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A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation
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A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation Paulo Bastos , Raquel Barbosa , Marco Fernandes , Isabel Alonso PII: DOI: Reference:
S0960-8966(20)30035-3 https://doi.org/10.1016/j.nmd.2020.02.009 NMD 3803
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
23 December 2019 2 February 2020 17 February 2020
Please cite this article as: Paulo Bastos , Raquel Barbosa , Marco Fernandes , Isabel Alonso , A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation, Neuromuscular Disorders (2020), doi: https://doi.org/10.1016/j.nmd.2020.02.009
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Highlights
Congenital Myasthenic Syndromes (CMS) can present as late-onset disorders;
Heterozygotic mutations in classical CMS genes may allow for disease manifestation;
Heterozygotic DOK7 may benefit from the same treatments as classical patients;
1
A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation Running Title: Heterozygous Congenital Myasthenia
Paulo Bastos1,* , Raquel Barbosa2,* , Marco Fernandes2,, Isabel Alonso3,4,5
1
Institut Pasteur, Unité Biologie et Génétique de la Paroi Bactérienne, Paris, France
2
Department of Neurology, Hospital de Egas Moniz, Centro Hospitalar de Lisboa Ocidental, Lisboa,
Portugal 3
Center for Predictive and Preventive Genetics (CGPP), Institute of Molecular and Cellular Biology
(IBMC), Porto, Portugal 4
3S - Institute of Investigation and Innovation in Health, Universidade do Porto, Porto, Portugal
5
UnIGENe (Unit for Genetic and Epidemiological Research in Neurological Diseases), IBMC (Institute
for Molecular and Cell Biology), Universidade do Porto, Porto, Portugal
.
*
Corresponding authors e-mail addresses: [email protected] ; +33695205827 [email protected] ; 00351917115615
2
Abstract Congenital myasthenic syndromes are disorders of the neuromuscular junction resulting from genetic defects in its components. Clinical presentations are diverse and virtually always of early onset. We herein report a 67-year-old female patient first presenting with episodes of sudden respiratory failure. A diagnosis of seronegative myasthenia gravis was put forward based on the presence of a limb-girdle pattern of muscle weakness with pathological decremental responses on repetitive nerve stimulation. Lack
of
response
to
steroids,
intravenous
human
immunoglobulin
and
acetylcholinesterase inhibitors lead us to test for classical congenital myasthenic syndrome genes. A c.1378dup heterozygotic mutation in DOK7 was found, classically (albeit not exclusively) described as pathogenic only when inherited in a homozygotic fashion. Patients with such a single, heterozygous mutation have been previously described, but these have been left unexplained. Thus, under certain still poorly understood circumstances, an heterozygotic state may allow for disease manifestation. These patients may benefit from tailored therapies akin to those normally reserved to homozygotic/compound heterozygotic patients. Awareness for and recognition of such conditions are expected to allow for better provided care and improved quality of life.
Keywords:
Myasthenia
Gravis,
DOK7,
Congenital
Myasthenic
Syndromes,
Neuromuscular Junction, Repetitive Nerve Stimulation 3
Introduction Myasthenia Gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), with autoantibodies binding proteins in the NMJ detected in ~85% of the patients. However, 10-15% of MG patients present with no known antibodies [1,2]. Congenital myasthenic syndromes (CMS), in turn, are disorders of the NMJ due to various genetic defects in components essential for NMJ structure and function. Symptoms in CMS patients typically start shortly after birth or during childhood, with an adult onset being rarer. Clinically, they present with weakness in axial, respiratory, bulbar and extraocular muscles. While effort-dependent muscle weakness in the autoimmune condition MG is typically accompanied by rapid fluctuations that can change in a matter of minutes, CMS-associated fatigue is less prominent and displays fluctuations over longer periods of time (i.e. days to weeks to months) [3–5]. Several mutations have been implied in CMS and genes associated with it can be divided into five different groups depending on how they influence the NMJ function and development: i) those impairing the synthesis and release of acetylcholine (ACh), ii) those involved in information transmission between nerve and muscle cells in the synaptic cleft, iii) defected ACh receptor (AChR) assembly/functioning, iv) core proteins involved in the postsynaptic signalling and v) molecules of unknown function in the NMJ [6]. Docking Protein 7 (DOK7) is a cytoplasmic protein that activates MusK (Musclespecific tyrosine kinase) and it is essential for normal maturation of the postsynaptic membrane [7]. Mutations in the DOK7 gene underlie a big proportion of CMS (up to almost 20%) [8]. These can be found throughout the whole gene coding sequencing, with the most common (~65%) mutation being c.1124_1127dupTGCC [9–12]. While altered residues falling on DOK7 C-terminal may still allow for partial MuSK activation, 4
these may nonetheless lead to alterations in synaptic structure. Such mutations have been described as either i) homozygotic, supporting the classical view of CMS as an autosomal recessive condition (e.g. c.415G>C, p.V139L in Alleles 1 & 2), or ii) compound heterozygotic, in which both alleles are defective but due to different mutations (e.g. c.325G>T, p.G109C in Allele 1 & c.596delT, p.I198Tfs*47 in Allele 2) [8]. CMS-associated mutations in DOK7 are widely accepted as being inherited in a homozygotic dominant fashion. However, this is not necessarily always the case. In this manuscript, we report a patient carrier of an heterozygotic mutation in DOK7 presenting with a late-onset presentation of CMS, and discuss how heterozygotic mutations in genes responsible for CMS may also be pathogenic and benefit from therapy typically reserved for homozygotic and/or compound heterozygotic carriers.
Case Presentation A 67-year-old woman presented to our outpatient clinic with an history reporting back to two episodes of sudden unexplained respiratory failure requiring mechanical ventilation taking place 8 years earlier. Pulmonary or cardiac causes of acute respiratory failure were excluded. After the second episode, the patient was started on long-term oxygen therapy and non-invasive nocturnal ventilatory support with bi-level positive airway pressure. One-and-a-half years before presentation, the patient had started experiencing increased difficulty climbing stairs, reporting weakness on both legs and to a smaller extent lack of strength on both arms. The patient reported occasional episodes of choking and dysphagia with both solids and liquids, but dysphonia, dysarthria, diplopia and ptosis were denied. Perinatal history was 5
unremarkable and the patient had achieved all developmental milestones on time. Familial history of muscular or neurological diseases was denied. At neurological examination, the patient presented with normal strength on the upper limbs without fatigability, but with mild proximal lower limbs weakness (Medical Research Council MRC G4 on the right and G4+ on the left). There were no ptosis, facial palsy, ophthalmoparesis, muscle atrophy and fasciculations, and tendon reflexes were normal. Repetitive Nerve Stimulation (RNS) at low frequency showed a decremental response of 10% in the spinal accessory nerve. Antibodies typical of autoimmune myasthenia were negative on two different occasions. Creatine Kinase (CK) levels were normal and antiVCGG antibody analysis was negative. A thoracic CT scan showed no evidence of thymoma or thymic hyperplasia. Due to the important respiratory impairment, Pompe disease was also tested for. The activity of alfa-glucosidase using a Dried Blood Spot Essay was normal. During the years following initial contact with the patient the condition deteriorated, with the neurological examination two years later revealing mild weakness on i) arm abduction (MRC G4), ii) arm (MRC G4), hip (MRC G4) and neck (MRC G4+) flexion, and iii) neck extension (MRC G4). Generalized fatigue was not noticed. Ophtalmoparesis, ptosis and facial palsy were also absent. Tendon reflexes were normal and no fasciculations were observed. A slow (3Hz) RNS was done and revealed decremental responses of 25% in the spinal accessory nerve and 15% in the tibial nerve. Needle electromyography revealed no myopathic potentials or myotonic discharges. Deltoid muscle biopsy findings were unspecific, without inflammatory infiltrates or dystrophic changes. Ragged-red fibres and Cox-2 negative fibres were absent.
6
Assuming a diagnosis of probable seronegative MG, the patient was started on acetylcholinesterase inhibitors and steroids, but these proved not to be beneficial. A 5day cycle of intravenous immunoglobulin (IVIG) (2g/kg) was also tried, but without improvement. The presence of decremental responses in the RNS pointed to a myasthenic syndrome. However, the absence of a known antibody and lack of response to immunotherapy together with an atypical clinical presentation (pattern of limb girdle presentation without ocular symptoms or significant fatigability) lead us to test a panel of 29 genes associated with CMS (AGRN, ALG14, ALG2, CHAT, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DNM2, DOK7, DPAGT1, GFPT1, GMPPB, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, RAPSN, SCN4A, SLC25A1, SLC5A7, SNAP25, STIM1, SYT2). A known pathogenic mutation in heterozygosity on the DOK7 gene (c.1378dup ; p.Gln460Profs*59) falling within exon 7 was detected. Thereafter, a trial with salbutamol or fluoxetine was recommended, but the patient refused further treatment.
Discussion The differential diagnosis of seronegative MG patients is challenging and of foremost importance because it has decisive therapeutic implications. In patients as the one herein presented, some features lead us to question the diagnosis of MG and to pursue other possible aetiologies. First, the lack of response to immunotherapy and acetylcholinesterase inhibitors. Second, the absence of MG antibodies. Third, the presence of atypical features (e.g. absence of ocular symptoms, acute episodes of respiratory failure with subtle fatigability).
7
The diagnosis of NMJ dysfunction is largely facilitated by the use of RNS and single-fiber EMG. In general, a reproducible decrease of compound muscle action potential (CMAP) by more than 10% between the 1st and 4th amplitudes is evidence of a NMJ disorder. Although the study of RNS showed a decremental response typical of a myasthenic syndrome in our patient, CMS can also present with similar features in electrophysiologic studies. A deltoid muscular biopsy revealed no features of muscular dystrophy, inflammatory myopathies or mitochondrial disease, conditions to be taken into account in a patient with a limb-girdle pattern of weakness. While a late-onset presentation is atypical, it does not rule out CMS [5]. In fact, although late-onset CMS has been reported, it is probably still underdiagnosed to a very large extent. As in the case herein discussed, patients may end up being exposed to unnecessary immunomodulatory/immunosuppressive drugs or to cholinesterase inhibitors if alternative diagnoses are not put forward. The patient herein described had been under steroids, immunoglobulins and cholinesterase inhibitors and these have all failed to improve the clinical status. Differential clinical responses of CMS to medications are helpful in establishing a diagnosis and in orienting the therapeutic regimen. Even though most CMS are caused by defects in AChR subunits and associated with decreased expression of it, some subtypes result in changes in the channel kinetic properties instead and may require a different therapeutic approach [13]. In the patient herein discussed, trials with salbutamol and fluoxetine were recommended but refused by the patient. Anticholinesterase therapy is effective in most myasthenia cases but fails to provide any benefit in CMS cases resulting from endplate AChE or β2-laminin deficiency, slow-channel syndromes, DOK7 myasthenia 8
and faulty MuSK, agrin and plectin. The alternative use of selective β2 adrenergic agonists such as salbutamol has been proven beneficial in CMS patients, including those with DOK7 mutations, resulting in augmented walked distances and increased numbers of climbed stairs [14]. However, the mechanisms of action underlying these benefits are not fully elucidated. We know from early studies that salbutamol acts as short-lived open-channel blockers on the AChR channel [15]. Also, β2-adrenoceptors are the most common adrenoceptor subtypes in skeletal muscle and stimulation of presynaptic βadrenoceptors facilitates neuromuscular transmission via activation of the cAMP Protein kinase A [16], whose post-synaptic accumulation supports synaptic integrity and AChR stability [17]. Very recently, salbutamol has been shown to rescue morphologicald effects in the NMJ, increasing muscle strength, synaptic area, acetylcholine receptor area and density, and the extent of post-junctional folds [18]. Fluoxetine, in turn, binds to ACh receptors to reduce the channel opening time and opening frequency via stabilization of the receptors desensitized state. Thus, fluoxetine decreases ACh-evoked charge influx and limits excitotoxic damage typically resulting from cation overflow in defective postsynaptic membranes [19,20]. However, its beneficial effects in CMS cases other than slow-channel CMS are still unexplained and hard to pinpoint due to its pleiotropic effects. Nonetheless, even though fluoxetine is typically regarded as the therapy of choice for slow-channel CMS, it has also been previously proven as an effective agent in the case of DOK7 mutations that failed to respond to other agents [21]. The lack of response to immunotherapy and acetylcholinesterase inhibitors, associated with the atypical predominant limb-girdle weakness and the episodes of sudden respiratory failure (that can occur in some CMS) lead us to search for CMS and 9
the genetic tests have revealed a heterozygous mutation in DOK7 previously implicated in pathological phenotypes. However, such mutations are classically considered to produce a clinical phenotype only when present in the homozygotic (or compounded heterozygotic) state. CMS patients with single heterozygotic mutations affecting only one allele have been reported [8–11], but to date these have been left largely unexplained. It is possible for such mutation to be a mere incidental finding. But is it possible for such heterozygous mutations alone to result in a clinically relevant phenotype under certain conditions? Different mutations may lead to several different presentations even between affected siblings or between non-related carriers of the same homozygous mutations [22], reflecting that the genetic background in which a mutation appears is relevant for the manifestation of myasthenic syndromes. Something (with potentially profound implications) that should be kept in mind when trying to understand the genotype-phenotype relationship in the case of DOK7 expression is the fact that both alleles are expressed and that we therefore face a case of co-dominance. If a wild-type allele can or not compensate for a defective one depends on several factors including which specific genes, mutations and respective surroundings are under evaluation. The heterozygotic mutation described in the patient herein presented leaves intact the NES sequence and we suggest that it may induce an effect similar to that observed with the frameshift mutation 1124_1127dupTGCC, where the COOH-terminal peptide from Ala378 to Pro-504 is missing but reduced levels of MuSK activation and AChR clustering are still observed [11,23]. Thus, such frameshift mutations leading to truncation may still be accompanied by expression at the protein level, even though these may lead to impaired AChR clustering as previously demonstrated for other mutations [8]. 10
One is tempted to hypothesize that an heterozygotic state may allow for a dosage effect. Gene regulatory systems may operate in a dosage-dependent manner and such dosage effects operate at both the protein and mRNA level. In diploid organisms, the term haploinsufficiency describes the phenomenon through which a single copy of the wild-type allele may not be sufficient to allow for a “normal” phenotype. The result may be the contribution of defective, little or no gene product from that allele. Additionally, while a single “wild-type” allele is typically capable of (clinically but not necessarily biochemically) compensating for a defected counterpart, one can envision a possible competition between the “wild-type” and mutant allele products for binding sites. Thus, a heterozygous genotype may result in a (sub)standard (i.e. mild) but nonetheless deleterious phenotype. Haploinsufficiency could result, for instance, from the loss or mutation in one of the two copies of the gene. In addition, the fact that we have not sequenced the entire genome (i.e. only exosome sequencing for the genes of interest was preformed) should be taken in account. Mutations in introns or in regulatory regions outside the protein-coding sequence would not be detected by the methods currently employed in the clinical setting. All of these could be particularly relevant because they dictate the gene expression and may have pathological implications. Therefore, one cannot rule out that other mutations may also be present together with the one herein reported on exon 7. Likewise, a restricted number of genes was studied, and other genes may contribute for disease manifestation in the setting of heterozygotic mutations. Accordingly, the widespread use of more comprehensive sequencing techniques allows for the identification of “new” mutations associated with CMS [24]. In a recent paper [25], a CMS patient with a single classic heterozygotic mutation was presented. Faced with the 11
absence of further identifiable mutations, the authors preformed Whole Genome Sequencing (WGS) in order to screen for potential additional mutations and an intragenic DOK7 deletion not previously identified by conventional sequencing analytical approaches was found. This observation lead the authors to hypothesize that (in heterozygotic CMS patients) compound heterozygotic states may still be diagnosed and explain the observed phenotypes when employing more comprehensive screening methods. (e.g. WGS in order to identify copy number variations on other alleles). Similarly, copy number variations have been reported to account for a significant portion of myasthenic syndromes and may be missed by conventional screening approaches [26,27]. Thus, one may argue that the techniques currently available to the clinician may at times fail to provide complete explanations in turn required to support correct diagnoses and therapeutic interventions. In the specific case of the patient herein discussed, one cannot rule out the presence of copy number variations and/or mutations outside the gene-coding sequence that may in turn influence gene expression and disease manifestation. With the aforementioned, one is led to believe that a heterozygous state could theoretically be responsible for the clinical presentation of CMS we face. However, a question still arises pertaining to how could a patient present with symptoms of a CMS and a known mutation so late in life. We suggest that the most likely explanation, as previously shown for other conditions, is for a subclinical pre-existing biochemical deficit to be unmasked under certain stressful conditions and that one’s own ability to cope with such deficits diminishes with ageing. Additionally, one should also highlight the growing body of knowledge from preclinical studies regarding the pathophysiology
12
of myasthenic syndromes, which is expected to improve our understanding of its diverse range of manifestations. A number of CMS patients with a single, heterozygous mutation has been described in prior cohorts of patients [8–11]. Thus, under certain but still poorly understood circumstances, an heterozygotic state may allow for the manifestation of deleterious phenotypes. Recognizing that such patients may benefit from tailored therapies akin to those provided to homozygotic/compound heterozygotic CMS patients is expected to allow for better provided care and improved quality of life.
Disclosure of interest The authors report no potential conflicts of interest.
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17
A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation Paulo Bastos , Raquel Barbosa , Marco Fernandes , Isabel Alonso PII: DOI: Reference:
S0960-8966(20)30035-3 https://doi.org/10.1016/j.nmd.2020.02.009 NMD 3803
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
23 December 2019 2 February 2020 17 February 2020
Please cite this article as: Paulo Bastos , Raquel Barbosa , Marco Fernandes , Isabel Alonso , A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation, Neuromuscular Disorders (2020), doi: https://doi.org/10.1016/j.nmd.2020.02.009
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Highlights
Congenital Myasthenic Syndromes (CMS) can present as late-onset disorders;
Heterozygotic mutations in classical CMS genes may allow for disease manifestation;
Heterozygotic DOK7 may benefit from the same treatments as classical patients;
1
A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation Running Title: Heterozygous Congenital Myasthenia
Paulo Bastos1,* , Raquel Barbosa2,* , Marco Fernandes2,, Isabel Alonso3,4,5
1
Institut Pasteur, Unité Biologie et Génétique de la Paroi Bactérienne, Paris, France
2
Department of Neurology, Hospital de Egas Moniz, Centro Hospitalar de Lisboa Ocidental, Lisboa,
Portugal 3
Center for Predictive and Preventive Genetics (CGPP), Institute of Molecular and Cellular Biology
(IBMC), Porto, Portugal 4
3S - Institute of Investigation and Innovation in Health, Universidade do Porto, Porto, Portugal
5
UnIGENe (Unit for Genetic and Epidemiological Research in Neurological Diseases), IBMC (Institute
for Molecular and Cell Biology), Universidade do Porto, Porto, Portugal
.
*
Corresponding authors e-mail addresses: [email protected] ; +33695205827 [email protected] ; 00351917115615
2
Abstract Congenital myasthenic syndromes are disorders of the neuromuscular junction resulting from genetic defects in its components. Clinical presentations are diverse and virtually always of early onset. We herein report a 67-year-old female patient first presenting with episodes of sudden respiratory failure. A diagnosis of seronegative myasthenia gravis was put forward based on the presence of a limb-girdle pattern of muscle weakness with pathological decremental responses on repetitive nerve stimulation. Lack
of
response
to
steroids,
intravenous
human
immunoglobulin
and
acetylcholinesterase inhibitors lead us to test for classical congenital myasthenic syndrome genes. A c.1378dup heterozygotic mutation in DOK7 was found, classically (albeit not exclusively) described as pathogenic only when inherited in a homozygotic fashion. Patients with such a single, heterozygous mutation have been previously described, but these have been left unexplained. Thus, under certain still poorly understood circumstances, an heterozygotic state may allow for disease manifestation. These patients may benefit from tailored therapies akin to those normally reserved to homozygotic/compound heterozygotic patients. Awareness for and recognition of such conditions are expected to allow for better provided care and improved quality of life.
Keywords:
Myasthenia
Gravis,
DOK7,
Congenital
Myasthenic
Syndromes,
Neuromuscular Junction, Repetitive Nerve Stimulation 3
Introduction Myasthenia Gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), with autoantibodies binding proteins in the NMJ detected in ~85% of the patients. However, 10-15% of MG patients present with no known antibodies [1,2]. Congenital myasthenic syndromes (CMS), in turn, are disorders of the NMJ due to various genetic defects in components essential for NMJ structure and function. Symptoms in CMS patients typically start shortly after birth or during childhood, with an adult onset being rarer. Clinically, they present with weakness in axial, respiratory, bulbar and extraocular muscles. While effort-dependent muscle weakness in the autoimmune condition MG is typically accompanied by rapid fluctuations that can change in a matter of minutes, CMS-associated fatigue is less prominent and displays fluctuations over longer periods of time (i.e. days to weeks to months) [3–5]. Several mutations have been implied in CMS and genes associated with it can be divided into five different groups depending on how they influence the NMJ function and development: i) those impairing the synthesis and release of acetylcholine (ACh), ii) those involved in information transmission between nerve and muscle cells in the synaptic cleft, iii) defected ACh receptor (AChR) assembly/functioning, iv) core proteins involved in the postsynaptic signalling and v) molecules of unknown function in the NMJ [6]. Docking Protein 7 (DOK7) is a cytoplasmic protein that activates MusK (Musclespecific tyrosine kinase) and it is essential for normal maturation of the postsynaptic membrane [7]. Mutations in the DOK7 gene underlie a big proportion of CMS (up to almost 20%) [8]. These can be found throughout the whole gene coding sequencing, with the most common (~65%) mutation being c.1124_1127dupTGCC [9–12]. While altered residues falling on DOK7 C-terminal may still allow for partial MuSK activation, 4
these may nonetheless lead to alterations in synaptic structure. Such mutations have been described as either i) homozygotic, supporting the classical view of CMS as an autosomal recessive condition (e.g. c.415G>C, p.V139L in Alleles 1 & 2), or ii) compound heterozygotic, in which both alleles are defective but due to different mutations (e.g. c.325G>T, p.G109C in Allele 1 & c.596delT, p.I198Tfs*47 in Allele 2) [8]. CMS-associated mutations in DOK7 are widely accepted as being inherited in a homozygotic dominant fashion. However, this is not necessarily always the case. In this manuscript, we report a patient carrier of an heterozygotic mutation in DOK7 presenting with a late-onset presentation of CMS, and discuss how heterozygotic mutations in genes responsible for CMS may also be pathogenic and benefit from therapy typically reserved for homozygotic and/or compound heterozygotic carriers.
Case Presentation A 67-year-old woman presented to our outpatient clinic with an history reporting back to two episodes of sudden unexplained respiratory failure requiring mechanical ventilation taking place 8 years earlier. Pulmonary or cardiac causes of acute respiratory failure were excluded. After the second episode, the patient was started on long-term oxygen therapy and non-invasive nocturnal ventilatory support with bi-level positive airway pressure. One-and-a-half years before presentation, the patient had started experiencing increased difficulty climbing stairs, reporting weakness on both legs and to a smaller extent lack of strength on both arms. The patient reported occasional episodes of choking and dysphagia with both solids and liquids, but dysphonia, dysarthria, diplopia and ptosis were denied. Perinatal history was 5
unremarkable and the patient had achieved all developmental milestones on time. Familial history of muscular or neurological diseases was denied. At neurological examination, the patient presented with normal strength on the upper limbs without fatigability, but with mild proximal lower limbs weakness (Medical Research Council MRC G4 on the right and G4+ on the left). There were no ptosis, facial palsy, ophthalmoparesis, muscle atrophy and fasciculations, and tendon reflexes were normal. Repetitive Nerve Stimulation (RNS) at low frequency showed a decremental response of 10% in the spinal accessory nerve. Antibodies typical of autoimmune myasthenia were negative on two different occasions. Creatine Kinase (CK) levels were normal and antiVCGG antibody analysis was negative. A thoracic CT scan showed no evidence of thymoma or thymic hyperplasia. Due to the important respiratory impairment, Pompe disease was also tested for. The activity of alfa-glucosidase using a Dried Blood Spot Essay was normal. During the years following initial contact with the patient the condition deteriorated, with the neurological examination two years later revealing mild weakness on i) arm abduction (MRC G4), ii) arm (MRC G4), hip (MRC G4) and neck (MRC G4+) flexion, and iii) neck extension (MRC G4). Generalized fatigue was not noticed. Ophtalmoparesis, ptosis and facial palsy were also absent. Tendon reflexes were normal and no fasciculations were observed. A slow (3Hz) RNS was done and revealed decremental responses of 25% in the spinal accessory nerve and 15% in the tibial nerve. Needle electromyography revealed no myopathic potentials or myotonic discharges. Deltoid muscle biopsy findings were unspecific, without inflammatory infiltrates or dystrophic changes. Ragged-red fibres and Cox-2 negative fibres were absent.
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Assuming a diagnosis of probable seronegative MG, the patient was started on acetylcholinesterase inhibitors and steroids, but these proved not to be beneficial. A 5day cycle of intravenous immunoglobulin (IVIG) (2g/kg) was also tried, but without improvement. The presence of decremental responses in the RNS pointed to a myasthenic syndrome. However, the absence of a known antibody and lack of response to immunotherapy together with an atypical clinical presentation (pattern of limb girdle presentation without ocular symptoms or significant fatigability) lead us to test a panel of 29 genes associated with CMS (AGRN, ALG14, ALG2, CHAT, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DNM2, DOK7, DPAGT1, GFPT1, GMPPB, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, RAPSN, SCN4A, SLC25A1, SLC5A7, SNAP25, STIM1, SYT2). A known pathogenic mutation in heterozygosity on the DOK7 gene (c.1378dup ; p.Gln460Profs*59) falling within exon 7 was detected. Thereafter, a trial with salbutamol or fluoxetine was recommended, but the patient refused further treatment.
Discussion The differential diagnosis of seronegative MG patients is challenging and of foremost importance because it has decisive therapeutic implications. In patients as the one herein presented, some features lead us to question the diagnosis of MG and to pursue other possible aetiologies. First, the lack of response to immunotherapy and acetylcholinesterase inhibitors. Second, the absence of MG antibodies. Third, the presence of atypical features (e.g. absence of ocular symptoms, acute episodes of respiratory failure with subtle fatigability).
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The diagnosis of NMJ dysfunction is largely facilitated by the use of RNS and single-fiber EMG. In general, a reproducible decrease of compound muscle action potential (CMAP) by more than 10% between the 1st and 4th amplitudes is evidence of a NMJ disorder. Although the study of RNS showed a decremental response typical of a myasthenic syndrome in our patient, CMS can also present with similar features in electrophysiologic studies. A deltoid muscular biopsy revealed no features of muscular dystrophy, inflammatory myopathies or mitochondrial disease, conditions to be taken into account in a patient with a limb-girdle pattern of weakness. While a late-onset presentation is atypical, it does not rule out CMS [5]. In fact, although late-onset CMS has been reported, it is probably still underdiagnosed to a very large extent. As in the case herein discussed, patients may end up being exposed to unnecessary immunomodulatory/immunosuppressive drugs or to cholinesterase inhibitors if alternative diagnoses are not put forward. The patient herein described had been under steroids, immunoglobulins and cholinesterase inhibitors and these have all failed to improve the clinical status. Differential clinical responses of CMS to medications are helpful in establishing a diagnosis and in orienting the therapeutic regimen. Even though most CMS are caused by defects in AChR subunits and associated with decreased expression of it, some subtypes result in changes in the channel kinetic properties instead and may require a different therapeutic approach [13]. In the patient herein discussed, trials with salbutamol and fluoxetine were recommended but refused by the patient. Anticholinesterase therapy is effective in most myasthenia cases but fails to provide any benefit in CMS cases resulting from endplate AChE or β2-laminin deficiency, slow-channel syndromes, DOK7 myasthenia 8
and faulty MuSK, agrin and plectin. The alternative use of selective β2 adrenergic agonists such as salbutamol has been proven beneficial in CMS patients, including those with DOK7 mutations, resulting in augmented walked distances and increased numbers of climbed stairs [14]. However, the mechanisms of action underlying these benefits are not fully elucidated. We know from early studies that salbutamol acts as short-lived open-channel blockers on the AChR channel [15]. Also, β2-adrenoceptors are the most common adrenoceptor subtypes in skeletal muscle and stimulation of presynaptic βadrenoceptors facilitates neuromuscular transmission via activation of the cAMP Protein kinase A [16], whose post-synaptic accumulation supports synaptic integrity and AChR stability [17]. Very recently, salbutamol has been shown to rescue morphologicald effects in the NMJ, increasing muscle strength, synaptic area, acetylcholine receptor area and density, and the extent of post-junctional folds [18]. Fluoxetine, in turn, binds to ACh receptors to reduce the channel opening time and opening frequency via stabilization of the receptors desensitized state. Thus, fluoxetine decreases ACh-evoked charge influx and limits excitotoxic damage typically resulting from cation overflow in defective postsynaptic membranes [19,20]. However, its beneficial effects in CMS cases other than slow-channel CMS are still unexplained and hard to pinpoint due to its pleiotropic effects. Nonetheless, even though fluoxetine is typically regarded as the therapy of choice for slow-channel CMS, it has also been previously proven as an effective agent in the case of DOK7 mutations that failed to respond to other agents [21]. The lack of response to immunotherapy and acetylcholinesterase inhibitors, associated with the atypical predominant limb-girdle weakness and the episodes of sudden respiratory failure (that can occur in some CMS) lead us to search for CMS and 9
the genetic tests have revealed a heterozygous mutation in DOK7 previously implicated in pathological phenotypes. However, such mutations are classically considered to produce a clinical phenotype only when present in the homozygotic (or compounded heterozygotic) state. CMS patients with single heterozygotic mutations affecting only one allele have been reported [8–11], but to date these have been left largely unexplained. It is possible for such mutation to be a mere incidental finding. But is it possible for such heterozygous mutations alone to result in a clinically relevant phenotype under certain conditions? Different mutations may lead to several different presentations even between affected siblings or between non-related carriers of the same homozygous mutations [22], reflecting that the genetic background in which a mutation appears is relevant for the manifestation of myasthenic syndromes. Something (with potentially profound implications) that should be kept in mind when trying to understand the genotype-phenotype relationship in the case of DOK7 expression is the fact that both alleles are expressed and that we therefore face a case of co-dominance. If a wild-type allele can or not compensate for a defective one depends on several factors including which specific genes, mutations and respective surroundings are under evaluation. The heterozygotic mutation described in the patient herein presented leaves intact the NES sequence and we suggest that it may induce an effect similar to that observed with the frameshift mutation 1124_1127dupTGCC, where the COOH-terminal peptide from Ala378 to Pro-504 is missing but reduced levels of MuSK activation and AChR clustering are still observed [11,23]. Thus, such frameshift mutations leading to truncation may still be accompanied by expression at the protein level, even though these may lead to impaired AChR clustering as previously demonstrated for other mutations [8]. 10
One is tempted to hypothesize that an heterozygotic state may allow for a dosage effect. Gene regulatory systems may operate in a dosage-dependent manner and such dosage effects operate at both the protein and mRNA level. In diploid organisms, the term haploinsufficiency describes the phenomenon through which a single copy of the wild-type allele may not be sufficient to allow for a “normal” phenotype. The result may be the contribution of defective, little or no gene product from that allele. Additionally, while a single “wild-type” allele is typically capable of (clinically but not necessarily biochemically) compensating for a defected counterpart, one can envision a possible competition between the “wild-type” and mutant allele products for binding sites. Thus, a heterozygous genotype may result in a (sub)standard (i.e. mild) but nonetheless deleterious phenotype. Haploinsufficiency could result, for instance, from the loss or mutation in one of the two copies of the gene. In addition, the fact that we have not sequenced the entire genome (i.e. only exosome sequencing for the genes of interest was preformed) should be taken in account. Mutations in introns or in regulatory regions outside the protein-coding sequence would not be detected by the methods currently employed in the clinical setting. All of these could be particularly relevant because they dictate the gene expression and may have pathological implications. Therefore, one cannot rule out that other mutations may also be present together with the one herein reported on exon 7. Likewise, a restricted number of genes was studied, and other genes may contribute for disease manifestation in the setting of heterozygotic mutations. Accordingly, the widespread use of more comprehensive sequencing techniques allows for the identification of “new” mutations associated with CMS [24]. In a recent paper [25], a CMS patient with a single classic heterozygotic mutation was presented. Faced with the 11
absence of further identifiable mutations, the authors preformed Whole Genome Sequencing (WGS) in order to screen for potential additional mutations and an intragenic DOK7 deletion not previously identified by conventional sequencing analytical approaches was found. This observation lead the authors to hypothesize that (in heterozygotic CMS patients) compound heterozygotic states may still be diagnosed and explain the observed phenotypes when employing more comprehensive screening methods. (e.g. WGS in order to identify copy number variations on other alleles). Similarly, copy number variations have been reported to account for a significant portion of myasthenic syndromes and may be missed by conventional screening approaches [26,27]. Thus, one may argue that the techniques currently available to the clinician may at times fail to provide complete explanations in turn required to support correct diagnoses and therapeutic interventions. In the specific case of the patient herein discussed, one cannot rule out the presence of copy number variations and/or mutations outside the gene-coding sequence that may in turn influence gene expression and disease manifestation. With the aforementioned, one is led to believe that a heterozygous state could theoretically be responsible for the clinical presentation of CMS we face. However, a question still arises pertaining to how could a patient present with symptoms of a CMS and a known mutation so late in life. We suggest that the most likely explanation, as previously shown for other conditions, is for a subclinical pre-existing biochemical deficit to be unmasked under certain stressful conditions and that one’s own ability to cope with such deficits diminishes with ageing. Additionally, one should also highlight the growing body of knowledge from preclinical studies regarding the pathophysiology
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of myasthenic syndromes, which is expected to improve our understanding of its diverse range of manifestations. A number of CMS patients with a single, heterozygous mutation has been described in prior cohorts of patients [8–11]. Thus, under certain but still poorly understood circumstances, an heterozygotic state may allow for the manifestation of deleterious phenotypes. Recognizing that such patients may benefit from tailored therapies akin to those provided to homozygotic/compound heterozygotic CMS patients is expected to allow for better provided care and improved quality of life.
Disclosure of interest The authors report no potential conflicts of interest.
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