Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington's disease patients

Abstract

Background: Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously.

Methods: We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts.

Results: This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT.

Conclusions: We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.

Trial registration: Not applicable.

Funding: CHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.

Figure 4. CSF concentrations of mHTT and of the neuronal proteins tau and NFL are significantly associated.

Significant positive linear associations were seen between CSF concentrations of mHTT and tau in (A) London (n = 9) and (B) Vancouver (n = 30) cohorts. A strikingly close association was seen between CSF mHTT concentration and NFL concentration in the (C) London (n = 9) and (D) Vancouver (n = 20) cohorts. Data were calculated by linear regression analysis.

Figure 2. mHTT is detectable in CSF from HTT mutation carriers but not from control participants in 2 populations.

(A) London population (n = 12) and (B) Vancouver population (n = 30). CSF mHTT concentration was significantly elevated in manifest HD subjects compared with that in premanifest HD subjects in both populations. Horizontal bars indicate the group means. Data were calculated by weighted-least-squares ANOVA.

Figure 3. CSF mHTT concentration correlates significantly with disease stage.

mHTT concentration was positively associated with the disease burden score in both (A) London (n = 9) and (B) Vancouver (n = 30) cohorts. Significant positive associations were also seen with (C) 5-year conditional onset probability in premanifest participants (n = 13) and (D) in UHDRS motor scores (n = 39). Data were calculated by linear regression analysis.

Figure 1. The mHTT SMC immunoassay is specific and sensitive.

(A) Schematic of mHTT (not to scale) indicating the epitopes bound by the 2B7 and MW1 antibodies used for the assay. 2B7 binds residues 1–17 of the N terminus (11), while MW1 conveys specificity for the mutant protein by binding preferentially to the expanded polyglutamine tract (13). (B) Detection of purified recombinant HTT proteins with the SMC immunoassay shows specificity for mutant (mHTT) over WT human HTT protein. (C) Determination of the assay’s LLoQ and ULoQ shows a low femtomolar detection threshold and broad dynamic range.