TCIRG1-dependent recessive osteopetrosis : mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA

Susani, Lucia; Pangrazio, Alessandra; Sobacchi, Cristina; Mortier, Geert; et al.

doi:10.1002/HUMU.20076

Title

TCIRG1-dependent recessive osteopetrosis : mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA

Author

Susani, Lucia

Pangrazio, Alessandra

Sobacchi, Cristina

Mortier, Geert

et al.

Abstract

Human malignant infantile osteopetrosis (arOP) is a genetically heterogeneous autosomal recessive disorder of bone metabolism. The TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, is responsible for more than one-half of the arOP patients. We performed genetic analysis of TCIRG1 in 55 arOP patients including 25 new cases and identified nine novel mutations. The two most frequent mutations, c.16741G>A (aberrant splicing: r.1674_1884del) and c.2005C>T (protein variation: p.Arg669X), found in 17 and 16 alleles, respectively, constituted 30% of all TCIRG1 abnormalities. They both originated in Northern Europe, p.Arg669X quite recently from West Flanders, Belgium. As substitutions in splicing regulatory sequences represented a large portion (40%; 44 alleles) of the TCIRG1 variations, we developed a functional splicing assay to distinguish between polymorphic variants and disease-causing mutations. Three intronic nucleotide substitutions flanking the splice sites (c.117+4A>T; c.1673+5G>A; and c.5048G>A) were studied using hybrid minigenes and an abnormal processing of the transcripts was demonstrated in all cases. Cotransfection experiments with complementary U1 snRNAs performed in c.117+4A>T and c.1673+5G>A mutations showed that only in the first case was the defect at the 5′ splice site corrected, indicating that mutations near the invariant GT donor sites are mechanistically different. These findings indicate the feasibility of the hybrid minigene approach to detect splicing defects, particularly in patients in whom the RNA is not available. In addition, the present results suggest that modified U1 snRNAs may represent a new therapeutic strategy for arOP patients with a U1 snRNP-dependent splicing defect.

Language

English

Source (journal)

Human mutation. - New York, N.Y.

Publication

New York, N.Y. : 2004

ISSN

1059-7794

DOI

10.1002/HUMU.20076

Volume/pages

24 :3 (2004) , p. 225-235