Fig. 2
The two variants of NOS3 detected in M035 and M084 probands are loss-of-function mutations. Panel A: The c.1502 + 1G > C splice-site variant identified in M084 causes a total loss of eNOS protein. (a) The c.1502 + 1G > C variant disrupts the canonical donor GT splice-site and causes intron 12 retention into the mRNA, resulting in a frameshift and in a premature termination codon in exon 13; Light grey arrows represent the primers used for cDNA amplification. Abbreviations: nt = nucleotides; PTC = premature termination codon. (b) Agarose gel electrophoresis migration of cDNA PCR amplicons from control (lane 2) and M084 (lane 3) using the primers shown in figure A (sequences provided on request). Lane 1: 100 base pairs ladder. The size of the amplicons obtained for M084 cDNA is about 200 nucleotides longer that those got from wild-type cDNA. Sequencing of amplicons showed a 202 nucleotides insert corresponding to the intron 12 retention into the mRNA. Detection of the mutated mRNA in M084 blood cells suggests that this mutant mRNA is spared by nonsense mRNA-mediated decay. (c) Western-blot performed on lysates form EPC derived from M084 proband and controls. Labelling with the monoclonal B-5 antibody directed against the N-terminal part of human and murine eNOS (Santa Cruz) showed a total loss of expression of eNOS in M084 proband (absence of signal on lane 5). Lane 1: NOS3 -/- knock-out mouse. Lane 2: wild-type mouse. Lane 3: EPC from healthy control 1. Lane 4: EPC from healthy control 2. Lane 5: EPC from M084 proband. Lane 6: EPC from healthy control 3. Panel B: The mutated p.C648R eNOS protein (c.1942 T > C variant) is unstable. HEK-293 cells were transfected with vectors containing the WT and mutated M035 full-length NOS3 cDNA. Western-blot performed on lysates from transfected cells showed a strong reduction of eNOS protein amount in three independent clones (lanes 1–3) in comparison to the clone transfected with the wild-type cDNA (lane 4). The primary antibody used is the monoclonal B-5 (Santa-Cruz). Lane 1: c.1942 T > C mutated overexpression clone 1. Lane 2: c.1942 T > C mutated overexpression clone 2. Lane 3: c.1942 T > C mutated overexpression clone 3. Lane 4: wild-type overexpression clone