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

• Published in: Human mutation Vol. 24; no. 3; pp. 225 - 235
• Main Authors: Susani, Lucia, Pangrazio, Alessandra, Sobacchi, Cristina, Taranta, Anna, Mortier, Geert, Savarirayan, Ravi, Villa, Anna, Orchard, Paul, Vezzoni, Paolo, Albertini, Alberto, Frattini, Annalisa, Pagani, Franco
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-09-2004; Hindawi Limited
• Subjects: Alternative Splicing; Amino Acid Substitution; Cell Line; Defects; DNA Mutational Analysis; Exons - genetics; Genes; Genes, Recessive; Genes, Synthetic; Genetic engineering; HeLa Cells; Humans; Introns - genetics; Molecular Sequence Data; Mutation; Mutation, Missense; Organ Specificity; Osteoclasts - metabolism; osteopetrosis; Osteopetrosis - genetics; Point Mutation; Polymerase Chain Reaction; Protein Conformation; Protein Subunits - chemistry; Protein Subunits - genetics; RNA Splice Sites - genetics; RNA Splicing - genetics; RNA, Small Nuclear - genetics; snRNP; splicing defects; TCIRG1; Transfection; U1 snRNA; Vacuolar Proton-Translocating ATPases - chemistry; Vacuolar Proton-Translocating ATPases - genetics; Belgium; Italy
• ISSN: 1059-7794; 1098-1004
• DOI: 10.1002/humu.20076

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.1674–1G>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.504–8G>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. Hum Mutat 24:225–235, 2004. © 2004 Wiley‐Liss, Inc.