Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI1
Molecular analysis of oral pathogens and
saliva, dental caries
Jana Mrázková, PhD
Department of Pathophysiology MED MUNI
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI2
Topics
̶ Factors involved in development of dental caries
̶ Molecular analysis of saliva
̶ Molecular analysis of oral microbiome
̶ Genetic basis of dental caries
̶ Genetic association studies related to dental caries
3
Dental caries
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
Dental Health Services Victoria
(www.betterhealth.vic.gov.au)
and factors affecting
their development
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI4
Dental caries
̶ most common chronic disease
̶ 3.5 billion people worldwide (530 million children, according to WHO)
̶ non-communicable disease (according to FDI World Dental Federation and WHO)
̶ shares similar risk factors with other chronic/systemic diseases
̶ Infectious/transmissible
̶  Streptococcus mutans transmission from parents to infants (Early childhood caries), and from
one person to another (kissing couples)
̶ complex disease
̶ multifactorial (endogenous and exogenous factors), multiple genes are involved in
̶ interaction of several factors contributes to formation of caries
̶ oral microflora composition (main factor)
̶ enamel and dentin properties (tooth surface quality)
̶ saliva composition and physical effects
̶ genetic predisposition
̶ overall health condition (immune system disorders, systemic disease affecting IS)
̶ behavioral and environmental factors
̶ time for which the factors act / interact
Puwadol Jaturawutthichai
(www.shutterstock.com)
https://www.ncbi.nlm.nih.gov/books/NBK551699/
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI5
Dental caries
DOI:10.5005/jp-journals-10047-0051
Diagrammatic representation of the determining (risk factors) and confounding factors (risk
indicators/predictors) in dental caries disease.
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI6
Dental caries
̶ caries dentium
disruption of dynamic process of cyclic alteration between demineralization and
remineralization of enamel  ↑ demineralization  caries formation begins
̶ enamel  cca 97 % of inorganic matter (apatites – cationic complexes = ligands Ca2+ a (PO4)2- + counter-ions
 Ca10(PO4)6CO3 (carbonate apatite), Ca10(PO4)6(OH)2 (hydroxyapatite), Ca10(PO4)6F2 (fluoroapatite)
̶ organic acids (bacteria, diet)  apatite counter-ions neutralization  disintegration of crystal structure units  dissolution of
mineral part of the enamel  caries formation
̶ bacterial proteolytic enzymes degradation of organic matrix (collagens and proteoglycans)
̶ carious lesion  dentin  dentinal tubules  pulp  pulpitis, periodontitis
Puwadol Jaturawutthichai
(www.shutterstock.com)
Color Atlas of Biochemistry
(3rd edition, 2013)
diet
medicaments
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI7
Factors involved in caries development
̶ Saliva:
̶ complex carioprotective factor
̶ homeostasis maintenance
̶ physical factor
saliva flow (washing and lubrication of oral cavity tissues)
oral cavity clearance (washing of harmful substances,
unadhered microorganisms)
̶ „chemical“ factor
gustin (Carbonic anhydrase VI buffering capacity),
calcium, phosphate, fluoride ions
lysozyme, lactoferrin,
proteins of specific (IgA, IgG)
and non-specific immunity (defensins, cathelicidins, histatins, statherin),
proline rich proteins (PRPs),
mucins
https://doi.org/10.1371/journal.ppat.1008058
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI8
Factors involved in caries development
www.dentalcare.com/en-us/professional-education/ce-
courses/ce410/fluoride-s-mechanism-of-action
Demineralization (F- ion presence)
Remineralization (F- ion presence)
Acid
(pH < 5,5)
Saliva
pH
6 – 7
̶ Saliva:
̶ effects of saliva:
 ↑ balance between re- and demineralization
 ↓ food remnants, ↓ microorganisms,
↓ environment acidity (dilution, buffer systems
- bicarbonate, hydrogen phosphate, proteins)
 ↑ substances with antibacterial, antifungal and
antiviral properties
̶ problem  reduced production of saliva
 dehydratation, anxiety, obstruction / hypofunction of
salivary glands (DM, Sjögren‘s syndrome, AIDS,
tumors and radioteraphy, acute infection)
 medicaments (beta blockers, antidepressants, antihistamines)
 drugs (methamphetamine, THC)
 promotion of carious lesions formation
9
Factors involved in caries development
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
https://periobasics.com/dental-plaque/
̶ Oral microbiome:
̶ oral cavity  unique microbiological habitat  separate
ecological niches (non/desquamating surfaces, saliva) 
colonized by specific species of microorganisms
̶ second most diverse (up to 1000 species of microorganisms)
 homeostasis maintenance (competition and displacement of
exogenous pathogens  maintaining ecosystem stability)
 immunomodulation
̶ dental plaque = microbial biofilm
 matrix of extracellular polymeric substances (EPS)
 aerobic bacteria (Streptococcus sanguinis), facultative anaerobes (S. mutans, S. sobrinus, Lactobacillus sp.),
anaerobes (Actinomyces sp., Veillonella sp.), fungi (Candida sp.)
 saliva  proteins with charged surfaces (acidic PRPs, statherin, histatins)  electrostatic interaction with phosphate and
calcium ions of apatite  acelular pellicle formation (mucins, cystatins, albumin, IgA, IgG, lysozyme, alpha-amylase,
carbohydrates, neutral lipids, phospho- and glycolipids, glucosyltransferase)  protection against demineralization, partial
reduction of microbial adhesion (proteins on the surface are also present in saliva  competition of bacterial binding receptors)
 substrate for bacteria   biofilm formation
https://doi.org/10.3389/fmicb.2018.03323
̶ Dental plaque
̶ problem: oral microbiome dysbiosis
 homeostasis disruption  eubiotic balance shift 
from mutualism/commensalism to unbalanced
parasitic/pathogenic state  disease onset and
development
̶ dental plaque  ↑ cariogenic species (ferment carbohydrates to organic acids + tolerate low pH
environment)  predominate Streptococcus mutans a Streptococcus sobrinus, Lactobacillus sp.,
Candida sp.
̶ factors supporting cariogenic species predominance
 ↑ intake of sugars / acids  acidification; ↓ immunity, inflammation,…
 ↓ saliva, ↓ oral hygiene  ↑ plaque thickness
 ↑ dental plaque  lack of oxygen  ↑ anaerobic metabolism metabolism of fermentable carbohydrates  organic
acids  ↓ pH  demineralization
 ↑ dental plaque  protects cariogenic bacteria from host defense mechanisms
S. mutans  dextran (α-1,6-D-glucan)  extracellular insoluble polysaccharide  ↑ protection of bacteria
against adverse environment (low pH, antimicrobial factors), ↑ co-adhesion of other species, ↑ plaque adhesion
10
Factors involved in caries development
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI11
Factors involved in caries development
̶ External factors:
 poor oral hygiene
 poor eating habits (excessive intake of fermentable carbohydrates)
 smoking (e-cigarettes – filling has a high sugar content)
 alcohol consumption
 medicaments (salivary glands function impairment, acidification of oral cavity, antibiotics)
 poor access to quality food, drinking water, hygiene supplies, medical care
̶ Time
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI12
Factors involved in caries development
̶ Genetic predisposition:
̶ complex disease (genetic, epigenetic and exogenous factors)
• multiple genes
• genetic heterogeneity – locus heterogeneity (mutations in genes at different loci), allelic heterogeneity (different
mutations in one gene)
• incomplete penetrance – pathological phenotype is not manifested in all individuals carrying disease-causing gene
(positive effects of other alleles or exogenous factors)
• phenocopy – pathological phenotype is manifested by individuals who are not carrying disease-causing gene
• high frequency of risk alleles in population
• ethnic variability (disease-causing genes can vary among populations, variant alleles can have different impact on
phenotype in different populations)
 it is possible to determine only genes (alleles) that act as risk factors  predisposition (predisposing genotype can
increase probability of disease development, but does not determine the disease)
13
Molecular analysis
of saliva
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
(Salivaomics)
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI14
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
̶ rich reservoir of peptides and proteins
̶ saliva components demonstrably change in
response to certain diseases and conditions
̶ more than 100 molecules detected in saliva
samples are evaluated as potential
diagnostic or prognostic biomarkers for
various diseases (eg. tooth decay,
periodontitis, cancer, diabetes)
Saliva is composed of biomolecules and fluids from different sources. Saliva is mainly
secreted by salivary glands, and its informative biomolecules (DNA, RNA, proteins,
metabolites and microbiota) are obtained from salivary glands, oral mucosa cells, oral
microbiota and gingival crevicular fluid.
https://doi.org/10.1111/prd.12099
https://doi.org/10.1371/journal.ppat.1008058
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI15
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
̶ Salivaomics
 the term introduced in 2008
 combines knowledge of various "omic" components
of saliva
(proteome, transcriptome, metabolome, microbiome,…)
 utilizes high-throughput technologies (genomics,
transcriptomics, proteomics, metabolomics, lipidomics and
microbiomics,…)
 saliva analysis  identification of biomarkers
The different and complementary components of salivaomics
https://doi.org/10.1007/978-3-030-37681-9_4
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI16
Saliva molecular
analysis
̶ Saliva as a diagnostic fluid
Advantages:
̶ saliva collection is non-invasive, easy, painless, repeatable (permanent availability of the material),
usable for all age categories, untrained staff can do the collection
̶ large sample volume, stable in time, processing is fast, cheap
̶ promising potential to replace blood in screening, diagnosis and prognosis of disease
https://doi.org/10.3390/ijms17060846
https://doi.org/10.1371/journal.ppat.1008058
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI17
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
Limitations:
̶ serum / saliva biomarker levels correlation
↓↓↓ analyte concentration when compared to serum  ↑ saliva sample volume, detection limit of the method, depletion
of abundant proteins (PRPs, α-amylase, albumin, mucins and secretory IgA can form up to 80%), osmolality
̶ high variability  worse reproducibility of results
• technical (sampling, processing, used method)
• inter- (age, sex, physiological status) and intraindividual (circadian, circulatory)
• biological (influence of oral condition, circadian rhythm, systemic diseases (Sjögren's syndrome), drugs, chemo /
radiotherapy)  saliva volume and composition
• saliva flow rate and its stimulation  concentration of salivary biomarkers
• proteolytic enzymes (microbiome / host)  stability of certain biomarkers
̶ the issue of standardization
 correlation of protein markers to total saliva protein concentration (same person as sample and control)
 standardization of used methods, validation of protocols
 considering all variables (saliva composition variability, sampling, saliva flow rate, sample volume,
stimulation, blood contamination, collection kits, analyte integrity)
https://doi.org/10.1371/journal.ppat.1008058
https://doi.org/10.1007/978-3-030-37681-9
https://www.aacc.org/cln/articles/2013/january/saliva
 the issue of biomarkers validation for
clinical applications
• verification  determination of the biomarker by
various techniques  achieving similar results
• validation  preclinical  definitive academic
(prospective sampling, retrospective evaluation 
multicenter studies
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI18
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
Spectrophotometric methods
̶ UV/Vis spectrophotometry
(enzymes, metabolites, proteins, anti / oxidants)
̶ Atomic absorption / emission spectrometry
(atoms and ions – Ca, Mg, Cr, Mn, Ni, Pb / Na, K)
̶ NIR (near infrared) spectroscopy
(transition metal ions and rare earth metal ions, molecules
containing bonds C-H, N-H, S-H, O-H – thyocyanate, IgA, cortisol,
salivary α-amylase, urea, phosphates, total protein)
https://doi.org/10.1007/978-3-030-37681-9
Table describing examples of commonly analyzed biomarkers in whole mouth saliva;
CRP – C-reactive protein; HPLC – high performance liquid chromatography; IC – ion
chromatography; LC-MS – liquid chromatography mass spectrometry; MALDI-TOF MS
- matrix assisted laser desorption ionization-time of flight mass spectrometry; RT-LAMP
– reverse transcriptase loop-mediated isothermal amplification; AOPP – Advanced
Oxidation Protein Products; TBARS – Thiobarbituric Acid Reactive Substances; TAC –
Total Antioxidant Capacity; FRAS – Free Radical Analytical Systém.
Janšáková et at.,
Klin. Biochem. Metab., 26 (47), 2018, No. 1, p. 21–26
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI19
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
Immunoassays
̶ Enzyme-linked immunosorbent assay (ELISA)
(direct, indirect or sandwich-type – adiponectin, cortisone, cortisol,
C-reactive protein, D-dimer, lactoferrin, IgA, IgM, IgG, IgE, myoglobin)
̶ Chemiluminescence immunoassay (cortisol, testosterone, lactate)
̶ Fluoroimmunoassay (salivary α-amylase, Haptoglobin, C-reactive protein)
̶ Radioimmunoassay (cortisol, estradiol, oxytocin)
̶ Unlabeled immunoassays (nephelometry, turbidimetry,
immunochromatography, biosensors / chips)
https://doi.org/10.1007/978-3-030-37681-9
Table describing examples of commonly analyzed biomarkers in whole
mouth saliva; CRP – C-reactive protein; HPLC – high performance liquid
chromatography; IC – ion chromatography; LC-MS – liquid chromatography
mass spectrometry; MALDI-TOF MS - matrix assisted laser desorption
ionization-time of flight mass spectrometry; RT-LAMP – reverse transcriptase
loop-mediated isothermal amplification; AOPP – Advanced Oxidation Protein
Products; TBARS – Thiobarbituric Acid Reactive Substances; TAC – Total
Antioxidant Capacity; FRAS – Free Radical Analytical Systém.
Janšáková et at.,
Klin. Biochem. Metab., 26 (47), 2018, No. 1, p. 21–26
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI20
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
Liquid biopsy (fluid biopsy)
̶ cancer, tumors
̶ simple, non-invasive
̶ efforts to replace tissue biopsy
̶ tests that detect circulating tumor cells, exomas, tumor
DNA, tumor RNA, and proteins that were released to
bloodstream or saliva from the primary lesion
https://doi.org/10.1007/978-3-030-37681-9
Table describing examples of commonly analyzed biomarkers in whole
mouth saliva; CRP – C-reactive protein; HPLC – high performance liquid
chromatography; IC – ion chromatography; LC-MS – liquid chromatography
mass spectrometry; MALDI-TOF MS - matrix assisted laser desorption
ionization-time of flight mass spectrometry; RT-LAMP – reverse transcriptase
loop-mediated isothermal amplification; AOPP – Advanced Oxidation Protein
Products; TBARS – Thiobarbituric Acid Reactive Substances; TAC – Total
Antioxidant Capacity; FRAS – Free Radical Analytical Systém.
Janšáková et at.,
Klin. Biochem. Metab., 26 (47), 2018, No. 1, p. 21–26
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI21
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
„Omics“ methods
̶ DNA (genomics and epigenomics - cancer)
̶ RNA (transcriptomics - biomarkers for chronic periodontitis,
Sjögren's syndrome, lung, ovarian, breast and pancreatic cancer)
̶ proteins (proteomes for Sjögren's syndrome, Down's syndrome, schizophrenia)
̶ metabolites metabolomics - oral cancer, hepatocellular and colorectal cancers, periodontitis, chronic kidney disease)
̶ lipids and microbiome (lipidomics, microbiomics) and others
̶ simultaneous analysis of hundreds of analytes  precise detection of small changes
̶ high sensitivity, quantitative results
̶ analysis of a set of biomarkers for particular disease
̶ none in clinical application yet  „point-of-care“ testing
https://doi.org/10.1007/978-3-030-37681-9
https://doi.org/10.3390/bios11100396
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI22
Saliva molecular analysis
̶ Saliva as a diagnostic fluid – dental caries
̶ no diagnostic test
̶ caries susceptibility tests  microbiological laboratory
 determination of presence of cariogenic microflora
 quantitative determination of the presence of fermenting microorganisms (acidifying environment) in saliva
 determination of saliva buffering capacity and saliva secretion rate
 colony quantification of fungus Candida albicans
̶ efforts to relate the prevalence of tooth decay to the saliva phenotype  ambiguous results
https://doi.org/10.1007/978-3-030-37681-9
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI23
Saliva molecular analysis
̶ Saliva as a diagnostic fluid – dental caries susceptibility
̶ salivary protein biomarkers associated with dental caries susceptibility:
↑ total protein, total antioxidant activity
↑ alpha-amylase, mucins (MUC1 a MUC5B)
↓ arginine deiminase system, albumin, proteinase 3, PRP1/3, statherin, histatin 1
↓ concentrations of calcium and bicarbonate ions
↓ urease activity
̶ salivary protein biomarkers associated with susceptibility to ECC:
↑ PRPs, histatins, IgA, IgG
↓ statherin
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI24
Saliva molecular analysis
̶ Saliva as a diagnostic fluid – periodontitis, oral cancer
̶ periodontitis
 ↑ salivary biomarkers – IL-1β, MMP-8, MMP-9, TNF-α, AST, ligand for receptor activator of nuclear factor κB
(RANKL), osteoprotegerin, prostaglandin E2
– they can be used to detect beginning stages of periodontitis, to distinguish between
periodontitis and gingivitis, to predict the progression of periodontitis and monitor the prognosis
 ↑ red complex bacteria (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola)
 ↑ Aggregatibacter actinomycetemcomitans, Prevotella intermedia (G- anaerobic bacteria)
 genetic predisposition to susceptibility  IL-1 gene polymorphism
̶ cancer (oral squamous cell carcinoma – OSCC)
↑ salivary biomarkers – IL-6, IL-8, IL-1α, IL-1β, CD59, MRP14 (myeloid related protein), profilin 1 protein, catalase, Mac-2
binding protein (M2BP)
 other potential salivary markers  telomerase, Cytokeratin 19 fragment (Cyfra21-1), tissue polypeptide antigen (TPA), cancer
antigen CA 125, CD44, glutathione, transferrin, mRNA (IL8, IL-1β, phosphatase DUSP1, hemagglutinin HA3, enzyme OAZ1, S100 calcium-binding
protein P, acetyltransferase SAT), levels of some amino acids, lactate, extracellular DNA, microRNA, carcinoma cells
doi: 10.3390/bios11100396
https://www.mdpi.com/2075-4418/7/1/7/htm
https://doi.org/10.1016/j.tibtech.2018.05.013
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI25
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
̶ point-of-care testing, chair-side kits:
̶ improving individualized care
• caries risk determination
• periodontitis onset risk and progression assesment
• oral cancer screening
̶ caries
• saliva physical parameters: volume, flow rate, viscosity, consistency
pH and buffering capacity of saliva
• lactate
• determination of cariogenic bacteria S. mutans a Lactobacillus sp.
commercial kits (visual or
colorimetric detection)
commercial kits
(immunochromatographic detection
of antigen, cultivation kit)
commercial kits
(colorimetric detection)
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI26
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
̶ point-of-care testing, chair-side kits:
̶ periodontitis
• detection in saliva – active MMP-8  PerioSafe® PRO DRS (immunochromatography) a ORALyzer® (analyzer)
• detection in gingival crevicular fluid – aMMP-8 (ImplantSafe DR®), AST (PerioGard, PocketWatch)
̶ cancer (OSCC)
screening
https://doi.org/10.1016/j.bios.2021.112995
https://doi.org/10.3390/diagnostics11060932
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI27
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
̶ periodontitis
https://doi.org/10.3390/diagnostics11060932
https://decisionsindentistry.com/article/diagnostic-utility-oral-fluid-biomarkers/
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI28
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
– diseases and examples of biomarkers
̶ autoimmune diseases
Sjögren's syndrome – α-amylase, carbonic anhydrase VI,
lactoferrin, β2-microglobulin
̶ neurodegenerative diseases
Alzheimer's disease – total tau protein, phosphorylated tau
protein, amyloid-β and α-synuclein
̶ genetic diseases
cystic fibrosis – Ca, PO4
2-, Na, K, Cl, ↓ saliva volume, urea, uric acid, prostaglandin E2
̶ cancer
squamous cell carcinoma – IL-8, IL-6, IL-1β, IL-4, IL-1, VEGF, HER2, tissue polypeptide antigen (TPA) and EGFR,
LDH, N-α-acetyltransferase 10 protein (Naa10p), carcinoembryonic antigen (CEA) protein, serum basic fibroblast
growth factor (bFGF), transferrin, cyclin D, Maspin, specific mRNAs ………….
breast cancer - HER2/neu (C-erbB-2), VEGF, EGF, specific mRNAs, autoantibodies against HER2 and MUC-1
pancreas cancer – transcriptomic markers of mRNAs (KRAS, MBD3L2, ACRV1 and DPM1), specific miRNA,
lactoperoxidase, Cyclophilin B, Cytokeratins (14, 16 a 17)
̶ allergy
food allergies - IgE and IgG1 doi:10.7759/cureus.7708
https://doi.org/10.1016/j.medntd.2022.100115
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI29
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
– diseases and examples of biomarkers
̶ cardiovascular diseases
CK-MB, myoglobin, troponin I, myeloperoxidase,
inflammation markers (CRP, TNF-α, MMP-9),
cellular adhesion molecules (soluble CD40 and ICAM-1)
̶ metabolism
diabetes mellitus type 2 – 1,5-anhydroglucitol, CRP,
leptin, IL-6, TNF-α
̶ infectious diseases
HIV – antibodies against HIV
viruses – IgM / IgA antibodies, viral RNA
Candidiasis, amebiasis – presence of Candida sp., Entamoeba histolytica (antibodies)
Hepatitis – presence of DNA of HBV virus
Peptic ulcer disease, gastritis – presence of Helicobacter pylori (IgG antibodies, H. pylori DNA)
̶ endocrine diseases
Cushing's syndrome and Addison's disease - cortisol
sex hormones - polycystic ovary syndrome, menopause / andropause, anovulation, hypogonadism,
hyperestrogenism
doi:10.7759/cureus.7708
https://doi.org/10.1016/j.medntd.2022.100115
30
Saliva molecular analysis
̶ Saliva as a diagnostic fluid – biomarkers analyzed in labs
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI31
Saliva molecular analysis
̶ Saliva as a diagnostic fluid
– biomarkers analyzed in labs
https://www.oraldna.com/trends-in-salivary-testing/
https://www.ada.org/en/member-center/oral-health-topics/salivary-diagnostics
32
Molecular analysis
of oral microbiome
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
(„Oralome“)
https://www.jorthodsci.org/viewimage.asp?img=
JOrthodontSci_2014_3_4_125_143233_f6.jpg
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI33
Molecular analysis of OM
̶ Oral microbiome
̶ community of up to 1000 different microbial species  bacteria, fungi, viruses, archaea, protozoa
 bacterial species predominate
̶ „oralome“
 the summary of the dynamic interactions orchestrated between the ecological
community of oral microorganism that live in the oral cavity and the host
(For example, oral microflora is important for the maturation and development of an appropriate oral immune
response  the host's immune system must defend itself against pathogenic microbes, but at the same time it must
harmonize and protect commensal oral microbes)
https://doi.org/10.1016/j.csbj.2021.02.010
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI34
Molecular analysis of OM
̶ Oral microbiome
̶ dysbiosis  oral diseases
 host diet
 inflammatory reactions
 systemic diseases (DM 2, hyperglycemia)
 host habits (smoking, alcohol, chronic stress)
̶ caries  ↑ dietary carbohydrate intake  ↑ acid production  ↓ saliva pH and buffering capacity
 ↑ production of ECM of biofilm  ↑ acid concentration on the enamel surface  ↑ growth
support of aciduric and acidogenic species  dysbiosis
̶ periodontitis  dysbiosis of subgingival microbial communities (biofilm)  formation and
maintaining of gingival and periodontal inflammation  adverse effect on the host IS  blocking
of IS subversion and tissue regeneration
 some types of OM biofilm  etiological agents (red complex bacteria)
 Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola  show the ability to manage processes involved
in the pathogenesis of periodontal disease by controlling microbiota restructuring and promoting inflammation
 oral virome may be as important in the pathogenesis of the disease as oral bacteriome
https://doi.org/10.1111/prd.12393
https://doi.org/10.1038/s41579-018-0089-x
Microbial colonization occurs on
all available surfaces, and
microorganisms can also
penetrate epithelial tissues and
cells. The microbiota assembles
into biofilm communities on the
abiotic and biotic surfaces. In
health (left), eubiotic biofilms
maintain a homeostatic balance
with the host. In disease (right),
caries and periodontitis ensue
when biofilms become dysbiotic,
resulting in increased levels and
duration of low pH challenge and
the induction of destructive
inflammatory responses,
respectively. EPS, extracellular
polymeric substance; GCF,
gingival crevicular fluid.
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI35
Molecular analysis of OM
̶ Oral microbiome
̶ dysbiosis  oral microbes can affect the immune system
response and pathogenesis of diseases in the system
(reservoir of pathobionts)
 oral cavity  high degree of vascularization,
entry into the respiratory and digestive systems
̶ systemic diseases  primary mechanisms linking oral infection to systemic pathologies
 spread of infection from the oral cavity due to transient bacteremia
 circulation of microbial toxins
 systemic inflammation caused by adverse immunological reactions to oral microbes
̶ microbes associated with the oral cavity detected in many distant organs (small intestine, lungs,
heart, brain, placenta)  colonization depends on the health status of the tissue
̶ proven association between microbes involved in periodontitis and chronic conditions
(cardiovascular diseases, hypertension, inflammatory diseases)
 subgingival biofilm  a source of bacteria and pro-inflammatory mediators  blood circulation
https://doi.org/10.1111/prd.12393
https://doi.org/10.1038/s41579-018-0089-x
Microbial colonization occurs on
all available surfaces, and
microorganisms can also
penetrate epithelial tissues and
cells. The microbiota assembles
into biofilm communities on the
abiotic and biotic surfaces. In
health (left), eubiotic biofilms
maintain a homeostatic balance
with the host. In disease (right),
caries and periodontitis ensue
when biofilms become dysbiotic,
resulting in increased levels and
duration of low pH challenge and
the induction of destructive
inflammatory responses,
respectively. EPS, extracellular
polymeric substance; GCF,
gingival crevicular fluid.
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI36
Molecular analysis of OM
̶ Oral microbiome – a potential biomarker of systemic diseases
https://doi.org/10.1111/prd.12393
https://doi.org/10.3390/microorganisms8020308
Oral and systemic diseases associated with the oral microbiome. A representation of the associations found between diseases with
increases or decreases of the abundances of organisms in the oral cavity. Organisms listed in blue have been shown to be increased in
abundance in the oral cavity in individuals presenting with the noted disease, and organisms listed in red have been shown to be
decreased. Those in purple may be either increased or decreased depending on the conditions or progression of the disease.
oral
microbiome
as a non-
invasive
biomarker
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI37
Molecular analysis of OM
̶ Oral microbiome
https://doi.org/10.3390/microorganisms8020308
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI38
Molecular analysis of OM
̶ Oral microbiome – methods of analysis
̶ sampling place – sample location
 OM at various sites of the oral cavity (saliva, tongue, palate, buccal mucosa, tooth surfaces, gums,
supra- / subgingival plaque, tonsils, throat) shows an overall similarity, but with some differences 
ecological niches
 general microbial screening for diagnosis is performed from saliva
or site-specifically from gingival crevicular fluid or dental biofilm
https://doi.org/10.1111/prd.12393
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI39
Molecular analysis of OM
̶ Oral microbiome – methods of analysis
̶ microbiological cultivations
 250 species isolated and characterized
 OM is complex  some species have not been cultivated
̶ single-cell sequencing NGS
 originally developed for immune profiling
 modified to allow evaluation of individual microbial cells
 ↑ detection rate of non-cultivable organisms
̶ 16S rRNA sequencing
 sequencing of the conserved gene for 16S rRNA (bacteria) or ITS (internal transcribed spacer) DNA region (fungi)
 most common sequencing method, cheap
 taxonomic data only (limited differentiation of phylogenetically related species / strains)
̶ whole genome shotgun sequencing (WGS)
 DNA is randomly fragmented and then subjected to Sanger sequencing or NGS
 tool for metagenomic analysis, parallel evaluation of all kingdoms (bacteria, fungi, viruses) in one sample
 not only taxonomic data but also biological functional profiles of the microbial community
 evolutionary analysis of specific organisms associated with a particular disease or environment
https://doi.org/10.1111/prd.12393
genomics
qPCR
 not only 16S rRNA gene, but also
other genes, also allows quantification
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI40
Molecular analysis of OM
̶ Oral microbiome – methods of analysis
̶ transcriptomics  defining the expression of microbial and host genes in the context of oral
health and pathology
̶ mRNA sequencing (metatranscriptomics)
 utilizing random hexamer primers
 summary of viable and transcriptionally active microbes
 parallel evaluation of microbial and host transcriptomes  examined to assess the interactome
̶ metabolomics  metabolic and functional activities of the host and its microbiome  view of
complex interspecies interactions
 low molecular weight metabolites, proteins
 liquid / gas chromatography with mass spectrometry, NMR
 periodontitis  a characteristic shift in the composition of oral bacteria, which is partly mediated by bacterial
metabolites  metabolomics  how and why this shift occurs
 it can offer guidance for critical time points at which therapeutic interventions could be beneficial
https://doi.org/10.1111/prd.12393
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI41
Molecular analysis of OM
̶ Oral microbiome – methods of analysis
̶ proteomics  profile of all proteins in an organism, tissue, cell or biological fluid, or subcomponent
of any of them  view on health / illness
 proteins in a sample  yes/no, amount, posttranslational modifications, isoforms, molecular interactions
 1-D/2-D gel electrophoresis with mass spectrometry, liquid chromatography with mass spectrometry
 to characterize changes in gingivitis, or mild, moderate and chronic periodontitis
̶ high resolution techniques + clinical data + longitudinal studies  understanding the interactions
of microbes and hosts from the species level to the molecular level and their implications for oral
health
https://doi.org/10.1111/prd.12393
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI42
Molecular analysis of OM
https://doi.org/10.3390/microorganisms8020308
Schematics of standard techniques used in microbiome studies. (A) Marker gene sequencing techniques can use primers to target certain conserved regions of a genome to capture intermittent variable regions, which can then be used
to identify organisms in a sample rapidly and inexpensively. The 16S rRNA gene is the most commonly used marker gene in bacteria and archaea, and in the figure, primers are used to capture the V3 and V4 variable regions together, a
common approach for 16S sequencing. The internal transcribed spacer (ITS) region of the nuclear rRNA cistron in fungi is made of two segments, which can be captured with primers targeting the 18S, 5.8S, and 28S rRNA sections that
surround them. (B–D) Instead of targeting one small segment of the genome, these techniques capture the entirety of the genetic material from an organism. (B) Single virus genomics (SVG) uses a fluorescent stain to isolate individual
virus particles in a sample by fluorescence-activated virus sorting (FAVS), wherein they are embedded in an agarose bead before undergoing whole genome amplification and sequencing. (C) Whole metagenome shotgun sequencing
(WMS) involves the fragmentation of all DNA in a sample, sequencing of the fragments, and assembly of the sequences, which can then be mapped to reference genomes, or de novo assembly can be performed. (D)
Metatranscriptomics also involves a shotgun sequencing approach, but it is performed after mRNA extraction. The outputs then allow for differential gene expression analysis. (E) Metabolomics and metaproteomics allow for
quantification of the metabolites and proteins produced by the microbiome in a sample, respectively. Mass spectrometry is a common approach to quantification.
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI43
Molecular analysis of OM
https://doi.org/10.3390/app11094050
̶ Oral microbiome
 clinical applications
 prediction of susceptibility to oral diseases
 microbial screening for systemic diseases
 early diagnosis of a disease (before onset of symptoms)
 monitoring of disease process and effectiveness of treatment (shift of microbiota from dysbiosis to eubiosis),
targeted treatment
 effective tool for disease prevention (evidence of a patient's dental care)
 development of new therapeutic approaches, personalized dental treatment
 research – an effort to fully characterize a "healthy" microbiome
(Which components of the microbiome should be monitored to evaluate the return of the microbiome from dysbiosis to
a state compatible with health? Is it sufficient to monitor only selected key species or is it necessary
to use multispecies assays?)
̶ Oral microbiome – methods of analysis
̶ point-of-care testing (chair-side diagnostics):
caries susceptibility – device CariScreen Susceptibility Testing Meter (Oral BioTech LLC)
 bacterial aktivity of S. mutans
 after wiping the plaque off the tooth surface, a bioluminescent reaction ATP occurs in a special brush,
which is measured by the device
periodontitis – BANA-Enzymatic test™ kit, Evalusite kit (immunoassay)
̶ laboratory analysis
commercial tests - example
periodontitis – kit MyPerioPath® (OralDNA Lab)
 salivary test for the presence and amount of 11 bacteria species that contribute
to periodontitis (quantitative real-time PCR analysis)
ELISA test  antigens of P. gingivalis
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI44
Molecular analysis of OM
https://www.creative-bioarray.com/support/atp-
cell-viability-assay.htm
https://carifree.com/product/pro-cariscreen-testing-meter/
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI45
Molecular analysis of OM
https://doi.org/10.3390/diagnostics11060932
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI46
Molecular analysis of OM
̶ Oral microbiome
̶ databasis
47
Genetic association
studies - candidate
gene approach
(case-control studies)
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI
http://www.discoveryandinnovation.com/BIOL20
2/notes/lecture25.html
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI48
Genetic association studies
̶ Candidate genes
̶ Selection of suitable candidate genes
 based on known biological, physiological or functional significance in relation to the disease
 search for new potential genes (alleles) in the whole genome (GWAS, QTL - quantitative trait loci)
̶ Suitable candidate genes for caries risk association studies
 genes participating in tooth development and affecting its morphology
 genes related to immune response
 genes related to production and composition of saliva
 genes related to taste preferences
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI49
Genetic association studies
̶ Candidate genes
̶ Selection of alleles (polymorphisms)
 SNP (Single Nucleotide Polymorhism), CNV (Copy Number Variation), VNTR (Variable Number of Tandem Repeat)
 based on studies that have been performed in other populations, GWAS, QTL
 minor allele frequency is sufficient in chosen population
(↓ frequency of allele in the population  ↑ number of cases / controls)
 linkage disequilibrium among SNPs  tagSNP
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI50
Genetic association studies
̶ Genotyping methods
̶ selection of an appropriate methodical approach
 number of polymorphisms to be determined
 total number of samples to be genotyped
 quality of analyzed DNA sample (genomic DNA - blood, saliva, buccal swab)
 costs of instruments, equipment, chemicals and consumables
 availability of commercial genotyping services
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI51
Genetic association studies
̶ Genotyping methods
̶ PCR+RFLP (restriction fragment lenght polymorphism)
 PCR amplification followed by specific restriction digestion
polymorphism is part of a palindromic sequence
Hinf I
5´… G A N T C … 3´
3´… C T N A G … 5´
II. alela  5´… G A G T C … 3´
I. alela  5´… G A G C C … 3´
Huang, BCM Cancer (2008)
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI52
Genetic association studies
̶ Genotyping methods
̶ allele-specific PCR
 primers that are specific for particular allele
 if the allele is present  amplification product is generated  detection
https://doi.org/10.1016/j.jim.2004.10.007
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI53
Genetic association studies
̶ Genotyping methods
̶ real-time PCR  fluorescently labeled hybridization probes
commercial TaqMan probes
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI54
Genetic association studies
̶ Genotyping methods
̶ Sanger sequencing  sequence of a part of DNA with polymorphism
heterozygote
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI55
Genetic association studies
̶ Genotyping methods
̶ Single Nucleotide Polymorphism
Detection with the iPLEX® Assay
and the MassARRAY® System
Genetika v zubním lékařství, Jaro/2021 – Zubní lékařství, Ústav patologické fyziologie LF MU56
Genetic association studies
̶ Metody genotypizace
̶ výběr vhodného metodického přístupu – podle čeho vybírat
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI57
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Proteins involved in development of enamel
AMELX – Amelogenin gene
ENAM – Enamelin gene
TUFT1 – Tuftelin gene
KLK4 – gene encoding Kallikrein-related peptidase 4
AMBN – Ameloblastin gene
TFIP11 – gene encoding Tuftelin-interacting protein 11
MMPs (MMP20) – genes encoding Matrix Metalloproteinases
Schematic diagram of histological changes in amelogenesis. The histological development of enamel crystals goes
hand in hand with changes in ameloblast morphology. Undifferentiated epithelial cells receive signals to transform into
secretory ameloblast cells of some 75 μm tall and ∼5 μm in diameter with a specialized distal cell process (Tomes’
process) which plays an important role in matrix exocytosis. These same cells will retransform into shorter cells
(∼35 μm tall) during maturation devoid of the Tomes’ process. In maturation stage, ameloblasts undergo cyclical
changes from a cell with a distal ruffled border, the ruffled‐ameloblast (RA), to a cell with a smooth distal border, the
smooth‐ameloblast (SA). Tight junctions are found at the basal and apical pole of secretory ameloblasts. The apical or
distal pole is closest to the enamel crystals. In RA cells, tight junctions are found only at the apical pole but in SA cells
they are located at the basal pole. Organellar distribution differs in cells at each stage (see text for details).
SI = stratum intermedium, PL = papillary layer, EMPs = enamel matrix proteins. MMP20 and KLK4 are the main
proteases in AMEL processing. See also organellar distribution at each stage.
https://doi.org/10.1016/j.sjbs.2020.11.071
https://doi.org/10.1113/JP272775
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI58
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Taste receptors  associated with ↑ preference for sweet taste  ↑ sugar intake
TAS2R38 – gene encoding Taste receptor 2 member 38  G protein-coupled receptor, responsible for sensitivity to bitter
taste
TAS1R1/TAS1R3 – genes encoding Taste receptor 1 member 2 and 3  G protein-coupled heterodimeric receptor,
responsible for sensitivity to sweet taste
https://doi.org/10.3390/s100403411
https://doi.org/10.1080/00016357.2020.1832253
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI59
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Glucose transporter  associated with ↑ preference for sweet taste  ↑ sugar intake
GLUT2 – gene encoding Glucose transporter 2 – required for glucose-stimulated insulin secretion (pancreatic β-cells),
controls perception of glucose (nervous system) control of food intake
- expression is required for the physiological control of glucose-sensitive genes, its inactivation in the liver leads
to impaired glucose-stimulated insulin secretion
https://doi.org/10.1080/00016357.2020.1832253
https://doi.org/10.3390/s100403411
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI60
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Proteins of immune system
MBL2 – gene encoding Mannose-binding lectin (AKA Mannose-binding protein, Mannan-binding protein/lectin,
Collectin 1, MBP1, or Mannose-binding protein C)
– soluble serum lectin recognizing specific carbohydrates on bacterial surfaces  ↓ complement activation
Schematic representation of the lectin pathway of the complement system. The lectin pathway (LP) is
triggered by five pattern recognition receptors (PRR): mannose-binding lectin (MBL), ficolin-1, -2, and -3,
and collectin 11 (CL11 or CL-K1). The LP is initiated when these PRRs bind to pathogen-associated
molecular patterns (PAMPs) on the surface of pathogens or to apoptotic or necrotic cells (damageassociates
molecular patterns, DMAPs). Circulating MBL, CL11, and ficolins form complexes with MASP-1
and MASP-2. After the binding of MBL, ficolins, and CL-11 to their targets, MASP-1 auto-activates and
triggers MASP-2. Activated MASP-2 cleaves C4 and C2 allowing the assembly of the C3 (C4bC2a) and C5
(C4bC2a(C3)n)convertases and the subsequent activation of the terminal pathway. Activated MASP-1 also
cleaves C2. MAC = membrane attack complex.
https://doi.org/10.1007/978-1-4614-9209-2_7-1
https://doi.org/10.1080/08927014.2020.1856821
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI61
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Proteins in saliva
DEFB1 – gene encoding β-Defensin 1 – an antimicrobial peptide from family of Defensins (alpha, beta), which includes
cysteine-rich cyclic cationic peptides. They are part of innate immunity, create channels in the cytoplasmic
membrane of bacteria, stimulate the immune system incl. complement (classical pathway), act as chemoattractants.
LTF – Lactoferrin gene – transport globular glycoprotein, binds free iron. Part of innate immunity, antibacterial (peroxides are
formed when interacting with bacterial membranes), antiviral (competition of adhesion of viral particles to host cells,
binding to particles of certain types of viruses), antifungal (against C. albicans) activity, stimulation of phagocytosis.
LYZL2 – gene encoding Lysozyme-like protein 2 – part of C-type Lysozyme family.
Hydrolyzes glycosidic bonds in peptidoglycans (breaking down the cell wall of G+ bacteria).
https://doi.org/10.1590/1807-3107bor-2017.vol31.0041
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI62
Genetic association studies
̶ Candidate genes that have been associated
with increased risk of dental carries
̶ Proteins in saliva
CA6 – gene encoding Carbonic anhydrase VI – enzyme also called ‚gustin‘, catalyzes the hydration of carbon dioxide to form
bicarbonate ions and protons. Saliva pH maintenance (bicarbonate buffer system)
MUC7 – gene encoding Mucin 7 – low molecular weight glycoprotein (MG2), participates in the formation of acquired pellicle
and thus in bacterial adhesion and biofilm formation
MUC5B – gene encoding Mucin 5B – glycoprotein (MG2), participates in the formation of acquired pellicle and thus in
bacterial adhesion and biofilm formation
PRH1 – gene encoding salivary acidic proline-rich phosphoprotein 1, participates in the formation of acquired
pellicle and thus in bacterial adhesion and biofilm formation
https://doi.org/10.1590/1807-3107bor-2017.vol31.0041
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI63
Genetic association studies
̶ Pitfalls of genetic association studies of dental caries
 too many factors playing role in etiopathogenesis  set of patients (cases) can never be perfectly categorized 
defined set of cases cannot be created  maximally defined set of cases as far as possible
 most studies do not confirm the association, only suggests (some studies even give conflicting results)  further
studies are needed to understand the correlations found
 further association studies (more samples)  studies of individual polymorphisms (but their effect may be small),
but also genes and loci (gene-based and gene-cluster analysis)  strengthening of the results
 meta-analysis – a combination of data obtained by an exhaustive search of published and unpublished worldwide
data  increasing the consistency of the results (by increasing the strength of the result). Many primary studies
are too small to demonstrate an important clinical effect (not strong enough). A combination of all studies that
answer the same clinical question  increase in statistical power or significance level
 detailed questionnaires for evaluating the influence of psychological, sociological, economic and behavioral
factors  fragmentation of set of cases to too many groups of too few patients
Genetics in Dentistry, Spring/2022 – Dentistry, Department of Pathophysiology MED MUNI64
https://doi.org/10.1111/adj.12262