Bernhard O. Boehm

Increased DNA methylation variability in type 1 diabetes across three immune effector cell types

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.

Influence of TCF7L2 gene variants on the therapeutic response to the dipeptidylpeptidase-4 inhibitor linagliptin

Aims/hypothesisIndividuals carrying variants of the transcription factor 7-like 2 gene (TCF7L2) are at increased risk for type 2 diabetes. These metabolic genetic risk factors have been linked to diminished pancreatic islet-cell responsiveness to incretins, thus pharmacological interventions aimed at amplifying endogenous incretin biology may be affected. However, clinical evidence from randomised controlled trials so far is lacking. We investigated the influence of TCF7L2 risk alleles on the response to treatment with the dipeptidylpeptidase-4 (DPP-4) inhibitor linagliptin from four 24xa0week, phase III, placebo-controlled trials.MethodsPharmacogenomic samples and clinical data were available from 961 patients with type 2 diabetes. Whole-blood DNA samples were genotyped for TCF7L2 single-nucleotide polymorphisms in conjunction with assessments of 24xa0week changes in HbA1c.ResultsLinagliptin lowered HbA1c meaningfully in all three genotypes of rs7903146 (non-risk variant carriers CC [nu2009=u2009356]: −0.82% [−9.0xa0mmol/mol], pu2009<u20090.0001; heterozygous CT [nu2009=u2009264]: −0.77% [−8.4xa0mmol/mol], pu2009<u20090.0001; homozygous risk variant carriers TT [nu2009=u200973]: −0.57% [−6.2xa0mmol/mol], pu2009<u20090.0006). No significant treatment differences were seen between CC and CT patients, although HbA1c response was reduced in TT compared with CC patients (~0.26% [~2.8xa0mmol/mol], pu2009=u20090.0182).Conclusions/interpretationLinagliptin significantly improved hyperglycaemia in patients with type 2 diabetes both with and without the TCF7L2 gene diabetes risk alleles. However, differences in treatment response were observed, indicating that diabetes susceptibility genes may be an important contributor to the inter-individual variability of treatment response.

Genome-wide association study on dimethylarginines reveals novel AGXT2 variants associated with heart rate variability but not with overall mortality

AIMSnThe purpose of this study was to identify novel genetic variants influencing circulating asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) levels and to evaluate whether they have a prognostic value on cardiovascular mortality.nnnMETHODS AND RESULTSnWe conducted a genome-wide association study on the methylarginine traits and investigated the predictive value of the new discovered variants on mortality. Our meta-analyses replicated the previously known locus for ADMA levels in DDAH1 (rs997251; P = 1.4 × 10(-40)), identified two non-synomyous polymorphisms for SDMA levels in AGXT2 (rs37369; P = 1.4 × 10(-40) and rs16899974; P = 1.5 × 10(-38)) and one in SLC25A45 (rs34400381; P = 2.5 × 10(-10)). We also fine-mapped the AGXT2 locus for further independent association signals. The two non-synonymous AGXT2 variants independently associated with SDMA levels were also significantly related with short-term heart rate variability (HRV) indices in young adults. The major allele (C) of the novel non-synonymous rs16899974 (V498L) variant associated with decreased SDMA levels and an increase in the ratio between the low- and high-frequency spectral components of HRV (P = 0.00047). Furthermore, the SDMA decreasing allele (G) of the non-synomyous SLC25A45 (R285C) variant was associated with a lower resting mean heart rate during the HRV measurements (P = 0.0046), but not with the HRV indices. None of the studied genome-wide significant variants had any major effect on cardiovascular or total mortality in patients referred for coronary angiography.nnnCONCLUSIONSnAGXT2 has an important role in SDMA metabolism in humans. AGXT2 may additionally have an unanticipated role in the autonomic nervous system regulation of cardiac function.

New insights into the architecture of the islet of Langerhans: a focused cross-species assessment

The human genome project and its search for factors underlying human diseases has fostered a major human research effort. Therefore, unsurprisingly, in recent years we have observed an increasing number of studies on human islet cells, including disease approaches focusing on type 1 and type 2 diabetes. Yet, the field of islet and diabetes research relies on the legacy of rodent-based investigations, which have proven difficult to translate to humans, particularly in type 1 diabetes. Whole islet physiology and pathology may differ between rodents and humans, and thus a comprehensive cross-species as well as species-specific view on islet research is much needed. In this review we summarise the current knowledge of interspecies islet cytoarchitecture, and discuss its potential impact on islet function and future perspectives in islet pathophysiology research.

Relative contribution of type 1 and type 2 diabetes loci to the genetic etiology of adult-onset, non-insulin-requiring autoimmune diabetes

BackgroundIn adulthood, autoimmune diabetes can present as non-insulin-requiring diabetes, termed as ‘latent autoimmune diabetes in adults’ (LADA). In this study, we investigated established type 1 diabetes (T1D) and type 2 diabetes (T2D) genetic loci in a large cohort of LADA cases to assess where LADA is situated relative to these two well-characterized, classic forms of diabetes.MethodsWe tested the association of T1D and T2D GWAS-implicated loci in 978 LADA cases and 1057 non-diabetic controls of European ancestry using a linear mixed model. We then compared the associations of T1D and T2D loci between LADA and T1D and T2D cases, respectively. We quantified the difference in genetic risk between each given disease at each locus, and also calculated genetic risk scores to quantify how genetic liability to T1D and T2D distinguished LADA cases from controls.ResultsOverall, our results showed that LADA is genetically more similar to T1D, with the exception of an association at the T2D HNF1A locus. Several T1D loci were associated with LADA, including the major histocompatibility complex region, as well as at PTPN22, SH2B3, and INS. Contrary to previous studies, the key T2D risk allele at TCF7L2 (rs7903146-T) had a significantly lower frequency in LADA cases, suggesting that this locus does not play a role in LADA etiology. When constrained on antibody status, the similarity between LADA and T1D became more apparent; however, the HNF1A and TCF7L2 observations persisted.ConclusionLADA is genetically closer to T1D than T2D, although the genetic load of T1D risk alleles is less than childhood-onset T1D, particularly at the major histocompatibility complex region, potentially accounting for the later disease onset. Our results show that the genetic spectrum of T1D extends into adult-onset diabetes, where it can clinically masquerade as T2D. Furthermore, T2D genetic risk plays a small role in LADA, with a degree of evidence for the HNF1A locus, highlighting the potential for genetic risk scores to contribute towards defining diabetes subtypes.

Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus.

Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100u2009μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (Pu2009<u20090.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

Subclinical and clinical hypothyroidism and non-alcoholic fatty liver disease: a cross-sectional study of a random population sample aged 18 to 65 years.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most common disorders of the liver worldwide. Recently, a correlation between thyroid dysfunction and NAFLD has been discussed. Objective of the present study was to investigate the association between thyroid dysfunction and hepatic steatosis.MethodsData from 2,445 subjects (51.7xa0% females) aged 18 to 65xa0years participating in a population-based cross-sectional study were assessed based on a standardized questionnaire and documentation of physical, biochemical and ultrasonographic findings. After application of exclusion criteria, a total of 1,276 subjects were included in the study collective. The influence of potential factors on the development of hepatic steatosis was assessed using multivariate logistic regression.ResultsThe prevalence of hepatic steatosis in the study collective was 27.4xa0% (nu2009=u2009349). The serum thyroxin (TT4) concentration in subjects with hepatic steatosis was reduced (pu2009=u20090.0004). Adjusting for age, or BMI, there was an increased prevalence of hepatic steatosis in subjects with reduced TT4 concentrations (pu2009=u20090.0143; pu2009=u2009<.0001).ConclusionsThe findings of the present study confirm an association between both subclinical and clinical hypothyroidism and hepatic steatosis

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.

Aims/hypothesisA strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM.MethodsTwo groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4xa0weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements.ResultsPAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death.Conclusions/interpretationThe coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.

A randomised controlled trial evaluating the impact of targeted vitamin D supplementation on endothelial function in type 2 diabetes mellitus: The DIMENSION trial

We sought to determine if vitamin D supplementation, to target 25(OH)D concentrations of 30–40u2009ng/mL, improves endothelial function in Singapore’s multi-ethnic type 2 diabetes mellitus population. We randomised 64 type 2 diabetes mellitus patients with hypovitaminosis D to cholecalciferol 4000u2009International Unit/matching placebo [baseline 25(OH)Du2009<u200920u2009ng/mL] or cholecalciferol 2000u2009International Unit/matching placebo [baseline 25(OH)D: 20–30u2009ng/mL] daily for 16u2009weeks with a down titration at 8u2009weeks if 25(OH)Du2009>u200930u2009ng/mL. Endothelial function was assessed by peripheral tonometry (reactive hyperaemia index–endothelial peripheral arterial tonometry) and vascular biomarkers: E-selectin, von-Willebrand factor and high-sensitivity C-reactive protein. We compared the change from baseline parameters in the two groups using Student’s t-test or Kruskal–Wallis test. A log-normal multivariate regression analysis was used to adjust for relevant baseline variables. The median reactive hyperaemia index in the vitamin D group increased from 0.65 (interquartile range: 0.42) to 0.73 (interquartile range: 0.36), whereas it decreased from 0.73 (interquartile range: 0.65) to 0.65 (interquartile range: 0.38) (pu2009=u20090.02) in the placebo group. After adjustment for baseline variables, the change was not statistically significant for reactive hyperaemia index (pu2009=u20090.07) and for other vascular biomarkers (pu2009>u20090.05). Targeted vitamin D supplementation for 16u2009weeks resulted in a small but non-significant improvement in endothelial function in a type 2 diabetes mellitus cohort.

Coffee consumption and NAFLD: a community based study on 1223 subjects

AbstractBackgroundnObjective of the present cross-sectional study was to investigate the impact of caffeine consumption on fatty liver and serum alanine aminotransferase (ALT) concentrations in a random population sample.MethodsnAll subjects (nxa0=xa01452; 789 women, 663 men; average age 42.3xa0±xa012.8xa0years) underwent ultrasonographic examination of the liver and completed a standardized questionnaire regarding personal and lifestyle data, in particular relating to coffee consumption and past medical history. In addition, anthropometric data were documented and laboratory examinations performed. Statistical interpretation of the data was performed descriptively and by means of bivariate and multivariate analysis.ResultsnData of the present study demonstrated a significant association between hepatic steatosis male gender (pxa0<xa00.0001), advanced age (pxa0<xa00.0001) and elevated body-mass index (BMI; pxa0<xa00.0001). No association between caffeine consumption and fatty liver was identified. An association between caffeine consumption and elevated serum ALT concentrations was not identified.ConclusionsThe findings of the present study provide no evidence for an association between caffeine consumption and either the prevalence of hepatic steatosis or serum ALT concentrations.