Miriam Moscovitch-Lopatin1, Rachel E Goodman, Shirley Eberly, James J Ritch, H Diana Rosas, Samantha Matson, Wayne Matson, David Oakes, Anne B Young, Ira Shoulson, Steven M Hersch. 1. From the Massachusetts General Hospital (M.M.-L., R.E.G., J.J.R., H.D.R., S.M., A.B.Y., S.M.H.), MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Charlestown, MA; University of Rochester Medical Center (S.E., D.O.), Department of Biostatistics and Computational Biology, Rochester, NY; Veterans Administration Hospital (W.M.), Bedford, MA; and Program for Regulatory Science & Medicine (I.S.), Georgetown University, Washington, DC.
Abstract
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin.
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