To further clarify, the two mutations were present in seven out of ~1300 AD families screened. Only two unaffecteds from two different families were mutations carriers (one for each mutation). Thus, we concluded that these mutations are partially penetrant. However, it is still possible that the affection status of either of those those two subjects could change over time. Within the seven families, the two mutations increased risk by 3.5 to ~5-fold. While these O.R.'s are a little greater than that conferred by one copy of ApoE4, these are exceedingly rare familial mutations, not common polymorphisms. Thus, they exert a smaller overall effect on the genetic variance of late-onset AD than ApoE4.
I wonder what these mutations do to Notch processing/signaling? ADAM10-knockout in mice is embryonic-lethal due the loss of proper Notch function. It's a bit difficult to understand how a human could survive with a 70 percent loss of Notch signaling; but maybe there are compensatory changes in this pathway.
REAGENTS/MATERIAL: Western blot analysis:
For primary antibodies, mouse monoclonal anti-Aβ (6E10) (Covance) was used to
detect sAPPα levels in conditioned medium.
rabbit anti-APP C-terminal (A8717) (Sigma) was utilized for full-length APP and CTFs in cell lysates
and mouse monoclonal anti-HA (6E2) (Cell Signaling) for HA-tagged ADAM10.
Mouse monoclonal Anti-actin, pan (C-4) (Lab Vision Neo-Markers) was used as control signal for equal loading. Aβ ELISA:
Levels of Aβx-40 in cell culture media were determined using a
human/rat Aβ-specific chemiluminescence ELISA kit (Wako Chemicals USA, Inc., VA, USA).
Briefly, samples and Aβ standards were aliquoted to 96 well plates coated with monoclonal antibody against Aβ1–28 region. After overnight incubation,
anti-Aβ40 specific antibody conjugated with horseradish peroxidase (HRP) was added to the captured Aβ in each well.