R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Identification of nuclear localization signals in the human homeoprotein MSX1.

MSX1 is one of the homeoproteins with the homeodomain (HD) sequence, which regulates proliferation and differentiation of mesenchymal cells. In this study, we investigated the nuclear localization signal (NLS) in the MSX1 HD by deletion and amino acid substitution analyses. The web-based tool NLStradamus predicted 2 putative basic motifs in the N- and C-termini of the MSX1 HD. Green fluorescent protein (GFP) chimera studies revealed that NLS1 (161RKHKTNRKPR170) and NLS2 (216NRRAKAKR223) were independently insufficient for robust nuclear localization. However, they can work cooperatively to promote nuclear localization of MSX1, as was shown by the 2 tandem NLS motifs partially restoring functional NLS, leading to a significant nuclear accumulation of the GFP chimera. These results demonstrate a unique NLS motif in MSX1, which consists of an essential single core motif in helix-I, with weak potency, and an auxiliary subdomain in helix-III, which alone does not have nuclear localization potency. Additionally, other peptide sequences, other than predicted 2 motifs in the spacer, may be necessary for complete nuclear localization in MSX1 HD.

Characterisation of novel RUNX2 mutation with alanine tract expansion from Japanese cleidocranial dysplasia patient.

Cleidocranial dysplasia (CCD; MIM 119600) is an autosomal dominant skeletal dysplasia characterised by hypopalstic and/or aplastic clavicles, midface hypoplasia, absent or delayed closure of cranial sutures, moderately short stature, delayed eruption of permanent dentition and supernumerary teeth. The molecular pathogenesis can be explained in about two-thirds of CCD patients by haploinsufficiency of the RUNX2 gene. In our current study, we identified a novel and rare variant of the RUNX2 gene (c.181_189dupGCGGCGGCT) in a Japanese patient with phenotypic features of CCD. The insertion led an alanine tripeptide expansion (+3Ala) in the polyalanine tract. To date, a RUNX2 variant with alanine decapeptide expansion (+10Ala) is the only example of a causative variant of RUNX2 with polyalanine tract expansion to be reported, whilst RUNX2 (+1Ala) has been isolated from the healthy population. Thus, precise analyses of the RUNX2 (+3Ala) variant were needed to clarify whether the tripeptide expanded RUNX2 is a second disease-causing mutant with alanine tract expansion. We therefore investigated the biochemical properties of the mutant RUNX2 (+3Ala), which contains 20 alanine residues in the polyalanine tract. When transfected in COS7 cells, RUNX2 (+3Ala) formed intracellular ubiquitinated aggregates after 24h, and exerted a dominant negative effect in vitro. At 24h after gene transfection, whereas slight reduction was observed in RUNX2 (+10Ala), all of these mutants significantly activated osteoblast-specific element-2, a cis-acting sequence in the promoter of the RUNX2 target gene osteocalcin. The aggregation growth of RUNX2 (+3Ala) was clearly lower and slower than that of RUNX2 (+10Ala). Furthermore, we investigated several other RUNX2 variants with various alanine tract lengths, and found that the threshold for aggregation may be RUNX2 (+3Ala). We conclude that RUNX2 (+3Ala) is the cause of CCD in our current case, and that the accumulation of intracellular aggregates in vitro is related to the length of the alanine tract.

A novel in-frame deletion of the RUNX2 gene causes a classic form of cleidocranial dysplasia.

The runt-related transcription factor 2 (RUNX2) is a physiological regulatory gene implicated in the development of cleidocranial dysplasia (CCD). Molecular analysis of the RUNX2 gene in a 2-year-old boy with a diagnosis of CCD demonstrated a heterozygous in-frame 9-bp deletion (c.593_601delCCTTGACCA, corresponding to the amino-acid deletion p.ΔTLT198_200). Transcription activity of the ΔTLT198_200 mutant decreased in a similar degree to that of the L199F mutant, which was previously reported in the case with classic CCD. Atomic model assessment demonstrated that the ΔTLT198_200 mutation abolished the heterodimerization of the RUNX2 protein with the partner subunit, polyomavirus enhancer-binding protein 2ß (PEBP2ß). Destruction of RUNX2/PEBP2ß heterodimerization activity appears to impair the function of the RUNX2 protein and cause the disease.

Novel nonsense mutation in MSX1 in familial nonsyndromic oligodontia: subcellular localization and role of homeodomain/MH4.

Nonsyndromic tooth agenesis is one of the most common anomalies in human development. Part of the malformation is inherited and is associated with paired box 9 (PAX9), msh homeobox 1 (MSX1), and axin 2 (AXIN2) mutations. To obtain a comprehensive understanding of the genetic and molecular mechanisms that underlie this genetic disease, we investigated six familial and seven sporadic Japanese cases of nonsyndromic tooth agenesis. Searches for mutations in these candidate genes detected a novel nonsense mutation (c.416G>A) in exon 1 of MSX1 from a family with oligodontia. This mutation co-segregated in the affected family members. Moreover, this mutation produced a termination codon in the first exon and therefore the gene product (W139X) was truncated at the C terminus, hence, the entire homeodomain/MH4, which has many functions, such as DNA binding, protein-protein interaction, and nuclear localization, was absent. We characterized the properties of this truncated MSX1 by investigating the subcellular localization of the mutant gene product in transfected cells. The wild-type MSX1 localized exclusively at the nuclear periphery of transfected cells, whereas the mutant MSX1 was stable but localized diffusely throughout the whole cell. These results indicate that W139X MSX1 is responsible for tooth agenesis.

Novel frameshift variant of WNT10A in a Japanese patient with hypodontia.

Congenital tooth agenesis is caused by the impairment of crucial genes related to tooth development, such as Wnt signaling pathway genes. Here, we investigated the genetic causes of sporadic congenital tooth agenesis. Exome sequencing, followed by Sanger sequencing, identified a novel single-nucleotide deletion in WNT10A (NC_000002.12(NM_025216.3):c.802del), which was not found in the healthy parents of the patient. Thus, we concluded that the variant was the genetic cause of the patient's agenesis.

Novel WNT10A variant in a Japanese case of nonsyndromic oligodontia.

Congenital tooth agenesis is one of the most common anomalies in humans. Many genetic factors are involved in tooth development, including MSX1, PAX9, WNT10A, and LRP6. Thus, mutations in these genes can cause congenital tooth agenesis in humans. In this study, we identified a novel nonsense WNT10A variant, NM_025216.3(WNT10A_v001):c.1090A > T, which produces a C-terminal truncated gene product, p.(Lys364*), in a sporadic form of congenital tooth agenesis. The variant was not found in the healthy parents and thus was considered to cause congenital tooth agenesis in the case.

Novel MSX1 frameshift mutation in a Japanese family with nonsyndromic oligodontia.

Congenital tooth agenesis is a common anomaly in humans. We investigated the etiology of human tooth agenesis by exome analysis in Japanese patients, and found a previously undescribed heterozygous deletion (NM_002448.3(MSX1_v001):c.433_449del) in the first exon of the MSX1 gene. The deletion leads to a frameshift and generates a premature termination codon. The truncated form of MSX1, namely, p.(Trp145Leufs*24) lacks the homeodomain, which is crucial for transcription factor function.

A novel LRP6 variant in a Japanese family with oligodontia.

Congenital tooth agenesis is a common anomaly in human development. We performed exome sequence analysis of genomic DNA collected from Japanese patients with tooth agenesis and their relatives. We found a novel single-nucleotide insertion in the LRP6 gene, the product of which is involved in Wnt/ß-catenin signaling as a coreceptor for Wnt ligands. The single-nucleotide insertion results in a premature stop codon in the extracellular region of the encoded protein.

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice.

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, interacts with pleiotrophin, a heparin-binding growth factor.

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.

Development of tooth regenerative medicine strategies by controlling the number of teeth using targeted molecular therapy.

Analysis of various genetically modified mice, with supernumerary teeth, has revealed the following two intrinsic molecular mechanisms that increase the number of teeth. One plausible explanation for supernumerary tooth formation is the rescue of tooth rudiments. Topical application of candidate molecules could lead to whole tooth formation under suitable conditions. Congenital tooth agenesis is caused by the cessation of tooth development due to the deletion of the causative gene and suppression of its function. The arrest of tooth development in Runx2 knockout mice, a mouse model of congenital tooth agenesis, is rescued in double knockout mice of Runx2 and Usag-1. The Usag-1 knockout mouse is a supernumerary model mouse. Targeted molecular therapy could be used to generate teeth in patients with congenital tooth agenesis by stimulating arrested tooth germs. The third dentition begins to develop when the second successional lamina is formed from the developing permanent tooth in humans and usually regresses apoptotically. Targeted molecular therapy, therefore, seems to be a suitable approach in whole-tooth regeneration by the stimulation of the third dentition. A second mechanism of supernumerary teeth formation involves the contribution of odontogenic epithelial stem cells in adults. Cebpb has been shown to be involved in maintaining the stemness of odontogenic epithelial stem cells and suppressing epithelial-mesenchymal transition. Odontogenic epithelial stem cells are differentiated from one of the tissue stem cells, enamel epithelial stem cells, and odontogenic mesenchymal cells are formed from odontogenic epithelial cells by epithelial-mesenchymal transition. Both odontogenic epithelial cells and odontogenic mesenchymal cells required to form teeth from enamel epithelial stem cells were directly induced to form excess teeth in adults. An approach for the development of targeted therapeutics has been the local application of monoclonal neutralizing antibody/siRNA with cationic gelatin for USAG-1 or small molecule for Cebpb.

A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death.

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.

Differential effects of two ROCK inhibitors, Fasudil and Y-27632, on optic nerve regeneration in adult cats.

A ROCK inhibitor Fasudil is widely administered to relieve vasospasm in patients after subarachnoid hemorrhage in Japan. We investigated the difference of Fasudil and Y-27632, a common ROCK inhibitor, on neurite regeneration in culture and axonal regeneration after injuring the optic nerve (OpN) in cats. The optimal dose of Y-27632, determined by counting the number and length of neurites in retinal explants, was found to be 100 microM: the only effect of Fasudil was to promote extension of glial processes. We next examined the effects of Fasudil (10 microM-100 microM) and Y-27632 (10 microM-300 microM) on axonal regeneration in the crushed OpN model in vivo. Immediately after crushing the left OpN, Fasudil or Y-27632 was injected into the vitreous and the crushed site. Injection of 10 microM and 100 microM Y-27632 induced extension of the optic axons beyond the crush site, with the latter dosage giving stronger regeneration. Very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection, and no axons elongated in the OpN with Fasudil injection.

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb-/-Runx2+/- Murine Incisors.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Neuroprotective effect of nipradilol, an NO donor, on hypoxic-ischemic brain injury of neonatal rats.

Hypoxia-ischemia is a common cause of neonatal brain injuries. Nitric oxide (NO) is upregulated in the brain after hypoxia-ischemia and generally believed to exert a paradoxical effect on neurons, neurodestruction and neuroprotection, but it has not been demonstrated that NO is actually neuroprotective in neonatal hypoxic-ischemic encephalopathy. We evaluated the effect of intracerebroventricular administration of nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-nitroxy-2H-1-benzopyran), a potent NO donor, at various concentrations (0.1 muM to 1 mM in 5 mul PBS/brain) to neonatal rats with hypoxic-ischemic treatment. The extent of the infarct area in the brain was significantly reduced by injection of the 1 muM nipradilol solution. However, denitro-nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)-propoxy-3-hydroxy-2H-1-benzopyran), that does not release NO, did not show the neuroprotective effect, suggesting that NO released from nipradilol exerts a neuroprotective effect on neonatal neurons.

Disparities in visuo-spatial constructive abilities in Williams syndrome patients with typical deletion on chromosome 7q11.23.

BACKGROUND: Williams syndrome (WS) is known for its uneven cognitive abilities, especially the difficulty in visuo-spatial cognition, though there are some inter-individual phenotypic differences. It has been proposed that the difficulty in visuo-spatial cognition of WS patients can be attributed to a haploinsufficiency of some genes located on the deleted region in 7q11.23, based on an examination of atypical deletions identified in WS patients with atypical cognitive deficits. According to this hypothesis, the inter-individual differences in visuo-spatial cognitive ability arise from variations in deletion. METHODS: We investigated whether there were inter-individual differences in the visuo-spatial constructive abilities of five unrelated WS patients with the typical deletion on chromosome 7q11.23 that includes the candidate genes contributing visuo-spatial difficulty in WS patients. We used tests with three-dimensional factors such as Benton's three-dimensional block construction test, which are considered to be more sensitive than those with only two-dimensional factors. RESULTS: There were diverse inter-individual differences in the visuo-spatial constructive abilities among the present participants who shared the same typical genomic deletion of WS. One of the participants showed almost equivalent performances to typically developing adults in those tests. CONCLUSION: In the present study, we found a wide range of cognitive abilities in visuo-spatial construction even among the patients with a common deletion pattern of WS. The findings suggest that attributing differences in the phenotypes entirely to genetic factors such as an atypical deletion may not be always correct.

WNT10A variants isolated from Japanese patients with congenital tooth agenesis.

It has been reported that dozens of WNT10A variants are associated with human isolated tooth agenesis, however, little is known about the precise phenotypes. In 50 Japanese patients with severe congenital tooth agenesis, we identified 11 patients with WNT10A variants. Comparing phenotypes between the tooth agenesis patients carrying the wild-type and variants of WNT10A, we revealed that the development of lateral incisors is relatively susceptive to insufficiency of WNT/ß-catenin signaling.

Fates of Cdh23/CDH23 with mutations affecting the cytoplasmic region.

BUS/Idr mice carrying a mutant waltzer allele (vbus) are characterized by splayed hair bundles in inner ear sensory cells, providing a mouse homolog of USH1D/DFNB12. RT-PCR-based screening for the presence of mutations in mouse Cdh23, the gene responsible for the waltzer phenotype, has identified a G>A mutation in the donor splice site of intron 67 (Cdh23:c.9633+1G>A: GenBank AF308939.1), indicating that two altered Cdh23 molecules having intron-derived COOH-terminal structures could be generated in BUS mouse tissues. Immunochemical analyses with anti-Cdh23 antibodies showed, however, no clear Cdh23-related proteins in vbus/vbus tissues, while the antibodies immunoreacted with approximately 350 kDa proteins in control mice. Immunofluorescent experiments revealed considerable weakening of Cdh23 signals in sensory hair cell stereocilia and Reissner's membrane in the vbus/vbus inner ear, and transmission electron microscopy demonstrated abundant autophagosome/autolysosome vesicles, suggesting aberrant Cdh23:c.9633+1G>A-derived protein-induced acceleration of lysosomal bulk degradation of proteins. In transfection experiments, signal sequence-preceded FLAG-tagged transmembrane plus cytoplasmic regions (TMCy) of tissue-specific Cdh23(+/-68) isoforms were localized to filamentous actin-rich protrusions and the plasma membrane of cultured cells, whereas FLAG-TMCy:c.9633+1G>A proteins were highly insoluble and retained in the cytoplasm. In contrast, FLAG-tagged TMCy:p.Arg3175His and human TMCy:c.9625_9626insC forms were both localized to the plasma membrane in cultured cells, allowing prediction that USH1D-associated CDH23:p.Arg3175His and CDH23:c.9625_9626insC proteins could be transported to the plasma membrane in vivo. The present results thus suggest different fates of CDH23/Cdh23 with mutations affecting the cytoplasmic region.