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Molecular Screening and Association Analyses of the Interleukin 6 Receptor Gene Variants with Type 2 Diabetes, Diabetic Nephropathy, and Insulin Sensitivity

Division of Endocrinology and Metabolism (H.W., Z.Z., W.C., T.H., J.J.C., S.C.E.), Department of Internal Medicine, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Endocrinology Section (T.H., J.J.C., S.C.E.), Medicine and Research Services, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Steven C. Elbein, M.D., Division of Endocrinology and Metabolism, Central Arkansas Veterans Healthcare System, Endocrinology 111J-1/LR, Little Rock, Arkansas 72205. E-mail: elbeinstevenc{at}uams.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IL-6 levels and polymorphisms have been implicated in type 2 diabetes mellitus (T2DM) and insulin resistance. The IL-6 receptor (IL-6R) comprises two subunits, IL-6R and gp130, of which IL-6R confers specificity to IL-6 action and is located in a region of replicated linkage to T2DM on chromosome 1q21. We screened this gene for variation in Northern European Caucasian and African-American ethnic groups. We identified 11 variants with a minor allele frequency over 5%, including two amino acid changes (D358A and V385I) and four variants in the 3' untranslated region. No variant was associated with obesity or measures of insulin sensitivity, but two single nucleotide polymorphisms in the 3' untranslated region showed a trend to an association with T2DM in all Caucasians, and three single nucleotide polymorphisms, including D358A, showed a trend (P < 0.06) to an association with T2DM among the subset of Northern European Caucasians. Variant V385I was unique to African-Americans and was significantly associated with diabetes and diabetic nephropathy (P < 0.05). Among individuals heterozygous for the four variants in the transcribed sequence, one allele was significantly overrepresented, thus suggesting the existence of a regulatory variant controlling mRNA stability or expression. IL-6R is not likely to explain the linkage to diabetes in this region, but our work supports a minor role of variants in T2DM risk and suggests that sequence variants may alter IL-6R mRNA levels and possibly levels of soluble IL-6R.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
TYPE 2 DIABETES MELLITUS (T2DM) is characterized by insulin resistance and progressive pancreatic ß-cell failure. Increasing evidence suggests that chronic activation of the innate immune system reduces insulin sensitivity and hence may precede the development of T2DM (1, 2). Molecular markers of inflammation in T2DM have included the acute-phase response proteins sialic acid, -1 acid glycoprotein, serum amyloid A, C-reactive protein, cortisol, and the cytokine IL-6 (3).

IL-6, a key mediator of the inflammatory response, is expressed widely, including expression in adipocytes. Although immune cells, fibroblasts, endothelial cells, and monocytes have been considered the major sources, adipocytes contribute significantly to circulating IL-6 (4, 5). Accordingly, IL-6 levels correlate with several measures of obesity, including body mass index (BMI), waist to hip ratio, and percent body fat (4), and are increased in T2DM and insulin-resistant states (6). Hence, elevated IL-6 levels and increased IL-6 action may contribute to the development of T2DM (7). In support of this hypothesis, polymorphisms of the IL-6 gene have been associated with T2DM in several studies (8, 9, 10). Furthermore, IL-6 levels and polymorphisms have been associated with the risk of diabetic nephropathy (11).

IL-6 acts through a receptor comprising two subunits, IL-6 receptor (IL-6R) and gp130. IL-6 first binds to the IL-6R, and this complex in turn binds subsequently to the ubiquitous gp130 subunit (12, 13). Specificity in the IL-6 response is conferred by the IL-6R subunit, which is expressed in its membrane-bound form in hepatocytes and leukocytes. Although most IL-6 action may be through membrane-bound IL-6R, membrane-bound IL-6R is cleaved proteolytically to a circulating, soluble form of IL-6R (sIL-6R), which may activate cells that express the gp130 subunit but not IL-6R (13). Increased levels of IL-6/sIL-6R have been reported in both T2DM (14) and diabetic nephropathy (15).

The IL-6R gene maps to human chromosome 1q21, a region of well-replicated linkage to T2DM described by our laboratory in both Northern European Caucasians (16, 17) and African-Americans (18) and in multiple other diverse populations (19). Thus, IL-6R must be considered a strong functional and positional candidate gene for T2DM. Based on these arguments, we tested the hypothesis that sequence variation in IL-6R was associated with T2DM, insulin resistance, and diabetic nephropathy in Caucasian and African-American populations. We have extensively screened the IL-6R gene for sequence variation, we have evaluated the role of these variants in T2DM and insulin sensitivity in Caucasians and in T2DM and diabetic nephropathy in African-Americans, and we have tested for possible regulatory polymorphisms by searching for an imbalance in IL-6R mRNA levels between parental alleles.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Experimental subjects

Caucasian individuals were ascertained in Utah for Northern European ancestry, as described previously (20). Additional Caucasian individuals and African-American individuals were ascertained from Arkansas for similar criteria. All diabetic individuals had at least one other first-degree relative with T2DM. Nondiabetic control individuals had no known family history of diabetes and had either a normal 75-g oral glucose tolerance test or a fasting glucose of less than 5.6 mmol/liter (100 mg/dl). Screening for mutations was conducted on DNA from 32 unrelated subjects with T2DM, consisting of 16 Caucasian individuals who were selected from families with the greatest evidence for linkage to chromosome 1q21-q24 and 16 African-American subjects from families ascertained in Arkansas for at least two diabetic siblings.

The association of variants with T2DM was assessed in both Caucasian and African-American populations. Studies of Caucasians were conducted in 192 unrelated, nondiabetic individuals and 192 unrelated individuals with T2DM requiring therapy, of whom 69 were drawn from families used in our linkage studies (16). Analyses of samples only ascertained in Utah included 191 cases and 118 control individuals. Association of single nucleotide polymorphisms (SNPs) among African-American individuals was tested in 129 unrelated nondiabetic individuals who had no family history of T2DM and 275 T2DM subjects who had at least one diabetic first-degree relative. The diabetic cases included 150 individuals who had diabetes and diabetic nephropathy (end-stage renal disease or a spot albumin to creatinine ratio > 300 mg/g creatinine) and 125 individuals with T2DM but no nephropathy (albumin to creatinine ratio < 30 mg/g creatinine). Alternatively, the association of IL-6R variants with diabetes and diabetic nephropathy in African-Americans was tested in samples of DNA pools constructed in triplicate from each of the three African-American subject groups (nondiabetic controls, individuals with T2DM and diabetic nephropathy, and individuals with T2DM but without nephropathy).

Insulin sensitivity was tested in 126 Caucasian nondiabetic members from 26 families for whom measurements of insulin sensitivity were available, as described previously (20). All subjects provided written informed consent under protocols approved by either the University of Utah or the University of Arkansas for Medical Sciences Institutional Review Boards.

Detection of sequence variation

IL-6R genomic sequence was obtained from the public database (accession no. NT_021933) and compared with the mRNA sequence (accession no. NM_000565) to determine the intron-exon boundaries. We designed 11 sets of primers to amplify each of the 10 exons, 100–200 bp of each flanking intron, and 1023 bp of the 5' flanking region using Primer 3.0 software (Whitehead Institute for Biomedical Research, Boston, MA). Each fragment of 480–580 bp was amplified in the presence of ³²P-deoxycytidine triphosphate and digested with the appropriate restriction enzyme to generate fragments of 100–280 bp before single-strand conformation polymorphism analysis, as described previously (21). Conformation variants were characterized using bidirectional sequence analysis (22) on LI-COR GR-4200 sequencers (LI-COR Biotech, Lincoln, NE). Primer sequences and detailed methods are available from the authors.

Genetic analysis

Biallelic SNPs were genotyped by pyrosequencing on a PSQ-96 machine (Pyrosequencing, Inc., Uppsala, Sweden) according to the manufacturer methods but with the modification that each reaction was coamplified with a universal biotinylated primer and the complementary sequence was added to each allele-specific primer. SNP2 (rs4845617) could not be assayed accurately using pyrosequencing and was typed using an oligonucleotide ligation assay as described previously (23). In African-Americans, noncoding and synonymous SNPs were typed initially in pools using Pyrosequencing and Allele Quantification software (Pyrosequencing, Inc.). Each pool was constructed in triplicate and analyzed in duplicate. SNPs were typed in individual African-American samples if they altered the coding sequence, showed an association in Caucasians, showed a 5% difference in allele frequency estimated from pooled samples, or if pooled typing failed quality control checks. Finally, DNA from at least 92 unrelated individuals was tested for each ethnic group to determine linkage disequilibrium and Hardy Weinberg equilibrium. All reported SNPs were in Hardy Weinberg equilibrium.

Allele-specific expression

Differential expression of the two IL-6R alleles was examined in individuals heterozygous for SNPs located within the mRNA sequence. Total RNA (1 µg) from Epstein-Barr virus-transformed lymphocytes was reverse transcribed using random primers and Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA). Allele-specific expression of SNPs 11, 12, 13, and 16 was assayed using pyrosequencing assays as described earlier, and expression of each allele was quantified using Allele Quantification software (Pyrosequencing, Inc.). The ratio of the alleles was compared with genomic DNA using the same assay. Primer sequences are available upon request.

Statistical analysis

The significance of differences in allele frequencies at SNPs was assessed by allelic association using Fisher’s exact test. The effect of each SNP on quantitative traits was evaluated using general linear regression models and tested in SPSS for Windows, version 12.0 software (SPSS, Inc., Chicago, IL). Skewed variables (BMI and insulin sensitivity) were natural logarithmically transformed to normality before analysis. All models included diagnosis, genotype, and gender as fixed factors. Family studies included age as a covariate and family membership as a random factor. Nominal significance was considered to be P < 0.05, and results are presented without correction for multiple tests given the correlations between SNPs. Linkage disequilibrium was assessed by allele counting and the expectation maximization algorithm, as implemented in the 2LD program (24) to assess D' and r² values. Differences in haplotype distributions between cases and controls were tested using Arlequin version 2.0 (Genetics and Biometry Laboratory, Department of Anthropology, University of Geneva. Geneva, Switzerland) (25). Allele-specific expression was tested by comparing the major allele frequency estimated from Allele Quantification software (Pyrosequencing, Inc.) in the cDNA from transformed lymphocytes to that in at least 97 DNA samples from heterozygous individuals using a Mann-Whitney U test. Reported P values are based on the comparison of the ratio in cDNA to that in genomic DNA, but the reported allelic ratios for mRNA were corrected by dividing the mRNA ratio by the mean ratio estimated from at least 100 DNA samples from heterozygous individuals. Distortion of allelic ratios in mRNA was also tested by comparing the number of individuals with 50% increased expression between mRNA and DNA samples using Fisher’s exact test.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Sequence variation in the IL-6R gene

We screened the exons, adjacent intronic sequences, and 5' flanking region of the IL-6R gene spanning more than 60 kb of genomic DNA in both Caucasian and African-American individuals. We identified 17 SNPs and the previously examined microsatellite polymorphism (17), including two nonsynonymous SNPs (D358A, SNP11, or rs8192284; V385I, SNP12, or rs2228146) and two synonymous SNPs (SNP3, A31, or rs8192282; SNP4, H70, or rs2229237; Table 1; Fig. 1). In total, five SNPs were unique to African-American subjects, including V385I (SNP12), and two SNPs were observed only in Caucasian subjects. We identified one additional SNP from the public database (www.ncbi.nih.gov/SNP) in a region of intron 1 that was not screened (rs1386821; SNP17).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Map of IL-6R gene. Exons are shown as black boxes; the 5' and 3' untranslated regions are shown as gray boxes. Each SNP is shown in the approximate location, as detailed in Table 1.

Linkage disequilibrium and haplotype analyses

Among Caucasians, SNPs from exon 2 (SNP3) to the 3' untranslated region (SNP16) were in strong linkage disequilibrium when measured by D' values (D' > 0.88; Table 3). We selected seven tag SNPs using r² > 0.8 for the 10 SNPs that were over 10% minor allele frequency using the LDSelect program (26) (Table 3). Among Caucasians, only eight haplotypes comprising SNPs 2, 17, 3, 6, 7, 15, and 16 were predicted with a frequency of over 5%, thus accounting for 70% of all haplotypes (Table 4). Global comparison showed no significant difference in the distribution of the haplotypes between case and control populations (P = 0.35). Among African-American individuals, the block structure was similar with SNPs 3 through 12 showing strong linkage disequilibrium by D' (D' > 0.83), but no SNP pair exceeded the r² threshold of 0.8 (Table 5). Only SNPs 5 and 7 were associated (r² = 0.73). Although 10 tag SNPs are predicted based on the lower levels of linkage disequilibrium, our primary strategy in African-Americans was to test pooled DNA samples. Hence, we tested haplotypes comprising the seven SNPs that were typed in individual samples (Table 5). Again, we observed only seven of 128 possible haplotypes with a predicted frequency of more than 5%, and these seven haplotypes accounted for 72% of all predicted haplotypes (Table 6). In contrast to individual SNP data, the global haplotype distribution between cases and controls was not significantly different (P = 0.15; Table 6), although two rare haplotypes not shown in Table 5 were significantly different between cases and controls (GTCAACT; frequency 0.041 vs. 0.009, P = 0.035; GGCAGCT; frequency 0.008 vs. 0.029, P = 0.03). Notably, the rare A allele of SNP12 (V385I) was present on only one common haplotype (GTCAACC; Table 6), where a trend to an association was noted (case frequency, 0.107; control frequency, 0.165), but the same allele was found on two rare haplotypes, including GTCAACT noted earlier, that were present in 3.3% and 1.3% of the full population. The presence of this allele on three haplotypes is consistent with low levels of linkage disequilibrium among the seven SNPs typed in African-American individuals (Table 5).

IL-6 levels are reported to correlate well with insulin resistance, and data support an association of IL-6 polymorphisms with insulin sensitivity. To address the hypothesis that IL-6R polymorphisms might alter insulin sensitivity, we tested SNPs 3, 6, 11, 16, and 17, which were both common and not in strong linkage disequilibrium, in 126 Caucasian individuals for whom insulin sensitivity measures were available. No SNP showed any trend to influence insulin sensitivity when age, gender, BMI, and family membership were included as covariates in a general linear model (P > 0.5 for all five SNPs). A comparable African-American population was not available for this study.

A previous study suggested that IL-6R variants might influence BMI (27). To test this hypothesis, we examined the following three populations: the 126 subjects who had undergone careful phenotyping, the Caucasian case control population, and our African-American case control population. Among the carefully phenotyped samples, we again found no trend for SNPs 3, 6, 11, or 16 to influence BMI (P > 0.5 for all analyses) when age, gender, glucose tolerance status, and family membership were included in the model. Similarly, among 360 Caucasian individuals with or without diabetes, we found no trend for SNPs 17, 3, 5, 6, 9, 11, 13, or 16 to influence BMI, neither when tested using a general linear model with gender, diagnosis, and IL-6R genotype as covariates nor when BMI was compared by genotype without considering covariates (data not shown). Among African-American subjects, SNP7 was associated with BMI whether genotypes were compared by ANOVA without consideration of diabetes status (P = 0.030) or when age, gender, and diagnosis were included in the model (P = 0.015). However, no other SNP, including V385I and D358A, showed any association with obesity (data not shown). Notably, most subjects with end-stage renal disease were not included in these analyses because accurate height and weight could not be obtained. Among those individuals for whom BMI was available, we would anticipate that advanced nephropathy would result in weight loss, but this was not included in our analysis.

Allele-specific expression

Several recent studies have demonstrated that differences in allele-specific expression for polymorphisms in the coding region of genes are common and inherited in transformed lymphocytes (28). SNPs in coding regions offer the possibility of comparing the ratio of the levels between two alleles among heterozygous individuals. Hence, we tested whether sequence variation in IL-6R might alter the mRNA ratio between the alleles by comparing the ratio of the two alleles for SNPs 11 (D358A), 12 (V1385I), 13 (3' untranslated region), and 16 (3' untranslated region) (Table 7). To correct for possible unequal amplification of the alleles, we used the same assay for both DNA and cDNA, and we corrected the raw measured ratio in cDNA using the ratio in genomic DNA. Significance was judged by comparing the uncorrected ratio in cDNA prepared from transformed lymphocyte to that observed in genomic DNA using a nonparametric Mann-Whitney U test. The results (Table 4) suggested that some but not all heterozygous individuals had increased expression of one allele by 50–250%, for mean levels that showed 30–60% excess expression from one allele.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Considerable data support a role for adipocyte-derived cytokines (adipokines), such as TNF-, leptin, adiponectin, resistin, and IL-6, in insulin sensitivity and possibly T2DM. The metabolic pathways for these factors are thus potential candidates for diabetes pathogenesis. We examined the IL-6R based on considerable data supporting IL-6 in the pathogenesis of insulin resistance in obesity and T2DM (14, 29), the key role of IL-6R in IL-6 action, and the location of the IL-6R gene on chromosome 1q21 in a region of widely replicated linkage to T2DM. We identified 17 SNPs, including two that alter amino acids in the transmembrane domain. D358A is predicted to alter the splicing of IL-6R to sIL-6R and thus might alter the response to IL-6 in nonhepatic tissues (30). Despite the putative functional consequences, we only found a trend to an association of D358A among the subset of our Caucasian population that was ascertained in Utah for Northern European ancestry. Among all Caucasians, we could not show an association of this variant with T2DM. Similarly, SNPs 13 and 16, which were associated with unequal allelic mRNA levels, showed a trend to an association with T2DM among all Caucasians, but no other sequence variation in the IL-6R gene or haplotypes comprising tag SNPs were associated with T2DM among Caucasians. Additionally, no SNP was associated with measures of insulin sensitivity or obesity among our Caucasian population.

Our findings differ from the recent report of Wolford et al. (27) in Pima Indians, in which only two SNPs were identified from screening and only five SNPs were evaluated. These authors also reported no association of IL-6R SNPs with T2DM or prediabetic measures, but they reported an association of genotypes for all five SNPs, including D358A, with BMI. Escobar-Morreale et al. (31) examined the association of a microsatellite polymorphism of the IL-6R gene with obesity in Spanish women, but they did not test other variation in the gene. They reported an increased frequency of the 149-bp allele with obesity defined as a BMI of more than 25 kg/m². Pedersen et al. (32) recently typed the single D358A variant in a large Danish cohort of 1393 subjects with T2DM and 4596 glucose-tolerant subjects. In contrast to Wolford et al. (27) and in support of our results in Caucasian Americans of Northern European ancestry, they found a modest association of D358A with T2DM (P = 0.02) but not with obesity or surrogate measures of insulin sensitivity (Pedersen, O., personal communication). We have 80% power to detect a 10% difference in allele frequencies for common variants, which translates into an OR of 1.5. Hence, the modest risk of the D358A allele (OR = 1.14) in the Danish study would likely not have reached statistical significance in our smaller study, and thus, our finding of a trend to an association is consistent with the Danish study.

Among African-American subjects, we identified a previously unreported amino acid change, V385I, that was associated with both T2DM and diabetic nephropathy. This variant, which was not seen in Caucasians in our study, not observed in Pima Indians in the study by Wolford et al. (27), and not reported among the seven SNPs detected in Koreans (33), is predicted to lie within the transmembrane domain of the IL-6R gene. However, the functional consequences of this conservative substitution are unknown. The apparent association of different alleles of V385I with diabetes and diabetic nephropathy was unexpected. The I385 variant was more common among diabetic individuals without nephropathy than among either controls or diabetic individuals with nephropathy, whereas the frequency was similar among control individuals and individuals with diabetic nephropathy. This finding may represent a spurious association. Alternatively, I385 may predispose to diabetes but protect against diabetic nephropathy, whereas the V385 allele may impart a modest protection against diabetes but increase the risk of nephropathy among those with diabetes and hyperglycemia. Given the complicated association and the modest P values, most of which would not be significant with a Bonferroni correction, this finding must be confirmed in other African-American populations. Nonetheless, a previous study supported a role for IL-6 polymorphisms in the progression of diabetic nephropathy (11). We also found a nominally significant association of SNP7 (intron 5) with BMI among African-American individuals. Again, considering the seven SNPs examined, the significance of this finding is uncertain.

In addition to the evidence for an association of V385I with diabetes and diabetic nephropathy in African-Americans and the trend to an association of SNPs 11 (D358A), 13, and 16 in Caucasians, we found evidence for possible cis-acting regulatory SNPs in the IL-6R gene through altered ratios of mRNA between two alleles from heterozygous individuals. We anticipated that either regulatory SNPs that altered mRNA expression or SNPs that stabilized the mRNA, such as 3' untranslated region SNPs, would alter the ratio between two alleles. Two scenarios for cis-acting SNPs are possible; there is either a direct effect from the SNP responsible for the difference or an indirect effect through partial linkage disequilibrium with an upstream regulatory SNP. The former scenario should give uniform ratios of one allele to the other, whereas incomplete linkage disequilibrium would result in some individuals with an altered ratio, and other individuals may be homozygous for the regulatory variant and would not show an altered mRNA ratio. Our data are most compatible with the latter scenario. Hence, modest increases in the mean ratio, in fact, reflect a large number of individuals in whom the mRNA ratio between alleles is 1.5 or greater. Furthermore, this observation applied to all four SNPs tested and to both ethnic groups. We propose that the altered allelic expression reflects an upstream polymorphism that altered IL-6R mRNA expression in individuals who carried the regulatory SNP. The apparently incomplete linkage disequilibrium between this undetected SNP and our transcribed SNPs suggests that the variant is likely in the 5' region of the gene, beyond the haplotype block and possibly outside of the region screened. Alternatively, the altered ratio might reflect epigenetic regulation, such as imprinting, although no imprinting in this region has been reported. Were the SNP in an unknown upstream regulatory region, low levels of linkage disequilibrium between the SNPs we tested and that putative regulatory SNP would likely mask any association with diabetes or metabolic traits.

In summary, we have identified a large number of SNPs in two populations in the IL-6R gene. Many of these SNPs were not identified in previous screens of the gene in Pima Indians or Koreans. We find the best evidence for an association of an amino acid change (V385I) with T2DM and diabetic nephropathy in African-Americans, and we find a trend to an association of several SNPs, including D358A, with T2DM in Caucasians. The latter finding corroborates unpublished data from Pedersen et al. (32). Finally, we demonstrate altered mRNA levels between the alleles of approximately 50% of individuals heterozygous for coding and 3' untranslated region SNPs, suggesting that, in addition to the amino acid variants (nonsynonymous SNPs), SNPs altering mRNA expression or perhaps stability are likely. Further studies are required to determine the identity of the regulatory SNP and to confirm the associations of D358A and V385I with T2DM.

	Acknowledgments

 
We thank Yiwen Jia and Xiaoqin Wang for RNA preparation and technical support, and the General Clinical Research Center nursing and laboratory staff for invaluable assistance.

	Footnotes

 
This work was supported by Grants DK039311 and DK054636 from the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, by the Research Service of the Department of Veterans Affairs, by grant support from the American Diabetes Association, and by General Clinical Research Center Grant M01RR14288 from National Center for Research Resources (National Institutes of Health) to the University of Arkansas for Medical Sciences.

First Published Online November 23, 2004

Abbreviations: BMI, Body mass index; IL-6R, IL-6 receptor; sIL-6R, soluble IL-6 receptor; SNP, single nucleotide polymorphism; T2DM, type 2 diabetes mellitus.

Received August 12, 2004.

Accepted November 10, 2004.

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