Luciane Martins

G-quadruplex formation enhances splicing efficiency of PAX9 intron 1

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon–intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

Correction of hypophosphatasia-associated mineralization deficiencies in vitro by phosphate/pyrophosphate modulation in periodontal ligament cells.

BACKGROUND Mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene in hypophosphatasia (HPP) reduce the function of tissue non-specific alkaline phosphatase (ALP), resulting in increased pyrophosphate (PP(i)) and a severe deficiency in acellular cementum. We hypothesize that exogenous phosphate (P(i)) would rescue the in vitro mineralization capacity of periodontal ligament (PDL) cells harvested from HPP-diagnosed patients, by correcting the P(i)/PP(i) ratio and modulating expression of genes involved with P(i)/PP(i) metabolism. METHODS Ex vivo and in vitro analyses were used to identify mechanisms involved in HPP-associated PDL/tooth root deficiencies. Constitutive expression of PP(i)-associated genes was contrasted in PDL versus pulp tissues obtained from healthy individuals. Primary PDL cell cultures from patients with HPP (monozygotic twin males) were established to assay ALP activity, in vitro mineralization, and gene expression. Exogenous P(i) was provided to correct the P(i)/PP(i) ratio. RESULTS PDL tissues obtained from healthy individuals featured higher basal expression of key PP(i) regulators, genes ALPL, progressive ankylosis protein (ANKH), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), versus paired pulp tissues. A novel ALPL mutation was identified in the twin patients with HPP enrolled in this study. Compared to controls, HPP-PDL cells exhibited significantly reduced ALP and mineralizing capacity, which were rescued by addition of 1 mM P(i). Dysregulated expression of PP(i) regulatory genes ALPL, ANKH, and ENPP1 was also corrected by adding P(i), although other matrix markers evaluated in our study remained downregulated. CONCLUSION These findings underscore the importance of controlling the P(i)/PP(i) ratio toward development of a functional periodontal apparatus and support P(i)/PP(i) imbalance as the etiology of HPP-associated cementum defects.

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities.

Hypophosphatasia-associated Deficiencies in Mineralization and Gene Expression in Cultured Dental Pulp Cells Obtained from Human Teeth

INTRODUCTION Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PP(i)) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (P(i)) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes. METHODS Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous P(i) was provided to the correct P(i)/PP(i) ratio in cell culture. RESULTS HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PP(i) regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of P(i) provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PP(i)-associated genes. CONCLUSIONS These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP.

Novel ALPL genetic alteration associated with an odontohypophosphatasia phenotype

Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism caused by mutations in ALPL, encoding tissue non-specific alkaline phosphatase (TNAP). Here, we report the molecular findings from monozygotic twins, clinically diagnosed with tooth-specific odontohypophosphatasia (odonto-HPP). Sequencing of ALPL identified two genetic alterations in the probands, including a heterozygous missense mutation c.454C>T, leading to change of arginine 152 to cysteine (p.R152C), and a novel heterozygous gene deletion c.1318_1320delAAC, leading to the loss of an asparagine residue at codon 440 (p.N440del). Clinical identification of low serum TNAP activity, dental abnormalities, and pedigree data strongly suggests a genotype-phenotype correlation between p.N440del and odonto-HPP in this family. Computational analysis of the p.N440del protein structure revealed an alteration in the tertiary structure affecting the collagen-binding site (loop 422-452), which could potentially impair the mineralization process. Nevertheless, the probands (compound heterozygous: p.[N440del];[R152C]) feature early-onset and severe odonto-HPP phenotype, whereas the father (p.[N440del];[=]) has only moderate symptoms, suggesting p.R152C may contribute or predispose to a more severe dental phenotype in combination with the deletion. These results assist in defining the genotype-phenotype associations for odonto-HPP, and further identify the collagen-binding site as a region of potential structural importance for TNAP function in the biomineralization.

Computational analysis for GNAQ mutations: New insights on the molecular etiology of Sturge-Weber syndrome

Somatic activating mutations in the GNAQ have been recently associated with several congenital genetic disorders and tumors; however, the molecular mechanism/etiology that leads to GNAQ somatic mosaic mutation are unknown. Here, we reported a case of Sturge-Weber Syndrome (SWS) manifesting cutaneous vascular malformations (hemifacial Port-wine stain), cerebral and ocular vascular abnormalities (including epilepsy and glaucoma) and harboring a c.548G>A (p.R183Q) somatic mosaic mutation in GNAQ. Computational modeling studies were performed to assistant with the comprehension of the functional impact of p.R183Q and p.Q209L mutations in GNAQ, which encodes a G protein subunit alpha q (Gαq). The p.R183Q mutation was predicted to abolish hydrogen bonds between R183 residue and GDP molecule, destabilizing the inactive GDP-bound conformation of the Gαq mutants. Furthermore, replacement of R183 by Q183 residue was predicted to promote conformation changes in protein surface features affecting the switch I region, a key region that undergoes conformational changes triggered by receptor binding during signal transduction. In addition, replacement of Q209 by L209 residue was predicted to affect the molecular interaction between Gαq and Gβ subunit, impairing formation of the inactive heterotrimeric complex. These findings, in association with PPI network analysis, indicate that p.R183Q and p.Q209L mutations result in the over-activation of different downstream effectors, which in turn will determine the distinct cell responses and phenotype. These findings bring new insights on molecular etiology of vascular malformations associated to SWS and on different mechanisms underlying hyperactivation of downstream pathways to Gαq.

EDA mutation by exome sequencing in non-syndromic X-linked oligodontia

To the Editor: Oligodontia is a severe form of selective tooth agenesis (STHAG), a human condition characterized by failure of development of multiple teeth during organogenesis, which can be inherited as an isolated trait or as part of a recognized genetic syndrome. STHAG exhibits high genetic heterogeneity, as result of a number of mutations in genes known to regulate tooth development (1), and many aspects underlying its genetic causes remain elusive. Here, we used a whole-exome sequencing (WES) strategy followed by in silico prediction tools and 3D structure analysis to determine genomic variants associate with non-syndromic oligodontia in an 8-year-old Brazilian Caucasian boy. This study was approved by the Piracicaba Dental School – Institutional Review Board. Detailed clinical and WES information are available at PhenomeCentral (P0003267; https://phenomecentral.org/). Candidate variants were identified after filtering for rare variants (MAF< 1%), protein-coding regions (including non-sense, non-synonymous variants and Indels), splice-site variants absent from public datasets, and putative inheritance pattern (autosomal dominant and X-linked). Next, considering the role of EDA (MIM#300451) and TBX22 (MIM#300307) in craniofacial and tooth development anomalies, we performed mutational analysis by Sanger sequencing to validate segregation patterns for variants in EDA c.1013C>T (p.T338M) and TBX22 c.1033T>C (p.S345P) in 10 related family members. A positive association between STHAG phenotype and segregation patterns for EDA and TBX22 mutations was observed in this family, confirming an X-linked dominant inheritance pattern (Fig. 1a). However, in silico prediction only supported the variant in EDA (c.1013C>T; dbSNP ID rs132630321) as pathogenic (ClinVar ID 11048), deleterious (SIFT score= 0), probably damaging (PolyPhen= 0.974) and probably deleterious (MutationTaster= 0.99997), whereas the variant in TBX22 was found to be tolerated (SIFT score= 1), benign (PolyPhen= 0) and probably harmless (MutationTaster= 0.00005). Phylogenetic analysis supported these findings (Fig. 1b,c). Here, clinical findings are similar to a previous study on a Chinese family, where the affected male, who bearing only one copy of c.1013C>T mutation (hemizygous), showed a more severe phenotype (2). In addition, however, we found that heterozygous female carriers for this EDA mutation (Fig. 1a) may present a highly variable and milder dental phenotype. Against our hypothesis, the novel variant in TBX22 c.1033 T>C (p.S345P), identified in this study, was not critical for the broad phenotypic heterogeneity displayed by heterozygous female carriers, pointing to EDA as the solely causative gene of the congenitally missing teeth in this family. Recent studies have provided evidence that mutation in the TNF homology domain of ectodysplasin A (EDA) is implicated in familial non-syndromic STHAG, and heterozygous female carriers of EDA mutations may present STHAG with a considerable variation in clinical expression as result of differential pattern of X-chromosome inactivation (3), or still as result of modifier effects in other signaling pathways regulated by EDA-1A (4). The EDA gene is located on chromosome Xq13.1 and encodes isoforms of EDA. Among the isoforms, EDA-A1 (Q92838) is a trimeric type II membrane protein, and a collagen domain-containing member of the superfamily of the TNF ligands, involved in the early epithelial–mesenchymal interactions, and playing a critical role in tooth development. Here, analysis of the tertiary structure of EDA-A1 protein showed that the T338 residue is located in coil structure (corresponding to 337–342 amino acid) within the phylogenetically conserved TNF homology domain, on the outer surface of the molecule (Fig. 1c-e). Replacement of T338 native (hydrophilic) by an M338 mutated residue (hydrophobic) was predicted to alter the conformation of the 337–342 loop leading to a potential impact on EDA-A1 receptor binding properties (Fig. 1d-e). Furthermore, residue interaction analysis indicated that p.T338M mutation might lead to an additional hydrogen bond between chain B (T341) and chain C (D316) (Fig. 1g), suggesting that such mutation has the potential to impact on homotrimer stability. Moreover, because our in silico prediction analysis showed that residues within the 337–342 loop form hydrophobic interaction and hydrogen bonds, with other molecule regions (such as I312 and F314) critical for controlling receptor specificity (Fig. 1f-g) (5), our findings bring new evidences about the role of p.T338M mutation in the recognition/specificity of EDA to its receptor, resulting in a high heterogeneous STHAG phenotype.

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Variable expressivity and novel PTEN mutations in Cowden syndrome

Cowden syndrome (CS) is a phosphatase and tensin homolog gene (PTEN)-associated condition characterized by multiple mucocutaneous hamartomas and an increased risk of malignancies. We reported an isolated case and another of several individuals in one family affected by CS. The isolated case showed typical features, including fibrocystic breast disease, benign thyroid nodules, and multiple papillomatous lesions in the face and oral cavity, and the cause was a novel nonsense mutation-guanine (G) to thymine (T) transition at position 940 (c.940 G>T)-in PTEN. In the family, the proband showed erythema nodosum, duodenal ulcer, intestinal polyps, cervical lipoma, renal cysts, and glaucoma, whereas multiple members of her family were found to have intestinal polyps, and a sister had been diagnosed with breast cancer at early age. An intronic mutation-T>G transition at the +32 position of intron 8 (c.1026+32 T>G)-was found in this family, with in silico analysis revealing the creation of a new donor splice site. This study confirmed the involvement of PTEN in CS and the variable clinical expressivity of disease.

Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires Flotillin-1 Mediated Endocytosis.

Basic, pre‐clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine‐Rich Amelogenin Peptide—LRAP), which is supposed to act as a signaling molecule in epithelial–mesenchymal interactions. LRAP‐binding protein complexes from immortalized murine cementoblasts (OCCM‐30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin‐1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co‐precipitation and co‐localization assays as a protein‐binding partner for LRAP in OCCM‐30 cells. In addition, we found that exogenously added GST‐LRAP recombinant protein was internalized by OCCM‐30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1‐dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM‐30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt‐related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin‐assisted endocytosis, which suggests an involvement of LRAP in lipid‐raft‐dependent signaling pathways which are mediated by flotillin‐1. J. Cell. Physiol. 232: 556–565, 2017.