The inherited Neuromuscular Disorders (NMDs) is an umbrella term that encompasses a plethora of diseases that affect the functioning of the muscles and/or their underlying nervous system control. Although individually uncommon, NMDs collectively are no longer considered rare diseases. Only in Europe, approximately 300,000 people are yearly diagnosed with one NMD. The diagnosis of a neuromuscular condition –successful in less than 50% of the cases- is often devastating to patients and their relatives. No cure is available for most of these disorders, and the few available treatments will at best delay disease progression. To find treatments and to develop methods for the early diagnosis of NMDs is a goal of highest importance, and the recent advances in the NGS technologies are the platform to reach this objective. Particularly, the implementation of WES has not only minimized the time and costs of genetic diagnostics but also provided unique opportunities for the exploration of gene function in NMDs pathogenesis.
The uppermost goal of this PhD project was to identify and characterize novel NMDs-causative genes. Thereby, two novel NMD-causative genes were investigated in this thesis. First, CHP1 (Calcineurin Homologous Protein-1) was identified as a novel causing gene of Autosomal Recessive Cerebellar Ataxia (ARCA) and second, VAChT (Vesicular Acetylcholine Transporter, encoded by SLC18A3) was analysed in the context of distal Hereditary Motor Neuropathy (dHMN).
Following a combination of WES and linkage analysis, we identified a biallelic 3-bp deletion (p.K19del) in CHP1 that co-segregates with a complex ARCA in two siblings of a consanguineous family exhibiting motor neuropathy, cerebellar atrophy and spastic paraparesis. CHP1 was selected as a top disease candidate since: (I) the mutation affects an amino acid highly conserved across species, (II) a point mutation in murine Chp1, causing aberrant splicing and reduced full-length Chp1 transcripts, leads to Purkinje cells loss and ataxia, (III) CHP1 assists posttranscriptional glycosylation and membrane localization of NHE1, a major neuronal Na+/H+ exchanger, (IV) KO of mouse Nhe1 cause ataxia and loss-of-function mutation in NHE1 (encoded by SLC19A1) cause ataxia-deafness Lichtenstein-Knorr syndrome (LINKS). Therefore, we hypothesized that a mutation in CHP1, as a crucial regulator of NHE1, could impair expression and targeting of the exchanger resembling the pathogenesis in mice and humans. To further uncover other families carrying CHP1 mutations, we performed a focused screening for CHP1 variants in two large ARCA and NMD cohorts (approximately 1000 exomes). No additional variants fulfilling or selection criteria were found, which emphasizes on the scarcity of CHP1 variants and the reduced tolerability of CHP1 for mutations.
With the purpose to assess the functional consequences of the CHP1-K19del mutation on protein function, size exclusion chromatography (SEC), protein fractionation, 3D-protein modelling, fluorescence microscopy and in vivo zebrafish modelling were performed. We demonstrated that mutant CHP1 fails to integrate into functional protein complexes and is prone to aggregate, thereby leading to diminished levels of soluble CHP1 and reduced membrane targeting of NHE1 both in neuronal and non-neuronal cells. To analyze the pathogenic consequences of the hypomorphic CHP1-K19del mutation in vivo, we used morpholinos (MOs) to inhibit chp1 translation in zebrafish. Closely resembling the clinical features of the ARCA-affected siblings, chp1 downregulation in zebrafish led to cerebellar hypoplasia, Caudal Primary Motor Neuron (CaP-MN) defects and spastic trunk movements. All defects were ameliorated by co-injection with WT, but not mutant, human CHP1 mRNA, hence demonstrating both the specificity of the chp1-MO-induced phenotypes and validating the effect of CHP1-K19del on protein expression and/or function in vivo. Altogether, our results identified CHP1 as a novel ataxia-causative gene in humans, further expanding the spectrum of ARCA-causative loci, and highlight the crucial role of NHE1 within the pathogenesis of these disorders.
Moreover, we conducted functional analyses to ascertain the functional basis of a dHMN presented by a family with cranial nerves palsy and vocal cord paresis as an initial feature of a non-progressive infantile onset dominant dHMN. WES analysis of this family led to the identification of a de novo dominant missense mutation (c.439 G>A, p.D147N) in VAChT. The mutation occurred first in the affected mother and was inherited by her affected daughter. VAChT controls the storage of the neurotransmitter Acetylcholine (ACh) by synaptic vesicles, hence it plays a fundamental role in cholinergic neurotransmission and therefore, in the plethora of processes reliant on it, which include: neuronal development and maturation, synaptic transmission and plasticity, patterning of the neuromuscular junction (NMJ), among others. The potential effect of the D147N mutation on VAChT subcellular distribution was analysed in neuron-like NSC-34 cells transiently overexpressing WT or mutant VAChT-GFP tagged proteins. No significant differences were observed in protein expression or localization, thus a detrimental effect of VAChT-D147N mutation at this level was not possible. This prompted us to further examine potential defects either in MN development and axonal outgrowth. Capitalizing once again on the advantages of the zebrafish for the modelling of human neurodegenerative disorders and further considering the evolutionary conservation of both VAChT and the D147 residue across species, the effect of WT and VAChT-D147N OE on CaP-MN outgrowth was analysed in detail. Although our findings were not conclusive at discerning the pathogenicity of the VAChT–D147N in vivo, we observed an axonal migration phenotype that could potentially underlie impairments at the NMJ level. In the light of the novel association of VAChT mutations as causative of myasthenia syndromes, follow-up studies will be performed in order to conclusively confirm the pathogenicity of the VAChT-D147N in a CaP-MN-independent context.
The biological function of CHP1 was of further relevance within the scope of this doctoral project, since CHP1 is currently subject of study as potential modifier of Spinal Muscular Atrophy (SMA). Pre-existing evidence from our research group indicates that Chp1 downregulation –within a certain threshold- restores neurite outgrowth and impaired endocytosis (a key pathway disturbed in SMA) in Smn1-depleted NSC-34 cells. Thereupon, this thesis further aimed to validate reduction of Chp1 as potential SMA protective modifier in a zebrafish model of SMA, as a first in vivo approach. Here, we demonstrate that Chp1 downregulation ameliorates the CaP-MN axonal outgrowth defects of Smn-deficient fish larvae. These findings are in concordance with prior validation studies of two other human SMA modifiers –PLS3 and NCALD- in zebrafish, which despite their different function and mode of action (upregulation or downregulation, respectively) exert similar effects on CaP-MN morphology whereby restoring the CaP-MN phenotype of smn morphants in a highly comparable range. Altogether, our findings together with the preliminary findings aforementioned, strongly support CHP1 reduction as a promising therapeutic target for a combinatorial treatment, i.e. together with SMN restoration, counteracting SMA pathology
