Search

Dominant mutations in desmocollin-2 (DSC2) gene cause arrhythmogenic cardiomyopathy (ACM), a progressive heart muscle disease characterized by ventricular tachyarrhythmias, heart failure, and risk of juvenile sudden death. Recessive mutations are rare and are associated with a cardiac or cardiocutaneous phenotype. Here, we evaluated the impact of a homozygous founder DSC2 mutation on clinical expression of ACM. An exon-by-exon analysis of the DSC2 coding region was performed in 94 ACM index patients. The c.536A>G (p.D179G) mutation was identified in 5 patients (5.3%), 4 of which resulted to be homozygous carriers. The 5 subjects shared a conserved haplotype, strongly indicating a common founder. Genetic and clinical investigation of probands' families revealed that p.D179G homozygous carriers displayed severe forms of biventricular cardiomyopathy without hair or skin abnormalities. The only heterozygous proband, who carried an additional variant of unknown significance in αT-catenin gene, showed a mild form of ACM without left ventricular involvement. All heterozygous family members were clinically asymptomatic. In conclusion, this is the first homozygous founder mutation in DSC2 gene identified among Italian ACM probands. Our findings provide further evidence of the occurrence of recessive DSC2 mutations in patients with ACM predominantly presenting with biventricular forms of the disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations., Methods: HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively., Results: TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation., Conclusions: The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.

Background/aims: A non-enveloped single-stranded DNA virus (TTV) was detected in Japanese patients with fulminant hepatitis (47%) and chronic liver disease of unknown etiology (46%) more frequently than in blood donors (12%). Subsequent studies, however, questioned the association of TTV with liver disease. We further investigated the role of this novel virus in liver diseases., Methods: We tested 106 patients and 102 blood donors for TTV by polymerase chain reaction using conserved region primers., Results: TTV DNA was found in 19 of 102 volunteer blood donors (18.6%) and in 27 of 106 patients with liver disease (25.5%): 10 of 28 chronic hepatitis B (35.7%), 9 of 28 chronic hepatitis C (32.1%) and 8 of 50 (16%) cryptogenic liver disease patients. Previous interferon treatment was not associated with a significantly lower prevalence of TTV infection. TTV prevalence was higher in patients with blood exposure (42.8%, 6/14) than in patients without risk factors (21.4%, 18/84). Four of five patients (80%) with HBV familial infection and without blood exposure were also TTV positive. Partial nucleotide sequences from 3 Italian isolates diverged more than 30% from the 2 prototype genotypes G1 and G2 and were 88% homologous to the recently described genotype G4., Conclusions: G1 and G2 TTV are common in Italy and in the USA in liver disease patients and in blood donors. The prevalence is high in patients with blood exposure but also in subjects without risk factors; other routes of transmission should therefore be considered.

An Italian Committee for the quality assessment of steroid receptor assay was instituted in 1979; the number of laboratories participating in this program increased from 7 in 1979 to 43 in 1989. The Italian program in collaboration with EORTC (European Organization Research and Treatment of Cancer) initiatives, utilizes as working standards lyophilized samples that tolerate prolonged storage at 4 degrees C. The national representatives agreed, according to EORTC, that all laboratories would perform, for measurement of steroid receptors, the dextran coated charcoal (DCC) method using the same concentration of radioactive ligands (3-H-estradiol, 3-H-ORG-2058) and would determine the non-specific binding with the same compounds (diethylstilbestrol and ORG-2058). The computation method is a potential source of interlaboratory variation; the multipoint Scatchard analysis, though difficult to apply for receptor problems, is the most widely used approach at present. The standardized DCC method is the most common and recognized (Food and Drug Administration) procedure for quantifying hormone receptor in human cancer. Since new technologies are being introduced (receptor enzyme immunoassay, EIA), adequate programs of quality assessment are required; in general, it has demonstrated an excellent correlation between radioligand binding assay (DCC) and immunochemical assay (EIA). Other prognostic factors, such as proteins produced by oncogenes and growth factors of the malignant cells have become more important. Some of these factors, as well as estrogen receptor status, seem the major determinants of recurrence after the first treatment.

Since 1979 the quality control design proposed by the Italian ad hoc Committee has evaluated several lyophilized preparations with scalar receptor content; this permits the identification by linear regression analysis of systematic and non systematic errors. At present 41 laboratories from most of the national regions have joined the Italian Committee. The overall results of five years application of quality assurance in Italy show that there was a different pattern of imprecision with satisfactory indexes for intralaboratory performances but major variations in interlaboratory controls. There was also a remarkable difference of variability indices between the so-called "expert" and "new" laboratories; this problem can be reduced with practical seminars for new centers. On the basis of the results and experience achieved the Committee is starting another program of quality assurance for different new methodologies to provide guidelines for international working reference standards.

The importance of evaluating receptors for estrogen and progestin in human breast cancer has been pointed out by many authors. In the absence of a reference standard, receptor assays must be controlled by intra and interlaboratory quality control programs. Much interlaboratory variability exists due to non-uniform analytical protocols, non-uniform ligands, intrinsic errors and also errors in computation methods. The goals of our Italian Quality Control Program on Multicenter Trials are to standardize the analytical procedures and computation methods. Twenty Italian laboratories participated in the Quality Control Program. Each specimen was assayed for steroid receptor content according to the standardized dextran-coated-charcoal method. Data were subjected to computerized analyses by 5 different methods of calculation (Scatchard plot, direct plot, Lineweaver-Burk method, Brunauer-Emmet-Teller analysis, single-point approach). The results were than evaluated to identify intra- and inter-assay variation coefficients and to define other statistical parameters. The authors suggest different calculation methods depending on the specific experimental and/or physiopathological conditions.