Ph.D Arne S Schaefer
email: a.schaefer@ikmb.uni-kiel.de

Gesa M Richter

Henrik Dommisch

Markus Reinartz

Michael Nothnagel

Barbara Noack

Marja L Laine

Mathias Folwaczny

Groessner-Schreiber Birte

Bruno L Loos

Birte Groessner- Schreiber

Bruno G Loos

Søren Jepsen

Stefan Schreiber

CDKN2BAS is associated with periodontitis in different European populations and is activated

Keywords: CDKN2BAS, periodontitis, CHD, association, bacterial stimulation

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INTRODUCTION

Coronary heart disease (CHD) is the leading cause of death worldwide[1, 2] and is considered to be a complex multifactorial disease. Epidemiological studies have shown a significant correlation between periodontal health and the occurrence of cardiovascular events. [3,4] Periodontitis and CHD are both strongly promoted by very similar environmental and behavioral risk factors, with smoking and diabetes being the most prominent. [5,6,7,8,9,10,[START_REF] Gelskey | Cigarette smoking and periodontitis: methodology to assess the strength of evidence in support of a causal association[END_REF][START_REF] Stunkard | A twin study of human obesity[END_REF][START_REF] Saremi | Periodontal disease and mortality in type 2 diabetes[END_REF]. Furthermore, the same pathogenic bacterial strains were observed in both inflamed gingival tissue and atherosclerotic plaques. [10, [START_REF] Ott | Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease[END_REF][START_REF] Chiu | Multiple infections in carotid atherosclerotic plaques[END_REF] It has also been shown that periodontal treatment results in improvement of endothelial function, a marker for risk prediction of cardiovascular events. [START_REF] Tonetti | Treatment of periodontitis and endothelial function[END_REF] Recently, a major genetic susceptibility locus for CHD was identified by various genome-wide association studies (GWAS). [START_REF] Helgadottir | A common variant on chromosome 9p21 affects the risk of myocardial infarction[END_REF]18,[START_REF] Samani | Genomewide association analysis of coronary artery disease[END_REF][START_REF] Mcpherson | A common allele on chromosome 9 associated with coronary heart disease[END_REF] This locus is located within the chromosomal region 9p21.3, mapping to the large noncoding (nc) antisense RNA transcript CDKN2BAS, formerly called ANRIL. This association was verified by a subsequent meta-analysis [9] and by a largescale replication study, [START_REF] Samani | Large scale association analysis of novel genetic loci for coronary artery disease[END_REF] making this genetic region the best replicated CHD associated risk locus to date. In addition, variants within this region were independently found to be associated with type 2 diabetes, [START_REF] Zeggini | Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes[END_REF][START_REF] Saxena | Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels[END_REF][START_REF] Scott | A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants[END_REF] abdominal aortic and intracranial aneurysms, [START_REF] Helgadottir | The same sequence variant on 9p21 associates with myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm[END_REF] ischemic stroke, [START_REF] Matarin | Whole genome analyses suggest ischemic stroke and heart disease share an association with polymorphisms on chromosome 9p21[END_REF] Alzheimer's disease and vascular dementia, [START_REF] Emanuele | Chromosome 9p21.3 genotype is associated with vascular dementia and Alzheimer's disease[END_REF] and high-grade glioma susceptibility. [START_REF] Wrensch | Variants in the CDKN2B and RTEL1 regions are associated with high-grade glioma susceptibility[END_REF] Recently, we could demonstrate the association of a highly increased risk for aggressive periodontitis (AgP) with specific variants of CDKN2BAS. [START_REF] Schaefer | Identification of a shared genetic susceptibility locus for coronary heart disease and periodontitis[END_REF] However, despite extensive fine-mapping and functional studies, the causative variant and the functional role of CDKNBAS has remained unknown. [9,[START_REF] Jarinova | Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus[END_REF] In recent years, long noncoding RNAs like CDKN2BAS have been recognized for their role in regulating gene expression by contributing to the recruitment of protein complexes to target genes in trans, and to provide an architectural matrix to retain the proteins at the repression site of the target gene. Unprecedented, a role in cis regulation of CDKN2A expression was very recently described for a new transcript of CDKN2BAS that initiates upstream of the previously reported start site, [START_REF] Yap | Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a[END_REF] indicating a regulatory role of CDKN2BAS in the context of cellular proliferation.

In the present study, we aimed to identify genetic risk variants of CDKN2BAS that are shared by different European case-control populations of aggressive and chronic periodontitis. To this end, we covered >150 kb with 51 tagging single nucleotide polymorphisms (tagSNPs) mapping to CDKN2BAS and the adjacent cell cycle inhibitor genes CDKN2A and CDKN2B. We explored the SNP associations in two populations of AgP and CP of Dutch origin (n=313, and replicated the significant associations in two populations of AgP and CP of German origin (n=1,264).

Recently, differential gene expression of CDKN2BAS in a variety of CHD relevant tissues such as vascular endothelial cells, smooth coronary muscle cells, fibroblasts, and macrophages was shown. [START_REF] Jarinova | Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus[END_REF][START_REF] Liu | INK4/ARF transcript expression is associated with chromosome 9p21 variants linked to atherosclerosis[END_REF][START_REF] Broadbent | Susceptibility to coronary artery disease and diabetes is encoded by distinct, tightly linked SNPs in the ANRIL locus on chromosome 9p[END_REF] To study the tissue specific expression in the gingiva, we quantified the mRNA transcription pattern of CDKN2BAS, CDKN2A and CDKN2B in healthy and inflamed human gingival tissue samples. Additionally, we studied the gingival expression of CDK4, a cell cycle regulating kinase that is directly inhibited by CDKN2A and CDKN2B. To explore the possibility that both periodontitis and CHD share an inflammatory pathophysiology, we assessed the potential role of pathogenic bacteria in the transcriptional regulation of these genes in different gingival tissues.

MATERIALS & METHODS

Study population

The patient and control samples of the CP and AgP association studies were recruited across Germany and the Netherlands as previously described. [START_REF] Schaefer | A 3' UTR transition within DEFB1 is associated with chronic and aggressive periodontitis[END_REF] Only individuals of German and Dutch ethnicity were included, judged upon the location of both parental birthplaces. Prior to the study, the genetic sub-structure of the German population and of the Dutch population had been assessed, [START_REF] Lao | Correlation between genetic and geographic structure in Europe[END_REF][START_REF] Steffens | SNP-based analysis of genetic substructure in the German population[END_REF] indicating only negligible substructures and therefore allowing an analysis of the German and Dutch individuals in a single genetic study without the risk of substantial population stratification. A detailed description of the study population is given in Table 1 and in the supplementary informations. Informed written consent was obtained from all subjects recruited into this study. The study was approved by each institute's own ethical review board (Medical Ethical Committee, Universities of Bonn, Dresden, Kiel and Munich, and Medical Ethical Committee, Academic Medical Center, University of Amsterdam, The Netherlands).

DNA extraction and genotyping

Genomic DNA was extracted from blood (Invisorb Blood Universal Kit, Invitek, Berlin, Germany) or mouthwash samples, [START_REF] Laine | The mouthwash: a non-invasive sampling method to study cytokine gene polymorphisms[END_REF] and was amplified by whole genome amplification (GenomiPhi, Amersham, Uppsala, Sweden). TagSNPs were selected from the CEU data of the International HapMap project (http://www.hapmap.org, NCBI build 36, 2008-03) using the Tagger feature [START_REF] De Bakker | Efficiency and power in genetic association studies[END_REF] of Haploview 4.0. [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF] TagSNPs were defined by pair-wise linkage disequilibrium tagging (r 2 >0.8).Genotyping was carried out using the SNPlex and TaqMan Genotyping System (Applied Biosystems, Foster City, CA, USA) on an automated platform, using TECAN Freedom EVO and 96-well and 384-well TEMO liquid handling robots (TECAN, Männedorf, Switzerland). Genotypes were generated by automatic calling using the Genemapper 4.0 software (Applied Biosystems) with the following settings: sigma separation >6, angle separation for two cluster SNPs <1.2 rad, median cluster intensity >2.2 logs. Genotypes were further reviewed manually and call rates ≥90% in each sample set were required.

Gingival tissue samples and cell culture

For gene expression study, gingival tissue samples were obtained from individuals with healthy gingival conditions and no periodontitis (n=10) as well as from patients with advanced CP (n=10). All biopsies were collected from healthy patients (age 19-58 years) who underwent third-molar extraction or periodontal surgery and from patients with advanced CP. Healthy gingival conditions were defined as follows: no redness, no swelling, no bleeding on probing, and with periodontal pockets ≤3 mm. Patients with advanced CP showed all typical clinical signs of periodontal inflammation (redness, swelling, bleeding on probing) and a periodontal probing depth of ≥6 mm. For culture of GECs (n=3) and HGFs (n=3), gingival epithelial tissue samples were prepared as previously described. [START_REF] Krisanaprakornkit | Expression of the peptide antibiotic human beta-defensin 1 in cultured gingival epithelial cells and gingival tissue[END_REF] Epithelial cells were cultured in keratinocyte growth medium (KGM) using supplements provided with the medium (PAA, Cölbe, Germany) and 1% antibiotics (penicillin, streptomycin, amphotericin; PAA), whereas fibroblasts were cultured using Dulbecco`s modified Eagle`s Medium (DMEM, PAA) supplemented with 1% antibiotics (penicillin, streptomycin, amphotericin; PAA) and 10% foetal bovine serum (FBS; PAA). Both GECs and HGFs were cultured at 37 ºC in a humidified atmosphere (5% CO 2 ).

Bacterial cell culture and stimulation experiments

Wild-type Porphyromonas gingivalis (P. gingivalis ) strain ATCC 33277 was cultured under anaerobic conditions, whereas Streptococcus gordonii (S. gordonii) was grown under aerobic conditions as previously described. [START_REF] Chung | Expression of defensins in gingiva and their role in periodontal health and disease[END_REF][START_REF] Dommisch | Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis[END_REF] Bacterial numbers were determined by absorbance measurement using a spectro-photometer (Biochrom AG, Berlin, Germany). To mimic early stages of inflammation and to test the potential transcriptional response of these genes, the commensal oral bacterium S. gordonii and the pathogenic oral bacterium P. gingivalis, commonly present during severe periodontal infection, were chosen to create an in-vitro model to mimic periodontal infection of GECs and HGFs. For stimulation experiments, gingival epithelial cells (GECs) and human gingival fibroblasts (HGFs) were grown to 80% confluence and then incubated with P. gingivalis and S. gordonii, respectively, using an amount equivalent to a multiplicity of infection of 100:1, for either 6 or 24 hours. Experimental groups were: (1) unstimlated GECs and HGFs, (2) control cells (GECs and HGFs) treated with blank bacterial medium for S. gordonii not containing bacterial cells, (3) control cells (GECs and HGFs) treated with blank bacterial medium for P.

gingivalis not containing bacterial cells, (4) stimulation of GECs and HGFs with S. gordonii, (5) stimulation of GECs and HGFs with P. gingivalis, and (6) pre-incubation of GECs and HGFs with S. gordonii followed by medium change and stimulation with P. gingivalis. Each stimulation experiment was performed in triplicate, using cells from three different donors.

RNA extraction and PCR conditions

To extract mRNA from the epithelial and the connective tissue layer separately, biopsies were enzymatically dissected using 3 ml dispase (Cascade Biologics, Paisley, United Kingdom) for 6 hours on ice. After separation of gingival biopsies, total RNA was extracted from both epithelium and connective tissue (CT) using the RNeasy Protection Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Subsequent to in-vitro stimulation experiments on GECs and HGFs, total RNA was extracted as described above. The reverse transcription reaction with 500 ng of total RNA was performed using iScript ® (Bio-Rad, Munich, Germany) following the manufacturer's instructions.

Quantitative analysis of the cDNA was performed using the iCycler ® (Bio-Rad) and the QuantiFast SYBR Green PCR kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR reactions were carried out in 96-well plates in a total volume of 25 µl, including 10 ng of cDNA and 250 nM primers. At the end of each real-time PCR, melting curve analysis was performed to confirm that the amplified product was specific. Gene expression levels in the gingival epithelium and the gingival connective tissue were analyzed by normalization to the gene expression levels in healthy gingival epithelium. All sample values were normalized to the expression of the housekeeping gene glycerylaldehyd-3-dehydrogenase (GAPDH, GeneGlobe primer, Qiagen) and relative gene expression was calculated using the delta-delta Ct (threshold cycle) method. PCR controls were performed using water. Primer sequences are given in Supplementary Table 1.

Statistical analysis

Markers were checked for deviation from Hardy-Weinberg equilibrium (HWE) in controls to a significance level of α=0.05 before inclusion into the analysis. We assessed the significance of phenotypic association with single-marker alleles and genotypes using χ 2 and Fisher's exact tests for 2×2 and 2×3 contingency tables, respectively. Logistic regression analysis allowing for covariate adjustment was performed in the R statistical environment, version 2.8.1 [START_REF]Team TRDC[END_REF]. Significance was assessed by a likelihood-ratio test. P values ≤0.05 were considered nominally significant. Akaike's information criterion (AIC) was used to choose the model that best explained the underlying associations. LD measures were calculated and plotted using Haploview. qRT-PCR data were analyzed using the Mann-Whitney Test (SPSS, Version 18, Munich, Germany). The significance level was set at p ≤ 0.05.

RESULTS

Association Study with Aggressive Periodontitis

To analyze potential genetic associations within the entire CDKN2BAS region, we selected 51 tagSNPs covering 150 kb. These SNPs were genotyped in our combined population of 159 Dutch generalized and localized AgP patients and 421 Dutch healthy controls. In our previous study on the association of CDKN2BAS with AgP, [START_REF] Schaefer | Identification of a shared genetic susceptibility locus for coronary heart disease and periodontitis[END_REF] we demonstrated that the allele frequencies at this genetic locus were very similar between localized and generalized AgP cases, allowing the pooling of both sub-phenotypes to increase the statistical power for detecting associated variants. In a second step, we validated the significant SNPs in an independent panel of our 301 German patients with generalized or localized AgP and 963 German controls. [START_REF] Schaefer | Identification of a shared genetic susceptibility locus for coronary heart disease and periodontitis[END_REF] In a third step, we subsequently tested the selected tagSNPs in this genetic region for potential associations with chronic periodontitis in a panel of 154 Dutch CP cases, and replicated the significant associations in an independent panel of 740 German CP cases. To increase the statistical power, we used the same panels of Dutch and German controls for the association tests performed with the Dutch and German AgP and CP patients, respectively (Table 1).

Out of the 51 tagSNPs, eight were found to have a minor allele frequency (MAF) below 0.01 in the Dutch populations. These were excluded from the analysis due to their limited expected statistical power. Five further SNPs were excluded due to deviations from HWE in the Dutch controls (p<0.05). One SNP, rs564398, had poor genotyping quality (call rate<0.9) and was excluded from further analyses.In the explorative association study of the Dutch AgP case-control population, four SNPs were nominally significantly associated with AgP prior to adjustment for the common risk factors gender, smoking, and diabetes (Supplementary Table 2). Upon covariate adjustment, seven SNPs were nominally significantly associated (Table 2), with SNP rs3217992 being the most significant (p=0.007, OR=2.53 [CI 95% 1.29-5.13]). Two SNPs, rs1360590 and rs11790231, showed associations prior and after covariate adjustment (Supplementary Table 2, Supplementary Table 3).

These seven nominally significantly associated SNPs were then tested for their association in the German AgP population. One SNP, rs10965224, showed significant deviations from HWE in the German controls (p<0.05) and was excluded from the further analyses. Prior to the covariate adjustment, three of the associations remained significant (rs3217992, rs518394, rs1360590), with SNP rs1360590 being most significantly associated with p=0.0005 (multiplicative genetic model) and an OR of 1.41 (CI 95% 1.16-1.72, Supplementary Table 4). Upon covariate adjustment, these three SNPs remained significantly associated, with rs3217992 being most significant with p=0.0004 (Table 2, Supplementary Table 5) and an OR of 1.48 (95% CI 1.19-1.85). After Bonferroni correction for multiple testing, these three SNPs remained significant at the corrected significance threshold of p≤0.0083 for six independent tests. These three SNPs span a chromosomal region of 38 kb (NCBI build 36.3) and are located within (rs3217992) or downstream of CDKN2B, but were only in moderate LD to each other in our control population (Figure 1). Interestingly, none of the SNPs which tagged the large LD region at the 5' end of CDKN2BAS, and which was most significantly associated with CHD [9, [START_REF] Helgadottir | A common variant on chromosome 9p21 affects the risk of myocardial infarction[END_REF][START_REF] Samani | Genomewide association analysis of coronary artery disease[END_REF][START_REF] Mcpherson | A common allele on chromosome 9 associated with coronary heart disease[END_REF][START_REF] Wtccc | Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[END_REF] as well as with AgP in our German cases, [START_REF] Schaefer | Identification of a shared genetic susceptibility locus for coronary heart disease and periodontitis[END_REF] was significantly associated with the Dutch AgP patients in this study. Whereas the lead SNPs of this region were associated with CHD and the German AgP cases with e.g. for SNP rs1333048 a 10% increase of the rare allele in the homozygotes but showed similar allele frequencies for the heterozygotes between cases and controls, for the Dutch AgP cases we observed an enrichment of 10% in the heterozygotes but a decrease of 6% of the rare allele in the homozygotes (Supplementary Table 6).

Association Study with Chronic Periodontitis

In the next step, we tested the 37 tagSNPs that had passed the pre-assigned QC for their association with the phenotype chronic periodontitis in the explorative Dutch CP population.

Five SNPs were significant prior to covariate adjustment for gender, age, smoking, and diabetes, with SNP rs10965238 being the most significant (p=0.004, OR=0.57 [95% C.I. 0.38-0.84], Supplementary Table 7). Upon adjustment, four SNPs remained significant (rs1360590, rs10965224, rs10965238 and rs10811658), two of which showed stable associations prior to and upon covariate adjustment (rs10965238, rs10811658, Figure 1, Table 2, Supplementary Table 8).

We next tested these three SNPs for their association in the German CP population, again excluding SNP rs10965224 due to its significant deviations from HWE in this control population. Prior to covariate adjustment, SNP rs10811658 was significantly associated with CP. (Supplementary Table 9). Upon adjustment, this association remained significant in a similar fashion with p=0.025 (OR=0.66 [95% C.I. 0.46-0.95], Table 2). However, after Bonferroni correction for multiple testing, this association missed significance at the corrected significant threshold of p<0.0167 for three independent tests. None of the other SNPs tested in this case-control population showed significant associations upon covariate adjustment (Supplementary Table 10).

CDKN2BAS, CDKN2A, CDKN2B, and CDK4 mRNA expression in healthy and inflamed gingival tissues

To show in-vivo gene expression of CDKN2BAS, CDKN2A, CDKN2B, and CDK4, the specific mRNA levels of these genes within the epithelium (EPI) and the connective tissue (CT) of gingival biopsies were analyzed. We observed tissue specific expression of all four genes in both EPI and CT. In this explorative study, the expression of CDKN2BAS was siginifcantly higher in healthy CT compared to healthy EPI (p=0.004, Figure 2A), and this difference remained significant after Bonferroni correction for multiple comparisons.

Whereas for CDKN2A no difference in transcript levels was observed between CT and EPI (Figure 2B), higher mRNA levels, although not significant in our experiments, were observed for CDKN2B in healthy CT compared to healthy EPI (p=0.019, Figure 2C). A trend towards a lower gene expression for CDK4, inhibited by CDKN2A and 2B in vivo, was noticed in CT compared to EPI (Figure 2 D).

CDKN2BAS gene expression was up-regulated in S. gordonii pre-treated and P. gingivalis stimulated GECs

We next tested whether the genes were differentially regulated in cultured primary gingival epithelial cells (GECs) when exposed to commensal and/or pathogenic periodontal bacteria. We observed a significant increase of the CDKN2BAS gene expression after 24 hours of stimulation with pathogenic P. gingivalis in commensal S. gordonii pre-treated GECs with p=0.003 (4-fold increase), and this difference remained significant after Bonferroni correction for multiple comparisons (Figure 3A). In the same experiments, CDKN2A mRNA levels were significantly reduced after 24 hours of stimulation with pathogenic P. gingivalis in commensal S. gordonii pre-treated GECs with p=0.004, if compared to unstimulated GECs (Figure 3B). A similar reduction, however not significant in our experiments, was observed between 6 and 24 hours P. gingivalis stimulation after S.gordonii-pre-treatment. For CDKN2B, we observed a trend towards reduced transcript levels after 24h incubation with P. gingivalis in S. gordonii pre-treated GECs (Figure 3C).

CDK4 expression was significantly reduced after 24 hours of stimulation with pathogenic P. gingivalis in commensal S. gordonii pre-treated GECs compared to unstimulted GECs, with p=0.004 (Figure 3D). A similar reduction, however not significant in our experiments, was observed between 6 and 24 hours P. gingivalis stimulation after S.gordonii-pre-treatment.

CDKN2BAS gene expression was up-regulated in P. gingivalis stimulated HGFs

In the second gingival cell population of gingival fibroblasts (HGFs), 24h stimulation with P. gingivalis alone and after pre-treatment with the S. gordonii led to significant CDKN2BAS mRNA up-regulation after 24 hours with p=0.004, and p=0.003 (25-fold), respectively, and remained significant after Bonferroni correction for multiple comparisons (Figure 4A).

CDKN2A mRNA levels were significantly reduced (p=0.001) after 24h stimulation with P. gingivalis after pre-treatment with S. gordonii (Figure 4B). No clear trend in transcript levels could be observed for CDKN2B and CDK4 in any experimental condition (Figure 4 C-D).

DISCUSSION

The aim of this study was to identify genetic risk alleles within the genetic region of CDKN2BAS, CDKN2A and CDKN2B, which are shared by different European populations of the periodontal disease phenotypes AgP and CP. We showed three SNPs to be significantly associated with two AgP populations of different ancestry (rs3217992, rs518394, and rs1360590), and confirmed these associations to be independent of the traditional risk factors smoking, diabetes and gender. These SNPs were located within or closely downstream of the coding region of CDKN2B, but no SNP could be identified as a lead SNP that fully explained the associations with periodontitis. Interestingly, we could not replicate our previous finding of a statistical significant association of the large CHD associated LD region at the 5' end of CDKN2BAS with the Dutch AgP patients. As described earlier, [START_REF] Schaefer | Identification of a shared genetic susceptibility locus for coronary heart disease and periodontitis[END_REF] in the German AgP cases the frequency of the rare allele of the lead SNP from this study, rs1333048, was 10% higher compared to the controls and the frequencies for the heterozygotes was similar to the controls. This observation was similarly reported by association studies on CHD.[9] Interestingly, in the present study, the Dutch AgP cases showed an enrichment of 10% in the heterozygotes for this SNP, but the homozygotes of the risk allele were decreased. We ruled out potential genotyping errors of this assay by verifying the genotypes of SNP rs1333048 with two independent technologies, which gave identical results. Increased heterozygotes in the Dutch AgP cases were similarly observed for the significantly associated SNPs rs1360590, rs3217992 and rs518394. In contrast to the observation in the Dutch AgP patients, the Dutch CP cases, and to a lesser extend the German CP cases, showed a tendency towards an increased frequency of homozygosity for the risk allele, with a frequency of heterozygotes similar to the controls. A possible however speculative explanation for the observed increase of heterozygous Dutch AgP patients at this particular locus could be the presence of duplicated structural variations of this element in these cases, which were not present in the Dutch controls and CP cases.

The discovered associations, which were shared between different populations, did not follow a particular model of inheritance (genotypic, dominant, recessive or multiplicative). However, this observation is rather typical for association studies of our sample sizes, where it is practically impossible to confidently assess which model fits the data best, and were the specific models might reflect noise rather than a specific fit to a model. A future meta-analysis with data across many different data sets will improve this situation and might help to identify the best-fit model. [START_REF] Ioannidis | Validating, augmenting and refining genome-wide association signals[END_REF] The genetic effects that were estimated for the associated SNPs did also partly differ between the populations.

Corresponding to the variations in the genetic best-fit models, the genetic effects are statistical estimates which are liable to estimation errors, which are mostly attributed to effects of individual random samples which constitute each population analysed. If the causative variant has not been identified, the observed SNP associations reflect the genetic effect of the causative variant through shared LD, which can vary between populations of different ancestry.

Although this broad validation study of the previously reported association of CDKN2BAS

with periodontitis adds further evidence that variants at this locus increase the risk of periodontitis in individuals of Northern European origin, it is evident from the number and distribution of the significant SNP associations, which were observed in our current and our previous study, and similarly in other larger studies on CHD,[9, 33] that the causative variant(s) and the related disease mechanisms remain to be identified. Additionally, we observed nominally significant SNP associations in the CP populations. Considering that individuals who experienced early development of periodontitis are likely to have inherited a major genetic disease variant, it is conceivable that in larger populations of the rather moderate phenotype chronic periodontitis these SNPs may show their significance for CP as well as they did for the more severe AgP phenotype. To clarify this, replication experiments in further large populations of CP would be desirable.

The risk of periodontitis related to this locus may vary among different ethnic groups, which is suggested by frequency variations of the disease associated rs1333048-C allele from 21% in Yoruba to 51% in European and Japanese in HapMap. To identify the true causative variant(s), that may also be different between various ethnic groups, further genetic exploration of this genetic region in much larger analysis populations, including large analysis populations of various ethnic background, is necessary. A technical limitation of our study was the failure of genotyping 16 of the selected tagSNPs in all populations, especially when the moderate LD across most of the genetic region of CDKN2BAS is considered. Here, an LD region harbouring the potential true variant(s) might have been missed. Future studies that will apply deep re-sequencing of cases carrying the associated variants across CDKN2BAS will obtain the full spectrum of variation in this region, and will be able to identify the causal variant(s) that affect the genetic risk.

In the second part of our study, we analyzed the expression patterns of CDKN2BAS, CDKN2A, CDKN2B and CDK4 in healthy and inflamed gingiva, and assessed if these genes were regulated by a signal transduction cascade sensitive to bacterial infection. All analyzed genes were expressed in the gingiva, with CDKN2BAS and CDKN2B both showing the highest mRNA levels of the analyzed genes in CT. We did not observe different transcript levels between healthy or inflamed gingival tissues. This could indicate that these genes are not regulated by the inflammatory status of the gingiva. However, although healthy gingival samples were taken from areas with no clinical signs of inflammation, surfaces of the oral cavity are constantly exposed to both commensal and pathogenic microorganisms. Therefore, it can be speculated that the clinically healthy gingiva is subjected to challenges similar to those of early stages of infection. Likewise, gingival samples with periodontitis represent a tissue where constant infection and chronic inflammation occurred over years, and short-term responses on the transcriptional level are most likely missed in inflamed gingival tissue samples. Here, an experimental setting that precisely controls for pathogenic infection might be a better way to assess specific transcriptional responses. Because the functional variant(s) are yet unknown for both periodontitis as well as for CHD, we did not aim to study the putative biological effects of the associated variants. Thus, we did not distinguish between the allelic background in our cell cultures, but studied the transcription pattern of the selected genes in response to bacterial infection. We observed that after 24h of stimulation of healthy HGFs and GECs with the pathogenic bacterium P. gingivalis, CDKN2BAS was significantly up-regulated compared to the unstimulated controls. This real-time response of CDKN2BAS gene activity to a specific pathogenic inflammatory trigger indicates that CDKN2BAS is subjected to pathogen sensitive transcriptional regulation. Furthermore, the clear response after 24h of stimulation but the absence of elevated transcript levels after 6h of stimulation suggests that CDKN2BAS may be located at a rather downstream position of a putative pathogen sensitive signaling cascade. We further observed a minor but significant downregulation of CDKN2A after 24h of bacterial stimulation in both gingival epidermal tissue and gingival fibroblasts. These large quantitative differences in CDKN2BAS and CDKN2A transcript levels in response to 24h bacterial stimulation, possibly indicate that CDKN2A, unlike CDKN2BAS, is not directly regulated by bacterial infection on the transcriptional level, but on a later stage of this signal transduction cascade. Our observations were in accordance with a very recent study on CDKN2BAS function which showed that CDKN2BAS transcripts negatively regulate CDKN2A expression. [START_REF] Yap | Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a[END_REF] This recent study detected that CDKN2BAS transcript initiates in prostate cancer tissues at a start site several hundred bases upstream of the previously reported start site. An antisense construct designed to target this newly identified transcript caused a marked increase in the expression of CDKN2A, but had a much less profound effect on CDKN2B. [START_REF] Yap | Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a[END_REF] In conjunction with various other experiments, the authors concluded that the transcript encoded upstream of the previously reported start site of CDKN2BAS controls CDKN2A silencing by cis-recruitment of polycomb group complex proteins, and in addition functions as an architectural matrix to retain these proteins at the target site. Interestingly, a recent study which performed genome-wide expression profiling in healthy individuals who were homozygous for the common and the rare allele of rs1333049 showed that the nature of the genes and pathways linked to CDKN2BAS transcript levels comprised genes which control cellular proliferation. [START_REF] Jarinova | Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus[END_REF] These studies did not functionally analyse exon 1-19, but a second antisense construct designed to target exon 3 silenced CDKN2A to a much lesser extent than the antisense construct upstream of exon 1. [START_REF] Yap | Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a[END_REF] Although the function of this transcript was not studied in detail, it indicates that different CDKN2BAS transcripts may have different functions. Currently, ten alternative spliced variants were reported in different tissues as diverse as lung, brain, heart, vascular epithelial tissues, testis, and blood [START_REF] Jarinova | Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus[END_REF][START_REF] Folkersen | Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants[END_REF][START_REF] Pasmant | Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF[END_REF]. They may have specific functions in their tissue specific context, additional or alternative to the observed negative control of CDKN2A. Two studies measured the expression of CDKN2BAS spliced transcript NR_003529, spanning exons 17 to 18, and found slightly decreased expression which was associated with the risk allele in healthy individuals. [START_REF] Jarinova | Functional analysis of the chromosome 9p21.3 coronary artery disease risk locus[END_REF][START_REF] Liu | INK4/ARF transcript expression is associated with chromosome 9p21 variants linked to atherosclerosis[END_REF] Interestingly, a study on individuals with clinically suspected CHD revealed that NR_003529 expression was significantly increased in individuals with atherosclerotic plaque burden, and correlated ANRIL transcripts levels with severity of atherosclerosis. [START_REF] Holdt | ANRIL expression is associated with atherosclerosis risk at chromosome 9p21[END_REF] Our primers for the quantification of CDKN2BAS transcript levels were designed to detect NR_003529 transcripts, and we found NR_003529 up-regulation by bacterial infection. The correlation of CDKN2BAS expression upon bacterial infection and increased CDKN2BAS expression in individuals with atherosclerotic plaque burden add to the theory of inflammatory components in the pathophysiology of CHD. Considering the recent finding on the ability of the 5' transcript of CDKN2BAS to recruit and retain regulatory proteins, the functions of the proximal and considerable long sequences of CDKN2BAS might well have a similar mechanistic function, but may also be able to act in trans. Theories concerning the actual disease mechanism that is mediated by the causative risk variants of CDKN2BAS remain speculative as for the time being the exact nature of the causative variants and of the target proteins or genes await elucidation. They are possibly differing between CHD and periodontitis. Here, we present findings which link the pathogenicity of P. gingivalis and its contribution to rapid periodontal degradation to a biological mechanism that is mediated by CDKN2BAS, that may directly act in cis on the negative regulation of CDKN2A and the downstream lying genes of the CDKN2A regulation cascade, or additionally, may act on other yet unknown trans-acting regulatory proteins with unidentified genetic targets. Taking into consideration the correlation of increased ANRIL transcript levels with the severity of atherosclerosis, [START_REF] Holdt | ANRIL expression is associated with atherosclerosis risk at chromosome 9p21[END_REF] this link might also join bacterial infection to atherosclerotic plaque burden by a potentially regulatory mechanism that is mediated by ANRIL transcription.

Licence for Publication statement

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence. A. After 24 hours of bacterial infection, CDKN2BAS mRNA expression was significantly increased upon 24h P. gingivalis stimulation of S.gordonii-pre-treated GECs compared to 6h of stimulation (P = 0.003). B. CDKN2A gene expression was significantly reduced in response to 24h P. gingivalis stimulation after S.gordonii-pre-treatment compared to unstimulated GECs (P = 0.004). A similar reduction, however not significant in our experiments, was observed between 6h and 24h P. gingivalis stimulation after S.gordoniipre-treatment. C. For CDKN2B, no significant differences in mRNA transcript levels were observed in GECs upon bacterial stimulation. D. CDK4 mRNA levels were significantly reduced after S. gordonii-pre-treated followed by 24h stimulation with P. gingivalis compared to unstimulated GECs (P = 0.004). A similar reduction, however not significant in our experiments, was observed between 6h and 24h P. gingivalis stimulation after S.gordonii-pre-treatment. (Each boxplot respresents values derived from 9 independent experiments and cells from three different donors were tested. GEC = gingival epidermial cells, C = unstimulated cells; MSG = blank medium for S. gordonii; MPG = blank medium for P. gingivalis; SG = S. gordonii; PG = P. gingivalis; SG-PG = pre-treatment with S. gordonii for 1h followed by P. gingivalis-stimulation for 6h and 24h). A. CDKN2BAS mRNA levels were significantly increased in non S. gordonii-pre-treated (P = 0.004) and S. gordonii-pre-treated HGFs (P = 0.003) after 24h stimulation with P. gingivalis. B. CDKN2A mRNA levels were significantly reduced in S. gordonii-pre-treated and 24h P. gingivalis stimulated HGFs compared to unstimulated HGFs (P < 0.001). C-D. No significant differences of mRNA levels were observed for CDKN2B and CDK4 mRNA levels in neither condition. (Each boxplot respresents values derived from 9 independent experiments and cells from three different donors were tested (HGF = human gingival fibroblasts, C = unstimulated cells; MSG = blank medium for S. gordonii; MPG = blank medium for P. gingivalis; SG = S. gordonii; PG = P. gingivalis; SG-PG = pre-treatment with S. gordonii for 1h followed by P. gingivalis-stimulation for 6h and 24h).

Tables Table 1. Characteristics of the Study Populations

Values are given as mean (standard deviation [SD]) when appropriate. Affection status was based on reported history. Age was reported by the clinicians. Smoking was estimated by self-report of the individuals (n.s. = not specified). Given are the minor allele frequencies (MAF), the p values, and the odds ratios (OR) with the 95% confidence intervals (CI) for the different populations. The p values were obtained from a likelihood-ratio test and correspond to the genetic model with the lowest AIC value each. Values are given upon adjustment for the covariates smoking, diabetes, and gender for the AgP analyses and additionally for age for the CP analyses, in a logistic regression model.

Dutch German

Figure 1 .

 1 Figure 1. Genetic Region of CDKN2BASThe top diagram shows the nominal -log 10 P under the assumption of the different genetic models of those SNPs that passed the preassigned quality criteria. The -log 10 P values are plotted as a function of the genomic htSNP position (NCBI build 36). The bottom panel shows pairwise LD in the controls using metric r 2 . Geno, genotypic model; dom, dominant model; rec, recessive model; AgP, aggressive periodontitis; CP, chronic periodontitis; bp,basepares; 1, rs3731257; 2, rs3731239; 3, rs2811709; 4, rs7036656; 5, rs3218009; 6, rs3217992; 7, rs3217986; 8, rs495490; 9, rs518394; 10, rs1360590; 11, rs17694493; 12, rs11790231; 13, rs1011970; 14, rs10965224; 15, rs16905599; 16, rs10757270; 17, rs1412832; 18, rs10965227; 19, rs10965228; 20, rs1333040; 21, rs2891168; 22, rs10125231; 23, rs1333042; 24, rs13301964; 25, rs1333045; 26, rs1333048; 27, rs1333049; 28, rs1333050; 29, rs10757281; 30, rs10965238; 31, rs10811658; 32, rs12347779; 33, rs10965245; 34, rs2891169; 35, rs7045889; 36, rs10811661; 37, rs10757283

Figure 2 .

 2 Figure 2. mRNA levels of CDKN2BAS, CDKN2A, CDKN2B and CDK4 in healthy and inflamed gingival epithelium and connective tissue. (A) CDKN2BAS transcript levels were higher in healthy connective tissue compared to healthy gingival epithelium (P = 0.004); (B, C, D) CDKN2A, CDKN2B, and CDK4 mRNA showed no statistically significant differences in transcript levels between healthy or inflamed epithelial and connective gingival tissues. (Each boxplot respresents results derived from 10 different subjects. EPI = gingival epithelial tissue, CT = connective tissue, H = healthy, I = inflamed).

Figure 3 .

 3 Figure 3. mRNA levels of CDKN2BAS, CDKN2A, CDKN2B and CDK4 in gingival epidermial cells after stimulation with S. gordonii and P. gingivalis. A. After 24 hours of bacterial infection, CDKN2BAS mRNA expression was significantly increased upon 24h P. gingivalis stimulation of S.gordonii-pre-treated GECs compared to 6h of stimulation (P = 0.003). B. CDKN2A gene expression was significantly reduced in response to 24h P. gingivalis stimulation after S.gordonii-pre-treatment compared to unstimulated GECs (P = 0.004). A similar reduction, however not significant in our experiments, was observed between 6h and 24h P. gingivalis stimulation after S.gordoniipre-treatment. C. For CDKN2B, no significant differences in mRNA transcript levels were

Figure 4 .

 4 Figure 4. mRNA levels of CDKN2BAS, CDKN2A, CDKN2B and CDK4 in human gingival fibroblasts after stimulation with S. gordonii and P. gingivalis. A. CDKN2BAS mRNA levels were significantly increased in non S. gordonii-pre-treated (P = 0.004) and S. gordonii-pre-treated HGFs (P = 0.003) after 24h stimulation with P. gingivalis. B. CDKN2A mRNA levels were significantly reduced in S. gordonii-pre-treated and 24h P. gingivalis stimulated HGFs compared to unstimulated HGFs (P < 0.001). C-D. No significant differences of mRNA levels were observed for CDKN2B and CDK4 mRNA levels in neither condition. (Each boxplot respresents values derived from 9 independent experiments and cells from three different donors were tested (HGF = human gingival fibroblasts, C = unstimulated cells; MSG = blank medium for S. gordonii; MPG = blank medium for P. gingivalis; SG = S. gordonii; PG = P. gingivalis; SG-PG = pre-treatment with S. gordonii for 1h followed by P. gingivalis-stimulation for 6h and 24h).

  ). The bottom panel shows pairwise LD in the controls using metric r 2 . Geno, genotypic model; dom, dominant model; rec, recessive model; AgP,

	aggressive periodontitis; CP, chronic periodontitis; bp,basepares; 1, rs3731257; 2, rs3731239; 3,
	rs2811709; 4, rs7036656; 5, rs3218009; 6, rs3217992; 7, rs3217986; 8, rs495490; 9, rs518394; 10,
	rs1360590; 11, rs17694493; 12, rs11790231; 13, rs1011970; 14, rs10965224; 15, rs16905599; 16,
	rs10757270; 17, rs1412832; 18, rs10965227; 19, rs10965228; 20, rs1333040; 21, rs2891168; 22,
	rs10125231; 23, rs1333042; 24, rs13301964; 25, rs1333045; 26, rs1333048; 27, rs1333049; 28,
	rs1333050; 29, rs10757281; 30, rs10965238; 31, rs10811658; 32, rs12347779; 33, rs10965245; 34,
	rs2891169; 35, rs7045889; 36, rs10811661; 37, rs10757283

Table 2 .

 2 Minor Allele Frequencies (MAFs) and SNP Associations of the Significant SNPs in the Different Populations upon Adjustment for Covariates.

		Dutch Controls	German Controls			Dutch AgP			German AgP		Dutch CP			German CP
	SNP (Alleles)	MAF	MAF	p		OR (CI 95%)	MAF	p	OR (CI 95%)	MAF	p	OR (CI 95%)	MAF	p	OR (CI 95%)	MAF
	rs3217992 (G,A)	37.1	35.0	7.2x10	-3	2.53 (1.3-5.1)	34.0	4.4x10 -4	1.48 (1.2-1.9)	43.3	0.060	1.82 (1.0-3.4)	38.9	NA	NA	NA
	rs518394 (C,G)	44.5	46.1	0.045		2.32 (1.1-4.9)	50.3	7.8x10 -3	0.74 (0.6-0.9)	39.3	0.114	0.56 (0.3-1.2)	39.2	NA	NA	NA
	rs1360590 (A,G)	47.5	44.2	0.012		2.6 (1.3-5.2)	42.8	2.0x10 -3	1.41 (1.1-1.8)	52.9	0.029	2.1 (1.1-3.9)	50.0	0.119	1.22 (1.0-1.6)	46.4
	rs11790231 (G,A)	11.4	9.0	0.039		NA	9.1	0.08	1.35 (1.0-1.8)	10.7	0.504	0.5 (0.1-4.4)	8.9	NA	NA	NA
	rs10965238 (A,G)	28.5	24.2	0.024		0.30 (0.1-0.9)	24.9	0.367	1.12 (0.9-1.4)	25.4	3.0x10 -3	0.48 (0.3-0.8)	20.2	0.259	0.89 (0.7-1.1)	23.6
	rs10811658 (G,A)	33.8	30.8	0.047		0.40 (0.16-0.99)	29.7	0.055	1.2 (1.0-1.5)	34.0	0.024	0.59 (0.4-0.9)	26.2	0.025	0.89 (0.7-1.1)	28.3

Table 3 .

 3 Genotypes and Frequencies of the Significant SNPs in the Different Populations.

		Dutch Controls	German Controls		Dutch AgP		German AgP		Dutch CP			German CP
	SNP	11	12	22	11	12	22	11	12	22	11	12	22	11	12	22	11	12	22
	(Alleles)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
	rs3217992 (G,A)	171 (41.1)	182 (43.6)	64 (15.3)	281 (42.5)	298 (45.0)	83 (12.5)	66 (41.5)	78 (49.1)	15 (9.4)	96 (32.3)	145 (48.8)	56 (18.9)	60 (39.2)	67 (43.8)	26 (17.0)	NA	NA	NA
	rs518394 (C,G)	132 (31.5)	201 (48.0)	86 (20.5)	197 (29.7)	321 (48.4)	145 (21.9)	38 (23.9)	82 (51.6)	39 (24.5)	113 (37.7)	138 (46.0)	49 (16.3)	60 (39.3)	66 (43.1)	27 (17.6)	NA	NA	NA
	rs1360590	122	198	101	207	327	130	48	86	25	64	154	81	41	72	41	219	352	166
	(A,G)	(29.0)	(47.0)	(24.0)	(31.2)	(49.2)	(19.6)	(30.2)	(54.1)	(15.7)	(21.4)	(51.5)	(27.1)	(26.6)	(46.8)	(26.6)	(29.7)	(47.8)	(22.5)
	rs11790231 (G,A)	330 (78.6)	84 (20.0)	6 (1.4)	551 (83.2)	104 (15.7)	7 (1.1)	130 (81.8)	29 (18.2)	0 (0.0)	238 (79.9)	56 (18.8)	4 (1.3)	129 (84.3)	21 (13.7)	3 (2.0)	NA	NA	NA
	rs10965238	202	158	33	363	216	42	86	58	9	160	105	20	97	44	8	420	232	50
	(A,G)	(51.4)	(40.2)	(8.4)	(58.4)	(34.8)	(6.8)	(56.2)	(37.9)	(5.9)	(56.2)	(36.8)	(7.0)	(65.1)	(29.5)	(5.4)	(59.9)	(33.0)	(7.1)
	rs10811658	181	193	45	324	266	70	77	68	13	130	135	34	84	58	11	374	310	53
	(G,A)	(43.2)	(46.1)	(10.7)	(49.1)	(40.3)	(10.6)	(48.8)	(43.0)	(8.2)	(43.4)	(45.2)	(11.4)	(54.9)	(37.9)	(7.2)	(50.7)	(42.1)	(7.2)

Conflict of interest and source of funding statement:

The authors declare that they have no conflict of interests. This study was supported by a research grant of the "Research Center for Inflammation Medicine" of the Medical Faculty, Christian-Albrechts-University, University Medical Center Schleswig-Holstein, Campus Kiel, (Arne S.