Studio sul ruolo dei fattori genetici coinvolti nella Valvola Aortica Bicuspide e/o nell'Aneurisma dell'Aorta Toracica

La Valvola Aortica Bicuspide (BAV) rappresenta la più comune anomalia cardiaca congenita, con un’incidenza dello 0,5%-2% nella popolazione generale. Si caratterizza per la presenza di due cuspidi valvolari anziché tre e comprende diverse forme. La BAV è frequentemente associata agli aneurismi dell’aorta toracica (TAA). La dilatazione dell’aorta espone al rischio di sviluppare le complicanze aortiche acute. Materiali e metodi Sono stati reclutati 20 probandi consecutivi sottoposti a chirurgia della valvola aortica e dell'aorta ascendente presso l'Unità di Chirurgia Cardiaca di Policlinico S.Orsola-Malpighi di TAA associata a BAV. Sono stati esclusi individui con una condizione sindromica predisponente l’aneurisma aortico. Ciascun familiare maggiorenne di primo grado è stato arruolato nello studio. L’analisi di mutazioni dell’intero gene ACTA2 è stata eseguita con la tecnica del “bidirectional direct sequencing”. Nelle forme familiari, l’intera porzione codificante del genoma è stata eseguita usando l’exome sequencing. Risultati Dopo il sequenziamento di tutti i 20 esoni e giunzioni di splicing di ACTA2 nei 20 probandi, non è stata individuata alcuna mutazione. Settantasette familiari di primo grado sono stati arruolati. Sono state identificate cinque forme familiari. In una famiglia è stata trovata una mutazione del gene MYH11 non ritenuta patogenetica. Conclusioni La mancanza di mutazioni, sia nelle forme sporadiche sia in quelle familiari, ci suggerisce che questo gene non è coinvolto nello sviluppo della BAV e TAA e, l’associazione che è stata riportata deve essere considerata occasionale. L’architettura genetica della BAV verosimilmente dovrebbe consistere in svariate differenti varianti genetiche che interagiscono in maniera additiva nel determinare un aumento del rischio.

Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects during the conversion of muscle to meat, due to their influence on meat texture and sensory characteristics, mainly in seasoned products. Their contribution is fundamental in flavour development of dry-curing hams. However, several authors have demonstrated that high cathepsin activity, in particular of cathepsin B, is correlated to defects of these products, such as an excessive meat softness together with abnormal free tyrosine content, astringent or metallic aftertastes and formation of a white film on the cut surface. Thus, investigation of their genetic variability could be useful to identify DNA markers associated with these dry cured hams parameters, but also with meat quality, production and carcass traits in Italian heavy pigs. Unfortunately, no association has been found between cathepsin markers and meat quality traits so far, in particular with cathepsin B activity, suggesting that other genes, besides these, affect meat quality parameters. Nevertheless, significant associations were observed with several carcass and production traits in pigs. A recent study has demonstrated that different single nucleotide polymorphisms (SNPs) localized in cathepsin D (CTSD), F (CTSF), H and Z genes were highly associated with growth, fat deposition and production traits in an Italian Large White pig population. The aim of this thesis was to confirm some of these results in other pig populations and identify new cathepsin markers in order to evaluate their effects on cathepsin activity and other production traits. Furthermore, starting from the data obtained in previous studies on CTSD gene, we also analyzed the known polymorphism located in the insulin-like growth factor 2 gene (IGF2 intron3-g.3072G>A). This marker is considered the causative mutation for the quantitative trait loci (QTL) affecting muscle mass and fat deposition in pigs. Since IGF2 maps very close to CTSD on porcine chromosome (SSC) 2, we wanted to clarify if the effects of the CTSD marker were due to linkage disequilibrium with the IGF2 intron3-g.3072G>A mutation or not. In the first chapter, we reported the results from these two SSC2 gene markers. First of all, we evaluated the effects of the IGF2 intron3-g.3072G>A polymorphism in the Italian Large White breed, for which no previous studies have analysed this marker. Highly significant associations were identified with all estimated breeding values for production and carcass traits (P<0.00001), while no effects were observed for meat quality traits. Instead, the IGF2 intron3-g.3072G>A mutation did not show any associations with the analyzed traits in the Italian Duroc pigs, probably due to the low level of variability at this polymorphic site for this breed. In the same Duroc pig population, significant associations were obtained for the CTSD marker for all production and carcass traits (P < 0.001), after excluding possible confounding effects of the IGF2 mutation. The effects of the CTSD g.70G>A polymorphism were also confirmed in a group of Italian Large White pigs homozygous for the IGF2 intron3-g.3072G allele G (IGF2 intron3-g.3072GG) and by haplotype analysis between the markers of the two considered genes. Taken together, all these data indicated that the IGF2 intron3-g.3072G>A mutation is not the only polymorphism affecting fatness and muscle deposition in pigs. In the second chapter, we reported the analysis of two new SNPs identified in cathepsin L (CTSL) and cathepsin S (CTSS) genes and the association results with meat quality parameters (including cathepsin B activity) and several production traits in an Italian Large White pig population. Allele frequencies of these two markers were evaluated in 7 different pig breeds. Furthermore, we mapped using a radiation hybrid panel the CTSS gene on SSC4. Association studies with several production traits, carried out in 268 Italian Large White pigs, indicated positive effects of the CTSL polymorphism on average daily gain, weight of lean cuts and backfat thickness (P<0.05). The results for these latter traits were also confirmed using a selective genotype approach in other Italian Large White pigs (P<0.01). In the 268 pig group, the CTSS polymorphism was associated with feed:gain ratio and average daily gain (P<0.05). Instead, no association was observed between the analysed markers and meat quality parameters. Finally, we wanted to verify if the positive results obtained for the cathepsin L and S markers and for other previous identified SNPs (cathepsin F, cathepsin Z and their inhibitor cystatin B) were confirmed in the Italian Duroc pig breed (third chapter). We analysed them in two groups of Duroc pigs: the first group was made of 218 performance-tested pigs not selected by any phenotypic criteria, the second group was made of 100 Italian Duroc pigs extreme and divergent for visible intermuscular fat trait. In the first group, the CTSL polymorphism was associated with weight of lean cuts (P<0.05), while suggestive associations were obtained for average daily gain and backfat thickness (P<0.10). Allele frequencies of the CTSL gene marker also differed positively among the visible intermuscular extreme tails. Instead, no positive effects were observed for the other DNA markers on the analysed traits. In conclusion, in agreement with the present data and for the biological role of these enzymes, the porcine CTSD and CTSL markers: a) may have a direct effect in the biological mechanisms involved in determining fat and lean meat content in pigs, or b) these markers could be very close to the putative functional mutation(s) present in other genes. These findings have important practical applications, in particular the CTSD and CTSL mutations could be applied in a marker assisted selection (MAS) both in the Italian Large White and Italian Duroc breeds. Marker assisted selection could also increase in efficiency by adding information from the cathepsin S genotype, but only in the Italian Large White breed.