A de novo G+1 → a mutation at the α2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI Type IV proband

Abstract

We have investigated the procollagen, collagen, alpha 2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two alpha 2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast alpha 2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of alpha,alpha'-dipyridyl. The alpha 2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating alpha 2(I) chain was reduced 6 degrees C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to alpha 2(I) nt 236-1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+1-->A mutation at the exon 16 donor site in one allele. The mutation eliminates a StyI site.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisense oligodeoxynucleotides selectively suppress expression of the mutant alpha 2(I) collagen allele in type IV osteogenesis imperfecta fibroblasts. A molecular approach to therapeutics of dominant negative disorders.

“…At the nuclear level, the antisense oligonucleotide is complementary to the primary unspliced transcript of the mutant allele (13) and, thus, to the nontranscribed strand. The missense oligonucleotide is complementary to only 3 nt of the mutant splice donor site in the unspliced transcript.…”

“…Here, we have treated cultured fibroblasts from a patient with moderately severe OI type IV with antisense oligonucleotides to target the naturally occurring mutation in ␣ 2(I). Since the point mutation in this ␣ 2(I) gene occurs at a splice donor site (13) and causes splicing out of the adjacent exon, these cells allow us to independently target the structures present in the nucleus and mRNA of the mutant allele. We have investigated the selectivity and efficiency of antisense suppression of a human mutation in its natural structural context.…”

Mutation producing alternative splicing of exon 26 in the COL1A2 gene causes type IV osteogenesis imperfecta with intrafamilial clinical variability

“…maintain a sort of gradient of phenotypic expression and the transition lethal/nonlethal occurs N-terminally to exon 28 [Tromp and Prockop, 1988]. Skipping exon 16 causes moderately severe (type IV/III) OI [Filie et al, 1993;Zolezzi et al, 1995], and skipping exons 20 [Mottes et al, 1994] and 21 [Superti-Furga et al, 1993] causes mild OI. In all of these cases, the substitutions were in either of the universally conserved splicing sites and were efficiently expressed.…”

“…Furthermore, the varying efficiency of exon skipping can play a role in determining the clinical outcome. Substitutions at any of the universally conserved splicing sites invariably result in the production of abnormal mRNA molecules from the mutant allele [Tromp and Prockop, 1988;Filie et al, 1993;Zolezzi et al, 1995]. On the other hand, substitutions at the moderately conserved G +5 position can still support to some extent normal splicing activity, possibly in a temperature-dependent way [Bonadio et al, 1990;Lee et al, 1991;Wu et al, 1993;Nicholls et al, 1996].…”

Type I Collagen Triplet Duplication Mutation in Lethal Osteogenesis Imperfecta Shifts Register of α Chains throughout the Helix and Disrupts Incorporation of Mutant Helices into Fibrils and Extracellular Matrix

“…Delayed cross-linking of mutant molecules is probably related to misalignment of residues on opposite molecules, which would be expected from propagation of the register shift. The delay in cross-linking of the triplet duplication is not seen with ␣2(I)⌬E16 collagen (36), which causes a larger six-triplet register shift and is more likely to realign by "looping out" of the normal chains than to propagate the register shift along the full helix. 4 Proband mature matrix has a turnover that is comparable with normal, reflecting both its predominantly normal collagen composition and also the stability of the cross-links formed by helices with one mutant chain that have become fully incorporated.…”