Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease with the age at which characteristic symptoms manifest strongly influenced by inherited HTT CAG length. Somatic CAG expansion occurs throughout life and understanding the impact of somatic expansion on neurodegeneration is key to developing therapeutic targets. In 57 HD gene expanded (HDGE) individuals, ~23 years before their predicted clinical motor diagnosis, no significant decline in clinical, cognitive or neuropsychiatric function was observed over 4.5 years compared with 46 controls (false discovery rate (FDR) > 0.3). However, cerebrospinal fluid (CSF) markers showed very early signs of neurodegeneration in HDGE with elevated neurofilament light (NfL) protein, an indicator of neuroaxonal damage (FDR = 3.2 × 10−12), and reduced proenkephalin (PENK), a surrogate marker for the state of striatal medium spiny neurons (FDR = 2.6 × 10−3), accompanied by brain atrophy, predominantly in the caudate (FDR = 5.5 × 10−10) and putamen (FDR = 1.2 × 10−9). Longitudinal increase in somatic CAG repeat expansion ratio (SER) in blood was a significant predictor of subsequent caudate (FDR = 0.072) and putamen (FDR = 0.148) atrophy. Atypical loss of interruption HTT repeat structures, known to predict earlier age at clinical motor diagnosis, was associated with substantially faster caudate and putamen atrophy. We provide evidence in living humans that the influence of CAG length on HD neuropathology is mediated by somatic CAG repeat expansion. These critical mechanistic insights into the earliest neurodegenerative changes will inform the design of preventative clinical trials aimed at modulating somatic expansion. ClinicalTrials.gov registration: NCT06391619 . A comprehensive longitudinal analysis of individuals with preclinical Huntington’s disease identifies biomarkers of neurodegeneration and somatic expansion in blood DNA, detectable years before symptom onset.

Huntington's Disease Integrated Staging System (HD-ISS) stages are likely inclusion criteria in future clinical trials. Stage 1 volumetric cut-offs were derived using the FreeSurfer longitudinal stream (LG). However, trials will require cross-sectional stream (CS) application with one MRI. Volumetric outputs are not robust to software type or version. T1-weighted images from 88 participants with MRIs from baseline and follow-up HD-YAS visits were segmented using both streams. CS calculated smaller caudate and putamen volumes adjusted for total intracranial volume, with greater reduction for larger volumes, shifting towards HD-ISS stage 1. CS-specific cut-offs need to be established before application to clinical trials.

Promising blood-based biomarkers of neuropathology have emerged with potential for therapeutic development and disease monitoring. However, these tools will require specialist tertiary services for integration into clinical management. Remote sampling for biomarker assessment could ease the burden of in-person clinical visits for such tests and allow for frequent sampling. Here we evaluated a capillary finger-prick collection for remote quantification of blood neurofilament light (NfL), a common blood-based biomarker evident in various neurological disorders, and other exploratory markers of neuronal injury and neuroinflammation (GFAP, tau). Matched samples from venepuncture and finger-prick were collected and processed into plasma and/or serum to directly compare NfL levels across four different neurological conditions (HD, MS, ALS, PD). Two delayed processing conditions were compared, three- and seven-day delay, simulating ambient shipment. Capillary NfL and GFAP concentrations were equivalent to those in venous blood serum and plasma. Only NfL remained stable after seven-day processing delay. Capillary NfL replicated disease group differences displayed in venous blood. This data supports our finger-prick method for remote collection and quantification of NfL. With the widespread applications for NfL across the spectrum of neurological disorders, this has the potential to transform disease monitoring, prognosis, and therapeutic development within clinical practice and research. Graphical abstract: Figure 1 ### Competing Interest Statement LMB holds consultancy contracts with Annexon Biosciences, Remix Therapeutics, PTC Therapeutics, Alchemab Therapeutics, and LoQus23 Therapeutics Ltd via UCL Consultants Ltd. HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). AN received consultancy fees during the design phase of AccessPD and reports consultancy and personal fees from AstraZeneca, AbbVie, Profile, Roche, Biogen, UCB, Bial, Charco Neurotech, Alchemab, Sosei Heptares and Britannia, outside the submitted work. AN is an Associate Editor for the Journal of Parkinsons Disease. In the previous 12 months SJT has received research grant funding from the CHDI Foundation, the National Institute for Health Research, and the UK Medical Research Council. Through the offices of UCL Consultants Ltd, a wholly owned subsidiary of University College London, SJT has undertaken consultancy services in the past 12 months for Alnylam Pharmaceuticals, Annexon, Ascidian Therapeutics, Arrowhead Pharmaceuticals, Atalanta Therapeutics, Design Therapeutics, F. Hoffman-La Roche, Iris Medicine, Latus Bio, LifeEdit, Novartis Pharma, Pfizer, Prilenia Neurotherapeutics, PTC Therapeutics, Rgenta Therapeutics, Takeda Pharmaceuticals, UniQureBiopharma, Vertex Pharmaceuticals. In the past 12 months, University College London Hospitals NHS Foundation Trust, Professor Tabrizis host clinical institution, received funding to run clinical trials for F. Hoffman-La Roche, Novartis Pharma, PTC Therapeutics, and UniQure Biopharma. ### Funding Statement This work and the salaries of LMB, AC, and AT were supported by a Medical Research Council Career Development Award (MR/W026686/1). HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#202 01018 and #2019 02397), the European Unions Horizon Europe research and innovation programme under grant agreement No 101053962, Swedish State Support for Clinical Research (#ALFGBG 71320), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809 2016862), the AD Strategic Fund and the Alzheimers Association (#ADSF 21 831376 C, #ADSF 21 831381 C, and #ADSF 21 831377 C), the Bluefield Project, the Olav Thon Foundation, the Erling Persson Family Foundation, Stiftelsen for Gamla Tjanarinnor, Hjarnfonden, Sweden (#FO2022 0270), the European Unions Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No 860197 (MIRIADE), the European Union Joint Programme Neurodegenerative Disease Research (JPND2021 00694), the National Institute for Health and Care Research University College London Hospitals Biomedical Research Centre, and the UK Dementia Research Institute at UCL (UKDRI 1003). AN reports grants from Parkinsons UK, Barts Charity, Cure Parkinsons, National Institute for Health and Care Research, Innovate UK, Virginia Keiley benefaction, Solvemed, the Medical College of Saint Bartholomews Hospital Trust, Alchemab, Aligning Science Across Parkinsons Global Parkinsons Genetics Program (ASAP GP2) and the Michael J Fox Foundation. SJT received research grant funding from the Wellcome Trust (223082/Z/21/Z), and the UK Dementia Research Institute that receives its funding from DRI Ltd., funded by the UK MRC, Alzheimers Society, and Alzheimers Research UK. We are grateful to the United to End MND (U2EM) and UK MND Research Institute consortium for their contributions for the recruitment of the ALS patients in this study. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Hospital for Neurology and Neurosurgery and the Institute of Neurology Joint Research Ethics Committee of University College London gave ethical approval for this work. London, City & East Research Ethics committee of Queen Mary University of London gave ethical approval for this work. South West, Central Bristol Research Ethics Committee of Queen Mary University of London gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Background The Huntington’s Disease Young Adult Study (HD-YAS) provided a detailed analysis of early premanifest Huntington’s disease (preHD) participants along with age and sex-matched controls. We previously reported slightly smaller putamen volumes in the preHD cohort versus controls with no evidence of motor, psychiatric or cognitive impairment at 23·6 years from predicted disease onset. Aims We aim to quantify cortical layer-specific neuronal loss and breakdown of cortico-striatal and inter-hemispheric white matter connectivity using 7T qMRI and 3T diffusion MRI. We will also evaluate struc- ture-function relationships using magnetoencephalography (MEG). Finally, we will use 2-year follow-up data to determine change over time. Methods We will perform MEG and 7T MRI on 20 participants with premanifest HD and 20 matched controls from the HD-YAS 2.0 cohort. Results We report on our 7T-MRI and MEG methodology in the 7/40 assessments completed to date. In addition, we will present our 7T-MRI pilot data and outline our planned analysis for 7T-MRI and MEG. Conclusion High-field MRI and MEG will allow us to seek early pathogenic changes in a far-from-onset preHD cohort in unprecedented detail. In addition, performing this study as part of HD-YAS 2.0 will allow us to integrate our findings within an extensive deep phenotyping dataset of participants using clinical, cognitive, neuropsychiatric and biofluid analysis as part of HD-YAS 2.0.

Background Huntington’s disease (HD) is an autosomal dominant neurodegenerative condition. The Young Adult Study (HD-YAS) characterised the earliest pathological manifestations in premanifest HD gene carriers (preHD) ~24 years from predicted clinical onset. HD-YAS 2.0 will provide 5- and 7-year follow up to identify change over time in key biomarkers and facilitate further deep phenotyping of preHD. Methods Participants recruited from the baseline HD-YAS cohort (64 preHD and 67 matched controls) will undergo cognitive and neuropsychiatric assessments, plasma and CSF biofluid marker testing and high-resolution 3T MRI over two study visits. Results At baseline, compared to controls, the preHD cohort showed no evidence of motor, cognitive or neuropsychiatric differences. Putaminal volume reduction was significant but not related to age-by-CAG interaction. Pre-HD had elevated NfL and YKL-40 but only CSF NfL showed significant age-by-CAG inter- action. NfL levels were within the 95th percentile of controls in 53% and 87% of the preHD cohort in CSF and plasma, respectively. We will report on early data collection and participant retention in HD-YAS 2.0. Conclusion Longitudinal follow up will help understanding of early HD pathogenesis dynamics. Identifying reliable, sensitive and stable biomarkers of change in preHD is paramount to inform early preventative treatment trials.

Abstract Upregulation of functional network connectivity in the presence of structural degeneration is seen in the premanifest stages of Huntington’s disease (preHD) 10–15 years from clinical diagnosis. However, whether widespread network connectivity changes are seen in gene carriers much further from onset has yet to be explored. We characterized functional network connectivity throughout the brain and related it to a measure of disease pathology burden (CSF neurofilament light, NfL) and measures of structural connectivity in asymptomatic gene carriers, on average 24 years from onset. We related these measurements to estimates of cortical and subcortical gene expression. We found no overall differences in functional (or structural) connectivity anywhere in the brain comparing control and preHD participants. However, increased functional connectivity, particularly between posterior cortical areas, correlated with increasing CSF NfL level in preHD participants. Using the Allen Human Brain Atlas and expression-weighted cell-type enrichment analysis, we demonstrated that this functional connectivity upregulation occurred in cortical regions associated with regional expression of genes specific to neuronal cells. This relationship was validated using single-nucleus RNAseq data from post-mortem Huntington’s disease and control brains showing enrichment of neuronal-specific genes that are differentially expressed in Huntington’s disease. Functional brain networks in asymptomatic preHD gene carriers very far from disease onset show evidence of upregulated connectivity correlating with increased disease burden. These changes occur among brain areas that show regional expression of genes specific to neuronal GABAergic and glutamatergic cells.

Background Neurofilament light protein (NfL) has emerged as the leading biomarker candidate for disease progression and early detection of adult-onset Huntington’s Disease (HD). Juvenile-onset Huntington’s disease (JOHD) is a rare and particularly devastating form of Huntington’s disease (HD) for which clinical diagnosis is challenging and robust outcome measures are lacking. Aim 1) Evaluate plasma NfL as a biomarker of HD in children 2) develop a method for remote NfL quantification to facilitate frequent monitoring, using at-home finger-prick blood collections. Methods We performed a retrospective analysis of samples and data collected between 2009 and 2020 from the Kids-HD and Kids-JHD studies. Matched blood plasma and serum was collected from both venipuncture and finger-prick. NfL concentrations were quantified using ultrasensitive immunoassay. Results We report elevated plasma NfL concentrations in JOHD and premanifest HD mutation-carrying children. In pediatric HD mutation carriers who were within 20 years of their predicted onset and patients with JOHD, plasma NfL level was associated with caudate and putamen volumes. Preliminary data of this novel finger-prick blood collection method for NfL quantification shows strong agreement with NfL levels from the venous blood gold standard. Conclusions Quantifying plasma NfL concentration may assist clinical diagnosis and therapeutic trial design in the pediatric population. Remote blood collection may be used to study NfL in large numbers of HD mutation carriers and Juvenile-onset HD patients at-home with frequent sampling.

Background: Pathological processes in Huntington's disease (HD) begin many years prior to symptom onset. Recently we demonstrated that in a premanifest cohort approximately 24 years from predicted disease onset, despite intact function, there was evidence of subtle neurodegeneration. Here, we use novel imaging techniques to determine whether macro- and micro-structural changes can be detected across the whole-brain in the same cohort. Methods: 62 premanifest HD (PreHD) and 61 controls from the HD Young Adult Study (HD-YAS) were included. Grey and white matter volume, diffusion weighted imaging (DWI) measures of white matter microstructure, multiparametric maps (MPM) estimating myelin and iron content from magnetization transfer (MT), proton density (PD), longitudinal relaxation (R1) and effective transverse relaxation (R2*), and myelin g-ratio were examined. Group differences between PreHD and controls were assessed; associations between all imaging metrics and disease burden and CSF neurofilament light (NfL) were also performed. Volumetric and MPM results were corrected at a cluster-wise value of familywise error (FWE) 0.05. Diffusion and g-ratio results were corrected via threshold-free cluster enhancement at FWE 0.05. Findings: We showed significantly increased R1 and R2*, suggestive of increased iron, in the putamen, globus pallidum and external capsule of PreHD participants. There was also a significant association between lower cortical R2*, suggestive of reduced myelin or iron, and higher CSF NfL in the frontal lobe and the parieto-occipital cortices. No other results were significant at corrected levels. Interpretation: Increased iron in subcortical structures and the surrounding white matter is a feature of very early PreHD. Furthermore, increases in CSF NfL were linked to microstructural changes in the posterior parietal-occipital cortex, a region previously shown to undergo some of the earliest cortical changes in HD. These findings suggest that disease related process are occurring in both subcortical and cortical regions more than 20 years from predicted disease onset.

BACKGROUND:Disease-modifying treatments are in development for Huntington's disease; crucial to their success is to identify a timepoint in a patient's life when there is a measurable biomarker of early neurodegeneration while clinical function is still intact. We aimed to identify this timepoint in a novel cohort of young adult premanifest Huntington's disease gene carriers (preHD) far from predicted clinical symptom onset. METHODS:We did the Huntington's disease Young Adult Study (HD-YAS) in the UK. We recruited young adults with preHD and controls matched for age, education, and sex to ensure each group had at least 60 participants with imaging data, accounting for scan fails. Controls either had a family history of Huntington's disease but a negative genetic test, or no known family history of Huntington's disease. All participants underwent detailed neuropsychiatric and cognitive assessments, including tests from the Cambridge Neuropsychological Test Automated Battery and a battery assessing emotion, motivation, impulsivity and social cognition (EMOTICOM). Imaging (done for all participants without contraindications) included volumetric MRI, diffusion imaging, and multiparametric mapping. Biofluid markers of neuronal health were examined using blood and CSF collection. We did a cross-sectional analysis using general least-squares linear models to assess group differences and associations with age and CAG length, relating to predicted years to clinical onset. Results were corrected for multiple comparisons using the false discovery rate (FDR), with FDR <0·05 deemed a significant result. FINDINGS:Data were obtained between Aug 2, 2017, and April 25, 2019. We recruited 64 young adults with preHD and 67 controls. Mean ages of participants were 29·0 years (SD 5·6) and 29·1 years (5·7) in the preHD and control groups, respectively. We noted no significant evidence of cognitive or psychiatric impairment in preHD participants 23·6 years (SD 5·8) from predicted onset (FDR 0·22-0·87 for cognitive measures, 0·31-0·91 for neuropsychiatric measures). The preHD cohort had slightly smaller putamen volumes (FDR=0·03), but this did not appear to be closely related to predicted years to onset (FDR=0·54). There were no group differences in other brain imaging measures (FDR >0·16). CSF neurofilament light protein (NfL), plasma NfL, and CSF YKL-40 were elevated in this far-from-onset preHD cohort compared with controls (FDR<0·0001, =0·01, and =0·03, respectively). CSF NfL elevations were more likely in individuals closer to expected clinical onset (FDR <0·0001). INTERPRETATION:We report normal brain function yet a rise in sensitive measures of neurodegeneration in a preHD cohort approximately 24 years from predicted clinical onset. CSF NfL appears to be a more sensitive measure than plasma NfL to monitor disease progression. This preHD cohort is one of the earliest yet studied, and our findings could be used to inform decisions about when to initiate a potential future intervention to delay or prevent further neurodegeneration while function is intact. FUNDING:Wellcome Trust, CHDI Foundation.

Background: Huntington's disease (HD) results in motor, cognitive, and psychosocial impairments.Little is known about the specific relationships among cognitive, psychosocial, and simple and complex motor task performance.Objective: The objective of this pilot study was to explore performance on motor, cognitive, and behavioral measures between individuals with HD and healthy controls, and to determine the relationships among specific domains of cognitive function and motor function and pain.Methods: Individuals with HD and healthy controls performed a battery of cognitive, motor, and survey measures (i.e., fall reports, quality of life, and pain) in a single session.We examined differences between individuals with HD and controls, as well as relationships among motor, cognitive, and survey measures.Results: Four individuals with HD (mean(SD) age: 62.5(14.5);symptom duration: 5.8(7.1); 1 female) and six healthy controls (age: 50.7(9.7);6 females) completed this study.There was no significant difference between groups for age, gender, or years of education.As expected, individuals with HD performed significantly worse on motor and cognitive testing, as evidenced by slower walking (p = 0.019), poorer lower extremity coordination (p = 0.038), poorer working memory (p = 0.010), and worse pain severity (p = 0.010).Poorer working memory was significantly related to poorer performance timed walking (r = -0.705;p = 0.023); lower extremity coordination (r = -0.657;p = 0.039); and dual-tasks (r = -0.760;p = 0.011), as well as reports of more falls (r = -0.709;p = 0.022).Poorer cognitive processing speed was specifically related to worse dual-task performance (r = -0.733;p = 0.016).Worse pain severity was significantly related to slower timed walking (r = 0.679; p = 0.031); poorer lower extremity coordination (r = 0.743; p = 0.014); and worse dual-task performance (r = 0.731; p = 0.016), as well as poorer working memory (r > -0.811; p < 0.004) and reports of more falls (r = 0.713; p = 0.021).Conclusions: Individuals with HD experience declines in motor and cognitive performance, as well as increased pain, compared to healthy controls.Quick and easily administered motor tests, such as timed walking and lower extremity coordination, as well as surveys to assess pain, may be useful in determining underlying cognitive impairments in working memory and processing speed.Assessment of these factors may help clinicians to tailor rehabilitation protocols to improve outcomes in persons with HD.