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Biochemical screening of type I collagen in osteogenesis imperfecta: detection of glycine substitutions in the amino end of the alpha chains requires supplementation by molecular analysis
  1. W A Cabral,
  2. S Milgrom,
  3. A D Letocha,
  4. E Moriarty,
  5. J C Marini
  1. Bone and Extracellular Matrix Branch, NICHD, NIH, Bethesda, MD, USA
  1. Correspondence to:
 Dr Joan C Marini
 Section on Connective Tissue Disorders, Bone and Extracellular Matrix Branch, NICHD, NIH, 9000 Rockville Pike 10/9S241, Bethesda, MD 20892, USA;oidoc{at}


Background: The biochemical test for osteogenesis imperfecta (OI) detects structural abnormalities in the helical region of type I collagen as delayed electrophoretic migration of alpha chains on SDS-urea-PAGE. Sensitivity of this test is based on overmodification of alpha chains in helices with a glycine substitution or other structural defect. The limits of detectability have not been reported.

Methods: We compared the collagen electrophoretic migration of 30 probands (types III or IV OI) with known mutations in the amino half of the α1(I) and α2(I) chains. Differences in sensitivity were examined by 5% and 6% SDS-urea-PAGE, and with respect to alpha chain, location along the chain, and substituting amino acid.

Results: Sensitivity was enhanced on 5% gels, and by examination of intracellular and secreted collagen. In α1(I), substitutions in the first 100 residues were not detectable; 7% of cases in the current Mutation Consortium database are in this region. α1(I) substitutions between residues 100 and 230 were variably detectable, while those after residue 232 were all detected. In α2(I), variability of electrophoretic detection extended through residue 436. About a third of cases in the Consortium database are located in the combined variable detection region. Biochemical sensitivity did not correlate with substituting residue.

Conclusions: Complete testing of probands with normal type I collagen biochemical results requires supplementation by molecular analysis of cDNA or gDNA in the amino third of α1(I) and amino half of α2(I). Mutation detection in OI is important for counselling, reproductive decisions, exclusion of child abuse, and genotype-phenotype correlations.

  • CSGE, confirmation-sensitive gel electrophoresis
  • dHPLC, denaturing high performance liquid chromatography
  • DMEM, Dulbecco’s modified eagle medium
  • OI, osteogenesis imperfecta
  • RT-PCR, reverse transcription polymerase chain reaction
  • collagen biochemistry
  • osteogenesis imperfecta
  • overmodification
  • sensitivity limitations

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  • Competing interests: none declared

  • Ethics approval: all specimens were obtained from individuals enrolled in IRB approved protocols which included provisions for skin biopsies and molecular testing