Genetic Approaches to Understanding Periodontal Disease Risk

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Genetic Approaches to Understanding Periodontal
Disease Risk Susceptability

• Naheed Mohamed

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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.

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• 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)

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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 individuals immune response is protective or
  destructive (Malo and Skamene, Trends in
  Genetics, 1994)

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• 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)

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• 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).

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Introduction to Genetics
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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

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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).

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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.

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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.

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Sources of Variation

• Mutation alteration of the genomic sequence
  compared to a
• reference state. Not all mutations have
  harmful events
• (silent mutation). (lt1 of population)
• Polymorphism a region on the genome that varies
  between
• individual members of a population present in
  a
• significant number of individuals. (gt1 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.

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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 gt
  1 .

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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 TTTPhe
• 
  
  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

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Gene Expression
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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)

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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.

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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 (gt1),
  do not follow simple pattern of familial
  distribution, and are the result of interaction
  of multiple different gene loci as well as
  environmental factors.

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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.

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Methods of Genetic Analysis

• Familial Aggregation
• Twin Studies
• Segregation Analysis
• Linkage Studies
• Association Studies

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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. (Revd
  by Hassell Harris 1995)
• Note Important consider shared environment in
  these types of studies, due to interaction of
  genotypic expression and environment.

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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.

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Evidence Studies

• Segregation analysis in North American families
  performed by Marazita et al (1994).
• Studied gt100 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.

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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.

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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.

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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.

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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

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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

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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

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Association between CTSC gene alterations and PLS
patients 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 101421-424)
• 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 101421-424)

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Polymorphism Studies on Periodontitis

• Host response is predominantly influenced by
  genetic make-up.
• Several features of hosts 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.

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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.

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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.

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Genetic control of IL-1 Genes and Locus of SNPs
associated with controlling IL-1 biological
activity
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.
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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 MMPs that degrades
  proteins of the ECM.
• Forms IL-1a and IL-1B

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IL-1a

• IL-1a 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.

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IL-1ß

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

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IL-1 as modulator for Periodontitis
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• 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.

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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)

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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.

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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 (N333).
• - non-smokers genotype positive demonstrated
  significantly more bleeding (when compared to
  non-smokers genotype-negative).

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• Attachment loss
• Kornman et al. (1997)
• Reported 18.9 times greater risk (OR18.9) 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.

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• 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.

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• Genotype and healing after surgery
  De Sanctis and Zucchelli (2000)
• Evaluated the impact of patients genotype on
  maintenance of gained clinical attachment after
  guided tissue regeneration (GTR)
• At base line no difference between
  genotype-positive (N14) and genotype-negative
  (N26) 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)

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• 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.

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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.

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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)

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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.

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Instructions for Use
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Sample Report
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THE END