Late presentations of congenital myasthenic syndromes: How many do we miss?

Late presentations of congenital myasthenic syndromes – how many do we miss? Nidhi Garg, MBBS (Hons), FRACP1, Con Yiannikas, MBBS (Hons), FRACP2, Todd A. Hardy, PhD, FRACP1, Katsiaryna Belaya, PhD3, Jonathan Cheung, MA3, David Beeson, PhD3, Stephen W. Reddel, PhD, FRACP 1 1

Neuroimmunology Clinic, Concord Hospital and University of Sydney, NSW, Australia Department of Neurology, Concord and Royal North Shore Hospitals, University of Sydney, NSW, Australia 3 The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK 2

Characters in title: 76 Words in abstract: 149 Words in manuscript: 2978 Figures: 2 Tables: 3

Corresponding Author S. W. Reddel Departments of Neurology and Molecular Medicine, University of Sydney, Concord Hospital, Sydney, New South Wales 2139, Australia E-mail: [email protected] P: +61 2 9767 6416 F: +61 2 9767 7807 Full Names and Email Addresses of Authors Nidhi Garg: [email protected] Con Yiannikas: [email protected] Todd A. Hardy: [email protected] Katsiaryna Belaya: [email protected] Jonathan Cheung: [email protected] David Beeson: [email protected] Stephen Reddel: [email protected] Running Title: Late presentations of CMS Disclosures: All authors declare that they have no financial disclosures or conflicts of interest relevant to the submitted manuscript. Ethics Approval and Patient Consent: The project was approved by Sydney Local Health District Human Research Ethics Committee – Concord Repatriation General Hospital. Written patient consent was obtained for the use of de-identified medical details in a published case report. Acknowledgements: We would like to thank Doctors Denis Crimmins, Jamie Gordon, John King, and Carolyn Sue for referring patients and providing clinical details, and particularly, Dr Mark Thieben for referring the first family and suggesting the paper.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/mus.25085 This article is protected by copyright. All rights reserved.

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ABSTRACT Introduction: Congenital myasthenic syndrome (CMS) usually presents neonatally or in early childhood. When it presents later, it may be mistaken for seronegative autoimmune myasthenia, and unnecessary immunosuppressive treatment may be administered. Methods: Patients who met criteria for seronegative generalized myasthenia without congenital or early childhood onset, but with an affected sibling were tested for CMS associated genes using exome and Sanger sequencing. Results: Four sibling pairs from non-consanguineous families were identified. Three had mutations in the RAPSN gene, and 1 had a mutation in CHRNA1. One sibling of a pair with symptoms of fatigue but no convincing features of neuromuscular dysfunction tested negative on genetic studies. The definite CMS cases comprised 7 of 25 seronegative patients with definite generalized myasthenia in the clinic, and over half had been treated for autoimmune myasthenia. Discussion: CMS is probably underdiagnosed in seronegative myasthenic disorders and should be considered in the differential diagnosis. (note that I have altered the wording of the abstract to get it under the 150 word limit) Keywords: Congenital myasthenic syndrome, seronegative, RAPSN, CHRNA1, genetics, myasthenia gravis

John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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INTRODUCTION The congenital myasthenic syndromes (CMS) are a varied group of genetic disorders characterised by fluctuating weakness due to defective neuromuscular transmission1. They typically present shortly after birth or in early childhood, and post-childhood onset is uncommon2. Classification traditionally divides the syndromes into presynaptic, synaptic, and postsynaptic forms on the basis of the site of the defect, and postsynaptic mechanisms are most common. Recessive inheritance due to a loss of function mutation is typical for the vast majority of syndromes,2,3 and hence a family history of the disorder is commonly absent. Furthermore, although some patients with CMS may have distinct electrophysiological features, many will have a typical decremental response to slow frequency repetitive nerve stimulation,4 which makes electrophysiological differentiation from acquired autoimmune myasthenia gravis (MG) difficult. The prevalence of genetically confirmed CMS is 9.2 per million children under the age of 18 in the UK5. However, there are no reliable data on the true incidence or prevalence of CMS, and underdiagnosis is likely3.

MG is an autoimmune disorder of neuromuscular transmission. Approximately 80 – 90% of patients with generalized MG have serum antibodies to the acetylcholine receptor (AChR), and 40-50% of the remaining patients have antibodies to muscle-specific receptor tyrosine kinase (MuSK). Seronegative myasthenia gravis (SNMG) refers to generalized autoimmune disease without detectable autoantibodies to known antigens by standard assays, at least including AChR and MuSK6. Twothirds of seronegative patients have antibodies that only bind clustered AChR that are not detected by standard methods7. Others have antibodies against low-density lipoprotein receptor-related protein 4 (LRP4) or Agrin (AGRN), although the pathogenic role of these antibodies is not yet clear.

In this paper, we report that a proportion of late childhood or adult onset patients with apparent acquired SNMG in fact have a late presentation of CMS rather than autoimmune MG.

METHODS Patient cohort and selection criteria:


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Patients were drawn from the Neuroimmunology clinic of Concord Repatriation General Hospital, Sydney, Australia, where a significant number of patients are referred with possible or apparent SNMG. The majority of these patients do not have a myasthenic disorder on further assessment and instead have other eyelid or ocular motility disorders, and/or other causes of generalized weakness, fatigue, or asthenia. We excluded patients with probable or definite ocular MG without generalized features, as this is relatively common, often not fully investigated, and often seronegative.

Patients with a generalized myasthenic disorder without congenital or early onset were identified on the basis of a clinically consistent generalized myasthenic syndrome with either positive antibodies, electrophysiological confirmation of a neuromuscular junction disorder [abnormal repetitive nerve stimulation (RNS) study and/or single fiber electromyography (SFEMG)], or both. Cases were defined as definite seronegative if they were clinically typical with positive electrophysiology but AChR and MuSK antibodies on more than 1 sample and in 2 separate laboratories were negative. Patients with Lambert Eaton Myasthenic Syndrome (LEMS) were identified and excluded from this group.

Patients were tested for genes associated with CMS if they met criteria for SNMG, without congenital or early childhood onset, and with a definite or possibly affected sibling. All patients tested gave written informed consent to participate in the study.

Exome and Sanger sequencing: We used high throughput whole exome sequencing to identify potential CMS mutations in the sibling pairs. The exome libraries were captured from 3µg of genomic DNA using Agilent SureSelect XT Human All Exon v.4 kit. The libraries were sequenced by 100nt paired-end reads on the Illumina HiSeq platform. The obtained sequences were mapped to human genome build hg19 by using Novoalign software (Novocraft Technologies). Variants were called using the Samtools program8. Variants were filtered out if their population frequency was 0.01 or more according to 1000 Genomes Project9. We used ANNOVAR software to annotate and separate nonsynonymous substitutions,


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splicing mutations, and mutations in 3’ or 5’ UTRs10. We further filtered the obtained variants against an in-house database of 14 exomes from patients with unrelated disorders. The sequencing revealed mutations in known CMS-associated genes RAPSN and CHRNA1. The presence of mutations was then confirmed by PCR amplification of respective exons and Sanger sequencing.

Mutation RAPSN p.Glu162Lys has previously been shown to be pathogenic and to reduce AChR clustering11, however pathogenicity for RAPSN p.Ser201Asp was uncertain. We therefore looked for an effect of this mutation on AChR clustering by introducing cDNA harboring the RAPSN mutation into rapsn-/- myotubes as described by Cossins et al.12 (Figure 1).

RESULTS A total of 162 patients were identified with a generalized myasthenic syndrome without congenital or early onset (Table 1). Thirty-six patients were seronegative for AChR and MuSK antibodies. Ten of these patients had LEMS, and 1 had autoimmune MG with antibodies to LRP4. Of the remaining 25 patients, whom we classified as having late-onset SNMG, 7 patients were identified who had a definite or possibly affected sibling. One pair had 1 sibling with symptoms of fatigue but no definite clinical feature or positive test for neuromuscular dysfunction and was also negative on genetic studies. These 7 CMS patients comprise the remaining affected single sibling and 3 sibling pairs, all from non-consanguineous families. One sibling in a pair did not undergo genetic testing, but the clinical presentation and electrophysiology were consistent with a diagnosis of CMS, which was confirmed in the other sibling. A further 3 patients likely to have CMS have been identified within the generalized seronegative cohort and are yet to be tested.

Case Reports Case presentations are outlined below, and further clinical data are summarized in Tables 2 and 3.

Sibling Pair 1 (RAPSN N88K homozygous – case 1) Patient 1


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The diagnosis of a myasthenic syndrome was made after this patient developed respiratory failure and difficulty being weaned from a ventilator following a general anesthetic at age 55. He had a history of weakness since mid-childhood and struggled to keep up with his peers at sport. He had limb-girdle and tibialis anterior weakness. Eye abduction was limited to 30º. He was treated initially for autoimmune MG with plasma exchange which was not beneficial. He had initial symptomatic improvement with pyridostigmine, but it was not sustained. The addition of 3,4-diaminopyridine (3,4DAP) resulted in persistent improvement in strength. Patient 2 This patient developed significant symptoms of fatigue at age 50, although mild symptoms could be dated to childhood. A mood disorder complicated the history. She had generalized give-way type weakness on examination, but did not clearly fit into a myasthenic syndrome. There was no decrement on RNS, and SFEMG and muscle biopsy were normal. She tested negative for CMS on exome sequencing.

Sibling Pair 2 (RAPSN N88K homozygous) Patient 3 Patient 3 was first examined in his teens for bilateral ptosis. In his twenties, he found he was unable to hold his head up after 15 minutes of go-cart racing and was noted to have generalized weakness on review. Pyridostigmine was mildly effective for ptosis. He had a good response to 3,4-DAP. Patient 4 This patient became symptomatic at age 19 with fatigable proximal lower limb weakness. An edrophonium test was positive. Pyridostigmine was mildly effective. The addition of 3,4-DAP provided additional benefit.

Sibling Pair 3 (RAPSN S201N + E162K – only case 5 tested) Patient 5 Patient 5 first noticed symptoms at age 15 with frequent falls while playing sport and difficulty keeping up with his peers. He had limb-girdle and tibialis anterior weakness plus prominent deltoid


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wasting. He had a poor response to pyridostigmine. The addition of 3,4-DAP resulted in marked improvement in limb strength and improvement in electrodiagnostic parameters (Figure 2: A, B). Patient 6 Patient 6 was first noted to have weakness at age 8. Several years later she was diagnosed with MG and underwent a thymectomy at age 12. She also received treatment with corticosteroids. Neither resulted in any significant improvement. She had limb-girdle and tibialis anterior weakness. She was maintained on pyridostigmine for 35 years before the diagnosis of CMS was made on the assumption of a shared diagnosis with her brother. 3,4-DAP was added with additional effect. DNA on this patient was not available for testing at the time.

Pair 4 (CHRNA1 F256L + R55H) Patient 7 Patient 7 presented at age 27 years with a 6 month history of fluctuating lower limb weakness. In retrospect, symptoms were probably present earlier, as both siblings were repeatedly reprimanded in their teens for being unable to carry heavy packs during school camps. Initial treatment with pyridostigmine resulted in marked improvement in symptoms. Approximately 10 years later, symptoms worsened, and a trial of plasma exchange was given with no benefit. She responded to the addition of 3,4-DAP with a repair of decrement on RNS (Figure 2: C, D). Patient 8 Patient 8 presented at age 21 years with an 18 month history of fatigable proximal weakness. Two years previously there was a history of prolonged neuromuscular blockade following general anesthesia. Shortly after presentation, he underwent thymectomy followed by prolonged oral prednisone therapy in addition to pyridostigmine with some effect. He still feels better on treatment and therefore prefers to continue corticosteroids.

Results of exome and Sanger sequencing/Mutations: Three of the 7 CMS patients were homozygous for the RAPSN N88K (c.264C>A) mutation (Patients 1, 3, and 4). Patient 2 had suggestive symptoms but did not fulfill criteria for a myasthenic syndrome.


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No disease-causing mutation was identified on genetic studies. Patient 5 was compound heterozygous for mutations in the RAPSN gene (S201N + E162K). His sibling (Patient 6) has not yet been tested.

One sibling pair (Patients 7 and 8) were compound heterozygous for mutations CHRNA1 F256L + R55H. Analysis of the binding of α-bungarotoxin to the cell surface of cells expressing the AChR αsubunit harboring p.Arg55His shows a modest reduction in cell-surface AChR expression to approximately 60% of wild type. The modest reduction in expression of this second allele may be sufficient to exacerbate the borderline pathogenic effect of the F256L mutation on the first allele.

Frequency of CMS in the clinic: These 7 patients with CMS comprised 4% of the 162 patients in the clinic with a generalized myasthenic disorder. Furthermore, they comprise 7 of the 25 patients without a definitely positive antibody or LEMS (i.e., SNMG). A further 3 patients in this group have late-onset very slowly progressive myasthenia responding to pyridostigmine and 3,4-DAP, but not to plasma exchange or other immunotherapy, and have not been tested yet for CMS.

Clinical Data: The average age at presentation of the 7 patients was 26 years (range 8 – 55 years) with an average age of 15 years (range 6 – 26 years) at symptom onset. All patients had proximal weakness, and only 1 patient had involvement of extraocular muscles. One patient had bulbar involvement. Most of the RAPSN patients had weakness of tibialis anterior. The 2 CHRNA1 patients had facial weakness and ptosis. All affected patients tested had decrement on RNS of the accessory nerve. Four of the 7 patients were treated for autoimmune MG prior to being seen in our clinic, and 2 patients had undergone thymectomy. Almost all patients had some response to pyridostigmine, although it was only a mild response and was also not sustained over time. All patients who were given 3,4-DAP had a very good response to the drug either alone or in combination with pyridostigmine.

DISCUSSION


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Late-onset CMS may be difficult to differentiate from seronegative autoimmune MG on clinical and electrophysiological grounds and it appears to us that in many cases the possibility of late-onset CMS is not being considered. Difficulty in identification of late-onset CMS has previously been noted in a French cohort in which some patients were initially diagnosed as having SNMG or myopathy13. Later diagnosis or initial alternative diagnosis of milder CMS has also been recognized in a recent UK review14.

We present 7 patients with CMS who presented later in childhood or adulthood, many of whom were initially mistaken for SNMG. Our experience was informed by chance, as the presence of an affected or symptomatic sibling provided a clue to a genetic diagnosis. However, as the vast majority of CMS are recessively inherited, the probability of an affected offspring to carrier parents is only 1 in 4 and thus, in an average sized family, having 2 or more affected individuals is unlikely. We therefore suggest that late presentations of CMS should be considered more widely in sporadic cases of apparently acquired but repeatedly SNMG even in the absence of family history.

There are no accurate data on the incidence of CMS in apparently acquired and electrically definite SNMG, and a wide coverage study of CMS genes in this setting has not been performed. Furthermore, a genetic diagnosis is not established in a significant proportion of patients with clinically suspected CMS, adding to diagnostic uncertainty. In a large cohort study of European patients, a disease causing mutation was identified in only 44% of 680 patients with suspected CMS15.

Postsynaptic mechanisms account for the vast majority of CMS cases, and approximately 88% of UK cases result from mutations affecting the AChR, rapsyn, or DOK714. In a large European study, the most frequent mutation identified was in the CHRNE gene, which codes for the ε-subunit of the AChR, accounting for 50% of identified mutations. RAPSN, COLQ, and DOK7 were the next most frequently involved genes, accounting for 15%, 12%, and 10% of mutations, respectively. Mutations in the α-subunit of the AChR (CHRNA1) were rare, accounting for only 1% of all mutations15.


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Three families in our series had mutations in the RAPSN gene with 3 patients homozygous for the N88K mutation. Acetylcholine Receptor-Associated Protein of the SYNapse (rapsyn) is involved in co-clustering of the AChR at the postsynaptic membrane, and mutations result in postsynaptic CMS due to endplate AChR deficiency16. Rapsyn mutations have been reported to occur in 6-15% of genetically confirmed cases of CMS in the largest cohort studies4,15. The N88K mutation is most frequently identified, and most patients are either homozygous for N88K or heteroallelic for N88K and another mutation12. The carrier frequency is reported to be 1.7% in the European population,17 and it has been suggested that this mutation derives from an ancient Indo-European founder17-19. In a Norwegian cohort of 74 SNMG patients with late onset symptoms, 1 patient was found to be homozygous for the N88K mutation, and another was a carrier6. Although the authors concluded that the overall frequency of this mutation in SNMG is low, the cohort was not limited to generalized cases and included seronegative cases with purely ocular symptoms, differentiating it from our patient cohort.

Late childhood and adult-onset cases of rapsyn CMS have been reported, but are less common than onset in the first few years of life19,20. A late-onset phenotype resembling SNMG has been described with limb weakness starting in late childhood or adulthood21. Ankle dorsiflexion weakness was described as a feature of this phenotype. This is said to be extremely rare in autoimmune MG14, although this is not entirely in accord with our clinical experience. In contrast, the early-onset phenotype tends to present with bulbar and respiratory symptoms at birth associated with joint contractures and episodic crises19,21.

The fourth sibling pair we describe were compound heterozygous for rare mutations involving CHRNA1 (F256L + R55H). The α-subunit mutation F256L has been described previously as a single missense mutation that causes a rare fast-channel syndrome due to a dominant “loss of function” mutation in a father-son pair, resulting in very brief opening of the AChR ion channel. There was marked phenotypic variation with the son having respiratory and bulbar weakness from birth. In contrast, the father had no clinical weakness but had electrodiagnostic evidence of a neuromuscular


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transmission defect evident on SFEMG, suggesting variable penetrance22. The F256L mutation in our CHRNA1 family was inherited from the father. He had no clinical symptoms or signs of a neuromuscular junction defect on formal examination, but he has not had electrodiagnostic testing.

There are a number of clinical features which when present should suggest late onset CMS: 1. A slow onset, including but not limited to long standing ptosis on inspection of prior photographs 2. Symmetrical ocular presentation - genetic forms tend not to have the asymmetry often seen in autoimmune cases 3. Stability on examination over time, excepting exacerbating features such as temperature or medications 4. Failure to respond objectively to immunomodulatory medications (noting frequent general feelings of well-being from corticosteroids). If necessary response can be tested with a shortterm treatment such as plasmapheresis.

From a practical standpoint, how one should test for CMS genes when this diagnosis is considered remains difficult. Screening is available at various centers, but there is a charge. These Australian patients were tested genetically as part of a generous UK research project. However this service is not routinely available in Australia and may not be in other countries. Nevertheless, the cost of an exome screen with associated bioinformatics (now ~$2000USD) is similar to the cost of a single daily treatment with intravenous immunoglobulin or plasmapheresis and less than the cost of a therapeutic thymectomy. As the cost of next generation sequencing further declines and becomes more widely available, it will be feasible for genetic testing to be more broadly applied to seronegative cases.

Distinguishing CMS from autoimmune generalized MG is critical to prevent the unnecessary risks and costs of immunosuppression and thymectomy. In our series, 2 patients were treated with thymectomy and steroid therapy, and another 2 received plasma exchange prior to a genetic diagnosis being made. Furthermore, CMS is a treatable disorder with therapeutic options varying depending on


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the underlying molecular defect and include anticholinesterase therapy, 3,4-DAP, and salbutamol. Many of the patients in our series responded better to 3,4-DAP than to pyridostigmine.

CONCLUSION Late childhood or adult onset seronegative generalized myasthenic syndromes should not be assumed to be autoimmune, as late presentations of CMS can occur. Given the typically recessive inheritance pattern, a family history is commonly absent. Recognition of CMS and differentiation from seronegative autoimmune MG is imperative for optimization of treatment and prevention of unnecessary immunosuppression and surgery.


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Late Presentations of CMS ABBREVIATIONS Abs: antibodies AChR: acetylcholine receptor CMS: Congenital myasthenic syndrome LEMS: Lambert Eaton Myasthenic Syndrome LRP4: low-density lipoprotein receptor-related protein 4 MG: myasthenia gravis MuSK: muscle-specific receptor tyrosine kinase RNS: repetitive nerve stimulation SNMG: seronegative myasthenia gravis SFEMG: single fiber electromyography VGCC: voltage gated calcium channel 3,4-DAP: 3,4-diaminopyridine


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myasthenic syndromes, 24-26 September 2004, Naarden, the Netherlands. Neuromuscul Disord 2005;15(7):498-512. 3.

Engel AG. The therapy of congenital myasthenic syndromes. Neurotherapeutics

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Alseth EH, Maniaol AH, Elsais A, Nakkestad HL, Tallaksen C, Gilhus NE et al. Investigation

for RAPSN and DOK-7 mutations in a cohort of seronegative myasthenia gravis patients. Muscle Nerve 2011;43(4):574-577. 7.

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human genome variation from population-scale sequencing. Nature 2010;467(7319):1061-1073. 10.

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high-throughput sequencing data. Nucleic Acids Res 2010;38(16):e164. 11.

Maselli R, Dris H, Schnier J, Cockrell J, Wollmann R. Congenital myasthenic syndrome

caused by two non-N88K rapsyn mutations. Clin Genet 2007;72(1):63-65.


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mechanisms involved in AChR deficiency due to rapsyn mutations. Brain 2006;129(Pt 10):27732783. 13.

Eymard B, Stojkovic T, Sternberg D, Richard P, Nicole S, Fournier E et al. [Congenital

myasthenic syndromes: difficulties in the diagnosis, course and prognosis, and therapy--The French National Congenital Myasthenic Syndrome Network experience]. Rev Neurol (Paris) 2013;169 Suppl 1:S45-55. 14.

Finlayson S, Beeson D, Palace J. Congenital myasthenic syndromes: an update. Pract Neurol

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Abicht A, Dusl M, Gallenmuller C, Guergueltcheva V, Schara U, Della Marina A et al.

Congenital myasthenic syndromes: achievements and limitations of phenotype-guided gene-aftergene sequencing in diagnostic practice: a study of 680 patients. Hum Mutat 2012;33(10):1474-1484. 16.

Ohno K, Engel AG, Shen XM, Selcen D, Brengman J, Harper CM et al. Rapsyn mutations in

humans cause endplate acetylcholine-receptor deficiency and myasthenic syndrome. Am J Hum Genet 2002;70(4):875-885. 17.

Richard P, Gaudon K, Andreux F, Yasaki E, Prioleau C, Bauche S et al. Possible founder

effect of rapsyn N88K mutation and identification of novel rapsyn mutations in congenital myasthenic syndromes. J Med Genet 2003;40(6):e81. 18.

Muller JS, Abicht A, Burke G, Cossins J, Richard P, Baumeister SK et al. The congenital

myasthenic syndrome mutation RAPSN N88K derives from an ancient Indo-European founder. J Med Genet 2004;41(8):e104. 19.

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Table 1. Concord clinic cohort – patients with generalized myasthenic disorders Seropositive myasthenia gravis (127) AChR antibody positive (110) MuSK antibody positive (16) LRP4 antibody positive (1) Seronegative myasthenic syndromes (25) Seronegative GMG (15) AChR assay negative (5) AChR assay low-binding or equivocal, but above background* (10) Late onset CMS (10) Confirmed CMS gene as described in this paper (7) Recent referrals, clinically suggestive of CMS, not yet tested (3) Other myasthenic syndromes: LEMS (10) VGCC antibody positive (9) Seronegative (1) Total patients (162)

*Patients with GMG with consistent AChR antibodies above the background population and in the 0.1 – 0.4nM/l negative or equivocal range, generally responsive to immunotherapies, suspected low avidity AChR patients7.

GMG: generalized myasthenia gravis; AChR: acetylcholine receptor; MuSK: muscle-specific receptor tyrosine kinase; LRP4: low-density lipoprotein receptor-related protein 4; CMS: Congenital myasthenic syndrome. LEMS: Lambert Eaton Myasthenic Syndrome; VGCC: voltage gated calcium channel.


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Table 2. Clinical features of CMS cases

Patie

Sympto

Pattern of weakness

nt/

matic

Gend

Age/

Prox

er

Presenta

limbs

TA

Facial

Ptosis

EOM

Bulbar

Treatm

Treatment

ent for

Response *

SNMG

Pyrid

3,4-DAP

ostig

tion Age

mine

Pair

1/M

6/55

+

+

-

+

+

-

PEX

+

++ †

1

2/F ‡

50/60

+

-

-

-

-

-

-

-

-

Pair

3/M

14/27

+

+

-

+

-

+

-

+

++

2

4/F

19/29

+

-

-

-

-

-

-

+

++ †

Pair

5/M

15/15

+

+

-

-

-

-

-

-

++ †

3

6/F

8/8

+

+

-

-

-

-

THY,

+

++ †

CS Pair

7/F

26/27

+

-

+

+

-

-

PEX

++

++ †

4

8/M

19/21

+

-

+

+

-

-

THY,

+

Not tried

CS

* no response ( - ); mild/moderate response (+); very good response (++) † 3,4-DAP in combination with pyridostigmine ‡ somewhat suggestive symptoms but clinical features not consistent with a myasthenic syndrome

TA: tibialis anterior; EOM: extraocular muscles; 3,4-DAP: 3,4 diaminopyridine; SNMG: seronegative myasthenia gravis; THY: thymectomy; CS: corticosteroids.


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Late Presentations of CMS Table 3: Investigations and mutation analysis

Pair 1

Patie

Edrophonium

Repetitive

nt

Test

Stimulation

1

+

abN (trap)

SFEMG

Mutation

N (EDC)

RAPSN N88K homozygous

Pair 2

2

ND

N

N

Negative

3

-

abN

abN


4

+

abN

abN


Pair 3

Pair 4

5

-

abN

abN

RAPSN S201N + E162K

6

-

abN

ND

Not tested

7

+

abN

abN

CHRNA1 F256L + R55H

8

+

abN

ND

CHRNA1 F256L + R55H

N: Normal; abN: abnormal; ND: not done; SFEMG: single fiber electromyography; trap: trapezius; EDC: extensor digitorum communis; +: positive; -: negative


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Figure Legends

Figure 1. Agrin-induced AChR clusters on rapsn -/- myotubes expressing either wild type (WT) or mutant (S201N) RAPSN cDNA. AChR clusters were labelled with tetramethylrhodamine αbungarotoxin (α-BuTx), and visualized with an Axion 200 inverted Zeiss fluorescence microscope as described in Cossins et al., 200612. The number of clusters > 3 µm in length in 40 fields were counted and expressed as average number of clusters/field. (A) Example panels of WT clusters and (B) S201N clusters. Insets at higher magnification demonstrate a relative reduction in AChR labelling of S201N clusters compared with WT clusters. (C) number of clusters per field demonstrating a significant reduction in AChR clusters in myotubes expressing mutant (S201N) RAPSN cDNA (P < 0.05, 1-way ANOVA).

Figure 2. Electrodiagnostic testing. A, B (Patient 5): 3 Hz stimulation of accessory nerve to trapezius demonstrating: (A) decrement in compound muscle action potential (CMAP) amplitude pre3,4-diaminopyridine (3,4-DAP) and (B) increase in CMAP amplitude and partial repair of decrement post-3,4-DAP. C, D (Patient 7): 3 Hz stimulation of right ulnar nerve to abductor digit minimi shows: (C) decrement in CMAP amplitude pre-3,4-DAP and (D) repair of decrement post-3,4-DAP.


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Muscle & Nerve

Agrin induced AChR clusters on rapsn -/- myotubes expressing either wild type (WT) or mutant (S201N) RAPSN cDNA. AChR clusters were labelled with tetramethylrhodamine α-bungarotoxin (α-BuTx), and visualised using an Axion 200 inverted Zeiss fluorescence microscope as described in Cossins et al., 200612. The number of clusters > 3 um in length in 40 fields were counted and expressed as average number of clusters/field. (A) Example panels of WT clusters; (B) S201N clusters; (C) number of clusters per field demonstrating a significant reduction in AChR clusters in myotubes expressing mutant (S201N) RAPSN cDNA (p < 0.05, one way ANOVA). 219x55mm (300 x 300 DPI)


Muscle & Nerve

A, B (Case 5): 3 Hertz (Hz) stimulation of accessory nerve to trapezius demonstrating decrement in compound muscle action potential (CMAP) amplitude pre-3,4-diaminopyridine (3,4-DAP) (A) and increase in CMAP amplitude and partial repair of decrement post-3,4-DAP (B). C, D (Case 7): 3 Hz stimulation of right ulnar nerve to abductor digit minimi with decrement in CMAP amplitude pre-3,4-DAP (C) and repair of decrement post-3,4-DAP (D). 131x70mm (300 x 300 DPI)


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