Systematic evaluation of cell-type deconvolution pipelines for sequencing-based bulk DNA methylomes.

DNA methylation analysis by sequencing is becoming increasingly popular, yielding methylomes at single-base pair and single-molecule resolution. It has tremendous potential for cell-type heterogeneity analysis using intrinsic read-level information. Although diverse deconvolution methods were developed to infer cell-type composition based on bulk sequencing-based methylomes, systematic evaluation has not been performed yet. Here, we thoroughly benchmark six previously published methods: Bayesian epiallele detection, DXM, PRISM, csmFinder+coMethy, ClubCpG and MethylPurify, together with two array-based methods, MeDeCom and Houseman, as a comparison group. Sequencing-based deconvolution methods consist of two main steps, informative region selection and cell-type composition estimation, thus each was individually assessed. With this elaborate evaluation, we aimed to establish which method achieves the highest performance in different scenarios of synthetic bulk samples. We found that cell-type deconvolution performance is influenced by different factors depending on the number of cell types within the mixture. Finally, we propose a best-practice deconvolution strategy for sequencing data and point out limitations that need to be handled. Array-based methods-both reference-based and reference-free-generally outperformed sequencing-based methods, despite the absence of read-level information. This implies that the current sequencing-based methods still struggle with correctly identifying cell-type-specific signals and eliminating confounding methylation patterns, which needs to be handled in future studies.

AllergoOncology: Biomarkers and refined classification for research in the allergy and glioma nexus-A joint EAACI-EANO position paper.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Effect of Chitosan on the Number of Streptococcus mutans in Saliva: A Meta-Analysis and Systematic Review.

We conducted a meta-analysis and systematic review to investigate the efficacy of chitosan-containing chewing gums, and to test their inhibitory effects on Streptococcus mutans. The systematic search was performed in three databases (Cochrane Library, EMBASE, and PubMed) and included English-language randomized-controlled trials to compare the efficacy of chitosan in reducing the number of S. mutans. To assess the certainty of evidence, the GRADE tool was used. Mean differences were calculated with a 95% confidence interval for one outcome: bacterial counts in CFU/mL. The protocol of the study was registered on PROSPERO, registration number CRD42022365006. Articles were downloaded (n = 6758) from EMBASE (n = 2255), PubMed (n = 1516), and Cochrane (n = 2987). After the selection process, a total of four articles were included in the qualitative synthesis and three in the quantitative synthesis. Our results show that chitosan reduced the number of bacteria. The difference in mean quantity was -4.68 × 105. The interval of the random-effects model was [-2.15 × 106; 1.21 × 106] and the prediction interval was [1.03 × 107; 9.40 × 106]. The I2 value was 98% (p = 0.35), which indicates a high degree of heterogeneity. Chitosan has some antibacterial effects when used as a component of chewing gum, but further studies are needed. It can be a promising antimicrobial agent for prevention.

Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.

OBJECTIVE: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options. DESIGN: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling. RESULTS: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin. CONCLUSION: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

Methrix: an R/Bioconductor package for systematic aggregation and analysis of bisulfite sequencing data.

MOTIVATION: Whole-genome bisulfite sequencing (WGBS) measures DNA methylation at base pair resolution resulting in large bedGraph like coverage files. Current options for processing such files are hindered by discrepancies in file format specification, speed, and memory requirements. RESULTS: We developed methrix, an R package, which provides a toolset for systematic analysis of large datasets. Core functionality of the package includes a comprehensive bedGraph or similar tab-separated text file reader-which summarizes methylation calls based on annotated reference indices, infers and collapses strands and handles uncovered reference CpG sites while facilitating a flexible input file format specification. Additional optimized functions for quality control filtering, subsetting and visualization allow user-friendly and effective processing of WGBS results. Easy integration with tools for differentially methylated region (DMR) calling and annotation further eases the analysis of genome-wide methylation data. Overall, methrix enriches established WGBS workflows by bringing together computational efficiency and versatile functionality. AVAILABILITY AND IMPLEMENTATION: Methrix is implemented as an R package, made available under MIT license at https://github.com/CompEpigen/methrix and can be installed from the Bioconductor repository. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Astrocyte Ca2+ Waves and Subsequent Non-Synchronized Ca2+ Oscillations Coincide with Arteriole Diameter Changes in Response to Spreading Depolarization.

Spreading depolarization (SD) is a wave of mass depolarization that causes profound perfusion changes in acute cerebrovascular diseases. Although the astrocyte response is secondary to the neuronal depolarization with SD, it remains to be explored how glial activity is altered after the passage of SD. Here, we describe post-SD high frequency astrocyte Ca2+ oscillations in the mouse somatosensory cortex. The intracellular Ca2+ changes of SR101 labeled astrocytes and the SD-related arteriole diameter variations were simultaneously visualized by multiphoton microscopy in anesthetized mice. Post-SD astrocyte Ca2+ oscillations were identified as Ca2+ events non-synchronized among astrocytes in the field of view. Ca2+ oscillations occurred minutes after the Ca2+ wave of SD. Furthermore, fewer astrocytes were involved in Ca2+ oscillations at a given time, compared to Ca2+ waves, engaging all astrocytes in the field of view simultaneously. Finally, our data confirm that astrocyte Ca2+ waves coincide with arteriolar constriction, while post-SD Ca2+ oscillations occur with the peak of the SD-related vasodilation. This is the first in vivo study to present the post-SD astrocyte Ca2+ oscillations. Our results provide novel insight into the spatio-temporal correlation between glial reactivity and cerebral arteriole diameter changes behind the SD wavefront.

ERp57 is protective against mutant SOD1-induced cellular pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.

Integrative Analysis Defines Distinct Prognostic Subgroups of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. It is defined by cholangiocytic differentiation and has poor prognosis. Recently, epigenetic processes have been shown to play an important role in cholangiocarcinogenesis. We performed an integrative analysis on 52 iCCAs using both genetic and epigenetic data with a specific focus on DNA methylation components. We found recurrent isocitrate dehydrogenase 1 (IDH1) and IDH2 (28%) gene mutations, recurrent arm-length copy number alterations (CNAs), and focal alterations such as deletion of 3p21 or amplification of 12q15, which affect BRCA1 Associated Protein 1, polybromo 1, and mouse double minute 2 homolog. DNA methylome analysis revealed excessive hypermethylation of iCCA, affecting primarily the bivalent genomic regions marked with both active and repressive histone modifications. Integrative clustering of genetic and epigenetic data identified four iCCA subgroups with prognostic relevance further designated as IDH, high (H), medium (M), and low (L) alteration groups. The IDH group consisted of all samples with IDH1 or IDH2 mutations and showed, together with the H group, a highly disrupted genome, characterized by frequent deletions of chromosome arms 3p and 6q. Both groups showed excessive hypermethylation with distinct patterns. The M group showed intermediate characteristics regarding both genetic and epigenetic marks, whereas the L group exhibited few methylation changes and mutations and a lack of CNAs. Methylation-based latent component analysis of cell-type composition identified differences among these four groups. Prognosis of the H and M groups was significantly worse than that of the L group. Conclusion: Using an integrative genomic and epigenomic analysis approach, we identified four major iCCA subgroups with widespread genomic and epigenomic differences and prognostic implications. Furthermore, our data suggest differences in the cell-of-origin of the iCCA subtypes.

Polymorphisms in the Angiogenesis-Related Genes EFNB2, MMP2 and JAG1 Are Associated with Survival of Colorectal Cancer Patients.

An individual's inherited genetic variation may contribute to the 'angiogenic switch', which is essential for blood supply and tumor growth of microscopic and macroscopic tumors. Polymorphisms in angiogenesis-related genes potentially predispose to colorectal cancer (CRC) or affect the survival of CRC patients. We investigated the association of 392 single nucleotide polymorphisms (SNPs) in 33 angiogenesis-related genes with CRC risk and survival of CRC patients in 1754 CRC cases and 1781 healthy controls within DACHS (Darmkrebs: Chancen der Verhütung durch Screening), a German population-based case-control study. Odds ratios and 95% confidence intervals (CI) were estimated from unconditional logistic regression to test for genetic associations with CRC risk. The Cox proportional hazard model was used to estimate hazard ratios (HR) and 95% CIs for survival. Multiple testing was adjusted for by a false discovery rate. No variant was associated with CRC risk. Variants in EFNB2, MMP2 and JAG1 were significantly associated with overall survival. The association of the EFNB2 tagging SNP rs9520090 (p < 0.0001) was confirmed in two validation datasets (p-values: 0.01 and 0.05). The associations of the tagging SNPs rs6040062 in JAG1 (p-value 0.0003) and rs2241145 in MMP2 (p-value 0.0005) showed the same direction of association with overall survival in the first and second validation sets, respectively, although they did not reach significance (p-values: 0.09 and 0.25, respectively). EFNB2, MMP2 and JAG1 are known for their functional role in angiogenesis and the present study points to novel evidence for the impact of angiogenesis-related genetic variants on the CRC outcome.

Genome-Wide Interaction Analyses between Genetic Variants and Alcohol Consumption and Smoking for Risk of Colorectal Cancer.

Genome-wide association studies (GWAS) have identified many genetic susceptibility loci for colorectal cancer (CRC). However, variants in these loci explain only a small proportion of familial aggregation, and there are likely additional variants that are associated with CRC susceptibility. Genome-wide studies of gene-environment interactions may identify variants that are not detected in GWAS of marginal gene effects. To study this, we conducted a genome-wide analysis for interaction between genetic variants and alcohol consumption and cigarette smoking using data from the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Interactions were tested using logistic regression. We identified interaction between CRC risk and alcohol consumption and variants in the 9q22.32/HIATL1 (Pinteraction = 1.76×10-8; permuted p-value 3.51x10-8) region. Compared to non-/occasional drinking light to moderate alcohol consumption was associated with a lower risk of colorectal cancer among individuals with rs9409565 CT genotype (OR, 0.82 [95% CI, 0.74-0.91]; P = 2.1×10-4) and TT genotypes (OR,0.62 [95% CI, 0.51-0.75]; P = 1.3×10-6) but not associated among those with the CC genotype (p = 0.059). No genome-wide statistically significant interactions were observed for smoking. If replicated our suggestive finding of a genome-wide significant interaction between genetic variants and alcohol consumption might contribute to understanding colorectal cancer etiology and identifying subpopulations with differential susceptibility to the effect of alcohol on CRC risk.

Large-conductance Ca2+-activated potassium channels are potently involved in the inverse neurovascular response to spreading depolarization.

Recurrent spreading depolarizations occur in the cerebral cortex from minutes up to weeks following acute brain injury. Clinical evidence suggests that the immediate reduction of cerebral blood flow in response to spreading depolarization importantly contributes to lesion progression as the wave propagates over vulnerable tissue zones, characterized by potassium concentration already elevated prior to the passage of spreading depolarization. Here we demonstrate with two-photon microscopy in anesthetized mice that initial vasoconstriction in response to SD triggered experimentally with 1 M KCl is coincident in space and time with the large extracellular accumulation of potassium, as shown with a potassium indicator fluorescent dye. Moreover, pharmacological manipulations in combination with the use of potassium-sensitive microelectrodes suggest that large-conductance Ca2+-activated potassium (BK) channels and L-type voltage-gated calcium channels play significant roles in the marked initial vasoconstriction under elevated baseline potassium. We propose that potassium efflux through BK channels is a central component in the devastating neurovascular effects of spreading depolarizations in tissue at risk.

Associations Between Dietary Patterns and Longitudinal Quality of Life Changes in Colorectal Cancer Patients: The ColoCare Study.

Quality of life (QoL) is an important clinical outcome in cancer patients. We investigated associations between dietary patterns and QoL changes in colorectal cancer (CRC) patients. The study included 192 CRC patients with available EORTC QLQ-C30 data before and 12 months post-surgery and food frequency questionnaire data at 12 months post-surgery. Principal component analysis was used to identify dietary patterns. Multivariate regression models assessed associations between dietary patterns and QoL changes over time. We identified four major dietary patterns: "Western" dietary pattern characterized by high consumption of potatoes, red and processed meat, poultry, and cakes, "fruit&vegetable" pattern: high intake of vegetables, fruits, vegetable oils, and soy products, "bread&butter" pattern: high intake of bread, butter and margarine, and "high-carb" pattern: high consumption of pasta, grains, nonalcoholic beverages, sauces and condiments. Patients following a "Western" diet had lower chances to improve in physical functioning (OR = 0.45 [0.21-0.99]), constipation (OR = 0.30 [0.13-0.72]) and diarrhea (OR: 0.44 [0.20-0.98]) over time. Patients following a "fruit&vegetable" diet showed improving diarrhea scores (OR: 2.52 [1.21-5.34]. A "Western" dietary pattern after surgery is inversely associated with QoL in CRC patients, whereas a diet rich in fruits and vegetables may be beneficial for patients' QoL over time.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study.

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10-5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Natural diversity in daily rhythms of gene expression contributes to phenotypic variation.

Daily rhythms of gene expression provide a benefit to most organisms by ensuring that biological processes are activated at the optimal time of day. Although temporal patterns of expression control plant traits of agricultural importance, how natural genetic variation modifies these patterns during the day and how precisely these patterns influence phenotypes is poorly understood. The circadian clock regulates the timing of gene expression, and natural variation in circadian rhythms has been described, but circadian rhythms are measured in artificial continuous conditions that do not reflect the complexity of biologically relevant day/night cycles. By studying transcriptional rhythms of the evening-expressed gene gigantea (GI) at high temporal resolution and during day/night cycles, we show that natural variation in the timing of GI expression occurs mostly under long days in 77 Arabidopsis accessions. This variation is explained by natural alleles that alter light sensitivity of GI, specifically in the evening, and that act at least partly independent of circadian rhythms. Natural alleles induce precise changes in the temporal waveform of GI expression, and these changes have detectable effects on phytochrome interacting factor 4 expression and growth. Our findings provide a paradigm for how natural alleles act within day/night cycles to precisely modify temporal gene expression waveforms and cause phenotypic diversity. Such alleles could confer an advantage by adjusting the activity of temporally regulated processes without severely disrupting the circadian system.

Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves.

BACKGROUND: Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants. RESULTS: ABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves. CONCLUSIONS: Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.

Evening expression of arabidopsis GIGANTEA is controlled by combinatorial interactions among evolutionarily conserved regulatory motifs.

Diurnal patterns of gene transcription are often conferred by complex interactions between circadian clock control and acute responses to environmental cues. Arabidopsis thaliana GIGANTEA (GI) contributes to photoperiodic flowering, circadian clock control, and photoreceptor signaling, and its transcription is regulated by the circadian clock and light. We used phylogenetic shadowing to identify three evolutionarily constrained regions (conserved regulatory modules [CRMs]) within the GI promoter and show that CRM2 is sufficient to confer a similar transcriptional pattern as the full-length promoter. Dissection of CRM2 showed that one subfragment (CRM2-A) contributes light inducibility, while another (CRM2-B) exhibits a diurnal response. Mutational analysis showed that three ABA RESPONSE ELEMENT LIKE (ABREL) motifs in CRM2-A and three EVENING ELEMENTs (EEs) in CRM2-B are essential in combination to confer a high amplitude diurnal pattern of expression. Genome-wide analysis identified characteristic spacing patterns of EEs and 71 A. thaliana promoters containing three EEs. Among these promoters, that of FLAVIN BINDING KELCH REPEAT F-BOX1 was analyzed in detail and shown to harbor a CRM functionally related to GI CRM2. Thus, combinatorial interactions among EEs and ABRELs confer diurnal patterns of transcription via an evolutionarily conserved module present in GI and other evening-expressed genes.

Distribution Characteristics and Combined Effect of Polymorphisms Affecting Alcohol Consumption Behaviour in the Hungarian General and Roma Populations.

AIMS: Harmful alcohol drinking habits, even among Roma children and adolescents, are more common than in the majority population. The aim of the study was to evaluate the genetic susceptibility of Roma to hazardous alcohol consumption compared to the Hungarian general population. METHODS: A total of 1273 samples from the population of segregated Hungarian Roma colonies and 2967 samples from the Hungarian general population were genotyped for 25 polymorphisms. Differences in genotype and allele distributions were investigated. Genetic risk scores (GRS) were generated to estimate the joint effect of individual single-nucleotide polymorphisms (SNPs). After unweighted and weighted GRS were calculated the distribution of scores in study populations was compared. RESULTS: The allele frequencies differed significantly between the study populations for 17 SNPs (P < 0.002), but the genetic alterations that predispose to or protect against harmful alcohol consumption were not overrepresented in the Roma population. The distribution of unweighted GRS in Roma population was left shifted compared to general population (P = 0.0013). The median weighted genetic risk score was lower among the subjects of Roma population compared to the subjects of general population (0.53 vs 0.65, P = 3.33 × 10-27) even after adjustment for confounding factors. CONCLUSIONS: Differences in alcohol consumption habits between the Hungarian Roma and Hungarian general populations do not appear to be linked to genetic constitution, this behaviour may occur as a result of different cultural values and environmental exposures. Population-based measures to tackle the fundamental drivers of consumption, which take account of cultural acceptability, are needed to reduce harmful alcohol consumption in the Roma population.

High Prevalence of Smoking in the Roma Population Seems to Have No Genetic Background.

INTRODUCTION: The prevalence of smoking in Romani of both genders is significantly higher than in the general population. Our aim was to determine whether a genetic susceptibility contributes to the high prevalence of smoking among Roma in a study based on data collected from cross-sectional surveys. METHODS: Twenty single nucleotide polymorphisms known to be closely related to smoking behavior were investigated in DNA samples of Hungarian Roma (N = 1273) and general (N = 2388) populations. Differences in genotype and allele distribution were investigated. Genetic risk scores (GRSs) were generated to estimate the joint effect of single nucleotide polymorphisms in genes COMT, CHRNA3/4/5, CYP2A6, CTNNA3, DRD2, MAOA, KCNJ6, AGPHD1, ANKK1, TRPC7, GABRA4, and NRXN1. The distribution of scores in study populations was compared. Age, gender, and body mass index were considered as confounding factors. RESULTS: Difference in allele frequencies between the study populations remained significant for 16 polymorphisms after multiple test correction (p < .003). Unexpectedly, the susceptible alleles were more common in the general population, although the protective alleles were more prevalent among Roma. The distribution of unweighted GRS in Roma population was left shifted compared to general population (p < .001). Furthermore, the median weighted GRS was lower among the subjects of Roma population compared to the subjects of general population (p < .001) even after adjustment for confounding factors. CONCLUSIONS: The harmful smoking behavior of the Roma population could not be accounted for by genetic susceptibility; therefore, interventions aimed at smoking prevention and cessation should focus on cultural and environmental factors. IMPLICATIONS: This is the first study designed to determine whether genetic background exists behind the harmful behavior of the smoking of the Roma population. Although the frequencies of susceptible and protective alleles strongly differ between the Hungarian Roma and general populations, it is shown that calculated GRSs being significantly higher in the general population, which do not support the hypothesis on the genetic susceptibility of the Roma population. Interventions aimed at smoking cessation in the Roma population should preferentially target cultural and environmental factors.

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.