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Molecular analysis of NPC1 and NPC2 gene in 34 Niemann–Pick C Italian Patients: identification and structural modeling of novel mutations

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Abstract

Niemann–Pick C, the autosomal recessive neuro-visceral disease resulting from a failure of cholesterol trafficking within the endosomal–lysosomal pathway, is due to mutations in NPC1 or NPC2 genes. We characterized 34 unrelated patients including 32 patients with mutations in NPC1 gene and two patients in NPC2 gene. Overall, 33 distinct genotypes were encountered. Among the 21 unpublished NPC1 alleles, 15 were due to point mutations resulting in 13 codon replacements (p.C100S, p.P237L, p.R389L, p.L472H, p.Y634C, p.S636F, p.V780G, p.Q921P, p.Y1019C, p.R1077Q, p.L1102F, p.A1187V, and p.L1191F) and in two premature stop codons (p.R934X and p.Q447X); a new mutant carried two in cis mutations, p.[L648H;M1142T] and four other NPC1 alleles were small deletions/insertions leading both to frame shifts and premature protein truncations (p.C31WfsX26, p.F284LfsX26, p.E1188fsX54, and p.T1205NfsX53). Finally, the new intronic c.464-2A>C change at the 3′ acceptor splice site of intron 4 affected NPC1 messenger RNA processing. We also found a new NPC2 mutant caused by a change of the first codon (p.M1L). The novel missense mutations were further investigated by two bioinformatics approaches. Panther proein classification system computationally predicted the detrimental effect of all new missense mutations occurring at evolutionary conserved positions. The other bioinformatics approach was based on prediction of structural alterations induced by missense mutations on the NPC1 atomic models. The in silico analysis predicted protein malfunctioning and/or local folding alteration for most missense mutations. Moreover, the effects of the missense mutations (p.Y634C, p.S636F, p.L648H, and p.V780G) affecting the sterol-sensing domain (SSD) were evaluated by docking simulation between the atomic coordinates of SSD model and cholesterol.

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Acknowledgments

The samples were obtained from the “Cell Line and DNA Biobank from Patients Affected by Genetic Diseases” (G. Gaslini Institute)—Telethon Genetic Biobank Network (project no. GTB07001A). The study was supported by a grant from Fondazione Cassa di Risparmio di Modena to P.T. and by the Italian Association of Niemann–Pick. We also thank Dr. A. Donati (Meyer Hospital, Florence, Italy), Dr. O. Gabrielli and Dr. A. Ficcadenti (Department of Pediatrics, University of Ancona, Italy), Dr. M. Spada for their contribution of clinical information and patient samples, and Sarah Tripepi (Winteringham, BA MCIL), for assistance in manuscript revision.

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Authors and Affiliations

  1. Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Modena, Italy

    Tatiana Fancello, Patrizia Tarugi, Elisa Pinotti & Sebastiano Calandra

  2. Unità di Malattie Metaboliche, IRCCS Burlo Garofolo, Trieste, Italy

    Andrea Dardis, Stefania Zampieri & Bruno Bembi

  3. Bioinformatica, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy

    Camillo Rosano

  4. Laboratorio Diagnosi Pre-Postnatale Malattie Metaboliche, IRCCS G. Gaslini, Largo G. Gaslini 5, 16147, Genoa, Italy

    Barbara Tappino, Fabio Corsolini & Mirella Filocamo

  5. Dipartimento di Pediatria, Università Federico II” di Napoli, Naples, Italy

    Simona Fecarotta

  6. Unità di Medicina Molecolare, IRCCS Bambino Gesù, Rome, Italy

    Adele D’Amico

  7. U.O. Pediatria II, IRCCS G. Gaslini, Genoa, Italy

    Maja Di Rocco

  8. Dipartimento di Neuroscienze Pediatriche, IRCCS Istituto Neurologico “C. Besta”, Milan, Italy

    Graziella Uziel

  9. Centro di Coordinamento Regionale per le Malattie Rare, Ospedale Universitario “Santa Maria della Misericordia”, Udine, Italy

    Bruno Bembi

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  1. Tatiana Fancello
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  2. Andrea Dardis
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Correspondence to Bruno Bembi or Mirella Filocamo.

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Fancello, T., Dardis, A., Rosano, C. et al. Molecular analysis of NPC1 and NPC2 gene in 34 Niemann–Pick C Italian Patients: identification and structural modeling of novel mutations. Neurogenetics 10, 229–239 (2009). https://doi.org/10.1007/s10048-009-0175-3

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