Sequencing of NOTCH1, GATA5, TGFBR1 and TGFBR2genes in familial cases of bicuspid aortic valve

BACKGROUND: The purpose of our study was to investigate the potential contribution of germline mutations in NOTCH1, GATA5 and TGFBR1 and TGFBR2 genes in a cohort of Italian patients with familial Bicuspid Aortic Valve (BAV). METHODS: All the coding exons including adjacent intronic as well as 5(′) and 3(′) untranslated (UTR) sequences of NOTCH1, GATA5, TGFBR1 and TGFBR2 genes were screened by direct gene sequencing in 11 index patients (8 males; age = 42 ± 19 years) with familial BAV defined as two or more affected members. RESULTS: Two novel mutations, a missense and a nonsense mutation (Exon 5, p.P284L; Exon 26, p.Y1619X), were found in the NOTCH1 gene in two unrelated families. The mutations segregated with the disease in these families, and they were not found on 200 unrelated chromosomes from ethnically matched controls. No pathogenetic mutation was identified in GATA5, TGFBR1 and TGFBR2 genes. CONCLUSIONS: Two novel NOTCH1 mutations were identified in two Italian families with BAV, highlighting the role of a NOTCH1 signaling pathway in BAV and its aortic complications. These findings are of relevance for genetic counseling and clinical care of families presenting with BAV. Future studies are needed in order to unravel the still largely unknown genetics of BAV

Analysis of genetic and epigenetic alterations in candidate genes in thoracic aortic aneurysm

Aortic aneurysm and dissections account for 1% to 2% of all deaths in the Western countries, representing the 15th leading cause of death in individuals older than 65 years. Despite progressive dilatation, thoracic aortic aneurysm (TAA) usually remain asymptomatic until dissection or rupture occurs. Because of this, the detection and monitoring of TAAs are absolutely crucial, and studies on potential biomarkers are underway. While size is currently the main aortic parameter/criterion by which to predict complications, other characteristics of TAAs, such as mechanical properties and genes and molecular aspects are being investigated as potential additional criteria for the future. Gene defect identification in human thoracic aortic aneurysm conditions is proceeding at a rapid pace and the integration of pathogenesis-based management strategies in clinical practice is an emerging reality. Despite the remarkable progress of the past decade, the pathogenesis of TAA remains unclear. The main molecular hypothesis of TAA formation appears to be an altered signaling pathway of the transforming growth factor-β (TGF-β). Several studies showed that mutations in transforming growth factor-β receptor 1 (TGFBR1), receptor 2 (TGFBR2) and (SMAD3) genes play a key role in the onset of syndromic and familial TAA. Moreover, an altereted expression of this signaling components, caused by microRNA (miRNAs), could lead to TAA formation. The purpose of this study was to investigate the potential contribution of germline mutations in TGFBR1, TGFBR2 and SMAD3 genes in a cohort of Italian patients with familial TAA. Moreover, we evaluated the different expression levels of latent transforming growth factor-β binding proteins (LTBPs) and the role of miRNAs, in the development and progression of the TAA in patients with bicuspid (BAV) or tricuspid aortic valve (TAV). We performed a direct sequencing of all coding regions and untranslated reregions of TGFBR1, TGFBR2 and SMAD3 genes in 10 Italian patients with familial TAA. The LTBPs levels were examinated by qRT-PCR, while the miRNA profile using next generation sequencing platform (MiSeq). As regards the genetic screening, a novel TGFBR2 mutation in the 5’ untranslated region (c.-59 C>T) was identified in a 31-year-old male who referred for an emergent operative repair of Stanford type A aortic dissection. Bioinformatics tools showed that c.-59 C>T variant was predicted to affect exonic splicing enhancer and was validated by quantitative real-time RT-PCR, revealing a six-fold increase of TGFBR2 mRNA level in aneurysmatic aortic tissue of patient harboring mutation, compared to aortic samples from patients without the mutation. Moreover, we found a previously described missense mutation, p.E239K, in the MH2 domain of the SMAD3 gene in a 60-year-old man who presented diffuse aneurysms involving various arteries. As regard gene expression pattern of LTBP-isoforms, we found a significantly down expression of LTBP4L mRNA levels in BAV group compared with TAV. Conversely, we found any significant difference in the expression of other TGF-β-related factors. The miRNAs profile revealed several miRNAs differentially expressed between BAV and TAV, in particular 2 miRNAs were up-regulated and 10 were down-regulated in BAV compared to TAV. A preliminary analysis with DIANA tool revealed the role of 4 specific miRNAs (hsa-miR-424-3p, hsa-miR-3158-3p, hsa-miR-3688-3p, hsa-miR-486-3p) in the regulation of 12 genes involved in several cellular mechanisms previously described to be important in the pathogenesis of TAA, such as apoptosis, angiogenesis extracellular matrix neogenesis, and osteoblast differentiation. In conclusion, our results confirm the key role played by TGF-β pathway in the etiopathogenesis of TAA and support the hypothesis that TAA in BAV and TAV patients is linked to different molecular mechanisms

Screening genetico di TGFBR1 e TGFBR2 in casi familiari di aneurisma dell'aorta toracica

Gli aneurismi dell’aorta toracica rappresentano un’importante causa di morbosità e mortalità nei paesi occidentali. Studi in letteratura hanno dimostrato come disordini genetici, vedi la sindrome di Marfan, la bicuspidia aortica e altri disordini del tessuto connettivo, incrementino il rischio di sviluppare la patologia aneurismatica soprattutto in individui di giovane età. Tuttavia la maggior parte dei casi tendono ad essere sporadici, asintomatici e spesso non vengono diagnosticati prima della comparsa della rottura. I meccanismi genetici e molecolari alla base della patologia aneurismatica sono ancora poco chiari, sebbene negli ultimi anni sia stato osservato un ruolo chiave svolto dal TGF-beta. Infatti studi di sequenziamento genico in casi familiari di aneurisma aortico hanno riscontrato la presenza di mutazioni a livello dei geni codificanti i recettori di tipo 1 e 2 del TGF-beta (TGFBR1 e TGFBR2). Pertanto lo scopo della tesi è quello di verificare, mediante screening genetico, la presenza di mutazioni a carico dei geni TGFBR1 e TGFBR2 in casi familiari di aneurisma dell’aorta toracica. Abbiamo arruolato 2 pazienti, afferenti presso l’ospedale Pasquinucci di Massa, i quali presentavano una storia familiare positiva di aneurisma toracico. L’analisi mutazionale è stata effettuata mediante sequenziamento genico sia delle regioni codificanti, 9 esoni per il gene TGFBR1 e 7 esoni per il gene TGFBR2 che delle vicine regioni introniche, importanti siti di splicing. Il sequenziamento genico è stato ottenuto mediante l’impiego della piattaforma ad alta processività “GenomeLab GeXP Genetic Analysis System” (CEQ8800, Beckman) e le sequenze ottenute sono state analizzate con un software specifico, che ci ha permesso di confrontarle con sequenze ottenute dalla GenBank. Lo screening genetico ha rilevato la presenza di varianti polimorfiche già precedentemente descritte in letteratura. L’assenza di mutazioni patologiche nei nostri pazienti non esclude tuttavia il possibile ruolo dei geni TGFBR1 e TGFBR2 nell’eziologia aneurismatica. Studi futuri si propongono di chiarire il meccanismo molecolare alla basa di tale patologia ed il potenziale effetto di mutazioni a carico del gene TGF-beta

Hypothesis-free secretome analysis of thoracic aortic aneurysm reinforces the central role of TGF-b cascade in patients with bicuspid aortic valve

Background: Ascending thoracic aortic aneurysm (ATAA) is a major cause of morbidity and mortality worldwide. The pathogenesis of medial degeneration of the aorta remains undefined. High-throughput secretome analysis by mass spectrometry may be useful to elucidate the molecular mechanisms involved in aneurysm formation as well as to identify biomarkers for early diagnosis or targets of therapy. The purpose of the present study was to analyze the secreted/released proteins from ATAA specimens of both tricuspid aortic valve (TAV) and bicuspid aortic valve (BAV) patients. Methods: Aortic specimens were collected from patients undergoing elective surgery and requiring graft replacement of the ascending aorta. Each sample of the ascending aortic aneurysm, 4 BAV (3 males; aged 53.5 11.4 years) and 4 TAV (1 male; 78 7.5 years), was incubated for 24 h in serum-free medium. Released proteins were digested with trypsin. Peptide mixtures were fractioned by nano-high performance liquid chromatography and analyzed by mass spectrometry. Following identification of differentially expressed proteins, quantitative real time polymerase chain reaction (qRT-PCR) analysis was performed. Results: The comparison between the proteins released from BAV and TAV aneurysmatic tissues showed significantly diverging expression fingerprints in the two groups of patients. Bioinformatics analysis revealed 38 differentially released proteins; in particular 7 proteins were down-regulated while 31 were up-regulated in BAV with respect to TAV. Most of the proteins that were up-released in BAV were related to the activation of transforming growth factor (TGF)-b signaling. Latent TGF-b binding protein 4 (LTBP4) exhibited one of the highest significant under-expressions (10-fold change) in BAV secretomes with respect to TAV. qRT-PCR analysis validated this significant difference at LTBP4 gene level (BAV: 1.03 0.9 vs TAV: 3.6 3.2; p < 0.05). Conclusion: Hypothesis-free secretome profiling clearly showed diverging expression fingerprints in the ATAA of TAV and BAV patients, confirming the crucial role of TGF-b signaling in modulating ATAA development in bicuspid patients

Homenaje a Ana María Barrenechea

Este número de Cuadernos LIRICO está dedicado a Ana María Barrenechea, destacada profesora e investigadora argentina que fue maestra de varias generaciones de críticos de las literaturas hispánicas y de estudiosos de la lengua. Nuestro volumen se propone recordar la importancia de su obra escrita y el aura de su magisterio y de su personalidad excepcional, evocando su figura y recorriendo algunos de entre los tantos caminos críticos abiertos por ella. De esta manera, el Homenaje pretende ser a la vez un recordatorio de su presencia y una puesta en perspectiva de la amplitud y la persistencia de sus aportes, de la fertilidad y los desafíos de su trabajo. Colaboran en él tanto una serie de especialistas que trabajó con ella o recibió directamente su estímulo y su influencia, como un grupo de otros que se abocó ulteriormente a sus temas dentro de las líneas de lectura que ella contribuyó a enriquecer. El número incluye una Introducción dedicada a su memoria y una serie de veinticuatro artículos, que abarcan estudios sobre prosa y poesía ríoplatense y literaturas extranjeras, historia y literatura del siglo XIX hispanoamericano, poética y teoría literaria, así como de crítica genética