Human salivary Raman fingerprint as biomarker for the diagnosis of Amyotrophic Lateral Sclerosis

www.nature.com/scientificreports OPEN Human salivary Raman fingerprint as biomarker for the diagnosis of Amyotrophic Lateral Sclerosis C. Carlomagno1, P. I. Banfi1, A. Gualerzi 1, S. Picciolini1, E. Volpato1,2, M. Meloni1, A. Lax1, E. Colombo3,4, N. Ticozzi 3,4, F. Verde3, V. Silani 3,4,5 & M. Bedoni 1 ✉ Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to progressive and irreversible muscle atrophy. The diagnosis of ALS is time-consuming and complex, with the clinical and neurophysiological evaluation accompanied by monitoring of progression and a long procedure for the discrimination of similar neurodegenerative diseases. The delayed diagnosis strongly slows the potential development of adequate therapies and the time frame for a prompt intervention. The discovery of new biomarkers could improve the disease diagnosis, as well as the therapeutic and rehabilitative effectiveness and monitoring of the pathological progression. In this work saliva collected from 19 patients with ALS, 10 affected by Parkinson’s disease, 10 affected by Alzheimer’s disease and 10 healthy subjects, was analysed using Raman spectroscopy, optimizing the parameters for detailed and reproducible spectra. The statistical multivariate analysis of the data revealed a significant difference between the groups, allowing the discrimination of the disease onset. Correlation of Raman data revealed a direct relationship with paraclinical scores, identifying multifactorial biochemical modifications related to the pathology. The proposed approach showed a promising accuracy in ALS onset discrimination, using a fast and sensitive procedure that can make more efficient the diagnostic procedure and the monitoring of therapeutic and rehabilitative processes in ALS. Amyotrophic Lateral Sclerosis (ALS) is a complex and lethal neurodegenerative disease that progressively leads to irreversible muscle atrophy due to the death of motoneurons replaced by gliosis, with a life expectation from the onset of first symptoms between 2 and 5 years, depending on the cases1. This disorder affects both lower and upper motoneurons with symptoms including generalized muscle weakness, possible cognitive dysfunction, cramps, fasciculations, spasticity, serious functional limitations with parallel and progressive paralysis leading to death, typically resulting from ventilatory failure2. An American study showed that there are 223,000 people affected by ALS worldwide with an incidence of 1.75/100,000 and a predicted increase of 69% in 2040 due to the population aging3. The causes for ALS disease are still unclear with different mechanisms proposed including genetic, environmental, viral, immunological and epidemiological factors4. Compared to other neurodegenerative diseases, the identification of potential biomarkers in ALS has been hampered by the long lag-time between symptoms onset and diagnosis (approximately 12 months) and to the low annual incidence that makes general screening strategies not feasible5. Nowadays, no diagnostic test can specifically detect ALS at onset and discriminate ALS from other motoneuron and similar neurodegenerative diseases, thus hindering the diagnosis, prognosis, patients’ stratification, treatment monitoring or the objective evaluation of the effects of new possible therapies. Currently, the diagnosis of ALS is achieved by the combination of clinical data and neurophysiological evidence together with the monitoring of the symptoms progression in a time-consuming process that limits the time frame for a prompt intervention and the choice of a personalized therapy6. The discovery of a new biomarker easily accessible and quickly detectable represents a priority for ALS early diagnosis, stratification and evaluation of the therapeutic and rehabilitative effectiveness. In recent years, several potential biomarkers were isolated from different tissues and highly specific techniques have been proposed. The road taken by researchers regards the analysis of biofluids, whose molecular 1 IRCCS Fondazione Don Carlo Gnocchi, Milan, 20148, Italy. 2Department of Psychology, Università Cattolica del Sacro Cuore, Milan, 20123, Italy. 3Istituto Auxologico Italiano, IRCCS Department of Neurology and Laboratory of Neuroscience, 20149, Milan, Italy. 4Department of Pathophysiology and Transplantation, “Dino Ferrari” Center, Università degli Studi di Milano, 20122, Milan, Italy. 5“Aldo Ravelli” Center for Neurotechnology and Experimental Brain Therapeutics, Università degli Studi di Milano, 20122, Milan, Italy. ✉e-mail: Scientific Reports | (2020) 10:10175 | https://doi.org/10.1038/s41598-020-67138-8 1 www.nature.com/scientificreports/ www.nature.com/scientificreports composition (i.e. proteins, lipids, nucleic acids, carbohydrates, metabolites, hormones) is representative of the physiological/pathological state. The research for biomarkers related to ALS has been performed principally on Cerebrospinal Fluid (CSF), with a large variety of molecules associated with ALS including neurofilament proteins, components of the inflammation process, C9orf72 dipeptide repeat proteins, TAR DNA-Binding protein (43 kDa), cystatin C, specific microRNA (miRNA181a-5p, miRNA-143-5p, miRNA-338-3p) and the mutated Superoxide Dismutase enzyme type 1 (SOD1)7. Some of the listed molecules have been detected also in serum and plasma, that share a less invasive collection procedure compared to CSF, making periodical collection feasible. Overall, no reliable and repeatable data have been obtained, so far8–11. Up to now, one of the most promising genetic biomarkers is the mutation of C9orf72 repeat expansion, which has been attributed to the onset of familial ALS, frontotemporal lobar degeneration and a small part of sporadic ALS12. Clinical studies are under development to validate the C9-based therapies, but its restriction to familial ALS (5–10% of the total13) and the continuous need for the CSF collection to monitor the disease progression using the C9orf72 dipeptide repeat proteins are limiting the development of this approach7. Other specific biomarkers are under investigation for the detection and discrimination of sporadic ALS (~90% of the cases) from the familial onset, although the two forms possess a comparable pathological mechanism with common biomarkers (e.g. TAR DNA-binding protein)14. Similarly, also mutations of SOD1 (~20% of familial ALS) and TAR DNA-binding protein (2–5% of familial ALS) genes, show the same limitations of C9-based proteins regarding the invasiveness of biofluid collection procedures13,15. In the same way, neurofilament proteins have been studied as indicator of neurodegeneration in ALS and other neurodegenerative diseases. Different studies reported high levels of neurofilament in CSF and blood of ALS patients and related pathological controls, respect to the healthy counterparts highlighting a neurodegenerative process in progress8,16–18. Despite some promising results, th (...truncated)