TY - JOUR
T1 - TCIRG1-dependent recessive osteopetrosis
T2 - Mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA
AU - Susani, Lucia
AU - Pangrazio, Alessandra
AU - Sobacchi, Cristina
AU - Taranta, Anna
AU - Mortier, Geert
AU - Savarirayan, Ravi
AU - Villa, Anna
AU - Orchard, Paul
AU - Vezzoni, Paolo
AU - Albertini, Alberto
AU - Frattini, Annalisa
AU - Pagani, Franco
PY - 2004/7/26
Y1 - 2004/7/26
N2 - 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: 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.
AB - 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: 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.
KW - Osteopetrosis
KW - SnRNP
KW - Splicing defects
KW - TCIRG1
KW - U1 snRNA
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U2 - 10.1002/humu.20076
DO - 10.1002/humu.20076
M3 - Article
C2 - 15300850
AN - SCOPUS:4143077123
VL - 24
SP - 225
EP - 235
JO - Human Mutation
JF - Human Mutation
SN - 1059-7794
IS - 3
ER -