Studio del possibile ruolo del complesso polycomb nel determinare la schisi del labbro e del palato

La labioschisi con o senza palatoschisi non-sindromica (NSCL/P) è tra le più frequenti alterazioni dello sviluppo embrionale, causata dall’interazione di fattori genetici e ambientali, moti dei quali ancora ignoti. L'obiettivo del mio progetto di Dottorato consiste nell’identificazione di fattori di rischio genetico in un processo a due stadi che prevede la selezione di geni candidati e la verifica del loro coinvolgimento nella determinazione della malformazione mediante studi di associazione. Ho analizzato alcuni polimorfismi a singolo nucleotide (SNPs) dei geni RFC1 e DHFR, appartenenti alla via metabolica dell’acido folico, evidenziando una debole associazione tra alcuni degli SNPs indagati e la NSCL/P nella popolazione italiana. Presso il laboratorio della Dott.ssa Mangold dell’Università di Bonn, ho valutato il ruolo di 15 diverse regioni cromosomiche nel determinare la suscettibilità alla malattia, evidenziando una significativa associazione per i marcatori localizzati in 8q24 e 1p22. Ho quindi rivolto la mia attenzione al ruolo del complesso Polycomb nell’insorgenza della schisi. Nell’uomo i due complessi Polycomb, PRC1 e PRC2, rimodellano la cromatina agendo da regolatori dei meccanismi trascrizionali alla base della differenziazione cellulare e dello sviluppo embrionale. Ho ipotizzato che mutazioni a carico di geni appartenenti a PRC2 possano essere considerati potenziali fattori di rischio genetico nel determinare la NSCL/P. Il razionale consiste nel fatto che JARID2, una proteina che interagisce con PRC2, è associata all’insorgenza della NSCL/P ed espressa a livello delle cellule epiteliali delle lamine palatine che si approssimano alla fusione. L’indagine condotta analizzando i geni di elementi o partner dei due complessi Polycomb, ha evidenziato un’associazione significativa con alcuni polimorfismi dei geni indagati, associazione ulteriormente confermata dall’analisi degli aplotipi. Le analisi condotte sui geni candidati mi hanno permesso di raccogliere dati interessanti sull’eziologia della malformazione. Studi indipendenti saranno necessari per poter validare l'associazione tra le varianti genetiche di questi geni candidati e la NSCL/P.

Evidenza del coinvolgimento di geni nell'insorgenza delle schisi labio-palatine

The CL/P are the most common and easily recognizable craniofacial malformations with a complex etiology that requires the involvement of genetic and environmental components. The analysis of the genetic component shows more than 14 loci and genes involved in the onset of the disease. I’ve selected and investigated some of the possible candidate genes for CL/P. MYH14 gene, that maps on chromosome 19, on the OFC3 locus, and shows a strong homology with MYH9 gene. I’ve also investigated TP63 and MID1 genes, that are responsible respectively for EEC syndrome and Opitz syndrome, both of them presenting cleft. I’ve also decided to investigate JAG2 because TP63 product regulates the this gene, and both of them are component of the Notch signalling pathway. I’ve, also, studied the MKX and LMO4 genes. MKX is an important development regulator that is highly expressed in palatal mesenchyme, and map in the region responsible for Twirler mutation that cause cleft in mouse. LMO4 is necessary for neural tube development and cooperating with Grhl3, promotes cellular migration during morphogenetic events like “in utero” cleft healing. Low folate levels and high levels of homocysteine increase the risk of cleft, genes involved in their metabolism may be of interest in cleft occurrence. I’ve decided to investigate BHMT and CBS genes coding for enzymes involved in homocysteine metabolism. I’ve also investigated BHMT2 gene that maps close to BHMT and presents with him a 73% of homology. I’ve performed a linkage analysis using SNPs mapping in the genes and their boundaries, for each gene, for MKX and LMO4 I’ve also performed a sequencing analysis. My results for MID1 and CBS genes support the hypothesis of a possible role of these genes in cleft. I’ve found borderline association values for JAG2, MKX and LMO4 genes.

Characterization of the structure and iron uptake mechanisms of the Staphylococcus aureus ferric hydroxamate-binding lipoprotein FhuD2

The Reverse Vaccinology (RV) approach allows using genomic information for the delineation of new protein-based vaccines starting from an in silico analysis. The first powerful example of the application of the RV approach is given by the development of a protein-based vaccine against serogroup B Meningococcus. A similar approach was also used to identify new Staphylococcus aureus vaccine candidates, including the ferric hydroxamate-binding lipoprotein FhuD2. S. aureus is a widespread human pathogen, which employs various different strategies for iron uptake, including: (i) siderophore-mediated iron acquisition using the endogenous siderophores staphyloferrin A and B, (ii) siderophore-mediated iron acquisition using xeno-siderophores (the pathway exploited by FhuD2) and (iii) heme-mediated iron acquisition. In this work the high resolution crystal structure of FhuD2 in the iron (III)-siderophore-bound form was determined. FhuD2 belongs to the Periplasmic Binding Protein family (PBP ) class III, and is principally formed by two globular domains, at the N- and C-termini of the protein, that make up a cleft where ferrichrome-iron (III) is bound. The N- and C-terminal domains, connected by a single long α-helix, present Rossmann-like folds, showing a β-stranded core and an α-helical periphery, which do not undergo extensive structural rearrangement when they interact with the ligand, typical of class III PBP members. The structure shows that ferrichrome-bound iron does not come directly into contact with the protein; rather, the metal ion is fully coordinated by six oxygen donors of the hydroxamate groups of three ornithine residues, which, with the three glycine residues, make up the peptide backbone of ferrichrome. Furthermore, it was found that iron-free ferrichrome is able to subtract iron from transferrin. This study shows for the first time the structure of FhuD2, which was found to bind to siderophores ,and that the protein plays an important role in S. aureus colonization and infection phases.