Clinical and Genetic Aspects of Juvenile Amyotrophic Lateral Sclerosis: A Promising Era Emerges

Juvenile Amyotrophic Lateral Sclerosis is a genetically heterogeneous neurodegenerative disorder, which is frequently misdiagnosed due to low clinical suspicion and little knowledge about disease characteristics. More than 20 different genetic loci have been associated with both sporadic and familial juvenile Amyotrophic Lateral Sclerosis. Currently, almost 40% of cases have an identifiable monogenic basis; type 6, associated with FUS gene variants, is the most prevalent globally. Despite several upper motor neuron-dominant forms being generally associated with long-standing motor symptoms and slowly progressive course, certain subtypes with lower motor neuron-dominant features and early bulbar compromise lead to rapidly progressive motor handicap. For some monogenic forms, there is a well-established genotypic-phenotypic correlation. There are no specific biochemical and neuroimaging biomarkers for the diagnosis of juvenile Amyotrophic Lateral Sclerosis. There are several inherited neurodegenerative and neurometabolic disorders which can lead to the signs of motor neuron impairment. This review emphasizes the importance of high clinical suspicion, assessment, and proper diagnostic work-up for juvenile Amyotrophic Lateral Sclerosis.


Introduction
Amyotrophic Lateral Sclerosis (ALS) represents the main form of sporadic and inherited adult-onset Motor Neuron Disease (MND) [1].Despite most cases of ALS occurring between the fifth and seventh decade of life, at least 10% of cases present as young-onset forms, with motor symptoms starting before age 45 [2][3][4].A rare and expanding subgroup of such cases includes juvenile ALS (JALS), with onset of motor compromise before age 25.JALS is commonly misdiagnosed and underrecognized in clinical practice, mainly due to low disease awareness and several diagnostic misconceptions [5].
Global prevalence and incidence of JALS are still largely unknown [5].A rare multicentric European study including data from 46 specialized ALS centers estimated a prevalence of 0.008 cases per 100,000 inhabitants with onset of symptoms before age 18, representing less than 0.1% of all ALS cases [6].In a Portuguese cohort of patients with young-onset ALS, 14.3% of cases were related to JALS [7].Since the introduction of Whole-exome sequencing (WES) and next-generation sequencing (NGS)-based tests in routine diagnostic work-up in the last two decades, there has been a significant increase in knowledge about pathophysiological mechanisms and a better understanding of the natural history and clinical complications of each monogenic form of JALS.Although only 40% of JALS cases have an identifiable genetic basis, more than 20 distinct genetic loci have been associated with Genes 2024, 15, 311 2 of 14 JALS [5].Most JALS cases occur as sporadic ALS (sALS) presentations but can be associated with autosomal recessive or dominant and, rarely, X-linked patterns of inheritance [5].
Given the expansion of available diagnostic methods and the development of new therapeutic modalities based on antisense oligonucleotides and viral vector platforms in gene therapy, it is essential to organize current knowledge and review the state of the art in this topic.This review article presents the main clinical, pathophysiological, genetic, and therapeutic aspects related to JALS and its main genetic subtypes.

Pathophysiology
The neuropathological involvement of JALS is not limited to upper and lower motor neuron compromise [5].Multiple neural pathways have been associated as well as, less frequently, cognitive and affective areas and, rarely, sensory cortical regions.Several genetic subtypes related to different neuronal and glial dysfunctions have been identified [8] (Table 1).Motor neuron loss in this context results from several pathophysiological mechanisms, similarly to typical sporadic and familial ALS (fALS), including the following: (i) abnormal protein misfolding and aggregation; (ii) disturbances of autophagy; (iii) disorders of the ubiquitin-proteosome system; (iv) defects in retrograde and anterograde axonal transport; (v) mitochondrial dysfunction; and (vi) abnormal neuronal metabolic function with accumulation of neurotoxic compounds, such as reactive oxygen species, sorbitol, or other toxic intermediates [1,[8][9][10].There is important pathophysiological and genetic overlap of JALS with several other inherited neurological disorders, including Hereditary Spastic Paraplegia (HSP), axonal Charcot-Marie-Tooth disease (CMT), non-5q Spinal Muscular Atrophy (SMA), autosomal recessive cerebellar ataxia, and inherited metabolic disorders [11][12][13][14].Glial cell dysfunction seems also to contribute in JALS pathogenesis, especially in rare cases associated with SOD1 variants [5].However, the scientific evidence base obtained through the observation of the monogenic basis seen in patients with JALS indicates a possible lesser importance of neuroinflammatory and neurotoxic mechanisms, and less pronounced role of microglial activation in the pathogenesis of JALS [5,8,9].Toxic (i.e., radiation, pesticides, solvents, β-methylamino-L-alanine, methylphenyltetrahydropyridine, heavy metals, immunization), infectious (i.e., retroviruses, herpesviruses), and environmental and behavioral factors (i.e., dietary factors, low polyunsaturated fatty acid consumption, strenuous physical activity, athleticism, repeated head trauma, electromagnetic field exposure in occupation, military veterans) have been associated with ALS [1,3,9], and may potentially participate in JALS as potential trigger mechanisms when associated with genetic predisposition [1,5].

Clinical Presentation
The clinical picture in typical JALS presentation is dominated by symmetric or asymmetric upper and lower motor neuron signs with variable rates of motor progression and bulbar compromise.Almost pure upper motor neuron compromise may also be observed, presenting with marked spasticity, brisk tendon reflexes, clonus, and extensor plantar responses [9], mimicking features of HSP and Primary Lateral Sclerosis [11,12,15] (Table 2).Pure lower motor neuron involvement may also be identified, presenting with amyotrophy, weakness, fasciculation, hypotonia, and reduced or absent tendon reflexes [9], similarly to 5q and non-5q SMA and Progressive Muscular Atrophy [12,[14][15][16]] (Table 2).Significant cognitive compromise and autonomic disturbances are rare, and Frontotemporal Dementia (FTD) has not been directly correlated with JALS.Ben Hamida's classification of the JALS phenotype (1990) includes the three main groups of patients in clinical practice: (i) Group 1: distal upper limb amyotrophy form, evolving with bilateral pyramidal signs, spastic paraparesis and bulbar involvement; (ii) Group 2: late childhood-or juvenile-onset spastic paraparesis form with peroneal amyotrophy, sparing the bulbar region; and (iii) Group 3: childhood-onset spastic pseudobulbar form with spastic paraparesis [17].
The genetic basis associated with the identifiable monogenic forms of JALS differs from the pattern observed in sporadic and familial forms of ALS globally and regionally.Sporadic and familial forms of typical ALS are related to the greater occurrence of hexanucleotide repeat expansions in the C9orf72, and pathogenic variants in the SOD1, TARDBP and FUS genes [1,[8][9][10].There is, however, the influence of local epidemiological factors and founder effects related, respectively, to the lower and higher frequency of distinct genetic basis in specific populations.Therefore, the high frequency of the pathogenic variant p.Pro56Ser in the VAPB gene in the Brazilian population stands out, as well as the low frequency of expansions in C9orf72 in populations of Asian origin [1,8,9].The potential role of founder effects in the context of JALS is not well established [5].Clinical course is generally associated with the monogenic basis involved with each JALS subtype [5,8,13].Rapidly progressive clinical course is more commonly observed in patients with specific pathogenic variants in FUS and SOD1 genes [47,48], in patients with lower motor neuron-dominant phenotypes [49,50], and in cases with prominent early bulbar compromise or in bulbar-onset JALS [35].Slowly progressive presentation is commonly identified in cases with upper motor neuron-dominant phenotypes, mimicking HSP features, and in specific variants in SOD1, ERLIN1, and TARDBP genes [27,28,51,52].A heat map with the main neurological features related to each genetic subtype of JALS is presented in Figure 1.Cognitive compromise is not a hallmark of JALS, despite the occurrence of intellectual disability and autism in FUS variants [53,54].Pseudobulbar affect is most observed in ALS2 and SPG11 variants [33].Cerebellar ataxia may be observed in SETXand SYNE1-related JALS [31,41,43], while myoclonus and tremor have been described in FUS variants [54,55].Diplopia and ophthalmoparesis may also be observed with FUS variants [56].Furthermore, homozygous pathogenic variants in SOD1 have recently been associated with SOD1 deficiency and a very early-onset upper motor neuron-dominant phenotype, called Progressive Spastic Tetraplegia and Axial Hypotonia (STAHP) [57].Au-tonomic disturbances and variable sensory neuropathy may be identified in VRK1-related JALS and SPG11 variants [5,33,34,58].

Diagnostic Work-Up and Differential Diagnosis
Clinical examination disclosing features suggestive of lower and motor neuron dysfunction is a key step during the initial assessment.Several clinical conditions share similar clinical signs with JALS and frequently represent diagnostic challenges for a

Diagnostic Work-Up and Differential Diagnosis
Clinical examination disclosing features suggestive of lower and motor neuron dysfunction is a key step during the initial assessment.Several clinical conditions share similar clinical signs with JALS and frequently represent diagnostic challenges for a definite diagnosis [9,11,12,14-16] (Table 2).There are no fully specific diagnostic biomarkers for the diagnostic definition of ALS and JALS.Neuroimaging studies are generally unremarkable and do not disclose specific findings.The classic "wine glass sign" associated with abnormal bilateral signal changes (hyperintensity in FLAIR and T2-weighted imaging) involving the corticospinal tracts (especially identified in coronal views) is commonly observed in JALS, especially in upper motor neuron-dominant phenotypes.Cortical and cerebellar atrophy, thin corpus callosum, and leukoencephalopathy are also detected in specific genetic subtypes (Figure 1).Needle electromyography discloses chronic denervation and reinnervation involving different spinal segments, commonly with asymmetric patterns and variable signs of abnormal spontaneous activity and acute axonal denervation (fasciculation, fibrillation potentials, positive sharp waves).Split-hand index, ALS diagnostic index, Motor Unit Number Index (MUNIX), and Motor Unit Number Estimation (MUNE) measures represent additional neurophysiological biomarkers which raise clinical suspicion of MND [59-62].There were no previous studies which specifically evaluated the role of these neurophysiological biomarkers and indices in the diagnosis and follow-up of patients with JALS.The El Escorial criteria, the revised Airlie House criteria, the Awaji-shima criteria, and more recently the Gold Coast criteria, have been used in sporadic and familial ALS to establish levels of diagnostic certainty, following specific criteria [63][64][65][66].In summary, the presence of signs of chronic multisegmental denervation associated with elements of acute denervation (fibrillation, positive sharp waves, fasciculations) in patients with clinical suspicion of MND allows the strengthening of diagnostic suspicion.Technical aspects relating to the application of diagnostic criteria from a laboratory and neurophysiological point of view are detailed in the original works in the literature related to the propositions of these criteria [63][64][65][66].These criteria are currently applied also in JALS to define the presence of MND, being complemented by the onset of motor symptoms and signs before the age of 25 years, regardless of clinical severity.Nerve conduction studies may also demonstrate chronic sensory axonal polyneuropathy in cases of JALS associated with SPG11, VRK1, and SPTLC1 genes [29,33,34,58].
Genetic testing is routinely recommended in JALS, both in sporadic and familial cases.Whole-exome sequencing or large next-generation sequencing-based multigene panels may be used in diagnostic work-up, looking for a specific monogenic basis [67,68].Negative genetic testing results do not rule out the diagnosis of JALS.A broad genetic evaluation is essential for the following reasons: (i) providing individual and familial genetic counseling; (ii) stopping and shortening the diagnostic odyssey of patients with JALS; (iii) indicating the potential need to look for alternative differential diagnosis; (iv) providing patients and family with more reliable data about prognostic factors based on genetic aspects; and (v) including patients with specific genetic subtypes in current and future clinical trials related to viral vector-based gene therapies and antisense oligonucleotide-based therapies [6,69,70].

Treatment
Clinical trials in rare diseases during childhood and adolescence represent a great challenge due to difficulties regarding patient recruitment periods, control and placebo groups, ethical and legal aspects, outcomes, and trial designs.These are exactly the most difficult steps involved in JALS treatment [6,71].Non-pharmacological treatment based on a specialized multidisciplinary team approach, involving physical, speech, and occupational therapists, nutritionists, psychologists, and nurses, represents the core therapy of patients with JALS.Proper management of symptomatic therapies improves the quality of life and well-being of patients in more advanced stages of the disease [1,9,72].
There are still very limited specific data regarding disease-modifying therapies for JALS.Most drugs used to treat ALS were evaluated previously in Phase 2 and 3 studies mainly for typical and sporadic forms of the disease, with a different clinical course from that observed in most cases of JALS.Thus, data on efficacy, safety, and dosage aspects of drugs potentially used in the treatment of JALS are essentially derived from clinical studies related to typical ALS, such as in the cases of Riluzole, Edaravone, and sodium phenylbutyrate with taurursodiol (tauro-ursodeoxycholic acid, TUDCA) [72] (Table 3).In such cases, for most MND-specialized centers the therapeutic indication is based on a caseby-case expert decision.In cases related to the defined single-gene base of JALS, new genetic target therapies represent great hope in the current stage of development, especially for antisense oligonucleotide therapies related to SOD1, FUS, and ATXN2 genes [69][70][71][72][73][74][75].Other monogenic forms of therapeutic interest include SPTLC1-related JALS which is amenable to oral L-serine supplementation therapy [29,76], and the rare SORD-related JALS which is potentially treatable with use of selective aldose reductase inhibitors [32] (Table 3).H-dependent degradation of mRNA.I IV [69] ALS: familial Amyotrophic Lateral Sclerosis; JALS: Juvenile Amyotrophic Lateral Sclerosis; L-BMAA: L-β-N-methylamino-L-alanine; MND: Motor Neuron Disease; N/A: not applicable; NMDA: N-methyl-D-aspartate; RWE: Real-world Evidence; sALS: sporadic Amyotrophic Lateral Sclerosis; TUDCA: Tauroursodeoxycholic acid.* Currently underway.

Prognosis
Prognosis represents a complex subject during ALS management.Despite many individuals presenting with typical ALS with survival time from symptom-onset to death ranging from 20 to 48 months [9,78], more than 10% of individuals present with longstanding ALS course with more than 10 years of survival [78].Few studies regarding the natural history of each genetic subtype of JALS are available, and most reliable data derive from several case series studies.Most JALS cases present as long-standing ALS, despite being associated with severe compromise of quality of life, marked functional capacity decline, and frequently with the need for nutritional enteral support, percutaneous gastrostomy, and permanent dependence on mechanical ventilation [52,79].Childhoodonset, bulbar-onset, and JALS associated with complex neurological pictures are commonly more severe and have generally poor prognosis [5].Rapidly progressive clinical course has been typically observed both in JALS types 6 (FUS) and 1 (SOD1) [5,47,48].

Conclusions
JALS represents a rare neurodegenerative disorder with several needs still unmet in clinical practice.Diagnosis is based mainly on clinical and neurophysiological aspects and supported by genetic testing, although a definite monogenic basis is not necessary for definite diagnosis.Identification of both sporadic and familial JALS cases and their involved genetic basis is essential so that genetic counseling can be provided in a timely fashion and potentially treatable etiologies can be identified earlier.Several presentations have gene therapies under investigation in current clinical trials.

Figure 1 .
Figure 1.Heat map of neurological and neuroimaging features associated with JALS.Frequencies of features reported in the literature are represented by different colors, according to the specific analyzed feature and each genetic subtype.

Figure 1 .
Figure 1.Heat map of neurological and neuroimaging features associated with JALS.Frequencies of features reported in the literature are represented by different colors, according to the specific analyzed feature and each genetic subtype.
* The designation of the forms of JALS follows the numbering established in the classification updated annually by the Gene MuscleTable of the World Muscle Society (WMS; available at: URL: https://www.musclegenetable.fr)(accessed on 20 January 2024) and the nomenclature of the Online Mendelian Inheritance in Man (OMIM; available at: https://www.omim.org)(accessed on 20 January 2024).Forms that have not yet been assigned specific numbers were classified based on the official name of the related gene.** Subtypes with unidentified monogenic basis.Legend: AD: autosomal dominant; ALS: Amyotrophic Lateral Sclerosis; AR: autosomal recessive; CMT: Charcot-Marie-Tooth disease; FTD: Frontotemporal dementia; HSAN: Hereditary Sensory and Autonomic Neuropathy; IAHSP: Infantile-onset Ascending Hereditary Spastic Paralysis; LMN: Lower Motor Neuron; PLS: Primary Lateral Sclerosis; SMA: Spinal Muscular Atrophy; SMALED: Spinal Muscular Atrophy with lower extremity dominance; SPG: Spastic Paraplegia; STAHP: Progressive Spastic Tetraplegia and Axial Hypotonia; UMN: Upper Motor Neuron; XLD: X-linked inheritance (dominant).