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
✍ Scribed by Jane D. Filie; Bonnie M. Orrison; Qin Wang; Mary Beth Lewis; Joan C. Marini
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 839 KB
- Volume
- 2
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
Communicaced by Chrles 1. Epsrein
We have investigated the procollagen, collagen, a2(I) mRNA, and DNA of a proband with type IV 01. The proband synthesized two a 2 ( I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast a 2 ( I ) chain was relatively retained within the cell and was present in collagens synthesized in the presence of a,a'-dipyridyl. The a 2 ( I ) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating a 2 ( I) chain was reduced 6°C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total R N A and antisense riboprobe complementary to a 2 ( I ) nt 236-1390 were digested with RNase A and TI. 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 R N N D N A hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G+ '-+A mutation at the exon 16 donor site in one allele. The mutation eliminates a Sty1 site. Digestion of PCR fragments amplified from the proband and parental WBC D N A revealed that only the proband had the undigested mutant fragment.