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Introduction
Recent research has indicated a connection between amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD), an inherited neurological condition caused by a trinucleotide CAG repeat expansion within exon 1 of the Huntingtin gene (HTT; MIM:613004).1 The same pathogenic CAG repeat expansions (40 or more CAG repeats) observed in patients with HD have been identified in patients with frontotemporal dementia (FTD)/ALS.1 Similarly, non-pathogenic intermediate-length CAG repeats in the ATXN2 gene are a well-established factor associated with increased ALS risk and faster disease progression.2 Given these observations, we investigated the impact of intermediate HTT alleles on survival in two cohorts of patients diagnosed with ALS.
Methods
Discovery cohort
The study population consisted of 1181 patients with ALS identified through the Piemonte and Valle d’Aosta Register for ALS (PARALS) (online supplemental etable 1). Among these, 996 samples were part of the original report of HTT repeat expansions in individuals with FTD/ALS.3 The characteristics of the PARALS register are described in online supplemental materials. These subjects were negative for pathogenic mutations in C9orf72, SOD1, TARDBP and FUS.
Supplemental material
Replication cohort
As a replication cohort, we used clinical and genomic data from the Answer ALS database (https://dataportal.answerals.org/home). We retrieved whole-genome sequence data from 376 patients with ALS who did not have mutations in the four major ALS genes (online supplemental etable 1). The data processing pipeline is described in online supplemental materials.
HTT CAG repeat analysis
We used ExpansionHunter (V.5.0.0) to estimate repeat lengths of HTT expansions from the whole-genome sequence data. HTT CAG alleles were deemed ‘fully penetrant’ if they carried 40 or more repeats, ‘incompletely penetrant’ if they carried between 36 and 39 repeats and were designated as ‘intermediate’ if they carried between 27 and 35 repeats. Healthy subjects typically have 26 or fewer CAG repeats.
Statistical methods
Survival was calculated from symptom onset to death, tracheostomy or censoring date (31 December 2021 in the PARALS cohort; last available follow-up in the Answer ALS cohort). Survival times were calculated using the Kaplan-Meier method. Multivariate survival analysis was performed using the Cox proportional hazards model, modelling the presence of intermediate expansion as a binary variable. In a separate additional analysis, we included the number of HTT CAG repeats as a continuous variable. All models were adjusted for relevant clinical predictors; sensitivity analyses were conducted to evaluate the effect of population structure, haplogroups or genetic modifiers of HTT alleles. Further details on statistical methods and analysis are given in Supplementary Materials.
Data availability
The individual-level sequence data are available on the dbGaP web portal, accession number phs001963.3 The clinical data are available on reasonable request by interested researchers. The programming code used to analyse the data is available at https://github.com/maurigrassano/CAG_HTT_in_ALS.
Results
PARALS cohort
In the PARALS cohort, we discovered one patient with ALS carrying a pathogenic HTT allele (40 repeats) (online supplemental etable 2); this sample was not reported in the Dewan et al paper.3 In addition, we identified four ALS cases carrying incomplete penetrance CAG alleles (36–39 repeats, representing 0.33% of the cohort). These five subjects were excluded from the analyses.
We identified 79 (6.67%) ALS cases in the discovery cohort who carried an intermediate-length HTT CAG allele (ie, 27–35 repeats). None of these patients manifested atypical symptoms or clinical features distinctive of HD; their clinical characteristics are reported in online supplemental etable 3. Patients carrying intermediate HTT alleles had a median survival time of 29.3 months compared with the 34.5 months of those without (HR = 1.37, 95% CI 1.03 to 1.82, p=0.0318, figure 1A). There was no relationship between the number of HTT CAG repeats (8–35 repeats as continuous variables) and survival time (p=0.2597). The effect of HTT alleles on survival was unrelated to population structure or haplogroups (online supplemental efigure 1).
Survival from disease onset according to HTT CAG intermediate number of repeats in the Piemonte and Valle d’Aosta Register for ALS (PARALS) (A) and Answer amyotrophic lateral sclerosis (ALS) (B) cohort. (A) PARALS cohort (n = 1181). The blue line represents the survival of 79 patients with ALS carrying 27–35 CAG repeats (ie, intermediate length), and the red line represents the survival of 1102 patients with ALS carrying 26 or fewer CAG repeats (ie, normal length). (B) Answer ALS cohort (n = 476). The green line represents the survival of 30 patients with ALS carrying 27–35 CAG repeats (ie, intermediate length), and the orange line represents the survival of 346 patients with ALS carrying 26 or fewer CAG repeats (ie, normal length).
Answer ALS cohort
Within the replication cohort, we detected 30 (7.98%) ALS cases carrying HTT expansion in the 27–35 range (online supplemental etable 2). Their clinical characteristics were comparable to the rest of the cohort (online supplemental etable 4). Survival analysis confirmed that these patients had a worse prognosis, with a median survival time of 29.5 months compared with the 56.4 months observed in patients without intermediate HTT alleles (HR=1.85, 95% CI 1.06 to 3.25, p=0.0310, figure 1B). No correlation was observed between the number of HTT CAG repeats and survival duration (p=0.1048).
Discussion
This study found that patients with ALS carrying intermediate-length HTT expansions had a more severe disease course characterised by reduced survival rates. Our study underscores the importance of genetic factors in determining the natural history of ALS and supports the notion that additional studies in this area should be prioritised.
Our study has limitations, primarily the small number of cases carrying the intermediate HTT expansions available for phenotype analysis. Nevertheless, we replicated our findings in an independent cohort, suggesting that the HTT gene does influence ALS survival. Thus, this study is an additional example of the link connecting HTT with multiple neurodegenerative phenotypes and longevity (see online supplemental etable 5). It remains unclear whether the detrimental effects of CAG expansions in the HTT gene on ALS result from a direct interaction between poly-Q residues and TDP-43, or from other potential mechanisms.3 4 Further research is needed to elucidate the precise pathways involved. Notably, an enhancement of TDP-43 aggregation and toxicity has been hypothesised for polyQ expansions within ataxin 2 (encoded by the ATXN2 gene). Intriguingly, therapeutic lowering of ataxin 2 reduces TDP-43 aggregation, improving survival and motor function in a disease model.5 This observation led to the development of gene-directed therapy for ALS targeting polyQ repeats (ClinicalTrials.gov Identifier: NCT04494256).
In conclusion, our study contributes to the growing evidence linking HTT expansions to ALS pathology. It emphasises the crucial role of genetics in shaping disease progression and opens new avenues for intervention and treatment strategies, offering hope for improved outcomes for patients with ALS.
Ethics statements
Patient consent for publication
Ethics approval
This study was approved by Comitato Etico Azienda Ospedaliero-Universitaria Città della Salute e della Scienza, Torino (protocol number #0038876). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors thank the Laboratory of Neurogenetics (NIH) and ALS Centers staff for their collegial support and technical assistance.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
MG and AC contributed equally.
BT and AC contributed equally.
Contributors Concept and design: MG, ACanosa, BT, and AChio. Acquisition, analysis, or interpretation of data: all authors. Drafting of the manuscript: MG, ACanosa, SS, ACalvo, BT, and AChio. Critical review of the manuscript for important intellectual content and administrative, technical, or material support: SD, LC, GB, EK, PC, UM, RV, FP, LM, SG, CM, RD, RC, JD, CD and RJG. Statistical analysis: MG and EK. Obtained funding: SS, BT and AChio. Supervision: BT and AChio.
Funding This work was in part supported by the Italian Ministry of Health (Ministero della Salute, Ricerca Sanitaria Finalizzata, grant RF-2016-02362405), the European Commission's Health Seventh Framework Programme (FP7/2007-2013 under grant agreement 259867), the Italian Ministry of Education, University and Research (Progetti di Ricerca di Rilevante Interesse Nazionale [PRIN] grant 2017SNW5MB), the Joint Programme–Neurodegenerative Disease Research (ALS-Care, Strength and Brain-Mend projects), granted by the Italian Ministry of Education, University and Research, the Horizon 2020 Programme (project Brainteaser under grant agreement 101017598), the Fondazione Mario e Anna Magnetto and the Thierry Latran Foundation (INSPIRED project). This work was also supported in part by the Intramural Research Programs of the National Institute on Aging (grant Z01-AG000933) and the National Institute of Neurological Disorders and Stroke (grant ZIANS003154). This study was performed under the Department of Excellence grant of the Italian Ministry of Education, University and Research to the 'Rita Levi Montalcini' Department of Neuroscience, University of Torino, Italy, and to the Department of Health Sciences, University of Eastern Piedmont, Novara, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.
Competing interests AChio serves on scientific advisory boards for Mitsubishi Tanabe, Roche, Biogen, Denali Pharma, AC Immune, Biogen, Lilly, and Cytokinetics and has received a research grant from Biogen. BT holds the US, Canadian and European patents on the clinical testing and therapeutic intervention for the hexanucleotide repeat expansion in C9orf72. BT and SS received research support from Cerevel Therapeutics. ACalvo has received a research grant from Cytokinetics. MG has received grants from the American Academy of Neurology, the American Brain Foundation and the ALS Association.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.