The impact of polymorphisms in P-gp, DNA repair and folic acid metabolism genes in newly diagnosed multiple myeloma patients treated with thalidomide plus dexamethasone, with or without bortezomib

The principle aim of this study was to investigate biological predictors of response and resistance to multiple myeloma treatment. Two hypothesis had been proposed as responsible of responsiveness: SNPs in DNA repair and Folate pathway, and P-gp dependent efflux. As a first objective, panel of SNPs in DNA repair and Folate pathway genes, were analyzed. It was a retrospective study in a group of 454, previously untreated, MM patients enrolled in a randomized phase III open-label study. Results show that some SNPs in Folate pathway are correlated with response to MM treatment. MTR genotype was associated with favorable response in the overall population of MM patients. However, this relation, disappear after adjustment for treatment response. When poor responder includes very good partial response, partial response and stable/progressive disease MTFHR rs1801131 genotype was associated with poor response to therapy. This relation - unlike in MTR – was still significant after adjustment for treatment response. Identification of this genetic variant in MM patients could be used as an independent prognostic factor for therapeutic outcome in the clinical practice. In the second objective, basic disposition characteristics of bortezomib was investigated. We demonstrated that bortezomib is a P-gp substrate in a bi-directional transport study. We obtain apparent permeability rate values that together with solubility values can have a crucial implication in better understanding of bortezomib pharmacokinetics with respect to the importance of membrane transporters. Subsequently, in view of the importance of P-gp for bortezomib responsiveness a panel of SNPs in ABCB1 gene - coding for P-gp - were analyzed. In particular we analyzed five SNPs, none of them however correlated with treatment responsiveness. However, we found a significant association between ABCB1 variants and cytogenetic abnormalities. In particular, deletion of chromosome 17 and t(4;14) translocation were present in patients harboring rs60023214 and rs2038502 variants respectively.

Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

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.