Salivary Proteomic Patterns in Children Affected by Different Severity Degrees of Molar Incisor Hypomineralization

Salivary Proteomic Patterns in Children Affected by Different Severity Degrees of Molar Incisor Hypomineralization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Salivary Proteomic Patterns in Children Affected by Different Severity Degrees of Molar Incisor Hypomineralization Elora Silva Toledo¹, Karina Ferreira Rizzardi¹, Fabíola Galbiatti de Carvalho, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2309540/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Saliva is a rich-bodily fluid with recognized clinical diagnosis roles and this research aimed at investigating if there is any change in the salivary proteome signatures of MIH children with distinct degrees of severity. Fifty schoolers (6–10 years) were equally assigned into the following groups: G1 (Control group - Healthy teeth), G2 (Mild MIH with white/creamy opacity and free of caries), G3 (Mild MIH with yellow/brown opacity and free of caries), G4 (Severe MIH with white/creamy, yellow/brown opacities including post-eruptive fracture and free of caries), G5 (Severe MIH with white/creamy, yellow/brown opacities, post-eruptive fracture, and caries). Unstimulated saliva samples were collected and later explored using mass spectrometry analysis. In total, 6,471 proteins were found, 5,073 exclusively from MIH children, and 778 overlapping among the different degrees of the disturb. The Biological Pathways displayed distinct patterns among the groups, being different according to the degrees of MIH. Gene-Odontology differences might not be verified regarding the biological processes and cellular components. Conversely, with respect to molecular function, alterations among groups were evident, with the presence of proteins that would contribute to MIH in children with the severe condition ( i.e , calcium ion binding, microtubule binding, platelet-derived growth factor binding). In conclusion, the results of this study support important salivary proteomic changes in MIH children, according to distinct degrees of severity, reinforcing the interplay between the clinical characteristics and changes in the salivary proteome. Dental hypomineralization Salivary proteins Dental enamel hypoplasia Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Pediatric Dentistry is facing a new disease in this century, known as Molar Incisor Hypomineralization (MIH - Weerheijm et al. 2001). MIH affects the enamel of the first permanent molars, important teeth for grinding food (GAISER, 2012), together or not with the permanent incisors, which are responsible for biting and cutting dietary substrates, as well as for phonetics (ABDALLA, 2020). Recent systematic reviews revealed a constantly increasing pooled prevalence, with a 13,5-14.2% overall average (LOPES, 2021; ZHAO, 2019; DAVE, 2018). Teeth with MIH show morphologically normal enamel; however, its structure is deficient (TAYLOR, 2017). Since there is a decrease in the quantity and quality of minerals, the enamel is more porous, softened, and shows white, yellow, or brownish opacities (WEERHEIJIM, 2003). This way, the defective tooth becomes more fragile and susceptible to caries lesions and erosion, usually with exacerbated sensitivity (WEERHEIJIM, 2003); leading to challenging dental procedures (SCHWENDICKE, 2018; TAYLOR, 2017; WEERHEIJIM, 2003). Of interest, the treatment of this condition is not definitive (MAST, 2013), but palliative seeking to prolong the vitality of the tooth, and to improve the oral health-related quality of life of the children. According to the enamel hypomineralization rate, MIH was classified into different degrees of severity. White, yellow, or brown demarcated opacities, were assigned as a mild degree of MIH; and post-eruptive enamel breakdown jointly with opacities, carious lesions, or complex restorations disconnected to caries pattern, was defined as severe (NEGRE-BARBER, 2018). Although the exact etiology of MIH is unknown (WHATILING, 2008), some hypotheses have been suggested, including systemic health disturbances during the permanent teeth mineralization period (SALEM, 2016). Among these disorders, early childhood diseases, i.e. asthma (RIZZARDI, 2022), frequent use of antibiotics (GHAMIM, 2013), as well as genetic influences (VIEIRA 2016, ALALUUSUA, 2010) (considering that enamel-forming cells are genetically controlled) should be highlighted. In summary, over the last decade, more than 30 systemic etiological hypotheses have been identified; some are well-established, and others are more contemporary (GAROT, 2022). Outstandingly, saliva is a body fluid with complex composition, capable of playing lots of roles connected with oral and systemic health (KHURSHID 2016; JAVAID 2016). It contains bicarbonate ions, responsible for the buffering capacity, neutralizing organic acids produced by bacterial food substrates fermentation (de SOUZA, 2020). Hence, teeth demineralization could be minimized. Several saliva components or proteins can interact with pathogenic bacteria and may be involved in the protection of dental tissues, such as statins, mucins, lactoferrin, defensins, and peroxidases (PAPPA, 2019). Although saliva contains numerous peptides, many of them have not yet been characterized (PAPPA, 2019). With scientific advances, state-of-the-art techniques, such as mass spectrometry analysis, for example, have provided a better understanding of the course of pathologies, identifying biomarkers at a molecular level (VITORINO, 2004). For the last 10 years, salivary proteomics have been tested aiming to disclose pathologically mechanisms on protein expression levels for several oral and systemic conditions (WANG, 2015). As changes in saliva composition are associated with oral pathologies, it is reasonable to assume that the saliva proteome of MIH-affected children might be altered compared to healthy children. Although a considerable amount of studies has attempted to identify proteomic profiles in oral diseases (PAPPA, 2020), there are only scarce data in the literature about the proteome in MIH pathology, indicating differential expression of proteins in MIH patients’ saliva (BEKES, 2020; PAPPA, 2022). Furthermore, we were not able to find studies investigating how salivary proteomic changes as MIH turns from mild to more severe. Caring-out investigation at the systems biology level, additional insights by connecting the disease phenotype to specific biological processes may be brought. Given this background, the main purpose of this research was to investigate if there is any change in the salivary proteome signatures of MIH children with distinct degrees of severity. 2. Results Patient characteristics The control group (G1) comprised 4 girls and 6 boys, with 9.2 years mean age. The MIH groups (G2, G3, G4, G5) comprised 10 girls and 30 boys (mean age of 10.2 years), accounting for 118 affected teeth with different degrees of severity (84.8% molars and 15.2% incisors). The number of decayed, missing, or filled teeth in the G1, G2, G3, and G4 was 0, while in G5 this number was 2,55. Proteomic profile Proteomic analysis was capable of identifying a total of 6,471 proteins, 15% belonging to the control group (G1) and 85% to the MIH groups (G2, G3, G4, G5) (Fig. 1A). Of the nine hundred ninety-two and 5,479 proteins found in the Control and the MIH groups, respectively, 406 overlapped among them. Regarding the 4 MIH groups together, 778 proteins were shown in common (Fig. 1B). 2.1 Proteins overlapping among the MIH groups Overlapping proteins identified among the disease groups were shown according to the degree of severity (Fig. 2). Seven hundred and seventeen, 905, 554, and 1,150 proteins were only detected in groups 2, 3, 4, and 5, respectively. Thirty proteins were common among all MIH groups, with the highest number (451) between the moderate group (G3) and the most severe group including the presence of caries (G5) (Fig. 2). No protein was concomitantly found concerning all MIH groups without decayed, missing, or filled teeth (G2, G3, G4). 2.2 Biological Pathway and Gene-Ontology The proteins found in each group were displayed in Biological Pathways (Fig. 3A, B, C, D, and E) and Gene-Ontology (Fig. 4A, B, C, D, and E). Figures 3F and 4F show the Biological Pathway and Gene-Ontology of the overlapping proteins among the disease groups (778), and Figs. 3G and 4G refers to proteins exclusively from healthy children (G1). In general, the Biological Pathways of the studied groups showed different protein-binding patterns. However, G2 and G3 (Mild MIH degree – Fig. 3B and Fig. 3C) are closer to each other, as well as G4 and G5 (Severe MIH degree – Fig. 3D and Fig. 3E), which presented fewer connections, compared to healthy children’s group. A striking protein-binding pattern difference was identified between Figs. 3F and 3G, accounting respectively for common proteins among the disease groups (G2, G3, G4, G5) and exclusive proteins to the control group (G1). The analysis of the biological process patterns (GOBP) demonstrated intense similarity among all studied groups (G1, G2, G3, G4, G5) (Fig. 4A-4E). On the other hand, GOBP regarding common proteins among the disease groups (G2, G3, G4, G5) versus exclusive proteins to the control group (G1) (Fig. 4F and 4G) were quite different. Assessment of the cellular components patterns (GOCC) revealed close similarities among G1, G2, G3, and G4 (Fig. 4A-4D). Conversely, Fig. 4E, representing the severe degree of MIH including the presence of caries lesions, changed in terms of GOCC. Figures 4F and G also show some discrepancies. The greatest distinctions between the clusters were found concerning the molecular function (GOMF) of each of the studied groups, including the common proteins among the MIH groups (G2, G3, G4, G5 – Fig. 4F) and the exclusive proteins of the control group (G1 – Fig. 4G). 3. Discussion It is well established that saliva is a rich full biological fluid and possibly a biomarker source. Results of the present study highlight a notable difference in the proteomic profile according to distinct degrees of MIH severity. In addition, it seems that the most important difference occurs in the gene-ontology molecular function of the salivary proteins. When exploring the molecular function, the presence of metallopeptidase and metalloendopeptidase activity (GO:0008237 and GO:0004222; Fig. 4) was observed since health condition until MIH degree involving post-eruptive breakdown, without carious lesions (G1-G4). At the most severe degree, involving post-eruptive fracture together with dental caries (G5), the peptidases mentioned above disappeared. Both enzymes catalyze the hydrolysis of bound peptides by a mechanism in which water acts as a nucleophile (BINNS, 2009). Intriguingly, calcium ion binding protein (GO:0005509), was only detected in the most MIH severe group (G5; Fig. 4), as well as when common proteins among the MIH groups were considered (Fig. 4F). This molecular function is connected to calcium ion storage activity (BINNS, 2009). Calcium phosphate is the main component of dental enamel, providing the hardness characteristic of the element (ELFRINK, 2012), composed of 97% minerals. Maybe the calcium ion binding protein interferes with the availability of calcium ion, contributing to the disturbance in the mineralization process, which favor enamel defects. Similarly, it has already been reported that hypocalcemia can stimulate biological mechanisms linked to hypoplasia and hypomineralization development (ALALUUSUA, 2010; VAN AMEROGEN, 1995). These defects imply color alterations (white/creamy to brownish-yellow) and breakdown susceptibility during food grinding (de OLIVEIRA, 2015), and provide a convenient niche for pathogenic bacteria/biofilm accumulation, failure in enamel layers, exposing the dentin layer to the oral environment (SUNDFELD 2020). Regarding the healthy group (G1) and the groups with mild MIH (G2 and G3), endopeptidase activity (GO:0004175) is the second or third most prevalent molecular function (Fig. 4A-4C). When common proteins among the MIH groups were considered, and also in G4 (severe degree), this molecular function drops to the penultimate position (Fig. 4D and 4F), and in G5 (most severe degree) it could not be found (Fig. 4E). Specifically, endopeptidase activity plays a role in catalyzing the hydrolysis of internal alpha-peptide bindings in a polypeptide chain (BINNS, 2009). Proteolytic enzymes are important in the process of enamel formation (amelogenesis), which is highly sensitive. After the secretion of the organic matrix during amelogenesis, proteolytic enzymes reabsorbed certain proteins, providing adequate space for mineral deposition (FARAH, 2010), and normal hydroxyapatite crystal development, which is crucial for healthy enamel. Interestingly, the molecular function of Alpha-amylase and Amylase activity (GO: 0004556 and 0016160) was only identified in the control/healthy group (G1 - Fig. 4A). This enzyme is responsible for starch carbohydrate digestion in the oral cavity and is released during chewing, by the salivary glands. The pancreas is also able to produce Alpha-amylase, which is released in the pancreatic juice, accounting for starch digestion in the upper part of the small intestine. Salivary alpha-amylase has been found bound to microorganisms for bacterial clearance, important for health conditions in impairing caries formation (SCANNAPIECO, 1993). This way, the hypothesis that starch digestion might be prejudiced in the mouth of MIH - children could be raised. The identification of Alpha-amylase and Amylase activity only in G1 emphasizes the salivary proteomic changes in affected children. Conversely, platelet-derived growth factor binding (GO:0048407) could not be detected in the healthy group (G1 -Fig. 4A). However, it appeared in all MIH - children, particularly in G4 (G2, G3, G4, G5 - Fig. 4B-4F). One supposition would be that hypomineralized enamel, particularly with fractures, possibly potentializes molecular interplay between the pulp organ (the innermost portion of the tooth) and the oral environment, exposing blood components to the mouth. When the fractures were connected with carious lesions, maybe there was some stimulation of reactionary dentin, to protect and isolate the pulp from infectious agents. Perhaps that is the reason why platelet-derived growth factor binding (GO:0048407) was especially identified in G4. The transmembrane receptor protein tyrosine kinase (GO:0004714) and syndecan binding protein (GO:0045545) were identified in G3 (Fig. 4C). Tyrosine kinase belongs to a large multigene family accountable for cell functions regulation, i.e . cell-to-cell signaling, differentiation, growth, motility, and adhesion (DIAZ GALICIA, 2019). More specifically, the transmembrane domains of receptor tyrosine kinases are responsible for signaling, which could be related to the regulation of pain transmission. Outstandingly, MIH teeth usually show hypersensitivity. The nerve fibers of the pulp organ are mostly of two types: fast-conducting A-fibres and unmyelinated C-fibres, with slower conduction speeds (NAIR, 1995). A-fibres respond to stimuli such as direct mechanical activation of exposed dentine, desiccation (NARHI, 1982), fluid motion within dentinal tubules due to osmotic pressure changes, air drying, and temperature variations. These stimuli are believed to induce action potentials in the A-fibres in the innermost part of the dentine, and the signals transmitted by these fibers commonly lead to pain (ALLISON, 2020). On the other hand, C-fibres, which are polymodal, react to exacerbate cold or heat, as well as to inflammatory mediators like bradykinin and histamine (NARHI, 1992; ALLISON, 2020). C-fibres activation leads to a deeper, duller, poorly localized aching sensation. Furthermore, those fibers are less capable to respond to direct mechanical stimuli (ALLISON, 2020). Regarding syndecan binding protein (GO:0045545), its molecular function is associated with epithelial cells, binding to structural molecules of the extracellular matrix, such as collagens I, III, and V (BINNS, 2009). It should be emphasized that type I collagen is responsible for the process of bone mineralization (deposition of hydroxyapatite in bone tissue) and also odontogenesis (the process of tooth formation) (BARBIRATO, 2009). In the most severe MIH condition together with carious lesions (G5) the microtubule-binding protein (GO: 0008017) and the motor microtubule protein (GO:0003777) were clearly identified (Fig. 4E). The function of these proteins is linked to microtubules binding and movement along a microtubule, driven by ATP hydrolysis (BINNS, 2009). The microtubule-binding protein (GO: 0008017) could be also found commonly in the disease groups (Fig. 4E and 4F). These functions could be better understood in the context of dentin structure, composition, and mineralization. Particular data and experiments with microtubule inhibitors pointed to the flux of forces in predentin, and active collagen fibrils transportation from the proximal to distal area of predentin, where the process of mineralization takes place (GOLDBERG, 2011; GOLDBERG, 1987). This way, we supposed that microtubule proteins could interfere in the mineralization process. When analyzing Gene - Ontology of proteins found in the control group only (G1) (Fig. 4.2 -G), a great difference was identified when compared to common and exclusive proteins among the disease groups (Fig. 4.2 - F). The biological process and the molecular function are totally distinct, as well as the String Biological Pathway pattern (Fig. 3F versus G). In general, regarding the Venn diagram (Fig. 2) the number of exclusive proteins in the MIH groups (G2, G3, G5) increases as the severity of the MHI increases, suggesting that the worse the defect, the greater the number of exclusive proteins. The present study has limitations. Due to the transverse design, it could not be affirmed if changes occurring in the different degrees of MIH are a result of the disturbance or even one of the reasons. Moreover, we explored only saliva as a protein source of proteomic signatures, and maybe, if a more informative niche has been used, such as the dental biofilm accumulated on the defective MIH teeth, important biomarkers for MIH pathophysiology, could be revealed. Biomarkers will certainly help in the early detection of MIH, improving the non-invasive therapeutic monitoring and prognosis, of prime importance for the child population. Further research should overcome these lacunes and bring insights into a deeper understanding of this issue. In conclusion, the present study supports important proteomic alterations in the saliva of MIH schoolers, according to distinct degrees of severity, reinforcing the interplay between the clinical findings or characteristics and changes in the salivary proteome. 4. Material And Methods Study design and population Sampling characteristics All research was performed in accordance with the Declaration of Helsinki. After ethical approval (Ethics Committee of the University São Francisco - protocol number: 10408119.0.0000.5514) and obtention of informed consent of all legal guardians’, this cross-sectional observational study recruited children, without distinction of sex, from public schools in the central area of Bragança Paulista. This city is located in the most developed state of Brazil, São Paulo, account with ≅ 172.346 inhabitants, a 0.776 human development index, and optimally fluoridated tap water (0.69mg F/L). The age of 6 to 8 years was chosen because, at this age range, the first permanent molars have newly erupted in the oral cavity. As part of a larger investigation including 566 children, and after caries and MIH diagnosis, fifty children were conveniently assigned into the following groups: G1- Control - Healthy teeth (n = 10); G2 - Mild MIH with white/creamy opacity and free of caries (n = 10), G3 - Mild MIH with yellow/brown opacity and free of caries (n = 10), G4 - Severe MIH with white/creamy, yellow/brown opacities including post-eruptive fracture and free of caries (n = 10); G5 - Severe MIH with white/creamy, yellow/brown opacities, post- eruptive fracture, and presence of caries (n = 10). Enrolled children were from low to mid-socioeconomic backgrounds. At schools, standardized meals were provided and toothbrushing was performed at least once a day with 1.100 ppm F dentifrice. Children taking medicines, such as antibiotics or corticoids, at the time of the study, or 30 days preceding the study, were dismissed. In addition, children with syndromes, who were not able to cooperate with the examinations were excluded, as well as children with enamel defects that did not characterize MIH. Assessment of MIH and dental caries The diagnosis of MIH and dental caries was made by visual inspection, in the schools, using headset light, gauze (for cleaning and drying teeth), clinical mirror, and ball-ended probe. MIH detection was performed according to the European Academy of Paediatric Dentistry (EAPD) (WEERHEIJM 2003 b .) criteria and caries detection following the World Health Organization (WHO, 2013) scores, modified by the inclusion of active white chalky spot lesions (ASSAF, 2006). Initially, the dentist (K.F.R) who performed the examinations received theoretical and practical instructions about the clinical scores to be used, given by a gold-standard examiner. Around 10 children were re-examined within 7 days between the examinations. Inter-examiner Kappa values were: 0,82 for MIH and 0,99 for caries. Saliva samples collection and preparation At least 500 microliters of unstimulated saliva were collected by passive drooling (3 minutes) in plastic cups and transferred to microcentrifuge tubes using sterilized disposable syringes. During the entire collection period, saliva was kept under refrigeration and was later frozen at -80 o C. The collection was performed at least one hour after food intake and teeth brushing, usually in the morning. In the Laboratory of Clinical and Molecular Microbiology, the samples were thawed and to 100 µl of saliva, 50 µl 8M urea solution (8 M) was added. After vortexed, the microcentrifuge tubes were transferred to a dry bath heated block (80°C) for 15 minutes, followed by centrifugation (spin). Then, 5 microliters of Dithiothreitol (DTT − 100 mM) were added. Again, tubes were vortexed and transferred to a dry bath heated block (60°C) for 30 minutes. After reaching room temperature, centrifugation (spin) was performed and 5 µl of Iodoacetamide (300 mM) was added. Considering that Iodoacetamide is a light-sensitive compound, the tubes were transferred to a dark box at room temperature, where they remained for 30 minutes. After that, 200 µl of Tris-HCl (50 mM, pH 8) and 20 µl of Trypsin solution in NH₄HCO₃ (50 mM) were added. The samples were vortexed and incubated overnight at digestion temperature (37°C). The enzymatic reaction was stopped by adding 20 µl of Trifluoroacetic Acid (TFA, 5%). Then, samples were frozen (-80°C), subsequently freeze-dried for 2 hours, and stored (-20°C) until spectrometry analysis. Mass spectrometry analysis Ten µl aliquots of the samples described above were inserted in a C18 column (130Å, 1.7 µm, 2.1 mm X 50 mm, ACQUITY UPLC BEH, Waters Co) and the eluted content was automatically inserted into a Q-ToF Xevo G2-XS mass spectrometer (Waters Co, USA). Peptides were eluted in a linear gradient (1–40% B; A = 0.1% formic acid; B: 90% acetonitrile in 0.1% formic acid), at 0.2 µl/min for 90 minutes. Spectra were acquired in positive ionization (200–1800 m/z) and FWHM 40 000 resolution at 500 m/z. The capillary voltage was set at 3 kV, source temperature of 150 o C, desolvation temperature of 350 o C, and desolvation gas flow of 600 L/h. For the MS/MS assessment, argon collision energy was exercised, with intensity according to the peptide charge. The instrument control and data obtention were performed by MassLinx 4.2. Data analysis and visualization For protein identification, the raw files were loaded in the Peaks Studio V.7.0 (Bioinformatics Solutions Inc. BSI, Waterloo, ON, Canada), using IPI and a database constructed by retrieving proteins from Uniprot using the term 'secreted proteins. The following parameters were considered: MS and MS / MS tolerance set to 2 ppm, deamination at N and Q, N-terminal acetylation, and oxidation at M, as variable modifications. The identified proteins were assessed according to Venn Diagrams (OLIVEROS, 2007), along with the expressed data combination using the CNB-CSIC Venny 4 (online tool) (OLIVEROS, 2007; CHAWADE, 2014) (Figs. 1 and 2). Gene-odontology was performed throughout the "Enricher" database (CHEN, 2013; KULESHOV, 2016) (Fig. 4). Biological Pathway enrichment analysis (Fig. 3) was performed employing the String program (version 11.5, online tool) (CHAWADE, 2014; SZKLARCZYK, 2021). Declarations Data availability Data will be available upon request to the corresponding author. Competing interests The authors declare that they have no competing interests. Funding Funding institutions did not support this work. Authors’ contributions Conceptualization: TMP; Acquisition of data: EST and KFR; Methodology: JMC, EST; Data Analysis: EST; Project administration: TMP; Supervision: TMP; Writing—review & editing: EST, KFR, JMS, FGC, MNS, and TMP; All authors have contributed to the final manuscript. All of them have also read and approved the final version. Acknowledgments We thank the Secretary of Education of Bragança Paulista-SP/Brazil and all children who took part in this research. We particularly thank Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for EST master's scholarship. References Abdalla R. Teaching dental anatomy & morphology: An updated clinical- & digital-based learning module. Eur J Dent Educ . 2020 Nov; 24 (4):650-659. doi: 10.1111/eje.12552. Epub 2020 Jul 1. PMID: 32531077. Alaluusua S. Aetiology of Molar-Incisor Hypomineralisation: A systematic review. 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Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016 Jul 8; 44 (W1):W90-7. doi: 10.1093/nar/gkw377. Epub 2016 May 3. PMID: 27141961; PMCID: PMC4987924. Lopes LB, Machado V, Mascarenhas P, Mendes JJ, Botelho J. The prevalence of molar-incisor hypomineralization: a systematic review and meta-analysis. Sci Rep. 2021 Nov 17; 11 (1):22405. doi: 10.1038/s41598-021-01541-7. PMID: 34789780; PMCID: PMC8599453. Mast P, Rodrigueztapia MT, Daeniker L, Krejci I. Understanding MIH: definition, epidemiology, differential diagnosis and new treatment guidelines. Eur J Paediatr Dent . 2013 Sep; 14 (3):204-8. PMID: 24295005. Närhi MV, Hirvonen TJ, Hakumäki MO. Responses of intradental nerve fibres to stimulation of dentine and pulp. Acta Physiol Scand. 1982 Jun; 115 (2):173-8. doi: 10.1111/j.1748-1716.1982.tb07062.x. PMID: 7136809. Närhi M, Jyväsjärvi E, Virtanen A, Huopaniemi T, Ngassapa D, Hirvonen T. Role of intradental A- and C-type nerve fibres in dental pain mechanisms. Proc Finn Dent Soc. 1992; 88 Suppl 1:507-16. PMID: 1508908. Negre-Barber A, Montiel-Company JM, Catalá-Pizarro M, Almerich-Silla JM. Degree of severity of molar incisor hypomineralization and its relation to dental caries. Sci Rep. 2018 Jan 19; 8 (1):1248. doi: 10.1038/s41598-018-19821-0. PMID: 29352193; PMCID: PMC5775201. Oliveros, J.C. (2007-2015) Venny. An Interactive Tool for Comparing Lists with Venn’s Diagrams. https://bioinfogp.cnb.csic.es/tools/venny/index.html Pappa E, Kousvelari E, Vastardis H. Saliva in the “Omics” era: A promising tool in paediatrics. Oral Dis . 2019; 25 :16–25. https://doi.org/10.1111/odi.12886. Pappa E, Vastardis H, Makridakis M, Zoidakis J, Vougas K, Stamatakis G, Samiotaki M, Rahiotis C. Analysis of Human and Microbial Salivary Proteomes in Children Offers Insights on the Molecular Pathogenesis of Molar-Incisor Hypomineralization. Biomedicines. 2022 Aug 24; 10 (9):2061. doi: 10.3390/biomedicines10092061. PMID: 36140166; PMCID: PMC9495719. Pappa E, Vougas K, Zoidakis J, Vastardis H. Proteomic advances in salivary diagnostics. Biochim Biophys Acta Proteins Proteom . 2020 Nov; 1868 (11):140494. doi: 10.1016/j.bbapap.2020.140494. Epub 2020 Jul 12. PMID: 32663525. Rizzardi KF, da Silva Toledo E, Ferraz LFC, Darrieux M, Girardello R, de Lima Marson FA, Parisotto TM. Association between asthma and enamel defects in primary and young permanent teeth - A systematic review. Pediatr Pulmonol . 2022 Jan; 57 (1):26-37. doi: 10.1002/ppul.25737. Epub 2021 Nov 8. PMID: 34698451. Salem K, Aziz D, Asadi M. Prevalence and Predictors of Molar Incisor Hypomineralization (MIH) among Rural Children in Northern Iran. Iran J Public Health . 2016 Nov; 45 (11):1528-1530. PMID: 28032070; PMCID: PMC5182269. Scannapieco FA, Torres G, Levine MJ. Salivary alpha-amylase: role in dental plaque and caries formation. Crit Rev Oral Biol Med. 1993; 4 (3-4):301-7. doi: 10.1177/10454411930040030701. PMID: 8373987. Schwendicke F, Elhennawy K, Reda S, Bekes K, Manton DJ, Krois J. Global burden of molar incisor hypomineralization. J Dent . 2018 Jan; 68 :10-18. doi: 10.1016/j.jdent.2017.12.002. Epub 2017 Dec 6. Erratum in: J Dent. 2019 Jan;80:89-92. PMID: 29221956. Sundfeld D, da Silva L, Kluppel OJ, Santin GC, de Oliveira R, Pacheco RR, Pini N. Molar Incisor Hypomineralization: Etiology, Clinical Aspects, and a Restorative Treatment Case Report. Oper Dent . 2020 Jul 1; 45 (4):343-351. doi: 10.2341/19-138-T. PMID: 32053452. Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, Doncheva NT, Legeay M, Fang T, Bork P, Jensen LJ, von Mering C. The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res . 2021 Jan 8; 49 (D1):D605-D612. doi: 10.1093/nar/gkaa1074. Erratum in: Nucleic Acids Res. 2021 Oct 11;49(18):10800. PMID: 33237311; PMCID: PMC7779004. Taylor GD. Molar incisor hypomineralisation. Evid Based Dent . 2017 Mar; 18 (1):15-16. doi: 10.1038/sj.ebd.6401219. PMID: 28338027. van Amerongen WE, Kreulen CM. Cheese molars: a pilot study of the etiology of hypocalcifications in first permanent molars. ASDC J Dent Child . 1995 Jul-Aug; 62 (4):266-9. PMID: 7593885. Vieira AR, Kup E. On the Etiology of Molar-Incisor Hypomineralization. Caries Res . 2016; 50 (2):166-9. doi: 10.1159/000445128. Epub 2016 Apr 26. PMID: 27111773. Vitorino R, Lobo MJ, Ferrer-Correira AJ, Dubin JR, Tomer KB, Domingues PM, Amado FM. Identification of human whole saliva protein components using proteomics. Proteomics. 2004 Apr; 4 (4):1109-15. doi: 10.1002/pmic.200300638. PMID: 15048992. Wang Q, Yu Q, Lin Q, Duan Y. Emerging salivary biomarkers by mass spectrometry. Clin Chim Acta. 2015 Jan 1; 438 :214-21. doi: 10.1016/j.cca.2014.08.037. Epub 2014 Sep 3. PMID: 25195008. Weerheijm KL, Duggal M, Mejàre I, Papagiannoulis L, Koch G, Martens LC, Hallonsten AL. Judgement criteria for molar incisor hypomineralisation (MIH) in epidemiologic studies: a summary of the European meeting on MIH held in Athens, 2003. Eur J Paediatr Dent. 2003 b Sep; 4 (3):110-3. PMID: 14529329. Weerheijm KL, Jälevik B, Alaluusua S. Molar-incisor hypomineralisation. Caries Res. 2001 Sep-Oct; 35 (5):390-1. doi: 10.1159/000047479. PMID: 11641576. Weerheijm KL. Molar incisor hypomineralisation (MIH). Eur J Paediatr Dent. 2003 Sep; 4 (3):114-20. PMID: 14529330. Whatling R, Fearne JM. Molar incisor hypomineralization: a study of aetiological factors in a group of UK children. Int J Paediatr Dent . 2008 May; 18 (3):155-62. doi: 10.1111/j.1365-263X.2007.00901.x. PMID: 18384347. World Health Organization. Oral health surveys: basic methods 2013.https://apps.who.int/iris/bitstream/handle/10665/97035/9789241548649_eng.pdf Accessed April 17, 2020. Zhao D, Dong B, Yu D, Ren Q, Sun Y. The prevalence of molar incisor hypomineralization: evidence from 70 studies. Int J Paediatr Dent. 2018 Mar; 28 (2):170-179. doi: 10.1111/ipd.12323. Epub 2017 Jul 21. PMID: 28732120. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1Figure3.1A.png SupplementaryFigure2Figure3.1B.png SupplementaryFigure3Figure3.1C.png SupplementaryFigure4Figure3.1D.png SupplementaryFigure5Figure3.1E.png SupplementaryFigure6Figure3.2F.png SupplementaryFigure7Figure3.2G.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2309540","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":155876768,"identity":"582e1197-dcc8-4d2b-b3cd-3b43c2e39018","order_by":0,"name":"Elora Silva Toledo¹","email":"","orcid":"","institution":"University São Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elora","middleName":"Silva","lastName":"Toledo¹","suffix":""},{"id":155876769,"identity":"c6d61e83-bd93-464b-ab8c-d5f277361074","order_by":1,"name":"Karina Ferreira Rizzardi¹","email":"","orcid":"","institution":"University São Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karina","middleName":"Ferreira","lastName":"Rizzardi¹","suffix":""},{"id":155876770,"identity":"ed574f82-58a5-471d-935a-7a2419a332b0","order_by":2,"name":"Fabíola Galbiatti de Carvalho","email":"","orcid":"","institution":"Federal University of Juiz de Fora – Governador Valadares","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fabíola","middleName":"Galbiatti","lastName":"de Carvalho","suffix":""},{"id":155876771,"identity":"5b1315b8-781f-4869-bfb2-f53c8dbc15e7","order_by":3,"name":"Marinês Nobre-dos-Santos","email":"","orcid":"","institution":"University of Campinas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marinês","middleName":"","lastName":"Nobre-dos-Santos","suffix":""},{"id":155876772,"identity":"919db75e-095a-4c22-96d1-1b6aabe00261","order_by":4,"name":"Juliana Mozer Sciani","email":"","orcid":"","institution":"University São Francisco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"Mozer","lastName":"Sciani","suffix":""},{"id":155876773,"identity":"915649ab-9678-4559-918c-3ee0f88298b0","order_by":5,"name":"Thaís Manzano Parisotto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie2SsWrCUBSG/3DBLLd1FSz6CkcCDdIUXyUSSBYLncSxUGgX6XwLfQihL3DlQLuIrg4OCQVn3bIUvKmUIiRx7XC/6fzDd+9/4AAWy3+kBeifQRxzB3BSoAFQhSFPFB3CMxOdVf4wyvDhnDJoP2bzHJuu/+xmX/s8SGYrBnZjht/W5b9cfRBLbHtvLD3SYXw3W0dw1JLRfwkrioVkXmVHCdlo6ZCN0tTi4olBi6pdkp0pxgMl3G2hJGSKie9aZURagodK4LpQQtIRhFOnrEf3LGkbmWIeLeK492p2mU+XiexPyxVXJe/7fLK5Vc3PLJ0EQfdyxU6aj286vixXjpA+zUWsFfB7ABaLxWIp5QC6T19cjooiDQAAAABJRU5ErkJggg==","orcid":"","institution":"University São Francisco","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Thaís","middleName":"Manzano","lastName":"Parisotto","suffix":""}],"badges":[],"createdAt":"2022-11-24 14:29:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2309540/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2309540/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29825544,"identity":"d1e5d3af-22e1-4579-865e-05b646ff5f41","added_by":"auto","created_at":"2022-12-02 15:47:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61323,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of identified proteins in the children`s saliva by mass spectrometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSector graph representing the total amount of proteins identified in the control group (G1) and the MIH groups (G2, G3, G4, G5). \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eVenn\u003c/em\u003e diagrams produced using the \u003cem\u003eCNB-CSIC Venny 4 online tool.\u003c/em\u003e The outer numbers indicate the total amount of proteins found in the control and MIH groups and the inner values of each circle, the number of proteins exclusively from them. The intersection represents the number of proteins found in common among all groups (G1, G2, G3, G4, G5). The small circle (light brown) represents the total number of proteins found in common among the MIH groups only.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/9a7317e200020de5a1877e94.png"},{"id":29825547,"identity":"5b7bf157-b2c1-4ce7-8d48-274e23d2138e","added_by":"auto","created_at":"2022-12-02 15:47:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVenn \u003c/em\u003ediagrams of the number of the identified proteins exclusive or common among the MIH groups (G2, G3, G4, G5).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/4873a7bc16cdbf84e22dd90b.png"},{"id":29825562,"identity":"7a8ff206-6700-43f9-9c13-0dc7543a346f","added_by":"auto","created_at":"2022-12-02 15:47:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":239596,"visible":true,"origin":"","legend":"\u003cp\u003eSTRING biological pathway separated by the studied groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Biological pathway of the interaction of proteins identified in the control group (G1). The number of nodes: 283; the number of edges: 1552; average node degree: 11; avg. local clustering coefficient: 0.465.\u003cstrong\u003e (B)\u003c/strong\u003eBiological pathway of the interaction of proteins identified in G2 (white opacities group). The number of nodes: 303; the number of edges: 1670; average node degree: 11; avg. local clustering coefficient: 0.462.\u003cstrong\u003e (C)\u003c/strong\u003e Biological pathway of the interaction of proteins identified in G3 (yellow opacities group). The number of nodes: 335; the number of edges: 1715; average node degree: 10.2; avg. local clustering coefficient: 0.443. \u003cstrong\u003e(D)\u003c/strong\u003e Biological pathway of the interaction of the proteins identified in G4 (yellow opacities with post-eruptive breakdown and caries-free. The number of nodes: 187; the number of edges: 731; average node degree: 7.82; avg. local clustering coefficient: 0.455. \u003cstrong\u003e(E)\u003c/strong\u003e Biological pathway of the interaction of the proteins identified in the yellow opacities with post-eruptive breakdown and caries (G5). The number of nodes: 194; the number of edges: 471; average node degree: 4.86; avg. local clustering coefficient: 0.438. High-resolution images are available in supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 (F)\u003c/strong\u003e Biological pathway of the interaction of the children identified commonly among the MIH groups (G2, G3, G4, G5).\u003cstrong\u003e (G) \u003c/strong\u003eBiological pathway of the interaction of the proteins identified in the control group only (G1). High-resolution images are available in supplementary files.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/194060d1894520a2e442a518.png"},{"id":29825545,"identity":"bfda7c82-980e-4457-ae7f-45f9bbf75d48","added_by":"auto","created_at":"2022-12-02 15:47:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222368,"visible":true,"origin":"","legend":"\u003cp\u003eGene-Odontology (GO) considering biological process, molecular function, and cellular component of the identified proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1(A)\u003c/strong\u003e GO of the control group. \u003cstrong\u003e(B)\u003c/strong\u003e GO of G2 (white opacities). \u003cstrong\u003e(C)\u003c/strong\u003e GO of G3 (yellow opacities). \u003cstrong\u003e(D)\u003c/strong\u003e GO of G4 (opacities with post-eruptive breakdown). \u003cstrong\u003e(E)\u003c/strong\u003eGO of G5 (opacities with fracture and caries).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 (F)\u003c/strong\u003e GO of the identified proteins in common among the MIH groups (G2, G3, G4, G5). \u003cstrong\u003e(G)\u003c/strong\u003e GO of the identified proteins exclusive to the control group (G1).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/2a55aba211a0f721063e6965.png"},{"id":35921248,"identity":"b3b080ac-8e6b-47a3-8671-3d39ab63363c","added_by":"auto","created_at":"2023-04-18 08:29:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":902593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/a7b4bc4d-9d78-4bff-bfe6-9b0050e907e0.pdf"},{"id":29825553,"identity":"6d9e8359-4ffa-407d-98b0-17c42c1f81ea","added_by":"auto","created_at":"2022-12-02 15:47:23","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2779350,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1Figure3.1A.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/3ae16225b1d6a9cb2394852a.png"},{"id":29825559,"identity":"3b315855-4048-4c76-b2c0-29c73c8485c9","added_by":"auto","created_at":"2022-12-02 15:47:23","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1940466,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2Figure3.1B.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/c2fdbded50013a69de80a061.png"},{"id":29825530,"identity":"39ffcd56-528f-4673-8d00-7ef41ca9a438","added_by":"auto","created_at":"2022-12-02 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15:47:22","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1738797,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure5Figure3.1E.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/1109129d9fa1fff958a18a98.png"},{"id":29825543,"identity":"2a541422-57ef-4a76-ba43-af64fd15c2de","added_by":"auto","created_at":"2022-12-02 15:47:21","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2242501,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure6Figure3.2F.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/5112f2c8e2f882eb559bfffb.png"},{"id":29825556,"identity":"5e61db3b-0290-4048-857c-80abe9477358","added_by":"auto","created_at":"2022-12-02 15:47:23","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":360764,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure7Figure3.2G.png","url":"https://assets-eu.researchsquare.com/files/rs-2309540/v1/cb26f12ce988d2231e390688.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSalivary Proteomic Patterns in Children Affected by Different Severity Degrees of Molar Incisor Hypomineralization\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePediatric Dentistry is facing a new disease in this century, known as Molar Incisor Hypomineralization (MIH - Weerheijm et al. 2001). MIH affects the enamel of the first permanent molars, important teeth for grinding food (GAISER, 2012), together or not with the permanent incisors, which are responsible for biting and cutting dietary substrates, as well as for phonetics (ABDALLA, 2020). Recent systematic reviews revealed a constantly increasing pooled prevalence, with a 13,5-14.2% overall average (LOPES, 2021; ZHAO, 2019; DAVE, 2018).\u003c/p\u003e \u003cp\u003eTeeth with MIH show morphologically normal enamel; however, its structure is deficient (TAYLOR, 2017). Since there is a decrease in the quantity and quality of minerals, the enamel is more porous, softened, and shows white, yellow, or brownish opacities (WEERHEIJIM, 2003). This way, the defective tooth becomes more fragile and susceptible to caries lesions and erosion, usually with exacerbated sensitivity (WEERHEIJIM, 2003); leading to challenging dental procedures (SCHWENDICKE, 2018; TAYLOR, 2017; WEERHEIJIM, 2003). Of interest, the treatment of this condition is not definitive (MAST, 2013), but palliative seeking to prolong the vitality of the tooth, and to improve the oral health-related quality of life of the children.\u003c/p\u003e \u003cp\u003eAccording to the enamel hypomineralization rate, MIH was classified into different degrees of severity. White, yellow, or brown demarcated opacities, were assigned as a mild degree of MIH; and post-eruptive enamel breakdown jointly with opacities, carious lesions, or complex restorations disconnected to caries pattern, was defined as severe (NEGRE-BARBER, 2018).\u003c/p\u003e \u003cp\u003eAlthough the exact etiology of MIH is unknown (WHATILING, 2008), some hypotheses have been suggested, including systemic health disturbances during the permanent teeth mineralization period (SALEM, 2016). Among these disorders, early childhood diseases, \u003cem\u003ei.e.\u003c/em\u003e asthma (RIZZARDI, 2022), frequent use of antibiotics (GHAMIM, 2013), as well as genetic influences (VIEIRA 2016, ALALUUSUA, 2010) (considering that enamel-forming cells are genetically controlled) should be highlighted. In summary, over the last decade, more than 30 systemic etiological hypotheses have been identified; some are well-established, and others are more contemporary (GAROT, 2022).\u003c/p\u003e \u003cp\u003e Outstandingly, saliva is a body fluid with complex composition, capable of playing lots of roles connected with oral and systemic health (KHURSHID 2016; JAVAID 2016). It contains bicarbonate ions, responsible for the buffering capacity, neutralizing organic acids produced by bacterial food substrates fermentation (de SOUZA, 2020). Hence, teeth demineralization could be minimized. Several saliva components or proteins can interact with pathogenic bacteria and may be involved in the protection of dental tissues, such as statins, mucins, lactoferrin, defensins, and peroxidases (PAPPA, 2019). Although saliva contains numerous peptides, many of them have not yet been characterized (PAPPA, 2019). With scientific advances, state-of-the-art techniques, such as mass spectrometry analysis, for example, have provided a better understanding of the course of pathologies, identifying biomarkers at a molecular level (VITORINO, 2004).\u003c/p\u003e \u003cp\u003eFor the last 10 years, salivary proteomics have been tested aiming to disclose pathologically\u003c/p\u003e \u003cp\u003emechanisms on protein expression levels for several oral and systemic conditions (WANG, 2015). As changes in saliva composition are associated with oral pathologies, it is reasonable to assume that the saliva proteome of MIH-affected children might be altered compared to healthy children. Although a considerable amount of studies has attempted to identify proteomic profiles in oral diseases (PAPPA, 2020), there are only scarce data in the literature about the proteome in MIH pathology, indicating differential expression of proteins in MIH patients\u0026rsquo; saliva (BEKES, 2020; PAPPA, 2022). Furthermore, we were not able to find studies investigating how salivary proteomic changes as MIH turns from mild to more severe. Caring-out investigation at the systems biology level, additional insights by connecting the disease phenotype to specific biological processes may be brought. Given this background, the main purpose of this research was to investigate if there is any change in the salivary proteome signatures of MIH children with distinct degrees of severity.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cem\u003ePatient characteristics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe control group (G1) comprised 4 girls and 6 boys, with 9.2 years mean age. The MIH groups (G2, G3, G4, G5) comprised 10 girls and 30 boys (mean age of 10.2 years), accounting for 118 affected teeth with different degrees of severity (84.8% molars and 15.2% incisors). The number of decayed, missing, or filled teeth in the G1, G2, G3, and G4 was 0, while in G5 this number was 2,55.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProteomic profile\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eProteomic analysis was capable of identifying a total of 6,471 proteins, 15% belonging to the control group (G1) and 85% to the MIH groups (G2, G3, G4, G5) (Fig.\u0026nbsp;1A). Of the nine hundred ninety-two and 5,479 proteins found in the Control and the MIH groups, respectively, 406 overlapped among them. Regarding the 4 MIH groups together, 778 proteins were shown in common (Fig.\u0026nbsp;1B).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003ch2\u003e2.1 Proteins overlapping among the MIH groups\u003c/h2\u003e\n\u003cp\u003eOverlapping proteins identified among the disease groups were shown according to the degree of severity (Fig. 2). Seven hundred and seventeen, 905, 554, and 1,150 proteins were only detected in groups 2, 3, 4, and 5, respectively. Thirty proteins were common among all MIH groups, with the highest number (451) between the moderate group (G3) and the most severe group including the presence of caries (G5) (Fig. 2). No protein was concomitantly found concerning all MIH groups without decayed, missing, or filled teeth (G2, G3, G4).\u003c/p\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Biological Pathway and Gene-Ontology\u003c/h2\u003e\n \u003cp\u003eThe proteins found in each group were displayed in Biological Pathways (Fig.\u0026nbsp;3A, B, C, D, and E) and Gene-Ontology (Fig.\u0026nbsp;4A, B, C, D, and E). Figures\u0026nbsp;3F and 4F show the Biological Pathway and Gene-Ontology of the overlapping proteins among the disease groups (778), and Figs.\u0026nbsp;3G and 4G refers to proteins exclusively from healthy children (G1).\u003c/p\u003e\n \u003cp\u003eIn general, the Biological Pathways of the studied groups showed different protein-binding patterns. However, G2 and G3 (Mild MIH degree \u0026ndash; Fig. 3B and Fig. 3C) are closer to each other, as well as G4 and G5 (Severe MIH degree \u0026ndash; Fig. 3D and Fig. 3E), which presented fewer connections, compared to healthy children\u0026rsquo;s group.\u003c/p\u003e\n \u003cp\u003eA striking protein-binding pattern difference was identified between Figs.\u0026nbsp;3F and 3G, accounting respectively for common proteins among the disease groups (G2, G3, G4, G5) and exclusive proteins to the control group (G1).\u003c/p\u003e\n \u003cp\u003eThe analysis of the biological process patterns (GOBP) demonstrated intense similarity among all studied groups (G1, G2, G3, G4, G5) (Fig. 4A-4E). On the other hand, GOBP regarding common proteins among the disease groups (G2, G3, G4, G5) \u003cem\u003eversus\u003c/em\u003e exclusive proteins to the control group (G1) (Fig. 4F and 4G) were quite different.\u003c/p\u003e\n \u003cp\u003eAssessment of the cellular components patterns (GOCC) revealed close similarities among G1, G2, G3, and G4 (Fig.\u0026nbsp;4A-4D). Conversely, Fig.\u0026nbsp;4E, representing the severe degree of MIH including the presence of caries lesions, changed in terms of GOCC. Figures\u0026nbsp;4F and G also show some discrepancies.\u003c/p\u003e\n \u003cp\u003eThe greatest distinctions between the clusters were found concerning the molecular function (GOMF) of each of the studied groups, including the common proteins among the MIH groups (G2, G3, G4, G5 \u0026ndash; Fig.\u0026nbsp;4F) and the exclusive proteins of the control group (G1 \u0026ndash; Fig.\u0026nbsp;4G).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIt is well established that saliva is a rich full biological fluid and possibly a biomarker source. Results of the present study highlight a notable difference in the proteomic profile according to distinct degrees of MIH severity. In addition, it seems that the most important difference occurs in the gene-ontology molecular function of the salivary proteins.\u003c/p\u003e\n\u003cp\u003eWhen exploring the molecular function, the presence of metallopeptidase and metalloendopeptidase activity (GO:0008237 and GO:0004222; Fig.\u0026nbsp;4) was observed since health condition until MIH degree involving post-eruptive breakdown, without carious lesions (G1-G4). At the most severe degree, involving post-eruptive fracture together with dental caries (G5), the peptidases mentioned above disappeared. Both enzymes catalyze the hydrolysis of bound peptides by a mechanism in which water acts as a nucleophile (BINNS, 2009).\u003c/p\u003e\n\u003cp\u003eIntriguingly, calcium ion binding protein (GO:0005509), was only detected in the most MIH severe group (G5; Fig.\u0026nbsp;4), as well as when common proteins among the MIH groups were considered (Fig.\u0026nbsp;4F). This molecular function is connected to calcium ion storage activity (BINNS, 2009). Calcium phosphate is the main component of dental enamel, providing the hardness characteristic of the element (ELFRINK, 2012), composed of 97% minerals. Maybe the calcium ion binding protein interferes with the availability of calcium ion, contributing to the disturbance in the mineralization process, which favor enamel defects. Similarly, it has already been reported that hypocalcemia can stimulate biological mechanisms linked to hypoplasia and hypomineralization development (ALALUUSUA, 2010; VAN AMEROGEN, 1995). These defects imply color alterations (white/creamy to brownish-yellow) and breakdown susceptibility during food grinding (de OLIVEIRA, 2015), and provide a convenient niche for pathogenic bacteria/biofilm accumulation, failure in enamel layers, exposing the dentin layer to the oral environment (SUNDFELD 2020).\u003c/p\u003e\n\u003cp\u003eRegarding the healthy group (G1) and the groups with mild MIH (G2 and G3), endopeptidase activity (GO:0004175) is the second or third most prevalent molecular function (Fig. 4A-4C). When common proteins among the MIH groups were considered, and also in G4 (severe degree), this molecular function drops to the penultimate position (Fig. 4D and 4F), and in G5 (most severe degree) it could not be found (Fig. 4E). Specifically, endopeptidase activity plays a role in catalyzing the hydrolysis of internal alpha-peptide bindings in a polypeptide chain (BINNS, 2009). Proteolytic enzymes are important in the process of enamel formation (amelogenesis), which is highly sensitive. After the secretion of the organic matrix during amelogenesis, proteolytic enzymes reabsorbed certain proteins, providing adequate space for mineral deposition (FARAH, 2010), and normal hydroxyapatite crystal development, which is crucial for healthy enamel.\u003c/p\u003e\n\u003cp\u003eInterestingly, the molecular function of Alpha-amylase and Amylase activity (GO: 0004556 and 0016160) was only identified in the control/healthy group (G1 - Fig.\u0026nbsp;4A). This enzyme is responsible for starch carbohydrate digestion in the oral cavity and is released during chewing, by the salivary glands. The pancreas is also able to produce Alpha-amylase, which is released in the pancreatic juice, accounting for starch digestion in the upper part of the small intestine. Salivary alpha-amylase has been found bound to microorganisms for bacterial clearance, important for health conditions in impairing caries formation (SCANNAPIECO, 1993). This way, the hypothesis that starch digestion might be prejudiced in the mouth of MIH - children could be raised. The identification of Alpha-amylase and Amylase activity only in G1 emphasizes the salivary proteomic changes in affected children.\u003c/p\u003e\n\u003cp\u003eConversely, platelet-derived growth factor binding (GO:0048407) could not be detected in the healthy group (G1 -Fig.\u0026nbsp;4A). However, it appeared in all MIH - children, particularly in G4 (G2, G3, G4, G5 - Fig.\u0026nbsp;4B-4F). One supposition would be that hypomineralized enamel, particularly with fractures, possibly potentializes molecular interplay between the pulp organ (the innermost portion of the tooth) and the oral environment, exposing blood components to the mouth. When the fractures were connected with carious lesions, maybe there was some stimulation of reactionary dentin, to protect and isolate the pulp from infectious agents. Perhaps that is the reason why platelet-derived growth factor binding (GO:0048407) was especially identified in G4.\u003c/p\u003e\n\u003cp\u003eThe transmembrane receptor protein tyrosine kinase (GO:0004714) and syndecan binding protein (GO:0045545) were identified in G3 (Fig.\u0026nbsp;4C). Tyrosine kinase belongs to a large multigene family accountable for cell functions regulation, \u003cem\u003ei.e\u003c/em\u003e. cell-to-cell signaling, differentiation, growth, motility, and adhesion (DIAZ GALICIA, 2019). More specifically, the transmembrane domains of receptor tyrosine kinases are responsible for signaling, which could be related to the regulation of pain transmission. Outstandingly, MIH teeth usually show hypersensitivity. The nerve fibers of the pulp organ are mostly of two types: fast-conducting A-fibres and unmyelinated C-fibres, with slower conduction speeds (NAIR, 1995). A-fibres respond to stimuli such as direct mechanical activation of exposed dentine, desiccation (NARHI, 1982), fluid motion within dentinal tubules due to osmotic pressure changes, air drying, and temperature variations. These stimuli are believed to induce action potentials in the A-fibres in the innermost part of the dentine, and the signals transmitted by these fibers commonly lead to pain (ALLISON, 2020). On the other hand, C-fibres, which are polymodal, react to exacerbate cold or heat, as well as to inflammatory mediators like bradykinin and histamine (NARHI, 1992; ALLISON, 2020). C-fibres activation leads to a deeper, duller, poorly localized aching sensation. Furthermore, those fibers are less capable to respond to direct mechanical stimuli (ALLISON, 2020). Regarding syndecan binding protein (GO:0045545), its molecular function is associated with epithelial cells, binding to structural molecules of the extracellular matrix, such as collagens I, III, and V (BINNS, 2009). It should be emphasized that type I collagen is responsible for the process of bone mineralization (deposition of hydroxyapatite in bone tissue) and also odontogenesis (the process of tooth formation) (BARBIRATO, 2009).\u003c/p\u003e\n\u003cp\u003eIn the most severe MIH condition together with carious lesions (G5) the microtubule-binding protein (GO: 0008017) and the motor microtubule protein (GO:0003777) were clearly identified (Fig.\u0026nbsp;4E). The function of these proteins is linked to microtubules binding and movement along a microtubule, driven by ATP hydrolysis (BINNS, 2009). The microtubule-binding protein (GO: 0008017) could be also found commonly in the disease groups (Fig.\u0026nbsp;4E and 4F). These functions could be better understood in the context of dentin structure, composition, and mineralization. Particular data and experiments with microtubule inhibitors pointed to the flux of forces in predentin, and active collagen fibrils transportation from the proximal to distal area of predentin, where the process of mineralization takes place (GOLDBERG, 2011; GOLDBERG, 1987). This way, we supposed that microtubule proteins could interfere in the mineralization process.\u003c/p\u003e\n\u003cp\u003eWhen analyzing Gene - Ontology of proteins found in the control group only (G1) (Fig.\u0026nbsp;4.2 -G), a great difference was identified when compared to common and exclusive proteins among the disease groups (Fig.\u0026nbsp;4.2 - F). The biological process and the molecular function are totally distinct, as well as the String Biological Pathway pattern (Fig.\u0026nbsp;3F \u003cem\u003eversus\u003c/em\u003e G). In general, regarding the Venn diagram (Fig.\u0026nbsp;2) the number of exclusive proteins in the MIH groups (G2, G3, G5) increases as the severity of the MHI increases, suggesting that the worse the defect, the greater the number of exclusive proteins.\u003c/p\u003e\n\u003cp\u003eThe present study has limitations. Due to the transverse design, it could not be affirmed if changes occurring in the different degrees of MIH are a result of the disturbance or even one of the reasons. Moreover, we explored only saliva as a protein source of proteomic signatures, and maybe, if a more informative niche has been used, such as the dental biofilm accumulated on the defective MIH teeth, important biomarkers for MIH pathophysiology, could be revealed. Biomarkers will certainly help in the early detection of MIH, improving the non-invasive therapeutic monitoring and prognosis, of prime importance for the child population. Further research should overcome these lacunes and bring insights into a deeper understanding of this issue.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the present study supports important proteomic alterations in the saliva of MIH schoolers, according to distinct degrees of severity, reinforcing the interplay between the clinical findings or characteristics and changes in the salivary proteome.\u003c/p\u003e"},{"header":"4. Material And Methods","content":"\u003cp\u003e \u003cb\u003eStudy design and population\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSampling characteristics\u003c/em\u003e \u003c/p\u003e \u003cp\u003e All research was performed in accordance with the Declaration of Helsinki. After ethical approval (Ethics Committee of the University S\u0026atilde;o Francisco - protocol number: 10408119.0.0000.5514) and obtention of informed consent of all legal guardians\u0026rsquo;, this cross-sectional observational study recruited children, without distinction of sex, from public schools in the central area of Bragan\u0026ccedil;a Paulista. This city is located in the most developed state of Brazil, S\u0026atilde;o Paulo, account with \u0026cong;\u0026thinsp;172.346 inhabitants, a 0.776 human development index, and optimally fluoridated tap water (0.69mg F/L). The age of 6 to 8 years was chosen because, at this age range, the first permanent molars have newly erupted in the oral cavity.\u003c/p\u003e \u003cp\u003eAs part of a larger investigation including 566 children, and after caries and MIH diagnosis, fifty children were conveniently assigned into the following groups: G1- Control - Healthy teeth (n\u0026thinsp;=\u0026thinsp;10); G2 - Mild MIH with white/creamy opacity and free of caries (n\u0026thinsp;=\u0026thinsp;10), G3 - Mild MIH with yellow/brown opacity and free of caries (n\u0026thinsp;=\u0026thinsp;10), G4 - Severe MIH with white/creamy, yellow/brown opacities including post-eruptive fracture and free of caries (n\u0026thinsp;=\u0026thinsp;10); G5 - Severe MIH with white/creamy, yellow/brown opacities, post- eruptive fracture, and presence of caries (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e \u003cp\u003eEnrolled children were from low to mid-socioeconomic backgrounds. At schools, standardized meals were provided and toothbrushing was performed at least once a day with 1.100 ppm F dentifrice. Children taking medicines, such as antibiotics or corticoids, at the time of the study, or 30 days preceding the study, were dismissed. In addition, children with syndromes, who were not able to cooperate with the examinations were excluded, as well as children with enamel defects that did not characterize MIH.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAssessment of MIH and dental caries\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe diagnosis of MIH and dental caries was made by visual inspection, in the schools, using headset light, gauze (for cleaning and drying teeth), clinical mirror, and ball-ended probe. MIH detection was performed according to the European Academy of Paediatric Dentistry (EAPD) (WEERHEIJM 2003\u003csup\u003eb\u003c/sup\u003e.) criteria and caries detection following the World Health Organization (WHO, 2013) scores, modified by the inclusion of active white chalky spot lesions (ASSAF, 2006).\u003c/p\u003e \u003cp\u003eInitially, the dentist (K.F.R) who performed the examinations received theoretical and practical instructions about the clinical scores to be used, given by a gold-standard examiner. Around 10 children were re-examined within 7 days between the examinations. Inter-examiner Kappa values were: 0,82 for MIH and 0,99 for caries.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSaliva samples collection and preparation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAt least 500 microliters of unstimulated saliva were collected by passive drooling (3 minutes) in plastic cups and transferred to microcentrifuge tubes using sterilized disposable syringes. During the entire collection period, saliva was kept under refrigeration and was later frozen at -80\u003csup\u003eo\u003c/sup\u003eC. The collection was performed at least one hour after food intake and teeth brushing, usually in the morning.\u003c/p\u003e \u003cp\u003eIn the Laboratory of Clinical and Molecular Microbiology, the samples were thawed and to 100 \u0026micro;l of saliva, 50 \u0026micro;l 8M urea solution (8 M) was added. After vortexed, the microcentrifuge tubes were transferred to a dry bath heated block (80\u0026deg;C) for 15 minutes, followed by centrifugation (spin). Then, 5 microliters of Dithiothreitol (DTT \u0026minus;\u0026thinsp;100 mM) were added. Again, tubes were vortexed and transferred to a dry bath heated block (60\u0026deg;C) for 30 minutes. After reaching room temperature, centrifugation (spin) was performed and 5 \u0026micro;l of Iodoacetamide (300 mM) was added. Considering that Iodoacetamide is a light-sensitive compound, the tubes were transferred to a dark box at room temperature, where they remained for 30 minutes. After that, 200 \u0026micro;l of Tris-HCl (50 mM, pH 8) and 20 \u0026micro;l of Trypsin solution in NH₄HCO₃ (50 mM) were added. The samples were vortexed and incubated overnight at digestion temperature (37\u0026deg;C). The enzymatic reaction was stopped by adding 20 \u0026micro;l of Trifluoroacetic Acid (TFA, 5%). Then, samples were frozen (-80\u0026deg;C), subsequently freeze-dried for 2 hours, and stored (-20\u0026deg;C) until spectrometry analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMass spectrometry analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTen \u0026micro;l aliquots of the samples described above were inserted in a C18 column (130\u0026Aring;, 1.7 \u0026micro;m, 2.1 mm X 50 mm, ACQUITY UPLC BEH, Waters Co) and the eluted content was automatically inserted into a Q-ToF Xevo G2-XS mass spectrometer (Waters Co, USA). Peptides were eluted in a linear gradient (1\u0026ndash;40% B; A\u0026thinsp;=\u0026thinsp;0.1% formic acid; B: 90% acetonitrile in 0.1% formic acid), at 0.2 \u0026micro;l/min for 90 minutes. Spectra were acquired in positive ionization (200\u0026ndash;1800 m/z) and FWHM 40 000 resolution at 500 m/z. The capillary voltage was set at 3 kV, source temperature of 150\u003csup\u003eo\u003c/sup\u003eC, desolvation temperature of 350\u003csup\u003eo\u003c/sup\u003eC, and desolvation gas flow of 600 L/h. For the MS/MS assessment, argon collision energy was exercised, with intensity according to the peptide charge. The instrument control and data obtention were performed by MassLinx 4.2.\u003c/p\u003e \u003cp\u003e \u003cem\u003eData analysis and visualization\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor protein identification, the raw files were loaded in the Peaks Studio V.7.0 (Bioinformatics Solutions Inc. BSI, Waterloo, ON, Canada), using IPI and a database constructed by retrieving proteins from Uniprot using the term 'secreted proteins. The following parameters were considered: MS and MS / MS tolerance set to 2 ppm, deamination at N and Q, N-terminal acetylation, and oxidation at M, as variable modifications.\u003c/p\u003e \u003cp\u003eThe identified proteins were assessed according to Venn Diagrams (OLIVEROS, 2007), along with the expressed data combination using the CNB-CSIC Venny 4 (online tool) (OLIVEROS, 2007; CHAWADE, 2014) (Figs.\u0026nbsp;1 and 2). Gene-odontology was performed throughout the \"Enricher\" database (CHEN, 2013; KULESHOV, 2016) (Fig.\u0026nbsp;4). Biological Pathway enrichment analysis (Fig.\u0026nbsp;3) was performed employing the String program (version 11.5, online tool) (CHAWADE, 2014; SZKLARCZYK, 2021).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding institutions did not support this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: TMP; Acquisition of data: EST and KFR; Methodology: JMC, EST; Data Analysis: EST; Project administration: TMP; Supervision: TMP; Writing\u0026mdash;review \u0026amp; editing: EST, KFR, JMS, FGC, MNS, and TMP; All authors have contributed to the final manuscript. All of them have also read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Secretary of Education of Bragan\u0026ccedil;a Paulista-SP/Brazil and all children who took part in this research. We particularly thank Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for EST master\u0026apos;s scholarship.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdalla R. Teaching dental anatomy \u0026amp; morphology: An updated clinical- \u0026amp; digital-based learning module. \u003cem\u003eEur J Dent Educ\u003c/em\u003e. 2020 Nov;\u003cstrong\u003e24\u003c/strong\u003e(4):650-659. doi: 10.1111/eje.12552. Epub 2020 Jul 1. PMID: 32531077.\u003c/li\u003e\n\u003cli\u003eAlaluusua S. 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PMID: 28732120. \u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dental hypomineralization, Salivary proteins, Dental enamel hypoplasia","lastPublishedDoi":"10.21203/rs.3.rs-2309540/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2309540/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSaliva is a rich-bodily fluid with recognized clinical diagnosis roles and this research aimed at investigating if there is any change in the salivary proteome signatures of MIH children with distinct degrees of severity. Fifty schoolers (6\u0026ndash;10 years) were equally assigned into the following groups: G1 (Control group - Healthy teeth), G2 (Mild MIH with white/creamy opacity and free of caries), G3 (Mild MIH with yellow/brown opacity and free of caries), G4 (Severe MIH with white/creamy, yellow/brown opacities including post-eruptive fracture and free of caries), G5 (Severe MIH with white/creamy, yellow/brown opacities, post-eruptive fracture, and caries). Unstimulated saliva samples were collected and later explored using mass spectrometry analysis. In total, 6,471 proteins were found, 5,073 exclusively from MIH children, and 778 overlapping among the different degrees of the disturb. The Biological Pathways displayed distinct patterns among the groups, being different according to the degrees of MIH. Gene-Odontology differences might not be verified regarding the biological processes and cellular components. Conversely, with respect to molecular function, alterations among groups were evident, with the presence of proteins that would contribute to MIH in children with the severe condition (\u003cem\u003ei.e\u003c/em\u003e, calcium ion binding, microtubule binding, platelet-derived growth factor binding). In conclusion, the results of this study support important salivary proteomic changes in MIH children, according to distinct degrees of severity, reinforcing the interplay between the clinical characteristics and changes in the salivary proteome.\u003c/p\u003e","manuscriptTitle":"Salivary Proteomic Patterns in Children Affected by Different Severity Degrees of Molar Incisor Hypomineralization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-02 15:45:36","doi":"10.21203/rs.3.rs-2309540/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aef4c191-04c0-407a-96bc-0b9ba97d854a","owner":[],"postedDate":"December 2nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-18T08:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-02 15:45:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2309540","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2309540","identity":"rs-2309540","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}