Esther Manor

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.

Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1

In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD.

Lethal Contractural Syndrome Type 3 (LCCS3) Is Caused by a Mutation in PIP5K1C, Which Encodes PIPKIγ of the Phophatidylinsitol Pathway

Lethal congenital contractural syndrome (LCCS) is a severe form of arthrogryposis. To date, two autosomal recessive forms of the disease (LCCS and LCCS2) have been described and mapped to chromosomes 9q34 and 12q13, respectively. We now describe a third LCCS phenotype (LCCS3)--similar to LCCS2 yet without neurogenic bladder. Using 10K single-nucleotide-polymorphism arrays, we mapped the disease-associated gene to 8.8 Mb on chromosome 19p13. Further analysis using microsatallite markers narrowed the locus to a 3.4-Mb region harboring 120 genes. Of these genes, 30 candidates were sequenced, which identified a single homozygous mutation in PIP5K1C. PIP5K1C encodes phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIPKI gamma ), an enzyme that phophorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate (PIP(2)). We demonstrate that the mutation causes substitution of aspartic acid with asparagine at amino acid 253 (D253N), abrogating the kinase activity of PIPKI gamma . Thus, a defect in the phosphatidylinositol pathway leading to a decrease in synthesis of PIP(2), a molecule active in endocytosis of synaptic vesicle proteins, culminates in lethal congenital arthrogryposis.

Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

SCN9Aencodes the voltage‐gated sodium channel Nav1.7, a protein highly expressed in pain‐sensing neurons. Mutations in SCN9A cause three human pain disorders: bi‐allelic loss of function mutations result in Channelopathy‐associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non‐truncating mutations in families with CIP: a homozygously‐inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7‐R896Q) and a five amino acid in‐frame deletion found in a sporadic compound heterozygote (Nav1.7‐ΔR1370‐L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant‐transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7.

Familial neonatal isolated cardiomyopathy caused by a mutation in the flavoprotein subunit of succinate dehydrogenase

Cardiomyopathies are common disorders resulting in heart failure; the most frequent form is dilated cardiomyopathy (DCM), which is characterized by dilatation of the left or both ventricles and impaired systolic function. DCM causes considerable morbidity and mortality, and is one of the major causes of sudden cardiac death. Although about one-third of patients are reported to have a genetic form of DCM, reported mutations explain only a minority of familial DCM. Moreover, the recessive neonatal isolated form of DCM has rarely been associated with a mutation. In this study, we present the association of a mutation in the SDHA gene with recessive neonatal isolated DCM in 15 patients of two large consanguineous Bedouin families. The cardiomyopathy is presumably caused by the significant tissue-specific reduction in SDH enzymatic activity in the heart muscle, whereas substantial activity is retained in the skeletal muscle and lymphoblastoid cells. Notably, the same mutation was previously reported to cause a multisystemic failure leading to neonatal death and Leighs syndrome. This study contributes to the molecular characterization of a severe form of neonatal cardiomyopathy and highlights extreme phenotypic variability resulting from a specific missense mutation in a nuclear gene encoding a protein of the mitochondrial respiratory chain.

Primary intraosseous squamous cell carcinoma arising in an odontogenic cyst - a clinicopathologic analysis of 116 reported cases

PURPOSE To review the literature on primary intraosseous squamous cell carcinoma (PIOSCC) associated with odontogenic cyst. METHODS All well-documented cases of PIOSCC published between 1938 and 2010 were collected. Only cases of PIOSCC arising from the lining of an odontogenic cyst, including the keratocystic odontogenic tumor, were selected. Age, sex, signs and symptoms, affected jaw, cyst type, treatment, histopathology, and outcome were recorded. RESULTS The mean age was 60.2 years (range 1.3-90). There were 80 (69%) men and 36 (31%) women. Mass and pain were the most common presenting symptoms. The mandible was affected in 92 (79%) patients and the maxilla in 24 (21%). It was a residual/radicular cyst in 70 (60%) patients and a dentigerous cyst or a keratocystic odontogenic tumor in the remaining 40%. The histopathology was well-differentiated SCC in 53 (46%) patients and moderately differentiated SCC in 47 (40%) patients. Fifty-three (46%) patients were treated with surgery alone and 44 (38%) with surgery and radiotherapy. Fifty-eight (62%) patients survived 2 years and 36 (38%) survived 5 years. CONCLUSION PIOSCC has a predilection for men (M/F ratio of 2.22:1), affects mainly adults in their 6-8th decades, occurs most frequently (79%) in the mandible, and is associated mainly with a residual/radicular cyst. Histologically, the well-to-moderately differentiated SCC was the most common. Surgery alone or combined therapy of surgery and radiation was the most common approach. The prognosis is 62% surviving 2 years and 38% 5 years.

Oral lipoma: analysis of 58 new cases and review of the literature

Lipomas are common soft-tissue tumors but occur infrequently in the oral region. Here, we present 58 new cases of oral lipoma (OL), their clinical features, diagnostic workup, and management. The records of 58 patients with OL were reviewed. Demographic data, presenting symptoms, primary tumor site, histopathology, and clinical management were analyzed. Extraoral and intraosseous lesions were excluded. The patients were 29 men and 29 women with mean age of 59.7 years, range, 11 to 98 years. Tumor sites included the buccal mucosa (n = 31), tongue (n = 10), lip (n = 6), floor of the mouth (n = 6), and vestibule (n = 5). The mean tumor size was 2.1 cm (range, 0.3-5.0 cm). The mean tumor duration before excision was 2.6 years (31.2 months), ranging from 2 months to 12 years. Most common presenting symptom was an asymptomatic, circumscribed mass. Histologically, the tumors were classified as classic lipoma (n = 28), fibrolipoma (n = 19), intramuscular lipoma (n = 4), minor salivary gland lipoma (n = 2), angiolipoma (n = 2), and spindle cell lipoma (n = 3). A case of spindle cell lipoma affecting the lower lip that has not been described previously is presented. The provided treatment was surgical excision. Although uncommon, OL are most common in adults and very uncommon in children. No sex predilection was found. The buccal mucosa region is the most common site followed by the tongue. Classic lipomas are most common in the oral region and comprise 48% of the tumors. A case of spindle cell lipoma of the lower lip is reported. Its clinical, histologic, and cytogenetic workup is the first well-documented case. Further cytogenetic studies should be undertaken to learn more about the pathogenesis and tumorigenesis of OL.

O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome

Background: O-GlcNAcylation regulates cellular processes such as transcription, signal transduction, metabolism, and cell cycle. Results: O-GlcNAcylation is elevated in CRC metastatic clone. OGA silencing resulted in the acquisition of a fibroblast-like morphology, growth retardation, and alteration of gene expression. Conclusion: O-GlcNAcylation affects gene transcription, metabolism, and proliferation. Significance: This research supports O-GlcNAcylation involvement in various aspects of tumor cell physiology. O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer.

Lethal Congenital Contractural Syndrome Type 2 (LCCS2) Is Caused by a Mutation in ERBB3 (Her3), a Modulator of the Phosphatidylinositol-3-Kinase/Akt Pathway

Lethal congenital contractural syndrome type 2 (LCCS2) is an autosomal recessive neurogenic form of arthrogryposis that is associated with atrophy of the anterior horn of the spinal cord. We previously mapped LCCS2 to 6.4 Mb on chromosome 12q13 and have now narrowed the locus to 4.6 Mb. We show that the disease is caused by aberrant splicing of ERBB3, which leads to a predicted truncated protein. ERBB3 (Her3), an activator of the phosphatidylinositol-3-kinase/Akt pathway--regulating cell survival and vesicle trafficking--is essential for the generation of precursors of Schwann cells that normally accompany peripheral axons of motor neurons. Gain-of-function mutations in members of the epidermal growth-factor tyrosine kinase-receptor family have been associated with predilection to cancer. This is the first report of a human phenotype resulting from loss of function of a member of this group.

Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type 1 infected Vero cells

N-methanocarbathymidine ((N)-MCT), a thymidine analog incorporating a pseudosugar with a fixed Northern conformation, exhibits potent antiherpetic activity against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). This study contrasts the metabolic pathway of (N)-MCT and the well-known antiherpetic agent ganciclovir (GCV) in HSV-1-infected and uninfected Vero cells. Treatment of HSV-1 infected Vero cells immediately after viral infection with (N)-MCT profoundly inhibited the development of HSV-1 infection. Using standard plaque reduction assay to measure viral infection, (N)-MCT showed a potency greater than that of ganciclovir (GCV), the IC50s were 0.02 and 0.25 microM for (N)-MCT and GCV, respectively. (N)-MCT showed no cytotoxic effect on uninfected Vero cells (CC50>100 microM). Dose and time dependence studies showed high levels of (N)-MCT-triphosphate ((N)-MCT-TP), and GCV-triphosphate (GCV-TP) in HSV-1-infected cells incubated with (N)-MCT or GCV, respectively. In contrast, uninfected cells incubated with (N)-MCT showed elevated levels of (N)-MCT-monophosphate only, while low levels of mono, di- and triphosphates of GCV were found following incubation with GCV. Although the accumulation rate of (N)-MCT and GCV phosphates in HSV-1-infected cells were similar, the decay rate of (N)-MCT-TP was slower than that of GCV-TP. These results suggest that: (1) the antiviral activity of (N)-MCT against herpes viruses is mediated through its triphosphate metabolite; (2) in contrast to GCV, the diphosphorylation of (N)-MCT in HSV-1- infected cells is the rate limiting step; (3) (N)-MCT-TP accumulates rapidly and has a long half-life in HSV-1-infected cells; and (4) HSV-tk catalyzed the mono, and diphosphorylation of (N)-MCT while monophosphorylating GCV only. These results provide a biochemical rational for the highly selective and effective inhibition of HSV-1 by (N)-MCT.