Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

Mutation in SSUH2 Causes Autosomal-Dominant Dentin Dysplasia Type I.

Dentin dysplasia type I (DDI) is an autosomal-dominant genetic disorder resulting from dentin defects. The molecular basis of DDI remains unclear. DDI exhibits unique characteristics with phenotypes featuring obliteration of pulp chambers and diminutive root, thus providing a useful model for understanding the genetics of tooth formation. Using a large Chinese family with 14 DDI patients, we mapped the gene locus responsible for DDI to 3p26.1-3p24.3 and further identified a missense mutation, c.353C>A (p.P118Q) in the SSUH2 gene on 3p26.1, which co-segregated with DDI. We showed that SSUH2 (p.P118Q) perturbed the structure and significantly reduced levels of mutant (MT) protein and mRNA compared with wild-type SSUH2. Furthermore, MT P141Q knock-in mice (+/- and -/-) had a unique partial obliteration of the pulp cavity and upregulation or downregulation of six major genes involved in odontogenesis: Dspp, Dmp1, Runx2, Pax9, Bmp2, and Dlx2. The phenotype of missing teeth was determined in zebrafish with morpholino gene knockdowns and rescued by injection of normal human mRNA. Taken together, our observations demonstrate that SSUH2 disrupts dental formation and that this novel gene, together with other odontogenesis genes, is involved in tooth development.

A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia.

BACKGROUND: Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations. METHODS: Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies. RESULTS: We identified 2 complex heterozygous mutations involving a c.398-399 insACAGCAGCAGCAGCA insertion and a c.411-412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus. CONCLUSIONS: Our findings demonstrated that the novel c.398-399insACAGCAGCAGCAGCA mutation occurred alongside the c.411-412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.

A splicing mutation in VPS4B causes dentin dysplasia I.

BACKGROUND: Dentin dysplasia I (DDI) is a genetically heterogeneous autosomal-dominant disorder characterised by rootless teeth with abnormal pulpal morphology, the aetiology of which presents as genetically heterogeneous. METHODS AND RESULTS: Using a cohort of a large Chinese family with 10 patients with DDI, we mapped to a 9.63 Mb candidate region for DDI on chromosome 18q21.2-q21.33. We then identified a mutation IVS7+46C>G which resulted in a novel donor splice site in intron 7 of the VPS4B gene with co-segregation of all 10 affected individuals in this family. The aberrant transcripts encompassing a new insert of 45 bp in size were detected in gingival cells from affected individuals. Protein structure prediction showed that a 15-amino acid insertion altered the ATP-binding cassette of VPS4B. The mutation resulted in significantly reduced expression of mRNA and protein and altered subcellular localisation of VPS4B, indicating a loss of function of VPS4B. Using human gingival fibroblasts, the VPS4B gene was found to act as an upstream transducer linked to Wnt/ß-catenin signalling and regulating odontogenesis. Furthermore, knockdown of vps4b in zebrafish recapitulated the reduction of tooth size and absence of teeth similar to the tooth phenotype exhibited in DDI index cases, and the zebrafish mutant phenotype could be partially rescued by wild-type human VPS4B mRNA. We also observed that vps4b depletion in the zebrafish negatively regulates the expression of some major genes involved in odontogenesis. CONCLUSIONS: This study identifies VPS4B as a disease-causing gene for DDI, which is one of the important contributors to tooth formation, through the Wnt/ß-catenin signalling pathway.