Clinical spectrum in three families with familial hemiplegic migraine type 2 including a novel mutation in the ATP1A2 gene.

INTRODUCTION: Familial hemiplegic migraine (FHM) is a rare subtype of migraine with transient hemiplegic aura. PATIENTS AND METHODS: We describe three unrelated families with familial hemiplegic migraine type II (FHM2). Retrospectively, information on 47 family members could be obtained, 15 by personal examination and 32 by indirect anamnesis from relatives. Genetic analyses were performed in 13 patients. RESULTS: One family had a novel missense mutation in the ATP1A2 gene (c.659C>T, p.Ser220Leu) that segregated with the phenotype in three generations. Two further unrelated families with different ethnic backgrounds (one from Germany and one from Russia) had a missense mutation that has not been described as yet in FHM, but occurred in only a single patient with sporadic hemiplegic migraine (c.2723G>A, p.Arg908Gln). Clinically the patients had severe attacks lasting up to several weeks as well as epileptic seizures. Three patients with a proven mutation in the ATP1A2 gene clinically presented without hemiparesis. Furthermore, there was a possible relation of FHM2 to mental retardation in another two patients. CONCLUSION: Clinical symptoms may last for several weeks in some patients. Patients with FHM2 may also present without hemiplegia. Therefore, the full family history has to be taken into account to establish the diagnosis of FHM.

Increased expression of zinc finger protein 267 in non-alcoholic fatty liver disease.

Hepatocellular lipid accumulation is a hallmark of non-alcoholicfatty liver disease (NAFLD), which encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and ultimately cirrhosis. Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulate diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 expression is up-regulated in liver cirrhosis and is further increased in hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in tissue specimens of NAFLD patients and found a significant up-regulation compared to normal liver tissue. Noteworthy, ZNF267 mRNA was already significantly increased in steatotic liver tissue without inflammation. In line with this, incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation and corresponding dose-dependent ZNF267 induction in vitro. Furthermore, hepatocellular lipid accumulation induced formation of reactive oxygen species (ROS), and also chemically induced ROS formation increased ZNF267 mRNA expression. In summary with previous findings, which revealed ZNF267 as pro-fibrogenic and pro-cancerogenic factor in chronic liver disease, the present study further suggests ZNF267 as promising therapeutic target particularly for NAFLD patients. In addition, it further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.

Zinc finger protein 267 is up-regulated in hepatocellular carcinoma and promotes tumor cell proliferation and migration.

Zinc finger protein 267 (ZNF267) belongs to the family of Kruppel-like transcription factors, which regulates diverse biological processes that include development, proliferation, and differentiation. We have previously demonstrated that ZNF267 mRNA is up-regulated in liver cirrhosis, which is the main risk factor for hepatocellular carcinoma (HCC). Here, we analyzed the expression of ZNF267 in human HCC cells and tissue specimens and found a significant up-regulation compared to primary human hepatocytes and corresponding non-tumorous liver tissue. Over-expression of the transcription factor Ets-1 further enhanced ZNF267 expression, and reporter gene assays revealed that mutation of the Ets-1 binding site to the ZNF267 promotor markedly inhibited ZNF267 promotor activity. Hypoxic conditions induced Ets-1 in HCC cells via HIF1alpha activation, and hypoxia induced ZNF267 expression while HIF1alpha inhibition significantly reduced both hypoxia-induced as well as basal ZNF267 expression in HCC cells. It is known that hypoxic conditions in tumorous tissues induce the formation of reactive oxygen species (ROS), and ROS have been identified as important factor in the regulation of Ets-1 expression in tumor cells. Here, we found that ROS induction induced and ROS scavenging reduced ZNF267 expression in HCC cells, respectively. Loss and gain of function analysis applying siRNA directed against ZNF267 or transient transfection revealed that ZNF267 promotes proliferation and migration of HCC cells in vitro. These findings indicate Ets-1 and HIF1alpha as critical regulators of basal and hypoxia- or ROS-induced ZNF267 expression in HCC, and further suggest that the pro-tumorigenic effect of these factors is at least in part mediated via increased ZNF267 expression in HCC. Since ZNF267 is already elevated in cirrhosis, ZNF267 appears as promising target for both prevention as well as treatment of HCC in patients with chronic liver disease.

Elevated systemic monocyte chemoattractrant protein-1 in hepatic steatosis without significant hepatic inflammation.

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity and the metabolic syndrome. It encompasses a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine monocyte chemoattractant protein 1 (MCP-1) plays an important role in the progression of hepatic inflammation and fibrosis, and both increased hepatic expression and circulating serum levels have been described in NASH. Here, we aimed to investigate MCP-1 expression in simple hepatic steatosis. Upon feeding a high-fat diet mice developed hepatic steatosis in the absence of significant hepatic inflammation, but elevated hepatic MCP-1 expression compared to control mice fed a standard chow. Interestingly, high-fat diet fed mice had significantly higher MCP-1 serum levels, and MCP-1 mRNA expression was significantly increased in visceral adipose tissue. Furthermore, MCP-1 serum levels were also elevated in patients with ultrasound-diagnosed NAFLD and correlated with the body-mass index and fasting glucose. In conclusion, our data indicate both the liver and adipose tissue as cellular sources of elevated circulating MCP-1 levels already in the early phase of hepatic steatosis. Since MCP-1 derived from visceral adipose tissue reaches the liver via portal circulation at high concentrations it may significantly contribute to the progression of simple steatosis to NASH.

Down-regulation of methylthioadenosine phosphorylase (MTAP) induces progression of hepatocellular carcinoma via accumulation of 5'-deoxy-5'-methylthioadenosine (MTA).

Recently, we have shown that down-regulation of methylthioadenosine phosphorylase (MTAP) in hepatocellular carcinoma (HCC) cells enhances the invasive potential and the resistance against cytokines. Here, we aimed at investigating the molecular mechanism underlying this tumor-promoting effect and expanded the analysis to a large series of human HCC tissues. Liquid chromatography tandem mass spectrometry revealed that reduced MTAP expression resulted in higher intra- and extracellular concentrations of 5'-deoxy-5'-methylthioadenosine (MTA) in cultivated HCC cells and, concordantly, higher levels of MTA in HCC tissue. MTA induced matrix metalloproteinase (MMP) and interleukin-8 transcription in HCC cells in vitro, accompanied by enhanced proliferation and activation of the transcription factor NFκB. In addition, MTA secreted by HCC cells induced expression of fibroblast growth factor-2 and MMP1 in stromal myofibroblasts. In human HCC tissues, MTAP mRNA correlated inversely with MTA levels, and immunohistochemical analysis of a tissue microarray of 140 human HCCs revealed that low MTAP protein expression correlated with advanced tumor stages. In conclusion, MTAP deficiency results in accumulation of MTA, which is associated with increased tumorigenicity. These data further indicate MTAP as a tumor suppressor in HCC, and MTA as a potential biomarker for HCC progression.

Analysis of a promoter polymorphism of the GLUT1 gene in patients with hepatocellular carcinoma.

The glucose transporter isoform 1 (GLUT1) is a key rate-limiting factor in the transport and metabolism of glucose in cancer cells. Recently, we found that GLUT1 expression is increased in hepatocellular carcinoma (HCC) and promotes tumorigenicity of HCC cells. Hypoxia further increased GLUT1 expression in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor (HIF)-1alpha. The promoter region of the GLUT1 gene harbors a single nucleotide polymorphism (SNP; Rs710218; A to T at -2841) closely positioned to a putative HIF-1alpha binding site, and recently, this SNP was found to be more frequent in patients with renal cell carcinoma. In the present study, the A-2841T genotype distribution did not differ significantly between HCC patients (n = 95; AA: 60%; AT 36% and TT: 4%) and healthy controls (n = 127; AA: 50%; AT 41% and TT: 9%). However and noteworthy, non-carriers of the T allele had higher GLUT1 expression levels in cancerous hepatic tissue, and tended to reveal a more aggressive tumour growth. These data indicate that the SNP Rs710218 is not associated with a higher risk for HCC but rather for HCC progression, potentially via HIF-1alpha mediated increased GLUT1 expression.

Prevalence of ultrasound-diagnosed non-alcoholic fatty liver disease in a hospital cohort and its association with anthropometric, biochemical and sonographic characteristics.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) is considered as the most common liver disease in Western countries with still rising prevalence due to a lifestyle favoring the development of the metabolic syndrome. AIM: To investigate the prevalence of ultrasound-diagnosed NAFLD in patients with referral for sonographic examination of the abdomen, and to determine risk factors. METHODS: After exclusion of patients with known liver disease or risk factors for secondary NAFLD, a total of 155 arbitrarily selected patients (mean age 53.6±17.4 years; 52.6% male) from the interdisciplinary ultrasound department of a German University Hospital were included in this prospective study. Each patient underwent a standardized ultrasound, anthropometric and biochemical examination. RESULTS: The prevalence of ultrasound-diagnosed NAFLD was 40.0%. NAFLD-patients had significantly higher body mass index (BMI) and waist-to-hip ratio, higher rates of reported hypertension and diabetes mellitus, and lower HDL cholesterol serum levels. Furthermore, NAFLD-patients revealed significantly higher serum ALT levels (23.2±22.1 U/l vs. 15.0±8.2 U/l; p=0.001), lower AST/ALT ratio (1.76±0.79 vs. 2.11±0.94; p=0.019), and notably, decreased flow in the portal vein (22.9±6.3 cm/s vs. 26.7±10.5 cm/s; p=0.011). Multivariate analysis revealed BMI (odds ratio (OR): 14.05; 95% Confidence interval (CI): 3.3-59.8), AST/ALT ratio (OR: 0.39; CI: 0.18-0.82), and HDL-C (OR: 4.33; CI: 1.6-11.9) as independent risk factors. CONCLUSIONS: Ultrasound-diagnosed NAFLD is frequent in patients with referral for ultrasound examination of the abdomen, and our findings further support that NAFLD is the hepatic manifestation of the metabolic syndrome with obesity being the most important risk factor.

Hepatic steatosis causes induction of the chemokine RANTES in the absence of significant hepatic inflammation.

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to cirrhosis. Hepatocellular lipid accumulation is a hallmark of both nonalcoholic steatosis and steatohepatitis (NASH). The latter develops upon pro-inflammatory cell infiltration and is widely considered as the first relevant pathophysiological step in NAFLD-progression. The chemokine CCL5/RANTES plays an important role in the progression of hepatic inflammation and fibrosis. We here aimed to investigate its expression in NAFLD. Incubation of primary human hepatocytes with palmitic acid induced a dose-dependent lipid accumulation, and corresponding dose-dependent RANTES induction in vitro. Furthermore, we observed significantly elevated hepatic RANTES expression in a dietary model of NAFLD, in which mice were fed a high-fat diet for 12 weeks. This diet induced significant hepatic steatosis but only minimal inflammation. In contrast to the liver, RANTES expression was not induced in visceral adipose tissue of the group fed with high-fat diet. Finally, RANTES serum levels were elevated in patients with ultrasound-diagnosed NAFLD. In conclusion, our data indicate hepatocytes as cellular source of elevated hepatic as well as circulating RANTES levels in response to hepatic steatosis. Noteworthy, upregulation of RANTES in response to lipid accumulation occurs in the absence of relevant inflammation, which further indicates that hepatic steatosis per se has pathophysiological relevance and should not be considered as benign.

Expression of fatty acid synthase in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD.

Epidemiology and survival of patients with hepatocellular carcinoma in Southern Germany.

Hepatocellular carcinoma (HCC) belongs to the most frequent tumors worldwide with an incidence still rising. Patients with cirrhosis are at the highest risk for cancerogenesis and are candidates for surveillance, and here, as well as for the choice of potential forms of treatment, identification of suitable parameters for estimating the prognosis is of high clinical importance. The aim of this study was to describe the etiology of underlying liver disease and to identify predictors of survival in a large single center cohort of HCC patients in Southern Germany. Clinicopathologi-cal characteristics and survival rates of 458 patients (83.6% male; mean age: 62.5+/-11.2 years) consecutively admitted to a University Hospital between 1994 and 2008 were retrospectively analyzed. The results indicate that chronic alcohol abuse was the most common risk factor (57.2%), followed by infection with hepatitis B and C viruses (HBV: 10.9% and HCV: 20.5%). Overall median survival was 19.0 months, and higher OKUDA, CHILD and CLIP scores correlated negatively with prognosis. Of these, only the CLIP Score was an independent predictor in multivariate analysis. We conclude that chronic alcohol abuse is frequently associated with HCC in low hepatitis virus endemic areas, such as Germany. Our study suggests the CLIP score as a valuable prognostic marker for patients' survival, particularly of patients with alcohol related HCC.

Quantification of intermediates of the methionine and polyamine metabolism by liquid chromatography-tandem mass spectrometry in cultured tumor cells and liver biopsies.

By means of liquid chromatography-tandem mass spectrometry we showed recently, that the chromosomal deletion or inactivation of the methylthioadenosine phosphorylase (MTAP) gene led to the accumulation of 5'-deoxy-5'-(methylthio)adenosine (MTA) in cancer cells. Here, we expanded the method to other key intermediates of the methionine and polyamine pathways to further elucidate the molecular consequences of a lack of MTAP activity. Employing multiple-reaction monitoring, limits of detection and lower limits of quantification in the range of 2.5-100 and 5.0-500 nM, respectively, were achieved according to the guidelines of the FDA, thus enabling the direct measurement of the metabolites in biological samples without prior enrichment and derivatization with an analytical repeatability of 1-3%. Relative standards deviations for quadruplicate 80% methanol extractions of metabolites from cultured tumor cells ranged from 1.1 to 25.5%, while the combined methodological and biological variability in metabolite concentrations in 10 liver biopsies was 11.8-51.4%. The method enabled the demonstration of changes in the concentration of intermediates of the methionine and polyamine metabolism other than MTA in hepatocellular carcinoma specimens lacking the enzyme MTAP compared to normal liver tissue.