Giuseppe Castaldo

Increased BDNF Promoter Methylation in the Wernicke Area of Suicide Subjects

CONTEXT Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. OBJECTIVES To test the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. DESIGN Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. SETTING Academic medical center. PATIENTS OR OTHER PARTICIPANTS Forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity. MAIN OUTCOME MEASURES The DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brains Wernicke area. RESULTS Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P < .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. The reported data reveal a novel link between epigenetic alteration in the brain and suicidal behavior.

Genetic Modifiers of Liver Disease in Cystic Fibrosis

CONTEXT A subset (approximately 3%-5%) of patients with cystic fibrosis (CF) develops severe liver disease with portal hypertension. OBJECTIVE To assess whether any of 9 polymorphisms in 5 candidate genes (alpha(1)-antitrypsin or alpha(1)-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor beta1 [TGFB1]) are associated with severe liver disease in patients with CF. DESIGN, SETTING, AND PARTICIPANTS Two-stage case-control study enrolling patients with CF and severe liver disease with portal hypertension (CFLD) from 63 CF centers in the United States as well as 32 in Canada and 18 outside of North America, with the University of North Carolina at Chapel Hill as the coordinating site. In the initial study, 124 patients with CFLD (enrolled January 1999-December 2004) and 843 control patients without CFLD were studied by genotyping 9 polymorphisms in 5 genes previously studied as modifiers of liver disease in CF. In the second stage, the SERPINA1 Z allele and TGFB1 codon 10 genotype were tested in an additional 136 patients with CFLD (enrolled January 2005-February 2007) and 1088 with no CFLD. MAIN OUTCOME MEASURES Differences in distribution of genotypes in patients with CFLD vs patients without CFLD. RESULTS The initial study showed CFLD to be associated with the SERPINA1 Z allele (odds ratio [OR], 4.72; 95% confidence interval [CI], 2.31-9.61; P = 3.3 x 10(-6)) and with TGFB1 codon 10 CC genotype (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 x 10(-3)). In the replication study, CFLD was associated with the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 x 10(-3)) but not with TGFB1 codon 10. A combined analysis of the initial and replication studies by logistic regression showed CFLD to be associated with SERPINA1 Z allele (OR, 5.04; 95% CI, 2.88-8.83; P = 1.5 x 10(-8)). CONCLUSIONS The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, approximately 5) of developing severe liver disease with portal hypertension.

Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

ABSTRACT Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype

This study reports a wide spectrum of factor 8 mutations in the large Italian database. Findings of the study indicate hat the type of mutations is a strong predictor of the clinical phenotype. Background The high mutational heterogeneity of hemophilia A is a challenge for the provision of genetic services. We plan to identify the mutation in patients with hemophilia A in order to create a confidential national database of mutations for the optimization of genetic services in Italy. Design and Methods The factor VIII gene (F8) was analyzed in 1296 unrelated patients with hemophilia A using screening methods for intron 22 and 1 inversions and rare mutations (denaturing high performance liquid chromatography, conformation sensitive gel electrophoresis) and/or direct sequencing. Results F8 mutations were identified in 874 (89%), 146 (89%), and 133 (94%) families with severe, moderate, or mild hemophilia A, respectively. Mutations predicting a null allele were responsible for 80%, 15%, and less than 1% of cases of severe, moderate, or mild hemophilia A, respectively. About 40% of missense and nonsense mutations occurred at a CpG site, arginines being most frequently affected. Of the small deletions or insertions, 29% occurred at one of two stretches of adenines, codons 1191–1194 (8As) and 1439–1441 (9As). Overall, these “hotspots” accounted for 31% of the point mutations in the patients with hemophilia A. Inhibitors developed in 22% of the patients with severe hemophilia A, 8% of those with moderate disease and in 4% of patients with mild hemophilia A. Patients who had severe hemophilia A and mutations predicting a null allele developed inhibitors more frequently (22 to 67%) than patients with missense mutations (5%). Conclusions We report a wide spectrum of mutations in a large national database. The type of mutation was a strong predictor of the clinical phenotype. This database is expected to considerably improve the genetic counselling and medical care of families with hemophilia A in Italy.

Genetic history of cystic fibrosis mutations in italy. I. Regional distribution

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre‐Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, ΔF508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non‐DNA ‘classical’ genetic markers. The northern regions are well differentiated from the central‐southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north‐western regions of Italy, characterized by the mutation 1717‐1G→A, were under Celtic influence, while the north‐east regions, characterized by the mutations R1162X, 2183AA→G and 711 + 5G→A, were under the influence of the Venetic culture.

Congenital diarrheal disorders: Improved understanding of gene defects is leading to advances in intestinal physiology and clinical management

Congenital diarrheal disorders (CDD, Online Mendelian Inheritance in Man [OMIM] 251850) represent one of the most challenging clinical conditions for pediatric gastroenterologists because of the severity of the clinical picture and the broad range of disorders in its differential diagnosis. The number of conditions included within CDD has gradually increased. Recent advances made in the pathophysiology of these conditions have led to a better understanding of the more common diarrheal diseases. Based on the body of data accumulated in recent years, we suggest that CDD be classified in 4 categories depending on the alteration in absorption and transport of nutrients and electrolytes, enterocyte differentiation and polarization, enteroendocrine cell differentiation, and modulation of the intestinal immune response. Our knowledge of the genes responsible for CDD is also rapidly increasing, thanks to linkage studies based on genome-wide analysis of polymorphisms. In this context, the identification of disease genes is a step forward in the diagnostic approach to a patient in whom CDD is strongly suspected. However, it is conceivable that faster, less expensive molecular procedures will, in the near future, become available. This approach could spare the patient invasive procedures and limit complications associated with a delay in diagnosis. Furthermore, carrier and prenatal molecular diagnosis may help pediatricians better manage the condition in the early stages of life.

The efficacy and safety of telaprevir – a new protease inhibitor against hepatitis C virus

Importance of the field: Hepatitis C virus (HCV) is the main agent of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in the western world. It affects more than 170 million people worldwide. HCV treatment, based on the combination of Peg-interferon and ribavirin, is effective in about 50% of treated patients. Therefore, there is a need to develop new drugs active against HCV. Areas covered in this review: Data were obtained by searching for all full articles on Medline and abstracts presented at major international congresses on viral hepatitis. What the reader will gain: A review of clinical data about the efficacy and safety of telaprevir (VX-950), the HCV protease inhibitor that is in the most advanced phase of clinical development. Take home message: Telaprevir has an acceptable pharmacokinetic profile and seems to be a potent antiviral drug against HCV, although, owing to a low genetic barrier, resistant variants emerge within a few days when used in monotherapy, thereby decreasing its efficacy. Consequently, telaprevir has been combined with pegylated-interferon and ribavirin in clinical trials. This triple combination is more effective but has a higher rate of adverse events (notably rash) than the standard of care, despite the shorter duration of therapy.

Lung cancer metastatic cells detected in blood by reverse transcriptase-polymerase chain reaction and dot-blot analysis.

PURPOSE We analyzed the blood of patients with lung cancer at different stages of presentation for the presence of carcinoembryonic antigen (CEA) mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) combined with the dot-blot procedure as an indicator of micrometastatic malignant cells. PATIENTS AND METHODS We studied 24 lung cancer patients (10 with distant metastases and 14 with no evidence of distant metastases), eight age- and sex-matched patients affected by nonneoplastic respiratory diseases (four smokers), and eight healthy subjects. We used immunohistochemistry and RT-PCR dot-blot analysis to evaluate CEA expression in the neoplastic tissue, and the RT-PCR dot-blot procedure to analyze CEA mRNA in circulating cells. RESULTS The RT-PCR dot-blot procedure was highly sensitive aspecific: it detected CEA mRNA in samples of RNA from lung cancer diluted 10(6)-fold with RNA extracted from normal blood cells, and sequence analysis confirmed that the amplified product was CEA. CEA mRNA was found in circulating cells from eight of 10 lung cancer patients with distant metastases (diagnostic sensitivity, 80%) and in four of 14 patients with no evidence of distant metastases. Two of the latter had distant metastases within 6 months of analysis. Thus, the diagnostic specificity of the analysis toward lung cancer without distant metastases was 86%. The analysis was negative in the eight nonneoplastic patients and in the eight healthy controls. CONCLUSION The RT-PCR dot-blot analysis of CEA mRNA in blood cells seems to be a promising tool for the early detection of micrometastatic circulating cells in patients with lung cancer.

Gene Mutation in MicroRNA Target Sites of CFTR Gene: A Novel Pathogenetic Mechanism in Cystic Fibrosis?

Cystic fibrosis (CF) is the most frequent lethal genetic disorder among Caucasians. It depends on alterations of a chloride channel expressed by most epithelial cells and encoded by CFTR gene. Also using scanning techniques to analyze the whole coding regions of CFTR gene, mutations are not identified in up to 10% of CF alleles, and such figure increases in CFTR-related disorders (CFTR-RD). Other gene regions may be the site of causing-disease mutations. We searched for genetic variants in the 1500 bp of CFTR 3′ untranslated region, typical target of microRNA (miRNA) posttranscriptional gene regulation, in either CF patients with the F508del homozygous genotype and different clinical expression (n = 20), CF (n = 32) and CFTR-RD (n = 43) patients with one or none mutation after CFTR scanning and in controls (n = 50). We identified three SNPs, one of which, the c.*1043A>C, was located in a region predicted to bind miR-433 and miR-509-3p. Such mutation was peculiar of a CFTR-RD patient that had Congenital Bilateral Absence of Vas Deferens (CBAVD), diffuse bronchiectasis, a borderline sweat chloride test and the heterozygous severe F508del mutation on the other allele. The expression analysis demonstrated that the c.*1043A>C increases the affinity for miR-509-3p and slightly decreases that for the miR-433. Both miRNAs cause in vitro a reduced expression of CFTR protein. Thus, the c.*1043A>C may act as a mild CFTR mutation enhancing the affinity for inhibitory miRNAs as a novel pathogenetic mechanism in CF.

A Novel Promising Therapeutic Option Against Hepatitis C Virus: An Oral Nucleotide NS5B Polymerase Inhibitor Sofosbuvir

Hepatitis C virus (HCV) infection is one of the main causes of liver disease worldwide. Its treatment is currently based on the combination of peg-interferon, ribavirin, and, for patients with genotype 1, a protease inhibitor (telaprevir or boceprevir). However, interferon-based combinations are not effective in all patients. Moreover, they are contraindicated in patients who cannot receive interferon (e.g. those with decompensated cirrhosis), and are frequently associated with adverse events. Consequently, there is a need to develop new drugs to treat HCV infection. This review focuses on preclinical and clinical data regarding sofosbuvir (GS-7977), a uridine nucleotide analogue inhibitor of HCV NS5 B polymerase that is effective against HCV genotypes 1,2, 3,4 and 6. Thanks to its excellent pharmacokinetic profile, sofosbuvir can be administered in an oral single daily dose. In vitro it exerts a potent antiviral effect against HCV. Clinical data show that combined with peg-interferon and ribavirin for 12 weeks it yields SVR of about 90% in subjects with HCV genotype 1 and about 100% in patients with HCV genotype 2 or 3. Moreover, sofosbuvir and ribavirin administered for 12 weeks yield similar high SVR rate (84% for genotype 1 and 100% for genotype 2/3 patients) as well as sofosbuvir and daclatasvir (an inhibitor of NS5A) which produce SVR rate of about 100% regardless of genotype or of ribavirin employment. Safety and tolerability of sofosbuvir appear to be excellent. In conclusion, sofosbuvir especially in interferon-free combinations represents a very promising option in the treatment of chronic hepatitis C.