Arne S. Schaefer

Genome-wide association study of biologically-informed periodontal complex traits offers novel insights into the genetic basis of periodontal disease

Genome-wide association studies (GWAS) of chronic periodontitis (CP) defined by clinical criteria alone have had modest success to-date. Here, we refine the CP phenotype by supplementing clinical data with biological intermediates of microbial burden (levels of eight periodontal pathogens) and local inflammatory response (gingival crevicular fluid IL-1β) and derive periodontal complex traits (PCTs) via principal component analysis. PCTs were carried forward to GWAS (∼2.5 million markers) to identify PCT-associated loci among 975 European American adult participants of the Dental ARIC study. We sought to validate these findings for CP in the larger ARIC cohort (n = 821 participants with severe CP, 2031—moderate CP, 1914—healthy/mild disease) and an independent German sample including 717 aggressive periodontitis cases and 4210 controls. We identified six PCTs with distinct microbial community/IL-1β structures, although with overlapping clinical presentations. PCT1 was characterized by a uniformly high pathogen load, whereas PCT3 and PCT5 were dominated by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. We detected genome-wide significant signals for PCT1 (CLEC19A, TRA, GGTA2P, TM9SF2, IFI16, RBMS3), PCT4 (HPVC1) and PCT5 (SLC15A4, PKP2, SNRPN). Overall, the highlighted loci included genes associated with immune response and epithelial barrier function. With the exception of associations of BEGAIN with severe and UBE3D with moderate CP, no other loci were associated with CP in ARIC or aggressive periodontitis in the German sample. Although not associated with current clinically determined periodontal disease taxonomies, upon replication and mechanistic validation these candidate loci may highlight dysbiotic microbial community structures and altered inflammatory/immune responses underlying biological sub-types of CP.

A genome-wide association study identifies nucleotide variants at SIGLEC5 and DEFA1A3 as risk loci for periodontitis.

Abstract 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%. The disease is characterized by destruction of the alveolar bone due to an aberrant host inflammatory response to a dysbiotic oral microbiome. Previous genome‐wide association studies (GWAS) have reported several suggestive susceptibility loci. Here, we conducted a GWAS using a German and Dutch case‐control sample of aggressive periodontitis (AgP, 896 cases, 7,104 controls), a rare but highly severe and early‐onset form of periodontitis, validated the associations in a German sample of severe forms of the more moderate phenotype chronic periodontitis (CP) (993 cases, 1,419 controls). Positive findings were replicated in a Turkish sample of AgP (223 cases, 564 controls). A locus at SIGLEC5 (sialic acid binding Ig‐like lectin 5) and a chromosomal region downstream of the DEFA1A3 locus (defensin alpha 1‐3) showed association with both disease phenotypes and were associated with periodontitis at a genome‐wide significance level in the pooled samples, with P = 1.09E‐08 (rs4284742,‐G; OR = 1.34, 95% CI = 1.21‐1.48) and P = 5.48E‐10 (rs2738058,‐T; OR = 1.28, 95% CI = 1.18‐1.38), respectively. SIGLEC5 is expressed in various myeloid immune cells and classified as an inhibitory receptor with the potential to mediate tyrosine phosphatases SHP‐1/‐2 dependent signaling. Alpha defensins are antimicrobial peptides with expression in neutrophils and mucosal surfaces and a role in phagocyte‐mediated host defense. This study identifies the first shared genetic risk loci of AgP and CP with genome‐wide significance and highlights the role of innate and adaptive immunity in the etiology of periodontitis.

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.

Integration of Murine and Human Studies for Mapping Periodontitis Susceptibility

Periodontitis is one of the most common inflammatory human diseases with a strong genetic component. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWASs) of chronic periodontitis (CP) have largely been unsuccessful in identifying common susceptibility factors. A combination of quantitative trait loci (QTL) mapping in mice with association studies in humans has the potential to discover novel risk loci. To this end, we assessed alveolar bone loss in response to experimental periodontal infection in 25 lines (286 mice) from the Collaborative Cross (CC) mouse population using micro–computed tomography (µCT) analysis. The orthologous human chromosomal regions of the significant QTL were analyzed for association using imputed genotype data (OmniExpress BeadChip arrays) derived from case-control samples of aggressive periodontitis (AgP; 896 cases, 7,104 controls) and chronic periodontitis (CP; 2,746 cases, 1,864 controls) of northwest European and European American descent, respectively. In the mouse genome, QTL mapping revealed 2 significant loci (–log P = 5.3; false discovery rate = 0.06) on chromosomes 1 (Perio3) and 14 (Perio4). The mapping resolution ranged from ~1.5 to 3 Mb. Perio3 overlaps with a previously reported QTL associated with residual bone volume in F2 cross and includes the murine gene Ccdc121. Its human orthologue showed previously a nominal significant association with CP in humans. Use of variation data from the genomes of the CC founder strains further refined the QTL and suggested 7 candidate genes (CAPN8, DUSP23, PCDH17, SNORA17, PCDH9, LECT1, and LECT2). We found no evidence of association of these candidates with the human orthologues. In conclusion, the CC populations enabled mapping of confined QTL that confer susceptibility to alveolar bone loss in mice and larger human phenotype-genotype samples and additional expression data from gingival tissues are likely required to identify true positive signals.

GWAS for Interleukin-1β levels in gingival crevicular fluid identifies IL37 variants in periodontal inflammation

There is no agnostic GWAS evidence for the genetic control of IL-1β expression in periodontal disease. Here we report a GWAS for “high” gingival crevicular fluid IL-1β expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10−22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12–2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09–1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01–1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1β in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1β and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10−7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.IL-1β in gingival crevicular fluid (GCF) is a marker of inflammation in periodontal disease. Here, Offenbacher et al. identify genetic variants in the IL37 locus associated with GCF-IL-1β and show that the IL-1β-increasing allele at rs3811046 leads to an enhanced inflammatory response in vitro and in vivo.

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.