USP37 Deubiquitinates CDC73 in HPT-JT Syndrome.

The CDC73/HRPT2 gene, a defect which causes hyperparathyroidism-jaw tumor (HPT-JT) syndrome, encodes CDC73/parafibromin. We aimed to investigate whether CDC73 would be a target for ubiquitin-proteasome degradation. We cloned full-length cDNAs encoding a family of 58 ubiquitin-specific deubiquitinating enzymes (DUBs), also known as ubiquitin-specific proteases (USPs). Use of the yeast two-hybrid system then enabled us to identify USP37 as interacting with CDC73. The biochemical interaction between the USP37 and CDC73 and their reciprocal binding domains were studied. Co-localization of CDC73 and USP37 was observed in cells. CDC73 was found to be polyubiquitinated, and polyubiquitination of CDC73 was prominent in mutants. CDC73 was deubiquitinated via K48-specific ubiquitin chains by USP37, but not by the catalytically inactive USP37C350S mutant. Observation of the binding between deletion mutants of CDC73 and USP37 revealed that the ß-catenin binding site of CDC73 and the ubiquitin-interacting motifs 2 and 3 (UIM2 and 3) of USP37 were responsible for the interaction between the two proteins. Moreover, these two enzymes co-existed within the nucleus of COS7 cells. We conclude that USP37 is a DUB for CDC73 and that the two proteins interact through specific domains, suggesting that USP37 is responsible for the stability of CDC73 in HPT-JT syndrome.

Clinical features and signaling effects of RET D631Y variant multiple endocrine neoplasia type 2 (MEN2).

BACKGROUND/AIMS: Germline mutations of the rearranged during transfection (RET) gene cause multiple endocrine neoplasia type 2 (MEN2). About 85% of RET mutations in MEN2 occur in codon Cys634. The RET D631Y mutation has recently been discovered, and we have studied its molecular expression and clinical consequences. METHODS: We analyzed the clinical characteristics of a total of 34 D631Y variant MEN2 individuals from seven families. We also constructed wild-type and mutant C630Y, D631Y, and C634R/W expression vectors and investigated their effects on signaling pathways and ability to correct the phenotypes of RET mutant cells. RESULTS: The median ages at diagnosis of pheochromocytoma and medullary thyroid carcinoma (MTC) were higher in patients with RET D631Y variant MEN2 than in those with the C634R/W variant (49:53.5 years vs. 33.5:27 years, respectively), and the penetration of the D631Y mutation with respect to MTC was lower than that of the C634R/W mutation (32.3% vs. 90%). The effects of the mutant vectors on phosphorylation of RET signaling molecules and focus formation were significantly different from those of wild type, but there were no significant differences between the mutants. D631Y scored significantly higher for chemotaxis and wound healing than C630Y, but lower than C634R and C634W. CONCLUSION: We suggest that the tumorigenic potential conferred by the D631Y mutation is lower than that conferred by the C634R/W mutation, but higher than that conferred by C630Y. Thus, the risk level of the RET D631Y variant appears to be higher than that of C630Y and lower than that of C634R/W.

Comparison of enteroendocrine cells and pancreatic ß-cells using gene expression profiling and insulin gene methylation.

Various subtypes of enteroendocrine cells (EECs) are present in the gut epithelium. EECs and pancreatic ß-cells share similar pathways of differentiation during embryonic development and after birth. In this study, similarities between EECs and ß-cells were evaluated in detail. To obtain specific subtypes of EECs, cell sorting by flow cytometry was conducted from STC-1 cells (a heterogenous EEC line), and each single cell was cultured and passaged. Five EEC subtypes were established according to hormone expression, measured by quantitative RT-PCR and immunostaining: L, K, I, G and S cells expressing glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, cholecystokinin, gastrin and secretin, respectively. Each EEC subtype was found to express not only the corresponding gut hormone but also other gut hormones. Global microarray gene expression profiles revealed a higher similarity between each EEC subtype and MIN6 cells (a ß-cell line) than between C2C12 cells (a myoblast cell line) and MIN6 cells, and all EEC subtypes were highly similar to each other. Genes for insulin secretion-related proteins were mostly enriched in EECs. However, gene expression of transcription factors crucial in mature ß-cells, such as PDX1, MAFA and NKX6.1, were remarkably low in all EEC subtypes. Each EEC subtype showed variable methylation in three cytosine-guanosine dinucleotide sites in the insulin gene (Ins2) promoter, which were fully unmethylated in MIN6 cells. In conclusion, our data confirm that five EEC subtypes are closely related to ß-cells, suggesting a potential target for cell-based therapy in type 1 diabetes.

Association of serum calcium concentrations with fibrinogen and homocysteine in nondiabetic Korean subjects.

Considerable evidence shows that increased serum calcium levels are associated with metabolic disorders, cardiovascular disease, and increased mortality. This study investigated whether serum calcium, within a normal range, is significantly associated with serum fibrinogen and homocysteine, markers of increased cardiovascular disease risk in nondiabetic Korean subjects.A cross-sectional analysis was performed on 1096 subjects (mean age, 55.1 ±â€Š11.1 years; 36.1% women) undergoing a general health checkup. Serum biochemistry was analyzed including serum albumin-corrected calcium (Cac), insulin resistance (IR, using homeostasis model assessment [HOMA]), fibrinogen, and homocysteine.Compared with patients within the lowest Cac quartile, those with higher Cac levels had increased fibrinogen and homocysteine levels as well as an increased proportion of smoking, dyslipidemia, and HOMA-IR. Correlation analyses revealed linear relationships for Cac with fibrinogen and homocysteine in both genders. After adjustment for confounding factors, serum Cac was significantly associated with high fibrinogen (odds ratio [OR] for the highest vs the lowest quartile = 1.76, 95% confidence interval [CI] = 1.09-2.83, P = 0.02) and homocysteine (OR = 1.83, 95% CI = 1.07-3.11, P = 0.027). Multivariate regression models showed that Cac was linearly associated with fibrinogen (standardized ß = 0.14, P < 0.001) and homocysteine (standardized ß = 0.07, P = 0.009).High normal calcium concentrations were independently associated with increased levels of fibrinogen and homocysteine. Further investigation is needed to validate whether slightly increased calcium levels within the normal range indicate a higher risk of cardiovascular disease.

Association between serum calcium and phosphorus concentrations with non-alcoholic fatty liver disease in Korean population.

BACKGROUND AND AIM: Growing evidence suggests that non-alcoholic fatty liver disease (NAFLD) is interrelated with renal dysfunction and disturbed bone metabolism, both of which play a key role in calcium and phosphorus homeostasis. We investigated the association between NAFLD and serum calcium and phosphorus levels in Korean subjects. METHODS: We performed a cross-sectional analysis of 16,592 subjects undergoing a general health checkup. NAFLD was assessed based on ultrasonographically detected fatty liver in the absence of excessive alcohol consumption and other causes of liver disease. RESULTS: The proportion of the population with fatty liver detected by ultrasonography was 43.2% for males and 17.6% for females. We observed that a higher serum albumin-corrected calcium (Ca(c)) level was associated with smoking, hypertension, and unfavorable metabolic parameters in both genders, but the serum phosphorus levels showed an inconsistent correlation with metabolic abnormalities. After adjusting for age, gender, waist circumference, body mass index, smoking status, exercise, diabetes, hypertension, lipid profiles, and renal function, serum Cac , phosphorus, and Cac -phosphorus products were independent risk factors for fatty liver (odds ratio [OR]: 1.71, 95% confidence interval [CI]: 1.49-1.95, P < 0.001; OR: 1.34, 95% CI: 1.22-1.48, P < 0.001; and OR: 1.20, 95% CI: 1.14-1.26, P < 0.001, respectively), and the risk of fatty liver increased in a graded manner over the quartiles. CONCLUSION: Serum calcium and phosphorus levels are significantly associated with NAFLD. Further investigation is needed to verify whether calcium and phosphorus levels indicate a higher risk of NAFLD.

Upregulation of FGFR1 expression is associated with parathyroid carcinogenesis in HPT-JT syndrome due to an HRPT2 splicing mutation.

Mutations of the HRPT2 gene, which are responsible for hyperparathyroidism-jaw tumor (HPT-JT) syndrome, have been implicated in the development of a high proportion of parathyroid carcinomas. The aim of this study was to investigate differences in expression of the most important genes connected with parathyroid carcinoma between HPT-JT syndrome due to an HRPT2 splicing mutation, normal parathyroid tissue and sporadic parathyroid adenoma. Total RNAs were extracted from parathyroid carcinoma in HPT-JT syndrome harbouring HRPT2 splicing mutation or sporadic parathyroid adenoma and normal parathyroid gland, and subjected to Illumina DASL-based gene expression assay. Unsupervised hierarchical clustering analysis was used to compare gene expression in HPT-JT syndrome, sporadic parathyroid adenoma and normal parathyroid glands. We identified differentially regulated genes in HPT-JT syndrome and sporadic parathyroid adenoma relative to normal parathyroid glands using a combination of Welch's t-test and fold-change analysis. Quantitative PCR, RT-PCR and IHC were used for validation. Sixteen genes differentially regulated in the parathyroid carcinoma were associated with signal pathways, MAPK, regulation of actin cytoskeleton, prostate cancer and apoptosis. FGFR1 expression was confirmed to be significantly upregulated by validation experiments. Our gene expression profiling experiments suggest that upregulated FGFR1 expression appears to be associated with parathyroid carcinoma in HPT-JT syndrome due to an HRPT2 splicing mutation.

Novel germline SDHD mutation in a patient with recurrent familial carotid body tumor and concomitant pheochromocytoma.

BACKGROUND: Recent advances in genetics revealed that 25% to 30% of head and neck paragangliomas (PGLs) are inherited tumors associated with germline mutation, mainly in the succinate dehydrogenase (SDH) gene. METHODS: DNA was isolated from whole blood and polymerase chain reaction (PCR) products were sequenced with an ABI3730 × 1 Genetic Analyzer. RESULTS: A 30-year-old Korean woman underwent resection of a carotid PGL. Fourteen years later, she was readmitted for a cervical mass. (18) F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/CT revealed a hot spot in the adrenal gland, besides the carotid mass. Surgical pathology confirmed recurrence of the carotid PGL and a concomitant pheochromocytoma. Genetic analysis revealed SDHD c.119del T (p.I40TfsX46) mutation. One daughter has been identified as a carrier. CONCLUSION: We found a novel SDHD mutation from a Korean family that shows similar clinical features to those in other SDHD mutations, mostly from Western countries. Further studies are needed to determine whether similar genotype-phenotype correlations exist in the Asian patients with familial PGLs.

Reprogramming of enteroendocrine K cells to pancreatic ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic ß-cells in a process called cell reprogramming. Enteroendocrine cells and ß-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in ß-cells. Thus, K cells could be reprogrammed to ß-cells under certain conditions. However, there is no clear evidence on whether these cells convert to ß-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1(+)-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1(+)-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1(+)-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

A case of Dyke-Davidoff-Masson syndrome associated with central hypothyroidism and secondary adrenal insufficiency.

A diagnosis of central hypothyroidism (CH) can be missed easily or delayed without a high index of suspicion due to normal or slightly altered thyroid stimulating hormone (TSH) levels during the initial screening test for thyroid dysfunction. A correct diagnosis of CH is very important for safely treating patients. Specifically, doctors must ensure a proper evaluation of combined adrenal insufficiency to prevent a fatal adrenal crisis. Here we report a case of CH combined with secondary adrenal insufficiency in a 42-year-old woman with Dyke-Davidoff-Masson syndrome, which is a rare neurological disease.

Association between low serum free thyroxine concentrations and coronary artery calcification in healthy euthyroid subjects.

BACKGROUND: Considerable evidence suggests that hypothyroidism could promote atherosclerotic vascular changes. We planned this study to investigate whether serum free thyroxine (FT4) or thyroid-stimulating hormone (TSH) levels are associated with coronary artery calcification measured in healthy euthyroid subjects. METHODS: A cross-sectional analysis was performed among subjects who visited our hospital for a health checkup. Among 1849 subjects, 669 (mean age 55.3±8.8 years; 392 men) with FT4 and TSH in the normal ranges were included after excluding those with diabetes, a history of current smoking and cardiovascular disease (CVD), or the use of drugs for hypertension, antithyroid drugs, or thyroid hormone preparations. Coronary artery calcium scores (CACS) were measured by multi-detector computed tomography. RESULTS: Subjects with a CACS >100 had lower FT4 levels than those with a lower CACS (p=0.017), whereas no difference was observed in the TSH levels among CACS categories. FT4 levels had an odds ratio of 0.06 for high CACS (95% confidence interval=0.01-0.74; p=0.028) after the adjustment for CVD risk factors. In multivariate regression analysis, CACS was negatively correlated with FT4 levels (ß=-0.823, p=0.032), and the inverse association between FT4 and CACS remained significant only in men (p=0.011). CONCLUSION: FT4 levels were inversely associated with coronary artery calcification in euthyroid healthy subjects, especially in men independent of conventional CVD risk factors. Further studies are needed to validate whether subjects with decreased FT4 levels within the normal reference range are at a high CVD risk and have poor cardiovascular outcomes.

Absence of galectin-3 immunostaining in fine-needle aspiration cytology specimens from papillary thyroid carcinoma is associated with favorable pathological indices.

BACKGROUND: Galectin-3 (G3) immunostaining of fine-needle aspiration (FNA) samples from thyroid nodules is very useful for the diagnosis of malignancy. The objective of the present study was to determine whether the absence of G3 immunostaining in preoperative FNA samples is associated with favorable clinicopathological parameters, including lymph node (LN) metastasis, in papillary thyroid carcinoma (PTC). METHODS: The records of 868 patients with PTC who had prethyroidectomy ultrasonography-guided FNA with G3 immunostaining between January 2006 and December 2009 were retrospectively reviewed. G3 immunostaining was considered positive if the percentage of tumor cells showing definite cytoplasmic immunostaining exceeded 5%. Patients who had negative G3 immunostaining of FNA samples were assigned to the G3-negative (G3N) group; whereas those who had positive G3 immunostaining were assigned to the G3-positive (G3P) group. RESULTS: There were 92 patients who were assigned to the G3N group (10.6%) because of the negative staining for G3 in the preoperative FNA samples from their thyroid nodules. The proportion of PTC subtypes in the G3N and G3P groups was similar (p=0.376). There was less frequent thyroid capsular invasion (46.7% vs. 66.5%, p<0.001), extrathyroidal extension (28.3% vs. 48.5%, p<0.001), and LN metastasis (22.2% vs. 48.7%, p<0.001) in the G3N group than the G3P group. In multivariate regression analysis, G3N expression predicted a lower risk of LN metastasis (odds ratio=0.37, 95% confidence interval 0.18-0.78) after adjustment for other clinicopathological parameters. Over a median follow-up of 33 months, no association was observed between G3N and disease-free survival. CONCLUSION: The absence of G3 expression in FNA samples from PTC is associated with pathological parameters considered less aggressive than is the case for PTCs with G3 expression, including being a negative predictor of negative LN involvement. Long-term follow-up studies, however, are needed to verify whether G3N patients have lower recurrence and mortality rates.

Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus.

Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked recessive disorder associated with germ-line mutations of the arginine vasopressin (AVP) receptor type 2 (AVPR2) gene. Recent molecular studies have demonstrated that insensitivity of renal tubule cells to AVP is associated with AVPR2 mutations. We identified a novel deletion mutation at nucleotide position 302 (302delC), in a Korean NDI family, that results in a frameshift and a truncated receptor protein. To identify the mutant AVPR2 protein we developed an expression vector for the AVPR2 mutation by a PCR-based restriction fragment replacement strategy. COS-7 cells were transiently transfected with expression vectors for the wild-type and mutant genes, and we analyzed AVP-induced cyclic adenosine monophos-phate (cAMP) responses, and assessed the localization of AVPR2 receptors, in the transfected COS-7 cells. In the cells expressing the mutant gene, the maximum AVP-induced cAMP response was reduced and the truncated receptor proteins were retained within the cytoplasmic compartment. These results suggest that the novel frameshift AVPR2 (302delC) mutation is responsible for the AVP resistance in the family with congenital NDI.

A Novel IVS2-1G>A mutation causes aberrant splicing of the HRPT2 gene in a family with hyperparathyroidism-jaw tumor syndrome.

HRPT2, the gene associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome, was previously mapped to 1q24-q32. It was recently cloned, and several germline mutations were found to predispose to HPT-JT syndrome. We sequenced the complete HRPT2 coding sequence and splice-junctional regions in a Korean family with HPT-JT syndrome and identified a novel germline mutation, IVS2-1G>A in intron 2, that caused the autosomal dominant trait of HPT-JT syndrome in this family. RT-PCR and sequencing of the transcripts revealed that this splicing mutation generated alternative splicing errors leading to the formation of two different transcripts, one with exon 3 deleted, the other lacking the first 23 bp of exon 3 due to the use of an internal splice acceptor in exon 3. Translation of both transcripts results in premature termination. In addition, we detected two novel somatic mutations of HRPT2 in malignant parathyroid tumors from the affected individuals. One, 85delG, causes premature termination; the other, an 18 bp in-frame deletion of 13_30delCTTAGCGTCCTGCGACAG, suggests that this region may be important in the development of the parathyroid carcinomas in HPT-JT syndrome. These findings provide further evidence that mutation of HRPT2 is associated with the formation of parathyroid tumors in HPT-JT syndrome.