Characterization of the genomic structure of the human neuronal nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster and identification of novel intragenic polymorphisms

Genes coding for the alpha5, alpha3, and beta4 subunits (CHRNA5, CHRNA3, and CHRNB4) of the neuronal nicotinic acetylcholine receptors (nAChRs) are clustered on chromosome 15q24. Linkage of this chromosomal region to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), an idiopathic partial epilepsy, was reported in one family. Moreover, mutations in other neuronal nAChR subunit genes coding for the alpha4 (CHRNA4) and the beta2 (CHRNB2) subunits were associated with ADNFLE. Apart from the exon-intron structure of CHRNA3, the genomic organization of this gene cluster was unknown, making comprehensive mutational analyses impossible. The genomic structure of CHRNA5 and CHRNB4 is here reported. Moreover, two hitherto unknown introns were identified within the 3' untranslated region of CHRNA3, causing a partial tail-to-tail overlap with CHRNA5. Four novel intragenic polymorphisms were identified and characterized in the cluster

A type mutation II (Glu117stop), induction of allele-specific mRNA degradation and FXI deficiency

The Glu117stop mutation in the factor XI (FXI) gene is the most common cause of FXI deficiency and might cause the disease either by poor secretion/stability of the truncated protein or by decreased mRNA levels. Platelet- and lymphocyte-derived mRNA from three Glu117stop heterozygotes were analyzed by reverse-transcriptase polymerase chain reaction and sequencing, demonstrating allele-specific reduction of FXI Glu117stop mRNA

Type II mutation (Glu117stop) causes factor XI deficiency by inducing allele specific mRNA degradation

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. The disease has been described in most populations but is particularly common in Jews, where a heterozygous frequency of 9% has been reported. Among known genetic defects in the FXI gene, the type II mutation (Glu117stop) is highly frequent in Ashkenazi and Iraqi Jews, and was repeatedly found also in other populations, always associated with the same founder haplotype. This nonsense mutation, which creates a premature termination codon (PTC) in exon 5, is predicted to cause the synthesis of a truncated protein, which is absent from patients\u2019 plasma. However, transcripts harboring PTCs are also known to frequently undergo an allele specific degradation through the nonsense-mediated mRNA decay mechanism, which prevents cells from the deleterious effects (i.e. dominant negative or gain-of-function) of truncated protein possibly originating from stable nonsense transcripts. So far, no evidence has been reported on the effect of the Glu117stop mutation on FXI mRNA metabolism. In this study, platelet-derived mRNAs from three Italian patients carrying the type II mutation in the heterozygous state were analyzed by RT-PCR and sequencing, and compared to their corresponding genomic sequence. In all patients, only transcripts originating from the wild-type allele were detected, suggesting the selective degradation of FXI mRNAs harboring the PTC. Moreover, no evidence of alternative splicing of FXI exon 5, previously reported to occur in platelets, was found in these patients. It will be interesting to extend analysis to other FXI null mutations in order to evaluate the effect of premature termination codons on FXI mRNA metabolism

La mutazione tipo II (Glu117stop) causa carenza di fattore XI della coagulazione mediante degradazione allele specifica del corrispondente mRNA

La carenza grave di fattore XI (FXI) della coagulazione \ue8 una malattia emorragica ereditata generalmente come un tratto autosomico recessivo e caratterizzata da una sintomatologia emorragica principalmente associata a traumi o interventi chirurgici. La malattia \ue8 stata descritta in molte popolazioni, ma \ue8 particolarmente comune fra gli ebrei, nei quali \ue8 stata riportata una frequenza degli eterozigoti del 9%. Tra i difetti genetici identificati nel gene per il FXI, la mutazione di tipo II (Glu117stop) \ue8 la pi\uf9 frequente fra gli ebrei ashkenazi ed iracheni, ed \ue8 stata ripetutamente riscontrata in altre popolazioni, sempre associata con lo stesso aplotipo fondatore. Questa mutazione, che crea un codone di stop prematuro (PTC) nell\u2019esone 5, causa la sintesi di una proteina tronca, assente nel plasma dei pazienti. Tuttavia, \ue8 noto che trascritti recanti PTC sono frequentemente soggetti a degradazione allele specifica mediante il meccanismo del Nonsense-Mediated mRNA Decay, che protegge le cellule dagli effetti deleteri (sia dominanti negativi che dovuti a gain of function) delle proteine tronche sintetizzate a partire da trascritti contenenti mutazioni nonsense. A tutt\u2019oggi non \ue8 stata fornita una dimostrazione diretta dell\u2019effetto della mutazione Glu117stop sul metabolismo dell\u2019mRNA per il FXI. In questo studio, l\u2019mRNA piastrinico di tre pazienti italiani eterozigoti per la mutazione di tipo II \ue8 stato analizzato mediante RT-PCR e sequenziamento. Dal confronto con le sequenze genomiche corrispondenti \ue8 stato possibile dimostrare la presenza del solo allele wild-type, suggerendo la degradazione selettiva dell\u2019mRNA recante il PTC. Inoltre, non \ue8 stato possibile evidenziare alcun evento di splicing alternativo dell\u2019esone 5 del FXI, che potrebbe risultare dal meccanismo del Nonsense-Mediated Altered Splicing. Sar\ue0 interessante estendere l\u2019analisi ad altre mutazioni che determinano l\u2019inserzione di PTC per valutarne l\u2019effetto sul metabolismo dell\u2019mRNA del FXI

Differential expression of microRNAs in peripheral blood mononuclear cells of multiple sclerosis patients

MicroRNAs (miRNAs) are a class of short single-stranded RNAs that act as post-transcriptional regulators of gene expression and represent a major group of regulators potentially associated with human multifactorial diseases. Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. As miRNA-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response, their deregulation may result in defects in immune tolerance. In this frame, we sought to explore the possible involvement of miRNAs in MS pathogenesis by monitoring the differential expression of 22 miRNAs (expressed in the immune system or transcribed from previously reported MS susceptibility loci) in peripheral blood mononuclear cells of MS patients and healthy controls by using a microbead-based technology. Four miRNAs resulted >3 folds up-regulated in MS vs controls, whereas only 1 resulted down-regulated. Interestingly, 2 of the most up-regulated miRNAs (mir-155, mir-146a) have been reported to be altered also in rheumatoid arthritis and systemic lupus erythematosus, suggesting shared pathogenic mechanisms with MS. To confirm mir-155 and mir-146a up-regulation, qPCR experiments will be performed. Moreover, the role of these miRNAs in MS susceptibility will be investigated by genotyping single nucleotide polymorphisms mapping in mir-155 and mir-146a regions

Natriuretic peptide fragments as possible biochemical markers of hypertension in the elderly.

AIMS: To study the relationship between C-type natriuretic peptide (NT-proCNP) and other natriuretic peptides, such as pro-atrial natriuretic peptide [proANP(1-98)] and N-terminal pro-brain natriuretic peptide (NT-proBNP), in the elderly, investigating also their correlation with other traditional clinical markers of the hypertensive condition. METHODS: NT-proCNP, NT-proBNP and proANP(1-98) were measured in 57 elderly patients. They were hypertensive patients (n\u200a=\u200a36) and normotensive controls (n\u200a=\u200a21). Their anthropometric parameters, including Winsor's index and total and high-density lipoprotein cholesterol, were determined. RESULTS: A diagnostic role of NT-proBNP in hypertensive patients was detected by a model of logistic regression, which gave a significant result [odds ratio (OR) 1.0115, P\u200a=\u200a0.0184]. By this model the area (AUC) under the receiver-operating characteristic (ROC) curve was 0.69\u200a\ub1\u200a0.071 (P\u200a=\u200a0.0075). On the basis of the ROC curve, the calculated serum NT-proBNP cut-off for the prediction of hypertension was greater than 164 pmol/l - the value being provided with a sensitivity of 89% coupled with a specificity of 55%. NT-proCNP and proANP(1-98) did not predict the hypertensive condition, although significant correlations were detected with serum lipid profile and creatinine levels. CONCLUSIONS: By using the logistic regression analysis, NT-proBNP was identified as a significant predictor of hypertension, whereas NT-proCNP and proANP circulating levels were not shown to reliably predict the hypertensive condition. Further validation by means of larger cohort studies is undoubtedly needed to assess the use of all three peptides to increase the performance of a possible test for the prediction of the hypertensive condition in humans

A new class of non-coding RNAs associated with 3\u2019 untranslated regions of mRNAs

The importance of non-coding RNAs (ncRNAs) in controlling gene expression is becoming increasingly evident. However, except for some well characterized examples, such as miRNAs, Xist and Air, the function of most non-coding transcripts is still to be determined. Moreover, while small regulatory RNAs can be relatively easily classified on the basis of their length, secondary structure, and biochemical pathway, the classification of long \u201cmRNA-like\u201d ncRNAs has been problematic. Here we identify a large class of non-coding transcripts that originate within the 3\u2019UTR of at least one third of all genes in the mouse genome. We have several lines of evidence from genome-wide bioinformatic analyses (EST coverage, CAGE data, chromatin state maps of active promoters) and from invitro studies (strand-specific RT-PCR, 5\u2019RACE, Northern blot) showing that these 3\u2019UTR-associated ncRNAs (uaRNAs) can be either linked or transcribed separately to the upstream protein-coding sequences. In addition, expression profiles obtained by custom-designed microarrays on three different developmental systems (myoblast differentiation, male gonadal ridge formation, embryonic stem cell differentiation) showed that uaRNA expression is highly regulated and tissue-specific, and might be either concordant or discordant with respect to the upstream coding region depending on the cell type and on the developmental stage. This observation is confirmed by in-situ hybridization experiments, which evidenced that uaRNA and the associated coding transcript might have different subcellular locations. Our results highlight a further level of complexity at 3\u2019UTRs, suggesting the presence of new regulatory mechanisms that control gene expression during embryonic development. Our data have also important implications for the design of in-situ hybridization and microarray probes as well as for the interpretation of gene expression dat

Maintenance therapy in epithelial ovarian cancer: from chemotherapy to targeted agents.

Over the past years, although no increase in the cure rate for advanced epithelial ovarian cancer patients has been achieved, a slow prolongation in patients survival has been observed, thanks to the introduction of effective second line or salvage therapies. Attempts to disease chronicization seem therefore of value in this setting. A major effort has been pursued to establish the role of maintenance therapies for epithelial ovarian cancer patients. Although chemotherapy does not seem to have an effective role, promising results are coming from trials investigating maintenance targeted treatments, especially with antiangiogenic agents or PARP inhibitors for selected patients. The aim of this article is to review current evidences on maintenance therapy for epithelial ovarian cancer and put the results in perspective

Molecular characterization of two novel mutations causing factor XI deficiency : a splicing defect and a missense mutation responsible for a CRM+ defect

Severe factor XI (FXI) deficiency is a bleeding disorder generally inherited as an autosomal recessive trait and characterized by haemorrhagic symptoms mainly associated with injury or surgery. So far, more than 150 causative molecular defects have been identified throughout the F11 gene. In the present study, we investigated the molecular basis of FXI deficiency in two Italian patients. Mutational screening of the F11 gene disclosed a novel missense substitution (Arg184Gly) in exon 7 and two splicing mutations: a novel G>A transition affecting the last nucleotide of exon 4 (325G>A), and the already known IVS6+3A>G. RT-PCR assays were performed on total RNA extracted from platelets and lymphocytes of each patient. Sequencing of RT-PCR products demonstrated that both 325G>A and IVS6+3A>G mutations abolish the corresponding donor splice site, causing the skipping of the affected exon; this in turn results in a frameshift introducing a premature termination codon. Expression of recombinant FXI-Arg184Gly revealed a 70% reduction in FXI activity, suggesting that the Arg184Gly mutation might cause a cross-reactive material positive (CRM+) deficiency. In conclusion, the functional consequences of two splicing mutations leading to FXI deficiency have been elucidated. Moreover, we report a novel missense mutation in the FIX-binding region of the FXI A3 domain leading to a CRM+ deficiency