Short Communication Optimization of DNA extraction from dental remains

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor-intensive and time-consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100-year-old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies

A novel nonsense PTH1R variant shows incomplete penetrance of primary failure of eruption: a case report

Background: Aim of this work was to describe a rare inheritance pattern of Primary Failure of Eruption (PFE) in a small family with incomplete penetrance of PFE and a novel nonsense PTH1R variant. Case presentation: The proband, a 26 year-old man with a significant bilateral open-bite, was diagnosed with PFE using clinical and radiographic characteristics. DNA was extracted from the proband and his immediate family using buccal swabs and the entire PTH1R coding sequence was analyzed, revealing a novel heterozygous nonsense variant in exon 7 of PTH1R (c.505G > T). This variant introduces a premature stop codon in position 169, predicted to result in the production of a truncated and non-functional protein. This variant has never been reported in association with PFE and is not present in the Genome Aggregation Database (gnomAD). Interestingly, the c.505G > T variant has also been identified in the unaffected mother of our proband, suggesting incomplete penetrance of PFE. Conclusions: In this study, we report a new PTH1R variant that segregates in an autosomal dominant pattern and causes PFE with incomplete penetrance. This underlines the diagnostic value of a thorough clinical and genetic analysis of all family members in order to estimate accurate recurrence risks, identify subtle clinical manifestations and provide proper management of PFE patients

Association between Vitamin D Receptor Gene Polymorphisms and Periodontal Bacteria: A Clinical Pilot Study

Abstract: Background: Periodontitis is an inflammatory disease caused by microorganisms involving the supporting tissues of the teeth. Gene variants may influence both the composition of the biofilm in the oral cavity and the host response. The objective of the study was to investigate the potential correlations between the disease susceptibility, the presence and the quantity of periodontopathogenic oral bacterial composition and the VDR gene polymorphisms. Methods: Fifty (50) unrelated periodontal patients and forty-one (41) healthy controls were selected for genomic DNA extraction. DNA concentration was measured and analyzed. The periodontopathogenic bacterial species were identified and quantified using a Real Time PCR performed with species-specific primers and probes. Results: Genotype distribution showed a different distribution between the groups for BsmI rs1544410 genotypes (p = 0.0001) with a prevalence of the G(b) allele in periodontal patients (p = 0.0003). Statistical significance was also found for VDR TaqI rs731236 (p ≤ 0.00001) with a prevalence of the T(T) allele in periodontal patients (p ≤ 0.00001). The average bacterial copy count for the periodontitis group was significantly higher than that of control group. Dividing patients into two groups based on high or low bacterial load, FokI rs2228570 T allele (f) was statistically more represented in patients with high bacterial load. Conclusions: The findings of the study suggest the involvement of the VDR gene BsmI and TaqI polymorphisms in periodontal disease, while FokI and BsmI may be involved in determining an increased presence of periodontopathogens

Comment on Kopańska et al. Disorders of the Cholinergic System in COVID-19 Era—A Review of the Latest Research. Int. J. Mol. Sci. 2022, 23, 672

We read the recent review article by Marta Kopańska et al. [...

Exfoliative Cytology and Genetic Analysis for a Non-Invasive Approach to the Diagnosis of White Sponge Nevus: Case Series

Background: White Sponge Nevus (WSN) is a rare benign disorder associated with mutations in genes coding for cytokeratin 4 (KRT4) and 13 (KRT13) characterized by dyskeratotic hyperplasia of mucous membranes. This study was aimed at examining different approaches (cytology, pathology and genetic analysis) to WSN diagnosis. Methods: A series of four patients with asymptomatic white diffuse oral lesions were evaluated and, before performing an incisional biopsy for pathology, an oral brush Thin Prep was collected for exfoliative liquid-based cytology (LBC). DNA for genetic analysis was also obtained from patients and both their parents, using buccal swabs. Results: Pathology and cytology showed similar results, leading to the same diagnosis of hyperkeratotic epithelium with acanthosis and spongiosis, without atypia, demonstrating the efficiency of LBC for the differential diagnosis. Sequencing analysis revealed at least 6 rare variants in the KRT4 and KRT13 genes in each patient, contributed in part by both unaffected parents. Conclusions: Thin Prep for oral exfoliative cytology and genetic analysis are sufficient for an accurate diagnosis of WSN. The combination of cytological and genetic analyses could substitute the histologic exam, providing a non-invasive alternative for incisional biopsy

Exfoliative Cytology and Genetic Analysis for a Non-Invasive Approach to the Diagnosis of White Sponge Nevus: Case Series

Background: White Sponge Nevus (WSN) is a rare benign disorder associated with mutations in genes coding for cytokeratin 4 (KRT4) and 13 (KRT13) characterized by dyskeratotic hyperplasia of mucous membranes. This study was aimed at examining different approaches (cytology, pathology and genetic analysis) to WSN diagnosis. Methods: A series of four patients with asymptomatic white diffuse oral lesions were evaluated and, before performing an incisional biopsy for pathology, an oral brush Thin Prep was collected for exfoliative liquid-based cytology (LBC). DNA for genetic analysis was also obtained from patients and both their parents, using buccal swabs. Results: Pathology and cytology showed similar results, leading to the same diagnosis of hyperkeratotic epithelium with acanthosis and spongiosis, without atypia, demonstrating the efficiency of LBC for the differential diagnosis. Sequencing analysis revealed at least 6 rare variants in the KRT4 and KRT13 genes in each patient, contributed in part by both unaffected parents. Conclusions: Thin Prep for oral exfoliative cytology and genetic analysis are sufficient for an accurate diagnosis of WSN. The combination of cytological and genetic analyses could substitute the histologic exam, providing a non-invasive alternative for incisional biopsy

Microbiota revolution: How gut microbes regulate our lives

The human intestine is a natural environment ecosystem of a complex of diversified and dynamic microorganisms, determined through a process of competition and natural selection during life. Those intestinal microorganisms called microbiota and are involved in a variety of mechanisms of the organism, they interact with the host and therefore are in contact with the organs of the various systems. However, they play a crucial role in maintaining host homeostasis, also influencing its behaviour. Thus, microorganisms perform a series of biological functions important for human well-being. The host provides the microorganisms with the environment and nutrients, simultaneously drawing many benefits such as their contribution to metabolic, trophic, immunological, and other functions. For these reasons it has been reported that its quantitative and qualitative composition can play a protective or harmful role on the host health. Therefore, a dysbiosis can lead to an association of unfavourable factors which lead to a dysregulation of the physiological processes of homeostasis. Thus, it has pre-viously noted that the gut microbiota can participate in the pathogenesis of autoimmune diseases, chronic intestinal inflammation, diabetes mellitus, obesity and atherosclerosis, psychic disorders (e.g., neurological diseases, autism, etc.) colorectal cancer, and more

Primary failure of eruption: Clinical and genetic findings in the mixed dentition

ABSTRACT Objective: To test the hypothesis that mutations in the parathyroid hormone 1 receptor (PTH1R) include effects in both primary and permanent teeth. Materials and Methods: DNA was extracted from saliva samples of 29 patients (8 familial and 21 sporadic) who presented with clinical evidence of infraoccluded teeth, and their unaffected relatives (N \ubc 22). Sequencing followed by mutational analysis of the coding regions of PTH1R gene was completed for all individuals (N \ubc 29). Results: Eight of 29 cases revealed a heterozygous pathogenic variant in the PTH1R gene; five of eight variants represented distinct mutations based on comparison with the dbSNP, HGMD, and ESP databases. One mutation (c.1765 T.C p.Trp89Arg) was found to segregate within a family (n \ubc3). In silico analyses for all variants revealed a putative pathogenic effect. A genotype-phenotype correlation was reported as defined by a functional mutation in PTH1R and corresponding effects on one or more posterior teeth only; unilateral or bilateral involvement, infraoccluded primary teeth. Conclusions: Novel mutations were reported in the PTH1R gene that included PFE-affected primary molars, thus providing the basis for using a genetic diagnostic tool for early diagnosis leading to proper management

Primary Failure of Eruption: Clinical and Genetic Findings in the Mixed Dentition

CONTROL ID: 2639647 TITLE: Primary Failure of Eruption: Clinical and Genetic Findings in the Mixed Dentition AUTHORS (FIRST NAME INITIAL LAST NAME): C. Grippaudo1, I. D'Apolito1, C. Cafiero1, B. Ricci1, S. A. Frazier- Bowers2 AUTHORS/INSTITUTIONS: C. Grippaudo, I. D'Apolito, C. Cafiero, B. Ricci, Dental Institute, Universit\ue0 Cattolica, Rome, ITALY|S.A. Frazier-Bowers, Department of Orthodontics, University of North Carolina, Chapel Hill, North Carolina, UNITED STATES| PREFERRED PRESENTATION TYPE: Oral CURRENT SCIENTIFIC GROUPS & NETWORKS: Craniofacial Biology ABSTRACT BODY: Objectives: Eruption disorders represent an enigmatic aspect of dental and orthodontic diagnosis. Since the discovery that Primary Failure of Eruption (PFE, MIM #125350) is due to a genetic defect, several mutations of the PTH1R gene have been identified as causative. This study aimed to refine our understanding of the phenotype:genotype correlation of PFE and PTH1R mutations in the mixed dentition. This characterization may lead to improved diagnostic approaches and provide the foundation for downstream mechanistic studies to understand the pathogenesis of PTH1R mutations. Methods: DNA was extracted from saliva samples of 29 patients (3 families and 23 unrelated individuals) who presented with clinical evidence of infraoccluded teeth. Mutational analysis was completed for the coding regions of PTH1R gene following PCR amplification and direct sequencing. Results: Eight of 29 cases revealed a heterozygous pathogenic variant in the PTH1R gene; 5 of 8 variants represent distinct mutations within the cohort. One mutation (c.1765 T>C p.Trp89Arg) was found to segregate within a family (n=3) represented by two generations. Mutational analysis using the dbSNP, HGMD and the ESP databases identified the mutations were previously unreported. In silico analyses of all variants further predicted a putative pathogenic effect. Extended clinical analysis of the cohrot verified that all the novel mutations co-segregated with the PFE phenotype that included affection of the mixed dentition. Six of the 8 patients carrying a functional PTH1R mutation were children in mixed dentition with one or more primary teeth affected. Conclusions: We report that PFE in the mixed and permanent dentition positively correlates with pathogenic mutations in the PTH1R gene. Further studies to identify additional genes and correlate the pathogenesis of PFE from mixed to permanent dentition are ongoing and forthcoming. (no table selected) TABLE FOOTER: (No Tables) (No Image Selected) KEYWORDS: Primary failure of eruption (PFE), orthodontics, Dental eruption, genetics, mixed dention. Support Funding Agency/Grant Number - Abstracts: Financial Interest Disclosure: NONE AWARDS: Group Author Abstracts - Abstract: Session Chair Volunteers - Abstracts: Not Interested Special Scheduling Needs - Abstracts: Student Status - Abstracts: No Student Other Designation - Abstracts: Abstract Submission - Track Selection: Clinician Trac

TLR2 and TLR4 Are Expressed in Epiretinal Membranes: Possible Links with Vitreous Levels of Complement Fragments and DAMP-Related Proteins

Previous studies reported the expression of toll-like receptors (TLRs), merely TLR2 and TLR4, and complement fragments (C3a, C5b9) in vitreoretinal disorders. Other than pathogens, TLRs can recognize endogenous products of tissue remodeling as damage-associated molecular pattern (DAMPs). The aim of this study was to confirm the expression of TLR2 and TLR4 in the fibrocellular membranes and vitreal fluids (soluble TLRs) of patients suffering of epiretinal membranes (ERMs) and assess their association with disease severity, complement fragments and inflammatory profiles. Twenty (n = 20) ERMs and twelve (n = 12) vitreous samples were collected at the time of the vitrectomy. Different severity-staged ERMs were processed for: immunolocalization (IF), transcriptomic (RT-PCR) and proteomics (ELISA, IP/WB, Protein Chip Array) analysis. The investigation of targets included TLR2, TLR4, C3a, C5b9, a few selected inflammatory biomarkers (Eotaxin-2, Rantes, Vascular Endothelial Growth Factor (VEGFA), Vascular Endothelial Growth Factor receptor (VEGFR2), Interferon-γ (IFNγ), Interleukin (IL1β, IL12p40/p70)) and a restricted panel of matrix enzymes (Matrix metalloproteinases (MMPs)/Tissue Inhibitor of Metallo-Proteinases (TIMPs)). A reduced cellularity was observed as function of ERM severity. TLR2, TLR4 and myD88 transcripts/proteins were detected in membranes and decreased upon disease severity. The levels of soluble TLR2 and TLR4, as well as C3a, C5b9, Eotaxin-2, Rantes, VEGFA, VEGFR2, IFNγ, IL1β, IL12p40/p70, MMP7 and TIMP2 levels were changed in vitreal samples. Significant correlations were observed between TLRs and complement fragments and between TLRs and some inflammatory mediators. Our findings pointed at TLR2 and TLR4 over-expression at early stages of ERM formation, suggesting the participation of the local immune response in the severity of disease. These activations at the early-stage of ERM formation suggest a potential persistence of innate immune response in the early phases of fibrocellular membrane formation