Congenital myasthenic syndromes: increasingly complex

Purpose of review Congenital myasthenia syndromes (CMS) are treatable, inherited disorders affecting neuromuscular transmission. We highlight that the involvement of an increasing number of proteins is making the understanding of the disease mechanisms and potential treatments progressively more complex. Recent findings Although early studies identified mutations of proteins directly involved in synaptic transmission at the neuromuscular junction, recently, next-generation sequencing has facilitated the identification of many novel mutations in genes that encode proteins that have a far wider expression profile, some even ubiquitously expressed, but whose defective function leads to impaired neuromuscular transmission. Unsurprisingly, mutations in these genes often causes a wider phenotypic disease spectrum where defective neuromuscular transmission forms only one component. This has implications for the management of CMS patients. Summary Given the widening nonneuromuscular junction phenotypes in the newly identified forms of CMS, new therapies need to include disease-modifying approaches that address not only neuromuscular weakness but also the multisystem involvement. Whilst the current treatments for CMS are highly effective for many subtypes there remains, in a proportion of CMS patients, an unmet need for more efficacious therapies.


INTRODUCTION
Congenital myasthenia syndromes (CMS) are caused by genetic defects that impair neuromuscular transmission.Efficient synaptic transmission at the neuromuscular junction (NMJ) incorporates many different proteins involved both in the signal transmission itself and in generating the prerequisite specialized synaptic structures.As a consequence, there are multiple pathogenic mechanisms that can underlie CMS.A recent comprehensive review of CMS highlights many of these mechanisms [1 && ].There are currently 35 genes reported to be associated with CMS (Fig. 1 and Table 1).The prevalence reported varies considerably between regions, 2.8-14.8 per million (UK), 1.8 per million (Brazil), and 22.2 per million (Slovenia) [2].Differences are likely to occur because of mutation founder effects in certain regions and historically because of different pick-up rates as new detection technologies are adopted.The clinical use of next-generation sequencing greatly facilitates detection of mutations in larger genes, such as AGRN, LRP4, MUSK, COLQ, and thus increases in reported prevalence is likely to continue [3][4][5][6][7].
Whilst symptom onset at birth or infancy is the commonest presentation, later onset in childhood and adult life is now increasingly recognized.The clinical phenotypes can vary in the subtypes, but the hallmark is fatigable weakness predominantly affecting axial and proximal limb muscle, ocular, bulbar and or respiratory involvement.A suspected diagnosis is based on typical clinical features, decremental responses of 10% or more on repetitive nerve stimulation (RNS) and/or abnormal jitter and block on single-fibre EMG.It is prudent to note that RNS may show incremental response on high-frequency stimulation in some presynaptic (VAMP1, UNC13A, SYT2, RPH3A) and one synaptic (LAMA5) CMS subtype [8,9].The inheritance patten in CMS is autosomal recessive except for dominant pattern seen in slow channel syndromes, SNAP25, PURA and some SYT2 patients [1

&&
]. Here, we highlight the widening phenotype spectrum associated with gene mutations that can not only have an effect of the NMJ but also show systemic and brain involvement (Table 2), and on current and emerging therapeutic strategies for CMS.
Historically, CMS has been broadly classified, based on the location of the affected protein or the site of functional impairments, into presynaptic, synaptic and postsynaptic CMS subtypes.Identification of mutations in ubiquitously expressed proteins that potentially affect functions at all these locations, or proteins that are derived from the presynaptic side of the NMJ but have their effect on the postsynaptic side can confuse these categories, but nevertheless they remain useful for understanding characteristics of the different CMS subtypes.

KEY POINTS
There is an expanding clinical phenotype with multisystem involvement in many of the newer CMS genes.
The current CMS treatments do not target multisystem involvements.
New treatments are under development, and these will need multicentre collaboration for effective clinical trials.

PRESYNAPTIC CONGENITAL MYASTHENIA SYNDROMES
Presynaptic CMS were reported to be the least common CMS subtype but there is now a growing number of gene mutations in proteins associated with release of acetylcholine (Ach) from the motor nerve terminal [10 & ].The clinical phenotype is usually early onset at birth/neonatal or infancy with varying severity of respiratory involvement (recurrent apnoea and or respiratory dependence), bulbar weakness, hypotonia, with or without muscle weakness.They can be divided into two broad categories, those involved in the synthesis and recycling of ACh (choline acetyltransferase CHAT), vesicular acetylcholine transporter SLC18A3, the high-affinity choline uptake transporter SLC5A7, and the mitochondrial citrate transporter, SLC25A1; and those involved in vesicular exocytosis, largely made up of soluble N-ethylmaleimide-sensitive factor attachment (SNARE) protein complex [synaptosomal-associated protein 25, SNAP25B, synaptobrevin 1 (also known as vesicle-associated membrane protein 1, VAMP1)], MUNC13-1 (UNC13A) and synaptotagmin 2 (SYT2).SYT2 acts as a calcium sensor to trigger vesicular release and dominant mutations in SYT2 can cause Lambert-Eaton-like features at the NMJ but are mainly seen to have a mild distal neuropathy phenotype, whereas biallelic mutations in SYT2 usually result in early-onset severe phenotype.As vesicular exocytosis is a CMS common process within many organs, and especially within the central nervous system, it is not surprising that gene defects that affect presynaptic neurotransmitter release at the NMJ can cause a wider phenotypic spectrum.Other proteins where mutations are thought to have presynaptic effects include PREPL, which encodes for the protein prolyl-oligopeptidase, and is proposed to be involved in trafficking of the vesicular ACh transporter at the NMJ and MYO9A, encoding myosin-IXA, that is involved in neuronal branching and axonal transport.RPH3A encodes for Rabphilin 3a, recently reported in a single case and is considered to be involved in synaptic vesicle regulation [9].Phenotypic features distinct from myasthenia for mutations in many of these genes are highlighted in Table 2.

Synaptic CONGENITAL MYASTHENIA SYNDROMES
The arrangement of proteins located in the synaptic cleft of the NMJ is poorly understood but mutations have been identified in a number that can have functional and/or structural roles.COLQ encodes for collagenic tail and binding domains, which anchors the asymmetric form of acetylcholinesterase to the basal lamina.Views on the exact binding partners for COLQ vary, but the most recent target put forward is LRP4 [11], which would tie in with a role for COLQ in influencing postsynaptic structures as well as ensuring (through acetylcholinesterase) the efficient breakdown of ACh.Reduced COLQ results in the persistence of ACh within the synaptic cleft, causing desensitization of endplate AChR with consequently reduced end-plate potentials, restricting the activation of the postsynaptic sodium channels (SCN4A).COLQ CMS also leads to prolonged and excessive AChR signalling, leading to AChR  [18].The phenotype in slow channel CMS is more varied, though in early years, they frequently respond well to treatment.Onset can be from birth, childhood to adulthood and weakness is generalized or may selectively involve of scapular, cervical and distal upper limb muscles with or without ocular involvement [19].AChR deficiency also results from mutations in RAPSN, the protein that anchors the tight aggregation of AChR molecules in the postsynaptic membrane, although the resulting phenotype differs from mutations within the AChR itself or from within the AGRN-signalling pathway.
RAPSN mutations present at birth or in infancy with hypotonia, weakness, ptosis, bulbar difficulties and recurrent respiratory crisis, which can be life-threatening.These crises tend to be frequent in infancy and early childhood and then improve, overall patients tend to have minimal or mild weakness in adult life.Agrin-induced AChR clustering pathway related CMS: the Agrin (AGRN)-LRP4-MUSK-DOK7 signalling pathway is crucial for the formation and maintenance of the NMJ.Agrin, which is secreted from the nerve terminal into the synaptic cleft binds to the LRP4 (low-density lipoprotein receptor-related protein 4) on the postsynaptic membrane and activates MUSK (muscle skeletal receptor tyrosine kinase).Activated MUSK signalling is amplified by the interaction with DOK7 (downstream of tyrosine kinase 7) and leads to the clustering and stabilization of AChR in the postsynaptic membrane.Mutations in AGRN, LRP4, MUSK and DOK7 present with limb-girdle pattern of weakness with varying severity, often with some ptosis but minimal eye movement abnormality, though there is marked bulbar/respiratory involvement in some cases.

CONGENITAL MYASTHENIA SYNDROMES DUE TO GLYCOSYLATION DEFECTS
Glycosylation is a ubiquitous process that is involved in protein folding, solubility, stability, assembly and intracellular transport and involves O-glycosylation and N-glycosylation.Glycosylation defects usually cause multiorgan disorders and include congenital disorders of glycosylation, congenital and limb girdle muscular dystrophy, and congenital myasthenic syndromes.Abnormal glycosylation results in reduced AChRs at the muscle endplates, leading to impaired neuromuscular transmission but it is still unclear as to why defects in this ubiquitous process cause a primary NMJ dysfunction.There are currently five genes known to cause CMS because of glycosylation defects all of whom are involved in the N-glycosylation pathway, they are GFPT1, DPAGT1, ALG2, ALG14 and GMPPB.It is of note that these genes code for proteins at the early stages or feed in to the early stages of the N-linked pathway and so presumably the NMJ is highly sensitive to disruption at this stage.Clinical phenotype is of onset in childhood to adulthood with limb-girdle weakness without usually significant eye, respiratory or bulbar involvement.Other features include raised creatine kinase, muscle biopsy in some cases showing dystrophic changes (GMPPB) and tubular aggregates (GFPT1, DPAGT1 and ALG14).Mild-to-moderate cognitive difficulties have been reported in some cases.

OTHER CONGENITAL MYASTHENIA SYNDROME SUBTYPES
Several other genes are reported to associate with a CMS phenotype.TOR1AIP1 (torsinA-interacting protein 1 gene) encodes the inner nuclear membrane protein lamin-associated protein 1 (LAP1).Whereas nuclear membrane proteins are associated with a number of muscle disorders, this is the first where mutations impair NMJ function, although the pathogenic molecular mechanism has yet to be determined.The CMS phenotype described is of childhood-onset fatigable limb-girdle pattern muscle weakness without facial or bulbar involvement [20].PLEC1, which encodes for plectin is a ubiquitously expressed intermediate filament-linking protein concentrated at sites of mechanical stress.PLEC1 mutations result epidermolysis bullosa in isolation or in association with limb-girdle muscular dystrophy.SCN4A encodes the skeletal muscle voltage-gated sodium channel Nav1.4 and is a well recognized cause of channelopathies, including hyperkalemic and hypokalemic periodic paralysis, paramyotonia congenita and sodium channel myotonia with rare cases of SCN4A-related CMS reported.PURA gene encodes Pur-alpha, which is a ubiquitous protein that has RNA and DNA binding properties and is known to be associated with neurodevelopmental problems, a CMS phenotype has now been reported in some cases [21,22].

TREATMENT
Currently there are no licensed drugs specifically for CMS treatment and all the drugs used are off-label, the doses and formulations used in CMS can be different to the licensed uses.Given that CMS is a heterogeneous disorder with many different underlying molecular mechanisms, the treatment response depends upon the subtype of CMS and treatment needs to be tailored for disease severity and patient response and can involve utilizing different combinations of the drugs.It is important to note that drugs that benefit some CMS types can worsen other subtypes (COLQ, DOK7 and Slow channel CMS worsen with acetylcholine esterase inhibitors) and hence in those awaiting genetic confirmation, empirical treatment must be with caution.The common drugs used are acetylcholinesterase inhibitors (Pyridostigmine), 3,4 diaminopyridine (3,4-DAP), Salbutamol and Ephedrine.Fluoxetine and Quinidine are used in slow channel CMS.
Acetylcholinesterase inhibitors slow the breakdown of acetylcholine in the synaptic cleft and hence prolong its activity.Pyridostigmine (Mestinon) is most commonly prescribed and is the firstline treatment in presynaptic CMS, AChR deficiency CMS, fast channel CMS and CMS due to glycosylation defects.The main side effects include nausea, abdominal cramps, diarrhoea, increased sweating and respiratory secretions.Side effects are well controlled by antimuscarinic agents like propantheline.Intramuscular neostigmine may be used during apnoeic episodes in infants and children.
3,4-DAP is a potassium channel blocker and works on the presynaptic nerve terminal by prolonging the motor nerve action potential, and hence increasing the release of acetylcholine into the synaptic cleft.Common side effect is perioral and peripheral paraesthesia.Seizures have been reported as a rare side effect of 3,4-DAP when used in the treatment of Lambert-Eaton myasthenic syndrome though rarely seen in clinical practice.It is used as add on treatment in AChR deficiency, Fast channel CMS and RAPSN among the common CMS subtypes.
Fluoxetine and Quinidine act as blockers of the AChR ion channel when in the open state and are used only in slow-channel CMS.Quinidine can cause cardiac conduction defects and both Quinidine and Fluoxetine can prolong QT interval at high doses.Fluoxetine side effects reportedly include suicidal ideation so patients should be monitored.
Beta 2-adrenergic receptor agonists include Ephedrine, and Salbutamol (Albuterol in the USA) are treatment of choice in DOK 7 and COLQ CMS.They are proposed to stabilize the synaptic structure and hence the effects can take several months to have an optimal effect.They also can have role as add-on treatment to pyridostigmine in AChR deficiency CMS and RAPSN CMS.Baseline ECG, BP and potassium level is recommended prior to initiation, after each dose increase and during intercurrent illness.Side effects can include exacerbating muscle cramps (especially in DOK7), hypotension, tachycardia and hypokalaemia particularly at high doses.High doses should be prescribed with caution because of possible adverse effects, such as hypertension, anxiety, insomnia and palpitations.

Recent developments in therapeutics
Several studies have published promising data from preclinical trials on novel therapeutics for CMS (Fig. 2).These include using adeno-associated viruses (AAV) to deliver gene replacement therapies for treating deficiencies in DOK7, COLQ and CHAT, as well as a MUSK agonist antibody therapy that rescued a DOK7-CMS mouse model.Both the AAV9-DOK7 gene therapy and the MUSK agonist antibody effectively rescued mouse models that harbour the most common mutation found in patients DOK7 c.1124_1127dupTGCC, making the mice as strong as wildtype littermates [23,24].Similarly, an AAV8-COLQ gene therapy restored ColQ-deficient mice to [25]; and an AAV9-CHAT treatment rescued a mouse model CHAT deficiency to normal strength and neuromuscular signalling [26 && ].New therapeutic strategies aimed at improving neuromuscular transmission in myasthenia gravis by either inhibiting chloride channels to lower the safety factor [27 && ], or by positive allosteric modulation of nAChR activity [28 & ] are intriguing as these strategies may also be applicable for treating a variety of subtypes of CMS.Natural history studies are now crucial to determine the most appropriate outcome measures and design for clinical trials.

Looking to the future
Improved DNA-sequencing technology has made it cheaper and easier to conduct whole-exome and whole-genome sequencing, which quickly expanded the number of genes associated with CMS.However, it remains challenging to experimentally determine whether genetic variants of unknown significance (VUS) are pathogenic, particularly missense variants and variants in noncoding regions of genes.Recent developments in bioinformatics, particularly using artificial intelligence and machine learning, have produced a number of tools that look to evaluate variants based on their position within the gene structure (such as CADD and DANN), and how well conserved the change is in evolution (EVE) and their effect on splicing (SpliceAI).Nu ´ñez-Carpintero et al. [29  & ] recently published a multilayered workflow combining genetics and metabolomics data to elucidate the molecular determinates of disease severity.A notable alternative approach recently published by Deepmind is Alphamissense, which uses their Alphafold2 structural prediction software and boasts a 90% accuracy in predicting the pathogenicity of missense variants deposited in ClinVar [30  & ].Others have combined sequenced-based approaches with the 3D position information to predict protein stability [31].Several recent publications of near atomic resolution structures of electric ray muscle type nAChR in resting and desensitized conformations have improved our understanding of the structure-function relationship of these channels, helping us understand how mutations in the channel change its function [32][33][34].

CONCLUSION
The spectrum of CMS disorders is still expanding with multisystem involvement in many of the newly identified CMS-associated genes, particularly presynaptic gene defects.This has important implications on treatment strategies as the current CMS treatments are unlikely to affect the systemic symptoms.The newer treatments in the pipeline are likely to be in clinical trials before long and there is a need to ensure that there are robust reliable outcome measures.Finally, as many CMS are treatable, expanding newborn screening programmes globally would be ideal for the early diagnosis of CMS and reducing the mortality and long-term morbidity that can results from diagnostic delays.

FIGURE 1 .
FIGURE 1. Schematic diagram of how the different congenital myasthenia syndromes-associated gene products are involved in neuromuscular junction biology, based on our current understanding.Gene names are in bold italicised capitals.Where the protein names are different to the gene names, they are in brackets beneath the gene name.

FIGURE 2 .
FIGURE 2. Therapeutic mechanisms of potential new treatments of congenital myasthenia syndromesadenoassociated viral DOK7 replacement therapy, humanized MUSK agonist antibody therapy, ClC-1 chloride channel inhibitor treatment, muscle nicotinic acetylcholine receptor-positive allosteric modulator treatment.

Table 1 .
Summary of congenital myasthenia syndrome genes

Table 2 .
Nonneuromuscular junction involvement in congenital myasthenia syndrome genes reported in the literature