Figure 2
Biochemical and molecular results. A. Biochemical collagen analysis was performed on collagens produced by the patients dermal fibroblasts, which were grown for 16 hrs in the presence of 14C-Proline. Radioactively labelled intracellular and secreted fibrillar collagen proteins were isolated and mature collagens were obtained by pepsin digestion. Foetal secreted (left panel) as well as intracellular (right panel) mature type I collagen revealed a normal electrophoretic pattern when compared to a control (C) sample. Also for the unprocessed, secreted type I procollagen a normal electrophoretic migration pattern was observed (data not shown). B. ArrayCGH analysis on a 1M SurePrint G3 Human CGH Microarray revealed a homozygous deletion of the entire CREB3L1 gene in the affected foetus III:4. C. Expression level analysis by RT-qPCR was performed in duplicate on total RNA extracted from three biological replicates of the fibroblast cell lines from foetus III:4 and three controls (C1, C2 and C3) (LightCycler480 and RealTime ready DNA Probe Master Mix, Roche). The expression level of each investigated gene was quantified using qbasePLUS (Biogazelle)[27]. HPRT1, RLP13a and YWHAZ were applied as reference targets. RT-qPCR for foetus III:4 confirmed the total absence of CREB3L1 expression when compared to control samples (C1, C2 and C3). DGKZ has two alternative (tissue-specific) isoforms[28].