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.

Jody Corey-Bloom1, Alaina M Aikin2, Sungmee Park1, Ameera Haque1, Ajay S Nathan1, Douglas A Granger3, Steven W Granger4, Elizabeth A. Thomas2

1Department of Neurosciences, University of California San Diego, La Jolla, CA; 2Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, CA; 3Institute for Interdisciplinary Salivary Bioscience Research, UCI, Irvine, CA; 4Salimetrics, Carlsbad, CA

Figure 2. Salivary Htt levels are elevated in human HD patients

CONCLUSIONS

Biomarkers for HD: The mutation responsible for Huntington’s disease (HD) is an abnormal CAG repeat expansion in the HTT gene that encodes huntingtin (Htt), a large protein of 3,144 amino acids. Pathogenesis in HD arises mainly from the expression of mutant Htt, which leads to the formation of toxic soluble protein oligomers as well as insoluble aggregates 4, and contributes to the disruption of many intracellular pathways. While insoluble mHtt aggregates in the CNS is the primary pathological hallmark of HD, the formation of polyglutamine inclusions also occurs in non-CNS tissues 5. Further, patients with HD also exhibit multiple peripheral changes, including skeletal muscle abnormalities and peripheral immune system dysfunction 6.

Measurements of Htt in the brains of patients are not possible, hence several studies have turned to measuring Htt in CSF7 and blood8, 9 from human patients in order to assess the utility of Htt to serve as a biomarker. However, CSF collection requires a lumbar puncture, which is highly invasive, and CSF levels of Htt are known to be very low7. Measurements of Htt in blood have been successful, however levels can vary depending on the blood cell type, large starting volumes (i.e. 50 ml) are often required and blood drawing itself is also an invasive technique. In this study, we explored whether Htt and other disease proteins could be reliably measured in saliva from HD patients and normal individuals.

Saliva as a biospecimen: Saliva is a composite of oral fluids secreted from many different glands, including the major and minor salivary glands, as well as the buccal, lingual, and palatal tissues 10. Like blood, saliva contains a wealth of hormones, proteins and nucleic acid molecules that reflect physiological status. Importantly, several disease proteins have been identified in human saliva, such as amyloid beta 11 and tau 12, which are associated with Alzheimer’s disease, and alpha-synuclein 13 and DJ-1 14 15 , which are associated with Parkinson’s disease. Salivary levels of these proteins are thought to represent useful biomarkers for these diseases. In this study, we report that significant and reproducible levels of Htt can be detected in saliva from human subjects and that salivary Htt is correlated with HD symptoms, suggesting utility as a biomarker for symptom onset and, perhaps disease severity.

Figure 4. Correlations of salivary Htt to clinical measures

METHODS

REFERENCES

SalivarybiomarkersforHun3ngton’sdisease

Participants: Patients were recruited through the UCSD HDSA Center of Excellence under an IRB-approved protocol. Manifest HD inclusion criteria included a definitive diagnosis of HD with expanded trinucleotide CAG repeat of >36. Controls have no reported history of neurological or psychiatric disorders, and no use of psychoactive substances or medications.

Sample collection: All donors refrained from smoking, eating, drinking, or oral hygiene procedures for at least 1 hr prior to sample collection; then rinsed their mouths thoroughly with water. Samples were collected between 9 am and 12 noon using the passive drool method. At the time of use, saliva samples are thawed and centrifuged at 10,000 rpm for 10 min at 4C to remove insoluble material and cell debris. Supernatants were collected and used for all assays. Total protein in the supernatants was determined using the BCA protein assay kit (Pierce).

Western Blots: Human saliva supernatants were concentrated 4-fold by vacuum centrifugation or used un-concentrated (“neat”). Samples were separated by 7% SDS-PAGE and blotted onto nitrocellulose membranes using standard techniques. For immunodetection, the anti-Htt antibody MAB2166 (Millipore, 1:2000) was used.

ELISAs: Total Htt protein levels in saliva samples were quantified using a commercially available ELISA kit (LifeSpan BioSciences, Inc.) according to the manufacturers protocol. The recombinant protein standard in this kit corresponds to amino acids 802-940 of the human Htt protein, with antibodies corresponding to proteins including this region. Samples were assayed for CRP, cortisol, alpha-amylase, and urate using immunoassay kits optimized for saliva (Salimetrics, LLC in State College, PA, USA) following the manufacturers recommended protocol.

Statistics: Htt values obtained from ELISA in each diagnostic group were tested for outliers using the Grubbs test (Graphpad outlier calculator). After this test, 1 sample in the control group and 1 sample in the HD group were removed. The distribution of the values was tested for normality using the Kolmogorov-Smirnov normality test. Htt values in all groups, except the AR(+) group, showed a normal distribution. Differences between diagnostic groups were determined using One-way ANOVA followed by Dunnett’s post-test comparing all groups to the normal control group. Linear regression analysis (Spearman correlation) was used to compare Htt and other disease protein levels against clinical variables, as these measures were not normally distributed. Differences between males and females were determined using Student’s t test (unpaired; two-tailed). All analyses were performed using Graphpad software (Prism).

INTRODUCTION

ABSTRACT RESULTS

Figure 1. Western blot of full-length Htt protein in saliva of normal individuals.

Figure 3. Salivary Htt levels do not vary with gender, age, time of day or over different days

Htt levels in saliva measured by ELISA kit for total Htt (LSBio), following directions by the manufacturer. Differences in Htt levels were determined by one-way ANOVA followed by Dunnett’s post-test . NC, normal controls; AR, at-risk; Transitional. Bar graph on right shows the difference between all gene (+) individuals (AR+, transitional and HD), with the control group including gene (-) individuals. *, p<0.05.

In addition, salivary Htt in the same subjects did not vary over the course of the day (Fig. 3A). Consistent with this finding, salivary levels in a subset of n=40 patients were not correlated with time of day of sampling (Fig. 3B). Further, in patients who gave samples on four different days, there was remarkable consistency in the Htt measurements.

Figure 5. Other salivary analytes in HD patients

1. Arrasate M, Finkbeiner S. 2012; Exp Neurol 238:1-11.; 2. van der Burg JM, Bjorkqvist M, Brundin P. 2009; Lancet Neurol 8:765-774; 3.Wild EJ, Boggio R, Langbehn D, Robertson N, Haider S, Miller JR, et al. 2015; J Clin Invest 125:1979-1986; 4. Massai L, Petricca L, Magnoni L, Rovetini L, Haider S, Andre R, et al. 2013; BMC biochemistry 14:34; 5. Weiss A, Trager U, Wild EJ, Grueninger S, Farmer R, Landles C, et al. 2012; J Clin Invest 122:3731-3736; 6. Baum BJ. 1993; Annals of the New York Academy of Sciences 694:17-23; 7. Yan W, Apweiler R, Balgley BM, Boontheung P, Bundy JL, Cargile BJ, et al. 2009; Proteomics. Clinical applications 3:116-134; 8. Lenander-Lumikari M, Ihalin R, Lahteenoja H. 2000; Arch Oral Biol 45:347-354; 9. Helenius LM, Meurman JH, Helenius I, Kari K, Hietanen J, Suuronen R, et al. 2005; Acta Odontol Scand 63:284-293; 10. Holmstrom P, Syrjanen S, Laine P, Valle SL, Suni J. 1990; J Med Virol 30:245-248; 11. Matsuda S, Oka S, Honda M, Takebe Y, Takemori T. 1993; Scand J Immunol 38:428-434; 12. Belazi MA, Galli-Tsinopoulou A, Drakoulakos D, Fleva A, Papanayiotou PH. 1998; Int J Paediatr Dent 8:29-33; 13. Lopez ME, Colloca ME, Paez RG, Schallmach JN, Koss MA, Chervonagura A. 2003; Braz Dent J 14:26-31; 14. Streckfus C, Bigler L. 2005; Adv Dent Res 18:17-24; 15. Ryu OH, Atkinson JC, Hoehn GT, Illei GG, Hart TC. 2006; Rheumatology (Oxford) 45:1077-1086; 16. Drobitch RK, Svensson CK. 1992; Clin Pharmacokinet 23:365-379; 17. Granger DA, Fortunato CK, Beltzer EK, Virag M, Bright MA, Out D. 2012; J Adolesc 35:1081-1095; 18. Want EJ, O'Maille G, Smith CA, Brandon TR, Uritboonthai W, Qin C, et al. 2006; Analytical chemistry 78:743-752; 19. Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G. 2006; Analytical chemistry 78:779-787.

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Table1.HDPa3entSamples(n=178) HD Transi3onal AR(+) AR(-) NC

Ini3alCohort N 31 9 11 21 45 Gender,F:M 20:11 6:3 6:5 13:8 19:26 Meanage,yrs 55.3 52.4 44.3 46.3 52.1 Valida3onCohort N 10 1 17 13 20 Gender,F:M 7:3 0:1 9:8 6:7 11:9 Meanage,yrs 57.7 66.1 41.7 49.5 55.3

SUMMARY

•  Full-length Htt and other disease proteins can be detected in human saliva. •  Total Htt levels are elevated in saliva from HD patients; mHtt levels are elevated in pre-

symptomatic patients (data not shown). These could represent indicators of disease symptoms and markers for Htt-lowering therapies.

•  Salivary Htt levels do not vary with gender, age, time of day, or over different days •  Salivary Htt showed a significant positive correlation with the UHDRS Total Motor score and a

significant negative correlation with the UHDRS TFC score. •  Non-affected family members show elevated salivary cortisol, possibly indicative of an

abnormal stress response. •  CRP levels are elevated in pre-symptomatic patients, possibly suggestive of a pathological

inflammatory or metabolic state in HD and an early marker for disease.

To determine first whether Htt protein was present in human saliva, we performed Western blotting using the MAB2166 anti-Htt antibody on samples from four normal individuals. We detected a single band just above the 250 kDa marker, corresponding to the full-length Htt protein (Fig. 1A). In samples that had been concentrated four-fold, this signal increased approximately proportionally (3.06 fold increase) (Fig. 1B).

Salivary Htt showed a significant positive correlation with the UHDRS Motor score (Spearman’s r=0.264; p=0.049) and a significant negative correlation to the TFC score (Spearman’s r=-0.283; p=0.032). No significant correlations were observed for the other clinical measures.

Non-affected family members showed elevated salivary cortisol, possibly indicative of an abnormal stress response. CRP levels were elevated in pre-symptomatic patients, possibly indicating a pathological inflammatory or metabolic state in HD and an early marker for disease.

•  Measurements of salivary Htt, and possibly other salivary proteins, offer significant promise as relevant, non-invasive biomarkers of disease onset and severity in HD.

ACKNOWLEDGEMENTS

This study was supported by the UCSD Huntington’s Disease Society of America Center of Excellence and the UCSD Shiley-Marcos Alzheimer’s Disease Research Center NIH P50 AG005131.

MDS Annual Meeting 2017, Vancouver BC

Salivary Htt levels did not vary with gender or age.