Utilizzo di minigeni ibridi per la validazione di mutazioni di splicing

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for “unclassified variant”) found in genetic screenings or patients represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. The most direct approach to determine whether disease-causing mutations are associated with splicing is to perform a reverse transcription PCR (RT-PCR) analysis on RNA from the relevant tissues of affected individuals. However, tissue samples are often not available because the expression of these genes are tissue specific (for example only in brain or heart) or patients are too far from laboratory. An alternative approach is to test the effects of the splicing mutations using minigenes. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patients genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the mutated constructs are compared by reverse transcription-PCR analysis and sequencing. With classical minigenes is possible have difficulties to test mutations because is difficult clone the fragment that contains this mutation in the minigene. Many times the DNA fragment containing restriction sites incompatible with restriction sites of minigene, or the ligation reaction between the fragment and minigene is hardly for the size of the fragment. For bypass this problem we cloned a Gateway cassette that allow fast and easy cloning. Whit this assay we tested 16 various splicing mutation in different genes (NF1, CFTR, AIP, COQ6, STK11) with conclusive result

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients

Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with sco2 mutations

Background: Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients’ cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. Methods: We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. Results: Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 μM BZF, and peaked at 400 μM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 μM CuCl2 and 200 μM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. Conclusions: These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined

Utilizzo di minigeni ibridi per la validazione di mutazioni di splicing

The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for “unclassified variant”) found in genetic screenings or patients represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. The most direct approach to determine whether disease-causing mutations are associated with splicing is to perform a reverse transcription PCR (RT-PCR) analysis on RNA from the relevant tissues of affected individuals. However, tissue samples are often not available because the expression of these genes are tissue specific (for example only in brain or heart) or patients are too far from laboratory. An alternative approach is to test the effects of the splicing mutations using minigenes. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patients genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the mutated constructs are compared by reverse transcription-PCR analysis and sequencing. With classical minigenes is possible have difficulties to test mutations because is difficult clone the fragment that contains this mutation in the minigene. Many times the DNA fragment containing restriction sites incompatible with restriction sites of minigene, or the ligation reaction between the fragment and minigene is hardly for the size of the fragment. For bypass this problem we cloned a Gateway cassette that allow fast and easy cloning. Whit this assay we tested 16 various splicing mutation in different genes (NF1, CFTR, AIP, COQ6, STK11) with conclusive resultsL’ interpretazione di numerose varianti di sequenza di significato biologico e clinico sconosciuto (UV) trovate in screening di popolazione o pazienti rappresenta una sfida nella diagnosi molecolare delle malattie genetiche, inclusa la predisposizione al cancro. Una parte di UV potrebbe avere effetto deleterio perchè possono interessare lo splicing dell’mRNA. L’approccio diretto per determinare se la malattia è associata alle mutazioni di splicing è attuare un’analisi dell’mRNA dei tessuti dei pazienti affetti dalla patologia tramite retrotrascrizione (RT-PCR). Tuttavia, i campioni di tessuto non sono sempre disponibili perchè l’espressione di questi geni possono essere tessuto-specifica (per esempio in cervello o cuore) o i pazienti sono troppo lontani dal laboratorio di analisi. Un approccio alternativo è testare la mutazione di splicing con l’uso dei minigeni. Qui, descriviamo un metodo funzionale di studio di splicing basato sull’utilizzo di minigeni che valutano l’impatto sullo splicing di varianti di sequenza. Un segmento genomico che comprende la variante di splicing di interesse comprese le zone introniche fiancheggianti, amplificata dal paziente mediante PCR è clonata nel minigene. Dopo una trasfezione transiente in culture cellulari umane, il pattern dei trascritti generati dal wild-type e dal mutato sono confrontati mediante PCR da retrotrascrizione e successivo sequenziamento. Con l’utilizzo di minigeni classici è possibile incontrare delle difficoltà in quanto si possono trovare ostacoli nel clonaggio del frammento che contiene la mutazione nel minigene. Molte volte il frammento di DNA contiene dei siti di restrizione incompatibili con quelli del minigene, o la reazione di ligazione può essere complicata data la grandezza dell’inserto. Per superare questi problemi abbiamo clonato una cassetta Gateway per permettere un veloce e facile clonaggio. Con questo metodo abbiamo testato 16 mutazioni in differenti geni (NF1, CFTR, AIP, COQ6, STK11) con risultati conclusiv

Il bezafibrato è in grado di ripristinare la normale attività della citocromo c ossidasi in fibroblasti di paziente con mutazione in SCO2

Somministrazione di bezafibrato a fibroblasti con mutazione in SCO2 per valutare il ripristino dell'attività della citocromo C ossidasi. Associazione di bezafibrato e CuCl2 per osservare se c'è il completo ripristino dell'attività della COX. Saggi biochimici e western blot. Osservazione dell'espressione genica dei geni coinvolti nella catena respiratoria

Prevalence of AIP mutations in a large series of sporadic Italian acromegalic patients and evaluation of CDKN1B status in acromegalic patients with multiple endocrine neoplasia.

BACKGROUND: Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene and the p27(KIP1) encoding gene CDKN1B have been associated with two well-defined hereditary conditions, familial isolated pituitary adenoma (FIPA) and multiple endocrine neoplasia type 4 (MEN4). Somatotropinomas are present in most AIP mutated FIPA kindreds, as well as in two-thirds of MEN4 patients who carry pituitary tumors. METHODS: Germline DNA samples of 131 Italian sporadic acromegalic patients including 38 individuals with multiple tumors, and of six FIPA families (four homogeneous for prolactinomas and two heterogeneous with prolactin/nonfunctioning pituitary adenomas) were collected in a multicentric collaborative study. The prevalence of AIP and CDKN1B gene point mutations and copy number variations were evaluated. RESULTS: Two novel (IVS3+1G>A and c.871G>A) and one previously described (c.911G>A) AIP mutations were detected in four apparently sporadic cases (3.1%) with relatively high age at diagnosis (49+/-18, range 30-67). No mutations/rearrangements were detected in FIPA families. The highly conserved c.871G>A substitution was detected in a patient who also carried a MEN1 mutation suggesting that she is a double heterozygote. The possible pathogenic effect on AIP splicing of the silent substitution c.144G>A found in another patient was ruled out using a minigene-based approach. CDKN1B mutations/rearrangements were neither identified in patients with multiple neoplasia nor in FIPA families. CONCLUSION: AIP is mutated in about 3% of apparently sporadic acromegalic patients. The relatively high age at diagnosis, as well as its sporadic presentation, suggests that these patients are carriers of mutations with reduced pathogenicity. p27(KIP1) is unlikely to represent the common unifying nonendocrine etiology for acromegaly and cancer

Synthesis of an Original Oxygenated Taxuspine X Analogue: a Versatile “Non-Natural” Natural Product with Remarkable P-gp Modulating Activity

An efficient synthetic strategy has been developed to prepare an oxygenated analog of Taxuspine X. Macrocycle formation through Yamaguchi macrolactonization approach gave access to an original compound (18) showing remarkable P-gp modulating activity. Further functionalization of this versatile scaffold could lead to potential anticancer and/or MDR reversing agents

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA

Copper and bezafibrate cooperate to rescue cytochrome <it>c</it> oxidase deficiency in cells of patients with <it>sco2</it> mutations

Abstract Background Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients’ cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. Methods We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. Results Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 μM BZF, and peaked at 400 μM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 μM CuCl2 and 200 μM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. Conclusions These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.</p