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Note

Note

To perform a collagen study in patients with Osteogenesis Imperfecta(OI) or Ehlers Danlos syndrome(EDS) we need a skin fibroblast culture from the proband to perform a biochemical and molecular study of the respective collagens a-chains/collagen genes. The skin fibroblast culture is used for a double purpose:

• Fibroblasts are metabolically labelled with 14C Proline and studied electrophoretically by SDS-PAGE.
• Another part of the culture is used for the isolation of mRNA that is converted to cDNA prior to analysis. Genomic DNA is isolated from leukocytes or from cultured fibroblasts, amniocytes or chorionic villi.

Each patient is first investigated at the biochemical level by the evaluation of radioactively labelled type I (pro)collagen molecules by SDS-PAGE.

Based on the biochemical results, molecular analysis is performed:

• Patients with evidence for the presence of a structurally abnormal collagen(types I, III or V) are subjected to cDNA mutation screening of the respective genes(COL1A1 and COL1A2 for OI; COL3A1 for vascular EDS; and COL5A1 and COL5A2 for classical EDS).
• In the patients presenting reduced collagen, screening for the presence of a null-allele is the first step in the molecular investigations. If a null allele is present, a genomic mutation screening is performed. In patients with two transcripts, a mutation screening on cDNA is started.
• In patients with a normal collagen profile the clinical history is used to direct further molecular work: in patients with clinically mild OI the possibility of a COL1A1 null-allele is investigated prior to genomic DNA mutation screening, while in patients with a more severe clinical phenotype a cDNA- based mutation analysis is performed.

A turn around time of 6 to 12 months must be taken into account before the first results can be expected. This period is indeed necessary to perform the biochemical and molecular studies.