Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

The impact of craniofacial and dental osteogenesis imperfecta manifestations on oral health-related quality of life of children and adolescents.

OBJECTIVE: Craniofacial and oral manifestations of Osteogenesis Imperfecta (OI) can affect the functioning of the stomatognathic system and impact the patient's quality of life. The objective of the study was to evaluate the relationship between craniofacial and oral manifestations and the Oral Health-related Quality of Life (OHRQoL) of OI children and adolescents. MATERIAL AND METHODS: A total of 30 OI patients aged eight to fourteen years old followed up at the Oral Care Center for Inherited Diseases were enrolled in the research. OHRQoL was assessed using the short form of the Child Perceptions Questionnaire (CPQ) for eight to ten-year-olds (CPQ8-10) and 11 to 14-year-olds (CPQ11-14). The relationship between the OHRQoL index and its assessment domains, OI types, and the presence of dentinogenesis imperfecta (DI), class III malocclusion, and dental agenesis were evaluated. RESULTS: The median CPQ score of patients was 5, and there was no significant difference in OHRQoL between children and adolescents, nor associated with the disease severity or the presence of DI. The oral manifestations evaluated did not directly impact the patients' OHRQoL. CONCLUSIONS: The study demonstrated that the perception of OHRQoL is similar for both adolescents and children. The oral symptom was the most relevant domain for the index among patients aged eight to fourteen years while the emotional well-being was the most impacted. CLINICAL RELEVANCE: this study makes contributions by indicating that addressing dental care for children and adolescents with OI is important in clinical management and better OHRQoL for this population.

Oro-dental phenotyping and report of three families with RELT-associated amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of rare genetic conditions characterized by quantitative and/or qualitative tooth enamel alterations. AI can manifest as an isolated trait or as part of a syndrome. Recently, five biallelic disease-causing variants in the RELT gene were identified in 7 families with autosomal recessive amelogenesis imperfecta (ARAI). RELT encodes an orphan receptor in the tumor necrosis factor (TNFR) superfamily expressed during tooth development, with unknown function. Here, we report one Brazilian and two French families with ARAI and a distinctive hypomineralized phenotype with hypoplastic enamel, post-eruptive enamel loss, and occlusal attrition. Using Next Generation Sequencing (NGS), four novel RELT variants were identified (c.120+1G>A, p.(?); c.120+1G>T, p.(?); c.193T>C, p.(Cys65Arg) and c.1260_1263dup, p.(Arg422Glyfs*5)). Our findings extend the knowledge of ARAI dental phenotypes and expand the disease-causing variants spectrum of the RELT gene.

Carbonic anhydrase II deficiency syndrome with amelogenesis imperfecta linked to a homozygous CA2 deletion.

We performed a study to present a phenotypic and genotypic characterization of a patient clinically diagnosed with carbonic anhydrase II (CAII) deficiency syndrome. Medical records were reviewed, and oral examination was performed. Sanger sequencing was undertaken for molecular diagnosis. The patient presented with osteopetrosis, renal tubular acidosis, cerebral calcification, blindness, deafness, and development delay. The oral manifestations included anterior open bite, posterior crossbite, tooth eruption impairment, and hypoplastic amelogenesis imperfecta (AI). Molecular analysis revealed a CA2 homozygous deletion (c.753delG, p.Asn252Thrfs*14) and confirmed the clinical diagnosis. This study suggests that AI can be another feature of CAII deficiency syndrome. For the first time, a CA2 disease-causing variant is reported to be associated with syndromic AI.

Pamidronate Therapy Increases Trabecular Bone Complexity of Mandibular Condyles in Individuals with Osteogenesis Imperfecta.

Patients with Osteogenesis Imperfecta (OI) present extra-skeletal manifestations, including important orodental and craniofacial features as dentinogenesis imperfecta, dental agenesis, failure of maxilla growth and hypotonia of masticatory muscles. These features may compromise vital functions speech and mastication. Studies have demonstrated that cyclic pamidronate infusion, the standard therapy for patients with moderate to severe OI, influences the histomorphometric pattern of different body bones. The present study aimed to investigate the condyle trabecular bone pattern in OI patients. We used fractal dimension (FD) analysis on dental panoramic radiographic images to characterize the mandibular condyle trabecular bone in adolescents diagnosed with OI and treated with pamidronate. Imaging exam of 33 adolescents of both sexes, aged between 12 and 17 years, were analyzed and compared with 99 age- and sex-matched healthy adolescents. FD in patients was significantly lower (1.23 ± 0.15) than in healthy controls (1.29 ± 0.11; p < 0.01). Type of OI, age at treatment onset, and the duration of therapy were variables that showed a statistically significant effect on the FD results. This study demonstrated that the bone architecture of mandibular condyles may be altered in pediatric patients with moderate and severe forms of OI. Also, pamidronate treatment seems to have a positive effect on condyle trabecular bone in these patients. This is supported by our finding that FD values were positively influenced by the length of cyclic pamidronate treatment at the time of imaging, as well as by the age of the individual at treatment onset.

Phenotypic dento-osseous characterization of a Brazilian family with Familial Adenomatous Polyposis.

OBJECTIVE: To perform a phenotypic characterization of the dento-osseous anomalies in a Brazilian family with Familial Adenomatous Polyposis (FAP) and to investigate the adenomatous polyposis coli (APC) causative variant. DESIGN: The study included a family of 14 individuals (Group A: affected; Group B: non-affected). The frequency of radiographic findings in both groups was evaluated according to the Dental Panoramic Radiograph Score (DPRS) diagnostic method. The accuracy and reproducibility of DPRS were tested. The DNA was isolated from the index patient's saliva and submitted to whole-exome and Sanger sequencing approach. RESULTS: DPRS ≥ 7 was observed in 80 % of Group A but in none of Group B. The most common findings in Group A were dense bone islands (60 %), hazy sclerosis (40 %), osteomas (40 %), and supernumerary tooth (20 %). DPRS has proved to be a reliable method while DPRS ≥ 5 and DPRS ≥ 7 were taken as positive for FAP, and reproducible diagnosis test considering that the evaluators correctly identified the affected patients (Kappa agreement>0.8, p = 0.002). A nonsense heterozygous mutation in the APC gene (c.1370C > G; p.Ser457*) of the index case was detected. CONCLUSION: FAP patients have a higher frequency of dento-osseous anomalies (p = 0.005). Bone abnormalities were more prevalent than dental anomalies (p = 0.001). Thus, FAP patients should be referred for dental examination and genetic counseling to perform early diagnosis of dento-osseous anomalies and evaluate the implications of the molecular findings in each particular family.

Restorations after selective caries removal: 5-Year randomized trial.

OBJECTIVE: To compare the survival of restorations placed in deep caries lesions after selective caries removal to soft dentin (SCRSD) over a 5-year period. A secondary aim was to investigate whether the material (amalgam or resin composite) affected the survival of restorations. METHODS: This study used data derived from a multicenter randomized controlled clinical trial (Clinical trials registration NCT00887952). Inclusion criteria were: patients with permanent molars presenting occlusal or proximal deep caries lesions (≥1/2 of the dentin thickness on radiographic examination), positive response to a cold test, absence of spontaneous pain, negative sensitivity to percussion, and absence of periapical lesions. The teeth were randomized into SCRSD and restoration in a single visit or stepwise excavation (SW). Each of these groups was divided according to the filling material: amalgam (AM) or resin composite (RC). Survival analyses were performed to estimate therapy success rates over 5 years (adjusted Weibull regression model). RESULTS: 172 restorations were evaluated, 95 from SCRSD group and 77 from SW group, being 61 AMG and 111 RC. The 5-year survival analysis showed similar success rates for SW (76 %) and SCRSD (79 %) as well as for AM and RC (p > 0.05). CONCLUSION: This study showed that, after a 5-year follow-up period, the presence of decayed tissue beneath restorations in deep caries lesions did not seem to affect restoration survival. Amalgam and resin composite restorations had similar survival rates, irrespective of the caries removal technique used - SCRSD or SW. CLINICAL SIGNIFICANCE: Selective caries removal to soft dentin can be used in the management of deep caries to avoid pulp exposure and preserve tooth structure without affecting restoration longevity.

Amelogenesis imperfecta in familial hypomagnesaemia and hypercalciuria with nephrocalcinosis caused by CLDN19 gene mutations.

BACKGROUND: Amelogenesis imperfecta (AI) is a group of genetic diseases characterised by tooth enamel defects. AI was recently described in patients with familial hypercalciuria and hypomagnesaemia with nephrocalcinosis (FHHNC) caused by CLDN16 mutations. In the kidney, claudin-16 interacts with claudin-19 to control the paracellular passage of calcium and magnesium. FHHNC can be linked to mutations in both genes. Claudin-16 was shown to be expressed during amelogenesis; however, no data are available on claudin-19. Moreover, the enamel phenotype of patients with CLDN19 mutations has never been described. In this study, we describe the clinical and genetic features of nine patients with FHHNC carrying CLDN19 mutations and the claudin-19 expression profile in rat ameloblasts. METHODS: Six FHHNC Brazilian patients were subjected to mutational analysis. Three additional French patients were recruited for orodental characterisation. The expression profile of claudin-19 was evaluated by RT-qPCR and immunofluorescence using enamel epithelium from rat incisors. RESULTS: All patients presented AI at different degrees of severity. Two new likely pathogenic variations in CLDN19 were found: p.Arg200Gln and p.Leu90Arg. RT-qPCR revealed low Cldn19 expression in ameloblasts. Confocal analysis indicated that claudin-19 was immunolocalised at the distal poles of secretory and maturing ameloblasts. CONCLUSIONS: For the first time, it was demonstrated that AI is associated with FHHNC in patients carrying CLDN19 mutations. The data suggest claudin-19 as an additional determinant in enamel formation. Indeed, the coexistence of hypoplastic and hypomineralised AI in the patients was consistent with claudin-19 expression in both secretory and maturation stages. Additional indirect systemic effects cannot be excluded.

Identification of the first large deletion in the CLDN16 gene in a patient with FHHNC and late-onset of chronic kidney disease: case report.

BACKGROUND: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is a rare autosomal recessive renal disease characterized by tubular disorders at the thick ascending limb of Henle's loop. It is caused by mutations in the tight junction structural proteins claudin-16 or claudin-19, which are encoded by the CLDN16 and CLDN19 genes, respectively. Patients exhibit excessive wasting of calcium and magnesium, nephrocalcinosis, chronic kidney disease, and early progression to end-stage renal failure during infancy. CASE PRESENTATION: We here report the phenotype and molecular analysis of a female Brazilian patient with a novel large homozygous deletion in the CLDN16 gene. The proband, born from consanguineous parents, presented the first symptoms at age 20. Clinical examination revealed hypocalcemia, hypomagnesemia, nephrocalcinosis, mild myopia, high serum levels of uric acid and intact parathyroid hormone, and moderate chronic kidney disease (stage 3). She and her mother were subjected to CLDN16 and CLDN19 mutational analysis. In addition, the multiplex ligation-dependent probe amplification method was used to confirm a CLDN16 multi-exon deletion. Direct sequencing revealed a normal CLDN19 sequence and suggested a large deletion in the CLDN16 gene. Multiplex ligation-dependent probe amplification showed a homozygous CLDN16 multi-exon deletion (E2_E5del). The patient initiated conventional treatment for familial hypomagnesemia with hypercalciuria and nephrocalcinosis and progressed to end-stage kidney disease after five years. CONCLUSIONS: This study provides the first report of a large homozygous deletion in the CLDN16 gene causing familial hypomagnesemia with hypercalciuria and nephrocalcinosis with late onset of the first symptoms. This description expands the phenotypic and genotypic characterization of the disease. The late-onset chronic kidney disease in the presence of a homozygous deletion in the CLDN16 gene reinforces the great variability of genotype-phenotype manifestation in patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations.

BACKGROUND: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. METHODS: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. RESULTS: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. CONCLUSIONS: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations.

Pathognomonic oral profile of Enamel Renal Syndrome (ERS) caused by recessive FAM20A mutations.

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects. Commonly described as an isolated trait, it may be observed concomitantly with other orodental and/or systemic features such as nephrocalcinosis in Enamel Renal Syndrome (ERS, MIM#204690), or gingival hyperplasia in Amelogenesis Imperfecta and Gingival Fibromatosis Syndrome (AIGFS, MIM#614253). Patients affected by ERS/AIGFS present a distinctive orodental phenotype consisting of generalized hypoplastic AI affecting both the primary and permanent dentition, delayed tooth eruption, pulp stones, hyperplastic dental follicles, and gingival hyperplasia with variable severity and calcified nodules. Renal exam reveals a nephrocalcinosis which is asymptomatic in children affected by ERS. FAM20A recessive mutations are responsible for both syndromes. We suggest that AIGFS and ERS are in fact descriptions of the same syndrome, but that the kidney phenotype has not always been investigated fully in AIGFS. The aim of this review is to highlight the distinctive and specific orodental features of patients with recessive mutations in FAM20A. We propose ERS to be the preferred term for all the phenotypes arising from recessive FAM20A mutations. A differential diagnosis has to be made with other forms of AI, isolated or syndromic, where only a subset of the clinical signs may be shared. When ERS is suspected, the patient should be assessed by a dentist, nephrologist and clinical geneticist. Confirmed cases require long-term follow-up. Management of the orodental aspects can be extremely challenging and requires the input of multi-disciplinary specialized dental team, especially when there are multiple unerupted teeth.

In vitro study of laser diode 655 nm diagnosis of occlusal caries.

The aim of this in vitro study was to evaluate the performance of a new laser method (DIAGNOdent, KaVo) by comparing it to the visual inspection, conventional bitewing radiography and digital bitewing radiography. Fifty fresh permanent teeth with clinically sound or suspicious fissures were selected. The teeth were submitted to the diagnostic tests by 2 examiners independently after calibration. The laser examination was repeated to assess the reproducibility. The teeth were sectioned right in the spot that showed the highest value by the laser device. Histological examination of sections (40x magnification) served as gold standard. The results showed that: 1) the intra and interobserver agreement for the DIAGNOdent was excellent (values over 0.87); 2) the sensitivity, specificity and predictive values were very high; 3) and the Spearman correlation coefficient of DIAGNOdent was the best one (R = 0.81), followed by visual inspection (R = 0.68).