Abstract
Background
A genetic diagnosis in ALS can inform genetic counselling, prognosis and, in the light of incoming gene-targeted therapy, management. However, conventional genetic testing strategies are often costly and time-consuming.
Objective
To evaluate the diagnostic yield and advantages of whole-genome sequencing (WGS) as a standard diagnostic genetic test for ALS.
Methods
In this population-based cohort study, 1043 ALS patients from the Piemonte and Valle d’Aosta Register for ALS (PARALS) and 755 healthy individuals were screened by WGS for variants in 42 ALS-related genes and for repeated-expansions in C9orf72 and ATXN2.
Results
A total of 279 ALS cases (26.9%) received a genetic diagnosis, namely 75.2% of patients with a family history of ALS and 21.5% of sporadic cases. The mutation rate among early-onset ALS patients was 43.9%, compared to 19.7% of late-onset patients. An additional 14.6% of the cohort carried a genetic factor that worsen prognosis.
Conclusions
Our results suggest that, because of its high diagnostic yield and increasingly competitive costs, along with the possibility of retrospectively reassessing newly described genes, WGS should be considered as standard genetic testing for all ALS patients. Additionally, our results provide a detailed picture of the genetic basis of ALS in the general population.
Introduction
Considerable progress has been made in unravelling the complex molecular mechanisms underlying ALS1. Nevertheless, it is not easy to gauge the impact of these genetic advances across the ALS space. Estimates of mutation carrier rate among ALS patients seen in specialist centers may be skewed due to referral bias2,3. This knowledge gap hinders efforts to develop therapies and to counsel patients effectively. For this reason, we systematically analyzed a population-based cohort of ALS patients using whole-genome sequencing. Our comprehensive approach provides a snapshot of what we currently know about the genetic architecture of ALS. In the process. The current study builds on our previous work, where we showed that whole-genome sequencing is a reliable tool to assess gene burden effects4.
Methods
Patients and controls cohort
We analyzed ALS patients who had been enrolled in Piemonte and Valle d'Aosta Register for ALS (PARALS). All samples have been previously analyzed for mutations in the SOD1, TARDBP, FUS and C9orf72 genes (eMaterials). An additional 755 healthy individuals underwent whole genome sequencing and were used as control data for mutation filtering.
Sequencing and bioinformatics analysis
Whole-genome sequencing methodology and quality control filters have been detailed elsewhere4-6 and are described in eMaterials. We extracted variant information for 46 genes previously implicated in ALS pathogenesis (eFigure 1). We estimated the repeat lengths of the C9orf72 and ATXN2 repeat expansions using ExpansionHunter – Targeted (version 0.3)7. For variants classification, we employed a framework based on the 2015 ACMG-AMP guidelines8. Loss-of-function and previously reported ALS variants were considered pathogenic unless present in the control cohort. The remaining rare variants (defined as minor allele frequency less than 0.0001 in the non-Finnish European population) were then classified based on computational prediction and expert review (eMaterials) and reported if deemed to be potentially pathogenic (eFigure 1).
Statistical analysis
A two-tailed Fisher exact test was used to evaluate the genetic association between ATXN2 CAG-repeat sizes and ALS. The burden of multiple variant carriers was assessed by a binomial test9. The analyses were performed in R (version 3.6.0).
Results
We analyzed a population-based cohort of 1,043 patients who had been diagnosed with ALS and enrolled in Piemonte and Valle d'Aosta Register for ALS (PARALS). The 1,043 patients represented 71.3% of incident cases during the 2007–2016 period. We found 96 mutations known to cause ALS among 203 cases, representing 19.5% of our cohort. An additional 76 patients (7.4%) carried potentially pathogenic variants based on our classification pipeline, bringing the total number of ALS cases with disease-causing variants to approximately a quarter of ALS cases (26.9%, n=279, Figure 1.A and eTable 1). The distribution of variants across ALS genes is reported in Figure 1.B. The most common mutation type was C9orf72 (present in 7.7% of ALS cases), followed by SOD1 (2.0%), NEK1 (1.8%), TARDBP (1.4%), and KIF5A (0.8%). All mutations were detected by both traditional Sanger sequencing and whole-genome sequencing screening, confirming the ability of whole-genome sequencing to identify relevant mutations correctly.
Figure 1. Distribution of mutated genes across our cohort.
(A)The frequency of mutations in all ALS cases (out band), sporadic cases (middle band), and familial cases (inner circle). (B) The number of potentially pathogenic variants identified per gene.
Diagnostic yield in familial and early-onset ALS cases
Subdividing our cohort, we detected disease-causing mutations in 88 (75.2%) familial ALS cases. We also observed a high mutation rate among early-onset ALS patients (age at onset<50 years, n=43, 43.9%) compared to late-onset patients (age at onset>75 years, n=41, 19.7%). Nearly half of the mutations observed in the elderly cohort were due to the C9orf72 repeat expansion, illustrating the reduced penetrance observed with this mutation10. Furthermore, 21.5% of apparently sporadic ALS patients carried a disease-causing variant (Figure 1.A).
Risk factor and prognostic variants
Intermediate-length ATXN2 CAG expansion (30-33 repeats) was the only high-risk genetic factor (defined as odds ratio≥2.0) identified in our cohort (odds ratio=2.84, 95% confidence interval=1.45-5.57, p=0.0023). The ATXN2 expansions were present in 41 (3.9%) patients. The prognostic UNC13A rs12608932 CC genotype was observed in 10.7% (n=112) of our ALS cases.
Oligogenic cases
Our cohort’s rate of oligogenic ALS cases was 1.3% (n=13) (eTable 2A). However, the proportion of oligogenic patients was not higher than expected (binomial p=0.98) based on the frequency of monogenic (25.6%) and non-mutated cases (73.1%) (eTable 2B).
Discussion
We detected a high rate of patients carrying pathogenic mutations (26.9%) in our population-based ALS cohort. This represents the largest percentage explained by genetic causes for any cohort reported to date2,11,12. As we sequenced an ALS cohort from a population-based registry, our data also represent a detailed audit of what is currently known about the genetic architecture of this fatal neurodegenerative condition in the general population. Our findings reflect both our increasing knowledge of the genetic architecture of ALS and the power of whole-genome sequencing to identify these variants. For example, the C9orf72 repeat expansion accounted for nearly one-third of all cases where the mutation was known.
Our results support the use of whole-genome sequencing in ALS patients at the time of their diagnosis, irrespective of family history, age at onset or clinical phenotype13. Whole-genome sequencing is a flexible tool that does not rely on a pre-determined set of genes and a variant prioritization process but can be adapted to different types of mutations as the collection of ALS-related genes evolves. The continuous improvement in variant calling and interpretation, along with our improving understanding of ALS genetics, will facilitate those endeavors. Ideally, periodic reevaluations of the whole-genome sequence data will become a feed-forward loop that enhances our knowledge with each iteration.
Our data do not support the oligogenicity theory of ALS, as it occurs very infrequently within the Italian population. The future discovery of more genes involved in ALS pathogenesis may reveal true oligogenicity. However, the existing data do not currently uphold a role for this disease mechanism.
Our primary reason for championing whole-genome sequencing as a routine test lies in the therapeutic implications for ALS patients. For example, we detected ATXN2 intermediate trinucleotide expansion in 41 ALS subjects (3.9%) and confirmed that it increased ALS risk. This result is clinically relevant since antisense oligonucleotide therapy targeting ATXN2 is in human trials at present14. Considering ATXN2, C9orf72, SOD1 and FUS, all genes currently under study for the treatment with ASOs15, at least 144 patients (13.8% of the cohort) could be candidates for gene-based therapy (Figure 1.A). Whole-genome sequencing allows those variants to be screened in a low-cost, rapid manner compared to traditional methodologies.
Our study is not without limitations. Above all, the lack of definite criteria for variant interpretation increases the risk of misclassifying ALS patients as wild-type or mutated. While our internal framework was reasonably robust, we do not maintain that it is definitive. Over time, the availability of updated mutation databases based on ever-larger cohort sizes and improved interpretation algorithms will enhance the analysis of these variants. Ambitious research programs are collecting whole-genome sequencing data from millions of human genomes to pair with phenotypical and long-term clinical data.
To summarize, our population-based evaluation sheds light on the complex genetics of ALS and provides a valuable benchmark of where the field currently stands. We show that whole-genome sequencing increases diagnostic yield and facilitates the assessment of the pathogenic role of involved genes. This information will be crucial for clinical care as precision therapeutics emerge as effective treatments. In anticipation of that, we consider whole-genome sequencing as the first-tier genetic test on all ALS patients.
Supplementary Material
Acknowledgments
This work was supported by the Italian Ministry of Health (Ministero della Salute, Ricerca Sanitaria Finalizzata, grant RF-2016-02362405); the Progetti di Rilevante Interesse Nazionale program of the Ministry of Education, University and Research (grant 2017SNW5MB); Horizon 2020 (grant RF H2020-SC1-DTH-2020-1, Grant agreement ID: 101017598); and the Joint Programme–Neurodegenerative Disease Research (Strength, ALS-Care and Brain-Mend projects), granted by Italian Ministry of Education, University and Research. This work was also supported by the Intramural Research Program of the NIH, National Institute on Aging (Z01-AG000949-02) and by the National Institute of Neurological Disorders and Stroke (1ZIANS003154). 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. The funders had no role in data collection or analysis and did not participate in writing or approving the manuscript. The Authors thank the Laboratory of Neurogenetics (NIH) staff for their collegial support and technical assistance
Footnotes
Search terms: Amyotrophic Lateral Sclerosis, Whole-genome sequencing. Population-based
Disclosures
Maurizio Grassano, Cristina Moglia, Luca Sbaiz, Maura Brunetti, Marco Barberis, Federico Casale, Umberto Manera, Rosario Vasta, Antonio Canosa, Sandra D’Alfonso, Lucia Corrado, Letizia Mazzini, Clifton Dalgard, Ramita Karra and Ruth Chia report no disclosures.
Andrea Calvo has received research grant from Cytokinetics.
Bryan J. Traynor holds European, Canadian and American patents on the clinical testing and therapeutic intervention for the hexanucleotide repeat expansion of C9orf72.
Adriano Chiò serves on scientific advisory boards for Mitsubishi Tanabe, Roche, and Cytokinetics, and has received a research grant from Italfarmaco.
Contributor Information
Maurizio Grassano, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; Neuromuscular Diseases Research Section, Laboratory of Neurogenetics, National Institute on Aging, NIH, Porter Neuroscience Research Center, Bethesda, MD 20892, USA.
Andrea Calvo, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; S.C. Neurologia 1U, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Cristina Moglia, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; S.C. Neurologia 1U, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Luca Sbaiz, Laboratory of Genetics, Department of Clinical Pathology, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Maura Brunetti, Laboratory of Genetics, Department of Clinical Pathology, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Marco Barberis, Laboratory of Genetics, Department of Clinical Pathology, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Federico Casale, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy.
Umberto Manera, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; S.C. Neurologia 1U, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Rosario Vasta, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy.
Antonio Canosa, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; S.C. Neurologia 1U, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy.
Sandra D’Alfonso, Department of Health Sciences, Interdisciplinary Research Center of Autoimmune Diseases, “Amedeo Avogadro” University of Eastern Piedmont, Novara, Italy.
Lucia Corrado, Department of Health Sciences, Interdisciplinary Research Center of Autoimmune Diseases, “Amedeo Avogadro” University of Eastern Piedmont, Novara, Italy.
Letizia Mazzini, ALS Center, Department of Neurology, Azienda Ospedaliera Universitaria Maggiore della Carità, Novara, Italy.
Clifton Dalgard, Department of Anatomy, Physiology & Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA; The American Genome Center, Collaborative Health Initiative Research Program, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Ramita Karra, Neuromuscular Diseases Research Section, Laboratory of Neurogenetics, National Institute on Aging, NIH, Porter Neuroscience Research Center, Bethesda, MD 20892, USA.
Ruth Chia, Neuromuscular Diseases Research Section, Laboratory of Neurogenetics, National Institute on Aging, NIH, Porter Neuroscience Research Center, Bethesda, MD 20892, USA.
Bryan J. Traynor, Neuromuscular Diseases Research Section, Laboratory of Neurogenetics, National Institute on Aging, NIH, Porter Neuroscience Research Center, Bethesda, MD 20892, USA; Reta Lila Weston Institute, UCL Queen Square Institute of Neurology, University College London, London WC1N 1PJ, UK; Department of Neurology, Johns Hopkins University Medical Center, Baltimore, MD 21287, USA; National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA; ASO Rapid Development Laboratory, Therapeutics Development Branch, National Center for Advancing Translational Sciences, NIH, Rockville, MD, USA.
Adriano Chiò, ALS Center, “Rita Levi Montalcini” Department of Neuroscience, University of Turin, Turin, Italy; S.C. Neurologia 1U, Azienda Ospedaliero Universitaria Città della Salute e della Scienza di Torino, Turin, Italy; Institute of Cognitive Sciences and Technologies, National Council of Research, Rome, Italy.
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