Clinical and molecular analysis of a novel variant responsive to salbutamol monotherapy during COVID-19 outbreak related to congenital and late-onset of myasthenic syndrome in large kindred

Clinical and molecular analysis of a novel variant responsive to salbutamol monotherapy during COVID-19 outbreak related to congenital and late-onset of myasthenic syndrome in large kindred | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical and molecular analysis of a novel variant responsive to salbutamol monotherapy during COVID-19 outbreak related to congenital and late-onset of myasthenic syndrome in large kindred Zahra Nouri, Javad Saffari-Chaleshtori, Akram Sarmadi, Mohammadreza Sehhati, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3924937/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Congenital myasthenic syndromes (CMSs) are a group of clinically and genetically heterogeneous disorders. Mutations in the DOK7 gene underlie CMS with fatigue and muscle weakness, which would worsen with some conventional treatments but show excellent response to special drugs. Here, we applied exome sequencing (ES) to investigate the etiology of CMS in several patients with congenital and late-onset presentations of the disease. Methods: We ascertained a big pedigree including 3 homozygous and 5 carriers, primary proband was subjected to ES, following immunological, biochemical and neurological evaluations. Molecular dynamics (MD) simulation studies were conducted to investigate the DOK7 proteins' stability. The variant’s pathogenicity was assessed using bioinformatics tools and co-segregation analysis. We adapted the American College of Medical Genetics and Genomics (ACMG) guidelines for variant interpretation. Results: ES results showed a novel homozygous variant (c.1139-1140delinsA:p.Ala380AspfsTer76) in the DOK7 gene. Co-segregating analysis confirmed the pathogenicity of it based on the ACMG guidelines. Interestingly, the identified variant has shown partial autosomal dominant inheritance. The MD simulation analysis revealed this variant to result in the protein function impairment. Effective treatment with salbutamol was obtained in an 18-months follow-up. Remarkably, therapeutic doses of salbutamol in severe COVID-19 patients prevented recurrence of paralysis or muscle weaknesses that occurred with a mild cold. Conclusion: We found a novel variant in the DOK7 gene, with the newly identified partial autosomal dominant inheritance. The findings were used to administer suitable drugs to the patients with maximum efficiency. Thus, ES creates a unique opportunity to promote personalized medicine. Congenital myasthenic syndromes DOK7 gene Pathogenic variant Exome sequencing Late-onset Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Congenital myasthenic syndromes (CMS) represent a heterogeneous group of inherited disorders with the clinical features of fluctuating, weakness in axial, respiratory, bulbar and extraocular muscles and easy fatigability ( 1 ). CMSs are disorders of the neuromuscular junction (NMJ) resulting from genetic defects in pre-synaptic, synaptic and post-synaptic proteins of the NMJ ( 2 , 3 ). The age of onset of CMS greatly varies, from the most common presentations shortly after birth or during childhood, to the rare occurrence of the adult onset manifestations ( 4 ). Different mutations in genes related to the NMJ formation, function, development and maintenance could cause different types of CMS with autosomal dominant or recessive pattern of inheritance ( 5 ). The deficient protein includes DOK-7, muscle-specific kinase (MUSK), AChR, GFPT1, b2-Laminin and Plectin-1 ( 6 , 7 ). Neuromuscular synapse formation and maintenance needs an extremely professional assembly of presynaptic and postsynaptic membranes ( 8 , 9 ). A key molecule is AGRIN, a protein released from the terminals of motor nerve which binds to the lipoprotein receptor-related protein 4 (LRP4) in the muscle. This coupling will stimulate the formation of a complex between LRP4 and MUSK, which is a tyrosine kinase receptor and has a vital regulator role in synaptic differentiation ( 9 – 11 ). After activation of MUSK in the postsynaptic membrane, LRP4 will be clustered stimulating presynaptic differentiation by signals directly back to motor axons ( 12 ). Mutations in AGRIN , LRP4 and MUSK , as well as in the genes that encode subunits of acetylcholine receptors (AChRs), also cause CMS ( 13 ). Docking Protein 7 (DOK7) is a cytoplasmic protein that mediates the activation of MUSK and has important role in postsynaptic specialization of the NMJ ( 14 ). This protein is encoded by the DOK7 gene on chromosome 4p16.2 ( 15 , 16 ). Clinical presentations in patients with DOK7 mutations include ptosis, hypotonia, and bulbar symptoms such as dysphagia and dysarthria ( 17 ). Weakness in limb, neck and facial muscles are common and proximal muscle weakness is more prominent that distal muscles. Spinal deformity can be caused by truncal weakness, which is usually seen in these patients ( 18 ). Three main functional domains of DOK-7 include: a pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain and a large C-terminal domain following by multiple tyrosine residues ( 19 ). The recently developed next generation sequencing (NGS) technologies such as exome sequencing (ES) provide an opportunity to identify causative mutations in different types of monogenic and heterogeneous disorders and discovery of novel disease-causing genes and variants ( 20 – 22 ). In this study, we describe in detail the clinical features of a 10-year-old CMS patient and two of her father’s second cousins due to a DOK7 gene mutation. In addition to these 3 cases with homozygous variant, there are 5 other cases in the family who are heterozygous and have presented the late-onset form of CMS. In this family, variable clinical presentations are reported, while the genetic defect is similar. We performed ES to provide molecular diagnosis of this disorder in the primary proband. This variant was found to be co-segregating in the family. Remarkable good response to salbutamol was noted in this family. Methods Subject and clinical evaluations The primary proband (case VI-4), who is currently a 10-year-old girl, belongs to a consanguineous couple. She was born through normal delivery. Muscle weakness and muscle atrophy were noted in the age of 6 months. In that age, she was able to crawl but floppy phenotype warned the parents to consult a specialist. She was unable to sit without any support and experienced intermittent worsening lasting from days to weeks. The first common immunological and biochemical tests (anti-acetylcholin receptor and anti-Musk antibodies) were done followed by testing creatine kinase (CK) and lactate dehydrogenase (LDH), Nerve conduction velocity (NCV) and Electromyography (EMG) tests at the age of 1 year. Sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) tests were performed when she was 3 years old and analysis of deletion of exon 7 and 8 of the SMN1 gene and NAIP4 and NAIP5 gene deletions and alpha-1,4 Glucosidase enzyme activity were also done at the age of 6 years old (using PCR-RLFP technique and tandem mass spectrometry, respectively) for checking spinal muscular atrophy (SMA) and pompe disease. Her blood sample was taken for DNA extraction and doing ES when she was 7 years. After identifying the pathogenic variant, co-segregation analysis and detailed clinical evaluation were performed in other patients in the family. The study was approved by the Review Board of Isfahan University of Medical Sciences (grant no: 2400173 and ethics code: IR.ARI.MUI.REC.1400.011). Immunological and metabolic panel tests The thyroid function and the level of CK and LDH and also anti-acetylcholin receptor and anti-Musk antibodies were measured in all the patients before doing any molecular testing. Molecular study The peripheral blood lymphocytes (5 ml) was received from the studied patient. Extraction of the genomic DNA was conducted using Prime Prep Genomic DNA Extraction kit from blood (CTGA, Iran), according to the manufacturer’s instruction. Quality and quantity of DNA were assessed by agarose gel and Nanodrop 2000 instrument (Thermo Fisher Scientific Inc, USA), respectively and the patient’s sample was applied for ES. ES and bioinformatics analyses The Sample of 300 ng Genomic DNA was sent to Macrogen (South Korea) and was subjected to ES using the NovaSeq 6000 platform (Illumina, US). Briefly, genomic DNA was fragmented and fragments were captured to target all exons, splicing sites, and flanking intronic sequences of all genes. After amplification of fragments, sequencing was performed with a 150X mean depth of coverage for more than 92% of the sequences. The released row data were converted to the FASTQ file and clean reads were then aligned with the reference human genome sequence (hg19, NCBI Build 38) using the Burrows-Wheeler Aligner (BWA). All the steps were the same as we explained in our previous articles ( 20 , 21 , 23 ). Finally, the suspected pathogenic variant was evaluated by different in silico software tools such as Mutation-Taster and CADD to predict its deleterious effect on protein in terms of function. The American College of Medical Genetics and Genomics (ACMG) guidelines were used to classify the variants ( 24 ). Variant confirmation Co-segregation analysis was performed using exon-specific custom primers to examine segregation of the genotype and phenotype among the parents using direct Sanger sequencing. The forward primer sequence was 5´-ACTCCTCTTACTCCAGCAGC-3´ and the reverse primer sequence was 5´- ACATGCCGAAAAGAAAGCCA-3´ in exon 7. Electropherograms were compared with the reference sequence (NM_173660) encoding 504 residues using the SeqMan software version 5.00© (DNASTAR, Madison, WI, USA). The next step was to investigate in the Human Gene Mutation Database (HGMD) and the literature to seek the novelty of the variant or its association with CMS. Preparation of PDB files and proteins modeling The FASTA sequence of DOK-W (wild type of the DOK7 protein) was obtained from NCBI server and the FASTA sequence of DOK-M (Mutant form of DOK protein) was designed manually. Also, the homology modeling of their 3 dimensions (3D) were produced by I-TASSER server ( https://zhanggroup.org/I-TASSER/ ). Then, to confirmation of the stability of PDB files/3D structures, the ramachandran plot was designed by Ramachandran Plot Server ( https://zlab.umassmed.edu/bu/rama ). Molecular dynamics (MD) simulation of the DOK-W and DOK-M proteins The molecular dynamics simulation of the DOK-W and DOK-M protein structures was done in pure water and the force field of G43A1 with SPC216 model using the GROMACS 2018 simulation package. The grid box with 4.3×4.3×3.5 nm was built for each protein. The suitable amounts of chloride and sodium ions were used to neutralize the molecules to provide 140 mM concentration of Na + and Cl − in water for each protein, through assessment of the electrical charge of each protein. The energy of the systems was minimized throughout 70000 steps by using steepest descent method and equilibrated for 2 ns in NVT ensemble. The simulations were run for 200 nanoseconds (ns) and at a constant temperature of 300ºK for all the simulation intervals. Then, the output results were analyzed using Grapher V.10 software. Pharmacologic Treatment Patients (VI-1, VI-2 and VI-4) started being treated with different dosages of ephedrine, based on the recommendations by a neurologist). However, the symptoms of the disease got even worsened, thus, they stopped taking the medicine. Salbutamol was an alternative drug for treatment, which was prescribed after the identification of the genetic mutation based on the opinion of our multidisciplinary team. The effects of treatment were measured using the quantitative myasthenia gravis (QMG) score for 3 patients and Myasthenia Gravis Activities of Daily Living Scale (MG-ADL) was used for all the cases during the 18 months follow-up ( 25 – 28 ). We also performed a literature search in the MEDLINE, PubMed Embase, Web of Science, and Google Scholar databases using the following headings: DOK7, DOK7 mutations, DOK7 treatment and myasthenic syndrome to find all publications describing the treatment response and drug effects in patients with DOK7-CMS. The last search was performed in April, 2023. If more than one article had been published by the same group, a comparison of the participants in the studies was made, and duplicated data were excluded. Results Clinical findings A 10-year-old girl and two of her father’s second cousins were visited with a history of progressive limb weakness. Upon further genetic counseling sessions, we noticed 5 other cases with late-onset presentations of the disease, with less severe symptoms. The clinical findings of all cases are provided in Supplementary Table 1. Case VI-4 The primary proband was a 10-year-old girl who had experienced mild weakness in the 6 months of age. Progressive bilateral ptosis was notable at birth and facial and neck weaknesses were appeared. Bilateral proximal limb weakness and disability to stand without support were reported when she was 1-year-old. She started walking at the age of 2 years’, but she had difficulty walking independently. She only could walk for about 4–5 steps without support and stairs climbing was very difficult for her. She had waddling gait, facial weakness, significant proximal muscle weakness of both upper and lower extremities and the ptosis was aggravated during ages but cognitive and language milestones were normal. Mild swallowing and chewing difficulties happened latter. Vocal cord paralysis (VCP) was started when she was 5 years old. She became very tired when walking or playing with her friends. She could hardly keep a glass of water, hold a pencil in her hands and experienced a decline in academic performance. During a mild cold, if the fever rose a little, she would be completely paralyzed and could not stand up or walk unaided. The first common biochemical tests were in normal ranges. The results of NCV and EMG tests which were done at the same age suggested CMS to be the probable explanation. In the age of 3 years, the results of SNAPs and CMAPs of nerves and F-waves were normal and as myopathic motor unit action potentials (MUAPs) were seen in most muscles. The possibility of the SMA and pompe diseases was ruled out by performing the analysis of deletion of exon 7 and 8 of SMN1 gene and NAIP4 and NAIP5 genes deletion at the age of 6 years, along with the evaluation of the alpha-1,4 Glucosidase enzyme activity. Cases VI-1 and VI-2 These two cases are two brothers who are second cousins of the proband’s father. They are 16 and 11 years old. They were both healthy at birth with normal motor milestones, except showing mild congenital unilateral ptosis. Walking, sitting and standing were normal and independently until the age of six. By the age of 6 years, they experienced muscle weakness and rapid fatigue while playing and running (exercise induced weakness) and they were getting worse through time. At the age of about 10 years, both brothers had severe motor weakness and it was very difficult for them to walk for more than a few steps, run and climb stairs and unilateral ptosis was aggravated. Cases V-2, V-8, V-10 and V-11 These cases are the proband's father, aunt and uncle and their second cousins. All of these patients have late-onset symptoms of CMS. The most obvious presentation of the disease was facial weakness, which was often revealed after the age of 40. Also, mild weakness on proximal limbs and lack of strength on arms and shoulder were evident, which sometimes became very annoying. While ptosis was obvious in all cases, dysphonia, dysarthria and diplopia were not present. Facial weakness and the ptosis were exacerbated during the years. Motor nerve conduction study (NCS) and EMG were normal in these cases and the result of Repetitive Nerve Stimulation (RNS) test showed decremental response in facial muscles indicating NMJ defects. Case IV-4 This case is the proband's grandmother (paternal grandmother). The first clinical feature was fatigue and proximal limb weakness, that was followed by episodes of sudden respiratory failure in her 50s, which caused the patient to start a long-term oxygen therapy and non-invasive nocturnal ventilatory support. She suffered from weakness on proximal limbs and fatigability which were progressive during times. All these symptoms started after the age of 40 years old. (The pedigree of the family is shown in Fig. 1 ). Immunological and metabolic panel tests results There was no abnormality in the immunological and metabolic profiles of the patients. All laboratory evaluations were within normal ranges, including thyroid function, CK and LDH levels and anti-Musk and AChR antibodies. Molecular findings As a result of ES, a novel homozygous deletion/insertion (c.1139-1140delinsA:p.Ala380AspfsTer76) in the DOK7 gene was found in the proband. This variant results in a truncated protein with 454 residues (versus 504 residues in the intact protein). This frameshift variant was not found in the literature and also was absent from 1000 genomes project phase 3, HGMD, dbSNP version 147, Clinvar, NHLBI GO ESP, ExAC and Iranome databases, as well as, our GTaC local database. Variant confirmation and co-segregation analysis Co-segregation analysis of the variant in the family shows heterozygous status in the parents (V-11 & V-12), but homozygous in their affected girl and also in the cases VI-1 & VI-2 (Fig. 1 ). The other cases with late-onset presentations of the disease (cases IV-4, V-2/8/10/11) were also heterozygous for the variant. According to the ACMG guideline, this variant is classified as a pathogenic variant (1 very strong, 2 Moderate and 2 Supporting criteria): Molecular dynamics simulation of the DOK-W and DOK-M proteins results: The ramachandran plot showed that more than 96.64% of DOK-M amino acid residues and 95.01% of DOK-W amino acid residues are in the preferred and highly preferred regions (Fig. 2 ). The root-mean-square deviation (RMSD) was stable after 30 ns of simulation time for both proteins. Also, they were stable until the end of simulation time. Although, the mean RMSD of DOK-M was decreased during the simulation (Fig. 3 A). The stability of simulation system was confirmed by radius of gyration (Rg) results. Figure 3 B shows that Rg parameters are stable after 30 ns of simulation time. However, there is a significant decrease in the Rg of DOK-M compared with the Rg of DOK-W. The results of root-mean-square-fluctuation (RMSF) and total energy (TE) are shown in the Fig. 3 C & 3 D. There are increases in the amount of RMSF for 45 to 90 and 345 to 370 residue amino acid regions of DOK-M in comparison of the DOK-W. RMSF graphs revealed that the residue amino acid regions of 140 to 145, 190 to 210, and 370 to 400 of the DOK-W were decreased during the simulation. Furthermore, an increment in TE for DOK-M in comparison with the mean of TE for DOK-W during the simulation time was identified (Fig. 3 D). The mean of the TE for DOK-W was about − 1.45×10 6 kj/mol while, the TE for DOK-M increased significantly and it was about − 1.35×10 6 kj/mol. The amounts of hydrogen bonds (H-bonds) are displayed in Fig. 3 E. The mean of H-bonds decreased remarkably in the DOK-M in comparison to the mean of H-bonds in the DOK-W during the simulation time. The variation in the secondary structure parameters such as α-Helix, β-Sheet, Coil, Bend, and Turn between the DOK-M and DOK-W during the 200 ns of simulation time is shown in Fig. 3 F. There is a significant change between the secondary structure parameters of DOK-M as compared to DOK-W. The Coil, Turn, and α –Helix secondary structures were increased while, the β-Sheet, and Bend secondary structures were decreased during the simulation time. Monitoring response to therapy: In the primary proband, a short-term therapy of 7 days with ephedrine was not successful, the symptoms got worse in such a way that she was not able to walk and became unable to perform daily activities. Thus, the treatment was discontinued. After the genetic test was performed and it was found that the pathogenic variant in the DOK7 gene is the cause of the disease, our multidisciplinary team suggested starting treatment with salbutamol (at a dose of a quarter of a 2 mg tablet 4 times daily, total of 2mg/daily for 5 months). After only 3 days of receiving it, improvement of strengths of the legs was seen in her and she became more active and movement of extremities improved significantly. She was able to sit and stand without support, and walk and run long distances and her ptosis and VCP got improved gradually (the dosage of the drug was changed after 5 months, and she was ordered to increase one meal of the drug to a half of a tablet for 5 months, and then increased other 3 times in one complete 2 mg tablet). An 18-month follow-up was performed by our multidisciplinary team (3, 6, 9, 12 and 18 months after starting salbutamol therapy). This medication was well tolerated in this patient, she did not show any side effects and the progress was notable. Even when the patient (proband) contracted COVID-19 and had a high fever of 39.8°C, she did not lose her ability to walk without any support. She used to be completely paralyzed by a cold with milder fever in the past. Both brothers (cases VI-1 & VI-2) also start treatment with ephedrine. Their daily function deteriorated and facial and muscle weakness were aggravated. However, the condition improved significantly as soon as they stopped taking ephedrine and started treatment with salbutamol (one 2 mg tablet 3 times daily for the elder brother and half of a tablet 3 times daily for the other). Only after a short time, their ptosis, VCP and dysphagia were improved and their muscle were strengthened to the degree they were able to play football. Also, in the past, when they had a cold, they became very weak and could hardly walk, but after taking salbutamol, they did not have any movement problems even when they were infected with COVID-19. Since the remarkable effective signs of salbutamol therapy was observed in this family, treatment was started in heterozygous patients (2 mg four times daily) and the improvement of facial weakness and strengthening of the muscle was reported. Furthermore, sudden episodes of respiratory failure in case IV-4 were completely resolved after using salbutamol spray. Salbutamol was also orally admitted for her and the permanent fatigue was relieved in addition to increased muscle strength. Thus, her muscle weakness was also improved. In the literature review we have done; in 136 DOK7-CMS patients, positive effects of salbutamol were observed in 40 of 41 patients who received it. It was also found that salbutamol did not have any negative effects on patients and it had no effect only in one patient (Fig. 4 ). Strength measures included the QMG (severity) score and MG-ADL showed significant improvement from baseline to the 3- to 18-month follow-up in our cases (Fig. 5 A & B). Discussion Congenital Myasthenic Syndromes (CMS) are a group of inherited disorders resulting from abnormal signal transmission of NMJ at different levels of the presynaptic, synaptic and postsynaptic apparatus. Up to now, more than 30 genes have been found to underlie CMS and the differentiation of CMS subtypes, usually clinically heterogenous, can be performed by molecular genetic tests ( 29 , 30 ). DOK7-CMS is reported to occur in up to almost 20% of CMSs ( 19 ). The DOK7 protein, which is encoding by DOK7 gene, is involved in the normal development and maintenance of the NMJ ( 18 , 31 ). DOK7-associated CMS mutations are widely accepted as being inherited in an autosomal recessive pattern. However, this is not always the cases ( 32 ). A variety of mutations in the DOK7 gene are reported to be the cause of CMS with the most common (more than 65%) mutation being c.1124_1127dupTGCC in exon 7, which is in homozygous or compound heterozygous form ( 16 , 33 ). This variant results in a truncated protein and leads to the loss of two tyrosine residues that are phosphorylated and recruits CRK proteins, which are important for anchoring acetylcholine receptors at synapses. In this study, in addition to reporting a novel disease-causing variant in DOK7 gene, we describe a late-onset form of CMS in 5 carriers of this variant. Whereas a late-onset presentation of CMS is uncommon, it does not rule out CMS (Supplementary Table 2&3). Due to the overlap of clinical features, during the initial diagnosis process, it may be misdiagnosed as limb-girdle weakness or myasthenia gravis ( 34 ). Thus, late-onset CMS is probably still underdiagnosed in plenty of cases ( 35 ). Despite the fact that our homozygous patients have the same genotype, their manifestations of the disease were not the same. This may be due to their different genetic background. Congenital bilateral ptosis and facial and muscle weakness were observed in the primary proband, while in the cases VI-1 and VI-2, unilateral ptosis in different eyes (Fig. 5 C) and muscle weakness onset were noticeable during childhood (at the age of about 6 years). However, all of them had VCP. This feature is reported in CMS patients with mutations in COLQ and DOK7 genes, as well as patients with MUSK deficiency ( 36 – 38 ). Therefore, genetic testing is essential to identify the etiology and could have a role in the proper management of the disease. Furthermore, the severity of muscle weakness in all homozygous patients was mostly variable. Thus, there was no consistent genotype-phenotype correlations. Moreover, the clinical features in the heterozygous cases with late-onset manifestations were somewhat different. The facial weakness, mild ptosis and lack of strength in arms and shoulders with different severity after the age of 40 were mostly common, while, the respiratory failure was only seen in the case IV-4 after the age of 50 years (none of other carriers have reached this age yet). Meanwhile, there are reports of several cases of DOK7-CMS with neonatal or childhood respiratory failure ( 4 , 18 , 33 ), none of our homozygous patients showed this feature. However, it was the first report of the late-onset CMS in patients with a heterozygous pathogenic variant in DOK7 gene in Iran, there are a few reports of late-onset presentations with clinical heterogeneity in carriers, but the precise mechanism is unexplained ( 2 , 39 ). The phenotypic presentations and intra-and inter-familial variable expressivity of CMS in carries of this study may be related to the dominant-negative effects of the identified variant or haploinsufficiency mechanism as a consequence of partial autosomal dominant inheritance. A late-onset CMS patient caused by heterozygous mutation in the DOK7 gene, which was previously described by Bastos et al, represented the symptoms of the disease after the age of 65 and the first features were increased difficulty in climbing stairs, weakness on both legs, lack of strength in both arms and finally sudden respiratory failure without ptosis and facial palsy ( 2 ). Thus, the clinical presentations of late-onset CMS would not be similar, even if the mutated gene is the same. Simulation analysis of this study showed that mutation in the DOK-W induces the variations in the structures and molecular dynamic parameters and subsequently instability and dysfunction of the protein. The mean of RMSD indicated that simulation system is stabled in the 200 ns of simulation time. However, decrease in RMSD for DOK-M compared to DOK-W demonstrated that this mutation affects the protein structure (40). Also, a comparison of Rg data of both proteins revealed the more compactness in DOK-M which might lead to decreased stability and functional impairment of the DOK-M ( 41 ). The different variations in the RMSF parameter of amino acid residues in the DOK-M in comparison of DOK-W shows the impact of this mutation on amino acids’ fluctuation. Given that increment of TE in the mutant form of the protein increases the intermolecular energy, molecular instability could ensue. Moreover, changes in the DOK-M structure decrease the mean of H-bonds compared with DOK-W. Decrease in the intermolecular hydrogen bonds can cause the protein stability reduction ( 42 ). Finally, the variation of secondary structure of the protein and induction of changes in the mean of α-Helix, β-Sheet, Coil, Bend, and Turn parameters between the DOK-M and DOK-W during the simulation time showed that this mutation induced remarkable conformational changes in the wild type of DOK protein, subsequently influencing the folding and function of the protein ( 43 ). A variety of therapeutic interventions are available, which are beneficial in treating some kinds of CMSs, but accurate genotype diagnosis is important for the disease management ( 28 , 31 , 44 ). Ephedrine (a sympathomimetic with α- and β-adrenergic effects) and salbutamol (a selective β2 agonist) were very effective therapeutic agents in some CMS patients, especially those with COLQ, laminin-β2, DOK7, MUSK, Agrin and plectin-1 deficiency, and provided more benefit in types of CMS, which are not effectively treated by acetylcholinesterase inhibitors ( 2 , 45 – 48 ). β2-Adrenergic agonists, partially compensate disrupted postsynaptic structures. Therefore, a remarkable response is seen in CMS subtypes with mutations in genes encoding proteins that are involved in formation and stability of the NMJ within the LRP4-MUSK-DOK7 signaling pathway ( 49 , 50 ). In an in vivo study on DOK7-CMS model mice, salbutamol treatment increased the number of active NMJs as well as increasing muscle strength and prolonged the survival of these myasthenic mice ( 51 ). The precise selection of drug therapy is very important, as the same drug can be effective, ineffective, or even worsen the clinical features in different types of CMS ( 52 )(Supplementary Table 1). In the present study, when the clinical symptoms of the disease were obvious, these patients experienced a short period of ephedrine. This treatment not only did not improve their symptoms but also worsened the condition of all homozygous patients in a short time. Therefore, ephedrine treatment was discontinued after 7 days. After that, when the pathogenic variant in the DOK7 gene was identified by ES, the treatment with salbutamol was started, and the dramatical improvement of the patients' condition was observed in a very short period of time. The peak of treatment effect with ephedrine and salbutamol was reported to be about 6–8 months after start using these drugs ( 53 ), in contrast to anticholinesterases, full effects of which were not immediate ( 25 ). Only after 3 days of using salbutamol, our homozygous patients started doing their daily tasks. Walking and climbing up the stairs got better and their ptosis gradually improved. Eventually during our 18 months follow-up a significant improvement was observed. While headache, tremor and occasional palpitations were reported as side effects of salbutamol in some cases ( 24 ), no side effects were observed in our patients after using it. We also started salbutamol therapy (in a different dose) in 5 carriers, who were showing late-onset form of CMS (supplementary table 1 ), and its very beneficial effects were obtained. In cases IV-4, V-2, V-8, V-10 and V-11, there was a significant increase in the strength of the muscles, a decrease in the facial weakness, and a prominent improvement in their ptosis in an 18-month follow-up. Also, no recurrence of respiratory failure was observed in case IV-4, after receiving salbutamol. It has been proven that in the treatment with salbutamol, the age of the disease onset, the age at treatment start and the drug dosage have no effect on the treatment outcome ( 53 ). Our literature review showed that in addition to the aforementioned factors, gender is also ineffective in responding to treatment with this drug, and both sexes, regardless of the age of onset of the disease, responded well to salbutamol therapy ( 48 , 54 – 56 )(Fig. 6) (Supplementary Table 2&3). Interestingly, although the mechanism of action of ephedrine and salbutamol are probably similar, in our patients, ephedrine had a negative effect on the disease, while salbutamol had completely positive consequences. There are some reports of other studies which the ephedrine therapy in DOK7-CMS was not efficient, but herein we are reporting negative effects of ephedrine in our patients (Supplementary Table 1). Conclusion DOK7-CMS often presents with fatigues and muscle weakness, ptosis and facial weakness, which without proper treatment could be worsen. Recent achievements of the exome sequencing in combination with computational studies and pharmacogenetics have provided additional insights on the treatment and management of different diseases. Furthermore, in heterozygous individuals for a pathogenic variant, follow-up of their condition is recommended because they may subsequently develop the late-onset form of this disease. Thus, personalized medicine is being advanced through data obtained from patient’s genetic profile. Declarations Conflicts of interest: The authors declare no conflict of interest. Consent to Participate: Written informed consent was obtained from all of the participants in the study and a written consent to participate was obtained from the parents of the patient (younger than the age of 16). Consent for publication: Written informed consent for publication of clinical details and clinical images was obtained from the all of the participants and from the parents the participant under the age of 18. Ethics approval: The study was approved by the Review Board of Isfahan University of Medical Sciences (ethics code: IR.ARI.MUI.REC.1400.011). Funding: This work was financially supported by Isfahan University of Medical Sciences grant NO. 2400173. MAT has received this research support. Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Author contributions: Study design: MAT; Data collection, analysis, and interpretation: MAT, ZN, JSC, AS and MS; Clinical evaluation: MAT; manuscript preparation: ZN, JSC and AS; critically reviewed by MAT. All authors have read and approved the manuscript. Acknowledgments: We take this opportunity to express our special thanks to all individuals who cooperated in this study, and also to the patient and family. References Engel AG. Congenital myasthenic syndromes in 2018. Current neurology and neuroscience reports. 2018;18(8):1-11. Bastos P, Barbosa R, Fernandes M, Alonso I. A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation. Neuromuscular Disorders. 2020;30(4):331-5. Engel AG, Sine SM. Current understanding of congenital myasthenic syndromes. Current opinion in pharmacology. 2005;5(3):308-21. Ammar AB, Petit F, Alexandri N, Gaudon K, Bauché S, Rouche A, et al. Phenotype genotype analysis in 15 patients presenting a congenital myasthenic syndrome due to mutations in DOK7. Journal of neurology. 2010;257(5):754-66. Beeson D, Hantaï D, Lochmüller H, Engel AG. 126th international workshop: congenital myasthenic syndromes, 24–26 September 2004, Naarden, the Netherlands. Neuromuscular Disorders. 2005;15(7):498-512. Tsujino A, Maertens C, Ohno K, Shen X-M, Fukuda T, Harper CM, et al. Myasthenic syndrome caused by mutation of the SCN4A sodium channel. Proceedings of the National Academy of Sciences. 2003;100(12):7377-82. Engel AG. Congenital myasthenic syndromes in 2012. Current neurology and neuroscience reports. 2012;12(1):92-101. Tintignac LA, Brenner H-R, Rüegg MA. Mechanisms regulating neuromuscular junction development and function and causes of muscle wasting. Physiological reviews. 2015;95(3):809-52. Burden SJ, Yumoto N, Zhang W. The role of MuSK in synapse formation and neuromuscular disease. Cold Spring Harbor Perspectives in Biology. 2013;5(5):a009167. Kim N, Stiegler AL, Cameron TO, Hallock PT, Gomez AM, Huang JH, et al. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell. 2008;135(2):334-42. Zhang B, Luo S, Wang Q, Suzuki T, Xiong WC, Mei L. LRP4 serves as a coreceptor of agrin. Neuron. 2008;60(2):285-97. Yumoto N, Kim N, Burden SJ. Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses. Nature. 2012;489(7416):438-42. McMacken G, Abicht A, Evangelista T, Spendiff S, Lochmüller H. The increasing genetic and phenotypical diversity of congenital myasthenic syndromes. Neuropediatrics. 2017;48(04):294-308. Okada K, Inoue A, Okada M, Murata Y, Kakuta S, Jigami T, et al. The muscle protein Dok-7 is essential for neuromuscular synaptogenesis. Science. 2006;312(5781):1802-5. Hamuro J, Higuchi O, Okada K, Ueno M, Iemura S-i, Natsume T, et al. Mutations causing DOK7 congenital myasthenia ablate functional motifs in Dok-7. Journal of Biological Chemistry. 2008;283(9):5518-24. Müller JS, Herczegfalvi A, Vilchez JJ, Colomer J, Bachinski LL, Mihaylova V, et al. Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes. Brain. 2007;130(6):1497-506. Anderson JA, Ng JJ, Bowe C, Mcdonald C, Richman DP, Wollmann RL, et al. Variable phenotypes associated with mutations in DOK7. Muscle & nerve. 2008;37(4):448-56. Klein A, Pitt MC, McHugh JC, Niks EH, Sewry CA, Phadke R, et al. DOK7 congenital myasthenic syndrome in childhood: early diagnostic clues in 23 children. Neuromuscular Disorders. 2013;23(11):883-91. Cossins J, Liu WW, Belaya K, Maxwell S, Oldridge M, Lester T, et al. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Human molecular genetics. 2012;21(17):3765-75. Sarmadi A, Nasrniya S, Narrei S, Nouri Z, Abtahi H, Tabatabaiefar MA. Whole exome sequencing identifies novel compound heterozygous pathogenic variants in the MYO15A gene leading to autosomal recessive non-syndromic hearing loss. Molecular Biology Reports. 2020;47(7):5355-64. Sarmadi A, Nasrniya S, Farsani MS, Narrei S, Nouri Z, Sepehrnejad M, et al. A novel pathogenic variant in the LRTOMT gene causes autosomal recessive non-syndromic hearing loss in an Iranian family. BMC Medical Genetics. 2020;21(1):1-9. Azuma Y, Töpf A, Evangelista T, Lorenzoni PJ, Roos A, Viana P, et al. Intragenic DOK7 deletion detected by whole-genome sequencing in congenital myasthenic syndromes. Neurology Genetics. 2017;3(3). Nouri Z, Sarmadi A, Narrei S, Sehhati M, Tabatabaiefar MA. Whole exome sequencing identified a novel LAMA2 frameshift variant causing merosin-deficient congenital muscular dystrophy in a patient with cardiomyopathy, and autism-like behavior. Neuromuscular Disorders. 2022;32(9):776-84. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in medicine. 2015;17(5):405-23. Lashley D, Palace J, Jayawant S, Robb S, Beeson D. Ephedrine treatment in congenital myasthenic syndrome due to mutations in DOK7. Neurology. 2010;74(19):1517-23. Della Marina A, Wibbeler E, Abicht A, Kölbel H, Lochmüller H, Roos A, et al. Long term follow-up on pediatric cases with congenital myasthenic syndromes—A retrospective single centre cohort study. Frontiers in human neuroscience. 2020;14:560860. Wolfe GI, Herbelin L, Nations S, Foster B, Bryan W, Barohn R. Myasthenia gravis activities of daily living profile. Neurology. 1999;52(7):1487-. Lorenzoni PJ, Scola RH, Kay CS, Filla L, Miranda AP, Pinheiro JM, et al. Salbutamol therapy in congenital myasthenic syndrome due to DOK7 mutation. Journal of the Neurological Sciences. 2013;331(1-2):155-7. Rodríguez Cruz PM, Palace J, Beeson D. The neuromuscular junction and wide heterogeneity of congenital myasthenic syndromes. International journal of molecular sciences. 2018;19(6):1677. Durmus H, Shen X-M, Serdaroglu-Oflazer P, Kara B, Parman-Gulsen Y, Ozdemir C, et al. Congenital myasthenic syndromes in Turkey: clinical clues and prognosis with long term follow-up. Neuromuscular Disorders. 2018;28(4):315-22. Engel AG, Shen X-M, Selcen D, Sine SM. Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment. The Lancet Neurology. 2015;14(4):420-34. Palace J, Lashley D, Newsom-Davis J, Cossins J, Maxwell S, Kennett R, et al. Clinical features of the DOK7 neuromuscular junction synaptopathy. Brain. 2007;130(6):1507-15. Beeson D, Higuchi O, Palace J, Cossins J, Spearman H, Maxwell S, et al. Dok-7 mutations underlie a neuromuscular junction synaptopathy. Science. 2006;313(5795):1975-8. Tsao C-Y. Effective treatment with albuterol in DOK7 congenital myasthenic syndrome in children. Pediatric Neurology. 2016;54:85-7. Engel AG, Lambert EH, Mulder DM, Torres CF, Sahashi K, Bertorini TE, et al. A newly recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine‐induced ion channel. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society. 1982;11(6):553-69. Al-Shahoumi R, Brady LI, Schwartzentruber J, Tarnopolsky MA. Two cases of congenital myasthenic syndrome with vocal cord paralysis. Neurology. 2015;84(12):1281-2. Chevessier F, Faraut B, Ravel-Chapuis A, Richard P, Gaudon K, Bauche S, et al. MUSK, a new target for mutations causing congenital myasthenic syndrome. Human molecular genetics. 2004;13(24):3229-40. Murali C, Li D, Grand K, Hakonarson H, Bhoj E. Isolated vocal cord paralysis in two siblings with compound heterozygous variants in MUSK: expanding the phenotypic spectrum. American Journal of Medical Genetics Part A. 2019;179(4):655-8. Alsallum MS, Alshareef A, Abuzinadah AR, Bamaga AK, Dallol A. A novel DOK7 mutation causing congenital myasthenic syndrome with limb-girdle weakness: case series of three family members. Heliyon. 2021;7(5):e06869. Gupta S, Singh AK, Kushwaha PP, Prajapati KS, Shuaib M, Senapati S, et al. Identification of potential natural inhibitors of SARS-CoV2 main protease by molecular docking and simulation studies. Journal of Biomolecular Structure and Dynamics. 2021;39(12):4334-45. MIu L, Bogatyreva N, Galzitskaia O. Radius of gyration is indicator of compactness of protein structure. Molekuliarnaia biologiia. 2008;42(4):701-6. Kushwaha PP, Singh AK, Bansal T, Yadav A, Prajapati KS, Shuaib M, et al. Identification of natural inhibitors against SARS-CoV-2 drugable targets using molecular docking, molecular dynamics simulation, and MM-PBSA approach. Frontiers in cellular and infection microbiology. 2021:728. Schaefer C, Rost B, editors. Predict impact of single amino acid change upon protein structure. BMC genomics; 2012: BioMed Central. Selcen D, Milone M, Shen XM, Harper CM, Stans AA, Wieben ED, et al. Dok‐7 myasthenia: phenotypic and molecular genetic studies in 16 patients. Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society. 2008;64(1):71-87. Liewluck T, Selcen D, Engel AG. Beneficial effects of albuterol in congenital endplate acetylcholinesterase deficiency and Dok‐7 myasthenia. Muscle & nerve. 2011;44(5):789-94. Burke G, Hiscock A, Klein A, Niks EH, Main M, Manzur AY, et al. Salbutamol benefits children with congenital myasthenic syndrome due to DOK7 mutations. Neuromuscular disorders. 2013;23(2):170-5. Khadilkar S, Bhutada A, Nallamilli B, Hegde M. Limb girdle weakness responding to salbutamol: An Indian family with DOK7 mutation. Indian Pediatrics. 2015;52(3). Nishikawa A, Mori-Yoshimura M, Okamoto T, Oya Y, Nakata T, Ohno K, et al. Beneficial effects of 3, 4-diaminopyridine in a 26-year-old woman with DOK7 congenital myasthenic syndrome who was originally diagnosed with facioscapulohumeral dystrophy. Rinsho Shinkeigaku= Clinical Neurology. 2014;54(7):561-4. Inoue A, Setoguchi K, Matsubara Y, Okada K, Sato N, Iwakura Y, et al. Dok-7 activates the muscle receptor kinase MuSK and shapes synapse formation. Science signaling. 2009;2(59):ra7-ra. Yamanashi Y, Higuchi O, Beeson D. Dok-7/MuSK signaling and a congenital myasthenic syndrome. Acta Myologica. 2008;27(1):25. Webster RG, Vanhaesebrouck AE, Maxwell SE, Cossins JA, Liu W, Ueta R, et al. Effect of salbutamol on neuromuscular junction function and structure in a mouse model of DOK7 congenital myasthenia. Human Molecular Genetics. 2020;29(14):2325-36. Schara U, Barisic N, Deschauer M, Lindberg C, Straub V, Strigl-Pill N, et al. Ephedrine therapy in eight patients with congenital myasthenic syndrome due to DOK7 mutations. Neuromuscular Disorders. 2009;19(12):828-32. Witting N, Vissing J. Pharmacologic treatment of downstream of tyrosine kinase 7 congenital myasthenic syndrome. JAMA neurology. 2014;71(3):350-4. Mihaylova V, Scola R, Gervini B, Lorenzoni P, Kay C, Werneck L, et al. Molecular characterisation of congenital myasthenic syndromes in Southern Brazil. Journal of Neurology, Neurosurgery & Psychiatry. 2010;81(9):973-7. Mahjneh I, Bushby K, Anderson L, Muntoni F, Tolvanen-Mahjneh H, Bashir R, et al. Merosin-positive congenital muscular dystrophy: a large inbred family. Neuropediatrics. 1999;30(01):22-8. Jadhav T, Shah P, Karnavat PK, Hegde AU. Intrafamilial variation in clinical manifestations and response to salbutamol in siblings with congenital myasthenic syndrome caused by DOK7 mutations. Journal of the International Child Neurology Association. 2019. Supplementary Files Supplementary.13.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3924937","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297631745,"identity":"57b2391b-af60-4390-9d06-2262c4a6e0bf","order_by":0,"name":"Zahra Nouri","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Nouri","suffix":""},{"id":297631746,"identity":"dac0c1f1-441e-4083-9fa1-905571b9aa85","order_by":1,"name":"Javad Saffari-Chaleshtori","email":"","orcid":"","institution":"Shahrekord University of Medical Science","correspondingAuthor":false,"prefix":"","firstName":"Javad","middleName":"","lastName":"Saffari-Chaleshtori","suffix":""},{"id":297631747,"identity":"d482a36f-8e7f-4b27-9254-0e79d9dbcf25","order_by":2,"name":"Akram Sarmadi","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Akram","middleName":"","lastName":"Sarmadi","suffix":""},{"id":297631749,"identity":"d8aa1f6c-ffe3-44fe-814e-1594bfae7bee","order_by":3,"name":"Mohammadreza Sehhati","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammadreza","middleName":"","lastName":"Sehhati","suffix":""},{"id":297631750,"identity":"6c6bef7b-52d7-4617-9952-7642f771a555","order_by":4,"name":"Mohammad-Amin Tabatabaiefar","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0730-750X","institution":"Isfahan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Mohammad-Amin","middleName":"","lastName":"Tabatabaiefar","suffix":""}],"badges":[],"createdAt":"2024-02-03 18:21:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3924937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3924937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56195354,"identity":"47f23386-5dcc-4040-82d5-3f78505e1fa5","added_by":"auto","created_at":"2024-05-09 18:01:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78610,"visible":true,"origin":"","legend":"\u003cp\u003eIn the left side, the pedigree of the family is shown. The proband is marked by an arrow. Homozygous cases filled in black, but heterozygotes cases are marked with a dot in the center. In the right side, the electropherogram of the muatation in the family is shown: the father (A) and mother (B) of the proband (C) are heterozygous for the variant, while the proband (case VI-4), case VI-1 (D) and case VI-2 (E) are homozygous.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/cb859f66c86172eb36f5cce1.jpg"},{"id":56195376,"identity":"edf0070d-d227-4042-a30f-a670645420c3","added_by":"auto","created_at":"2024-05-09 18:01:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149025,"visible":true,"origin":"","legend":"\u003cp\u003eThe ramachandran plot show that more than 96.64% of DOK-M amino acid residues and 95.01% of DOK-W amino acid residues are in the preferred and highly preferred regions.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/7fa16c139fe7a701f919c570.jpg"},{"id":56195352,"identity":"6103d22a-0882-46ca-95c0-efb7f73a09e6","added_by":"auto","created_at":"2024-05-09 18:01:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86522,"visible":true,"origin":"","legend":"\u003cp\u003eConformational changes in proteins: \u003cstrong\u003eA:\u003c/strong\u003e The root-mean-square deviation (RMSD), \u003cstrong\u003eB:\u003c/strong\u003eradius of gyration (Rg), \u003cstrong\u003eC:\u003c/strong\u003e root-mean-square-fluctuation (RMSF), \u003cstrong\u003eD:\u003c/strong\u003ethe total energy (TE). The red line is DOK-M and the black line is DOK-W, \u003cstrong\u003eE:\u003c/strong\u003eThe hydrogen bonds (H-bonds) and \u003cstrong\u003eF:\u003c/strong\u003e The secondary structure parameters (α-Helix, β-Sheet, Coil, Bend, and Turn) between the DOK-M and DOK-W during the 200 ns of simulation time.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/c7d91195a9288fbd8d6b833f.jpg"},{"id":56195383,"identity":"bd0a2340-3c9c-491e-8d74-1bf861c5eccb","added_by":"auto","created_at":"2024-05-09 18:01:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60432,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of four conventional Drugs using in CMS vs age at onset in female and male.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/d55043e9391e80506eee25a2.jpg"},{"id":56195378,"identity":"bb98e5fc-6730-4761-a013-f0228f4ce085","added_by":"auto","created_at":"2024-05-09 18:01:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eQMG is a severity score with a maximum of 39. Changes in QMG score of 3 patients (cases VI-1 \u0026amp; VI-2 \u0026amp; VI-4) during 18 months’ follow-up. \u003cstrong\u003eB:\u003c/strong\u003e Change in MG-ADL score of all cases during 18 months’ follow-up. \u003cstrong\u003eC:\u003c/strong\u003e Ptosis in 3 patients, it showed bilateral petosis in case VI-4, unilateral ptosis in right eye in case VI-1 and unilateral ptosis in left eye in case VI-2.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/97399e70f1761f17f98d9cd8.jpg"},{"id":66505917,"identity":"56394ed5-342e-4179-bf09-011dec9afea5","added_by":"auto","created_at":"2024-10-13 17:32:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1034627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/85b33f7f-dfc8-4804-96f9-7e82b0040a86.pdf"},{"id":56195379,"identity":"ffbe5b7a-042e-4d2d-a62b-8a06c91fa302","added_by":"auto","created_at":"2024-05-09 18:01:30","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":51792,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.13.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3924937/v1/c76f4549c0edf82b5a5f45f8.xlsx"}],"financialInterests":"","formattedTitle":"Clinical and molecular analysis of a novel variant responsive to salbutamol monotherapy during COVID-19 outbreak related to congenital and late-onset of myasthenic syndrome in large kindred","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital myasthenic syndromes (CMS) represent a heterogeneous group of inherited disorders with the clinical features of fluctuating, weakness in axial, respiratory, bulbar and extraocular muscles and easy fatigability (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). CMSs are disorders of the neuromuscular junction (NMJ) resulting from genetic defects in pre-synaptic, synaptic and post-synaptic proteins of the NMJ (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The age of onset of CMS greatly varies, from the most common presentations shortly after birth or during childhood, to the rare occurrence of the adult onset manifestations (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Different mutations in genes related to the NMJ formation, function, development and maintenance could cause different types of CMS with autosomal dominant or recessive pattern of inheritance (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The deficient protein includes DOK-7, muscle-specific kinase (MUSK), AChR, GFPT1, b2-Laminin and Plectin-1 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Neuromuscular synapse formation and maintenance needs an extremely professional assembly of presynaptic and postsynaptic membranes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). A key molecule is AGRIN, a protein released from the terminals of motor nerve which binds to the lipoprotein receptor-related protein 4 (LRP4) in the muscle. This coupling will stimulate the formation of a complex between LRP4 and MUSK, which is a tyrosine kinase receptor and has a vital regulator role in synaptic differentiation (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). After activation of MUSK in the postsynaptic membrane, LRP4 will be clustered stimulating presynaptic differentiation by signals directly back to motor axons (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Mutations in \u003cem\u003eAGRIN\u003c/em\u003e, \u003cem\u003eLRP4\u003c/em\u003e and \u003cem\u003eMUSK\u003c/em\u003e, as well as in the genes that encode subunits of acetylcholine receptors (AChRs), also cause CMS (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDocking Protein 7 (DOK7) is a cytoplasmic protein that mediates the activation of MUSK and has important role in postsynaptic specialization of the NMJ (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This protein is encoded by the \u003cem\u003eDOK7\u003c/em\u003e gene on chromosome 4p16.2 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Clinical presentations in patients with \u003cem\u003eDOK7\u003c/em\u003e mutations include ptosis, hypotonia, and bulbar symptoms such as dysphagia and dysarthria (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Weakness in limb, neck and facial muscles are common and proximal muscle weakness is more prominent that distal muscles. Spinal deformity can be caused by truncal weakness, which is usually seen in these patients (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Three main functional domains of DOK-7 include: a pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain and a large C-terminal domain following by multiple tyrosine residues (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe recently developed next generation sequencing (NGS) technologies such as exome sequencing (ES) provide an opportunity to identify causative mutations in different types of monogenic and heterogeneous disorders and discovery of novel disease-causing genes and variants (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we describe in detail the clinical features of a 10-year-old CMS patient and two of her father\u0026rsquo;s second cousins due to a \u003cem\u003eDOK7\u003c/em\u003e gene mutation. In addition to these 3 cases with homozygous variant, there are 5 other cases in the family who are heterozygous and have presented the late-onset form of CMS. In this family, variable clinical presentations are reported, while the genetic defect is similar. We performed ES to provide molecular diagnosis of this disorder in the primary proband. This variant was found to be co-segregating in the family. Remarkable good response to salbutamol was noted in this family.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubject and clinical evaluations\u003c/h2\u003e \u003cp\u003eThe primary proband (case VI-4), who is currently a 10-year-old girl, belongs to a consanguineous couple. She was born through normal delivery. Muscle weakness and muscle atrophy were noted in the age of 6 months. In that age, she was able to crawl but floppy phenotype warned the parents to consult a specialist. She was unable to sit without any support and experienced intermittent worsening lasting from days to weeks. The first common immunological and biochemical tests (anti-acetylcholin receptor and anti-Musk antibodies) were done followed by testing creatine kinase (CK) and lactate dehydrogenase (LDH), Nerve conduction velocity (NCV) and Electromyography (EMG) tests at the age of 1 year. Sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) tests were performed when she was 3 years old and analysis of deletion of exon 7 and 8 of the \u003cem\u003eSMN1\u003c/em\u003e gene and \u003cem\u003eNAIP4\u003c/em\u003e and \u003cem\u003eNAIP5\u003c/em\u003e gene deletions and alpha-1,4 Glucosidase enzyme activity were also done at the age of 6 years old (using PCR-RLFP technique and tandem mass spectrometry, respectively) for checking spinal muscular atrophy (SMA) and pompe disease. Her blood sample was taken for DNA extraction and doing ES when she was 7 years. After identifying the pathogenic variant, co-segregation analysis and detailed clinical evaluation were performed in other patients in the family.\u003c/p\u003e \u003cp\u003eThe study was approved by the Review Board of Isfahan University of Medical Sciences (grant no: 2400173 and ethics code: IR.ARI.MUI.REC.1400.011).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunological and metabolic panel tests\u003c/h2\u003e \u003cp\u003eThe thyroid function and the level of CK and LDH and also anti-acetylcholin receptor and anti-Musk antibodies were measured in all the patients before doing any molecular testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMolecular study\u003c/h2\u003e \u003cp\u003eThe peripheral blood lymphocytes (5 ml) was received from the studied patient. Extraction of the genomic DNA was conducted using Prime Prep Genomic DNA Extraction kit from blood (CTGA, Iran), according to the manufacturer\u0026rsquo;s instruction. Quality and quantity of DNA were assessed by agarose gel and Nanodrop 2000 instrument (Thermo Fisher Scientific Inc, USA), respectively and the patient\u0026rsquo;s sample was applied for ES.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eES and bioinformatics analyses\u003c/h2\u003e \u003cp\u003eThe Sample of 300 ng Genomic DNA was sent to Macrogen (South Korea) and was subjected to ES using the NovaSeq 6000 platform (Illumina, US). Briefly, genomic DNA was fragmented and fragments were captured to target all exons, splicing sites, and flanking intronic sequences of all genes. After amplification of fragments, sequencing was performed with a 150X mean depth of coverage for more than 92% of the sequences. The released row data were converted to the FASTQ file and clean reads were then aligned with the reference human genome sequence (hg19, NCBI Build 38) using the Burrows-Wheeler Aligner (BWA). All the steps were the same as we explained in our previous articles (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Finally, the suspected pathogenic variant was evaluated by different \u003cem\u003ein silico\u003c/em\u003e software tools such as Mutation-Taster and CADD to predict its deleterious effect on protein in terms of function. The American College of Medical Genetics and Genomics (ACMG) guidelines were used to classify the variants (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eVariant confirmation\u003c/h2\u003e \u003cp\u003e Co-segregation analysis was performed using exon-specific custom primers to examine segregation of the genotype and phenotype among the parents using direct Sanger sequencing. The forward primer sequence was 5\u0026acute;-ACTCCTCTTACTCCAGCAGC-3\u0026acute; and the reverse primer sequence was 5\u0026acute;- ACATGCCGAAAAGAAAGCCA-3\u0026acute; in exon 7. Electropherograms were compared with the reference sequence (NM_173660) encoding 504 residues using the SeqMan software version 5.00\u0026copy; (DNASTAR, Madison, WI, USA). The next step was to investigate in the Human Gene Mutation Database (HGMD) and the literature to seek the novelty of the variant or its association with CMS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of PDB files and proteins modeling\u003c/h2\u003e \u003cp\u003eThe FASTA sequence of DOK-W (wild type of the DOK7 protein) was obtained from NCBI server and the FASTA sequence of DOK-M (Mutant form of DOK protein) was designed manually. Also, the homology modeling of their 3 dimensions (3D) were produced by I-TASSER server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/I-TASSER/\u003c/span\u003e\u003cspan address=\"https://zhanggroup.org/I-TASSER/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Then, to confirmation of the stability of PDB files/3D structures, the ramachandran plot was designed by Ramachandran Plot Server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zlab.umassmed.edu/bu/rama\u003c/span\u003e\u003cspan address=\"https://zlab.umassmed.edu/bu/rama\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics (MD) simulation of the DOK-W and DOK-M proteins\u003c/h2\u003e \u003cp\u003eThe molecular dynamics simulation of the DOK-W and DOK-M protein structures was done in pure water and the force field of G43A1 with SPC216 model using the GROMACS 2018 simulation package. The grid box with 4.3\u0026times;4.3\u0026times;3.5 nm was built for each protein. The suitable amounts of chloride and sodium ions were used to neutralize the molecules to provide 140 mM concentration of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e in water for each protein, through assessment of the electrical charge of each protein. The energy of the systems was minimized throughout 70000 steps by using steepest descent method and equilibrated for 2 ns in NVT ensemble. The simulations were run for 200 nanoseconds (ns) and at a constant temperature of 300\u0026ordm;K for all the simulation intervals. Then, the output results were analyzed using Grapher V.10 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePharmacologic Treatment\u003c/h2\u003e \u003cp\u003ePatients (VI-1, VI-2 and VI-4) started being treated with different dosages of ephedrine, based on the recommendations by a neurologist). However, the symptoms of the disease got even worsened, thus, they stopped taking the medicine. Salbutamol was an alternative drug for treatment, which was prescribed after the identification of the genetic mutation based on the opinion of our multidisciplinary team. The effects of treatment were measured using the quantitative myasthenia gravis (QMG) score for 3 patients and Myasthenia Gravis Activities of Daily Living Scale (MG-ADL) was used for all the cases during the 18 months follow-up (\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). We also performed a literature search in the MEDLINE, PubMed Embase, Web of Science, and Google Scholar databases using the following headings: DOK7, DOK7 mutations, DOK7 treatment and myasthenic syndrome to find all publications describing the treatment response and drug effects in patients with DOK7-CMS. The last search was performed in April, 2023. If more than one article had been published by the same group, a comparison of the participants in the studies was made, and duplicated data were excluded.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical findings\u003c/h2\u003e \u003cp\u003eA 10-year-old girl and two of her father\u0026rsquo;s second cousins were visited with a history of progressive limb weakness. Upon further genetic counseling sessions, we noticed 5 other cases with late-onset presentations of the disease, with less severe symptoms. The clinical findings of all cases are provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase VI-4\u003c/strong\u003e \u003cp\u003eThe primary proband was a 10-year-old girl who had experienced mild weakness in the 6 months of age. Progressive bilateral ptosis was notable at birth and facial and neck weaknesses were appeared. Bilateral proximal limb weakness and disability to stand without support were reported when she was 1-year-old. She started walking at the age of 2 years\u0026rsquo;, but she had difficulty walking independently. She only could walk for about 4\u0026ndash;5 steps without support and stairs climbing was very difficult for her. She had waddling gait, facial weakness, significant proximal muscle weakness of both upper and lower extremities and the ptosis was aggravated during ages but cognitive and language milestones were normal. Mild swallowing and chewing difficulties happened latter. Vocal cord paralysis (VCP) was started when she was 5 years old. She became very tired when walking or playing with her friends. She could hardly keep a glass of water, hold a pencil in her hands and experienced a decline in academic performance. During a mild cold, if the fever rose a little, she would be completely paralyzed and could not stand up or walk unaided.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe first common biochemical tests were in normal ranges. The results of NCV and EMG tests which were done at the same age suggested CMS to be the probable explanation. In the age of 3 years, the results of SNAPs and CMAPs of nerves and F-waves were normal and as myopathic motor unit action potentials (MUAPs) were seen in most muscles. The possibility of the SMA and pompe diseases was ruled out by performing the analysis of deletion of exon 7 and 8 of \u003cem\u003eSMN1\u003c/em\u003e gene and \u003cem\u003eNAIP4\u003c/em\u003e and \u003cem\u003eNAIP5\u003c/em\u003e genes deletion at the age of 6 years, along with the evaluation of the alpha-1,4 Glucosidase enzyme activity.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCases VI-1 and VI-2\u003c/strong\u003e \u003cp\u003eThese two cases are two brothers who are second cousins of the proband\u0026rsquo;s father. They are 16 and 11 years old. They were both healthy at birth with normal motor milestones, except showing mild congenital unilateral ptosis. Walking, sitting and standing were normal and independently until the age of six. By the age of 6 years, they experienced muscle weakness and rapid fatigue while playing and running (exercise induced weakness) and they were getting worse through time. At the age of about 10 years, both brothers had severe motor weakness and it was very difficult for them to walk for more than a few steps, run and climb stairs and unilateral ptosis was aggravated.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCases V-2, V-8, V-10 and V-11\u003c/strong\u003e \u003cp\u003eThese cases are the proband's father, aunt and uncle and their second cousins. All of these patients have late-onset symptoms of CMS. The most obvious presentation of the disease was facial weakness, which was often revealed after the age of 40. Also, mild weakness on proximal limbs and lack of strength on arms and shoulder were evident, which sometimes became very annoying. While ptosis was obvious in all cases, dysphonia, dysarthria and diplopia were not present. Facial weakness and the ptosis were exacerbated during the years. Motor nerve conduction study (NCS) and EMG were normal in these cases and the result of Repetitive Nerve Stimulation (RNS) test showed decremental response in facial muscles indicating NMJ defects.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCase IV-4\u003c/strong\u003e \u003cp\u003eThis case is the proband's grandmother (paternal grandmother). The first clinical feature was fatigue and proximal limb weakness, that was followed by episodes of sudden respiratory failure in her 50s, which caused the patient to start a long-term oxygen therapy and non-invasive nocturnal ventilatory support. She suffered from weakness on proximal limbs and fatigability which were progressive during times. All these symptoms started after the age of 40 years old. (The pedigree of the family is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunological and metabolic panel tests results\u003c/h2\u003e \u003cp\u003eThere was no abnormality in the immunological and metabolic profiles of the patients. All laboratory evaluations were within normal ranges, including thyroid function, CK and LDH levels and anti-Musk and AChR antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMolecular findings\u003c/h2\u003e \u003cp\u003eAs a result of ES, a novel homozygous deletion/insertion (c.1139-1140delinsA:p.Ala380AspfsTer76) in the \u003cem\u003eDOK7\u003c/em\u003e gene was found in the proband. This variant results in a truncated protein with 454 residues (versus 504 residues in the intact protein). This frameshift variant was not found in the literature and also was absent from 1000 genomes project phase 3, HGMD, dbSNP version 147, Clinvar, NHLBI GO ESP, ExAC and Iranome databases, as well as, our GTaC local database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVariant confirmation and co-segregation analysis\u003c/h2\u003e \u003cp\u003eCo-segregation analysis of the variant in the family shows heterozygous status in the parents (V-11 \u0026amp; V-12), but homozygous in their affected girl and also in the cases VI-1 \u0026amp; VI-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The other cases with late-onset presentations of the disease (cases IV-4, V-2/8/10/11) were also heterozygous for the variant.\u003c/p\u003e \u003cp\u003eAccording to the \u003cb\u003eACMG\u003c/b\u003e guideline, this variant is classified as a pathogenic variant (1 very strong, 2 Moderate and 2 Supporting criteria):\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics simulation of the DOK-W and DOK-M proteins results:\u003c/h2\u003e \u003cp\u003eThe ramachandran plot showed that more than 96.64% of DOK-M amino acid residues and 95.01% of DOK-W amino acid residues are in the preferred and highly preferred regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The root-mean-square deviation (RMSD) was stable after 30 ns of simulation time for both proteins. Also, they were stable until the end of simulation time. Although, the mean RMSD of DOK-M was decreased during the simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe stability of simulation system was confirmed by radius of gyration (Rg) results. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows that Rg parameters are stable after 30 ns of simulation time. However, there is a significant decrease in the Rg of DOK-M compared with the Rg of DOK-W.\u003c/p\u003e \u003cp\u003eThe results of root-mean-square-fluctuation (RMSF) and total energy (TE) are shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. There are increases in the amount of RMSF for 45 to 90 and 345 to 370 residue amino acid regions of DOK-M in comparison of the DOK-W. RMSF graphs revealed that the residue amino acid regions of 140 to 145, 190 to 210, and 370 to 400 of the DOK-W were decreased during the simulation.\u003c/p\u003e \u003cp\u003eFurthermore, an increment in TE for DOK-M in comparison with the mean of TE for DOK-W during the simulation time was identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The mean of the TE for DOK-W was about \u0026minus;\u0026thinsp;1.45\u0026times;10\u003csup\u003e6\u003c/sup\u003e kj/mol while, the TE for DOK-M increased significantly and it was about \u0026minus;\u0026thinsp;1.35\u0026times;10\u003csup\u003e6\u003c/sup\u003e kj/mol. The amounts of hydrogen bonds (H-bonds) are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE. The mean of H-bonds decreased remarkably in the DOK-M in comparison to the mean of H-bonds in the DOK-W during the simulation time.\u003c/p\u003e \u003cp\u003eThe variation in the secondary structure parameters such as α-Helix, β-Sheet, Coil, Bend, and Turn between the DOK-M and DOK-W during the 200 ns of simulation time is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF. There is a significant change between the secondary structure parameters of DOK-M as compared to DOK-W. The Coil, Turn, and α \u0026ndash;Helix secondary structures were increased while, the β-Sheet, and Bend secondary structures were decreased during the simulation time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMonitoring response to therapy:\u003c/h2\u003e \u003cp\u003eIn the primary proband, a short-term therapy of 7 days with ephedrine was not successful, the symptoms got worse in such a way that she was not able to walk and became unable to perform daily activities. Thus, the treatment was discontinued. After the genetic test was performed and it was found that the pathogenic variant in the \u003cem\u003eDOK7\u003c/em\u003e gene is the cause of the disease, our multidisciplinary team suggested starting treatment with salbutamol (at a dose of a quarter of a 2 mg tablet 4 times daily, total of 2mg/daily for 5 months). After only 3 days of receiving it, improvement of strengths of the legs was seen in her and she became more active and movement of extremities improved significantly. She was able to sit and stand without support, and walk and run long distances and her ptosis and VCP got improved gradually (the dosage of the drug was changed after 5 months, and she was ordered to increase one meal of the drug to a half of a tablet for 5 months, and then increased other 3 times in one complete 2 mg tablet). An 18-month follow-up was performed by our multidisciplinary team (3, 6, 9, 12 and 18 months after starting salbutamol therapy). This medication was well tolerated in this patient, she did not show any side effects and the progress was notable. Even when the patient (proband) contracted COVID-19 and had a high fever of 39.8\u0026deg;C, she did not lose her ability to walk without any support. She used to be completely paralyzed by a cold with milder fever in the past.\u003c/p\u003e \u003cp\u003eBoth brothers (cases VI-1 \u0026amp; VI-2) also start treatment with ephedrine. Their daily function deteriorated and facial and muscle weakness were aggravated. However, the condition improved significantly as soon as they stopped taking ephedrine and started treatment with salbutamol (one 2 mg tablet 3 times daily for the elder brother and half of a tablet 3 times daily for the other). Only after a short time, their ptosis, VCP and dysphagia were improved and their muscle were strengthened to the degree they were able to play football. Also, in the past, when they had a cold, they became very weak and could hardly walk, but after taking salbutamol, they did not have any movement problems even when they were infected with COVID-19. Since the remarkable effective signs of salbutamol therapy was observed in this family, treatment was started in heterozygous patients (2 mg four times daily) and the improvement of facial weakness and strengthening of the muscle was reported. Furthermore, sudden episodes of respiratory failure in case IV-4 were completely resolved after using salbutamol spray. Salbutamol was also orally admitted for her and the permanent fatigue was relieved in addition to increased muscle strength. Thus, her muscle weakness was also improved.\u003c/p\u003e \u003cp\u003eIn the literature review we have done; in 136 DOK7-CMS patients, positive effects of salbutamol were observed in 40 of 41 patients who received it. It was also found that salbutamol did not have any negative effects on patients and it had no effect only in one patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Strength measures included the QMG (severity) score and MG-ADL showed significant improvement from baseline to the 3- to 18-month follow-up in our cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u0026amp; B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCongenital Myasthenic Syndromes (CMS) are a group of inherited disorders resulting from abnormal signal transmission of NMJ at different levels of the presynaptic, synaptic and postsynaptic apparatus. Up to now, more than 30 genes have been found to underlie CMS and the differentiation of CMS subtypes, usually clinically heterogenous, can be performed by molecular genetic tests (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). DOK7-CMS is reported to occur in up to almost 20% of CMSs (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The DOK7 protein, which is encoding by \u003cem\u003eDOK7\u003c/em\u003e gene, is involved in the normal development and maintenance of the NMJ (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). DOK7-associated CMS mutations are widely accepted as being inherited in an autosomal recessive pattern. However, this is not always the cases (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). A variety of mutations in the \u003cem\u003eDOK7\u003c/em\u003e gene are reported to be the cause of CMS with the most common (more than 65%) mutation being c.1124_1127dupTGCC in exon 7, which is in homozygous or compound heterozygous form (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This variant results in a truncated protein and leads to the loss of two tyrosine residues that are phosphorylated and recruits CRK proteins, which are important for anchoring acetylcholine receptors at synapses.\u003c/p\u003e \u003cp\u003eIn this study, in addition to reporting a novel disease-causing variant in \u003cem\u003eDOK7\u003c/em\u003e gene, we describe a late-onset form of CMS in 5 carriers of this variant. Whereas a late-onset presentation of CMS is uncommon, it does not rule out CMS (Supplementary Table\u0026nbsp;2\u0026amp;3). Due to the overlap of clinical features, during the initial diagnosis process, it may be misdiagnosed as limb-girdle weakness or myasthenia gravis (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Thus, late-onset CMS is probably still underdiagnosed in plenty of cases (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Despite the fact that our homozygous patients have the same genotype, their manifestations of the disease were not the same. This may be due to their different genetic background. Congenital bilateral ptosis and facial and muscle weakness were observed in the primary proband, while in the cases VI-1 and VI-2, unilateral ptosis in different eyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and muscle weakness onset were noticeable during childhood (at the age of about 6 years). However, all of them had VCP. This feature is reported in CMS patients with mutations in \u003cem\u003eCOLQ\u003c/em\u003e and \u003cem\u003eDOK7\u003c/em\u003e genes, as well as patients with MUSK deficiency (\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Therefore, genetic testing is essential to identify the etiology and could have a role in the proper management of the disease. Furthermore, the severity of muscle weakness in all homozygous patients was mostly variable. Thus, there was no consistent genotype-phenotype correlations. Moreover, the clinical features in the heterozygous cases with late-onset manifestations were somewhat different. The facial weakness, mild ptosis and lack of strength in arms and shoulders with different severity after the age of 40 were mostly common, while, the respiratory failure was only seen in the case IV-4 after the age of 50 years (none of other carriers have reached this age yet). Meanwhile, there are reports of several cases of DOK7-CMS with neonatal or childhood respiratory failure (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), none of our homozygous patients showed this feature. However, it was the first report of the late-onset CMS in patients with a heterozygous pathogenic variant in \u003cem\u003eDOK7\u003c/em\u003e gene in Iran, there are a few reports of late-onset presentations with clinical heterogeneity in carriers, but the precise mechanism is unexplained (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The phenotypic presentations and intra-and inter-familial variable expressivity of CMS in carries of this study may be related to the dominant-negative effects of the identified variant or haploinsufficiency mechanism as a consequence of partial autosomal dominant inheritance. A late-onset CMS patient caused by heterozygous mutation in the \u003cem\u003eDOK7\u003c/em\u003e gene, which was previously described by \u003cem\u003eBastos\u003c/em\u003e et al, represented the symptoms of the disease after the age of 65 and the first features were increased difficulty in climbing stairs, weakness on both legs, lack of strength in both arms and finally sudden respiratory failure without ptosis and facial palsy (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Thus, the clinical presentations of late-onset CMS would not be similar, even if the mutated gene is the same.\u003c/p\u003e \u003cp\u003eSimulation analysis of this study showed that mutation in the DOK-W induces the variations in the structures and molecular dynamic parameters and subsequently instability and dysfunction of the protein. The mean of RMSD indicated that simulation system is stabled in the 200 ns of simulation time. However, decrease in RMSD for DOK-M compared to DOK-W demonstrated that this mutation affects the protein structure (40). Also, a comparison of Rg data of both proteins revealed the more compactness in DOK-M which might lead to decreased stability and functional impairment of the DOK-M (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The different variations in the RMSF parameter of amino acid residues in the DOK-M in comparison of DOK-W shows the impact of this mutation on amino acids\u0026rsquo; fluctuation. Given that increment of TE in the mutant form of the protein increases the intermolecular energy, molecular instability could ensue. Moreover, changes in the DOK-M structure decrease the mean of H-bonds compared with DOK-W. Decrease in the intermolecular hydrogen bonds can cause the protein stability reduction (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Finally, the variation of secondary structure of the protein and induction of changes in the mean of α-Helix, β-Sheet, Coil, Bend, and Turn parameters between the DOK-M and DOK-W during the simulation time showed that this mutation induced remarkable conformational changes in the wild type of DOK protein, subsequently influencing the folding and function of the protein (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA variety of therapeutic interventions are available, which are beneficial in treating some kinds of CMSs, but accurate genotype diagnosis is important for the disease management (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Ephedrine (a sympathomimetic with α- and β-adrenergic effects) and salbutamol (a selective β2 agonist) were very effective therapeutic agents in some CMS patients, especially those with COLQ, laminin-β2, DOK7, MUSK, Agrin and plectin-1 deficiency, and provided more benefit in types of CMS, which are not effectively treated by acetylcholinesterase inhibitors (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). β2-Adrenergic agonists, partially compensate disrupted postsynaptic structures. Therefore, a remarkable response is seen in CMS subtypes with mutations in genes encoding proteins that are involved in formation and stability of the NMJ within the LRP4-MUSK-DOK7 signaling pathway (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In an \u003cem\u003ein vivo\u003c/em\u003e study on DOK7-CMS model mice, salbutamol treatment increased the number of active NMJs as well as increasing muscle strength and prolonged the survival of these myasthenic mice (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The precise selection of drug therapy is very important, as the same drug can be effective, ineffective, or even worsen the clinical features in different types of CMS (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e)(Supplementary Table\u0026nbsp;1). In the present study, when the clinical symptoms of the disease were obvious, these patients experienced a short period of ephedrine. This treatment not only did not improve their symptoms but also worsened the condition of all homozygous patients in a short time. Therefore, ephedrine treatment was discontinued after 7 days. After that, when the pathogenic variant in the \u003cem\u003eDOK7\u003c/em\u003e gene was identified by ES, the treatment with salbutamol was started, and the dramatical improvement of the patients' condition was observed in a very short period of time. The peak of treatment effect with ephedrine and salbutamol was reported to be about 6\u0026ndash;8 months after start using these drugs (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e), in contrast to anticholinesterases, full effects of which were not immediate (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Only after 3 days of using salbutamol, our homozygous patients started doing their daily tasks. Walking and climbing up the stairs got better and their ptosis gradually improved. Eventually during our 18 months follow-up a significant improvement was observed. While headache, tremor and occasional palpitations were reported as side effects of salbutamol in some cases (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), no side effects were observed in our patients after using it. We also started salbutamol therapy (in a different dose) in 5 carriers, who were showing late-onset form of CMS (supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and its very beneficial effects were obtained. In cases IV-4, V-2, V-8, V-10 and V-11, there was a significant increase in the strength of the muscles, a decrease in the facial weakness, and a prominent improvement in their ptosis in an 18-month follow-up. Also, no recurrence of respiratory failure was observed in case IV-4, after receiving salbutamol. It has been proven that in the treatment with salbutamol, the age of the disease onset, the age at treatment start and the drug dosage have no effect on the treatment outcome (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Our literature review showed that in addition to the aforementioned factors, gender is also ineffective in responding to treatment with this drug, and both sexes, regardless of the age of onset of the disease, responded well to salbutamol therapy (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)(Fig.\u0026nbsp;6) (Supplementary Table\u0026nbsp;2\u0026amp;3). Interestingly, although the mechanism of action of ephedrine and salbutamol are probably similar, in our patients, ephedrine had a negative effect on the disease, while salbutamol had completely positive consequences. There are some reports of other studies which the ephedrine therapy in DOK7-CMS was not efficient, but herein we are reporting negative effects of ephedrine in our patients (Supplementary Table\u0026nbsp;1).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDOK7-CMS often presents with fatigues and muscle weakness, ptosis and facial weakness, which without proper treatment could be worsen. Recent achievements of the exome sequencing in combination with computational studies and pharmacogenetics have provided additional insights on the treatment and management of different diseases. Furthermore, in heterozygous individuals for a pathogenic variant, follow-up of their condition is recommended because they may subsequently develop the late-onset form of this disease. Thus, personalized medicine is being advanced through data obtained from patient\u0026rsquo;s genetic profile.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all of the participants in the study and a written consent to participate was obtained from the parents of the patient (younger than the age of 16).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication: \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication of clinical details and clinical images was obtained from the all of the participants and from the parents the participant under the age of 18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Review Board of Isfahan University of Medical Sciences (ethics code: IR.ARI.MUI.REC.1400.011).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Isfahan University of Medical Sciences grant NO. 2400173. MAT has received this research support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Author contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: \u0026nbsp; MAT; Data collection, analysis, and interpretation: MAT, ZN, JSC, AS and MS; Clinical evaluation: MAT; manuscript preparation: ZN, JSC and AS; critically reviewed by MAT. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe take this opportunity to express our special thanks to all individuals who cooperated in this study, and also to the patient and family.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEngel AG. Congenital myasthenic syndromes in 2018. Current neurology and neuroscience reports. 2018;18(8):1-11.\u003c/li\u003e\n\u003cli\u003eBastos P, Barbosa R, Fernandes M, Alonso I. A late-onset congenital myasthenic syndrome due to a heterozygous DOK7 mutation. Neuromuscular Disorders. 2020;30(4):331-5.\u003c/li\u003e\n\u003cli\u003eEngel AG, Sine SM. Current understanding of congenital myasthenic syndromes. Current opinion in pharmacology. 2005;5(3):308-21.\u003c/li\u003e\n\u003cli\u003eAmmar AB, Petit F, Alexandri N, Gaudon K, Bauch\u0026eacute; S, Rouche A, et al. 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Ephedrine therapy in eight patients with congenital myasthenic syndrome due to DOK7 mutations. Neuromuscular Disorders. 2009;19(12):828-32.\u003c/li\u003e\n\u003cli\u003eWitting N, Vissing J. Pharmacologic treatment of downstream of tyrosine kinase 7 congenital myasthenic syndrome. JAMA neurology. 2014;71(3):350-4.\u003c/li\u003e\n\u003cli\u003eMihaylova V, Scola R, Gervini B, Lorenzoni P, Kay C, Werneck L, et al. Molecular characterisation of congenital myasthenic syndromes in Southern Brazil. Journal of Neurology, Neurosurgery \u0026amp; Psychiatry. 2010;81(9):973-7.\u003c/li\u003e\n\u003cli\u003eMahjneh I, Bushby K, Anderson L, Muntoni F, Tolvanen-Mahjneh H, Bashir R, et al. Merosin-positive congenital muscular dystrophy: a large inbred family. Neuropediatrics. 1999;30(01):22-8.\u003c/li\u003e\n\u003cli\u003eJadhav T, Shah P, Karnavat PK, Hegde AU. Intrafamilial variation in clinical manifestations and response to salbutamol in siblings with congenital myasthenic syndrome caused by DOK7 mutations. Journal of the International Child Neurology Association. 2019.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Congenital myasthenic syndromes, DOK7 gene, Pathogenic variant, Exome sequencing, Late-onset","lastPublishedDoi":"10.21203/rs.3.rs-3924937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3924937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Congenital myasthenic syndromes (CMSs) are a group of clinically and genetically heterogeneous disorders. Mutations in the \u003cem\u003eDOK7\u003c/em\u003e gene underlie CMS with fatigue and muscle weakness, which would worsen with some conventional treatments but show excellent response to special drugs. Here, we applied exome sequencing (ES) to investigate the etiology of CMS in several patients with congenital and late-onset presentations of the disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We ascertained a big pedigree including 3 homozygous and 5 carriers, primary proband was subjected to ES, following immunological, biochemical and neurological evaluations. Molecular dynamics (MD) simulation studies were conducted to investigate the DOK7 proteins' stability. The variant’s pathogenicity was assessed using bioinformatics tools and co-segregation analysis. We adapted the American College of Medical Genetics and Genomics (ACMG) guidelines for variant interpretation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e ES results showed a novel homozygous variant (c.1139-1140delinsA:p.Ala380AspfsTer76) in the \u003cem\u003eDOK7\u003c/em\u003e gene. Co-segregating analysis confirmed the pathogenicity of it based on the ACMG guidelines. Interestingly, the identified variant has shown partial autosomal dominant inheritance. The MD simulation analysis revealed this variant to result in the protein function impairment. Effective treatment with salbutamol was obtained in an 18-months follow-up. Remarkably, therapeutic doses of salbutamol in severe COVID-19 patients prevented recurrence of paralysis or muscle weaknesses that occurred with a mild cold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e We found a novel variant in the \u003cem\u003eDOK7\u003c/em\u003e gene, with the newly identified partial autosomal dominant inheritance. The findings were used to administer suitable drugs to the patients with maximum efficiency. Thus, ES creates a unique opportunity to promote personalized medicine.\u003c/p\u003e","manuscriptTitle":"Clinical and molecular analysis of a novel variant responsive to salbutamol monotherapy during COVID-19 outbreak related to congenital and late-onset of myasthenic syndrome in large kindred","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-09 18:01:01","doi":"10.21203/rs.3.rs-3924937/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d104925e-1717-4b81-8f2d-b6221486c299","owner":[],"postedDate":"May 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-13T17:24:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-09 18:01:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3924937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3924937","identity":"rs-3924937","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}