The multi-factorial modes of action of urease in the pathogenesis of incontinence associated dermatitis.

Background: Incontinence Associated Dermatitis (IAD) is a type of skin inflammation caused by chronic exposure to urine and/or faeces. Current treatment strategies involve creating a barrier between the skin and urine/faeces rather than targeting specific irritants. Urease expressing pathogens catalyse the conversion of urea, present in urine, into ammonia. The accumulation of ammonia causes an elevation in skin pH which is believed to activate faecal enzymes which damage skin, and opportunistic pathogens, which lead to secondary infections. Objectives: To develop a better, multi-factorial model of IAD pathogenesis, including the effect of urease-expressing bacteria on skin, mechanism of damage of urease and urease-triggered activity of faecal enzymes and secondary pathogens. To study the effect of urease inhibition on preventing IAD skin damage. Methods: Five separate studies were made using ex vivo porcine skin and in vivo human skin models. Measurements of the change in skin barrier function were made using skin impedance, trans-epidermal water loss (TEWL), stratum corneum moisture and pH. Skin was exposed to artificial urine, inoculated with various microbes, enzymes and chemicals to examine the influence of: 1) urease-positive Proteus mirabilis 2) ammonia, 3) combination of P. mirabilis and a faecal enzyme, trypsin, 4) combination of P. mirabilis and opportunistic pathogens, Candida albicans and Staphylococcus aureus, 5) inhibition of urease using acetohydroxamic acid (AHA) on barrier function. Results: The urease-mediated production of ammonia had two principal effects: it elevated skin pH and caused inflammation, leading to significant breakdown in skin (stratum corneum) barrier function. Urease was found to further increase the activity of faecal enzymes and opportunistic pathogens, due to elevated skin pH. The urease inhibitor, AHA, was shown to have significantly reduced damage to skin barrier function, measured as its electrical resistance. Conclusions: Targeted therapeutic strategies should be developed to prevent the manifestation of IAD, rather than creating a generic barrier between skin and urine/faeces. Urease has been identified as a crucial component in the manifestation of IAD, due to its role in the production of ammonia. Urease inhibition provides a promising therapeutic target to halt the progression of IAD.

The hazards of genotype imputation when mapping disease susceptibility variants.

BACKGROUND: The cost-free increase in statistical power of using imputation to infer missing genotypes is undoubtedly appealing, but is it hazard-free? This case study of three type-2 diabetes (T2D) loci demonstrates that it is not; it sheds light on why this is so and raises concerns as to the shortcomings of imputation at disease loci, where haplotypes differ between cases and reference panel. RESULTS: T2D-associated variants were previously identified using targeted sequencing. We removed these significantly associated SNPs and used neighbouring SNPs to infer them by imputation. We compared imputed with observed genotypes, examined the altered pattern of T2D-SNP association, and investigated the cause of imputation errors by studying haplotype structure. Most T2D variants were incorrectly imputed with a low density of scaffold SNPs, but the majority failed to impute even at high density, despite obtaining high certainty scores. Missing and discordant imputation errors, which were observed disproportionately for the risk alleles, produced monomorphic genotype calls or false-negative associations. We show that haplotypes carrying risk alleles are considerably more common in the T2D cases than the reference panel, for all loci. CONCLUSIONS: Imputation is not a panacea for fine mapping, nor for meta-analysing multiple GWAS based on different arrays and different populations. A total of 80% of the SNPs we have tested are not included in array platforms, explaining why these and other such associated variants may previously have been missed. Regardless of the choice of software and reference haplotypes, imputation drives genotype inference towards the reference panel, introducing errors at disease loci.

Characterization of missing data patterns and mechanisms in longitudinal composite outcome trial in rheumatoid arthritis.

Background: Composite measures, like the Disease Activity Score for 28 joints (DAS28), are key primary outcomes in rheumatoid arthritis (RA) trials. DAS28 combines four different components in a continuous measure. When one or more of these components are missing the overall composite score is also missing at intermediate or trial endpoint assessments. Objectives: This study examined missing data patterns and mechanisms in a longitudinal RA trial to evaluate how best to handle missingness when analysing composite outcomes. Design: The Tumour-Necrosis-Factor Inhibitors against Combination Intensive Therapy (TACIT) trial was an open label, pragmatic randomized multicentre two arm non-inferiority study. Patients were followed up for 12 months, with monthly measurement of the composite outcome and its components. Active RA patients were randomized to conventional disease modifying drugs (cDMARDs) or Tumour Necrosis Factor-α inhibitors (TNFis). Methods: The TACIT trial was used to explore the extent of missing data in the composite outcome, DAS28. Patterns of missing data in components and the composite outcome were examined graphically. Longitudinal multivariable logistic regression analysis assessed missing data mechanisms during follow-up. Results: Two hundred and five patients were randomized: at 12 months 59/205 (29%) had unobserved composite outcome and 146/205 (71%) had an observed DAS28 outcome; however, 34/146 had one or more intermediate assessments missing. We observed mixed missing data patterns, especially for the missing composite outcome due to one component missing rather than patient not attending thier visit. Age and gender predicted missingness components, providing strong evidence the missing observations were unlikely to be Missing Completely at Random (MCAR). Conclusion: Researchers should undertake detailed evaluations of missing data patterns and mechanisms at the final and intermediate time points, whether or not the outcome variable is a composite outcome. In addition, the impact on treatment estimates in patients who only provide data at milestone assessments need to be assessed. Trial Registration ISRCTN Number: 37438295.

Multi-ancestry genome-wide association study of gestational diabetes mellitus highlights genetic links with type 2 diabetes.

Gestational diabetes mellitus (GDM) is associated with increased risk of pregnancy complications and adverse perinatal outcomes. GDM often reoccurs and is associated with increased risk of subsequent diagnosis of type 2 diabetes (T2D). To improve our understanding of the aetiological factors and molecular processes driving the occurrence of GDM, including the extent to which these overlap with T2D pathophysiology, the GENetics of Diabetes In Pregnancy Consortium assembled genome-wide association studies of diverse ancestry in a total of 5485 women with GDM and 347 856 without GDM. Through multi-ancestry meta-analysis, we identified five loci with genome-wide significant association (P < 5 × 10-8) with GDM, mapping to/near MTNR1B (P = 4.3 × 10-54), TCF7L2 (P = 4.0 × 10-16), CDKAL1 (P = 1.6 × 10-14), CDKN2A-CDKN2B (P = 4.1 × 10-9) and HKDC1 (P = 2.9 × 10-8). Multiple lines of evidence pointed to the shared pathophysiology of GDM and T2D: (i) four of the five GDM loci (not HKDC1) have been previously reported at genome-wide significance for T2D; (ii) significant enrichment for associations with GDM at previously reported T2D loci; (iii) strong genetic correlation between GDM and T2D and (iv) enrichment of GDM associations mapping to genomic annotations in diabetes-relevant tissues and transcription factor binding sites. Mendelian randomization analyses demonstrated significant causal association (5% false discovery rate) of higher body mass index on increased GDM risk. Our results provide support for the hypothesis that GDM and T2D are part of the same underlying pathology but that, as exemplified by the HKDC1 locus, there are genetic determinants of GDM that are specific to glucose regulation in pregnancy.

Maternal Glycemic Dysregulation During Pregnancy and Neonatal Blood DNA Methylation: Meta-analyses of Epigenome-Wide Association Studies.

OBJECTIVE: Maternal glycemic dysregulation during pregnancy increases the risk of adverse health outcomes in her offspring, a risk thought to be linearly related to maternal hyperglycemia. It is hypothesized that changes in offspring DNA methylation (DNAm) underline these associations. RESEARCH DESIGN AND METHODS: To address this hypothesis, we conducted fixed-effects meta-analyses of epigenome-wide association study (EWAS) results from eight birth cohorts investigating relationships between cord blood DNAm and fetal exposure to maternal glucose (Nmaximum = 3,503), insulin (Nmaximum = 2,062), and area under the curve of glucose (AUCgluc) following oral glucose tolerance tests (Nmaximum = 1,505). We performed lookup analyses for identified cytosine-guanine dinucleotides (CpGs) in independent observational cohorts to examine associations between DNAm and cardiometabolic traits as well as tissue-specific gene expression. RESULTS: Greater maternal AUCgluc was associated with lower cord blood DNAm at neighboring CpGs cg26974062 (ß [SE] -0.013 [2.1 × 10-3], P value corrected for false discovery rate [PFDR] = 5.1 × 10-3) and cg02988288 (ß [SE]-0.013 [2.3 × 10-3], PFDR = 0.031) in TXNIP. These associations were attenuated in women with GDM. Lower blood DNAm at these two CpGs near TXNIP was associated with multiple metabolic traits later in life, including type 2 diabetes. TXNIP DNAm in liver biopsies was associated with hepatic expression of TXNIP. We observed little evidence of associations between either maternal glucose or insulin and cord blood DNAm. CONCLUSIONS: Maternal hyperglycemia, as reflected by AUCgluc, was associated with lower cord blood DNAm at TXNIP. Associations between DNAm at these CpGs and metabolic traits in subsequent lookup analyses suggest that these may be candidate loci to investigate in future causal and mediation analyses.

Improving the Interpretation of Afternoon Cortisol Levels and SSTs to Prevent Misdiagnosis of Adrenal Insufficiency.

BACKGROUND: Adrenal Insufficiency (AI), especially iatrogenic-AI, is a treatable cause of mortality. The difficulty in obtaining 9 am cortisol levels means samples are taken at suboptimal times, including a substantial proportion in the afternoon. Low afternoon cortisol levels often provoke short Synacthen tests (SSTs). It is important that this does not lead to patients misdiagnosed with AI, exposing them to the excess mortality and morbidity of inappropriate steroid replacement therapy. METHODS: This retrospective study collected 60 178 cortisol results. Medical records, including subsequent SSTs of initial cortisol results measured after midday were reviewed. RESULTS: Receiver operating characteristic analysis (area under the curve: 0.89) on 6531 suitable cortisol values showed that a limit of <201.5 nmol/L achieved a sensitivity and specificity of 95.6% and 72.6%, while a limit of <234 nmol/L had a sensitivity of 100% and a specificity of 59.5%. Out of 670 SSTs, 628 patients passed. Of these, 140 would have otherwise failed if only their 30-min cortisol was assessed without the 60-min value. A 30- and 60-min SST cortisol cutoff of 366.5 nmol/L and 418.5 nmol/L, respectively, can achieve a sensitivity of >95% on the Abbott analyser platform. CONCLUSION: An afternoon cortisol >234 nmol/L excludes AI on Abbott analyser platforms. In patients who have an afternoon cortisol <234 nmol/L, including both 30- and 60-min SST cortisol values prevents unnecessary glucocorticoid replacement therapy in 22.3% of individuals in this study. The Abbott analyser SST cortisol cutoffs used to define AI should be 366.5 nmol/L and 418.5 nmol/L at 30 and 60 min, respectively. All patients remained well subsequently with at least 1-year longitudinal follow-up.

New Insights Into Mitochondrial Dysfunction at Disease Susceptibility Loci in the Development of Type 2 Diabetes.

This study investigated the potential genetic mechanisms which underlie adipose tissue mitochondrial dysfunction in Type 2 diabetes (T2D), by systematically identifying nuclear-encoded mitochondrial genes (NEMGs) among the genes regulated by T2D-associated genetic loci. The target genes of these 'disease loci' were identified by mapping genetic loci associated with both disease and gene expression levels (expression quantitative trait loci, eQTL) using high resolution genetic maps, with independent estimates co-locating to within a small genetic distance. These co-locating signals were defined as T2D-eQTL and the target genes as T2D cis-genes. In total, 763 cis-genes were associated with T2D-eQTL, of which 50 were NEMGs. Independent gene expression datasets for T2D and insulin resistant cases and controls confirmed that the cis-genes and cis-NEMGs were enriched for differential expression in cases, providing independent validation that genetic maps can identify informative functional genes. Two additional results were consistent with a potential role of T2D-eQTL in regulating the 50 identified cis-NEMGs in the context of T2D risk: (1) the 50 cis-NEMGs showed greater differential expression compared to other NEMGs and (2) other NEMGs showed a trend towards significantly decreased expression if their expression levels correlated more highly with the subset of 50 cis-NEMGs. These 50 cis-NEMGs, which are differentially expressed and associated with mapped T2D disease loci, encode proteins acting within key mitochondrial pathways, including some of current therapeutic interest such as the metabolism of branched-chain amino acids, GABA and biotin.

Epigenome-Wide Association Study Reveals Methylation Loci Associated With Offspring Gestational Diabetes Mellitus Exposure and Maternal Methylome.

OBJECTIVE: Gestational diabetes mellitus (GDM) is associated with an increased risk of obesity and insulin resistance in offspring later in life, which might be explained by epigenetic changes in response to maternal hyperglycemic exposure. RESEARCH DESIGN AND METHODS: We explored the association between GDM exposure and maternal blood and newborn cord blood methylation in 536 mother-offspring pairs from the prospective FinnGeDi cohort using Illumina MethylationEPIC 850K BeadChip arrays. We assessed two hypotheses. First, we tested for shared maternal and offspring epigenetic effects resulting from GDM exposure. Second, we tested whether GDM exposure and maternal methylation had an epigenetic effect on the offspring. RESULTS: We did not find any epigenetic marks (differentially methylated CpG probes) with shared and consistent effects between mothers and offspring. After including maternal methylation in the model, we identified a single significant (false discovery rate 1.38 × 10-2) CpG at the cg22790973 probe (TFCP2) associated with GDM. We identified seven additional FDR-significant interactions of maternal methylation and GDM status, with the strongest association at the same cg22790973 probe (TFCP2), as well as cg03456133, cg24440941 (H3C6), cg20002843 (LOC127841), cg19107264, and cg11493553 located within the UBE3C gene and cg17065901 in FAM13A, both susceptibility genes for type 2 diabetes and BMI, and cg23355087 within the DLGAP2 gene, known to be involved in insulin resistance during pregnancy. CONCLUSIONS: Our study reveals the potential complexity of the epigenetic transmission between mothers with GDM and their offspring, likely determined by not only GDM exposure but also other factors indicated by maternal epigenetic status, such as maternal metabolic history.

The feasibility of the PAM intervention to support treatment-adherence in people with hypertension in primary care: a randomised clinical controlled trial.

The PAM intervention is a behavioural intervention to support adherence to anti-hypertensive medications and therefore to lower blood pressure. This feasibility trial recruited 101 nonadherent patients (54% male, mean age 65.8 years) with hypertension and high blood pressure from nine general practices in the UK. The trial had 15.5% uptake and 7.9% attrition rate. Patients were randomly allocated to two groups: the intervention group (n = 61) received the PAM intervention as an adjunct to usual care; the control group (n = 40) received usual care only. At 3 months, biochemically validated medication adherence was improved by 20% (95% CI 3-36%) in the intervention than control, and systolic blood pressure was reduced by 9.16 mmHg (95% CI 5.69-12.64) in intervention than control. Improvements in medication adherence and reductions in blood pressure suggested potential intervention effectiveness. For a subsample of patients, improvements in medication adherence and reductions in full lipid profile (cholesterol 1.39 mmol/mol 95% CI 0.64-1.40) and in glycated haemoglobin (3.08 mmol/mol, 95% CI 0.42-5.73) favoured the intervention. A larger trial will obtain rigorous evidence about the potential clinical effectiveness and cost-effectiveness of the intervention.Trial registration Trial date of first registration 28/01/2019. ISRCTN74504989. https://doi.org/10.1186/ISRCTN74504989 .

Evaluating the Diagnostic Value of a Combined Indicator of Vitamin B12 Status (cB12) Throughout Pregnancy.

BACKGROUND: Inadequate provision of vitamin B12 during pregnancy is associated with a number of adverse maternal and fetal outcomes. We set out to (1) suggest pregnancy-specific reference ranges for a range of biomarkers of vitamin B12; (2) assess the temporal behaviors of these markers over the course of pregnancy; and (3) test whether any biomarkers, including the genetic marker HIBCH rs291466 strongly associated with MMA measured early in pregnancy could reliably and significantly predict future B12 status within a healthy UK population of pregnant women. MATERIALS AND METHODS: We used existing biobank samples from the placebo arm of the UK Selenium in PRegnancy Intervention (SPRINT) study, to generate biochemical data for serum folate, B12, holotranscobalamin (HoloTC), total homocysteine (tHcy), and MMA, calculate cB12, and genotyped the polymorphism rs291466 in gene HIBCH on a total of n=114 women across trimesters 1-3 of their pregnancy. We performed a series of exploratory cross-sectional and longitudinal analyses to investigate levels at each trimester, suggest references ranges, evaluate changes and correlations between the B12 biomarkers, and assess the predictive capabilities of each biomarker from 12-weeks to 35-weeks of gestation. RESULTS: Significant changes in all vitamin B12 biomarker values were observed over the three trimesters (P < 0.05). Our study shows that cB12 values were largely constant and stable throughout trimester 1 (T1) and T2 (i.e., up to week 20), but declined significantly in T3 (-66% | P < 0.001). Yet, cB12 generally remained within the normal boundaries. We identified pregnancy and trimester-specific reference ranges for each biomarker at each trimester, notably for total serum B12. This marker fell below the recommended cut-offs in 1/3 of the cohort at the third trimester, contrasting other markers (mostly normal). Our multivariate analyses indicated that none of the biomarkers could reliably and accurately predict any other biomarkers than themselves later in pregnancy. Yet, HoloTC seems to be a promising predictor within the limitations of our cohort, constituted of B12-replete individuals. Most notably, cB12 did not significantly predict itself between trimesters. Finally, we show that the HIBCH variant has little predictive power for MMA or cB12 as it does not explain the significant increase in MMA concentrations nor the decline of cB12 throughout pregnancy. CONCLUSION: Trimester-specific reference ranges for biomarkers of vitamin B12 in normal pregnancy are suggested. However, these biomarkers have limited predictive value in identifying mothers at elevated risk of vitamin B12 insufficiency/deficiency during pregnancy.

Histone deacetylase 9 promoter hypomethylation associated with adipocyte dysfunction is a statin-related metabolic effect.

BACKGROUND: Adipogenesis, the process whereby preadipocytes differentiate into mature adipocytes, is crucial for maintaining metabolic homeostasis. Cholesterol-lowering statins increase type 2 diabetes (T2D) risk possibly by affecting adipogenesis and insulin resistance but the (epi)genetic mechanisms involved are unknown. Here, we characterised the effects of statin treatment on adipocyte differentiation using in vitro human preadipocyte cell model to identify putative effective genes. RESULTS: Statin treatment during adipocyte differentiation caused a reduction in key genes involved in adipogenesis, such as ADIPOQ, GLUT4 and ABCG1. Using Illumina's Infinium '850K' Methylation EPIC array, we found a significant hypomethylation of cg14566882, located in the promoter of the histone deacetylase 9 (HDAC9) gene, in response to two types of statins (atorvastatin and mevastatin), which correlates with an increased HDAC9 mRNA expression. We confirmed that HDAC9 is a transcriptional repressor of the cholesterol efflux ABCG1 gene expression, which is epigenetically modified in obesity and prediabetic states. Thus, we assessed the putative impact of ABCG1 knockdown in mimicking the effect of statin in adipogenesis. ABCG1 KD reduced the expression of key genes involved in adipocyte differentiation and decreased insulin signalling and glucose uptake. In human blood cells from two cohorts, ABCG1 expression was impaired in response to statins, confirming that ABCG1 is targeted in vivo by these drugs. CONCLUSIONS: We identified an epigenetic link between adipogenesis and adipose tissue insulin resistance in the context of T2D risk associated with statin use, which has important implications as HDAC9 and ABCG1 are considered potential therapeutic targets for obesity and metabolic diseases.

Comparative Healthcare Research Outcomes of Novel Surgery in prostate cancer (IP4-CHRONOS): A prospective, multi-centre therapeutic phase II parallel Randomised Control Trial.

INTRODUCTION: Focal therapy (FT) targets individual areas of cancer within the prostate, providing oncological control with minimal side-effects. Early evidence demonstrates encouraging short-medium-term outcomes. With no randomized controlled trials (RCT) comparing FT to radical therapies, Comparative Healthcare Research Outcomes of Novel Surgery in prostate cancer (CHRONOS) will compare the cancer control of these two strategies. PATIENTS AND METHODS: CHRONOS is a parallel phase II RCT for patients with clinically significant non-metastatic prostate cancer, dependent upon clinician/patient decision, patients will enrol into either CHRONOS-A or CHRONOS-B. CHRONOS-A will randomize patients to either radical treatment or FT. CHRONOS-B is a multi-arm, multistage RCT comparing focal therapy alone to FT with neoadjuvant agents that might improve the current focal therapy outcomes. An internal pilot will determine the feasibility of, and compliance to, randomization. The proposed definitive study plans to recruit and randomize 1190 patients into CHRONOS-A and 1260 patients into CHRONOS-B. RESULTS: Primary outcome in CHRONOS-A is progression-free survival (transition to salvage local or systemic therapy, development of metastases or prostate-cancer-related mortality) and in CHRONOS-B is failure-free survival (includes the above definition and recurrence of clinically significant prostate cancer after initial FT). Secondary outcomes include adverse events, health economics and functional outcomes measured using validated questionnaires. CHRONOS is powered to assess non-inferiority of FT compared to radical therapy in CHRONOS-A, and superiority of neoadjuvant agents with FT in CHRONOS-B. CONCLUSION: CHRONOS will assess the oncological outcomes after FT compared to radical therapy and whether neoadjuvant treatments improve cancer control following one FT session.

Enhanced Colorimetric Differentiation between Staphylococcus aureus and Pseudomonas aeruginosa Using a Shape-Encoded Sensor Hydrogel.

Herein, we demonstrate a combined fluorescent probe/shape-encoded hydrogel strategy for the fast, sensitive, and selective detection of bacterial species via their characteristic enzymes. A poly(vinyl alcohol) (PVA) hydrogel loaded with the fluorescent probe N,N'-(3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)bis(2,2,3,3,3-pentafluoropropanamide) (ACS-HNE) was designed for the detection of elastase, an enzyme produced by Pseudomonas aeruginosa. Likewise, a chitosan-derived hydrogel was loaded with the fluorescent probe 4-methylumbelliferyl-α-d-glucopyranoside (MUD) by entrapment for the selective detection of α-glucosidase, an enzyme produced by Staphylococcus aureus. For an observation time of 60 min, limits of detection (LODs) of ≤20 nM for elastase and ≤30 pM for α-glucosidase were obtained, which in the latter case is 3 orders of magnitude better than related chitosan systems with covalently coupled substrate. To illustrate the potential utility of these highly sensitive sensor hydrogels as a simple point-of-care test system, shaped hydrogel slabs representing the letters P and S were manufactured to detect P. aeruginosa and S. aureus, respectively. These shapes were shown to provide an additional unique color code under UV illumination corresponding to the characteristic enzyme produced by the corresponding bacteria. This study shows potential for the future development of an effective and simple point-of-care test for the rapid identification of bacterial species that can be operated by nonspecialists.

NUMT Confounding Biases Mitochondrial Heteroplasmy Calls in Favor of the Reference Allele.

Homology between mitochondrial DNA (mtDNA) and nuclear DNA of mitochondrial origin (nuMTs) causes confounding when aligning short sequence reads to the reference human genome, as the true sequence origin cannot be determined. Using a systematic in silico approach, we here report the impact of all potential mitochondrial variants on alignment accuracy and variant calling. A total of 49,707 possible mutations were introduced across the 16,569 bp reference mitochondrial genome (16,569 × 3 alternative alleles), one variant at-at-time. The resulting in silico fragmentation and alignment to the entire reference genome (GRCh38) revealed preferential mapping of mutated mitochondrial fragments to nuclear loci, as variants increased loci similarity to nuMTs, for a total of 807, 362, and 41 variants at 333, 144, and 27 positions when using 100, 150, and 300 bp single-end fragments. We subsequently modeled these affected variants at 50% heteroplasmy and carried out variant calling, observing bias in the reported allele frequencies in favor of the reference allele. Four variants (chrM:6023A, chrM:4456T, chrM:5147A, and chrM:7521A) including a possible hypertension factor, chrM:4456T, caused 100% loss of coverage at the mutated position (with all 100 bp single-end fragments aligning to homologous, nuclear positions instead of chrM), rendering these variants undetectable when aligning to the entire reference genome. Furthermore, four mitochondrial variants reported to be pathogenic were found to cause significant loss of coverage and select haplogroup-defining SNPs were shown to exacerbate the loss of coverage caused by surrounding variants. Increased fragment length and use of paired-end reads both improved alignment accuracy.

A genetic epidemiological study in British adults and older adults shows a high heritability of the combined indicator of vitamin B12 status (cB12) and connects B12 status with utilization of mitochondrial substrates and energy metabolism.

Vitamin B12 deficiency is common among older adults. However, the most commonly used marker of deficiency, total serum vitamin B12 (B12), is not sensitive enough to diagnose true deficiency in a significant proportion of the population. The combined indicator of B12 status (cB12), formulated as a composite score of various biomarkers of vitamin B12 status (which also accounts for low folate status and age) has been shown to offer a more robust and powerful test to diagnose B12 deficiency. There are no epidemiological studies of cB12 variability in older adults. We carried out a twin study to characterize the relative contribution of heritable (h2) and environmental factors to the observed variability in cB12 score in an adult and older adult population (n=378). Furthermore, we tested for association between variability in cB12 and candidate polymorphisms and genes previously associated with B12 biomarker levels characterized in silico the mechanism linking the genetic variants and cB12 variability. We found the variability in cB12 and its constituents to be highly heritable (h2=55%-64%). The single nucleotide polymorphism rs291466 in HIBCH, previously associated with variation in MMA, was significantly associated with cB12 (R2=5%, P=5E-04). Furthermore, variants in MTRR, MMAB and MUT, underlying inborn errors of B12 metabolism, were nominally associated with variation in cB12. Pathway accompanied by expression quantitative trait loci analysis revealed that HIBCH rs291466 influences the concentration of MMA via the valine degradation pathway. Our study provides etiological insight into how B12 deficiency can manifest into impaired mitochondrial function through perturbations in mitochondrial "fuel" usage.

Interaction between plasma homocysteine and the MTHFR c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans.

One-carbon metabolism provides a direct link among dietary folate/vitamin B12 exposure, the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR), and epigenetic regulation of the genome via DNA methylation. Previously, it has been shown that the common c.677C > T polymorphism in MTHFR influences global DNA methylation status through a direct interaction with folate status and (indirectly) with total homocysteine (tHcy) levels. To build on that and other more recent observations that have further highlighted associations among MTHFR c.677C > T, tHcy, and aberrations in DNA methylation, we investigated whether the interaction between mildly elevated plasma tHcy and the c.677C > T polymorphism is associated with site-specific changes in DNA methylation in humans. We used data on plasma tHcy levels, c.677C > T polymorphism, and site-specific DNA methylation levels for a total of 915 white women and 335 men from the TwinsUK registry ( n = 610) and the Rotterdam study ( n = 670). We performed methylome-wide association analyses in each cohort to model the interaction between levels of tHcy and c.677C > T genotypes on DNA methylation ß values. Our meta-analysis identified 13 probes significantly associated with rs1801133 × tHcy levels [false-discovery rate (FDR) < 0.05]. The most significant associations were with a cluster of probes at the AGTRAP-MTHFR-NPPA/B gene locus on chromosome 1 (FDR = 1.3E-04), with additional probes on chromosomes 2, 3, 4, 7, 12, 16, and 19. Our top 2 hits on chromosome 1 were functionally associated with variability in expression of the TNF receptor superfamily member 8 ( TNFRSF8) gene/locus on that chromosome. This is the first study, to our knowledge, to provide a direct link between perturbations in 1-carbon metabolism, through an interaction of tHcy and the activity of MTHFR enzyme on epigenetic regulation of the genome via DNA methylation.-Nash, A. J., Mandaviya, P. R., Dib, M.-J., Uitterlinden, A. G., van Meurs, J., Heil, S. G., Andrew, T., Ahmadi, K. R. Interaction between plasma homocysteine and the MTHFR c.677C>T polymorphism is associated with site-specific changes in DNA methylation in humans.

Association of maternal prenatal smoking GFI1-locus and cardio-metabolic phenotypes in 18,212 adults.

BACKGROUND: DNA methylation at the GFI1-locus has been repeatedly associated with exposure to smoking from the foetal period onwards. We explored whether DNA methylation may be a mechanism that links exposure to maternal prenatal smoking with offspring's adult cardio-metabolic health. METHODS: We meta-analysed the association between DNA methylation at GFI1-locus with maternal prenatal smoking, adult own smoking, and cardio-metabolic phenotypes in 22 population-based studies from Europe, Australia, and USA (n = 18,212). DNA methylation at the GFI1-locus was measured in whole-blood. Multivariable regression models were fitted to examine its association with exposure to prenatal and own adult smoking. DNA methylation levels were analysed in relation to body mass index (BMI), waist circumference (WC), fasting glucose (FG), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), diastolic, and systolic blood pressure (BP). FINDINGS: Lower DNA methylation at three out of eight GFI1-CpGs was associated with exposure to maternal prenatal smoking, whereas, all eight CpGs were associated with adult own smoking. Lower DNA methylation at cg14179389, the strongest maternal prenatal smoking locus, was associated with increased WC and BP when adjusted for sex, age, and adult smoking with Bonferroni-corrected P < 0·012. In contrast, lower DNA methylation at cg09935388, the strongest adult own smoking locus, was associated with decreased BMI, WC, and BP (adjusted 1 × 10-7 < P < 0.01). Similarly, lower DNA methylation at cg12876356, cg18316974, cg09662411, and cg18146737 was associated with decreased BMI and WC (5 × 10-8 < P < 0.001). Lower DNA methylation at all the CpGs was consistently associated with higher TG levels. INTERPRETATION: Epigenetic changes at the GFI1 were linked to smoking exposure in-utero/in-adulthood and robustly associated with cardio-metabolic risk factors. FUND: European Union's Horizon 2020 research and innovation programme under grant agreement no. 633595 DynaHEALTH.

High-Resolution Genetic Maps Identify Multiple Type 2 Diabetes Loci at Regulatory Hotspots in African Americans and Europeans.

Interpretation of results from genome-wide association studies for T2D is challenging. Only very few loci have been replicated in African ancestry populations and the identification of the implicated functional genes remain largely undefined. We used genetic maps that capture detailed linkage disequilibrium information in European and African Americans and applied these to large T2D case-control samples in order to estimate locations for putative functional variants in both populations. Replicated T2D locations were tested for evidence of being regulatory hotspots using adipose expression. We validated a sample of our co-location intervals using next generation sequencing and functional annotation, including enhancers, transcription, and chromatin modifications. We identified 111 additional disease-susceptibility locations, 93 of which are cosmopolitan and 18 of which are European specific. We show that many previously known signals are also risk loci in African Americans. The majority of the disease locations appear to confer risk of T2D via the regulation of expression levels for a large number (266) of cis-regulated genes, the majority of which are not the nearest genes to the disease loci. Sequencing three cosmopolitan locations provided candidate functional variants that precisely co-locate with cell-specific chromatin domains and pancreatic islet enhancers. These variants have large effect sizes and are common across populations. Results show that disease-associated loci in different populations, gene expression, and cell-specific regulatory annotation can be effectively integrated by localizing these effects on high-resolution genetic maps. The cis-regulated genes provide insights into the complex molecular pathways involved and can be used as targets for sequencing and functional molecular studies.

A systematic review of randomised controlled trials in rheumatoid arthritis: the reporting and handling of missing data in composite outcomes.

BACKGROUND: Most reported outcome measures in rheumatoid arthritis (RA) trials are composite, whose components comprise single measures that are combined into one outcome. The aims of this review were to assess the range of missing data rates in primary composite outcomes and to document the current practice for handling and reporting missing data in published RA trials compared to the Consolidated Standards of Reporting Trials (CONSORT) recommendations. METHODS: A systematic search for randomised controlled trials was conducted for RA trials published between 2008 and 2013 in four rheumatology and four high impact general medical journals. RESULTS: A total of 51 trials with a composite primary outcome were identified, of which 38 (75 %) used the binary American College of Rheumatology responder index and 13 (25 %) used the Disease Activity Score for 28 joints (DAS28). Forty-four trials (86 %) reported on an intention-to-treat analysis population, while 7 trials (14 %) analysed according to a modified intention-to-treat population. Missing data rates for the primary composite outcome ranged from 2-53 % and were above 30 % in 9 trials, 20-30 % in 11 trials, 10-20 % in 18 trials and below 10 % in 13 trials. Thirty-eight trials (75 %) used non-responder imputation and 10 (20 %) used last observation carried forward to impute missing composite outcome data at the primary time point. The rate of dropout was on average 61 % times higher in the placebo group compared to the treatment group in the 34 placebo controlled trials (relative rate 1.61, 95 % CI: 1.29, 2.02). Thirty-seven trials (73 %) did not report the use of sensitivity analyses to assess the handling of missing data in the primary analysis as recommended by CONSORT guidelines. CONCLUSIONS: This review highlights an improvement in rheumatology trial practice since the revision of CONSORT guidelines, in terms of power calculation and participant's flow diagram. However, there is a need to improve the handling and reporting of missing composite outcome data and their components in RA trials. In particular, sensitivity analyses need to be more widely used in RA trials because imputation is widespread and generally uses single imputation methods, and in this area the missing data rates are commonly differentially higher in the placebo group.

ABCC5 transporter is a novel type 2 diabetes susceptibility gene in European and African American populations.

Numerous functional studies have implicated PARL in relation to type 2 diabetes (T2D). We hypothesised that conflicting human association studies may be due to neighbouring causal variants being in linkage disequilibrium (LD) with PARL. We conducted a comprehensive candidate gene study of the extended LD genomic region that includes PARL and transporter ABCC5 using three data sets (two European and one African American), in relation to healthy glycaemic variation, visceral fat accumulation and T2D disease. We observed no evidence for previously reported T2D association with Val262Leu or PARL using array and fine-map genomic and expression data. By contrast, we observed strong evidence of T2D association with ABCC5 (intron 26) for European and African American samples (P = 3E-07) and with ABCC5 adipose expression in Europeans [odds ratio (OR) = 3.8, P = 2E-04]. The genomic location estimate for the ABCC5 functional variant, associated with all phenotypes and expression data (P = 1E-11), was identical for all samples (at Chr3q 185,136 kb B36), indicating that the risk variant is an expression quantitative trait locus (eQTL) with increased expression conferring risk of disease. That the association with T2D is observed in populations of disparate ancestry suggests the variant is a ubiquitous risk factor for T2D.