Digital archive of theses discussed at the University of Pisa


Thesis etd-03042015-200353

Thesis type
Tesi di dottorato di ricerca
Thesis title
Analysis of genetic and epigenetic alterations in candidate genes in thoracic aortic aneurysm
Academic discipline
Course of study
tutor Dott. Andreassi, Maria Grazia
tutor Dott. Foffa, Ilenia
  • real-time pcr
  • next generation sequencing
  • LTBP protein
  • miRNA
  • SMAD3
  • TGFBR2
  • TGFBR1
  • thoracic aortic aneurysm
Graduation session start date
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.