Presynaptic Congenital Myasthenic Syndromes: Understanding Clinical Phenotypes through In vivo Models

SLC5A7

Solute Carrier Family 5 Member 7 gene (SLC5A7), also called Choline Transporter (CHT), is located on chromosome 2q12.3, and encodes a high-affinity choline transporter (CHT). CHT is involved in the resynthesis of ACh, taking up the choline from the extracellular space into presynaptic terminals (GC02P107969, GeneCards). (Fig. 2).

Fig. 2

–The synthesis and recycling of acetylcholine: 1. Synthesis of ACh requires the influx of glucose through a glucose transporter (GLUT) into the cell. 2. Intracellular glucose is then metabolized by glycolysis to produce pyruvate 3. Pyruvate then enters the mitochondria where it is further metabolized to Acetyl-CoA. 4. Choline acetyltransferase (CHAT) combines Acetyl-CoA with choline to produce acetylcholine (ACh). 5. ACh is packaged into vesicles through Vesicular Acetylcholine Transporter (SLC18A3), a 12-membrane protein located on the synaptic vesicle. Prolyl Endopeptidase (PREPL), plays a role in the packaging of ACh and other pre-synaptic machinery such as SNAREs into the synaptic vesicle, although the exact mechanism remains unknown. 6. Vesicles travel towards the pre-synaptic membrane and undergo exocytosis. 7. ACh is released from the pre-synaptic membrane through vesicle exocytosis. ACh diffuses across the synaptic cleft and binds to acetylcholine receptors (AChR). Acetylcholine esterase (AChE) breaks down ACh. 8. SLC5A7 is a Na⁺ and Cl^- dependent high-affinity transporter that mediates the uptake of choline at the motor neuron terminal. Genes implicated in CMS are in red.

SLC5A7 CMS is typically inherited in an autosomal recessive manner presenting at birth, however, prenatal signs like antenatal hydramnios, reduced foetal movements, and arthrogryposis have been described [8, 9, 18–20]. Respiratory dysfunction with life-threatening episodic apnoeas is a constant feature, in variable association with hypotonia, fatigable weakness, oculo-bulbar symptoms and gastrointestinal alterations [8, 9, 18–21]. RNS reveals a decremental response, and brain MRI may show brain atrophy with hypoxic ischaemic encephalopathy [8, 9, 18, 20, 21]. Cognitive defects and neurodevelopmental delay are not unusual features in SLC5A7 CMS, underlying the link between cortical cholinergic neurotransmission and CHT [9, 20, 21] (Table 1).

SLC5A7 CMS can be referred to as a multisystem disorder, encompassing the central, peripheral, and possibly the autonomic nervous system. This multisystemic involvement was apparent in animal models prior to the first case reports, confirming a causal link between clinical features and the CHT mutation.

The presence of CHT in both the central [22, 23] and peripheral nervous system was apparent when Ferguson., et al. created Cht-knockout (KO) mice (Cht^-/-) which became paralyzed and cyanotic a few minutes after birth and died of hypoxia probably due to failure of diaphragm neurotransmission [24]. When these mice were later transfected with the Cht gene under control of the motor neuron promoter Hb9, they were able to move and breathe for about 24 hours [25]. Additionally, intracellular recordings from muscle fibres revealed only initial evoked and spontaneous responses, supporting the hypothesis that Cht^-/- mice are not able to synthetize ACh after depleting initial stores. The attempt to rescue the phenotype, by limiting the degradation of ACh with AChEI physostigmine and neostigmine, failed in these animals [24]. In humans, the response to AChEI is variable, resulting in some improvements in most cases, sometimes in addition with ephedrine or salbutamol [8, 9, 20, 21]. However, in one patient, presenting with a delayed motor development and MRI brain atrophy, pyridostigmine was not efficient after 3-days neonatally [21]. This was also the case in an older 14-year-old patient with ocular and bulbar involvement [8]. Finally, three patients died despite treatment with AchEI [19, 20].

Heterozygous Cht^+/- mice survive and exhibit lower, but functionally acceptable CHT levels compared with WT [24]. They present normal performance on multiple sensorimotor, anxiety and cognitive tasks, except under physical or pharmacological stress [26]. Similar results have been obtained by Paolone G. et al., who tested cognitive performance in mice to examine cortical cholinergic neurotransmission. They postulated the presence of mechanisms that could compensate for reduced cholinergic activity in Cht^+/– mice during moderate cognitive challenges, but not in conditions requiring a sudden ACh increase. Some behavioural and mood disorders in humans were correlated with CHT gene mutations, especially the p.Ile89Val variant [27]. Indeed, a new mouse model expressing the Cht –p.Ile89Val variant has been generated, resulting in a reduction of choline clearance in the striatum and cortex, decreased ACh release after prolonged neuronal depolarization, and altered attentional performance [28]. These attentional and behavioural deficits reflect cognitive alterations found in humans. Indeed, a few patients have presented with global developmental delay, resulting in speech, learning and social interaction impairment with no awareness of surroundings [20, 21].

Cht^+/– mice also exhibited diminished parasympathetic heart effects with basal resting tachycardia and hypertension, because of depleted cardiac ACh levels [29]. There is no evidence of cardiac involvement in patients harbouring mutations in CHT, but autonomic dysfunction has been noted with core temperatures as low as 32 °C [20].

In contrast to mice, homozygous null alleles of C. elegans CHT (cho-1) are viable but only release around 40% ACh compared to WT animals. However, this is enough to permit thrashing under normal conditions, but with marked motor alterations when stressed [16] (Table 2).

CHAT

The Choline-O-Acetyltransferase (CHAT) gene encodes the homonym enzyme which catalyses ACh synthesis from acetyl CoA and choline at cholinergic synapses. It is expressed both in the central and peripheral nervous system. (GC10P049609, GeneCards) (Fig. 2).

CHAT CMS is the most common presynaptic form, accounting for 4–5% of all CMS cases. It is inherited in an autosomal recessive manner, and also known as CMS with episodic apnoea (CMS-EA) [30, 31]. Sudden episodes of respiratory crises and bulbar weakness may be provoked by infection, fever, or other stressful conditions in birth or early infancy, and in several cases this has resulted in death [32–34]. Occasionally EA is accompanied by loss of consciousness and tonic–clonic seizures [35]. Other reported symptoms comprise ptosis, fatigable weakness, proximal muscle weakness, aggravated by exposure to cold, delayed motor milestones and intellectual disability [11, 36–41]. RNS usually detects no decrement at low-frequency stimulation, but it appears after prolonged high-frequency stimulation [11]. AChEI are effective in moderating respiratory crises and improving other symptoms in some cases [11] (Table 1).

Mice with targeted deletion of the Chat gene at the NMJ appear normal in a heterozygous state, while homozygous pups die at birth, displaying flaccid paralysis. Both spontaneous and nerve-evoked postsynaptic potentials are absent in mutants, and several morphology and developmental abnormalities have been noted at the NMJ (i.e., marked excess of nerve terminal branching, altered synapse distribution with widened endplate, thinner muscles, and precocious appearance of larger AChR-rich sites at the postsynaptic membrane) [42, 43].

Similar results came from two mutant zebrafish lines, bajan and chata^tk64. Bajan zebrafish have a single point mutation (A to C) at the splice acceptor site of Chat intron 2, while chata^tk64 carry a missense mutation changing a highly conserved serine to an arginine (p.S102 R). Both exhibited no coiling movements and reduced touch response in a homozygous state [44, 45]. Spontaneous synaptic currents as well as evoked synaptic responses were markedly decreased [44] (Table 2).

Finally, an example of a naturally occurring CHAT mutant animal is the Old Danish Pointing Dog, carrying a valine to methionine substitution at position 29 of the CHAT exon 6. Affected dogs can walk and run for only 5–30 min before manifesting fore- and hind leg fatigability. Moreover, electrophysiology shows a decrement at 3 Hz induced by a long stimulation train [46].

SLC18A3

Solute Carrier Family 18 Member A3 (SLC18A3) mapped to chromosome 10q11.2, encodes for vesicular acetylcholine transporter (VAChT) that mediates ACh internalization into vesicles in the presynaptic terminal (600336, OMIM). Mutations of this gene leads to an autosomal recessive inherited CMS form (Fig. 2).

Clinical onset of SLC18A3 CMS occurs in the neonatal period, and is often associated with apnoeic episodes, hypotonia, variable degrees of joint contractures, and feeding difficulties [10, 47, 48]. However, four patients also displayed prenatal alterations [49, 50]. Ptosis, ophthalmoplegia, mild facial weakness and generalized fatigable weakness are other common symptoms. Hakoken A.H. et al. described two cases of severe and lethal CMS characterized by foetal akinesia, arthrogryposis, generalized body edema and dysmorphic features [51].

Cognitive function, especially learning capacity, attention and working memory, may also be affected [10, 47]. A decremental response to RNS is usually detected. Pyridostigmine improves clinical manifestations, and 3,4-Diaminopyridine (3,4-DAP) and ephedrine may be added in some cases [10, 48, 50]. Intriguingly, a mild reduction of left ventricular systolic function was rescued after treatment with AChEI in one patient [10] (Table 1).

One of the first animal models of the VAChT mutation was generated in Drosophila melanogaster (common fruit fly). Homozygous mutants were lethal, and those that survived till the larval stage showed reduced motility. Moreover, heterozygous flies exhibited electrophysiological defects in the giant fibre pathway after high-frequency stimulation [52]. More recently, Drosophila models have been tested with an overexpression of VAChT, demonstrating its negative impact on longevity, locomotor ability in an age-dependent way, learning and memory [53]. Surprisingly, reduced VAChT function produced a similar effect on lifespan and locomotive behavior [54]. There are currently no specific reports on VAChT mutations and life expectancy of patients, but clinical and electrophysiological features of affected Drosophila resemble those recognized in patients.

The effects of a reduced expression of VAChT in the central and peripheral nervous systems were later confirmed by Prado V.F. et al. in a Slc18a3 knockdown (KD) mouse model. They detected a reduction in VAChT levels in several central nervous system regions both in heterozygous KD mice and more markedly in homozygous ones. Moreover, miniature end-plate potentials (MEPPs) recorded at NMJs were smaller and less frequent in homozygous mutants.

Homozygous KD mice were significantly limited in physical activity, while both mutant genotypes showed difficulty in learning or remembering features for discriminating a novel object or recognizing a littermate [55]. The reduced cholinergic tone due to a slow refilling of synaptic vesicles in homozygous KD mice was displayed through an alteration in basic cognitive capacity, especially functions requiring a prolonged cholinergic involvement [56].

In addition, homozygous KD mice displayed impaired cardiac function related to autonomic imbalance between parasympathetic and sympathetic tone that was rescued by pyridostigmine [57, 58].

Further proof that VAChT is rate-limiting for neurotransmission is derived from Slc18a3 KO mice who died of respiratory insufficiency within 2 to 5 minutes following birth. However, small-amplitude and low-frequency MEPPs were detected in embryonic NMJs, probably due to a spontaneous ACh entry into presynaptic vesicles. Indeed, a lack of VAChT leads to the increase of CHAT and CHT expression, and subsequently of cytoplasmic ACh levels. Although, they demonstrated alterations in neuromuscular development like an absence of ACh, proving that passive ACh uptake by vesicles cannot compensate for VAChT deficiency [59]. Similar feedback mechanisms were detected in C. elegans by Zhu H. et al [60].

Conversely, heterozygous KO mice presented no locomotor activity dysfunction, no alteration in MEPPs or in NMJ morphology [59].

To avoid the interference from impaired peripheral cholinergic function, Martin-Silva C. et al. generated novel mutant lines with intact VAChT expression in the somatomotor cholinergic neurons, but significant reductions in the other cholinergic neurons. This technique allowed them to alter only brain cholinergic activity, resulting in the hyperactive behaviour of mice [61]. Deleting Slc18a3 from their forebrain impaired spatial memory, cognitive flexibility, and sustained attention [62, 63]; and knocking it out of the striatum caused different degrees of short-term memory deficit [64]. These studies provide evidence that ACh is fundamental to control locomotor function and to modulate hippocampal memory.

Finally, Cre-loxP technology has allowed for the deletion of Slc18a3 in select groups of motoneurons (mnVAChT-KD). Mutant mice have atrophy in muscle fibres innervated by mnVAChT-KD and appear hypoactive, leaner and with kyphosis and less muscle strength [65] (Table 2).

Findings from mice models trace once again the human phenotype from a motor, cardiac and cognitive point of view. Notably, a compromised cardiac pump function was found both in homozygous KD mice and in an affected patient, benefiting from AchEI [10]; as well as compromised hippocampal and other superior functions, that were identified as significant deficits in attention span, working memory, and immediate and delayed visual memory in a formal psychometric assessment of the same patient [10].

VAMP1/SYB1

The vesicle-associated membrane protein (VAMP), also called synaptobrevin (SYB) is part of the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complex, in association with syntaxin1 and synaptosomal-associated protein 25 kilodalton (kDa). The SNARE complex is involved in the fusion of synaptic vesicles with the presynaptic plasma membrane for the subsequent ACh release into the synaptic cleft [66] (Fig. 3).

Fig. 3

–The mechanism of calcium-dependent synaptic vesicle exocytosis at the NMJ: The ready releasable pool (RRPs) of synaptic vesicles containing ACh. When activated, the synaptic vesicle moves towards the pre-synaptic membrane terminal where calcium influx releases the synaptic vesicle bound to actin and other cytoskeletal elements (not shown in image). The “Active Zone” is the area at the pre-synaptic terminal membrane where exocytosis occurs. 1. The pool of synaptic vesicles is termed the “readily releasable pool” (RRP) and is necessary to respond to an electrical impulse. Synaptotagmin II (SytII) and Synaptobrevin/VAMP1 (SYB1/VAMP1) proteins attach to the outer synaptic vesicle membrane forming the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex. The cell membrane contains SNAP-25 and syntaxin components essential to the fusion of the vesicle and the cell membrane. 2. Docking –Here as the synaptic vesicle moves closer to the cell membrane, the SNARE complex begins to form 3. When an action potential depolarizes the pre-synaptic membrane, this causes the excitation of voltage-gated calcium channels and calcium entry. Calcium binds to both C2-domains (C2a and C2b) of SytII which induces a simultaneous binding of the vesicle to the cell membrane while completing the formation of the SNARE complex. Calcium induced binding of the SNARE complex requires additional proteins for vesicle exocytosis. MUNC18-1 inhibits the formation of the SNARE complex, locking syntaxin in a closed conformation, upon calcium induction MUNC18-1 interacts with MUNC13-1 opening the syntaxin conformation enabling the formation of the SNARE complex. This change in syntaxin conformation enables the binding of SYB1/VAMP1 initiating the fusion of the synaptic vesicle and cell membrane. In addition, MUNC13-1 contains C1, C2A, C2B, and C2 C domains which sequester calcium. MUNC13-1 also contains a central MUN domain. Importantly, Rab3-interacting molecule (RIMS1), which serves as an active zone scaffolding protein has been shown to interact with proteins ELKs and RIM-BPs. RIMS1 also interacts with MUNC13-1 by preventing homodimerization and recruiting MUNC13 s towards the active zone. RIMS1 also serves as a key regulator as it acts upon MUNC13 and 18, which are essential in determining the number of release sites and in the fusion of competent vesicles. RIMS1 can interact directly with the voltage-gated calcium channels at the presynaptic membrane, but altering the density of calcium channels present at the active zone, hereby effecting the size of the RRP. 4. The vesicle and cell membrane fuse together, the SNARE complex “zippers” the vesicle tightly to the cell membrane releasing ACh and other proteins into the synaptic cleft. Genes implicated in CMS are in red.

SYB1/VAMP1 CMS is an autosomal recessive disorder, occurring at birth. To date, nine patients have been described with a typical phenotype characterized by generalized hypotonia and muscle atrophy, bulbar dysfunction, feeding difficulties, delayed motor milestones, joint contractures or laxity, kyphoscoliosis and respiratory distress, perhaps caused by pulmonary infections [67–69]. Elongated myopathic face with ophthalmoparesis, frontal bossing, low set ears and high arched palate have also been reported [69, 70]. RNS revealed a decremental response in one case, with synaptic facilitation indicating a presynaptic defect [67] and an incremental response in others [68, 70]. Therapy with pyridostigmine led to a variable degree of improvement. In one of the most recently reported patients, salbutamol was administered in addition with pyridostigmine with some benefit [69] (Table 1).

Several VAMPs have been targeted in mice and different phenotypes elicited depending on the mutated isoform. Two highly homologous isoforms, VAMP1/SYB1 and VAMP2/SYB2, have been detected in neurons and in nerve terminals of the NMJ [71].

A nonsense mutation in Vamp1/Syb1 truncated nearly half the protein, resulting in a lethal-wasting mutant phenotype (lew) in mice. Vamp1/Syb1^lew/lew mice displayed altered movements of paws and legs, becoming progressively immobile before death within three weeks [72]. While motor endplates developed with no delay in Vamp1/Syb1^lew/lew mice, spontaneous and evoked synaptic activities were reduced [71]. The minimal registered activity was probably due to the presence of SYB2, which can partially compensate for the absence of SYB1, but its presence in the spinal cord is not enough to guarantee Vamp1/Syb1^lew/lew mice survival. However, SYB2 plays a critical role in the sensitivity to calcium-triggered vesicle release [71]. Indeed, the complete lack of SYB2 drastically reduced evoked synaptic activity in hippocampal neurons of Syb2^-/- mouse embryos, while spontaneous miniature excitatory currents had normal amplitude and rise time. Mice died immediately after birth, exhibiting body shape dysmorphism [66].

To examine the link between Syb1 and Syb2, Liu Y. et al. compared Syb2^-/- to Syb1^lew/lewSyb2^-/- mice. Both presented with increased intramuscular nerve branching. Moreover, in the latter, evoked synaptic transmission was completely abolished and spontaneous ACh release was insensitive to calcium [73] (Table 2).

Human clinical features are relatively milder than the mouse model. Salpietro V. et al. suggested the possibility of a species-specific compensation of vesicle fusion and release at the nerve terminal to explain this difference [68]. Electrophysiological results in mouse models can explain the synaptic facilitation detected in some patients, but there is no evidence of increased intramuscular nerve branching. Since the molecular pathways involving VAMP1 are still not clear, we need research to deeply understand its role at the NMJ.

SNAP25

The Synaptosomal-associated protein 25 kDa (SNAP25) gene is located on chromosome 20p12.2 and encodes for a protein that is part of the SNARE complex (SNAP25). SNAP25 and syntaxin1 mediate the fusion of synaptic vesicles to presynaptic plasma membrane where they are docked. (600322, OMIM). (Fig. 3) An obligate alternative splicing of two exon 5 sequences in SNAP25 can result in two isoforms of SNAP25 (SNAP25A and SNAP25B), which are identical except for 9 residues. In the mouse brain, SNAP25B replaces SNAP25A by the second postnatal week, as the adult-specific isoform [74].

The first report of SNAP25 CMS dates to 2014, when a de novo dominant mutation of the isoform SNAP25B (c.200T>A; p.Ile67Asn) was identified in a patient who presented with foetal hypomotility, cyanosis, and joint contractures at birth. During childhood, she exhibited developmental delay with epileptic absence episodes, confirmed by EEG, and developed eyelid ptosis, fatigable weakness, dysarthria, and ataxic gait. The response to RNS was decremental. Therapy with pyridostigmine was ineffective, while 3,4-DAP along with levetiracetam for seizures, resulted in some improvements [74] (Table 1).

While not in our original literature search time frame (from January 2000 to March 2022), a recent publication has reported another de novo SNAP25B mutation (c.529 C>T; p.Gln177X). The patient died in the sixth day of life due to respiratory failure. Prenatal ultrasound revealed multiple anomalies including severe polyhydramnios and multiple findings consistent with arthrogryposis such as micrognathia, right clubfoot, left rocker-bottom foot, diffuse skin thickening, and hypomobility [75] (Table 1).

Like other forms of presynaptic CMS, the phenotype observed in the mouse models was more pronounced than in the patient. Heterozygous Snap25 null mutant mice were phenotypically indistinguishable from WT, while homozygous mice were embryologically lethal with smaller body size and no spontaneous movements. Evoked ACh release was abolished at NMJs, proving that the SNARE complex is crucial for Ca²⁺-triggered synaptic transmission and explaining the lethality as linked to failure of diaphragm contraction [76].

As well as the peripheral nervous system, SNAP25 is highly expressed by neurons in the central nervous system. Which may explain the presence of central neurologic manifestations such as seizures and ataxic gait in patients.

The Snap25 mouse model obtained by creating a missense mutation in a highly conserved domain of the SNAP25b isoform has been termed the “blind-drunk mouse”. These mice display ataxic gait with behavioural impairment resembling that of schizophrenia. They also have a lower frequency of evoked cortical excitatory postsynaptic potentials [77].

A brain-specific Snap25 KO mouse also demonstrated behaviour signs related to schizophrenia. The reduction of SNAP25 expression in the cortex caused a remarkable elevation of extracellular glutamate levels, resulting in abnormal hyperactivity and stereotypical behavior [78]. Hoerder-Suabedissen et al. produced a transgenic mouse with cell-specific loss of Snap25 from cortical projection neurons (cKO). cKO mice were viable at birth with normal neuronal development, but axonal projections became fragmented and deteriorated three weeks later [79].

In 2010, McKee A.G. et al. over-expressed Snap25 in the adult rat dorsal hippocampus by infusion of a recombinant adeno-associated virus vector. They noticed a defect in synaptic function resulting in cognitive deficits, such as delayed acquisition of spatial and fear memory consolidation. Hence, both reduced and excessive Snap25 activity has been implicated in memory and cognitive function impairment [80] (Table 2).

The only reported patient did not suffer from cognitive and behavioural alterations. However, we should consider these features as part of SNAP25 CMS clinical pictures in order to reduce misdiagnoses of this type of CMS.

SYT2

The Synaptotagmin 2 (SYT2) gene encodes for a synaptic vesicle membrane protein (SYT2) that works as a calcium sensor in vesicular trafficking and exocytosis (GC01M202559, GeneCards) (Fig. 3).

SYT2 CMS can be inherited in either an autosomal dominant or a recessive manner. The former pattern was described in patients mainly with a childhood onset [81–83], although not exclusively [84]. The latter, was associated with early onset and a more severe phenotype, characterized by reduced foetal movements, hypotonia at birth, joint laxity, and delayed motor milestone [85–87]. Typical manifestations include muscle weakness and fatigability, variable muscle atrophy and reduced or absent deep tendon reflexes, partially reversible after exercise [81]. Ocular involvement has been reported in a few families [82]. Other common features are foot deformities, such as pes cavus and hammer toe, and lower limbs sensitivity alterations [81, 82]. Sometimes electromyography of distal muscles detects a slight reinnervation, that is consistent with a motor neuropathy [81, 86]. Electrophysiological studies revealed low-amplitude CMAPs and a certain degree of post exercise amplitude facilitation [81, 83]. Therapy with 3,4-DAP ameliorated both clinical and electrophysiological manifestations, as did pyridostigmine and salbutamol in some patients [82, 84–87] (Table 1).

The role of SYT2 was first examined in Drosophila, in which a decrease of evoked transmitter release and its calcium-affinity was obtained through mutating two aspartate residues in the C₂B calcium-binding pocket of SYT2 [88]. More recently, Shields. et al. reproduced the human substitution of a leucine with a proline in position 308 (syt^L - P), in the same domain of Drosophila SYT2, via pan-neuronal expression of syt transgenes in the syt^null background. SYT2 is substituted by other isoforms in flies, but syt^L - P results in lethality in the absence of WT syt. Heterozygous syt^L - P were viable and showed marked fatigability, less motor capacity and reduced evoked transmitter release, similar to humans [89].

Analogous results were obtained from mice with a point mutation in Syt2, resulting in a decreased neurotransmitter calcium-dependent release in the Calyx of Held. Mice were viable but infertile, presenting smaller and severely ataxic in a homozygous state [90]. These results were further confirmed in Syt2 homozygous KO mice, which displayed severe motor dysfunction two weeks after birth and died after three weeks [91].

Finally, experiments on syt2 MO KD zebrafish showed a reduction in synchronous release that was responsible for around 90% of the neuromuscular transmission [92] (Table 2).

UNC13A

UNC13A encodes the protein MUNC13-1 which is a protein of the presynaptic active zone in cholinergic neuromuscular synapses and glutamatergic brain synapses that is involved in synaptic vesicle priming [93]. It binds the SNARE protein syntaxin 1B, shifting in closed conformation the position of Munc18-1, that normally stabilizes syntaxin 1B. In this way, syntaxin 1B is active and contributes to form the SNARE complex for vesicles exocytosis [94] (Fig. 3).

UNC13A CMS has been reported in only one patient with a homozygous nonsense mutation. The patient was born prematurely, presenting with hypotonia, feeding difficulties and respiratory distress with cyanotic episodes. She developed some dysmorphisms (marked frontotemporal narrowing, high-arched palate, small jaw, thoracic kyphoscoliosis, clinodactyly and joint contractures), delay in motor function acquisition, eyelid ptosis and reduced oculomotion. Clinical features comprised microcephaly and cortical hyperexcitability. EMG revealed low-amplitude CMAPs at rest, decremental response to low-frequency RNS and an incremental response to high-frequency. Therapy with pyridostigmine and 3,4-DAP slightly improved her symptoms, but the child died around 4 years old [94] (Table 1).

This type of CMS looks like a syndromic disorder, comprising a marked involvement of the central nervous system supported by the fact that MUNC13-1 has been abundantly shown in rat brain. Two other minor isoforms of the protein are MUNC13-2 and MUNC13-3 that are expressed in the rostral brain region and cerebellum, respectively. MUNC13-1 mice died postnatally because of a deficiency in synaptic vesicle exocytosis in the central nervous system. While Munc13-3 mice presented parallel fibre–Purkinje cell synapses of normal morphology but altered function, with a negative impact on motor learning [93]. Varoqueaux et al. created mice with a double deletion of Munc13-1/2 and triple deletion of Munc13-1/2/3 and, once more, the absence of MUNC13-1 resulted in lethality for both. Experiments on embryos, presenting paralyzed, showed morphological alterations in NMJs such as abnormal nerve terminal branching, larger endplates and improper alignment of muscle fibres. Evoked synaptic transmission was reduced while spontaneous release persisted [95].

UNC13A was also evaluated in C. elegans as the ortholog unc-13, which shares similar C1 and C2B domains that are essential for neurotransmission regulation. Removal of either the C1 or C2B domain enhanced calcium-triggered synaptic transmission, while deletion of both domains stopped synaptic transmission. Indeed, unc-13 null mutants were paralyzed, but the rescue of a single copy of unc-13 or of the protein lacking only C2B domain reverted to normal locomotion [96].

Motor impairments, altered learning, and reduced synaptic transmission from mouse and C. elegans models reflect UNC13A CMS, resulting in lethality in a homozygous state.

MYO9A

The Myosin IXA (MYO9A) gene encodes for an unconventional myosin that is an actin-based motor molecule that inhibits RhoA by stimulating its GTPase activity (MYO9A) (GeneCards, GC15M071822) (Fig. 4A).

Fig. 4

–MYO9A and LAMA5 in CMS: A) Loss of MYO9A has been shown to impair actin cross-linking, causing an alteration in the growth of axons and terminal structure. In addition, RhoGAP activity is impeded, increasing the activation of the RhoA pathway, impairing the nerve cytoskeleton and intracellular transport resulting in compromised secretion of agrin. A reduction in agrin secretion results in less activation of the Agrin-MuSK-LRP4 AChR clustering pathway. B) Laminins are the most abundant non-collagenous protein in the basal lamina. There are five extracellular matrix heterotrimeric glycoproteins composed of α, β, and γ subunits. Laminins are involved in cell signalling, development and maintenance of the NMJ, and regeneration of the nerve terminal. While Laminin α5 (LAMA5) function has not been fully elucidated, CMS patients with mutations in LAMA5 have a reduction in the number of synaptic vesicles present for release due to the small size in nerve terminals and a reduction in the number of active zones. In addition, specific mutation to the coiled-coil domain LAMA5 may play a role in preventing interaction with other laminins causing dysfunctional agrin secretion.

To date, three patients of German and Turkish origin have been described in a single publication [15]. They variably presented with ptosis and ophthalmoplegia, bulbar dysfunction, generalized muscle weakness, hypotonia, episodic apnoea and respiratory failure at birth. During follow-up, developmental delay and some cognitive abnormalities were evident. SFEMG detected abnormal jitter and RNS revealed a decremental response in one patient. The best treatment approach remains ambiguous as the German patient responded to pyridostigmine and 3,4-DAP, but the Turkish siblings suffered from a respiratory crisis after oral intake of 3.4-DAP and fluoxetine [15] (Table 1).

MYO9A CMS presents a complex clinical picture, probably due to the ubiquitous expression of this protein. Rho activity is involved in morphology and differentiation of ciliated epithelial cells and renal proximal tubules, reflecting different aspects of MYO9A mutated animal models.

Myo9a deficient mice developed severe hydrocephalus with enlargement of lateral and third ventricles and stenosis of the Sylvian aqueduct. As a result, they displayed hopping and a trembling gait in adulthood [97]. They also developed bilateral renal disease, characterized by dilated renal calyces and parenchyma fibrosis, leading to polyuria and proteinuria [98]. Folci et al. demonstrated that MYO9A plays a role in hippocampal synapses and cognitive behaviour. Myo9a heterozygous KO mice presented with structural and molecular changes in hippocampal synapses, affecting learning and memory tasks as in reported patients [99].

Recently, the function of MYO9A at the mammalian NMJ was examined and while its exact location has still not been determined, several pieces of evidence suggest a presynaptic role [15]. A zebrafish model, in which the MYO9A orthologues, myo9aa and myo9ab, were knocked down using morpholinos developed curly tails, cardiac oedema, and abnormal swimming in response to tactile stimulation. The axons were aberrant, branching to the adjacent myotome beyond their target [15].

Based on the results from Myo9a-depleted NSC-34 cells (mouse motor neuron-derived cells), the same group demonstrated an impaired trafficking and release of proteins in the absence of Myo9a. One of these proteins, agrin, is crucial for the proper development and function of the postsynaptic endplate. It was hypothesized that some of the aberrations in the myo9a morphant fish were due to its reduction. This theory was confirmed in deficient myo9aa/ab zebrafish, obtained through a CRISPR/Cas9 approach where injection of morphants and crispants with agrin improved their locomotion and NMJ morphology [100, 101].

Animal models’ motor and behaviour alterations partly match the human phenotype. Patients’ hypotonia and muscle weakness assume the same pathophysiological mechanism of zebrafish abnormal swimming. In addition, patients present with cognitive impairment, similar to mice who display learning and memory defects. However, there is no evidence of hydrocephalus in reported patients, whose brain MRIs are normal, and there is no evidence of renal dysfunction.

PREPL

Prolyl Endopeptidase-Like (PREPL) is a ubiquitous serine protease, highly expressed in the brain, kidney and muscle. Its function remains partly elusive, but it is probably involved in the control of the trans-Golgi network morphology and sorting, as well as in the regulation of synaptic vesicle exocytosis (GC02M044281, GeneCards; 609557, OMIM) (Fig. 2).

Deletions of PREPL in combination with other genes at the 2p21 chromosomal region are associated with different variants of the hypotonia-cystinuria syndrome (HCS), characterized by cystinuria, neonatal hypotonia, eyelid ptosis, occasional bulbar symptoms, and growth hormone deficiency [102]. Some of these features suggest a neuromuscular transmission defect, but the first case diagnosed with a myasthenic syndrome related to PREPL deficiency was reported by Régal L. et al. The patient presented with marked hypotonia and feeding difficulties at birth, later associated with ptosis and daily fluctuating proximal muscle weakness. Electrophysiology studies revealed low amplitude MEPPs. Additionally, growth hormone blood levels were deficient. Genetic analysis detected compound heterozygosity for a paternal nonsense mutation in PREPL (p.Met270Ter) and a maternal deletion involving PREPL and SLC3A1 (one of the genes causing HCS). The patient improved with pyridostigmine treatment and did not worsen after weaning off the medication [103]. Other patients have been recently reported with similar features and various combinations of facial dysmorphism, developmental delay, hypergonadotropic hypogonadism, and childhood obesity [104–108]. One patient with a splicing mutation in the PREPL gene was characterized by the absence of a uterus and ovaries, microcephaly and short neck [109] (Table 1).

PREPL is a unique protein, different from the others in the family, because it plays a role in protein-protein interactions rather than in peptide proteolysis [110]. It shows homology with prolyl endopeptidase (PREP), whose knock-down in mice produced an impairment in spatial learning and memory, due to its expression in hippocampus and entorhinal cortex [111]. Deletion of Prepl in mice led to hypotonia in five-day old pups and impaired growth, as reported in patients. Therefore, it could be also hypothesized an underlying reduced growth hormone signalling, remarking patients’ phenotype [110] (Table 2).

LAMA5

Laminin alpha-5 (LAMA5) is part of the laminins family, a group of extracellular matrix glycoproteins that participate in several biological processes. LAMA5 is widely expressed in vertebrate tissues and mediates cellular attachment and migration during embryonic development (GC20M062307, GeneCards) (Fig. 4b).

Currently, the only described LAMA5 CMS was caused by a homozygous missense mutation, resulting in a deficient number of releasable SVs. Indeed, the mutant LAMA5 reduces cell adhesion and neurite outgrowth, and influences agrin attachment to the extracellular matrix. Furthermore, the reported mutation impairs the interaction between LAMA5 and the synaptic vesicle glycoprotein 2A (SV2A), which in turn binds to synaptotagmin, regulating the Ca2 + dependent SVs exocytosis.

LAMA5 CMS patient presented with a multi-faceted phenotype. Clinical features comprised congenital hypotonia and respiratory failure, scoliosis, chronic inflammatory bowel disease, neck and limb muscles weakness. Dysmorphic features, myopia, bilateral ptosis, facial muscles weakness and tics were also observed. RNS showed a marked decremental response and facilitation occurred after maximal muscle activation. The patient responded well to pyridostigmine and 3,4 DAP [14, 112] (Table 1).

A more complex phenotype occurred in mouse models, which also demonstrated the role of LAMA5 in pulmonary [113, 114] and renal development [115]. For instance, Lama5 KO mice died at the embryonic stage. Embryos presented with a defective anterior neural tube closure, syndactyly, aberrant genesis of kidneys and an absence of visceral pleura basement membrane with reduced lobar lung septation [113]. To avoid early lethality, LAMA5 deficiency was restricted to lung epithelial cells but mice still died perinatally of respiratory failure due to marked abnormalities in lung development [114].

However, a specific neuromuscular phenotype was obtained by creating a muscle specific KO Lama5 mouse, showing a delayed pre- and postsynaptic differentiation in NMJs [116].

In addition, a missense mutation of the Lama5 gene was generated in mouse models via CRISPR-Cas9 genome editing, further elucidating the importance of the role of laminin in tissue development and homeostasis. Mutant mice showed defective foetal growth and developmental abnormalities. Similar alterations occurred in the reported patient, such as elongated face, elevated hard palate, and eye defects, reflecting the role of LAMA5 in embryonic development [117].

Finally, comparable results were obtained from zebrafish, in which LAMA5 deficiency led to an altered formation of pectoral fins and a lack of fin folds, highlighting its role in basement membrane integrity [118] (Table 2).

RPH3A

Rabphilin 3a (RPH3A) encodes for the protein RPH3A, which is involved in docking and fusion steps for neurotransmitter release. Specifically, at the presynaptic level, RPH3A is recruited to the synaptic vesicle membrane binding 14-3-3 protein, Ca2+, phosphatidylinositol 4,5-bisphosphate (PIP2) and SNAP25 to modulate vesicle trafficking and calcium-triggered exocytosis (GC12P112570, GeneCards) [119].

An 11-year-old girl with a heterozygous mutation in RPH3A was described by Maselli R.A. et al. During early childhood, she presented with limb weakness and fatigability, later followed by hand tremors, hands incoordination, postural imbalance, and nasal speech. She also had learning disabilities with no abnormalities at brain MRI and EEG. High frequency RNS revealed and increment of 30% of CMAP areas and SFEMG detected an increased jitter. A favourable response to treatment with albuterol sulfate was shown. The authors demonstrated a synaptic vessicle density reduction, an increase in nonvesicular synaptic membranes, and the presence of degenerative lamellar bodies in the patient NMJ [119] (Table 1).

The exact function of RPH3A in the NMJ has not been completely elucidated. A null mutation in rbf-1, the RPH3A homolog in C. elegans, did not alter NMJ general morphology, nor defecation, pharyngeal pumping, and mating assays. Rbf-1 mutants appeared lethargic in absence of exogenous stimulation. Indeed, RPH3A alterations seemed synergic with SNARE complex’s ones, but rbf-1 mutants did not enhance the behavioural defects of synaptotagmin mutants. Thus, the role of RPH3A in the Ca²⁺-sensitive SVs release is still controversial [120] (Table 2).

Mitochondrial genes

It has long been thought that mitochondria play a critical role at the NMJ, partly due to their clustering at the synapse. Solute carrier Family 25 Member 1 (SLC25A1) encodes the mitochondrial citrate carrier across the inner mitochondrial membrane. (GC22M019407, GeneCards) Hence, SLC25A1 mutations result in an impaired citrate/isocitrate export from mitochondria, and subsequently increased excretion of 2-hydroxyglutaric acid. This condition was reported in an 18-month-old female manifesting poor sucking, hypotonia, and prolonged apnoeas at birth, followed by psychomotor retardation, epilepsy, sensorineural deafness, and radiological evidence of corpus callosum agenesia and optic nerves hypoplasia [121]. Other patients with a variable associations of speech and motor developmental delay, learning difficulties, fatigable weakness, ptosis, and diplopia were later described, recognizing a link between SLC25A1 and altered NMJ transmission [122–126]. Indeed, SFEMG examination showed increased jitter or jitter with blocking and RNS detected a decremental response with some clinical improvement after therapy with 3,4-DAP or AChEI. Interestingly, reflexes were reduced at rest with post-exercise potentiation in one patient, suggesting a presynaptic alteration [123, 124].

This hypothesis was corroborated by the SLC25A1 zebrafish orthologues knocking down via antisense MOs injection. The animal model presented a motor impairment similarly to humans, displaying altered tail morphology, swimming, and touch-evoked escape responses. Furthermore, histological NMJ evaluation revealed short motor axon terminals with incomplete synapse formation [123]. Indeed, mitochondria are crucial for the presynaptic nerve terminal function since it requires a high energy demand [124] (Table 2) [127–129]. To date the SLC25A1 mutation is the only mitochondrial mutation described to cause CMS. However, given the importance of mitochondria to neurotransmission it is likely future mutations will be described necessitating the creation of a fifth ’mitochondrial CMS’ group.