Estimating the health impact of nicotine exposure by dissecting the effects of nicotine versus non-nicotine constituents of tobacco smoke: A multivariable Mendelian randomisation study.

The detrimental health effects of smoking are well-known, but the impact of regular nicotine use without exposure to the other constituents of tobacco is less clear. Given the increasing daily use of alternative nicotine delivery systems, such as e-cigarettes, it is increasingly important to understand and separate the effects of nicotine use from the impact of tobacco smoke exposure. Using a multivariable Mendelian randomisation framework, we explored the direct effects of nicotine compared with the non-nicotine constituents of tobacco smoke on health outcomes (lung cancer, chronic obstructive pulmonary disease [COPD], forced expiratory volume in one second [FEV-1], forced vital capacity [FVC], coronary heart disease [CHD], and heart rate [HR]). We used Genome-Wide Association Study (GWAS) summary statistics from Buchwald and colleagues, the GWAS and Sequencing Consortium of Alcohol and Nicotine, the International Lung Cancer Consortium, and UK Biobank. Increased nicotine metabolism increased the risk of COPD, lung cancer, and lung function in the univariable analysis. However, when accounting for smoking heaviness in the multivariable analysis, we found that increased nicotine metabolite ratio (indicative of decreased nicotine exposure per cigarette smoked) decreases heart rate (b = -0.30, 95% CI -0.50 to -0.10) and lung function (b = -33.33, 95% CI -41.76 to -24.90). There was no clear evidence of an effect on the remaining outcomes. The results suggest that these smoking-related outcomes are not due to nicotine exposure but are caused by the other components of tobacco smoke; however, there are multiple potential sources of bias, and the results should be triangulated using evidence from a range of methodologies.

Epigenetic Regulation of F2RL3 Associates With Myocardial Infarction and Platelet Function.

BACKGROUND: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. METHODS: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3, and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. RESULTS: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2-2.4, P=0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. CONCLUSIONS: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Association Between MR Elastography Liver Stiffness and Histologic Liver Fibrosis in Children and Young Adults With Autoimmune Liver Disease.

BACKGROUND. Liver fibrosis is an important clinical endpoint of the progression of autoimmune liver disease (AILD); its monitoring would benefit from noninvasive imaging tools. OBJECTIVE. The purpose of this study was to assess the relationship between MR elastography (MRE) liver stiffness measurements and histologic liver fibrosis, as well as to evaluate the performance of MRE and biochemical-based clinical markers for stratifying histologic liver fibrosis severity, in children and young adults with AILD. METHODS. This retrospective study used an existing institutional registry of children and young adults diagnosed with AILD (primary sclerosing cholangitis [PSC], autoimmune sclerosing cholangitis [ASC], or autoimmune hepatitis [AIH]). The registry was searched to identify patients who underwent both a research abdominal 1.5-T MRI examination that included liver MRE (performed for registry enrollment) and a clinically indicated liver biopsy within 6 months of that examination. MRE used a 2D gradient-recalled echo sequence. One analyst measured mean liver shear stiffness (in kilopascals) for each examination. Laboratory markers of liver fibrosis (aspartate aminotransferase-to-platelet ratio index [APRI] and fibrosis-4 [FIB-4] score) were recorded. For investigational purposes, one pathologist, blinded to clinical and MRI data, determined histologic Metavir liver fibrosis stage. The Spearman rank order correlation coefficient was calculated between MRE liver stiffness and Metavir liver fibrosis stage. ROC analysis was used to evaluate diagnostic performance for identifying advanced fibrosis (i.e., differentiating Metavir F0-F1 from F2-F4 fibrosis), and sensitivity and specificity were calculated using the Youden index. RESULTS. The study included 46 patients (median age, 16.6 years [IQR, 13.7-17.8 years]; 20 female patients, 26 male patients); 12 had PSC, 10 had ASC, and 24 had AIH. Median MRE liver stiffness was 2.9 kPa (IQR, 2.2-4.0 kPa). MRE liver stiffness and Meta-vir fibrosis stage showed strong positive correlation (ρ = 0.68). For identifying advanced liver fibrosis, MRE liver stiffness had an AUC of 0.81, with sensitivity of 65.4% and specificity of 90.0%; APRI had an AUC of 0.72, with sensitivity of 64.0% and specificity of 80.0%; and FIB-4 score had an AUC of 0.71, with sensitivity of 60.0% and specificity of 85.0%. CONCLUSION. MRE liver stiffness measurements were associated with histologic liver fibrosis severity. CLINICAL IMPACT. The findings support a role for MRE in noninvasive monitoring of liver stiffness, a surrogate for fibrosis, in children and young adults with AILD. TRIAL REGISTRATION. ClinicalTrials.gov NCT03175471.

Long-term cardiometabolic health in people born after assisted reproductive technology: a multi-cohort analysis.

AIMS: To examine associations of assisted reproductive technology (ART) conception (vs. natural conception: NC) with offspring cardiometabolic health outcomes and whether these differ with age. METHODS AND RESULTS: Differences in systolic (SBP) and diastolic blood pressure (DBP), heart rate (HR), lipids, and hyperglycaemic/insulin resistance markers were examined using multiple linear regression models in 14 population-based birth cohorts in Europe, Australia, and Singapore, and results were combined using meta-analysis. Change in cardiometabolic outcomes from 2 to 26 years was examined using trajectory modelling of four cohorts with repeated measures. 35 938 (654 ART) offspring were included in the meta-analysis. Mean age ranged from 13 months to 27.4 years but was <10 years in 11/14 cohorts. Meta-analysis found no statistical difference (ART minus NC) in SBP (-0.53 mmHg; 95% CI:-1.59 to 0.53), DBP (-0.24 mmHg; -0.83 to 0.35), or HR (0.02 beat/min; -0.91 to 0.94). Total cholesterol (2.59%; 0.10-5.07), HDL cholesterol (4.16%; 2.52-5.81), LDL cholesterol (4.95%; 0.47-9.43) were statistically significantly higher in ART-conceived vs. NC offspring. No statistical difference was seen for triglycerides (TG), glucose, insulin, and glycated haemoglobin. Long-term follow-up of 17 244 (244 ART) births identified statistically significant associations between ART and lower predicted SBP/DBP in childhood, and subtle trajectories to higher SBP and TG in young adulthood; however, most differences were not statistically significant. CONCLUSION: These findings of small and statistically non-significant differences in offspring cardiometabolic outcomes should reassure people receiving ART. Longer-term follow-up is warranted to investigate changes over adulthood in the risks of hypertension, dyslipidaemia, and preclinical and clinical cardiovascular disease.

Corrected T1 Mapping in Children and Young Adults With Autoimmune Liver Disease: Correlation With Histology.

The purpose of this study was to assess relationships between liver-corrected T1 (cT1) values (adjusted for T2* effect, MRI system manufacturer, and field strength) and histologic inflammation and fibrosis in 35 participants (15 women and girls, 20 boys and men; median age, 16.0 years) with autoimmune liver disease. At multivariable analysis, inflammation score (ß = 15.5) and sex (ß = 56.0 [female]) were independent predictors of cT1, and fibrosis score (ß = 32.3) and age (ß = 5.5) were independent predictors of cT1 IQR. Liver T1 may have relevance for assessing liver inflammatory activity and fibrosis stage.

Association of genetic liability to smoking initiation with e-cigarette use in young adults: A cohort study.

BACKGROUND: Tobacco smoking and e-cigarette use are strongly associated, but it is currently unclear whether this association is causal, or due to shared factors that influence both behaviours such as a shared genetic liability. The aim of this study was to investigate whether polygenic risk scores (PRS) for smoking initiation are associated with ever use of e-cigarettes. METHODS AND FINDINGS: Smoking initiation PRS were calculated for young adults (N = 7,859, mean age = 24 years, 51% male) of European ancestry in the Avon Longitudinal Study of Parents and Children, a prospective birth cohort study initiated in 1991. PRS were calculated using the GWAS & Sequencing Consortium of Alcohol and Nicotine use (GSCAN) summary statistics. Five thresholds ranging from 5 × 10-8 to 0.5 were used to calculate 5 PRS for each individual. Using logistic regression, we investigated the association between smoking initiation PRS and the main outcome, self-reported e-cigarette use (n = 2,894, measured between 2016 and 2017), as well as self-reported smoking initiation and 8 negative control outcomes (socioeconomic position at birth, externalising disorders in childhood, and risk-taking in young adulthood). A total of 878 young adults (30%) had ever used e-cigarettes at 24 years, and 150 (5%) were regular e-cigarette users at 24 years. We observed positive associations of similar magnitude between smoking initiation PRS (created using the p < 5 × 10-8 threshold) and both smoking initiation (odds ratio (OR) = 1.29, 95% CI 1.19 to 1.39, p < 0.001) and ever e-cigarette use (OR = 1.24, 95% CI 1.14 to 1.34, p < 0.001) by the age of 24 years, indicating that a genetic predisposition to smoking initiation is associated with an increased risk of using e-cigarettes. At lower p-value thresholds, we observed an association between smoking initiation PRS and ever e-cigarette use among never smokers. We also found evidence of associations between smoking initiation PRS and some negative control outcomes, particularly when less stringent p-value thresholds were used to create the PRS, but also at the strictest threshold (e.g., gambling, number of sexual partners, conduct disorder at 7 years, and parental socioeconomic position at birth). However, this study is limited by the relatively small sample size and potential for collider bias. CONCLUSIONS: Our results indicate that there may be a shared genetic aetiology between smoking and e-cigarette use, and also with socioeconomic position, externalising disorders in childhood, and risky behaviour more generally. This indicates that there may be a common genetic vulnerability to both smoking and e-cigarette use, which may reflect a broad risk-taking phenotype.

Investigating the impact of cigarette smoking behaviours on DNA methylation patterns in adolescence.

Smoking usually begins in adolescence, and early onset of smoking has been linked to increased risk of later life disease. There is a need to better understand the biological impact of cigarette smoking behaviours in adolescence. DNA methylation profiles related to smoking behaviours and cessation in adulthood have been previously identified, but alterations arising from smoking initiation have not been thoroughly investigated. We aimed to investigate DNA methylation in the Avon Longitudinal Study of Parents and Children in relation to (1) different smoking measures, (2) time since smoking initiation and frequency of smoke exposure and (3) latent classes of smoking behaviour. Using 2620 CpG sites previously associated with cigarette smoking, we investigated DNA methylation change in relation to own smoking measures, smoke exposure duration and frequency, and using longitudinal latent class analysis of different smoking behaviour patterns in 968 adolescents. Eleven CpG sites located in seven gene regions were differentially methylated in relation to smoking in adolescence. While only AHRR (cg05575921) showed a robust pattern of methylation in relation to weekly smoking, several CpGs showed differences in methylation among individuals who had tried smoking compared with non-smokers. In relation to smoke exposure duration and frequency, cg05575921 showed a strong dose-response relationship, while there was evidence for more immediate methylation change at other sites. Our findings illustrate the impact of cigarette smoking behaviours on DNA methylation at some smoking-responsive CpG sites, even among individuals with a short smoking history.

The effect of body mass index on smoking behaviour and nicotine metabolism: a Mendelian randomization study.

Given clear evidence that smoking lowers weight, it is possible that individuals with higher body mass index (BMI) smoke in order to lose or maintain their weight. We performed Mendelian randomization (MR) analyses of the effects of BMI on smoking behaviour in UK Biobank and the Tobacco and Genetics Consortium genome-wide association study (GWAS), on cotinine levels and nicotine metabolite ratio (NMR) in published GWAS and on DNA methylation in the Avon Longitudinal Study of Parents and Children. Our results indicate that higher BMI causally influences lifetime smoking, smoking initiation, smoking heaviness and also DNA methylation at the aryl-hydrocarbon receptor repressor (AHRR) locus, but we do not see evidence for an effect on smoking cessation. While there is no strong evidence that BMI causally influences cotinine levels, suggestive evidence for a negative causal influence on NMR may explain this. There is a causal effect of BMI on smoking, but the relationship is likely to be complex due to opposing effects on behaviour and metabolism.

Mendelian randomisation for mediation analysis: current methods and challenges for implementation.

Mediation analysis seeks to explain the pathway(s) through which an exposure affects an outcome. Traditional, non-instrumental variable methods for mediation analysis experience a number of methodological difficulties, including bias due to confounding between an exposure, mediator and outcome and measurement error. Mendelian randomisation (MR) can be used to improve causal inference for mediation analysis. We describe two approaches that can be used for estimating mediation analysis with MR: multivariable MR (MVMR) and two-step MR. We outline the approaches and provide code to demonstrate how they can be used in mediation analysis. We review issues that can affect analyses, including confounding, measurement error, weak instrument bias, interactions between exposures and mediators and analysis of multiple mediators. Description of the methods is supplemented by simulated and real data examples. Although MR relies on large sample sizes and strong assumptions, such as having strong instruments and no horizontally pleiotropic pathways, our simulations demonstrate that these methods are unaffected by confounders of the exposure or mediator and the outcome and non-differential measurement error of the exposure or mediator. Both MVMR and two-step MR can be implemented in both individual-level MR and summary data MR. MR mediation methods require different assumptions to be made, compared with non-instrumental variable mediation methods. Where these assumptions are more plausible, MR can be used to improve causal inference in mediation analysis.

Metabolic characterisation of disturbances in the APOC3/triglyceride-rich lipoprotein pathway through sample-based recall by genotype.

INTRODUCTION: High plasma triacylglyceride levels are known to be associated with increased risk of atherosclerotic cardiovascular disease. Apolipoprotein C-III (apoC-III) is a key regulator of plasma triacylglyceride levels and is associated with hypertriglyceridemia via a number of pathways. There is consistent evidence for an association of cardiovascular events with blood apoC-III level, with support from human genetic studies of APOC3 variants. As such, apoC-III has been recognised as a potential therapeutic target for patients with severe hypertriglyceridaemia with one of the most promising apoC-III-targeting drugs, volanesorsen, having recently progressed through Phase III trials. OBJECTIVES: To exploit a rare loss of function variant in APOC3 (rs138326449) to characterise the potential long-term treatment effects of apoC-III targeting interventions on the metabolome. METHODS: In a recall-by-genotype study, 115 plasma samples were analysed by UHPLC-MS to acquire non-targeted metabolomics data. The study included samples from 57 adolescents and 33 adults. Overall, 12 985 metabolic features were tested for an association with APOC3 genotype. RESULTS: 161 uniquely annotated metabolites were found to be associated with rs138326449(APOC3). The highest proportion of associated metabolites belonged to the acyl-acyl glycerophospholipid and triacylglyceride metabolite classes. In addition to the anticipated (on-target) reduction of metabolites in the triacylglyceride and related classes, carriers of the rare variant exhibited previously unreported increases in levels of a number of metabolites from the acyl-alkyl glycerophospholipid class. CONCLUSION: Overall, our results suggest that therapies targeting apoC-III may potentially achieve a broad shift in lipid profile that favours better metabolic health.

Prescribing Prevalence, Effectiveness, and Mental Health Safety of Smoking Cessation Medicines in Patients With Mental Disorders.

OBJECTIVE: We conducted a prospective cohort study of the Clinical Practice Research Database to estimate rates of varenicline and nicotine replacement therapy (NRT) prescribing and the relative effects on smoking cessation, and mental health. METHODS: We used multivariable logistic regression, propensity score matched regression, and instrumental variable analysis. Exposure was varenicline or NRT prescription. Mental disorders were bipolar, depression, neurotic disorder, schizophrenia, or prescriptions of antidepressants, antipsychotics, hypnotics/anxiolytics, mood stabilizers. Outcomes were smoking cessation, and incidence of neurotic disorder, depression, prescription of antidepressants, or hypnotics/anxiolytics. Follow-ups were 3, 6, and 9 months, and at 1, 2, and 4 years. RESULTS: In all patients, NRT and varenicline prescribing declined during the study period. Seventy-eight thousand four hundred fifty-seven smokers with mental disorders aged ≥18 years were prescribed NRT (N = 59 340) or varenicline (N = 19 117) from September 1, 2006 to December 31, 2015. Compared with smokers without mental disorders, smokers with mental disorders had 31% (95% CI: 29% to 33%) lower odds of being prescribed varenicline relative to NRT, but had 19% (95% CI: 15% to 24%) greater odds of quitting at 2 years when prescribed varenicline relative to NRT. Overall, varenicline was associated with decreased or similar odds of worse mental health outcomes than NRT in patients both with and without mental disorders, although there was some variation when analyses were stratified by mental disorder subgroup. CONCLUSIONS: Smoking cessation medication prescribing may be declining in primary care. Varenicline was more effective than NRT for smoking cessation in patients with mental disorders and there is not clear consistent evidence that varenicline is adversely associated with poorer mental health outcomes. IMPLICATIONS: Patients with mental disorders were less likely to be prescribed varenicline than NRT. We triangulated results from three analytical techniques. We found that varenicline was more effective than NRT for smoking cessation in patients with mental disorders. Varenicline was generally associated with similar or decreased odds of poorer mental health outcomes (ie, improvements in mental health) when compared with NRT. We report these findings cautiously as our data are observational and are at risk of confounding.

Is e-cigarette use in non-smoking young adults associated with later smoking? A systematic review and meta-analysis.

OBJECTIVE: The aim of this review was to investigate whether e-cigarette use compared with non-use in young non-smokers is associated with subsequent cigarette smoking. DATA SOURCES: PubMed, Embase, Web of Science, Wiley Cochrane Library databases, and the 2018 Society for Research on Nicotine and Tobacco and Society for Behavioural Medicine conference abstracts. STUDY SELECTION: All studies of young people (up to age 30 years) with a measure of e-cigarette use prior to smoking and an outcome measure of smoking where an OR could be calculated were included (excluding reviews and animal studies). DATA EXTRACTION: Independent extraction was completed by multiple authors using a preprepared extraction form. DATA SYNTHESIS: Of 9199 results, 17 studies were included in the meta-analysis. There was strong evidence for an association between e-cigarette use among non-smokers and later smoking (OR: 4.59, 95% CI: 3.60 to 5.85) when the results were meta-analysed in a random-effects model. However, there was high heterogeneity (I2 =88%). CONCLUSIONS: Although the association between e-cigarette use among non-smokers and subsequent smoking appears strong, the available evidence is limited by the reliance on self-report measures of smoking history without biochemical verification. None of the studies included negative controls which would provide stronger evidence for whether the association may be causal. Much of the evidence also failed to consider the nicotine content of e-liquids used by non-smokers meaning it is difficult to make conclusions about whether nicotine is the mechanism driving this association.

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.

Evidence for Genetic Correlations and Bidirectional, Causal Effects Between Smoking and Sleep Behaviors.

INTRODUCTION: Cigarette smokers are at increased risk of poor sleep behaviors. However, it is largely unknown whether these associations are due to shared (genetic) risk factors and/or causal effects (which may be bidirectional). METHODS: We obtained summary-level data of genome-wide association studies of smoking (smoking initiation [n = 74 035], cigarettes per day [n = 38 181], and smoking cessation [n = 41 278]) and sleep behaviors (sleep duration and chronotype, or "morningness" [n = 128 266] and insomnia [n = 113 006]). Using linkage disequilibrium (LD) score regression, we calculated genetic correlations between smoking and sleep behaviors. To investigate causal effects, we employed Mendelian randomization (MR), both with summary-level data and individual-level data (n = 333 581 UK Biobank participants). For MR with summary-level data, individual genetic variants were combined with inverse variance-weighted meta-analysis, weighted median regression, MR-Robust Adjusted Profile Score, and MR Egger methods. RESULTS: We found negative genetic correlations between smoking initiation and sleep duration (rg = -.14, 95% CI = -0.26 to -0.01) and smoking cessation and chronotype (rg = -.18, 95% CI = -0.31 to -0.06), and positive genetic correlations between smoking initiation and insomnia (rg = .27, 95% CI = 0.06 to 0.49) and cigarettes per day and insomnia (rg = .15, 95% CI = 0.01 to 0.28). MR provided strong evidence that smoking more cigarettes causally decreases the odds of being a morning person, (RAPS) and weak evidence that insomnia causally increases smoking heaviness and decreases smoking cessation odds. CONCLUSIONS: Smoking and sleep behaviors show moderate genetic correlation. Heavier smoking seems to causally affect circadian rhythm and there is some indication that insomnia increases smoking heaviness and hampers cessation. Our findings point to sleep as a potentially interesting smoking treatment target. IMPLICATIONS: Using LD score regression, we found evidence that smoking and different sleep behaviors (sleep duration, chronotype (morningness), and insomnia) are moderately genetically correlated-genetic variants associated with less or poorer sleep also increased the odds of smoking (more heavily). MR analyses suggested that heavier smoking causally affects circadian rhythm (decreasing the odds of being a morning person) and there was some indication that insomnia increases smoking heaviness and hampers smoking cessation. Our findings indicate a complex, bidirectional relationship between smoking and sleep behaviors and point to sleep as a potentially interesting smoking treatment target.

Diagnostic performance of quantitative magnetic resonance imaging biomarkers for predicting portal hypertension in children and young adults with autoimmune liver disease.

BACKGROUND: Primary sclerosing cholangitis, autoimmune hepatitis and autoimmune sclerosing cholangitis are forms of chronic, progressive autoimmune liver disease (AILD) that can affect the pediatric population. OBJECTIVE: To determine whether quantitative MRI- and laboratory-based biomarkers are associated with conventional imaging findings of portal hypertension (radiologic portal hypertension) in children and young adults with AILD. MATERIALS AND METHODS: Forty-four patients with AILD enrolled in an institutional registry underwent a research abdominal MRI examination at 1.5 tesla (T). Five quantitative MRI techniques were performed: liver MR elastography, spleen MR elastography, liver iron-corrected T1 mapping, liver T2 mapping, and liver diffusion-weighted imaging (DWI, quantified as apparent diffusion coefficients). Two anatomical sequences were used to document splenomegaly, varices and ascites. We calculated aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores - laboratory-based biomarkers of liver fibrosis. We used receiver operating characteristic (ROC) curve analyses to establish the diagnostic performance of quantitative MRI and laboratory biomarkers for indicating the presence of radiologic portal hypertension. RESULTS: Twenty-three (52%) patients were male; mean age was 15.2±4.0 years. Thirteen (30%) patients had radiologic portal hypertension. Liver and spleen stiffness demonstrated the greatest diagnostic performance for indicating the presence of portal hypertension (area-under-the-ROC-curve [AUROC]=0.98 and 0.96, respectively). The APRI and FIB-4 scores also demonstrated good diagnostic performance (AUROC=0.87 and 0.88, respectively). CONCLUSION: MRI-derived measures of liver and spleen stiffness as well as laboratory-based APRI and FIB-4 scores are highly associated with imaging findings of portal hypertension in children and young adults with AILD and thus might be useful for predicting portal hypertension impending onset and directing personalized patient management.

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin ß1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

Investigating genetic correlations and causal effects between caffeine consumption and sleep behaviours.

Observationally, higher caffeine consumption is associated with poorer sleep and insomnia. We investigated whether these associations are a result of shared genetic risk factors and/or (possibly bidirectional) causal effects. Summary-level data were available from genome-wide association studies on caffeine intake (n = 91 462), plasma caffeine and caffeine metabolic rate (n = 9876), sleep duration and chronotype (being a "morning" versus an "evening" person) (n = 128 266), and insomnia complaints (n = 113 006). First, genetic correlations were calculated, reflecting the extent to which genetic variants influencing caffeine consumption and those influencing sleep overlap. Next, causal effects were estimated with bidirectional, two-sample Mendelian randomization. This approach utilizes the genetic variants most robustly associated with an exposure variable as an "instrument" to test causal effects. Estimates from individual variants were combined using inverse-variance weighted meta-analysis, weighted median regression and MR-Egger regression. We found no clear evidence for a genetic correlation between caffeine intake and sleep duration (rg = 0.000, p = .998), chronotype (rg = 0.086, p = .192) or insomnia complaints (rg = -0.034, p = .700). For plasma caffeine and caffeine metabolic rate, genetic correlations could not be calculated because of the small sample size. Mendelian randomization did not support causal effects of caffeine intake on sleep, or vice versa. There was weak evidence that higher plasma caffeine levels causally decrease the odds of being a morning person. Although caffeine may acutely affect sleep when taken shortly before bedtime, our findings suggest that a sustained pattern of high caffeine consumption is more likely to be associated with poorer sleep through shared environmental factors. Future research should identify such environments, which could aid the development of interventions to improve sleep.

Investigating the possible causal role of coffee consumption with prostate cancer risk and progression using Mendelian randomization analysis.

Coffee consumption has been shown in some studies to be associated with lower risk of prostate cancer. However, it is unclear if this association is causal or due to confounding or reverse causality. We conducted a Mendelian randomisation analysis to investigate the causal effects of coffee consumption on prostate cancer risk and progression. We used two genetic variants robustly associated with caffeine intake (rs4410790 and rs2472297) as proxies for coffee consumption in a sample of 46,687 men of European ancestry from 25 studies in the PRACTICAL consortium. Associations between genetic variants and prostate cancer case status, stage and grade were assessed by logistic regression and with all-cause and prostate cancer-specific mortality using Cox proportional hazards regression. There was no clear evidence that a genetic risk score combining rs4410790 and rs2472297 was associated with prostate cancer risk (OR per additional coffee increasing allele: 1.01, 95% CI: 0.98,1.03) or having high-grade compared to low-grade disease (OR: 1.01, 95% CI: 0.97,1.04). There was some evidence that the genetic risk score was associated with higher odds of having nonlocalised compared to localised stage disease (OR: 1.03, 95% CI: 1.01, 1.06). Amongst men with prostate cancer, there was no clear association between the genetic risk score and all-cause mortality (HR: 1.00, 95% CI: 0.97,1.04) or prostate cancer-specific mortality (HR: 1.03, 95% CI: 0.98,1.08). These results, which should have less bias from confounding than observational estimates, are not consistent with a substantial effect of coffee consumption on reducing prostate cancer incidence or progression.

Pharmacological inhibition of apical sodium-dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice.

UNLABELLED: Deficiency of multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leads to excretion of low-phospholipid "toxic" bile causing progressive cholestasis. We hypothesize that pharmacological inhibition of the ileal, apical sodium-dependent bile acid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2(-/-) mice. Thirty-day-old, female mdr2(-/-) mice were fed high-fat chow containing 0.006% SC-435, a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/day of compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry. Compared with untreated mdr2(-/-) mice, SC-435 treatment for 14 days increased fecal BA excretion by 8-fold, lowered total BA concentration in liver by 65%, reduced total BA and individual hydrophobic BA concentrations in serum by >98%, and decreased plasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levels by 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved, and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenic gene expression. Biliary BA concentrations significantly decreased and phospholipids remained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratio in treated mice corrected toward a ratio of 0.28 found in wild-type mice, indicating decreased bile toxicity. Hepatic RNA sequencing studies revealed up-regulation of putative anti-inflammatory and antifibrogenic genes, including Ppara and Igf1, and down-regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated in leukocyte recruitment. Flow cytometric analysis revealed significant reduction of frequencies of hepatic CD11b(+) F4/80(+) Kupffer cells and CD11b(+) Gr1(+) neutrophils, accompanied by expansion of anti-inflammatory Ly6C(-) monocytes in treated mdr2(-/-) mice. CONCLUSION: Inhibition of ASBT reduces BA pool size and retention of hydrophobic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic transcriptome, attenuates recruitment of leukocytes, and abrogates progression of murine sclerosing cholangitis.