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San Donato Milanese, Italy

Cardani R.,Laboratory of Muscle Histopathology and Molecular Biology | Bugiardini E.,University of Milan | Renna L.V.,University of Milan | Rossi G.,University of Rome Tor Vergata | And 6 more authors.
PLoS ONE | Year: 2013

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested. © 2013 Cardani et al.

Renna L.V.,University of Milan | Cardani R.,Laboratory of Muscle Histopathology and Molecular Biology | Botta A.,University of Rome Tor Vergata | Rossi G.,University of Rome Tor Vergata | And 5 more authors.
European Journal of Histochemistry | Year: 2014

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic disorders linked to two different genetic loci and characterized by several features including myotonia, muscle weakness and atrophy, cardiac dysfunctions, cataracts and insulin-resistance. In both forms, expanded nucleotide sequences cause the accumulation of mutant transcripts in the nucleus deregulating the activity of some RNAbinding proteins and providing an explanation for the multisystemic phenotype of DM patients. However this pathogenetic mechanism does not explain some histopathological features of DM skeletal muscle like muscle atrophy. It has been observed that DM muscle shares similarities with the ageing muscle, where the progressive muscle weakness and atrophy is accompanied by a lower regenerative capacity possibly due to the failure in satellite cells activation. The aim of our study is to investigate if DM2 satellite cell derived myoblasts exhibit a premature senescence as reported for DM1 and if alterations in their proliferation potential and differentiation capabilities might contribute to some of the histopathological features observed in DM2 muscles. Our results indicate that DM myoblasts have lower proliferative capability than control myoblasts and reach in vitro senescence earlier than controls. Differentely from DM1, the p16 pathway is not responsible for the premature growth arrest observed in DM2 myoblasts which stop dividing with telomeres shorter than controls. During in vitro senescence, a progressive decrease in fusion index is observable in both DM and control myotubes with no significant differences between groups. Moreover, myotubes obtained from senescent myoblasts appear to be smaller than those from young myoblasts. Taken together, our data indicate a possible role of DM2 premature myoblast senescence in skeletal muscle histopathological alterations i.e., dystrophic changes and type 2 fibre atrophy. © L.V. Renna et al., 2014.

Valaperta R.,Research Laboratories Molecular Biology | Rizzo V.,University of Pavia | Lombardi F.,Research Laboratories Molecular Biology | Verdelli C.,Research Laboratories Molecular Biology | And 9 more authors.
BMC Nephrology | Year: 2014

Background: Adenine phosphoribosyltransferase deficiency (APRTD) is an under estimated genetic form of kidney stones and/or kidney failure, characterized by intratubular precipitation of 2,8-dihydroxyadenine crystals (2,8-DHA). Currently, five pathologic allelic variants have been identified as responsible of the complete inactivation of APRT protein. Case presentation. In this study, we report a novel nonsense mutation of the APRT gene from a 47- year old Italian patient. The mutation, localized in the exon 5, leads to the replacement of a cytosine with a thymine (g.2098C > T), introducing a stop codon at amino acid position 147 (p.Gln147X).This early termination was deleterious for the enzyme structural and functional integrity, as demonstrated by the structure analysis and the activity assay of the mutant APRT protein. Conclusion: These data revealed that the p.Gln147X mutation in APRT gene might be a new cause of APRT disease. © 2014 Valaperta et al.; licensee BioMed Central Ltd.

Valaperta R.,Research Laboratories Molecular Biology | Sansone V.,Stroke Unit and Center for Neuromuscular Disease | Lombardi F.,Research Laboratories Molecular Biology | Verdelli C.,Research Laboratories Molecular Biology | And 6 more authors.
BioMed Research International | Year: 2013

The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, "Myotonic Dystrophy SB kit," to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%). Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the "classical" form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results. © 2013 Rea Valaperta et al.

Lombardi F.,Research Laboratories Molecular Biology | Gaia P.,Clinical Microbiology Laboratory | Valaperta R.,Research Laboratories Molecular Biology | Cornetta M.,Clinical Microbiology Laboratory | And 8 more authors.
BioMed Research International | Year: 2015

Frequent use of carbapenems has contributed to the increase to K. pneumoniae strains resistant to this class of antibiotics (CRKP), causing a problem in the clinical treatment of patients. This investigation reports the epidemiology, genetic diversity, and clinical implication of the resistance to drugs mediated by CRKP in our hospital. A total of 280 K. pneumoniae strains were collected; in particular 98/280 (35%) were CRKP. Sequencing analysis of CRKP isolated strains showed that 9/98 of MBL-producing strains carried the blaVIM-1 gene and 89/98 of the isolates were positive for blaKPC-2. Antimicrobial susceptibility tests revealed a complete resistance to third-generation cephalosporins and a moderate resistance to tigecycline, gentamicin, and fluoroquinolones with percentages of resistance of 61%, 64%, and 98%, respectively. A resistance of 31% was shown towards trimethoprim-sulfamethoxazole. Colistin was the most active agent against CRKP with 99% of susceptibility. Clonality was evaluated by PFGE and MLST: MLST showed the same clonal type, ST258, while PFGE analysis indicated the presence of a major clone, namely, pulsotype A. This finding indicates that the prevalent resistant isolates were genetically related, suggesting that the spread of these genes could be due to clonal dissemination as well as to genetic exchange between different clones. © 2015 Fortunata Lombardi et al.

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