Unraveling metagenomics through long-read sequencing: a comprehensive review.

The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics. However, a new era has emerged with the advent of long-read sequencing (LRS), which offers substantial improvements over its predecessor, short-read sequencing (SRS). LRS produces reads that are several kilobases long, enabling researchers to obtain more complete and contiguous genomic information, characterize structural variations, and study epigenetic modifications. The current leaders in LRS technologies are Pacific Biotechnologies (PacBio) and Oxford Nanopore Technologies (ONT), each offering a distinct set of advantages. This review covers the workflow of long-read metagenomics sequencing, including sample preparation (sample collection, sample extraction, and library preparation), sequencing, processing (quality control, assembly, and binning), and analysis (taxonomic annotation and functional annotation). Each section provides a concise outline of the key concept of the methodology, presenting the original concept as well as how it is challenged or modified in the context of LRS. Additionally, the section introduces a range of tools that are compatible with LRS and can be utilized to execute the LRS process. This review aims to present the workflow of metagenomics, highlight the transformative impact of LRS, and provide researchers with a selection of tools suitable for this task.

Tooth agenesis related to a novel KDF1 variant: A case report and literature review.

OBJECTIVES: To investigate the role of Keratinocyte Differentiation Factor 1 (KDF1) in ectodermal dysplasia (ED) and nonsyndromic tooth agenesis (NSTA) and perform a literature review. METHODS: Genome sequencing was used to identify genetic variants in a Thai, NSTA proband and validated through Sanger sequencing. Pathogenicity was assessed using ACMG guidelines, MetaRNN and AlphaMissense. A comprehensive review of KDF1/NSTA cases informed genotype-phenotype analysis of the proband. RESULTS: The proband revealed multiple missing teeth, caries and extensive periodontal disease. Deep phenotyping showed no signs of ED beyond tooth agenesis. The identified novel KDF1 variant, p.Ile243Leu, was classified as 'likely pathogenic' by ACMG and predicted as 'detrimental' by MetaRNN and AlphaMissense analyses. A total of 14 reviewed KDF1 cases revealed ED-associated variants (3 variants in 8 patients) clustering in the region of amino acids 251-275, within the DUF4656 domain, while NSTA-causing variants (4 variants in 6 patients) were typically found in amino- or carboxy-termini to this region. KDF1/NSTA cases exhibited an average of 15 missing teeth, with a higher prevalence in the mandible. CONCLUSION: This study identifies a novel KDF1 variant-related NSTA in Thai people. The genotype-phenotype correlates suggest a distinctive pattern and tooth agenesis of KDF1-related NSTA.

Deep dental phenotyping and a novel FAM20A variant in patients with amelogenesis imperfecta type IG.

OBJECTIVES: To identify etiologic variants and perform deep dental phenotyping in patients with amelogenesis imperfecta (AI). METHODS: Three patients of two unrelated families were evaluated. Genetic variants were investigated by exome and Sanger sequencing. An unerupted permanent third molar (AI1) from Patient1 and a deciduous first molar (AI2) from Patient2, along with three tooth-type matched controls for each were characterized. RESULTS: All three patients harbored biallelic pathogenic variants in FAM20A, indicating AI1G. Of the four identified variants, one, c.1231C > T p.(Arg411Trp), was novel. Patient1 possessed the largest deletion, 7531 bp, ever identified in FAM20A. In addition to hypoplastic enamel, multiple impacted teeth, intrapulpal calcification, pericoronal radiolucencies, malocclusion, and periodontal infections were found in all three patients, gingival hyperplasia in Patient1 and Patient2, and alveolar bone exostosis in Patient3. Surface roughness was increased in AI1 but decreased in AI2. Decreased enamel mineral density, hardness, and elastic modulus were observed in AI1 enamel and dentin and AI2 dentin, along with decreased phosphorus, increased carbon, and increased calcium/phosphorus and carbon/oxygen ratios. Severely collapsed enamel rods and disorganized dentin-enamel junction were observed. CONCLUSIONS: We report a novel FAM20A variant and, for the first time, the defective mineral composition and physical/mechanical properties of AI1G teeth.

In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

AIM: Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. METHODOLOGY: DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. RESULTS: The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1ß and TGF-ß1, whereas IL-6 and NFκB1 expression was significantly reduced. CONCLUSION: The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

A germline STAT6 gain-of-function variant is associated with early-onset allergies.

BACKGROUND: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. OBJECTIVE: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. METHODS: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. RESULTS: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. CONCLUSIONS: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.

Genetic Variants in KCTD1 Are Associated with Isolated Dental Anomalies.

KCTD1 plays crucial roles in regulating both the SHH and WNT/ß-catenin signaling pathways, which are essential for tooth development. The objective of this study was to investigate if genetic variants in KCTD1 might also be associated with isolated dental anomalies. We clinically and radiographically investigated 362 patients affected with isolated dental anomalies. Whole exome sequencing identified two unrelated families with rare (p.Arg241Gln) or novel (p.Pro243Ser) variants in KCTD1. The variants segregated with the dental anomalies in all nine patients from the two families. Clinical findings of the patients included taurodontism, unseparated roots, long roots, tooth agenesis, a supernumerary tooth, torus palatinus, and torus mandibularis. The role of Kctd1 in root development is supported by our immunohistochemical study showing high expression of Kctd1 in Hertwig epithelial root sheath. The KCTD1 variants in our patients are the first variants found to be located in the C-terminal domain, which might disrupt protein-protein interactions and/or SUMOylation and subsequently result in aberrant WNT-SHH-BMP signaling and isolated dental anomalies. Functional studies on the p.Arg241Gln variant are consistent with an impact on ß-catenin levels and canonical WNT signaling. This is the first report of the association of KCTD1 variants and isolated dental anomalies.

Novel CNNM4 variant and clinical features of Jalili syndrome.

The study identifies a non-consanguineous multigenerational family of the Lua ethnic group in Northern Thailand with three members affected with hypoplastic-hypocalcified amelogenesis imperfecta, cone-rod dystrophy, and harboring a novel homozygous missense variant, c.1475G>A p.(Gly492Asp), in CNNM4, indicating Jalili syndrome. We report features including advanced dental age, crossbite, developmental delay, expanding genotypic and phenotypic spectra of Jalili syndrome, and perform the prenatal genetic testing that helps avoid unnecessary pregnancy termination.

PTEN regulates proliferation and osteogenesis of dental pulp cells and adipogenesis of human adipose-derived stem cells.

OBJECTIVE: To investigate the role of phosphatase and tensin homolog (PTEN) in dental pulp cells (hDPs) and adipose-derived mesenchymal stem cells (hADSCs). MATERIALS AND METHODS: Genetic variant was identified with exome sequencing. The hDPs isolated from a patient with Cowden syndrome were investigated for their proliferation, osteogenesis, adipogenesis, and gene expression compared with controls. The normal hDPs and hADSCs were treated with the PTEN inhibitor, VO-OHpic trihydrate (VOT), to investigate the effect of PTEN inhibition. RESULTS: A heterozygous nonsense PTEN variant, c.289C>T (p.Gln97*), was identified in the Cowden patient's blood and intraoral lipomas. The mutated hDPs showed significantly decreased proliferation, but significantly upregulated RUNX2 and OSX expression and mineralization, indicating enhanced osteogenic ability in mutated cells. The normal hDPs treated with VOT showed the decreases in proliferation, colony formation, osteogenic marker genes, alkaline phosphatase activity, and mineral deposition, suggesting that PTEN inhibition diminishes proliferation and osteogenic potential of hDPs. Regarding adipogenesis, the VOT-treated hADSCs showed a reduced number of cells containing lipid droplets, suggesting that PTEN inhibition might compromise adipogenic ability of hADSCs. CONCLUSIONS: PTEN regulates proliferation, enhances osteogenesis of hDPs, and induces adipogenesis of hADSCs. The gain-of-function PTEN variant, p.Gln97*, enhances osteogenic ability of PTEN in hDPs.

Dental characteristics of patients with four different types of skeletal dysplasias.

OBJECTIVE: Skeletal dysplasia (SD) comprises more than 450 separate disorders. We hypothesized that their dental features would be distinctive and investigated the tooth characteristics of four patients with different SDs. MATERIAL AND METHODS: Four SD patients with molecularly confirmed diagnoses, Pt-1 acromicric dysplasia, Pt-2 hypophosphatasia and hypochondroplasia, Pt-3 cleidocranial dysplasia, and Pt-4 achondroplasia, were recruited. A tooth from each patient was evaluated for mineral density (micro-computerized tomography), surface roughness (surface profilometer), microhardness, mineral contents (energy-dispersive X-ray), and ultrastructure (scanning electron microscopy and histology), and compared with three tooth-type matched controls. RESULTS: Pt-1 and Pt-3 had several unerupted teeth. Pt-2 had an intact-root-exfoliated tooth at 2 years old. The lingual surfaces of the patients' teeth were significantly smoother, while their buccal surfaces were rougher, than controls, except for Pt-1's buccal surface. The patients' teeth exhibited deep grooves around the enamel prisms and rough intertubular dentin. Pt-3 demonstrated a flat dentinoenamel junction and Pt-2 had an enlarged pulp, barely detectable cementum layer, and ill-defined cemento-dentinal junction. Reduced microhardnesses in enamel, dentin, and both layers were observed in Pt-3, Pt-4, and Pt-1, respectively. Pt-1 showed reduced Ca/P ratio in dentin, while both enamel and dentin of Pt-2 and Pt-3 showed reduced Ca/P ratio. CONCLUSION: Each SD has distinctive dental characteristics with changes in surface roughness, ultrastructure, and mineral composition of dental hard tissues. CLINICAL RELEVANCE: In this era of precision dentistry, identifying the specific potential dental problems for each patient with SD would help personalize dental management guidelines.

Orodental health status of patients with inborn errors of immunity.

BACKGROUND: Various orodental problems affect patients with inborn errors of immunity (IEI), but there are limited studies on these issues. AIM: To study orodental status and its confounding factors in patients with IEI. DESIGN: Caries, enamel defects, gingival, and soft tissue conditions were examined. Data on patient characteristics, dental hygiene habits, dental attendance, and household income were collected. Statistical analysis and logistic regression were performed. RESULTS: Forty-five participants with a mean age of 9.20 ± 6.41 years were included. Almost all participants had gingivitis (42 of 45; 93.3%), whereas a small number had periodontitis (five of 45; 11.1%). Calculus was found in 33 (73.3%) and caries in 30 (66.7%). Mucosal ulcers, enamel defects, and candidiasis were observed in 23 of 45 (51.1%), 16 of 43 (37.2%), and six of 43 (14.0%), respectively. Chances of having caries, moderate-to-severe gingivitis, periodontitis, calculus, and ulcers increased with age. Taking antibiotics in the last two months increased the risk of caries by five times. Lower income increased the risk of calculus deposit by nine times. CONCLUSION: Gingivitis, calculus, caries, and mucosal ulcers were the most common orodental findings in patients with IEI. Antibiotics increased the risk of caries, and low-income children had higher calculus accumulation.

Patterns of nonsyndromic tooth agenesis and sexual dimorphism.

BACKGROUND: Sex dimorphism has been implicated in oral health differences and the pathogenesis of oral diseases, such as tooth agenesis, periodontal disease, dental caries, and tooth loss. Tooth agenesis (TA) is one of the most common developmental anomalies in humans, and its prevalence and patterns are different across ethnic groups. The aim of this study was to investigate the phenotypes and sex-associated patterns of nonsyndromic tooth agenesis (TA) in Thai dental patients. METHODS: One thousand ninety panoramic radiographs were examined. One hundred and one subjects (37 males, 64 females, 15-20 years-old) with nonsyndromic TA were evaluated. Differences in TA prevalence between groups were analyzed using the chi-square or Fisher exact test. RESULTS: The TA prevalence, excluding third molars, was 9.3% and more frequently found in the mandible compared with the maxilla. The maxilla demonstrated a higher prevalence of first premolar agenesis than the mandible (P = 0.012), while the mandible had a higher prevalence of second premolar agenesis than the maxilla (P = 0.031). There were significantly more males missing one tooth than females, however, there were more females missing two or more teeth than males (P = 0.042). A missing maxillary left lateral incisor was significantly more frequent in males (P = 0.019), while a missing mandibular right lateral incisor was more frequent in females (P = 0.025). In females, the pattern of two mandibular lateral incisors agenesis was the most common and significantly present in females more than males (P = 0.015). In contrast, the pattern of one mandibular left lateral incisor agenesis was only observed in males and significantly found in males more than females (P = 0.047). CONCLUSIONS: We demonstrate sex-associated differences in nonsyndromic tooth agenesis. The prevalence of single tooth agenesis was higher in males, while that of two or more teeth agenesis was higher in females. We found different patterns of lateral incisor agenesis between males and females.

Patterns of molar agenesis associated with p.P20L and p.R77Q variants in PAX9.

Nonsyndromic tooth agenesis is associated with variants in several genes. There are numerous genotype-phenotype publications involving many patients and kindreds. Here, we identified six Thai individuals in two families with nonsyndromic tooth agenesis, performed exome sequencing, and conducted functional experiments. Family 1 had four affected members carrying the heterozygous PAX9 variant, c.59C>T (p.Pro20Leu). The p.Pro20Leu was previously reported in two families having four and three affected members. These seven cases and Proband-1 had agenesis of at least three third molars. Family 2 comprised two affected members with agenesis of all 12 molars. Both individuals were heterozygous for c.230G>A (p.Arg77Gln) in PAX9, which has not been reported previously. This variant is predicted to be damaging, evolutionarily conserved, and resides in the PAX9 linking peptide. The BMP4 RNA levels in Proband-1's leukocytes were not significantly different from those in the controls, whereas BMP4 levels observed in Proband-2 were significantly increased. Moreover, the p.Arg77Gln variant demonstrated nuclear localization similar to the wild-type but resulted in significantly impaired transactivation of BMP4, a PAX9 downstream gene. In conclusion, we demonstrate that the PAX9 p.Pro20Leu is highly associated with absent third molars, while the novel PAX9 p.Arg77Gln impairs BMP4 transactivation and is associated with total molar agenesis.

Age-related dental phenotypes and tooth characteristics of FAM83H-associated hypocalcified amelogenesis imperfecta.

OBJECTIVES: Autosomal-dominant hypocalcified amelogenesis imperfecta (ADHCAI) shows phenotypic heterogeneity. Our aim was to characterise the ADHCAI phenotypes, tooth properties and genotypes. METHODS: Three unrelated ADHCAI probands and seven additional affected members of the three families were recruited. Mutations were identified by exome and Sanger sequencing, and haplotypes by SNP array. Tooth colour, roughness, density, nanohardness, minerals and ultrastructure were investigated. RESULTS: Ten participants were heterozygous for the FAM83H mutation c.1387C>T (p.Gln463*). All shared a 3.43 Mbp region on chromosome 8q24.3 encompassing the FAM83H variant, indicating a common ancestry. The c.1387C>T was estimated to be 23.8 generations or 600 years. The FAM83H enamel had higher roughness and lower lightness, density, nanohardness, and calcium and phosphorus levels than controls. Blunted enamel rods, wide interrod spaces and disorganised dentinoenamel junctions were observed. Evaluating the patients with the same mutation and reviewing others with different mutations in FAM83H revealed that the FAM83H heterogeneous phenotypes are age-influenced. Tooth colour and surface texture change with ageing. CONCLUSIONS: FAM83H enamel demonstrated decreased lightness, density, hardness, calcium, phosphorus and defective ultrastructure. We have identified that the phenotypic variation in FAM83H-associated ADHCAI is age-related. Awareness of the correlation between age and clinical features of FAM83H-ADHCAI can help dentists make an accurate diagnosis.

MBTPS2, a membrane bound protease, underlying several distinct skin and bone disorders.

The MBTPS2 gene on the X-chromosome encodes the membrane-bound transcription factor protease, site-2 (MBTPS2) or site-2 protease (S2P) which cleaves and activates several signaling and regulatory proteins from the membrane. The MBTPS2 is critical for a myriad of cellular processes, ranging from the regulation of cholesterol homeostasis to unfolded protein responses. While its functional role has become much clearer in the recent years, how mutations in the MBTPS2 gene lead to several human disorders with different phenotypes including Ichthyosis Follicularis, Atrichia and Photophobia syndrome (IFAP) with or without BRESHECK syndrome, Keratosis Follicularis Spinulosa Decalvans (KFSD), Olmsted syndrome, and Osteogenesis Imperfecta type XIX remains obscure. This review presents the biological role of MBTPS2 in development, summarizes its mutations and implicated disorders, and discusses outstanding unanswered questions.

The Thai reference exome (T-REx) variant database.

To maximize the potential of genomics in medicine, it is essential to establish databases of genomic variants for ethno-geographic groups that can be used for filtering and prioritizing candidate pathogenic variants. Populations with non-European ancestry are poorly represented among current genomic variant databases. Here, we report the first high-density survey of genomic variants for the Thai population, the Thai Reference Exome (T-REx) variant database. T-REx comprises exome sequencing data of 1092 unrelated Thai individuals. The targeted exome regions common among four capture platforms cover 30.04 Mbp on autosomes and chromosome X. 345 681 short variants (18.27% of which are novel) and 34 907 copy number variations were found. Principal component analysis on 38 469 single nucleotide variants present worldwide showed that the Thai population is most genetically similar to East and Southeast Asian populations. Moreover, unsupervised clustering revealed six Thai subpopulations consistent with the evidence of gene flow from neighboring populations. The prevalence of common pathogenic variants in T-REx was investigated in detail, which revealed subpopulation-specific patterns, in particular variants associated with erythrocyte disorders such as the HbE variant in HBB and the Viangchan variant in G6PD. T-REx serves as a pivotal addition to the current databases for genomic medicine.

Expanding the genotypic spectrum of PYCR2 and a common ancestry in Thai patients with hypomyelinating leukodystrophy 10.

PYCR2 pathogenic variants lead to an autosomal recessive hypomyelinating leukodystrophy 10 (HLD10), characterized by global developmental delay, microcephaly, facial dysmorphism, movement disorder, and hypomyelination. This study identified the first two unrelated Thai patients with HLD10. Patient 1 harbored the novel compound heterozygous variants, c.257T>G (p.Val86Gly) and c.400G>A (p.Val134Met), whereas patient 2 possessed the homozygous variant, c.400G>A (p.Val134Met), in PYCR2. Haplotype analysis revealed that the two families' members shared a 2.3 Mb region covering the c.400G>A variant, indicating a common ancestry. The variant was estimated to age 1450 years ago. Since the c.400G>A was detected in three out of four mutant alleles and with a common ancestry, this variant might be common in Thai patients. We also reviewed the phenotype and genotype of all 35 previously reported PYCR2 patients and found that majorities of cases were homozygous with a consanguineous family history, except patient 1 and another reported case who were compound heterozygous. All patients had microcephaly and developmental delay. Hypotonia and peripheral spasticity were common. Hypomyelination or delayed myelination was a typical radiographic feature. Here, we report the first two Thai patients with HLD10 with the novel PYCR2 variants expanding the genotypic spectrum and suggest that the c.400G>A might be a common mutation in Thai patients.

Tooth ultrastructure of a novel COL1A2 mutation expanding its genotypic and phenotypic spectra.

OBJECTIVES: To investigate tooth ultrastructure and mutation of two patients in a family affected with osteogenesis imperfecta (OI) type IV and dentinogenesis imperfecta (DGI). METHODS: Mutations were detected by whole exome and Sanger sequencing. The permanent second molar obtained from the proband (DGI1) and the primary first molar from his affected son (DGI2) were studied for their color, roughness, mineral density, hardness, elastic modulus, mineral content, and ultrastructure, compared to the controls. RESULTS: Two novel missense COL1A2 variants, c.752C > T (p.Ser251Phe) and c.758G > T (p.Gly253Val), were identified in both patients. The c.758G > T was predicted to be the causative mutation. Pulp cavities of DGI1 (permanent teeth) were obliterated while those of DGI2 (primary teeth) were wide. The patients' teeth had darker and redder colors; reduced dentin hardness; decreased, disorganized, and scattered dentinal tubules and collagen fibers; and irregular dentinoenamel junction (DEJ), compared to controls. Lacunae-like structures were present in DGI2. CONCLUSIONS: We reported the novel causative mutation, c.758G > T (p.Gly253Val), in COL1A2 for OI type IV and DGI. The DGI dentin demonstrated inferior mechanical property and ultrastructure, suggesting severe disturbances of dentin formation. These could contribute to fragility and prone to infection of DGI teeth. This study expands phenotypic and genotypic spectra of COL1A2 mutations.

Human dental pulp stem cell responses to different dental pulp capping materials.

BACKGROUND: Direct pulp capping is a vital pulp therapy for a pin-point dental pulp exposure. Applying a pulp capping material leads to the formation of a dentin bridge and protects pulp vitality. The aim of this study was to compare the effects of four dental materials, DyCal®, ProRoot® MTA, Biodentine™, and TheraCal™ LC in vitro. METHODS: Human dental pulp stem cells (hDPs) were isolated and characterized. Extraction medium was prepared from the different pulp capping materials. The hDP cytotoxicity, proliferation, and migration were examined. The odonto/osteogenic differentiation was determined by alkaline phosphatase, Von Kossa, and alizarin red s staining. Osteogenic marker gene expression was evaluated using real-time polymerase chain reaction. RESULTS: ProRoot® MTA and Biodentine™ generated less cytotoxicity than DyCal® and TheraCal™ LC, which were highly toxic. The hDPs proliferated when cultured with the ProRoot® MTA and Biodentine™ extraction media. The ProRoot® MTA and Biodentine™ extraction medium induced greater cell attachment and spreading. Moreover, the hDPs cultured in the ProRoot® MTA or Biodentine™ extraction medium migrated in a similar manner to those in serum-free medium, while a marked reduction in cell migration was observed in the cells cultured in DyCal® and TheraCal™ LC extraction media. Improved mineralization was detected in hDPs maintained in ProRoot® MTA or Biodentine™ extraction medium compared with those in serum-free medium. CONCLUSION: This study demonstrates the favorable in vitro biocompatibility and bioactive properties of ProRoot® MTA and Biodentine™ on hDPs, suggesting their superior regenerative potential compared with DyCal® and TheraCal™.

Four novel mutations of FAM20A in amelogenesis imperfecta type IG and review of literature for its genotype and phenotype spectra.

Amelogenesis imperfecta type IG (AI1G) is caused by mutations in FAM20A. Genotypic and phenotypic features of AI1G are diverse and their full spectra remain to be characterized. The aim of this study was to identify and summarize variants in FAM20A in a broad population of patients with AI1G. We identified a Thai female (Pt-1) and a Saudi male (Pt-2) affected with AI1G. Both had hypoplastic enamel, gingival hyperplasia, and intrapulpal calcification. Pt-1 also had rapidly progressive embedding of unerupted teeth, early eruption of permanent teeth, and spontaneous dental infection. Uniquely, Pt-2 had all permanent teeth erupted which was uncommon in AI1G patients. Whole exome sequencing (WES) identified that Pt-1 was heterozygous for FAM20A, c.758A > G (p.Tyr253Cys), inherited from her father. The mutation on maternal allele was not detected by WES. Pt-2 possessed compound heterozygous mutations, c.1248dupG (p.Phe417Valfs*7); c.1081C > T (p.Arg361Cys) in FAM20A. Array comparative genomic hybridization (aCGH), cDNA sequencing, and whole genome sequencing successfully identified 7531 bp deletion on Pt-1's maternal allele. This was the largest FAM20A deletion ever found. A review of all 70 patients from 50 independent families with AI1G (including two families in this study) showed that the penetrance of hypoplastic enamel and gingival hyperplasia was complete. Unerupted permanent teeth were found in all 70 patients except Pt-2. Exons 1 and 11 were mutation-prone. Most mutations were frameshift. Certain variants showed founder effect. To conclude, this study reviews and expands phenotypic and genotypic spectra of AI1G. A large deletion missed by WES can be detected by WGS. Hypoplastic enamel, gingival hyperplasia, and unerupted permanent teeth prompt genetic testing of FAM20A. Screening of nephrocalcinosis, early removal of embedded teeth, and monitoring of dental infection are recommended.

Genotype-phenotype correlation and expansion of orodental anomalies in LTBP3-related disorders.

The latent transforming growth factor-beta-binding protein 3 (LTBP3), encoding extracellular matrix proteins, plays a role in skeletal formation. Mutations in LTBP3 have been associated with various types of skeletal dysplasia. We aimed to characterize clinical and molecular features of more patients with mutations in the gene, which may help suggest genotype-phenotype correlation. The first two East Asian patients with short stature, heart defects, and orodental anomalies having LTBP3 mutations were identified. Whole exome and Sanger sequencing revealed that the one with a novel heterozygous missense (c.2017G>T, p.Gly673Cys) mutation in LTBP3 had clinical features consistent with acromicric dysplasia (ACMICD). The variant was located in the highly conserved EGF-like calcium-binding domain adjacent to the single reported LTBP3 variant associated with ACMICD. This finding supports that LTBP3 is a disease gene for ACMICD. Another patient with a novel homozygous splice site acceptor (c.1721-2A>G) mutation in LTBP3 was affected with dental anomalies and short stature (DASS). Previously undescribed orodental features included multiple unerupted teeth, high-arched palate, and microstomia found in our patient with ACMICD, and extensive dental infection, condensing osteitis, and deviated alveolar bone formation in our patient with DASS. Our results and comprehensive reviews suggest a genotype-phenotype correlation: biallelic loss-of-function mutations cause DASS, monoallelic missense gain-of-function mutations in the EGF-like domain cause ACMICD, and monoallelic missense gain-of-function mutations with more drastic effects on the protein functions cause geleophysic dysplasia (GPHYSD3). In summary, we expand the phenotypic and genotypic spectra of LTBP3-related disorders, support that LTBP3 is a disease gene for ACMICD, and propose the genotype-phenotype correlation of LTBP3 mutations.