, D’Adamo E, Dykas DJ, Zhang CK, Bale AE, Calı´ AM, Narayan D, Shaw MM, Pierpont B, Savoye M, Lartaud D, Eldrich S, Cushman SW, Zhao H, Shulman GI, Caprio S (2010) A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology 52:1281–1290 Lin YC, Chang PF, Hu FC, Yang WS, Chang MH, Ni YH (2011) A common variant in the PNPLA3 gene is a risk factor for nonalcoholic fatty liver disease in obese Taiwanese children. J Pediatr 158:740–744 Larrieta-Carrasco E, Leo´n-Mimila P, Villarreal-Molina T, Villamil-Ramı´rez H, Romero-Hidalgo S, Jacobo-Albavera L, Gutie´rrez-Vidal R, Lo´pez-Contreras BE, Guillen-Pineda LE, Sa´nchez-Mun˜oz F, Bojalil R, Mejı´a Domı´nguez AM, Me´ndezSa´nchez N, Domı´nguez-Lo´pez A, Aguilar-Salinas CA, CanizalesQuinteros S (2013) Association of the I148M/PNPLA3 variant with elevated alanine transaminase levels in normal-weight and overweight/obese Mexican children. Gene 520:185–188
123
