Citations

Abstract Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. Methods: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen®, and the high-resolution melter, HR-1™, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. Results: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

ABSTRACT The amyloid β‐protein (Aβ) deposited in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid β‐protein precursor (APP). Generation of Aβ from the APP requires two sequential proteolytic events: an initial β‐secretase cleavage at the amino terminus of the Aβ sequence followed by γ ‐secretase cleavage at the carboxyl terminus of Aβ. We describe the development of a robust in vitro assay for γ ‐secretase cleavage by showing de novo Aβ production in vitro and establish that this assay monitors authentic gamma‐secretase activity by documenting the production of a cognate γ ‐CTF, confirming the size of the Aβ produced by mass spectrometry, and inhibiting cleavage in this system with multiple inhibitors that alter γ‐ secretase activity in living cells. Using this assay, we demonstrate that the γ‐secretase activity 1 ) is tightly associated with the membrane, 2 ) can be solubilized, 3 ) has a pH optimum of 6.8 but is active from pH 6.0 to pH >8.4, and 4 ) ascertain that activities of the γ ‐40 and γ ‐42 are indeed pharmacologically distinct. These studies should facilitate the purification of the protease or proteases that are responsible for this unusual activity, which is a major therapeutic target for the treatment of AD.