Association between a common missense variant in LOXL3 gene and the risk of non-syndromic cleft palate

To investigate possible association between functional common variants in the lysyl oxidase like 3 gene and non-syndromic cleft palate we selected a common missense variant p.Ile615Phe (rs17010021), which was predicted to have a probably damaging effect on the lysyl oxidase like 3 enzyme. We genotyped 258 non-syndromic cleft palate case-parent triads of European origin and tested genetic association using the transmission disequilibrium test and log-linear regression analyses of genotypic relative risks and of parent-of-origin effects. The observed genotype frequency in parents was in Hardy-Weinberg equilibrium. Compared with wild-type Ile/Ile homozygotes, the relative risks for Phe/Phe homozygote infants was 6.87 (P value 3.0 × 10-3 ), while that for Ile/Phe heterozygotes was not significant. Assuming an autosomal recessive model, the relative risks for Phe/Phe genotype resulted 10.54 (P value 2.9 × 10-5 ), with a 3.6% population attributable risk. No parent-of-origin effect was observed. The identification in lysyl oxidase like 3 of a missense variant which under a recessive model associates with 10-fold increased risk of non-syndromic cleft palate supports the hypothesis that the genetic etiology of this congenital anomaly includes relatively uncommon recessive variants with moderate penetrance and located in genes which are also involved in syndromes that include cleft palate as part of the phenotype. Our findings require functional validation and replication in a larger independent genetic association study

Evaluating LINE-1 methylation in cleft lip tissues and its association with early pregnancy exposures

Aim: To pilot investigation of methylation of long interspersed nucleotide element-1 in lip tissues from infants with nonsyndromic cleft lip, and its association with maternal periconceptional exposures. Methods: The lateral and medial sides of the cleft lips of 23 affected infants were analyzed for long interspersed nucleotide element-1 methylation by bisulfite conversion and pyrosequencing. Results: The medial side showed 1.8% higher methylation compared with the lateral side; p = 0.031, particularly in male infants (2.7% difference; p = 0.011) or when the mothers did not take folic acid during periconceptional period (2.4% difference; p = 0.011). These results were not statistically significant when Bonferroni adjustment was used. Conclusion: The observed differences in DNA methylation, although nonsignificant after correction for multiple comparisons, suggest that differential regulation of the two sides may impact lip fusion and warrant larger-scale replication

MULTIDISCIPLINARY APPROACHES AND INTERACTION NETWORK IN THE DIAGNOSIS AND TREATMENT OF RHEUMATOID ARTHRITIS

The multidisciplinary approaches described and reported in the present thesis were performed with the aim to gain new insight into Rheumatoid Arthritis (RA), multifactorial, progressive and autoimmune disease. The case-control study, focused on the genetic occurrence risk of RA in a complete Italian casuistry, reported the HLA-DQA2 rs9275595 T>C variant strongest associated to RA susceptibility. SNP × SNP investigation revealed significant synergic combined effect between HLA-DQA2 rs9275595 and HLA-DRB1 rs660895 A>G variants, suggesting that, beyond single variant association, the powerful gain arises from interaction between variants belonging to HLA complex. The predictive biomarkers identification of occurrence, progression and therapy efficacy has been carried out in Undifferentiated Arthritis/ Early Rheumatoid Arthritis patients (UA/ERA). Follow-up after 6 months from the first visit has allowed to stratify subjects accordingly to disease evolution (from UA to RA) and to disease progression (from ERA to RA) using DAS28 variation (Disease Activity Score). Interaction study between genetics and serological parameters reported a significant combined effect of Rheumatoid Factor (RF), resistin and HLA-DRB1 rs6910071 A>G, playing a role together to predict "best responders", the group of patients with higher DAS28 improvement. Focusing on the influence of sexual dimorphism, a statistical significant gender-dependent effect has been shown in the context of Methtotrexate (MTX) pharmacogenetics: RA women D/I for HLA-G 14bp D>I variant reported significant higher MTX inefficacy, whereas male trend of association was completely opposite. Investigating on women previous obstetric history, it came out the association between the ascertained pregnancies, HLA-G 14bp D/I genotype and MTX inefficacy. Thus, we focused the approach on the particular women-view, underlying how pregnancy and miscarriage could exert an important and discriminant role in the disease pathogenesis. Pregnancy represents a physiological context requiring immune tolerance toward the fetus and, especially during the first trimester, exists a bidirectional trafficking of cells and DNA in the fetus-maternal interface that can results in naturally acquired microchimerism in both mother (fMC) and fetus. Male fMC could be detected in women bloodstream even after many years from the childbirth and in fact, our findings revealed the presence of male fMC in SLE patients' bloodstream. The widespread symptoms of SLE could justify the higher presence of fMC than which has detected in RA patients' bloodstream and the investigation on localized RA affected synovium might be crucial to understating fMC role. Exploring women synovial biopsies, it failed to reveal positive detection of fMC, empathizing that microchimeric cells can exert a rescue role, acting as stem cells repairing tissues injuries; thus, it has possible to detect fMC cells only in slight or mild RA cases. Finally, we explored the epigenetic profile, through the methylation analysis of LINE-1 and of specific genes HLA-G and MTHFR. Study has been carried out on healthy subjects, passing through early symptoms, to established RA diseased patients. HLA-G gene was significant hypermethylated in female RA patients, respect to RA males. Significant hypomethylation was identified in MTHFR gene, with a progressive rising to the increase of disease establishment, suggesting that epigenetic state persists beyond RA stages. In conclusion, the evidences presented in this thesis underlined the interaction network as the important causative factor of RA susceptibility and therapy efficacy. In addition, results on RA affected women emphasized the gender medicine application. Lastly, the epigenetic study is the first to explore methylation status over the time and progression of RA disease, highlighting the environmental contribution and opening new directional window in the RA knowledge.I differenti approcci multidisciplinari affrontati e descritti nella presente tesi sono stati effettuati con l'intento di arricchire le conoscenze in ambito di Artrite Reumatoide (AR), patologia a eziologia multifattoriale, progressiva ed autoimmune. Lo studio caso-controllo, focalizzato sull'individuazione di un profilo genetico di rischio in pazienti italiani con diagnosi di AR, ha riportato la variante HLA-DQA2 rs9275595 T>C significativamente associata ad insorgenza. L'analisi delle interazioni SNP × SNP ha rivelato un significativo effetto sinergico della combinazione fra le varianti HLA-DQA2 rs9275595 e HLA-DRB1 rs660895 A>G, suggerendo che, oltre al singolo effetto, il risultato maggiore si ha dall'interazione fra varianti appartenenti al complesso HLA. L'identificazione di biomarkers predittivi di suscettibilità, di progressione e di efficacia del trattamento impiegato è stata condotta in pazienti con Artrite Indifferenziata/Artrite Reumatoide Precoce (UA/ERA). Il follow-up a 6 mesi ha consentito la stratificazione dei soggetti UA in base all'insorgenza o meno della patologia, mentre per i pazienti ERA in base alla variazione del DAS28 (Disease Activity Score). I risultati delle interazioni genetiche e sierologiche relativamente alla progressione di AR, hanno individuato un algoritmo predittivo di efficacia terapeutica, evidenziando una relazione sinergica fra il Fattore Reumatoide (RF), la Resistina e la variante HLA-DRB1 rs6910071 A>G. Focalizzandosi sullo studio del dimorfismo sessuale, è stato riportato un significativo effetto genere-dipendente nel contesto della farmacogenetica del Methotrexate (MTX): infatti la variante HLA-G 14bp D>I costituisce un fattore predittivo dell'inefficacia del MTX solo nelle pazienti femmine. Approfondendo l'indagine su peculiari caratteristiche del genere femminile che possano interagire con il genotipo HLA-G, è risultata una forte associazione fra la presenza di precedenti gravidanze accertate, il genotipo HLA-G 14bp D/I e l'inefficacia al MTX. Considerando l'elevata espressione di HLA-G a livello del trofoblasto e il suo coinvolgimento nelle poliabortività, è stato indagato il microchimerismo fetale (fMC), già noto per essere presente nel sangue di donne affette da patologie autoimmuni, come AR e Lupus Eritematoso Sistemico (LES), anche dopo molti anni dalla gravidanza o dall'evento abortivo. Lo studio caso-controllo ha rilevato significativa presenza di fMC maschile nel campione ematico periferico di pazienti lupiche. La sintomatologia sistemica di LES potrebbe giustificare l'elevata presenza di fMC maschile nelle pazienti lupiche rispetto alle pazienti AR. Indagando le biopsie sinoviali di donne affette da AR, non è stata rivelata presenza di fMC maschile, suggerendo il possibile ruolo delle cellule microchimeriche come elementi di supporto e di rescue nel miglioramento della patologia. Sarebbe, dunque, possibile individuare cellule fMC solo nei casi di pazienti con AR stabile o in fase di remissione. Infine, è stata esplorato il profilo epigenetico attraverso analisi di metilazione di LINE-1 e dei geni HLA-G e MTHFR, su soggetti sani di controllo, su pazienti con artrite agli esordi e su pazienti con diagnosi accertata di AR. HLA-G ha evidenziato una significativa ipermetilazione nelle pazienti donne con AR rispetto agli uomini; mentre MTHFR ha rivelato una significativa ipometilazione, gradualmente maggiore a seconda della progressione di AR. In conclusione, i risultati esposti nella presente tesi hanno evidenziato il network di interazioni come importante elemento causativo di insorgenza e risposta terapeutica di AR. Inoltre, il focus sulle pazienti donne affette da AR ha enfatizzato l'applicazione della medicina di genere; infine, lo studio di epigenetica è il primo ad indagare lo stato di metilazione e la progressione di AR, sottolineando l'importanza del contributo ambientale e consentendo una nuova direzione esplorativa

Analysis of cytotoxic activity of peripheral blood natural killer cells in women with recurrent miscarriage

Around 50% of recurrent spontaneous abortions (RSA) remain unexplained. Immunological etiology has been proposed, supported by evidence of lower count of natural killer (NK) cells in peripheral blood of RSA women compared to women with normal delivery history. However, studies concerning the cytotoxic activity of NK cells in women with RSA are still controversial. We performed an observational case-control study assaying peripheral blood NK (pNK) cells cytotoxic activity in non-pregnant RSA women compared to non-pregnant women with normal delivery history. Twelve RSA and nine control women were recruited and blood samples were drawn during the luteal phase of ovarian cycle. pNK cells were incubated with target CFSE-labeled K562 cells and cytotoxicity was measured by cytofluorimetry. In non-pregnant RSA women pNK cytotoxic activity was not significantly altered compared to control women. In luteal phase of ovarian cycle the level of cytotoxic activity of pNK cells is not a marker for predicting RSA, and clinicians should not use pNK activity as a systematic recurrent pregnancy loss examination

Antitubulinic effect of new fluorazone derivatives on melanoma cells

Microtubules are composed by α- and β-tubulin polypeptides. α-tubulin undergoes a reversible post-translational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyr-tubulin). Recent studies have shown that α-tubulin tyrosine residues can be nitrated and the incorporation of NO2Tyr into the C-terminus of Glu-tubulin forms a complex that blocks the tyrosination/detyrosination cycle, an event that can compromise protein/enzyme functions, such as cell division. Since many studies demonstrated that Glu-tubulin levels increase in cancer, the aim of the present study was to investigate the effect of new drugs, fluorazone derivatives (K1-K2-K9-K10-K11), on the proliferation of melanoma cells. Our results demonstrated that these drugs, except for K2, were able to inhibit cellular proliferation without exhibiting cytotoxicity. The anti-proliferative effect was accompanied by the decrease of Glu-tubulin levels and the increase of its nitration. This effect seems to be a consequence of NO2 induction and NO2Tyr ligation to Glu-tubulin. Collectively, these results, showing that the fluorazone derivatives, by promoting NO2Tyr incorporation into α-tubulin, are able to arrest the cycle of detyrosination/tyrosination and to inhibit cell proliferation, offer new perspectives for the possible usage of these drugs, alone or in combination, as non-toxic, anti-proliferative agents in melanoma