Dissertations / Theses on the topic 'Identificazione di geni malattia'

Duchenne muscular dystrophy (DMD) is a lethal childhood muscular disorder characterized clinically by progressive muscle wasting. The onset of the muscular weakness is usually between 3-5 years of age leading to loss ambulation by a mean age of 10.5 years [Allsop & Ziter, 1981] and death is usually due to respiratory or cardiac insufficiency in the early twenties [Dubowitz, 1995]. To date, the only effective pharmacological therapy shown to delay progression of disease is treatment with steroids, that may prolongs ambulation by up to 18 months. Even if the steroids efficacy in DMD is proved, interpatient variability in pharmacological response and a broad variation in the age at which loss of ambulation occurs among steroid-treated DMD patients are also reported. Several analysis of the standard deviation shown that this parameter is often greater than the mean, suggesting a wide distribution of values presumably due to the existence of patients that loss the ambulation either extremely early or late [Silva et al, 1987]. The molecular basis of this phenotypic variability has to be cleared. The long term goal of this work is the dissection of the molecular mechanisms underlying steroid response in DMD patients to identify the predisposing factors to a more favourable disease course. The study relied upon the DMD database maintained by the Neuromuscular Center of the University of Padua and 8 patients with a molecular diagnosis of DMD were selected to enrol in the analysis. All of them have been treated with steroids (deflazacort and prednisolone) until the loss of ambulation. Based upon an arbitrary criteria, these patients were classified as “responder” (R) (n=5) when the loss of ambulation occurs after 13 years of age and as “no-responder” (NR) (n=3) when it occurs before 10 years of age [Bonifati et al, 2000]. To identify the transcripts significantly dysregulated in the two groups of patients, muscle biopsies of the selected patients are used for the expression profiling, performing a microarray analysis using a genome-wide platform (Operon; Huntsville, AL, USA) and a following statistical analysis. After valuation of differentially expressed genes, the study proceeds with the identification of discriminant genes belonging to different groups, responder and no-responder. The analysis led to the identification of 47 deregulated genes in the steroids-responder patients group matched up to no-responder patients group: 10 genes down-regulated, among which several structural genes expressed during development, immune response signals and extracellular matrix genes and 37 genes up-regulated, many of which encoding transcription factors. Subsequently, each of these genes was investigated in order to individuate the molecular pathways involved. Evaluating the interaction networks between the identified genes, two pathways were recognized: the interferon (IFN)-signaling pathway and the NF-kappaB transduction signal cascade. The Nuclear Factor kappa-B (NF-kB) is an ubiquitary transcriptional factor implicated in the regulation of expression of a number of cellular genes involved in immune, inflammatory and anti-apoptotic responses, and found to be increased in mdx (modello murino di DMD) muscle. Several reports suggest that inhibition of NF-kB is a valuable strategy for the treatment of DMD [Acharyya et al, 2007; Mozzetta et al, 2008]. Almost 4 genes among those considerate (IFIT3, IFIT1, STAT1, TFF3) are involved in the NF-kB activation pathway. In order to validate the differential expression of these genes a quantitative real time-PCR test was performed; the analysis has spotlighted expression variability between the patients of the single groups and the existence of an outlier patient. To counter this trouble, a second statistical analysis was carried out comparing each single responder patient with a control patient and the differentially expressed genes were evaluated considering function, physiological expression and gene interaction characteristics. In this list of genes we identified the presence of many members of TNF superfamily (TNF, TNFSF10, LTA, LTB) and a gene coding a putative NF-kB inhibitor, as well as other 5 genes (USMG5, SPP1, S100A9, ICOSLG, LILRA2) selected because of their homogeneous expression profiling. After Real Time-PCR analysis, the gene that survives the test is USMG5 (Up-regulated during Skeletal Muscle Growth 5), subsequently investigated in order to perform a SNP association studies with other two genes chosen through the literature: ACTN3 that encodes the protein ?-actinin 3, a fast-twitch-specific isoform expressed only in type II myofibers, and AKT1 that encodes the serine-threonine protein kinase, involved in the growth factor-I (IGF-1) transduction signal cascade and the NF-kB activation pathway. 110 DMD patients were genotyped for different SNPs, sought out in the existing SNP database, one in each of the candidate gene: R577X in ACTN3, +G205T in AKT1 and S49P in USMG5. The genotype distribution was analysed for the association with the muscle strength using chi squared methods, but statistical significance was not observed. In conclusion, our study has demonstrated the possibility of a priori clustering patients with different clinical progression. The results of this study provides therapeutic alternatives to correct the single gene defect with gene therapy. Further studies are needed to better define the molecular mechanisms underlying the modulation of the phenotype in DMD.
La distrofia muscolare di Duchenne (DMD) e’ una malattia neuromuscolare dell’eta’ infantile invariabilmente letale caratterizzata da un deficit di forza progressivo. L’esordio della debolezza muscolare e’ usualmente verso i 3-5 anni e progredisce fino alla perdita della deambulazione in media intorno ai 10 anni e mezzo. L’exitus, nella seconda-terza decade, e’ dovuta alla comparsa di insufficienza respiratoria e/o cardiaca. La sola terapia palliativa efficace nel rallentare la progressione di malattia e’ la terapia steroidea. La somministrazione di steroidi nella DMD risulta nel prolungamento della deambulazione di circa 18 mesi nei pazienti trattati rispetto ai non trattati. Tuttavia l’eta’ alla perdita della deambulazione presenta una ampia deviazione standard dovuta alla presenza sia di pazienti che perdono la deambulazione molto precocemente ed altri molto tardivamente sia nei pazienti trattati che non trattati. Le basi molecolari di questa variabilita’ fenotipica non sono. Nel tentativo di identificare i fattori modulanti favorevolmente il fenotipo clinico, abbiamo studiato 8 pazienti con diagnosi molecolare di DMD. Tutti i pazienti erano stati trattati con steroidi a dosaggio standard fino alla perdita della deambulazione. I pazienti sono stati arbitrariamente classificati come responsivi (R) (n=5) se la perdita della deambulazione e’ avvenuta dopo i 13 anni e non-responsivi (NR) (n=3) se la perdita della deambulazione e’ avvenuta prima dei 10 anni di età. Allo scopo di identificare gruppi di trascritti genici differenzialmente espressi tra i due gruppi di pazienti è stata eseguita un’analisi dei profili d’espressione (Microarray) utilizzando una piattaforma di oligonucleotidi genome-wide (Operon), seguita da un’elaborazione statistica dei dati. Una volta identificati i geni regolati in modo differenziale, lo studio ha mirato all’identificazione di un sottogruppo di geni differenzialmente espressi nei pazienti R e NR. Con tale criterio sono stati individuati nel pool dei pazienti R 47 geni significativamente deregolati rispetto ai pazienti NR: 37 geni sovraespressi, molti dei quali codificanti per fattori di trascrizione, e 10 geni sottoespressi, tra cui geni strutturali normalmente espressi durante lo sviluppo, geni coinvolti nella risposta immunologica e altri che codificano per proteine presenti nella matrice extracellulare. I geni differenzialmente espressi identificati sono stati sottoposti ad analisi funzionale allo scopo di individuare cascate biochimiche in cui essi fossero coinvolti. Valutando l’interazione tra i geni identificati, sono risultati particolarmente significativi la cascata di signaling dell’interferone (IFN) e la cascata di trasduzione del segnale del Fattore Nucleare kappa B (NF-kB), un fattore di trascrizione ubiquitario che regola l’espressione di geni coinvolti nei processi infiammatori e nella risposta acuta allo stress. Dati sperimentali suggeriscono che la regolazione dell’attività di NF-kB possa favorevolmente modulare la progressione della distrofia muscolare nel modello animale di DMD (topo mdx). Almeno 4 tra i geni considerati (IFIT3, IFIT1, STAT1, TFF3) hanno un ruolo nel processo di attivazione di NF-kappaB. Al fine di validare la significativa sovraespressione di questi trascritti nel pool di pazienti “steroido-responsivi” rispetto al pool di pazienti “steroido non-responsivi”, sono stati condotti degli esperimenti di Real Time-PCR. Dall’analisi è emersa una variabilità all’interno dei singoli gruppi. Per ovviare a tale problema, una seconda analisi statistica dei dati è stata eseguita confrontando singolarmente ogni paziente con un controllo scelto in base all’età alla perdita della deambulazione. I geni differenzialmente espressi identificati sono stati valutati in base alle loro caratteristiche di funzione, espressione fisiologica ed interazioni. In questa seconda analisi, si è evidenziato in particolare un coinvolgimento di alcuni membri della superfamiglia del TNF (TNF, TNFS10, LTA, LTB) ed un gene codificante per un putativo inibitore di NF-kappaB (NFKBIL1). Sono stati presi in considerazione anche altri 5 geni (USMG5, SPP1, S100A9, ICOSLG, LILRA2), selezionati perché la loro espressione risultava uniforme all’interno dei due gruppi di pazienti, così da poter eliminare la variabilità intergruppo. Anche in questo caso i dati sono stati necessariamente validati con esperimenti di Real Time-PCR. Il gene che, superato il controllo di validazione, è risultato significativo è USMG5 (Up-regulated during the skeletal muscle growth 5), successivamente studiato allo scopo di identificare polimorfismi di singolo nucleotide (SNP). È stata valutata l’esistenza di SNP noti, consultando il database di SNP, nel gene USMG5 ed in altri 2 geni scelti dalla letteratura: ACTN3 che codifica per la proteina alpha-actinina 3, espressa esclusivamente nel disco Z delle fibre di tipo II del muscolo scheletrico, e AKT1 che codifica per la serina-treonina protein chinase, coinvolta nella trasduzione del segnale dei fattori di crescita come IGF1, oltre ad avere un ruolo nello sviluppo e nella sopravvivenza cellulare e nell’attivare NF-kB. I pazienti sono stati analizzati per il polimorfismo R577X nel gene ACTN3, +G205T nel gene AKT1 e S49P nel gene USMG5. La distribuzione genotipica è stata messa a confronto con la forza muscolare, ma i test non hanno evidenziato differenze statisticamente significative. In conclusione, il nostro studio dei profili di espressione nei pazienti affetti da DMD ha dimostrato la possibilita’ di clusterizzare a priori pazienti con progressione clinica diversa. Questo risultato e’ rilevante considerando che implica la possibilita’ che alla base della progressione di malattia nella DMD vi sia una regolazione genica differenziale. Questo dato offre strategie terapeutiche alternative alla sola correzione del difetto genico con terapia genica o cellulare. Ulteriori studi sono necessari per definire meglio i meccanismi molecolari alla base della modulazione del fenotipo nella DMD.

The genetics of most common single-gene neurological disorders has been dissected in the last twenty years. However, the aetiology of several rare Mendelian neurological conditions is still unknown, with significant implications for diagnosis, genetic counselling and therapy. So far the identification of genes for these rare conditions have been constrained by methodological issues, such as the shortage of informative families for linkage analysis, the lack of very dense maps of polymorphic markers and the availability of efficient and low-cost platforms for genotyping and sequencing. Recently, the high-throughput sequencing of the coding DNA (i.e. exome) has been successfully applied to identify causative mutations by comparing the genome-wide profile of non-synonymous variants among unrelated patients affected by the same disease. Thus, exome sequencing may not be readily applicable to patients affected by rare unclassified conditions who cannot be grouped into genetically homogenous pools. To dissect the heterogenous genetic etiology of rare neurological phenotypes, we decided to combine exome sequencing with homozygosity mapping, a robust approach to localize disease genes involved in autosomal recessive (AR) Mendelian disorders in discrete genomic regions. We focused our attention on 7 phenotypic heterogeneity families that they have not been genetically diagnosed of the our Pediatric Neurological Unit. We analyzed variants with standard bioinformatic pipeline and databases of populations as ExAC, GnomaAD, and in-house database (200 normal controls). We confirmed the selected variants by Sanger Sequencing in probands and parents. We excluded the negative variants by Sanger sequencing in affected subjects and parents. In more cases, we demonstrated the pathogenetic role for these variants with functional studies as WesternBlot, staning of tissue and immunofluorescence of the patients’ cells. We generated the Zebrafish model to evaluate the pathogenetic role of some variants. We highlighted the importance of the bioinformatic analysis, which selected the effective genetic causes of pathology in these families. We identified different genes, adding important genotype-phenotype correlations to literature data. In conclusion, this study highlighted a powerful approach for clarify the genetic basis of rare neurological recessive diseases. Furthermore, this genome-wide approach allowed to highlight new genotype-phenotype correlations and expanded the clinical phenotypic spectrum in specificgenetic conditions.

Genome wide association studies have identified two quantitative trait loci outside of the β-globin cluster associated with fetal hemoglobin (HbF) levels, number of F cell and β-thalassemia severity: the HBS1L-MYB intergenic region and the BCL11A gene. In order to understand the functional role of the associated variants at these loci we applied “Genome Wide Chromosome Conformation Capture” (Hi-C), followed by a novel technique for a selective enrichment at these target regions, to characterize whether they are involved in long range physical interactions able to modulate HBS1L-MYB and BCL11A expression. As a first step we optimized a Hi-C protocol in the K562 fetal erythroid cell line and we set up the conditions for the new method based on the selective enrichment of target regions. We were able to validate the new capture system analyzing the β-globin locus, as a control model, detecting all the interactions found by other “3C-like” technologies with a higher resolution. We then analyzed the data at the HBS1L-MYB and BCL11A loci; the most significant detected interactions involved four HBS1L-MYB intergenic regions, three of which contain the GWAS SNPs, the HBS1L and MYB genes. We hypothesized a contact model where the associated variants could exert their regulatory role likely altering transcription factors binding sites and thus DNA long-range interactions resulting in different levels of MYB expression. Indeed, although we can not exclude the implication of HBS1L gene in this mechanism, MYB represents the best candidate in modulating HbF levels given its role in the erythropoiesis kinetics. Our results highlighted the power of the new capture system, able to identify chromatin interactions with very high resolution simultaneously at different loci. Finally, we discovered new chromatin interactions that support the transcription factor MYB as a potential good candidate to develop new targeted therapeutic strategies to treat β-thalassemia patients.

I disturbi dello spettro autistico (DSA) ed il ritardo mentale (RM) sono caratterizzati da un’eziologia genetica complessa ed eterogenea. Grazie ai recenti sviluppi nella ricerca genomica, è stato possibile dimostrare il ruolo di numerose copy number variants (CNVs) nella patogenesi di questi disturbi, anche se nella maggior parte dei casi l’eziologia rimane ancora sconosciuta. Questo lavoro riguarda l’identificazione e la caratterizzazione dei CNVs in famiglie con DSA e RM. E’ stata studiata una microdelezione in 7q31 che coinvolge i geni IMMP2L e DOCK4, trasmessa dalla madre con dislessia a due figli con autismo ed una figlia con dislessia. Nella stessa famiglia segrega una seconda microdelezione in 2q14 che inattiva il gene CNTNAP5 ed è trasmessa dal padre (con tratti autistici) ai due figli con autismo. Abbiamo quindi ipotizzato che i geni DOCK4 e CNTNAP5 potessero essere implicati, rispettivamente, nella suscettibilità a dislessia e DSA. Lo screening di numerosi individui affetti ha supportato la nostra ipotesi, con l’identificazione di una nuova microdelezione di DOCK4 che segrega con la dislessia, e 3 nuove varianti missenso in CNTNAP5 in individui con autismo. Dall’analisi genomica comparativa su array (aCGH) di individui con RM, è stata identificata una delezione nella regione 7q31.32, che coinvolge il gene CADPS2, in due fratelli con RM e tratti autistici, probabilmente ereditata dalla madre. Lo screening di mutazione di questo gene in individui con autismo o RM, ha portato all’identificazione di 3 varianti non sinonime, assenti nei controlli, ed ereditate per via materna. Poiché CADPS2 risiede in una regione genomica che contiene loci soggetti ad imprinting, abbiamo ipotizzato che il gene CADPS2 possa essere anch’esso caratterizzato da imprinting, con espressione monoallelica materna. Lo studio di espressione di CADPS2 in cellule del sangue ha avvalorato questa ipotesi, implicando perciò CADPS2 come un nuovo gene di suscettibilità per il RM e DSA.
Autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by a complex and heterogeneous genetic etiology. Recent developments in genomic research have enabled the discovery of numerous copy number variants (CNVs) in the pathogenesis of these disorders, although their etiology remains unknown in the majority of cases. This work concerns the identification and characterization of specific CNVs in families with ASD and ID. I studied a microdeletion in 7q31 encompassing the two genes DOCK4 and IMMP2L, transmitted from the mother (who has dylsexia) to two children with autism and to a daughter with dyslexia. In the same family we identified a second microdeletion in 2q14, that inactivates CNTNAP5, and is transmitted by the father (with ASD) to the two children with autism. We therefore hypothesized that DOCK4 and CNTNAP5 could be implicated in susceptibility to dyslexia and ASD, respectively. Screening of numerous affected individuals supported our hypothesis, leading to the identification of a new DOCK4 microdeletion segregating with dyslexia, and 3 new missense variants in CNTNAP5 in individuals with autism.Through array comparative genomic hybridization (aCGH) of individuals with ID, we also identified a 7q31.32 microdeletion involving the CADPS2 gene in two brothers with ID and autistic features, probably inherited from the mother. Screening for mutations in this gene in individuals with autism or ID, has led to the identification of 3 maternally inherited nonsynonymous variants, absent in controls. Since CADPS2 is located in a genomic region containing imprinted loci, we hypothesized that CADPS2 itself could be subjected to imprinting, with maternal monoallelic expression. Expression analysis of CADPS2 in blood cells supported this hypothesis, therefore suggesting CADPS2 as a new susceptibility gene for ID and ASD, and as possible new imprinted gene .

I disturbi dello spettro autistico (DSA) ed il ritardo mentale (RM) sono caratterizzati da un’eziologia genetica complessa ed eterogenea. Grazie ai recenti sviluppi nella ricerca genomica, è stato possibile dimostrare il ruolo di numerose copy number variants (CNVs) nella patogenesi di questi disturbi, anche se nella maggior parte dei casi l’eziologia rimane ancora sconosciuta. Questo lavoro riguarda l’identificazione e la caratterizzazione dei CNVs in famiglie con DSA e RM. E’ stata studiata una microdelezione in 7q31 che coinvolge i geni IMMP2L e DOCK4, trasmessa dalla madre con dislessia a due figli con autismo ed una figlia con dislessia. Nella stessa famiglia segrega una seconda microdelezione in 2q14 che inattiva il gene CNTNAP5 ed è trasmessa dal padre (con tratti autistici) ai due figli con autismo. Abbiamo quindi ipotizzato che i geni DOCK4 e CNTNAP5 potessero essere implicati, rispettivamente, nella suscettibilità a dislessia e DSA. Lo screening di numerosi individui affetti ha supportato la nostra ipotesi, con l’identificazione di una nuova microdelezione di DOCK4 che segrega con la dislessia, e 3 nuove varianti missenso in CNTNAP5 in individui con autismo. Dall’analisi genomica comparativa su array (aCGH) di individui con RM, è stata identificata una delezione nella regione 7q31.32, che coinvolge il gene CADPS2, in due fratelli con RM e tratti autistici, probabilmente ereditata dalla madre. Lo screening di mutazione di questo gene in individui con autismo o RM, ha portato all’identificazione di 3 varianti non sinonime, assenti nei controlli, ed ereditate per via materna. Poiché CADPS2 risiede in una regione genomica che contiene loci soggetti ad imprinting, abbiamo ipotizzato che il gene CADPS2 possa essere anch’esso caratterizzato da imprinting, con espressione monoallelica materna. Lo studio di espressione di CADPS2 in cellule del sangue ha avvalorato questa ipotesi, implicando perciò CADPS2 come un nuovo gene di suscettibilità per il RM e DSA.
Autism spectrum disorders (ASD) and intellectual disability (ID) are characterized by a complex and heterogeneous genetic etiology. Recent developments in genomic research have enabled the discovery of numerous copy number variants (CNVs) in the pathogenesis of these disorders, although their etiology remains unknown in the majority of cases. This work concerns the identification and characterization of specific CNVs in families with ASD and ID. I studied a microdeletion in 7q31 encompassing the two genes DOCK4 and IMMP2L, transmitted from the mother (who has dylsexia) to two children with autism and to a daughter with dyslexia. In the same family we identified a second microdeletion in 2q14, that inactivates CNTNAP5, and is transmitted by the father (with ASD) to the two children with autism. We therefore hypothesized that DOCK4 and CNTNAP5 could be implicated in susceptibility to dyslexia and ASD, respectively. Screening of numerous affected individuals supported our hypothesis, leading to the identification of a new DOCK4 microdeletion segregating with dyslexia, and 3 new missense variants in CNTNAP5 in individuals with autism.Through array comparative genomic hybridization (aCGH) of individuals with ID, we also identified a 7q31.32 microdeletion involving the CADPS2 gene in two brothers with ID and autistic features, probably inherited from the mother. Screening for mutations in this gene in individuals with autism or ID, has led to the identification of 3 maternally inherited nonsynonymous variants, absent in controls. Since CADPS2 is located in a genomic region containing imprinted loci, we hypothesized that CADPS2 itself could be subjected to imprinting, with maternal monoallelic expression. Expression analysis of CADPS2 in blood cells supported this hypothesis, therefore suggesting CADPS2 as a new susceptibility gene for ID and ASD, and as possible new imprinted gene .

Many fruit species bear an abundance of flowers producing a surplus of fruits that the tree is unable to support. In anticipation of this, the major fruit species developed an immature fruit (fruitlet) physiological drop as a self-regulatory mechanism. This process is, at least in part, a consequence of the competition among fruits and between fruits and shoots for carbon assimilates. The self-regulatory mechanism responsible for the immature apple fruit shedding may be magnified by chemicals such as naphthaleneacetic acid (NAA) and its amide (NAD), and benzylaminopurine (BA) sprayed within 5-6 weeks after full bloom. The thinning action of bioregulators is quite variable and depends on environmental conditions and genotypes. In apple, there are varieties easy to thin and others difficult even though different chemicals or combinations of them are used. Understanding the molecular mechanisms and processes involved in abscission might help in finding new approaches and new chemical thinners to control abscission in fruit, or new self-thinning varieties. The described research was aimed to elucidate the molecular events underlying the in planta fruitlet abscission, taking into account the characteristics of this system and the practical importance of thinning in apple. Fruit drop is due to the activation of specific abscission zones (AZs). It is accepted that abscission is a highly regulated developmental process that is both influenced and activated in response to internal cues and/or environmental conditions. Nevertheless, the identity of the signals responsible for the activation of the AZ is as yet unknown. Among phytohormones, ethylene enhances abscission in several species and systems as well as in apple, while auxins produced by seeds are thought to desensitize AZs to ethylene and prevent abscission. In apple trees, the fruitlet physiological drop is due to the activation of the AZ located at the junction of the peduncle into the twig. In this region four lateral (LF) and one central (CF) fruitlets and the shoot are inserted. The CF comes from the pollinated king flower (KF) that, since it blooms earlier within the cluster, originates a fruitlet larger than the lateral ones. During the physiological drop, the shoot at cluster side, is thought to be a sink in competition with fruitlets for assimilate supply. Considering that seeds and/or fruits are involved in determining the shedding signal while the morphogenetic response occurs always at the AZ level, it is crucial to analyse the whole fruitlet system involved in abscission that should include concurrently seed, cortex, peduncle, and AZ. It is generally believed that the interaction between ethylene and auxin plays a major regulatory role in abscission. Starting from this, a mass gene approach was used in this work to identify genes regulating or involved in abscission. The cDNA-AFLP technique was adopted for transcriptional profiling of differentially expressed genes during apple fruitlet abscission. This allowed the isolation of 278 differential clones by comparing expression profiles of abscising (AF) versus non-abscising (NAF) fruitlet populations. AFs were obtained from lateral fruitlets of trees sprayed with benzylaminopurine (BA) at 200 ppm, 17 days after petal fall (APF) when the fruit cross diameter was about 10-12 mm. NAF originated from central flowers grown in clusters where all the lateral flowers had been removed at bloom. All ESTs (expressed sequence tags) obtained have been annotated with the Gene Ontology vocabulary and grouped according to cellular components, biological processes and molecular functions. Considering the cellular components, the most affected genes in the cortex were related to mitochondrion, plastid and membranes. Concerning the molecular functions, the mostly affected ones were the binding and the transferase activities in the cortex, the hydrolase and transport activities in the seed, and the binding activity in the peduncle. Considering the biological process, in the cortex the most abundant genes were those controlling transport, protein and carbohydrate metabolism. As a general remark, taking into account all the three ontology criteria, it appeared that a prominent up-regulation occurred in the seed. This might be consistent with the determinant role attributed to the seed in the regulation of fruit abscission. The expression and functional analyses of the most interesting clones were carried out by semiquantitative RT-PCR on agarose gel on cDNA obtained from seeds, cortex, peduncles and AZs of AFs and NAFs. Expression analyses confirmed the efficacy of the cDNA-AFLP approach to find a large amount of differentially expressed ESTs and the involvement of the studied genes in regulating the abscission and senescence processes. In particular the differential expression of sugar-metabolism and signalling related genes confirmed the importance of carbohydrates, together with hormones, in controlling the induction of AZs. Since functional studies through silencing or overexpression approaches cannot be easily performed on trees, additional experiments were carried out in Arabidopsis thaliana to investigate the participation of these and other genes in abscission. To this end, Arabidopsis genes putatively homologous to those differentially expressed in relation to fruitlet abscission in apple were identified. A dual approach was chosen to study their function in abscission. In a first attempt, insertional (T-DNA) homozygous mutants were obtained and scored for the presence of abscission-related phenotypes. Probably due to gene redundancy, no phenotypes were detected. Therefore, expression analyses were carried out on the same genes with real time RT-PCR on abscission zones of known Arabidopsis mutants with delayed (dab4-1, dab5-1) or no petal abscission (ida). The results showed a different pattern of expression in comparison to that found in wild type and confirmed an involvement of these genes in abscission. Current work is devoted to further characterise the putative role played by these genes in regulating the abscission of flower organs in Arabidopsis. In addition, a "systematic" approach for the analysis of the whole apple fruitlet abscission transcriptome is needed. To this end an apple microarray is being developed from the large number of already available ESTs, to be used for screening of new chemical thinners and for marker assisted selection of self thinning genotypes.

Introduction. Cardiovascular disease (CVD) represents the main cause of morbidity and mortality in kidney recipients. This study was undertaken to assess the impact of functional polymorphisms located in cytokine and apoptosis genes on CVD after kidney transplantation. Cytokine polymorphisms, generally located in gene regulatory regions, are associated with high and low cytokine production and are likely to modulate the magnitude of inflammatory responses following transplantation, depending on the balance between the levels of pro-inflammatory and antiinflammatory cytokines. The role of apoptosis in atherosclerosis has not been completely elucidated, and here we explored the hypothesis that the heterogeneity in cardiovascular risk in kidney recipients may also be linked to functional polymorphisms involved in apoptosis induction. Purpose. In the search for relevant genetic markers of predisposition to CVD after renal transplant, the present investigation was undertaken to identify the clinical impact of polymorphisms of cytokines TNF-α, TGF-β, IL-10, IL-6, IFN-γ and IL-8 and of apoptosis genes Fas and Caspase 9 in a population of kidney transplant recipients. Materials and methods. The study involved 167 patients who received cadaveric kidney transplantation at our centre between 1997 and 2005 (minimum follow-up of 12 months); 35 of them had experienced cardiovascular events (CVD group) and 132 had no cardiovascular complications (non-CVD group). Genotyping was performed using RFLP (Restriction Fragment Length Polymorphism) for RFLP per IL-8/T-251A, Fas/G-670A e Casp9/R221Q polymorphism and SSP (Sequence Specific Primer) for TNF-α/G-308A, TGF-β/L10P, TGF-β/R25P, IL-10/G-1082A, IL- 10/C-819T, IL-10/C-592A, IL-6/G-174C, IFN-γ/T+874A polymorphisms.Results. We found a significant difference in TNF-α and IL-10 genotype frequencies between the patients who had suffered cardiovascular events and those with no CVD history. The high producer genotype for proflogistic cytokine TNF-α appeared to have a significantly superior prevalence in the CVD group compared to the non-CVD group (40.0% vs 21.2%) and it resulted in a 2.4-fold increased cardiovascular risk (OR=2.361; p=0.0289). On the other hand, the high producer genotype for the antiinflammatory cytokine IL-10 was found in 2.8% of the CVD group and in 16.7% of non-CVD group; logistic regression showed a 0.3-fold reduced risk of CVD associated with genetically determined high IL-10 production (OR=0.278; p<0.0001). The other polymorphisms did not prove to have any impact on CVD. Conclusions. TNF-α and IL-10 gene polymorphisms might represent cardiovascular risk markers in renal transplant recipients.

Introduction. Cardiovascular disease (CVD) represents the main cause of morbidity and mortality in kidney recipients. This study was undertaken to assess the impact of functional polymorphisms located in cytokine and apoptosis genes on CVD after kidney transplantation. Cytokine polymorphisms, generally located in gene regulatory regions, are associated with high and low cytokine production and are likely to modulate the magnitude of inflammatory responses following transplantation, depending on the balance between the levels of pro-inflammatory and antiinflammatory cytokines. The role of apoptosis in atherosclerosis has not been completely elucidated, and here we explored the hypothesis that the heterogeneity in cardiovascular risk in kidney recipients may also be linked to functional polymorphisms involved in apoptosis induction. Purpose. In the search for relevant genetic markers of predisposition to CVD after renal transplant, the present investigation was undertaken to identify the clinical impact of polymorphisms of cytokines TNF-α, TGF-β, IL-10, IL-6, IFN-γ and IL-8 and of apoptosis genes Fas and Caspase 9 in a population of kidney transplant recipients. Materials and methods. The study involved 167 patients who received cadaveric kidney transplantation at our centre between 1997 and 2005 (minimum follow-up of 12 months); 35 of them had experienced cardiovascular events (CVD group) and 132 had no cardiovascular complications (non-CVD group). Genotyping was performed using RFLP (Restriction Fragment Length Polymorphism) for RFLP per IL-8/T-251A, Fas/G-670A e Casp9/R221Q polymorphism and SSP (Sequence Specific Primer) for TNF-α/G-308A, TGF-β/L10P, TGF-β/R25P, IL-10/G-1082A, IL- 10/C-819T, IL-10/C-592A, IL-6/G-174C, IFN-γ/T+874A polymorphisms.Results. We found a significant difference in TNF-α and IL-10 genotype frequencies between the patients who had suffered cardiovascular events and those with no CVD history. The high producer genotype for proflogistic cytokine TNF-α appeared to have a significantly superior prevalence in the CVD group compared to the non-CVD group (40.0% vs 21.2%) and it resulted in a 2.4-fold increased cardiovascular risk (OR=2.361; p=0.0289). On the other hand, the high producer genotype for the antiinflammatory cytokine IL-10 was found in 2.8% of the CVD group and in 16.7% of non-CVD group; logistic regression showed a 0.3-fold reduced risk of CVD associated with genetically determined high IL-10 production (OR=0.278; p<0.0001). The other polymorphisms did not prove to have any impact on CVD. Conclusions. TNF-α and IL-10 gene polymorphisms might represent cardiovascular risk markers in renal transplant recipients.

E' stato dimostrato che le basse temperature incrementano la concentrazione di antocianine nel succo di arance rosse, fattore accompagnato da una maggiore espressione di geni coinvolti sia nei primi stadi della biosintesi delle antocianine (Fenilalaninaammonio liasi), sia negli steps successivi (Calcone sintasi, diidroflavanolo 4-reduttasi, UDP-Glucosio3-O-glucosiltransferasi e glutatione transferasi). In base a questi cambiamenti nel pattern di espressione genetico, la biosintesi delle antocianine puo' essere considerata un percorso cold-regulated e potrebbe essere utilizzato per la caratterizzazione delle risposte molecolari nelle piante ai segnali di stress. Uno studio basato sulla ibridizzazione sottrattiva ha avuto lo scopo di isolare geni differenzialmente espressi in condizioni di stress da freddo (4 gradi per 77 giorni) in arance Tarocco Sciara. La espressione dei geni isolati e' stata monitorata mediante Real Time RT-PCR. In seguito si e'analizzato il cambiamento del pattern di espressione genica in arance Tarocco Sciara sottoposte ad un breve periodo di cold stress (4 gradi per 15 giorni). Le analisi effettuate, sia per l'esperimento nel lungo che nel breve periodo, si sono incentrate sul monitoraggio della espressione di tutti i geni coinvolti nella biosintesi delle antocianine, nella biosintesi dell'acido abscissico e dei fattori di trascrizione implicati nella risposta allo stress.

Alzheimer’s disease (AD) and Frontotemporal Lobar Degeneration (FTLD) are two neurodegenerative and multifactorial diseases with complex etiology in which several genes involved in inflammation, oxidative damage and neuronal survival have been proposed to be candidate susceptibility factors. Given these premises, aims of this study have been to further analyze the association of candidate functional and positional genes in a population of 374 patient with AD, 291 with FTLD and 344 age matched controls. The first candidates studied have been the cell-dependent kinase inhibitor (CDKN) 2A and 2B, involved in cell cycle G1 phase progression, because abnormal cell cycle re-entry has been hypothesized to play a role in neurodegenerative disease, primarily AD. Allelic and genotypic frequencies of three tagging Single Nucleotide Polymorphisms (SNPs), namely rs3731239 T/C, rs2811710 C/T and rs3217992 G/A, spanning CDKN2A and CDKN2B, and covering 100% gene variability, were analyzed. According to our data, CDKN2A and CDKN2B don’t likely act as risk factors for AD. Given the potential importance of kinesin KIF24, protein expressed in neurons and involved in axonal transport and neuron development, we studied the distribution of five Single Nucleotide Polymorphism (SNPs) located in the chromosome 9 haplotype identified via linkage analysis, including UBAP1 rs7018487, UBAP2 rs1785506 and rs307658, and KIF24 rs17350674 and rs10814083. A statistically significant increased frequency of the KIF24 rs17350674 AA genotype was observed in FTLD patients compared with controls, particularly in bvFTD and female patients, showing that KIF24 rs17350674 polymorphism may act as a risk factor for sporadic FTLD. BCL2-associated athanogene 1 (BAG1) is an anti-apoptotic factor interacting with tau and regulating its proteasomal degradation. A statistically significant decreased allelic frequency of the BAG-1 rs706118 SNP was observed in patients with FTLD, but not in AD. BAG-1 rs706118 SNP likely acts as protective factor for sporadic FTLD, but not for AD. Finally we studied the Heterogeneous nuclear ribonucleoprotein hnRNP-A1, which may be linked to AD, because it could influence the maturation of the Amyloid Precursor Protein (APP) mRNA, but also to FTLD, because it contains a glycine-rich consensus domain included in TAR DNA binding protein (TDP)43. In our study, hnRNP-A1 rs7967622 C/C genotype acts as risk factor only for FTLD in male population, while hnRNP-A1 relative expression levels were increased in AD patients, togheter with decreased levels of its transcription regulatory factor hsa-miR-590-3p. We confirmed the importance of genes influencing neuronal transport, metabolism and survival in both neurodegenerative diseases AD and FTLD; the observed differences are probably related to specific roles of these genes in pathogenic events occurring in FTLD only.

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare.
Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare.
Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

To identify the regions of recurrent copy number abnormality in osteosarcoma and their effect on gene expression, we performed an integrated genome-wide high-resolution array CGH (aCGH) and gene expression profiling analysis on 40 human OS tissues and 12 OS cell lines. This analysis identified several recurrent chromosome regions that contain genes that show a gene dosage effect on gene expression. A further search, performed on those genes that were over-expressed and localized in the frequently amplified chromosomal regions, greatly reduced the number of candidate genes while their characterization using gene ontology (GO) analysis suggests the importance of the deregulation of the G1-to-S phase in the development of the disease. We also identified frequent deletions on 3q in the vicinity of LSAMP and performed a fine mapping analysis of the breakpoints. We precisely mapped the breakpoints in several instances and demonstrated that the majority do not involve the LSAMP gene itself, and that they appear to form by a process of non-homologous end joining. In addition, aCGH analysis revealed frequent gains of IGF1R that were highly correlated with its protein level. Blockade of IGF1R in OS cell lines with high copy number gain led to growth inhibition suggesting that IGF1R may be a viable drug target in OS, particularly in patients with copy number driven overexpression of this receptor.

To identify the regions of recurrent copy number abnormality in osteosarcoma and their effect on gene expression, we performed an integrated genome-wide high-resolution array CGH (aCGH) and gene expression profiling analysis on 40 human OS tissues and 12 OS cell lines. This analysis identified several recurrent chromosome regions that contain genes that show a gene dosage effect on gene expression. A further search, performed on those genes that were over-expressed and localized in the frequently amplified chromosomal regions, greatly reduced the number of candidate genes while their characterization using gene ontology (GO) analysis suggests the importance of the deregulation of the G1-to-S phase in the development of the disease. We also identified frequent deletions on 3q in the vicinity of LSAMP and performed a fine mapping analysis of the breakpoints. We precisely mapped the breakpoints in several instances and demonstrated that the majority do not involve the LSAMP gene itself, and that they appear to form by a process of non-homologous end joining. In addition, aCGH analysis revealed frequent gains of IGF1R that were highly correlated with its protein level. Blockade of IGF1R in OS cell lines with high copy number gain led to growth inhibition suggesting that IGF1R may be a viable drug target in OS, particularly in patients with copy number driven overexpression of this receptor.

This PhD study is intended to perform a genetic screening on a population of AD and FTLD patients in order to identify pathogenetic causal mutations (PSEN1, PSEN2 and APP for AD; MAPT, GRN and the GGGGCC repeat expansion one the C9orf72 gene for FTLD) and to investigate the role of several candidate genes (GRN,TMEM106b and OLR1) considered to be risk factors for the two disease. Eighteen patients were carriers of pathogenetic causal mutations: 1 carrier of Ala260Val (g.49964C>T) situated in exon 8 of PSEN1,16 carriers of GRN gene mutations and 1 carrier of a new variant, Gly304Ser (g.123789G>A), located in exon 10 of MAPT gene. The presence of GGGGCC repeat expansion, positioned on the first intron of C9orf72 gene, was analyzed in a larger population (651 FTLD patients, 21 CBD and 31 PSP patients). Thirty nine patients with FTLD were carriers of pathogenetic repeat expansion, whereas none of CBD and PSP patients as well as 222 controls carried the mutation. Several associations studies were performed in the remaining sporadic population of AD and FTLD patients. Regarding the influence of GRN genetic variability on susceptibility to AD, two SNPs rs9897526G>A and rs5848 were investigated. A tendency to an increased frequency of rs5848T allele was found in AD patients as compared with controls, whereas for the rs9897526 SNP, in patients carrying the rs9897526A variant was observed a significant earlier age at disease onset compared with patients carrying the G allele. The case-control study carried out on a populations of FTLD patients was focused on four Tagging SNPs (rs2879096, rs3785817, rs4792938 and rs9897526) as well as on rs5848 SNP, localized in the 3’UTR of GRN gene. A statistically significant association of the rs4792938 CC genotype was observed in FTLD patients compared with healthy controls. Concerning the role of TMEM106b gene on susceptibility to AD, an association analysis was performed on three SNP, rs1020004 A/G, rs6966915 C/T and rs1990622 A/G, but no significant differences in allelic and genotype frequencies were found for all polymorphisms between AD patients and controls. The possible functional importance of genetic variability associated with this gene was tested by plasmatic ELISA detection of GRN on eighty AD patients. Stratifying the results according to rs1990622 SNP status, no significant differences in progranulin plasma levels were found in AD patients. Regarding OLR1, in particular it was analyzed the SNP rs1050283 T/C, located in 3’UTR of the gene. Logistic regression analysis, adjusted for gender and ApoE status, showed a statistically significant association of OLR1 rs1050283 under the assumption of a dominant and a genotypic model. Therefore this SNP could be considered a susceptibility factor for sporadic AD. Given that the SNP rs1050283 is also located in a predicted binding site of the miRNA has-miR369-3p, a preliminary expression analysis was performed on the two transcripts in the PBMC in order to clarify a possible functional role of individual genetic variability on the expression of OLR1 gene. Stratifying the results according to the presence of rs1050283C allele, a significant decrease of relative expression levels of OLR1 was observed in patients carrying at least one polymorphic C allele, despite the normal expression levels of has-miR369-3p. These data suggest that the presence of the polymorphic allele could influence the binding of has-miR369-3p to its 3’UTR consensus sequence, in which the SNP is located.

As defined by European Union, rare diseases are a broad range of disorders with prevalence ≤ 5/10000 in general population. Some rare diseases are the result of bacterial or viral infections, allergies and environmental causes, or are degenerative and proliferative, but the most have a genetic etiology. Discovering alleles underlying genetic conditions is essential for the comprehension of the pathogenesis and for the prevention of the disease through identification of carriers and prenatal diagnosis. Conventional strategies for disease gene discovery, like positional cloning and Sanger sequencing of candidate genes, have led, to date, to detect the genetic detrminants of about 3000 Mendelian phenotypes, representing about the 50% of genetic phenotypes known. In recent years many genes causing human genetic disorders have been identified through the analysis of the variants revealed by whole-genome or exome sequencing by “Next-Generation” sequencing technologies (NGS). Herein, we report the results of an exome sequencing project applied to the identification of the gene responsible for a rare and genetically heterogenous ciliopathy, the Jeune Syndrome (Asphyxiating thoracic dystrophy, JATD, MIM 208500). Exome sequencing has been performed on the affected and respective parents of two unrelated Sardinian families in which genetic basis of the disease was not yet elucidated. Exome variants analysis strategies led us to identify a missense substitution in DYNC2H1 (MIM 603297), a gene involved in intraflagellar transport pathways of cilia that has been previously associated to the disease (Dagoneau et al., 2009; Merrill et al., 2009). The substiution has been found in homozygosity in the probands of one of the two families JATD and in heterozygosity in their parents. To date, exome analysis is still in progress in the second family JATD. For this family, there was only one gene that has been considered a good candidate for the disease, MAP1S (MIM 607573), that codifies for a microtubule associated protein. Unfortunately, the compound heterozygous state for two variants, revealed by exome sequencing in the proband, was not validated by variants resequencing with Sanger method. No deleterious variants have been found in DYNC2H1 but the involvement of this gene in disease pathogenesis cannot be excluded because of the failed capture of some exons, that will be resequenced by NGS technologies. Studies in vivo and in vitro could explain the functional impact of the DYNC2H1 missense substitution identified by exome sequencing on the protein function.

Obiettivi: Studiare la presenza, i contesti genetici e la trasferibilità dei geni di resistenza agli oxazolidinoni in enterococchi di origine suina. Materiali e Metodi: Sono stati raccolti 255 campioni fecali da 76 allevamenti di suini della regione Marche. Tutti gli enterococchi resistenti al florfenicolo sono stati analizzati, mediante PCR, per la presenza dei geni optrA, cfr e poxtA. Gli isolati con almeno un determinante di resistenza al linezolid sono stati saggiati mediante test di sensibilità (MIC e E-test). E’ stato inoltre studiato: (i) il trasferimento dei geni di oxazolidinone-resistenza mediante saggi di coniugazione su filtro; (ii) la localizzazione dei determinanti di resistenza tramite S1-PFGE, Southern blotting ed esperimenti di ibridazione utilizzando sonde specifiche; (iii) i contesti genetici e la clonalità mediante analisi genomica dei ceppi [Whole Genome Sequencing (WGS)]. Risultati: Nello studio sono stati isolati 145 enterococchi resistenti al florfenicolo da campioni fecali di suini. Trenta enterococchi resistenti al florfenicolo, provenienti da 23 allevamenti, avevano almeno un gene di resistenza al linezolid. optrA è risultato essere il gene di resistenza al linezolid più diffuso (23/30 ceppi), mentre cfr e poxtA sono stati rilevati rispettivamente in 6/30 e 7/30 isolati enterococcici. L'analisi WGS ha anche mostrato la presenza del gene cfr(D) in Enterococcus faecalis (n=2 isolati) e in Enterococcus avium (n=1 isolato). Saggi di ibridazione hanno mostrato che i geni di resistenza al linezolid erano localizzati sia sul cromosoma che su plasmidi di diverse dimensioni (range ~ 25 - ~ 240 kb). Dodici isolati erano in grado di trasferire geni di resistenza al linezolid ai ceppi riceventi E. faecalis JH2-2 e/o E. faecium 64/3. L'analisi WGS ha evidenziato una ampia variabilità dei contesti genetici optrA identici o correlati a trasposoni (Tn6628 e Tn6674), plasmidi (pE035 e pWo27-9) e regioni cromosomiche. Gli ambienti genetici di cfr mostravano alti livelli di identità nucleotidica sia con il trasposone Tn6644 che con una regione dal plasmide p12-2300; i contesti genetici del gene cfr(D) erano correlati alla regione corrispondente del plasmide 4 di E. faecium E8014; il gene poxtA era sempre localizzato sul trasposone Tn6657. Le forme circolari sono state ottenute solo per i contesti genetici di optrA e poxtA. Le analisi delle relazioni filogenetiche hanno rivelato la presenza di cloni di E. faecalis (ST16, ST27, ST476 e ST585) ed E. faecium (ST21) di origine umana. Conclusione: I risultati di questo studio evidenziano un’ampia distribuzione dei geni di oxazolidinone-resistenza negli enterococchi di origine suina nell'Italia centrale e confermano la diffusione della resistenza al linezolid negli ambienti animali.
Objectives: To investigate the occurrence, the genetic environments and the transferability of oxazolidinone resistance genes in enterococci of swine origin. Materials and Methods: A total of 255 faecal samples were collected from 76 pig farms of Marche region. Selected florfenicol-resistant enterococci were screened for optrA, cfr, and poxtA genes by PCR. Isolates with at least one linezolid resistance determinant were tested for their susceptibility. Resistance genes transfer (filter mating), localization (S1-PFGE/hybridization), genetic contexts and clonality (WGS) were analyzed. Results: One hundred forty-five florfenicol-resistant enterococci were isolated from swine fecal samples. Thirty florfenicol-resistant enterococci from 23 farms had at least one linezolid resistance gene. optrA was found to be the most widespread linezolid resistance gene (24/31), while cfr and poxtA were detected in 6/31 and 7/31 enterococcal isolates, respectively. WGS analysis also showed the presence of the cfr(D) gene in Enterococcus faecalis (n = 2 isolates) and in Enterococcus avium (n = 1 isolate). The linezolid resistance genes hybridized both on chromosome and plasmids ranging from ~25 to ~240 kb. Twelve isolates were able to transfer linezolid resistance genes to enterococci recipients. WGS analysis showed a great variability of optrA genetic contexts identical or related with transposons (Tn6628 and Tn6674), plasmids (pE035 and pWo27-9) and chromosomal regions. cfr genetic environments showed identities with Tn6644-like transposon and a region from p12-2300 plasmid; cfr(D) genetic contexts were related to the corresponding region of the plasmid 4 of Enterococcus faecium E8014; poxtA was always found on Tn6657 transposon. Circular forms were obtained only for optrA- and poxtA-carrying genetic contexts. Clonality analysis revealed the presence of clones of E. faecalis (ST16, ST27, ST476, and ST585) and E. faecium (ST21) previously isolated from humans. Conclusions: These results demonstrate a dissemination of linezolid resistance genes in enterococci of swine origin in Central Italy and confirm the spread of linezolid resistance in animal settings

Objective: To correlate neuropsychological testing with cerebrospinal fluid Amyloid beta (AD), tau and Ptau levels in patients with Mild Cognitive Impairment (MCI) Background: Principal diagnostic instrument to define global cognitive state is the Mini Mental State Examination (MMSE). Cognitive domains that best predict progression from amnestic MCI to AD are episodic memory and executive functioning. Were selected two neuropsychological tests for both domains: Story Recall Test (SRT) and Paired-Associate Learning (PAL) to examine episodic verbal memory, and Coloured Progressive Matrices of Raven (CPMR) and Clock Drawing Test (CDT) to investigate executive functioning. Methods: Forty subjects with amnestic MCI were recruited. All of them underwent neurological exam, neuropsychological testing and lumbar puncture at time of diagnosis. Cognition were explored by MMSE (global cognitive functioning), CPMR and CDT (executive functions), SRT and PAL (episodic verbal memory). Statistical analysis was carried out by using t-test and Spearman test for correlations. Results: In the whole population, no significant correlation between cognitive and biological markers was observed. Considering CSF biomarker level of amiloidβ, thirteen subjects showed an altered pattern and converted to AD after few months, the other subjects whit a normal profile did not convert. MMSE show a significant difference between converters/no converters groups (26.4 versus 27.6 p=0.066). Also CDT and SRT had a significant difference between two groups (3 versus 5 p= 0.003 and 5.85 versus 8.32, p=0.03). Conclusion: According to these results, MMSE value is lower in converters, in according to patological biomarkers profile. PAL and CDT are more specific to predict conversion from MCI to AD.

IDENTIFYING NEW MOLECULAR PATHWAYS AND PREDICTOR BIOMARKERS OF GVHD AFTER ALLOGENEIC HSCT Background and rationale Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Strategies to control GVHD are often associated with suppression of the immune system leading to the impairment of the beneficial graft versus tumor (GVT) effect. The ideal approach to prevent and treat GVHD would limit alloantigen-specific reactivity while preserving immunity against malignant cells and pathogens. Chemokines are well known inducers of leukocyte trafficking and activation. Stimulation of the chemokine receptor signaling pathway leads to initiation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway that contributes to the pathogenesis of GVHD. The key role of JAK signaling in normal and abnormal lymphocyte development and function prompted us to hypotesize that the inhibition of the JAK/STAT signaling could prevent GVHD from developing. We therefore evaluated the efficacy of two distinct JAK inhibitors, CP-690,550 and INC-424, in the allogeneic setting. Moreover, we tested whether the treatment with JAK inhibitors preserved the GVT effect in a mouse model of aGVHD. However, it would be a great advantage to predict aGVHD before its onset in order to tailor the immunosuppressive therapy in a patient-specific manner. To date, noninvasive, diagnostic and prognostic tests for aGVHD are lacking but necessary to predict aGVHD and improve the safety of allo-HSCT. We therefore performed a prospective analysis of miRNA expression profile in plasma of allografted patients to detect specific miRNAs with predictive role for aGVHD. Methods A major histocompatibility complex (MHC) mismatched HSCT mouse model was set up. Lethally irradiated BALB/c mice received spleen (SC) and bone marrow (BM) cells from donors C57BL/6 (B6) mice, and were treated with CP690,550 or INCB18424 for 14 days at 15mg/Kg/day or 45mg/Kg/day respectively. Syngeneic transplants (B6-B6) and BALB/c recipients treated with B6 BM cells only were also used. To determine the GVT activity, allo-HSCT recipients were co-injected with either a B-cell lymphoma cell line (A20) or a myeloid leukemia cell line (RMB-1) and treated or not with INC-424 at the dose of 45mg/kg/day. Mice were monitored for overall survival (OS) and weight loss. GVHD was histologically scored in tissues harvested on day 14, 30 and 60 and cytokine production by ELISA. Immune reconstitution and tumor cells were monitored by flow cytometry. For biomarker discovery, plasma samples from 24 patients who received unmanipulated allo-HSCT were collected weekly. MicroRNAs were isolated from plasma and miRNA expression profile examined using quantitative Real Time PCR. Results JAK/STAT inhibition, especially by INC-424 treatment, caused significantly less GVHD when compared to untreated animals, as determined by survival, weight loss, and histopathology of GVHD target organs. Plasma levels of inflammatory cytokines (IL-6 and IL-12) were significantly reduced in all treated animals when compared to controls. Daily administration of INC-424 post allo-HSCT did not alter hematologic parameters and did not impair immune reconstitution but shifted the CD4+ Treg to CD8+ effector cell ratio because of a relative decrease in the latter population and a relative increase in Treg in GVHD treated mice. A reduction in the Th17/Treg ratio was also observed. When allo-HSCT recipients were challenged with tumor cells, the GVT activity in INCB-424 treated mice was intact in the absence of GVHD, resulting in significantly improved survival compared to untreated animals (p<0.001) and reduced the presence of tumor cells. On the other hand, circulating miRNA profiling before aGVHD onset was able to identify patients at high risk of developing the disease. Two unique miRNAs (miR-194 and miR-518f) were upregulated in samples of patients that would lately develop aGVHD. Pathway prediction analysis indicated that these miRNAs are critical in GVHD pathogenesis. Conclusions The inhibition of JAK/STAT signaling using the sensitive and specific inhibitor of JAK1/JAK2 INC-424 at the optimal dose of 45mg/kg/day, conferred effective protection from lethal acute GVHD in a MHC mismatched HSCT mouse model. Mice retain the ability to mount aggressive graft-versus-tumor (GVT) effects and to generate complete donor T-cell reconstitution. Taken together, these data suggest that INC-424, recently approved for the treatment of myelofibrosis, might also be tested for GVHD prophylaxis and treatment in clinical trials. In addition, considering the non-invasive characteristics of plasma sampling and the feasibility of detecting miRNAs after allo-HSCT, our results indicate that circulating miRNAs represent a promising tool for the early diagnosis of aGVHD. However, inconsistencies with prior published data highlight the need to develop standards for circulating miRNA studies to move their discovery from the molecular biology laboratory to the clinic.

Il morbo di Parkinson è una delle malattie neurodegenerative più diffuse ed è estremamente invalidante perché comporta deficit psicologici e motori. Uno dei sintomi più problematici è il freezing degli arti inferiori che compromette la deambulazione dei soggetti che ne sono affetti ed è la causa più frequente di cadute nei Parkinsoniani. Data la gravità del fenomeno e grazie al crescente uso di sensoristica indossabile in campo clinico, molteplici algoritmi sono stati implementati per identificare automaticamente gli istanti di freezing a partire da misure inerziali. Tuttavia, nessuno studio ha comparato le performance dei diversi algoritmi al fine di fornire istruzioni pratiche di utilizzo. L’obiettivo di questa tesi è quello di individuare i lavori presenti in letteratura per la detezione del freezing del cammino su soggetti Parkinsoniani tramite sensori inerziali, implementarli e confrontarne tra loro le performance. Per questo lavoro sono stati considerati 5 pazienti, cui è stato chiesto di camminare per 6 minuti con 5 sensori inerziali applicati su tronco, caviglie e piedi. A valle di una revisione sistematica della letteratura inerente agli algoritmi di identificazione del freezing a partire da misure inerziali sono stati identificati e implementati 8 algoritmi. Le performance degli algoritmi sono state valutate in termini di sensitività, falsi positivi, accuratezza e ripetibilità in relazione al diverso criterio implementativo, ovvero posizione del sensore, variabile analizzata e dominio. Dai risultati si evince che, nell’identificazione degli eventi di FOG il posizionamento dei sensori posti su caviglia e piede danno performance migliori, rispetto a quando vengono posizionati sul tronco. Per quanto riguarda la variabile analizzata, invece, gli algoritmi che sfruttano l’accelerazione sono da preferire a quelli che usano la velocità angolare. In base al dominio, performance differenti si osservano nel dominio del tempo e in quello della frequenza.

It is well established that several forms of inheritable idiopathic epilepsy sindrome are ion channelopathies, that is pathologies associated with dysfunctional ion channels, even if the functional link between channel dysfunction and clinical phenotype is often unresolved. Hyperpolarization-activated, Cyclic-Nucleotide gated (HCN) channels are a class of voltage- and cAMP-dependent channels. They mediate the hyperpolarization-activated Ih current, which control synaptic integration and intrinsic excitability in various brain areas. Ih is pathologically altered after experimentally-induced seizures and has been proposed to have a role in different forms of epileptogenesis. Hcn1 and Hcn2 genes variants have been identified in patients with febrile seizure or GEFS+. While existing data therefore clearly show a link between HCN channel dysfunction and epileptogenesis, no specific mutation-induced HCN channel modification has so far been correlated functionally with increased neuronal excitability. To investigate this we used a candidate gene approach and screened a panel of idiopathic generalized epilepsy patients and related families for mutations in the Hcn1 and Hcn2 genes. We found a form of sporadic IGE associated with a recessive point mutation in the gene coding for the HCN2 channel. The protein mutation E515K is located in the C-linker region (exon 5 of the Hcn2 gene). Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/E515K channels, have a negative shift in the activation kinetics. Furthermore, the time-constant curve for homomeric E515K channels was also shifted to the negative direction. Moreover, omomeric mutant, but not heteromeric wild type/mutant channels, showed a lowering of the threshold of action potential firing and a strongly increased cell excitability and firing frequency when compared to wild-type channels. In conclusion, our results show that the homozygous E515K mutation in human HCN2 channels is a loss-of-function mutation causing a large negative shift of the activation curve and slowing of activation, and a consequent strong reduction of Ih availability near resting voltages. These changes cause a substantial increase of neuronal excitability, a condition predisposing to epileptogenesis, and are associated with a recessive type of inheritance compatible with the idiopathic generalized epilepsy of the proband in the family pedigree.

La malattia di Parkinson è una malattia neurodegenerativa che colpisce attualmente 10 milioni di persone in tutto il mondo. Circa il 50% delle persone con il morbo di Parkinson sperimenta il freezing dell'andatura (FoG), una breve, episodica assenza o marcata riduzione della progressione in avanti dei piedi, nonostante l'intenzione di camminare. Il freezing rappresenta una delle principali cause di cadute e scarsa qualità della vita in suddetti soggetti. L'occorrenza episodica e piuttosto imprevedibile degli eventi di freezing, insieme alla risposta variabile alla terapia farmacologica, rende la valutazione obiettiva della gravità del FoG una delle principali sfide cliniche nella gestione terapeutica dei pazienti con malattia di Parkinson. Per l’identificazione del FoG vengono principalmente proposti metodi basati su unità di misura inerziali (IMU) indossabili, soluzioni ottimali per il monitoraggio oggettivo a lungo termine dei pazienti data l’indossabilità, il basso costo e il facile utilizzo. In questa tesi è stata eseguita una revisione sistematica della letteratura degli algoritmi per l’identificazione automatica del FoG in soggetti con la malattia di Parkinson, basati sui dati registrati dai sensori inerziali.

Idiopatic Central Hypogonadism (ICH) is a rare pathology with a strong genetic component, in which hypothalamic and pituitary dysfunctions involving development and/or functionality of GnRH neurons, cause a reduced or absent gonads functionality. This disease can occur in association with anosmia or hyposmia, (Kallmann Syndrome, KS) o with a normal sense of smell (normosmic idiopathic hypogonadotropic hypogonadism, nIHH) and it shows an extreme phenotypic variability. Despite the identification of 14 genes implicated in the pathogenesis of the disease, approximatively 70% of ICH cases remains idiopathic. Among the causative genes, a role of particular importance is covered by the the Prokineticin pathway, in particular the Prokineticin-2 (PROK2) and its receptor (PROKR2). In fact, in approximately 10% of cases of ICH is possible to identify a genetic variant in one of these two genes such as pathogenetic event of the disease. The receptor PROKR2 belongs to the family of G-protein coupled receptor (GPCR). Its activation, through the binding with PROK2, determines the activation of protein Gq, Gs and Gi, a consequent production of IP3, cAMP and subsequently the mobilization of intracellular calcium. To date in literature have been described 27 PROKR2 mutations and the functional studies performed on a minority of them have only evaluated the effects on the Gq-IP3 signal transduction pathway. Nevertheless a growing number of works in the field of GPCRs demonstrates the importance of the functional studies of all the possible pathways related to a single receptor in order to interpret the functional consequences of genetic variants identified. In the present work we have developed two main lines of research starting from the wider availability of Italian cohort of ICH patients. In the first part of this work we have carried out studies of genetic screening of a cohort of 217 patients, considering the main causative known genes for that pathology, including PROKR2. Genetic variants identified in PROKR2 were then characterized by a functional point of view to test their potential pathogenic role. This screening allowed the identification of seven PROKR2 missense variants (V158I, L173R,T260M, R268C, V274D, V331M and V334M) of which 3 have not yet been described in the literature; in addition to 2 variants nonsense (15fsX45, 20fsX43). The variants identified have been inserted by site-directed mutagenesis into vectorsSPRT-PROKR2-pcDNA3, characterized by the presence of a Rhodopsin tag at the N-terminus of the receptor. This allows the display of the cellular localization of the mutants by binding with an anti-rhodopsin antibody. The constructs thus generated were transfected into HEK 293 cells and CHO for the following functional studies. The FACS analysis revealed that all variants have a reduced membrane expression (reduction of 11-55%), with the exception of the mutation V334M, which shows an expression slightly exceeding that of the wild-type receptor. Functional assays were then performed with the generation of concentration-effectcurves for both IP1 that for cAMP. The results obtained show how the mutation T260M, R268C, V274D, V331M and V334M cause a strong reduction of the signal mediated by the Gq protein , while the signal of cAMP mediated by the Gs protein is significantly reduced in the mutant L173R andV334M. The V334M and V274D mutations are characterized by a marked inactivation of both pathways. Finally analyzing the homology model of PROKR2 it appears evident that the variant V331M is localized at the level of a highly conserved domain (the motif NPXXY), involved in signal transduction. These are the first experiments that analyze both transduction pathways activated by PROKR2 receptor and showing how the different variants associated with ICH can affect signal transduction pathways in a very variable manner. In particular, some variants causing inability to stimulate the two pathways, suggesting that the integrity of both is necessary for normal development and function of GnRH-secreting neurons. The second part of this thesis, it was instead intended to further clarify the genetic mechanisms (and eventually epigenetic) about the ethiopathogenesis underlying ICH. To conduct these studies we used the techniques of SNPs and CNVs genotyping , on a selected series of familial cases of ICH. For each patient were analyzed 660,000 SNPs and CNVs 100,000, then comparing them with a large database of apparently healthy controls in our possession, in a case / control analysis. The analyzes focused on the identification of SNPs and on the presence of CNVs significantly correlated with ICH and on an family type analysis to detect extended regions of homozygosity in patients (LOH = Loss of Heterozigosity regions). A first macroscopic analysis of the data obtained shows that the number and extent of the deletions in single copy is significantly higher in cases of ICH, compared to controls. The analysis of SNPs and CNVs showed, among the 30 identified loci four genes (CNTNAP2, GPC, RAB39B and PPFIA2) that for expression and molecular function seem to be good candidates for direct sequencing screening in ICH patients. Furthermore we have identified three clusters of microRNA (mir4275, mir507/508/509,mir320D2), suggesting for the first time a potential involvement of these molecules in ICH pathogenesis. Analyzing instead the genes that reside in LOH areas , it is possible to observe an enrichment for certain pathways or protein families, such as: the FGFR pathway; cadherins and cell adhesion molecules (CAM); receptors and ligands involved in the differentiation of the central nervous system (CNS), hypothalamus and pituitary; genes associated with midline defects or with Prader-Willi and Angelmann syndrome; plexins and RAB proteins.

ABSTRACT IN ENGLISH Objectives: to elucidate the natural history of senile myoclonic epilepsy, a type of myoclonic epilepsy associated with Alzheimer’s disease in adult Down syndrome patients. Methods: Twelve Down syndrome patients over the age of 40 years with myoclonic epilepsy and Alzheimer’s disease underwent clinical, neuropsychological, neurophysiological and neuroradiological study. The kariotypes, APOE polymorphisms, all exons in the PSEN1 and PSEN2 genes and exons 16 and 17 in the APP gene were determined for all patients. CSF Aβ42, p-tau181, and t-tauAg were determined for two patients. Results: Three main stages appeared during the course of the syndrome. The first stage was characterized by dementia onset (mean age: 51±6.63 years), diffuse EEG abnormalities during sleep, and cerebral atrophy determined using neuroimaging. During the second stage, myoclonic epilepsy manifested (mean age: 51.4±7.29 years) with myoclonic jerks time-locked to diffuse epileptiform abnormalities upon awakening, which was controlled with antiepileptic drugs. During the third stage (mean age: 54.8±7.61 years), myoclonic seizures were replaced with myoclonus beyond seizure events, and cerebellar signs, severe dementia and photosensitivity developed. All patients showed complete trisomy 21. Mutations were ruled out on the APP, PSEN1 and PSEN2 genes, and APOE analysis revealed ε3/ε3 homozygosity. CSF biomarkers showed a decrease in Aβ42 and an increase in p-tau181. Conclusions: The natural history of senile myoclonic epilepsy is consistent with progressive myoclonus epilepsy. Chromosome 21 is implicated in its pathophysiology; however, other genetic and/or environmental risk factors cannot be excluded. The absence of the APOE type 4 allele may predict its progression. Key-words: Down syndrome, video-EEG/polygraphy, senile myoclonic epilepsy, Alzheimer’s disease, progressive myoclonus epilepsy, APOE, CSF biomarkers, PSEN 1 gene, PSEN2 gene, APP gene. ABSTRACT IN ITALIANO Obiettivi: definire la storia naturale dell’Epilessia Mioclonica Senile, un forma di epilessia mioclonica associata alla Malattia di Alzheimer in soggetti adulti con Sindrome di Down. Materiali e Metodi: 12 pazienti con sindrome di Down di età > 40 anni con epilessia mioclonica e malattia di Alzheimer sono stati sottoposti ad un completo studio clinico, neuropsicologico, neurofisiologico e neuroradiologico. Il cariotipo, lo studio dei polimorfismi dell’APOE, di tutti gli esoni dei geni della presenilina 1 e 2 e degli esoni 16 e 17 del gene della proteina precursore dell’amiloide sono stati determinati in tutti i pazienti. Sono stati anche valutati in due pazienti i livelli liquorali di Aβ42, p-tau181, e t-tauAg.. Risultati: tre principali stadi compaiono durante il decorso del quadro sindromico. La prima fase si caratterizza per la comparsa della demenza (età media di insorgenza: 51±6.63 anni), con anomalie diffuse nel corso del sonno e l’evidenza di atrofia cerebrale alle neuroimmagini. Durante la seconda fase, si apprezza l’insorgenza della epilessia mioclonica (età media di insorgenza: 51.4±7.29 anni), con scosse miocloniche, di solito al risveglio e controllate dal trattamento con farmaci anti-epilettici, correlate a diffuse anomalie epilettiformi. La terza fase (età media di insorgenza 54.8±7.61 anni) si caratterizza per la comparsa di mioclono associato ad una sindrome cerebellare con demenza di grado severo e fotosensibilità. Tutti i pazienti mostravano una trisomia completa del cromosoma 21 e un genotipo ε3/ε3 dell’APOE. Non sono state documentate mutazioni a carico dei geni della presenilina 1 e 2 e della proteina precursore dell’amiloide. I biomarcatori liquorali hanno documentato un decremento della Aβ42 e un incremento della p-tau181, suggestivi per una patologia neurodegenerativa di tipo alzhemeriana. Conclusioni: la storia naturale dell’epilessia mioclonica senile è compatibile con una epilessia mioclonica progressiva. Il cromosoma 21 è implicato nella sua fisiopatologia, sebbene non si possa escludere il ruolo di altri fattori di rischio genetici e/o ambientali. La mancata documentazione dell’allele di tipo 4 dell’APOE potrebbe rappresentare un fattore predittivo di progressione della malattia.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a heart muscle disease characterized by myocardial atrophy and fibrofatty replacement, mainly involving he right ventricle (RV). However also the left ventricle (LV) can be involved in a significant number of cases. The pathologic substrate constitutes the anatomic basis both for re-entry phenomena, that can lead to onset of ventricular arrhythmias, and for morphological alterations of the RV. The clinical manifestations of disease usually appear between 10 and 20 years of age. ARVC/D has been proved to have a genetic origin with autosomic dominant transmission in the majority of cases. Moreover, several disease-genes have been identified so far. Genetic screening enables to identify subjects carrying a gene mutation and thus to early identify patients at risk of develop the disease. In these subjects, including children, a clinical follow-up (FU) is necessary to detect promptly the clinical onset of disease. AIM OF THE STUDY: we analysed a series of subjects carrying an ARVC-gene mutation evaluated for the first time at a young age (< 18 yrs), with the purpose to describe the clinical phenotype and the natural history of the disease. METHODS: a total of 62 subjects (38 males, 24 famales, mean age at first evaluation 12.4?3.9 years) examined for the first time below the age of 18 and in whom a mutation of a know-ARVC gene has been detected were analysed. Thirtheen subjects (21%) were probands and 49 (79%) were family-members. This population was divided into 3 groups according to the age at the first evaluation: 1] 1-10 years old; 2] 11-14 years old; 3] 15-18 years old. The study protocol included 12-lead ECG, signal-averaged ECG (SAECG), 24-hour Holter monitoring and 2D-echocardiogram with Doppler analysis After first clinical evaluation subjects were divided in “affected”, “not-affected” and “border-line”, then they entered in a annual or 6 months FU program. a Cardiac magnetic resonance (CMR) with contrast agent gadolinium was performed in border-line patients and in non-affected subjects older than 14 years. RESULTS: At first evaluation none subjects belonging to group A fulfilled the diagnostic criteria, that were reached in 8 pts (40%) of group B and in 7 pts (30,5%) of group C. During FU, lasting 8.8+6.7 yrs, 7 subjects previously considered unaffected were diagnosed with the disease (12,9%, mean age at diagnosis 19.5+5.3 anni) and 10 pts already affected showed a progression of cardiomyopathy. Moreover, among the 12 subjects who did not fulfilled the diagnostic criteria who underwent CMR, 6 (50%, mean age 15.5+5.6 yrs, range 9-22 yrs) were diagnosed with an ARVC/D form . CONCLUSION: Our data confirm that ARVC/D is not present at birth and is characterized by clinical onset during adolescence and young adulthood. A significant number of subjects carrying a genetic mutation does not fulfill the diagnostic criteria and in this group CMR with gadolinium injection is a fundamental diagnostic tool.
INTRODUZIONE: La cardiomiopatia aritmogena del ventricolo destro (ARVC) è una malattia primitiva del muscolo cardiaco dovuta ad una necrosi miocitaria con successiva sostituzione fibro-adiposa. Tale sostituzione, dal punto di vista clinico, si traduce in instabilità elettrica e in alterazioni morfologiche in particolare a carico del ventricolo destro (VD), anche se il ventricolo sinistro può essere coinvolto in un numero significativo di casi. L’ARVC si manifesta clinicamente fra la seconda e la terza decade di vita con la presenza soprattutto di aritmie ventricolari che possono portare anche a morte improvvisa. La malattia riconosce un’origine genetica, con modalità di trasmissione autosomica dominante nella maggior parte dei casi, con un substrato genetico alquanto eterogeneo dato che sono stati fino ad ora identificati numerosi geni correlati alla malattia. Lo screening genetico permette attualmente l’identificazione dei soggetti portatori di una mutazione ritenuta causativa all’interno delle famiglie affette. Questo comporta l’identificazione precoce, anche in età pediatrica, di soggetti potenzialmente a rischio di sviluppare la malattia, nei quali è necessario eseguire uno stretto follow-up (FU) clinico-strumentale, al fine di rintracciare tempestivamente l’eventuale comparsa di segni fenotipici di malattia. OBIETTIVI: Scopo dello studio è la valutazione clinico-strumentale di una serie di soggetti portatori di mutazioni causative legate all’ARVC giunti alla nostra osservazione ad un’età inferiore a 18 anni e seguiti in un programma di FU, allo scopo di descrivere gli aspetti clinici precoci della malattia. MATERIALI E METODI: Sono stati studiati 62 soggetti (38 maschi, 24 femmine, età media alla prima osservazione 12.4?3.9 anni) nei quali era stata identificata una mutazione causativa di un gene-malattia legato all’ARVC. Di questi 13 (21%) erano probandi e 49 (79%) familiari di soggetti affetti. I soggetti sono stati divisi in 3 gruppi in base all’età alla prima osservazione: 1] 1-10 anni; 2] 11-14 anni; 3] 15-18 anni. Il protocollo di studio comprendeva: visita cardiologia, ECG a 12 derivazioni, signal-averaged ECG, ECG-Holter delle 24 ore ed Ecocardiogramma mono e bidimensionale con analisi Color-Doppler. In base ai risultati degli esami strumentali i soggetti, dopo la prima visita, venivano suddivisi in: affetti, non affetti, soggetti con alcuni segni clinici di malattia ma che non soddisfacevano i criteri di diagnosi; venivano inseriti, quindi, in un programma di FU con intervalli variabili dai 6 ai 12 mesi. Il protocollo di studio, a questo punto, prevedeva l’esecuzione di risonanza magnetica cardiaca (RMC) con gadolinio nei soggetti senza controindicazioni all’esame, che avessero età ?14 anni o diagnosi dubbia. RISULTATI: Dei 19 soggetti esaminati prima dei 10 anni nessuno era risultato affetto alla prima visita. Fra i 20 pazienti del secondo gruppo, in 8 (40%) è stata fatta diagnosi di ARVC. Dei 23 soggetti appartenenti al terzo gruppo, in 7 (30.5%) erano presenti criteri sufficienti per la diagnosi di ARVC. Durante il FU altri 8 soggetti (12.9%, età media 19.5+5.3 anni, FU medio 8.8+6.7 anni) hanno mostrato la comparsa della malattia e 11 soggetti con diagnosi alla prima visita hanno mostrato un’evoluzione della patologia. Infine 12 soggetti che non raggiungevano i criteri di diagnosi sono stati sottoposti a RMC, che in 6 (50%, età media 15.5+5.6 anni, range 9-22 anni) ha rivelato la presenza di alterazioni compatibili con la malattia. CONCLUSIONI: La ARVC si conferma come malattia non presente alla nascita e con esordio clinico nell’adolescenza e prima giovinezza. Già in età adolescenziale, comunque, si possono rilevare forme importanti ed estese e casi di forte instabilità elettrica, per cui la diagnosi precoce è fondamentale. Numerosi soggetti con mutazione genetica non soddisfano i criteri di diagnosi ed in questa particolare popolazione la RMC con gadolinio è un mezzo diagnostico efficace per aggiungere informazioni morfo-funzionali e di caratterizzazione tissutale.

Over the years many different approaches and techniques have been employed to get insight genetic data of family and patients. The first approach for genetic studies and gene discovery was the linkage analysis, but to be efficient it required large family or large numbers of patients sharing the same disease phenotype. The advent of sequencing technology made the genetic analysis more handy but still it was time consuming and not cost effective when a large number of genes needed to be screened , for example in case of diseases with a known genetic heterogeneity as the neuromuscular disorders (NMDs). The high throughput molecular diagnostics tools such as Comparative Genomic Hybridization (CGH) and next Generation Sequencing (NGS) technology are changing medical genomics by accelerating new disease causing mutations discovery; these techniques could enable quick, reliable and cost-effective analysis of numerous NMD genes in parallel. The NGS methods promise to speed up the discovery of the genetic causes of diseases both in the research and the clinical setting. We performed whole exome sequencing analysis (WES) through NGS technology on a family with a Bethlem phenotype (BM) orphan of mutations in COLVI genes and a coohort of patients with a clinical diagnosis of myofibrillar myopathy (MFM). We performed the linkage analysis on BM family; the linkage regions identified were used as filters in WES output data. We selected four components (two affected and two unaffected) of this family and performed Whole Exome Sequencing by Illumina GAIIe platform obtaining a few candidate genes. Regarding the MFMs patients, we identified a large rearrangements in laminin alpha 2 (LAMA2) gene through CGH; while WES identified small variations in five patients: mutations in a known gene, and two variations in two novel genes previously unreported as involved in MFMs.
Over the years many different approaches and techniques have been employed to get insight genetic data of family and patients. The first approach for genetic studies and gene discovery was the linkage analysis, but to be efficient it required large family or large numbers of patients sharing the same disease phenotype. The advent of sequencing technology made the genetic analysis more handy but still it was time consuming and not cost effective when a large number of genes needed to be screened , for example in case of diseases with a known genetic heterogeneity as the neuromuscular disorders (NMDs). The high throughput molecular diagnostics tools such as Comparative Genomic Hybridization (CGH) and next Generation Sequencing (NGS) technology are changing medical genomics by accelerating new disease causing mutations discovery; these techniques could enable quick, reliable and cost-effective analysis of numerous NMD genes in parallel. The NGS methods promise to speed up the discovery of the genetic causes of diseases both in the research and the clinical setting. We performed whole exome sequencing analysis (WES) through NGS technology on a family with a Bethlem phenotype (BM) orphan of mutations in COLVI genes and a coohort of patients with a clinical diagnosis of myofibrillar myopathy (MFM). We performed the linkage analysis on BM family; the linkage regions identified were used as filters in WES output data. We selected four components (two affected and two unaffected) of this family and performed Whole Exome Sequencing by Illumina GAIIe platform obtaining a few candidate genes. Regarding the MFMs patients, we identified a large rearrangements in laminin alpha 2 (LAMA2) gene through CGH; while WES identified small variations in five patients: mutations in a known gene, and two variations in two novel genes previously unreported as involved in MFMs.

L’Encefalopatia Epilettica (EE) è una condizione clinica severa che causa un grave ritardo cognitivo e neurologico. Recentemente, sono state identificate mutazioni associate ad EE nei geni kcnq2 e kcnq3 che codificano rispettivamente per le subunità del potassio voltaggio-dipendenti Kv7.2 e Kv7.3. Ciascuna subunità è formata da 6 segmenti transmembrana e da un lungo dominio C-terminale a cui possono legarsi diverse molecole regolatorie quali il fostatidil-inositolo-(4,5)-bisfosfato (PIP2), un importante attivatore dei canali Kv7, e la calmodulina (CaM). I canali maturi sono formati dall’assemblaggio eteromerico delle subunità Kv7.2 e Kv7.3 e sottendono una corrente del potassio detta “corrente M” che inibisce l’eccitabilità neuronale. Lo scopo del presente lavoro è stato quello di investigare le conseguenze funzionali e la sensibilità farmacologica delle seguenti mutazioni: • Kv7.2 R325G identificata in tre soggetti con EE; • due varianti in eterozigosi composta nel gene kcnq3 riscontrate in un soggetto con EE (Kv7.3 V359L/Kv7.3 D542N) e la mutazione Kv7.2 D535N, corrispondente alla variante Kv7.3 D542N, descritta in tre casi con epilessia neonatale; • Kv7.2 G310S riscontrata in un paziente con EE. Al fine di studiare tali mutazioni cellule CHO sono state trasfettate con le subunità di interesse e le correnti espresse dalle cellule sono state registrate mediante la tecnica del patch-clamp in configurazione whole-cell. Dagli esperimenti di elettrofisiologia è emerso che i canali omomerici mutanti non sono funzionali rispetto ai controlli rappresentati dai canali Kv7.2 o Kv7.3 wild-type (wt). Al fine di riprodurre il bilancio genico dei probandi in studio, le subunità mutanti sono state espresse in configurazione eteromerica con le subunità Kv7.2 e Kv7.3 wt ed è stata osservata una significativa riduzione della densità di corrente dei canali eteromerici mutanti rispetto al canale eteromerico wt. Sulla base di questi risultati, è stato testato il farmaco attivatore retigabina che ha consentito di ripristinare, ai livelli del wt, la corrente elicitata dai canali eteromerici mutanti. Per comprendere il possibile meccanismo responsabile dell’effetto indotto dalle mutazioni è stato utilizzato un modello strutturale delle subunità Kv7.2 o Kv7.3 da cui è emerso che i residui di interesse sono localizzati in un sito di legame per il PIP2 e adiacenti al sito di legame per la CaM. Pertanto, sono stati condotti ulteriori esperimenti di elettrofisiologia utilizzando la chinasi PIP5K che incrementa i livelli di PIP2 o la fosfatasi DrVSP che ne riduce i livelli. La co-espressione della PIP5K con i canali Kv7.2 omomerici mutanti ha consentito un significativo aumento della densità di corrente. Al contrario, tale effetto non è stato osservato per i canali Kv7.3 mutanti. Gli esperimenti con la DrVSP hanno mostrato un maggiore effetto di inibizione ed una più lenta cinetica di recupero della corrente espressa dai canali eteromerici mutanti rispetto a quella misurata per il canale wt. Tali risultati suggeriscono che le mutazioni in studio alterano la regolazione della corrente M mediata dal PIP2. Un significativo recupero della funzionalità del canale è stato anche osservato dalla co-espressione dei canali Kv7.2 D535N e Kv7.2 G310S con la CaM1234 (una calmodulina mutata che non lega il calcio), suggerendo che per tali canali mutanti risulta compromessa anche la regolazione dipendente dalla calmodulina. In conclusione, le mutazioni studiate causano una completa perdita di funzione dei canali probabilmente in seguito all’alterata regolazione operata dal PIP2 e, in alcuni casi, dalla calmodulina. Infine, tali risultati forniscono un razionale per l’uso di attivatori, quali la retigabina o suoi analoghi, per il trattamento di pazienti affetti da EE e portatori di tali mutazioni.
Epileptic Encephalopathy (EE) is a severe form of epilepsy in which epileptiform activity contributes to a progressive cerebral dysfunction. Recently, mutations in the kcnq2 or kcnq3 genes have been identified in patients affected by EE. These genes encode for neuronal Kv7.2 or Kv7.3 subunits characterized by the presence of six transmembrane segments and a long C-terminus domain to which several modulatory proteins are associated, such as the phosphatidylinositol-4-5-bis-phosphate (PIP2), that is a know Kv7 activator, and the calmodulin (CaM). The heteromeric channels underlie the neuronal M current, a potassium current which inhibits neuronal excitability. The aim of this work is to study the functional consequences and the pharmacological sensitivity of Kv7.2 or Kv7.3 channels incorporating the following mutations: • Kv7.2 R325G identified in three patients affected by EE; • two mutations in the kcnq3 gene in compound heterozygosis identified in a patient affected by EE (Kv7.3 V359L/Kv7.3 D542N) and the Kv7.2 D535N corresponding to the Kv7.3 D542N variant and described in three cases with EE; • Kv7.2 G310S identified in a patient with EE. To study this mutation channel subunits were expressed in CHO cells by transient transfection. One day after transfection, we recorded the currents by whole cell patch-clamp. Patch-clamp recordings revealed that homomeric mutant channel are not functional when compared to homomeric Kv7.2 or Kv7.3 wild-type (wt) channels. To reproduce the genetic balance of EE-affected patients, mutant subunits were co-expressed with Kv7.2 or Kv7.3 wt subunits. The results obtained suggest that heteromeric mutant channels carried currents size smaller than those from heteromeric wt channels. Based on these results, we therefore tested the activator drug, called retigabine, that was able to restore, at wt levels, the currents carried by heteromeric mutant channels. To better understand the pathogenic mechanism induced by the mutations, we used a structural model in which was possible to reproduce a portion of Kv7.2 or Kv7.3 channels. The residues of our interest are involved in the binding-site of PIP2 and are near to the binding-site of CaM. To this aim, we used two experimental tools: a PIP2-synthesizing enzyme, called PIP5K, that increses PIP2 levels, or a phosphatase, like DrVSP, which reduces PIP2 levels. PIP5K co-expression with Kv7.2 homomeric mutant channels significantly increased current size. On the other end, this effect was not observed for Kv7.3 mutant channels. DrVSP experiments showed that heteromeric mutant currents is more easily inhibeted by DrVSP e more slowly recovered, when compared to heteromeric wt channels. These results suggest that the studied mutations interfere with the PIP2-dependent regulation. Furthermore, a significant current size increase was observed by the co-expression of CaM1234 (a mutated calmodulin that does not bind calcium) with Kv7.2 D535N and Kv7.2 G310S mutant channels, suggesting that these channels also interfere with CaM regulation. In conclusions, the mutations herein investigated causes a loss of function by interfering with the PIP2 regulation and, in some cases, also with the CaM regulation. Moreover, these results provide a rationale for the use of Kv7 channels activators, like retigabine or retigabine derivates, for the pharmacological treatment of patients affected by EE carrying Kv7 loss-of-function mutations.

Cluster headache (CH) and bipolar disorder (BD) are pathological conditions affecting 1% and 1.5% of the general population, respectively. Albeit the pathogenesis has not yet been completely elucidated, family and twin studies have suggested a genetic basis for both disorders, with an estimated heritability of 80% for BD and up to 60% for CH. Though BD and CH are very different diseases, they show important clinical similarities, such as a temporal pattern of disturbances, dysregulation of the wake-­‐sleep cycle, neuroendocrine derangements, and more important positive clinical response to lithium and valproate treatments in a significant proportion of treated patients. In the present study, we sought to explore whether BD and CH patients responders to lithium share common molecular pathways potentially involved in predisposing to positively respond to prophylactic lithium treatment. To this aim, we carried out a transcriptome study in lymphoblastoid cell lines from 10 BD type I patients, responders to lithium, 8 CH patients responders to lithium treatment and 10 healthy subjects (CO). Expression profiles were measured by Affymetrix GeneChip Human Gene ST 1.0 covering 36,079 transcripts. Expression levels of BD and CH patients were compared with CO using a t-­‐test, in order to identify commonly dysregulated genes. Pathway analysis was performed based on the hypergeometric test for over representation of specific Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 544 and 1172 genes were differentially expressed in BD versus CO and CH versus CO respectively. Filtering criteria were based on corrected p value < 0.05 and a Fold Change (FC) ≥ |1.3|. Among these genes, 314 were commonly altered both in CH and BD compared to CO. The most significant dysregulated gene in BD and CH was RNA binding motif (RNP1, RRM) protein 3 (RBM3), a gene implicated in sleep regulation and in the temperature entrained circadian gene expression (corrected p value of 6,30x 10-­‐09 in BD vs CO and 1,88x 10-­‐09 in CH vs CO). Pathway analysis showed that Protein processing in endoplasmic reticulum pathway was one of the most significantly enriched in BD and CH when compared to CO. In conclusion, data from this pilot microarray study may provide useful and relevant information for a better understanding of the molecular underpinnings of lithium response and on the neurobiology of BD and CH.

Recently single nucleotide polymorphisms (SNPs) of the genes NOD2-CARD15, IL23-R and TLR-4 have been showed to influence the risk for acute GvHD in patients who underwent to allogeneic hematopoietic stem cells (HSCs) transplantation. To investigate whether these genes play a role in the pathogenesis of GvHD also in the Sardinian population, 8 SNPs four for NOD2, two for TLR4 and two for IL23R in 86 recipients, their coupled donors and in 150 healthy Sardinians individualswere genotyped and the SNPs frequencies compared. The SNP rs2066842 of NOD2 gene was significantly increased in the group of patients who did not develop acute GvHD(p = 0.002). Our data, if confirmed in GvHD patients from other population, could suggest the inclusion of the non-HLA NOD2/CARD15 genes genotyping in the attribution of the immunological donor/recipient pre-transplant score.

E’ stata approfondita l’espressione genica complessiva in spighe di mais, in seguito all’ infezione fungina. Nella prima parte del lavoro, sono stati valutati un genotipo di mais resistente ed uno suscettibile a F. verticillioides, campionando le cariossidi 48 ore dopo l’infezione. Sono state identificate circa 800 sequenze differenzialmente espresse e circa il 10% è stato assegnato alla categoria della difesa. Nel genotipo resistente, i geni coinvolti nella difesa hanno mostrato un tipo di risposta basale, mentre in quello suscettibile tali geni rispondevano specificamente all’infezione. Nella seconda parte del lavoro, l’analisi di espressione è stata estesa a fasi precoci e tardive dell’infezione utilizzando un ceppo normale ed uno mutante di F. verticillioides. Numerosi geni risultavano differenzialmente regolati 48 ore dopo l’infezione con entrambi i ceppi. Il ceppo normale era in grado di attivare i meccanismi di difesa prima del mutante. Nella terza parte del lavoro, 10 linee resistenti e suscettibili sono state infettate con 4 specie fungine. In tutti i genotipi l’espressione dei geni coinvolti nella difesa era indotta in seguito all’infezione, ma le linee resistenti presentavano una risposta basale di difesa.
We investigated global gene expression in maize ears at several time points after fungal infection. In the first part of the work, resistant and susceptible genotypes were tested in kernels sampled 48 h after infection with a wild type strain of F. verticillioides. About 800 differentially expressed sequences were identified and nearly 10% assigned to the category cell rescue, defense and virulence. In the resistant genotype, defense-related genes provided basic defense against the fungus, while in the susceptible genotype defense genes responded specifically to pathogen infection. In the second part of the work the expression analysis was extended to early and late phases of infection with a wild type and a mutant strains of F. verticillioides. Kernels were sampled in the area around the point of infection. Most of genes were differentially regulated 48 h after infection with both fungal strains. The wild type strain was able to activate host defense genes before the mutant strain. In the third part of the work, ten resistant and susceptible lines were infected by different fungal species. All genotypes were able to induce the expression of defense genes upon infection, but the resistant lines showed a basal defense response.

E’ stata approfondita l’espressione genica complessiva in spighe di mais, in seguito all’ infezione fungina. Nella prima parte del lavoro, sono stati valutati un genotipo di mais resistente ed uno suscettibile a F. verticillioides, campionando le cariossidi 48 ore dopo l’infezione. Sono state identificate circa 800 sequenze differenzialmente espresse e circa il 10% è stato assegnato alla categoria della difesa. Nel genotipo resistente, i geni coinvolti nella difesa hanno mostrato un tipo di risposta basale, mentre in quello suscettibile tali geni rispondevano specificamente all’infezione. Nella seconda parte del lavoro, l’analisi di espressione è stata estesa a fasi precoci e tardive dell’infezione utilizzando un ceppo normale ed uno mutante di F. verticillioides. Numerosi geni risultavano differenzialmente regolati 48 ore dopo l’infezione con entrambi i ceppi. Il ceppo normale era in grado di attivare i meccanismi di difesa prima del mutante. Nella terza parte del lavoro, 10 linee resistenti e suscettibili sono state infettate con 4 specie fungine. In tutti i genotipi l’espressione dei geni coinvolti nella difesa era indotta in seguito all’infezione, ma le linee resistenti presentavano una risposta basale di difesa.
We investigated global gene expression in maize ears at several time points after fungal infection. In the first part of the work, resistant and susceptible genotypes were tested in kernels sampled 48 h after infection with a wild type strain of F. verticillioides. About 800 differentially expressed sequences were identified and nearly 10% assigned to the category cell rescue, defense and virulence. In the resistant genotype, defense-related genes provided basic defense against the fungus, while in the susceptible genotype defense genes responded specifically to pathogen infection. In the second part of the work the expression analysis was extended to early and late phases of infection with a wild type and a mutant strains of F. verticillioides. Kernels were sampled in the area around the point of infection. Most of genes were differentially regulated 48 h after infection with both fungal strains. The wild type strain was able to activate host defense genes before the mutant strain. In the third part of the work, ten resistant and susceptible lines were infected by different fungal species. All genotypes were able to induce the expression of defense genes upon infection, but the resistant lines showed a basal defense response.