Role of GALNT2 on Insulin Sensitivity, Lipid Metabolism and Fat Homeostasis.

O-linked glycosylation, the greatest form of post-translational modifications, plays a key role in regulating the majority of physiological processes. It is, therefore, not surprising that abnormal O-linked glycosylation has been related to several human diseases. Recently, GALNT2, which encodes the GalNAc-transferase 2 involved in the first step of O-linked glycosylation, has attracted great attention as a possible player in many highly prevalent human metabolic diseases, including atherogenic dyslipidemia, type 2 diabetes and obesity, all clustered on the common ground of insulin resistance. Data available both in human and animal models point to GALNT2 as a molecule that shapes the risk of the aforementioned abnormalities affecting diverse protein functions, which eventually cause clinically distinct phenotypes (a typical example of pleiotropism). Pathways linking GALNT2 to dyslipidemia and insulin resistance have been partly identified, while those for type 2 diabetes and obesity are yet to be understood. Here, we will provide a brief overview on the present knowledge on GALNT2 function and dysfunction and propose novel insights on the complex pathogenesis of the aforementioned metabolic diseases, which all impose a heavy burden for patients, their families and the entire society.

Morphological and molecular characterization of GALNT2-mediated adipogenesis.

3T3L1 mouse pre-adipocytes develop into adipocytes differently in response to GALNT2 overexpression or to stimulation with rosiglitazone, a reference inducer of adipogenesis. To investigate the biology of alternative pathways of adipogenesis, we studied lipid droplets (LD) morphology, chromatin organization, and gene expression in GALNT2- versus rosiglitazone-induced 3T3L1 adipogenesis. 3T3L1 overexpressing either GALNT2 (GALNT2) or GFP and treated with rosiglitazone (GFPR) were differentiated into adipocytes. LD and nuclei were profiled measuring their morphological features. The expression of adipogenesis-related genes was measured by RT-PCR. As compared to GFPR, GALNT2 showed smaller and more clustered LD, more nuclei with condensed chromatin and several gene expression changes (P < 0.001 for all). As compared to those stimulated by rosiglitazone, GALNT2 overexpressing cells show differences in the most established readouts of adipogenesis. Characterizing alternative pathways of adipogenesis may help tackle those diseases which are secondary to increased dysfunctional mass of adipose tissue.

GALNT2 as a novel modulator of adipogenesis and adipocyte insulin signaling.

BACKGROUND/OBJECTIVES: A better understanding of adipose tissue biology is crucial to tackle insulin resistance and eventually coronary heart disease and diabetes, leading causes of morbidity and mortality worldwide. GALNT2, a GalNAc-transferase, positively modulates insulin signaling in human liver cells by down-regulating ENPP1, an insulin signaling inhibitor. GALNT2 expression is increased in adipose tissue of obese as compared to that of non-obese individuals. Whether this association is secondary to a GALNT2-insulin sensitizing effect exerted also in adipocytes is unknown. We then investigated in mouse 3T3-L1 adipocytes the GALNT2 effect on adipogenesis, insulin signaling and expression levels of both Enpp1 and 72 adipogenesis-related genes. METHODS: Stable over-expressing GALNT2 and GFP preadipocytes (T0) were generated. Adipogenesis was induced with (R+) or without (R-) rosiglitazone and investigated after 15 days (T15). Lipid accumulation (by Oil Red-O staining) and intracellular triglycerides (by fluorimetric assay) were measured. Lipid droplets (LD) measures were analyzed at confocal microscope. Gene expression was assessed by RT-PCR and insulin-induced insulin receptor (IR), IRS1, JNK and AKT phosphorylation by Western blot. RESULTS: Lipid accumulation, triglycerides and LD measures progressively increased from T0 to T15R- and furthermore to T15R+. Such increases were significantly higher in GALNT2 than in GFP cells so that, as compared to T15R+GFP, T15R- GALNT2 cells showed similar (intracellular lipid and triglycerides accumulation) or even higher (LD measures, p < 0.01) values. In GALNT2 preadipocytes, insulin-induced IR, IRS1 and AKT activation was higher than that in GFP cells. GALNT2 effect was totally abolished during adipocyte maturation and completely reversed at late stage maturation. Such GALNT2 effect trajectory was paralleled by coordinated changes in the expression of Enpp1 and adipocyte-maturation key genes. CONCLUSIONS: GALNT2 is a novel modulator of adipogenesis and related cellular phenotypes, thus becoming a potential target for tackling the obesity epidemics and its devastating sequelae.

Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus.

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552(∗)] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3ß phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.

The rs12917707 polymorphism at the UMOD locus and glomerular filtration rate in individuals with type 2 diabetes: evidence of heterogeneity across two different European populations.

BACKGROUND: UMOD variability has been associated at a genome-wide level of statistical significance with glomerular filtration rate (GFR) in Swedish individuals with type 2 diabetes (T2D; n = 4888). Whether this finding is extensible also to diabetic patients from other populations deserves further study. Our aim was to investigate the relationship between UMOD variability and GFR in patients with T2D from Italy. METHODS: Genotyping of the single nucleotide polymorphism (SNP) rs12917707 at the UMOD locus has been carried out in 3087 individuals from four independent Italian cohorts of patients with T2D by TaqMan allele discrimination. RESULTS: In none of the four study cohorts was rs12917707 significantly associated with GFR (P > 0.05 for all). Similar results were obtained when the four samples were pooled and analyzed together (ß = 0.83, P = 0.19). Such effect was strikingly smaller than that previously reported in Swedish patients (P for heterogeneity = 1.21 × 10-7). CONCLUSIONS: The previously reported strong association between rs12917707 and GFR in diabetic patients from Sweden is not observed in Italian diabetic patients, thus clearly pointing to a heterogeneous effect across the two different samples. This suggests that UMOD is a strong genetic determinant of kidney function in patients with T2D in some, but not all, populations.

Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action: GALNT2: a novel modulator of insulin signaling.

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3'untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3'UTR binding proteins, and investigate their role on insulin signaling. By RNA pull-down, 49 proteins bound to ENPP1-3'UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA. GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase (PEPCK) expression, a key neoglucogenetic enzyme. Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cells.

Role of somatomedin-B-like domains on ENPP1 inhibition of insulin signaling.

The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR ß-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.

GALNT2 expression is associated with glucose control and serum metabolites in patients with type 2 diabetes.

AIMS: Aim of this study was to investigate in type 2 diabetes whether expression level of GALNT2, a positive modulator of insulin sensitivity, is associated with a metabolic signature. METHODS: Five different metabolite families, including acylcarnitines, aminoacids, biogenic amines, phospholipids and sphingolipids were investigated in fasting serum of 70 patients with type 2 diabetes, by targeted metabolomics. GALNT2 expression levels were measured in peripheral white blood cells by RT-PCR. The association between GALNT2 expression and serum metabolites was assessed using false discovery rate followed by stepwise selection and, finally, multivariate model including several clinical parameters as confounders. The association between GALNT2 expression and the same clinical parameters was also investigated. RESULTS: GALNT2 expression was independently correlated with HbA1c levels (P value = 0.0052), a finding that is the likely consequence of the role of GALNT2 on insulin sensitivity. GALNT2 expression was also independently associated with serum levels of the aminoacid glycine (P value = 0.014) and two biogenic amines phenylethylamine (P value = 0.0065) and taurine (P value = 0.0011). The association of GALNT2 expression with HbA1c was not mediated by these three metabolites. CONCLUSIONS: Our data indicate that in type 2 diabetes the expression of GALNT2 is associated with several serum metabolites. This association needs to be further investigated to understand in depth its role in mediating the effect of GALNT2 on insulin sensitivity, glucose control and other clinical features in people with diabetes.

Leveraging Genetics to Address the Role of GALNT2 on Atherogenic Dyslipidemia.

Several studies have shown that downregulation of GALNT2 (Polypeptide N-Acetylgalactosaminyltransferase 2), encoding polypeptide N-acetylgalactosaminyltransferase 2, decreases high-density lipoprotein cholesterol (HDL-C) and increases triglycerides levels by glycosylating key enzymes of lipid metabolism, such as angiopoietin like 3, apolipoprotein C-III, and phospholipid transfer protein. GALNT2 is also a positive modulator of insulin signaling and action, associated with in vivo insulin sensitivity and during adipogenesis strongly upregulates adiponectin. Thus, the hypothesis that GALNT2 affects HDL-C and triglycerides levels also through insulin sensitivity and/or circulating adiponectin, is tested. In 881 normoglycemic individuals the G allele of rs4846914 SNP at the GALNT2 locus, known to associate with GALNT2 downregulation, is associated with low HDL-C and high values of triglycerides, triglycerides/HDL-C ratio, and theHomeostatic Model Assessment of insulin resistance HOMAIR (p-values = 0.01, 0.027, 0.002, and 0.016, respectively). Conversely, no association is observed with serum adiponectin levels (p = 0.091). Importantly, HOMAIR significantly mediates a proportion of the genetic association with HDL-C (21%, 95% CI: 7-35%, p = 0.004) and triglyceride levels (32%, 95% CI: 4-59%, p = 0.023). The results are compatible with the hypothesis that, besides the effect on key lipid metabolism enzymes, GALNT2 alters HDL-C and triglyceride levels also indirectly through a positive effect on insulin sensitivity.

STAT1 overexpression triggers aplastic anemia: a pilot study unravelling novel pathogenetic insights in bone marrow failure.

We identified STAT1 gain of function (GOF) in a 32-year-old female with pallor, weakness, cough, and dyspnea admitted to our Division of Medicine. She had severe oral ulcers (OU), type 1 diabetes (T1DM), and pancytopenia. Bone marrow (BM) biopsy showed the absence of erythroid precursors. Peripheral blood parameters such as neutrophils < 500/mL, reticulocytes < 2%, and BM hypo-cellularity allowed to diagnose severe aplastic anemia. A heterozygous variant (p.520T>C, p.Cys174Arg) of STAT1 was uncovered. Thus, p.Cys174Arg mutation was investigated as potentially responsible for the patient's inborn immunity error and aplastic anemia. Although STAT1 GOF is rare, aplastic anemia is a more common condition; therefore, we explored STAT1 functional role in the pathobiology of BM failure. Interestingly, in a cohort of six patients with idiopathic aplastic anemia, enhanced phospho-STAT1 levels were observed on BM immunostaining. Next, the most remarkable features associated with STAT1 signaling dysregulation were examined: in both pure red cell aplasia and aplastic anemia, CD8+ T cell genetic variants and mutations display enhanced signaling activities related to the JAK-STAT pathway. Inborn errors of immunity may represent a paradigmatic condition to unravel crucial pathobiological mechanisms shared by common pathological conditions. Findings from our case-based approach and the phenotype correspondence to idiopathic aplastic anemia cases prompt further statistically powered prospective studies aiming to elucidate the exact role and theragnostic window for JAK/STAT targeting in this clinical context. Nonetheless, we demonstrate how a comprehensive study of patients with primary immunodeficiencies can lead to pathophysiologic insights and potential therapeutic approaches within a broader spectrum of aplastic anemia cases.

Role of Actionable Genes in Pursuing a True Approach of Precision Medicine in Monogenic Diabetes.

Monogenic diabetes is a genetic disorder caused by one or more variations in a single gene. It encompasses a broad spectrum of heterogeneous conditions, including neonatal diabetes, maturity onset diabetes of the young (MODY) and syndromic diabetes, affecting 1-5% of patients with diabetes. Some of these variants are harbored by genes whose altered function can be tackled by specific actions ("actionable genes"). In suspected patients, molecular diagnosis allows the implementation of effective approaches of precision medicine so as to allow individual interventions aimed to prevent, mitigate or delay clinical outcomes. This review will almost exclusively concentrate on the clinical strategy that can be specifically pursued in carriers of mutations in "actionable genes", including ABCC8, KCNJ11, GCK, HNF1A, HNF4A, HNF1B, PPARG, GATA4 and GATA6. For each of them we will provide a short background on what is known about gene function and dysfunction. Then, we will discuss how the identification of their mutations in individuals with this form of diabetes, can be used in daily clinical practice to implement specific monitoring and treatments. We hope this article will help clinical diabetologists carefully consider who of their patients deserves timely genetic testing for monogenic diabetes.

Early-Onset Diabetes in an Infant with a Novel Frameshift Mutation in LRBA.

We describe early-onset diabetes in a 6-month-old patient carrying an LRBA gene mutation. Mutations in this gene cause primary immunodeficiency with autoimmune disorders in infancy. At admission, he was in diabetic ketoacidosis, and treatment with fluid infusion rehydration and then i.v. insulin was required. He was discharged with a hybrid closed-loop system for insulin infusion and prevention of hypoglycemia (Minimed Medtronic 670G). He underwent a next-generation sequencing analysis for monogenic diabetes genes, which showed that he was compound heterozygous for two mutations in the LRBA gene. In the following months, he developed arthritis of hands and feet, chronic diarrhea, and growth failure. He underwent bone marrow transplantation with remission of diarrhea and arthritis, but not of diabetes and growth failure. The blood glucose control has always been at target (last HbA1c 6%) without any severe hypoglycemia. LRBA gene mutations are a very rare cause of autoimmune diabetes. This report describes the clinical course in a very young patient. The hybrid closed-loop system was safe and efficient in the management of blood glucose. This report describes the clinical course of diabetes in a patient with a novel LRBA gene mutation.

Gain of Function of Malate Dehydrogenase 2 and Familial Hyperglycemia.

CONTEXT: Genes causing familial forms of diabetes mellitus are only partially known. OBJECTIVE: We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. METHODS: Existing whole-exome sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the United States and Italy. Functional studies were carried out in vitro (transduced MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of 2 variants in the malate dehydrogenase 2 (MDH2) gene linked with hyperglycemia in 2 of the families. RESULTS: A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in 1 family from the United States. An infrequent MDH2 missense variant (p.Val160Met) also showed disease cosegregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio-a change known to affect insulin signaling and secretion. Stable expression of human wild-type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. CONCLUSION: Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes.

PPARγ2 P12A polymorphism and albuminuria in patients with type 2 diabetes: a meta-analysis of case-control studies.

BACKGROUND: Insulin resistance has a role in diabetic nephropathy. The A12 variant of the PPARγ2 P121A polymorphism has been firmly associated with reduced risk of insulin resistance, while its role on the risk of albuminuria in patients with type 2 diabetes is uncertain. This study investigated whether the PPARγ2 P12A polymorphism modulates the risk of albuminuria in these patients. METHODS: We tested the association between the A12 variant and albuminuria in three new case-control studies in diabetic patients from Italy (n = 841, n = 623 and n = 714 patients, respectively) and then performed a meta-analysis of all studies available to date. The nine studies we meta-analysed (six previously published and three presented here) comprised a total of 2376 cases and 4188 controls. RESULTS: In none of the three new studies was a significant association observed with odds ratio (OR) [95% confidence intervals (95% CI)] being 1.115, 0.799 and 0.849 (P = 0.603, 0.358 and 0.518, respectively). At meta-analysis, the overall OR (95% CI) for association between A12 and albuminuria was 0.694 (0.528-0.912). A significant heterogeneity of the genetic effect was observed (P = 0.026), which was totally explained by the different method of urine collection and albuminuria definition utilized across the studies. In fact, most of the effect was observed in the four studies determining albumin excretion rate rather than in those using albumin concentration in a single spot (OR, 95% CI: 0.529, 0.397-0.706, P = 0.0000164 and 0.919, 0.733-1.153, P = 0.47, respectively). CONCLUSION: The present study shows that the PPARγ2 Ala12 variant is significantly associated with a reduced risk of albuminuria among patients with type 2 diabetes.

A Serum Resistin and Multicytokine Inflammatory Pathway Is Linked With and Helps Predict All-cause Death in Diabetes.

CONTEXT: Type 2 diabetes (T2D) shows a high mortality rate, partly mediated by atherosclerotic plaque instability. Discovering novel biomarkers may help identify high-risk patients who would benefit from more aggressive and specific managements. We recently described a serum resistin and multicytokine inflammatory pathway (REMAP), including resistin, interleukin (IL)-1ß, IL-6, IL-8, and TNF-α, that is associated with cardiovascular disease. OBJECTIVE: We investigated whether REMAP is associated with and improves the prediction of mortality in T2D. METHODS: A REMAP score was investigated in 3 cohorts comprising 1528 patients with T2D (409 incident deaths) and in 59 patients who underwent carotid endarterectomy (CEA; 24 deaths). Plaques were classified as unstable/stable according to the modified American Heart Association atherosclerosis classification. RESULTS: REMAP was associated with all-cause mortality in each cohort and in all 1528 individuals (fully adjusted hazard ratio [HR] for 1 SD increase = 1.34, P < .001). In CEA patients, REMAP was associated with mortality (HR = 1.64, P = .04) and a modest change was observed when plaque stability was taken into account (HR = 1.58; P = .07). REMAP improved discrimination and reclassification measures of both Estimation of Mortality Risk in Type 2 Diabetic Patients and Risk Equations for Complications of Type 2 Diabetes, well-established prediction models of mortality in T2D (P < .05-< .001). CONCLUSION: REMAP is independently associated with and improves predict all-cause mortality in T2D; it can therefore be used to identify high-risk individuals to be targeted with more aggressive management. Whether REMAP can also identify patients who are more responsive to IL-6 and IL-1ß monoclonal antibodies that reduce cardiovascular burden and total mortality is an intriguing possibility to be tested.

ENPP1 Q121 variant, increased pulse pressure and reduced insulin signaling, and nitric oxide synthase activity in endothelial cells.

OBJECTIVE: Insulin resistance induces increased pulse pressure (PP), endothelial dysfunction (ED), and reduced bioavailability of endothelium-derived nitric oxide (NO). The genetic background of these 3 cardiovascular risk factors might be partly common. The ENPP1 K121Q polymorphism is associated with insulin resistance and cardiovascular risk. METHODS AND RESULTS: We investigated whether the K121Q polymorphism is associated with increased PP in white Caucasians and with ED in vitro. In 985 individuals, (390 unrelated and 595 from 248 families), the K121Q polymorphism was associated with PP (P=8.0 x 10(-4)). In the families, the Q121 variant accounted for 0.08 of PP heritability (P=9.4 x 10(-4)). This association was formally replicated in a second sample of 475 individuals (P=2.6 x 10(-2)) but not in 2 smaller samples of 289 and 236 individuals (P=0.49 and 0.21, respectively). In the individual patients' data meta-analysis, comprising 1985 individuals, PP was associated with the Q121 variant (P=1.2 x 10(-3)). Human endothelial cells carrying the KQ genotype showed, as compared to KK cells, reduced insulin-mediated insulin receptor autophosphorylation (P=0.03), Ser(473)-Akt phosphorylation (P=0.03), and NO synthase activity (P=0.003). CONCLUSIONS: Our data suggest that the ENPP1 Q121 variant is associated with increased PP in vivo and reduced insulin signaling and ED in vitro, thus indicating a possible pathogenic mechanism for the increased cardiovascular risk observed in ENPP1 Q121 carriers.

Association of a homozygous GCK missense mutation with mild diabetes.

BACKGROUND: Homozygous inactivating GCK mutations have been repeatedly reported to cause severe hyperglycemia, presenting as permanent neonatal diabetes mellitus (PNDM). Conversely, only two cases of GCK homozygous mutations causing mild hyperglycemia have been so far described. We here report a novel GCK mutation (c.1116G>C, p.E372D), in a family with one homozygous member showing mild hyperglycemia. METHODS: GCK mutational screening was carried out by Sanger sequencing. Computational analyses to investigate pathogenicity and molecular dynamics (MD) were performed for GCK-E372D and for previously described homozygous mutations associated with mild (n = 2) or severe (n = 1) hyperglycemia, used as references. RESULTS: Of four mildly hyperglycemic family-members, three were heterozygous and one, diagnosed in the adulthood, was homozygous for GCK-E372D. Two nondiabetic family members carried no mutations. Fasting glucose (p = 0.016) and HbA1c (p = 0.035) correlated with the number of mutated alleles (0-2). In-silico predicted pathogenicity was not correlated with the four mutations' severity. At MD, GCK-E372D conferred protein structure flexibility intermediate between mild and severe GCK mutations. CONCLUSIONS: We present the third case of homozygous GCK mutations associated with mild hyperglycemia, rather than PNDM. Our in-silico analyses support previous evidences suggesting that protein stability plays a role in determining clinical severity of GCK mutations.

Common haplotypes at the adiponectin receptor 1 (ADIPOR1) locus are associated with increased risk of coronary artery disease in type 2 diabetes.

Adiponectin, an adipokine facilitating insulin action, has antiatherogenic effects. This study investigated whether common polymorphisms in the adiponectin receptor 1 (ADIPOR1) gene mediating these effects influence the risk of coronary artery disease (CAD) in type 2 diabetes. Linkage disequilibrium analysis of 28 single nucleotide polymorphisms (SNPs) spanning the entire ADIPOR1 locus revealed two haplotype blocks that could be tagged by six SNPs. These six markers were typed in two populations of CAD-positive and -negative subjects with type 2 diabetes, one from Boston (n = 411) and the other from Italy (n = 533). In the Boston population, the three tags of the more 3' block were all significantly associated with CAD (P = 0.001-0.01). A similar trend, although not significant, was found in Italian subjects. Haplotype analysis of the combined populations revealed different haplotype distributions in case and control subjects (P = 0.0002), with one common haplotype being associated in homozygotes with a greater than threefold increase in cardiovascular risk (odds ratio 3.6 [95% CI 1.8-7.2]). Some of the genotypes associated with increased cardiovascular risk were associated with 30-40% lower ADIPOR1 mRNA levels in blood mononuclear cells (n = 60) and adipose tissue biopsies (n = 28) (P = 0.001-0.014). Our findings point to genetic variability at the ADIPOR1 locus as a strong determinant of CAD susceptibility in type 2 diabetes.