Skip to main content
Fig. 5 | Orphanet Journal of Rare Diseases

Fig. 5

From: A case of fatal Type I congenital disorders of glycosylation (CDG I) associated with low dehydrodolichol diphosphate synthase (DHDDS) activity

Fig. 5

Evaluation of the consequences of DHDDS mutations in fibroblasts. a Quantitation of DHDDS cDNA generated from mRNA derived from fibroblasts obtained from two cell populations from the patient (P and P’), a normal subject (N) and whole blood cell extracts from the father (F) and mother (M). QRT-PCR using either ribosomal protein S14 (S14) or hydroxymethylbilane synthase (HMBS) as housekeeping genes was performed. b DHDDS activity was assayed using farnesyl diphosphate (FPP) as acceptor. c After subtracting background values (− FPP) from the data points, the initial slopes of the curves shown in (b) and two other similar experiments were estimated. The values (SlopeP/SlopeN) × 100 are plotted (solid circles). SlopeN is set at 100 % for each experiment (open circles). d Dol-P-Man synthase (DPMS) activity was measured in the same microsome preparations as used in (b) using GDP-[14C]mannose and DolP (DP) as acceptor. e Measurement of endogenous DolP levels in microsomes prepared from fibroblasts derived from the patient (P) and a normal subject (N). Incubations were carried out with GDP-[14C]Man in the absence or presence of the glycosylation acceptor peptide (NYT) and bacitracin as indicated. The specific activity of the GDP-[14C]Man is used to calculate the pmoles Dol-P-Man recovered from the organic phase after stopping the reactions. f Extracts of fibroblasts from three control subjects, the patient (P), and patients diagnosed with ALG12-CDG (Patient 2) and DPM2-CDG (Patient 3) were subjected to SDS-PAGE and Western Blot. The blots were probed with antibodies directed towards intercellular adhesion molecule-1 (ICAM), calnexin (CNX), activating transcription factor 6 alpha (ATF6α) and actin

Back to article page