Self-assembled branched polypeptides as amelogenin mimics for enamel repair.

The regeneration of demineralized enamel holds great significance in the treatment of dental caries. Amelogenin (Ame), an essential protein for mediating natural enamel growth, is no longer secreted after enamel has fully matured in childhood. Although biomimetic mineralization based on peptides or proteins has made significant progress, easily accessible, low-cost, biocompatible and highly effective Ame mimics are still lacking. Herein, we construct a series of amphiphilic branched polypeptides (CAMPs) by facile coupling of the Ame's C-terminal segment and poly(γ-benzyl-L-glutamate), which serves to simulate the Ame's hydrophobic N-terminal segment. Among them, CAMP15 is the best biomimetic mineralization template with great self-assembly performance to guide the oriented crystallization of hydroxyapatite and is capable of inhibiting the adhesion of Streptococcus mutans and Staphylococcus aureus on the enamel surfaces. This work highlights the potential application of amphiphilic branched polypeptide as Ame mimics in repairing defected enamel, providing a promising strategy for prevention and treatment of dental caries.

AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

Roles of the histone methyltransferase SET domain bifurcated 1 in epithelial cells during tooth development.

OBJECTIVE: This study aimed to reveal the effects of SET domain bifurcated 1 (SETDB1) on epithelial cells during tooth development. DESIGN: We generated conditional knockout mice (Setdb1fl/fl,Keratin14-Cre+ mice), in which Setdb1 was deleted only in epithelial cells. At embryonic day 14.5 (E14.5), immunofluorescence staining was performed to confirm the absence of SETDB1 within the epithelium of tooth embryos from Setdb1fl/fl,Keratin14-Cre+ mice. Mouse embryos were harvested after reaching embryonic day 13.5 (E13.5), and sections were prepared for histological analysis. To observe tooth morphology in detail, electron microscopy and micro-CT analysis were performed at postnatal months 1 (P1M) and 6 (P6M). Tooth embryos were harvested from postnatal day 7 (P7) mice, and the epithelial components of the tooth embryos were isolated and examined using quantitative RT-PCR for the expression of genes involved in tooth development. RESULTS: Setdb1fl/fl,Keratin14-Cre+ mice exhibited enamel hypoplasia, brittle and fragile dentition, and significant abrasion. Coronal sections displayed abnormal ameloblast development, including immature polarization, and a thin enamel layer that detached from the dentinoenamel junction at P7. Electron microscopic analysis revealed characteristic findings such as an uneven surface and the absence of an enamel prism. The expression of Msx2, Amelogenin (Amelx), Ameloblastin (Ambn), and Enamelin (Enam) was significantly downregulated in the epithelial components of tooth germs in Setdb1fl/fl,Keratin14-Cre+ mice. CONCLUSIONS: These results indicate that SETDB1 in epithelial cells is important for tooth development and clarify the relationship between the epigenetic regulation of SETDB1 and amelogenesis imperfecta for the first time.

Role of amelogenin phosphorylation in regulating dental enamel formation.

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELXS16A KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

Ameloblastin and its multifunctionality in amelogenesis: A review.

Extracellular matrix proteins play crucial roles in the formation of mineralized tissues like bone and teeth via multifunctional mechanisms. In tooth enamel, ameloblastin (Ambn) is one such multifunctional extracellular matrix protein implicated in cell signaling and polarity, cell adhesion to the developing enamel matrix, and stabilization of prismatic enamel morphology. To provide a perspective for Ambn structure and function, we begin this review by describing dental enamel and enamel formation (amelogenesis) followed by a description of enamel extracellular matrix. We then summarize the established domains and motifs in Ambn protein, human amelogenesis imperfecta cases, and genetically engineered mouse models involving mutated or null Ambn. We subsequently delineate in silico, in vitro, and in vivo evidence for the amphipathic helix in Ambn as a proposed cell-matrix adhesive and then more recent in vitro evidence for the multitargeting domain as the basis for dynamic interactions of Ambn with itself, amelogenin, and membranes. The multitargeting domain facilitates tuning between Ambn-membrane interactions and self/co-assembly and supports a likely overall role for Ambn as a matricellular protein. We anticipate that this review will enhance the understanding of multifunctional matrix proteins by consolidating diverse mechanisms through which Ambn contributes to enamel extracellular matrix mineralization.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System.

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the 'separating' and 'merging' strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133 bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 ™ Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1 ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler ™ NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Effect of phosphate group on remineralization of early enamel caries regulated by amelogenin peptide.

OBJECTIVE: To show the effect of the phosphate group on the remineralization process of early enamel caries mediated by amelogenin peptide. METHODS: Freshly extracted, completed, and crack-free bovine teeth were used to create artificial early enamel caries, which were randomly divided into four groups: Group A: fluorination remineralized solution treatment group; Group B: pure remineralized solution treatment group. Group C: 100 g/ml recombinant Amelogenin peptide remineralized solution treatment group (with single phosphate group on N-terminus); Group D: 100 g/ml non-phosphorylated recombinant Amelogenin peptide remineralized solution treatment group (without single phosphate group on N-terminus). For 12 days, fresh remineralized solutions were replaced daily. Transverse microradiography (TMR) was used after remineralization to determine mineral loss and demineralization depth before and after each sample's remineralization. Each sample's depth of remineralization and mineral acquisition were then determined. RESULTS: The recombinant amelogenin peptide group significantly outperformed the non-phosphorylated amelogenin peptide group in terms of mineral acquisition and mineralization depth (P<0.05). CONCLUSIONS: The recombinant Amelogenin's solitary phosphate group at the N-terminus helps recombinant Amelogenin to encourage the remineralization process of early enamel caries.

A DNA typing panel of 201 genetic markers for degraded samples: development and validation.

With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.

Evaluation of the usefulness of insertion-null markers in critical skeletal remains.

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

A photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel promotes bone regeneration.

OBJECTIVES: In order to evaluate the effect of methacrylated hyaluronic acid (HAMA) hydrogels containing the recombinant human amelogenin (rhAm) in vitro and in vivo. BACKGROUND: The ultimate goal in treating periodontal disease is to control inflammation and achieve regeneration of periodontal tissues. In recent years, methacrylated hyaluronic acid (HAMA) containing recombinant human amyloid protein (rhAm) has been widely used as a new type of biomaterial in tissue engineering and regenerative medicine. However, there is a lack of comprehensive research on the periodontal regeneration effects of this hydrogel. This experiment aims to explore the application of photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel for periodontal tissue regeneration and provide valuable insights into its potential use in this field. MATERIALS AND METHODS: The effects of rhAm-HAMA hydrogel on the proliferation of human periodontal ligament cells (hPDLCs) were assessed using the CCK-8 kit. The osteogenic differentiation of hPDLCs was evaluated through ALP staining and real-time PCR. Calvarial parietal defects were created in 4-week-old Sprague Dawley rats and implanted with deproteinized bovine bone matrix in different treatment groups. The animals were euthanized after 4 and 8 weeks of healing. The bone volume of the defect was observed by micro-CT and histological analysis. RESULTS: Stimulating hPDLCs with rhAm-HAMA hydrogel did not significantly affect their proliferation (p > .05). ALP staining and real-time PCR results demonstrated that the rhAm-HAMA group exhibited a significant upregulation of osteoclastic gene expression (p < .05). Micro-CT results revealed a significant increase in mineralized tissue volume fraction (MTV/TV%), trabecular bone number (Tb.N), and mineralized tissue density (MTD) of the bone defect area in the rhAm-HAMA group compared to the other groups (p < .05). The results of hematoxylin and eosin staining and Masson staining at 8 weeks post-surgery further supported the results of the micro-CT. CONCLUSIONS: The results of this study indicate that rhAm-HAMA hydrogel could effectively promote the osteogenic differentiation of hPDLCs and stabilize bone substitutes in the defects that enhance the bone regeneration in vivo.

Developmental validation of the STRSeqTyper122 kit for massively parallel sequencing of forensic STRs.

Massively parallel sequencing allows for integrated genotyping of different types of forensic markers, which reduces DNA consumption, simplifies experimental processes, and provides additional sequence-based genetic information. The STRseqTyper122 kit genotypes 63 autosomal STRs, 16 X-STRs, 42 Y-STRs, and the Amelogenin locus. Amplicon sizes of 117 loci were below 300 bp. In this study, MiSeq FGx sequencing metrics for STRseqTyper122 were presented. The genotyping accuracy of this kit was examined by comparing to certified genotypes of NIST standard reference materials and results from five capillary electrophoresis-based kits. The sensitivity of STRseqTyper122 reached 125 pg, and > 80% of the loci were correctly called with 62.5 pg and 31.25 pg input genomic DNA. Repeatability, species specificity, and tolerance for DNA degradation and PCR inhibitors of this kit were also evaluated. STRseqTyper122 demonstrated reliable performance with routine case-work samples and provided a powerful tool for forensic applications.

AMELX gene association to early childhood caries in south-Indian children: a case-control study.

PURPOSE: Genetic variants of AMELX gene can affect the protein content, organization of enamel prisms, microstructure and microhardness of the enamel, thus altering the caries susceptibility. The present study aims to assess the association between polymorphisms rs17878486, rs5934997, and rs5933871 of AMELX gene and Early Childhood Caries (ECC). MATERIALS AND METHODS: This case-control study was conducted on 200 participants, aged 3-6 years, with 100 controls and 100 children with ECC. A questionnaire was used to collect demographic data, birth-weight, type of delivery, oral hygiene practices, feeding history and 24-h diet diary. DNA was isolated from blood and subjected to PCR followed by Sanger sequencing. RESULTS: The CC genotype of rs17878486 showed an OR of 1.93 (0.34-10.81; P = 0.73). In a recessive model, the CC genotype of rs17878486 reported an OR of 2.04 (0.36-11.40; P = 0.68); rs5593871 reported an OR of 1.00 (0.31-3.21). Statistically significant differences (P ≤ 0.05) between genotype and allele frequencies of rs17878486, rs5934997, and rs5933871 were not observed between children with ECC and the controls. CONCLUSION: Polymorphisms of AMELX gene did not show a significant association with ECC in this population. However, documentation of genetic data in a global context of ECC may be essential for the future.

Amelogenin-inspired peptide, calcium phosphate solution, fluoride and their synergistic effect on enamel biomimetic remineralization: an in vitro pH-cycling model.

BACKGROUND: Several methods were introduced for enamel biomimetic remineralization that utilize a biomimetic analogue to interact and absorb bioavailable calcium and phosphate ions and induce crystal nucleation on demineralized enamel. Amelogenin is the most predominant enamel matrix protein that is involved in enamel biomineralization. It plays a major role in developing the enamel's hierarchical microstructure. Therefore, this study was conducted to evaluate the ability of an amelogenin-inspired peptide to promote the remineralization potential of fluoride and a supersaturated calcium phosphate solution in treating artificially induced enamel carious lesions under pH-cycling regimen. METHODS: Fifty enamel slices were prepared with a window (4*4 mm2 ) on the surface. Five samples were set as control healthy enamel and 45 samples were subjected to demineralization for 3 days. Another 5 samples were set as control demineralized enamel and 40 enamel samples were assigned into 8 experimental groups (n=5) (P/I, P/II, P/III, P/AS, NP/I, NP/II, NP/III and NP/AS) according to peptide treatment (peptide P or non-peptide NP) and remineralizing solution used (I; calcium phosphate solution, II; calcium phosphate fluoride solution, III; fluoride solution and AS; artificial saliva). Samples were then subjected to demineralization/remineralization cycles for 9 days. Samples in all experimental groups were evaluated using Raman spectroscopy for mineral content recovery percentage, microhardness and nanoindentation as healthy, demineralized enamel and after pH-cycling. Data were statistically analysed using two-way repeated measures Anova followed by Bonferroni-corrected post hoc test for pairwise multiple comparisons between groups. Statistical significance was set at p= 0.05. Additionally, XRD, FESEM and EDXS were used for crystal orientation, surface morphology and elemental analysis after pH-cycling. RESULTS: Nanocrystals clumped in a directional manner were detected in peptide-treated groups. P/II showed the highest significant mean values in mineral content recovery (63.31%), microhardness (268.81±6.52 VHN), elastic modulus (88.74±2.71 GPa), nanohardness (3.08±0.59 GPa) and the best crystal orientation with I002/I300 (1.87±0.08). CONCLUSION: Despite pH changes, the tested peptide was capable of remineralizing enamel with ordered crystals. Moreover, the supplementary use of calcium phosphate fluoride solution with peptide granted an enhancement in enamel mechanical properties after remineralization.

Epigenetic Regulation of Ameloblast Differentiation by HMGN Proteins.

Dental enamel formation is coordinated by ameloblast differentiation, production of enamel matrix proteins, and crystal growth. The factors regulating ameloblast differentiation are not fully understood. Here we show that the high mobility group N (HMGN) nucleosomal binding proteins modulate the rate of ameloblast differentiation and enamel formation. We found that HMGN1 and HMGN2 proteins are downregulated during mouse ameloblast differentiation. Genetically altered mice lacking HMGN1 and HMGN2 proteins show faster ameloblast differentiation and a higher rate of enamel deposition in mice molars and incisors. In vitro differentiation of induced pluripotent stem cells to dental epithelium cells showed that HMGN proteins modulate the expression and chromatin accessibility of ameloblast-specific genes and affect the binding of transcription factors epiprofin and PITX2 to ameloblast-specific genes. Our results suggest that HMGN proteins regulate ameloblast differentiation and enamel mineralization by modulating lineage-specific chromatin accessibility and transcription factor binding to ameloblast regulatory sites.

Genetic sexing of subadult skeletal remains.

When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using morphological features is unreliable, and molecular genetic methods were applied in this study. Eighty-three ancient skeletons were used as models for poorly preserved DNA. Three sex-informative markers on the Y and X chromosome were used for sex identification: a qPCR test using the PowerQuant Y target included in PowerQuant System (Promega), the amelogenin test included in ESI 17 Fast STR kit (Promega), and a Y-STR amplification test using the PowerPlex Y-23 kit (Promega). Sex was successfully determined in all but five skeletons. Successful PowerQuant Y-target, Y-amelogenin, and Y-chromosomal STR amplifications proved the presence of male DNA in 35 skeletons, and in 43 subadults female sex was established. No match was found between the genetic profiles of subadult skeletons, and the elimination database and negative control samples produced no profiles, indicating no contamination issue. Our study shows that genetic sex identification is a very successful approach for biological sexing of subadult skeletons whose sex cannot be assessed by anthropological methods. The results of this study are applicable for badly preserved subadult skeletons from routine forensic casework.

Splicing mutations in AMELX and ENAM cause amelogenesis imperfecta.

BACKGROUND: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. METHODS: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. RESULTS: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. CONCLUSIONS: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation.

Amplification Failure of the Amelogenin X Gene Caused by a Rare Mutation in the Primer-Binding Region.

The study of gender markers is essential in forensic genetic analysis. Mutations in the X or Y homologs of the amelogenin gene can be misleading, resulting in serious mistakes in forensic genetic analysis. We recently discovered two male cases of the X homolog of the amelogenin (AMELX) allelic dropout while analyzing short tandem repeat genotypes obtained from crime scene evidence. Subsequently, we evaluated the molecular characteristics of AMELX allelic dropout in this study. We used two previously reported amelogenin primers to verify a half level of amelogenin gene amplification intensity in the two male cases, which we confirmed was caused by AMELX allelic dropout. We then characterized the point mutation using Sanger sequencing and designed mutation-specific primers that could overcome AMELX allelic dropout. Short tandem repeat genotyping analysis confirmed that the AMELX allelic dropout was recovered by the mutation-specific primer designed specifically for this case. The sequencing of the AMELX allele revealed a single-point variant from A→G at base position 7 downstream from the 3' end in the amelogenin forward primer-binding region. This point mutation was identically found in two different male cases, resulting in AMELX allelic dropout. To our knowledge, these mutations and the X homolog amplification failure of amelogenin have not been reported in the Korean population. Our study provides a reliable approach to AMELX allelic dropout due to rare case mutations and could enable the better interpretation of gender markers for forensic samples.

Triple Function of Amelogenin Peptide-Chitosan Hydrogel for Dentin Repair.

Biomimetic strategies like peptide-guided collagen mineralization promise to enhance the effectiveness of dentin remineralization. We recently reported that rationally designed amelogenin-derived peptides P26 and P32 promoted apatite nucleation, mineralized collagen, and showed potential in enamel regrowth and dentin remineralization. To facilitate the clinical application of amelogenin-derived peptides and to uncover their effectiveness in repairing dentin, we have now implemented a chitosan (CS) hydrogel for peptide delivery and have investigated the effects of P26-CS and P32-CS hydrogels on dentin remineralization using 2 in situ experimental models that exhibited different levels of demineralization. The efficacy of the peptide-CS hydrogels in dentin repair was evaluated by characterizing the microstructure, mineral density, mineral phase, and nanomechanical properties of the remineralized samples. The new strategy of atomic force microscopy PeakForce quantitative nanomechanical mapping was used for direct visualization and nanomechanical analysis of repaired dentin lesions across the lesion depth. Results from the 2 models indicated the potential triple functions of peptide-CS hydrogels for dentin repair: building a highly organized protective mineralized layer on dentin, occluding dentinal tubules by peptide-guided in situ mineralization, and promoting biomimetic dentinal collagen remineralization. Importantly, peptides released from the CS hydrogel could diffuse into the dentinal matrix and penetrate the dentinal tubules, leading to both surface and subsurface remineralization and tubule occlusion. Given our previous findings on peptide-CS hydrogels' potential for remineralizing enamel, we see further promise for hydrogels to treat tooth defects involving multiple hard tissues, as in the case of noncarious cervical lesions.

Unveiling the mechanism of an amelogenin-derived peptide in promoting enamel biomimetic remineralization.

Amelogenin and its derived peptides have exhibited excellent efficacy in promoting enamel biomimetic remineralization. However, little is known about their specific action mechanisms. Herein, by combining experiments and computer simulation, the mechanism of an amelogenin-derived peptide QP5 in regulating enamel biomimetic remineralization is unveiled for the first time. In experiments, peptide QP5 was separated into (QPX)5 and C-tail domains, the interactions of peptide-minerals in nucleation solution and the regulation of peptide on enamel biomimetic remineralization were explored. QP5 exhibited an unordered conformation when mineral ions existed, and it could adsorb on minerals through its two domains, thereby inhibiting spontaneous nucleation. The remineralized enamel regulated by C-tail showed better mechanical properties and formed more biomimetic crystals than that of (QPX)5, indicating the C-tail domain of QP5 played an important role in forming enamel-like crystals. The simulation results showed that the conformation of QP5 changed greatly, mainly exhibiting ß-bend, ß-turn, and coil structures, and it eventually adsorbed on enamel through negatively charged residues of the C-tail domain, then captured Ca2+ from solution to promote enamel remineralization. This study improved the evaluation methods of the mechanism of biomimetic peptides, and laid a theoretical basis for the amelioration and clinical transformation of peptide QP5.