# New Insights into the Neuromyogenic Spectrum of a Gain of Function Mutation in SPTLC1
## 4 Discussion
### Molecular Signature of p.(A20S)-SPTLC1-Mutant Lymphoblastoid Cells  
Proteomic profiling is a robust approach to obtain unbiased insights into the pathophysiology of different diseases, including neuromuscular disorders, by utilizing different tissues or in vitro models [21; 22] such as immortalized lymphoblastoid cells [15; 23]. Here, we investigated the proteomic signature of immortalized lymphoblastoid cells derived from an ALS patient with juvenile-onset based on the dominant p.(A20S) variant in SPTLC1 resulting in a splicing defect, predominantly causing exon 2 skipping. Among the dysregulated proteins, SPTLC1 itself was identified with a significant decrease to 0.48-fold expression. This is the first study indicating that the presence of a dominant SPTLC1-variant leading to juvenile ALS is associated with a decrease of SPTLC1 protein level. Further transcript studies revealed no alteration of _SPTLC1_ expression (Figure 6A). The expression level of the wildtype (wt) allele in the patient cells was approximately 0.5-fold compared to the healthy controls (Figure 6), indicating no compensation on RNA level for the mutated allele. Further single-molecule nanopore sequencing of the cDNA revealed, beside the skipping of exon 2 (Figure 6C), an activation of a cryptic splice site inside of exon 2 (Figure 6D,E) due to the pathogenic variant. The resulting transcript is missing the first 2 bases of exon 2 (c.58_85 del), which leads to a frameshift and immediate stop (p.A20 Gs*3). However, this transcript accounts only for 5% of the total transcript amount and is very likely degraded via nonsense-mediated decay (NMD). Nevertheless, the nanopore data indicated a comparable expression of both alleles, confirming data obtained by qPCR analysis. Hence, the reduction of protein level observed by the results of our proteomic studies might originate from the decreased stability of the aberrant protein. However, functional studies are needed to address this hypothesis. In 2013, [PERSON] and co-workers reported on a French girl presenting with a syndromic phenotype defined by severe growth restriction, cognitive impairment, amyotrophicrophy, hyperreflexia, vocal cord paralysis, and respiratory failure [24] similar to the patients described by [PERSON] and colleagues [7]. Although our patient did not present with cognitive impairment and had a normal brain MRI, proteomic signature of his lymphoblastoid cells unravelled a decrease of a variety of proteins associated with cognitive impairment (Table 1). Thus, one might speculate that the threshold of (i) the decrease of these proteins or (ii) the increase of compensatory proteins determines manifestation of cognitive symptoms. With respect to potential contributing proteins with decreased abundance, CTTN (decreased to 0.51-fold expression) plays a role in the regulation of neuron morphology, axon growth, and formation of neuronal growth cones [25], while DBN1 (decrease to 0.42-fold expression) is involved in memory-related synaptic plasticity in the hippocampus [26]. In addition, IQSEC1 (decrease to 0.23-fold expression) mediates the internalization of synaptic AMPAR receptors [27]. Ca\({}^{2+}\) dysfunction, a common noxious mechanism in familiar and sporadic ALS, impacts on \(\alpha\)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA), receptor (AMPAR)-mediated excitotoxicity [28], and dysregulation of AMPAR subunit expression was already demonstrated in sporadic ALS post-mortem brains [29]. One candidate protein with potential compensatory/rescue function is PTK2, demonstrating a 13.10-fold upregulation in lymphoblastoid cells of our patient. PTK2/FAK1 acts as a non-receptor protein-tyrosine kinase and regulates axon growth and neuronal cell migration, axon branching, synapse formation, and is required for normal development of the nervous system [30]. PTK2 was linked to the protection of neurons from apoptosis [31].