23289503, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51460 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [20/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BRIEF COMMUNICATION Interaction between sex and neurofilament light chain on brain structure and clinical severity in Huntington’s disease Frederic Sampedro1,2,3,4 , Saul Martinez-Horta1,2,3, Jesús Pérez-Pérez1,2,3, Rocio Perez-Gonzalez1,2,3, Andrea Horta-Barba1,2,3, Antonia Campolongo1,2,3, Ignacio Aracil-Bolaños1,2,3, Beatriz Gomez-Anson4 & Jaume Kulisevsky1,2,3 1 Movement Disorders Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 2 Biomedical Research Institute (IIB-Sant Pau), Barcelona, Spain 3 Centro de Investigación en Red-Enfermedades Neurodegenerativas (CIBERNED), Barcelona, Spain 4 Neuroradiology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Correspondence Abstract Jaume Kulisevsky, Movement Disorders Unit, Neurology Department, Hospital de la Santa Female Huntington’s disease (HD) patients have consistently shown a faster Creu i Sant Pau, Mas Casanovas 90, 08041 clinical worsening than male, but the underlying mechanisms responsible for Barcelona, Spain. Tel: +34 93 5565986; this observation remain unknown. Here, we describe how sex modifies the E-mail: jkulisevsky@santpau.cat impact of neurodegeneration on brain atrophy and clinical severity in HD. Cerebrospinal fluid neurofilament light chain (NfL) levels were used as a bio- logical measure of neurodegeneration, and brain atrophy was assessed by struc- Funding information This study was partially funded by a grant tural magnetic resonance imaging. We found that larger NfL values in women from the Huntington’s Disease Society of reflect higher brain atrophy and clinical severity than in men (p < 0.05 for an America and by the PI17/001885 grant from interaction model). This differential vulnerability could have important implica- the Fondo de Investigaciones Sanitarias, tions in clinical trials. Instituto de Salud Carlos III (ISCIII), Fondo Europeo de Desarrollo Regional (FEDER). Received: 13 July 2021; Revised: 30 August 2021; Accepted: 14 September 2021 Annals of Clinical and Translational Neurology 2021; 8(12): 2309–2313 doi: 10.1002/acn3.51460 In vivo biomarker-by-sex interactions on clinical sever- Introduction ity and brain atrophy have contributed to better under- Huntington’s disease (HD) is an autosomal-dominant neu- stand the origin of sex-related differences in other rological disorder caused by a CAG repeat expansion in the neurodegenerative diseases. Among the available biomark- HTT gene driving a cascade of pathological processes lead- ers of neurodegeneration, cerebrospinal fluid (CSF) neu- ing to massive neuronal death. HD patients will experience rofilament light chain (NfL) levels have shown the best progressive motor, cognitive, and behavioral symptoms monitoring and prognostic performance in HD.4 resulting in a total loss of functional independence. In this work, we hypothesize that sex differences in the Female HD patients have consistently shown a faster clinical worsening of HD patients could be driven by a clinical worsening than male.1–3 However, the origin of differential impact of the underlying neurodegenerative this clinical observation is not well understood and needs processes on brain atrophy. Women would be then more to be investigated further, as it could shed light on novel vulnerable to the naturally occurring neurodegeneration pathological mechanisms. Crucially, a well-established as measured by NfL. To test this hypothesis, we per- gender difference in HD should have important implica- formed an NfL-by-sex interaction model on structural tions for the design and interpretation of clinical trials. neuroimaging data. ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 2309 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 23289503, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51460 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [20/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neurofilament-by-Sex Interaction in HD F. Sampedro et al. Materials and Methods M ¼ β0 þ β1 ∗sex þ β2 ∗logNfL þ βint ∗½sex∗logNfL þβ4 ∗age þ β5 ∗DBS Sample and assessments Using this model, we investigated the presence of a A total of 41 HD patients were included in this study significant NfL-by-sex interaction term (βint) independent from the outpatient clinic of the Movement Disorders of age and disease burden. For clinical variables and stri- Unit at Hospital de la Santa Creu i Sant Pau (Barcelona, atal volumes, significance of βint was set at p < 0.05. For Spain). All of them were confirmed gene mutation carri- the vertexwise Cth analyses, to be consistent with our ers (CAG length ≥39). This study was approved by the previous works,8 surface maps were smoothed using a appropriate ethics committee and all participants pro- Gaussian kernel of 15 mm full-width-at-half-maximum vided signed informed consent. to increase the signal-to-noise ratio; and only clusters The following clinical indicators were considered in this surviving p < 0.05 and family-wise error (FWE) correc- study. The disease burden score (DBS), defined as age × ( tion for multiple-comparison by a Monte Carlo simula- CAG-35.5), as an index of pathological burden due to tion with 10,000 repeats were considered significant. lifetime exposure to mutant huntingtin. Total functional Effect sizes for the interaction model were assessed by capacity (TFC) and motor symptoms (TMS) were Cohen’s f2. recorded from the Unified Huntington’s Disease Rating Finally, aiming to investigate the clinical translation of Scale (UHDRS). The following cognitive and neuropsy- the imaging findings, we performed the following chiatric indicators, known to be sensitive to HD progres- exploratory analyses. In the set of brain regions showing a sion with minimal medication-related alterations,5,6 were significant interaction effect, we computed its Cth/volume included: Symbol Digit Modality Test (SDMT), Stroop- values. Then, using Pearson’s correlation coefficients, we word task, and the apathy score of the Problem Behavior studied the association of these imaging alterations with Assessment (PBA). Finally, composite UHDRS (cUHDRS) the worsening of clinical indicators, for which a value of scores were computed, given its improved performance as p < 0.05 was considered significant. a measure of disease progression in HD.6 Biomarker and neuroimaging procedures Results CSF samples were collected, processed, aliquoted in Table 1 summarizes the sample’s sociodemographic, clini- polypropylene tubes, and frozen at −80°C until analysis cal, and biomarker characteristics. No significant sex- according to international consensus recommendations.7 related differences in terms of these variables were NfL levels were measured with the NF-light Advantage kit observed in this sample. (Cat# 103186) using the Single Molecule Array (Simoa) However, significant NfL-by-sex interactions were technology (Simoa; Quanterix, Lexington, MA, USA) in observed on: cUHDRS (interaction β = 3.0, p = 0.026, the SR-X Biomarker detection system by following the f2 = 0.19; NfL-correlation: males r = −0.44, p = 0.050; manufacturer’s instructions. A logarithmic transformation females r = −0.74, p < 0.001), SDMT (interaction (base 10) was applied to work with normally distributed β = 3.2, p = 0.034, f2 = 0.16; NfL-correlation: males CSF NfL data. r = −0.36, p = 0.122; females r = −0.68, p = 0.001) and Given that tau accumulation rate is greater in females, we Stroop-word (interaction β = 4.1, p = 0.007, f2 = 0.25; also measured CSF total Tau levels in order to discard that NfL-correlation: males r = −0.23, p = 0.328; females sex differences in our sample were related to an increase in r = −0.68, p = 0.001). No significant tau-by-sex interac- Tau pathology in the female group. CSF Tau was measured tions on clinical severity were observed. with the Neurology 3-Plex Advantage kit (Cat# 101995) The right caudate volume also showed a significant NfL- using the Simoa technology as described above. by-sex interaction: interaction β = 3.0, p = 0.035, Specific details on the acquisition and preprocessing f2 = 0.15; NfL-correlation: males r = −0.50, p = 0.023; steps of 3-Tesla T1-MRI images are available in our previ- females r = −0.65, p = 0.001. This interaction remained ous work.8 It is important to note that cortical thickness significant after further controlling for total intracranial (Cth) analyses are not significantly affected by differences volume. The vertexwise NfL-by-sex interaction model on in total intracranial volume or morphology. Cth revealed two significant clusters where NfL-related cor- tical thinning was more pronounced in the female group (Figure 1). In the right hemisphere, the fronto-temporal Statistical analysis cluster mainly included middle-frontal, insular, and The following interaction model was applied for each of anterosuperior-temporal regions (interaction β = 0.34, the considered clinical or neuroimaging variables M: p = 0.006, f2 = 0.25). In the left hemisphere, the frontal 2310 ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 23289503, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51460 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [20/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F. Sampedro et al. Neurofilament-by-Sex Interaction in HD Table 1. Sample characteristics. HD HD-Female HD-Male p-value n 41 21 20 Age [years] 44.2  11.4 43.6  10.7 44.9  12.4 0.71 Education [years] 12.6  4.1 12.7  4.1 12.5  4.2 0.89 CAG length 43.4  2.6 43.5  3.0 43.3  2.2 0.70 DBS 419.1  103.6 416.4  102.9 421.8  107.0 0.87 TFC 12.2  1.6 12.0  2.1 12.3  1.1 0.51 cUHDRS 14.4  4.5 14.2  5.0 14.6  4.0 0.79 UHDRS-TMS 12.8  18.3 11.8  15.8 13.8  20.9 0.73 SDMT 43.6  20.0 42.5  22.8 44.6  17.2 0.74 Stroop-word 86.6  27.2 85.1  32.2 88.1  21.4 0.72 PBA – Apathy item 2.2  2.5 2.5  2.4 1.9  2.6 0.39 CSF NfL [pg/mL] 1934.9  1225.4 1810.3  1305.7 2065.8  1153.9 0.51 CSF Tau [pg/mL] 98.1  40.1 88.7  39.3 108.5  39.3 0.12 Values are expressed as mean  standard deviation. DBS, Disease Burden Score; UHDRS, Unified Huntington’s Disease Rating Scale; cUHDRS, composite UHDRS score; TFC, Total Functional Capacity; UHDRS-TMS, Total Motor Score; SDMT, Symbol Digit Modality Test; PBA, Problems Behaviors Assessment; CSF NfL, Cerebrospinal fluid Neurofilament Light Chain. cluster mainly included the orbitofrontal cortex (interac- important if longitudinal studies confirm that increases in tion β = 0.46, p = 0.006, f2 = 0.24). CSF NfL over time translate into a faster atrophy rate in Finally, the following set of exploratory clinical- women. neuroimaging associations were observed in the set of From a clinical perspective, the NfL-by-sex interaction brain regions showing a significant NfL-by-sex interac- on cUHDRS is noteworthy. This composite score is a tion. Both the right caudate volume and the average Cth multidimensional measure of progression in HD being in the right frontotemporal cluster correlated with all used as a primary outcome in clinical trials.6 Interestingly, the considered clinical measures: cUHDRS r = 0.54, direct NfL-by-sex interactions on motor or psychiatric p < 0.001, and r = 0.60, p < 0.001, respectively; TFC = symptoms were not observed in our sample. However, as 0.44, p = 0.004, and r = 0.49, p = 0.001, respectively; expected, in an indirect manner, the integrity in the brain UHDRS-TMS r = −0.39, p = 0.011, and r = −0.57, regions showing a significant NfL-by-sex interaction cor- p < 0.001, respectively; SDMT r = 0.51, p = 0.001 and related with motor, cognitive, and psychiatric symptoms. r = 0.47, p = 0.002, respectively; Stroop-word r = 0.48, These results highlight the clinical relevance of the p = 0.002, and r = 0.56, p < 0.001, respectively; PBA— observed interaction. Apathy item r = −0.34, p = 0.027, and r = −0.35, Importantly, the increased vulnerability observed in the p = 0.026, respectively. female group appear unrelated to possible differences in tau pathology. Therefore, further research is needed to elucidate the specific pathological mechanisms responsible Discussion for this phenomenon. Vascular or hormonal factors could We found a significant NfL-by-sex interaction on brain be involved. Sex-related neurodevelopmental differences structure and clinical severity in HD. The impact of related to the HTT gene could also underlie the observed increasing NfL levels on brain atrophy and clinical sever- effects.9 Finally, as similar sex-related clinical differences ity was more pronounced in women. This could explain, were observed in spinocerebellar ataxia,10 sex-related at least partially, their faster clinical progression previ- alterations in dynamic mutation mechanisms could also ously described in large longitudinal cohorts. be implicated. Notably, CSF NfL levels were not significantly increased Limitations of this work include cross-sectional and a in the female group compared to males. This suggests relative low sample size. As this study was motivated by that women brains are more vulnerable to the naturally an HD-specific clinical observation with potential implica- occurring neurodegeneration in HD, that is, the same tions in clinical trials involving HD patients, we did not amount of measured NfL reflects higher damage in include a control group. The inclusion of other risk factors women than in men. These findings open up the possibil- such as the APOE genotype, MAPT haplotype, or cardio- ity to consider different CSF NfL reference limits for men vascular profiles could have also contributed to a better and women in HD clinical trials. This would be especially understanding of the origin of the observed interaction. ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 2311 23289503, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51460 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [20/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Neurofilament-by-Sex Interaction in HD F. Sampedro et al. Figure 1. NfL-by-sex interaction on cUHDRS scores (A), right caudate volume (B) and Cth (C). (D) Scatter plot illustrating the direction of the Cth interaction in the right fronto-temporal cluster. * A p < 0.05 for the interaction model. To conclude, we found that sex modifies the impact of Author Contributions HD’s neurodegeneration on clinical severity and brain atrophy. Overall, these results suggest that future studies FS: Data analysis and manuscript writing. SMH, AHB: using CSF NfL as a marker of neurodegeneration in Project conception and clinical project execution. RPG, HD should take into consideration this NfL-by-sex AC, IAB: Project execution: blood sample collection and interaction. processing. BGA, Project execution - MRI acquisition. JP, Manuscript review and critique. JK: Project organization, manuscript review, and critique. Acknowledgments The authors wish to thank all those at the Hospital de la Data Availability Statement Santa Creu i Sant Pau involved in the study. The authors also wish to thank the study participants and their The data that support the findings of this study are available families. from the corresponding author upon reasonable request. References Conflict of Interest 1. Hentosh S, Zhu L, Patino J, Furr JW, Rocha NP, None of the authors of this manuscript have any potential Stimming EF. Sex differences in Huntington’s disease: conflict of interest related to the content of this study. FS, evaluating the enroll-HD database, movement disorders SMH, RPG, AHB, IAB, AC, BGA, JP, and JK have noth- clinical. Practice. 2021;8:420-426. https://doi.org/10.1002/ ing to disclose. mdc3.13178 2312 ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 23289503, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/acn3.51460 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [20/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License F. Sampedro et al. Neurofilament-by-Sex Interaction in HD 2. Zielonka D, Marinus J, Roos RAC, et al. The influence of 6. Estevez-Fraga C, Scahill RI, Durr A, et al. Composite gender on phenotype and disease progression in patients UHDRS correlates with progression of imaging biomarkers with Huntington’s disease. Parkinsonism Relat Disord. in Huntington’s disease. Mov Disord. 2021;36(5):1259- 2013;19:192-197. https://doi.org/10.1016/j.parkreldis.2012. 1264. https://doi.org/10.1002/mds.28489 09.012 7. Teunissen CE, Petzold A, Bennett JL, et al. A consensus 3. Zielonka D, Ren M, De Michele G, et al. The contribution protocol for the standardization of cerebrospinal fluid of gender differences in motor, behavioral and cognitive collection and biobanking. Neurology. 2009;73:1914-1922. features to functional capacity, independence and quality https://doi.org/10.1212/WNL.0b013e3181c47cc2 of life in patients with Huntington’s disease. Parkinsonism 8. Sampedro F, Pérez-Pérez J, Martı́nez-Horta S, et al. Relat Disord. 2018;49:42-47. https://doi.org/10.1016/j.pa Cortical microstructural correlates of plasma neurofilament rkreldis.2018.01.006 light chain in Huntington’s disease. Parkinsonism Relat 4. Rodrigues FB, Byrne LM, Tortelli R, et al. Mutant Disord. 2021;85:91-94. https://doi.org/10.1016/j.parkreldis. huntingtin and neurofilament light have distinct 2021.03.008 longitudinal dynamics in Huntington’s disease. Sci Transl 9. Lee JK, Ding Y, Conrad AL, et al. Sex-specific effects of Med. 2020;12(574):eabc2888. https://doi.org/10.1126/scitra the Huntington gene on normal neurodevelopment. J nslmed.abc2888 Neurosci Res. 2017;95:398-408. https://doi.org/10.1002/jnr. 5. Martinez-Horta S, Perez-Perez J, van Duijn E, et al. 23980 Neuropsychiatric symptoms are very common in 10. Klockgether T, Lüdtke R, Kramer B, et al. The natural premanifest and early stage Huntington’s disease. history of degenerative ataxia: a retrospective study in 466 Parkinsonism Relat Disord. 2016;25:58-64. https://doi.org/ patients. Brain. 1998;121(Pt 4):589-600. https://doi.org/10. 10.1016/j.parkreldis.2016.02.008 1093/brain/121.4.589 ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 2313