OTOP1: A New Candidate Gene for Non-syndromic Peg Lateralis

OTOP1: A New Candidate Gene for Non-syndromic Peg Lateralis | 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 OTOP1: A New Candidate Gene for Non-syndromic Peg Lateralis Jae-Hoon lee, junglim choi, Sungnam Kim, Hyunsoo Ahn, Donghyo Kim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3811797/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 Although peg-shaped lateral incisors are a common dental anomaly, the genetic mechanisms underlying peg-shaped lateral incisors are poorly understood, particularly in cases without associated anomalies. The present study aimed to identify potential candidate genes contributing to the development of non-syndromic peg lateralis, by performing whole-exome sequencing (WES). Saliva samples were collected from 20 cases of unrelated Korean individuals that were; not associated with other anomalies. WES was conducted on these samples, and variants were filtered using criteria of a p -value < 0.05, a false discovery rate 1. In silico mutation impact analysis was performed using Polymorphism Phenotyping v2, sorting intolerant from tolerant, and integrated score of co-evolution and conservation algorithms. We identified a heterozygous OTOP1 gene allele encoding the Otopetrin-1 protein, a proton channel, in all 20 individuals. Gene Ontology analysis revealed an association between OTOP1 and peg lateralis. We further confirmed that the peg lateralis candidate variant, rs199742451, of the same genotype was found in the family member of three subjects with the same phenotype. The results suggest a new possible function of OTOP1, which is yet to be studied, and identified it as a new candidate contributing to the development of peg lateralis. This study provides new insights into the genetic basis of non-syndromic peg lateralis and has important implications for further studies on the role of new genes in peg lateralis Biological sciences/Genetics/Sequencing/DNA sequencing Biological sciences/Genetics/Genetic markers Calcium flux Dental anomalies Otopetrin Peg-shaped maxillary lateral incisors Whole exome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction More than 350 genes, most of which encode signaling molecules that play crucial roles in molecular and cellular interactions, have been identified to be involved in odontogenesis [ 1 ]. The epithelial-mesenchymal interactions mediated by these proteins regulate the different stages of tooth formation, including initiation, morphogenesis, and differentiation [ 2 ]. Tooth anomalies, which appear as alterations in the number, size, morphology, or structure of a single tooth or multiple teeth, occur in isolation or in association with other syndromes [ 3 ]. Congenital tooth anomalies cause functional, esthetic, and psychological problems; thus, early diagnosis is required for long term treatment [ 4 , 5 ]. Peg lateralis is a microdontia that only occurs in maxillary lateral incisors and is tapered in shape, with incisal mesiodistal width shorter than cervical width (Fig. 1 ). The prevalence of peg lateralis varies from 0.6–9.9% depending on ethnicity, sex, and region; Mongolians have a significantly higher prevalence rate than other ethnic groups. However, the overall prevalence rate is 1.8% according to meta-analyses [ 6 ]. Although non-syndromic teeth abnormalities are inherited as an autosomal trait, the literature suggests that peg shaped teeth are 1.35 times more prevalent in women [ 7 ]. Both unilateral and bilateral peg lateralis seem to have a similar prevalence, but the frequency of left-side unilateral peg laterals is almost twice that of right-side ones [ 8 , 9 ]. Peg lateralis has a higher incidence of being associated with other tooth anomalies, such as hypodontia, palatal displacement of maxillary canines, dens invaginatus, tooth transposition, premolar rotations, and supernumerary teeth. These anomalies are considered as the same genotype exhibiting different phenotypes [ 10 ]. According to Brook’s etiological model, he suggested the relationship between alterations in number and size [ 11 ]. These associations have been considered as different phenotypes of an identical genotype, leading to variations in tooth number and size. Brook suggested an etiological model wherein the absence of tooth may be a quasi-continuous phenotype of tooth size distribution [ 11 , 12 ]. Mutations in genes, such as MSX1, AXIN2, FGF3, EDA, WNT5A, WNT10A , and Pax9 , are related to the occurrence of both microdontia and hypodontia [ 13 – 15 ]. This is because dental diseases are not determined by a single gene malfunction, but by the interaction of several genes and environmental factors. This pleiotropic and multifactorial nature of tooth anomalies, due to a complex mechanism of odontogenesis, makes it difficult to identify a specific single gene involved in a specific tooth anomaly. Because of these obstacles, genetic studies of non-syndromic isolated peg lateralis have rarely been reported. This study aimed to investigate the specific genotype associated with the phenotype of peg lateralis. We analyzed DNA from subjects with a peg-shaped incisor without any syndromes or other anomalies. Whole exome sequencing (WES) is a next-generation sequencing technique that analyzes the exon region, which codes for proteins and accounts for only 2% of the total genome [ 16 ]. Only approximately 23,000 genes in the human genome are protein-coding genes. Interestingly, about 85% of diseases-related genes are associated with these exon sites. Therefore, while whole genome sequencing (WGS) analyzes genes, including non-coding genes, WES is an easier and a more effective tool for investigating genes contributing to the pathogenesis of various diseases. In this study, we applied WES to identify potential candidate genes of peg lateralis under the hypothesis that the subjects had a specific genotype for the peg-shaped incisors. Materials and Methods Sample collection and DNA extraction The study protocol was approved by the Institutional Review Board of Yonsei University College of Dentistry (Yonsei IRB No. 2-2020-0045). The subjects were recruited from 2020 to 2022 at Yonsei University Dental Hospital and Dankook University Dental Hospital. The experimental group consisted of 20 individuals with peg lateralis who voluntarily provided consent to participate in this clinical trial. Subjects were limited to patients with a family member who had peg laterals; patients with systemic disease or other tooth anomalies were excluded. Consent was obtained from all participants before sample collection and was documented. The subject demographics are presented in Table 1. Additionally, saliva samples were collected from the family members of three subjects with peg lateralis, who voluntarily agreed to participate, to additionally validate the association between candidate variants and peg lateralis. For DNA analysis, saliva samples were collected from the subjects using a DNA self-collection kit (Genotek, Ottawa, Ontario, Canada). Each subject provided 2 mL saliva in the collection kit containing a 2 mL DNA-preserving solution in the lid. After collecting saliva, the lid on the kit was closed, and the DNA-preserving solution was mixed with the saliva. The genetic information of the control group was analyzed with the Ansan cohort’s information provided by the Korea Disease Control and Prevention Agency. This can be considered a standardization of Koreans as a population-based cohort, which was established for general population groups aged 40 or older, and a “gene-environment model cohort” to identify risk factors for genetic and environmental interaction in chronic diseases. WES data variant filtering using statistical analysis Variants were identified from the WES results, with only variants in protein-coding transcripts being considered. Variants that exhibited a p -value < 0.05, a false discovery rate (FDR) 1 were identified using Fisher's exact test when comparing them to those in the Ansan’s cohort. Variants with minor allele frequency (MAF) < 1% in the East Asian genome or with no reported MAF were subsequently filtered out using the genome aggregation database (gnomAD) v2.1.1 dataset [ 17 ]. Additional filters were applied to exclude low-mutational impact variants, such as those annotated as LOW and MODIFIER impact in SnpEff, and low-mutational impact variants predicted with in-silico mutation-impact analysis [ 18 ]. A list of candidate variants is summarized in Table 2. In silico mutation impact analysis To identify potentially pathogenic mutations in patients with peg lateralis, an in-silico mutation-impact analysis was performed. Missense variants in all possible non-redundant protein sequences of Ensembl GRCh37.p13 were analyzed using Polymorphism Phenotyping v2 (PolyPhen2), sorting intolerant from tolerant (SIFT), and an integrated score of co-evolution and conservation (CES) [ 19 – 21 ]. These methods predict the effects of mutations by analyzing evolutionary patterns in protein sequences. We obtained PolyPhen2 and SIFT scores for candidate variants from SnpEf, which uses pre-calculated scores from the DbNspf database [ 18 , 22 ]. The CES score was measured using pre-calculated scores provided at the CES website ( https://sbi.postech.ac.kr/w/CE ). The results of mutation-impact analysis are summarized in Table 2. In-silico analysis of mutation impact on protein structure To investigate the impact of the Arg598Pro (Arginine 598 to proline) mutation on the protein structure of Otopetrin-1 (OTOP1), we generated a 3D structure of OTOP1 using AlphaFold2, a state-of-the-art approach for predicting protein structure [ 23 ]. We selected interacting residues in which the shortest distance from Arg598 was less than 4 Å. Naccess was used to quantify the relative solvent accessibility (RSA) of the residues. Naccess calculates the atomically accessible surface by simulating the motion of a solvent molecule over a van der Waals surface [ 24 ]. Changes in the energy of residues (ΔΔG) upon mutation were assessed using the FoldX and PyRosetta computational methods [ 25 , 26 ]. For FoldX, we employed the BuildModel function in its mutation engine to predict stability changes, and the default scoring function was used for PyRosetta. Results Identification of candidate variants discovered by WES analysis We identified two candidate variants through WES analysis of 20 individuals with peg lateralis (Table 2). One of these variants was a rare variant, rs199742451, in OTOP1 , with a MAF of 0.0105 in the East Asian population. All 20 peg lateralis individuals had a heterozygous genotype for this variant, while none of the 100 control individuals carried this variant (Table 3). The P -value of fisher’s exact test was 1.18X10 − 18 and the FDR corrected p-value was 2.19X10 − 15 . The other candidate variant, a frameshift variant in RP11-131H24.4 , was not analyzed because of lack of studies. In silico mutation impact analysis of the OTOP1 variant In-silico mutation-impact analysis predicted that the missense variant in OTOP1 (c.1793G > C, p.Arg598Pro, rs199742451) is deleterious. Three different tools were used for the analysis: Polyphen2 [ 21 ], SIFT [ 19 ], and CES [ 20 ]. All three tools predicted that the OTOP1 variant is probably damaging and intolerant (Table 2). These predictions suggest that the variant is evolutionarily conserved and may be under a strong selective pressure. These evolutionary analyses imply that the observed variant will likely cause OTOP1 protein dysfunction. For instance, in Fig. 2 a, the missense variant is located in exon 1 of OTOP1 , and arginine at amino acid position 598 in this exon is well conserved in mammalian orthologs (Fig. 2 b). Potential effect of OTOP1 mutation on protein 3D structure In-silico structural analysis indicated that the Arg598Pro mutation is likely to have a deleterious impact on protein function. Analysis based on the tertiary structure of the protein showed that the Arg598 residue had limited exposure to solvents and interacted with 16 other amino acids (Fig. 3 a, yellow sticks). The RSA value of Arg598 was low (Fig. 3 b), suggesting that the residue is usually located in the inner region of the protein and has limited exposure to solvent. Therefore, the Arg598Pro substitution, where Arg is substituted by an amino acid with very different physicochemical properties at a low RSA position, may interfere with proper protein folding. In addition, the Arg598Pro variant was predicted to reduce the stability of the protein. Both Rosetta and FoldX, which calculate the stability change produced by mutations using the 3D structure of the protein, predicted that the Arg598Pro mutation makes OTOP1 unstable. The total stability change predicted by Rosetta and FoldX was 184.533 rosetta energy unit and 3.6573 kcal/mol, respectively (Fig. 3 d,e). Furthermore, the reduced protein stability caused by mutations can potentially interfere with protein function. The Arg598Pro substitution in OTOP1 disrupts a direct interaction with Glu435, which may impair the protein function. The interaction between the aligned sites with Arg598 and Glu435 was critical for the proton transport activity of zebrafish Otop1 [ 27 ]. A single mutation that interferes with the electrostatic interaction between Arg598 and Glu435 greatly diminished the current amplitude observed from Otop1. The tertiary structures of human OTOP1 and zebrafish Otop1 are well conserved, and the conformation and interactions of Arg598 and Glu435 are maintained in both species (Fig. 3 c). This suggests that changes in the interaction between Arg598 and Glu435 may also lead to the loss of protein function in human OTOP1. Functional analysis of OTOP1 Functional analysis of OTOP1 suggested a possible association with peg lateralis. The Gene Ontology (GO) terms associated with OTOP1 , obtained from the AmiGO database, included biomineral tissue development, which is critical for tooth development [ 28 , 29 ]. This suggests a possible relationship between OTOP1 and the development of peg lateralis. Validation of the OTOP1 variant in additional patients To further validate the relationship between the rs199742451 variant of OTOP1 and peg lateralis, the genotype of the loci was additionally identified in the family members of study participants, as shown in Fig. 4 . Sequencing the family members of three subjects (the sister of No. 8, cousin of No. 12, and mother of No. 18) revealed the presence of the rs199742451 variant in all three. The variant was expressed in a heterozygous condition similar to that in the 20 study participants. This finding indicated the possibility that the rs199742451 variant is associated with the occurrence of peg lateralis within the family. Known hypodontia variants in Korean peg lateralis patients The presence of peg lateralis is highly linked to other dental irregularities, including hypodontia, the genetics of which is relatively well studied [ 10 ]. None of the study participants in this study exhibited any association with variants known to cause hypodontia. Analysis of variants related to hypodontia obtained from the DisGeNet database did not show any significant association with this study’s participants (Table 4) [ 30 ]. This suggests that the peg lateralis in our cohort is not related to previously revealed hypodontia-related genes. Discussion In the present study, all patients with peg lateralis had a heterozygous missense mutation in exon 1 of OTOP1 (c.1793G > C, p.Arg598Pro, rs199742451) (Fig. 2 ). OTOP1 belongs to the Otopetrin family of multi-transmembrane domain proteins that are highly conserved in mammals [ 31 ]. OTOP1 is required for the formation of otoconia and otoliths, and mutations in OTOP1 in mice and zebrafish lead to non-syndromic otoconia agenesis. The Otopetrin proteins are a proton selective ion channel activated by purinergic stimuli in vestibule-supporting cells and play a crucial role in regulating intracellular calcium concentration [ 32 ]. Since otoconia are essential for mechanosensory transduction of linear acceleration and gravity in the inner ear, their degeneration or displacement can result in dizziness and progressive loss of balance. OTOP1 expression is continuously observed in adult mice and is involved in the maintenance and restoration of otoconia mineralization. However, whether OTOP1 expression persists in humans remains unclear [ 33 ]. Although the biochemical role of OTOP1 was recently identified, it has not been implicated in odontogenesis. Nevertheless, our results show that OTOP1 is likely to be involved in dental development. During dental development, ion channels regulate or control various physiological and biological activities, such as pH control, calcium flux, and gene expression [ 34 ]. One study found that mutations in CACNA1S , which encodes the voltage-dependent calcium channel, leads to the formation of multiple cusps in molars [ 35 ], this study analyzed the genes of five Thai families and demonstrated that defects in calcium signal interaction could cause variations in tooth morphology. Similarly, the loss-of-function mutations of OTOP1, identified in the present study, might cause an influx of calcium ion and disrupt calcium homeostasis, potentially leading to peg laterals (Fig. 3 ). CACNA1S is expressed in epithelial cells around the secondary enamel from the initial bud to the postnatal stage [ 36 ]. During tooth morphogenesis, the enamel knot serves as a signaling center that expresses several signals, including SHH, BMP, FGF, and WNT families, regulating tooth size and shape by controlling the growth and folding of the internal dental epithelium [ 37 ]. In addition, apoptosis in the enamel knot determines tooth size or morphology by regulating the duration of signaling interactions and is crucial in determining the size and patterning of cusps [ 38 ]. In Kim’s study, inhibiting apoptosis of enamel knots reduced the crown height [ 39 ]. This demonstrated that the final shape of the crown was already determined at the bell and cap stages. CACNA1S is also expressed in the Hertwig’s epithelial root sheath during postnatal development and is implicated in mal-shaped roots [ 36 ]. Thus, the morphology of teeth is affected by the time and location of gene expression. However, since the location or time of OTOP1 expression in odontogenesis is unknown, it is not easy to determine how its variants affect the pathogenesis of peg lateralis. As OTOP1 is a proton-selective ion channel, it is activated by acidic conditions and directly gated by protons [ 40 ]. In Tain’s study, it was activated by alkaline conditions, and mutations in OTOP1 affected its activation in alkaline conditions but had no influence in acidic conditions [ 41 ]. The alkali-activated OTOP1 is permeable to protons and cations but not calcium ions. The mechanism of pH regulation in odontogenesis remains unknown, but the characteristics of OTOP1 might affect abnormal tooth morphology by altering calcium ion concentration. Furthermore, previous twin-studies have suggested that environmental and epigenetic factors, as well as genetic factors, contribute to anomalies [ 42 ]. In this study, a few individuals did not inherit peg incisors directly from their parents, which is consistent with previous studies (Table 1, Fig. 4 ). Unlike previous studies, this study only analyzed the DNA of patients with peg lateralis unassociated with other anomalies or syndromes using WES. We identified OTOP1 as a candidate gene contributing to the development of peg lateralis. Notably, this finding differs from previous findings that suggested that dental anomalies within the same genotype have different phenotypes. Although the role of OTOP1 in dental development has not been clearly defined, our result suggesting that OTOP1 could affect tooth morphogenesis indicates the need for future research on ion channels in odontogenesis. Moreover, additional analysis within the same family identified that close family members carried the same genetic variant, which further supported the finding from the initial study cohort (Fig. 4 ). While the OTOP1 variant had significant associations in the 20 Korean patients, the sample size was relatively small to definitively confirm a significant association between the variant and peg-lateralis. Additionally, in-silico analysis had limitations in establishing a causal relationship. However, as this study presents the relationship between OTOP1 and peg lateralis for the first time, we hope that the findings herein will serve as a cornerstone for further experimental studies, such as animal mutation or molecular studies, to determine the cause and pathogenesis of peg-lateralis. Declarations Acknowledgment We also thank all of the members of the Kim laboratory for helpful discussions. Author contributions All authors gave final approval and agree to be accountable for all aspects of the work. Junglim Choi: Investigation; formal analysis; writing – original draft preparation; writing – review and editing Sungnam Kim: Data curation; formal analysis; visualization; table preparation; writing – original draft preparation; writing – review and editing Hyunsoo Ahn: Data curation; formal analysis; writing – review and editing Donghyo Kim: Methodology; Data curation Sung–Won Cho: Conceptualization Sanguk Kim: Conceptualization; supervision: methodology Jae-Hoon Lee: Conceptualization; supervision; investigation Funding This research was partially supported by a Co=professor research fund from the Yonsei University College of Dentistry (6-2019-0016). Ethical approval Not applicable Competing interests None to declare Sources of Support This research was partially supported by Co-professor research fund from the Yonsei University College of Dentistry (6-2019-0016). We also thank all of the members of the Kim laboratory for helpful discussions. Data Availability Statement The WES Data of 20 Korean peg lateralis patients are deposited in the Sequence Read Archive (SRA) (accession code PRJNA1013609). Code Availability Statement The Fisher's exact test was done by using python module SciPy (https://scipy.org/). The CES score can be obtained from CES website (https://sbi.postech.ac.kr/w/CE). The PyRosetta code for calculating Protein stability change is available at https://www.pyrosetta.org/. The FoldX code for calculating Protein stability change is available at https://foldxsuite.crg.eu/. References Townsend G, Bockmann M, Hughes T, Brook A. Genetic, environmental and epigenetic influences on variation in human tooth number, size and shape. Odontology. 2012;100:1–9. Tucker A, Sharpe P. The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet. 2004;5:499–508. Cobourne MT, Sharpe PT. Diseases of the tooth: the genetic and molecular basis of inherited anomalies affecting the dentition. Wiley Interdiscip Rev Dev Biol. 2013;2:183–212. Townsend GC, Richards L, Hughes T, Pinkerton S, Schwerdt W. Epigenetic influences may explain dental differences in monozygotic twin pairs. Aust Dent J. 2005;50:95–100. Suda N, Hattori M, Kosaki K, et al. Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia. Orthod Craniofac Res. 2010;13:197–202. Hua F, He H, Ngan P, Bouzid W. Prevalence of peg-shaped maxillary permanent lateral incisors: A meta-analysis. Am J Orthod Dentofacial Orthop. 2013;144:97–109. Polder BJ, Van't Hof MA, Van der Linden FP, Kuijpers-Jagtman AM. A meta-analysis of the prevalence of dental agenesis of permanent teeth. Community Dent Oral Epidemiol. 2004;32:217–26. Meskin LH, Gorlin RJ. Agenesis and Peg-Shaped Permanent Maxillary Lateral Incisors. J Dent Res. 1963;42:1476–9. Magnusson TE. Prevalence of hypodontia and malformations of permanent teeth in Iceland. Community Dent Oral Epidemiol. 1977;5:173–8. Baccetti T. A controlled study of associated dental anomalies. Angle Orthod. 1998;68:267–74. Brook AH. A unifying aetiological explanation for anomalies of human tooth number and size. Arch Oral Biol. 1984;29:373–8. Liuk IW, Olive RJ, Griffin M, Monsour P. Associations between palatally displaced canines and maxillary lateral incisors. Am J Orthod Dentofacial Orthop. 2013;143:622–32. Laurikkala J, Mikkola M, Mustonen T, et al. TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Dev Biol. 2001;229:443–55. Cai J, Mutoh N, Shin JO, et al. Wnt5a plays a crucial role in determining tooth size during murine tooth development. Cell Tissue Res. 2011;345:367–77. Brook AH, Jernvall J, Smith RN, Hughes TE, Townsend GC. The dentition: the outcomes of morphogenesis leading to variations of tooth number, size and shape. Aust Dent J. 2014;59 Suppl 1:131–42. Rabbani B, Tekin M, Mahdieh N. The promise of whole-exome sequencing in medical genetics. J Hum Genet. 2014;59:5–15. Karczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581:434–43. Cingolani P, Platts A, Wang le L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012;6:80–92. Vaser R, Adusumalli S, Leng SN, Sikic M, Ng PC. SIFT missense predictions for genomes. Nat Protoc. 2016;11:1–9. Kim D, Han SK, Lee K, Kim I, Kong J, Kim S. Evolutionary coupling analysis identifies the impact of disease-associated variants at less-conserved sites. Nucleic Acids Res. 2019;47:e94. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9. Liu X, Li C, Mou C, Dong Y, Tu Y. dbNSFP v4: a comprehensive database of transcript-specific functional predictions and annotations for human nonsynonymous and splice-site SNVs. Genome Med. 2020;12:103. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9. Lee B, Richards FM. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971;55:379–400. Chaudhury S, Lyskov S, Gray JJ. PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics. 2010;26:689–91. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33:W382-8. Saotome K, Teng B, Tsui CCA, et al. Structures of the otopetrin proton channels Otop1 and Otop3. Nat Struct Mol Biol. 2019;26:518–25. Moradian-Oldak J, George A. Biomineralization of Enamel and Dentin Mediated by Matrix Proteins. J Dent Res. 2021;100:1020–9. Gene Ontology C. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021;49:D325-D34. Pinero J, Ramirez-Anguita JM, Sauch-Pitarch J, et al. The DisGeNET knowledge platform for disease genomics: 2019 update. Nucleic Acids Res. 2020;48:D845-D55. Hurle B, Ignatova E, Massironi SM, et al. Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1. Hum Mol Genet. 2003;12:777–89. Hughes I, Saito M, Schlesinger PH, Ornitz DM. Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium. Proc Natl Acad Sci U S A. 2007;104:12023–8. Kim E, Hyrc KL, Speck J, et al. Regulation of cellular calcium in vestibular supporting cells by otopetrin 1. J Neurophysiol. 2010;104:3439–50. Duan X. Ion channels, channelopathies, and tooth formation. J Dent Res. 2014;93:117–25. Laugel-Haushalter V, Morkmued S, Stoetzel C, et al. Genetic Evidence Supporting the Role of the Calcium Channel, CACNA1S, in Tooth Cusp and Root Patterning. Front Physiol. 2018;9:1329. Kantaputra P, Butali A, Eliason S, et al. CACNA1S mutation-associated dental anomalies: A calcium channelopathy. Oral Dis. 2023; doi: 10.1111/odi.14551 . Jernvall J, Kettunen P, Karavanova I, Martin LB, Thesleff I. Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene. Int J Dev Biol. 1994;38:463–9. Jernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I. The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development. 1998;125:161–9. Kim JY, Cha YG, Cho SW, et al. Inhibition of apoptosis in early tooth development alters tooth shape and size. J Dent Res. 2006;85:530–5. Teng B, Kaplan JP, Liang Z, et al. Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels. Elife. 2022;11. Tian L, Zhang H, Yang S, et al. Vertebrate OTOP1 is also an alkali-activated channel. Nat Commun. 2023;14:26. Townsend G, Hughes T, Luciano M, Bockmann M, Brook A. Genetic and environmental influences on human dental variation: a critical evaluation of studies involving twins. Arch Oral Biol. 2009;54 Suppl 1:S45-51. Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files table1.xlsx table2.xlsx table3.xlsx table4.xlsx 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. 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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-3811797","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267198666,"identity":"86d18388-017a-4abc-9c6f-867c9dbaeede","order_by":0,"name":"Jae-Hoon lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACxgYGNhDNwyZ/+ACIR7wWGT4JtgTitAABWIuNnASPAXFamNsPH3vwoeIOD5t0zzdp3h11DPztBwg4rCct3XDGmWc8bDJnt0nznjnMIHEmgYCWhhwzad62wzxsDLlALW1AG24QcBhj//tvUC05z4CMOgZ5glpm5LBBtEiAGcwMBoS1PDOTnHEGqIXnmLHlXKBeQ0J+MexPfibxoeKwvXx788Mbb9vq5OSOHyCgpQHBZpEAEjwEnMXAII/EZv5AUPkoGAWjYBSMSAAA0aw/qgKVDh8AAAAASUVORK5CYII=","orcid":"","institution":"Yonsei University College of Dentistry","correspondingAuthor":true,"prefix":"","firstName":"Jae-Hoon","middleName":"","lastName":"lee","suffix":""},{"id":267198667,"identity":"74f2a5d2-7a36-428c-aa33-02ad905cae3b","order_by":1,"name":"junglim choi","email":"","orcid":"https://orcid.org/0000-0002-8157-8044","institution":"School of dentistry, Dankook University","correspondingAuthor":false,"prefix":"","firstName":"junglim","middleName":"","lastName":"choi","suffix":""},{"id":267198668,"identity":"df060b3b-4185-46d7-b39c-1836693692ee","order_by":2,"name":"Sungnam Kim","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sungnam","middleName":"","lastName":"Kim","suffix":""},{"id":267198669,"identity":"22528006-64fe-434b-9557-266677d66889","order_by":3,"name":"Hyunsoo Ahn","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hyunsoo","middleName":"","lastName":"Ahn","suffix":""},{"id":267198670,"identity":"b5e99691-8fcd-45f3-a8f8-4338c09cdc4b","order_by":4,"name":"Donghyo Kim","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Donghyo","middleName":"","lastName":"Kim","suffix":""},{"id":267198671,"identity":"15f012c6-6acc-4c57-81a6-81b62e8a67a6","order_by":5,"name":"Sung-Won Cho","email":"","orcid":"","institution":"Yonsei University college of dentistry","correspondingAuthor":false,"prefix":"","firstName":"Sung-Won","middleName":"","lastName":"Cho","suffix":""},{"id":267198672,"identity":"87a65da0-6b8c-4752-b2ae-54e4938cbe28","order_by":6,"name":"Sanguk Kim","email":"","orcid":"https://orcid.org/0000-0002-3449-3814","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sanguk","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2023-12-27 10:35:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3811797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3811797/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49769247,"identity":"c27f4ad8-e8ca-40fd-8edf-1a14ea09852e","added_by":"auto","created_at":"2024-01-17 17:26:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50022,"visible":true,"origin":"","legend":"\u003cp\u003ePeg lateralis on both maxillary lateral incisors.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/63fcd81a6ecac3c3ac5c3973.jpg"},{"id":49769246,"identity":"6b1ad65f-6590-4994-8c8b-7d1b9a082e45","added_by":"auto","created_at":"2024-01-17 17:26:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58751,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic view and multiple sequence alignments of \u003cem\u003eOTOP1\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ea. Coding exons are shown as red boxes and introns are shown as gray lines. The missense variant (c.1793G\u0026gt;C, p.Arg598Pro, rs199742451) identified in the present is shown above the exons. b. Multiple sequence alignments for \u003cem\u003eOTOP1\u003c/em\u003ehomologs in various mammal species. The mutated Arg598 residue is indicated using red font.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/ffb24a9f7df94bea5cd99c8d.png"},{"id":49767820,"identity":"8d4ec59f-f76f-4e4c-9ba7-8f2d4c895ded","added_by":"auto","created_at":"2024-01-17 17:18:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1637987,"visible":true,"origin":"","legend":"\u003cp\u003eProtein structural analysis of the OTOP1 Arg598Pro variant.\u003c/p\u003e\n\u003cp\u003ea. Three-dimensional structure of OTOP1 predicted by AlphaFold. The structure was rendered using PyMO (residues with pLDDT\u0026lt;50 are hidden). The red spheres and yellow sticks indicate arginine 598 (Arg598) and its interacting amino acid residues, respectively. The Arg598 residue interacts with 16 residues. b. Relative solvent accessibility (RSA) plot of residues in OTOP1. The black line depicts the mean RSA of -2 to +2 residues. c. Comparison of structural characteristics of the OTOP1 Arg598Pro variant and sequence alignment between human and zebrafish homologs. Gray structure is the three-dimensional protein structure of human OTOP1 predicted by AlphaFold. Blue structure is the three-dimensional protein structure of zebrafish OTOP1 (PDB ID: 6NF4). The red and yellow residues in human OTOP1 indicate arginine 598 (Arg598) and glutamic acid 435 (Glu435), respectively. The red and yellow residues in zebrafish OTOP1 are the corresponding sites of human OTOP1 Arg598 and Glu435, respectively. The structure was rendered using PyMOL. d. Energy changes calculated with Rosetta and broken down by each score term.e. Energy changes calculated with FoldX and broken down by each score term.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/b1d672e6e06ee74860ed979c.png"},{"id":49767817,"identity":"294abc02-a421-4ad8-a12d-5cb43617190b","added_by":"auto","created_at":"2024-01-17 17:18:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOTOP1\u003c/em\u003e genotype of family members of three subjects. a-c. Pedigrees of family No. 8 (a), No. 12 (b), and No. 18 (c). d \u003cem\u003eOTOP1 \u003c/em\u003egenotype of additional patients. All individuals had a reference allele and a c.1793G\u0026gt;C variant allele.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/e564740ef1c933900afeaceb.png"},{"id":55187664,"identity":"4e798b59-9645-4024-93b5-242c9d79ba64","added_by":"auto","created_at":"2024-04-23 18:33:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":919675,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/44a69c71-a0cc-47cd-bc80-2b4a5c1ff9b0.pdf"},{"id":49767815,"identity":"e8432600-2c13-4d25-b57f-dd076c854e91","added_by":"auto","created_at":"2024-01-17 17:18:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12555,"visible":true,"origin":"","legend":"","description":"","filename":"table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/453e258309bd6d5c9509bccb.xlsx"},{"id":49767818,"identity":"50cc3799-86ae-4254-9d46-fbaf6368354a","added_by":"auto","created_at":"2024-01-17 17:18:29","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10959,"visible":true,"origin":"","legend":"","description":"","filename":"table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/0653ce26aaceabfde9932867.xlsx"},{"id":49769248,"identity":"85ee48a9-ed4a-49c7-a61e-ae0523c164c3","added_by":"auto","created_at":"2024-01-17 17:26:29","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9549,"visible":true,"origin":"","legend":"","description":"","filename":"table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/274367f1c44cb30731175a28.xlsx"},{"id":49767822,"identity":"1156b961-85ad-4d8a-bc5b-afc30581f0a5","added_by":"auto","created_at":"2024-01-17 17:18:29","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10843,"visible":true,"origin":"","legend":"","description":"","filename":"table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3811797/v1/1d745b6d2d9264d30bb23b35.xlsx"}],"financialInterests":"There is no duality of interest","formattedTitle":"OTOP1: A New Candidate Gene for Non-syndromic Peg Lateralis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMore than 350 genes, most of which encode signaling molecules that play crucial roles in molecular and cellular interactions, have been identified to be involved in odontogenesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The epithelial-mesenchymal interactions mediated by these proteins regulate the different stages of tooth formation, including initiation, morphogenesis, and differentiation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Tooth anomalies, which appear as alterations in the number, size, morphology, or structure of a single tooth or multiple teeth, occur in isolation or in association with other syndromes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Congenital tooth anomalies cause functional, esthetic, and psychological problems; thus, early diagnosis is required for long term treatment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePeg lateralis is a microdontia that only occurs in maxillary lateral incisors and is tapered in shape, with incisal mesiodistal width shorter than cervical width (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The prevalence of peg lateralis varies from 0.6\u0026ndash;9.9% depending on ethnicity, sex, and region; Mongolians have a significantly higher prevalence rate than other ethnic groups. However, the overall prevalence rate is 1.8% according to meta-analyses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although non-syndromic teeth abnormalities are inherited as an autosomal trait, the literature suggests that peg shaped teeth are 1.35 times more prevalent in women [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Both unilateral and bilateral peg lateralis seem to have a similar prevalence, but the frequency of left-side unilateral peg laterals is almost twice that of right-side ones [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePeg lateralis has a higher incidence of being associated with other tooth anomalies, such as hypodontia, palatal displacement of maxillary canines, dens invaginatus, tooth transposition, premolar rotations, and supernumerary teeth. These anomalies are considered as the same genotype exhibiting different phenotypes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. According to Brook\u0026rsquo;s etiological model, he suggested the relationship between alterations in number and size [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These associations have been considered as different phenotypes of an identical genotype, leading to variations in tooth number and size. Brook suggested an etiological model wherein the absence of tooth may be a quasi-continuous phenotype of tooth size distribution [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mutations in genes, such as \u003cem\u003eMSX1, AXIN2, FGF3, EDA, WNT5A, WNT10A\u003c/em\u003e, and \u003cem\u003ePax9\u003c/em\u003e, are related to the occurrence of both microdontia and hypodontia [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This is because dental diseases are not determined by a single gene malfunction, but by the interaction of several genes and environmental factors. This pleiotropic and multifactorial nature of tooth anomalies, due to a complex mechanism of odontogenesis, makes it difficult to identify a specific single gene involved in a specific tooth anomaly. Because of these obstacles, genetic studies of non-syndromic isolated peg lateralis have rarely been reported. This study aimed to investigate the specific genotype associated with the phenotype of peg lateralis. We analyzed DNA from subjects with a peg-shaped incisor without any syndromes or other anomalies.\u003c/p\u003e \u003cp\u003eWhole exome sequencing (WES) is a next-generation sequencing technique that analyzes the exon region, which codes for proteins and accounts for only 2% of the total genome [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Only approximately 23,000 genes in the human genome are protein-coding genes. Interestingly, about 85% of diseases-related genes are associated with these exon sites. Therefore, while whole genome sequencing (WGS) analyzes genes, including non-coding genes, WES is an easier and a more effective tool for investigating genes contributing to the pathogenesis of various diseases. In this study, we applied WES to identify potential candidate genes of peg lateralis under the hypothesis that the subjects had a specific genotype for the peg-shaped incisors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSample collection and DNA extraction\u003c/p\u003e \u003cp\u003e The study protocol was approved by the Institutional Review Board of Yonsei University College of Dentistry (Yonsei IRB No. 2-2020-0045). The subjects were recruited from 2020 to 2022 at Yonsei University Dental Hospital and Dankook University Dental Hospital. The experimental group consisted of 20 individuals with peg lateralis who voluntarily provided consent to participate in this clinical trial. Subjects were limited to patients with a family member who had peg laterals; patients with systemic disease or other tooth anomalies were excluded. Consent was obtained from all participants before sample collection and was documented. The subject demographics are presented in Table\u0026nbsp;1. Additionally, saliva samples were collected from the family members of three subjects with peg lateralis, who voluntarily agreed to participate, to additionally validate the association between candidate variants and peg lateralis.\u003c/p\u003e \u003cp\u003eFor DNA analysis, saliva samples were collected from the subjects using a DNA self-collection kit (Genotek, Ottawa, Ontario, Canada). Each subject provided 2 mL saliva in the collection kit containing a 2 mL DNA-preserving solution in the lid. After collecting saliva, the lid on the kit was closed, and the DNA-preserving solution was mixed with the saliva. The genetic information of the control group was analyzed with the Ansan cohort\u0026rsquo;s information provided by the Korea Disease Control and Prevention Agency. This can be considered a standardization of Koreans as a population-based cohort, which was established for general population groups aged 40 or older, and a \u0026ldquo;gene-environment model cohort\u0026rdquo; to identify risk factors for genetic and environmental interaction in chronic diseases.\u003c/p\u003e \u003cp\u003eWES data variant filtering using statistical analysis\u003c/p\u003e \u003cp\u003eVariants were identified from the WES results, with only variants in protein-coding transcripts being considered. Variants that exhibited a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e, and an odds ratio\u0026thinsp;\u0026gt;\u0026thinsp;1 were identified using Fisher's exact test when comparing them to those in the Ansan\u0026rsquo;s cohort. Variants with minor allele frequency (MAF)\u0026thinsp;\u0026lt;\u0026thinsp;1% in the East Asian genome or with no reported MAF were subsequently filtered out using the genome aggregation database (gnomAD) v2.1.1 dataset [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additional filters were applied to exclude low-mutational impact variants, such as those annotated as LOW and MODIFIER impact in SnpEff, and low-mutational impact variants predicted with in-silico mutation-impact analysis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A list of candidate variants is summarized in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eIn silico mutation impact analysis\u003c/p\u003e \u003cp\u003eTo identify potentially pathogenic mutations in patients with peg lateralis, an in-silico mutation-impact analysis was performed. Missense variants in all possible non-redundant protein sequences of Ensembl GRCh37.p13 were analyzed using Polymorphism Phenotyping v2 (PolyPhen2), sorting intolerant from tolerant (SIFT), and an integrated score of co-evolution and conservation (CES) [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These methods predict the effects of mutations by analyzing evolutionary patterns in protein sequences. We obtained PolyPhen2 and SIFT scores for candidate variants from SnpEf, which uses pre-calculated scores from the DbNspf database [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The CES score was measured using pre-calculated scores provided at the CES website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sbi.postech.ac.kr/w/CE\u003c/span\u003e\u003cspan address=\"https://sbi.postech.ac.kr/w/CE\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results of mutation-impact analysis are summarized in Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003eIn-silico analysis of mutation impact on protein structure\u003c/p\u003e \u003cp\u003eTo investigate the impact of the Arg598Pro (Arginine 598 to proline) mutation on the protein structure of Otopetrin-1 (OTOP1), we generated a 3D structure of OTOP1 using AlphaFold2, a state-of-the-art approach for predicting protein structure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We selected interacting residues in which the shortest distance from Arg598 was less than 4 \u0026Aring;. Naccess was used to quantify the relative solvent accessibility (RSA) of the residues. Naccess calculates the atomically accessible surface by simulating the motion of a solvent molecule over a van der Waals surface [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Changes in the energy of residues (ΔΔG) upon mutation were assessed using the FoldX and PyRosetta computational methods [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For FoldX, we employed the BuildModel function in its mutation engine to predict stability changes, and the default scoring function was used for PyRosetta.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIdentification of candidate variants discovered by WES analysis\u003c/p\u003e \u003cp\u003eWe identified two candidate variants through WES analysis of 20 individuals with peg lateralis (Table\u0026nbsp;2). One of these variants was a rare variant, rs199742451, in \u003cem\u003eOTOP1\u003c/em\u003e, with a MAF of 0.0105 in the East Asian population. All 20 peg lateralis individuals had a heterozygous genotype for this variant, while none of the 100 control individuals carried this variant (Table\u0026nbsp;3). The \u003cem\u003eP\u003c/em\u003e-value of fisher\u0026rsquo;s exact test was 1.18X10\u003csup\u003e\u0026minus;\u0026thinsp;18\u003c/sup\u003e and the FDR corrected p-value was 2.19X10\u003csup\u003e\u0026minus;\u0026thinsp;15\u003c/sup\u003e. The other candidate variant, a frameshift variant in \u003cem\u003eRP11-131H24.4\u003c/em\u003e, was not analyzed because of lack of studies.\u003c/p\u003e \u003cp\u003eIn silico mutation impact analysis of the \u003cem\u003eOTOP1\u003c/em\u003e variant\u003c/p\u003e \u003cp\u003eIn-silico mutation-impact analysis predicted that the missense variant in \u003cem\u003eOTOP1\u003c/em\u003e (c.1793G\u0026thinsp;\u0026gt;\u0026thinsp;C, p.Arg598Pro, rs199742451) is deleterious. Three different tools were used for the analysis: Polyphen2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], SIFT [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and CES [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All three tools predicted that the \u003cem\u003eOTOP1\u003c/em\u003e variant is probably damaging and intolerant (Table\u0026nbsp;2). These predictions suggest that the variant is evolutionarily conserved and may be under a strong selective pressure. These evolutionary analyses imply that the observed variant will likely cause OTOP1 protein dysfunction. For instance, in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the missense variant is located in exon 1 of \u003cem\u003eOTOP1\u003c/em\u003e, and arginine at amino acid position 598 in this exon is well conserved in mammalian orthologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePotential effect of OTOP1 mutation on protein 3D structure\u003c/p\u003e \u003cp\u003eIn-silico structural analysis indicated that the Arg598Pro mutation is likely to have a deleterious impact on protein function. Analysis based on the tertiary structure of the protein showed that the Arg598 residue had limited exposure to solvents and interacted with 16 other amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, yellow sticks). The RSA value of Arg598 was low (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), suggesting that the residue is usually located in the inner region of the protein and has limited exposure to solvent. Therefore, the Arg598Pro substitution, where Arg is substituted by an amino acid with very different physicochemical properties at a low RSA position, may interfere with proper protein folding. In addition, the Arg598Pro variant was predicted to reduce the stability of the protein. Both Rosetta and FoldX, which calculate the stability change produced by mutations using the 3D structure of the protein, predicted that the Arg598Pro mutation makes OTOP1 unstable. The total stability change predicted by Rosetta and FoldX was 184.533 rosetta energy unit and 3.6573 kcal/mol, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e). Furthermore, the reduced protein stability caused by mutations can potentially interfere with protein function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Arg598Pro substitution in OTOP1 disrupts a direct interaction with Glu435, which may impair the protein function. The interaction between the aligned sites with Arg598 and Glu435 was critical for the proton transport activity of zebrafish Otop1 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A single mutation that interferes with the electrostatic interaction between Arg598 and Glu435 greatly diminished the current amplitude observed from Otop1. The tertiary structures of human OTOP1 and zebrafish Otop1 are well conserved, and the conformation and interactions of Arg598 and Glu435 are maintained in both species (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This suggests that changes in the interaction between Arg598 and Glu435 may also lead to the loss of protein function in human OTOP1.\u003c/p\u003e \u003cp\u003eFunctional analysis of \u003cem\u003eOTOP1\u003c/em\u003e\u003c/p\u003e \u003cp\u003eFunctional analysis of \u003cem\u003eOTOP1\u003c/em\u003e suggested a possible association with peg lateralis. The Gene Ontology (GO) terms associated with \u003cem\u003eOTOP1\u003c/em\u003e, obtained from the AmiGO database, included biomineral tissue development, which is critical for tooth development [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This suggests a possible relationship between \u003cem\u003eOTOP1\u003c/em\u003e and the development of peg lateralis.\u003c/p\u003e \u003cp\u003eValidation of the \u003cem\u003eOTOP1\u003c/em\u003e variant in additional patients\u003c/p\u003e \u003cp\u003eTo further validate the relationship between the rs199742451 variant of \u003cem\u003eOTOP1\u003c/em\u003e and peg lateralis, the genotype of the loci was additionally identified in the family members of study participants, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Sequencing the family members of three subjects (the sister of No. 8, cousin of No. 12, and mother of No. 18) revealed the presence of the rs199742451 variant in all three. The variant was expressed in a heterozygous condition similar to that in the 20 study participants. This finding indicated the possibility that the rs199742451 variant is associated with the occurrence of peg lateralis within the family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKnown hypodontia variants in Korean peg lateralis patients\u003c/p\u003e \u003cp\u003eThe presence of peg lateralis is highly linked to other dental irregularities, including hypodontia, the genetics of which is relatively well studied [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. None of the study participants in this study exhibited any association with variants known to cause hypodontia. Analysis of variants related to hypodontia obtained from the DisGeNet database did not show any significant association with this study\u0026rsquo;s participants (Table\u0026nbsp;4) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This suggests that the peg lateralis in our cohort is not related to previously revealed hypodontia-related genes.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, all patients with peg lateralis had a heterozygous missense mutation in exon 1 of \u003cem\u003eOTOP1\u003c/em\u003e (c.1793G\u0026thinsp;\u0026gt;\u0026thinsp;C, p.Arg598Pro, rs199742451) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). OTOP1 belongs to the Otopetrin family of multi-transmembrane domain proteins that are highly conserved in mammals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. OTOP1 is required for the formation of otoconia and otoliths, and mutations in OTOP1 in mice and zebrafish lead to non-syndromic otoconia agenesis. The Otopetrin proteins are a proton selective ion channel activated by purinergic stimuli in vestibule-supporting cells and play a crucial role in regulating intracellular calcium concentration [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Since otoconia are essential for mechanosensory transduction of linear acceleration and gravity in the inner ear, their degeneration or displacement can result in dizziness and progressive loss of balance. OTOP1 expression is continuously observed in adult mice and is involved in the maintenance and restoration of otoconia mineralization. However, whether OTOP1 expression persists in humans remains unclear [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although the biochemical role of OTOP1 was recently identified, it has not been implicated in odontogenesis. Nevertheless, our results show that \u003cem\u003eOTOP1\u003c/em\u003e is likely to be involved in dental development.\u003c/p\u003e \u003cp\u003eDuring dental development, ion channels regulate or control various physiological and biological activities, such as pH control, calcium flux, and gene expression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. One study found that mutations in \u003cem\u003eCACNA1S\u003c/em\u003e, which encodes the voltage-dependent calcium channel, leads to the formation of multiple cusps in molars [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], this study analyzed the genes of five Thai families and demonstrated that defects in calcium signal interaction could cause variations in tooth morphology. Similarly, the loss-of-function mutations of OTOP1, identified in the present study, might cause an influx of calcium ion and disrupt calcium homeostasis, potentially leading to peg laterals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCACNA1S\u003c/em\u003e is expressed in epithelial cells around the secondary enamel from the initial bud to the postnatal stage [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. During tooth morphogenesis, the enamel knot serves as a signaling center that expresses several signals, including SHH, BMP, FGF, and WNT families, regulating tooth size and shape by controlling the growth and folding of the internal dental epithelium [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, apoptosis in the enamel knot determines tooth size or morphology by regulating the duration of signaling interactions and is crucial in determining the size and patterning of cusps [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In Kim\u0026rsquo;s study, inhibiting apoptosis of enamel knots reduced the crown height [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This demonstrated that the final shape of the crown was already determined at the bell and cap stages. \u003cem\u003eCACNA1S\u003c/em\u003e is also expressed in the Hertwig\u0026rsquo;s epithelial root sheath during postnatal development and is implicated in mal-shaped roots [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, the morphology of teeth is affected by the time and location of gene expression. However, since the location or time of \u003cem\u003eOTOP1\u003c/em\u003e expression in odontogenesis is unknown, it is not easy to determine how its variants affect the pathogenesis of peg lateralis.\u003c/p\u003e \u003cp\u003eAs OTOP1 is a proton-selective ion channel, it is activated by acidic conditions and directly gated by protons [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In Tain\u0026rsquo;s study, it was activated by alkaline conditions, and mutations in OTOP1 affected its activation in alkaline conditions but had no influence in acidic conditions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The alkali-activated OTOP1 is permeable to protons and cations but not calcium ions. The mechanism of pH regulation in odontogenesis remains unknown, but the characteristics of OTOP1 might affect abnormal tooth morphology by altering calcium ion concentration. Furthermore, previous twin-studies have suggested that environmental and epigenetic factors, as well as genetic factors, contribute to anomalies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, a few individuals did not inherit peg incisors directly from their parents, which is consistent with previous studies (Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnlike previous studies, this study only analyzed the DNA of patients with peg lateralis unassociated with other anomalies or syndromes using WES. We identified \u003cem\u003eOTOP1\u003c/em\u003e as a candidate gene contributing to the development of peg lateralis. Notably, this finding differs from previous findings that suggested that dental anomalies within the same genotype have different phenotypes. Although the role of OTOP1 in dental development has not been clearly defined, our result suggesting that \u003cem\u003eOTOP1\u003c/em\u003e could affect tooth morphogenesis indicates the need for future research on ion channels in odontogenesis. Moreover, additional analysis within the same family identified that close family members carried the same genetic variant, which further supported the finding from the initial study cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). While the OTOP1 variant had significant associations in the 20 Korean patients, the sample size was relatively small to definitively confirm a significant association between the variant and peg-lateralis. Additionally, in-silico analysis had limitations in establishing a causal relationship. However, as this study presents the relationship between OTOP1 and peg lateralis for the first time, we hope that the findings herein will serve as a cornerstone for further experimental studies, such as animal mutation or molecular studies, to determine the cause and pathogenesis of peg-lateralis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also thank all of the members of the Kim laboratory for helpful discussions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave final approval and agree to be accountable for all aspects of the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJunglim Choi: Investigation; formal analysis; writing \u0026ndash; original draft preparation; writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003eSungnam Kim: Data curation; formal analysis; visualization; table preparation; writing \u0026ndash; original draft preparation; writing \u0026ndash; review and editing\u003c/p\u003e\n\u003cp\u003eHyunsoo Ahn: Data curation; formal analysis; writing \u0026ndash; review and editing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDonghyo Kim: Methodology; Data curation\u003c/p\u003e\n\u003cp\u003eSung\u0026ndash;Won Cho: Conceptualization\u003c/p\u003e\n\u003cp\u003eSanguk Kim: Conceptualization; supervision: methodology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJae-Hoon Lee: Conceptualization; supervision; investigation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by a Co=professor research fund from the Yonsei University College of Dentistry (6-2019-0016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone to declare\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSources of Support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by Co-professor research fund from the Yonsei University College of Dentistry (6-2019-0016). We also thank all of the members of the Kim laboratory for helpful discussions.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WES Data of 20 Korean peg lateralis patients are deposited in the Sequence Read Archive (SRA) (accession code PRJNA1013609).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Fisher\u0026apos;s exact test was done by using python module SciPy (https://scipy.org/). The CES score can be obtained from CES website (https://sbi.postech.ac.kr/w/CE). The PyRosetta code for calculating Protein stability change is available at https://www.pyrosetta.org/. The FoldX code for calculating Protein stability change is available at https://foldxsuite.crg.eu/.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTownsend G, Bockmann M, Hughes T, Brook A. Genetic, environmental and epigenetic influences on variation in human tooth number, size and shape. Odontology. 2012;100:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker A, Sharpe P. The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet. 2004;5:499\u0026ndash;508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCobourne MT, Sharpe PT. Diseases of the tooth: the genetic and molecular basis of inherited anomalies affecting the dentition. Wiley Interdiscip Rev Dev Biol. 2013;2:183\u0026ndash;212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTownsend GC, Richards L, Hughes T, Pinkerton S, Schwerdt W. Epigenetic influences may explain dental differences in monozygotic twin pairs. Aust Dent J. 2005;50:95\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuda N, Hattori M, Kosaki K, et al. Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia. Orthod Craniofac Res. 2010;13:197\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua F, He H, Ngan P, Bouzid W. Prevalence of peg-shaped maxillary permanent lateral incisors: A meta-analysis. Am J Orthod Dentofacial Orthop. 2013;144:97\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolder BJ, Van't Hof MA, Van der Linden FP, Kuijpers-Jagtman AM. A meta-analysis of the prevalence of dental agenesis of permanent teeth. Community Dent Oral Epidemiol. 2004;32:217\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeskin LH, Gorlin RJ. Agenesis and Peg-Shaped Permanent Maxillary Lateral Incisors. J Dent Res. 1963;42:1476\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagnusson TE. Prevalence of hypodontia and malformations of permanent teeth in Iceland. Community Dent Oral Epidemiol. 1977;5:173\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaccetti T. A controlled study of associated dental anomalies. Angle Orthod. 1998;68:267\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrook AH. A unifying aetiological explanation for anomalies of human tooth number and size. Arch Oral Biol. 1984;29:373\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiuk IW, Olive RJ, Griffin M, Monsour P. Associations between palatally displaced canines and maxillary lateral incisors. Am J Orthod Dentofacial Orthop. 2013;143:622\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaurikkala J, Mikkola M, Mustonen T, et al. TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Dev Biol. 2001;229:443\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai J, Mutoh N, Shin JO, et al. Wnt5a plays a crucial role in determining tooth size during murine tooth development. Cell Tissue Res. 2011;345:367\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrook AH, Jernvall J, Smith RN, Hughes TE, Townsend GC. The dentition: the outcomes of morphogenesis leading to variations of tooth number, size and shape. Aust Dent J. 2014;59 Suppl 1:131\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRabbani B, Tekin M, Mahdieh N. The promise of whole-exome sequencing in medical genetics. J Hum Genet. 2014;59:5\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarczewski KJ, Francioli LC, Tiao G, et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581:434\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCingolani P, Platts A, Wang le L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012;6:80\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaser R, Adusumalli S, Leng SN, Sikic M, Ng PC. SIFT missense predictions for genomes. Nat Protoc. 2016;11:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Han SK, Lee K, Kim I, Kong J, Kim S. Evolutionary coupling analysis identifies the impact of disease-associated variants at less-conserved sites. Nucleic Acids Res. 2019;47:e94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Li C, Mou C, Dong Y, Tu Y. dbNSFP v4: a comprehensive database of transcript-specific functional predictions and annotations for human nonsynonymous and splice-site SNVs. Genome Med. 2020;12:103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee B, Richards FM. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971;55:379\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhury S, Lyskov S, Gray JJ. PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics. 2010;26:689\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33:W382-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaotome K, Teng B, Tsui CCA, et al. Structures of the otopetrin proton channels Otop1 and Otop3. Nat Struct Mol Biol. 2019;26:518\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradian-Oldak J, George A. Biomineralization of Enamel and Dentin Mediated by Matrix Proteins. J Dent Res. 2021;100:1020\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGene Ontology C. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021;49:D325-D34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinero J, Ramirez-Anguita JM, Sauch-Pitarch J, et al. The DisGeNET knowledge platform for disease genomics: 2019 update. Nucleic Acids Res. 2020;48:D845-D55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHurle B, Ignatova E, Massironi SM, et al. Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1. Hum Mol Genet. 2003;12:777\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHughes I, Saito M, Schlesinger PH, Ornitz DM. Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium. Proc Natl Acad Sci U S A. 2007;104:12023\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim E, Hyrc KL, Speck J, et al. Regulation of cellular calcium in vestibular supporting cells by otopetrin 1. J Neurophysiol. 2010;104:3439\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan X. Ion channels, channelopathies, and tooth formation. J Dent Res. 2014;93:117\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaugel-Haushalter V, Morkmued S, Stoetzel C, et al. Genetic Evidence Supporting the Role of the Calcium Channel, CACNA1S, in Tooth Cusp and Root Patterning. Front Physiol. 2018;9:1329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKantaputra P, Butali A, Eliason S, et al. CACNA1S mutation-associated dental anomalies: A calcium channelopathy. Oral Dis. 2023; doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/odi.14551\u003c/span\u003e\u003cspan address=\"10.1111/odi.14551\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJernvall J, Kettunen P, Karavanova I, Martin LB, Thesleff I. Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene. Int J Dev Biol. 1994;38:463\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJernvall J, Aberg T, Kettunen P, Keranen S, Thesleff I. The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development. 1998;125:161\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JY, Cha YG, Cho SW, et al. Inhibition of apoptosis in early tooth development alters tooth shape and size. J Dent Res. 2006;85:530\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeng B, Kaplan JP, Liang Z, et al. Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels. Elife. 2022;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian L, Zhang H, Yang S, et al. Vertebrate OTOP1 is also an alkali-activated channel. Nat Commun. 2023;14:26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTownsend G, Hughes T, Luciano M, Bockmann M, Brook A. Genetic and environmental influences on human dental variation: a critical evaluation of studies involving twins. Arch Oral Biol. 2009;54 Suppl 1:S45-51.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e\n"}],"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":"Calcium flux, Dental anomalies, Otopetrin, Peg-shaped maxillary lateral incisors, Whole exome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-3811797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3811797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough peg-shaped lateral incisors are a common dental anomaly, the genetic mechanisms underlying peg-shaped lateral incisors are poorly understood, particularly in cases without associated anomalies. The present study aimed to identify potential candidate genes contributing to the development of non-syndromic peg lateralis, by performing whole-exome sequencing (WES). Saliva samples were collected from 20 cases of unrelated Korean individuals that were; not associated with other anomalies. WES was conducted on these samples, and variants were filtered using criteria of a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, a false discovery rate\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e, and an odds ratio\u0026thinsp;\u0026gt;\u0026thinsp;1. In silico mutation impact analysis was performed using Polymorphism Phenotyping v2, sorting intolerant from tolerant, and integrated score of co-evolution and conservation algorithms. We identified a heterozygous \u003cem\u003eOTOP1\u003c/em\u003e gene allele encoding the Otopetrin-1 protein, a proton channel, in all 20 individuals. Gene Ontology analysis revealed an association between \u003cem\u003eOTOP1\u003c/em\u003e and peg lateralis. We further confirmed that the peg lateralis candidate variant, rs199742451, of the same genotype was found in the family member of three subjects with the same phenotype. The results suggest a new possible function of OTOP1, which is yet to be studied, and identified it as a new candidate contributing to the development of peg lateralis. This study provides new insights into the genetic basis of non-syndromic peg lateralis and has important implications for further studies on the role of new genes in peg lateralis\u003c/p\u003e","manuscriptTitle":"OTOP1: A New Candidate Gene for Non-syndromic Peg Lateralis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 17:18:24","doi":"10.21203/rs.3.rs-3811797/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":"509411c2-0c3b-4df9-a870-993d6e0c36e3","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28147807,"name":"Biological sciences/Genetics/Sequencing/DNA sequencing"},{"id":28147808,"name":"Biological sciences/Genetics/Genetic markers"}],"tags":[],"updatedAt":"2024-04-23T18:25:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 17:18:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3811797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3811797","identity":"rs-3811797","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}