Single nucleotide variants of succinate dehydrogenase A gene in renal cell carcinoma.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is mainly associated with a mutation in the SDHB gene and sometimes with mutations in the SDHC or SDHD genes. However, only three cases of succinate dehydrogenase A (SDHA)-deficient RCC have been reported, and the relation between SDHA mutations and RCC has not been clarified. This study assessed the role of SDHA gene mutations in human RCC. We investigated SDHA/B/C/D gene mutations in 129 human RCCs. Targeted next-generation sequencing and direct Sanger sequencing revealed single nucleotide variants (SNVs) of the SDHA gene with amino acid sequence variations in 11/129 tumors, while no SDHB/C/D gene mutations were found. Tumor cells with SNVs of the SDHA gene were characterized by eosinophilic cytoplasm and various patterns of proliferation. Immunohistochemistry examination found that the 11 tumors with SNVs of the SDHA gene showed significant reduction of SDHA protein and SDHB protein expression compared to the 19 tumors without SDHA or SDHB mutations (both P < .0001). Western blotting showed a greater decrease in the expression of SDHA and SDHB proteins in the 11 tumors with SNVs of the SDHA gene than in the 19 tumors without (both P < .0001). There was a positive correlation between SDHA and SDHB protein levels (P < .0001). On immunohistochemistry and Western blotting, the 11 tumors with SNVs of the SDHA gene had higher protein expression for nuclear factor E2-related factor 2 (Nrf2) compared to the 19 tumors without the mutation (P < .01). These observations suggest that SDHA gene mutations might be associated with a subset of RCC.

Rise of oligodendroglioma hypermutator phenotype from a subclone harboring TP53 mutation after TMZ treatment.

Oligodendrogliomas characterized and defined by 1p/19q co-deletion are slowly growing tumors showing better prognosis than astrocytomas. TP53 mutation is rare in oligodendrogliomas while the vast majority of astrocytomas harbor the mutation, making TP53 mutation mutually exclusive with 1p/19q codeletion in lower grade gliomas virtually. We report a case of 51-year-old woman with a left fronto-temporal oligodendroglioma that contained a small portion with a TP53 mutation, R248Q, at the initial surgery. On a first, slow-growing recurrence 29 months after radiation and nitrosourea-based chemotherapy, the patient underwent TMZ chemotherapy. The recurrent tumor responded well to TMZ but developed a rapid progression after 6 cycles as a malignant hypermutator tumor with a MSH6 mutation. Most of the recurrent tumor lacked typical oligodendroglioma morphology that was observed in the primary tumor, while it retained the IDH1 mutation and 1p/19q co-deletion. The identical TP53 mutation observed in the small portion of the primary tumor was universal in the recurrence. This case embodied the theoretically understandable clonal expansion of the TP53 mutation with additional mismatch repair gene dysfunction leading to hypermutator phenotype. It thus indicated that TP53 mutation in oligodendroglioma, although not common, may play a critical role in the development of hypermutator after TMZ treatment.

Genetic alteration of ARMC5 in a patient diagnosed with meningioma and primary macronodular adrenal hyperplasia: a case report.

A monoallelic germline alteration of ARMC5 causes primary bilateral macronodular adrenal hyperplasia (PBMAH) with Cushing's syndrome via its subsequent somatic alteration on the other allele as the second hit. PBMAH is sometimes complicated with meningioma. Dependency of such a multi-organ disease on the second hit mechanism was reported before, but this finding has not been confirmed yet. We describe a case of a 65-year-old female with PBMAH, carrying a heterozygous germline alteration of ARMC5, p.R267*, complicated with meningioma associated with somatic loss of heterozygosity (LOH) of the unaffected allele. Pathogenic alterations of ARMC5 may also contribute to the development of meningioma by the two-hit mechanism.

Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates trefoil factor family 2 (TFF2) expression in gastric epithelial cells.

Although trefoil factor family 2 (TFF2) plays a critical role in the defense and repair of gastric mucosa, the regulatory mechanism of TFF2 expression is not fully understood. In this study, we investigated the regulation of TFF2 expression by peroxisome proliferator-activated receptor gamma (PPARgamma) in gastric epithelial cells. MKN45 gastric cells were used. TFF2 mRNA expression was analyzed by real-time quantitative RT-PCR. The promoter sequence of the human TFF2 gene was cloned into pGL3-basic vector for reporter gene assays. Ciglitazone was mainly used as a specific PPARgamma ligand. MKN45 cells expressed functional PPARgamma proteins. Endogenous TFF2 mRNA expression and TFF2 reporter gene transcription was significantly up-regulated by ciglitazone in a dose-dependent manner. Reporter gene assays showed that two distinct cis-elements are involved in the response to PAPRgamma activation. Within one of these element (nucleotides -558 to -507), we identified a functional peroxisome proliferator responsive element (PPRE) at -522 (5'-GGGACAAAGGGCA-3'). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay confirmed the binding of PPARgamma to this sequence. Another element (nucleotides -407 to -358) appeared to be a composite enhancer element indirectly regulated by PPARgamma and a combination of these two cis-elements was required for the full response of the human TFF2 gene expression to PPARgamma. These data demonstrate that human TFF2 gene is a direct target of PPARgamma in gastric epithelial cells. Since TFF2 is a critical gastroprotective agent, PPARgamma may be involved in the gastric mucosal defense through regulating TFF2 expression in humans.

Familial hypocalciuric hypercalcemia with a de novo heterozygous mutation of calcium-sensing receptor.

UNLABELLED: A de novo heterozygous inactivating mutation of calcium-sensing receptor (CASR) gene typically causes neonatal hyperparathyroidism (NHPT) with moderate hypercalcemia and hyperparathyroid bone disease. We present a case of asymptomatic hypocalciuric hypercalcemia with a de novo heterozygous mutation in CASR, S591C, which is primarily reported to be responsible for NHPT. A 54-year-old female was referred for investigation of asymptomatic hypercalcemia that was initially found in the 1980s but without a history of bone disease during the perinatal period. She had moderate hypercalcemia (12.4 mg/dl) and relative hypocalciuria (fractional extraction of calcium 1.07%) but normal intact parathyroid hormone and serum 1,25-dihydroxyvitamin D3. Pedigree analysis revealed that she carried a de novo heterozygous mutation of S591C, which she transmitted to an affected child with moderate hypercalcemia but not to other children, who had normal serum calcium levels. A de novo heterozygous CASR mutation that is responsible for NHPT may also present in individuals with asymptomatic hypocalciuric hypercalcemia. Caution is required when predicting course and outcome in a pedigree with CASR mutation, as well as incidental hypercalcemia, because of its variable phenotypes. LEARNING POINTS: The phenotype and severity of CASR mutations are thought to be dependent on genotypes.We report an asymptomatic case of the de novo heterozygous S591C mutation in CASR, which has previously been reported as a responsible mutation of NHPT with bone diseases.Variable phenotypes of CASR raise a cautionary note about predicting outcome by genotyping in a pedigree with CASR mutation.

PPARgamma mediates NSAIDs-induced upregulation of TFF2 expression in gastric epithelial cells.

Trefoil factor family (TFF) is a group of peptides that play critical roles in maintaining gastric mucosal integrity. In real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and reporter gene assays, we show that indomethacin and aspirin upregulate TFF2 expression in MKN45 gastric cells. These drugs also activated peroxisome proliferator-activated receptor gamma (PPARgamma) at concentration ranges that increase TFF2 expression, and upregulated TFF2 expression was suppressed by GW9662, a specific inhibitor of PPARgamma. These results suggest that indomethacin and aspirin upregulate gastric expression of TFF2 through activation of PPARgamma. This mechanism may be important in reducing the extent of gastric mucosal injury caused by the administration of non-steroidal anti-inflammatory drugs (NSAIDs).

TTF-2 stimulates expression of 17 genes, including one novel thyroid-specific gene which might be involved in thyroid development.

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.

Up-regulation of TFF1 (pS2) expression by TNF-alpha in gastric epithelial cells.

BACKGROUND AND AIM: TFF1 (pS2) is expressed at a high level in gastric epithelial cells and plays an important role in protecting the gastric mucosa. However, the regulatory mechanisms of TFF1 expression are not fully understood. The aim of this study was to investigate the effect of TNF-alpha, a representative proinflammatory cytokine, on TFF1 expression. METHODS: MKN45 and AGS cells, derived from human gastric carcinoma, were used. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RT-PCR. The sequences of the human TFF1 promoter were cloned into the pGL3-basic vector and reporter gene assays were performed. Nuclear factor (NF)-kappaB activity was monitored using a reporter vector that contained multiple copies of NF-kappaB responsive element upstream of the luciferase gene. Interaction between NF-kappaB and TFF1 cis-element was examined by electophoretic mobility shift assay (EMSA). RESULTS: TNF-alpha activated NF-kappaB and up-regulated endogenous TFF1 mRNA expression as well as the transcription of the TFF1 reporter genes in a dose-dependent manner. IL-1beta, another proinflammatory cytokine, also up-regulated TFF1 expression. TNF-alpha responsive element was mapped between -342 and -147 of the human TFF1 promoter and a putative NF-kappaB binding site was identified at -231. When this element was deleted, the reporter genes became almost insensitive to TNF-alpha treatment. EMSA showed binding of NF-kappaB to this element. CONCLUSIONS: Inflammatory stimuli that activate NF-kappaB appear to up-regulate TFF1 expression in gastric epithelial cells. This mechanism may aid in the protection of the gastric mucosa under inflammatory conditions.