# New Insights into the Neuromyogenic Spectrum of a Gain of Function Mutation in SPTLC1
## 4 Discussion
### Findings in the Quadriceps Biopsy of the p.(A205)-Sptlc1-Patient  
Microscopic studies revealed fiber-type grouping, a very well-known pathomorphological hallmark of denervated skeletal muscle, accompanied by subsarcolemmal increase of mitochondria in a proportion of muscle fibers. This latter finding is rather nonspecific and has already been described in the context of muscle cell denervation [45]. MyHC-neo and -developmental are predominantly expressed in developing muscle fibers [46] and are not increased in the muscle biopsy of our patient, suggesting absence of immature myofibers. Our immunohistochemical studies revealed irregular Troponin-T expression and Troponins have been postulated to serve as a good protein marker for muscle pathology based on loss of motor neuron function [47]. Although excess of sphingolipid synthesis is likely the major pathomechanism caused by the gain of the function variant identified in our patient [6], our CARS microscopic investigations did not indicate the presence of lipid aggregates (and Oil-red-O did not indicate increased lipid droplets). This might be based on a functional irrelevance of SPTLC1 in muscle (SPTLC1 expression is low in muscle compared to nervous tissue, blood, and cultured fibroblasts) and/or the activation of compensatory mechanisms. However, more functional studies on muscle cells are needed to prove this assumption and to delineate a potential functional role of SPTLC1 in muscle cells. In addition, more detailed studies on nervous tissue are needed to investigate if the gain of SPTLC1-function leads to lipid aggregation.  
Although immune system components may indeed play a role in ALS pathogenesis, detailed studies on their impact in early disease pathology are limited, and more focused studies examining the role of the immune system together with a characterized pathogenesis to determine when, where, and if immune and inflammatory processes are taking place are still needed [48]. Here, combined immunohistochemistry and transcript studies revealed increased antigen presentation via the major histocompatibility complex within a small area in which immune cells (macrophages) are also detectable, indicating an active (while still limited) immune response. HLA class II antigens play a central role in the immune system by presenting peptides derived from extracellular proteins and are expressed in antigen presenting cells, like macrophages. Although an elevated expression accords with our histological findings, the very high transcript level of the HLA-DRA and HLA-DRB, respectively, remain surprising, given that the overall immunoreactivity for these proteins was of minor increase in the patient-derived muscle biopsy. This finding indicates that increased expression of the _HLA-DRA_ and _HLA-DRB_ genes does not necessarily correlate with the same extension of an immune response on the protein level, and that combined transcript and protein studies are needed to draw a conclusion with respect to the intensity of respective protein contributions on muscle inflammation.  
Our confirmational studies focussing on phospho-FAK1 abundance and distribution in quadriceps muscle of the patient revealed increased expression within the perimysium. The focal adhesion kinase (FAK1) is known to act as a downstream non-receptor tyrosine kinase that translates cytoskeletal stress and strain signals transmitted across the cytoplasmic membrane to activate multiple anti-apoptotic and cell growth pathways. Notably, changes in FAK expression and phosphorylation correlate to specific developmental states in myoblast differentiation, muscle fiber formation and muscle size in response to loading and unloading [49]. However, the increase within indicates a role in fibrosis, and a model wherein hypoxia (as a secondary insult) caused exacerbated pulmonary fibrosis elevated expression of phosphorylated FAK1 was already described [50].