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Novel ITGB6 variants cause hypoplastic-hypomineralized amelogenesis imperfecta and taurodontism: characterization of tooth phenotype and review of literature

Novel ITGB6 variants cause hypoplastic-hypomineralized amelogenesis imperfecta and taurodontism:... www.nature.com/bdjopen ARTICLE OPEN Novel ITGB6 variants cause hypoplastic-hypomineralized amelogenesis imperfecta and taurodontism: characterization of tooth phenotype and review of literature 1 1 2 3 1✉ Kanokwan Sriwattanapong , Thanakorn Theerapanon , Lawan Boonprakong , Anucharte Srijunbarl , Thantrira Porntaveetus and 4,5 Vorasuk Shotelersuk © The Author(s) 2023 OBJECTIVES: To characterize phenotype and genotype of amelogenesis imperfecta (AI) in a Thai patient, and review of literature. MATERIALS AND METHODS: Variants were identified using trio-exome and Sanger sequencing. The ITGB6 protein level in patient’s gingival cells was measured. The patient’s deciduous first molar was investigated for surface roughness, mineral density, microhardness, mineral composition, and ultrastructure. RESULTS: The patient exhibited hypoplastic-hypomineralized AI, taurodontism, and periodontal inflammation. Exome sequencing identified the novel compound heterozygous ITGB6 mutation, a nonsense c.625 G > T, p.(Gly209*) inherited from mother and a splicing c.1661-3 C > G from father, indicating AI type IH. The ITGB6 level in patient cells was significantly reduced, compared with controls. Analyses of a patient’s tooth showed a significant increase in roughness while mineral density of enamel and microhardness of enamel and dentin were significantly reduced. In dentin, carbon was significantly decreased while calcium, phosphorus, and oxygen levels were significantly increased. Severely collapsed enamel rods and a gap in dentinoenamel junction were observed. Of six affected families and eight ITGB6 variants that have been reported, our patient was the only one with taurodontism. CONCLUSION: We report the hypoplasia/hypomineralization/taurodontism AI patient with disturbed tooth characteristics associated with the novel ITGB6 variants and reduced ITGB6 expression, expanding genotype, phenotype, and understanding of autosomal recessive AI. BDJ Open (2023) 9:15 ; https://doi.org/10.1038/s41405-023-00142-y INTRODUCTION taurodontism, and subdivided into fifteen subtypes (IA-IG, IIA-IID, IIIA- Dental enamel is an epithelial-derived tissue comprising of IIIB, and IVA-IVB) by phenotype and inheritance pattern [5]. The Online organized hydroxyapatite crystals configured into enamel rods [1]. Mendelian Inheritance in Man (OMIM) has associated specific genes Amelogenesis in mouse has been described in four defined stages: with phenotypes and expanded the AI designations into: AI type IA presecretory, secretory, transition, to maturation stages [2]. In the (LAMB3), IB and IC (ENAM), IE (AMELX), IF (AMBN), IG (FAM20A), IH presecretory stage, the ameloblasts acquire their characteristics and (ITGB6), IJ (ACP4), IK (SP6), IIA1 (KLK4), IIA2 (MMP20), IIA3 (WDR72), IIA4 begin secreting enamel matrix proteins. In the secretory stage, the (ODAPH), IIA5 (SLC24A4), IIA6 (GPR68), III1 (FAM83H), IIIB (AMTN), IIIC entire thickness of the enamel layer is established. In the transition (RELT), and IV (DLX3)[6]. and maturation stages, the ameloblasts restructure and transport The integrin beta-6 (ITGB6) gene (OMIM *147558) comprises mineral ions into the enamel fluid, promoting the inorganic content fifteen exons and encodes the β6 subunit of the integrin αvβ6 that and growth of enamel prisms [3]. An entire process of amelogenesis plays an important role in cell-cell and cell-matrix adhesion, is under genetic control. Alterations in genes responsible for cellular proliferation and migration, tissue repair, apoptosis, amelogenesis at different timings result in enamel malformations inflammation, and angiogenesis [7, 8]. It also activates extracellular with diverse phenotypes [4]. matrix protein and transcription factors such as fibronectin, Amelogenesis imperfecta (AI) is a heterogeneous group of genetic tenascin-C, vitronectin, latency-associated peptide of transforming conditions characterized by defects in enamel formation and affecting growth factor- β1 (TGF- β1), and TGF- β3[9, 10]. The integrin αvβ6 both deciduous and permanent teeth. The Witkop’sclassification is expressed in the ameloblasts and regulates enamel biominer- categorized AI into four main types: (1) hypoplastic, (2) hypomatura- alization, amelogenin deposition, and TGF-β1 that controls cell tion, (3) hypocalcification, and (4) hypomaturation-hypoplastic with growth and differentiation during early tooth development [11, 12]. 1 2 Center of Excellence in Genomics and Precision Dentistry, Department of Physiology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Office of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Dental Materials R&D Center, Faculty of Dentistry, Chulalongkorn University, Bangkok 4 5 10330, Thailand. Center of Excellence for Medical Genomics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Excellence Center for Genomics and Precision Medicine, King Chulalongkorn Memorial Hospital, the Thai Red Cross Society, Bangkok 10330, Thailand. email: thantrira.p@chula.ac.th Received: 11 January 2023 Revised: 11 March 2023 Accepted: 23 March 2023 1234567890();,: K. Sriwattanapong et al. The expression of Itgb6 is detected in the differentiating culture dish (Corning, New York, USA). Cells were maintained in growth medium composed of Dulbecco’s Modified Eagle Medium (DMEM), 10% ameloblasts, secretory ameloblasts, and maturation ameloblasts, −/− fetal bovine serum (Gibco, CA, USA), 1% L-glutamine (Gibco), and 1% indicating its involvement in amelogenesis [13]. In the Itgb6 penicillin and streptomycin (Gibco), and incubated in a humidified mice, an accumulation of amelogenin in enamel extracellular matrix environment at 37 °C and 5% CO . Cells from passages 5 were used in was disturbed, resulting in a reduction in enamel mineralization the experiments. [14], that mimics the human AI phenotype. The biallelic mutations in the ITGB6 gene lead to hypoplastic and/or hypomineralized AI (AI Western blot analysis type IH) (MIM #616221) [9, 13, 15]. Confluent monolayer cells were harvested, washed with ice-cold Currently, the knowledge of genotypes and phenotypes related phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation to the ITGB6 are still elusive. There have been only five families TM buffer (RIPA) (Thermo Fisher Scientific, MA, USA) containing the Halt and six different ITGB6 mutations reported. Only one study protease inhibitor cocktail (Thermo Fisher Scientific, MA, USA). Protein demonstrated the scanning electron microscopic images of one TM concentration was determined using Pierce BCA Protein Assay Kit affected tooth [9]. Identifying more patients and new ITGB6 (Thermo Fisher Scientific, MA, USA). A total protein (30 µg) was separated variants helps understand the biological role of ITGB6 in humans by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and and establish phenotype-genotype correlation benefiting disease transferred to a polyvinylidene difluoride membranes (PVDF) (Bio-Rad, CA, USA). The PVDF membranes were blocked with 5% BSA (Merck Millipore, management. Germany) in TBST buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% In this study, we identified a Thai family with the proband having Tween-20) for 1 h at room temperature, and probed with a primary AI, taurodontism, and periodontal inflammation. Trio-exome and antibody overnight at 4 °C. Mouse anti-human Integrin β6 monoclonal Sanger sequencing was performed to detect pathogenic variants. antibody (1:1000 dilution; Merck, Darmstadt, Germany, Cat No. MAB2076Z) The patient’s tooth was investigated for its physical and mechanical and mouse anti-human GAPDH (1:3000 dilution; Abcam, Cambridge, UK, properties and ultrastructure. The patient’s gingival fibroblasts were Cat No. ab8245) were used as the primary antibodies. The membranes investigated for the ITGB6 protein level. A review of literature of all were incubated with the HRP-conjugated secondary antibody; anti-mouse, reported ITGB6 cases and mutations were performed. (1:2500 dilution; R&D systems, MN, USA, Cat. No. HAF018) for 2 h at room TM temperature. They were treated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, MA, USA) and analyzed TM using the Amersham Imager 680 (GE Healthcare, Illinois, USA). The MATERIALS AND METHODS intensity of the bands was quantified using ImageJ software. Subjects This study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB 813/63) and in accordance with Tooth samples the 1964 Helsinki declaration and its later amendments. A written A deciduous maxillary first molar of the proband was extracted according to informed consent for participation in the study and publication of dental treatment plan. The cervical enamel was remained after stainless steel information was obtained from each participant or a legal guardian. crown was removed and used for further analyses, compared with three Medical examination and laboratory investigation were performed. tooth type-matched molars obtained from age-matched healthy individuals. Genetic variant analysis Micro-computerized tomography (micro-CT) Genomic DNA was extracted from peripheral blood leukocytes of patients Teeth were scanned with Specimen Micro-CT35 (SCANCO Medical AG). using Puregene Blood Kit (Qiagen, Hilden, Germany) and sent for whole Three spots (30 layers/ each spot) of enamel and dentin in the same exome sequencing using illumina Hiseq 2000 Sequencer (Macrogen, Seoul, location between the patient’s tooth and controls were selected to Korea). The sequences were aligned using University of California Santa Cruz quantify mineral density. The images were processed using Image (UCSC) hg19 and Burrows–Wheeler Aligner (http://bio-bwa.sourceforge.net/). Processing Language (Scanco Medical AG). Downstream process was performed by SAMtools (samtools.sourceforge.net/) and annotated against dbSNP and 1000 Genomes. The variants were Surface roughness, microhardness, ultrastructure, and mineral filtered out using the following criteria: (1) coverage < 10x; (2) minor allele composition frequency >1% in the 1000 Genomes Project, and Genome Aggregation The same locations of the patient’s tooth and controls were selected. Surface Database (gnomAD: gnomad.broadinstitute.org); (3) present in the Thai roughness was measured for thirty spots every 600 µm using the Talyscan reference exome (T-REx) variant database [16], (4) synonymous exonic 150 and TalyMap Universal program (Taylor Hobson Ltd). Five spots of variants; and (5) not located in the coding region and splice site of genes enamel and dentin were tested for microhardness using a microhardness related to AI (HP:0000705) (Table S1) [17]. The filtered variants were tester (FM-700e Type D, FUTURE-TECH, Kanagawa, Japan). For ultrastructural called novel if they were not detected by searching in Google scholar analysis, tooth sections were etched with 37% phosphoric acids, rinsed, dried (https://scholar.google.com/), Varsome [18] and Mastermind Genomic Search using a critical point dryer (Emitech K850), covered with gold powder (Jeol Engine (https://www.genomenon.com/mastermind). JFC-1200), and examined using the scanning electron microscope (SEM, The identified ITGB6 variants were confirmed by Sanger sequencing Quanta FEG-250). The amount of the patient’s remaining enamel was not using the primers (NM_000888.3) c.625 G > T: F′TAGGAGAATGTTGCTAAG sufficient for Energy Dispersive X-ray (EDX) measurement. In dentin, the CT and R′AGAGTGAAACAGCACTCCCT and c.1661-3 C > G: F’TGGAGAAAAG amount (%) of carbon (C), oxygen (O), phosphorus (P), and calcium (Ca) was CAGAGACATTACC and R′CTCATACTGCACCCCTCACAC. measured for five locations using EDX (ISIS 300). In silico analysis and pathogenicity prediction Statistical analysis Mutation taster, Human splicing finder (http://www.umd.be/HSF3/ Statistical analyses for tooth characterization and protein expression were credits.html), Combined Annotation Dependent Depletion (CADD) performed using Mann–Whitney U test (P < 0.05) in a GraphPad Prism8 v.16 software (https://cadd.gs.washington.edu/), and varSeak (www. Software Inc. varseak.bio) were used to predict the effects of the ITGB6 variants [19–21]. The pathogenicity of variants was classified according to the American College of Medical Genetics and Genomics (ACMG) standard RESULTS guidelines [22]. Patient phenotype A proband, 6-year-old girl, presented with discolored teeth and Cell isolation and culture anterior open bite. The patient had a history of frequent common Fibroblasts were isolated from gingival tissues of the proband and three cold since 3 months old and was admitted to the hospital due to sex-matched Thai healthy individuals of the same age range who did not pneumonia at the age of 13 years. Oral examination at age 8 years of have any systemic diseases and orodental abnormalities (controls). Briefly, age showed that the enamel on the erupting upper lateral incisors the gingival tissues were cut into 1 × 1 mm pieces and placed in a 35-mm BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 1 Clinical and radiographic feature, family pedigree, and chromatograms of the patient and family. A–C Orodental features of the proband at 8-year-old showed yellow teeth with rough and pitted enamel. The deciduous molars were restored with stainless steel crowns. D–F Orodental features of the proband at 15-year-old showed dark yellow teeth with rough enamel that was severely eroded. A band of enamel was observed along the cervical margin and cusp tips of the teeth. G, H Panoramic radiographs showed a reduction in thickness and radiodensity of enamel. Taurodontism was observed in the permanent maxillary second molars. The permanent first molars showed bone loss. I, J The parents did not have enamel defects. K Family pedigree showed that the proband (arrow) was the only person in the family with enamel malformation. and lower canines were rough, pitted, and yellowish. The permanent inherited from mother and the splicing variant c.1661-3 C > G incisors were restored using composite resin. The deciduous molars (SCV002058097) in the intron 11 was inherited from father and permanent lower left first molar were restored with stainless (Fig. 2A). No other variants in genes related to AI (HP:0000705) steel crowns. The permanent upper and lower right first molars were passed the filtering criteria. These indicate AI type IH (MIM covered with glass ionomer cement (GC Fuji VII) (Fig. 1A–C). At 15 #616221). Both identified variants have not been associated with years of age, the patient presented with severely eroded teeth with any clinical phenotype of defective enamel. In silico analysis using the remaining enamel along the cervical margin and cusp tips Mutation taster and Human Splicing Finder predicted that the of teeth. The incisors and upper premolars were restored ENST00000409872: c.1661-3 C > G resulted in the broken acceptor with composite resin and the first molars with stainless steel splice site of the intron 11 of ITGB6 that might affect splicing crowns. Generalized gingival inflammation was clinically observed (Table S2). The varSEAK predicted that the c.1661-3 C > G variant (Fig. 1D–F). Panoramic radiographs showed a reduction in thickness could affect mRNA splicing and cause exon skipping. The CADD and radiopacity of enamel. Hypertaurodontism was observed in the scores of the c.625 G > T and c.1661-3 C > G variants were 39 and permanent maxillary second molars. Alveolar bone loss associated 24.4, and, according to ACMG guideline, classified as pathogenic with the first molars was evidence. Dental development was within and likely pathogenic, respectively. Western blot results showed normal limit (Fig. 1G, H). The parents were healthy and not that the level of ITGB6 protein in the patient’s gingival cells was consanguineous. The father had a history of tooth extraction. Other significantly lower than that in controls (Fig. 2B, C). family members did not have AI (Fig. 1I–K). Properties and ultrastructure of the patient tooth Genetic variants and protein expression Micro-CT analysis showed that mineral density of the patient’s Trio-exome sequencing identified that the proband harbored the enamel was significantly reduced while its dentin mineral density compound heterozygous variants in ITGB6: the nonsense variant was comparable to those of controls (Fig. 3A). The surface roughness c.625 G > T, p.(Gly209*) (ClinVar: SCV002058096) in the exon 5 was of the patient’s tooth was significantly increased, compared with BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 2 Genetic analysis and protein expression. A Chromatogram demonstrated the compound heterozygous variants in the proband. The c.625 G > T, p.(Gly209*) was inherited from the mother and the c.1661-3 C > G from the father. B, C The ITGB6 level in the patient’s gingival cells was significantly lower than that in controls. those of controls (Fig. 3B). The microhardness of the patient’s enamel To the best of our knowledge, six affected families and eight and dentin was significantly reduced, compared with those of ITGB6 mutations (including a family in this study) have been controls (Fig. 3C). The patient’s dentin showed a significant decrease identified [9, 13, 15, 23]. Table 1 and Fig. 5 summarized the in carbon, but significantly increases in oxygen, phosphate, and genotype and phenotype of all ITGB6 affected individuals. Our calcium levels, compared with those in controls (Fig. 3D). patient was the only one who had taurodontism. All ITGB6 SEM demonstrated that the patient’s enamel rods were severely affected individuals shared common features of hypoplastic disorganized, coarse, and collapsed while controls showed typically and/or hypomineralized AI. The teeth were rough, pitted, and organized enamel rods (Fig. 4A, B). The patient’s tooth displayed a yellowish. These except the patients reported by Ansar et al., gap in the dentinoenamel junction (DEJ) while the control exhibited 2016 who showed only rough enamel surface, not a typical AI a smooth and continuous connection between enamel and dentin feature [23]. A Hutchinson/screwdriver tooth morphology was (Fig. 4C, D). Regarding the ultrastructure, the patient’s dentin was observed in a patient who was homozygous for the c.1846C > T, comparable to the controls (Fig. 4E, F). p.(Arg616*) and hemizygous for the c.1697T > C p.(Met566Thr) in Nance-Horan syndrome (NHS)[13]. Four families were found to be homozygous, and two families DISCUSSION were compound heterozygous for the variants. Eight identified Using trio-exome sequencing, the patient was found to be a ITGB6 mutations consist of 5 missense, 2 nonsense, and 1 splicing. compound heterozygote for the nonsense c.625 G > T p.(Gly209*) Of the six previously reported variants: three were in the exon 4, and the splicing c.1661-3 C > G in the ITGB6 gene while the parents two in the exon 6, and one in the exon 11. The p.(Gly209*) were heterozygous for each variant, indicating autosomal recessive detected in our patient was the first variant identified in the exon AI. The identified genetic mutations together with the feature of 5 and the c.1661-3 C > G was the first splicing variant near the hypoplastic and hypomineralized AI designates AI type IH. exon 11 of ITGB6. BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 3 Tooth characteristics. A The patient’s tooth showed a significant reduction in mineral density compared with controls. B Surface roughness values of the patient’s tooth was significantly higher than those of controls. C Microhardness values of the patient’s enamel and dentin were significantly reduced compared with those of controls. D The patient’s dentin demonstrated a significantly decrease in carbon content, but significant increases in oxygen, phosphorus, and calcium levels, compared with those in controls. Fig. 4 Ultrastructure of ITGB6 tooth. A, B SEM showed disorganized, flatted, and collapsed enamel rods in the patient’s tooth while an organized arrangement of enamel rods was observed in control. C, D A gap in the dentinoenamel junction of ITGB6 tooth was observed while control showed a smooth connection between enamel and dentin. E, F The dentinal ITGB6 was similar to that of the control teeth. BDJ Open (2023) 9:15 K. Sriwattanapong et al. BDJ Open (2023) 9:15 Table 1. The patients identified with enamel defects and ITGB6 variants. Patient Age (Y) Nation/ Consanguinity Inheritance Nucleotide change Amino Exon Clinical features Radiographic features Ethnic acid change This study Proband 8 Thai No Cpd Het c.625 G > T p.(Gly209*)- 5 - Pitted, rough, chalky and -Reduced radiopacity and c.1661-3 C > G Intron11 yellowish enamel thickness of enamel -Hypomineralized and -Taurodontism: maxillary hypoplastic enamel upper second molars -Generalized gingival inflammation -Anterior open bite Wang et al., 2014 Proband 8 Hispanic No Cpd Het c.427 G > A p.(Ala143Thr) 4 -Very thin/hypoplastic enamel, Thin and normal c.825 T > A p.(His275Gln) rough surface contrasting layer of enamel (Family1) 6 -Anterior open bite, Class III (unerupted teeth) malocclusion, attrition Proband 8 Hispanic No Homo c.1846C > T p.(Arg616*) 11 -Yellow-brown, rough, -Thin and contrasting layer hypoplastic enamel of enamel (Family2) -Thin enamel collar at the (Unerupted teeth) cervical margin -Missing mandibular left -Attrition second molar -Hutchinson/screwdriver maxillary incisors -A hemizygous c.1697T > C p.(Met566Thr) in the NHS Actin Remodeling Regulator or Nance-Horan Syndrome (NHS). Poulter et al., 2013 V:3 7 Pakistan Yes Homo c.586 C > A p.(Pro196Thr) 4 -Pitted, rough, hypomineralized Near-normal volume and enamel with pigmentation contrasting enamel -Remaining enamel on the (unerupted teeth) molar cusp tips and cervical margin -Reduced mineral density and voids in enamel -Disturbed orientation of enamel prisms Seymen et al., 2015 Proband 8 Turkish Yes Homo c.517 G > C p.(Gly173Arg) 4 -Thin, pitted, pigmented, Reduced thickness and hypoplastic and radiopacity of enamel hypomineralized enamel. -Thicker enamel in the cervical area of molars Ansar et al., 2016 IV-2 33 Pakistan Yes Homo c.898 G > A p.(Glu300Lys) 6 -Adolescent alopecia, Not available intellectual disability IV-3 39 -Rough enamel, yellowish- IV-6 40 brown staining, malocclusion, gingival recession, tooth loss -AI feature was not definite. Cpd Het compound heterozygous, Homo homozygous, Hemi hemizygous, Y Year. K. Sriwattanapong et al. Fig. 5 Schematic diagram of the ITGB6 gene and protein. In both schemes, all ITGB6 variants identified to date, which associated with enamel malformation, are shown. The variants detected in this study are in bold texts. The ITGB6 protein contains the extracellular, transmembrane, and been no reports of ITGB6 patients with pulmonary inflammation. cytoplasmic regions. The extracellular region consists of the VWA However, it is unclear whether a detailed medical examination of domain, integrin beta epidermal growth factor-like (I-EGF) domain 1, the reported patients was performed. AI may present with other and epidermal growth factor-like (EGF-like) domain. The nonsense medical problems such as skeletal anomalies, heart defects, p.(Gly209*) identified in our patient is located in the Von Willebrand nephrocalcinosis, and cone rod dystrophy [28–30]. Ansar et al., factor A (VWA) domain that is important for interacting with the α 2016 reported patients with ITGB6 mutation who presented rough subunit of ITGB. It is expected to be degraded by nonsense- enamel, periodontal disease, intellectual disability, early-onset skin mediated mRNA decay (NMD). Six out of eight identified variants are aging, and adolescent alopecia. Our patient also had periodontal detected in the VWA domain, confirmingthat thisdomainisvital for inflammation, frequent common cold, and a history of pneumo- the ITGB6 function. The splicing variant c.1661-3 C > G in the intron nia. These might suggest the possibility that ITGB6 mutations 11 is predicted to affect acceptor splice site and mRNA splicing, and might be the causes of other phenotypes of human disease not might cause exon skipping. The expression of ITGB6 in the patient’s limited to AI. gingival cells were significantly reduced, compared with that in Anterior open bite was observed in our patient and in the one controls, suggesting that the mutations were loss-of-function. This with ITGB6 variants reported by Wang et al., 2014 [13], indicating −/− finding is consistent with the Itgb6 mice that were negative for that open bite can be found in patients with ITGB6 mutations. the integrin beta 6 expression [14]. Clinical studies have shown that open bite is more commonly found −/− The Itgb6 mice showed chalky teeth with reduced mineraliza- in patients with AI than in the general population [31], particularly tion and severe attrition, mimicking hypomineralized AI [14]. those who carry mutations in the ENAM or AMELX gene [32–34]. The −/− Moreover, the Itgb6 mice developed periodontal disease exhibit- relationship between AI and open bite remains unknown; however, ing epithelial inflammation, pocket formation, and alveolar bone loss it has been suggested that the etiology of open bite in individuals [12]. The integrin αvβ6 is expressed in ameloblasts and junctional with AI may be due to a genetic factor that determines craniofacial epithelium and plays a crucial role in regulating amelogenin development and alveolar growth [31, 33]. Taurodontism is deposition in enamel mineralization as well as protecting periodontal classified into hypotaurodontism, mesotaurodontism, and hyper- tissue against inflammation through activation of TGF-β1[12, 14]. taurodontism [35]. It can occur as an isolated trait or as a part of The EGF-like domain of ITGB6 contains the RGD integrin-binding genetic syndromes such as amelogenesis imperfecta (AMELX or motif that interacts with extracellular matrix proteins and TGF-β1. DLX3), Down syndrome (Trisomy 21), ectodermal dysplasia (EDA) The TGF-β1 has been shown to involve not only in enamel and osteogenesis imperfecta (COL1A1 and COL1A2)[36–38]. mineralization but also periodontal inflammation [24, 25]. The Hypertaurodontism, which was observed in our patient, expands periodontal inflammation observed in our patient might be a the phenotypic spectrum of ITGB6-related disorder. consequence of altered ITGB6 functions or local factors such as Detailed analyses of the patient’s tooth showed a significant stainless steel crowns and poor oral hygiene, or both genetic and increase in surface roughness and significant decreases in enamel local involvements. Identification of more ITGB6 patients and further mineral density and microhardness of enamel and dentin. The functional studies should be performed to validate the relationship enamel findings are consistent with the clinical feature of between ITGB6 and periodontal disease. hypoplastic-hypomineralized AI and the hypomineralized enamel −/− Additionally, ITGB6 is highly expressed in the lungs and the in Itgb6 mice [14]. Unlike the study by Poulter et al., 2014 that Itgb6 knockout mice exhibited significant airway inflammation, showed the arrays of enamel prisms in the ITGB6 tooth sample, the age-related emphysema, and juvenile baldness, that are related to enamel rods of our patient’s tooth were severely collapsed. This is TGF-β1 deficiency [26, 27]. Unlike knockout mice, there have not explained by the expression of ITGB6 in the ameloblasts at the onset BDJ Open (2023) 9:15 K. Sriwattanapong et al. of enamel formation and similar to the Itgb6 null mice that failed to 18. Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35: form enamel rods [13]. In the patient’s dentin, the carbon level was 1978–80. significantly reduced while the oxygen, phosphate, and calcium 19. Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general levels were significantly increased, compared with those in controls. framework for estimating the relative pathogenicity of human genetic variants. Moreover, the patient’s tooth exhibited a gap along the DEJ, Nat Genet. 2014;46:310–5. suggesting the discontinuation between defective enamel and 20. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the dentin. During tooth development, ITGB6 is involved not only in deleteriousness of variants throughout the human genome. Nucleic Acids Res. enamel formation, but also in cell adhesion and cell-matrix 2019;47:D886–d94. interactions that are important in epithelial-mesenchymal interac- 21. Steinhaus R, Proft S, Schuelke M, Cooper DN, Schwarz Jana M, Seelow D. Muta- tions [13]. It is possible that the ITGB6 alterations might have a slight tionTaster2021. Nucleic Acids Res. 2021;49:W446–51. effect on dentin or the abnormal enamel formation consequently 22. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint affects dentin. The limitation of this study is a limited number of consensus recommendation of the American College of Medical Genetics patient and tooth sample. More diseased tooth samples would and Genomics and the Association for Molecular Pathology. Genet Med. clarify precise characteristics of the ITGB6 teeth 2015;17:405–24. 23. AnsarM,Jan A, Santos-CortezRLP,WangX,Suliman M, Acharya A, etal. Expansion of the spectrum of ITGB6-related disorders to adolescent alopecia, CONCLUSION dentogingival abnormalities and intellectual disability. Eur J Hum Genet. The study reports two novel ITGB6 variants in a patient with AI and 2016;24:1223–7. open bite, and, for the first time, hypertaurodontism and defective 24. Hidai C. EGF-like domains with a C-x-D-x(4)-Y-x-C motif. 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Shotelersuk V, Wichadakul D, Ngamphiw C, Srichomthong C, Phokaew C, Wilantho A, et al. The Thai reference exome (T-REx) variant database. Clin Genet. AUTHOR CONTRIBUTIONS 2021;100:703–12. KS: investigation, formal analysis, writing – original draft. TT, LB, AS, VS: validation, formal 17. Köhler S, Gargano M, Matentzoglu N, Carmody LC, Lewis-Smith D, analysis. TP: conceptualization, formal analysis, writing – original draft. All authors Vasilevsky NA, et al. The human phenotype ontology in 2021. Nucleic Acids critically revised manuscript, gave final approval, and agreed to be accountable for all Res. 2021;49:D1207–d17. aspects of the work. BDJ Open (2023) 9:15 K. Sriwattanapong et al. FUNDING Correspondence and requests for materials should be addressed to Thantrira This research is supported by the Health Systems Research Institute (66-101, 66-122), Porntaveetus. National Research Council of Thailand (NRCT) (N42A650229), Faculty of Dentistry (DRF 66_08), and Thailand Science Research and Innovation Fund Chulalongkorn Reprints and permission information is available at http://www.nature.com/reprints University (HEA663200060). KS is supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. Publisher’snote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. COMPETING INTERESTS The authors declare no competing interests. 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Novel ITGB6 variants cause hypoplastic-hypomineralized amelogenesis imperfecta and taurodontism: characterization of tooth phenotype and review of literature

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www.nature.com/bdjopen ARTICLE OPEN Novel ITGB6 variants cause hypoplastic-hypomineralized amelogenesis imperfecta and taurodontism: characterization of tooth phenotype and review of literature 1 1 2 3 1✉ Kanokwan Sriwattanapong , Thanakorn Theerapanon , Lawan Boonprakong , Anucharte Srijunbarl , Thantrira Porntaveetus and 4,5 Vorasuk Shotelersuk © The Author(s) 2023 OBJECTIVES: To characterize phenotype and genotype of amelogenesis imperfecta (AI) in a Thai patient, and review of literature. MATERIALS AND METHODS: Variants were identified using trio-exome and Sanger sequencing. The ITGB6 protein level in patient’s gingival cells was measured. The patient’s deciduous first molar was investigated for surface roughness, mineral density, microhardness, mineral composition, and ultrastructure. RESULTS: The patient exhibited hypoplastic-hypomineralized AI, taurodontism, and periodontal inflammation. Exome sequencing identified the novel compound heterozygous ITGB6 mutation, a nonsense c.625 G > T, p.(Gly209*) inherited from mother and a splicing c.1661-3 C > G from father, indicating AI type IH. The ITGB6 level in patient cells was significantly reduced, compared with controls. Analyses of a patient’s tooth showed a significant increase in roughness while mineral density of enamel and microhardness of enamel and dentin were significantly reduced. In dentin, carbon was significantly decreased while calcium, phosphorus, and oxygen levels were significantly increased. Severely collapsed enamel rods and a gap in dentinoenamel junction were observed. Of six affected families and eight ITGB6 variants that have been reported, our patient was the only one with taurodontism. CONCLUSION: We report the hypoplasia/hypomineralization/taurodontism AI patient with disturbed tooth characteristics associated with the novel ITGB6 variants and reduced ITGB6 expression, expanding genotype, phenotype, and understanding of autosomal recessive AI. BDJ Open (2023) 9:15 ; https://doi.org/10.1038/s41405-023-00142-y INTRODUCTION taurodontism, and subdivided into fifteen subtypes (IA-IG, IIA-IID, IIIA- Dental enamel is an epithelial-derived tissue comprising of IIIB, and IVA-IVB) by phenotype and inheritance pattern [5]. The Online organized hydroxyapatite crystals configured into enamel rods [1]. Mendelian Inheritance in Man (OMIM) has associated specific genes Amelogenesis in mouse has been described in four defined stages: with phenotypes and expanded the AI designations into: AI type IA presecretory, secretory, transition, to maturation stages [2]. In the (LAMB3), IB and IC (ENAM), IE (AMELX), IF (AMBN), IG (FAM20A), IH presecretory stage, the ameloblasts acquire their characteristics and (ITGB6), IJ (ACP4), IK (SP6), IIA1 (KLK4), IIA2 (MMP20), IIA3 (WDR72), IIA4 begin secreting enamel matrix proteins. In the secretory stage, the (ODAPH), IIA5 (SLC24A4), IIA6 (GPR68), III1 (FAM83H), IIIB (AMTN), IIIC entire thickness of the enamel layer is established. In the transition (RELT), and IV (DLX3)[6]. and maturation stages, the ameloblasts restructure and transport The integrin beta-6 (ITGB6) gene (OMIM *147558) comprises mineral ions into the enamel fluid, promoting the inorganic content fifteen exons and encodes the β6 subunit of the integrin αvβ6 that and growth of enamel prisms [3]. An entire process of amelogenesis plays an important role in cell-cell and cell-matrix adhesion, is under genetic control. Alterations in genes responsible for cellular proliferation and migration, tissue repair, apoptosis, amelogenesis at different timings result in enamel malformations inflammation, and angiogenesis [7, 8]. It also activates extracellular with diverse phenotypes [4]. matrix protein and transcription factors such as fibronectin, Amelogenesis imperfecta (AI) is a heterogeneous group of genetic tenascin-C, vitronectin, latency-associated peptide of transforming conditions characterized by defects in enamel formation and affecting growth factor- β1 (TGF- β1), and TGF- β3[9, 10]. The integrin αvβ6 both deciduous and permanent teeth. The Witkop’sclassification is expressed in the ameloblasts and regulates enamel biominer- categorized AI into four main types: (1) hypoplastic, (2) hypomatura- alization, amelogenin deposition, and TGF-β1 that controls cell tion, (3) hypocalcification, and (4) hypomaturation-hypoplastic with growth and differentiation during early tooth development [11, 12]. 1 2 Center of Excellence in Genomics and Precision Dentistry, Department of Physiology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Office of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Dental Materials R&D Center, Faculty of Dentistry, Chulalongkorn University, Bangkok 4 5 10330, Thailand. Center of Excellence for Medical Genomics, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Excellence Center for Genomics and Precision Medicine, King Chulalongkorn Memorial Hospital, the Thai Red Cross Society, Bangkok 10330, Thailand. email: thantrira.p@chula.ac.th Received: 11 January 2023 Revised: 11 March 2023 Accepted: 23 March 2023 1234567890();,: K. Sriwattanapong et al. The expression of Itgb6 is detected in the differentiating culture dish (Corning, New York, USA). Cells were maintained in growth medium composed of Dulbecco’s Modified Eagle Medium (DMEM), 10% ameloblasts, secretory ameloblasts, and maturation ameloblasts, −/− fetal bovine serum (Gibco, CA, USA), 1% L-glutamine (Gibco), and 1% indicating its involvement in amelogenesis [13]. In the Itgb6 penicillin and streptomycin (Gibco), and incubated in a humidified mice, an accumulation of amelogenin in enamel extracellular matrix environment at 37 °C and 5% CO . Cells from passages 5 were used in was disturbed, resulting in a reduction in enamel mineralization the experiments. [14], that mimics the human AI phenotype. The biallelic mutations in the ITGB6 gene lead to hypoplastic and/or hypomineralized AI (AI Western blot analysis type IH) (MIM #616221) [9, 13, 15]. Confluent monolayer cells were harvested, washed with ice-cold Currently, the knowledge of genotypes and phenotypes related phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation to the ITGB6 are still elusive. There have been only five families TM buffer (RIPA) (Thermo Fisher Scientific, MA, USA) containing the Halt and six different ITGB6 mutations reported. Only one study protease inhibitor cocktail (Thermo Fisher Scientific, MA, USA). Protein demonstrated the scanning electron microscopic images of one TM concentration was determined using Pierce BCA Protein Assay Kit affected tooth [9]. Identifying more patients and new ITGB6 (Thermo Fisher Scientific, MA, USA). A total protein (30 µg) was separated variants helps understand the biological role of ITGB6 in humans by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and and establish phenotype-genotype correlation benefiting disease transferred to a polyvinylidene difluoride membranes (PVDF) (Bio-Rad, CA, USA). The PVDF membranes were blocked with 5% BSA (Merck Millipore, management. Germany) in TBST buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.1% In this study, we identified a Thai family with the proband having Tween-20) for 1 h at room temperature, and probed with a primary AI, taurodontism, and periodontal inflammation. Trio-exome and antibody overnight at 4 °C. Mouse anti-human Integrin β6 monoclonal Sanger sequencing was performed to detect pathogenic variants. antibody (1:1000 dilution; Merck, Darmstadt, Germany, Cat No. MAB2076Z) The patient’s tooth was investigated for its physical and mechanical and mouse anti-human GAPDH (1:3000 dilution; Abcam, Cambridge, UK, properties and ultrastructure. The patient’s gingival fibroblasts were Cat No. ab8245) were used as the primary antibodies. The membranes investigated for the ITGB6 protein level. A review of literature of all were incubated with the HRP-conjugated secondary antibody; anti-mouse, reported ITGB6 cases and mutations were performed. (1:2500 dilution; R&D systems, MN, USA, Cat. No. HAF018) for 2 h at room TM temperature. They were treated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, MA, USA) and analyzed TM using the Amersham Imager 680 (GE Healthcare, Illinois, USA). The MATERIALS AND METHODS intensity of the bands was quantified using ImageJ software. Subjects This study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB 813/63) and in accordance with Tooth samples the 1964 Helsinki declaration and its later amendments. A written A deciduous maxillary first molar of the proband was extracted according to informed consent for participation in the study and publication of dental treatment plan. The cervical enamel was remained after stainless steel information was obtained from each participant or a legal guardian. crown was removed and used for further analyses, compared with three Medical examination and laboratory investigation were performed. tooth type-matched molars obtained from age-matched healthy individuals. Genetic variant analysis Micro-computerized tomography (micro-CT) Genomic DNA was extracted from peripheral blood leukocytes of patients Teeth were scanned with Specimen Micro-CT35 (SCANCO Medical AG). using Puregene Blood Kit (Qiagen, Hilden, Germany) and sent for whole Three spots (30 layers/ each spot) of enamel and dentin in the same exome sequencing using illumina Hiseq 2000 Sequencer (Macrogen, Seoul, location between the patient’s tooth and controls were selected to Korea). The sequences were aligned using University of California Santa Cruz quantify mineral density. The images were processed using Image (UCSC) hg19 and Burrows–Wheeler Aligner (http://bio-bwa.sourceforge.net/). Processing Language (Scanco Medical AG). Downstream process was performed by SAMtools (samtools.sourceforge.net/) and annotated against dbSNP and 1000 Genomes. The variants were Surface roughness, microhardness, ultrastructure, and mineral filtered out using the following criteria: (1) coverage < 10x; (2) minor allele composition frequency >1% in the 1000 Genomes Project, and Genome Aggregation The same locations of the patient’s tooth and controls were selected. Surface Database (gnomAD: gnomad.broadinstitute.org); (3) present in the Thai roughness was measured for thirty spots every 600 µm using the Talyscan reference exome (T-REx) variant database [16], (4) synonymous exonic 150 and TalyMap Universal program (Taylor Hobson Ltd). Five spots of variants; and (5) not located in the coding region and splice site of genes enamel and dentin were tested for microhardness using a microhardness related to AI (HP:0000705) (Table S1) [17]. The filtered variants were tester (FM-700e Type D, FUTURE-TECH, Kanagawa, Japan). For ultrastructural called novel if they were not detected by searching in Google scholar analysis, tooth sections were etched with 37% phosphoric acids, rinsed, dried (https://scholar.google.com/), Varsome [18] and Mastermind Genomic Search using a critical point dryer (Emitech K850), covered with gold powder (Jeol Engine (https://www.genomenon.com/mastermind). JFC-1200), and examined using the scanning electron microscope (SEM, The identified ITGB6 variants were confirmed by Sanger sequencing Quanta FEG-250). The amount of the patient’s remaining enamel was not using the primers (NM_000888.3) c.625 G > T: F′TAGGAGAATGTTGCTAAG sufficient for Energy Dispersive X-ray (EDX) measurement. In dentin, the CT and R′AGAGTGAAACAGCACTCCCT and c.1661-3 C > G: F’TGGAGAAAAG amount (%) of carbon (C), oxygen (O), phosphorus (P), and calcium (Ca) was CAGAGACATTACC and R′CTCATACTGCACCCCTCACAC. measured for five locations using EDX (ISIS 300). In silico analysis and pathogenicity prediction Statistical analysis Mutation taster, Human splicing finder (http://www.umd.be/HSF3/ Statistical analyses for tooth characterization and protein expression were credits.html), Combined Annotation Dependent Depletion (CADD) performed using Mann–Whitney U test (P < 0.05) in a GraphPad Prism8 v.16 software (https://cadd.gs.washington.edu/), and varSeak (www. Software Inc. varseak.bio) were used to predict the effects of the ITGB6 variants [19–21]. The pathogenicity of variants was classified according to the American College of Medical Genetics and Genomics (ACMG) standard RESULTS guidelines [22]. Patient phenotype A proband, 6-year-old girl, presented with discolored teeth and Cell isolation and culture anterior open bite. The patient had a history of frequent common Fibroblasts were isolated from gingival tissues of the proband and three cold since 3 months old and was admitted to the hospital due to sex-matched Thai healthy individuals of the same age range who did not pneumonia at the age of 13 years. Oral examination at age 8 years of have any systemic diseases and orodental abnormalities (controls). Briefly, age showed that the enamel on the erupting upper lateral incisors the gingival tissues were cut into 1 × 1 mm pieces and placed in a 35-mm BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 1 Clinical and radiographic feature, family pedigree, and chromatograms of the patient and family. A–C Orodental features of the proband at 8-year-old showed yellow teeth with rough and pitted enamel. The deciduous molars were restored with stainless steel crowns. D–F Orodental features of the proband at 15-year-old showed dark yellow teeth with rough enamel that was severely eroded. A band of enamel was observed along the cervical margin and cusp tips of the teeth. G, H Panoramic radiographs showed a reduction in thickness and radiodensity of enamel. Taurodontism was observed in the permanent maxillary second molars. The permanent first molars showed bone loss. I, J The parents did not have enamel defects. K Family pedigree showed that the proband (arrow) was the only person in the family with enamel malformation. and lower canines were rough, pitted, and yellowish. The permanent inherited from mother and the splicing variant c.1661-3 C > G incisors were restored using composite resin. The deciduous molars (SCV002058097) in the intron 11 was inherited from father and permanent lower left first molar were restored with stainless (Fig. 2A). No other variants in genes related to AI (HP:0000705) steel crowns. The permanent upper and lower right first molars were passed the filtering criteria. These indicate AI type IH (MIM covered with glass ionomer cement (GC Fuji VII) (Fig. 1A–C). At 15 #616221). Both identified variants have not been associated with years of age, the patient presented with severely eroded teeth with any clinical phenotype of defective enamel. In silico analysis using the remaining enamel along the cervical margin and cusp tips Mutation taster and Human Splicing Finder predicted that the of teeth. The incisors and upper premolars were restored ENST00000409872: c.1661-3 C > G resulted in the broken acceptor with composite resin and the first molars with stainless steel splice site of the intron 11 of ITGB6 that might affect splicing crowns. Generalized gingival inflammation was clinically observed (Table S2). The varSEAK predicted that the c.1661-3 C > G variant (Fig. 1D–F). Panoramic radiographs showed a reduction in thickness could affect mRNA splicing and cause exon skipping. The CADD and radiopacity of enamel. Hypertaurodontism was observed in the scores of the c.625 G > T and c.1661-3 C > G variants were 39 and permanent maxillary second molars. Alveolar bone loss associated 24.4, and, according to ACMG guideline, classified as pathogenic with the first molars was evidence. Dental development was within and likely pathogenic, respectively. Western blot results showed normal limit (Fig. 1G, H). The parents were healthy and not that the level of ITGB6 protein in the patient’s gingival cells was consanguineous. The father had a history of tooth extraction. Other significantly lower than that in controls (Fig. 2B, C). family members did not have AI (Fig. 1I–K). Properties and ultrastructure of the patient tooth Genetic variants and protein expression Micro-CT analysis showed that mineral density of the patient’s Trio-exome sequencing identified that the proband harbored the enamel was significantly reduced while its dentin mineral density compound heterozygous variants in ITGB6: the nonsense variant was comparable to those of controls (Fig. 3A). The surface roughness c.625 G > T, p.(Gly209*) (ClinVar: SCV002058096) in the exon 5 was of the patient’s tooth was significantly increased, compared with BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 2 Genetic analysis and protein expression. A Chromatogram demonstrated the compound heterozygous variants in the proband. The c.625 G > T, p.(Gly209*) was inherited from the mother and the c.1661-3 C > G from the father. B, C The ITGB6 level in the patient’s gingival cells was significantly lower than that in controls. those of controls (Fig. 3B). The microhardness of the patient’s enamel To the best of our knowledge, six affected families and eight and dentin was significantly reduced, compared with those of ITGB6 mutations (including a family in this study) have been controls (Fig. 3C). The patient’s dentin showed a significant decrease identified [9, 13, 15, 23]. Table 1 and Fig. 5 summarized the in carbon, but significantly increases in oxygen, phosphate, and genotype and phenotype of all ITGB6 affected individuals. Our calcium levels, compared with those in controls (Fig. 3D). patient was the only one who had taurodontism. All ITGB6 SEM demonstrated that the patient’s enamel rods were severely affected individuals shared common features of hypoplastic disorganized, coarse, and collapsed while controls showed typically and/or hypomineralized AI. The teeth were rough, pitted, and organized enamel rods (Fig. 4A, B). The patient’s tooth displayed a yellowish. These except the patients reported by Ansar et al., gap in the dentinoenamel junction (DEJ) while the control exhibited 2016 who showed only rough enamel surface, not a typical AI a smooth and continuous connection between enamel and dentin feature [23]. A Hutchinson/screwdriver tooth morphology was (Fig. 4C, D). Regarding the ultrastructure, the patient’s dentin was observed in a patient who was homozygous for the c.1846C > T, comparable to the controls (Fig. 4E, F). p.(Arg616*) and hemizygous for the c.1697T > C p.(Met566Thr) in Nance-Horan syndrome (NHS)[13]. Four families were found to be homozygous, and two families DISCUSSION were compound heterozygous for the variants. Eight identified Using trio-exome sequencing, the patient was found to be a ITGB6 mutations consist of 5 missense, 2 nonsense, and 1 splicing. compound heterozygote for the nonsense c.625 G > T p.(Gly209*) Of the six previously reported variants: three were in the exon 4, and the splicing c.1661-3 C > G in the ITGB6 gene while the parents two in the exon 6, and one in the exon 11. The p.(Gly209*) were heterozygous for each variant, indicating autosomal recessive detected in our patient was the first variant identified in the exon AI. The identified genetic mutations together with the feature of 5 and the c.1661-3 C > G was the first splicing variant near the hypoplastic and hypomineralized AI designates AI type IH. exon 11 of ITGB6. BDJ Open (2023) 9:15 K. Sriwattanapong et al. Fig. 3 Tooth characteristics. A The patient’s tooth showed a significant reduction in mineral density compared with controls. B Surface roughness values of the patient’s tooth was significantly higher than those of controls. C Microhardness values of the patient’s enamel and dentin were significantly reduced compared with those of controls. D The patient’s dentin demonstrated a significantly decrease in carbon content, but significant increases in oxygen, phosphorus, and calcium levels, compared with those in controls. Fig. 4 Ultrastructure of ITGB6 tooth. A, B SEM showed disorganized, flatted, and collapsed enamel rods in the patient’s tooth while an organized arrangement of enamel rods was observed in control. C, D A gap in the dentinoenamel junction of ITGB6 tooth was observed while control showed a smooth connection between enamel and dentin. E, F The dentinal ITGB6 was similar to that of the control teeth. BDJ Open (2023) 9:15 K. Sriwattanapong et al. BDJ Open (2023) 9:15 Table 1. The patients identified with enamel defects and ITGB6 variants. Patient Age (Y) Nation/ Consanguinity Inheritance Nucleotide change Amino Exon Clinical features Radiographic features Ethnic acid change This study Proband 8 Thai No Cpd Het c.625 G > T p.(Gly209*)- 5 - Pitted, rough, chalky and -Reduced radiopacity and c.1661-3 C > G Intron11 yellowish enamel thickness of enamel -Hypomineralized and -Taurodontism: maxillary hypoplastic enamel upper second molars -Generalized gingival inflammation -Anterior open bite Wang et al., 2014 Proband 8 Hispanic No Cpd Het c.427 G > A p.(Ala143Thr) 4 -Very thin/hypoplastic enamel, Thin and normal c.825 T > A p.(His275Gln) rough surface contrasting layer of enamel (Family1) 6 -Anterior open bite, Class III (unerupted teeth) malocclusion, attrition Proband 8 Hispanic No Homo c.1846C > T p.(Arg616*) 11 -Yellow-brown, rough, -Thin and contrasting layer hypoplastic enamel of enamel (Family2) -Thin enamel collar at the (Unerupted teeth) cervical margin -Missing mandibular left -Attrition second molar -Hutchinson/screwdriver maxillary incisors -A hemizygous c.1697T > C p.(Met566Thr) in the NHS Actin Remodeling Regulator or Nance-Horan Syndrome (NHS). Poulter et al., 2013 V:3 7 Pakistan Yes Homo c.586 C > A p.(Pro196Thr) 4 -Pitted, rough, hypomineralized Near-normal volume and enamel with pigmentation contrasting enamel -Remaining enamel on the (unerupted teeth) molar cusp tips and cervical margin -Reduced mineral density and voids in enamel -Disturbed orientation of enamel prisms Seymen et al., 2015 Proband 8 Turkish Yes Homo c.517 G > C p.(Gly173Arg) 4 -Thin, pitted, pigmented, Reduced thickness and hypoplastic and radiopacity of enamel hypomineralized enamel. -Thicker enamel in the cervical area of molars Ansar et al., 2016 IV-2 33 Pakistan Yes Homo c.898 G > A p.(Glu300Lys) 6 -Adolescent alopecia, Not available intellectual disability IV-3 39 -Rough enamel, yellowish- IV-6 40 brown staining, malocclusion, gingival recession, tooth loss -AI feature was not definite. Cpd Het compound heterozygous, Homo homozygous, Hemi hemizygous, Y Year. K. Sriwattanapong et al. Fig. 5 Schematic diagram of the ITGB6 gene and protein. In both schemes, all ITGB6 variants identified to date, which associated with enamel malformation, are shown. The variants detected in this study are in bold texts. The ITGB6 protein contains the extracellular, transmembrane, and been no reports of ITGB6 patients with pulmonary inflammation. cytoplasmic regions. The extracellular region consists of the VWA However, it is unclear whether a detailed medical examination of domain, integrin beta epidermal growth factor-like (I-EGF) domain 1, the reported patients was performed. AI may present with other and epidermal growth factor-like (EGF-like) domain. The nonsense medical problems such as skeletal anomalies, heart defects, p.(Gly209*) identified in our patient is located in the Von Willebrand nephrocalcinosis, and cone rod dystrophy [28–30]. Ansar et al., factor A (VWA) domain that is important for interacting with the α 2016 reported patients with ITGB6 mutation who presented rough subunit of ITGB. It is expected to be degraded by nonsense- enamel, periodontal disease, intellectual disability, early-onset skin mediated mRNA decay (NMD). Six out of eight identified variants are aging, and adolescent alopecia. Our patient also had periodontal detected in the VWA domain, confirmingthat thisdomainisvital for inflammation, frequent common cold, and a history of pneumo- the ITGB6 function. The splicing variant c.1661-3 C > G in the intron nia. These might suggest the possibility that ITGB6 mutations 11 is predicted to affect acceptor splice site and mRNA splicing, and might be the causes of other phenotypes of human disease not might cause exon skipping. The expression of ITGB6 in the patient’s limited to AI. gingival cells were significantly reduced, compared with that in Anterior open bite was observed in our patient and in the one controls, suggesting that the mutations were loss-of-function. This with ITGB6 variants reported by Wang et al., 2014 [13], indicating −/− finding is consistent with the Itgb6 mice that were negative for that open bite can be found in patients with ITGB6 mutations. the integrin beta 6 expression [14]. Clinical studies have shown that open bite is more commonly found −/− The Itgb6 mice showed chalky teeth with reduced mineraliza- in patients with AI than in the general population [31], particularly tion and severe attrition, mimicking hypomineralized AI [14]. those who carry mutations in the ENAM or AMELX gene [32–34]. The −/− Moreover, the Itgb6 mice developed periodontal disease exhibit- relationship between AI and open bite remains unknown; however, ing epithelial inflammation, pocket formation, and alveolar bone loss it has been suggested that the etiology of open bite in individuals [12]. The integrin αvβ6 is expressed in ameloblasts and junctional with AI may be due to a genetic factor that determines craniofacial epithelium and plays a crucial role in regulating amelogenin development and alveolar growth [31, 33]. Taurodontism is deposition in enamel mineralization as well as protecting periodontal classified into hypotaurodontism, mesotaurodontism, and hyper- tissue against inflammation through activation of TGF-β1[12, 14]. taurodontism [35]. It can occur as an isolated trait or as a part of The EGF-like domain of ITGB6 contains the RGD integrin-binding genetic syndromes such as amelogenesis imperfecta (AMELX or motif that interacts with extracellular matrix proteins and TGF-β1. DLX3), Down syndrome (Trisomy 21), ectodermal dysplasia (EDA) The TGF-β1 has been shown to involve not only in enamel and osteogenesis imperfecta (COL1A1 and COL1A2)[36–38]. mineralization but also periodontal inflammation [24, 25]. The Hypertaurodontism, which was observed in our patient, expands periodontal inflammation observed in our patient might be a the phenotypic spectrum of ITGB6-related disorder. consequence of altered ITGB6 functions or local factors such as Detailed analyses of the patient’s tooth showed a significant stainless steel crowns and poor oral hygiene, or both genetic and increase in surface roughness and significant decreases in enamel local involvements. Identification of more ITGB6 patients and further mineral density and microhardness of enamel and dentin. The functional studies should be performed to validate the relationship enamel findings are consistent with the clinical feature of between ITGB6 and periodontal disease. hypoplastic-hypomineralized AI and the hypomineralized enamel −/− Additionally, ITGB6 is highly expressed in the lungs and the in Itgb6 mice [14]. Unlike the study by Poulter et al., 2014 that Itgb6 knockout mice exhibited significant airway inflammation, showed the arrays of enamel prisms in the ITGB6 tooth sample, the age-related emphysema, and juvenile baldness, that are related to enamel rods of our patient’s tooth were severely collapsed. This is TGF-β1 deficiency [26, 27]. Unlike knockout mice, there have not explained by the expression of ITGB6 in the ameloblasts at the onset BDJ Open (2023) 9:15 K. Sriwattanapong et al. of enamel formation and similar to the Itgb6 null mice that failed to 18. Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35: form enamel rods [13]. In the patient’s dentin, the carbon level was 1978–80. significantly reduced while the oxygen, phosphate, and calcium 19. Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general levels were significantly increased, compared with those in controls. framework for estimating the relative pathogenicity of human genetic variants. Moreover, the patient’s tooth exhibited a gap along the DEJ, Nat Genet. 2014;46:310–5. suggesting the discontinuation between defective enamel and 20. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the dentin. During tooth development, ITGB6 is involved not only in deleteriousness of variants throughout the human genome. Nucleic Acids Res. enamel formation, but also in cell adhesion and cell-matrix 2019;47:D886–d94. interactions that are important in epithelial-mesenchymal interac- 21. Steinhaus R, Proft S, Schuelke M, Cooper DN, Schwarz Jana M, Seelow D. Muta- tions [13]. It is possible that the ITGB6 alterations might have a slight tionTaster2021. Nucleic Acids Res. 2021;49:W446–51. effect on dentin or the abnormal enamel formation consequently 22. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint affects dentin. The limitation of this study is a limited number of consensus recommendation of the American College of Medical Genetics patient and tooth sample. More diseased tooth samples would and Genomics and the Association for Molecular Pathology. Genet Med. clarify precise characteristics of the ITGB6 teeth 2015;17:405–24. 23. AnsarM,Jan A, Santos-CortezRLP,WangX,Suliman M, Acharya A, etal. Expansion of the spectrum of ITGB6-related disorders to adolescent alopecia, CONCLUSION dentogingival abnormalities and intellectual disability. Eur J Hum Genet. The study reports two novel ITGB6 variants in a patient with AI and 2016;24:1223–7. open bite, and, for the first time, hypertaurodontism and defective 24. Hidai C. EGF-like domains with a C-x-D-x(4)-Y-x-C motif. Open Access J Trans Med Res. 2018;2:67–71. physical/mechanical properties of teeth in hypoplastic- 25. Morandini AC, Sipert CR, Ramos-Junior ES, Brozoski DT, Santos CF. Periodontal hypomineralized AI. We expand the understanding of tooth ligament and gingival fibroblasts participate in the production of TGF-β, inter- defects and the genotypic and phenotypic spectra of autosomal leukin (IL)-8 and IL-10. Braz Oral Res. 2011;25:157–62. recessive AI. The irregularity, weakness, and defective ultrastruc- 26. Meecham A, Marshall JF. The ITGB6 gene: its role in experimental and clinical ture could cause ITGB6 teeth prone to severe deterioration. We biology. Gene. 2020;763:100023. suggest that detailed phenotyping of affected individuals is 27. Morris DG, Huang X, Kaminski N, Wang Y, Shapiro SD, Dolganov G, et al. Loss of important to delineate the phenotype of AI. integrin alpha(v)beta6-mediated TGF-beta activation causes Mmp12-dependent emphysema. Nature. 2003;422:169–73. 28. 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Shotelersuk V, Wichadakul D, Ngamphiw C, Srichomthong C, Phokaew C, Wilantho A, et al. The Thai reference exome (T-REx) variant database. Clin Genet. AUTHOR CONTRIBUTIONS 2021;100:703–12. KS: investigation, formal analysis, writing – original draft. TT, LB, AS, VS: validation, formal 17. Köhler S, Gargano M, Matentzoglu N, Carmody LC, Lewis-Smith D, analysis. TP: conceptualization, formal analysis, writing – original draft. All authors Vasilevsky NA, et al. The human phenotype ontology in 2021. Nucleic Acids critically revised manuscript, gave final approval, and agreed to be accountable for all Res. 2021;49:D1207–d17. aspects of the work. BDJ Open (2023) 9:15 K. Sriwattanapong et al. FUNDING Correspondence and requests for materials should be addressed to Thantrira This research is supported by the Health Systems Research Institute (66-101, 66-122), Porntaveetus. National Research Council of Thailand (NRCT) (N42A650229), Faculty of Dentistry (DRF 66_08), and Thailand Science Research and Innovation Fund Chulalongkorn Reprints and permission information is available at http://www.nature.com/reprints University (HEA663200060). KS is supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. Publisher’snote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. COMPETING INTERESTS The authors declare no competing interests. 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