Forme congenite di malattia di von Willebrand senza mutazioni nel gene del fattore von Willebrand: alla ricerca di un nuovo gene e di nuovi meccanismi patogenetici

The von Willebrand factor (VWF) is a multimeric glycoprotein that plays a key role in the early stages of hemostasis. Quantitative or functional defects of the molecule cause von Willebrand disease (VWD), the most common inherited bleeding disorder. Currently, VWF is the only gene involved in the VWD onset. However, VWF mutations are not identified in a substantial proportion of VWD cases (25-30%) by using the standard sequencing techniques. In this PhD work we elaborated a diagnostic flow-chart for the systematic study of families with no identified VWF gene mutations. The flow-chart core is the accomplishment of a linkage analysis with VNTR (variable number tandem repeat) markers within the VWF gene to assess the involvement of the gene itself: if the VNTR haplotype and the disease co-segregate, the investigation of VWF gene must be carried out with the analysis of mRNA or Real-Time PCR; otherwise, the VWF gene involvement is excluded and a whole-genome investigation will be performed to discover a new causative gene. In A Family, VNTR markers co-segregated with the disease and VWF mRNA analysis highlighted an aberrant splicing caused by a synonymous substitution (c.7056C>T). This substitution, which has been described previously, was incorrectly classified as a polymorphism. On the basis of expression experiments performed in BHK cell line, it was also possible to point out the typical loss-of-function mechanism of the mutation. The mRNA analysis allowed to identify the mutation responsible for VWD in B Family: a genomic deletion of 3411 base pairs involving three exons of the VWF gene (c.5456_5842del). In this case, the transient expression in HEK293T cell line showed the mutation has a dominant-negative effect. Furthermore, recombinant fragments of VWF comprising A2-B3 domains were synthesized in E.coli to better characterize the effect of the deletion. These fragments were subjected to in vitro proteolysis with recombinant ADAMTS13, in order to verify the susceptibility of mutated VWF to the metalloprotease. These experiments showed that the mutation identified makes VWF resistant to the proteolytic action of ADAMTS13, thus explaining the presence of abnormally large multimers observed in the patient’s plasma. In the C Family, linkage analysis excluded the involvement of VWF gene in the VWD onset. Then, the FVIII gene was also ruled out by direct sequencing, so the search for a new causative gene was carried out. We used two different strategies simultaneously: the whole-genome sequencing and the genome-wide linkage analysis. Data derived from the two strategies were integrated and the results allowed us to identify ~900 variations in 396 genes that were spread across 4 different linkage regions (chromosome 5, 7, 15, X). The involvement of the "candidate" genes in the linkage regions was assessed in detail, but it has not been possible to identify the one responsible for the disease so far. To complete the analysis of this Family C a new sequencing step is required. The D Family, whose small size made impossible to perform the linkage analysis, is characterized by a reduction of circulating VWF survival. The evidence that the exogenous hemoderivative VWF had the same behavior ruled out the involvement of the VWF gene. The intervention of the metalloprotease ADAMTS13 was excluded by direct sequencing, while the search for anti-VWF antibody, in a context of autoimmune disease, was negative. In conclusion, the use of the proposed flow-chart allowed the characterization of two new mutations in the VWF gene, responsible for VWD in Families A and B. It should be emphasized that both mutations described can’t be detected with standard sequencing techniques, due to inherent limitations, while the mRNA sequencing suggested in the flow-chart allowed the characterization of two unresolved cases of VWD. In families C and D, the study has definitively excluded the involvement of the VWF gene, confirming the existence of other factors/genes that may cause the VWD

Das tragédias alemãs às Crônicas de Nárnia

Trabalho de Conclusão de Curso (Graduação)No presente trabalho pretendo, primeiramente, explicar o que Nietzsche, Hegel e Schopenhauer pensam sobre a arte e a tragédia. Eles, seguindo Winckelmann, leem os gregos para a construção da identidade alemã ainda no século XIX. Assim como Kant, eles usam da arte para justificar suas posições filosóficas, mas diferente de Kant, não defendem que a razão é o suficiente para se conhecer a realidade. Também analisam a tragédia (grega e moderna) sob o pressuposto de que ela é capaz de comunicar como é a realidade. Isso gera consequências na Alemanha e na Europa no século XX, tais como o Nazismo, a relativização de valores morais e o surgimento da arte moderna, que não tem preocupações com técnicas ou beleza, mas apenas em fazer os horrores do mundo se tornarem aceitáveis. C.S. Lewis cresce com essa visão de mundo, mas se converte ao Cristianismo e passa a publicar diversas obras sobre os mais variados assuntos (ética, religiões, julgamentos, tradição, educação, literatura) como uma forma de combater as ideias dos alemães e a cultura que deixaram que, para ele, vão levar a abolição da humanidade. Ao mesmo tempo, como professor de literatura em Oxford e posteriormente em Cambridge, ele escreve as Crônicas de Nárnia, já velho e mais maduro e coloca muitas de suas ideias neste livro de fantasia infantil. E entre os seus propósitos, visa educar as crianças através de suas referências cristãs, literárias e históricas

The intermediate-age open cluster NGC 2112

We report on $BVI$ CCD photometry of a field centered on the region of the intermediate-age open cluster NGC 2112 down to V=21. Due to the smaller field coverage, we are able to limit the effect of field star contamination which hampered in the past precise determinations of the cluster age and distance. This way, we provide updated estimates of NGC 2112 fundamental parameters. Having extended the photometry to the $I$ pass-band, we are able to construct a colour-colour diagram, from which we infer a reddening $E_{B-V}= 0.63\pm0.14$ mag. The comparison of the Colour-Magnitude Diagram (CMD) with theoretical isochrones leads to a distance of $850 \pm 100$ pc, and an age of $2.0 \pm 0.3$ Gyr. While the distance is in agreement with previous determinations, the age turns out to be much better constrained and significantly lower than previous estimates.Comment: 7 pages, 7 eps figures, in press in MNRA

Forme congenite di malattia di von Willebrand senza mutazioni nel gene del fattore von Willebrand: alla ricerca di un nuovo gene e di nuovi meccanismi patogenetici

The von Willebrand factor (VWF) is a multimeric glycoprotein that plays a key role in the early stages of hemostasis. Quantitative or functional defects of the molecule cause von Willebrand disease (VWD), the most common inherited bleeding disorder. Currently, VWF is the only gene involved in the VWD onset. However, VWF mutations are not identified in a substantial proportion of VWD cases (25-30%) by using the standard sequencing techniques. In this PhD work we elaborated a diagnostic flow-chart for the systematic study of families with no identified VWF gene mutations. The flow-chart core is the accomplishment of a linkage analysis with VNTR (variable number tandem repeat) markers within the VWF gene to assess the involvement of the gene itself: if the VNTR haplotype and the disease co-segregate, the investigation of VWF gene must be carried out with the analysis of mRNA or Real-Time PCR; otherwise, the VWF gene involvement is excluded and a whole-genome investigation will be performed to discover a new causative gene. In A Family, VNTR markers co-segregated with the disease and VWF mRNA analysis highlighted an aberrant splicing caused by a synonymous substitution (c.7056C>T). This substitution, which has been described previously, was incorrectly classified as a polymorphism. On the basis of expression experiments performed in BHK cell line, it was also possible to point out the typical loss-of-function mechanism of the mutation. The mRNA analysis allowed to identify the mutation responsible for VWD in B Family: a genomic deletion of 3411 base pairs involving three exons of the VWF gene (c.5456_5842del). In this case, the transient expression in HEK293T cell line showed the mutation has a dominant-negative effect. Furthermore, recombinant fragments of VWF comprising A2-B3 domains were synthesized in E.coli to better characterize the effect of the deletion. These fragments were subjected to in vitro proteolysis with recombinant ADAMTS13, in order to verify the susceptibility of mutated VWF to the metalloprotease. These experiments showed that the mutation identified makes VWF resistant to the proteolytic action of ADAMTS13, thus explaining the presence of abnormally large multimers observed in the patient’s plasma. In the C Family, linkage analysis excluded the involvement of VWF gene in the VWD onset. Then, the FVIII gene was also ruled out by direct sequencing, so the search for a new causative gene was carried out. We used two different strategies simultaneously: the whole-genome sequencing and the genome-wide linkage analysis. Data derived from the two strategies were integrated and the results allowed us to identify ~900 variations in 396 genes that were spread across 4 different linkage regions (chromosome 5, 7, 15, X). The involvement of the "candidate" genes in the linkage regions was assessed in detail, but it has not been possible to identify the one responsible for the disease so far. To complete the analysis of this Family C a new sequencing step is required. The D Family, whose small size made impossible to perform the linkage analysis, is characterized by a reduction of circulating VWF survival. The evidence that the exogenous hemoderivative VWF had the same behavior ruled out the involvement of the VWF gene. The intervention of the metalloprotease ADAMTS13 was excluded by direct sequencing, while the search for anti-VWF antibody, in a context of autoimmune disease, was negative. In conclusion, the use of the proposed flow-chart allowed the characterization of two new mutations in the VWF gene, responsible for VWD in Families A and B. It should be emphasized that both mutations described can’t be detected with standard sequencing techniques, due to inherent limitations, while the mRNA sequencing suggested in the flow-chart allowed the characterization of two unresolved cases of VWD. In families C and D, the study has definitively excluded the involvement of the VWF gene, confirming the existence of other factors/genes that may cause the VWD .Il fattore von Willebrand (VWF) è una glicoproteina multimerica che svolge una funzione fondamentale nelle prime fasi dell’emostasi. Alterazioni quantitative o funzionali della molecola causano la malattia di von Willebrand (VWD), la più frequente malattia emorragica ereditaria. L’unico gene a tutt’oggi riconosciuto come responsabile della VWD è il gene del VWF. Tuttavia in una percentuale rilevante di casi di VWD (25-30%) non sono identificate mutazioni a carico del gene VWF. In questo lavoro di Dottorato è stata elaborata una flow-chart diagnostica per lo studio sistematico di famiglie con VWD che non presentano mutazioni nel gene VWF. Il punto focale della flow-chart è l’utilizzo dell’analisi di linkage con marcatori VNTR (variable number tandem repeat) interni al gene del VWF, per valutare il coinvolgimento del gene stesso: se aplotipo di VNTR e malattia co-segregano l’indagine del gene VWF deve essere approfondita con l’analisi del mRNA o con la Real-Time PCR, in caso contrario viene proposta un’indagine estesa a tutto il genoma per la ricerca di un nuovo gene causativo. Nella prima famiglia studiata (Famiglia A), i marker VNTR segregavano con la malattia e l’analisi del mRNA del VWF ha permesso di evidenziare uno splicing aberrante causato da una sostituzione sinonimo (c.7056C>T), già erroneamente classificata come polimorfismo. L’espressione in vitro del VWF mutato, condotta in linea cellulare BHK, ha permesso di dimostrare che la mutazione agisce con un tipico meccanismo di loss-of-function. L’analisi del mRNA si è rivelata utile anche per identificare la mutazione responsabile della VWD nella seconda famiglia (Famiglia B): una delezione genomica di 3411 paia di basi che coinvolge tre esoni del gene VWF (c.5456_5842del). In questo caso, l’espressione transiente in sistema cellulare HEK293T ha evidenziato un effetto dominante-negativo della mutazione nel rilascio del VWF. Lo studio funzionale è stato invece condotto attraverso l’espressione, in batteri E.coli, di frammenti ricombinanti di VWF mutato e normale (A2-B3). Questi frammenti sono stati sottoposti in vitro all’azione di ADAMTS13, una metalloproteasi che ha come unico substrato il VWF di cui regola la struttura multimerica. Questi esperimenti hanno dimostrato che la mutazione c.5456_5842del rende il VWF resistente all’azione proteolitica di ADAMTS13, offrendo così una spiegazione alla presenza di multimeri a peso molecolare abnormemente elevato in pazienti portatori di questa mutazione. Nella terza famiglia (Famiglia C) l’analisi di linkage ha escluso il coinvolgimento del gene VWF nell’insorgenza della VWD. Successivamente mediante sequenziamento diretto, è stato escluso anche un possibile ruolo per il gene del FVIII ed è stata quindi effettuata la ricerca di un nuovo gene causativo di VWD, utilizzando simultaneamente due diversi approcci: il sequenziamento dell’intero genoma e l’analisi di linkage familiare genome-wide. I dati derivati dalle due strategie sono stati integrati e il risultato ci ha permesso di identificare ~900 variazioni a carico di 396 geni distribuiti su 4 diverse regioni di linkage (cromosoma 5, 7, 15, X). Il contributo dei geni “candidati” presenti nelle regioni di linkage è stato valutato in maniera approfondita ma fino a questo momento non è stato possibile identificare quello responsabile della patologia emorragica. Il completamento dell’analisi di questa famiglia richiede una nuova fase di sequenziamento. La quarta e ultima famiglia (Famiglia D), le cui ridotte dimensioni non hanno consentito di eseguire l’analisi di linkage, è caratterizzata da una riduzione della sopravvivenza del VWF circolante. L’evidenza che anche il VWF esogeno (da emoderivato) presentava una ridotta sopravvivenza, ha escluso il coinvolgimento del gene VWF. Il sequenziamento genico ha permesso anche di escludere il coinvolgimento dell’ADAMTS13, così come la ricerca di anticorpi anti-VWF, in un quadro di malattia autoimmune, ha dato esito negativo. In conclusione, l’utilizzo della flow-chart proposta ha permesso di caratterizzare due nuove mutazioni sul gene VWF, responsabili per le forme di VWD delle Famiglie A e B. È opportuno sottolineare che entrambe le mutazioni descritte non potevano essere rilevate con il sequenziamento standard per limiti intrinseci della tecnica, mentre il sequenziamento del mRNA suggerito dalla flow-chart ha permesso la caratterizzazione di due casi irrisolti di VWD. Nelle famiglie C e D lo studio effettuato ha invece escluso definitivamente il coinvolgimento del gene VWF, confermando l’esistenza di altri fattori/geni responsabili di VWD

UBV(RI)_C photometry and spectroscopy of the young open cluster Haffner 18

Accepted....... Received.......; in original form.....

ARMC5 mutation analysis in patients with primary aldosteronism and bilateral adrenal lesions

Idiopathic hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia is the most common subtype of primary aldosteronism (PA). The pathogenesis of IHA is still unknown, but the bilateral disease suggests a potential predisposing genetic alteration. Heterozygous germline mutations of armadillo repeat containing 5 (ARMC5) have been shown to be associated with hypercortisolism due to sporadic primary bilateral macronodular adrenal hyperplasia and are also observed in African-American PA patients. We investigated the presence of germline ARMC5 mutations in a group of PA patients who had bilateral computed tomography-detectable adrenal alterations. We sequenced the entire coding region of ARMC5 and all intron/exon boundaries in 39 patients (37 Caucasians and 2 black Africans) with confirmed PA (8 unilateral, 27 bilateral and 4 undetermined subtype) and bilateral adrenal lesions. We identified 11 common variants, 5 rare variants with a minor allele frequency o1% and 2 new variants not previously reported in public databases. We did not detect by in silico analysis any ARMC5 sequence variations that were predicted to alter protein function. In conclusion, ARMC5 mutations are not present in a fairly large series of Caucasian patients with PA associated to bilateral adrenal disease. Further studies are required to definitively clarify the role of ARMC5 in the pathogenesis of adrenal nodules and aldosterone excess in patients with PA

Novel mutations in the L1CAM gene support the complexity of L1 syndrome

X-linked hydrocephalus, MASA syndrome, X-linked complicated Spastic Paraplegia Type I and X-linked partial agenesis of the corpus callosum are the four rare diseases usually referred to L1 syndrome, caused by mutations in the L1CAM gene. By direct sequencing of L1CAM in 16 patients, we were able to identify seven mutations, five of which were never described before. Patients' phenotype evaluation revealed a correlation between the number of clinical features typical of L1 syndrome and the chance to find causative mutation. Our findings support that L1CAM mutations are associated with widely heterogeneous phenotypes, however the occurrence of several clinical features remains the best criterion for planning molecular testing both in familial and apparently sporadic cases