ALTERNATIVE SPLICING IN COL1A1 MESSENGER-RNA LEADS TO A PARTIAL NULL ALLELE AND 2 IN-FRAME FORMS WITH STRUCTURAL DEFECTS IN NONLETHAL OSTEOGENESIS IMPERFECTA

We have identified a novel multiexon genomic deletion in one COL1A1 co llagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affecte d with severe type III osteogenesis imperfecta (OI). We previously rep orted detection of a mismatch in their alpha 1(I) amino acids 558-861 region by RNA/RNA hybrid analysis (Orange, D. K., Gottesman, G. S., Le wis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4286) . Single Strand Conformational Polymorphism further localized the mRNA mutation to the amino acids 579-679 coding region. At the gene level, polymerase chain reaction (PCR) amplification of patient leukocyte DN A from the exon 33-38 region yielded the normal 1004-base pair (bp) fr agment and an additional 442-bp fragment. Sequencing of the shorter ge nomic PCR product confirmed the presence of a 562-bp deletion, extendi ng from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3'-e nd of intron 36. The genomic deletion was also detected in the clinica lly normal grandmother, who was confirmed to be a mosaic carrier. PCR amplification and RNase protection experiments were used to investigat e the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical t o the genomic deletion. This results in a combination deletion/inserti on, with a deletion of amino acids 603-639 followed by an insertion of 156 nt from the 3'-end of intron 36. In addition, we found two altern atively spliced forms. One form uses a cryptic donor site in exon 34 a nd the exon 37 acceptor. The second form uses the normal exon 32 splic e donor and exon 37 acceptor. Use of the cryptic donor results in a co ding sequence that is out-of-frame. Both the retained intron form and the use of the exon 32 donor site result in coding sequences that are in-frame. This is the first report of a collagen defect in OI with alt ernative splicing generating both in-frame and out-of frame forms of m RNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collag en produced by cultured OI osteoblasts showed a significant increase i n the relative amount of type III collagen but no mutant alpha 1(I) ch ain.