Background: Huntington’s disease (HD) is an autosomal-dominant neurodegenerative movement disorder that presents with prominent cognitive and psychiatric dysfunction. Brain-derived neurotrophic factor (BDNF) plays an important role in the pathophysiology of HD, as well as other neurodegenerative and psychiatric disorders, and epigenetic alterations in the complex BDNF promoter have been associated with its deregulation in pathological conditions. BDNF has gained increased attention as a potential biomarker of disease; but currently, the conflicting results from measurements of BDNF in different biofluids difficult the assessment of its utility as a biomarker for HD. Here, we measured BDNF protein levels in plasma (n = 85) and saliva (n = 81) samples from premanifest and manifest HD patients and normal controls using ELISA assays. We further examined DNA methylation levels of BDNF promoter IV using DNA derived from whole blood of HD patients and healthy controls (n = 40) using pyrosequencing. BDNF protein levels were not significantly different in plasma samples across diagnostic groups. Plasma BDNF was significantly correlated with age in control subjects but not in HD patients, nor were significant gender effects observed. Similar to plasma, salivary BDNF was correlated with age only in control subjects, with no gender effects observed. Importantly, we detected significantly lower levels of salivary BDNF in premanifest and manifest HD patients compared to control subjects, with lower BDNF levels being observed in premanifest patients within a predicted 10 years to disease onset. Salivary and plasma BDNF levels were not significantly correlated with one another, suggesting different origins. DNA methylation at four out of the 12 CpG sites studied in promoter IV were significantly altered in HD patients in comparison to controls. Interestingly, methylation at three of these CpG sites was inversely correlated to the Hospital Anxiety and Depression Scale (HADS) scores. BDNF promoter methylation was not correlated with motor or cognitive scores in HD patients, and was not associated with sex or age in neither disease nor control groups. Conclusion: Our studies show that BDNF protein levels are decreased in saliva; and BDNF promoter methylation increased in blood in HD subjects when compared to controls. These findings suggest that salivary BDNF measures may represent an early marker of disease onset and DNA methylation at the BDNF promoter IV, could represent a biomarker of psychiatric symptoms in HD patients.
OBJECTIVE: The objective of the current study was to assess the potential for saliva to serve as a biospecimen for accessible biomarkers for Huntington’s disease (HD).
BACKGROUND: Peripheral biomarkers are greatly needed in the field of neurodegenerative disorders to anticipate onset of disease symptoms, monitor disease progression, and track potential therapeutic effects. HD is a fatal, inherited neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the protein, huntingtin (Htt). Pathogenesis is associated with expression of the mutant Htt (mHtt) protein in the CNS; however, HD is also associated with abnormalities in peripheral tissues. The Htt protein is the most significant molecular target for disease modifying therapies, and several therapeutic approaches directed at its production, processing, and/or turnover are under development for impending clinical trials. Measuring Htt and other disease proteins in peripheral cells represents an essential step in biomarker discovery for HD.
DESIGN/METHODS: In the current study, we measured Htt protein in saliva from manifest HD patients, gene-positive premanifest HD patients, and age- and sex-matched normal individuals (total n=178) using Western blots and ELISA methods. Additional salivary analytes, including alpha amylase, cortisol, C-reactive protein (CRP), and uric acid, were also measured using standardized ELISAs.
RESULTS: Salivary total Htt levels were significantly increased (p=0.0012) in saliva from HD individuals (mean=0.775 ng/ml) compared to normal controls (mean=0.359 ng/ml). Salivary total Htt did not vary over time of day or over different days, nor were there age or gender effects. Additionally, salivary mHtt levels were higher in gene positive premanifest HD subjects compared to normal controls (p<0.05). CRP, a widely used biomarker of systemic inflammation, was found to be significantly (p=0.025) elevated in premanifest HD subjects (9,548 pg/ml) compared to normal controls (3,399 pg/ml) and may be an early marker for disease onset. Levels of other salivary proteins, alpha amylase and uric acid, were not significantly different between HD patients, premanifest subjects, or normal controls.
CONCLUSIONS: Measurement of salivary Htt and other disease proteins offers significant promise as relevant, non-invasive biomarkers of disease onset and progression in HD.