14-3-3θ, AF4 and MLL-AF4 are targets of MiR-27a in t(4;11) acute lymphoblastic leukemia: implication for new targeted therapy options

The t(4;11)(q21;q23) chromosomal translocation results in the KMT2A/AFF1 fusion gene, which encodes the mixed-lineage leukemia (MLL)-AF4 oncogenic chimera, a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL). In t(4;11) ALL, MLL-AF4 recruits the endogenous co-activator AF4 and aberrantly triggers transcription of MLL target genes, including HOXA9 and MEIS1, thereby driving transformation of hematopoietic progenitors. We previously demonstrated that the scaffold protein 14-3-3θ is a direct interactor of AF4 and promotes AF4 binding to the HOXA9 promoter. Notably, 14-3-3θ is a target of MiR-27a, which acts as tumor suppressor in various human leukemias; moreover, expression levels of MiR-27a correlate with relapse free survival in childhood ALL. This PhD thesis aims to assess the potential role of MiR-27a in t(4;11) ALL pathogenesis. In different leukemia cell lines, we found an inverse correlation between levels of MiR-27a and 14-3-3θ, which was particularly relevant in t(4;11) cell lines. In t(4;11) leukemia cells, transient transfection of MiR-27a led to a decrease in 14-3-3θ protein amount and HOXA9, HOXA7 and MEIS1 transcription. Interestingly, our bioinformatic analysis predicted that AFF1-3’UTR, which is shared with KMT2A/AFF1, contains a putative MiR-27a seed sequence. Consistently, MiR-27a overexpression strongly reduced AF4 and MLL-AF4 protein levels, as well as protein level of RUNX1, a known target of MiR-27a with a key role in t(4;11) leukemia context. We therefore cloned AFF1-3’-UTR in an opportune plasmid vector and performed a luciferase reporter assay. The decreased luciferase activity we measured after co-transfection of MiR-27a and the recombinant plasmid proved that AFF1-3’UTR is a direct target of MiR-27a. Accordingly, transfection of anti-MiR-27a enhanced the expression of AF4 protein, in RS4;11 cells. Moreover, ChIP experiments gave direct proof that MiR-27a overexpression impaired MLL-AF4 binding to HOXA9 promoter. Consistently, MiR-27a overexpression decreased viability, proliferation and clonogenicity of t(4;11) cells, whereas enhanced their apoptotic rate. Lastly, we found that relative expression of MiR-27a was significantly lower in 9 patients affected by severe t(4;11) ALL compared with 9 patients affected by t(12;21) leukemia, which has a benign prognosis. Similarly to the t(4;11) cell lines, in our ALL patients we found an inverse relationship between MiR-27a and 14-3-3θ transcript levels. Overall, we demonstrate that MiR-27a has a pivotal role in t(4;11) ALL molecular pathogenesis and therefore it is a promising novel target for the therapy of this aggressive form of leukemia

En el centenario del premio Nobel. Golgi / Cajal (1906)

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The position of nonsense mutations can predict the phenotype severity : A survey on the DMD gene

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used theDMDgene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, theDMDgene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, becauseDMDmutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.Peer reviewe

Mystery(n) Phenotypic Presentation in Europeans: Report of Three Further Novel Missense RNF213 Variants Leading to Severe Syndromic Forms of Moyamoya Angiopathy and Literature Review

Moyamoya angiopathy (MMA) is a rare cerebral vasculopathy in some cases occurring in children. Incidence is higher in East Asia, where the heterozygous p.Arg4810Lys variant in RNF213 (Mysterin) represents the major susceptibility factor. Rare variants in RNF213 have also been found in European MMA patients with incomplete penetrance and are today a recognized susceptibility factor for other cardiovascular disorders, from extracerebral artery stenosis to hypertension. By whole exome sequencing, we identified three rare and previously unreported missense variants of RNF213 in three children with early onset of bilateral MMA, and subsequently extended clinical and radiological investigations to their carrier relatives. Substitutions all involved highly conserved residues clustered in the C-terminal region of RNF213, mainly in the E3 ligase domain. Probands showed a de novo occurring variant, p.Phe4120Leu (family A), a maternally inherited heterozygous variant, p.Ser4118Cys (family B), and a novel heterozygous variant, p.Glu4867Lys, inherited from the mother, in whom it occurred de novo (family C). Patients from families A and C experienced transient hypertransaminasemia and stenosis of extracerebral arteries. Bilateral MMA was present in the proband's carrier grandfather from family B. The proband from family C and her carrier mother both exhibited annular figurate erythema. Our data confirm that rare heterozygous variants in RNF213 cause MMA in Europeans as well as in East Asian populations, suggesting that substitutions close to positions 4118-4122 and 4867 of RNF213 could lead to a syndromic form of MMA showing elevated aminotransferases and extracerebral vascular involvement, with the possible association of peculiar skin manifestations

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications

PPFIBP1 encodes for the liprin-β1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications

A multi-element psychosocial intervention for early psychosis (GET UP PIANO TRIAL) conducted in a catchment area of 10 million inhabitants: study protocol for a pragmatic cluster randomized controlled trial

Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services

Momenti varesini nella vita di Camillo Golgi

Documenti d'archivio varesini relativi a Golgi e le sue relazioni con famiglie del luog