Method of detecting a biomarker for huntington's disease in a subject

Abstract

Method of detecting a biomarker for Huntington's disease in a subject, wherein the method comprises the step of detecting the biomarker for Huntington's disease, wherein the biomarker for Huntington's disease is mutant huntingtin, and wherein the biomarker for Huntington's disease is detected in a tear fluid sample obtained from the subject.

Description

[0001] Title: Method of detecting a biomarker for Huntington’s disease in a subject

[0002] Technical field

[0003] The present invention relates to a method of detecting a biomarker for Huntington’s disease in a subject. The present invention further relates to a method for diagnosing Huntington’s disease in a subject, a method for identifying premanifest or manifest Huntington’s disease in a subject and a method for monitoring the progression and severity of Huntington’s disease in a subject. The present invention also relates to the use of a tear fluid sample obtained from a subject in the detection of a biomarker for Huntington’s disease in the subject.

[0004] Huntington’s disease (HD) is an inherited disorder that causes nerve cells (neurons) in parts of the brain to gradually break down and die. The disease attacks areas of the brain that help to control voluntary (intentional) movement, as well as other areas. Huntington’s disease has a wide impact on a person's functional abilities and usually results in movement, thinking (cognitive) and psychiatric disorders.

[0005] To date, there is no treatment that can stop or reverse progression of Huntington’s disease. However, treatments and therapies are available to manage symptoms and improve the quality of life for affected individuals. The journey with Huntington’s disease is challenging for both patients and their families, but ongoing research aims to deepen our understanding of the disease and develop potential disease modifying treatments to slow down or prohibit onset and / or progression of the disease.

[0006] The clinical diagnosis of Huntington’s disease is predominantly made on the basis of characteristic motor signs often with a positive family history and / or confirmed by DNA analysis. Doctors conduct a thorough examination, looking for motor symptoms like chorea and cognitive changes. Imaging studies and genetic testing may be used to rule out other conditions. Since the disease is hereditary, family history is vital. However, genetic testing in persons at risk has important psychological and ethical implications, and counselling is recommended for informed decision-making.

[0007] Huntington’s disease is an inherited disorder. It is passed from parent to child through a mutation in a particular gene. When a parent has Huntington’s disease, each child has a 50% chance of inheriting the copy of chromosome 4 that carries the Huntington’s disease mutation. If a child does not inherit the Huntington’s disease mutation, he or she will not develop the disease and cannot pass it on to future generations.

[0008] Huntington’s disease is caused by a mutation in the gene for a protein called huntingtin (HTT). The defect causes the building blocks of DNA called cytosine, adenine, and guanine (CAG) to repeat many more times than they normally do.

[0009] Most people have fewer than 27 CAG repeats in their Huntington’s disease gene, so they are not at risk for the disease. People who have CAG repeats in the middle range (27 to 35) are not likely to develop the disease, but they could still pass it on to future generations. People with Huntington’s disease have 36 or more CAG repeats.

[0010] To the present, researchers are trying to better understand the cellular and molecular mechanisms involved in Huntington’s disease by investigating, for instance, how the huntingtin protein affects cell signalling and how its altered structure can contribute to disease. In addition, researchers are currently working to identify and validate biomarkers for Huntington’s disease. One goal of identifying biomarkers for Huntington’s disease is to determine disease-onset. Currently, the diagnosis is merely made on the basis of motor symptoms. However, early motor symptoms are variable in presentation and occurrence and may easily be missed during neurological examination. Furthermore, cognitive and behavioural changes may be present up to 10 years from estimated Huntington’s disease diagnosis. These personality and cognitive changes, such as irritability, depression or burnout, are not always disease specific. They, however, contribute to a decline in daily functioning and are often the most distressing aspects of Huntington’s disease for patients, families as well as their caregivers. Another goal is to find biomarkers that measure disease progression of Huntington’s disease. Especially, in the search of disease modifying treatments, this is of utmost importance to detect effectiveness of such treatments.

[0011] A related study aims to identify additional human genetic factors that influence the course of the disease. Finding genetic variants that slow or accelerate the pace of disease progression promise to provide important new targets for disease intervention and therapy. To date, biomarkers for Huntington’s disease that have been assessed in subjects are obtained from body fluids including cerebrospinal fluid, peripheral blood, and saliva. A succinct summary of the most interesting molecular biomarkers is, for example, provided by Marti-Martinez (Marti-Martinez, Silvia, and Luis M. Valor. “A glimpse of molecular biomarkers in Huntington’s disease.” International Journal of Molecular Sciences 23.10 (2022): 5411). An overview of blood-based biomarkers for Huntington’s disease is, for example, provided by Zhang (Zhang, Sirui, Yangfan Cheng, and Huifang Shang. “The updated development of blood-based biomarkers for Huntington’s disease.” Journal of Neurology (2023): 1-21).

[0012] Given the disclosure of Marti-Martinez and recent developments in diagnosing and monitoring Huntington’s disease, it is noted that an increasing number of biomarkers are proposed to predict the age of onset, relating to non-specific invalidating neuropsychiatric symptoms, and severity of the disease in Huntington’s disease. Validation of these candidates has usually been hampered due to the low prevalence of the disorder, making the recruitment of relatively homogeneous cohorts difficult. As it is concluded by Marti-Martinez, the detection of Neurofilament Light (NfL) in blood and cerebrospinal fluid seems to be one of the most suitable approaches to monitor the health status of Huntington’s disease patients. Also, Marti-Martinez concludes that more tightly linked to Huntington’s disease-specific dysfunction, examination of PDE10A offers the possibility for non-invasive imaging, but requires extensive preparation of individuals.

[0013] As it is concluded by Zhang, the monogenic mutation in Huntington’s disease provides an opportunity to identify premanifest Huntington’s disease before clinical disease-onset. However, to investigate the effect of potential disease-modified treatment strategies robust biomarkers that can objectively and quantitatively reflect pathophysiologic changes in Huntington’s disease patients are urgently required. It is noted by Zhang that although numerous potential peripheral biomarkers in bloodbased samples have been proposed and investigated, many of them demonstrated disappointing and conflicting results. Among those blood-based biomarkers, plasma NfL appeared to be the most promising and encouraging peripheral biomarker. Although it is a non-specific biomarker which can be confused with neuronal injuries caused by other diseases, the strength and practicability of blood-based NfL are highlighted in the case of Huntington’s disease since gene testing and mutant huntingtin (mHTT)-related neuropathology could provide additional information. The expression of mHTT, as a characteristic change in Huntington’s disease, has been investigated but, compared to NfL, it is not a reliable biomarker in plasma, so far. The phase III trial of the antisense oligonucleotide tominersen presented disappointing results, and some investigators suggested that allele-selective treatment strategies might be more reasonable, indicating the necessity of quantitative analysis of both mHTT and wild-type HTT in the future.

[0014] Given the current status of the developments in detecting and monitoring Huntington’s disease, as aptly described and explained in both Marti-Martinez and Zhang, there is a need of detecting biomarkers for Huntington’s disease in a reliable, objective and, preferably, quantitatively manner. It is noted that present techniques of identifying, diagnosing, and monitoring Huntington’s disease do require invasive sampling of cerebrospinal fluid (via lumbar puncture).

[0015] Detailed description

[0016] In order to provide a non-invasive, reliable and objective method for diagnosing and monitoring Huntington’s disease in a subject, the present invention provides hereto, in a first aspect, a method of detecting a biomarker for Huntington’s disease in a subject, wherein the method comprises the step of detecting the biomarker in a tear fluid sample obtained from the subject. It was found that tear fluid of a subject is a suitable medium for the detection of a biomarker for Huntington’s disease, such as mutant huntingtin (mHTT) protein.

[0017] At date, Huntington’s disease specific biomarkers, such as mHTT protein, are only detectable in cerebrospinal fluid resulting in a highly invasive sampling of cerebrospinal fluid (via lumbar puncture). Given the fact that the sampling of cerebrospinal fluid is highly invasive, sampling is performed often in patients diagnosed with Huntington’s disease, premanifest Huntington’s disease patients, or Huntington’s disease patients showing motor symptoms, like chorea, and cognitive changes indicating the onset of Huntington’s disease. By providing a simple, non- invasive sampling method, the present invention now provides for a method suitable for detecting, monitoring and diagnosing Huntington’s disease which method is not only relevant for patients already diagnosed with Huntington’s disease, but also for patients not diagnosed with Huntington’s disease and not showing motor symptoms, like chorea, and / or cognitive changes.

[0018] In fact, it was found that the signal of Huntington’s disease specific biomarkers, such as mHTT protein, in tear fluid is significantly stronger than that in cerebrospinal fluid. This newfound sensitivity in tear fluid allows for the detection of Huntington’s disease specific biomarkers signals even in premanifest patients, where Huntington’s disease specific biomarkers signals would typically be undetectable in cerebrospinal fluid.

[0019] As already noted, the tear fluid sampling process is far less invasive than the conventional lumbar puncture required for cerebrospinal fluid. The tear fluid sampling may involve a method wherein a tear sampling device, such as a simple and inexpensive paper strip, is placed in the lower or upper eyelid, where it absorbs the tear fluid. It is needless to note that patients generally prefer non-invasive diagnostic techniques over medium invasive diagnostic techniques (such as blood sampling) and highly invasive diagnostic techniques (such as cerebrospinal fluid sampling).

[0020] In addition, by providing the non-invasive and simple method of the present invention, the detection of a Huntington’s disease specific biomarker becomes readily available to any patient, in particular patients who cannot undergo cerebrospinal fluid sampling due to various reasons, such as contraindications, risk of complications, advanced dementia, old age, or immobility.

[0021] As such, the tear sampling method of the present invention presents an ideal alternative with minimal contraindications, complications, or side effects, making it possible to determine a Huntington’s disease specific biomarker, such as mHTT protein, and / or other related biomarker state for each patient. Unlike cerebrospinal fluid sampling, which is usually a one-time procedure due to its highly invasive nature, tear fluid sampling can be easily repeated during follow-up consultations, providing valuable new information on the disease's natural history and progression.

[0022] Furthermore, tear sampling is highly accessible as it does not require any specialized instruments or trained physicians, in contrast to cerebrospinal fluid sampling that is limited to specialized hospitals. The method of the present invention thus allows for direct implementation in existing clinical practice infrastructure, including local hospitals, general practices, retirement homes, and even in countries wherein patients having limited access to healthcare and / or no existing clinical practice infrastructure is in place.

[0023] Given the method of the present invention, it is noted that the biomarker may be mutant huntingtin (mHTT) or a combination of Neurofilament Light (NfL) and mutant huntingtin. It was found that the biomarker mutant huntingtin is a suitable and discriminating biomarker for Huntington’s disease.

[0024] In addition to the detection of the biomarker for Huntington’s disease, the method of the present invention also provides for the quantification of the biomarker for Huntington’s disease detected in the tear fluid sample obtained from the subject. To this end, the present invention provides for a method, wherein the method comprises the step of, after detecting the biomarker for Huntington’s disease in the tear fluid sample obtained from the subject, quantifying the biomarker for Huntington’s disease in the tear fluid sample obtained from the subject.

[0025] With the quantification of the biomarker for Huntington’s disease in the tear fluid sample obtained from the subject, the method of the present invention provides for a method for diagnosing Huntington’s disease in a subject, for identifying premanifest or manifest Huntington’s disease in a subject, and / or for monitoring the progression and severity of Huntington’s disease in a subject. In fact, it was found that the use of a tear fluid sample obtained from a subject provides for a highly efficient and highly specific method of detecting the biomarker for Huntington’s disease. As such, by detecting a biomarker for Huntington’s disease in a tear fluid sample, the method of the present invention now provides for a non-invasive method for diagnosing and monitoring patients with Huntington’s disease.

[0026] In particular, the present invention relates to a method for diagnosing Huntington’s disease in a subject, wherein the method comprises the steps of: i) detecting the biomarker for Huntington’s disease; ii) quantifying the biomarker for Huntington’s disease detected in step i); iii) comparing the concentration of the biomarker for Huntington’s disease quantified in step ii) with an optimal threshold value (Youden index) obtained from concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene; and iv) diagnosing the subject with Huntington’s disease in case the concentration of the biomarker for Huntington’s disease quantified in step ii) is above the optimal threshold value compared with in step iii).

[0027] By providing a method for diagnosing wherein the biomarker for Huntington’s disease is mutant huntingtin and wherein the biomarker is detected in a tear fluid sample obtained from the subject, a highly sensitive, specific and non-invasive method for diagnosing Huntington’s disease in a subject is obtained.

[0028] In an embodiment of the present invention, the method for diagnosing Huntington’s disease in a subject is suitable for diagnosing a subject with premanifest Huntington’s disease. In order to diagnose a subject with premanifest Huntington’s disease, the concentration of the biomarker for Huntington’s disease is compared with (and above) an optimal threshold value (Youden index) obtained from concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with premanifest Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene.

[0029] In another embodiment of the present invention, the method for diagnosing Huntington’s disease in a subject is suitable for diagnosing a subject with manifest Huntington’s disease. In order to diagnose a subject with manifest Huntington’s disease, the concentration of the biomarker for Huntington’s disease is compared with (and above) an optimal threshold value (Youden index) obtained from concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with manifest Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene.

[0030] In a second aspect of the present invention, the invention provides for a method for diagnosing Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the biomarker for Huntington’s disease detected in step a), wherein the biomarker is mutant huntingtin, and wherein the subject is diagnosed with Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 500 fM. It was observed that mutant huntingtin concentrations of above 500 fM are found in tear fluid samples obtained from subjects having 36 or more CAG repeats. It was further observed that mutant huntingtin concentrations of above 500 fM are found in tear fluid samples obtained from subjects identified with premanifest Huntington’s disease and manifest Huntington’s disease. In other words, a mutant huntingtin concentrations of above 500 fM in a tear fluid sample obtained from a subject, is a clear discriminator for diagnosing subjects with Huntington’s disease.

[0031] Although the lower threshold of 500 fM is already sufficient to make the diagnosis, it is further noted that subjects having 36 or more CAG repeats do have significant higher concentrations of mutant huntingtin in their tear fluid samples compared to subjects having 27 or less CAG repeats. As such, a subject may also be diagnosed with Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 750 fM. More preferably, the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 1000 fM. Even more preferably, the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 1250 fM.

[0032] In a third aspect of the present invention, the invention relates to a method for identifying premanifest Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the detected biomarker for Huntington’s disease detected in step a), wherein the biomarker is mutant huntingtin, and wherein the subject is identified with premanifest Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 500 fM and does not exceed 4000 fM.

[0033] It was observed that in premanifest Huntington’s disease subjects, the concentration of mutant huntingtin in the tear fluid sample is lower compared to the concentration of mutant huntingtin in the tear fluid sample obtained from subjects identified with manifest Huntington’s disease. Although an overlap in the mutant huntingtin concentration ranges between premanifest Huntington’s disease patients and manifest Huntington’s disease patients was observed, it was observed that a mutant huntingtin concentration in the range of 500 fM to 4000 fM is found in tear fluid samples obtained from subjects with premanifest Huntington’s disease only.

[0034] Preferably, the method of the present invention relates to a method for identifying premanifest Huntington’s disease in a subject wherein the mutant huntingtin concentration in the tear fluid sample obtained from the subject is in the range of 750 fM to 4000 fM, 1000 fM to 4000 fM, or 1250 fM to 4000 fM. More preferably, the mutant huntingtin concentration in the tear fluid sample obtained from the subject is in the range of 1500 fM to 4000 fM, 2000 fM to 3750 fM, or 2500 fM to 3500 fM. Even more preferably, the mutant huntingtin concentration in the tear fluid sample obtained from the subject is in the range of 2500 fM to 4000 fM.

[0035] In a fourth aspect of the present invention, the invention relates to a method for identifying manifest Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the detected biomarker for Huntington’s disease detected in step a), wherein the biomarker is mutant huntingtin, and wherein the subject is identified with manifest Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 10,500 fM.

[0036] It was observed that in manifest Huntington’s disease subjects, the concentration of mutant huntingtin in the tear fluid sample is higher compared to the concentration of mutant huntingtin in the tear fluid sample obtained from subjects identified with premanifest Huntington’s disease. Although an overlap in the mutant huntingtin concentration ranges between manifest Huntington’s disease patients and premanifest Huntington’s disease patients was observed, it was observed that a mutant huntingtin concentration above 10,500 fM is found in tear fluid samples obtained from subjects with manifest Huntington’s disease only.

[0037] Preferably, the method of the present invention relates to a method for identifying manifest Huntington’s disease in a subject wherein the mutant huntingtin concentration in the tear fluid sample obtained from the subject is in the range of 10,500 fM to 12,500 fM.

[0038] In a fifth aspect of the present invention, the invention relates to a method for monitoring the progression and severity of Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; b) quantifying the detected biomarker for Huntington’s disease detected in step a); and c) after a period of time, repeating steps a) and b), wherein the biomarker is mutant huntingtin, and wherein the progression and severity of Huntington’s disease in the subject is monitored by comparing the difference in concentration of the biomarker for Huntington’s disease as quantified in step b). It was found that a higher concentration of the biomarker for Huntington’s disease detected after the period of time can be used as an indicator for the progression and increasing severity of Huntington’s disease in the subject.

[0039] As such, the present invention provides for a method wherein the monitoring of patients diagnosed with Huntington’s disease is readily available without the need of subjecting the patient diagnosed with Huntington’s disease to highly invasive methods for monitoring purposes. By providing a readily available, non-invasive and highly specific monitoring method, the present invention now provides for opportunities to monitor the progression and severity of Huntington’s disease in a subject and the effectiveness of new and existing drug candidates.

[0040] Given the above, it is noted that the tear fluid sample may be obtained from the subject by using a tear sampling device, such as a paper strip. Typically (but not necessarily) such a tear sampling device is configured to absorb tear fluid of the subject. To this end, the tear sampling device may be placed in the lower or upper eyelid of the subject for a predefined amount of time, such as about 5 minutes, to absorb the tear fluid by the tear sampling device.

[0041] In an embodiment of the present invention, the tear fluid sample obtained from the subject may be diluted in an extraction buffer. Preferably, the tear fluid sample is diluted from 3-fold to 7-fold. More preferably, the tear fluid sample is diluted from 4- fold to 6-fold. Even more preferably, the tear fluid sample is diluted about 5-fold, however also other dilutions may be found suitable for detecting the biomarker for Huntington’s disease in a subject.

[0042] In case a paper strip is used as a tear sampling device, the volume of the tear fluid sample obtained from the subject mat be determined based on the migration length of the tear fluid on the paper strip.

[0043] In a sixth aspect of the present invention, the invention relates to the use of a tear fluid sample obtained from a subject in the detection of a biomarker for Huntington’s disease in the subject. The biomarker is mutant huntingtin. Optionally, the tear fluid sample obtained may also be used to detected mutant huntingtin in combination with Neurofilament Light.

[0044] In a seventh aspect of the present invention, the invention relates to the use of the tear fluid sample obtained from a subject according to the sixth aspect of the present invention for: diagnosing Huntington’s disease in the subject; identifying premanifest Huntington’s disease in the subject; identifying manifest Huntington’s disease in the subject; or monitoring the progression and severity of Huntington’s disease in the subject.

[0045] Brief description of the figures

[0046] Figure 1 shows a box plot of the concentration of mutant huntingtin (mHTT) detected in tears of manifest patients, premanifest patients and a control group.

[0047] Figure 2 shows the differences in tear mHTT levels per study group. (A) Tear mHTT levels differed significantly between manifest (n = 20) and premanifest (n = 13) patients as compared to age-matched controls (n = 20) (p < 0.0001 in both cases). (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) Within the premanifest group, there was a statistically significant difference in tear mHTT levels between early (n = 9) and late (n = 4) premanifest patients. (D) Within the premanifest group, there was no statistically significant difference in tear mHTT levels between pre-A en pre-B premanifest patients. (E) There was no correlation between tear mHTT levels and age. (F) Significant correlations were found between tear mHTT levels and CAG repeat length. Group differences were analysed by ANOVA with Tukey’ multiple comparisons test. Unpaired t-test was used to test the difference between early and late premanifest. All correlations were analysed by the Pearson’s correlation test. Horizontal bars indicate mean ± SD.

[0048] Figure 3 shows the tear mHTT level correlations with clinical parameters. (A) 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 score. Tear mHTT levels did not correlate with UHDRS TMS (D) and UHDRS TFC (E). All correlations were analysed by the Pearson’s correlation test.

[0049] Figure 4 shows the tear mHTT levels in relation to tear parameters. (A) Normalized tear mHTT levels differed significantly between manifest (n = 20) and premanifest (n = 13) patients as compared to age-matched controls (n = 20) (p = 0.0011 and p = 0.204, respectively). (B) There was no statistically significant difference in tear migration length across diagnostic groups. (C) Tear migration length did not correlate with tear mHTT levels. All group differences were analysed by ANOVA with Tukey’ multiple comparisons test. All correlations were analysed by the Pearson’s correlation test. Horizontal bars indicate mean ± SD.

[0050] Figure 5 shows the diagnostic ability of tear mHTT. The ROC curves discriminate between (A) control and manifest, (B) control and premanifest and (C) premanifest and manifest. Tear mHTT cut-off levels to distinguish patients from controls are represented by the dotted lines (D).

[0051] Experimental data 1

[0052] A first study was performed wherein mutant huntingtin (mHTT) protein was detected in (diluted) tears of manifest patients (n = 15), premanifest patients (n = 14) and control group (n = 15). The control group was based on subjects have fewer than 27 CAG repeats in their Huntington’s disease gene, i.e. , subjects not at risk for the disease. The results are shown in the box plot of Figure 1 , where the solid line represents the mean, and the whiskers represent the minimum and maximum value. As it can be derived from the results depicted in Figure 1 , the detected mHTT signals can easily distinguish patients from the control group.

[0053] In addition, it was found that the detection signals of mHTT in (diluted) tears (ranging from 3500 to 19,000 fM mHTT) of Huntington’s disease gene expansion carriers (HDGECs) are significantly higher compared to the detection signals of mHTT in undiluted cerebrospinal fluid (ranging from 8 to 150 fM mHTT) as reported in literature (see for example: Byrne, Lauren M., et al. “Evaluation of mutant huntingtin and neurofilament proteins as potential markers in Huntington’s disease.” Science translational medicine 10.458 (2018): eaat7108 and Wild, Edward J., et al. “Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients.” The Journal of clinical investigation 125.5 (2015): 1979-1986.). This opens opportunities to detect mHTT in premanifest HDGECs (i.e. , patients that carry the genetic defect but do not have symptoms yet). In cerebrospinal fluid, mHTT in premanifest patients is usually not detectable or close to the limit of detection (LOD = 8 fM mHTT). data 2

[0054] A second study was performed in order to detect and quantify Huntington’s Disease biomarkers in tear fluid of premanifest and manifest HDGECs compared to age-matched controls.

[0055] Study Design and participants

[0056] Participants were recruited from the MLIMC Huntington’s Expertise Centre and social media. Inclusion criteria for manifest Huntington’s disease patients were: age 18 years or older;

[0057] Unified Huntington’s Disease Rating Scale (UHDRS) Total Motor Score (TMS) > 5 (maximum score 124);

[0058] Diagnostic Confidence Level (DCL) of 4 (motor abnormalities that are unequivocal signs of HD, > 99 % confidence);

[0059] CAG repeat length > 36; and total functioning capacity (TFC) score > 2 (maximum score 13).

[0060] Inclusion criteria for premanifest Huntington’s disease patients were: age 18 years or older;

[0061] CAG repeat length > 36; and

[0062] UHDRS TMS <5.

[0063] Furthermore, age of onset Huntington’s disease and the duration of Huntington’s disease were documented.

[0064] Inclusion criteria for age-matched controls were: age 18 years or older; and no history of neurodegenerative disorders.

[0065] The CAG repeat was available from a subset of age-matched controls (n= 5 / 20).

[0066] From all participants general baseline characteristics were collected such as age, gender, comorbidity, and medication use. Exclusion criteria for all participants were mental incapacity and brain injury, e.g., brain tumour, epilepsy, encephalitis, or cerebrovascular accident (CVA) (<2 years before participation).

[0067] Clinical assessments

[0068] Participants were assessed using the UHDRS TMS being a reliable clinical rating scale to evaluate clinical performance and capacity in Huntington’s disease. Higher TMS indicate a higher degree of motor impairment. In addition, the TFC score was measured. This scale is a clinician checklist assessing overall functioning. Higher scores correspond with a better functional performance.

[0069] TFC scores were translated into a five-stage disease classification that corresponds with disease severity: stage 1 (11-13); stage 2 (7 to 10); stage 3 (3-6); stage 4 (1-2); and stage 5 (0). For each participant, the disease burden score ((CAG- 35.5)*age), 5-year onset probability, and estimated time to diagnosis were calculated.

[0070] Premanifest patients were subdivided into early (disease burden score < 250) and late (disease burden score > 250) and into pre-A (estimated time to diagnosis <17.5 years) and pre-B (estimated time to diagnosis >17.5 years). Assessments were conducted by an experienced and certified neurologist or trained and certified researcher.

[0071] Tear fluid sampling and pre-processing

[0072] Tear fluid was collected from both eyes via Schirmer’s paper strips (TEAR strips, Contacare Ophthalmics and diagnostics, Gujarat, India). The paper strips were placed at the junction of the middle and lateral thirds of the lower eyelid without topical anaesthesia. Exactly after 5 minutes, the strips were removed from the eyelid, the migration lengths (= part of the strip that is wetted with tear fluid) were recorded and samples were stored at -80°C.

[0073] Tear fluid was extracted from the Schirmer’s strips by agitating small cut pieces of these strips in 150 pL PBS, 1% Tween20 and complete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland) at 4°C for 1.5 hours. Subsequently, tear fluid was eluted by centrifugation and stored at -80°C until further use.

[0074] Tear samples were run blinded to the investigators and in duplicate. Tear fluid samples from one eye only (left or right) were used for mHTT analysis and randomized. A pilot experiment showed that undiluted and 5-times diluted tear fluid samples yield mHTT detection signals within the linear range of the standard curve (data not shown). Subsequently, all samples were diluted 5-fold and measured in technical duplicates.

[0075] Expanded Mut-HTT quantification on SMCxPRO

[0076] This assay employs the Single Molecule Counting (SMC™) technology which affords ultra-sensitivity and a wide linear detection range. Specific detection of mHTT in this assay occurs by immunoreaction with capture antibody 2B7 and detection antibody MW1 . The 2B7 antibody recognizes the N terminus of mouse and human HTT (residues 7-13) and the MW1 antibody binds to the polyQ-expanded domain of the mHTT protein. Prior to sample measurements, both capture and detection antibodies were labelled and stored until further use. The 2B7 antibody (Evotec batch) was labelled and coated to magnetic particles (MP) and stored at 2-8°C according to manufacturer’s instructions (SMC™ Capture Labelling Kit; #03-0077-02). The MW1 antibody (Evotec batch) was labelled and stored at 2-8°C according to manufacturer’s instructions (SMC™ Capture Labelling Kit; #3-0076-02).

[0077] Standard protein and samples were analysed as follows. 50 pl / well of D2X buffer + protease inhibitor (PI; 5X) was distributed into the assay plates (96-well V- plate). Then, 120 pL / well of standard (HTT-Q73, aa 1-573) or sample, diluted 1 :5 in artificial CSF (aCSF; 150 mM NaCI, 3 mM KCI, 1.4 mM CaCI2, 0.8 mM MgCI2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4) + 1% Tween-20 + PI, was distributed to the plates in required wells. As samples were analysed on two different plates, an inter-plate quality control (QC) was prepared by pooling 20 pl sample from six donors randomly selected. The QC sample was diluted 1 :5 similarly to the study samples and added to each plate in duplicate. A volume of 100 pL of 2B7 antibody (500 ng / mL; in assay buffer, Merck Millipore) was added to each well and the plates were sealed and incubated under shaking (400 rpm) at room temperature. After 1.5 hours, the plates were first placed on a magnetic stand and then washed once with 200 pL wash buffer (Merck Millipore).

[0078] All washing steps were performed with a HydroFlex TM microplate washer (Tecan). After washing, 20 pL / well of 0.22 pm filtered MW1 detection antibody (1000 ng / mL; in assay buffer, Merck Millipore) was immediately dispensed into each well and incubated under shaking (700 rpm) at room temperature for 1 hour. Each assay plate was washed, and MP were transferred to a new 96 well plate. After 4 washing rounds of the assay plates, 12 pL of elution buffer (Merck Millipore) was distributed in the wells. The plates were incubated for 6 minutes under shaking (700 rpm) followed by an incubation on the magnetic stand for about 2 minutes. The eluate was then transferred to a 384er read-out plate (containing neutralizing buffer D, Merck Millipore, 10 pL / well) and mixed for about 1-2 minutes on the shaker (450 rpm) and centrifuged for 2 minutes (about 1500 rpm). The plate was then sealed with an adhesive aluminium foil sheet and incubated in the SMCxPRO™ device for about 30 minutes for temperature adjustment before measurement. The assay was performed blinded to clinical state of the subjects.

[0079] The data was analysed using GraphPad Prism (version 9.5.1). The mut-HTT concentrations in the sample were obtained by interpolating signals measured in the samples using the five-parameter logistic (5 PL) calibration curve generated with the standards. The back-calculated concentrations obtained in the QC samples were compared and the variation between the two plates was calculated. A variation below 20% between the QCs was aimed for the analysis of the samples on the two plates together.

[0080] Statistical analyses

[0081] GraphPad Prism 10 was used to visualize data and calculate descriptives, grouped statistics and correlation analyses. Tear mHTT level duplicates were averaged and the mean was used for further analysis. Group differences were assessed via ANOVA with Tukey’s multiple comparisons test. Unpaired t-test was used to test the difference between early and late premanifest. Correlations between mHTT levels and continues variables were analysed by Pearson’ correlation test. Receiver operating curves (ROC), area under the curve (AUC) and associated values were generated using the Wilson / Brown method. The Youden's index was calculated by deducting 1 from the sum of test’s sensitivity and specificity expressed not as percentage but as a part of a whole number: (sensitivity + specificity) - 1. The statistical test used for calculating the significance of each graph is indicated in the figure legend. A p value < 0.05 was considered as significant.

[0082] Results

[0083] In all tear samples (n = 53) mHTT was detected. Consistent with previous reports using CSF, average tear mHTT levels in manifest (8092 ± 4149 fM) and premanifest patients (6615 ± 3864 fM) were significantly higher than in age-matched controls (208.2 ± 243.4 fM) (p < 0.0001 in both cases) (Figure 2A).

[0084] It was also observed that tear mHTT levels were higher in manifest patient as compared to premanifest patients, this difference was not statistically significant. For manifest patients, no differences were observed between disease stages 1 , 2 and 3 (Figure 2B). Significantly lower tear mHTT levels were found in early premanifest patients (4963 ± 3107 fM) as compared to late premanifest patients (10332 ± 2715 fM, p = 0.0127) (Figure 2C).

[0085] There was no statistically significant difference in tear mHTT levels between male (n = 21) and female (n = 32) participants (data not shown). Among all participants, there was no significant correlation between age and tear mHTT levels (Figure 2D). Significant positive correlations were observed between tear mHTT levels and CAG repeat length in the entire cohort (r = 0.6713, p < 0.0001) as well as in the manifest (r= 0.5267, p = 0.017) and premanifest (r = 0.6108, p = 0.0266) groups (Figure 2E).

[0086] The 5-year onset probability score did not correlate with tear mHTT levels in the premanifest group (Figure 3A). A negative correlation was found between the number of ‘estimated years to diagnosis’ and tear mHTT levels in the premanifest group (Figure 3B). The disease burden score correlated significantly with tear mHTT levels in the entire cohort (r = 0.4337, p = 0.0117) as well as in the premanifest (r = 0.6737, p = 0.016) group (Figure 3C). Tear mHTT levels did not correlate with LIHDRS TMS (Figure 3D) or UHDRS TFC (Figure 3E). Prior to the mHTT immunoassay, the collected tear fluid was being diluted in extraction buffer. Since the initial tear volume differs between participants, the dilution factor differs as well. In order to normalize for this, the obtained ‘raw’ concentrations are divided by the migration length (an approximate measure of tear volume) and multiplied by the extraction buffer volume. The resulting concentrations reflect ‘real’ individual tear mHTT levels. Figure 4A shows normalized tear mHTT levels per diagnostic group. The same trend could be observed as compared to un-normalized tear mHTT levels (Figure 1A): normalized tear mHTT levels were significantly higher in manifest (67,223 ± 80,360 fM) and premanifest (55,561 ± 45,931 fM) patients as compared to age-matched controls (1622 ± 2179 fM) (Figure 4A). There were no differences in tear migration length between manifest (27.1 ± 9.9 mm), premanifest (23.5 ± 10.5 mm) and age-matched controls (26.9 ± 10.4 mm) (Figure 4B), nor did tear migration length correlate with tear mHTT levels (Figure 4C).

[0087] The sensitivity and specificity of tear mHTT was analysed by examining its ability to discriminate between manifest and controls, premanifest and controls and between manifest and premanifest. The ROC curves and associated values are represented in Figure 5. The area under the ROC curve (AUC) between controls and manifest was 1.000, indicating a test that gives the correct answer every time (Figure 5A). The AUC between controls and premanifest was 0.9923, meaning a discriminatory ability of 99.23% or a 99.23% probability of the test giving the correct answer (Figure 5B). There was no significant difference between tear mHTT in discriminating between premanifest and manifest disease groups (Figure 5C). Further information regarding the sensitivity and specificity of tear mHTT is provided below in Table 1.

[0088] Table 1. Sensitivity and specificity test results analysis

[0089] The Youden index measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value (cutoff point) for the marker. The most optimal cut-off value (= highest Youden index) to distinguish between controls and manifest patients was 971.6 fM, whereas this was 1423 fM between controls and premanifest patients (Figures 5D, see also Table 2).

[0090] Table 2. Most optimal cut-off value

[0091] Summary

[0092] In summary, 20 manifest, 13 premanifest HDGECs and 20 age-matched controls were recruited. All patients underwent detailed assessments, including Unified Huntington’s Disease Rating Scale total motor score and total function capacity score. Tear fluid was collected using paper Schirmer’s strips. The level of tear mHTT was determined using the Expanded Mut-HTT Single Molecule Counting SMCxPRO technology.

[0093] Tear samples were obtained and mHTT at very high levels was detected in all HDGECs tear samples (n = 53). Average tear mHTT levels in manifest (8092 ± 4149 fM) and premanifest patients (6615 ± 3864 fM) were significantly higher than in age- matched controls (208.2 ± 243.4 fM). Significantly lower tear mHTT levels were found in early premanifest patients as compared to late premanifest patients. Positive correlations were noted between tear mHTT levels and CAG repeat length, and disease burden score. The area under the ROC curve (AUC) between controls and manifest was 1.000, indicating a test that gives the correct answer every time, and 0.9923 between controls and premanifest patients. The most optimal cut-off value (= highest Youden index) to distinguish between controls and manifest patients was 971.6 fM, whereas this was 1423 fM between controls and premanifest patients.

[0094] These findings provide evidence that tear mHTT levels have potential for early and non-invasive detection of alterations in Huntington’s disease and could be integrated into both clinical trials and the clinic.

Claims

CLAIMS1. Method of detecting a biomarker for Huntington’s disease in a subject, wherein the method comprises the step of detecting the biomarker for Huntington’s disease, characterized in that the biomarker for Huntington’s disease is mutant huntingtin, and in that the biomarker for Huntington’s disease is detected in a tear fluid sample obtained from the subject.

2. Method according to claim 1 , wherein method further comprises the step of detecting a further biomarker for Huntington’s disease in the tear fluid sample obtained from the subject, wherein the further biomarker for Huntington’s disease is Neurofilament Light.

3. Method according to claim 1 or 2, wherein the method comprises the step of, after detecting the biomarker for Huntington’s disease in the tear fluid sample obtained from the subject, quantifying the biomarker for Huntington’s disease in the tear fluid sample obtained from the subject.

4. Method for diagnosing Huntington’s disease in a subject, wherein the method comprises the steps of: i) detecting the biomarker for Huntington’s disease; ii) quantifying the biomarker for Huntington’s disease detected in step i); iii) comparing the concentration of the biomarker for Huntington’s disease quantified in step ii) with an optimal threshold value (Youden index) obtained from concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene; and iv) diagnosing the subject with Huntington’s disease in case the concentration of the biomarker for Huntington’s disease quantified in step ii) is above the optimal threshold value compared with in step iii), characterized in that the biomarker for Huntington’s disease is mutant huntingtin, and in that the biomarker for Huntington’s disease is detected in a tear fluid sample obtained from the subject.

5. Method according to claim 4, wherein the subject is diagnosed with premanifest Huntington’s disease in step iv) in case the concentration of the biomarker for Huntington’s disease is above an optimal threshold value (Youden index) obtainedfrom concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with premanifest Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene.

6. Method according to claim 4, wherein the subject is diagnosed with manifest Huntington’s disease in step iv) in case the concentration of the biomarker for Huntington’s disease is above an optimal threshold value (Youden index) obtained from concentration levels of the same biomarker for Huntington’s disease obtained from subjects diagnosed with manifest Huntington’s disease and subjects having fewer than 27 CAG repeats in their Huntington’s disease gene.

7. Method for diagnosing Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the biomarker for Huntington’s disease detected in step a), wherein: the biomarker is mutant huntingtin; and the subject is diagnosed with Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 500 fM, preferably above 750 fM, more preferably above 1000 fM, even more preferably above 1250 fM.

8. Method for identifying premanifest Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the detected biomarker for Huntington’s disease detected in step a), wherein: the biomarker is mutant huntingtin; and the subject is identified with premanifest Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 500 fM and does not exceed 4000 fM, preferably wherein the mutant huntingtin concentration is in the range of 2500 fM to 4000 fM.

9. Method for identifying manifest Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; and b) quantifying the detected biomarker for Huntington’s disease detected in step a), wherein: the biomarker is mutant huntingtin; and the subject is identified with manifest Huntington’s disease in case the mutant huntingtin concentration in the tear fluid sample obtained from the subject is above 10,500 fM, preferably wherein the mutant huntingtin concentration is in the range of 10,500 fM to 12,500 fM.

10. Method for monitoring the progression and severity of Huntington’s disease in a subject, wherein the method comprises the subsequent steps of: a) detecting the biomarker for Huntington’s disease in a tear fluid sample obtained from the subject; b) quantifying the detected biomarker for Huntington’s disease detected in step a); and c) after a period of time, repeating steps a) and b), wherein: the biomarker is mutant huntingtin; and the progression and severity of Huntington’s disease in the subject is monitored by comparing the difference in concentration of the biomarker for Huntington’s disease as quantified in step b), wherein a higher concentration of the biomarker for Huntington’s disease detected after the period of time is used as an indicator for the progression and increasing severity of Huntington’s disease in the subject.

11. Method according to any of the preceding claims, wherein the tear fluid sample from the subject is obtained by using a tear sampling device, such as a paper strip.

12. Method according to claim 11 , wherein the tear sampling device is configured to absorb tear fluid of the subject.

13. Method according to claim 11 or 12, wherein the tear sampling device is placed in the lower or upper eyelid of the subject for a predefined amount of time, preferably about 5 minutes.

14. Method according to any of the preceding claims, wherein the tear fluid sample obtained from the subject is diluted in an extraction buffer, preferably wherein the tear fluid sample is diluted from 3-fold to 7-fold, preferably from 4-fold to 6-fold, more preferably about 5-fold.

15. Method according to any of the preceding claims, wherein, in case a paper strip is used as a tear sampling device, the volume of the tear fluid sample obtained from the subject is determined based on the migration length of the tear fluid on the paper strip.

16. Use of a tear fluid sample obtained from a subject in the detection of a biomarker for Huntington’s disease in the subject, wherein the biomarker is mutant huntingtin and, optionally, Neurofilament Light.

17. Use of the tear fluid sample according to claim 16 for: diagnosing Huntington’s disease in the subject; identifying premanifest Huntington’s disease in the subject; identifying manifest Huntington’s disease in the subject; or monitoring the progression and severity of Huntington’s disease in the subject.

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