High Levels of Mutant Huntingtin Protein in Tear Fluid From Huntington's Disease Gene Expansion Carriers

Abstract

Objective: Huntington's disease (HD) is an autosomal dominant, fully penetrant, neurodegenerative disease that most commonly affects middle-aged adults. HD is caused by a CAG repeat expansion in the HTT gene, resulting in the expression of mutant huntingtin (mHTT). Our aim was to detect and quantify mHTT in tear fluid, which, to our knowledge, has never been measured before.

Methods: We recruited 20 manifest and 13 premanifest HD gene expansion carriers, and 20 age-matched controls. All patients underwent detailed assessments, including the Unified Huntington's Disease Rating Scale (UHDRS) total motor score (TMS) and total functional capacity (TFC) score. Tear fluid was collected using paper Schirmer's strips. The level of tear mHTT was determined using single-molecule counting SMCxPRO technology.

Results: The average tear mHTT levels in manifest (67,223 ± 80,360 fM) and premanifest patients (55,561 ± 45,931 fM) were significantly higher than those in controls (1,622 ± 2,179 fM). We noted significant correlations between tear mHTT levels and CAG repeat length, "estimated years to diagnosis," disease burden score and UHDRS TMS and TFC. The receiver operating curve demonstrated an almost perfect score (area under the curve [AUC] = 0.9975) when comparing controls to manifest patients. Similarly, the AUC between controls and premanifest patients was 0.9846. The optimal cutoff value for distinguishing between controls and manifest patients was 4,544 fM, whereas it was 6,596 fM for distinguishing between controls and premanifest patients.

Conclusion: Tear mHTT has potential for early and noninvasive detection of alterations in HD patients and could be integrated into both clinical trials and clinical diagnostics.

Keywords: Biomarker; Huntingtin; Huntington’s disease; Tear fluid.

Figure 1.

Tear mHTT group differences. A: Tear mHTT levels differed significantly between manifest (n = 20) and premanifest (n = 13) patients compared to healthy controls (n = 20). B: Within the manifest group, no significant differences were found between disease stage 1 (n = 6), stage 2 (n = 12), and stage 3 (n = 2). C and D: Within the premanifest group, there was no statistically significant difference in tear mHTT levels between early (n = 9) and late (n = 4) premanifest patients (C) or between preA and preB premanifest patients (D). E: There was no correlation between tear mHTT levels and age. F: Significant correlations were found between tear mHTT levels and CAG repeat length for all participants. G: There was no statistically significant difference in tear mHTT levels between carriers with reduced penetrance (36–39 CAG repeats, n = 4) and those with full penetrance (≥ 40 CAG repeats, n = 23). Group differences were analyzed by the Kruskal‒ Wallis test with Dunn’s multiple comparisons test. The Mann‒Whitney test was used to evaluate the difference between early and late premanifest HD and between preA and preB premanifest HD. All correlations were analyzed by Spearman correlation. The horizontal bars indicate the means ± standard deviations. ^*p < 0.001; ^†p < 0.0001. HD, Huntington’s disease; ns, not significant; preA, estimated time to diagnosis < 17.5 years; preB, estimated time to diagnosis > 17.5 years.

Figure 2.

Tear mHTT correlations with clinical parameters. A: The 5-year onset probability did not correlate with tear mHTT levels in premanifest patients. B: The number of “estimated years to diagnosis” correlated significantly with tear mHTT in premanifest patients. C: Significant correlations were found between tear mHTT levels and disease burden scores in premanifest patients. D and E: Tear mHTT levels strongly correlated with UHDRS TMS (D) and UHDRS TFC (E). All correlations were analyzed by Spearman correlation. ^*p < 0.05; ^†p < 0.01; ^‡p < 0.0001. HD, Huntington’s disease; UHDRS TMS, Unified Huntington’s Disease Rating Scale total motor score; UHDRS TFC, Unified Huntington’s Disease Rating Scale total functional capacity.

Figure 3.

Diagnostic ability of tear mHTT. The ROC curves discriminating between the control and manifest HD (A), control and premanifest HD (B), and premanifest and manifest HD groups (C). D: Tear mHTT cutoff levels used to distinguish patients from controls are represented by dotted lines. ROC, receiver operating curve; HD, Huntington’s disease; AUC, area under the curve.