ECTS Abstracts (2015) 1 OC6.2

Intronic mutations in the TCIRG1 gene cause human autosomal recessive osteopetrosis

Eleonora Palagano1,2, Harry Blair3, Alessandra Pangrazio1,2, Irina Tourkova3, Andrea Angius4,5, Gianmauro Cuccuru4, Manuela Oppo4, Paolo Uva4, Wim Van Hul6, Eveline Boudin6, Andrea Superti-Furga7, Flavio Faletra8, Agostino Nocerino9, Matteo Ferrari10, Guido Grappiolo10, Marta Monari11, Alessandro Montanelli11, Paolo Vezzoni1,2, Anna Villa1,2 & Cristina Sobacchi1,2

1UOS/IRGB, Milan Unit, National Research Council (CNR), Milan, Italy; 2Humanitas Clinical and Research Center, Rozzano, Italy; 3Veteran’s Affairs Medical Center and Department of Pathology, University of Pittsburgh, Pittsburgh, USA; 4CRS4, Science and Technology Park Polaris, Pula, Italy; 5IRGB-CNR, Monserrato, Italy; 6Department of Medical Genetics, University of Antwerp, Antwerp, Belgium; 7Department of Pediatrics, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; 8Institute for Maternal and Child Health-IRCCS, ‘urlo Garofolo’, Trieste, Italy; 9Clinica Pediatrica, Azienda Ospedaliero-Universitaria ‘S. Maria della Misericordia’, Udine, Italy; 10Hip and Prosthetic Replacement Unit, Humanitas Clinical and Research Center, Rozzano, Italy; 11Clinical Investigation Laboratory, Humanitas Clinical and Research Center, Rozzano, Italy.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment; in particular, intermediate cases often constitute a diagnostic challenge. Mutations in the TCIRG1 gene are responsible for more than 50% of ARO cases, and a wide range of molecular defects have been found. Here we describe the identification of four different single nucleotide changes in intron 15 in five patients from four unrelated families. These novel mutations were in the middle of a 368 nucleotide long intron, far from the canonical splice sites; therefore, they were missed by standard gene amplification and sequencing, focused on exons and exon-intron boundaries, and went ignored by exome sequencing. In three out of five patients, by cloning and sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated a reduced splicing efficiency, which did not completely abrogate the production of the normal transcript. In conclusion, we identified an intronic region in the TCIRG1 gene which seems to be prone to splicing mutations. These molecular defects allow the production of a small amount of protein sufficient to dampen the severe phenotype usually associated to TCIRG1 mutations. Indeed, the patients bearing these variants displayed a different level of severity of the disease, with three out of five reaching adulthood with a mild presentation. On this basis, we suggest the analysis of the TCIRG1 gene is appropriate not only in the molecular work up of severe patients, but also of intermediate cases. In addition, our results demonstrate that standard protocols for gene testing are likely to be revised. In particular, intron 15 should be included in the routine sequencing of the TCIRG1 gene; more in general, the effect of intronic changes in genes associated with osteopetrosis should be carefully evaluated.

Disclosure: The authors declared no competing interests. This work was supported by the Telethon Foundation [GGP12178], PRIN Project [20102M7T8X_003], Giovani Ricercatori from Ministero della Salute [GR-2011-02348266], Ricerca Finalizzata from Ministero della salute [RF-2009-1499,542], the European Community’s Seventh Framework Program [FP7/2007-2013, SYBIL Project], PNR-CNR aging Program 2012-2014, the Leenaards Foundation Lausanne and the Swiss National Foundation.

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