Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

Lu, Ting; Li, Meiyi; Xu, **angmin; **ong, Jun; Huang, Cheng; Zhang, Xuelian; Hu, Aiqin; Peng, Ling; Cai, Decheng; Zhang, Leitao; Wu, Buling; **ong, Fu

doi:10.1038/s41368-018-0027-9

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

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Published: 03 September 2018

Volume 10, article number 26, (2018)
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International Journal of Oral Science

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

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Ting Lu^1,2^na1,
Meiyi Li²^na1,
**angmin Xu^2,3,
Jun **ong⁴,
Cheng Huang²,
Xuelian Zhang²,
Aiqin Hu²,
Ling Peng¹,
Decheng Cai²,
Leitao Zhang¹,
Buling Wu¹ &
…
Fu **ong^2,3

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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.

The gain-of-function FAM83H mutation caused hypocalcification amelogenesis imperfecta in a Chinese family

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Introduction

Ameloblastin (AMBN: MIM *601259) expression was first detected in the enamel organ and is the second most abundant enamel matrix protein, after amelogenin, known to have key roles in enamel formation.^1,2 The extracellular matrix has a critical role in tissue development and homoeostasis by mediating cell growth, migration, differentiation, apoptosis, and gene expression.^3,4 In the enamel extracellular matrix, AMBN is believed to participate in ameloblast attachment to the underlying enamel matrix⁵ and in modulation of enamel crystal growth,⁶ and mutations in this gene are already known to cause amelogenesis imperfecta (AI: MIM104530).^7,8 AI is the term given to a heterogeneous group of disorders characterized by failure of normal amelogenesis, with a reported prevalence of between 1/700 and 1/14 000.^9,10 Genes associated with non-syndromic AI encode proteins involved in the formation and maintenance of the develo** enamel matrix, including amelogenin (AMELX), enamelin (ENAM), kallikrein-related peptidase 4 (KLK4), matrix metallopeptidase 20 (MMP20), Golgi-associated secretory pathway pseudokinase (FAM20A), chromosome 4 open reading frame 26 (C4ORF26), and amelotin (AMTN).^{8,11,12,13,14,15,16,17} Other genes associated with non-syndromic AI include solute carrier family 24 member 4 (SLC24A4) and G-protein-coupled receptor 68 (GPR68), which are involved in ion transport^18,19; laminin subunit beta 3 (LAMB3), integrin subunit beta 6 (ITGB6), collagen type XVII alpha 1 chain (COL17A1), and laminin subunit alpha 3 (LAMA3), which are involved in extracellular matrix adhesion^{20,21,22,23,24,25}; family with sequence similarity 83 member H (FAM83H) and WD repeat domain 72 (WDR72), which are associated with intracellular vesicles^26,27; acid phosphatase 4 (ACP4), which is a hydrolytic enzyme^28,29; dentin sialophosphoprotein (DSPP), which is associated with dentin development³⁰; and distal-less homeobox 3 (DLX3), which is associated with craniofacial development.³¹ Enamel abnormality is characteristic of mouse models with a deletion of Ambn exons 5 and 6 (Ambn ^{−5,6/−5,6})^32,33, and Ambn overexpression.³³ For many years, there were no known mutations of AMBN associated with AI phenotypes. Recently, hypoplastic AI was found to be associated with homozygous exon 6 deletion⁸ and another family was identified with a novel homozygous splice-site mutation (c.532–1G > C).³⁴ To date, no point mutations of AMBN have been found to be responsible for this disease.

Dentinogenesis is a highly ordered process in which the organic predentin matrix is progressively mineralized by ectomesenchymally derived cells called odontoblasts.³⁵ The odontoblasts differentiate at the bell stage of tooth development, forming a single layer of cells lining the pulp cavity, where they secrete the organic predentin matrix into the underlying space.³⁶ AMBN expression is also detected in pulpal mesenchymal cells, including preodontoblasts and young odontoblasts.³⁷ AMBN is known to be involved in the mesenchymal–ectodermal interaction that precedes dentin and enamel secretion, and the sequential expression pattern of AMBN also acts as a signalling molecule.³⁸ Currently, hereditary dentin disorders are divided into dentinogenesis imperfecta (DGI) and dentin dysplasia (DD).³⁹ Recent genetic studies have shown that mutations of collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), DSPP, secreted modular calcium-binding protein 2 (SMOC2), vacuolar protein sorting 4 homologue B (VPS4B), and ssu-2 homologue (SSUH2) cause dentin disorders.^{39,40,41,42,43,44,45,46,47,48,Full size image}

Three extracted disused mandibular third molar teeth from IV:8, IV:2, and IV:9 were available for phenotypic characterization after obtaining their informed consent (the donors were approximately 20–22 years old). High-resolution computed tomography scanning (Fig. 3a–c and Supplemental Videos 1-3) of control tooth 1 from IV:8 and teeth 2 and 3 from IV:2 and IV:9 showed that tooth 1 exhibited a normal enamel layer in terms of thickness and mineral density (enamel density = (2.91 ± 0.06) g·cm^‒3 compared with a range of (2.57–3.10) g·cm^‒3 previously reported for enamel)^{51,52,53,54,55} and a normal dentin mineral density, whereas teeth 2 and 3 exhibited reduced enamel and dentin mineral density (Fig. 3c–d). Enamel thickness also appeared to be reduced. The pulp chambers were narrower with pulp chamber calcification (Fig. 3b). The roots were shorter, with small or obliterated root canals (Fig. 3b).

When examined with scanning electron microscopy (SEM), the normal control teeth exhibited the prismatic structure characteristic of normal human enamel and dentin (Fig. 3e, g, i). SEM revealed that the mutation had little effect on the prismatic structure, but the numbers of enamel prisms of affected teeth were obviously reduced (Fig. 3f); the enamel also displayed distinct regions where the enamel structure was particularly disturbed (Fig. 3h). The outline of the dentin–enamel junction (DEJ) was changed: the width of the DEJ was increased and the structure of sigmoidal transitions within the DEJ zone, which firmly combine enamel and dentin together, became a straight line in appearance (Fig. 3h). Examination of the ultrastructure of dentin of affected teeth showed that the distribution of dentin tubules was not even and that the dentin tubular diameter was not consistent (Fig. 3j). This verified that there was an increase in the thickness of major peritubular dentin in mutant teeth, which reduced the size of or even obliterated the dentin tubules compared to control teeth (Fig. 3j).

Genetic analysis

To identify the pathogenic variant responsible for the phenotype, we first screened for mutations in the coding and splice regions of the DSPP gene (exons 1–5). However, no mutations were identified. To confirm this result, long-range PCR was conducted with primers designed to amplify all exons, including the highly repetitive sequence in exon 5 of DSPP. Sequencing of the resulting product revealed that there were no mutations present within the DSPP exons or the flanking splice regions. In addition, we also analysed the copy number variations (CNVs) in the DSPP gene via array comparative genomic hybridization. Data analysis revealed no pathogenic CNVs in the DSPP gene in affected individuals in this family. Next, we performed exome sequencing with genomic DNA for affected individuals IV:2 and IV:9, and control IV:1 in collaboration with Genesky-Shanghai (China). Sequence analysis of all exons showed that 47 potentially pathogenic variants were shared by the two affected individuals and not by the unaffected individual. None of these variants were within genes known to be involved in AI, except for AMBN, or in DGI as identified by function, expression or animal model studies, including AMELX, ENAM, KLK4, MMP20, DSPP, SMOC2, VPS4B, and SSUH2. However, a novel heterozygous missense mutation (c.1069C > T) was identified in exon 13 of AMBN (NM: 016519, chr4:71472172), causing a proline to serine mutation at the conserved amino acid position 357 of the protein (Fig. 4a). The pathogenic variant of AMBN was validated by Sanger sequencing followed by co-segregation analysis in all affected and unaffected family members (Fig. 4b). The candidate region was confirmed by additional genotype data from microsatellite markers on 4q, and linkage analysis gave a maximum logarithm of odds score of 4.515 between markers D4S1541 and D4S1558 (Figs. 1 and 4a). Further, this nucleotide change was not detected in DNA samples from 185 normal unrelated controls and 15 dominant AI patients matched for Chinese ethnicity.

Functional analysis of mutant AMBN

The c.1069C > T mutation in AMBN changes the three-dimensional (3D) structure of the AMBN protein predicted by I-TASSER, leading to a loss-of-function protein estimated by Polyphen-2 (Fig. 5b). To evaluate the effect of the mutation, the cellular localization of AMBN was analysed using wild type (WT) and mutant constructs in human embryonic kidney (HEK) 293 cells. The result showed that the mutant AMBN was localized in the cytoplasm, which was the same as WT AMBN (Fig. 5c). However, the mutant protein exhibited aggregation in transfected HEK293 cells (Fig. 5c). Moreover, we made two WT and mutant constructs that were transiently expressed in human gingival fibroblasts (HGFs) and HEK293 cells. There was no obvious difference in mRNA expression between cells transfected with the mutated transgenic vector (ΔcAMBN - GFP) or normal transgenic vector (cAMBN - GFP) in either HGF (P > 0.05) or HEK293 cells (P > 0.05) (Fig. 5d). However, we found that expression of the aberrant protein was significantly increased compared with the WT AMBN protein in both HGF (P < 0.001) and HEK293 cells (P < 0.01) (Fig. 5e, f).

Discussion

We have identified a large Chinese pedigree with segregating severe hypoplastic AI and dentin disorders in an autosomal-dominant manner. To predict the affected codon, we used whole exome sequencing and identified a rare variant at exon 13 of the AMBN gene on chromosome 4q shared by all affected individuals. Further investigation confirmed that this was a heterozygous missense mutation at position 1069 (c.1069C > T) of the coding region. Human AMBN is composed of 447 residues, many of which are unchanged or only substituted with a similar residue during more than 200 Ma of mammalian evolution.⁵⁶ It is worth noting that the average percentage of proline is 14.09% in the full-length AMBN sequence and 23.78% for the proline-rich region, compared with the 5.1% encountered in most proteins.⁵⁶ Estimation of the codon conservation index along the human AMBN sequence indicated that position P375 of exon 13 is highly conserved.⁵⁶ The evolutionary analyses performed on various secretory calcium-binding phosphoproteins, including AMELX, ENAM, matrix extracellular phosphoglycoprotein, AMTN, and dentin matrix acidic phosphoprotein 1 proteins, which are involved in tooth development, have shown that the patterns of long-term evolutionary conservation have a decisive influence on validating the potential pathogenicity of variants identified in human genetic diseases.⁵⁷ The conserved position sites are very important and such variants usually result in a genetic disorder.⁵⁶ In the present report, our results show that mutation of conserved position P375 of AMBN also causes severe dental defects.

In our study, we report an AMBN missense mutation causing severe autosomal-dominant AI. AMBN is an enamel matrix protein (EMP) that could determine the supramolecular enamel matrix assembly by the proline-rich region and is located on chromosome 4 in a region containing the locus for the autosomal hypoplastic form of AI (AIH2).⁵⁸ EMPs consist principally of AMELX, AMBN, and ENAM.⁵⁹ EMPs have a role in maintaining the prismatic structure of growing enamel crystals at the rod and inter-rod boundaries.^60,61 Heterozygous Ambn^+5,6/−5,6 mice had significantly less well-mineralized prismatic enamel of near-normal thickness compared with WT.⁶² Upon SEM examination, affected teeth exhibited a phenotype in which an ordered thinner prismatic structured layer of enamel was formed with fewer enamel rods and wider inter-rod distances (Fig. 3f). This patient phenotype is similar to that of heterozygous Ambn^{+5,6 /−5,6} mice. In addition, the mineral density of enamel was significantly decreased in affected individuals via deletion of AMBN exon 6. The mean mineral density of enamel is approximately 2.47 g/cm³. In our study, the mean mineral density of enamel was 2.45 g^.cm^‒3 in individuals IV:2 and IV:9, which is consistent with the enamel density values obtained from affected individuals with genomic deletion of AMBN exon 6.

We report the association of a genetic change in AMBN with dentin defects in the family identified in this study. In our case, some of the dentin features characteristic of DGI-II and DD-I were observed in the affected individuals (Fig. 2). They appeared similar to those associated with DGI-II, such as bulbous crowns with cervical constriction, mild discoloration, and pulp obliteration. Radiologically, permanent teeth of DD-I have short roots with a crescent-shaped pulpal remnant parallel to the cemento-enamel junction in the permanent dentition and total pulpal obliteration in the dentition.^{48,http://genetics.bwh.harvard.edu/pph2/).}

Cell transfection and subcellular localization

The coding region of AMBN was obtained by synthetic methods, as AMBN is not expressed in peripheral blood. The WT coding region was cloned into the Hind III and Bam HI sites of the pcDNA-3.1-flag plasmid (Life Technology, Thermo Fisher Scientific, Waltham, MA, USA) and used as the template for site-directed mutagenesis to clone the mutant form of AMBN, ΔcAMBN-pcDNA-3.1-FLAG. HEK293 cells were cultured in 24-well dishes and transfected with WT or mutant AMBN plasmids when cells were 50% confluent. Twenty-four hours after transfection, the cells were rinsed three times with phosphate-buffered saline (PBS, Sigma–Aldrich, St Louis, MO, USA) and fixed for 30 min with 4% paraformaldehyde (Sigma–Aldrich). After removing the paraformaldehyde, the dishes were washed with PBS three times, and then, the cells were permeabilized by incubating them in 0.1% Triton X-100 (Thermo Fisher Scientific). After blocking nonspecific reactive sites, the cells were incubated overnight in primary antibody (Sigma–Aldrich; 1:100) in the dark. The next day, the primary antibody was removed and the cells were washed with PBS three times and incubated in a species-specific secondary antibody for 2 h. Nuclei were stained with 4',6-diamidino-2-phenylindole (Sigma–Aldrich) and viewed under a fluorescence microscope (Nikon, Eclipse Ti-U, Tokyo, Japan).

RNA expression evaluation

HEK293 cells and HGF cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) at 37 °C and 5% CO₂ in a sterile incubator (Thermo Fisher Scientific). Cells that were 80% confluent were transfected with WT or mutant AMBN recombinant plasmids (ΔcAMBN-pcDNA-3.1-FLAG) using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were collected and total RNA was isolated. The total RNA was extracted with TRIzol (Thermo Fisher Scientific) and reverse-transcribed to cDNA using the GoScript Reverse Transcription System (Promega, Madison, WI, USA). GoTaqq PCR Master Mix (Promega) was used to measure the mRNA level of WT and mutant AMBN alleles. GAPDH was used as a reference gene to normalize the expression of the target gene. The transfection step was repeated three times, and all the real-time reverse transcription-PCRs were performed in triplicate to calculate mean values and SDs. The primers for amplifying AMBN were as follows: forward: 5′-TTTCTCACGGACCAATGCCA-3′ and reverse: 5′-GCCTCATGCCTCCAAATCCT-3′. Gene expression levels were calculated using the (2^–ΔΔCT) method. The results were used to calculate the mean values and SD shown in histograms.

Protein expression

HEK293 cells and HGF cells were cultured in six-well dishes at 37 °C in a 5% CO₂ atmosphere in a sterile incubator (Thermo Fisher Scientific). DMEM (Invitrogen) supplemented with 10% FBS (GIBCO, Thermo Fisher Scientific) was used for cell culture. To investigate differences in AMBN expression, cells were transfected with 2 μg of WT or mutant AMBN recombinant plasmid (ΔcAMBN-FLAG) or empty vector (negative control). After a further 24 h of culture, cells were collected and homogenized in radio-immunoprecipitation assay lysis buffer (Merck-Millipore, Darmstadt, Germany) with the protease inhibitor phenyl methyl sulfonyl fluoride (Beyotime Institute of Biotechnology, Jiangsu, China). Total protein was extracted and assessed using a standard bicinchoninic acid assay (Beyotime). Loading buffer was added before boiling for 5 min. For each sample, 20 μg of protein was loaded onto a 10% SDS-polyacrylamide gel electrophoresis gel (Bio-Rad, Hercules, CA, USA), transferred onto a polyvinylidene difluoride membrane (Millipore), and blocked with 5% skimmed milk (Thermo Fisher Scientific) and 0.1% Tris-buffered saline-Tween 20 (TBST) at room temperature for 1.5 h. The membranes were incubated overnight at 4 °C with mouse anti-FLAG (F1804, Sigma, USA). The membranes were then washed with TBST and incubated with goat anti-mouse IgG (F9006, Sigma) at room temperature for 2 h. SuperSignal West Pico ECL (Thermo Fisher Scientific) was freshly prepared just before detection of proteins. Protein expression was detected by exposure to an automatic chemiluminescence image analysis system (Tanon, Shanghai, China) and evaluated using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Statistical analyses

The significance of differences between two groups was determined using Student’s t-test followed by Bernoulli correction and adjusted P-values were used to identify significant differences. The quantified results are shown as the mean ± SD. P < 0.05 was considered statistically significant, *P < 0.05, **P < 0.01, or ***P < 0.001.

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Acknowledgements

We gratefully acknowledge the assistance of the patients and their family members for their participation in this research. This work was partially supported by a grant from the National Natural Science Foundation of China 31371279 (to Fu **ong), the National Natural Science Foundation of China 81371137 (to Bu-Ling Wu), and the Science and Technology Program of Guangzhou 201707010301 (to Fu **ong).

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These authors contributed equally: Ting Lu and Mei-Yi Li.

Authors and Affiliations

Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, Guangdong, China
Ting Lu, Ling Peng, Leitao Zhang & Buling Wu
Department of Medical Genetics, School of Basic Medicine Sciences, Southern Medical University, Guangzhou, Guangdong, China
Ting Lu, Meiyi Li, **angmin Xu, Cheng Huang, Xuelian Zhang, Aiqin Hu, Decheng Cai & Fu **ong
Guangdong Key Laboratory of Biological Chip, Guangzhou, Guangdong, China
**angmin Xu & Fu **ong
Department of Laboratory Medicine, ZhuJiang Hospital, Southern Medical University, Guangzhou, Guangdong, China
Jun **ong

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Ting Lu
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Meiyi Li
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**angmin Xu
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Jun **ong
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Cheng Huang
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Xuelian Zhang
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Aiqin Hu
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Ling Peng
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Decheng Cai
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Leitao Zhang
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Buling Wu
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Fu **ong
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Correspondence to Buling Wu or Fu **ong.

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The authors declare no competing interests.

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3D reconstruction vedio of normal tooth

3D reconstruction vedio of IV-2

3D reconstruction vedio of IV-9

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Lu, T., Li, M., Xu, X. et al. Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders. Int J Oral Sci 10, 26 (2018). https://doi.org/10.1038/s41368-018-0027-9

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Received: 18 January 2018
Revised: 15 May 2018
Accepted: 18 May 2018
Published: 03 September 2018
DOI: https://doi.org/10.1038/s41368-018-0027-9
Springer Nature Limited

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Editorial Summary

Gene mutation pinpointed in tooth development disorders

A mutation on a gene involved in healthy tooth development may cause both enamel and dentin disorders. The ameloblastin enamel protein, and its associated gene, AMBN, play vital roles in enamel formation and tooth remodelling. Mutations on AMBN can cause amelogenesis imperfecta (AI), a genetic and hereditory condition resulting in enamel defects and severe tooth decay. Now, Fu **ong and Bu-Ling Wu at Southern Medical University in Guangzhou, China, and co-workers have identified an AMBN mutation found in both enamel and dentin defect disorders. The researchers analyzed extracted teeth from a Chinese patient with both AI and a severe dentin disorder, along with teeth from affected and non-affected members of the same family, and compared the results with a control group. They identified a rare mutation on AMBN common to all affected individuals.

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

Abstract

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The gain-of-function FAM83H mutation caused hypocalcification amelogenesis imperfecta in a Chinese family

Amelogenesis imperfecta in a Chinese family resulting from a FAM83H variation and the effect of FAM83H on the secretion of enamel matrix proteins

Novel FAM83H mutations in patients with amelogenesis imperfecta

Introduction

Genetic analysis

Functional analysis of mutant AMBN

Discussion

Cell transfection and subcellular localization

RNA expression evaluation

Protein expression

Statistical analyses

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding authors

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Competing interests

Electronic supplementary material

3D reconstruction vedio of normal tooth

3D reconstruction vedio of IV-2

3D reconstruction vedio of IV-9

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About this article

Cite this article

This article is cited by

The gain-of-function FAM83H mutation caused hypocalcification amelogenesis imperfecta in a Chinese family

Effects of lipid metabolism on mouse incisor dentinogenesis

Genes in the pathway of tooth mineral tissues and dental caries risk: a systematic review and meta-analysis

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Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

Abstract

Similar content being viewed by others

Introduction

Genetic analysis

Functional analysis of mutant AMBN

Discussion

Cell transfection and subcellular localization

RNA expression evaluation

Protein expression

Statistical analyses

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding authors

Ethics declarations

Competing interests

Electronic supplementary material

Rights and permissions

About this article

Cite this article

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This article is cited by

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