lncRNA CDKN2B-AS1 regulates collagen expression.

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.

Oral microbiota of patients with phenylketonuria: A nation-based cross-sectional study.

AIM: The oral microenvironment contributes to microbial composition and immune equilibrium. It is considered to be influenced by dietary habits. Phenylketonuria (PKU) patients, who follow a lifelong low-protein diet, exhibit higher prevalence of oral diseases such as periodontitis, offering a suitable model to explore the interplay between diet, oral microbiota and oral health. MATERIALS AND METHODS: We conducted 16S rDNA sequencing on saliva and subgingival plaque from 109 PKU patients (ages 6-68 years) and 114 age-matched controls and correlated oral microbial composition and dental health. RESULTS: PKU patients exhibited worse dental health, reduced oral microbial diversity and a difference in the abundance of specific taxa, especially Actinobacteriota species, compared to controls. PKU patients with poor periodontal health exhibited higher alpha diversity than the orally healthy ones, marked by high abundance of the genus Tannerella. Notably, the observed taxonomic differences in PKU patients with normal indices of decayed/missing/filled teeth, plaque control record, gingival bleeding index and periodontal screening and recording index generally differed from microbial signatures of periodontitis. CONCLUSIONS: PKU patients' reduced microbial diversity may be due to their diet's metabolic challenges disrupting microbial and immune balance, thus increasing oral inflammation. Higher alpha diversity in PKU patients with oral inflammation is likely related to expanded microbial niches.

A genome-wide association study meta-analysis in a European sample of stage III/IV grade C periodontitis patients ≤35 years of age identifies new risk loci.

AIM: Few genome-wide association studies (GWAS) have been conducted for severe forms of periodontitis (stage III/IV grade C), and the number of known risk genes is scarce. To identify further genetic risk variants to improve the understanding of the disease aetiology, a GWAS meta-analysis in cases with a diagnosis at ≤35 years of age was performed. MATERIALS AND METHODS: Genotypes from German, Dutch and Spanish GWAS studies of III/IV-C periodontitis diagnosed at age ≤35 years were imputed using TopMed. After quality control, a meta-analysis was conducted on 8,666,460 variants in 1306 cases and 7817 controls with METAL. Variants were prioritized using FUMA for gene-based tests, functional annotation and a transcriptome-wide association study integrating eQTL data. RESULTS: The study identified a novel genome-wide significant association in the FCER1G gene (p = 1.0 × 10-9 ), which was previously suggestively associated with III/IV-C periodontitis. Six additional genes showed suggestive association with p < 10-5 , including the known risk gene SIGLEC5. HMCN2 showed the second strongest association in this study (p = 6.1 × 10-8 ). CONCLUSIONS: This study expands the set of known genetic loci for severe periodontitis with an age of onset ≤35 years. The putative functions ascribed to the associated genes highlight the significance of oral barrier tissue stability, wound healing and tissue regeneration in the aetiology of these periodontitis forms and suggest the importance of tissue regeneration in maintaining oral health.

In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment.

Background: Entamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro. Methods: Different strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall. Results: We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation. Discussion: We conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.

High-fat diet and oral infection induced type 2 diabetes and obesity development under different genetic backgrounds.

BACKGROUND: Type 2 diabetes (T2D) is an adult-onset and obese form of diabetes caused by an interplay between genetic, epigenetic, and environmental components. Here, we have assessed a cohort of 11 genetically different collaborative cross (CC) mouse lines comprised of both sexes for T2D and obesity developments in response to oral infection and high-fat diet (HFD) challenges. METHODS: Mice were fed with either the HFD or the standard chow diet (control group) for 12 weeks starting at the age of 8 weeks. At week 5 of the experiment, half of the mice of each diet group were infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria strains. Throughout the 12-week experimental period, body weight (BW) was recorded biweekly, and intraperitoneal glucose tolerance tests were performed at weeks 6 and 12 of the experiment to evaluate the glucose tolerance status of mice. RESULTS: Statistical analysis has shown the significance of phenotypic variations between the CC lines, which have different genetic backgrounds and sex effects in different experimental groups. The heritability of the studied phenotypes was estimated and ranged between 0.45 and 0.85. We applied machine learning methods to make an early call for T2D and its prognosis. The results showed that classification with random forest could reach the highest accuracy classification (ACC = 0.91) when all the attributes were used. CONCLUSION: Using sex, diet, infection status, initial BW, and area under the curve (AUC) at week 6, we could classify the final phenotypes/outcomes at the end stage of the experiment (at 12 weeks).

RSPO4 is a potential risk gene of stages III-IV, grade C periodontitis through effects on innate immune response and oral barrier integrity.

AIM: R-spondin 4 (RSPO4) is a suggestive risk gene of stage III-IV, grade C periodontitis and upregulated in gingiva of mice resistant to bacteria-induced alveolar bone loss. We aimed to replicate the association, identify and characterize the putative causal variant(s) and molecular effects, and understand the downstream effects of RSPO4 upregulation. MATERIALS AND METHODS: We performed a two-step association study for RSPO4 with imputed genotypes of a German-Dutch (896 stage III-IV, grade C periodontitis cases, 7104 controls) and Spanish sample (441 cases and 1141 controls). We analysed the allelic effects on transcription factor binding sites with reporter gene and antibody electrophoretic mobility shift assays. We used CRISPR/dCas9 activation and RNA sequencing to pinpoint RSPO4 as the target gene and to analyse downstream effects. RESULTS: RSPO4 was associated with periodontitis (rs6056178, pmeta  = 4.6 × 10-5 ). rs6056178 contains a GATA-binding motif. The rs6056178 T-allele abolished reporter activity (p = .004) and reduced GATA binding (-14.5%). CRISPRa of the associated region increased RSPO4 expression (25.8 ± 6.5-fold, p = .003). RSPO4 activation showed strongest induction of Gliomedin (439-fold) and Mucin 21 (178-fold) and of the gene set "response to interferon-alpha" (area under the curve [AUC] = 0.8, p < 5 × 10-6 ). The most repressed gene set was "extracellular matrix interactions" (AUC = 0.8, padj  = .00016). CONCLUSION: RSPO4 is a potential periodontitis risk gene and modifies host defence and barrier integrity.

Case-only design identifies interactions of genetic risk variants at SIGLEC5 and PLG with the lncRNA CTD-2353F22.1 implying the importance of periodontal wound healing for disease aetiology.

AIM: The basis of phenotypic variation of periodontitis is genetic variability. Disease relevant effects of individual risk alleles are considered to result from genetic interactions. We investigated gene × gene (G×G) interactions of suggestive periodontitis susceptibility alleles. MATERIALS AND METHODS: We used the case-only design and investigated single-nucleotide polymorphism (SNPs) that showed associations in our recent genome-wide association study (GWAS) and GWAS meta-analysis with p < 5 × 10-6 . CRISPR-dCas9 gene activation followed by RNA-sequencing and gene-set enrichment analyses elucidated differentially expressed genes and gene networks. With the databases of SNPInspector and Transfac professional, luciferase reporter gene assays and antibody electrophoretic mobility shift experiments, we analysed allele-specific effects on transcription factor binding. RESULTS: SNPs at the genes sialic acid binding Ig-like lectin 5 (SIGLEC5) and plasminogen (PLG) showed G×G interactions with rs1122900 at the long non-coding RNA (lncRNA) CTD-2353F22. Associated chromatin cis-activated CTD-2353F22.1 6.5-fold (p = .003), indicating CTD-2353F22.1 as target gene of this interaction. CTD-2353F22.1 regulated GADD45A (padj < 4.9 × 10-11 , log2 fold change (FC) = -0.55), THBS1, SERPINE1 and Tissue Factor F3 (padj < 5 × 10-7 , log2 FC ≥ -0.35) and the gene set "angiogenesis" (area under the curve = 0.71, padj  = 8.2 × 10-5 ). rs1122900 effect C-allele decreased reporter gene activity (5.5-fold, p = .0003) and PRDM14 binding (76%). CONCLUSIONS: CTD-2353F22.1 mediates interaction of SIGLEC5 and PLG, together with genes that function in periodontal wound healing.

Host prediction for disease-associated gastrointestinal cressdnaviruses.

Metagenomic techniques have facilitated the discovery of thousands of viruses, yet because samples are often highly biodiverse, fundamental data on the specific cellular hosts are usually missing. Numerous gastrointestinal viruses linked to human or animal diseases are affected by this, preventing research into their medical or veterinary importance. Here, we developed a computational workflow for the prediction of viral hosts from complex metagenomic datasets. We applied it to seven lineages of gastrointestinal cressdnaviruses using 1,124 metagenomic datasets, predicting hosts of four lineages. The Redondoviridae, strongly associated to human gum disease (periodontitis), were predicted to infect Entamoeba gingivalis, an oral pathogen itself involved in periodontitis. The Kirkoviridae, originally linked to fatal equine disease, were predicted to infect a variety of parabasalid protists, including Dientamoeba fragilis in humans. Two viral lineages observed in human diarrhoeal disease (CRESSV1 and CRESSV19, i.e. pecoviruses and hudisaviruses) were predicted to infect Blastocystis spp. and Endolimax nana respectively, protists responsible for millions of annual human infections. Our prediction approach is adaptable to any virus lineage and requires neither training datasets nor host genome assemblies. Two host predictions (for the Kirkoviridae and CRESSV1 lineages) could be independently confirmed as virus-host relationships using endogenous viral elements identified inside host genomes, while a further prediction (for the Redondoviridae) was strongly supported as a virus-host relationship using a case-control screening experiment of human oral plaques.

Sex-specific analysis in Behçet's disease reveals higher genetic risk in male patients.

OBJECTIVES: Behçet's disease tends to be more severe in men than women. This study was undertaken to investigate sex-specific genetic effects in Behçet's disease. METHODS: A total of 1762 male and 1216 female patients with Behçet's disease from six diverse populations were studied, with the majority of patients of Turkish origin. Genotyping was performed using an Infinium ImmunoArray-24 BeadChip, or extracted from available genotyping data. Following imputation and extensive quality control measures, genome-wide association analysis was performed comparing male to female patients in the Turkish cohort, followed by a meta-analysis of significant results in all six populations. In addition, a weighted genetic risk score for Behçet's disease was calculated and compared between male and female patients. RESULTS: Genetic association analysis comparing male to female patients with Behçet's disease from Turkey revealed an association with male sex in HLA-B/MICA within the HLA region with a GWAS level of significance (rs2848712, OR = 1.46, P = 1.22 × 10-8). Meta-analysis of the effect in rs2848712 across six populations confirmed these results. Genetic risk score for Behçet's disease was significantly higher in male compared to female patients from Turkey. Higher genetic risk for Behçet's disease was observed in male patients in HLA-B/MICA (rs116799036, OR = 1.45, P = 1.95 × 10-8), HLA-C (rs12525170, OR = 1.46, P = 5.66 × 10-7), and KLRC4 (rs2617170, OR = 1.20, P = 0.019). In contrast, IFNGR1 (rs4896243, OR = 0.86, P = 0.011) was shown to confer higher genetic risk in female patients. CONCLUSIONS: Male patients with Behçet's disease are characterized by higher genetic risk compared to female patients. This genetic difference, primarily derived from our Turkish cohort, is largely explained by risk within the HLA region. These data suggest that genetic factors might contribute to differences in disease presentation between men and women with Behçet's disease.

Complementary Experimental Methods in Genetics Open Up New Avenues of Research to Elucidate the Pathogenesis of Periodontitis.

A complex disease such as periodontitis is the sum of environmental and genetic effects. The personal genetic constitution interacts with the effects of internal and external risk factors like smoking, oral hygiene, malnutrition, emotional stress, and age. Accordingly, individuals who live in the same environmental context and share comparable lifestyle habits have different disease risks. Genetic research offers the identification of DNA sequence variants that have a causal role in disease etiology and allows the identification of disease relevant immune and metabolic pathways that contribute to disease susceptibility and pathogenesis in specific situations. Real advances have been made in genetic medical research in the last years. Starting from candidate gene association studies, new approaches were employed that have expanded the study design of genomewide association studies to genomewide meta-analyses and gene x environment interaction studies. Cost efficient whole-exome and whole-genome sequencing of patients with rare severe forms of periodontitis has the potential to identify genes and pathways with a direct role in the pathogenesis of common forms. In parallel, animal models were developed that use genetically highly diverse mouse lines to identify risk genes of human diseases. This chapter presents the main studies and the identified susceptibility genes that have clear statistical evidence. In addition, it describes pioneering studies that used advanced methods in experimental dental research, opening up new avenues of research. Although the knowledge of the genetic architecture of periodontitis is still in its infancy, genetic research is building the basis for future works with the potential to advance dental medicine in ways that will determine the various causes of periodontal diseases. This knowledge may eventually allow making predictions about disease risk for individual patients and leading to diagnosis and treatments that do not treat the symptoms but heal the disease.

Epigenetic adaptations of the masticatory mucosa to periodontal inflammation.

BACKGROUND: In mucosal barrier interfaces, flexible responses of gene expression to long-term environmental changes allow adaptation and fine-tuning for the balance of host defense and uncontrolled not-resolving inflammation. Epigenetic modifications of the chromatin confer plasticity to the genetic information and give insight into how tissues use the genetic information to adapt to environmental factors. The oral mucosa is particularly exposed to environmental stressors such as a variable microbiota. Likewise, persistent oral inflammation is the most important intrinsic risk factor for the oral inflammatory disease periodontitis and has strong potential to alter DNA-methylation patterns. The aim of the current study was to identify epigenetic changes of the oral masticatory mucosa in response to long-term inflammation that resulted in periodontitis. METHODS AND RESULTS: Genome-wide CpG methylation of both inflamed and clinically uninflamed solid gingival tissue biopsies of 60 periodontitis cases was analyzed using the Infinium MethylationEPIC BeadChip. We validated and performed cell-type deconvolution for infiltrated immune cells using the EpiDish algorithm. Effect sizes of DMPs in gingival epithelial and fibroblast cells were estimated and adjusted for confounding factors using our recently developed "intercept-method". In the current EWAS, we identified various genes that showed significantly different methylation between periodontitis-inflamed and uninflamed oral mucosa in periodontitis patients. The strongest differences were observed for genes with roles in wound healing (ROBO2, PTP4A3), cell adhesion (LPXN) and innate immune response (CCL26, DNAJC1, BPI). Enrichment analyses implied a role of epigenetic changes for vesicle trafficking gene sets. CONCLUSIONS: Our results imply specific adaptations of the oral mucosa to a persistent inflammatory environment that involve wound repair, barrier integrity, and innate immune defense.

Sex-specific genetic factors affect the risk of early-onset periodontitis in Europeans.

AIMS: Various studies have reported that young European women are more likely to develop early-onset periodontitis compared to men. A potential explanation for the observed variations in sex and age of disease onset is the natural genetic variation within the autosomal genomes. We hypothesized that genotype-by-sex (G × S) interactions contribute to the increased prevalence and severity. MATERIALS AND METHODS: Using the case-only design, we tested for differences in genetic effects between men and women in 896 North-West European early-onset cases, using imputed genotypes from the OmniExpress genotyping array. Population-representative 6823 controls were used to verify that the interacting variables G and S were uncorrelated in the general population. RESULTS: In total, 20 loci indicated G × S associations (P < 0.0005), 3 of which were previously suggested as risk genes for periodontitis (ABLIM2, CDH13, and NELL1). We also found independent G × S interactions of the related gene paralogs MACROD1/FLRT1 (chr11) and MACROD2/FLRT3 (chr20). G × S-associated SNPs at CPEB4, CDH13, MACROD1, and MECOM were genome-wide-associated with heel bone mineral density (CPEB4, MECOM), waist-to-hip ratio (CPEB4, MACROD1), and blood pressure (CPEB4, CDH13). CONCLUSIONS: Our results indicate that natural genetic variation affects the different heritability of periodontitis among sexes and suggest genes that contribute to inter-sex phenotypic variation in early-onset periodontitis.

hsa-miR-374b-5p regulates expression of the gene U2AF homology motif (UHM) kinase 1.

OBJECTIVE: We aimed to identify a microRNA (miRNA) that is significantly upregulated in blood and in cells of the oral mucosa upon exposure to the periodontitis main risk factors oral inflammation and tobacco smoke, to subsequently identify its target gene and to describe the molecular mechanism of gene regulation. BACKGROUND: miRNAs are associated with many disorders. Array-based miRNA expression studies indicated a number of differentially expressed miRNAs in the pathology of oral diseases. However, these miRNAs mostly lacked replication, and their target genes have remained unknown. METHODS: 863 miRNAs were analyzed in blood from 18 PD cases and 70 controls (Geniom Biochip). Selected miRNAs were analyzed for upregulation in the inflamed oral mucosa of PD patients using published miRNA expression profiling studies from gingival cells. hsa-miR-374b-5p mimic was overexpressed in primary gingival fibroblasts (pGFs) from 3 donors, and genome-wide mRNA expression was quantified (Clarion Array). Gene-specific regulation was validated by qRT-PCR and Luciferase activity in HeLa cells. RESULTS: hsa-miR-374b-5p showed >twofold change (FC) in 3 independent studies performed in blood, gingival tissues, and cells. After hsa-miR-374b-5p overexpression, genome-wide expression analysis showed UHMK1 as top 1 downregulated gene in pGFs (p = 2.5 × 10-04 , fold change = -1.8). Reporter genes demonstrated that hsa-miR-374b-5p downregulates mRNA levels (p = .02; FC = -1.5), leading to reduction in protein activity (p = .013, FC = -1.3). CONCLUSIONS: hsa-miR-374b-5p is upregulated in blood and ginvial cells exposed to oral inflammation and tobacco smoke and regulates UHMK1, which has a role in osteoclast differentiation.

Genetic Association of a Gain-of-Function IFNGR1 Polymorphism and the Intergenic Region LNCAROD/DKK1 With Behçet's Disease.

OBJECTIVE: Behçet's disease is a complex systemic inflammatory vasculitis of incompletely understood etiology. This study was undertaken to investigate genetic associations with Behçet's disease in a diverse multiethnic population. METHODS: A total of 9,444 patients and controls from 7 different populations were included in this study. Genotyping was performed using an Infinium ImmunoArray-24 v.1.0 or v.2.0 BeadChip. Analysis of expression data from stimulated monocytes, and epigenetic and chromatin interaction analyses were performed. RESULTS: We identified 2 novel genetic susceptibility loci for Behçet's disease, including a risk locus in IFNGR1 (rs4896243) (odds ratio [OR] 1.25; P = 2.42 × 10-9 ) and within the intergenic region LNCAROD/DKK1 (rs1660760) (OR 0.78; P = 2.75 × 10-8 ). The risk variants in IFNGR1 significantly increased IFNGR1 messenger RNA expression in lipopolysaccharide-stimulated monocytes. In addition, our results replicated the association (P < 5 × 10-8 ) of 6 previously identified susceptibility loci in Behçet's disease: IL10, IL23R, IL12A-AS1, CCR3, ADO, and LACC1, reinforcing the notion that these loci are strong genetic factors in Behçet's disease shared across ancestries. We also identified >30 genetic susceptibility loci with a suggestive level of association (P < 5 × 10-5 ), which will require replication. Finally, functional annotation of genetic susceptibility loci in Behçet's disease revealed their possible regulatory roles and suggested potential causal genes and molecular mechanisms that could be further investigated. CONCLUSION: We performed the largest genetic association study in Behçet's disease to date. Our findings reveal novel putative functional variants associated with the disease and replicate and extend the genetic associations in other loci across multiple ancestries.

Qtlizer: comprehensive QTL annotation of GWAS results.

Exploration of genetic variant-to-gene relationships by quantitative trait loci such as expression QTLs is a frequently used tool in genome-wide association studies. However, the wide range of public QTL databases and the lack of batch annotation features complicate a comprehensive annotation of GWAS results. In this work, we introduce the tool "Qtlizer" for annotating lists of variants in human with associated changes in gene expression and protein abundance using an integrated database of published QTLs. Features include incorporation of variants in linkage disequilibrium and reverse search by gene names. Analyzing the database for base pair distances between best significant eQTLs and their affected genes suggests that the commonly used cis-distance limit of 1,000,000 base pairs might be too restrictive, implicating a substantial amount of wrongly and yet undetected eQTLs. We also ranked genes with respect to the maximum number of tissue-specific eQTL studies in which a most significant eQTL signal was consistent. For the top 100 genes we observed the strongest enrichment with housekeeping genes (P = 2 × 10-6) and with the 10% highest expressed genes (P = 0.005) after grouping eQTLs by r2 > 0.95, underlining the relevance of LD information in eQTL analyses. Qtlizer can be accessed via https://genehopper.de/qtlizer or by using the respective Bioconductor R-package ( https://doi.org/10.18129/B9.bioc.Qtlizer ).

Detection of suspicious interactions of spiking covariates in methylation data.

BACKGROUND: In methylation analyses like epigenome-wide association studies, a high amount of biomarkers is tested for an association between the measured continuous outcome and different covariates. In the case of a continuous covariate like smoking pack years (SPY), a measure of lifetime exposure to tobacco toxins, a spike at zero can occur. Hence, all non-smokers are generating a peak at zero, while the smoking patients are distributed over the other SPY values. Additionally, the spike might also occur on the right side of the covariate distribution, if a category "heavy smoker" is designed. Here, we will focus on methylation data with a spike at the left or the right of the distribution of a continuous covariate. After the methylation data is generated, analysis is usually performed by preprocessing, quality control, and determination of differentially methylated sites, often performed in pipeline fashion. Hence, the data is processed in a string of methods, which are available in one software package. The pipelines can distinguish between categorical covariates, i.e. for group comparisons or continuous covariates, i.e. for linear regression. The differential methylation analysis is often done internally by a linear regression without checking its inherent assumptions. A spike in the continuous covariate is ignored and can cause biased results. RESULTS: We have reanalysed five data sets, four freely available from ArrayExpress, including methylation data and smoking habits reported by smoking pack years. Therefore, we generated an algorithm to check for the occurrences of suspicious interactions between the values associated with the spike position and the non-spike positions of the covariate. Our algorithm helps to decide if a suspicious interaction can be found and further investigations should be carried out. This is mostly important, because the information on the differentially methylated sites will be used for post-hoc analyses like pathway analyses. CONCLUSIONS: We help to check for the validation of the linear regression assumptions in a methylation analysis pipeline. These assumptions should also be considered for machine learning approaches. In addition, we are able to detect outliers in the continuous covariate. Therefore, more statistical robust results should be produced in methylation analysis using our algorithm as a preprocessing step.

A combined epigenome- and transcriptome-wide association study of the oral masticatory mucosa assigns CYP1B1 a central role for epithelial health in smokers.

BACKGROUND: The oral mucosa has an important role in maintaining barrier integrity at the gateway to the gastrointestinal and respiratory tracts. Smoking is a strong environmental risk factor for the common oral inflammatory disease periodontitis and oral cancer. Cigarette smoke affects gene methylation and expression in various tissues. This is the first epigenome-wide association study (EWAS) that aimed to identify biologically active methylation marks of the oral masticatory mucosa that are associated with smoking. RESULTS: Ex vivo biopsies of 18 current smokers and 21 never smokers were analysed with the Infinium Methylation EPICBeadChip and combined with whole transcriptome RNA sequencing (RNA-Seq; 16 mio reads per sample) of the same samples. We analysed the associations of CpG methylation values with cigarette smoking and smoke pack year (SPY) levels in an analysis of covariance (ANCOVA). Nine CpGs were significantly associated with smoking status, with three CpGs mapping to the genetic region of CYP1B1 (cytochrome P450 family 1 subfamily B member 1; best p = 5.5 × 10-8) and two mapping to AHRR (aryl-hydrocarbon receptor repressor; best p = 5.9 × 10-9). In the SPY analysis, 61 CpG sites at 52 loci showed significant associations of the quantity of smoking with changes in methylation values. Here, the most significant association located to the gene CYP1B1, with p = 4.0 × 10-10. RNA-Seq data showed significantly increased expression of CYP1B1 in smokers compared to non-smokers (p = 2.2 × 10-14), together with 13 significantly upregulated transcripts. Six transcripts were significantly downregulated. No differential expression was observed for AHRR. In vitro studies with gingival fibroblasts showed that cigarette smoke extract directly upregulated the expression of CYP1B1. CONCLUSION: This study validated the established role of CYP1B1 and AHRR in xenobiotic metabolism of tobacco smoke and highlights the importance of epigenetic regulation for these genes. For the first time, we give evidence of this role for the oral masticatory mucosa.

Protocols, Methods, and Tools for Genome-Wide Association Studies (GWAS) of Dental Traits.

Oral health and disease are known to be influenced by complex interactions between environmental (e.g., social and behavioral) factors and innate susceptibility. Although the exact contribution of genomics and other layers of "omics" to oral health is an area of active research, it is well established that the susceptibility to dental caries, periodontal disease, and other oral and craniofacial traits is substantially influenced by the human genome. A comprehensive understanding of these genomic factors is necessary for the realization of precision medicine in the oral health domain. To aid in this direction, the advent and increasing affordability of high-throughput genotyping has enabled the simultaneous interrogation of millions of genetic polymorphisms for association with oral and craniofacial traits. Specifically, genome-wide association studies (GWAS) of dental caries and periodontal disease have provided initial insights into novel loci and biological processes plausibly implicated in these two common, complex, biofilm-mediated diseases. This paper presents a summary of protocols, methods, tools, and pipelines for the conduct of GWAS of dental caries, periodontal disease, and related traits. The protocol begins with the consideration of different traits for both diseases and outlines procedures for genotyping, quality control, adjustment for population stratification, heritability and association analyses, annotation, reporting, and interpretation. Methods and tools available for GWAS are being constantly updated and improved; with this in mind, the presented approaches have been successfully applied in numerous GWAS and meta-analyses among tens of thousands of individuals, including dental traits such as dental caries and periodontal disease. As such, they can serve as a guide or template for future genomic investigations of these and other traits.

Linear isoforms of the long noncoding RNA CDKN2B-AS1 regulate the c-myc-enhancer binding factor RBMS1.

Variants in the long noncoding RNA (lncRNA) gene CDKN2B-AS1 (CDKN2B antisense RNA 1; ANRIL) are genome-wide associated with type 2 diabetes (T2D), atherosclerosis, and several forms of cancer. However, it is currently not understood how CDKN2B-AS1 transcripts translate into diabetes. We previously demonstrated trans-regulation of the proximal polyadenylated transcripts on several genes with functions in glucose and lipid metabolism. However, information on specific genes that are regulated at physiological concentrations by the distal polyadenylated CDKN2B-AS1 transcripts is lacking. To identify target genes of CDKN2B-AS1 trans-regulation, we designed inducible short hairpin RNA constructs and integrated them into the genome of T-Rex HEK293 cells. Changes of gene expression after induction were determined at defined time points by genome-wide mRNA expression analysis. We confirmed downregulation of RBMS1, located on chromosome 2 (RNA-binding motif, single-stranded interacting protein 1) at the transcript and protein level in stable-transfected, inducible HeLa cells, and demonstrated that the effect was independent of the cell type, known cis-regulatory effects, and regulation of the proximal polyadenylated CDKN2B-AS1 isoforms. Direct binding of CDKN2B-AS1 transcripts to RBMS1 was shown by RNA immunoprecipitation. RBMS1 encodes a cell cycle suppressor. We conclude that the distal and proximal polyadenylated CDKN2B-AS1 transcripts have separate functions in gene regulation, which are independent of the circular CDKN2B-AS1 isoforms and of the genes CDKN2A/2B.

Meta-analysis of genome-wide association studies of aggressive and chronic periodontitis identifies two novel risk loci.

Periodontitis is one of the most common inflammatory diseases, with a prevalence of 11% worldwide for the severe forms and an estimated heritability of 50%. It is classified into the widespread moderate form chronic periodontitis (CP) and the rare early-onset and severe phenotype aggressive periodontitis (AgP). These different disease manifestations are thought to share risk alleles and predisposing environmental factors. To obtain novel insights into the shared genetic etiology and the underlying molecular mechanisms of both forms, we performed a two step-wise meta-analysis approach using genome-wide association studies of both phenotypes. Genotypes from imputed genome-wide association studies (GWAS) of AgP and CP comprising 5,095 cases and 9,908 controls of North-West European genetic background were included. Two loci were associated with periodontitis at a genome-wide significance level. They located within the pseudogene MTND1P5 on chromosome 8 (rs16870060-G, P = 3.69 × 10-9, OR = 1.36, 95% CI = [1.23-1.51]) and intronic of the long intergenic non-coding RNA LOC107984137 on chromosome 16, downstream of the gene SHISA9 (rs729876-T, P = 9.77 × 10-9, OR = 1.24, 95% CI = [1.15-1.34]). This study identified novel risk loci of periodontitis, adding to the genetic basis of AgP and CP.