Alternative Splicing in COL1A1 mRNA Leads to a Partial Null Allele and Two In-frame Forms with Structural Defects in Non-lethal Osteogenesis Imperfecta

Qin Wang, Antonella Forlino, Joan C. Marini
1996 Journal of Biological Chemistry  
We have identified a novel multiexon genomic deletion in one COL1A1 collagen allele that results in three alternative forms of mutant mRNA. This mutation occurs in a 9-year-old girl and her father, both affected with severe type III osteogenesis imperfecta (OI). We previously reported detection of a mismatch in their ␣1(I) amino acids 558 -861 region by RNA/RNA hybrid analysis (Grange, D. K., Gottesman, G. S., Lewis, M. B., and Marini, J. C. (1990) Nucleic Acids Res. 18, 4227-4236). Single
more » ... -4236). 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 DNA from the exon 33-38 region yielded the normal 1004-base pair (bp) fragment and an additional 442-bp fragment. Sequencing of the shorter genomic PCR product confirmed the presence of a 562-bp deletion, extending from the last 3 nucleotides (nt) of exon 34 to 156 nt from the 3-end of intron 36. The genomic deletion was also detected in the clinically normal grandmother, who was confirmed to be a mosaic carrier. PCR amplification and RNase protection experiments were used to investigate the mRNA structure and occurrence of alternative splicing. One form of the mutant cDNA has a deletion with end points that are identical to the genomic deletion. This results in a combination deletion/insertion, 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 alternatively spliced forms. One form uses a cryptic donor site in exon 34 and the exon 37 acceptor. The second form uses the normal exon 32 splice donor and exon 37 acceptor. Use of the cryptic donor results in a coding 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 alternative splicing generating both in-frame and out-of-frame forms of mRNA. Although the in-frame forms constitute more than 60% of the mRNA from the mutant allele, no mutant protein chain was identified. Collagen produced by cultured OI osteoblasts showed a significant increase in the relative amount of type III collagen but no mutant ␣1(I) chain. Osteogenesis imperfecta (OI) 1 is a heritable disorder of connective tissue; its most significant clinical feature is fragile bones that are susceptible to fracture from minimal trauma (1). The phenotypic spectrum of OI is described by the Sillence classification and ranges from perinatal lethal to minimal skeletal involvement. The full clinical spectrum of OI has been demonstrated to be caused by defects in the structure or synthesis of type I collagen, the most abundant protein of bone and skin (for reviews, see Refs. 2 and 3). In the majority of instances, the mild OI type I is caused by defects that effectively create a null ␣1(I) allele (4), such as a frameshift resulting in premature chain termination. In these cases, all the secreted collagen is structurally normal. The decreased total production of type I collagen results in an increase of the ␣1(III)/␣1(I) ratio. In OI types II, III, and IV, there is an expressed structural defect in either the ␣1(I) or ␣2(I) chain (2). Over 100 of these defects have been molecularly delineated; 79% are point mutations resulting in the substitution of another amino acid for one of the glycine residues that occur in every third position of the chain, 11% are in-frame single exon splicing defects, and the small remainder comprises deletions, duplications, and insertions. Intronic inclusions and multiexon deletions are among the less common types of collagen mutations that have been demonstrated in OI. Intronic inclusions have been described in three cases. When these sequences are in-frame and contain no stop codons, a non-collagenous region is inserted into the helical portion of the collagen chain. Since the fibrillar collagens are defined by uninterrupted repeats of the Gly-X-Y triplet, such insertions disrupt the essential nature of the molecule. A patient with moderately severe type IV OI has a 6-amino acid intronic insertion from intron 33 in one ␣2(I) allele (5) resulting from activation of a cryptic splice site. Another patient with type IV OI (6) has a defect at the ϩ5 position of the intron 8 splice donor site in ␣1(I) that leads to a deletion of the 18 amino acids of exon 8 and insertion of 32 amino acids coded by intron 7. Finally, insertion of 75 bp of ␣1(I) intron 35 is associated with lethal type II OI phenotype (7). One multiexon deletion has been described in each of the type I collagen ␣ chains, both associated with a lethal phenotype. In ␣1(I), the well studied CRL 1262 has a deletion of exons 27, 28, and 29 resulting from an intron to intron recombination (8). The chains carrying the 84-amino acid deletion are detected in the cells but are poorly secreted (9). In ␣2(I), another intron to intron recombination results in the loss of the 180 amino acids encoded by exons 34 -40 (10). The short ␣2(I)
doi:10.1074/jbc.271.45.28617 pmid:8910493 fatcat:5ob3k37qcngrhjo7givogjf22q