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 Communitys Seventh Framework Program [FP7/2007-2013, SYBIL Project], PNR-CNR aging Program 2012-2014, the Leenaards Foundation Lausanne and the Swiss National Foundation.