HTRF analysis of soluble huntingtin in PHAROS PBMCs.

Abstract

We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker. Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenine-guanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures. From 363 assayed samples, 342 met quality control standards. Levels of mtHtt and mt/tHtt were higher in 114 subjects with expanded CAG repeats (CAG ≥ 37) compared with 228 subjects with nonexpanded CAG repeats (CAG <37) (p < 0.0001). Analysis of relationships to predicted time to onset or to phenoconversion suggested that the HTRF signal could mark changes during the Huntington disease prodrome or after clinical onset. The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.