**2. Amyotrophic lateral sclerosis: an overview**

**1. Introduction**

230 Update on Amyotrophic Lateral Sclerosis

[4].

challenging tasks [7].

The current diagnosis and classification of diseases are primarily based on physical signs and symptoms that, despite providing valuable information about clinical course, are often not sufficient to fully characterize the complex and heterogeneous nature of many disorders.

The completion of the human genome sequencing together with advances in high-throughput genomic, proteomic, imaging, and other diagnostic techniques in the past decades has provided a framework for developing a new, more accurate, and refined "molecular taxono‐ my" of human diseases which implies the use of molecular data (i.e., gene expression, copy number variants, single nucleotide polymorphisms, and haplotype analysis) to classify patients into distinct subgroups with differing diagnostic, prognostic, or therapeutic implica‐ tions. This new disease classification has profound implications not only providing new insights into studying mechanisms and environmental causes underpinning diseases but also facilitating the development of a more precise diagnosis and individualized treatment for optimal therapeutic efficacy [1–3]. One seminal example of how molecular data may translate into clinical practice is represented by the "drug repositioning "approach. In fact, many drugs that were abandoned at clinical stages because of their low efficacy and/or toxicity in a specific subtype of patients may be re-evaluated for their potential therapeutic role with the consequent possibility to reduce both the time and costs associated with drug discovery and development

Oncology offers multiple examples of how genomic medicine has changed disease under‐ standing and drove targeted therapeutic interventions. Numerous studies, in fact, have demonstrated the power and ability of gene expression profiling, and other molecular approaches, to classify and substratify patients with various types of cancer (e.g., glioblastoma, breast, and colon carcinoma) into selective clinically relevant subtypes characterized by similar clinicopathological features but different biological properties, prognostic biomarkers, and treatment options [5]. Based on these promising results, over the past years, this new molecular reclassification has been extended to other polygenic and multifactorial human disorders, including cardiovascular and rheumatic diseases and multiple sclerosis [6]. However, a lack of progress remains in the understanding of detailed molecular mechanisms of several neurological and neurodegenerative diseases mainly because of the limited access to human brain tissues. Thus, the patient-specific molecular diagnosis of many neurological disorders and the consequent translation of this into tailored clinical trials and specific treatments remain

Recently, by using an unsupervised hierarchical clustering analysis on motor cortex samples of patients with sporadic amyotrophic lateral sclerosis (SALS), our research group has identified two greatly divergent subtypes, each associated with differentially expressed genes and biological pathways [8]. These experiments highlight, for the first time, the genomic heterogeneity of SALS, revealing new clues for defining molecular signatures for this disease that were not put in evidence by considering SALS as a single entity. Moreover, the altered pathways of biological molecules in SALS also provided a number of potential biomarkers and

ALS is a neurodegenerative disease characterized by the progressive muscular paralysis reflecting the degeneration of upper and lower motor neurons which leads to respiratory insufficiency and death after three to five years. ALS is the commonest of the motor unit diseases in Europe and North America and its incidence ranges from 1.7 to 2.3 cases per 100,000 population per year worldwide [11]. Currently, there is no cure or prevention for ALS and Riluzole is the only disease-modifying medication presently approved by the US Food and Drug Administration (FDA) for the treatment of ALS [12]. Riluzole is largely symptomatic and prolongs survival but only with a modest effect. Many clinical trials have been performed but have unfortunately had limited success [13]. Thus, the development of novel treatments and diagnostic research strategies is a goal of increasing urgency.

Accurately understanding the etiopathogenic mechanisms underlying ALS is a crucial step for developing effective diagnostic–therapeutic strategies. Approximately 95% of the cases are isolated or sporadic (SALS), while about 10% are familial (FALS), showing autosomal domi‐ nant, recessive, or X-linked inheritance. Although genetic studies in FALS are rendered difficult by the late onset of disease, its incomplete penetrance and the short survival of affected family members, several familial ALS loci, and genes have been identified [14–16], such as *SOD1*, *ALSIN*, *SETX*, *SPG11*, *FUS*, *VAPB*, *ANG*, *TARDBP*, *FIG4*, *OPTN*, *ATXN2*, and *C9ORF72*. The contribution of genetic risk factors also seems to be considerable into the sporadic form of the disease [16]. Despite the identification of several disease-linked mutations, the etiology and pathogenesis of ALS remain largely unknown, supporting the multifactorial and complex nature of this disease, in which multiple genetic variants, each of the small effects, combine with a variety of environmental triggers and risk factors [14, 16–18].

The diagnosis of ALS is primarily based on the clinical observation of symptoms, physical signs, progression, and electrodiagnostic testing, in accordance with the "El Escorial" criteria. These represent a catalog of clinical and diagnostic features, specified by the World Federation of Neurology (www.wfneurology.org), that aim to exclude "ALS-mimic" syndromes (i.e., cervical spondylotic myelopathy, multifocal motor neuropathy, and Kennedy's disease) and permit to classify ALS patients for research studies [19–21]. The broad clinical spectrum of ALS comprehends distinct phenotypes ranging from pure upper motor neuron disease to pure lower motor neuron disease, with several different intermediate forms (classic, flail arm, flail leg, pyramidal, respiratory, and bulbar), each characterized by different degrees of involvment of Upper Motor Neurons (UMN) and Lower Motor Neurons (LMN), body regions that are affected, degrees of involvement of other systems especially cognition and behavior, and progression rates [22].

Although clinical neurophysiology in ALS plays a fundamental role in both diagnosis and assessment of its severity and progression, the initial symptoms of ALS are often subtle (limb or shoulder weakness and difficulty in walking), leading to a delay in the diagnosis as well as misdiagnosis and, consequently, restricting the possibilities for effective preventive and therapeutic strategies [23]. Thus, the adequate integration of neurophysiological techniques and advanced biological methods is essential in order to obtain a better understanding of disease pathogenesis, support earlier diagnosis, inform about prognosis, and monitor ALS progression in clinical trials.

The advent of high-throughput techniques—microarrays and next-generation sequencing has shed light on the pathophysiology of complex diseases, including ALS and oriented researchers from a single-molecule analysis toward a "system biology" approach, offering a better understanding of the molecular mechanisms that, interacting with each other, may contribute to ALS pathogenesis [4].
