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, 10 (3), 26

Whole Exome Sequencing Identifies an AMBN Missense Mutation Causing Severe Autosomal-Dominant Amelogenesis Imperfecta and Dentin Disorders

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Whole Exome Sequencing Identifies an AMBN Missense Mutation Causing Severe Autosomal-Dominant Amelogenesis Imperfecta and Dentin Disorders

Ting Lu et al. Int J Oral Sci.

Abstract

Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular, and cellular interactions. Ameloblastin (AMBN, also named "amelin" or "sheathlin") is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta (AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders. We recruited one kindred with autosomal-dominant amelogenesis imperfecta (ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Family pedigree and pedigree-based linkage study of affected and unaffected individuals. The segregating haplotype is indicated by a box with seven polymorphic microsatellite markers on 4q13.1-4q21.1. AMBN is flanked by D4S2969 and D4S1543. Affected subjects are denoted in black. The proband is indicated by an arrow.
Fig. 2
Fig. 2
Dental phenotype and panoramic radiograph of the patients. a Normal permanent teeth; b panoramic radiograph of normal teeth. Dental phenotype of IV:9 c,eh. An intraoral view of a male patient at the age of 21 shows brown crowns with thin and chipping enamel, especially in the cervical part of all teeth (black arrows), and short crowns of the permanent maxillary central incisors worn out by attrition (black arrow). d,j,l Panoramic radiographs of patients show bulbous crowns covered with thin enamel, cervical constriction (asterisk), short constricted roots, and apical radiolucencies in the second molars (white arrows). i An intraoral view of a patient at the age of 22 shows grey crowns with enamel chipped and absent in places. k,m Above the age of approximately 40, patients had lost almost all of their teeth or had several loose roots. l,n Patients were treated by long-span fixed partial denture repair, l implant prosthodontics, k removable partial denture, or o removable complete denture according to their family economic situation. Finally, o,p all patients lost all their teeth at an early age (generally by their 50s).
Fig. 3
Fig. 3
Tooth ultrastructural analyses. ad High-resolution X-ray CT analysis of exfoliated teeth from control individual IV:8 and affected individuals IV:2 and IV:9. a 3D reconstruction of the tooth CT data: individual IV:8 and individuals IV:2 and IV:9. b 3D reconstruction of pulp chambers. Teeth of IV:2 and IV:9 exhibit thistle-shaped pulp chambers. c Typical CT sections through the teeth are presented using false colour calibrated with respect to mineral density to generate mineral density maps. Scale bar is marked in g/cm3. d Mean enamel and dentin mineral density for each tooth is also shown graphically. The control tooth of IV:8 exhibits enamel and dentin apparently normal in structure and density. Affected teeth of IV:2 and IV:9 exhibit enamel and dentin significantly reduced in mineral density compared with the normal tooth (P < 0.001). *P < 0.05, **P < 0.01, or ***P < 0.001. Videos of 3D-rendered CT data showing surface detail and the internal structure of all teeth are available as Supplementary Material. ej SEM of representative exfoliated teeth. e Tooth of individual IV:8 exhibits normal enamel architecture comprising prisms (rods) of individual enamel crystallites. f Enamel of patient IV:9 tooth is in the normal range in terms of the order but characterized by fewer enamel prisms (rods) and wider inter-rod distances, h with occasional areas exhibiting a disturbed structure (asterisk). h The width of the DEJ of patient IV:9 is increased and the shapes of transitions within the DEJ zone are straight instead of sigmoidal. i SEM of the control tooth of individual IV:8 shows normal dentin structure. j The distribution of dentin tubules was not even and the size of the dentin tubular diameter was not consistent with the tooth of patient IV:9. j Most peritubular dentin is thicker and dentin tubes are smaller or obliterated completely compared to the control tooth.
Fig. 4
Fig. 4
Interval mapping and mutational analysis of AMBN. a Chromosomal location of the AMBN gene and the distribution of seven short tandem repeat (STR) markers. b Sanger sequencing result of the WT and the mutant. All the patients in this family carry a heterozygous mutation of AMBN (c.1069C > T). b This abnormal variation changes amino acid 357 of AMBN from a hydrophobic P to a hydrophilic S.
Fig. 5
Fig. 5
Effect of mutation on AMBN function. a Conservation analysis of this abnormal variation by Polyphen-2. The result showed that amino acid 357 of AMBN was highly conserved between different species. b The 3D structure of mutated AMBN was different from that of the wild-type predicted by I-TASSER. c Subcellular localization of AMBN in HEK293 cells. The mutant AMBN was localized in the cytoplasm similar to the wild-type protein, but the mutant protein exhibited aggregation in transfected HEK293 cells. d The mRNA expression level of AMBN in HEK293 and HGF cells. Mutant AMBN mRNA expression was no different than that of the wild type in either HEK293 cells (P > 0.05) or HGF cells (P > 0.05). e Western blot analysis of AMBN expression. The results showed that mutant AMBN was expressed at a higher level than the normal state. NC, negative control. f The protein expression level of AMBN in HEK293 and HGF cells. Compared with the wild type, the protein expression level of AMBN was higher in HEK293 cells (P < 0.01) and HGF cells (P < 0.001). Data in d and f are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, or ***P < 0.001

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