Two novelCOL1A1 mutations in patients with osteogenesis imperfecta (OI) affect the stability of the collagen type I triple-helix

Abstract

Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in the COL1A1 and COL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations in COL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34 degrees C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5 degrees C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39 degrees C (2 degrees C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

“…It is interesting that there is an apparent intrafamilial variability among individuals with the same mutation and interfamilial variability among individuals with the same type of OI (Basel and Steiner, 2009). The mutations at the C-terminus of alpha-2 chain appear to be more severe than those near the N-terminus, but mutations at N-terminal glycine residues of the alpha-1 chain caused more lethality (Bodian et al, 2008;Witecka et al, 2008).However, it remains difficult to establish correlation between a specific genotype and the resulting phenotype Basel and Steiner, 2009;Faqeih et al, 2009). Therefore, identification of new mutations will significantly facilitate further clinically genotypeephenotype correlation studies.…”

“…Type I collagen is the major structural protein of the bone, skin, and other tissues, and consisted of two alpha-1 chains and one alpha-2 chain (Witecka et al, 2008). Each chain contains 338 units of uninterrupted repeats of the Gly-X-Y triplet.…”

Cell therapy of a patient with type III <i>osteogenesis imperfecta</i> caused by mutation in <i>COL1A2</i> gene and unstable collagen type I

“…DNA fragments of the collagen COL1A1 and COL1A2 gene were amplified using primers described previously [22] and listed in Table 1. The primers were designed and verified using PrimerSelect in DNAStar programme package version 5.0.…”

“…Concentra-tions of primers were calculated based on the sample absorbance at the wave length 260 nm and adjusted to the primer compositions. PCRs were performed according to the conditions described elsewhere [22]. Briefly, the reaction mixture included 300 ng double stranded patient genomic DNA, 1 µl 10 mM dNTPs (PCR nucleotide Mix, Roche, Germany), 1 µl (5 pmol) of each primer, 2.5 µl 10x Taq polymerase buffer, 1U Taq polymerase and nuclease free water (all from FastStart Taq DNA Polymerase, Roche, Germany) to the total volume of 25 µl.…”

“…Harvested medium was processed as previously described [23] with subsequent modifications described elsewhere [22,24]. The volume of sample containing procollagen I was reduced to the appropriate collagen concentration using Amicon cell under nitrogen pressure fitted with the membrane with the size cut off 100,000 kDa, following double dialysis against 200 volumes of so called storage buffer containing 0.4 M NaCl, 0.1 M Trizma Base (pH 7.4) supplemented with 0.025 M EDTA and 0.04% NaN 3 .…”