Daniela Tavian

Prevalence of mutations in LEP, LEPR, and MC4R genes in individuals with severe obesity.

Obesity is a major public health concern; despite evidence of high heritability, the genetic causes of obesity remain unclear. In this study, we assessed the presence of mutations in three genes involved in the hypothalamic leptin-melanocortin regulation pathway (leptin, LEP; leptin receptor, LEPR; and melanocortin-4 receptor, MC4R), which is important for energy homeostasis in the body, in a group of patients with severe obesity. For this study, we selected 77 patients who had undergone bariatric surgery and had a pre-operative body mass index (BMI) >35 kg/m2, early onset and a family history of being overweight. Candidate genes were screened by direct sequence analysis to search for rare genetic variations. The common LEP -2548 G/A polymorphism was also evaluated for its influence on the BMI (in obesity patients) and for obesity risk, using a case-control study involving 117 healthy individuals. Two different non-synonymous alterations in MC4R were found in two patients: the p.(Thr112Met), previously described in the literature as a probable gene involved in the obesity phenotype, and the novel p.(Tyr302Asp) variant, predicted to be pathogenic by in silico evaluations and family segregation studies. The LEP -2548 G/A polymorphism was not associated with the BMI or obesity risk. In conclusion, we have reported a novel mutation in MC4R in a family of Italian patients with severe obesity. Screening for MC4R could be important for directing the carriers of mutations towards therapy including partial agonists of the MC4R that could normalize their appetite and inhibit compulsive eating. Next-generation sequencing could be used to clarify the genetic basis of obesity in the future.

u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma

Hepatocyte growth factor/scatter factor (HGF/SF) is one of the most important humoral mediators of liver regeneration. It is potentially related to molecular mechanisms of hepatocarcinogenesis via a paracrine system involving its cellular receptor, c‐met. In this study, the expression patterns of HGF and c‐met were evidenced by multiplex RT‐PCR in different specimens of human hepatic tissues (n = 71). A significant increase of c‐met mRNA expression was detected in hepatitis (P = 0.001), cirrhosis (P = 0.006), and hepatocellular carcinoma (HCC) tissue (P = 0.003) compared with normal parenchyma and steatosis. HGF mRNA expression was significantly higher only in hepatitis (P = 0.01). Over‐expression of c‐met mRNA and under‐expression of HGF mRNA were detected in the HCCs compared with the corresponding peri‐tumoral tissues. Neither HGF nor c‐met expression was related to age, sex, tumor size, grading, presence of pseudocapsula, and proliferative activity of the malignant hepatocytes. A significant inverse correlation was found between c‐met mRNA expression level and survival (in months) of patients (P = 0.007), as previously shown for urokinase‐type plasminogen activator (u‐PA) mRNA (P = 0.027). In addition, c‐met mRNA expression was strictly associated with u‐PA mRNA level in HCC samples (P = 0.001). These data show that a loss of balance concerning HGF, c‐met, and u‐PA mRNA expression occurs during hepatocarcinogenesis. Particularly, up‐regulation of c‐met and u‐PA mRNA transcription appears to be coordinately regulated, and their levels of expression are inversely correlated with survival; they must therefore play an important role in the development and progression of human HCC and may also be relevant prognostic markers. Int. J. Cancer 87:644–649, 2000.

Harm avoidance moderates the influence of serotonin transporter gene variants on treatment outcome in bipolar patients

Response to pharmacological treatments is moderated by both genetic and environmental factors. The contribution of such factors is relatively small and complex interactions are likely to be involved. Serotonin transporter gene (SLC6A4) is a major candidate gene associated to response to antidepressant treatment. Moreover, the 5-HTTLPR polymorphism has been associated with anxiety-related traits such as neuroticism and harm avoidance (HA), which are known to influence the risk to develop mood disorders and response to treatments. In the present study we aimed to investigate the interaction between 3 SLC6A4 variants and HA on medium term antidepressant response in a sample of depressed bipolar-spectrum patients followed for 12 months. Contrary to expectations, SLC6A4 variants did significantly influence neither the course of depressive symptoms nor HA scores. However, a significant interaction was observed between HA and 5-HTTLPR genotype. Indeed, a high HA impaired outcome in patients carrying the L(G)/S or the S/S genotype more than in L(A)/L(A) patients. Though a number of limitations characterize the present study, our results indicate HA as a potential moderator of the effect of 5-HTTLPR on the outcome of depression. Given that many factors may influence response to pharmacological treatments, studies that consider personality and other individual characteristics are warranted also in pharmacogenetic investigations.

Early Exposure to Alcohol Leads to Permanent Impairment of Dendritic Excitability in Neocortical Pyramidal Neurons

Exposure to alcohol in utero is a well known cause of mental retardation in humans. Using experimental models of fetal alcohol spectrum disorder, it has been demonstrated that cortical pyramidal neurons and their projections are profoundly and permanently impaired. Yet, how the functional features of these cells are modified and how such modifications impact cognitive processes is still unknown. To address this, we studied the intrinsic electrophysiological properties of pyramidal neurons in young adult rats (P30–P60) exposed to ethanol inhalation during the first week of postnatal life (P2–P6). Dual whole-cell recordings from the soma and distal apical dendrites were performed and, following the injection of depolarizing current into the dendrites, layer 5 neurons from ethanol-treated (Et) animals displayed a lower number and a shorter duration of dendritic spikes, attributable to a downregulation of calcium electrogenesis. As a consequence, the mean number of action potentials recorded at the soma after dendritic current injection was also lower in Et animals. No significant differences between Et and controls were observed in the firing pattern elicited in layer 5 neurons by steps of depolarizing somatic current, even though the firing rate was significantly lower in Et animals. The firing pattern and the firing rate of layer 2/3 neurons were not affected by alcohol exposure.

Contribution of novel ATGL missense mutations to the clinical phenotype of NLSD-M: a strikingly low amount of lipase activity may preserve cardiac function

The lack of adipose triglyceride lipase (ATGL), a patatin-like phospholipase domain-containing enzyme that hydrolyzes fatty acids from triacylglycerol (TAG) stored in multiple tissues, causes the autosomal recessive disorder neutral lipid storage disease with myopathy (NLSD-M). In two families of Lebanese and Italian origin presenting with NLSD-M, we identified two new missense mutations in highly conserved regions of ATGL (p.Arg221Pro and p.Asn172Lys) and a novel nonsense mutation (p.Trp8X). The Lebanese patients harbor homozygous p.Arg221Pro, whereas the Italian patients are heterozygotes for p.Asn172Lys and the p.Trp8X mutation. The p.Trp8X mutation results in a complete absence of ATGL protein, while the p.Arg221Pro and p.Asn172Lys mutations result in proteins with minimal lipolytic activity. Although these mutations did not affect putative catalytic residues or the lipid droplet (LD)-binding domain of ATGL, cytosolic LDs accumulated in cultured skin fibroblasts from the patients. The missense mutations might destabilize a random coil (p.Asn172Lys) or a helix (p.Arg221Pro) structure within or proximal to the patatin domain of the lipase, thereby interfering with the enzyme activity, while leaving intact the residues required to localize the protein to LDs. Overexpressing wild-type ATGL in one patients fibroblasts corrected the metabolic defect and effectively reduced the number and area of cellular LDs. Despite the poor lipase activity in vitro, the Lebanese siblings have a mild myopathy and not clinically evident myocardial dysfunction. The patients of Italian origin show a late-onset and slowly progressive skeletal myopathy. These findings suggest that a small amount of correctly localized lipase activity preserves cardiac function in NLSD-M.

Coordinated sumoylation and ubiquitination modulate EGF induced EGR1 expression and stability.

Background Human early growth response-1 (EGR1) is a member of the zing-finger family of transcription factors induced by a range of molecular and environmental stimuli including epidermal growth factor (EGF). In a recently published paper we demonstrated that integrin/EGFR cross-talk was required for Egr1 expression through activation of the Erk1/2 and PI3K/Akt/Forkhead pathways. EGR1 activity and stability can be influenced by many different post-translational modifications such as acetylation, phosphorylation, ubiquitination and the recently discovered sumoylation. The aim of this work was to assess the influence of sumoylation on EGF induced Egr1 expression and/or stability. Methods We modulated the expression of proteins involved in the sumoylation process in ECV304 cells by transient transfection and evaluated Egr1 expression in response to EGF treatment at mRNA and protein levels. Results We demonstrated that in ECV304 cells Egr1 was transiently induced upon EGF treatment and a fraction of the endogenous protein was sumoylated. Moreover, SUMO-1/Ubc9 over-expression stabilized EGF induced ERK1/2 phosphorylation and increased Egr1 gene transcription. Conversely, in SUMO-1/Ubc9 transfected cells, EGR1 protein levels were strongly reduced. Data obtained from protein expression and ubiquitination analysis, in the presence of the proteasome inhibitor MG132, suggested that upon EGF stimuli EGR1 sumoylation enhanced its turnover, increasing ubiquitination and proteasome mediated degradation. Conclusions Here we demonstrate that SUMO-1 modification improving EGR1 ubiquitination is involved in the modulation of its stability upon EGF mediated induction.

Severe steatohepatitis in a patient with a rare neutral lipid storage disorder due to ABDH5 mutation

Fatty liver disease is mainly caused by alcohol consumption, excessive body weight, dyslipidemia and impaired glucose tolerance, but inherited disorders can sometimes be involved. We report the case of a 40-year-old woman with steatohepatitis and severe portal hypertension, associated with ichthyosis, cataract and hypoacusia. The clinical, pathological and genetic findings were consistent with a diagnosis of Chanarin-Dorfman syndrome (CDS), a rare autosomal recessive inherited neutral lipid storage disorder, and genetic analysis showed that a novel ABHD5 mutation is responsible.

Improved cytochemical method for detecting Jordans’ bodies in neutral lipid storage diseases

We describe an improved cytochemical procedure for detecting the presence of cytoplasmic lipid droplets (Jordans’ bodies) in blood cells from patients suffering from neutral lipid storage diseases (NLSDs). The method employs Oil red O (ORO), Nile red (NR) or, preferably, Bodipy (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene) staining, coupled with DAPI (2-(4-amidinophenyl)-6-indolecarbamidine) staining of nuclei, to visualise clearly by fluorescence microscopy the presence of abundant neutral lipids (triacylglycerols and cholesterol esters) in granulocytes and monocytes. Using these reagents, we easily identified Jordans’ bodies in buffy coats from patients affected by Chanarin–Dorfman syndrome (NLSD with ichthyosis) and NLSD with progressive myopathy, a laboratory finding critical for the diagnosis of both inherited metabolic disorders. Due to their yellow-gold and green fluorescence arising selectively from neutral lipid binding and their water solubility that makes alcohol unnecessary for staining, NR and particularly Bodipy are superior to ORO for the specific detection of Jordans’ bodies in leucocytes. Neutral lipid storage diseases (NLSDs) are a clinically heterogeneous group of non-lysosomal inherited disorders characterized by a cytoplasmic accumulation of lipid droplets (LDs) in most tissues. Rather than being an inert lipid inclusion, an impression given prima facie by their simple morphological structure under the light microscope, LDs were recently shown to be distinct organelles consisting of a core of neutral lipids, predominantly triacylglycerols or cholesteryl esters, that are surrounded by a monolayer of phospholipids and associated proteins.1,2 LDs take active part in lipid metabolism as well as in membrane trafficking and other cell fuctions.3 Since the pioneering observation by Jordans of numerous LDs in the leucocytes of two Dutch brothers suffering from progressive muscular dystrophy,4 several reports have described the presence of abundant triacylglycerol deposits in the non-adipose cells of patients manifesting a variety of defects. Clinical phenotypes include myopathy (skeletal and heart muscle), liver damage, ataxia, neurosensory …

Nanotechnology advances in brain tumors: the state of the art.

Primary malignant central nervous system (CNS) tumors only represent about 2% of all cancers. However, they are very often associated with high morbidity and mortality. Despite current standard-of-care therapy, such as surgery, irradiation, and chemotherapy, neither cure nor any toxic therapy against malignant CNS tumors has been developed so far. Nanotechnology may alter this situation. It offers a new promise for cancer diagnosis and treatment. This emerging technology, by developing and manufacturing materials using atomic and molecular elements, can provide a platform for the combination of diagnostics, therapeutics and delivery to the tumor, with subsequent monitoring of the response. This review focuses on recent developments in cancer nanotechnology with particular attention to nanoparticle systems, important tools for the improvement of drug delivery in brain tumor. The latest advances in both the research sector and in recent patents for cancer imaging and therapy are discussed.

A new splicing site mutation of the ABCB4 gene in intrahepatic cholestasis of pregnancy with raised serum γ-GT

BACKGROUND Intrahepatic cholestasis of pregnancy is a liver disorder with a multifactorial etiology characterized by maternal pruritus, abnormal liver function tests and increased fetal risk. The main biochemical finding is the increase in total serum bile acid concentrations. In a subgroup of women, the serum gamma-glutamyl transpeptidase level is also increased. There is evidence that dysfunction of the ABCB4 gene might play a role in intrahepatic cholestasis of pregnancy development. AIM To investigate the role of the ABCB4 gene in Italian women with intrahepatic cholestasis of pregnancy and raised gamma-glutamyl transpeptidase by, analyzing the complete coding sequence and mRNA splicing products. METHODS Among 299 women with intrahepatic cholestasis of pregnancy, 10 showing raised gamma-glutamyl transpeptidase were enrolled in this study. DNA and RNA were extracted from peripheral blood mononuclear cells using standard procedures. The 27 coding exons and the promoter region were amplified by polymerase chain reaction and analyzed by sequencing. Reverse transcript-polymerase chain reaction analysis of ABCB4 mRNA and cDNA analysis were also performed. RESULTS A novel splicing mutation that causes a truncated protein of 249 amino acid was identified in a woman who had the highest serum levels of gamma-glutamyl transpeptidase, alkaline phosphatase, bile acids, and the highest pruritus score. We identified also one already described p.R590Q mutation in a woman who had significantly higher serum levels of alkaline phosphatase, aspartate, and alanine aminotransferase. CONCLUSIONS Our study demonstrates that splicing mutations in the ABCB4 gene can cause ICP in women with high gamma-glutamyl transpeptidase and thus a complete analysis of coding sequence and cDNA products is required.