Variable Number Of Tandem Repeats Class Research Articles

Both Polymorphic Variable Number of Tandem Repeats and Autoimmune Regulator Modulate Differential Expression of Insulin in Human Thymic Epithelial Cells

OBJECTIVEPolymorphic INS-VNTR plays an important role in regulating insulin transcript expression in the human thymus that leads to either insulin autoimmunity or tolerance. The molecular mechanisms underlying the INS-VNTR haplotype-dependent insulin expression are still unclear. In this study, we determined the mechanistic components underlying the differential insulin gene expression in human thymic epithelial cells, which should have profound effects on the insulin autoimmune tolerance induction.RESEARCH DESIGN AND METHODSA repetitive DNA region designated as a variable number of tandem repeats (VNTR) is located upstream of the human insulin gene and correlates with the incidence of type 1 diabetes. We generated six class I and two class III VNTR constructs linked to the human insulin basal promoter or SV40 heterologous promoter/enhancer and demonstrated that AIRE protein modulates the insulin promoter activities differentially through binding to the VNTR region.RESULTSHere we show that in the presence of the autoimmune regulator (AIRE), the class III VNTR haplotype is responsible for an average of three-fold higher insulin expression than class I VNTR in thymic epithelial cells. In a protein-DNA pull-down experiment, AIRE protein is capable of binding to VNTR class I and III probes. Further, the transcriptional activation of the INS-VNTR by AIRE requires the insulin basal promoter. The VNTR sequence loses its activation activity when linked to a heterologous promoter and/or enhancer.CONCLUSIONSThese findings demonstrate a type 1 diabetes predisposition encoded by the INS-VNTR locus and a critical function played by AIRE, which constitute a dual control mechanisms regulating quantitative expression of insulin in human thymic epithelial cells.

Risk of autoimmune diabetes in APECED: association with short alleles of the 5′insulin VNTR

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autoimmune disease causing a wide spectrum of autoimmune dysfunction potentially including diabetes of an autoimmune etiology. We have previously described a pair of discordant APECED siblings and pointed to a possible role of 5'insulin variable number of tandem repeats (VNTR) locus IDDM2 in the appearance of diabetes within this disease. In vitro studies have previously suggested that class I VNTR alleles were associated with decreased fetal thymic insulin expression. We genotyped the 5'INS VNTR locus and several flanking 11p15.5 markers in 50 Finnish APECED subjects and explored the possible contribution of IDDM2 in the development of diabetes. The shorter 5'INS VNTR class I alleles (<35 repeats) were more prevalent in the diabetic Finnish APECED subjects than in non-diabetic APECED subjects. Logistic regression analysis revealed that having 1 short (<35) VNTR allele did not increase the risk of developing diabetes (95% CI 0.6-27.0), whereas having 2 short alleles conferred a 43.5-fold increased risk (95% CI 3.0-634.6). We conclude that short 5'INS VNTR class I alleles play a role in susceptibility to autoimmune diabetes in the context of APECED.

Impact of IDDM2 on disease pathogenesis and progression in children with newly diagnosed type 1 diabetes: reduced insulin antibody titres and preserved beta cell function

The insulin-dependent diabetes mellitus 2 gene (IDDM2) is a type 1 diabetes susceptibility locus contributed to by the variable number of tandem repeats (VNTR) upstream of the insulin gene (INS). We investigated the association between INS VNTR class III alleles (-23HphIA/T) and both insulin antibody presentation and residual beta cell function during the first year after diagnosis in 257 children with type 1 diabetes. To estimate C-peptide levels and autoantibody presentation, patients underwent a meal-stimulated C-peptide test 1, 6, and 12 months after diagnosis. The insulin -23HphIA/T variant was used as a marker of class III alleles and genotyped by PCR-RFLP. The insulin antibody titres at 1 and 6 months were significantly lower in the class III/III and class I/III genotype groups than in the class I/I genotype group (p = 0.01). Class III alleles were also associated with residual beta cell function 12 months after diagnosis and independently of age, sex, BMI, insulin antibody titres, and HLA-risk genotype group (p = 0.03). The C-peptide level was twice as high among class III/III genotypes as in class I/I and class I/III genotypes (319 vs 131 and 166 pmol/l, p=0.01). Furthermore, the class III/III genotype had a 1.1% reduction in HbA(1)c after adjustment for insulin dose (p = 0.04). These findings suggest a direct connection in vivo between INS VNTR class III alleles, a decreased humoral immune response to insulin, and preservation of beta cell function in recent-onset type 1 diabetes.

INS VNTR Class Genotype and Indexes of Body Size and Obesity

The relevance of the insulin gene (INS) variable number tandem repeat (VNTR) polymorphism to indexes of body size and adult obesity is inconclusive. Given the equivocal reports on the association between the VNTR class genotype at the insulin gene locus and indexes of body size and obesity, we assessed these associations in a series of cohort studies based on 7,999 middle-aged men and women. We found no convincing evidence that INS VNTR class genotype was associated with indexes of body size and adult obesity. These data suggest that INS VNTR class is not an important determinant of size and body weight regulation in middle-aged men and women.

OAS1 Splice Site Polymorphism Controlling Antiviral Enzyme Activity Influences Susceptibility to Type 1 Diabetes

Both genetic and nongenetic factors contribute to the development of type 1 diabetes. Many investigations, including prospective studies of high-risk children, have implicated virus infections as predisposing environmental agents. We previously reported that basal activity of the key antiviral enzyme 2'5'-oligoadenylate synthetase (2'5'AS) was significantly elevated in type 1 diabetic patients compared with healthy control subjects. Recently, we showed that an A/G splice site single nucleotide polymorphism (SNP) in the OAS1 gene encoding 2'5'AS is strongly associated with basal 2'5'AS activity. Basal enzyme activity was highest in individuals with GG genotype and lowest in those with AA genotype. In the present study, we genotyped 835 type 1 diabetic and 401 healthy siblings at the OAS1 splice site polymorphism and (for comparison) at an A/C SNP of the insulin (IDDM2) locus. Results showed that OAS1 GG and GA were significantly increased in diabetic compared with healthy siblings (P = 0.0023). The strength of association was similar to that at IDDM2, where, as expected, the C/C (variable number tandem repeat class I homozygote) genotype was increased in affected compared with healthy siblings (P = 0.0025). The results suggest that host genetic response to virus infection could influence susceptibility to type 1 diabetes.

Insulin gene variable number of tandem repeats is associated with increased fat mass during adolescence in non‐obese girls

Obesity and related disorders have become a major health problem. Understanding the interaction between genetic and environmental factors influencing the susceptibility to develop obesity is important when pinpointing people at risk. In a longitudinal study of 256 non‐obese adolescents, the influence of the insulin gene (INS) variable number of tandem repeats (VNTR) on anthropometric measures and fat mass was investigated. The adolescents were examined at the age of 12.4 (2.3) (mean, SD) and 16.2 (2.3) years, and at follow‐up with dual x‐ray absorptiometry (DXA) for measurement of body composition. INS VNTR classes I and III alleles were investigated using the −23T/A single nucleotide polymorphism as a surrogate marker. There was a non‐significant trend towards increased body mass index (BMI) and fat mass with the class III allele in girls. Homozygotes for the INS VNTR class III allele had a greater increase in BMI compared with those that were homozygous or heterozygous for the class I allele (3.8 (1.6) versus 2.4 (1.9) kg/m2, p = 0.03), and they had higher fat mass (36.4 (3.9) versus 31.3 (6.8)%, p = 0.02) at follow‐up. Our finding that homozygosity of the INS VNTR class III allele seems to predispose to increased weight gain and fat mass raises the possibility that this genotype may be one of the important factors in the gene‐environment interaction that eventually results in overweight and insulin resistance.

Analysis of Multiple Data Sets Reveals No Association between the Insulin Gene Variable Number Tandem Repeat Element and Polycystic Ovary Syndrome or Related Traits

Variation at the insulin gene VNTR (variable number tandem repeat) minisatellite has been reported to be associated with polycystic ovary syndrome (PCOS), but findings have been inconsistent and all studies have featured small sample sizes. To gain a robust understanding of the role of the INS-VNTR in PCOS susceptibility. Case-control, family-based association and quantitative trait analyses. A UK population comprising 255 parent-offspring trios, 185 additional cases, and 1062 control subjects (cases and controls all British/Irish) as well as 1599 women from a northern Finland population-based birth cohort characterized for PCO symptomatology and testosterone levels. VNTR class was inferred from genotyping of the -23HphI variant. None. INS-VNTR genotype frequencies between subject groups, body mass index, and testosterone levels by genotype. Case-control analyses in both UK and Finnish samples failed to confirm previously reported class III allele associations with PCOS (UK, P = 0.43, Finnish, P = 0.31; Kruskal-Wallis chi2). Transmission analysis in trios showed no excess transmission of either allele (P = 0.62), regardless of parent of origin (maternal: P = 0.73; paternal: P = 0.66). No association between genotype and testosterone levels was seen in any sample (UK PCOS subjects, P = 0.95; Finnish symptomatic cases, P = 0.38; Finnish control women, P = 0.58). Despite the strong biological candidacy and supportive data from previous studies, we conclude that variation at the INS-VNTR has no major role in the development of PCOS.

Interaction between insulin (VNTR) and hepatic lipase (LIPC−514C&gt;T) variants on the response to an oral glucose tolerance test in the EARSII group of young healthy men

Insulin resistance is polygenic in origin, and can be observed at an early age. We have shown that variations in APOC3-482T>C and hepatic lipase (LIPC)-514C>T), individually (APOC3 alone) and interactively, modulate insulin and glucose levels after an OGTT in young healthy men participating in the European Atherosclerosis Research Study II (EARSII). Variation in the insulin gene (INS) variable number tandem repeat (VNTR) has been found to predispose to type 1 and type 2 diabetes. We have evaluated the HphI site 23 bp upstream of the INS gene (a surrogate marker for the VNTR) in EARSII (n=822), to determine if variation in INS contributes to insulin resistance. Carriers of the INS VNTR class III (HphI-) allele (frequency=0.29 (95%CI 0.27, 0.31)) had significantly higher 60-min insulin concentrations after the OGTT (P=0.014) and a marginally higher AUC insulin (P=0.07), compared to class I (HphI+) homozygotes. However, this effect on AUC insulin was modified by the level of physical activity, displaying significant gene:environment interaction (P=0.03). We tested for gene:gene interaction between the INS VNTR and both the LIPC-514C>T and APOC3-482T>C. While there was a significant interaction between INS VNTR and LIPC-514C>T on AUC glucose (P=0.013) and on AUC insulin (P=0.015), there was no interaction with APOC3-482T>C. Thus, despite a modest effect of the INS VNTR alone, the influence of this variant on insulin sensitivity was modified by gene:environment and gene:gene interactions, illustrating the biological complexity of insulin resistance.

The insulin gene variable number tandem repeat class I/III polymorphism is in linkage disequilibrium with birth weight but not Type 2 diabetes in the Pima population.

The insulin gene variable number tandem repeat (INS-VNTR) is proposed to exert pleiotropic genetic effects on birth weight and diabetes susceptibility. In our study, we examined the influence of a polymorphism in tight linkage disequilibrium with INS-VNTR (-23Hph1) on birth weight and type 2 diabetes in the Pima population. A parent-offspring "trio" design was used to assess parent-of-origin effects and population stratification. The presence of the -23Hph1 T-allele was associated with lower birth weight (n = 192; -140 g per copy of the T-allele; P = 0.04), even after adjustment for effects of population stratification (P = 0.03). The effects of paternally transmitted T-alleles were greater than those of maternally transmitted alleles (paternally transmitted: -250 g, P = 0.05; maternally transmitted: -111 g, P = 0.43), but this difference was not statistically significant (P = 0.50). The -23Hph1 T-allele was associated with an increased prevalence of type 2 diabetes (P = 0.009), which family-based association analysis suggested was attributable to population structure (P = 0.04) without significant evidence of linkage disequilibrium between diabetes prevalence and genotype (P = 0.86). Thus allelic variation of the INS gene is associated with lower birth weight and increased prevalence of type 2 diabetes. Significant linkage disequilibrium was found between -23Hph1 and birth weight but not type 2 diabetes, an observation that supports a potential functional role of INS polymorphisms in the regulation of birth weight.

Evidence of multiple causal sites affecting weight in the IGF2-INS-TH region of human chromosome 11.

The subtelomeric region of 11p harbours three closely linked genes, TH, INS and IGF2, that have been associated with obesity, size at birth, type I diabetes, polycystic ovary syndrome, overgrowth in Beckwith-Wiedemann syndrome and possibly hypertension. We have previously shown that the IGF2 ApaI single nucleotide polymorphism (SNP) associates with weight and body mass index in middle-aged Caucasian males but that there is no such association with the INS -23/ HphI site that marks INS 5' variable number of tandem repeats (VNTR) class I vs class III VNTR alleles. We report here the examination of three SNP markers in IGF2: 6815 A/T in the P1 promoter, AluI in exon 3 and ApaI in the 3' untranslated region (UTR), INS 5'VNTR class I alleles and the TH01 tetranucleotide microsatellite in a population sample. The analysis has taken into account the possibility that typing failure and the number of parameters required to model multiallelic loci could create spurious significance. We have exercised Hardy-Weinberg equilibrium tests, dichotomised multiallelic series to impose parsimony, and examined the data with failures modelled or excluded. Regression analysis infers that three markers, IGF2 ApaI, TH01 and subclasses of INS VNTR class I independently predict derived weight indices (combined P<10(-8) and accounting up to 2% of population weight variance), with no evidence of interaction. This establishes that there must be multiple causal sites impacting on weight in this genomic region.

Associations of IGF2 ApaI RFLP and INS VNTR class I allele size with obesity.

Body mass index (BMI) is an established epidemiological predictor of coronary disease, diabetes and hypertension. In a previous study of 2560 healthy British Caucasoid males aged 50-61 years (Northwick Park Heart Study II; NPHSII), we showed that IGF2 ApaI AA homozygotes display a mean body weight 3.3 kg lower than GG homozygotes (P = 0.0002) independent of height. Two RFLPs in the insulin (INS) gene, +1127/PstI shown previously and -23/HphI in this study, both of which are in strong linkage disequilibrium with class I/III alleles of the INS 5' variable number tandem repeat (VNTR), are not associated with weight or BMI. The IGF2 ApaI polymorphism therefore appears to mark an effect independent of INS VNTR class I vs class III. We now show by regression that there is a positive correlation of BMI with INS VNTR class I allele size, with an average 0.33% (95% CI = 0.13%, 0.50%) increase in BMI per extra tandem repeat (P < 0.0001) representing variation of 4.8% over the allele size range. However, an alternative interpretation is of 'step' rather than 'slope', the small class I subclass allele group (mode 669 bp) being lighter than the large subclass group (mode 814 bp). This small effect would not be evident as an association between INS VNTR class I/I1 genotype and BMI. The IGF2 ApaI association and INS VNTR class I subclass regression association account for at least 1.1% of population BMI variance. Neither, both, or a third site may be aetiological.