Genetic polymorphism

everything related to genetic polymorphism

1. GENETIC POLYMORPHISM Presented By Dr. M. Shiva Shanker Ist Year Post Graduate Student , Dept of Periodontics, Mamata Dental College.

2. contents Introduction Genetics Terminologies Types of genetic disorders Single gene defects Multifactorial diseases Methods of genetic analysis – genetic study designs Human gene polymorphisms Various type of polymorphisms and their relationship to the periodontal disease Proving a disease/polymorphism relationship Future application of genetic information Conclusion

3. introduction The science of genetics is concerned with the inheritance of traits, whether normal or abnormal, and with the interaction of genes and the environment. Individuals may respond differently to common environmental challenges, and this differential response is influenced by the individual’s genetic profile . The genetic origin of a disease is either partly or wholly due to abnormalities within the genetic code.

4. cell Cells are the basic building blocks of all living things. The human body is composed of trillions of cells. They provide structure for the body Cells consists of various parts

5. Nucleus The nucleus serves as the cell’s command center, sending directions to the cell to grow,mature , divide, or die. The nucleus is surrounded by a membrane called the nuclear envelope, which protects the DNA and separates the nucleus from the rest of the cell.

6. chromosome In the nucleus of each cell, the DNA molecule is packaged into thread-like structures called chromosomes . Each chromosome has a constriction point called the centromere. centromere divides the chromosome into two sections, or “arms .” The location of the centromere on each chromosome gives the chromosome its characteristic shape, and can be used to help describe the location of specific genes .

7. dna DNA consists of chemically linked sequences of nucleotides ; these are the "building blocks" of the DNA and always contain a nitrogenous base. Four nitrogenous bases exist: adenine (a), guanine (g), cytosine (q and thymine (t). The bases are linked to a sugar (2-deoxyribose), where a phosphate group is also added.

8. DNA contain the genetic code .The genetic code is read in groups of three nucleotide ,and called as “codon”. Coding region Promoter region

9. Variant forms of a gene that can occupy a specific chromosal site (locus) are called alleles . when different alleles of a given gene co-ex­ist in the human population, we speak about genetic polymorphisms . An alteration that changes in single point base pair is called a point mutation The most common class of point mu­tations is the transition, comprising the substitution of one nucleotide with another Termed as single nucleotide polymorphism

10. TERMINOLOGIES: Allele: alternate forms of a given gene differing in DNA sequence assumed to arise by mutation and affecting the function of a single product. Chromosome: a nuclear structure carrying genetic information arranged in a linear sequence. Genetic code: the consecutive nucleotide triplets that specify the sequence of aminoacids for protein synthesis. Genome: a term used to refer to all the genes carried by an individual or cell

11. Genotype: the genetic makeup of an organism or cell distinct from its expressed features. Locus: the physical location a gene occupies within a chromosome or portion of genomic DNA. Linkage: the tendency for certain genes to be inherited together due to their presence on the same chromosome. Linkage disequilibrium: occurrence of some genes together more often than would be expected by random distribution Polymorphism: when a mutation increases to a level involving more than 1% of the population, it is referred to as a polymorphism, which merely means that there are multiple forms of the specific nucleotide sequence that may be found at the genetic locus. Single nucleotide polymorphism ( snp ) : common, but minute, variations that occur in human DNA at a frequency of one in every 1,000 bases. These variations can be used to track inheritance in families. SNP is pronounced, "snip".

12. TYPES OF GENETIC DEFECTS Mendelian disorders (single-gene defects) Non- mendelian disorders (multifactorial disorders)

13. SIMPLE Vs MULTIFACTORIAL GENETIC TRAITS SIMPLE MENDELIAN TRAITS Mutations of critical genes alter gene function causing disease phenotype. Mutations of a specific gene are typically rare (<1%). Inherited in a predictable pattern-simple mendelian fashion. MULTIFACTORIAL GENETIC TRAITS   Complex diseases result from interactive effect of multiple gene products modulated by environmental agents. Genes responsible are not altered (mutated) instead they exist in multiple different forms(alleles) which differ in terms of function- functional polymorphism. Polymorphisms can be enhanced by environmental agents Difficult to estimate the degree of interaction between a range of genotypes & environment.

14. METHODS OF GENETIC ANALYSIS Evidence for a genetic predisposition to periodontitis comes from four areas of research: The study of inherited diseases and genetic syndromes Family studies Twin studies Population studies Linkage and association studies They are used to map disease alleles to specific regions on chromosomes.

15. Human gene polymorphism When a nucleotide change is very rare, and not present in many individuals, it is often called a mutation. In contrast to mutations, genetic polymorphisms are usually considered normal variants in population. When a specific allele occurs in at least 1% of the population, it is said to be a genetic polymorphism.

16. Polymorphism Polymorphisms of human genes occur at one or more of the following

17. Types of Polymorphisms Single nucleotide polymorphisms (SNP) The most common form of polymorphisms is the single nucleotide polymorphism, which is a change in a single base pair ( bp ) in the genomic DNA. Single nucleotide polymorphisms can affect gene function. For example, a single nucleotide polymorphism located in a promoter region may influence the amount of mrna produced. Another class of polymorphism is the simple sequence repeats, of which the common forms are the dinucleotide and trinucleotide repeats.

18. The variable number of tandem repeats (VNTR) can also influence the function of a gene, but the repeats are more likely to be linked with a functional polymorphism elsewhere in the gene. Another category of gene polymorphism involves insertions or deletions. Insertions and deletions can be as small as 1 base, in which case they may also be classified in the category of single nucleotide polymorphisms, but can also consist of a few bases, one or more exons, or even a whole gene.

19. Interleukin-1 gene polymorphism Interleukin-2 gene polymorphism Interleukin-4 gene polymorphism Interleukin-6 gene polymorphism Interleukin-10 gene polymorphism Interleukin-12 gene polymorphism Interleukin-18 gene polymorphism Tgf -β gene polymorphism Fc receptor polymorphism Tnf -α Human leukocyte antigen polymorphism CD14 gene polymorphism Toll like receptor gene polymorphism CARD15/NOD2 gene polymorphism N- formyl -l- methionyl -l- leucyl -l-phenylalanine receptor polymorphism MMP polymorphism Cathepsin C polymorphisms Vitamin D receptor polymorphism Calcitonin gene polymorphism RANK/ osteoprotegerin gene polymorphism Plasminogen activator gene polymorphism

20. Nat2(n- acetyltransferase ) gene polymorphism Fibrinogen gene polymorphism Estrogen receptor-α gene polymorphism Cytochrome P450 gene polymorphism Rage(receptor of advanced glycation end products) gene polymorphism Ccr5( crotonyl coenzyme reductase 5) gene polymorphism Lactoferrin gene polymorphism Interferon-γ gene polymorphism.

21. POLYMORPHISM IN RELATION TO PERIODONTAL DISEASES Cytokine gene polymorphisms IL-1 gene polymorphism Tnf -a gene polymorphism IL-10 gene polymorphism Receptor and other gene polymorphisms Fcγr gene polymorphisms FcγrIIa-131 H/R polymorphism FcγrIIIa-158 F/V polymorphism FcγrIIIb polymorphism Cytokine and chemokine receptor gene polymorphisms Immune receptor gene polymorphism FMLP receptor gene polymorphism

22. Metabolism – related gene polymorphism Vitamin D receptor gene polymorphism Calcitonin receptor gene polymorphism Antigen – recognition related gene polymorphism HLA gene polymorphism Polymorphisms in the innate immunity receptors TLR2 and TLR4 gene polymorphisms CD 14 gene polymorphism CARD 15 gene polymorphism Miscellaneous gene polymorphisms

23. IL-1 gene polymorphism Interleukin 1 is a multifunctional cytokine affecting most cell types with potent inflammatory and stimulatory properties. The il-1 b gene located on chromosome 2 juxtaposed to the genes which encode the other cytokines of the il-1 family and their receptors ( nicklin et al.1994). A cluster of genes regulating the production of these cytokines has been identified on the long arm of chromosome 2. (2q13). This cluster consists of the IL-1A, IL-1B and the I l- ra (receptor antagonist) genes.

24. In 1997, kornman et al found an association between polymorphisms in the genes encoding for il-1α and il-1β and an increased severity of periodontitis in caucasians . He reported 29.1% of northern european caucasian population evaluated as being genotype positive. In this study it was shown that individuals who carry a composite genotype (allele 2 of the il-1a polymorphism plus allele 2 of the il-1b polymorphism) are associated with a more severe periodontitis Genotype positive subjects have been described as presenting more rapid periodontal breakdown, increased bleeding on probing and an increased production of IL-1 by monocytes to the same bacterial challenge

25. Periodontal Susceptibility Test [PST ] It is modified to assess IL-1A +4845 & IL-1B +3954. Steps include:- Blood collection Genotyping Amplification and Cycling.

26. Meisel et al 2002 Chronic 154 subjects caucasian Smokers and non smokers Significant Quappe et al 2004 Aggressive 36 patients with AgP , 75 healthy controls, and 75 subjects of unknown periodontal status chile Smokers and non smokers Significant Meisel et al 2004 Chronic Randomly selected population based study caucasian Smokers and non smokers significant Lopez et al 2005 chronic 330 patients with periodontitis and 101 healthy controls Chile Smokers and non smokers Significant Wagner et al 2007 Chronic 194 individuals caucasian Non smokers Significant Ferreira et al 2008 chronic 292 individuals Brazilian Non smokers Significant Bernadette et al 2012 Chronic 49 with moderate 79 with severe chronic, 78 with aggressive periodontits Mixed Non smokers Significant Sasha et al 2013 55 individuals whites smokers significant

27. INTERLEUKIN-10 (IL-10) POLYMPRPHISM The biological activities of IL-10 comprise suppression of pro-inflammatory cytokines produced by Th-1 cells (IL-2, ifn -g, TNF). The promoter region of the IL10 gene exhibits high frequencies of single nucleotide polymorphisms ( snp ) and microsatellites. There are three single-nucleotide polymorphisms ( snps ) in the IL10 gene at positions – 1087, –819 and –592 from the transcriptional start site which have been associated with altered synthesis of IL10 in response to inflammatory stimuli (turner et al. 1997).

28. The –1087 SNP Is A G To A Substitution And Lies Within A Putative Ets Transcription Factor Binding Site. The –1087G Allele Is Known To Be Associated With High In Vitro IL10 Production. The –819 Snp Presents A Dimorphic Polymorphism, A C To T Substitution, And May Affect An Estrogen Responsive Element. The –592 Snp Is A C To A Substitution And Lies Within A Region With A Negative Regulatory Function. It may therefore be anticipated that ( i ) A homozygous GG –1087 genotype indicates considerably enhanced IL-10 production, (ii) A heterozygous AG genotype moderate production and, (Iii) A homozygous AA genotype reveals suppressed IL-10 production.

29. References Periodontitis Subjects Ethnicity Smoking Association Trevilato et al 2003   36 healthy individuals 24 with moderate and 24 with severe periodontitis Brazil Nonsmoking Significant Komatsu et al 2005 chronic 112 subjects with CP and 77 healthy subjects japanese Non smokers Significant Galicia et al 2006 Aggressive and Chronic 422 subjects 169nwith CP, 70 healthy, 43 AgP, 140 healthy Japanese Non smokers significant Tervonen et al 2007 Chronic 51 subjects with moderate to severe CP and 178 reference group Caucasian Smokers and non smokers Significant Guan et al 2008 Chronic 93 patients with periodontitis and 96 controls chinese Smokers and non smokers Significant Nibali et al 2009 Aggressive and chronic 765 subjects Mixed Smokers and non smokers Significant Qianfu zhong et al 2012 Aggressive and chronic 1438 patients and 1303 control Caucasians Non smokers Significant

30. TNF- α gene polymorphism TNF- α gene lies on chromosome 6 within MHC gene cluster. Gene polymorphisms are G to A transition. Chronic inflammatory bone diseases, such as rheumatoid arthritis, periodontal disease, and aseptic periprosthetic osteolysis , are characterized by bone loss around affected joints and teeth caused by increased osteoclastic bone resorption . This resorption is mediated largely by the increased local production of proinflammatory cytokines, such as T nf - α. Boyce et al 2005 Polymorphisms in the promoter region of the TNF- α gene at positions -238(G to A) and -308(G to A) have been reported A lack of association between TNF- α polymorphism and periodontitis has been consistently reported. Craandijk et al 2002; folwanczny et al 2004; M aria de freitas et al 2007.

31. Meta-analysis by nikolopoulos et al(2008) investigated the possible differential risk for periodontal disease of the tnf - α -308g/a allele. Study states the lack of association between the TNF- α-308g/A polymorphism and the susceptibility to CP.

32. References Periodontitis Subjects Ethnicity Smoking Association Craandijk et al 2002 Chronic 90 patients with periodontitis and 264 control reference subjects Caucasian and non Caucasian population Smokers and non smokers Non significant Soga et al 2003 Severe chronic 64 patients with severe adult periodontitis and 64 healthy subjects Japanese Non smokers Significant Shimada et al 2004 Chronic 58 patients with severe chronic periodontitis, 72 with moderate chronic, 52 controls Japanese Non smokers significant Babel et al 2006 Chronic 122 patients with chronic periodontitis and 114 controls Caucasian Smokers and non smokers Non significant Maria de frietas et al 2007 Aggressive 30 patients with G-AgP periodontitis and 70 healthy Brazilian Non smokers Non significant Guzeldemir et al 2008 Aggressive 31 patients with L-AgP and 31 healthy controls Turkish Non smokers Non significant Wenwei yang et al 2013 Chronic and aggressive CP180, AgP 180 and healthy controls180. chinese Non smokers significant

33. FCγR gene polymorphisms Leukocyte I gG receptors( fcγr ) serve as a link between the humoral and cellular branches of the immune system. Binding of the constant region of I gG to fcγr induces a plethora of cell type-specific pro and counterinflammatory functions. Fcγr polymorphisms influence the efficacy of cellular responses, and have been associated with inflammatory disease and disease severity Leukocyte fcγr belong to the ig superfamily. Divided into three classes, fcγri (cd64), fcγrii (cd32), and fcγriii (cd16), encompassing atleast 12 isoforms. Fcγr classes contain structurally and biochemically distinct molecules, and differ in cell distribution and affinity for igg subclasses.

34. FcgRII polymorphism A G to A transition polymorphism in the fcgrIIa gene results in the substitution of histidine (H) for arginine (R) at amino acid position 131 of the receptor. FcgrIIa-h131 binds igG2 immune complexes efficiently, whereas the fcgriia-r131 allotype cannot mediate this interaction. Due to efficient binding of igg2 to neutrophils of the H/H131 genotype, these neutrophils may become hyperreactive . Possibly over activated neutrophils release more tissue-destructing molecules (such as lysosomal enzymes or matrix metalloproteinases ) than less efficiently activated neutrophils ( i.E. Cells with the fcgriia-r1 genotype). This polymorphism has been associated with EOP and with phagocytic function of neutrophils in conjunction with more severe periodontitis in adults ( wilson & kalmar 1996)

35. FcgRIII polymorphism The G to T transition polymorphism in the fcgriiia gene results in an amino acid 158-valine (V) substitution for 158-phenylalanine (F). The fcgriiia-v158 has a higher affinity for igg1 and igg3 than fcgriiia-f158. Subjects bearing the fcgriiia-v1 genotype, monocytes may produce higher levels of (pro-)inflammatory cytokines or chemokines than monocytic cells of f/f158 genotypes, and consequently mediate increased inflammatory activity and periodontal tissue destruction Kobayashi et al. 1997 reported a positive correlation between the fcgriiib-na2 allotype and recurrence of adult periodontitis

36. The study by loos in 2003 in northern european caucasians reported that (1) the fcgriiia-v158 allele was over represented in the patient group and therefore may be regarded as a putative susceptibility factor for periodontitis. (2) the fcgriia -h/h131 genotype was associated with more periodontal bone loss than the r1 genotype, indicative that the h/h131 genotype can be regarded as a putative severity factor for periodontitis. (3) the fcgriiia-v1 genotype was not identified as a severity factor. (4) the fcgriiib-na21 genotype was neither a susceptibility factor nor a severity factor.

37. References Receptor Periodontitis Subjects Ethnicity Smoking Association Fu et al 2002 FCγRIIa FCγRIIIa Aggressive 48 patients with L-AgP, 67 healthy controls African-American population   Significant Loos et al 2003 FCγRIIa FCγRIIIa FCγRIIIb Aggressive 12 subjects with AP, 56 with CP, 61 controls Caucasian Smokers and non smokers Significant Yamamoto et al 2004 FCγRIIa Chronic 213 subjects with CP and 209 controls Caucasian Smokers and non smokers significant De souza and Colombo 2006 FCγRIIa FCγRIIIb Aggressive 31 patients with G-AgP and 49 healthy controls Brazilian Nonsmokers Significant Kobayashi et al 2007 FCγRIIa FCγRIIIa FCγRIIIb Chronic 100 rheumatoid arthritis, 100 periodontitis, 100 healthy controls Japanese Smokers and non smokers Non significant An et al 2009   Chronic and aggressive 30 AP, 131 CP, and 47 healthy patients Chinese   Significant for aggressive non significant for chronic Y wang et al 2012 Severe chronic 13 patients with periodontitis, 106 healthy patients japanese Non smokers Significant

38. FMLP RECEPTOR POLYMORPHISMS Human neutrophils play a key role in host defense against bacterial infections. Neutrophils are activated by the bacterial formyl peptide N- formyl-methionyl leucyl phenylalanine. It binds to specific formyl peptide receptor. These receptors are seven transmembrane pertussis toxin-sensitive G protein-coupled recptors . Ligand binding to fpr activates a number of downstream effector enzymes including phospholipase c, catalyzing the cleavage of phospotidylinositol 4,5-bisphosponate into secondary messengers inositol 1,4,5-triphosphate and diacylglycerol leading to calcium mobilization and activation of protein kinase c. Abnormal chemotaxis t owards FMLP in lagp has been correlated with altered FPR activity.

39. In 1999 gwinn et al discovered occurrence of two polymorphism in the FPR gene of lagp patients, the third transmembrane helix of FPR 110 phenylalanine- serie (F110S) or 126 custient - trytophan (126W), suggesting a molecular alteration in this region may contribute to abnormal chemotaxis in lagp patients. Gwinn et al noted that the f 110s mutation was much more common among F110s lagp patients than c126w.

40. VDR GENE POLYMORPHISM (VITAMIN D RECEPTOR GENE ) Human VDR gene is localized in chromosome 12. Mutations in functionally critical areas of vdr gene can have profound effect on mineral metabolism and bone mineral density ( bmd ). Vdr exerts its genomic action via the nuclear family receptor ( vdr ), which shows an extensive polymorphism. The vdr is an intracellular hormone receptor that specifically binds the active form of vit d and interacts with target cell nuclei to produce a variety of biologic effects Y amada et al 2003.

41. A silent mutation with in the codon 352 of the ninth exon alters a Taq1 site. Hennings 1977 showed a significant relationship between Taq1 VDR gene polymorphism and EOP in caucasians The T t gene might be a risk indicator for the susceptibility of EOP

42. References Periodontitis Subjects Ethnicity Smoking Association Inagaki et al 2003 Chronic 125 medically healthy middle aged men Caucasian Smokers and non smokers Significant De brito Jr et al 2004 Chronic 44 healthy individuals and 69 subjects with CP Mixed Non smokers Significant De souza et al 2007 Chronic 222 subjects with and without end stage renal disease were divided into with and without periodontitis Brazilian   Non significant Wang et al 2009 Chronic 107 patients with severe chronic periodontitis and 121 control subjects Chinese Non smokers significant Hui deng et al 2011 Chronic and aggressive 1338 patients and 1302 controls Mixed Smokers and non smokers significant Gurumoorthy karthikeyan et al 2013 Chronic 60 CP 60 healthy Dravidian Significant

43. CALCITONIN GENE POLYMORPHISM Calcitonin is a hormone produced in the thyroid that causes a reduction of calcium ions in the blood. Suzuki et al 2004 investigated genomic markers for periodontitis, genomic DNA was obtained from 19 healthy volunteers and 22 patients with severe periodontitis, all of whom were japanese . Statistically significant differences were found between healthy and periodontitis patients in calcitonin receptor

44. HUMAN LEUKOCYTE ANTIGEN Human leukocyte antigen also called MHC, are involved in predetermined humoral immune response via recognition of foreign antigens. The HLA complex plays an important role in immune responsiveness and may be involved in antigen recognition of periodontal pathogens. In human, the classical mhc class i molecules( hla-a,b,c ) are expressed on most nucleated cells, while mhc class ii molecules( hla-dp ,- dq ,- dr ) are expressed on cells that immunosurvey host cells including b and t cells, macrophages ( takashiba 2006) Evidence for association of DR4 HLA and periodontal disturbance has been reported by ( katz et al 1987). The HLA genotype HLA –DQBI * 0602 induced proliferation of T cells as an immune response against periodontal bacteria, P.Gingivalis . HLA –DRB1 *1501 was found to be responsible for this T cell proliferation. This accelerated T cell response led to increased susceptibility to EOP.

45. M eta-analysis performed by stein et al 2008 in caucasian population showed no significant HLA associations in patients with chronic periodontitis. Patients with aggressive periodontitis showed a positive relation with HLA-A9 and HLA-B15 as well as negative association with HLA-A2 and –B5. No significant associations were found with class II antigens stein et al. 2008.

46. References Periodontitis Subjects Ethnicity Smoking Association Machulla et al 2002 Chronic and aggressive 50 unrelated german Caucasian patients with RPP and a group of 102 unrelated german Caucasian patients with adult periodontitis are compared with a group of 102 healthy controls Caucasian   Significant Stein et al 2003 Chronic and aggressive 50 patients with generalized aggressive and 102 with CP, 102 healthy controls Caucasian Smokers and non smokers significant Roshna et al 2006 Aggressive 40 patients with G-AgP and 80 controls Indian population Smokers and non smokers Significant Reichert et al 2007 Chronic and aggressive 110 patients with juvenile idiopathic arthritis, 50 patients with G-AgP and 102 with CP, 102 with healthy Caucasian Smokers and non smokers significant

47. TOLL LIKE RECEPTOR, TLR2 and TLR4 GENE POLYMORPHISMS Toll was 1 st discovered as drosophila gene. The gene product is a transmembranous protein, functioning as a signal transduction receptor. There are 10 TLRs, named tlrs1-10 known in mammals, and each of these receptors recognizes molecules derived from a unique class of microbial agents. Four adapter molecules are known to be involved in signaling. These proteins are known as myd88, tirap , trif , tram. These adapters activate other molecules in the cell, including protein kinases that amplify the signal and lead to the induction or suppression of the genes that orchestrate the inflammatory response. All TLRs leads to activation of nfkb which produces proinflammatory cytokines, such as tnf -α, il-1 and il-2, there by regulating their expression.

48. Schroder et al 2005 reported that TLR-4 SNP were correlated with CP, but not with aggressive periodontitis. In a japanese population case control study, fukusaki et al 2007 revealed that the frequency of the c/c genotype in TLR4 3725G>C polymorphism was significantly higher in both the moderate and the severe periodontitis patient group than in the control group.

49. References Periodontitis Subjects Ethnicity Smoking Association D aluto et al 2004 Chronic 94 subjects with severe generalized periodontitis Mixed Smokers and non smokers Non significant Laine et al 2005 Chronic 100 patients with severe periodontitis and 99 healthy controls Caucasian Smokers and non smokers Non significant Berdeli et al 2007 Chronic 83 patients with CP and 106 periodontally healthy subjects Caucasian Smokers and non smokers Non significant Imamura et al 2008 Chronic 43 patients with periodontitis and 49 healthy controls Japanese   Non significant

50. CD 14 GENE POLYMORPHISM The CD14 receptor is a glycoprotein that is expressed primarily on the surface of monocytes , macrophages,neutrophils and gingival fibroblasts and is involved in the process of cellular response to bacterial LPS. The cd14 protein is expressed in two phenotypic forms .M cd 14 and sCD 14. Scd14 is present in the serum. LPS induced upregulation of sCD14 seems to elicit an increased production of il-2 by apc (antigen presenting cells). Systemic level of soluble cd14 increases in periodontal disturbances.

51. CD14 gene exists as a single copy gene. This polymorphism comprises a C to T substitution to position -159 to the 5’flanking region of the CD 14 gene. It has been demonstrated that C to T transformation at position -159 is related to the production of s cd14. Frequency of c allele carriage tends to be larger in subjects with severe periodontitis. The systemic level of the soluble form of cd14(scd14) is significantly increased in patients with periodontal disease( hayashi et al 1999) CD14 expression within the periodontal tissue was found to be negatively correlated with the amount of attachment loss( jin 2001). Reduced expression of cd14 on monocytes was suggested to an increasing susceptibility for EOP ( buduneli et al 2001).

52. References Periodontitis Subjects Ethnicity Smoking Association Yamazaki et al 2003 Chronic 163 subjects with periodontitis and in 104 healthy controls Japanese Non smokers Non significant Laine et al 2005 Chronic 100 patients with severe periodontitis and 99 controls Caucasian Smokers and non smokers Significant James et al 2007 Chronic and aggressive 73 subjects with AP, 95 subjects with CP, 95 healthy controls Caucasian Smokers and non smokers Non significant Donati et al 2008 Chronic 53 subjects with generalized and severe periodontitis Caucasian Smokers and non smokers Non significant

53. CARD15/NOD2 GENE POLYMORPHISM Nucleotide-binding o ligomerization domain receptors, including NOD1, NOD2, and ICE protease activating factor( ipaf ), are cytoplasmic receptors for recognition of microbial products. These receptors detect organisms that enter the cytoplasm of the cell, or products that may be released or transported into the cytoplasm by processes such as phagocytosis and degradation of microbes. Nod1 and Nod2 interact with receptor interacting protein 2(RIP2), also called RICK, a protein consisting of a kinase domain and a CARD.

54. RIP2 directly interacts with inhibitor of NFkB kinase γ (IKK γ), leading to ubiquitinylation of IKKγ and activation of the kinase activities of ikk α and ikkβ, this results in the activation of the proinflammatory responses mediated by nfkb (ferrero et al 2007 ) A clear mrna expression of NOD1 and NOD2 was revealed in gingival fibroblasts( uehera et al 2007) and in oral epithelial cells( sugawara et al 2006).

55. References Periodontitis Subjects Ethnicity Smoking Association Laine et al 2004 Chronic 104 dutch Caucasians with severe adult periodontitis and in 97 healthy controls Caucasian Smokers and non smokers Non significant Noack et al 2006 Aggressive 86 generaized aggressive periodontitis patients I comparison with 67 healthy controls Caucasian Smokers and non smokers Non significant

56. MISCELLANEOUS GENE POLYMORPHISMS INTERLEUKIN-2 (IL-2) GENE POLYMORPHISM IL-2 is a proinflammatory cytokine derived from Th 1 cells. Il-2 is involved in b-cell activation and stimulates macrophages, nk cells, t cell proliferation, and osteoclast activity. A polymorphism in the position - 330 (T - G ) of the il-2 gene promoter was identified by john et al. (1998). This polymorphism could be useful as a marker to diagnose susceptibility to inflammatory diseases .

57. INTERLEUKIN-6 (IL-6) GENe POLYMORPHISM Interleukin-6 (IL-6) plays a role in B cell differentiation and t-cell proliferation. It also stimulates hematopoiesis and accelerates bone resorption . High levels of il-6 in biological fluids and blood have been determined in chronic inflammatory diseases. Higher levels of il-6 were found in sites with gingivitis when compared to healthy sites. Polymorphism occurs due to transition of G to C at position 174 in the promoter region of IL6 gene The C allele was shown to alter the IL-6 gene transcription response to stimuli such as LPS and IL-1. Trevilatto 2003 first reported that the genotype g/g of the il-6_174 polymorphism was significantly associated with susceptibility to chronic periodontal disease

58. MMP POLYMORPHISMS The degradation of periodontal tissues is mainly mediated by matrix metalloproteinases (MMPs). Many types of MMPs have been identified in inflamed periodontal tissues and these enzymes are thought to play an important role in tissue destruction in periodontal diseases ( birkedal-hansen 1993).

59. MMP-1 PROMOTER POLYMORPHISM Fibroblast type collagenase (MMP-1) is the major type of proteolytic enzyme that can cleave native interstitial collagens type I and III, which are the most abundant protein components of periodontal extracellular matrix. Matrix metalloproteinase-1 seems to play an important role during the destruction of the extracellular matrix in periodontal disease. The MMP-1 gene is located in 11q22 and is translated in a wide variety of cells, e.G. Fibroblasts, macrophages, endothelial, and epithelial cells.

60. The level of MMP-1 expression can be influenced by the polymorphism in the promoter region of the MMP-1 gene resulting from a guanine insertion at –1607 bp ( i.E. , The GG versus G variants), which results in the creation of a binding site for the ets family of transcription factors as well as in the increased transcription of the MMP-1 gene and increased enzyme activity polymorphism in the promoter region of human MMP-1 gene has been described The two alleles (1G and 2G) are formed by an insertion/deletion of a guanine at position ª1607 Izakovic¡ova 2004 reported that the polymorphisms in the MMP-1 promoter may have only a small effect on the etiopathogenesis of chronic periodontitis. Astolfi 2006 investigated the relationship between polymorphisms in mmp-1 (-1607 1g/2g, -519 a/g) with chronic periodontitis in a brazilian population. And showed that no significant association is found for the MMP-1 polymorphisms with susceptibility of periodontitis.

61. MMP-3 PROMOTER GENE POLYMORPHISM Astolfi 2006 studied MMP-3 (-1612 5A/6A) gene promoter polymorphisms using PCR-RFLP methods and showed that the 5A/5A genotype was significantly more frequent in the periodontitis group and reported that MMP-3 gene polymorphism may contribute to periodontal tissue destruction during periodontitis in brazilian subjects.

62. MMP-9 PROMOTER GENE POLYMORPHISM Keles JC 2006 investigated the association between MMP-9 promoter polymorphism and severe generalized chronic periodontitis in a turkish population. The alleles of the C/T polymorphism at position -1562 in the promoter region of the MMP-9 gene showed a significant difference in MMP-9 genotypes between chronic periodontitis patients and healthy controls suggesting that MMP-9 promoter gene polymorphism seems to be associated with severe generalized chronic periodontitis.

63. RAGE GENE POLYMORPHISM AND PERIODONTITIS RAGE (receptor for advanced glycation end products) is a multiligent member of the ig super family of cell surface molecules, initially identified as a cellular interaction site for AGEs. RAGE gene is localized to the sixth chromosome region. Polymorphism of RAGE gene may contribute to individual’s susceptibility to develop chronic periodontitis, independently of diabetes .

64. PROVING A DISEASE/POLYMORPHISM RELATIONSHIP Polymorphism must influence the gene product. Biases in the study population should be recognized and controlled. Affected gene product should be the part of disease etio -pathology. Confounders such as smoking and socioeconomic class must be controlled.

65. CLINICAL UTILITY OF GENETIC KNOWLEDGE Polymorphisms associated with periodontitis illustrate a number of important points with respect to the clinical interpretation of this type of information. The associations between particular genes and disease may only (thus far) be apparent in certain populations and not in others. Thus, genetic tests based upon these genes may not apply to all patients. The associations between groups of interacting genes and disease may be stronger than those between individual genes and disease. Therefore, as more genetic risk factors are found, genetic tests for disease risk will continually evolve and merit scrutiny and evaluation. The associations between disease and genes may be indirect, that is, genetic factors may be associated with environmental risk factors for periodontitis (e.g. Smoking) and thereby influence disease only in those patients with the relevant biological exposure.

66. GENETIC SCREENING FOR PERIODONTITIS RISK The current practical clinical utility of genetic knowledge in periodontics is limited. However, performing clinical periodontal assessments of siblings of AgP probands is one of the most useful actions we can perform to ensure the early diagnosis of this disease. By careful clinical diagnostic procedures, we may detect susceptible patients early and instigate therapy, which may prevent the more significant disease aspects from occurring. In the pursuit of better genetic diagnostic tests for chronic and aggressive periodontitis, we must plan our research using plausible biological arguments and carefully avoid bias and misinterpretation of genetic associations with disease.

67. FUTURE APPLICATION OF GENETIC INFORMATION Prognostic tests: Genetic tests, should predict the likelihood of disease initiation before it occurs and ultimately will be linked to knowledge of the environmental factors that can initiate the inappropriate activation of genes that are problematic. Knowledge of the genetic profiles of individuals who are susceptible to periodontitis will identify those individuals whose environmental exposures must be modified in order to prevent disease.

68. DIAGNOSTIC TESTS A great deal of research has been performed in an attempt to detect active disease or subclinical initiation of disease but no universally accepted test has emerged for clinical application. Prognosis and diagnosis can become tightly linked because both a genetic profile would be available and a gene-expression profile could be monitored over time. Once the genetic risk and specific genetic risk factors for a given patient are known, tests of gene activation might be individually targeted and test intervals could be tailored to the susceptible patient. The specific environmental exposures leading to gene activation may be more clearly known.

69. CONCLUSION Periodontitis is clearly multifactorial and researchers need to design studies that examine the role of important environmental and genetic factors simultaneously. Given the large number of genes in the human genome and bacteria in the oral cavity, it is likely that genes and the environment interact in important yet unrecognized ways to alter disease risk. Identifying specific genetic risk factors may be academically appealing but is of little use unless it leads to improvements in the prevention/treatment of disease.

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