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Scientific Reports Nov 2023When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using...
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.
Topics: Male; Humans; Female; Body Remains; Amelogenin; Microsatellite Repeats; Forensic Medicine; DNA; DNA Fingerprinting; Chromosomes, Human, Y
PubMed: 37993531
DOI: 10.1038/s41598-023-47836-9 -
Journal of Dental Research Oct 2023Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin...
Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre-messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, and , related to multiple pathways. Given the mutations on the minigene, the expression of has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI.
Topics: Humans; Amelogenin; Amelogenesis Imperfecta; HEK293 Cells; Mutation; Dental Enamel Proteins; MicroRNAs; RNA, Messenger
PubMed: 37563801
DOI: 10.1177/00220345231180572 -
IScience Nov 2023Assignment of biological sex to skeletal remains is critical in the accurate reconstruction of the past. Analysis of sex-chromosome encoded AMELX and AMELY peptides from...
Assignment of biological sex to skeletal remains is critical in the accurate reconstruction of the past. Analysis of sex-chromosome encoded AMELX and AMELY peptides from the enamel protein amelogenin underpins a minimally destructive mass spectrometry (MS) method for sex determination of human remains. However, access to such specialist approaches limits applicability. As a convenient alternative, we generated antibodies that distinguish human AMELX and AMELY. Purified antibodies demonstrated high selectivity and quantitative detection against synthetic peptides by ELISA. Using acid etches of enamel from post-medieval skeletons, antibody determinations corrected osteological uncertainties and matched parallel MS, and for Bronze Age samples where only enamel was preserved, also matched MS analyses. Toward improved throughput, automated stations were applied to analyze 19th-century teeth where sex of individuals was documented, confirming MS can be bypassed. Our immunological tools should underpin development of routine, economical, high-throughput methods for sex determination, potentially even in a field setting.
PubMed: 37953951
DOI: 10.1016/j.isci.2023.108191 -
Forensic Science International. Synergy 2023The Amelogenin sex test included in forensic DNA typing kits has the potential to identify congenital conditions such as differences/disorders of sex development (DSD)....
The Amelogenin sex test included in forensic DNA typing kits has the potential to identify congenital conditions such as differences/disorders of sex development (DSD). It can also reveal mismatches between genotypic sex and gender marker in identity documents of transgender persons who obtained legal gender recognition. In a 13-year case history of paternity/kinship tests, involving n = 962 females and n = 1001 males, two mismatches between Amelogenin sex test (male) and gender marker (female), and three cases of chromosomal DSD (Klinefelter syndrome) were observed. The concrete risk of observing Amelogenin anomalies, their potential causes, and the context in which they occur (forensic, i.e. non-medical) mean that laboratory operators are called to strike a complex balance between privacy interests and individual health rights when providing preliminary information and reporting Amelogenin incidental findings. This case history argues for the need of a more responsible approach towards the Amelogenin sex test in the forensic community.
PubMed: 37840559
DOI: 10.1016/j.fsisyn.2023.100440 -
International Dental Journal Apr 2024The aim of this research was to compare the efficacy of the remineralising potential of self-assembling peptides (SAPs): Curodont Repair (P11-4), P26, and leucine-rich...
BACKGROUND
The aim of this research was to compare the efficacy of the remineralising potential of self-assembling peptides (SAPs): Curodont Repair (P11-4), P26, and leucine-rich amelogenin peptides (LRAP) with the standard 5% NaF varnish (Duraphat) on early enamel caries lesions (EECLs).
METHODS
A demineralising solution (DS) was used to create artificial EECLs in human dental enamel specimens, which were randomly allocated to treatment groups: P11-4; P26 solution; LRAP solution; 5% NaF varnish; and deionised water (DIW). Each specimen was subjected to 8 days of pH cycling. Specimens from each test group were subjected to microcomputed tomography (micro-CT) and nanomechanical testing to assess mineral density (MD), hardness (H), and elastic modulus (EM) properties of sound, demineralised, and treated enamel.
RESULTS
The mean MD percentage gain was highest in the P26 and P11-4 groups, followed by the LRAP, 5% NaF varnish, and DIW groups. There were statistically significant differences amongst groups. In the outer layer of EECLs, the EM and H were highest in P26 and P11-4 groups, followed by the LRAP and 5% NaF varnish. In the inner layer of EECLs, the EM and H were highest in P11-4 and P26 groups, indicative of enhanced penetration and remineralisation of the deeper parts of the artificial EECLs.
CONCLUSIONS
P26 and P11-4 SAPs are more effective than 5% NaF varnish in remineralising the depth of EECLs.
Topics: Humans; X-Ray Microtomography; Dental Enamel; Tooth Remineralization; Fluorides, Topical; Sodium Fluoride; Dental Caries; Peptides
PubMed: 37743135
DOI: 10.1016/j.identj.2023.07.003 -
BMC Genomics Apr 2024Previously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two...
Joint application of A-InDels and miniSTRs for forensic personal, full and half sibling identifications, and genetic differentiation analyses in two populations from China.
BACKGROUND
Previously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research.
RESULTS
The cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of F, D, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations.
CONCLUSIONS
This novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.
Topics: Humans; Phylogeny; Siblings; Genetics, Population; China; INDEL Mutation; Microsatellite Repeats; Forensic Genetics; Gene Frequency
PubMed: 38566035
DOI: 10.1186/s12864-024-10187-4 -
BMC Oral Health Nov 2023Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations...
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.
Topics: Humans; Female; Amelogenin; Amelogenesis Imperfecta; Dental Enamel Proteins; Mutation; Mutant Proteins; Extracellular Matrix Proteins
PubMed: 37985977
DOI: 10.1186/s12903-023-03508-8 -
PloS One 2024To show the effect of the phosphate group on the remineralization process of early enamel caries mediated 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.
Topics: Animals; Amelogenin; Dental Caries; Cattle; Tooth Remineralization; Dental Enamel; Phosphates; Peptides; Microradiography
PubMed: 38771806
DOI: 10.1371/journal.pone.0303147 -
International Journal of Molecular... Jun 2024mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness)...
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 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 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 -associated AI: While amorphic mutations, including large deletions and 5' truncations, of 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.
Topics: Amelogenesis Imperfecta; Humans; Amelogenin; Male; Female; Genetic Association Studies; Mutation; Pedigree; Phenotype; Child; Endoplasmic Reticulum Stress; Genotype; Exome Sequencing
PubMed: 38892321
DOI: 10.3390/ijms25116132 -
Cureus Feb 2024The purpose of the study was to compare and histologically investigate pulpal response and dentin bridge formation after direct pulp capping using recombinant amelogenin...
The purpose of the study was to compare and histologically investigate pulpal response and dentin bridge formation after direct pulp capping using recombinant amelogenin and mineral trioxide aggregate (MTA). Recombinant amelogenin protein and MTA were used as pulp capping materials in 120 teeth from eight mongrel dogs. Dogs were sacrificed at two different evaluation times. Regenerative changes were evaluated histologically. At two weeks, in contrast to the MTA group, most of the amelogenin group showed moderately formed hard tissue formation and the pulp tissue was completely filling the entire pulp chamber. These results were statistically significant. At two months, all the samples of the amelogenin group showed complete dentin bridge formation and the pulp chamber was filled entirely with tissue-mimicking the authentic pulp in all the specimens of the amelogenin group. These results were statistically significant. In conclusion, direct pulp capping by recombinant amelogenin protein resulted in significantly better regeneration of the dentin-pulp complex than MTA.
PubMed: 38516479
DOI: 10.7759/cureus.54560