Remapping the insulin gene/IDDM2 locus in type 1 diabetes

Type 1 diabetes susceptibility at the IDDM2 locus was previously mapped to a variable number tandem repeat (VNTR) 5' of the insulin gene (INS). However, the observation of associated markers outside a 4.1-kb interval, previously considered to define the limits of IDDM2 association, raised the possibility that the VNTR association might result from linkage disequilibrium (LD) with an unknown polymorphism. We therefore identified a total of 177 polymorphisms and obtained genotypes for 75 of these in up to 434 pedigrees. We found that, whereas disease susceptibility did map to within the 4.1-kb region, there were two equally likely candidates for the causal variant, -23HphI and +1140A/C, in addition to the VNTR. Further analyses in 2,960 pedigrees did not support the difference in association between VNTR lineages that had previously enabled the exclusion of these two polymorphisms. Therefore, we were unable to rule out -23HphI and +1140A/C having an etiological effect. Our mapping results using robust regression methods show how precisely a variant for a common disease can be mapped, even within a region of strong LD, and specifically that IDDM2 maps to one or more of three common variants in a approximately 2-kb region of chromosome 11p15.     

A polymorphic locus near the human insulin gene is associated with insulin-dependent diabetes mellitus

A polymorphic region flanking the human insulin gene on the short arm of chromosome 11, the insulin-gene-linked DNA polymorphism, can be described as a locus with at least three classes of alleles: a common small "class 1" allele averaging 570 base pairs, a rare intermediate "class 2" allele of about 1320 base pairs, and a large "class 3" allele averaging 2470 base pairs in size. We have determined the genotype at this locus of 393 unrelated diabetic and nondiabetic individuals. Differences were observed in the genotypic and allelic frequencies between groups of different races. Asians [17 nondiabetic, 2 with insulin-dependent diabetes mellitus (IDDM), and 8 with non-insulin-dependent diabetes mellitus (NIDDM)] exhibited the least variation in the size of this locus and 98% of the alleles in this group were class 1. A group of American blacks (32 nondiabetic, 5 with IDDM, and 40 with NIDDM) exhibited considerable variation in the size of this locus, and about 22% of the individuals examined had a genotype that included a rare class 2 allele. In neither of these two racial groups were the genotypic or allelic frequencies different between the nondiabetic and diabetic segments of these groups. However, in a group of Caucasians (83 nondiabetic, 113 with IDDM, and 76 with NIDDM), there was a significantly higher frequency of class 1 alleles and genotypes containing two class 1 alleles in the diabetic patients compared with nondiabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of association of the polymorphic locus in the 5'-flanking region of the human insulin gene and diabetes in American blacks

A polymorphic region 5' to the human insulin gene has been associated with diabetes in earlier studies. This polymorphic region is composed of tandem repeats that fall into 3 general size classes, designated class 1 (600 base pairs), class 2 (1300 base pairs), and class 3 (2500 base pairs). Frequencies of these classes of alleles vary among racial groups. American Blacks have been underrepresented in published studies of insulin gene polymorphism and diabetes. We undertook a cooperative study between two centers (San Francisco and St. Louis) to determine geno-types at the insulin locus in 313 unrelated American Blacks (132 nondiabetic, 27 with IDDM, and 154 with NIDDM). In both centers, nondiabetic individuals were younger and leaner than NIDDM patients. Allelic and genotypic frequencies at the insulin locus were not different between the two centers. Class 1 alleles represented 60% of all alleles, class 2 alleles 11%, and class 3 29%. No class of insulin allele was associated with NIDDM in this study. Subdivision of the study population by obesity, family history, or age at diagnosis failed to detect a subgroup for which the insulin allele was associated with NIDDM. Only 27 IDDM individuals were studied, and no significant association of class 1 alleles with this group was noted. However, examination of more IDDM individuals is required before a definitive statement can be made. Fasting serum triglyceride levels were determined retrospectively in 50 NIDDM individuals. No differences in triglyceride levels among genotypes were noted. The frequency of class 3 alleles in 13 hypertriglyceridemic NIDDM subjects was not different from that of the whole group.(ABSTRACT TRUNCATED AT 250 WORDS)     

A synonymous coding polymorphism in the alpha2-Heremans-schmid glycoprotein gene is associated with type 2 diabetes in French Caucasians

alpha2-Heremans-Schmid glycoprotein (AHSG) is an abundant plasma protein synthesized predominantly in the liver. The AHSG gene, consisting of seven exons and spanning 8.2 kb of genomic DNA, is located at chromosome 3q27, a susceptibility locus for type 2 diabetes and the metabolic syndrome. AHSG is a natural inhibitor of the insulin receptor tyrosine kinase, and AHSG-null mice exhibit significantly enhanced insulin sensitivity. These observations suggested that the AHSG gene is a strong positional and biological candidate for type 2 diabetes susceptibility. Direct sequencing of the AHSG promoter region and exons identified nine common single nucleotide polymorphisms (SNPs) with a minor allele frequency > or =5%. We carried out a detailed genetic association study of the contribution of these common AHSG SNPs to genetic susceptibility of type 2 diabetes in French Caucasians. The major allele of a synonymous coding SNP in exon 7 (rs1071592) presented significant evidence of association with type 2 diabetes (P = 0.008, odds ratio 1.27 [95% CI 1.06-1.52]). Two other SNPs (rs2248690 and rs4918) in strong linkage disequilibrium with rs1071592 showed evidence approaching significance. A haplotype carrying the minor allele of SNP rs1071592 was protective against type 2 diabetes (P = 0.014). However, our analyses indicated that rs1071592 is not associated with the evidence for linkage of type 2 diabetes to 3q27.     

Potential influence of IL1B haplotype and IL1A-IL1B-IL1RN extended haplotype on hand osteoarthritis risk

OBJECTIVE: To assess osteoarthritis (OA) association with the human interleukin-1 (IL-1) region. DESIGN: Sixty-four European-descent cases with radiographic hand OA and 48 European-descent controls were genotyped at nine single nucleotide polymorphism (SNP), one variable-number-of-tandem-repeat (VNTR), and one microsatellite marker extending across loci for IL-1alpha (IL1A), IL-1beta (IL1B), and IL-1 receptor antagonist (IL1RN). The genotype data were used to reconstruct individual locus haplotypes, and then locus haplotypes were used as superalleles for extended haplotype reconstruction. RESULTS: Nine different extended IL1A-IL1B-IL1RN haplotypes occurred at a frequency 0.05 or greater in either cases or controls. Only two IL1A-IL1B-IL1RN extended haplotypes were consistent with previously described extended risk haplotypes and totaled n=9 in cases and n=3 in controls [odds ratio (OR) 2.1, Haldane's chi(2) 1.67, one-sided P 0.1]. Our prior report showed hand OA association with homozygous IL1B rs1143633 minor allele genotype. All except one extended risk haplotype copy also had the IL1B rs1143633 minor allele. The rs1143633 genotype association was explained by one common six-SNP IL1B haplotype bearing rs1143633 minor allele and also risk alleles at rs1143634, rs1143627, and rs16944, component markers of the previously described extended risk haplotypes. The IL1B haplotype bearing all three risk alleles was found in 16 haplotype-homozygous hand OA cases and in four haplotype-homozygous controls and conferred OR 3.4 among homozygotes (nominal P value 0.006). CONCLUSION: Our evidence broadly supports the genetic association of OA phenotypes with an IL-1 region extended risk haplotype and specifically IL1B genotype. The extended risk haplotype previously associated with hip OA appears to be less frequent and has weaker genetic effect in hand OA. Hand OA risk is conferred by homozygous state for the IL1B haplotype characteristic of the extended risk haplotype.     

Analysis of parent-offspring trios provides evidence for linkage and association between the insulin gene and type 2 diabetes mediated exclusively through paternally transmitted class III variable number tandem repeat alleles

Variation at the variable number tandem repeat (VNTR) minisatellite 5' of the insulin gene (INS) is associated with several phenotypes, including type 1 diabetes, polycystic ovary syndrome, and birth weight. Case-control studies have suggested that class III VNTR alleles are also associated with type 2 diabetes, but results have been inconsistent and may reflect population stratification. To explore further the role of the INS-VNTR in type 2 diabetes susceptibility, we used family-based association methods in 155 parent-offspring trios from the British Diabetic Association-Warren Trios repository, each ascertained via a Europid proband with type 2 diabetes. Overall, there was no significant association between diabetes and the INS-VNTR genotype, with 65 of 119 heterozygous parents (55%) transmitting class III and 54 class I (P = 0.16, one-sided). However, whereas maternal transmissions followed Mendelian expectation, there was a marked excess of class III transmission from the 49 heterozygous fathers (34 [69%] vs. 15, P = 0.003 vs. 50% expectation, P = 0.003 vs. maternal transmission). These results confirm that variation within the TH-INS-IGF2 locus, most plausibly at the VNTR itself, influences type 2 diabetes susceptibility. By demonstrating that this effect is mediated exclusively by the paternally derived allele, these findings implicate imprinted genes in the pathogenesis of type 2 diabetes.     

HLA and genetics of IDDM. Holism vs. reductionism?

Analysis of HLA-associated susceptibility to insulin-dependent diabetes mellitus (IDDM) has largely focused on identifying the susceptibility gene. Adherents of a countertrend have long suggested the importance of analysis of HLA haplotypes (combinations of alleles on 1 chromosome) rather than individual genes. Accumulating data suggest that the relationship between IDDM susceptibility and HLA is much more complex than a single susceptibility gene. Consideration of this question should include the possibilities that 1) more than one HLA gene is involved in determining susceptibility or resistance; 2) different alleles of the same gene may be associated with different pathogenetic mechanisms; and 3) different susceptibility-associated haplotypes, even if they share an allele at an IDDM-relevant locus, may behave differently in IDDM. A better understanding of the genetics, and perhaps the pathogenesis, of IDDM may be obtained by following up the clues offered by analysis of the association of HLA haplotypes (rather than individual alleles) with one another, with clinical features of IDDM, and with possible non-HLA-linked susceptibility factors.     

Implication of specific DQB1 alleles in genetic susceptibility and resistance by identification of IDDM siblings with novel HLA-DQB1 allele and unusual DR2 and DR1 haplotypes

Genetic susceptibility to insulin-dependent diabetes mellitus (IDDM) is associated with the HLA-DR3 and DR4 haplotypes. The HLA-DR2 haplotype is negatively associated with IDDM, an association that has been interpreted as dominant protection. Here, we describe the molecular analysis of the HLA class II genes in an unusual family with three HLA-DR1/2 siblings, all of whom have IDDM. With polymerase chain reaction amplification and sequence analysis to characterize the class II alleles, we identified a novel DQB1 allele on the DR1 haplotype and an unusual DQB1 allele on the DR2 haplotype. However, the DRB1 alleles on these DR1 and DR2 haplotypes are the conventional alleles (*0101 and *1501, respectively). These results suggest that it is the conventional DQB1 allele (*0602) not the DRB1 allele (*1501) on the protective DR2 haplotype that confers protection in the general population and, furthermore, that these unusual DQB1 alleles may confer susceptibility to IDDM in this family. The unusual DQB1 allele on this DR2 haplotype encodes Asp at position 57, indicating that it is the allele DQB1*0602 and not simply the presence of this residue that is responsible for the protective effect.     

Susceptibility to large‐joint osteoarthritis (hip and knee) is associated with BAG6 rs3117582 SNP and the VNTR polymorphism in the second exon of the FAM46A gene on chromosome 6

Family with sequence similarity 46, member A (FAM46A) gene VNTR and BCL2‐Associated Athanogene 6 (BAG6) gene rs3117582 polymorphisms were genotyped in a case–control study with 474 large‐joint (hip and knee) osteoarthritis (OA) patients and 568 controls in Croatian population by candidate‐gene approach for association with OA. We found that BAG6 rs3117582 SNP genotypes were associated with protection (major allele homozygote) and susceptibility (major–minor allele heterozygote) to OA. BAG6 rs3117582 major allele (A) was associated with reduced risk to OA while the minor allele (C) was associated with increased risk to OA. We identified 6 alleles harboring 2 to 7 repeats making 20 genotypes for FAM46A. A rare FAM46A VNTR genotype comprising VNTR alleles with four and seven repeats (c/f) was associated with increased OA risk in both genders. The genotype with four and six repeats (c/e) was also associated with increased risk to OA in males. A polymorphic FAM46A allele with six repeats (e) was associated with reduced risk to OA in females. Our results suggest association between the FAM46A gene, BAG6 gene and OA in Croatian population, respectively. This is the first study to show associations between these genetic loci and OA. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:56–62, 2015.

     

A polymorphism in the AMPKalpha2 subunit gene is associated with insulin resistance and type 2 diabetes in the Japanese population

AMP-activated protein kinase (AMPK) acts as a fuel gauge for glucose and lipid metabolism. The gene encoding the alpha2 isoform of the catalytic subunit of AMPK (PRKAA2) is located at one of the Japanese type 2 diabetes loci mapped by our previous genome scan (1p36-32). PRKAA2 is, therefore, a good candidate gene for insulin resistance and type 2 diabetes. We screened all nine exons, their exon-intron boundaries, and the 5' and 3' flanking regions of PRKAA2 to identify single nucleotide polymorphisms (SNPs), and we genotyped 192 type 2 diabetic patients and 272 nondiabetic subjects to assess possible associations between genotypes or haplotypes and type 2 diabetes. None of the 10 SNPs genotyped was associated with type 2 diabetes, but the haplotype analysis, consisting of six representative SNPs, revealed one haplotype, with the A (minor) allele for rs2051040 and a major allele for the other five SNPs, to be associated with type 2 diabetes (P = 0.009). This finding was confirmed in two larger replication samples (657 case and 360 control subjects, P = 0.021; and 356 case and 192 control subjects from the same area in Japan, P = 0.007) and a significant P value was obtained in the joint haplotype analysis of all samples (1,205 case and 824 control subjects, P = 0.0001). Furthermore, insulin resistance was associated with rs2051040 in nondiabetic subjects, and those with the A (minor) allele had a higher homeostasis model assessment of insulin resistance index than those who did not (initial control subjects [n = 272], P = 0.002; and joint replication control subjects [n = 552], P = 0.037). We speculate that the PRKAA2 gene influences insulin resistance and susceptibility to type 2 diabetes in the Japanese population.     

Minor effect of GLUT1 polymorphisms on susceptibility to diabetic nephropathy in type 1 diabetes

Elevation of intracellular glucose in mesangial cells as mediated by GLUT1 may be important in initiating cellular mechanisms that cause diabetic nephropathy. To determine whether DNA sequence differences in GLUT1 confer susceptibility to this complication, single-nucleotide polymorphisms (SNPs) in this gene were examined using a large case-control study. SNPs examined included the known XbaI (intron 2) and HaeIII SNPs (exon 2). Four novel SNPs located in three putative enhancers were also investigated. Homozygosity for the XbaI(-) allele was associated with diabetic nephropathy (odds ratio 1.83 [95% CI 1.01-3.33]). Furthermore, homozygosity for the A allele for a novel SNP (enhancer-2 SNP 1) located in a putative insulin-responsive enhancer-2 was associated with diabetic nephropathy (2.38 [1.16-4.90]). Patients who were homozygous for risk alleles at both XbaI SNP and enhancer-2 SNP 1 [i.e., homozygosity for XbaI(-)/A haplotype] also had an increased risk of diabetic nephropathy (2.40 [1.13-5.07]). Because enhancer-2 SNP 1 may directly control GLUT1 expression, the strong linkage disequilibrium between the two SNPs likely accounts for XbaI SNP being associated with diabetic nephropathy. In conclusion, our study confirms that SNPs at the GLUT1 locus are associated with susceptibility to diabetic nephropathy in type 1 diabetes. Although these SNPs confer a considerable personal risk for diabetic nephropathy, they account for a limited proportion of cases among type 1 diabetic patients.     

The HLA-DPB1--associated component of the IDDM1 and its relationship to the major loci HLA-DQB1, -DQA1, and -DRB1

The major histocompatibility complex (MHC) HLA region on chromosome 6p21 contains the major locus of type 1 diabetes (IDDM1). Common allelic variants at the class II HLA-DRB1, -DQA1, and -DQB1 loci account for the major part of IDDM1. Previous studies suggested that other MHC loci are likely to contribute to IDDM1, but determination of their relative contributions and identities is difficult because of strong linkage disequilibrium between MHC loci. One prime candidate is the polymorphic HLA-DPB1 locus, which (with the DPA1 locus) encodes the third class II antigen-presenting molecule. However, the results obtained in previous studies appear to be contradictory. Therefore, we have analyzed 408 white European families (200 from Sardinia and 208 from the U.K.) using a combination of association tests designed to directly compare the effect of DPB1 variation on the relative predisposition of DR-DQ haplotypes, taking into account linkage disequilibrium between DPB1 and the DRB1, DQA1, and DQB1 loci. In these populations, the overall contribution of DPB1 to IDDM1 is small. The main component of the DPB1 contribution to IDDM1 in these populations appears to be the protection associated with DPB1*0402 on DR4-negative haplotypes. We suggest that the HLA-DP molecule itself contributes to IDDM1.     

Insulin expression levels in the thymus modulate insulin-specific autoreactive T-cell tolerance: the mechanism by which the IDDM2 locus may predispose to diabetes

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic beta-cells. Evidence from our laboratory and others has suggested that the IDDM2 locus determines diabetes susceptibility by modulating levels of insulin expression in the thymus: the diabetes-protective class III alleles at a repeat polymorphism upstream of the insulin gene are associated with higher levels than the predisposing class I. To directly demonstrate the effect of thymic insulin expression levels on insulin-specific autoreactive T-cell selection, we have established a mouse model in which there is graded thymic insulin deficiency in linear correlation with insulin gene copy numbers, while pancreatic insulin remains unaltered. We showed that mice expressing low thymic insulin levels present detectable peripheral reactivity to insulin, whereas mice with normal levels show no significant response. We conclude that thymic insulin levels play a pivotal role in insulin-specific T-cell self-tolerance, a relation that provides an explanation for the mechanism by which the IDDM2 locus predisposes to or protects from diabetes.     

Common variation in the LMNA gene (encoding lamin A/C) and type 2 diabetes: association analyses in 9,518 subjects

Mutations in the LMNA gene (encoding lamin A/C) underlie familial partial lipodystrophy, a syndrome of monogenic insulin resistance and diabetes. LMNA maps to the well-replicated diabetes-linkage region on chromosome 1q, and there are reported associations between LMNA single nucleotide polymorphisms (SNPs) (particularly rs4641; H566H) and metabolic syndrome components. We examined the relationship between LMNA variation and type 2 diabetes (using six tag SNPs capturing >90% of common variation) in several large datasets. Analysis of 2,490 U.K. diabetic case and 2,556 control subjects revealed no significant associations at either genotype or haplotype level: the minor allele at rs4641 was no more frequent in case subjects (allelic odds ratio [OR] 1.07 [95% CI 0.98-1.17], P = 0.15). In 390 U.K. trios, family-based association analyses revealed nominally significant overtransmission of the major allele at rs12063564 (P = 0.01), which was not corroborated in other samples. Finally, genotypes for 2,817 additional subjects from the International 1q Consortium revealed no consistent case-control or family-based associations with LMNA variants. Across all our data, the OR for the rs4641 minor allele approached but did not attain significance (1.07 [0.99-1.15], P = 0.08). Our data do not therefore support a major effect of LMNA variation on diabetes risk. However, in a meta-analysis including other available data, there is evidence that rs4641 has a modest effect on diabetes susceptibility (1.10 [1.04-1.16], P = 0.001).     

Identification of podocin (NPHS2) gene mutations in African Americans with nondiabetic end-stage renal disease

BACKGROUND: Podocin, encoded by NPHS2 and mapped to 1q25.2, is an integral membrane protein exclusively expressed in glomerular podocytes. Mutations in the NPHS2 gene cause autosomal-recessive nephrotic syndrome and have been associated with proteinuria in several populations. Evidence for linkage of end-stage renal disease (ESRD) to chromosome 1q25-31 in the region of NPHS2 has been identified in a genome-wide scan in African American (AA) siblings. METHODS: To investigate the potential role of this gene in ESRD, we sequenced all coding regions and approximately 2 kb of upstream promoter sequence of NPHS2 in 96 unrelated AA nondiabetic ESRD cases and 96 healthy population-based AA controls, and assessed several single nucleotide polymorphisms (SNPs) for association in a larger case-control sample. RESULTS: Fifty-five variants were identified with minor allele frequencies ranging from <1% to 44%. Twenty-three polymorphisms were located in the promoter region, 11 were exonic, 13 were intronic, and 8 were in the 5' and 3'- untranslated regions. Two novel nonsynonymous coding SNPs were identified (A44E and A61V). An insertion polymorphism in intron 3, IVS3+9insA, was detected in 6 ESRD patients and in no controls. This variant, and 4 other common SNPs, were evaluated in a larger sample of 288 AA ESRD cases and 278 AA controls. The overall minor allele frequencies for the insertion allele were 0.018 in cases and 0.002 in controls. Significant evidence of association of IVS3+9insA was observed (P= 0.012), and the haplotype containing the insertion allele in cases was also associated. CONCLUSION: These results suggest that uncommon variants of the NPHS2 gene may play a role in the development of nondiabetic ESRD in AAs.     

Genetic control of alternative splicing in the TAP2 gene: possible implication in the genetics of type 1 diabetes

The transporter 2, ATP-binding cassette, subfamily B (TAP2) is involved in the transport of antigenic peptides to HLA molecules. Coding TAP2 polymorphisms shows a strong association with type 1 diabetes, but it is not clear whether this association may be entirely due to linkage disequilibrium with HLA DR and DQ. Functionally, rat Tap2 nonsynonymous single-nucleotide polymorphisms (nsSNPs) confer differential selectivity for antigenic peptides, but this was not shown to be the case for human TAP2 nsSNPs. In the human, differential peptide selectivity is rather conferred by two splicing isoforms with alternative carboxy terminals. Here, we tested the hypothesis that alleles at the coding SNPs favor different splicing isoforms, thus determining peptide selectivity indirectly. This may be the basis for independent contribution to the type 1 diabetes association. In RNA from heterozygous lymphoblastoid lines, we measured the relative abundance of each SNP haplotype in each isoform. In isoform NM_000544, the G (Ala) allele at 665 Thr>Ala (rs241447) is more than twice as abundant as A (Thr) (GA = 2.2 +/- 0.4, P = 1.5 x 10(-4)), while isoform NM_018833 is derived almost exclusively from chromosomes carrying A (AG = 18.1 +/- 5.6, P = 2.04 x 10(-7)). In 889 Canadian children with type 1 diabetes, differential transmission of parental TAP2 alleles persisted (P = 0.011) when analysis was confined to chromosomes carrying only DQ*02 alleles, which mark a conserved DR-DQ haplotype, thus eliminating most of the variation at DR-DQ. Thus, we present evidence of TAP2 association with type 1 diabetes that is independent of HLA DR-DQ and describe a plausible functional mechanism based on allele dependence of splicing into isoforms known to have differential peptide selectivities.     

Determinants of IDDM and perinatal mortality in children of diabetic mothers

Offspring of women with insulin-dependent diabetes mellitus (IDDM) have a lower risk of developing IDDM than offspring of men with IDDM (1). To determine whether the risk of diabetes in offspring of diabetic mothers has changed after dramatic improvements in perinatal survival of these infants, we undertook a follow-up study of 1602 pregnancies of 739 women with IDDM who were patients at the Joslin Diabetes Center. Improvements in perinatal survival were abrupt rather than gradual. During the two decades before 1961, perinatal mortality was stable around 23%. After a sudden drop in 1961, it stabilized around 14% until 1975, when it was brought down to 4%, where it has remained. Of the 1391 offspring who survived the neonatal period, IDDM has developed in 21, a cumulative risk of 2.1 +/- 0.5% (SE) by age 20 yr. This is one-third the risk previously reported for offspring of fathers with IDDM and is independent of the calendar time of the births (1). The risk of diabetes in offspring of diabetic mothers is increased in young mothers and is otherwise independent of risk factors for perinatal mortality in this series. We conclude that there is no evidence that selective loss of diabetes-susceptible fetuses in perinatal deaths is a mechanism for the lower incidence of IDDM in the offspring of mothers with IDDM than in those of fathers with IDDM. The principal alternative mechanism is that exposure in utero to an affected mother can protect a fetus from developing IDDM later in life. Induction of immunologic tolerance to the autoantigens of the beta-cells is a plausible mechanism for this protective effect.