Investigating Maturity Onset Diabetes of the Young

Maturity Onset Diabetes of Young (MODY) is a monogenic and autosomal dominant form of diabetes mellitus with onset of the disease often before 25 years of age. It is due to dysfunction of pancreatic ß cells characterised by non-ketotic diabetes and absence of pancreatic auto-antibodies. It is frequently mistaken for type 1 or type 2 diabetes mellitus. Diagnosis of MODY is important as the GCK subtype has better prognosis and may not require any treatment. Subtypes HNF1A and HNF4A are sensitive to sulfonylureas, however diabetes complications are common if not treated early. Moreover, there is genetic implication for the patient and family. Rare MODY subtypes can be associated with pancreatic and renal anomalies as well as exocrine dysfunction of the pancreas. So far there are six widely accepted subtypes of MODY described but the list has grown to nine. Although the majority of diabetes mellitus in youth remains type 1 and the incidence of type 2 is rising, MODY should be considered in patients with non-ketotic diabetes at presentation, and in patients with a strong family history of diabetes mellitus without pancreatic auto-antibodies. Furthermore the diagnosis must be confirmed by molecular studies. With advancement in genomic technology, rapid screening for MODY mutations will become readily available in the future.     

Associations of Serum and Urinary Magnesium with the Pre-Diabetes,Diabetes and Diabetic Complications in the Chinese Northeast Population

The effect of magnesium (Mg) deficiency on the prevalence of diabetes and diabetic complications has received a great attention. The present study investigated the association of Mg level in the serum or urine of the patients, lived in the Northeast areas of China, with either pre-diabetes or diabetes with and without complications. From January 2010 to October 2011, patients with type 1 diabetes (T1D, n = 25), type 2 diabetes (T2D, n = 137), impaired fasting glucose (IFG, n = 12) or impaired glucose tolerance (IGT, n = 15), and age/gender matched control (n = 50) were enrolled in the First Hospital of Jilin University. In T2D group, there were 24, 34, and 50 patients with nephropathy, retinopathy or peripheral neuropathy. Serum Mg levels in the patients with IGT, IFG, T2D, and T1D were significantly lower than that of control. The urinary Mg levels were significantly increased only in T2D and T1D patients compared to control. There was no difference for these two changes among T2D with and without complications; In addition, there was a significantly positive correlation of serum Mg levels with serum Ca levels only in T2D patients, and also a significantly positive correlation of urinary Mg levels with urinary Ca levels in control, IGT patients, and T2D patients. Simvastatin treatment in T2D patients selectively reduced serum Ca levels and urinary Mg levels. These results suggest that the potential impact of Mg deficiency on metabolic syndrome, diabetes and diabetic complications needs to be received special attention.     

Urinary Collagen Fragments Are Significantly Altered in Diabetes: A Link to Pathophysiology

Background

The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D).

Methodology/Principal Findings

Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92–95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81–94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81–88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed.

Conclusions/Significance

These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.     

A Clinical Prediction Model to Distinguish Maturity-Onset Diabetes of the Young From Type 1 and Type 2 Diabetes in the Chinese Population

ObjectiveGenetic detection for the diagnosis of maturity-onset diabetes of the young (MODY) in China has low sensitivity and specificity. Better gene detection is urgently needed to distinguish testing subjects. We proposed to use numerous and weighted clinical traits as key indicators for reasonable genetic testing to predict the probability of MODY in the Chinese population.MethodsWe created a prediction model based on data from 306 patients, including 140 patients with MODY, 84 patients with type 1 diabetes (T1D), and 82 patients with type 2 diabetes (T2D). This model was evaluated using receiver operating characteristic curves.ResultsCompared with patients with T1D, patients with MODY had higher C-peptide levels and negative antibodies, and most patients with MODY had a family history of diabetes. Different from T2D, MODY was characterized by lower body mass index and younger diagnostic age. A clinical prediction model was established to define the comprehensive probability of MODY by a weighted consolidation of the most distinguishing features, and the model showed excellent discrimination (areas under the curve of 0.916 in MODY vs T1D and 0.942 in MODY vs T2D). Further, high-sensitivity C-reactive protein, glycated hemoglobin A1c, 2-h postprandial glucose, and triglyceride were used as indicators for glucokinase-MODY, while triglyceride, high-sensitivity C-reactive protein, and hepatocellular adenoma were used as indicators for hepatocyte nuclear factor 1-α MODY.ConclusionWe developed a practical prediction model that could predict the probability of MODY and provide information to identify glucokinase-MODY and hepatocyte nuclear factor 1-α MODY. These results provide an advanced and more reasonable process to identify the most appropriate patients for genetic testing.     

Adipokine pattern in subjects with impaired fasting glucose and impaired glucose tolerance in comparison to normal glucose tolerance and diabetes

Aim

Altered adipokine serum concentrations early reflect impaired adipose tissue function in obese patients with type 2 diabetes (T2D). It is not entirely clear whether these adipokine alterations are already present in prediabetic states and so far there is no comprehensive adipokine panel available. Therefore, the aim of this study was to assess distinct adipokine profiles in patients with normal glucose tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or T2D.

Methods

Based on 75 g oral glucose tolerance tests, 124 individuals were divided into groups of IFG (n = 35), IGT (n = 45), or NGT (n = 43). Furthermore, 56 subjects with T2D were included. Serum concentrations of adiponectin, chemerin, fetuin-A, leptin, interleukin (IL)-6, retinol-binding protein 4 (RBP4), monocyte chemoattractant protein (MCP)-1, vaspin, progranulin, and soluble leptin receptor (sOBR) were measured by ELISAs.

Results

Chemerin, progranulin, fetuin-A, and RBP4, IL-6, adiponectin and leptin serum concentrations were differentially regulated among the four investigated groups but only circulating chemerin was significantly different in patients with IGT compared to those with IFG. Compared to T2D the IFG subjects had higher serum chemerin, progranulin, fetuin-A and RBP4 levels which was not detectable in the comparison of the T2D and IGT group.

Conclusion

Alterations in adipokine serum concentrations are already detectable in prediabetic states, mainly for chemerin, and may reflect adipose tissue dysfunction as an early pathogenetic event in T2D development. In addition, distinct adipokine serum patterns in individuals with IFG and IGT suggest a specific role of adipose tissue in the pathogenesis of these prediabetic states.     

MRI discriminates thrombus composition and ST resolution after percutaneous coronary intervention in patients with ST-elevation myocardial infarction

Histological composition of material obtained by thrombus aspiration during percutaneous coronary intervention (PCI) in patients with ST-segment elevation acute myocardial infarction (STEMI) is highly variable. We aimed to characterize this material using magnetic resonance imaging (MRI) and to correlate MRI findings with the success of PCI in terms of ST-segment resolution. Thrombus aspiration during primary or rescue PCI was attempted in 100 consecutive STEMI patients, of whom enough material for MRI was obtained in 59. MR images were obtained at 9.4T and T1 and T2 values were measured. Patients with (n = 31) and without (n = 28) adequate ST resolution 120 min after PCI (≥70% of pre-PCI value) had similar baseline characteristics except for a higher prevalence of diabetes mellitus in the latter (10 vs. 43%, p = 0.003). T1 values were similar in both groups (1248±112 vs. 1307±85 ms, respectively, p = 0.7). T2 values averaged 31.2±10.3 and 36.6±12.2 ms; in thrombus from patients with and without adequate ST resolution (p = 0.09). After adjusting for diabetes and other baseline characteristics, lower T2 values were significantly associated with inadequate ST resolution (odds ratio for 1 ms increase 1.08, CI 95% 1.01–1.16, p = 0.027). Histology classified thrombus in 3 groups: coagulated blood (n = 38), fibrin rich (n = 9) and lipid-rich (n = 3). Thrombi composed mostly of coagulated blood were characterized as being of short (n = 10), intermediate (n = 15) or long evolution (n = 13), T2 values being 34.0±13.2, 31.9±8.3 and 31.5±7.9 ms respectively (p = NS). In this subgroup, T2 was significantly higher in specimens from patients with inadequate perfusion (35.9±10.3 versus 28.6±6.7 ms, p = 0.02). This can be of clinical interest as it provides information on the probability of adequate ST resolution, a surrogate for effective myocardial reperfusion.     

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: a multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10⁻⁸–1.2×10⁻⁴³). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10⁻⁴). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10⁻³, n = 22,044), increased triglycerides (p = 2.6×10⁻¹⁴, n = 93,440), increased waist-to-hip ratio (p = 1.8×10⁻⁵, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10⁻³, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10⁻¹³, n = 96,748) and decreased BMI (p = 1.4×10⁻⁴, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.     

The contrasting relationships between betaine and homocysteine in two clinical cohorts are associated with plasma lipids and drug treatments

Background

Urinary betaine excretion positively correlated with plasma homocysteine in outpatients attending a lipid disorders clinic (lipid clinic study). We aimed to confirm this in subjects with established vascular disease.

Methods

The correlation between betaine excretion and homocysteine was compared in samples collected from subjects 4 months after hospitalization for an acute coronary episode (ACS study, 415 urine samples) and from 158 sequential patients visiting a lipid disorders clinic.

Principal findings

In contrast to the lipid clinic study, betaine excretion and plasma homocysteine did not correlate in the total ACS cohort. Differences between the patient groups included age, non-HDL cholesterol and medication. In ACS subjects with below median betaine excretion, excretion correlated (using log transformed data) negatively with plasma homocysteine (r = −0.17, p = 0.019, n = 199), with no correlation in the corresponding subset of the lipid clinic subjects. In ACS subjects with above median betaine excretion a positive trend (r = +0.10) between betaine excretion and homocysteine was not significant; the corresponding correlation in lipid clinic subjects was r = +0.42 (p = 0.0001). In ACS subjects, correlations were stronger when plasma non-HDL cholesterol and betaine excretion were above the median, r = +0.20 (p = 0.045); in subjects above median non-HDL cholesterol and below median betaine excretion, r = −0.26 (p = 0.012). ACS subjects taking diuretics or proton pump inhibitors had stronger correlations, negative with lower betaine excretion and positive with higher betaine excretion.

Conclusions

Betaine excretion correlates with homocysteine in subjects with elevated blood lipids.     

Urinary bisphenol A and type-2 diabetes in U.S. adults: data from NHANES 2003-2008

Objective

Bisphenol A (BPA) is found in plastics and other consumer products; exposure may lead to insulin resistance and development of type-2 diabetes mellitus (T2DM) through over-activation of pancreatic β-cells. Previous studies using data from the National Health and Nutrition Examination Survey (NHANES) showed an inconsistent association between prevalence of self-reported T2DM and urinary BPA. We used a different diagnosis method of T2DM (hemoglobin A1c (HbA1c)) with a larger subset of NHANES.

Methods and Findings

We analyzed data from 4,389 adult participants who were part of a sub-study of environmental phenol measurements in urine from three NHANES cycles from 2003 to 2008. T2DM was defined as having a HbA1c ≥6.5% or use of diabetes medication. The weighted prevalence of T2DM was 9.2%. Analysis of the total sample revealed that a two-fold increase in urinary BPA was associated with an odds ratio (OR) of 1.08 of T2DM (95% confidence interval (CI), 1.02 to 1.16), after controlling for potential confounders. However, when we examined each NHANES cycle individually, we only found a statistically significant association in the 2003/04 cycle (n = 1,364, OR = 1.23 (95% CI, 1.07 to 1.42) for each doubling in urinary BPA). We found no association in either the NHANES cycle from 2005/06 (n = 1,363, OR = 1.05 (95% CI, 0.94 to 1.18)); or 2007/08 (n = 1,662, OR = 1.06 (95% CI, 0.91 to 1.23)). Similar patterns of associations between BPA and continuous HbA1c were also observed.

Conclusions

Although higher urinary BPA was associated with elevated HbA1c and T2DM in the pooled analysis, it was driven by data from only one NHANES cycle. Additional studies, especially of a longitudinal design with repeated BPA measurements, are needed to further elucidate the association between BPA and T2DM.     

Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human “stem cell cassette” containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1–60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.     

Structural basis of natural promoter recognition by a unique nuclear receptor, HNF4alpha. Diabetes gene product

HNF4alpha (hepatocyte nuclear factor 4alpha) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic beta-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4alpha is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4alpha recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 angstroms crystal structure of human HNF4alpha DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1alpha, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4alpha molecular function can cause significant effects in afflicted MODY patients.     

Phagocytic activity is impaired in type 2 diabetes mellitus and increases after metabolic improvement

Objective

1) To evaluate whether peripheral blood mononuclear cells (PBMCs) from type 2 diabetic patients present an impairment of phagocytic activity; 2) To determine whether the eventual impairment in phagocytic activity is related to glycemic control and can be reversed by improving blood glucose levels.

Methods

21 type 2 diabetic patients and 21 healthy volunteers were prospectively recruited for a case-control study. In addition, those patients in whom HbA1c was higher than 8% (n = 12) were hospitalized in order to complete a 5-day intensification treatment of blood glucose. Phagocytic activity was assessed by using a modified flow cytometry procedure developed in our laboratory based on DNA/RNA viable staining to discriminate erythrocytes and debris. This method is simple, highly sensitive and reproducible and it takes advantage of classic methods that are widely used in flow cytometry.

Results

Type 2 diabetic patients showed a lower percentage of activated macrophages in comparison with non-diabetic subjects (54.00±18.93 vs 68.53±12.77%; p = 0.006) Significant negative correlations between phagocytic activity and fasting glucose (r = −0.619, p = 0.004) and HbA1c (r = −0.506, p = 0.019) were detected. In addition, multiple linear regression analyses showed that either fasting plasma glucose or HbA1c were independently associated with phagocytic activity. Furthermore, in the subset of patients who underwent metabolic optimization a significant increase in phagocytic activity was observed (p = 0.029).

Conclusions

Glycemic control is related to phagocytic activity in type 2 diabetes. Our results suggest that improvement in phagocytic activity can be added to the beneficial effects of metabolic optimization.     

Low Frequency Variants in the Exons Only Encoding Isoform A of HNF1A Do Not Contribute to Susceptibility to Type 2 Diabetes

Background

There is considerable interest in the hypothesis that low frequency, intermediate penetrance variants contribute to the proportion of Type 2 Diabetes (T2D) susceptibility not attributable to the common variants uncovered through genome-wide association approaches. Genes previously implicated in monogenic and multifactorial forms of diabetes are obvious candidates in this respect. In this study, we focussed on exons 8–10 of the HNF1A gene since rare, penetrant mutations in these exons (which are only transcribed in selected HNF1A isoforms) are associated with a later age of diagnosis of Maturity onset diabetes of the young (MODY) than mutations in exons 1–7. The age of diagnosis in the subgroup of HNF1A-MODY individuals with exon 8–10 mutations overlaps with that of early multifactorial T2D, and we set out to test the hypothesis that these exons might also harbour low-frequency coding variants of intermediate penetrance that contribute to risk of multifactorial T2D.

Methodology and Principal Findings

We performed targeted capillary resequencing of HNF1A exons 8–10 in 591 European T2D subjects enriched for genetic aetiology on the basis of an early age of diagnosis (≤45 years) and/or family history of T2D (≥1 affected sibling). PCR products were sequenced and compared to the published HNF1A sequence. We identified several variants (rs735396 [IVS9−24T>C], rs1169304 [IVS8+29T>C], c.1768+44C>T [IVS9+44C>T] and rs61953349 [c.1545G>A, p.T515T] but no novel non-synonymous coding variants were detected.

Conclusions and Significance

We conclude that low frequency, nonsynonymous coding variants in the terminal exons of HNF1A are unlikely to contribute to T2D-susceptibility in European samples. Nevertheless, the rationale for seeking low-frequency causal variants in genes known to contain rare, penetrant mutations remains strong and should motivate efforts to screen other genes in a similar fashion.     

Proof-of-Concept, Randomized, Controlled Clinical Trial of Bacillus-Calmette-Guerin for Treatment of Long-Term Type 1 Diabetes

Background

No targeted immunotherapies reverse type 1 diabetes in humans. However, in a rodent model of type 1 diabetes, Bacillus Calmette-Guerin (BCG) reverses disease by restoring insulin secretion. Specifically, it stimulates innate immunity by inducing the host to produce tumor necrosis factor (TNF), which, in turn, kills disease-causing autoimmune cells and restores pancreatic beta-cell function through regeneration.

Methodology/Principal Findings

Translating these findings to humans, we administered BCG, a generic vaccine, in a proof-of-principle, double-blind, placebo-controlled trial of adults with long-term type 1 diabetes (mean: 15.3 years) at one clinical center in North America. Six subjects were randomly assigned to BCG or placebo and compared to self, healthy paired controls (n = 6) or reference subjects with (n = 57) or without (n = 16) type 1 diabetes, depending upon the outcome measure. We monitored weekly blood samples for 20 weeks for insulin-autoreactive T cells, regulatory T cells (Tregs), glutamic acid decarboxylase (GAD) and other autoantibodies, and C-peptide, a marker of insulin secretion. BCG-treated patients and one placebo-treated patient who, after enrollment, unexpectedly developed acute Epstein-Barr virus infection, a known TNF inducer, exclusively showed increases in dead insulin-autoreactive T cells and induction of Tregs. C-peptide levels (pmol/L) significantly rose transiently in two BCG-treated subjects (means: 3.49 pmol/L [95% CI 2.95–3.8], 2.57 [95% CI 1.65–3.49]) and the EBV-infected subject (3.16 [95% CI 2.54–3.69]) vs.1.65 [95% CI 1.55–3.2] in reference diabetic subjects. BCG-treated subjects each had more than 50% of their C-peptide values above the 95^th percentile of the reference subjects. The EBV-infected subject had 18% of C-peptide values above this level.

Conclusions/Significance

We conclude that BCG treatment or EBV infection transiently modified the autoimmunity that underlies type 1 diabetes by stimulating the host innate immune response. This suggests that BCG or other stimulators of host innate immunity may have value in the treatment of long-term diabetes.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00607230","term_id":"NCT00607230"}}NCT00607230     

Gene-Gene Interactions Lead to Higher Risk for Development of Type 2 Diabetes in an Ashkenazi Jewish Population

Background

Evidence has accumulated that multiple genetic and environmental factors play important roles in determining susceptibility to type 2 diabetes (T2D). Although variants from candidate genes have become prime targets for genetic analysis, few studies have considered their interplay. Our goal was to evaluate interactions among SNPs within genes frequently identified as associated with T2D.

Methods/Principal Findings

Logistic regression was used to study interactions among 4 SNPs, one each from HNF4A[rs1884613], TCF7L2[rs12255372], WFS1[rs10010131], and KCNJ11[rs5219] in a case-control Ashkenazi sample of 974 diabetic subjects and 896 controls. Nonparametric multifactor dimensionality reduction (MDR) and generalized MDR (GMDR) were used to confirm findings from the logistic regression analysis. HNF4A and WFS1 SNPs were associated with T2D in logistic regression analyses [P<0.0001, P<0.0002, respectively]. Interaction between these SNPs were also strong using parametric or nonparametric methods: the unadjusted odds of being affected with T2D was 3 times greater in subjects with the HNF4A and WFS1 risk alleles than those without either (95% CI = [1.7–5.3]; P≤0.0001). Although the univariate association between the TCF7L2 SNP and T2D was relatively modest [P = 0.02], when paired with the HNF4A SNP, the OR for subjects with risk alleles in both SNPs was 2.4 [95% CI = 1.7–3.4; P≤0.0001]. The KCNJ11 variant reached significance only when paired with either the HNF4A or WFSI SNPs: unadjusted ORs were 2.0 [95% CI = 1.4–2.8; P≤0.0001] and 2.3 [95% CI = 1.2-4.4; P≤0.0001], respectively. MDR and GMDR results were consistent with the parametric findings.

Conclusions

These results provide evidence of strong independent associations between T2D and SNPs in HNF4A and WFS1 and their interaction in our Ashkenazi sample. We also observed an interaction in the nonparametric analysis between the HNF4A and KCNJ11 SNPs (P≤0.001), demonstrating that an independently non-significant variant may interact with another variant resulting in an increased disease risk.     

Multicentric validation of proteomic biomarkers in urine specific for diabetic nephropathy

Background

Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82).

Methodology/Principal Findings

Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patient''s subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients.

Conclusions

These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.     

Esophageal cancer metabolite biomarkers detected by LC-MS and NMR methods

Background

Esophageal adenocarcinoma (EAC) is a rarely curable disease and is rapidly rising worldwide in incidence. Barret''s esophagus (BE) and high-grade dysplasia (HGD) are considered major risk factors for invasive adenocarcinoma. In the current study, unbiased global metabolic profiling methods were applied to serum samples from patients with EAC, BE and HGD, and healthy individuals, in order to identify metabolite based biomarkers associated with the early stages of EAC with the goal of improving prognostication.

Methodology/Principal Findings

Serum metabolite profiles from patients with EAC (n = 67), BE (n = 3), HGD (n = 9) and healthy volunteers (n = 34) were obtained using high performance liquid chromatography-mass spectrometry (LC-MS) methods. Twelve metabolites differed significantly (p<0.05) between EAC patients and healthy controls. A partial least-squares discriminant analysis (PLS-DA) model had good accuracy with the area under the receiver operative characteristic curve (AUROC) of 0.82. However, when the results of LC-MS were combined with 8 metabolites detected by nuclear magnetic resonance (NMR) in a previous study, the combination of NMR and MS detected metabolites provided a much superior performance, with AUROC = 0.95. Further, mean values of 12 of these metabolites varied consistently from healthy controls to the high-risk individuals (BE and HGD patients) and EAC subjects. Altered metabolic pathways including a number of amino acid pathways and energy metabolism were identified based on altered levels of numerous metabolites.

Conclusions/Significance

Metabolic profiles derived from the combination of LC-MS and NMR methods readily distinguish EAC patients and potentially promise important routes to understanding the carcinogenesis and detecting the cancer. Differences in the metabolic profiles between high-risk individuals and the EAC indicate the possibility of identifying the patients at risk much earlier to the development of the cancer.     

Urinary podocyte-associated mRNA profile in various stages of diabetic nephropathy

Background

Podocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We hypothesized that the urinary mRNA profile of podocyte-associated molecules may provide important clinical insight into the different stages of diabetic nephropathy.

Methods

DN patients (N = 51) and healthy controls (N = 13) were enrolled in this study. DN patients were divided into a normoalbuminuria group (UAE<30 mg/g, n = 17), a microalbuminuria group (UAE 30∼300 mg/g, n = 15), and a macroalbuminuria group (UAE>300 mg/g, n = 19), according to their urinary albumin excretion (UAE). Relative mRNA abundance of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined.

Results

The urinary mRNA levels of all genes studied were significantly higher in the DN group compared with controls (p<0.05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and BUN. The expression of podocalyxin, CD2-AP, α-actin4, and podocin mRNA correlated with serum creatinine (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021; r = 0.357, p = 0.006, respectively). Furthermore, podocalyxin mRNA was found to negatively correlate with eGFR (r = −0.349, p = 0.01).

Conclusion

The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, α-actin4, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN.     

Assessment of Urinary Betaine as a Marker of Diabetes Mellitus in Cardiovascular Patients

Abnormal urinary excretion of betaine has been demonstrated in patients with diabetes or metabolic syndrome. We aimed to identify the main predictors of excretion in cardiovascular patients and to make initial assessment of its feasibility as a risk marker of future diabetes development. We used data from 2396 patients participating in the Western Norway B-vitamin Intervention Trial, who delivered urine and blood samples at baseline, and in the majority at two visits during follow-up of median 39 months. Betaine in urine and plasma were measured by liquid-chromatography-tandem mass spectrometry. The strongest determinants of urinary betaine excretion by multiple regression were diabetes mellitus, age and estimated glomerular filtration rate; all p<0.001. Patients with diabetes mellitus (n = 264) had a median excretion more than three times higher than those without. We found a distinct non-linear association between urinary betaine excretion and glycated hemoglobin, with a break-point at 6.5%, and glycated hemoglobin was the strongest determinant of betaine excretion in patients with diabetes mellitus. The discriminatory power for diabetes mellitus corresponded to an area under the curve by receiver-operating characteristics of 0.82, and betaine excretion had a coefficient of reliability of 0.73. We also found a significant, independent log-linear relation between baseline betaine excretion and the risk of developing new diabetes during follow-up. The good discriminatory power for diabetes, high test-retest stability and independent association with future risk of new diabetes should motivate further investigation on the role of betaine excretion in risk assessment and long-term follow-up of diabetes mellitus.