Rogaeva EA, Fafel KC, Song YQ, Medeiros H, Sato C, Liang Y, Richard E, Rogaev EI, Frommelt P, Sadovnick AD, Meschino W, Rockwood K, Boss MA, Mayeux R, St George-Hyslop P. Screening for PS1 mutations in a referral-based series of AD cases: 21 novel mutations. Neurology. 2001 Aug 28;57(4):621-5. PubMed Abstract

REAGENTS/MATERIAL:
A total of 414 subjects from the United States, Germany, and Canada participated in the study (54% women and 46% men). Total RNA and genomic DNA were isolated from blood leukocytes. For the Leu235Pro, Gln222Arg, and Ile213Leu mutations, the 144-basepair PCR product was amplified with primers 974 (5'-TGTGGTGGGAATGA-3') and 892 (5'-TGAAATCACAGCCAAGATGAG-3'). A reaction volume of 20 uL containing 100 ng of DNA, 20 pmol of each primer, 2 uL of 10 X PCR reaction buffer (Qiagen, Missisauga, Canada), 1 U Taq polymerase, 250 mkM dNTPs, and 1 uCi a-32 P-deoxycytidine triphosphate was thermocycled for 35 cycles of 94° for 30 seconds, 57° for 20 seconds, and 72° for 20 seconds. The Gln222Arg PCR products were digested with 1 U MspI for 1 hour at 37°, and the resulting restriction fragments (wild-type 144 basepair; mutation 96 basepair, 48 basepair) were resolved on a 6% nondenaturing polyacrylamide gel and visualized by autoradiog-raphy. The Ile213Leu PCR product was digested with PflmI (wild-type 124 basepair and 22 basepair; mutation 144 basepair). The Leu235Pro PCR product was analyzed by direct sequencing approach.

FUTURE DIRECTION:
Analysis of Ab42 levels in cultured cells needed to provide biochemical support these PS1 mutations are likely to be pathogenic rather than innocent polymorphisms.