Genetic Approaches to Understanding Periodontal Disease Risk & Susceptability Naheed Mohamed

Introduction Periodontal diseases are initiated by microbial plaque, which accumulates in the sulcular region and induces an inflammatory response. This response may progress in certain susceptible individuals to chronic destructive inflammatory condition termed periodontitis.

While microbial and other environmental factors are believed to initiate and modulate periodontal disease progression, there now exists strong supporting evidence that genes play a role in the predisposition to and progression of periodontal diseases. (Sofaer, 1990; Hart, 1994; Michalowicz, 1994; Hassel and Harris, 1995; Hodge and Michalowicz, 2001)

Discuss how PD is a multifactorial disease.

Discuss the pathogenesis of PD how microbes cause host response, and how this relates to tissue destruction

Immune Response Production of inflammatory biochemical mediators, collectively known as “cytokines” (e.g., IL-1, TNF-alpha) Cytokines signal other cells, such as fibroblast, to produce PGE2 and MMPs, which have been associated with bone destruction and connective tissue degradation, respectively. Host response can be both protective and destructive. Specific genes may determine the degree to which an individual’s immune response is protective or destructive (Malo and Skamene, Trends in Genetics, 1994)

Discuss pathways of host response through cytokines and inflammatory mediators.

Show how genetic influence on certain host response mediators can have influence on course of PD.

Putative pathogens are essential to develop periodontitis, however, their mere presence is insufficient to initiate periodontitis. (Haffajee and Socransky, 1994) The primary etiology for periodontitis is bacteria, however the extent and severity of periodontal lesions can be influenced by environmental factors, acquired factors, and genetic predisposition. (Kornman et al., 1997 and Salvi et al., 1997)

Genetic factors influence inflammatory and immune responses in general Genetic factors influence inflammatory and immune responses in general. Individuals may respond differently to common environmental challenges due to their genetic profile. Specifically different forms of genes(allelic variants), can produce variations in tissue structure(innate immunity), and inflammatory mediators (non-specific inflammation). Allelic variants at multiple gene loci probably influence periodontitis susceptability. (Kinane 2003).

Introduction to Genetics

Basic Terminology Genome – refers to all the genes carried by an individual or cell. The human genome consists of more than 3 billion pairs of bases contained in 22 pairs of chromosomes, termed autosomes, and two sex chromosomes. Chromosome – a nuclear structure carrying genetic information arranged in a linear sequence. Gene – a functional and physical unit of inheritance that occupies a specific position (locus) within genome or chromosome. In other words, It is a sequence of nucleotides located at a particular position on a particular chromosome carrying a set of instructions usually directing the synthesis of proteins

Alternative form of a gene is ALLELE Humans carry two sets of chromosomes, one from each parent. Single nucleotide polymorphisms may render two sets of equivalent genes different. Allele – one of several possible alternative forms of a given gene at a particular locus of a chromosome differing in DNA sequence Different alleles are responsible for variation in inherited characteristics such as hair color or blood type. In an individual, the dominant form of an allele is expressed Homozygous – the presence of identical alleles of one or more specific genes (e.g. A/A). Heterozygous – the presence of differing alleles of one or more specific genes (e.g. A/B). 13

More Definitions Genotype – the genetic makeup of an organism or cell distinct from its expressed features or phenotype. Phenotype – the observable characteristics displayed by an organism as influenced by environmental factors and independent of the genotype of the organism. (Phenotype = genotype x environment) Gene expression – the process involving use of the information in a gene via transcription and translation leading to production of a protein affecting the phenotype of the organism determined by that gene. Autosomal dominant – the dominant effect of one gene located on an autosome regardless of the presence of the other normal copy. Autosomal recessive – A gene on an autosome that is required in two copies to be active in an individual. An individual who carries two such copies of the same abnormal gene will be subjected to effects from that gene.

Genetic Variance Different forms of genes are called allelic variants. Allelic variants differ in their nucleotide sequences. When a specific allele occurs in at least 1% of the population it is called a genetic polymorphism. When a nucleotide change is very rare and not present in many individuals it is often referred to as a mutation.

Sources of Variation Mutation : alteration of the genomic sequence compared to a reference state. Not all mutations have harmful events (silent mutation). (<1% of population) Polymorphism: a region on the genome that varies between individual members of a population present in a significant number of individuals. (>1% of population) Single nucleotide polymorphism (SNP): a polymorphism caused by the change in a single nucleotide believed to be the most common genetic variation between individual humans.

Single Nucleotide Polymorphism A Single Nucleotide Polymorphism, or SNP, is a small genetic change, or variation, that can occur within a person's DNA sequence An example of a SNP is the alteration of the DNA segment AAGGTTA to ATGGTTA, where the second “A” is replaced by a “T” On average, SNPs occur in the human population > 1 %.

SNP A change in nucleotide that occurs in the population with a frequency of  1% Reported Sequence AAGTACGGCTC SNP Sequence AAGTGCGGCTC Occurs approximately every 300 to 1000 bps. Coding Region Changes Synonymous TTT=Phe TTC = Phe Non-Synonymous GTT = Val GGT = Gly Stop codon TAC = Tyr TAA =Stop Changes that occur in the promoter region, 5’ and 3’ UTR(untranslated region), and intron may alter expression levels

Gene Expression Discuss process of how genetic information is used to make a functional protein. Discuss introns, exons, UTR’s, enhancer, silencer, genes that encode transcription factors, and promoter regions. Explain where mutations can occur and how the site of the mutation can affect expression and function of the protein.

Environmental Exposures Differences in physiologic functioning of proteins due to polymorphisms can be enhanced by certain environmental exposures (eg smoking, diabetes, microbes). If the protein functions in the inflammatory process then certain polymorphisms can increase or decrease risk for disease phenotype. Epigenomics (link between environment and its effects on gene expression)

Epigenetics study of heritable changes in genome function that occur without a change in DNA sequence. This includes; the study of how patterns of gene expression are passed from one cell to its descendants, how gene expression changes during the differentiation of one cell type into another, and how environmental factors can change the way genes are expressed. The discovery that enzymes can re-organise chromatin into accessible and inaccessible configurations revealed epigenetic mechanisms that considerably extend the information potential of the genetic code. Thus, one genome can generate many 'epigenomes', as the fertilised egg progresses through development and translates its information into a multitude of cell fates.

Genetic Basis of Disease Genetic variance and environmental exposures are the key determinants to phenotypic differences. Simple Mendelian Diseases follow predictable & simple patterns of transmission. In most cases a single gene locus is the major determinant of disease. Complex genetic disease are more prevalent (>1%), do not follow simple pattern of familial distribution, and are the result of interaction of multiple different gene loci as well as environmental factors.

Complex Genetic Diseases No correlation between presence of allele and occurrence of disease. Associated polymorphisms not directly causally linked. Each polymorphism contributes to a small part of the disease process, sometimes requiring multiple genes to develop disease phenotype. Environmental factors are also critical to etiology.

Methods of Genetic Analysis Familial Aggregation Twin Studies Segregation Analysis Linkage Studies Association Studies Discuss each method and how they are done.

Evidence for the Role of Genetic Variants in Periodontitis German studies of familial nature in the early 20th century have shown aggregation of chronic forms of periodontitis in families. This strongly suggested genetic predisposition. (Rev’d by Hassell & Harris 1995) Note: Important consider shared environment in these types of studies, due to interaction of genotypic expression and environment.

Evidence Studies Michalowicz et al. (1991) studied dizygous twins reared together and apart and monozygous twins reared together and apart. Mean probing depth and attachment level varied less for MZT than DZT. Alveolar bone ht. showed significant variations related to difference in genotype. Twin groups had similar OH and smoking hx. Concluded genetics plays a role in susceptibility to periodontal disease.

Evidence Studies Segregation analysis in North American families performed by Marazita et al (1994). Studied >100 families, segregating aggressive forms of periodontitis, and found support for autosomal dominant transmission. Concluded autosomal dominant inheritance with ~70% penetrance occurred in Blacks and non-Blacks.

Evidence Studies Boughman et al (1986) found an autosomal dominant form of LAgP in an extended family in Southern Maryland. In this family type III dentinogenesis imperfecta and a localized form of AgP were segregating as dominant traits. Gene for DGI-III had been previously localized to chromosome 4, performed linkage analysis and showed close linkage of gene for AgP to this DGI-III gene.

Evidence Studies Hart et al (1993) evaluated support for linkage of AgP near chromosome 4 in different population of families (14 AA and 4 caucasian). Results showed that in these populations no linkage existed . Results could mean that this population had a different form of AgP than the Maryland kindred.

Syndromic Forms of Periodontitis Severe periodontitis presents as part of the clinical manisfestations of several monogenetic syndromes. Significance of these conditions is that they clearly demonstrate that a genetic mutation at a single locus can impart susceptibility to periodontitis.

Papillon LeFevre Syndrome Clinically characterized by: Palmoplantar hyperkeratosis Severe early onset periodontitis that results in premature loss of the primary and secondary dentition (distinguishes PLS from other plamoplantar keratoderma) Prevalence 1/ 4million No gender or racial predilection 33

CTSC gene encodes for Cathepsin C protease CTSC gene lies on chromosone 11q14-q21; seven exons encoding for lysosomal protease cathepsin C. It is expressed at high levels in a variety of immune cells including polymorphonuclear leucocytes, macrophages, and in epithelial regions commonly affected by PLS, including the palms, soles, knees, and oral keratinized gingiva (RT-PCR) (Hart et al., 1999). Cathepsin C is a protease enzyme that processes and activates a number of granule serine proteases critical to immune and inflammatory responses of myeloid and lymphoid white blood cells 34

Mutations in CTSC gene Mutations in Cathepsin C (CTSC) gene are implicated for PLS For example: One exon 1 nonsense mutation (856CT): introduces a premature stop codon at amino acid 286. Three exon 2 mutations: single nucleotide deletion (2692delA) of codon 349: introduces a frameshift and premature termination codon, 2 bp deletion (2673-2674delCT): introduces a stop codon at amino acid 343, and GA substitution in codon 429 (2931GA): introduces a premature termination codon. Truncated or altered conformation of the protein may not be transported to the organelle and may not be able to activate protein kinases In other words, Cathepsin C activity in these patients is nearly absent 35

Association between CTSC gene alterations and PLS patient’s susceptibility to periodontal disease. Two possible explanations: CTSC protein is implicated in activation of proteases related to phagocytosis, antigen presentation, local activation, and deactivation of cytokines and other inflammatory markers (Toomes et al., Nature Genetics 1999 101:421-424) CTSC influences periodontal progression through its role in epithelial differentiation or desquamation. Aberrant differentiation of sulcular and junctional epithelium may alter the mechanical barrier to periodontal pathogens (Toomes et al., Nature Genetics 1999 101:421-424) 36

Polymorphism Studies on Periodontitis Host response is predominantly influenced by genetic make-up. Several features of host’s innate immune response may contribute to susceptibility to AgP and include epithelial, connective tissue, fibroblast, and PMN defects. Aspects of the host inflammatory response namely cytokines are crucial variants influencing host respone in periodontitis.

Immunological Polymorphisms MHC or HLA genes determine our response to particular antigens. Japanese study of AgP pts found a significant association for pts with atypical BamH1 restriction site in the HLA.DQB gene (Takashiba et al. 1994). Hodge & Kinane (1999) found no assoc. in caucasian AgP pts and this restriction site. MHC or HLA code for cell surface antigen presenting proteins. Found on chromosome 6.

IL-1 Gene Polymorphisms In 1997 Kornman et al found an association between polymorphisms in genes enconding for IL-1a(-889) and IL-1B(+3953) and an increased severity of periodontitis. The specific genotype of the polymorphic IL-1 cluster (called PST-periodontitis susceptibility trait) was associated with severity of PD in only non-smokers, and distinguished individuals with severe periodontitis from those with mild disease.

Genetic control of IL-1: Genes and Locus of SNPs associated with controlling IL-1 biological activity Genes Polymorphism Locus Current Locus assessed with test Controlled product IL-1A Allele 2 -889 Allele 2 IL-1A +4845 IL-1 alpha IL-1B Allele 2 +3953 Allele 2 IL-1B +3954 IL-1 beta IL-1RN Protein receptor antagonist (impedes IL-1 alpha and beta) Genetic Susceptibility Test for periodontitis: tests for the presence of at least one copy of allele 2 at the IL-1A +4845 loci and at least one copy of allele 2 at the IL-1B +3954 locus. *IL-1A +4845 is being used because it is easier to identify than IL-1A -889 and it is essentially concordant with it. ** IL-1B +3953 has been now renumbered as IL-1B +3954 because the current convention indicates that the numbering of the transcription should begin at +1 instead of zero. 40

Interleukin 1 A proinflammatory multifunctional cytokine. Enables ingress of inflammatory cells into sites of infection Promotes bone resoroption Stimulates eicosanoid (PGE2) release by monocytes and fibroblasts Stimulates release of MMP’s that degrades proteins of the ECM. Forms IL-1α and IL-1B

IL-1α IL-1α is a pleiotropic cytokine involved in various immune responses, inflammatory processes, and hematopoiesis This cytokine is produced by many cell types but is only secreted by monocytes and macrophages. produced as a proprotein, which is cleaved by calpain and released in a mechanism that is still not well studied.

IL-1β expressed in monocytes and macrophages Inactive precursor cleaved by caspase-1 enzyme Secreted to extracellular compartment Wide spectrum of immune functions

IL-1 as modulator for Periodontitis

Kornman et al. (1997) Genotype-positive non-smokers  18.9 times more likely to have severe periodontitis (when compared with genotype-negative non smokers) No significant association between periodontal status and genotype detected when smokers were included in the statistical analysis. 86% of the severe periodontitis patients were accounted for by either smoking or IL-1 genotype Presence of allele 2 at IL-1A -889 or IL-1B +3953 did not significantly increase the risk of periodontitis among smokers and non-smokers.

+IL-1 genotype and IL-1 protein The specific periodontitis-associated IL-1 genotype consists of a variant in the IL-B gene that is associated with high levels of IL-1 production.(Poiciot et al 1992) Patients positive for composite IL-1A (+4845) and IL-1B (+3954) periodontitis-associated genotype has higher level of IL-1B in GCF, but not in gingival tissue before and after treatment (Kornmann et al 1999) Carriage of allele 2 in the (-889) locus resulted in an almost four fold increase of IL-1 protein levels in chronic periodontitis patients (Shirodaria et al., 2000)

Contradictory Studies Meisel et al (2002) Stated that the composite genotype showed strong interaction with smoking, whereas non-smokers,even genotype-positive were not at any increased risk Papapanou (2001) 132 periodontitis pts who were age and sex matched with controls did not show any association with the composite genotype and periodontitis. Ehmeke (1999) Of 33 pts, 16 were genotype positive. Following 2yrs of maintanence tx, no difference in tooth or attachment loss between the two groups.

Clinical Significance of IL-1 Genotype Bleeding Upon Probing Lang et al. (2000) after periodontal surgery, maintenance patients were monitored for BOP and tested for the candidate genotype. no significant relationship between BOP and the candidate polymorphism when the entire study population was evaluated (N=333). - non-smokers genotype positive demonstrated significantly more bleeding (when compared to non-smokers genotype-negative).

Attachment loss Kornman et al. (1997) McDevitt et al. (2000) Reported 18.9 times greater risk (OR=18.9) of finding severe periodonitis among non-smokers genotype-positive. McDevitt et al. (2000) Among non-smokers or former smokers, genotype positive individuals had 3.75 greater odds of having moderate to advanced periodontitis than genotype-negative.

Genotype and tooth loss McGuire and Nunn (1999) Genotype-positive individuals had a 2.7 greater chance than genotype-negative patients of losing a tooth. Combined effect of being genotype-positive and heavy smoker increased the odds of tooth loss to 7.7 compared with genotype-negative non-smokers. PST can be helpful in treatment planning.

Genotype and healing after surgery De Sanctis and Zucchelli (2000) Evaluated the impact of patient’s genotype on maintenance of gained clinical attachment after guided tissue regeneration (GTR) At base line: no difference between genotype-positive (N=14) and genotype-negative (N=26) with reference to clinical parameters (BOP, PD, and CAL) At one year: no difference (gain of CAL 5.1 mm vs 5.2 mm; residual PD 6.3 mm vs 6.4 mm) At four years: genotype positive patients demostrated a significantly greater loss of clinical attachment (2.3 mm vs 1.0 mm) and increased probing depth (2.2 mm vs 0.8 mm)

Periodontal Pathogens Socransky et al (2000) Found that the mean counts of specific species were higher in general IL-1 positive genotypes compared to negative subjects. Species detected at higher levels were those frequently detected with periodontal inflammation.

Prevalence of genotype positive individuals in different ethnic groups Frequency of many genetic alleles varies between ethnic groups, therefore, it is necessary to establish allele frequencies in populations before genetic test can be evaluated and used. Caucasions: 29% of northern european caucasions were genotype positive (Kornman et al., 1997) African Americans: 14.5% of non-diseased individuals and 8% of patients with localized form of aggressive periodontitis were genotype-positive. (Walker et al., 2000) Chinese: 2.3% of sample of 132 mod-severe periodontitis cases were genotype-positive (Armitage et al., 2000) Hispanics: 26% of hispanic individuals with peridontitis were genotype-positive (Lopez et al., 2005) Take home message: Dissimilarity in the prevalence of genotypes in different ethnic groups precludes extrapolating data from one group to another. 55

Summary of IL-1 genotype in Periodontitis Unlikely to be relevant in AgP It is at best in linkage disequilibrium with the gene contributing susceptibility to chronic periodontitis It confers risk independent of that due to smoking The polymorphism is at best one of several involved. The polymorphism is a useful marker in only defined populations, relatively absent in some (Armitage et al 2000), and is too prevalent in others (Walker et al 2000). Demonstration of functional significance of this gene polymorphism is yet to be confirmed. Clinical utilization of these composite genotype polymorphisms for risk assessment and prognostic determination is currently premature. (Kinane, 2003)

GENETIC RISK DETERMINATION WITH GENOTYPE® PST® PLUS Mutations in the genes for interleukin-1 and its natural antagonist, the interleukin-1 receptor antagonist, may lead to an overproduction of interleukin-1, an important mediator of inflammation of the immune system. Even if only a few bacteria are present, an over activation of osteoclasts results in an increased degradation of periodontal soft and bone tissue. Patients with increased interleukin-1 levels require particularly intensive treatment strategies. In such cases, the GenoType PST™ plus test is a valuable tool for the optimization of follow-up and prophylaxis intervals and for the risk determination prior to extensive implant restorations.

Instructions for Use

Sample Report

THE END