Salivonomics:The Current Scenario

Abstract:

For generations, blood has been the medium of choice for diagnosing most diseases and conditions. The reason for this is mainly the limitations of technology. Concept of Oral diagnostics is preferred to more invasive methods. In recent years it has become evident that the salivary constituents become detectably altered in response to certain disease states. Even so, what is most impressive is that salivary biomarkers not only arise in correlation with oral disorders, but also those of distal tissues and organs. This suggests that oral fluids may represent a substantial reservoir of molecular and microbial information capable of communicating the onset or presence of disease throughout the body.   An initiative of the National Institute of Dental and Craniofacial Research (NIDCR) has created a roadmap to achieve these goals whereby, with  the use of oral fluids as the diagnostic medium,it would become possible to  scrutinize the health and/or disease status of patients.

The real promise of salivary analysis use is the ability of the patient or clinician to directly and continuously assess disease status, progression, and therapeutic efficacy. Sensitive analysis may even allow pre-symptomatic diagnosis. There are five major diagnostic alphabets available in saliva namely, proteins, mRNAs, mi-RNAs, metabolic compounds, and microbes which offer substantial advantages for salivary diagnostics because, the state of the disease may be associated with detectable changes in one, but not all, dimensions. Recently, the Salivaomics Knowledge Base (SKB) has been established by aligning the salivary biomarker discovery. The SKB constitutes data repository, management system, and web resource fabricated to support human salivary proteomics, transcriptomics, miRNA, metabolomics, and microbiome research.

Introduction

The diagnosis of systemic diseases in routine circumstances is via

(1) patient reported symptoms,

(2) examination and a medical history obtained by a physician or other medical professional,

(3) chemical analysis of blood and/or urine samples.

The patient’s samples are typically sent to a remote, clinical diagnostic laboratory for determination of the levels of a broad series of markers including ions, antibodies, hormone levels, and a variety of disease-specific biomarkers. (1)

For generations, blood has been the medium of choice for diagnosing most diseases and conditions. The reason for this is mainly the limitations of technology.

Despite the regular screenings and check-ups, many diseases are undetected until a late phase where morbid symptoms become apparent. To overcome these challenges, researchers are unravelling biomarkers. These biomarkers include genetic material (e.g. DNA, RNA) and protein molecules that reflect the current physiological state of an individual and hence help scientists to better understand the underlying cause of a disease. (2)Biomarkers in the blood are commonly measured in micrograms, which are one millionths of a gram.

Saliva is a biological fluid (bio-fluid) that could be a source of biochemical data capable of detecting certain diseases & so would be useful for novel approaches to prognosis, laboratory or clinical diagnosis, and monitoring and management of patients with both oral and systemic diseases. Besides, it is easily collected and stored and ideal for early detection of disease as it contains specific soluble biological markers (biomarkers). (4)

The various chemical components of saliva include water, inorganic compounds (ions), organic compounds (nonproteins and lipids), protein/polypeptides, and hormones, and an array of analytes.(5)

Saliva contains multiple biomarkers which make it useful for multiplexed assays that are being developed as point-of-care (POC) devices, rapid tests, or in more standardized formats for centralized clinical laboratory operations. Salivary diagnostics is a dynamic field that is being incorporated as part of disease diagnosis, clinical monitoring and for making important clinical decisions for patient care.(1)

Although, the discovery of  discriminatory  biomarkers in oral fluids epitomizes an almost incomparable breakthrough in clinical and translational science ,continuation in this area is required to  credentiate saliva as an acceptable diagnostic medium, an end point with the potential to bring about a paradigm shift in the discipline of molecular diagnostics.(4)

Besides, biomarkers in saliva are commonly measurable in picograms, one trillionth of a gram, or nanogram quantities, one billionth of a gram & so have to be very meticulously identified & assessed.

Thus, two prerequisites exist before the goal of SDs can be achieved:

  1. identification of specific biomarkers associated with a health or disease state and
  2. the development of technologies that can discriminate between the bio makers.

Although the utility and advantages of saliva as a screening tool for cystic fibrosis has been identified in the early 1960s, its full diagnostic potential was discovered three decades later when studies revealed distinct advantages of saliva over serum. Like serum, saliva also contains hormones, antibodies, growth factors, enzymes, microbes and their products.(2)

Today, since technology  is at a point where the minute quantities are readily detectable using a number of methodologies, including next-generation sequencing, proteomics, mass spectrometry, genome-wide association studies (GWAS), and other screening techniques so, the advantages of salivary testing are now obvious. Biomarkers in saliva have been identified for a wide variety of diseases and conditions including: HPV, oral cancer, dental caries, Sjogren’s syndrome, sleep apnea, Alzheimer’s disease, Parkinson’s disease, C-reactive protein, and the ulcer-causing bacteria—Helicobacter pylori (H. pylori)—among many others. In the near future, salivary samples may be able to distinguish between bacterial and viral infections and detect recent heart attacks, traumatic brain injury, even lung and gastric diseases.

Concept of Oral diagnostics is preferred to more invasive methods &   gets substantiated by the success of the oral thermometer to detect fever which has totally replaced its predecessor, the rectal thermometer. (4)

However, the ability to accurately assess biomarkers in samples obtained from the oral cavity depends on the biochemical nature of the marker, the source and type of sample being taken, and the mechanism by which the marker enters the oral cavity. The most widely used type of oral sample is a swab that collects a deoxyribonucleic acid (DNA) sample, particularly in Forensic odontology and more recently in single nucleotide polymorphisms (SNP) analyses for mutations associated with specific diseases .(1

Advantages of use of saliva for biomarker detection(6)

  1. It has real-time diagnostic values
  2. Safer for health professionals than blood sampling
  3. Ease of obtaining Multiple samples
  4. Collection & Screening can be done at home
  5. It is non-invasive, easy & inexpensive
  6. It has minimal risk of cross contamination
  7. More economical sampling, shipping &storage
  8. Manipulation required during diagnostic procedures is less as compared to that for serum
  9. There is commercial availability of screening assay
  10. saliva does not clot unlike blood

Limitations (6):

  1. Levels of certain markers in saliva are not always a reliable reflection of the levels of these markers in serum.
  2. Salivary composition can be influenced by the method of collection and degree of stimulation of salivary flow.
  3. Changes in salivary flow rate may affect the concentration of salivary markers and also their availability due to changes in salivary pH.
  4. Variability in salivary flow rate is expected between individuals and in the same individual under different conditions.
  5. In addition, many serum markers can reach whole saliva in an unpredictable way (i.e. gingival crevicular fluid flow and through oral wounds). These parameters will affect the diagnostic usefulness of many salivary constituents.
  6. Furthermore, certain systemic disorders, numerous medications and radiation may affect salivary gland function and consequently the quantity and composition of saliva.
  7. Whole saliva also contains proteolytic enzymes derived from the host and from oral microorganisms. These enzymes can affect the stability of certain diagnostic markers. Some molecules are also degraded during intracellular diffusion into saliva.

An initiative of the National Institute of Dental and Craniofacial Research (NIDCR) has created a roadmap to achieve these goals through the use of oral fluids as the diagnostic medium to scrutinize the health and/or disease status of patients. This is an ideal opportunity to optimize state-of-the-art saliva-based biosensors for salivary biomarkers that discriminate between diseases.(7)

In recent years it has become evident that the salivary constituents become detectably altered in response to certain disease states. Even so, what is most impressive is that salivary biomarkers not only arise in correlation with oral disorders, but also those of distal tissues and organs. This suggests that oral fluids may represent a substantial reservoir of molecular and microbial information capable of communicating the onset or presence of disease throughout the body.  Today, a growing number of studies are focused on elucidating and developing saliva-based biomarkers indicative of both local and systemic diseases. (4)

Some systemic diseases affect salivary glands directly or indirectly, and may influence the quantity and quality of saliva. These characteristic changes may contribute to the diagnosis and early detection of these diseases. Systemic disorders that may affect salivary glands and saliva are given below. (8)

SYSTEMIC DISEASES AFFECTING SALIVARY GLANDS AND SALIVA

Sjögren’s syndrome

Rheumatoid diseases

Myasthenia gravis

Cancer

Cirrhosis

Cystic fibrosis

HIV infection

Hypertension

Hormonal disorders

Malnutrition, dehydration, vitamin deficiency

Parkinsonism, Alzheimer’s disease

Renal disease

Sarcoidosis

Due to its diverse biological functions, salivary testing is rapidly growing as a practical and reliable means in clinics and research to recognize early signs of systemic illness and exposure to risk factors. Owing to the wide-spread use and growing acceptability of saliva as a diagnostic tool, various investigators have focused their efforts to establish its clinical usefulness. The data have proved to be of  significant utility for researchers, healthcare professionals,& community health program personals to detect  and monitor diseases & to improve general health of the public. (10)

The real promise of salivary analysis use is the ability of the patient or clinician to directly and continuously assess disease status, progression, and therapeutic efficacy. Sensitive analysis may even allow presymptomatic diagnosis. Thus, microfluidic and microelectromechanical systems (MEMS) and nanoelectrofluidic systems (NEMS) for salivary diagnostics have been

under development for at least the last decade. Microfluidics have the advantage of using low sample and reagent volumes; microelectronics facilitate the development of miniaturised  chairside and handheld devices suitable for use in the dental clinic and home in the absence of specialised laboratory facilities. In combination, they promise to provide simultaneous and rapid measurement of multiple biomarkers coupled with data storage and transmission. (11)

Process of biomarker discovery  is through definitive validation and approval. Biomarkers of oral and systemic diseases are discovered using one or more of the ‘omics’ libraries. Verified biomarkers are subjected to increasing scrutiny and larger independent cohorts until they reach a pivotal multicenter validation. Salivomics  includes: proteomics, epigenomics, metabolomics, immunomics, microbiome and transcriptomics.(4)

There are five major diagnostic alphabets available in saliva namely, proteins, mRNAs, mi-RNAs, metabolic compounds, and microbes which offer substantial advantages for salivary diagnostics because, the state of the disease may be associated with detectable changes in one, but not all, dimensions. The salivary biomarkers are also classified based on the mechanism of action.(5)

Mechanism of Action:

Level of Action

  1. Changes in the cellular DNA

Allelic loss on chromosomes 9p, mitochondrial DNA mutations, p53 gene mutations, promoter hypermethylation of genes (p16, MGMT, or DAPK), cyclin D1 gene amplification, increase of Ki67 markers, microsatellite alterations of DNA, presence of HPV and EBV viral genome.

2. Altered mRNA transcripts

Presence of IL8, presence of IL1B, DUSP1 (dual specificit phosphatase 1), H3F3A (H3 histone, family 3A), OAZ1 (ornithine decarboxylase antizyme 1), S100P (S100 calcium binding protein P), SAT (spermidine/spermine N1-acetyltransferase)

3. Altered protein markers

Elevated levels of defensin-1, elevated CD44, elevated IL-6 and IL-8, inhibitors of apoptosis (IAP), squamous cell carcinoma associated antigen (SCC-Ag), carcinoembryonic antigen (CEA) carcino-antigen (CA19-9), CA128 serum tumor marker (CA125), intermediate filament protein (Cyfra 21-1), tissue polypeptide specific antigen (TPS), reactive nitrogen species (RNS), 8-OHdG DNA damage marker, lactate dehydrogenase (LDH), immunoglobulin (IgG), s-IgA, insulin growth factor (IGF), metalloproteinases MMP-2 and MMP-11

Salivary Proteins & Proteomics:

In 2004 the National Institute of Dental and Craniofacial Research provided funding to comprehensively decipher, catalogue and annotate the human salivary proteome. These efforts revealed the salivary proteome as a sizeable collection of up to 1,166 diverse proteins , the majority of which are synthesized and subsequently secreted into the oral cavity by the acinar cells of the salivary gland.It could be indicative of both local and distant diseases. To evaluate the capacity of salivary proteomic entities as diagnostic metrics, scientists frequently employ traditional laboratory techniques including:liquid chromatography, gel and capillary electrophoresis, nuclear magnetic resonance, mass spectrography, immunoassays lectin probe analysis and 2-dimensional gel electrophoresis coupled with mass spectrometry(4)

More than 2300 minor proteins or peptides are present in saliva defining the first salivary biomarker alphabet, although ,the proteomic content of saliva was found to be only 30% that of blood. The most plentiful proteins includes α-amylase, albumin,cystatins, hystatins, secretory-IgA, lactoferrin, mucins, lysozymes, proline rich proteins, statherin and transferrin which together account for more than 98% of the total salivary proteins.

Functional use of salivary proteins:

Salivary proteins are involved in a number of metabolic pathways, including amino acid related metabolism, carbohydrate metabolism, energy metabolism, as well as glycan biosynthesis and metabolism. Therefore , the sequential amino acid in a protein provides a link between the proteins and their respective coding genes through genetic code, and the  protein complement of the genomics known as the proteome.(5)

The presence of these salivary proteomic markers in low concentration plays a major role in the discrimination of diseases. Researchers have reported unique proteomic profiles indicative of oral cancer, cystic fibrosis, endocrinological and connective tissue disorders, dental caries, periodontitis and even breast carcinoma in saliva.(3,4)

Analysis of the salivary proteomes may reveal morbidity signatures in the early stage and monitor disease progression .

Proteomic studies of human saliva constitutes four major salivary families of specific secretory proteins that differ significantly from other host defence salivary proteins, as the former group has specific functions in the oral environment . Aim of the proteomic study of saliva from mammals is to define the protein complex of whole human saliva in healthy subjects, the contribution of the different glands, the alterations related to pathological conditions, either systemic or restricted to the oral cavity, to understand the functions of each protein component in the oral cavity, to characterize new peptides and proteins displaying biological activity.(9)

Four major salivary families of specific secretory proteins:

Proline-rich proteins (PRPs), statherins, histatins and cystatins (can be distinguished in five cystatins of salivary origin (S, S1, S2, SA, SN) and two others common to various body fluids (C and D). They are multifunctional proteins playing various roles in the oral environment. cystatin C, was un-detectable in saliva)(9)

The difficulties encountered in the study of the peptidome (the complete set of peptides encoded by a particular genome or present within a particular cell type) are due to the high genetic polymorphisms, complicated by individual insertions/deletions and alternative splicing to the complex post-translational maturations including different proteolytic cleavages and glycosylation, phosphorylation and sulphation processes.(5)

Transcriptomics:

Composed of coding (messenger RNA) and noncoding RNAs (small nucleolar RNAs, microRNAs, i.e. miRNAs, and small nuclear RNAs), the transcriptome makes a significant source of potentially relevant diagnostic information with highly specific discriminatory indicators.

Whole saliva as well as saliva supernatant harbor both miRNAs and total RNA. Investigators probing the salivary transcriptome have identified over 1,000 miRNAs and more than 3,000 species of messenger RNAs in the human saliva, out of which 180 are common between different normal participants constituting the normal salivary transcriptome core.

In addition to the combined approach of transcriptomics and proteomics, miRNA constitutes the third diagnostic alphabet in saliva. This is because , any dysregulation in expressing these miRNA will adversely affect the cell growth and acts like a tumor suppressor or oncogene in many cancers. In oral cancer, miRNAs have been shown to affect cell proliferation, apoptosis, and even chemotherapy resistance in OSCC patients, miRNAs have also been observed in OSCC to be epigenetically regulated by DNA methylation.(3,5)

Salivary Metabolome

The collection of small molecules present in cells, tissues, organs, and biological fluids is known as metabolome, and the study of metabolome is metabolomics. The metabolome validates the parallel assessment of a group of endogenous and exogenous metabolites, including lipids, amino acids, peptides, nucleic acids, organic acids, vitamins, thiols, and carbohydrates and is a valuable tool for discovering biomarkers, monitoring physiological status, and making proper treatment decisions.For eg: Taurine and piperidine are considered as the oral cancer specific diagnostic marker.(5)

Salivary Microbiome

Oral microbiome is constituted by proportionately a minute number of bacterial phyla, of which the commonly reported abundant phyla are Firmictes, Proteobacteria, Bacteroidetes,

Actinobacteria, and Fusobacteria .The majority of interindividual variation has been personated because of diversity at the species or strain level. Streptococcus is routinely observed to be the dominant genus in the healthy oral microbiome and commonly Prevotella, Veillonella,

Neisseria, and Haemophilus dominate an individual’s oral microbiome. Variation is also observed in the microbial community composition of biofilms at each intraoral habitat(e.g., teeth surface, lateral and dorsal surface of tongue, etc.), most likely reflecting the different surface properties and microenvironments. Many individuals shelter only about 75 to 100 predominant species of bacteria which are known to inhabit the oral cavity from the reported 700 to 1,200 bacterial species that reside in the mouth.(5)

The identification of salivary biomarkers for oral, pancreatic, lung and gastric cancers along with Sjögren’s syndrome, diabetes and periodontal disease is being pursued by researchers. Gene chip arrays and quantitative polymerase chain reaction denote the most common methodologies for generating diagnostic values.(4)

Recently,the Salivaomics Knowledge Base (SKB) has been established by aligning the salivary biomarker discovery. The SKB constitutes data repository, management system, and web resource fabricated to support human salivary proteomics, transcriptomics, miRNA, metabolomics, and microbiome research. The SKB provides the first web resource dedicated to salivary “omics” studies. This comprises the major data and information required to explore the biology, diagnostic potentials, pharmacoproteomics, and pharmacogenomics of human saliva.(5)

References

  1. Daniel Malamud, Isaac R. Rodriguez-Chavez .Saliva as a diagnostic fluid.. Dent Clin North Am. 2011 Jan; 55(1): 159–178
  2. Mohammad A Javaid , Ahad S Ahmed et al.Saliva as a diagnostic tool for oral & systemic diseases. J Oral Biol Craniofac Res. 2016 Jan-Apr; 6(1): 66–75.
  3. Janice M. Yoshizawa, Christopher A. Schafer, Jason J. Schafer, James J. Farrell, Bruce J. Pasterand  David T. W. Wong .Salivary Biomarkers: Toward Future Clinical and Diagnostic Utilities. Microbiol. Rev. October 2013 vol. 26 no. 4 781-791
  4. Christopher A Schafer, Jason J Schafer et al. Saliva diagnostics: Utilising oral fluids to determine health status. Monogr Oral Sci. Basel, Karger, 2014, vol 24, pp 88–98
  5. Poornima G, Mahesh Kumar TS. Genomic Alphabets of Saliva as a Biomarker in Oral Cancer. J Indian Acad Oral Med Radiol 2017;29: 300-5.
  6. V B Madalli, Dr.S m Basavarradi, Dr. K Burde et al.Saliva-A diagnostic tool. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) ,Volume 11, Issue 6 (Nov.- Dec. 2013), PP 96-99
  7. S Arunkumar , Dr.Arunkumar J .S et al. Developments in diagnostic applications of saliva in oral and systemic diseases- A comprehensive review. Journal of Scientific and Innovative Research 2014; 3(3): 372-387
  8. Mittal S, Bansal V, Garg S, Atreja G, Bansal S. The diagnostic role of Saliva— A Review. J Clin Exp Dent. 2011;3(4):e314-20.
  9. E Scarano, A Fiorita, P M Piciotti et al.Proteomics of Saliva-Personal experience.Acta Otorhinolaryngol Ital.2010 June;30(3):125-130
  10. Shah FD, et al. A review on salivary genomics and proteomics biomarkers in oral cancer. Ind J Clin Biochem. 2011;26(4):326-334
  11. John J Taylor.Protein biomarkers of periodontitis in saliva(Review article) . ISRN Inflammation Volume 2014, Article ID 593151, 18 page

Dr. Sonalee Shah

Professor & HOD
Dept of Oral Pathology and Microbiology
Government Dental College & Hospital
Raipur, Chattisgarh, India

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