Moving into a new era of periodontal genetic studies: relevance of large case-control samples using severe phenotypes for genome-wide association studies.

Studies to elucidate the role of genetics as a risk factor for periodontal disease have gone through various phases. In the majority of cases, the initial 'hypothesis-dependent' candidate-gene polymorphism studies did not report valid genetic risk loci. Following a large-scale replication study, these initially positive results are believed to be caused by type 1 errors. However, susceptibility genes, such as CDKN2BAS (Cyclin Dependend KiNase 2B AntiSense RNA; alias ANRIL [ANtisense Rna In the Ink locus]), glycosyltransferase 6 domain containing 1 (GLT6D1) and cyclooxygenase 2 (COX2), have been reported as conclusive risk loci of periodontitis. The search for genetic risk factors accelerated with the advent of 'hypothesis-free' genome-wide association studies (GWAS). However, despite many different GWAS being performed for almost all human diseases, only three GWAS on periodontitis have been published - one reported genome-wide association of GLT6D1 with aggressive periodontitis (a severe phenotype of periodontitis), whereas the remaining two, which were performed on patients with chronic periodontitis, were not able to find significant associations. This review discusses the problems faced and the lessons learned from the search for genetic risk variants of periodontitis. Current and future strategies for identifying genetic variance in periodontitis, and the importance of planning a well-designed genetic study with large and sufficiently powered case-control samples of severe phenotypes, are also discussed.

miRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression.

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10-40 and Padj = 4 × 10-6], -miR-17-5p [Padj = 9.5 × 10-23], -miR-30e-5p [Padj = 8.2 × 10-18], -miR-130a-3p [Padj = 5 × 10-15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10-7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10-11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Periodontitis Risk Variants at SIGLEC5 Impair ERG and MAFB Binding.

Periodontitis is a common complex inflammatory disease of the oral cavity. It is characterized by inflammation of gingival tissues and alveolar bone loss. Recently, a genome-wide association study and 2 genome-wide association study meta-analyses found 2 associated regions (haplotype blocks) at the inhibitory immune receptor gene SIGLEC5 to increase the risk for periodontitis. The aims of the current study were the identification of the putative causal variants underlying these associations, characterization of their molecular biological effects, and validation of SIGLEC5 as the target gene. We mapped the associated single-nucleotide polymorphisms to DNA elements with predictive features of regulatory functions and screened the associated alleles for transcription factor (TF) binding sites. Antibody electrophoretic mobility shift assays (EMSAs) with allele-specific probes were used to identify TF binding and to quantify allele-specific effects on binding affinities. Luciferase reporter assays were used to quantify the effect directions and allele-specific strength of the associated regulatory elements. We used CRISPR-dCas9 gene activation to validate SIGLEC5 as a target of the association. EMSA in peripheral blood mononuclear cells showed that E-26 transformation-specific TF-related gene (ERG) binds at rs11084095, with almost complete loss of binding at the minor A-allele. Allele-specific reporter genes showed enhancer function of the DNA sequence at rs11084095, which was abrogated in the background of the A-allele. EMSA in B lymphocytes showed that TF MAF bZIP (MAFB) binds at the common G-allele of rs4284742, whereas the minor A-allele reduced TF binding by 69%, corresponding to 9-fold reduction of luciferase reporter gene activity by the A-allele. Using CRISPR-dCas9, we showed that the enhancer at rs4284742 strongly activated SIGLEC5 expression, validating this gene as the target gene of the association. We conclude that rs11084095 and rs4284742 are putatively causal for the genome-wide significant associations with periodontitis at SIGLEC5 that impair ERG and MAFB binding, respectively.

BACH1 Binding Links the Genetic Risk for Severe Periodontitis with ST8SIA1.

Genome-wide association studies identified various loci associated with periodontal diseases, but assigning causal alleles remains difficult. Likewise, the generation of biological meaning underlying a statistical association has been challenging. Here, we characterized the genetic association at the gene ST8SIA1 that increases the risk for severe periodontitis in smokers. We used CRISPR/dCas9 activation and RNA-sequencing to identify genetic interaction partners of ST8SIA1 and to determine its function in the cell. We used reporter gene assays to identify regulatory elements at the associated single-nucleotide polymorphisms (SNPs) and to determine effect directions and allele-specific changes of enhancer activity. Antibody electrophoretic mobility shift assays proved allele-specific transcription factor binding at the putative causal SNPs. We found the reported periodontitis risk gene ABCA1 as the top upregulated gene following ST8SIA1 activation. Gene set enrichment analysis showed highest effects on integrin cell surface interactions (area under the curve [AUC] = 0.85; q = 4.9 × 10-6) and cell cycle regulation (AUC = 0.89; q = 1.6 × 10-5). We identified 2 associated repressor elements in the introns of ST8SIA1 that bind the transcriptional repressor BACH1. The putative causative variant rs2012722 decreased BACH1 binding by 40%. We also pinpointed ST8SIA1 as the target gene of the association. ST8SIA1 inhibits cell adhesion with extracellular matrix proteins, integrins, and cell cycle, as well as enhances apoptosis. Likewise, tobacco smoke reportedly results in an inhibition of cell adhesion and a decrease in integrin-positive cells and cell growth. We conclude that impaired ST8SIA1 repression, independently caused by reduced BACH1 binding at the effect T allele, as well as by tobacco smoke, contributes to higher ST8SIA1 levels, and in smokers who carry the effect T allele, both factors would be additive with damaging effects on the gingival barrier integrity. The activity of ST8SIA1 is also linked with the periodontitis risk gene ABCA1.

Exome Sequencing of 5 Families with Severe Early-Onset Periodontitis.

Periodontitis is characterized by alveolar bone loss leading to tooth loss. A small proportion of patients develop severe periodontitis at the juvenile or adolescent age without exposure to the main risk factors of the disease. It is considered that these cases carry rare variants with large causal effects, but the specific variants are largely unknown. In this study, we performed exome sequencing of 5 families with children who developed stage IV, grade C, periodontitis between 3 and 18 y of age. In 1 family, we found compound heterozygous variants in the gene CTSC (p.R272H, p.G139R), 1 of which was previously identified in a family with prepubertal periodontitis. Subsequent targeted resequencing of the CTSC gene in 24 patients <25 y of age (stage IV, grade C) identified the known mutation p.I453V (odds ratio = 4.06, 95% CI = 1.6 to 10.3, P = 0.001), which was previously reported to increase the risk for adolescent periodontitis. An affected sibling of another family carried a homozygous deleterious mutation in the gene TUT7 (p.R560Q, CADD score >30 [Combined Annotation Dependent Depletion]), which is implicated in regulation of interleukin 6 expression. Two other affected siblings shared heterozygous deleterious mutations in the interacting genes PADI1 and FLG (both CADD = 36), which contribute to the integrity of the environment-tissue barrier interface. Additionally, we found predicted deleterious mutations in the periodontitis risk genes ABCA1, GLT6D1, and SIGLEC5. We conclude that the CTSC variants p.R272H and p.I453V have different expressivity and diagnostic relevance for prepubertal and adolescent periodontitis, respectively. We propose additional causal variants for early-onset periodontitis, which also locate within genes that carry known susceptibility variants for common forms. However, the genetic architecture of juvenile periodontitis is complex and differs among the affected siblings of the sequenced families.

Phenotype Harmonization in the GLIDE2 Oral Health Genomics Consortium.

Genetic risk factors play important roles in the etiology of oral, dental, and craniofacial diseases. Identifying the relevant risk loci and understanding their molecular biology could highlight new prevention and management avenues. Our current understanding of oral health genomics suggests that dental caries and periodontitis are polygenic diseases, and very large sample sizes and informative phenotypic measures are required to discover signals and adequately map associations across the human genome. In this article, we introduce the second wave of the Gene-Lifestyle Interactions and Dental Endpoints consortium (GLIDE2) and discuss relevant data analytics challenges, opportunities, and applications. In this phase, the consortium comprises a diverse, multiethnic sample of over 700,000 participants from 21 studies contributing clinical data on dental caries experience and periodontitis. We outline the methodological challenges of combining data from heterogeneous populations, as well as the data reduction problem in resolving detailed clinical examination records into tractable phenotypes, and describe a strategy that addresses this. Specifically, we propose a 3-tiered phenotyping approach aimed at leveraging both the large sample size in the consortium and the detailed clinical information available in some studies, wherein binary, severity-encompassing, and "precision," data-driven clinical traits are employed. As an illustration of the use of data-driven traits across multiple cohorts, we present an application of dental caries experience data harmonization in 8 participating studies (N = 55,143) using previously developed permanent dentition tooth surface-level dental caries pattern traits. We demonstrate that these clinical patterns are transferable across multiple cohorts, have similar relative contributions within each study, and thus are prime targets for genetic interrogation in the expanded and diverse multiethnic sample of GLIDE2. We anticipate that results from GLIDE2 will decisively advance the knowledge base of mechanisms at play in oral, dental, and craniofacial health and disease and further catalyze international collaboration and data and resource sharing in genomics research.

Entamoeba gingivalis Exerts Severe Pathogenic Effects on the Oral Mucosa.

The protozoan Entamoeba gingivalis colonizes the healthy oral mucosa with a prevalence of 15%. Colonization can be asymptomatic, and it is considered not pathogenic. However, it is able to invade lacerated oral mucosa, where it ingests fragments of live cells, suggesting pathogenous potential. Here, we characterized the transcriptomes of gingival cells after infection with E. gingivalis using RNA sequencing and observed pathogen interaction with the epithelial monolayer barrier by scanning electron microscopy. In epithelial and fibroblast cells, strongest differential expression showed gene set "chemokines and inflammatory molecules in myeloid cells" (area under the curve [AUC] = 0.9, effect size 5.15, adjusted P = 3.1 × 10-19) and "cell cycle and growth arrest" (AUC = 0.91, effect size = 4.56, adjusted P = 4.8 × 10-9), respectively. The most upregulated genes were TNF (fold change 430) and IL8 (fold change 359) in epithelial cells and ZN331 (fold change 18) in fibroblasts. We showed that E. gingivalis killed live epithelial cells by trogocytosis, demonstrating strong pathogenic potential.

A 3' UTR transition within DEFB1 is associated with chronic and aggressive periodontitis.

Periodontal diseases are complex inflammatory diseases and affect up to 20% of the worldwide population. An unbalanced reaction of the immune system toward microbial pathogens is considered as the key factor in the development of periodontitis. Defensins have a strong antimicrobial function and are important contributors of the immune system toward maintaining health. Here, we present the first systematic association study of DEFB1. Using a haplotype-tagging single nucleotide polymorphism (SNP) approach, including described promoter SNPs of DEFB1, we investigated the associations of the selected variants in a large population (N=1337 cases and 2887 ethnically matched controls). The 3' untranslated region SNP, rs1047031, showed the most significant association signal for homozygous carriers of the rare A allele (P=0.002) with an increased genetic risk of 1.3 (95% confidence interval: 1.11-1.57). The association was consistent with the specific periodontitis forms: chronic periodontitis (odds ratio=2.2 (95% confidence interval: 1.16-4.35), P=0.02), and aggressive periodontitis (odds ratio=1.3 (95% confidence interval 1.04-1.68), P=0.02). Sequencing of regulatory and exonic regions of DEFB1 identified no other associated variant, pointing toward rs1047031 as likely being the causative variant. Prediction of microRNA targets identified a potential microRNA-binding site at the position of rs1047031.

Entamoeba gingivalis Causes Oral Inflammation and Tissue Destruction.

A metagenomics analysis showed a strongly increased frequency of the protozoan Entamoeba gingivalis in inflamed periodontal pockets, where it contributed the second-most abundant rRNA after human rRNA. This observation and the close biological relationship to Entamoeba histolytica, which causes inflammation and tissue destruction in the colon of predisposed individuals, raised our concern about its putative role in the pathogenesis of periodontitis. Histochemical staining of gingival epithelium inflamed from generalized severe chronic periodontitis visualized the presence of E. gingivalis in conjunction with abundant neutrophils. We showed that on disruption of the epithelial barrier, E. gingivalis invaded gingival tissue, where it moved and fed on host cells. We validated the frequency of E. gingivalis in 158 patients with periodontitis and healthy controls by polymerase chain reaction and microscopy. In the cases, we detected the parasite in 77% of inflamed periodontal sites and 22% of healthy sites; 15% of healthy oral cavities were colonized by E. gingivalis. In primary gingival epithelial cells, we demonstrated by quantitative real-time polymerase chain reaction that infection with E. gingivalis but not with the oral bacterial pathogen Porphyromonas gingivalis strongly upregulated the inflammatory cytokine IL8 (1,900 fold, P = 2 × 10-4) and the epithelial barrier gene MUC21 (8-fold, P = 7 × 10-4). In gingival fibroblasts, we showed upregulation of the collagenase MMP13 (11-fold, P = 3 × 10-4). Direct contact of E. gingivalis to gingival epithelial cells inhibited cell proliferation. We indicated the strong virulence potential of E. gingivalis and showed that the mechanisms of tissue invasion and destruction are similar to the colonic protozoan parasite E. histolytica. In conjunction with abundant colonization of inflamed periodontal sites and the known resistance of Entamoeba species to neutrophils, antimicrobial peptides, and various antibiotics, our results raise the awareness of this protozoan as a potential and, to date, underrated microbial driver of destructive forms of periodontitis.

Smoking Modifies the Genetic Risk for Early-Onset Periodontitis.

Periodontitis has low-prevalence, highly severe disease manifestations with an early onset and rapid progression. The diagnosis is based on severe destruction of the alveolar bone in adolescents and young adults. Genetic susceptibility variants and smoking are well-established risk factors, but their interactions in modifying disease susceptibility have not been studied. We aimed to identify genetic risk variants of early-onset periodontitis that unmask their effects on tobacco smoke exposure. To this end, we analyzed 79,780,573 common variants in 741 northwest Europeans diagnosed to have >30% bone loss at >2 teeth before 35 y of age, using imputed genotypes of the OmniExpress BeadChip. Never versus ever smokers were compared in a logistic regression analysis via a case-only approach. To explore the effect of tobacco smoke on the expression of the G×S-associated genes, cultures of primary gingival fibroblasts (n = 9) were exposed to cigarette smoke extract, and transcripts were quantified by reverse transcription polymerase chain reaction. We identified 16 loci for which our analysis suggested an association with G×S increased disease risk (P < 5 × 10-5). Nine loci had previously been reported to be associated with spirometric measures of pulmonary function by an earlier G×S genome-wide association study. Genome-wide significant cis expression quantitative trait loci were reported for G×S-associated single-nucleotide polymorphisms at ST8SIA1 and SOST, indicating a causal role of these genes in tobacco-related etiopathology. Notably, SOST is a negative regulator of bone growth, and ST8SIA1 has a role in tissue remodeling. Cigarette smoke extract significantly altered the expression of 2 associated genes: SSH1 (P = 5 × 10-07), which is required for NF-κB activation and innate immune responses to bacterial invasion, and ST8SIA1 (P = 0.0048). We conclude that the genetic predisposition to early-onset periodontitis is in part triggered by smoking and that tobacco smoke directly affects the expression of genes involved in bone homeostasis, tissue repair, and immune response.

Genetic Susceptibility Contributing to Periodontal and Cardiovascular Disease.

Periodontal disease (PD) and coronary artery disease (CAD) are common diseases characterized by an overaggressive inflammatory response to diverse stimuli. Whereas PD leads to destruction of the tooth-supporting structures, CAD is a chronic inflammatory condition ultimately causing myocardial infarction via narrowing and occluding of blood vessels. Classical twin studies led to the conclusion that both complex diseases have a similar degree of heritability and that a significant fraction of the genetic factors accounting for this heritability is shared. Recent genome-wide association and large-scale candidate gene studies highlight that variations in >50 genes are associated with premature CAD, while variations in only 4 genes showing nominally significant associations with aggressive periodontitis and/or chronic periodontitis have so far been identified. Remarkably, 3 of the PD loci (75%) show shared associations with CAD ( ANRIL/CDKN2B-AS1, PLG, CAMTA1/VAMP3), suggesting involvement of common pathogenic mechanisms. In this critical review, we highlight recent progress in identifying genetic markers and variants associated with PD, present their overlap with CAD, and discuss functional aspects. In addition, we answer why a significant fraction of the heritability of PD is still missing, and we suggest approaches that may be taken to close the gap.

The PF4/PPBP/CXCL5 Gene Cluster Is Associated with Periodontitis.

Periodontitis is a common dysbiotic inflammatory disease with an estimated heritability of 50%. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWAS) of chronic periodontitis (CP) have been unsuccessful in discovering susceptibility factors. A strategy that combines agnostic GWAS with a well-powered candidate-gene approach has the potential to discover novel loci. We combined RNA-seq data from gingival tissues with quantitative trait loci (QTLs) that were identified in a F2-cross of mice resistant and susceptible to infection with oral bacterial pathogens. Four genes, which were located within the mapped QTLs, showed differential expression. The chromosomal regions across the human orthologous were interrogated for putative periodontitis-associated variants using existing GWAS data from a German case-control sample of aggressive periodontitis (AgP; 651 cases, 4,001 controls), the most severe and early onset form of periodontitis. Two haplotype blocks, one upstream to the coding region of UGT2A1 (rs146712414, P = 9.1 × 10-5; odds ratio [OR], 1.34; 95% confidence interval [CI], 1.16-1.56) and one downstream of the genes PF4/PPBP/CXCL5 (rs1595009, P = 1.3 × 10-4; OR, 1.32; 95% CI, 1.15-1.52), were associated with AgP. The association of rs1595009 was validated in an independent cohort of CP of European Americans (1,961 cases and 1,864 controls; P = 0.03; OR, 1.45; 95% CI, 1.01-1.29). This association was further replicated in another sample of 399 German CP cases (disease onset <60 y of age) and 1,633 controls ( P = 0.03; OR, 1.75; 95% CI, 1.06-2.90). The combined estimates of association from all samples were P = 2.9 × 10-5 (OR, 1.2; 95% CI, 1.1-1.3). This study shows the strength of combining QTL mapping and RNA-Seq data from a mouse model with association studies in human case-control samples to identify genetic risk variants of periodontitis.

COX-2 is associated with periodontitis in Europeans.

COX-2 plays an important role in periodontitis by mediating inflammatory reactions in periodontal tissues, and the COX-2 polymorphisms rs20417 and rs689466 have been reported to be associated with periodontitis in populations of Taiwanese and Chinese ethnicity. To test whether these variants were associated with periodontitis in populations of European ethnicity, we genotyped the single-nucleotide polymorphisms (SNPs) rs689466 and rs6681231, the latter a haplotype tagging SNP (htSNP) for rs20417 (r2>0.95), in our large-analysis population of individuals with aggressive (n = 532) and chronic periodontitis (n = 1052), and 2873 healthy control individuals. The rare G allele of htSNP rs6681231 was associated with aggressive periodontitis prior to and after adjustment for the covariates smoking, diabetes, and gender, with an odds ratio of 1.57 (95% confidence interval 1.18-2.08; p = 0.002). The validation of the association of rs20417 by the htSNP rs6681231 provides evidence for a general genetic risk of COX-2 variants in the pathogenesis of periodontitis.