Constantinos D. Constantinou

Structure of cDNAs Encoding the Triple-Helical Domain of Murine α2 (VI) Collagen Chain and Comparison to Human and Chick Homologues. Use of Polymerase Chain Reaction and Partially Degenerate Oligonucleotides for Generation of Novel cDNA Clones

Type VI collagen cDNAs of human and avian origin were recently obtained and characterized by screening cDNA libraries in lambda phage. Based on the published sequences of these cDNAs, we constructed partially degenerate oligonucleotide primers that we used in polymerase chain reactions for the generation of alpha 2(VI) collagen clones of murine origin. As template, we used cDNA derived from murine total RNA. We amplified, cloned and sequenced a 1043-bp fragment that contains the coding sequence for the entire triple-helical domain except for the first two amino acids that are Gly-Pro in both man and chicken. Comparison of the nucleotide and derived amino acid sequences revealed 84.6% and 92.5% identity between mouse and man at the DNA and protein levels, respectively. Comparison with chicken sequences showed 72% and 79.1% identity. The third base usage showed a distinct preference for A in the glycine codons for the three species; whereas, U is preferred in all human fibrillar collagen genes previously defined. The preference for third base codon in Y position prolines is U for the alpha 2(VI) collagen as it is for the human fibrillar collagen genes. A single cysteine at position 89, two Arg-Gly-Asp sequences, and one triple helix-interruption are conserved in mouse, man and chicken. Comparison of hydropathy plots showed great similarity between those of murine and human alpha 2(VI) collagen chains and to a lesser extent between murine and chick. Northern blot hybridization of murine poly A+ RNA with a nick-translated radiolabeled alpha 2(VI) collagen probe detected one major transcript of 3.7 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in Type I Procollagen Genes That Cause Osteogenesis Imperfecta

Recent data from several laboratories have demonstrated that most forms of osteogenesis imperfecta (OI) are caused by mutations in one of the two structural genes for type I procollagen. Few, if any, are in the many other genes expressed in bone. This surprising conclusion has several implications for other genetic diseases that involve connective tissues as well as several more common diseases.

A Substitution of Cysteine for Glycine 904 in COL1A1 in a Proband with Lethal Osteogenesis Imperfecta and in Her Asymptomatic Mother

A number of lethal and nonlethal cases of osteogenesis imperfecta (01) have been fully characterized to date. All of them were shown to harbor mutations in the genes for either the a1 or a2 chains of type I procollagen, a major component of bone, skin, tendon, and other connective tissues.’s2 From all variants thus far studied, there seems to be a correlation between the position of the mutation along the molecule and the severity of clinical phenotype. In particular, point mutations in the COOHterminal region of the al(1) chain have been associated with lethal or severe variants of OI.’*2 Previously, we studied a lethal case of 01: The proband was a female stillborn at 31 weeks of gestation to phenotypically normal unrelated parents who subsequently had two normal children. Radiographic examination of the proband showed multiple fractures of the long bones and ribs. The skull was not ossified, and consequently it was membranous and soft. Biochemical and molecular studies revealed that the mutation was a single base substitution that converted Gly to Cys at position 904 of the a1 chain of type I procollagen. Due to the mutation, the type I procollagen molecules were grossly overmodified, had decreased melting temperature (T,,,), and had impaired secretion.’ We subsequently examined skin fibroblasts from the proband’s mother. At low passage numbers they synthesized normal and overmodified collagen. The overmodified collagen had a decreased T,as assayed by brief protease digestion. Also, the fibroblasts synthesized little of what appeared to migrate as an al(1) dimer on SDS polyacrylamide gels, suggesting the presence of a new cysteine in the al(1) chain. In order to investigate the possibility of the mother being a carrier of the same mutation as the proband, we had two allele-specific oligonucleotides ( 1Pmers) synthesized that spanned the mutated site at position 904. One oligonucleotide, ASO-GlyW, was a wild type, and the other, ASO-Cysm, had a T instead of a G in the middle of the sequence providing for the Gly to Cys substitution. Either genomic DNA or cDNA