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There is increasing evidence to suggest that type 2 diabetes mellitus (T2D), a pandemic metabolic disease, may be caused by β-cell dedifferentiation (βCD). However, there is currently no universal definition of βCD, and the underlying mechanism is poorly understood. We hypothesise that a high-glucose in vitro environment mimics hyperglycaemia in vivo and that β cells grown in this milieu over a long period will undergo dedifferentiation. In the present study, we report that the pancreatic β cell line mouse insulinoma 6 (MIN6) grown under a high-glucose condition did not undergo massive cell death but exhibited a glucose-stimulated insulin-secreting profile similar to that of immature β cells. The expression of insulin and the glucose-sensing molecule glucose transporter 2 (Glut2) in late passage MIN6 cells was significantly lower than the early passage at both the RNA and protein levels. Mechanistically, these cells also expressed significantly less of the ‘pancreatic and duodenal homebox1’ (Pdx1) β-cell transcription factor. Finally, passaged MIN6 cells dedifferentiated to demonstrate some features of β-cell precursors, as well as neuroendocrine markers, in addition to expressing both glucagon and insulin. Thus, we concluded that high-glucose passaged MIN6 cells passaged MIN6 cells. provide a cellular model of β-cell dedifferentiation that can help researchers develop a better understanding of this process. These findings provide new insights that may enhance knowledge of the pathophysiology of T2D and facilitate the establishment of a novel strategy by which this disease can be treated. [ABSTRACT FROM AUTHOR]

Objective: Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1 diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions.Research Design and Methods: To address this issue, the Type 1 Diabetes Genetics Consortium recruited 3,892 families, including 4,422 affected sib-pairs. After genotyping 6,090 markers, linkage analyses of these families were performed, using a novel method and taking into account factors such as genotype at known susceptibility loci.Results: Evidence for linkage was robust at the HLA and INS loci, with logarithm of odds (LOD) scores of 398.6 and 5.5, respectively. There was suggestive support for five other loci. Stratification by other risk factors (including HLA and age at diagnosis) identified one convincing region on chromosome 6q14 showing linkage in male subjects (corrected LOD = 4.49; replication P = 0.0002), a locus on chromosome 19q in HLA identical siblings (replication P = 0.006), and four other suggestive loci.Conclusions: This is the largest linkage study reported for any disease. Our data indicate there are no major type 1 diabetes subtypes definable by linkage analyses; susceptibility is caused by actions of HLA and an apparently random selection from a large number of modest-effect loci; and apart from HLA and INS, there is no important susceptibility factor discoverable by linkage methods. [ABSTRACT FROM AUTHOR]

As part of its efforts to identify genes affecting the risk of type I diabetes (T1D), the Type I Diabetes Genetics Consortium commissioned an extensive survey of variants associated with genes reported earlier to have an association with disease susceptibility. In this report, we present the analysis of a set of single-nucleotide polymorphisms (SNPs) within and flanking the IL12B gene, which encodes the p40 subunit of the cytokines interleukin (IL)-12 and IL-23. No SNP showed individually significant association in the population as a whole. Nevertheless, subjects stratified according to genotype at the earlier reported SNP in the IL12B 3′UTR, rs3212227, confirmed small, but significant, differences in age of disease onset with a relative hazard=0.88 (P=0.005). The protective effect of rs3212227 allele 2 was gender specific (P=0.004 overall and P=0.0003 when unaffected siblings were considered). Among females, the 2.2 genotype was more protective, with relative hazard=0.75. We conclude that while there was no major effect of IL12B polymorphisms on T1D susceptibility in the entire study group, they have an impact on a subset of at-risk individuals. [ABSTRACT FROM AUTHOR]

Type I diabetes (T1D) results from interactions between environmental exposures and genetic susceptibility leading to immune dysfunction and destruction of the insulin-producing β cells of the pancreas. Vitamin D deficiency is likely to be one of the many environmental factors influencing T1D development and diagnosis, and, hence, the hormone receptor gene, VDR, was examined for association with T1D risk. The Type I Diabetes Genetics Consortium genotyped 38 single nucleotide polymorphisms (SNPs) in 1654 T1D nuclear families (6707 individuals, 3399 affected). Genotypes for 38 SNPs were assigned using the Illumina (ILMN) and Sequenom (SQN) technology. The analysis of data release as of July 2008 is reported for both platforms. No evidence of association of VDR SNPs with T1D at P<0.01 was obtained in the overall sample set, nor in subgroups analyses of the parent-of-origin, sex of offspring and HLA risk once adjusted for multiple testing. [ABSTRACT FROM AUTHOR]

Aim: Type 1 diabetes susceptibility is influenced by a number of genes, of which those with the strongest effects map to the human leucocyte antigen (HLA) complex. Evidence for linkage of non-HLA genes in several affected sib-pair analyses was increased if the HLA or gender status of the sibs was considered independently. We investigated whether linkage status at the HLA complex differentially affected transmission of alleles to sibs depending on their HLA or gender linkage status. Methods: Genotypes of 2437 markers typed on 11 279 individuals from 2363 families, stratified according to HLA and gender, were examined using the transmission disequilibrium test. Results: Several significant single nucleotide polymorphisms (SNPs) loci were found near the class II genes; no significant SNPs were found by these analyses near the class I or class II genes. Other significant effects were found when the gender of the sibs or the parents was considered. There was not a significant difference in HLA-DRB genotypes between the stratified sets. Conclusions: These results suggest the presence of novel recessive susceptibility gene(s) within the HLA complex. [ABSTRACT FROM AUTHOR]

Aim: The Major Histocompatibility Complex (MHC) is a highly polymorphic region on chromosome 6 encompassing the human leucocyte antigen ( HLA) -DQ/DR loci most predictive of susceptibility to type 1 diabetes (T1D). To assess the contribution of other MHC genes, in this exploratory analysis of Type 1 Diabetes Genetics Consortium (T1DGC) family data we characterize association between susceptibility and MHC single nucleotide polymorphism (SNP) genotype, with an emphasis on effects of genetic variation additional to carriage of predisposing or protective MHC haplotypes. Methods: We use Cox regression analyses of age of onset, stratified by family, to jointly test both linkage and association. Analysis is restricted to children from families having both affected and unaffected siblings and is conducted with and without adjustment for known HLA class I and II effects. Model fits provide scores for each individual that are based on estimates of the probability of being affected by the age of 35, given the individual’s SNP genotype. The mean within-family variation in these scores provides a measure that closely reflects the relative size of the likelihood ratio test statistics, and their covariation provides a means of mapping patterns of association that incorporate both effect size and commonality of effect that is attributable to the strong linkage disequilibrium (LD) extending across the region. Results: Univariate analyses yielded strong associations with T1D susceptibility that are dominated by SNPs in the class II HLA-DR/ DQ region but extend across the MHC. Similar effects are frequently observed across SNPs within multiple genes, sometimes spanning hundreds of kilobases. SNPs within a region at the telomeric end of the class II gene HLA-DRA yielded significant associations with and without adjustment for carriage of the predictive DR3, DR4, DR2 and DR7 HLA haplotypes, and remained highly prominent in a secondary analysis that was restricted to 66 families in whom at least one of the affected siblings carried neither the DR3 nor DR4 haplotype. Conclusions: While many of the associations can be attributed to LD between the SNPs and the dominant HLA-DRB/DQA/DQB loci, there is also evidence of additional modifying effects. [ABSTRACT FROM AUTHOR]

The cytokine interleukin (IL)-12 is bound by a heterodimeric receptor and mediates a range of immunological activities, in particular, favouring the development of uncommitted T cells to the Th1 phenotype. Genes encoding elements of the IL-12 pathway are therefore good candidates for mediating susceptibility or resistance to a range of immune disorders, including Type I diabetes. We made a systematic search for variants in the human gene encoding the low-affinity IL-12 receptor, IL12RB1. Four single-nucleotide polymorphisms and two microsatellite polymorphisms were defined. We also tested these IL12RB1 alleles for involvement in Type I diabetes susceptibility, testing 131 families. Although suggestive evidence for linkage to a susceptibility gene was found, none of the IL12RB1 variants we defined demonstrated preferential transmission in these families.Genes and Immunity (2003) 4, 222-227. doi:10.1038/sj.gene.6363948 [ABSTRACT FROM AUTHOR]

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type I diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population. [ABSTRACT FROM AUTHOR]

Summary Transgenic mice were created in which a sheep keratin promoter directed the expression of IL-2 into the dermis. These KIL-2 transgenic mice were used to investigate the effects of localized IL-2 dysregulation on immune responses. Peripheral tolerance to skin antigens was not broken by in situ IL-2 expression because syngeneic KIL-2 skin grafts were not rejected. However, MHC Class I-disparate skin grafts from KIL-2 donors were rejected faster (median survival time (MST) 12 days) than grafts of non-transgenic littermate skin (MST 18 days). In contrast, the kinetics of KIL-2 H-Y-disparate skin graft rejection (MST 14 days) did not differ significantly from controls (MST 16 days), suggesting that upregulation of IL-2 at the effector site could affect CD4+ T cell- independent, but not CD4+ T cell-dependent, responses. No effect on rejection kinetics was observed when wild type allogeneic skin was grafted onto transgenic mice that expressed bcl2 constitutively in their lymphocytes (MST of 14 days, both sets), indicating that this was not simply due to increased longevity of T cells within the IL-2 expressing graft. We therefore suggest that aberrant expression of IL-2 can accelerate helper-independent CD8+ T cell responses by increasing proliferation and/or differentiation of cytolytic T cells at the effector site. [ABSTRACT FROM AUTHOR]

Aims/Background: Graft versus host disease (GVHD) following histoincompatible bone marrow transplantation may be modelled experimentally using irradiated metallothionein promoter-H-2Kb transgenic mice (MET-Kb mice), reconstituted with syngeneic non-transgenic spleen and lymph node (LN) cells. In this model, inflammation peaks at 3 weeks post-reconstitution, but resolves by 3 months, and is focussed on portal tracts and bile ducts (BD). The aim of this study was to determine if transgene-expressing hepatocytes play a role in the immune response, why portal tracts are selectively targeted, and which cell types are involved. Methods: Intrahepatic BD (IHBD) with attached hepatocytes, or extrahepatic BD (EHBD) devoid of hepatoctyes, were isolated from MET-Kb mice and implanted under the kidney capsule of transgenic (syngeneic) and congenic (allogeneic) mice. Three weeks post-implantation, BD were scored histologically for rejection or survival, and stained for various cell-surface molecules. Results: Generally, IHBD survived better than EHBD, and T cells were the predominant infiltrating cell type in both implants. Both types of implants undergoing rejection expressed intercellular adhesion molecule-1 (ICAM-1) and leukocyte function antigen-1 (LFA-1) at high density; BD and the underlying kidney parenchyma also expressed class I and II major histocompatibility complex (MHC). Conclusions: The rejection of both groups of implants by congenic recipients suggests that BD from MET-Kb mice express the transgene, but the reason for the selective targeting of portal tracts rather than transgene-expressing hepatocytes remains unclear. One possible explanation is that dendritic cells/antigen-presenting cells (DC/APC) in portal tracts, which express high levels of MHC and co-stimulatory molecules, are the primary targets, and that BD are infiltrated and destroyed as ‘bystanders’. [ABSTRACT FROM AUTHOR]

Examines the hypothesis that aberrant expression of major histocompatibility complex (MHC) molecules in oligodendrocytes may be involved in multiple sclerosis (MS). Experimentation of wonky mice; Lack of lymphocytic infiltration in tissues of the transgenic mice; Methods; Results; Conclusions.

There is a great range in outcomes after mycobacterial infections, and this is probably due to individual variation in immune responses. One of the key cytokine regulators of the immune response is interleukin (IL-) 12. The IL12B gene encodes the p40 chain of both IL-12 and IL-23 and it has two major variant sites at which different alleles are associated with increased levels of gene expression and with susceptibility to a range of immune-related diseases. We hypothesized that IL12B variants associated with increased expression would be as associated with susceptibility to persistent mycobacterial infection. We tested this hypothesis by genotyping Indian subjects, having either leprosy or tuberculosis (TB), as well as ethnically matched controls. Subjects with leprosy were less likely to have the 3’UTR genotype associated with lower IL12B expression ( P= 0.001). Subjects with TB were not only more likely to have the high-expressing IL12B promoter genotype ( P= 0.01) but also more likely to have this in the same haplotype with the high expressing 3’UTR allele ( P= 0.0009). These results suggest these infectious diseases may be improved by modulating IL-l2p40 production. [ABSTRACT FROM AUTHOR]

Insulin dependent diabetes mellitus (type I DM) is caused by an autoimmune process which culminates in destruction of pancreatic beta cells with resultant loss of insulin production. Preceding the clinical diagnosis of type I DM is a preclinical stage characterized by autoantibodies to insulin, glutamic acid decarboxylase (GAD) and a tyrosine phosphatase-like molecule (IA-2). We have studied both HLA class I and class 2 allele distributions in diabetic probands and autoantibody positive individuals in members of 452 families recruited for the Australian type I diabetes DNA repository. The results demonstrate that progression to autoimmunity as measured by the appearance of autoantibodies is strongly associated with the class 2 alleles DRB1*03 and DRB*04 and with DRB1*03/04 heterozygosity. In contrast, the progression to clinical disease appears associated with class I alleles A24, A30 and B18 while A1, A28, B14 and B56 appear negatively associated. The class 2 alleles appear to have a minimal role in the progression from autoantibody positivity to clinical disease. These results are consistent with the view that CD4+ T cells responding to peptides in the context of class 2 molecules are responsible for initiating autoantibody production, while the destruction of islet cells leading to clinical expression of the disease is the function of CD8+ T cells recognizing relevant peptides in the context of class I molecules. [ABSTRACT FROM AUTHOR]

Owing to the importance of interleukin (IL)-12 in regulating immune responses, we have determined the complete genomic sequence and organization of the gene encoding its p40 subunit. The genomic sequence was determined and was compared to cDNA sequences to derive exon/intron boundaries. Unusually, both the first and last of the eight exons of this gene are not translated. An extensive search identified several polymorphisms in IL-12p40, including repeat elements in introns 2 and 4 and a polymorphic Taql site in the 3' UTR. However, no polymorphisms were found which could result in amino acid substitutions. This finding places constraints on any preferential involvement of alleles of IL-12p40 in contributing to autoimmune, inflammatory or infectious diseases. [ABSTRACT FROM AUTHOR]

Hypertriglyceridemia ( hTG) is a lipid disorder, resulting from an elevation in triglyceride levels, with a strong genetic component. It constitutes a significant risk factor for coronary artery disease ( CAD), a leading cause of death worldwide. In this study, we performed a common variant association study for hTG in ethnic Saudi Arabs. We genotyped 5501 individuals in a two-phase experiment using Affymetrix Axiom® Genome-Wide CEU 1 Array (Affymetrix, Santa Cruz, CA) that contains a total of 587,352 single nucleotide polymorphisms ( SNPs). The lead variant was the rs1558861 [1.99 (1.73-2.30); p = 7.37 × 10−22], residing on chromosome (chr) 11 at the apolipoprotein A-I/A-5 ( APOA1 / APOA5) locus. The rs780094 [1.34 (1.21-1.49); p = 8.57 × 10−8] on chr 2 at the glucokinase regulatory protein ( GCKR) locus was similarly significantly associated, while the rs10911205 [1.29 (1.16-1.44); p = 3.52 × 10−6] on chr1 at the laminin subunit gamma-1 ( LAMC1) locus showed suggestive association with disease. Furthermore, the rs17145738 [0.68 (0.60-0.77); p = 6.69 × 10−9] on chr7 at the carbohydrate-responsive element-binding protein-encoding ( MLXIPL) gene locus displayed significant protective characteristics, while another variant rs6982502 [0.76 (0.68-0.84); p = 5.31 × 10−7] on chr8 showed similar but weaker properties. These findings were replicated in 317 cases vs 1415 controls from the same ethnic Arab population. Our study identified several variants across the human genome that are associated with hTG in ethnic Arabs. [ABSTRACT FROM AUTHOR]

The genetic susceptibility to acquiring low high density lipoprotein-cholesterol ( LHDLC) levels is not completely elucidated yet. In this study, we performed a common variant association study for harboring this trait in ethnic Arabs. We employed the Affymetrix high-density Axiom Genome-Wide ASI Array (Asian population) providing a coverage of 598,000 single nucleotide variations ( SNPs) to genotype 5495 individuals in a two-phase study involving discovery and validation sets of experiments. The rs1800775 [1.31 (1.22-1.42); p = 3. 41E-12] in the CETP gene and rs359027 [1.26 (1.16-1.36); p = 2. 55E-08] in the LMCD1 gene were significantly associated with LHDLC levels. Furthermore, rs3104435 [1.26 (1.15-1.38); p = 1. 19E-06] at the MATN1 locus, rs9835344 [1.16 (1.08-1.26); p = 8. 75E-06] in the CNTN6 gene, rs1559997 [1.3 (1.14-1.47); p = 9. 48E-06] in the SDS gene and rs1670273 [1.2 (1.1-1.31); p = 4. 81E-06] in the DMN/ SYNM gene exhibited suggestive association with the disorder. Seven other variants including rs1147169 in the PLCL1 gene, rs10248618 in the DNAH11, rs476155 in the GLIS3, rs7024300 in the ABCA1, intergenic rs10836699, rs11603691 in P2RX3 and rs750134 in CORO1C gene exhibited borderline protective properties. Validation and joint meta-analysis resulted in rs1800775, rs3104435 and rs359027 retaining their predisposing properties, while rs10836699 and rs11603691 showed protective properties. Our data show several predisposing variants across the genome for LHDLC levels in ethnic Arabs. [ABSTRACT FROM AUTHOR]

Susceptibility to most common cancers is likely to involve interaction between multiple low risk genetic variants. Although there has been great progress in identifying such variants, their effect on phenotype and the mechanisms by which they contribute to disease remain largely unknown. We have developed a mouse melanoma model harboring two mutant oncogenes implicated in human melanoma, CDK4R24C and NRASQ61K. In these mice, tumors arise from benign precursor lesions that are a recognized strong risk factor for this neoplasm in humans. To define molecular events involved in the pathway to melanoma, we have for the first time applied the Collaborative Cross (CC) to cancer research. The CC is a powerful resource designed to expedite discovery of genes for complex traits. We characterized melanoma genesis in more than 50 CC strains and observed tremendous variation in all traits, including nevus and melanoma age of onset and multiplicity, anatomical site predilection, time for conversion of nevi to melanoma and metastases. Intriguingly, neonatal ultraviolet radiation exposure exacerbated nevus and melanoma formation in most, but not all CC strain backgrounds, suggesting that genetic variation within the CC will help explain individual sensitivity to sun exposure, the major environmental skin carcinogen. As genetic variation brings about dramatic phenotypic diversity in a single mouse model, melanoma-related endophenotype comparisons provide us with information about mechanisms of carcinogenesis, such as whether melanoma incidence is dependent upon the density of pre-existing nevus cells. Mouse models have been used to examine the functional role of gene mutations in tumorigenesis. This work represents their next phase of development to study how biological variation greatly influences lesion onset and aggressiveness even in the setting of known somatic driver mutations. [ABSTRACT FROM AUTHOR]

Exome sequencing is being increasingly used to identify disease-associated gene mutations. We used whole exome sequencing to determine the genetic basis of a syndrome of diabetes and renal disease affecting a mother and her son. We identified a mutation in the hepatocyte nuclear factor 1-b ( HNF1B) gene that encoded a methionine to valine amino acid change ( M160 V) in the HNF1B protein. This leads us to the previously unappreciated diagnosis of maturity-onset diabetes of the young type 5 and provided a basis for genetic counselling of other family members. [ABSTRACT FROM AUTHOR]

The Type I Diabetes Genetics Consortium (T1DGC) is an international collaboration whose primary goal is to identify genes whose variants modify an individual's risk of type I diabetes (T1D). An integral part of the T1DGC's mission is the establishment of clinical and data resources that can be used by, and that are fully accessible to, the T1D research community (http://www.t1dgc.org). The T1DGC has organized the collection and analyses of study samples and conducted several major research projects focused on T1D gene discovery: a genome-wide linkage scan, an intensive evaluation of the human major histocompatibility complex, a detailed examination of published candidate genes, and a genome-wide association scan. These studies have provided important information to the scientific community regarding the function of specific genes or chromosomal regions on T1D risk. The results are continually being updated and displayed (http://www.t1dbase.org). The T1DGC welcomes all investigators interested in using these data for scientific endeavors on T1D. The T1DGC resources provide a framework for future research projects, including examination of structural variation, re-sequencing of candidate regions in a search for T1D-associated genes and causal variants, correlation of T1D risk genotypes with biomarkers obtained from T1DGC serum and plasma samples, and in-depth bioinformatics analyses. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is the third major locus affecting risk of type I diabetes (T1D), after HLA-DR/DQ and INS. The most associated single-nucleotide polymorphism (SNP), rs2476601, has a C->T variant and results in an arginine (R) to tryptophan (W) amino acid change at position 620. To assess whether this, or other specific variants, are responsible for T1D risk, the Type I Diabetes Genetics Consortium analyzed 28 PTPN22 SNPs in 2295 affected sib-pair (ASP) families. Transmission Disequilibrium Test analyses of haplotypes revealed that all three haplotypes with a T allele at rs2476601 were overtransmitted to affected children, and two of these three haplotypes showed statistically significant overtransmission (P=0.003 to P=5.9E-12). Another haplotype had decreased transmission to affected children (P=3.5E-05). All haplotypes containing the rs2476601 T allele were identical for all SNPs across PTPN22 and only varied at centromeric SNPs. When considering rs2476601 ‘C’ founder chromosomes, a second haplotype (AGGGGC) centromeric of PTPN22 in the C1orf178 region was associated with protection from T1D (odds ratio=0.81, P=0.0005). This novel finding requires replication in independent populations. We conclude the major association of PTPN22 with T1D is likely due to the recognized non-synonymous SNP rs2476601 (R620W). [ABSTRACT FROM AUTHOR]

The Type I Diabetes Genetics Consortium (T1DGC) is an international, multicenter research program with two primary goals. The first goal is to identify genomic regions and candidate genes whose variants modify an individual's risk of type I diabetes (T1D) and help explain the clustering of the disease in families. The second goal is to make research data available to the research community and to establish resources that can be used by, and that are fully accessible to, the research community. To facilitate the access to these resources, the T1DGC has developed a Consortium Agreement (http://www.t1dgc.org) that specifies the rights and responsibilities of investigators who participate in Consortium activities. The T1DGC has assembled a resource of affected sib-pair families, parent–child trios, and case–control collections with banks of DNA, serum, plasma, and EBV-transformed cell lines. In addition, both candidate gene and genome-wide (linkage and association) studies have been performed and displayed in T1DBase (http://www.t1dbase.org) for all researchers to use in their own investigations. In this supplement, a subset of the T1DGC collection has been used to investigate earlier published candidate genes for T1D, to confirm the results from a genome-wide association scan for T1D, and to determine associations with candidate genes for other autoimmune diseases or with type II diabetes that may be involved with β-cell function. [ABSTRACT FROM AUTHOR]

The Type I Diabetes Genetics Consortium (T1DGC) has collected thousands of multiplex and simplex families with type I diabetes (T1D) with the goal of identifying genes involved in T1D susceptibility. These families have all been genotyped for the HLA class I and class II loci and a subset of samples has been typed for an major histocompatibility complex (MHC) single-nucleotide polymorphism (SNP) panel. In addition, the T1DGC has genotyped SNPs in candidate genes to evaluate earlier reported T1D associations. Individual SNPs and SNP haplotypes in IL4R, which encodes the α-chain of the IL4 and IL13 receptors, have been associated with T1D in some reports, but not in others. In this study, 38 SNPs in IL4R were genotyped using the Sequenom iPLEX Gold MassARRAY technology in 2042 multiplex families from nine cohorts. Association analyses (transmission-disequilibrium test and parental-disequilibrium test) were performed on individual SNPs and on three-SNP haplotypes. Analyses were also stratified on the high-risk HLA DR3/DR4-DQB1*0302 genotype. A modest T1D association in HBDI families (n=282) was confirmed in this larger collection of HBDI families (n=424). The variant alleles at the non-synonymous SNPs (rs1805011 (E400A), rs1805012 (C431R), and rs1801275 (Q576R)), which are in strong linkage disequilibrium, were negatively associated with T1D risk. These SNPs were more associated with T1D among non-DR3/DR4-DQB1*0302 genotypes than DR3/DR4-DQB1*0302 genotypes. This association was stronger, both in terms of odds ratio and P-values, than the initial report of the smaller collection of HBDI families. However, the IL4R SNPs and the three-SNP haplotype containing the variant alleles were not associated with T1D in the total data. Thus, in the overall families, these results do not show evidence for an association of SNPs in IL4R with T1D. [ABSTRACT FROM AUTHOR]

To reassess earlier suggested type I diabetes (T1D) associations of the insulin receptor substrate 1 (IRS1) and the paired domain 4 gene (PAX4) genes, the Type I Diabetes Genetics Consortium (T1DGC) evaluated single-nucleotide polymorphisms (SNPs) covering the two genomic regions. Sixteen SNPs were evaluated for IRS1 and 10 for PAX4. Both genes are biological candidate genes for T1D. Genotyping was performed in 2300 T1D families on both Illumina and Sequenom genotyping platforms. Data quality and concordance between the platforms were assessed for each SNP. Transmission disequilibrium testing neither show T1D association of SNPs in the two genes, nor did haplotype analysis. In conclusion, the earlier suggested associations of IRS1 and PAX4 to T1D were not supported, suggesting that they may have been false positive results. This highlights the importance of thorough quality control, selection of tagging SNPs, more than one genotyping platform in high throughput studies, and sufficient power to draw solid conclusions in genetic studies of human complex diseases. [ABSTRACT FROM AUTHOR]

Candidate gene studies have long been the principal method for identification of susceptibility genes for type I diabetes (T1D), resulting in the discovery of HLA, INS, PTPN22, CTLA4, and IL2RA. However, many of the initial studies that relied on this strategy were largely underpowered, because of the limitations in genomic information and genotyping technology, as well as the limited size of available cohorts. The Type I Diabetes Genetic Consortium (T1DGC) has established resources to re-evaluate earlier reported genes associated with T1D, using its collection of 2298 Caucasian affected sib-pair families (with 11 159 individuals). A total of 382 single-nucleotide polymorphisms (SNPs) located in 21 T1D candidate genes were selected for this study and genotyped in duplicate on two platforms, Illumina and Sequenom. The genes were chosen based on published literature as having been either ‘confirmed’ (replicated) or not (candidates). This study showed several important features of genetic association studies. First, it showed the major impact of small rates of genotyping errors on association statistics. Second, it confirmed associations at INS, PTPN22, IL2RA, IFIH1 (earlier confirmed genes), and CTLA4 (earlier confirmed, with distinct SNPs) loci. Third, it did not find evidence for an association with T1D at SUMO4, despite confirmed association in Asian populations, suggesting the potential for population-specific gene effects. Fourth, at PTPN22, there was evidence for a novel contribution to T1D risk, independent of the replicated effect of the R620W variant. Fifth, among the candidate genes selected for replication, the association of TCF7-P19T with T1D was newly replicated in this study. In summary, this study was able to replicate some genetic effects, reject others, and provide suggestions of association with several of the other candidate genes in stratified analyses (age at onset, HLA status, population of origin). These results have generated additional interesting functional hypotheses that will require further replication in independent cohorts. [ABSTRACT FROM AUTHOR]

Aim: The absence or ‘missingness’ of single nucleotide polymorphism (SNP) assay values because of genotype or related factors of interest may bias association and other studies. Missingness was determined for the Type 1 Diabetes Genetics Consortium (T1DGC) Major Histocompatibility Complex (MHC) data and was found to vary across the region, ranging up to 11.1% of the non-null proband SNPs, with a median of 0.3%. We consider factors related to missingness in the T1DGC data and briefly assess its possible influence on association studies. Methods: We assessed associations of missingness in the SNP assay data with human leucocyte antigen (HLA) genotype of the individual and with SNP genotypes of the parents. Within-cohort analyses were combined (over all cohorts) using (i) Mantel–Haenszel tests for two-by-two tables or (ii) by combining test statistics for larger tables and regression models. Mixed effect regression models were used to assess association of the SNP genotypes with affected status of the offspring after adjustment for parental SNP genotypes, cohort membership and HLA genotypes. Log-linear models were used to assess association of missingness in the unaffected sib assays with SNP genotypes of the probands. Results: Missingness of SNP values near the HLA class I (A, B and C) and class II (DR, DQ and DP) loci is strongly associated with carriage of corresponding HLA genotypes within these groups. Similar associations pertain to missing values among the microsatellite data. In at least some of these cases, regions of missingness coincided with known deletion regions corresponding to the associated HLA haplotype. We conjecture that other regions of associated missingness may point to similar haplotypic deletions. Analysis of association patterns of SNP genotypes with affected status of offspring does not indicate strong informative missingness. However, association of missingness in proband data with parental SNP genotypes may impact transmission disequilibrium test (TDT)-type analyses. Comparisons of affected and unaffected siblings point to possible susceptibility regions additional to the classical HLA-DR3/4 alleles near BAT4-LY6G5B-BAT5 and NOTCH4. Conclusions: Potentially informative missingness in SNP assay values in the MHC region may impact on association and related analyses based on the T1DGC data. These results suggest that it would be prudent to assess the degree to which missingness may abrogate assessed SNP disease markers in such studies. Initial analyses based on comparison of affected and unaffected status in offspring suggest that at least these may be little affected. [ABSTRACT FROM AUTHOR]

Objective: The rising incidence of type 1 diabetes has been attributed to environment, implying a lesser role for genetic susceptibility. However, the rise could be accounted for by either more cases with classic high-risk genes or by cases with other risk genes. Separately, for any degree of genetic susceptibility, age at presentation may decrease in a permissive environment. To examine these possibilities, human leukocyte antigen (HLA) class II DRB1 genes known to confer risk for type 1 diabetes were analyzed in relation to year of birth and age at diagnosis over the last five decades.Research Design and Methods: Caucasoid subjects (n = 462) diagnosed with type 1 diabetes before age 18 between 1950 and 2005 were DRB1 genotyped.Results: Mean +/- SD age at diagnosis, 8.5 +/- 4.5 years, did not differ across decades. Recent diagnosis was associated with a lower proportion but unchanged incidence of the highest-risk DRB1 genotype DR3,4 (2000-2005, 28% vs. 1950-1969, 79%; P < 0.0001) and a higher proportion of lower-risk genotypes DR4,X and DR3,X (2000-2005, 48% vs. 1950-1969, 20%; P = 0.0002). The frequency of the DRX,X genotype was low (Conclusions: The rising incidence and decreasing age at diagnosis of type 1 diabetes is accounted for by the impact of environment on children with lower-risk HLA class II genes, who previously would not have developed type 1 diabetes in childhood. [ABSTRACT FROM AUTHOR]

Interleukin-12 (IL-12), a heterodimeric cytokine, is important in the generation of a Th1-biased immune response. Several polymorphisms have been described in IL12B, the gene encoding the p40 subunit of IL-12. A bi-allelic polymorphism within the IL12B promoter region has been reported to show association with diseases as diverse as severe childhood asthma and fatal cerebral malaria. In order to define the molecular basis for these disease associations, we investigated the secretion of IL-12 by human monocyte-derived dendritic cells. Homozygotes for the IL12B promoter polymorphism showed a 10-fold difference in median p70 secretion in response to CD40 ligation. Remarkably, this difference resulted from the inability of most allele 1 homozygotes to secrete heterodimeric IL-12. In contrast, most of the donors homozygous for allele 2 had detectable secretion. These findings are important for the understanding of the highly complex regulation of IL-12 secretion, and its consequent impact on disease susceptibility, in humans.Genes and Immunity (2004) 5, 431-434. doi:10.1038/sj.gene.6364102 Published online 3 June 2004 [ABSTRACT FROM AUTHOR]

Interleukin (II)-12 is a heterodimeric cytokine composed of 35 and 40 kD chains that plays a key role in the induction of Th1 cells, a T cell subset involved in many autoimmune diseases. We report here the cDNA sequence encoding the IL-12 p40 subunit from the autoimmune-prone non-obese diabetic (NOD) mouse, which spontaneously develops type 1 diabetes. Compared with the C57BL/6 sequence, there are two base changes that lead to amino acid replacements. Other autoimmune-prone strains, but not the diabetes-resistant NOR strain, share the same allele as NOD. We found both trans- and cis-allele-dependent effects on levels of basal and induced IL-12p40 expression. Furthermore, we show that one of these changes results in a structural change in the p40 molecule, as evidenced by the failure of a monoclonal antibody to bind NOD IL-12. These findings have implications for the predisposition to autoimmune responses in NOD and other autoimmune-prone mouse strains. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The extent to which leptin protects against obesity is unknown. By intercrossing New Zealand obese mice with lean C57BL/6J mice, we have separated the genes controlling leptin and other weight-related phenotypes. This has allowed us to determine whether hyperleptinaemia is associated with reduced food intake and increased physical activity in mice spanning a large range in body weight. METHODS: Plasma leptin, glucose and insulin, body weight, food intake, running wheel activity, and four adipose depots were measured in 587 adult F2 and backcross mice RESULTS: When mice were categorized by adiposity, a plot of food intake vs leptin illustrated a U-shaped curve. Food intake decreased as leptin levels rose to ∼15 ng/ml, beyond which the relationship reversed. A negative relationship was observed between activity and leptin with a maximal decrease in activity once leptin reached ∼15 ng/ml. CONCLUSION: Leptin has differential responses to food intake and activity, suggesting that it has limited potential to defend against obesity. A genetic defect in leptin sensitivity is unlikely to be the primary cause of obesity in these mice, since hyperleptinaemia was not coinherited with both hyperphagia and inactivity as body weight increased. [ABSTRACT FROM AUTHOR]

Cardiac actin ( ACTC ) is the fetal homologue of skeletal muscle actin ( ACTA1 ), and is switched off at birth in skeletal muscle but remains highly expressed in adult heart and in regenerating muscle. ACTC is 99% identical to ACTA1 protein, differing at only 4 amino acids. We previously showed an ACTC transgene could functionally replace ACTA1 in postnatal skeletal muscle of Acta1 knockout mice that normally die within a few days of birth, indicating that ACTC could have a therapeutic role in skeletal muscle actin diseases. We harnessed the power of two different recombinant inbred (RI) mouse models including BXD and the more recently developed Collaborative Cross (CC), to identify genetic elements controlling Actc expression. RI mice are genetic reference populations consisting of large numbers of inbred mouse strains descended from either 2 (BXD) or 8 (CC) founder strains. Characterization of these strains for a trait of interest allows for the mapping of gene(s) mediating that trait to genomic intervals called quantitative trait loci (QTL). We have identified in both RI sets a highly significant QTL regulating Actc expression in skeletal muscle. This QTL also regulates Actc expression in particular non-skeletal muscle tissues (eye and lung). Identification of the regulatory genetic element may provide a target for modifying expression of cardiac actin by therapeutic intervention. [ABSTRACT FROM AUTHOR]

The article provides information on a research on Type 1 diabetes (T1D). It focuses on the major histocompatibility complex (MHC) fine mapping workshop in which the genetic aspect of diabetes is explored. It is stated that the complexity of the genetic basis of the disorder shows that even bigger sample sets maybe required for the complete resolution of the effects of MHC genes on T1D risk.

Analysis of voluntary running wheel activity in mice is a useful phenotypic measure of skeletal muscle function. We have collected voluntary running wheel activity and total body weight data from >50 strains of mice belonging to the ‘Collaborative Cross’ (aka The Gene Mine), and compared these to known models of neuromuscular disease. The Gene Mine is a mouse reference population derived from eight genetically diverse founder strains, with the genome of each offspring strain being a mosaic of chromosomal segments inherited randomly from the founders. The Gene Mine is designed specifically for complex trait analysis and the identification of quantitative trait loci (QTL). Our analyses of voluntary running wheel activity has involved measuring distance, maximum speed, average speed and time spent on the wheel over a six day consecutive period in male and female Gene Mine mice at two different ages (6 weeks and 6–9 months). We also recorded real-time activity levels and are imaging selected mouse strains by magnetic resonance imaging and computed tomography. Our results indicate marked phenotypic variation across all traits measured, including comparisons of young and older Gene Mine mice. We are now mapping QTL for the various phenotypes and comparing these with skeletal muscle gene expression. We previously crossed skeletal muscle actin knockout mice with Gene Mine strains to identify strains capable of extending the usual early lethal phenotype of this model. The phenotype resource we and others are building can now be used to search for strains, and from there QTL, that modify voluntary running wheel activity, weight and muscle bulk in various models of neuromuscular diseases. [ABSTRACT FROM AUTHOR]