Devices, kits, and methods for determining increased susceptibility to and treatment and prevention of periodontitis, alzheimer's disease, and other conditions

Abstract

The invention relates to a diagnostic microarray device comprising: a substrate, preferably formed of a glass material; wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one allele of a single nucleotide polymorphism (SNP), said SNR being selected from the group consisting of IL1B+3877, IL1B-511, and IL6-1363, and preferably IL10-592, CD14-260 and COX2+8473.

Description

DEVICES, KITS, AND METHODS FOR DETERMINING INCREASED SUSCEPTIBILITY TO AND TREATMENT AND PREVENTION OF PERIODONTITIS, ALZHEIMER'S DISEASE, AND OTHER CONDITIONSFIELD OF THE INVENTION

[0001] The invention relates to a diagnostic microarray device comprising: a substrate, preferably formed of a glass material; wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one allele of a single nucleotide polymorphism (SNP), said SNP being selected from the group consisting of / / .1B+3877, IL1B-511, and IL6-1363, and preferably IL10-592, CD14-2B0 and COX2+8473.

[0002] Preferably, the invention also relates to diagnostic methods e.g. a diagnostic assay for determining susceptibility to periodontal disease in a subject, by determining whether the subject is a strong or weak GCF responder to biofilm lysine.

[0003] The invention discloses a set of SNP alleles in the genes whose alleles were investigated herein along with their relationship to SNP alleles of the tooth loss genotype. On Figure 3, Column i identifies each participant. Columns ia, ib, ic, and id indicate sex, lysine contents, GCF exudation rates, and PI values, respectively. Column ii indicates the Strong or Weak (S / W) phenotype. Columns iia and iib indicate the genes of the tooth loss phenotype ILIA-889 and / / .1B+3954 as in FIG 2, and Columns ill - viii indicate the alleles of / / .1B+3877, IL1B-511, / L6-1363, / / . ID-592, CD14-260, and COX2+8473, respectively. The ninth allele (Column ix) was the A A P8-799 gene; this gene did not appear to associate with either strong or weak phenotype in this Example. Columns left to right are numbered as in FIG. 2, but here the white boxes are filled in to display allelic variants not associated with strong or weak response.BACKGROUND

[0004] Rapid advances in technology are bringing medicine close to tailoring genetic information into therapies for optimal health. Periodontal disease (PD), an inflammatory disease that destroys tooth attachments, is mediated by dentally adherent bacterial biofilms. Mastication produces traces of an inflammatory exudate called gingival crevicular fluid (GCF), and this fluid transudes into crevices between the teeth and gingiva where it provides nutrients for the basal layer of the dental epithelial attachment so that it maintains a proliferative phenotype and behaves as a barrier to dentally adherent bacteria. The saliva of most young adults contains traces of periodontopathic bacteria picked up from their social environment. These bacteria include a keystone pathogen, Porphorymonas gingivalis, along with Tannerella forsythia, Treponema denticola, and some others. These strictly anaerobic, protease-dependent bacteria, commonly known as the red complex, increase when the salivary biofilm is incubated in a medium simulating GCF in vitro, or when oral hygiene is inadequate, and GCF exudation remains increased in vivo.

[0005] The red complex normally takes many years to become established within dental biofilms, but once it does, the keystone bacterium, P. gingivalis, is unique in possessing a group of powerful secreted proteases called gingipains (gp) that can destroy the periodontium. Gingipains are divided into two major groups depending on whether they cleave after lysine (K) or arginine (R) residues in proteins (Kgp, or Rgp). These and other proteases in the red complex degrade collagen, fibronectin, various cytokines that disturb the host cytokine network, host defense mechanisms, interactions between host cells, the extracellular matrix, and the viability of fibroblasts and endothelial cells. The gingipain hypothesis proposes that the destruction of the periodontium allows P. gingivalis to break into the local systemic blood flow and spread to many different parts of the body, including the brain.

[0006] P. gingivalis is not normally detectable in the oral cavity prior to age 13, and this bacterium first appears with other red bacteria in young adults (>age 19), especially where pocket depth exceeds 2.0 mm. Dentally adherent bacteria are mostly viridans streptococci and actinomyces species, but other bacteria normally present in saliva appear within a week of discontinuing oral hygiene. One of these other bacteria is E. corrodens which produces lysine decarboxylase (LdcE), an enzyme, that depletes the GCF of its lysine by converting it to cadaverine and carbon dioxide. Lysine is an essential amino acid and its decreased availability impairs the weekly complete cell-turnover that maintains an epithelial barrier to bacteria at the crevice base. The epithelial attachment becomes exposed to microbial products that interact with receptors in the host epithelium to enhance GCF exudation, and the bacterial composition starts changing 2 -3 weeks of discontinued oral hygiene.

[0007] The red complex develops through many intermediate stages as gingival inflammation (gingivitis) persists, and its component bacteria catabolize enough GCF proteins to precipitate calcium phosphate around biofilm bacteria (dental calculus). The calcified attachment to teeth surfaces requires assistance to remove the calculus and underlying biofilm. Otherwise, gingival margins start to recede (periodontitis), cementum becomes exposed in the oral cavity (moderate periodontitis), and eventually the teeth may loosen and exfoliate, or require to be removed (severe periodontitis). Surprisingly, moderate and severe periodontitis develop in only about 38% of the population aged 30 years and older. In these individuals, periodontitis associates with common chronic inflammatory diseases such as cardiovascular diseases, rheumatoid arthritis, and Alzheimer's disease (AD). Although cardiovascular and rheumatoid arthritis can be diagnosed and controlled, periodontal and Alzheimer's diseases are neitherpredictable nor easily treated. Dental implants or dentures are options for treating moderate or severe periodontal disease, but only palliative therapies are available for Alzheimer's disease. At present, a major consideration revolves around studies indicating that P. gingivalis and its gingipain products in the brain promote Alzheimer's disease (Nara, et al., Journal of Alzheimer's Disease: JAD (2021) 82(4):1417-1450).

[0008] Studies of twins and epidemiological investigations of the natural history of periodontitis suggest that genetics play an important role in determining an individual's clinical presentation. The pro-inflammatory cytokine interleukin-1 ( I LI) is a key regulator of host inflammatory responses to microbial infections. There are two IL1 genes, ILIA and IL1B. The former synthesizes protein IL-la, and the latter synthesizes protein IL-10. The respective amino acid sequences are only 34% homologous. IL-la is constantly expressed within cells but only secreted after intrinsic or bacterial damage by bacteria or other foreign agents contacting the cell surface or invading the cytosol. These processes activate innate immunity which releases ILla into the interstitial fluid. ILla then binds to a receptor on surrounding cells through which it activates the synthesis and secretion of I Lip. from the surrounding epithelial, fibroblast, and other cells of the gingiva. The conventional wisdom is that the longer the initially adherent dental plaque (biofilm) remains, the more I Lip is secreted, and the greater is gingival inflammation (gingivitis) and the faster is the development of the 'red' complex, and its downstream cytokines are activated by microbial products.

[0009] A single nucleotide polymorphism (SNP) upstream of the protein encoding site at residue 889 ( / / .1A-889) increases IL-la expression 3-fold, and associates with severe periodontitis (Kiani, et al., Iran J Allergy Asthma Immunol (2009) 8(2):95-98). Unlike IL-la, IL-ip appears only after IL-la is activated and released into the interstitial fluid, in the presence of microbial products. The IL1B gene can possess an SNP at residue +3877 in an intron that interacts with some or all of 3 SNPs in the IL1B promoter region (-511, -1464, and -3737 upstream from the protein encoding region). Despite the possibility of many such potential interactions, only 5 such interactions, all containing / / .1B+3877 (rsll43633), associate with periodontitis, of which only one was strongly and significantly detected in multiple ethnicities residing in the US and elsewhere (Wu, X., et al., J. Periodontal Res (2015) 50(l):52-61). The function of IL1B+3877 was reported as unknown in the Wu et al. study.

[0010] In addition, a nearby SNP at ILB+3954, (rsll43634), lies (77 nucleotides downstream from IL1B+3877, lies within the I Lip polypeptide-encoding region without changing the encoded amino acid sequence. Greater levels of this other I L-i protein secreted by this SNP / / .1B+3954 are related to the presence of the red microbial complex and periodontal tissue destruction (Ferreira, et al., Infect Immun (2008) 76(8):3725-3734). Three independent predictors of a severe disease phenotype (tooth-loss) arerecognized: a) smoking tobacco; b) type 2 (adult onset) diabetes; and c) an / / .1A-889(GA) / / / .1B+3954(GA) genotype. The specified (major) nucleotide for both ILIA-889 (rsl800587) and / / .1B+3954 (rsll43634) is guanine (G) and the alternate (minor) nucleotide is adenine (A). Strand orientation of the DNA double helix indicates that the / / .1B+3954 major / minor nucleotides are not C / T as previously reported by Giannobile, et al., J Dent. Res (2013) 92(8):694-701. The genotype / / .1A-889(A) / / / .1B+3954(A) is present in over 60% of smokers, but only in 7% of the US population. This estimate derived from the minor (alternate) SNP frequencies, in both genes validated in a large European population (Karimbux et al., J Periodontal (2012):83:( 11); 1407-1419). Nevertheless, moderate plus severe cases of periodontitis account for 38% of individuals aged 30 through 65 years in the US (Genco and Borgnakke, Periodontol 2000 (2013):62(l);59-94). The tooth-loss genotype, / / .1A-889(A) / / / .1B+3954(A), does not possess a clinical phenotype that is broad enough to indicate differences in disease susceptibility. Such a phenotype should be capable of predicting the 38% of individuals susceptible to developing P. gingivalis in deepened crevices associated with periodontitis. This bacterium has powerful proteases that may cause other diseases and conditions, such as (but not limited to) late onset Alzheimer's and vascular dementia known to associate with periodontitis (Guo et al., Int. J. Environ. Res. Public. Health (2021): 18(13);6823).

[0011] Therefore, there is a need in the art for new and improved devices, kits, and methods for prediction, identification, diagnosis, and treatment of various disorders associated with severe periodontitis. It is to such devices, kits, and methods, which utilize a phenotype for a weak inflammatory response to periodontitis, that the present disclosure is directed.BRIEF DESCRIPTION OF THE INVENTION

[0012] The invention relates in particular to embodiments under the following paragraphs.The invention may relate to the following numbered paragraphs:1. The invention relates to a diagnostic microarray device, comprising:a substrate formed of a solid material, in particular a glass material;device comprises at least sixteen probes attached or otherwise associated with the substrate, wherein each probe is associated with the substrate at a defined location for detection thereof, wherein each probe is specific to one allele of a single nucleotide polymorphism (SNP), and wherein the at least sixteen probes comprise:a probe that detects a C allele of / / .1B+3877;a probe that detects a T allele of / / .1B+3877;a probe that detects a G allele of IL1B-511;a probe that detects an A allele of IL1B-511;a probe that detects a G allele of IL6-1363;a probe that detects a T allele of / / .6-1363;a probe that detects a G allele of IL10-592;a probe that detects a T allele of IL10-592;a probe that detects a G allele of CD14-260;a probe that detects an A allele of CD14-260;a probe that detects an A allele of COX2+8473; anda probe that detects a G allele of COX2+8473.In a particular embodiment the device comprises at least sixteen probes attached or otherwise associated with the substrate, wherein each probe is associated with the substrate at a defined location for detection thereof, wherein each probe is specific to one allele of a single nucleotide polymorphism (SNP), and wherein the at least sixteen probes comprise: a probe that detects a C allele of / / .1B+3877;a probe that detects a T allele of / / .1B+3877;a probe that detects a G allele of IL1B-511;a probe that detects an A allele of IL1B-511;a probe that detects a G allele of IL6-1363;a probe that detects a T allele of / / .6-1363;a probe that detects a G allele of IL10-592;a probe that detects a T allele of IL10-592;a probe that detects a G allele of CD14-260;a probe that detects an A allele of CD14-260;a probe that detects an A allele of COX2+8473;a probe that detects a G allele of COX2+8473, and optionallya probe that detects a G allele of ILIA-889;a probe that detects an A allele of ILIA-889.2. The diagnostic microarray device of claim 1, further defined as a diagnostic microarray device for detecting susceptibility to periodontal disease.3. The diagnostic microarray device of claim 1, further defined as a diagnostic microarray device for detecting susceptibility to Alzheimer's disease.4. The diagnostic microarray device of claim 1, further defined as a diagnostic microarray device for detecting susceptibility to cardiovascular disease.5. The diagnostic microarray device of claim 1, further defined as a diagnostic microarray device for detecting susceptibility to arthritis.6. The diagnostic microarray device of claim 1, further defined as a diagnostic microarray device for detecting susceptibility to adverse pregnancy outcomes.7. The diagnostic microarray device of claim 1, wherein the substrate is formed of a glass ionomer material.8. The diagnostic microarray device of claim 1, further defined as comprising at least the following probes: a probe that detects a C allele of / / .1B+3877;a probe that detects a T allele of / / .1B+3877;a probe that detects a G allele of IL1B-511;a probe that detects an A allele of IL1B-511;a probe that detects a G allele of IL6-1363;a probe that detects a T allele of / / .6-1363;9. A method of treating or reducing the occurrence of periodontal disease in a subject, the method comprising the steps of:contacting the diagnostic microarray device of claim 1 with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device;determining the alleles present for each of the SNPs IL1B+3877, IL1B-511, IL6-1363, IL10-592, CD14-260, and COX2+8473 to define a genotype for the biological sample; administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when:(i) the genotype for the biological sample comprises / / .1B+3877CT and / L1B-511GG; (ii) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GA, and wherein the genotype does not contain any of the following alleles: IL6- 1363T, and COX2+8473G; or(iii) the genotype for the biological sample comprises / / .1B+3877CC, / / .1B-511GA, and / / .6-1363GT.10. The method of claim 9, wherein the at least one therapeutic agent against periodontal disease, in particular periodontitis.11. The method of claim 9, wherein the at least one therapeutic agent comprises at least one of a furzinc oxide / zinc citrate toothpaste and a stannous fluoride toothpaste.il.12. The method of claim 9, wherein the at least one therapeutic protocol comprises performing dental scaling and root planing at least about once every three months.13. A method of treating or reducing the occurrence of Alzheimer's disease in a subject, the method comprising the steps of:contacting the diagnostic microarray device of claim 1 with a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes;determining the alleles present for each of the SNPs / / .1B+3877, IL1B-511, IL6-1363, IL10-592, CD14-260, C0X2 +8473 to define a genotype for the biological sample; administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when:(i) the genotype for the biological sample comprises / / .1B+3877CT and / L1B-511GG; (ii) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GA, and wherein the genotype does not contain any of the following alleles: IL6- 1363T, and COX2+8473G; or(iii) the genotype for the biological sample comprises / / .1B+3877CC, / / .1B-511GA, and / / .6-1363GT.14. The method of claim 13, wherein the at least one therapeutic agent against periodontal disease, in particular periodontitis.15. The method of claim 13, wherein the at least one therapeutic agent comprises at least one of a furzinc oxide / zinc citrate toothpaste and a stannous fluoride toothpaste.16. The method of claim 13, wherein the at least one therapeutic protocol comprises performing dental scaling and root planing at least about once every three months.17. A method of treating or reducing the occurrence of at least one of cardiovascular disease, arthritis, and adverse pregnancy outcomes in a subject, the method comprising the steps of:contacting the diagnostic microarray device of claim 1 with a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes;determining the alleles present for each of the SNPs ILIA-889, IL1B+3877, IL1B-511, IL6-1363, IL10-592, CD14-260, COX2+8473 to define a genotype for the biological sample;administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when:(i) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GG; (ii) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GA, and wherein the genotype does not contain any of the following alleles: IL6- 1363T, and COX2+8473G; or(iii) the genotype for the biological sample comprises / / .1B+3877CC, / / .1B-511GA, and / / .6-1363GT.Alternatively, a diagnostic assay is a diagnostic assay described below in the third aspect or in the appended claims.18. The method of claim 17, wherein the at least one therapeutic agent against periodontal disease, in particular periodontitis.19. The method of claim 17, wherein the at least one therapeutic agent comprises at least one of a furzinc oxide / zinc citrate toothpaste and a stannous fluoride toothpaste.20. The method of claim 17, wherein the at least one therapeutic protocol comprises performing dental scaling and root planing at least about once every three months.In further aspect the invention also relates to the embodiments defined in the following numbered paragraphs.1. A diagnostic microarray device comprising:a substrate, preferably formed of a glass material;wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one allele of a single nucleotide polymorphism (SNP),said SNP being selected from the group consisting of / / .1B+3877, IL1B-511, / / .6-1363 and, preferably, IL1O-5B2 and COX2+8473, and optionally CD14-260. 2. The diagnostic microarray device of claim 1wherein the plurality of probes further comprises the further probes being specific to one allele of a further SNP selected from the group consisting of: / / .10-592, CD14-260, and optionally ILIA-889.3. The diagnostic microarray device of claim 1 or 2,said device comprising:a substrate formed of a glass material;wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one allele of a single nucleotide polymorphism (SNP),said plurality of probes comprising at least the following probes:a probe that detects a C allele of / / .1B+3877;a probe that detects a T allele of IL1B+3877;a probe that detects a G allele of IL1B-511;a probe that detects an A allele of IL1B-511;and preferablya probe that detects a G allele of IL6-1363;a probe that detects a T allele of / / .6-1363;a probe that detects an A allele of COX2+8473; anda probe that detects a G allele of COX2+8473.4. A diagnostic microarray device of claim 3 further comprisinga probe that detects a G allele of ILIA-889;a probe that detects an A allele of ILIA-889;a probe that detects a G allele of IL10-592;a probe that detects a T allele of IL10-592;a probe that detects a G allele of CD14-260;a probe that detects an A allele of CD14-260.5. The diagnostic microarray device of any of claims 1 to 4, further defined as comprising at least eighteen probes, and wherein the at least eighteen probes further comprise at least two of:a probe that detects an A allele of MMP8-799;a probe that detects a G allele of MMP8-799;a probe that detects a C allele of IL10-W82; anda probe that detects a T allele of IL10-1082.6. The diagnostic microarray device of any of claims 1 to 4, wherein the plurality of probes further comprises the further probes being specific to one allele of a further SNP selected from the group consisting of:a probe that detects a G allele of ILIA-889;a probe that detects an A allele of ILIA-889;a probe that detects a G allele of IL10-592a probe that detects a T allele of IL10-592;a probe that detects a G allele of CD14-260;a probe that detects an A allele of CD14-260.a probe that detects an A allele of MMP8-799;a probe that detects a G allele of MMP8-799;a probe that detects a C allele of / / .10-1082; anda probe that detects a T allele of / / .10-1082.a probe that detects a G allele of IL6-1363;a probe that detects a T allele of IL6-1363.7. The diagnostic microarray device of claim 7, wherein the plurality of probes further comprises the probes being specific to one allele of a further SNP selected from the group consisting of:a probe that detects an A allele of COX2+8473a probe that detects a G allele of COX2+8473.8. The diagnostic microarray device of any of claims 1 to 7, wherein the substrate is formed of a glass ionomer material.9. Use of the diagnostic microarray device of any of claims 1 to 7, for detecting susceptibility of a subject to periodontal disease.10. Use of the diagnostic microarray device of any of claims 1 to 7, for detecting susceptibility of a subject to Alzheimer's disease.11. Use of the diagnostic microarray device of any of claims 1 to 7, for detecting susceptibility of a subject to cardiovascular disease.12. Use of the diagnostic microarray device of any of claims 1 to 7, for detecting susceptibility of a subject to arthritis.13. Use of the diagnostic microarray device of any of claims 1 to 7, for detecting susceptibility of a subject to adverse pregnancy outcomes.14. A method of diagnosing the susceptibility to at least one of periodontal disease, Alzheimer's disease cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject, the method comprising the steps of:contacting the diagnostic microarray device of any of claims 1 to 8 with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device;determining the alleles present for each of the SNPs IL1B+3877, IL1B-511, IL6-1363 and COX2+8473 to define a genotype for the biological sample;considering the subject as being susceptible to periodontal disease, andsuitable for administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when:(i) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GG;(ii) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GA, and wherein the genotype does not contain any of the following alleles: / / .6-1363T, and COX2+8473G; or(iii) the genotype for the biological sample comprises / / .1B+3877CC, / / .1B-511GA, and / / .6-1363GT.15. The method of diagnosing according to claim 14, wherein, in addition, the presence of one or more of / £1A-889(GA), / £M-889(AA), / / .10-592(TT), CD14-260(AA), and / or COX2+8473(AG) in the genotypes of (i), (ii), and / or (iii), indicate a condition selected from the group consisting of severe disease, early tooth loss, and increased susceptibility to periodontitis-associated diseases and conditions, such as (but not limited to) Alzheimer's disease, cardiovascular disease, arthritis and adverse pregnancy outcomes.16 The method of diagnosing the susceptibility to at least one of periodontal disease, Alzheimer's disease cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject, the method further comprising the steps of:contacting the diagnostic microarray device of any of claims 1 to 8, preferably 2 to 4 with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device;determining the alleles present for each of the SNPs / / .1A-889, IL10-592, CD14-260,considering the subject as being more susceptible to periodontal disease, andsuitable for administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when:the presence in the genotypes of (i), (ii), and / or (iii) of one or more of / / .1A-889(GA), / / .1A-889(AA), IL10-592(TT), CD14-260(AA), and / or COX2+8473(AG) can additionally indicate severe disease, early tooth loss, and increased susceptibility to periodontitis-associated diseases and conditions,in particular Alzheimer's disease, cardiovascular disease, arthritis or adverse pregnancy outcome.17. A furzinc oxide / zinc citrate toothpaste or a stannous fluoride toothpaste for use in the treatment or reducing occurrence of a disease selected from the group consisting of periodontal disease, Alzheimer's disease, a cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject diagnosed to be susceptible to said disease by a use of any of claim 8 to 13 or a method of any of claims 14 to 16.18. Atuzaginstat (COR388) for use in the treatment or reducing occurrence of a disease selected from the group consisting of periodontal disease, Alzheimer's disease, a cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject diagnosed to be susceptible to said disease by a use of any of claim 8 to 13 or a method of any of claims 14 to 16.19. A chemically modified curcumin, preferably CMC2.24 for use in the treatment or reducing occurrence of a disease selected from the group consisting of periodontal disease, Alzheimer's disease, a cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject diagnosed to be susceptible to said disease by a use of any of claim 8 to 13 or a method of any of claims 14 to 16.20. The toothpaste according to claim 17, the Atuzaginstat (COR388) according to claim 18 or the for use according to claim 19 wherein the treatment or reducing occurrence of a disease comprises regular review, preferably at least one therapeutic protocol comprises performing dental scaling and root planing at least about once every three months.In a further aspect the invention relates to diagnostic methods as defined in the following numbered paragraphs.1. A diagnostic assay for determining susceptibility to periodontal disease in a subject, by determining whether the subject is a strong or weak GCF responder to biofilm lysine, said method comprising the steps of:• examining, in a biological sample obtained from said subject, SNPs in the following genes associated with periodontitis: IL1B and IL6, and preferably IL10, CD14 and COX2• determining the presence of the following SNPs:o an SNP at IL1B+3877, an SNP at IL1B-511, and an SNP at IL6-1363, and preferably o one or more SNP(s) selected from the group consisting of an SNP at / / .10-592 and an SNP at CD14-260 and an SNP at COX2+8473,• considering the subject a weak GCF responder and thereby periodontal disease susceptible, if said subject has an SNP genotype selected from the group of the following SNP genotypes: o / / .1B+3877(CT)+ / / .1B-511(GG),o / / .! B+3877(CC)+ / / .lB-511(AG)+ / / .6-1363(GT), and, if preferably an SNP at IL10-592 and an SNP at CD14-260 is also determined,o IL1B+3877(CT)+IL1B-511(AG)+IL10-592(TT)o IL1B+3877(CT)+IL1B-511(AG)+CD14-260(AA)• and optionally• considering the subject a strong GCF responder and thereby periodontal disease protected, if said subject has an SNP genotype selected from the group of the following SNP genotypes: o / ilB+3877(CC)+ / / .lB-511(AG)o / £1B+3877(CC)+ / £1B-511(AA)o IL1B+3877(CT)+IL1B-511(AG)+IL6-1363(GT), oro IL1B+3877(CT)+IL1B-511(AA)+IL6-1363(GT), and if preferably an SNP at IL10-592 and an SNP at COX2+8473 is also determined,o IL1B+3877(CT)+IL1B-511(AG)+IL10-592(GG)o IL1B+3877(CT)+IL1B-511(AG)+COX2+8473(AG).2. The diagnostic assay for determining susceptibility to periodontal disease in a subject according to claim 1, wherein periodontal disease is selected from the group consisting of: periodontitis, gingivitis, periimplantitis, and preferably any associated comorbidity or disease thereof, or at any age to predict periodontitis susceptibilityPreferably the periodontal disease is at least periodontitis.Preferably the weak responders, i.e. who are periodontal disease susceptible, a treated as disclosed herein to prevent periodontal disease, preferably periodontitis, gingivitis, periimplantitis.Preferably the associated comorbidity is selected from the group consisting of Alzheimer's disease, cardiovascular disease, metabolic syndrome, arthritis, adverse pregnancy outcomes, etc.Preferably the weak responders, i.e. who are periodontal disease susceptible, a treated as disclosed herein to prevent any or more of these associated comorbidities.3. The diagnostic assay according to any of claims 1 to 2, wherein the presence of the SNPs is determined bycontacting a diagnostic microarray device of claim 1 with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray deviceIn a particular embodiment the presence of the SNPs is determined by sequencing, e.g. by shortread sequencing, e.g. by a "sequencing-by-synthesis method, like Illumina sequencing, alsoknown as Solexa sequencing, this is a high-throughput method that uses a "sequencing-by- synthesis" approach with reversible terminators;Ion Torrent sequencing, a method detects the release of hydrogen ions during nucleotide incorporation; a "sequencing-by-ligation" method that uses ligase with fluorescent probes, or a DNA Nanoball sequencing that self-assembling DNA nanoballs for sequencing,diseases. Other examples are well-known in the art.4. Certain non-limiting embodiments of the present disclosure are directed to method of treating or reducing the occurrence of at least one condition / disease in a subject, wherein the at least one condition / disease is selected from periodontal disease, Alzheimer's disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and the like. The method includes the steps of: contacting any of the diagnostic microarray devices disclosed or otherwise contemplated herein with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 graphically illustrates that, following a week of experimental gingivitis, host genotypes were separated into two clusters of gingival exudation rate phenotype, into two clusters. The upper curve indicates strong GCF responders to lysine in their biofilm, and the lower curve indicates weak GCF responders (the circle with a small gray spot above the lower of the two curves indicates a participant unavailable for genotyping). Unfilled circles indicate nine participants assigned only by different combinations of IL1B-511 and / / .1B+3877 alleles. The phenotypes of another two participants were changed by epistasis (i.e., reversal of the normal phenotype expression) in the presence of the T allele of / / .6-1363, and have a central dark spot. The association of these 11 participants with the appropriate response gave a Fisher exact test statistic = 0.007, p <0.01. The circles designated 1 through 4 indicate participants possessing both / / . B-511(A) and / / .1B+3877(T) alleles, a confounding genotype that required a second gene's SNP, indicated by specifies (s) in FIGs 2 and 3 to determine their GCF response. Weak responders 1 and 2 on FIG 1 had the uncommon, homozygous SNPs from IL10 and CD14, whereas the two strong responders numbered 3 and 4 in the upper curve had the common homozygous SNP from IL10 (FIGs 2 and 3). Host #3 indicated as s* (s1) had a second specifying SNP, the hemizygous SNP from COX2.

[0014] FIG. 3 contains genotypes that identify the strong (S) and weak (W) GCF responses shown in FIG 2. By convention, the gene name is italicized, and the site of a single polymorphism location isindicated by the number of nucleotides upstream (-) or downstream (+) from the translational start site of the encoded polypeptide. The National Center for Biotechnology Information (NCBI) has a Reference SNP (rs) portal that provides an ID number beginning with 'rs' for all known SNPs. The reference or common nucleotide is often but not always named first, A=Adenine, G=Guanine, C=Cytidine, or T=Thymidine, and the alternate nucleotide is named following a >sign to indicate that it is the minor allele frequency (MAF) for that site. The MAF in European or other large populations is also available and was used in this study (Table 1).

[0015] In FIGs 2 and 3, Hosts are identified by row numbers in lane i. Lanes iia and iib contain the SNPs that identify hosts whose genome possesses a long-studied genotype associated with severe periodontitis and tooth loss. Lanes ill through ix identify other SNPs from genes known to associate with moderate or severe periodontitis and were related to strong or weak GCF in this study. The reference SNP allele of each gene is shown first, and if the SNP alternate allele is also present (heterozygous), the background of the nucleotide is colored green. Otherwise, if both copies on the chromosome contain the reference SNP (homozygous) and the gene is translated from the forward (5') DNA strand, the reference SNP is colored red and the alternate homozygous SNP is colored blue as in lane ill, last line). If the reference SNP is translated from the complementary (3' strand), the homozygous box is colored blue, and the homozygous alternate SNP box is colored red as in lane iv. SNPs in boxes with a white background are unrelated to the study. Epistasiseand its dominancedindicate presence of the alternate T SNP at / / .6-1363 in hosts from rows 1 and 11. Specifiessindicates a second gene's SNP required to differentiate hosts possessing a 'conflicted' genotype based on the relationship of groups of SNPs to strong or weak GCF response. Square brackets around a gene name and location bearing the symbol 's*1(only present in host 3) indicates an SNP from an additional gene that may specify the same strong or weak GCF response. Surprisingly, none of the 15 participants possessed both the uncommon allele at both IL1B-511 and the common allele at / / .1B+3877.

[0016] FIG. 2 contains a complete list of the SNP alleles in the genes whose alleles were investigated herein along with their relationship to SNP alleles of the tooth loss genotype. FIG.3 shows only the SNP alleles related to the strong and weak responses. Unrelated responses are in white, except for the 1L10 column in which there are numerous instances of the same SNP not associating with the strong or weak response because it was unnecessary. Column i identifies each participant (Host). Columns ia, ib, ic, and id indicate sex, lysine contents, GCF exudation rates, and PI values, respectively. Column ii indicates the Strong or Weak (S / W) GCF phenotype. Columns iia and iib indicate the genes of the tooth loss (Perio Predict) phenotype / / .1A-889 and / / .1B+3954 as in FIG 2, and Columns iii - viii indicate the allelesof / / .1B+3877, IL1B-511, IL6-1363, IL10-592, CD14-260, and COX2+8473, respectively. The ninth allele (Column ix) was the MMP8-799 gene; this gene did not appear to associate with either strong or weak phenotype in this Example.DETAILED DESCRIPTION

[0017] Before explaining at least one embodiment of the inventive concept(s) in detail by way of exemplary language and results, it is to be understood that the inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0018] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0019] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0020] All of the compositions and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the inventive concept(s) have been described in terms of particular embodiments, it will be apparent to thoseof skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the inventive concept(s). All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the inventive concept(s) as defined by the appended claims.

[0021] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0022] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0023] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0024] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition " A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0025] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0026] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0027] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0028] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, " A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0029] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0030] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another as well as indirect association / binding of two moieties to one another. Non-limiting examples of associations / couplings include covalent binding of one moietyto another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.

[0031] The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects such as (but not limited to) toxicity, irritation, and / or allergic response commensurate with a reasonable benefit / risk ratio.

[0032] The term "pharmaceutically-acceptable excipient" refers to any carrier, vehicle, and / or diluent known in the art or otherwise contemplated herein that may improve solubility, deliverability, dispersion, stability, and / or conformational integrity of the compositions disclosed herein.

[0033] The term "patient" as used herein includes human and veterinary subjects. " Mammal" for purposes of treatment refers to any animal classified as a mammal, including (but not limited to) humans, domestic and farm animals, nonhuman primates, and any other animal that has mammary tissue.

[0034] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition / disease / infection as well as individuals who are at risk of acquiring a particular condition / disease / infection (e.g., those needing prophylactic / preventative measures). The term "treating" refers to administering an agent / element / method to a patient for therapeutic and / or prophylactic / preventative purposes.

[0035] A "therapeutic composition" or "pharmaceutical composition" refers to an agent that may be administered in vivo to bring about a therapeutic and / or prophylactic / preventative effect.

[0036] Administering a therapeutically effective amount or prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific amount that is therapeutically effective can be readily determined by the ordinary medical practitioner, and can vary depending on factors known in the art, such as (but not limited to) the type of condition / disease / infection, the patient's history and age, the stage of the condition / disease / infection, and the co-administration of other agents.

[0037] The term "effective amount" refers to an amount of a biologically active molecule or conjugate or derivative thereof, or an amount of a treatment protocol (i.e., an alternating electric field), sufficient to exhibit a detectable therapeutic effect without undue adverse side effects (such as (but not limited to) toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of the inventive concept(s). The therapeutic effect may include, for example butnot by way of limitation, preventing, inhibiting, or reducing the occurrence of at least one tumor and / or cancer. The effective amount for a subject will depend upon the type of subject, the subject's size and health, the nature and severity of the condition / disease / infection to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0038] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjunct therapy," and will be understood to mean that the patient in need of treatment is treated or given another drug for the condition / disease / infection in conjunction with the treatments of the present disclosure. This concurrent therapy can be sequential therapy, where the patient is treated first with one treatment protocol / pharmaceutical composition and then the other treatment protocol / pharmaceutical composition, or the two treatment protocols / pharmaceutical compositions are given simultaneously.

[0039] The terms "administration" and "administering," as used herein, will be understood to include all routes of administration known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, and including both local and systemic applications. In addition, the compositions of the present disclosure (and / or the methods of administration of same) may be designed to provide delayed, controlled, or sustained release using formulation techniques which are well known in the art.

[0040] Turning now to the inventive concept(s), certain non-limiting embodiments of the present disclosure are directed to diagnostic microarray devices, kits, and methods for use in determining increased susceptibility to and treatment and prevention of periodontitis as well as other systemic conditions that have been shown to be associated therewith, such as (but not limited to) Alzheimer's Disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and others.

[0041] Certain non-limiting embodiments are directed to a diagnostic microarray device that includes a substrate formed of a glass material and a plurality of probes attached or otherwise associated with the substrate. Each probe is associated with the substrate at a defined location that is spatially separated from the other probes so as to allow for detection of a signal generated by each probe. Each probe is specific to one allele of a single nucleotide polymorphism (SNP).

[0042] In certain non-limiting embodiments, the probes comprise one or more of the following: a probe that detects a G allele of / / .1A-889; a probe that detects an A allele of / / .1A-889; a probe that detectsa C allele of / / .1B+3877; a probe that detects a T allele of / / .1B+3877; a probe that detects a G allele of IL1B-511; a probe that detects an A allele of IL1B-511; a probe that detects a G allele of IL6-1363; a probe that detects a T allele of / / .6-1363; a probe that detects a G allele of IL10-592; a probe that detects a T allele of IL10-592; a probe that detects a G allele of CD14-260 a probe that detects an A allele of CD14-260 a probe that detects an A allele of COX2+8473; a probe that detects a G allele of COX2+8473; a probe that detects an A allele of MMP8-799; a probe that detects a G allele of MMP8-799; a probe that detects a C allele of / / .10-1082; a probe that detects a T allele of / / .10-1082; and / or a probe that detects a TREM2 R47H mutation.

[0043] In certain particular (but non-limiting) embodiments, the probes comprise two or more of the above, three or more of the above, four or more of the above, five or more of the above, six or more of the above, seven or more of the above, eight or more of the above, nine or more of the above, 10 or more of the above, 11 or more of the above, 12 or more of the above, 13 or more of the above, 14 or more of the above, 15 or more of the above, 16 or more of the above, 17 or more of the above, 18 or more of the above, or all 19 of the above probes.

[0044] The diagnostic microarray device can be utilized for detecting susceptibility to (for example, but not by way of limitation), periodontal disease, periimplantitis, Alzheimer's disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and the like.

[0045] The diagnostic microarray device may be formed in any manner and of any materials that allow the device to function in the manner described or otherwise contemplated herein. Methods of forming such microarray devices are known in the art and commercially available. Non-limiting examples of device platforms that can be utilized in accordance with the present disclosure include Axiom Biobank Genotyping Arrays (ThermoFisher Scientific, Waltham, MA), Illumina Microarrays (Illumina, Inc., San Diego, CA), TaqMan® Arrays (ThermoFisher Scientific, Waltham, MA), and the like. These types of arrays are typically produced to determine a genotype and are often genome wide or restricted to only one or two genes to help identify known genetic diseases. The present disclosure utilizes these microarray device platforms in a new and unique manner, by restricting the array to a group of up to about eight specific genotypes to detect a specific disease-associated phenotype.

[0046] The substrate of the diagnostic microarray device may be formed of any glass material that will allow the device to function as described herein. In a particular (but non-limiting) embodiment, the substrate is formed of a glass ionomer material. The various probes utilized are then formed on or otherwise attached to / associated with the glass material.

[0047] The various microarray devices known in the art detect binding of a nucleic acid moleculefrom a biological sample to each of the probes in different manners. For example (but not by way of limitation), nucleotide sequences may be isolated from the biological sample (and optionally treated to one or more fragmentation, amplification, and / or cDNA / cRNA preparation steps) and then labeled (such as, but not limited to, with a fluorescent dye) prior to passing the sample over the microarray device; in this manner, binding / hybridization of a labeled target nucleic acid to a probe of the device yields a positive result for the target allele to which the probe is directed. In another alternative, fragmented nucleotide sequences isolated from the biological sample are subjected to one or more amplification and / or cDNA / cRNA preparation steps that utilize labeled nucleotides in the preparation of the amplification / cDNA / cRNA products. For example (but not by way of limitation), one or more biotinylated nucleotides may be utilized in the sample preparation steps, and binding / hybridization of a target allele to a probe of the microarray device can be detected using a biotin binding agent (i.e., avidin, streptavidin, etc.); alternatively, one or more fluorescent dyes may be utilized in place of the biotin to label the nucleotides (i.e., up to four fluorescent dyes may be utilized, one for each nucleotide). In another alternative, fragmented nucleotide sequences isolated from the biological sample are allowed to first hybridize to a probe of the microarray device, and then labeled nucleotides (wherein each nucleotide is labeled with a different fluorescent dye or other label) are added to extend any single stranded portion of the hybridized complex.

[0048] In addition, each of the probes may be modified in any manner described herein or otherwise known in the art that will further aid in detection of the particular alleles / SNPs. For example, but not by way of limitation, each of the probes may have an enhancer of hybridization and / or fluorescence attached thereto.

[0049] One particular (but non-limiting) embodiment of the microarray devices utilizes the TaqMan® genotyping system. TaqMan® genotyping assays, considered the gold standard for allele detection, consist of pre-optimized PCR primer forward and reverse pairs and two probes for allelic discrimination, one to detect the allele (for example G or A) in forward sequence, and the other (C or T) in reverse sequence. The two TaqMan® probes have different fluorescent dyes at the 5' ends (for example, but not by way of limitation, one probe with a fluorescein derivative (FAM) dye and the other with a different fluorescent dye (VIC) at the 5' end). On the 3' end of both probes are minor groove binders (MGB) and nonfluorescent quenchers (NFQ). These assays are used to amplify and detect specific alleles in genomic DNA (gDNA). Genomic DNA is introduced into a reaction mixture consisting of TaqMan® Genotyping Master Mix, the forward and reverse primers and the two TaqMan® MGB Probes. Each TaqMan® MGB Probe anneals specifically to a complementary sequence, if present, between the forward and reverse primer sites.When the probe is intact, the proximity of the quencher dye to the reporter dye suppresses the reporter fluorescence. The exonuclease activity of AmpliTaq Gold® DNA Polymerase (ThermoFisher Scientific, Waltham, MA) cleaves only probes hybridized to the target. Cleavage separates the reporter dye from the quencher dye, increasing fluorescence by the reporter. The increase in fluorescence occurs only if the amplified target sequence is complementary to the probe. Thus, the FAM or VIC fluorescence signal generated by PCR amplification indicates which alleles are in the sample.

[0050] Certain non-limiting embodiments of the present disclosure are directed to kits for performing any of the methods disclosed or otherwise contemplated herein. The kits include one or more of any of the diagnostic microarray devices disclosed or otherwise contemplated herein. The kits may further include one or more of any reagents utilized in sample preparation and / or conductance of the assay. For example, but not by way of limitation, the kits may include one or more reagents utilized in the fragmentation of the DNA of the biological sample, amplification of the DNA of the biological sample, preparation of cDNA / cRNAfrom the biological sample, fluorescent dyes for attachment to the fragmented nucleotide sequences isolated from the biological sample, labeled nucleotides (such as, but not limited to, biotinylated nucleotides or fluorescently- or otherwise labeled nucleotides), and the like, as well as any combinations thereof.

[0051] In addition to the diagnostic microarray device(s) and sample preparation / assay reagent(s) disclosed herein above, the kits disclosed herein may further contain any other component(s) or reagent(s) for use when conducting any of the particular assays described or otherwise contemplated herein. For example (but not by way of limitation), the kit may further include positive and / or negative control reagents, wash solutions, etc. The nature of additional reagent(s) present in the kits will depend upon the particular microassay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary.

[0052] Also, the compositions / reagents present in the kits may each be in separate containers / compartments, or various compositions / reagents can be combined in one or more containers / compartments, depending on the reactivity and stability of the compositions / reagents. In addition, the kit may further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.

[0053] Certain non-limiting embodiments of the present disclosure are directed to method of treating or reducing the occurrence of at least one condition / disease in a subject, wherein the at least one condition / disease is selected from periodontal disease, Alzheimer's disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and the like. The method includes the steps of: contacting any ofthe diagnostic microarray devices disclosed or otherwise contemplated herein with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device; determining the alleles present for each of the SNPs ILIA-889, IL1B+3877, IL1B-511, / / .6-1363, IL10-592, IL10-1082, CD14-260, and / or COX2+8473 to define a genotype for the biological sample; and administering at least one therapeutic agent and / or recommending at least one therapeutic protocol to the subject when: (i) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GG; (ii) the genotype for the biological sample comprises / / .1B+3877CT and / / .1B-511GA, and wherein the genotype does not contain any of the following alleles: / / .6-1363T, IL10-1082C, and / or COX2+8473G; or (iii) the genotype for the biological sample comprises / / .1B+3877CC, IL1B-511GA, and / / .6-1363GT. In addition, the presence in the genotypes of (i), (ii), and / or (iii) of one or more of / £1A-889(GA), / £1A-889(AA), / £10-592(TT), CD14-260(AA), and / or COX2+8473(AG) can indicate severe disease, early tooth loss, and increased susceptibility to periodontitis-associated diseases and conditions, such as (but not limited to) Alzheimer's disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and the like.

[0054] The methods of the present disclosure may include any additional steps necessary for isolation and / or preparation of the biological sample prior to contact with the diagnostic microarray device. Non-limiting examples of additional steps that can be performed before contacting the biological sample with the diagnostic microarray include isolating nucleotide sequences from the biological sample; treating the isolated nucleotide sequences to one or more amplification and / or cDNA / cRNA preparation steps (with or without labeled nucleotides); and / or labeling the isolated nucleotide sequences. In addition, the incubating and determining steps may involve one or more particular (but non-limiting) steps specific for a particular microarray platform, as described in detail herein above.

[0055] Any therapeutic agent(s) effective in reducing the occurrence and / or severity of periodontitis may be utilized as the at least one therapeutic agent administered in accordance with the methods of the present disclosure. Non-limiting examples of therapeutic agents that may be utilized in accordance with the present disclosure include a furzinc oxide / zinc citrate toothpaste, a stannous fluoride toothpaste, an antimicrobial rinse, an oral antibiotic, an antibiotic gel, antibiotic microspheres, an antiseptic chip, Atuzaginstat (COR388), and the like, as well as any combinations thereof. Particular (but non-limiting) examples of antibiotics, antimicrobials, and antiseptics that may be utilized in accordance with the present disclosure include doxycycline, minocycline, chlorhexidine, carbamide peroxide, and the like, as well as any combinations thereof.

[0056] The therapeutic protocol(s) utilized in accordance with the present disclosure include any therapeutic protocols effective in reducing the occurrence and / or severity of periodontitis. Non-limiting examples thereof include performing dental scaling and root planing at an increased rate (i.e., increasing professional cleanings from once every six months to at least about once every month, every two months, every three months, etc.), as well as periodontal surgical options known in the art.EXAMPLE

[0057] An Example is provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein. Rather, the Example is simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[0058] Summary:

[0059] Susceptibility to periodontal disease (periodontitis) is unpredictable. This common, chronic inflammatory disease begins when inadequate oral hygiene allows salivary bacteria to adhere at and beneath the free gingival margin of teeth where they activate innate immunity. The epithelial attachment is irritated by mastication, and to repair itself it must keep replacing its cells to stay attached. This irritation activates traces of an inflammatory exudate called gingival crevicular fluid (GCF) to enter a crevice beneath the free gingival margin. Unlike saliva, GCF contains the essential amino acid, lysine. Moreover, the major proteins in saliva but not GCF are protected by glycoproteins covalently attached to surface side-chains of amino acids. These glycoproteins maintain a healthy microbiome in saliva, but once this microbiome adheres to teeth surfaces beneath gingival margins, bacteria can grow on the GCF intended for attachment turnover. Some of these bacteria, notably Eikenella corrodens, possess a constitutive surface enzyme called lysine decarboxylase (LdcE) which decarboxylates GCF lysine to form cadaverine. The loss of lysine retards the host mediated epithelial attachment from being repaired. The gingival crevice becomes impaired (leaky) to bacteria which can induce innate immunity and produce proinflammatory cytokines, initially interleukins 1A and IB, ILIA and IL1B (Levine and Lohinai, Adv Exp Med Biol (2026) 1492:485-507). I LI B is the primary form of IL1 associated with inflammation mediated by bacteria. Bacterial inflammation can develop into periodontitis or other microbially induced diseases.

[0060] As described herein, a curve of GCF / min against biofilm lysine concentration after a week of no oral hygiene was interpreted to show two parallel curves, strong or weak GCF responses (Figure 1).

[0061] Although the function of II1B+3877 was reported as unknown, the genetic patterns used in the Wu study (Wu et al., J Periodontal Res (2015) 50(l):52-61) include functional gene variations (SingleNucleotide Polymorphisms, SNPs) in the I LI B promoter region (20). The gene IL1B (+3877) rsll43633 was included in all haplotype patterns despite its uncertain function. Wu et al., provide studies indicating that IL1B (+3877) itself, or a locus in strong linkage disequilibrium, is important in differentiating multiple inflammatory disease phenotypes, especially in Asian populations. Indeed, some phenotypes have been validated as predictive of disease severity and treatment response. Wu et al., also cite combinations of an IL-1 genotype called PerioPredict that predict smoking associates with poor patient responses to periodontal therapy and complications of dental implants following placement. Other studies using Genome-Wide Association Studies (GWAS) have implicated independent markers of inflammation associated with periodontitis. Unfortunately, these markers occur as SNPs in genes that seem independent of each other. These genes NIN, NPY, WNT5A, NCR2, EMR1 and 10pl5, respond to periodontitis independently. In short, the field is currently constrained by the limited availability of adequate databases to fully explore and validate gene-gene interactions in periodontitis that may also differ between ethnic populations

[0062] Here we disclose a system called experimental gingivitis (EG) in which we examined SNP alleles of nine genes associated with periodontitis (Table 1). Surprisingly, a clear separation of the 15 participants was obtained by the inventors. Those possessing the reference A allele of IL1B-511 (A), or and the T dizygotic C allele (i.e. CC) of / / .1B+3877 were strong responders (Figs 2 and 3 and Tables 2 and 3). In two other individuals, the T allele of / / .6-1363 was epistatic; its presence changed the strong phenotype of / L1B+3877(T) from weak to strong in a host 5 with the conflicted genotype, and that of homozygous IL1B+3877(C) with heterozygous / / .1B-511A with from strong to weak (epistasis) in host 11. with the proviso that, If both IL1B-511(A) and / L1B+3877(T) occurred together, a conflicted genotype, was present, and one of the following responses occurred, epistasis occurred as described above for hosts 5 and 11. and Homozygous CD14-260(A) or homozygous IL10-592(T) assigned weak response, and homozygous I LIO-592592 (G) or a heterozygous COX2-8473(A) assigned strong response (Fig. 2 and Table 2). Together, these genotypes correctly identify strong or weak innate immune responses to microbial biofilms in all 15 of the participants available to provide a DNA sample (Figs 2 and 3).

[0063] It has long been recognized that cleaning teeth prevents gingival inflammation (gingivitis), but what has not been recognized is how the initial development of gingivitis activates innate immunity to bacteria. Innate immunity is a genetically controlled system of events in response to oral bacteria or any bacterial infection, but in the case of gingival inflammation (gingivitis), the initial host response was found to be caused by a complex mixture of bacteria from saliva. The weak innate immunity response phenotype identified herein increases susceptibility to periodontitis and other associated chronic diseases such asJ(but not limited to) Alzheimer's diseases.

[0064] Introduction:

[0065] Current therapy for preventing periodontal disease is primarily self-administered oral care (tooth brushing twice daily), supplemented with professionally administered care twice annually to remove precipitated calcium deposits (dental calculus), along with ensuring no smoking and controlling type 2 diabetes. The overarching need for lifelong oral hygiene indicates the presence in dentally attached biofilms of bacterial agents that induce gingival inflammation. The bacterial enzyme lysine decarboxylase from E. corrodens (LdcE) was identified as one of these agents (Lohinai, et al., J Periodontal. (2015) 86(10):1176-1184; and Lohinai, et al., J Periodontol. (2012) 83(8):1048-56). Other studies indicate that antibodies to this enzyme retard gingivitis development in the beagle dog model (Peters, et al., Vaccine (2012) 30(47):6706-12), and that its inhibition by zinc ions explains the well-known efficacy of zinc toothpastes in controlling human periodontal disease (Levine, et al., J Dent (2021) 104:103533). Unlike acid-induced lysine decarboxylases secreted from Escherichia coli and other gastro-intestinal bacteria (Ldcl), LdcE is constitutively present on the E. corrodens outer surface and is active in alkali in addition to dilute acid. The cadaverine produced by LdcE also contributes to changes in the microbiome of gingivodental biofilms and enhances growth on GCF instead of saliva within the gingival crevice.[0D66] The initiation of inflammation and its exacerbation by bacteria that adhere to teeth is called innate immunity, an initial, non-specific inflammatory response to an internal or external injury or foreign invader. Persistence of innate immunity induces acquired immunity, a highly specific response to its foreign antigens or the damage they produce. Host products of masticatory damage (DAMPs) and related microbial or pathogen activated microbial products (MAMPs or PAMPs) bind to pattern recognition receptors, PRRs. PRRs are mostly Toll-like receptors (TLRs) on the outer surface of epithelial cells such as the junctional epithelial attachment (JE), or nucleotide oligomerization domain receptors (NLRs) in the JE cytosol. Once bound, the DAMP-, MAMP-, or PAMP-PRR complexes activate alarmins such as ILIA that encodes a protein IL-1A which activates a related gene, IL1B in many bacterial diseases. The IL1B protein (IL-10) is strongly proinflammatory and induces neutrophils programmed to attract macrophages (M) that enhance (Ml) or stop the inflammation (M2). How this connects to IL1B is not clear. In this example, we will indicate that the difference in response may depend on a combination of upstream and downstream alleles of IL1B, at -511 and +3877 (Wu et al, J Periodontal Res, 2015: 15 (1): 52-61), not as previously reported at -511 and +3954 (Levine et al., IntJ Mol Sci, 2023, Vol 24(18). Neutrophils activated to enhance the damage and bring Ml macrophages to the infected site will strongly enhance GCF exudation, making the environment alkaline and causing itscalcium to precipitate as calculus. ¥et Once calculus has developed, calcified biofilms remain dentally attached or redevelop despite professional care.

[8067] As noted above, ILIA produces a protein (IL-la) that induces the expression of gene IL1B and activates its protein (IL-10). The amount of IL-10 is strongly associated with gingival and periodontal inflammation (Levine and Lohinai, J Clin Med (2021) 10(11); and Chapple, et al., J Clin Periodontol. (2015) 42 Suppl 16: S71-S76). Subsequently cleavage of the IL-10 protein by an extracellular serine protease, granzyme B, enhances IL-10 proinflammatory cytokine production (Afonina, et al., Mol Cell (2011) 44(2):265-78). During three weeks of EG, IL-la is secreted first, but IL- 10 expression catches up and then increases further as gingivitis persists and periodontitis develops (Tsalikis, et al., J. Int. Acad. Periodontol. (2002) 4(1):5-11; Offenbacher, et al., J Clin. Periodontol. (2010) 37(4):324-333). Therapy and the re-institution of oral hygiene downregulate IL-10 expression, and the removal of MAMPs reduces IL-la activity (Kaushik and Cuervo, Nat Rev Mol Cell Biol (2018) 19(6):365- 381). Nevertheless, mastication often maintains enough DAMPs to maintain a low-level activation of IL-6 (Dutzan, et al., Immunity. (2017) 46(1):133-147), and oral hygiene keeps the epithelial attachment intact by preventing LdcE from removing lysine.

[0068] Because the division of microbial response to LdcE activity in gingivitis and in periodontitis is significant, this Example initially investigated whether strong and weak inflammatory responses were genetically determined phenotypes after a week of experimental gingivitis (EG) in a population of healthy adults, ages 20 - 40. The strong (fast) or weak (slow) phenotype can provide a basis for deciding who requires drugs that prevent severe periodontal disease and its associated systemic chronic infections, such as (but not limited to) Alzheimer's disease, cardiovascular disease, and the like. The results presented in this Example demonstrate that an increased susceptibility to tooth loss should predominate in the weak phenotype.

[0069] Materials and Methods:

[0070] The EG procedures utilized in this Example are outlined in Lohinai et al. (2012).

[0071] The human protocol described below was reviewed and approved by the Ethics Committee of the Hungarian Medical Research Council (Approval no. 11878-1 / 2006-1017EKL), and Semmelweis University, Budapest, Hungary (Approval no. TUKEB 140 / 2006) as reported previously (Lohinai, et al., J Periodontol. (2012) 83(8):1048-56). At a follow-up visit, 15 of the 16 individuals who had participated in the prior EG study agreed to provide buccal cheek scrapings from which genomic DNA was purified by column separation. The 16th individual had emigrated from Hungary and was unavailable. SNP genotyping analysis used the TaqMan® assay method described by ThermoFisher Scientific (Waltham, Mass., USA).Each assay kit contained two probes, one in which the SNP of interest was present and one in which it was absent. One end of each probe had a covalently attached fluorescent molecule, and the other end an attached enhancer of both hybridization and fluorescence. Fluorescence measurements during genomic DNA amplification in the presence of both probes were graphed over time. From this graph, the intensity of fluorescence indicated whether the SNP was present in all of a host's genomic DNA, or in half or none of the DNA.

[0072] The SNP variants (alleles) were from 1 SNP site of ILIA, 3 of I LI B, 1 of IL6, 2 of I LIO, and 1 each of CD14, COX2 and MMP8. All are reported to influence periodontitis. These genes are listed in Table 1 (below) along with their Reference SNP (rs) numbers and MAFs. Each SNP is numbered by the position of the SNP-altered nucleotide before (minus) or after (plus) the first protein coding nucleotide. Significant associations of strong or weak phenotype with genotype were determined using Fisher's Exact Test (Table 2) below.Table 1 - Selected genes associated with periodontitis.Name&siteaAllelesbMAFacrs numberdLiterature Reference61. IL1A-BB9 G> A, C, T 0.285 rsl800587 (Kornman et al., 1997)2. IL1B+3877 OA, G, T 0.350 rsll43633 (Wu et al., 2015)3. / / .1B+3954 G> A 0.228 rsll43634 (Ferreira et al., 2008)4. IL1B-511 A> G 0.357 rsl6944 (Rogus et al., 2008)5. / / .6-1363 G> C, T 0.076 rs2069827 (Nibali et al., 2013)6.a IL10-592 T> G 0.294 rsl800872 (Wong et al., 2018)6.b IL10-1082 T> C 0.453 rsl800896 (Wong et al., 2018))7. CD14-260 G > A, C, T 0.485 rs2569190 (Nicu et al., 2009)8. COX2+8473 A> G, T 0.349 rs5275 (Prakash et al., 2015)9. MMP8-799 A> C, G 0.479 rsll225395 (Izakovicova Holla et al., 2012)aGene name and location of the allelic site are indicated by number of nucleotides before (+), or after (-) the translation start site.bReference nucleotide is first, the alternate SNP nucleotide(s) follow the > sign and the alternate nucleotide used is underlined if a choice is possible.cMinor SNP allele frequency (MAFa) validated in a large European population.dReference SNP cluster ID number.eLiterature references listed by line number on the left of the Table and detailed below.1. Kornman, K. S. et al. J Clin. Periodontol, 24(1), 72-77, 1977.2. Wu, X. et al. J Periodontal Res, 50(1), 52-61, 2015.3. Ferreira, S. B., Jr. et al. Infect Immun, 76(8), 3725-3734, 2008.4. Rogus, et al., Hum Genet (2008) 123(4):387-98, 2008.5. Nibali, L. et al. Clin Oral Investig, 17(1), 1235-42, 2013G. Wong, et al. BMC Oral Health Vol. 18(1) 171, 20187. Nicu, E. A. et al. Innate Immunity, 15(2), 121-1288. Prakash, G. et al. Oral Diseases, 21(1), 38-459.lzakovicova Holla, L. et al. Arch Oral Biol, 57(2), 188-196.Table 2: IL1B Alleles at -511 and +3194, Respectively, Indicate Strong or Weak Phenotypes IL1B+511 No A Wi AStrong 0 9Weak 3 3Fisher exact test statistic = 0.044, p <0.05.IL1B+3877 No Tor EpaWi T or EpStrong 7 2Weak 0 6Fisher exact test statistic = 0.007, p <0.01.aT allele or CC allele with epistatic (Ep) / / .6-1363T in the genome (original).

[0073] In FIGS. 2 and 3, each participant is listed by ID number, their strong or weak response, and the presence or absence of selected alleles from each of the nine genes. A medium gray background indicates that the SNP was present in only one of the two DNA chromosomes (heterozygous), a dark gray background with white letters indicates that the reference SNP allele was present on both chromosomes (homozygous reference SNP), and a light gray background with black type indicates that the alternate SNP was present on both chromosomes (homozygous alternate SNP). Data not relevant to the findings is evident as an empty white box or a white box containg the same data not required to distinguish a strong from a weak response in another participant. These changes in FIG 3 simplified understanding the underlying pattern of genetic associations reported in the Tables.

[0074] Statistical analysis: Using GCF exudation rate as the outcome, strong and weak GCF responses were examined using a quadratic polynomial regression model using the JMP Pro 13.1 computer program(SAS Institute Inc., Cary, NC.). Significance of the GCF response (main effect), and its interactions with lysine content and lysine content-squared (independent variables) were determined using an F-ratio test with 3 degrees of freedom to compare the sum of squares explained by the two independent terms (Lys and Lys-squared) relative to the sum of squares error. Differentiation of strong and weak responses by genes and genotypes in Table 2 was initially determined using the Fisher exact statistic (Social Science Statistics website).

[0075] Results:

[0076] In the population of 15 Hungarian young adults, the occurrence of five hosts with the tooth loss genotype, / / .1B+3954(A) plus / / .1B-511(A), was 33% which approximated that predicted from their minor allele frequencies (MAFs) in a large population (Karimbux, et al., J Periodontal. (2012) 83(ll):1407-1419). The preference of / / .1B-511(A) for strong response was significant (Table 2). The (T) allele of / / .1B+3877 for weak response was not significant unless the T allele of / / .6-1363 was also present. This allele changed the phenotype of a homozygous normal IL1B allele, / / .1B+3877(CC), from strong to weak, and that of a / / .1B+3877(CT) allele from weak to strong, a genetic event called epistasis (Wei, et al., Nat Rev Genet (2014) 15(ll):722-33). Because the / / .1B+3877 phenotype was subordinate to / / .6-1363(T) in the genome, it is referred to as the hypostatic gene and made the association of / / .1B+3877 with weak phenotype significant (Table 2). The epistatic / hypostatic activity is dominant, and identified in FIGS. 2 and 3 by a superscript 'ed.'

[0077] The current results are shown in FIG. 2, and a more complete rendition in FIG. 3. Table 3 shows the results: 1) 6 strong responders, the genotype of which is the reference SNP at / / .1B+3877(CC) plus the alternative SNP at / / . B-511(A); 2) 3 weak responders the genotype of which is the alternative SNP at / / .1B+3877(T) and the reference SNP at / / .1B-511(GG). Two hosts exhibited epistasis by the alternate T allele of / / .6-1363: a 7thhost with [ / / .1B+3877(CC) plus / MB-511(A)] was converted to weak, and a second host with a conflicted phenotype [( / / .1B+3877(T) plus / / .1B-511(A)] was converted to strong. There remained four hosts whose genotype was also conflicted, / / .1B+3877(CT), potentially weak, and IL1B-511(GA), potentially strong (Table 2).

[0078] FIG. 1 shows the relationship of genotype to strong and weak GCF response graphically by adding a number to indicate their respective positions on the graph. Two of these hosts exhibited a weak phenotype in the presence of the homozygous (A) allele of CD14-2601or the homozygous (T) allele of / / . ID-5922. The other two exhibited a strong phenotype if the second gene was the heterozygous (G) allele of COX2-8473(AG)3, or the major (C) homozygous allele of IL10-5924. FIG. 1 also indicates that the two individuals who possessed the epistatic / / .6-1363(Te) gene had the two smallest biofilm lysine contents ofall 15 participants in this Example. The most strong GCF responders were identified by the presence of the A allele of IL1B-511 along with the homozygous common (C) allele of IL1B +3877 (six responders), and weak GCF responders by the T allele of IL1B+3877 along with presence of the homozygous common (G) allele of IL1B-511 (three responders). In addition, one weak and one strong responder were determined by epistasis of / / .1B+3877(CC) and (CT) by the T allele of / / .6-1363, and the remaining four, who exhibited both / / .1B-511A and / / .1B+3877T, were resolved as strong or weak responders with unique third alleles as indicated in Table 3.Table 3: Summary of Genotypes That Detect Strong and Weak PhenotypesHost Phenotype Genotype1M S IL1B+3877CTedIL1B-511GA IL6-1363GTed2F S IL1B+3877CT IL1B-511GA IL10-592CCs3M S IL1B+3877CTs* IL1B-511GA COX2+8473AGs* 4FM S IL1B+3877CC 1B-511GA5F S IL1B+3877CC 1B-511GAF S IL1B+3877CC 1B-511GA7F S IL1B+3877CC 1B-511GA8F S IL1B+3877CC IL1B-511AA9M S IL1B+3877CC IL1B-511AA10M W IL1B+3877CT IL1B-511GA IL10-592TTs11M W IL1B+3877CCedIL1B-511GA IL6-1363GTed12F W IL1B+3877CT IL1B-511GA CD14-260AAs13 M W IL1B+3877CT IL1B-511GG14M W IL1B+3877CT IL1B-511GG15 F W IL1B+3877CT IL1B-511GGBold font indicates individuals whose phenotype depends only on alleles of / / .1B+3877 and IL1B-511. Underlining indicates individuals whose phenotype depends on epistasis of / / .1B+3877 alleles by IL6-1363T. No highlighting indicates individuals whose phenotype depends on a third second gene other than / / .6-1363GT Third Second genes with superscript 's' can make / / .1B-511GA dominant (strong phenotype), or / / .1B+3877CT dominant (weak phenotype). The superscript s* indicates the alternative 3rd gene to IL10-592CC for host 3.The rows of Table 4 summarize all 4 combinations of SNPs at / / .1B+3877 and IL1B-511 and relate them to GCF trait and periodontitis susceptibility. In row 1, the combination of two reference SNPs (CC) at / / .1B+3877 and the homozygous or heterozygous reference (A) allele at IL1B-511 was observed in 7 hosts. All but one produced the strong GCF phenotype which retards periodontitis development by removing bacteria from the biofilm. Traces of pathobionts are effectively removed from the biofilm, keepingperiodontitis susceptibility low. The seventh host was unique in also possessing / / .6-1363(T) which converted the strong GCF phenotype of / / .1B+3877(CC) to weak by epistasis. The reduced GCF flow permits pathobionts to persist longer and proliferate in GCF, enhancing the susceptibility to established periodontitis.In row 2, the alternate SNPs (T) at / / .1B+3877 and (G) at IL1B-511 were present in 3 hosts, all of whom had the weak GCF response that enhances pathobiont colonization and susceptibility to established periodontitis. The group in Row 3 had a conflicted genotype, featuring the alternate T allele at / / .1B+3877 with the homozygous or heterozygous reference (A) allele at IL1B-511. One / / .1B+3977(T) allele was converted from weak to strong by epistasis due to the presence of / / .6-1363(T). Two hosts exhibited a strong GCF response due to the presence of the reference SNP at / / .10-592(GG). In contrast, the presence of the alternate SNP (TT) at IL10-592, or (AA) at CD14-260 converted the GCF response from strong to weak. There was no representative for row 4, including a conflicted SNP pair, the homozygous reference SNP at / / .1B+3877(CC), potentially strong GCF response, with the homozygous alternate allele at IL1B -511(GG), potentially weak GCF response.Table 4. Summary of IL1B gene combinations, GCF trait, and susceptibility to periodontitis.# of Hosts IL1B+3877 IL1B-5111GCF trait2Periodontitis 7 47% / / .1B+3877(CC) IL1B-511(AG) 6S+lWeMild33 20% IL1B+3877 (CT) IL1B-511(GG) 3W Established45 33% IL1B+3877 (CT) IL1B-511(AG) 2S+lSe+2W52ndGene0 0 IL1B+3877 (CC) IL1B-511(GG) Not present6Unknown1GCF trait: S = Strong; W = Weak;eepistasis.2Periodontitis susceptibility:3No disease or Mild;4Moderate or Severe;52ndgene SNP determines periodontitis susceptibility;6due to absence.

[0079] GCF responses to biofilm lysine are therefore strong or weak, and governed by genotypes that are mostly based on one of two alleles of IL1B. The site located at IL1B-511 upstream of the encoded protein transcription site is protective, and that of / / .1B+3877 well downstream is destructive except in presence of / / .6-1363(T), in which the destructive and protective alleles of IL1B are reversed. These three genes indicate the strong or weak susceptibility to moderate and severe periodontitis of our Hungarian population with 73% accuracy. The various other third second SNP alleles of genesidentified in FIG. 3 increase the prediction to 100% accuracy, but additional third second alleles could be present in a largerpopulation.

[0080] Significance:

[0081] As noted above, the GCF response to biofilm lysine measures the strength of innate immunity to a mixture of bacteria as two parallel curves. Nine of the 16 tested participants possessed an innate immune response that was strong (upper curve), and seven participants possessed a response that was weak (lower curve). Fifteen of these individuals agreed to provide a sample of their DNA, but the 16th had emigrated and was unavailable. A strong response was associated with the SNP / / .1B-511(A), and a weak response with / / .1B+3877(T), unless / L6-1363(T) was present, and changed the response of / / .1B+3877(T) to be strong and that of its absence to be weak. Others have indicated that / / .1B+3877(T) and / L6-1363(T) indicate moderate to severe periodontitis, greater gingival inflammation, and bacterial dysbiosis (i.e., presence of the 'red' complex) in periodontal pockets (Wu, X., et al., J. Periodontal Res (2015) 50(l):52-61) (Nibali, et al., J Clin Periodontol (2008) 35(3):193-198). The remaining four individuals were characterized as strong or weak by the presence of a third second gene in the absence of / L6-1363(T).

[0082] Although the development of gingivitis during EG is thoughtto lead to periodontitis, observing that transition would cause an irreversible loss of periodontal attachment, making the experiment unethical. Nevertheless, the strong outward flow of GCF removes particles and bacteria from deepened crevices called periodontal pockets, suggesting less incubation time for red complex and periodontitis development. Likewise, the inhibition of GCF exudation by repeated tobacco smoking results in more incubation time for 'red' bacterial complex development, and a 5-fold greater likelihood of developing periodontitis. Indeed, smoking is a major risk factor for periodontitis. That a strong GCF response to gingivitis protects from dysbiosis, and a weak GCF response enhances dysbiosis, is supported in vitro (Naginyte et al., Sci Rep (2019) 9( 1):5491-5500). As noted above, a dysbiotic 'red' bacterial complex grows out from salivary bacteria in an in vitro fluid resembling GCF after the growth medium is changed twice weekly for three weeks. Thus, changing the medium more frequently would retard 'red' complex development and periodontitis. The rate of GCF exudation in FIG. 1 was about twice as great in strong responders than in weak responders. Therefore, the presence of genotypes associated with strong GCF exudation indicate that periodontitis and its related diseases should be controlled more easily than those with weak GCF exudation.

[0083] Others have reported statistically coherent subgroups of gingivitis development during EG, 36% fast and 64% slow (Nascimento, et al., European Journal of Oral Sciences (2019) 127(l):33-39) or 42% fast, 28% intermediate, and 28% slow (Bamashmous, et al., PNAS USA (2021) 118(27)). Compared with the 56-60% of strong responders in this Example, the separation of strong from weak responders in theseother studies appears incomplete, although saliva or GCF in fast responders exhibited greater levels of other pro-inflammatory cytokines than slow responders in both studies (Nascimento, et al., Cytokine (2019) 115:135-141; Bamashmous, et al., PNAS USA (2021) 118(27)). It was We concluded that the use of biofilm accumulation (PI) instead of biofilm lysine identified only the strongest and fastest of the GCF responders. Also, the use of clinical gingivitis to measure inflammation requires at least two weeks of EG, whereas GCF exudation can measure inflammation accurately at one week (Trombelli et al., 2004 J Clin Periodontal (2004) 3 l(4):239-252).

[0084] It has been noted herein that there are two levels of response to periodontitis, with a slightly smaller fraction of the population developing more disease. In another study, middle-aged, non-smoker patients from the University of Connecticut with periodontitis were enrolled between 2010 and 2012 (Hong, et al., PloS one (2015) 10(5):l-14:e0127077; Hong, et al., PloS one (2016) ll(2):e0148893). Nineteen had few deep pockets (crevices <5 mm deep) and a microbiome cluster, in which P. gingivalis accounted for about 12% of the biofilm bacteria. The remaining 15 participants (44% of the total) exhibited significantly more periodontal inflammation, deeper pockets (crevices >5 mm), and dysbiotic biofilms. Moreover, P. gingivalis accounted for 56% of the bacteria along with greater amounts of the accompanying red complex, suggesting that and therefore the a weak GCF responder phenotype (Hong, et aL, PloS one (2015) 10(5):l-14:e0127077). The 44% of patients possessing the cluster-enriched red bacteria have were also been indicated by the presence of the / / .1B+3954(GA) genotype in another study (Pani, et al., J Periodontal Res (2021) 56(3):501-511). However, testing for / / .1B+3954 would only be positive at some point after dysbiosis had developed, not at the level of experimental gingivitis.

[0085] Application to periodontal and Alzheimer's disease susceptibility: Taken together, the results demonstrate that moderate to severe periodontitis is caused by weak innate immunity that enhances susceptibility to biofilms possessing P. gingivalis, the keystone bacterium of the red microbial complex. As already noted, P. gingivalis is present in the brain of more than 90% of Alzheimer's patients after autopsy (Dominy, et al., Sci Adv. (2019) 5(l):eaau3333), and patients exhibiting moderate periodontal disease for 10 years have a nearly two-fold greater risk of developing Alzheimer's disease.; however, Nevertheless, whether periodontal disease is the cause or effect of cognitive decline on oral hygiene is still uncertain (Chen, et al., Alzheimers Res. Ther. (2017) 9(1):56). When 135 Chinese patients with Alzheimer's disease were classified into 108 G and 27 AA (i.e., non-G) IL1B-511, individuals differed only in the entorhinal-cingulum axis. This axis modulates the critical, direct white matter connections between the entorhinal cortex and the cingulate cortex via the cingulum bundle. All three components form a crucial neural circuit for memory and emotion. Interleukin-1 beta C-511A polymorphism modulates the structural covariancestrength on the anterior brain network and entorhinal-interconnected network independently of white matter tract integrity. Depending on the specific C-511 A genotype i.e. AA, GG, or AG, different network clusters could predict the cognitive test results.

[0086] Our Example reported that strong or weak innate immunity to the initial accumulation of biofilm in 73% of young adults is genetically determined by different SNP alleles of IL1B alone or by epistasis in the presence of / / .6-1363(GT). The remaining 4 individuals possess contradictory alleles of IL1B resolved by a third second gene's SNP allele which varies between individuals. Another SNP ( / / .1B+3954) associates with a greater presence of the red complex bacteria (Pani, et al., J Periodontal Res (2021) 56(3):501-511) and greater levels of P. gingivalis in the oral cavity, but not with the weak trait described in this Example. Therefore, based on the report that all patients exhibiting moderate periodontal disease for 10 years have a nearly two-fold greater risk of Alzheimer's disease, separating them into strong and weak responders indicates a four to five-fold greater risk for weak responders.

[0087] The results of this study could determine individual susceptibility to established periodontitis and its numerous comorbidities. Personalized therapy is a major aim of modern periodontal and medical research, although a proof-of-concept study is required to validate these our results. Our approach is illustrated in Table 5 which shows how a strong or weak GCF trait can be determined as strong or weak based on the SNP genotypes identified in Table 5 below. 4? Potential genetic screening methods could include a microarray-based approach that can be used to detect specific SNPs associated with periodontal susceptibility. In this method, the known SNP sequence(s) are pre-loaded on a glass slide, where they serve as a probes to detect matching sequences in a DNA sample from patients. A cheek scraping from which DNA could be obtained is also needed. To prepare a human DNA sample for detection in this method, the DNA is first extracted from the sample and fragmented and labeled with a fluorescent dye. The last step is to denature the DNA, which makes it single-stranded for hybridization to complementary probes previously attached on the glass slide (Conzone et al., Materials Today, (2004) 7(3), 20-26.). However, in practice, SNP testing could be performed through a genetics company, and the focus should be on encouraging patients to maintain a strong GCF phenotype and reserve intensive therapy and prevention for patients with a weak phenotype. Because periodontitis is uncommon before age 30 (Eke et aL, J Dent. Res (2012), 91(10), 914-920), it should be recommended that all individuals be tested as proposed in Table 5 below to individualize therapy towards prevention in strong responders or therapy as necessary in weak responders.Table 5. Determination of GCF trait from SNP allelic genotypes.Strong GCF Trait (Periodontitis Protected) Host%SIL1B+3877(CC)+IL1B-511(AG)1a40.00%IL1B+3877(CT)+IL1B-511(AG)+IL6-1363(GT)1b6.67%IL1B+3877(CT)+IL1B-511(AG)+IL10-592(GG)1c13.33%Weak GCF Trait (Periodontitis Susceptible) Host%SIL1B+3877(CT)+IL1B-511(GG)2a20.00%IL1B+3877(CC)+IL1B-511(AG)+IL6-1363(GT)2b6.67%IL1B+3877(CT)+IL1B-511(AG)+IL10-592(TT)2c6.67%IL1B+3877(CT)+IL1B-511(AG)+CD14-260(AA)2d6.67%1aStrong GCF trait;2aWeak GCF trait.1bBold=gene IL6-1363(GT) acts on IL1B+3877(CT) to produce a strong GCF trait and on2bIL1B+3877(CC) to produce a weak GCF trait.1cIL10-592(CC) or COX2+8473(AG) identifies a conflicted genotype as strong GCF trait, and2cIL10-592(TT) or2dCD14-260(AA) as weak trait.

[0088] The SNPs of IL1 and other genes identified in this study are a consequence of evolutionary processes that have shaped host immune responses. A strong or weak GCF response depends primarily on what SNPs occur at different sites on gene IL1B at residues +3877 and -511 (see rows 1 and 2 of Table 4). Protection from periodontitis depends on signals that activate acquired immunity. A phenotype that promotes a strong inflammatory GCF trait from the predominant symbionts can inhibit the development of pathobionts prior to age 30. The inflammasomes in response to LdcE-mediated loss of the epithelial barrier at the crevice base activate two IL1 proteins to attract PMNs and monocytes. Monocytes are pre-immune macrophages that differentiate by interacting with neutrophils. The macrophages adopt a proinflammatory (Ml) or reparative (M2-type) response. Nevertheless, an environmental factor such as persistent smoking heats the oral cavity and promotes weak GCF exudation and the development of 'red' complex pathobionts unrelated to the genetic information from our study.

[0089] The weak GCF trait leaves the GCF fluid stagnant, creating a more anaerobic environment and a better nutrient source for pathobionts than saliva. Symbionts such as Actinomyces and viridans-group streptococci cannot survive compete, and anaerobic pathobionts such as the 'red' complex gradually increase and eventually appear in saliva. These bacteria secrete proteolytic enzymes that break the crevice barrier open, and more inflammasomes result in more IL1 production, more GCF containing more activated PMNs, and more Ml macrophages to help remove the ever-increasing bacterial invaders andhost cell debris. In smokers and weak responders, gingivitis and periodontitis likely appear earlier than in strong responders.

[0090] Thus, the difference in GCF trait likely provides an environment where inflammation is more controlled (strong GCF) and one that is less controlled (weak GCF). The strength of the initial inflammatory exudate (GCF), invoked by innate immune IL1B SNP variation, determines a GCF trait that either prevents or facilitates pathobiont colonization. This means that weak responders are genetically predisposed to the development of established periodontitis in the absence of an environmental factor such as adequate oral hygiene.The invention also relates to the treatment of a patient identified as a weak responder. In particular, Alternatively, the patient is susceptible to any of the diseases defined herein. In preferred embodiments the invention relates to an anti-inflammatory compound, preferably a Zn or Sn providing toothpaste, preferably a furzinc oxide / zinc citrate toothpaste or a stannous fluoride toothpaste, or a chemically modified curcumin as defined herein, for use in the treatmentor reducing occurrence of a disease selected from the group consisting of periodontal disease, Alzheimer's disease, a cardiovascular disease, arthritis and adverse pregnancy outcomes in a subject diagnosed to be susceptible for a disease or being a weak responder as defined herein. Preferably, the invention relates to any of these compounds for use according to the invention wherein the treatment or reducing occurrence of a disease comprises regular review. Preferably at least one therapeutic protocol comprises first performing dental scaling and root planing at least about once every three months to control the natural response in the patient. In a particular embodiment the treatment is interrupted if the natural response of the patient is impaired, especially in response to chemically modified curcumin described below.

[0091] Therapy for periodontitis has long been aimed at inhibiting the collagenase activity caused by the IL1 mediated activation that produces excessive amounts of collagenolytic enzymes, especially the PMN matrix metalloproteinases (MMP-8, MMP-9, MMP-12 and MMP-13). This cascade led to a discovery that some tetracycline antibiotics and their derivatives can also inhibit these MMPS by mechanisms unrelated to their antibiotic activity (Golub et al., Periodontol 2000, 82(1), 186-204; Golub et al., Advances in Dental Research, (1998) 12(2), 12-26.). However, a new agent, a chemical modification of curcumin called CMC2.24 was tested in canine models over a 3-month period. Compared to placebo controls, CMC2.24 treatment significantly reduced gingival inflammation (gingival index, GCF flow), pocket depth (PD), and the numbers of pockets (PD>4mm), compared to placebo. CMC2.24 also significantly reduced active MMP-9 and MMP-2 in gingival tissue, alveolar bone loss and GCF IL-1β (Deng et al., Journal of Experimental Pharmacology, (2020) 12, 47-60.). It is plausible that a chemically modified curcumin (IUPACname: (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione), preferably an arylaminocarbonyl-comprising derivative of curcumin, in particular a phenylaminocarbonyl-comprising derivative thereof, highly preferably a 4-phenylaminocarbonyl derivative, is useful in a subject diagnosed to be susceptible according to the present invention. In a particular embodiment the derivative is a 4-arylaminocarbonyl-comprising derivative of curcumin.

[0092] A subsequent in vitro study using rat macrophages showed that CMC2.24 acts as a potent inhibitor of the pro-inflammatory Ml phenotype, while promoting the pro-resolving M2 phenotype, thus acting as a critical "switch" that reduces inflammation (Deng et al., Scientific Reports, (2023) 13(1), 15513). CMC2.24 is a derivative of curcumin, a natural spice that was also reported to prevent periodontitis. If CMC2.24 is used like doxycycline, as a supplement to standard periodontal therapy and continued for no more than a month, CMC2.24 may be a better alternative if approved by the US government. However, without long-term continued periodontal supervision from the periodontist, this drug may disrupt the natural immune response that slows the rate of destruction, potentially leading to disease recurrence worse than before. On the other hand, patients who have the weak GCF trait, under the age of 40, and without established periodontitis might, under monthly supervision by a periodontist, be given CMC2.24 to delay or prevent disease development.

[0093] While not wishing to be bound by a particular theory, once periodontal pathogens develop in dental biofilms and enough P. gingivalis is present, these bacteria might enter the bloodstream through a periodontal pocket (deepened crevice) whose epithelial barrier is inflamed and compromised by gingipains, powerful proteases that may eventually cause a gingipain-mediated brain infection. The ensuing disruption of equilibrium between pro- and anti-inflammatory mediators within the brain likely results in chronic neuroinflammation that eventually promotes Alzheimer's disease (Dominy et al. (2019) 5:1 eaau3333).

[0094] Lipoprotein E4 (apoE4) is a genetic variant of the apolipoprotein E protein that is a well-established major risk factor for Alzheimer's disease and other neurodegenerative disorders. It plays a role in lipid transport but is structurally unstable, leading to a higher risk of atherosclerosis and an increased risk of heart disease. The apoE4 variant can disrupt cellular functions, including mitochondrial metabolism and transport, and is associated with an increased risk of coronary artery disease and Alzheimer's. The ApoE4 gene is therefore a genetic factor associated with both vascular and Alzheimer's dementia and periodontitis (Cichonska Int J Mol Sci (2024) 25:5), the weak responders should be especially likely to develop this disease without adequate therapy along the suggestions discussed above. Although a similar situation is caused by mutations of genes encoding immune receptors, especially the triggeringreceptor expressed on myeloid cells type 2 (TREM2). TREM2 deficiency reduces clustering of the microglia that digest the plaques associated with Alzheimer's disease development. These plaques are accumulated protein fragments between neurons. If not continuously removed, they disrupt the brain's normal disposal processing and eventually impact cognition. Unfortunately, The frequency of TREM2 mutations is very low, with the most significant risk variant, R47H, having a minor allele frequency (MAF) of approximately (0.26%) in European American populations. By comparison, The frequency of the apoE4 allele varies significantly by ancestry, with the highest rates in certain indigenous populations and the lowest in East Asian and Mediterranean populations. In the general U. S. population, the allele frequency is approximately 14%, and it is present in about 25% of people in this population. This allele is the strongest genetic risk factor for late-onset Alzheimer's disease, but having does not guarantee a diagnosis. These and other substantial genetic and functional findings point to a central role for weak innate immunity in neurodegenerative diseases (Nara, etal., Journal of Alzheimer's Disease: JAD (2021) 82(4):1417-1450; Shi, et al., Nat. Rev. Immunol. (2018) 18(12):759-772) and are therefore relevant to this disclosure. Therefore, the present disclosure includes the addition of apoE4 and perhaps the major TREM2 variants such as (but not limited to) R47H to the devices, kits, and methods of the present disclosure to better account for Alzheimer's disease risk.

[0095] Atuzaginstat (COR388) is an orally administered brain-penetrating small molecule gingipain inhibitor, and a potentially new drug candidate that inhibits Kgp action in vitro and in mice (Dominy, et al., Sci Adv. (2019) 5(l):eaau3333). In a study conducted by Cortexzyme (San Francisco, CA), the efficacy of Atuzaginstat on clinical endpoints of periodontitis was measured in 233 volunteers. According to their disclosure, antibody measurements were expected to exhibit biofilms that had a high content of P. gingivalis determined by quantitative antibody measurements. Unfortunately, this approach indicates immune responsiveness, not antigen load. Excessive amounts of antigen can cause immune-suppression, and weak innate immunity can prevent a strong antibody response from developing. Consequently, the weak innate immunity response genotypes disclosed herein could solve this problem. The genotypes could determine which participants in that study were likely to have small amounts of P. gingivalis in their biofilm (strong genotype) as well as those likely to have much larger amounts (weak genotype). As the latter group would be most likely to show an effect of the drug Atuzaginstat, it would determine clearly whether this or a related drug can provide significant protection from P. gingivitis periodontitis in adults 10 - 20 years prior to developing Alzheimer's disease.

[0096] Conclusion:

[0097] The percentage of the US population diagnosed with Alzheimer's dementia increases to 35%by age 85, similar to the fraction of those over aged 30-65 with moderate to severe periodontal disease, and to the fraction of individuals at age 50 exhibiting excessive biofilm colonization with P. gingivalis. Thus, testing at least individuals of European descent for the genotypes described herein improves efforts to predict and prevent not only periodontal disease, but also Alzheimer's Disease, cardiovascular disease, and other periodontitis-associated diseases.

[0098] The two host genotypes described in this Example may explain why human biofilm microbiomes are expressed as two distinct clusters. One group's genotype is associated with allele IL1B-511(A). Their GCF is strong enough to remove bacteria from the gingival crevice and retard P. gingivalis-mediated periodontitis and Alzheimer's disease. The other group's genotype is associated with allele / / .1B+3877(T) and epistasis of its non-T allele by / / .6-1363(T). These alleles promote a weaker GCF exudate that lets more bacteria remain in the gingival crevice where they promote much more P. gingivalis colonization, and more disease (Hong, et al., PloS one (2015) 10(5):e0127077; Hong, et aL, PloS one (2016) ll(2):e0148893).

[0099] Thus, in accordance with the present disclosure there have been we have provided compounds, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure, specifically the ability to determine periodontitis susceptibility at any age by applying the genetic test of Table 5.

Claims

CLAIMS1. A diagnostic assay for determining susceptibility to periodontal disease in a subject, by determining whether the subject is a strong or weak GCF responder to biofilm lysine, said method comprising the steps of:• examining, in a biological sample obtained from said subject, SNPs in the following genes associated with periodontitis: IL1B and IL6, and preferably IL10, CD14 and C0X2• determining the presence of the following SNPs:o an SNP at IL1B+3877, an SNP at IL1B-511, and an SNP at IL6-1363, and preferably o one or more SNP(s) selected from the group consisting of an SNP at IL10-592 and an SNP at CD14-260 and an SNP at COX2+8473,• considering the subject a weak GCF responder and thereby periodontal disease susceptible, if said subject has an SNP genotype selected from the group of the following SNP genotypes: o IL1B+3877(CT)+IL1B-511(GG),o IL1B+3877(CC)+IL1B-511(AG)+IL6-1363(GT), and, if preferably an SNP at IL10-592 and an SNP at CD14-260 is also determined,o IL1B+3877(CT)+IL1B-511(AG)+IL10-592(TT)o IL1B+3877(CT)+IL1B-511(AG)+CD14-260(AA)• and optionally• considering the subject a strong GCF responder and thereby periodontal disease protected, if said subject has an SNP genotype selected from the group of the following SNP genotypes: o IL1B+3877(CC)+IL1B-511(AG)o IL1B+3877(CC)+IL1B-511(AA)o IL1B+3877(CT)+IL1B-511(AG)+IL6-1363(GT), oro IL1B+3877(CT)+IL1B-511(AA)+IL6-1363(GT), and if preferably an SNP at IL10-592 and an SNP at COX2+8473 is also determined,o IL1B+3877(CT)+IL1B-511(AG)+IL10-592(GG)o IL1B+3877(CT)+IL1B-511(AG)+COX2+8473(AG).

2. The diagnostic assay for determining susceptibility to periodontal disease in a subject according to claim 1, wherein periodontal disease is selected from the group consisting of: periodontitis, gingivitis, periimplantitis, and preferably any associated comorbidity or disease thereof, or at any age to predict periodontitis susceptibility3. The diagnostic assay according to any of claims 1 to 2, wherein the presence of the SNPs is determined by contacting a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one or two allele(s) of a single nucleotide polymorphism (SNP),said SNP being selected from the group consisting ofIL1B+3877, IL1B-511, IL6-1363 and, preferably, and IL10-592, COX2+8473, and optionally CD14-260.

4. Certain non-limiting embodiments of the present disclosure are directed to method of treating or reducing the occurrence of at least one condition / disease in a subject, wherein the at least one condition / disease is selected from periodontal disease, Alzheimer's disease, cardiovascular disease, arthritis, adverse pregnancy outcomes, and the like. The method includes the steps of: contacting any of the diagnostic microarray devices disclosed or otherwise contemplated herein with at least a portion of a biological sample from the subject and incubating the diagnostic microarray device under conditions that allow for detection of nucleic acid bound to any of the probes associated with the substrate of the diagnostic microarray device.

5. A diagnostic microarray device comprising: a substrate, preferably formed of a glass material; wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one or two allele(s) of a single nucleotide polymorphism (SNP),said SNP being selected from the group consisting ofIL1B+3877, IL1B-511, IL6-1363 and, preferably, and IL10-592 COX2+8473, and optionally CD14-260.

6. The diagnostic microarray device of claim 5,said device comprising:a substrate preferably formed of a glass material;wherein a plurality of probes attached or otherwise associated with the substrate, each probe being specific to one allele of a single nucleotide polymorphism (SNP),said plurality of probes comprising at least the following probes:a probe that detects a C allele of IL1B+3877;a probe that detects a T allele of IL1B+3877;a probe that detects a G allele of IL1B-511;a probe that detects an A allele of IL1B-511;a probe that detects a G allele of IL6-1363;a probe that detects a T allele of IL6-1363.

7. A diagnostic microarray device of any of claims 5 to 5 further comprisinga probe that detects an A allele of COX2+8473; anda probe that detects a G allele of COX2+8473.a probe that detects a G allele of IL10-592;a probe that detects a T allele of IL10-592;a probe that detects a G allele of CD14-260a probe that detects an A allele of CD14-260.

8. The diagnostic microarray device of any of claims 5 to 7, wherein the substrate is formed of a glass ionomer material.

9. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to periodontal disease, preferably gingivitis, periodontitis and / or periimplantitis.

10. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to Alzheimer's disease.

11. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to cardiovascular disease.

12. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to arthritis.

13. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to adverse pregnancy outcomes.

14. Use of the diagnostic microarray device of any of claims 5 to 8, for detecting susceptibility of a subject to metabolic syndrome.

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