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Low Prevalence of Interleukin-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes in Mexican Subjects with a Family History of Type 2 Diabetes
Archives of Medical Research 44 (2013) 529e534
ORIGINAL ARTICLE
Low Prevalence of Interleukin-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes in Mexican Subjects with a Family History of Type 2 Diabetes Irma Zamora-Ginez,a Alejandra Guadalupe Garcıa-Zapien,b Silvia Esperanza Flores-Martınez,b,c Jose Sanchez-Corona,b,c Blanca G. Baez-Duarte,a Enrique Torres-Rasgado,a Jose R. Romero,d Ricardo Perez-Fuentes,a,c,e Francisco Mendoza-Carrera,b and The Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social a
Facultad de Medicina, Benemerita Universidad Autonoma de Puebla (BUAP), Puebla, Mexico Divisi on Medicina Molecular, Centro de Investigacion Biomedica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Mexico c Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social, Mexico, D.F., Mexico d Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Boston, Massachusetts e Laboratorio de Investigacion en Fisiopatologıa de Enfermedades Cronicas, CIBIOR, IMSS, Atlixco, Puebla, Mexico
b
Received for publication March 20, 2013; accepted August 26, 2013 (ARCMED-D-13-00158).
Background and Aims. There is evidence that family history of type 2 diabetes (FHT2D) and single nucleotide polymorphisms (SNP) on the IL-6 gene promoter region are separately associated with the risk of developing type 2 diabetes. However the relationship between adult Mexican subjects with FHT2D and genotypes/haplotypes for IL-6 gene has not been explored. The aim of the present work was to study the prevalence of IL-6 598G OA572GOC174GOC haplotypes among subjects with FHT2D and to determine whether their presence influences the relationship between FHT2D and risk factors for diabetes. Methods. Two hundred fifty eight nondiabetic subjects participated in this study; 153 with and 105 without FHT2D. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) was used for genotyping. Logistic regression analysis was employed to assess the impact of IL-6 haplotypes on FHT2D per se and hyperinsulinemia and insulin resistance as risk factors for diabetes. Results. Subjects with FHT2D showed a higher prevalence of hyperinsulinemia and insulin resistance (IR) than those without FHT2D (14.4 vs. 5.7%, p 5 0.029, and 14.2 vs. 7.0% p 5 0.050, respectively). Lower prevalence of 598 -572-174 (AGC)-haplotype (19%) in subjects with FHT2D was observed as well as a lower prevalence of hyperinsulinemia and IR among AGC haplotype carriers (12 and 14%, respectively). The relationship between FHT2D and IR was modified by the presence of AGC haplotype (from OR, 2.70; 95% CI, 0.99e7.36; p 5 0.050 OR, 30.08; 95% CI, 0.58e1,568.06; p 5 0.092). Conclusions. IL-6 598/572/174 (AGC) haplotype has a low prevalence among firstdegree relatives of subjects with type 2 diabetes. Our results suggest that this haplotype is associated with decreased risk of type 2 diabetes in Mexican subjects with FHT2D. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: IL-6 haplotypes, Decreased risk, Mexican subjects, Family history.
Introduction Address reprint requests to: Irma Zamora-Ginez, PhD, Facultad de Medicina, Benemerita Universidad Autonoma de Puebla, Av. 13 Sur 2702 Col. Volcanes, Puebla, Puebla, 72410 Mexico; Phone: (þ52) (22) 2891-2728; E-mail: [email protected]
Family history of type 2 diabetes (FHT2D) is a well-established risk factor for developing diabetes. The presence of FHT2D has been associated with decreased
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.09.003
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insulin sensitivity (1), insulin resistance (IR) (2) and decreased insulin secretion (3). Similar observations have been reported among the Mexican population in whom FH2TD has been associated with hyperinsulinemia (4), decreased insulin sensitivity and pre-diabetes (5). According to the American Diabetes Association (6), type 2 diabetes (T2D) includes individuals with IR and usually those who have relative insulin deficiency. Clinical and experimental data suggest that inflammatory mechanisms mediated in part by increased pro-inflammatory cytokines such as interleukin-6 (IL-6) (7,8) contribute to IR. Indeed, several population-based studies have proposed a relationship between IL-6 and IR (9,10). IL-6 is a cytokine involved in the regulation of acutephase inflammatory responses. IL-6 plays an important role in growth control and regulating energy balance through its effects on glucose metabolism by regulating the effects of insulin (11). Single nucleotide polymorphisms (SNPs) (12,13) have been reported in the IL-6 gene promoter region: 598GOA (rs1800797), 572GOC (rs1800796) and 174GOC (rs1800795). These polymorphisms have been associated with IL-6 levels (13). However, few studies have reported on the role of the 598GOA SNP. For example, Illig et al. (14) demonstrated that the 598G allele was significantly associated with T2D in German participants. With respect to the 572-SNP, a study among Caucasian Danish subjects showed a significant increase for the risk of developing T2D that was associated with C allele carriers (15). The 174GOC SNP lies in an area that exerts a negative effect on gene transcription (12,16). Consistent with these reports, Fishman et al. (13) demonstrated that the 174GOC SNP affects reporter gene transcription rates. In addition, IL-6 adipose tissue gene expression was reported to be significantly higher in non-obese carriers of the G allele (17). Furthermore, obese women who are homozygous for the C allele have significantly lower circulating IL-6 levels when compared to non-obese women (18). Studies in Mexican subjects show that the 174C allele is associated with low C-reactive protein (CRP) levels in overweight adolescents (19). Of importance, Huth et al. (20) showed that 174 (GC) and 174 (CC) genotypes were associated with decreased risk of T2D following an analysis of O20,000 participants. In terms of the 598/174 haplotypes, previous reports showed that, in Caucasians, IL-6 levels were lower in individuals that were homozygous for haplotype 598/174 (AC) (21) and that 598/174 (GG) is significantly associated with diabetes in Mexican subjects (22). A recent report in adolescents from Mexico showed significant associations between 597/572/174 (GCG/ GCG) haplotype and hyperglycemia; between AGC/AGC and obesity; between GGG/GCG and low HDL-C; and between GCG/GCG and high hs-CRP (23). However the relationship among Mexican subjects with FHT2D and IL-6
gene SNPs is unclear. The aim of this study was to determine the prevalence of IL-6 598/572/174 haplotypes in Mexican subjects with FHT2D and to assess the influence of these haplotypes on the relationship between FHT2D and risk factors for diabetes.
Materials and Methods All participants provided signed informed consent to participate in the study, which was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Scientific Research Committee of the Mexican Social Security Institute (IMSS). Subjects and Setting The Mexican Diabetes Prevention Study (MexDiab) is a population-based study to evaluate strategies for preventing T2D in high-risk individuals and to provide information on the prevalence of metabolic disorders among the entire Mexican population (24). We designed a cross-sectional study that included 258 adult Mexican subjects from Puebla City, the Puebla state capital located in central Mexico, consisting of a population of males and non-pregnant females. Family history was defined by previous diagnosis of T2D in at least one first-degree relative (mother, father, sibling, or children) and was verified by review of clinical records. Diagnoses of T2D or another acute or chronic inflammatory disease comprised criteria for study exclusion. Eligible subjects were allocated into groups according to their FHT2D status: 1) group FHT2Dþ, subjects with FHT2D (n 5 153), and 2) group FHT2D, subjects without FHT2D (n 5 105). Additionally, blood samples from the general population (n 5 106) were selected among unrelated blood donors to have a reference for the allele and haplotype distribution in the population, the frequencies for IL-6 598GOA and 174GOC have been previously reported by our group (22). Clinical Characterization A standardized clinical history was performed. With the subject under fasting condition, wearing light clothing and without shoes, the subject’s height (0.01 m) and weight (0.1 kg) were measured (TBF-215; Tanita, Tokyo, Japan). Body mass index (BMI) was calculated as weight (kg)/ height (m2) (25,26). Waist circumference (WC) was measured midpoint between the highest point of the iliac crest and the lowest point of the costal margin at the midaxillary line using a non-elastic anthropometric measuring tape. Measurements were performed at the end of a normal inhalation using minimal pressure with tape to avoid compression of the skin. Hip circumference (HC) was measured at the widest part of the buttocks (27).
IL-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes
Assays Whole blood samples were collected from the antecubital vein following a 10e12 h overnight fast. Blood samples were centrifuged within 30e45 min of collection. Samples were frozen at 20 C and sent for analysis on a weekly basis. All assays were performed at the Central Laboratory of the Multidisciplinary Research Group on Diabetes. Serum glucose was determined with the Synchron CX4 System using a timed endpoint method (Beckman Coulter, Fullerton, CA). Intra- and interassay coefficients of variation were 1.1% for both assays. Plasma insulin was measured by immunoassay utilizing the Synchron CX4 System (Beckman Coulter). All subjects had an oral glucose tolerance test (1.75 g oral glucose solution/kg of body weight (BW) to a maximum of 75 g). Homeostasis model assessmenteinsulin resistance (HOMA-IR) was used to estimate IR. HOMA-IR was calculated based on ( fasting glucose [mmol/l] fasting insulin [mU/ml])/22.517 using the HOMA2 calculator (28). Definitions Obesity was defined by either BMI $30 kg/m2 or waist/hip ratio (WHR) O0.9 for males and O0.8 for females (25). Hyperinsulinemia was defined by fasting insulin O15 mU/ml and IR by HOMA2-IR $2.0 (29,30). PCR Restriction Fragment-length Polymorphism IL-6 Gene Analysis DNA was extracted from leukocytes (31). A 533-bp fragment including all three polymorphisms was amplified from the promoter region of the IL-6 gene (32). FokI, BsrBI, and NlaIII restriction endonucleases were utilized for 598GOA, 572GOC, and 174GOC genotyping, respectively, employing a previously reported method (22,32). Statistical Analyses Differences between groups were estimated using the unpaired two-tailed Student t test for numerical variables and the c2 Pearson test for non-numerical variables. All numerical data were logn-transformed to obtain symmetrical distribution. The Kolmogorov-Smirnov test was employed to test for normality of the distribution. Prevalence was expressed as a percentage. c2 analyses were utilized to determine the difference among prevalence of obesity, hyperinsulinemia, and IR. Hardy-Weinberg equilibrium (HWE) was performed with the c2 goodness-of-fit test (33). These results were compared with an HWE from a Mexican population from central Mexico by Pearson c2 test. Maximum-likelihood tri-locus haplotypes were determined utilizing the Arlequin v.3.3 software package for genetic data analysis with
531
10,000 iterations (34). Differences among genotypes and haplotypes were evaluated by the Pearson c2 test. To discard bias due to genetic substructure or inbreeding, F statistics (FST) was calculated according to the formula: F5PAA =PA þ Paa =Pa 1; where PAA and Paa are genotype frequencies and PA and Pa are allele frequencies (35). Multivariable logistic regression analysis adjusted for age, gender, obesity, and smoking was utilized to evaluate the relationship between FHT2D (independent variable) and each of the following dependent variables: hyperinsulinemia, and IR; p !0.05 was considered statistically significant. Data were analyzed with the SPSS v.15.0 software (SPSS, Inc., Chicago, IL).
Results Two hundred fifty eight subjects were enrolled (184 females and 74 males) in this study. Of these subjects, 153 were subjects with a FHT2D and 105 were subjects without FHT2D. The characteristics of our study population according to FHT2D status are shown in Table 1. Subjects with FHT2D showed higher prevalence of obesity, hyperinsulinemia and IR when compared to subjects without FHT2D (Table 1). In subjects without FHT2D group, genotype proportions observed for all SNP were in agreement with those expected for HWE ( p 5 0.73 for 598 and 174 SNP, and p 5 0.21 for 572 SNP) without significant differences between these and those expected for HWE in a Mexican general population ( p 5 0.10 for 598 and 174 SNP, and p 5 0.59 for 572 SNP). Inbreeding coefficient F was 0.03 and 0.19 for study population and general population, respectively. Table 1. Characteristics and prevalences according to the FHT2DM status FHT2D n 5 105 FHT2Dþ n 5 153 Age (years SD) Gender (F/M) BMI (kg/m2 SD) Obesity (%)a Hyperinsulinemia (%)b Insulin resistance (%)c
37.91 14.55 74/31 26.83 4.58 21.0 5.7 7.0
44.71 12.77 111/43 28.88 4.77 34.0 14.4 14.2
p 0.000 0.779 0.096 0.023 0.029 0.050
FHT2Dþ, subjects with family history of type 2 diabetes; FHT2D, subjects without HFT2D; BMI, body mass index; SD, standard deviation. a Obesity, body mass index (BMI) O30 kg/m2. b Hyperinsulinemia, fasting insulin O15 mU/mL. c Insulin resistance, homeostasis model assessment 2 (HOMA2-IR) $2.0. Differences between groups were estimated using the unpaired twotailed Student t test for numerical variables and the c2 Pearson test for non-numerical variables.
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Zamora-Ginez et al./ Archives of Medical Research 44 (2013) 529e534
Genotype prevalence in the studied groups is depicted in Table 2. Complete linkage disequilibrium was observed between the 598 and 174 sites (D’ 5 1.00) and nearly complete disequilibrium with 572 (D’ 5 0.98) in both groups. Consequently, only three haplotypes (locus order: 598/572/174) were observed: GGG; GCG; and AGC. There was no significant difference of haplotype prevalence between those with and without FHT2D (c2 5 2.72; p 5 0.251) (Table 2). Due to the low prevalence of IL-6 598/572/174 (AGC/AGC) haplogenotype (1.95% of the entire study population), individuals carrying the (AGC) haplotype were compared with those individuals that do not carry the haplotype (dominant model) (Table 2). Prevalence of obesity, hyperinsulinemia and IR among AGC- haplotype carriers are shown in Table 3. AGC haplotype was less prevalent among subjects with FHT2D than those without FHT2D (0.19 vs. 0.29; p 5 0.049) (Table 2). The association of FHT2D with hyperinsulinemia and IR did not change following adjustments for gender, age, smoking and obesity (OR, 3.37; 95% CI, 1.17e9.70; p 5 0.024 and OR, 2.70; 95% CI, 1.00e7.36; p 5 0.050; respectively). After adjustment for AGC haplotype-carrier status, FHT2Dþ remained associated with hyperinsulinemia (OR, 3.50; 95% CI, 1.20e10.13; p 5 0.021), but the relationship between FHT2Dþ and IR was lost (OR, 30.08; 95% CI, 0.58e1568.06; p 5 0.092) (Table 4).
Subjects (%) n 5 59
Obesity (%)a Hyperinsulinemia (%)b Insulin resistance (%)c
With
Without
c2
p
17 (29%) 7 (12%) 8 (14%)
42 (71%) 52 (88%) 51 (86%)
9.93 33.37 30.41
0.002 0.000 0.000
Obesity, body mass index (BMI) O30 kg/m2. Hyperinsulinemia, fasting insulin O15 mU/mL. c Insulin resistance, homeostasis model assessment (HOMA-IR) $2.0. a
b
Discussion
2 (0.02) 28 (0.27) 75 (0.71)
3 (0.02) 26 (0.17) 124 (0.81)
c2 5 3.59 p 5 0.166
In this study we describe the prevalence of IL-6 gene haplotypes in subjects with FHT2D and their association among a cohort of Mexican subjects. To the best of our knowledge, the influence of the IL-6 598GOA 572GOC174GOC haplotypes on the relationship between FHT2D and risk factors for diabetes in Mexican subjects has not yet been reported. We now show evidence for low prevalence of IL-6 598/572/174 (AGC) haplotype in Mexican subjects with FHT2D and that AGC haplotype carriers show a low prevalence of risk factors for T2D. Additionally we show that the relationship between FHT2D and IR is modified by the presence of the 598/572/174 (AGC) haplotype. In the current study, subjects with FHT2D showed a higher prevalence of obesity, hyperinsulinemia, and IR than subjects without FHT2D; an association that was maintained following adjustment for obesity among our Mexican cohort. The impact of FHT2D on the relationship between insulin-dependent and insulin-independent glucose disposal may be important in modulating the risk for the development of diabetes (36). Relatives of patients with T2D show lower insulin-stimulated glucose disposal, increased insulin-stimulated glucose oxidation and reduced insulin-stimulated non-oxidative glucose metabolism (37).
44 (0.42) 43 (0.41) 18 (0.17)
65 (0.43) 53 (0.35) 35 (0.23)
c2 5 1.67 p 5 0.434
Table 4. Association between family history of type 2 diabetes (FHT2D) and hyperinsulinemia or insulin resistance
32 (0.15) 108 (0.51) 70 (0.33)
32 (0.10) 162 (0.53) 112 (0.37)
c2 5 2.72 p 5 0.251
30 (0.29) 75 (0.71)
29 (0.19) 124 (0.81)
p 5 0.049
Table 2. IL-6 598/572/174 genotype and haplotype prevalence in subjects with and without family history of type 2 diabetes (FHT2D) from Central Mexico FHT2D FHT2Dþ n 5 105 (%) n 5 153 (%) 598 and 174 genotypes AA/CC AG/CG GG/GG 572 genotypes CC CG GG Haplotypes AGC GGG GCG AGC carriers AGC carriers Non-AGC haplotype carriers
Table 3. Prevalence of hyperinsulinemia and IR in AGC carrier haplotypes
FHT2Dþ, subjects with family history of type 2 diabetes; FHT2D, subjects without FHT2D; AGC carrier haplotype: ([AGC/AGC]) homozygote plus [AGC/GCG] heterozygote plus [AGC/GGG] heterozygote-haplogenotypes); non-(AGC)-carrier haplotype ([GCG/GCG] homozygote plus [GCG/GGG] heterozygote plus [GGG/GGG] homozygote haplogenotypes). Significance was evaluated by c2 test with a 2 3 contingency table (genotypes) and Fisher exact test in 2 2 tables (groups).
OR Hyperinsulinemia (dependent variable) 3.37 FHT2Da Adjusteda for AGC carriers 3.50 Non-AGC carriers 3.47 Insulin resistance (dependent variable) 2.70 FHT2Da Adjusteda AGC carriers 30.08 Non-AGC carriers 2.85
95% CI
p
1.17e9.70
0.024
1.20e10.13 1.19e10.05
0.021 0.022
1.00e7.36
0.050
0.58e1,568.06 1.03e7.86
0.092 0.043
OR, odds ratio; 95% CI, 95% confidence interval. Adjusted for age, gender, smoking, and obesity; (AGC) carriers: AGC/ AGC þAGC/GCG þ AGC/GGG haplo-genotypes.
a
IL-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes
In addition, many studies in non-diabetic relatives of families with T2D show that impaired insulin secretion, IR, and an adverse cardiovascular risk factor profile exist well before the development of diabetes (38). FHT2D is an independent risk factor for the development of diabetes (39,40). It has been reported that in first-degree relatives of patients with T2D there is a higher incidence of risk factors for diabetes such as hyperglycemia and IR (2,41). In agreement with this, the Framingham Offspring Study showed that the risk of developing T2D among offspring is greater than in those without diabetic parents (42). In Mexico, the Mexican National Health Survey showed that the prevalence of T2D in the general population is 10.9% and changes from 6.1% in the absence of FHT2De19.5% when FHT2D is present (43). Increasing evidence supports the contention that there is genetic susceptibility for T2D; however, there have been no reports on the association between 598/572/174 haplotypes with risk factors for T2D in Mexican subjects with FHT2D. Interestingly, our study shows lower prevalence of hyperinsulinemia and IR, both risk factors for T2D, among 598/572/174 (AGC) haplotype carrier subjects and lower prevalence of the 598/572/174 (AGC) haplotype in subjects with FHT2D. Moreover, the initial significant association found between FHT2D and IR seems to be lost after adjustment by the presence of the IL-6 598/ 572/174 (AGC) haplotype. However, and despite our results showing high OR, our overly wide confidence interval in the associations between FHT2D and IR as well as adjustment by the presence of IL-6 598/572/174 (AGC)-haplotype, suggest that we cautiously interpret these results. Also, given the low frequency of the AGC haplotype among the Mexican population in our study cohort, these results should likewise be cautiously interpreted. Consequently, our results provide the basis for additional larger studies that are needed to clarify these apparent trends among ethnically defined populations. Published evidence shows an association between 598 and 174 haplotypes with T2D (22) and between 598G and 572C alleles with T2D (14,15). In our study, the 598 (GOA) SNP exhibited complete linkage disequilibrium with the 174 (GOC) SNP, results that are consistent with that of others (21), whereas the 572 (GOC) SNP was not associated with T2D (20). In addition, there is evidence that the 174GOC SNP apparently regulates IL-6 transcription and expression depending on the allele present, probably because it is located near the negative regulatory domain (12,16). Consequently, the presence of the 174GOC SNP may explain the association between 598/572/174 haplotypes and T2D shown in our population and among adolescents from central Mexico (23). Our observations support previous findings of decreased T2D risk in 174C allele-carrier subjects (22). In addition, others have associated the IL-6 promoter polymorphism at
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position 174GOC with obesity (44), insulin sensitivity (45), and IR in T2D (46) and with HOMA-IR in subjects with and without T2D (14, 47). Furthermore, evidence shows that the 174C allele correlates with decreased IL-6 activity, secondary to modification of IL-6 gene transcription (13). In conclusion, the lower prevalence of IL-6 598/572/ 174 (AGC) haplotype in subjects with FHT2D compared to subjects without FHT2D and its association with a decreased risk for T2D suggests that this haplotype may represent a genetic marker for reduced risk of T2D among Mexican subjects. However further studies are needed to clarify the precise role of IL-6 gene polymorphisms and IL-6 levels and to confirm our observations in ethnically defined populations. Acknowledgments This study was supported by grants from the Consejo Nacional de Ciencia y Tecnologıa de Mexico, SALUD 2004-01-023, Fundaci on IMSS, A.C. IZ-G was supported by grants from Consejo Nacional de Ciencia y Tecnologıa de Mexico, CVU/Becario 165584. We gratefully acknowledge the commitment and dedication of physicians, chemists, nurses and participating personnel in the Mexican Diabetes Prevention Study. Investigators of the Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social participating in the Mexican Diabetes Prevention Study: Fernando Guerrero-Romero, MD, PhD, FACP; Martha Rodrıguez-Moran, MD, MSc, PhD; Jorge Escobedo de la Pe~ na, MD, MSc; Niels Wacher, MD, PhD; Adolfo Chavez-Negrete, MD, MSc, PhD; Cristina Revilla-Monsalve, MD, MSc, PhD; Esperanza Martınez-Abundis, MD, MSc, PhD; Javier E. Garcıa de Alva, MD, MSc, PhD; Jose Sanchez-Corona, MD, MSc, PhD; Silvia E. Flores Martınez MSc, PhD; Manuel Gonzalez-Ortız, MD, MSc, PhD; Sergio Islas-Andrade, MD, MSc, PhD, FACP. Special thanks should be given to Maggie Brunner, M.A. for helpful suggestions and critical reading of the manuscript.
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27. Verdalet-Guzman I, Silva-Hernandez ER. Elementos antropometricos para evaluar el estado nutricional. 1st ed Mexico: Universidad Veracruzana; 2001. pp. 15e27. 28. Levy JC, Matthews DR, Hermans MP. Correct homeostasis model assessment (HOMA) evaluation uses the computer program. Diabetes Care 1998;21:2191e2192. Date accessed: November 2011. University of Oxford. Diabetes Trials Unit. Homa Calculator Version 2.2.1. [Computer program]. http://www.dtu.ox.ac.uk/homacalculator/index.php. 29. Acosta AM, Escalona M, Maız A, et al. Determinacion del ındice de resistencia insulınica mediante HOMA en una poblacion de la Region Metropolitana de Chile. Rev Med Chile 2002;130:1227e1231. 30. Wallace TM, Levy JC, Matthews DR. Use and abuse of HOMA modeling. Diabetes Care 2004;27:1487e1495. 31. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells (abstract). Nucleic Acid Res 1998;16:1215. 32. Latkovskis G, Licis N, Kalnins U. C-reactive protein levels and common polymorphisms of the interleukin-1 gene cluster and interleukin6 gene in patients with coronary heart disease. Eur J Immunogenet 2004;31:207e213. 33. Salanti G, Amountza G, Ntzani EE. Hardy-Weinberg equilibrium in genetic association studies: an empirical evaluation of reporting, deviations, and power. Eur J Hum Genet 2005;13:840e848. 34. Excoffier L, Lischer HEL. Arlequin suite ver. 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molec Ecol Resour 2010;10:564e567. 35. Li CC, Horvitz DG. Some methods of estimating the inbreeding coefficient. Am J Hum Genet 1953;5:107e117. 36. Lopez X, Bouche C, Tatro E, et al. Family history of diabetes impacts on interactions between minimal model estimates of insulin sensitivity and glucose effectiveness. Diabetes Obes Metab 2009;11:123e130. 37. Vaag A, Lehtovirta M, Thye-R€onn P, et al. Metabolic impact of a family history of type 2 diabetes. Results from a European multicentre study (EGIR). Diabetic Med 2001;18:533e540. 38. Tripathy D, Lindholm E, Isomaa B, et al. Familiality of metabolic abnormalities is dependent on age at onset and phenotype of the type 2 diabetic proband. Am J Physiol Endocrinol Metab 2003;285: E1297eE1303. 39. Mitchell B, Valdez R, Hazuda H, et al. Differences in prevalence of diabetes and impaired glucose tolerance according to maternal and paternal history of diabetes. Diabetes Care 1993;16:1262e1267. 40. Bjornholt JV, Erikssen G, Liestol K, et al. Type 2 diabetes and maternal family history: an impact beyond slow glucose removal rate and fasting hyperglycemia in low-risk individuals? Results from 22.5 years of follow-up of healthy nondiabetic men. Diabetes Care 2000; 23:1255e1259. 41. Siewert S, Filipuzzi S, Codazzi L, et al. Impact of metabolic syndrome risk factors in first-degree relatives of type 2 diabetic patients. Rev Diabet Stud 2007;4:177e184. 42. Meigs JB, Cupples LA, Wilson PW. Parental transmission of type 2 diabetes: the Framingham Offspring study. Diabetes 2000;49: 2201e2207. 43. Olaiz G, Rojas R, Barquera S, et al. Diabetes Mellitus. Encuesta Nacional de Salud 2000 (ENSA 2000). Tomo 2. La salud de los adultos. Mexico: Mex Press; 2003. pp. 93e104. 44. Dedoussis GV, Manios Y, Choumerianou DM. The IL-6 gene G-174C polymorphism related to health indices in Greek primary school children. Obes Res 2004;12:1037e1041. 45. Cardellini M, Perego L, D’Adamo M, et al. C174G polymorphism in the promoter of the interleukin-6 gene is associated with insulin resistance. Diabetes 2005;28:2007e2012. 46. Testa R, Olivieri F, Bonfigli AR, et al. Interleukin-6e174 GOC polymorphism affects the association between IL-6 plasma levels and insulin resistance in type 2 diabetic patients. Diabetes Res Clin Pract 2006;71:299e305.
ORIGINAL ARTICLE
Low Prevalence of Interleukin-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes in Mexican Subjects with a Family History of Type 2 Diabetes Irma Zamora-Ginez,a Alejandra Guadalupe Garcıa-Zapien,b Silvia Esperanza Flores-Martınez,b,c Jose Sanchez-Corona,b,c Blanca G. Baez-Duarte,a Enrique Torres-Rasgado,a Jose R. Romero,d Ricardo Perez-Fuentes,a,c,e Francisco Mendoza-Carrera,b and The Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social a
Facultad de Medicina, Benemerita Universidad Autonoma de Puebla (BUAP), Puebla, Mexico Divisi on Medicina Molecular, Centro de Investigacion Biomedica de Occidente (CIBO), Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Mexico c Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social, Mexico, D.F., Mexico d Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Boston, Massachusetts e Laboratorio de Investigacion en Fisiopatologıa de Enfermedades Cronicas, CIBIOR, IMSS, Atlixco, Puebla, Mexico
b
Received for publication March 20, 2013; accepted August 26, 2013 (ARCMED-D-13-00158).
Background and Aims. There is evidence that family history of type 2 diabetes (FHT2D) and single nucleotide polymorphisms (SNP) on the IL-6 gene promoter region are separately associated with the risk of developing type 2 diabetes. However the relationship between adult Mexican subjects with FHT2D and genotypes/haplotypes for IL-6 gene has not been explored. The aim of the present work was to study the prevalence of IL-6 598G OA572GOC174GOC haplotypes among subjects with FHT2D and to determine whether their presence influences the relationship between FHT2D and risk factors for diabetes. Methods. Two hundred fifty eight nondiabetic subjects participated in this study; 153 with and 105 without FHT2D. Polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) was used for genotyping. Logistic regression analysis was employed to assess the impact of IL-6 haplotypes on FHT2D per se and hyperinsulinemia and insulin resistance as risk factors for diabetes. Results. Subjects with FHT2D showed a higher prevalence of hyperinsulinemia and insulin resistance (IR) than those without FHT2D (14.4 vs. 5.7%, p 5 0.029, and 14.2 vs. 7.0% p 5 0.050, respectively). Lower prevalence of 598 -572-174 (AGC)-haplotype (19%) in subjects with FHT2D was observed as well as a lower prevalence of hyperinsulinemia and IR among AGC haplotype carriers (12 and 14%, respectively). The relationship between FHT2D and IR was modified by the presence of AGC haplotype (from OR, 2.70; 95% CI, 0.99e7.36; p 5 0.050 OR, 30.08; 95% CI, 0.58e1,568.06; p 5 0.092). Conclusions. IL-6 598/572/174 (AGC) haplotype has a low prevalence among firstdegree relatives of subjects with type 2 diabetes. Our results suggest that this haplotype is associated with decreased risk of type 2 diabetes in Mexican subjects with FHT2D. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: IL-6 haplotypes, Decreased risk, Mexican subjects, Family history.
Introduction Address reprint requests to: Irma Zamora-Ginez, PhD, Facultad de Medicina, Benemerita Universidad Autonoma de Puebla, Av. 13 Sur 2702 Col. Volcanes, Puebla, Puebla, 72410 Mexico; Phone: (þ52) (22) 2891-2728; E-mail: [email protected]
Family history of type 2 diabetes (FHT2D) is a well-established risk factor for developing diabetes. The presence of FHT2D has been associated with decreased
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.09.003
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insulin sensitivity (1), insulin resistance (IR) (2) and decreased insulin secretion (3). Similar observations have been reported among the Mexican population in whom FH2TD has been associated with hyperinsulinemia (4), decreased insulin sensitivity and pre-diabetes (5). According to the American Diabetes Association (6), type 2 diabetes (T2D) includes individuals with IR and usually those who have relative insulin deficiency. Clinical and experimental data suggest that inflammatory mechanisms mediated in part by increased pro-inflammatory cytokines such as interleukin-6 (IL-6) (7,8) contribute to IR. Indeed, several population-based studies have proposed a relationship between IL-6 and IR (9,10). IL-6 is a cytokine involved in the regulation of acutephase inflammatory responses. IL-6 plays an important role in growth control and regulating energy balance through its effects on glucose metabolism by regulating the effects of insulin (11). Single nucleotide polymorphisms (SNPs) (12,13) have been reported in the IL-6 gene promoter region: 598GOA (rs1800797), 572GOC (rs1800796) and 174GOC (rs1800795). These polymorphisms have been associated with IL-6 levels (13). However, few studies have reported on the role of the 598GOA SNP. For example, Illig et al. (14) demonstrated that the 598G allele was significantly associated with T2D in German participants. With respect to the 572-SNP, a study among Caucasian Danish subjects showed a significant increase for the risk of developing T2D that was associated with C allele carriers (15). The 174GOC SNP lies in an area that exerts a negative effect on gene transcription (12,16). Consistent with these reports, Fishman et al. (13) demonstrated that the 174GOC SNP affects reporter gene transcription rates. In addition, IL-6 adipose tissue gene expression was reported to be significantly higher in non-obese carriers of the G allele (17). Furthermore, obese women who are homozygous for the C allele have significantly lower circulating IL-6 levels when compared to non-obese women (18). Studies in Mexican subjects show that the 174C allele is associated with low C-reactive protein (CRP) levels in overweight adolescents (19). Of importance, Huth et al. (20) showed that 174 (GC) and 174 (CC) genotypes were associated with decreased risk of T2D following an analysis of O20,000 participants. In terms of the 598/174 haplotypes, previous reports showed that, in Caucasians, IL-6 levels were lower in individuals that were homozygous for haplotype 598/174 (AC) (21) and that 598/174 (GG) is significantly associated with diabetes in Mexican subjects (22). A recent report in adolescents from Mexico showed significant associations between 597/572/174 (GCG/ GCG) haplotype and hyperglycemia; between AGC/AGC and obesity; between GGG/GCG and low HDL-C; and between GCG/GCG and high hs-CRP (23). However the relationship among Mexican subjects with FHT2D and IL-6
gene SNPs is unclear. The aim of this study was to determine the prevalence of IL-6 598/572/174 haplotypes in Mexican subjects with FHT2D and to assess the influence of these haplotypes on the relationship between FHT2D and risk factors for diabetes.
Materials and Methods All participants provided signed informed consent to participate in the study, which was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Scientific Research Committee of the Mexican Social Security Institute (IMSS). Subjects and Setting The Mexican Diabetes Prevention Study (MexDiab) is a population-based study to evaluate strategies for preventing T2D in high-risk individuals and to provide information on the prevalence of metabolic disorders among the entire Mexican population (24). We designed a cross-sectional study that included 258 adult Mexican subjects from Puebla City, the Puebla state capital located in central Mexico, consisting of a population of males and non-pregnant females. Family history was defined by previous diagnosis of T2D in at least one first-degree relative (mother, father, sibling, or children) and was verified by review of clinical records. Diagnoses of T2D or another acute or chronic inflammatory disease comprised criteria for study exclusion. Eligible subjects were allocated into groups according to their FHT2D status: 1) group FHT2Dþ, subjects with FHT2D (n 5 153), and 2) group FHT2D, subjects without FHT2D (n 5 105). Additionally, blood samples from the general population (n 5 106) were selected among unrelated blood donors to have a reference for the allele and haplotype distribution in the population, the frequencies for IL-6 598GOA and 174GOC have been previously reported by our group (22). Clinical Characterization A standardized clinical history was performed. With the subject under fasting condition, wearing light clothing and without shoes, the subject’s height (0.01 m) and weight (0.1 kg) were measured (TBF-215; Tanita, Tokyo, Japan). Body mass index (BMI) was calculated as weight (kg)/ height (m2) (25,26). Waist circumference (WC) was measured midpoint between the highest point of the iliac crest and the lowest point of the costal margin at the midaxillary line using a non-elastic anthropometric measuring tape. Measurements were performed at the end of a normal inhalation using minimal pressure with tape to avoid compression of the skin. Hip circumference (HC) was measured at the widest part of the buttocks (27).
IL-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes
Assays Whole blood samples were collected from the antecubital vein following a 10e12 h overnight fast. Blood samples were centrifuged within 30e45 min of collection. Samples were frozen at 20 C and sent for analysis on a weekly basis. All assays were performed at the Central Laboratory of the Multidisciplinary Research Group on Diabetes. Serum glucose was determined with the Synchron CX4 System using a timed endpoint method (Beckman Coulter, Fullerton, CA). Intra- and interassay coefficients of variation were 1.1% for both assays. Plasma insulin was measured by immunoassay utilizing the Synchron CX4 System (Beckman Coulter). All subjects had an oral glucose tolerance test (1.75 g oral glucose solution/kg of body weight (BW) to a maximum of 75 g). Homeostasis model assessmenteinsulin resistance (HOMA-IR) was used to estimate IR. HOMA-IR was calculated based on ( fasting glucose [mmol/l] fasting insulin [mU/ml])/22.517 using the HOMA2 calculator (28). Definitions Obesity was defined by either BMI $30 kg/m2 or waist/hip ratio (WHR) O0.9 for males and O0.8 for females (25). Hyperinsulinemia was defined by fasting insulin O15 mU/ml and IR by HOMA2-IR $2.0 (29,30). PCR Restriction Fragment-length Polymorphism IL-6 Gene Analysis DNA was extracted from leukocytes (31). A 533-bp fragment including all three polymorphisms was amplified from the promoter region of the IL-6 gene (32). FokI, BsrBI, and NlaIII restriction endonucleases were utilized for 598GOA, 572GOC, and 174GOC genotyping, respectively, employing a previously reported method (22,32). Statistical Analyses Differences between groups were estimated using the unpaired two-tailed Student t test for numerical variables and the c2 Pearson test for non-numerical variables. All numerical data were logn-transformed to obtain symmetrical distribution. The Kolmogorov-Smirnov test was employed to test for normality of the distribution. Prevalence was expressed as a percentage. c2 analyses were utilized to determine the difference among prevalence of obesity, hyperinsulinemia, and IR. Hardy-Weinberg equilibrium (HWE) was performed with the c2 goodness-of-fit test (33). These results were compared with an HWE from a Mexican population from central Mexico by Pearson c2 test. Maximum-likelihood tri-locus haplotypes were determined utilizing the Arlequin v.3.3 software package for genetic data analysis with
531
10,000 iterations (34). Differences among genotypes and haplotypes were evaluated by the Pearson c2 test. To discard bias due to genetic substructure or inbreeding, F statistics (FST) was calculated according to the formula: F5PAA =PA þ Paa =Pa 1; where PAA and Paa are genotype frequencies and PA and Pa are allele frequencies (35). Multivariable logistic regression analysis adjusted for age, gender, obesity, and smoking was utilized to evaluate the relationship between FHT2D (independent variable) and each of the following dependent variables: hyperinsulinemia, and IR; p !0.05 was considered statistically significant. Data were analyzed with the SPSS v.15.0 software (SPSS, Inc., Chicago, IL).
Results Two hundred fifty eight subjects were enrolled (184 females and 74 males) in this study. Of these subjects, 153 were subjects with a FHT2D and 105 were subjects without FHT2D. The characteristics of our study population according to FHT2D status are shown in Table 1. Subjects with FHT2D showed higher prevalence of obesity, hyperinsulinemia and IR when compared to subjects without FHT2D (Table 1). In subjects without FHT2D group, genotype proportions observed for all SNP were in agreement with those expected for HWE ( p 5 0.73 for 598 and 174 SNP, and p 5 0.21 for 572 SNP) without significant differences between these and those expected for HWE in a Mexican general population ( p 5 0.10 for 598 and 174 SNP, and p 5 0.59 for 572 SNP). Inbreeding coefficient F was 0.03 and 0.19 for study population and general population, respectively. Table 1. Characteristics and prevalences according to the FHT2DM status FHT2D n 5 105 FHT2Dþ n 5 153 Age (years SD) Gender (F/M) BMI (kg/m2 SD) Obesity (%)a Hyperinsulinemia (%)b Insulin resistance (%)c
37.91 14.55 74/31 26.83 4.58 21.0 5.7 7.0
44.71 12.77 111/43 28.88 4.77 34.0 14.4 14.2
p 0.000 0.779 0.096 0.023 0.029 0.050
FHT2Dþ, subjects with family history of type 2 diabetes; FHT2D, subjects without HFT2D; BMI, body mass index; SD, standard deviation. a Obesity, body mass index (BMI) O30 kg/m2. b Hyperinsulinemia, fasting insulin O15 mU/mL. c Insulin resistance, homeostasis model assessment 2 (HOMA2-IR) $2.0. Differences between groups were estimated using the unpaired twotailed Student t test for numerical variables and the c2 Pearson test for non-numerical variables.
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Genotype prevalence in the studied groups is depicted in Table 2. Complete linkage disequilibrium was observed between the 598 and 174 sites (D’ 5 1.00) and nearly complete disequilibrium with 572 (D’ 5 0.98) in both groups. Consequently, only three haplotypes (locus order: 598/572/174) were observed: GGG; GCG; and AGC. There was no significant difference of haplotype prevalence between those with and without FHT2D (c2 5 2.72; p 5 0.251) (Table 2). Due to the low prevalence of IL-6 598/572/174 (AGC/AGC) haplogenotype (1.95% of the entire study population), individuals carrying the (AGC) haplotype were compared with those individuals that do not carry the haplotype (dominant model) (Table 2). Prevalence of obesity, hyperinsulinemia and IR among AGC- haplotype carriers are shown in Table 3. AGC haplotype was less prevalent among subjects with FHT2D than those without FHT2D (0.19 vs. 0.29; p 5 0.049) (Table 2). The association of FHT2D with hyperinsulinemia and IR did not change following adjustments for gender, age, smoking and obesity (OR, 3.37; 95% CI, 1.17e9.70; p 5 0.024 and OR, 2.70; 95% CI, 1.00e7.36; p 5 0.050; respectively). After adjustment for AGC haplotype-carrier status, FHT2Dþ remained associated with hyperinsulinemia (OR, 3.50; 95% CI, 1.20e10.13; p 5 0.021), but the relationship between FHT2Dþ and IR was lost (OR, 30.08; 95% CI, 0.58e1568.06; p 5 0.092) (Table 4).
Subjects (%) n 5 59
Obesity (%)a Hyperinsulinemia (%)b Insulin resistance (%)c
With
Without
c2
p
17 (29%) 7 (12%) 8 (14%)
42 (71%) 52 (88%) 51 (86%)
9.93 33.37 30.41
0.002 0.000 0.000
Obesity, body mass index (BMI) O30 kg/m2. Hyperinsulinemia, fasting insulin O15 mU/mL. c Insulin resistance, homeostasis model assessment (HOMA-IR) $2.0. a
b
Discussion
2 (0.02) 28 (0.27) 75 (0.71)
3 (0.02) 26 (0.17) 124 (0.81)
c2 5 3.59 p 5 0.166
In this study we describe the prevalence of IL-6 gene haplotypes in subjects with FHT2D and their association among a cohort of Mexican subjects. To the best of our knowledge, the influence of the IL-6 598GOA 572GOC174GOC haplotypes on the relationship between FHT2D and risk factors for diabetes in Mexican subjects has not yet been reported. We now show evidence for low prevalence of IL-6 598/572/174 (AGC) haplotype in Mexican subjects with FHT2D and that AGC haplotype carriers show a low prevalence of risk factors for T2D. Additionally we show that the relationship between FHT2D and IR is modified by the presence of the 598/572/174 (AGC) haplotype. In the current study, subjects with FHT2D showed a higher prevalence of obesity, hyperinsulinemia, and IR than subjects without FHT2D; an association that was maintained following adjustment for obesity among our Mexican cohort. The impact of FHT2D on the relationship between insulin-dependent and insulin-independent glucose disposal may be important in modulating the risk for the development of diabetes (36). Relatives of patients with T2D show lower insulin-stimulated glucose disposal, increased insulin-stimulated glucose oxidation and reduced insulin-stimulated non-oxidative glucose metabolism (37).
44 (0.42) 43 (0.41) 18 (0.17)
65 (0.43) 53 (0.35) 35 (0.23)
c2 5 1.67 p 5 0.434
Table 4. Association between family history of type 2 diabetes (FHT2D) and hyperinsulinemia or insulin resistance
32 (0.15) 108 (0.51) 70 (0.33)
32 (0.10) 162 (0.53) 112 (0.37)
c2 5 2.72 p 5 0.251
30 (0.29) 75 (0.71)
29 (0.19) 124 (0.81)
p 5 0.049
Table 2. IL-6 598/572/174 genotype and haplotype prevalence in subjects with and without family history of type 2 diabetes (FHT2D) from Central Mexico FHT2D FHT2Dþ n 5 105 (%) n 5 153 (%) 598 and 174 genotypes AA/CC AG/CG GG/GG 572 genotypes CC CG GG Haplotypes AGC GGG GCG AGC carriers AGC carriers Non-AGC haplotype carriers
Table 3. Prevalence of hyperinsulinemia and IR in AGC carrier haplotypes
FHT2Dþ, subjects with family history of type 2 diabetes; FHT2D, subjects without FHT2D; AGC carrier haplotype: ([AGC/AGC]) homozygote plus [AGC/GCG] heterozygote plus [AGC/GGG] heterozygote-haplogenotypes); non-(AGC)-carrier haplotype ([GCG/GCG] homozygote plus [GCG/GGG] heterozygote plus [GGG/GGG] homozygote haplogenotypes). Significance was evaluated by c2 test with a 2 3 contingency table (genotypes) and Fisher exact test in 2 2 tables (groups).
OR Hyperinsulinemia (dependent variable) 3.37 FHT2Da Adjusteda for AGC carriers 3.50 Non-AGC carriers 3.47 Insulin resistance (dependent variable) 2.70 FHT2Da Adjusteda AGC carriers 30.08 Non-AGC carriers 2.85
95% CI
p
1.17e9.70
0.024
1.20e10.13 1.19e10.05
0.021 0.022
1.00e7.36
0.050
0.58e1,568.06 1.03e7.86
0.092 0.043
OR, odds ratio; 95% CI, 95% confidence interval. Adjusted for age, gender, smoking, and obesity; (AGC) carriers: AGC/ AGC þAGC/GCG þ AGC/GGG haplo-genotypes.
a
IL-6 Haplotypes Associated with a Decreased Risk of Type 2 Diabetes
In addition, many studies in non-diabetic relatives of families with T2D show that impaired insulin secretion, IR, and an adverse cardiovascular risk factor profile exist well before the development of diabetes (38). FHT2D is an independent risk factor for the development of diabetes (39,40). It has been reported that in first-degree relatives of patients with T2D there is a higher incidence of risk factors for diabetes such as hyperglycemia and IR (2,41). In agreement with this, the Framingham Offspring Study showed that the risk of developing T2D among offspring is greater than in those without diabetic parents (42). In Mexico, the Mexican National Health Survey showed that the prevalence of T2D in the general population is 10.9% and changes from 6.1% in the absence of FHT2De19.5% when FHT2D is present (43). Increasing evidence supports the contention that there is genetic susceptibility for T2D; however, there have been no reports on the association between 598/572/174 haplotypes with risk factors for T2D in Mexican subjects with FHT2D. Interestingly, our study shows lower prevalence of hyperinsulinemia and IR, both risk factors for T2D, among 598/572/174 (AGC) haplotype carrier subjects and lower prevalence of the 598/572/174 (AGC) haplotype in subjects with FHT2D. Moreover, the initial significant association found between FHT2D and IR seems to be lost after adjustment by the presence of the IL-6 598/ 572/174 (AGC) haplotype. However, and despite our results showing high OR, our overly wide confidence interval in the associations between FHT2D and IR as well as adjustment by the presence of IL-6 598/572/174 (AGC)-haplotype, suggest that we cautiously interpret these results. Also, given the low frequency of the AGC haplotype among the Mexican population in our study cohort, these results should likewise be cautiously interpreted. Consequently, our results provide the basis for additional larger studies that are needed to clarify these apparent trends among ethnically defined populations. Published evidence shows an association between 598 and 174 haplotypes with T2D (22) and between 598G and 572C alleles with T2D (14,15). In our study, the 598 (GOA) SNP exhibited complete linkage disequilibrium with the 174 (GOC) SNP, results that are consistent with that of others (21), whereas the 572 (GOC) SNP was not associated with T2D (20). In addition, there is evidence that the 174GOC SNP apparently regulates IL-6 transcription and expression depending on the allele present, probably because it is located near the negative regulatory domain (12,16). Consequently, the presence of the 174GOC SNP may explain the association between 598/572/174 haplotypes and T2D shown in our population and among adolescents from central Mexico (23). Our observations support previous findings of decreased T2D risk in 174C allele-carrier subjects (22). In addition, others have associated the IL-6 promoter polymorphism at
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position 174GOC with obesity (44), insulin sensitivity (45), and IR in T2D (46) and with HOMA-IR in subjects with and without T2D (14, 47). Furthermore, evidence shows that the 174C allele correlates with decreased IL-6 activity, secondary to modification of IL-6 gene transcription (13). In conclusion, the lower prevalence of IL-6 598/572/ 174 (AGC) haplotype in subjects with FHT2D compared to subjects without FHT2D and its association with a decreased risk for T2D suggests that this haplotype may represent a genetic marker for reduced risk of T2D among Mexican subjects. However further studies are needed to clarify the precise role of IL-6 gene polymorphisms and IL-6 levels and to confirm our observations in ethnically defined populations. Acknowledgments This study was supported by grants from the Consejo Nacional de Ciencia y Tecnologıa de Mexico, SALUD 2004-01-023, Fundaci on IMSS, A.C. IZ-G was supported by grants from Consejo Nacional de Ciencia y Tecnologıa de Mexico, CVU/Becario 165584. We gratefully acknowledge the commitment and dedication of physicians, chemists, nurses and participating personnel in the Mexican Diabetes Prevention Study. Investigators of the Multidisciplinary Research Group on Diabetes of the Instituto Mexicano del Seguro Social participating in the Mexican Diabetes Prevention Study: Fernando Guerrero-Romero, MD, PhD, FACP; Martha Rodrıguez-Moran, MD, MSc, PhD; Jorge Escobedo de la Pe~ na, MD, MSc; Niels Wacher, MD, PhD; Adolfo Chavez-Negrete, MD, MSc, PhD; Cristina Revilla-Monsalve, MD, MSc, PhD; Esperanza Martınez-Abundis, MD, MSc, PhD; Javier E. Garcıa de Alva, MD, MSc, PhD; Jose Sanchez-Corona, MD, MSc, PhD; Silvia E. Flores Martınez MSc, PhD; Manuel Gonzalez-Ortız, MD, MSc, PhD; Sergio Islas-Andrade, MD, MSc, PhD, FACP. Special thanks should be given to Maggie Brunner, M.A. for helpful suggestions and critical reading of the manuscript.
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