Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α–ε subunit interface

Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management.     

Severe congenital myasthenic syndrome due to homozygosity of the 1293insG epsilon-acetylcholine receptor subunit mutation

Recently, a congenital myasthenic syndrome (CMS) with end-plate acetylcholine receptor (AChR) deficiency due to missense mutations in the genes for the AChR subunit was described. The first observed patient with this CMS was heteroallelic for the two epsilon-AChR subunit mutations epsilon1101insT and epsilon1293insG. This patient had only a moderate phenotype with mild muscle weakness and abnormal fatigue. We have now found homozygosity for the epsilon1293insG mutation in a severely affected CMS patient, who lost the ability to walk in midchildhood and shows profound weakness and muscle wasting. Our observation allows a genotype-phenotype correlation illustrating how differences in the AChR mutation haplotype can profoundly influence disease severity.     

Congenital myasthenic syndromes: Genetic defects of the neuromuscular junction

Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or the resynthesis of ACh. Insufficient resynthesis of ACh is now known to be caused by mutations that reduce the expression, catalytic efficiency, or both of choline acetyltransferase. The synaptic CMS are caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent ColQ from associating with catalytic subunits or from insertion into the synaptic basal lamina. With one exception, postsynaptic CMS identified to date are associated with a kinetic abnormality or decreased expression of the acetylcholine receptor (AChR). Numerous mutations have now been identified in subunits of AChR that alter the kinetics or surface expression of the receptor. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow- and fast-channel syndromes, respectively. Most mutations that reduce surface expression of AChR reside in the receptor’s εsubunit and are partially compensated by residual expression of the fetal-type γ subunit. Null mutations in both alleles of other AChR subunits are likely lethal, owing to absence of a substituting subunit.     

A β-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the β subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.     

End-plate acetylcholine receptor deficiency due to nonsense mutations in the ε subunit

We describe a congenital myasthenic syndrome associated with severe end-plate (EP) acetylcholine receptor (AChR) deficiency not associated with an EP myopathy, and with evidence of immature AChR, containing the γ instead of the epsiv; subunit (γ-AChR) at the EPs. Molecular genetic analysis of AChR-subunit genes revealed two mutations in the ε-subunit gene: insertion of a thymine after ε nucleotide 1101 (ε1101insT) that generates a nonsense condon directly, and insertionof a guanine after ε nucleotide 1293 (ε1293insG) that generates three missense codons followed by a nonsense codon. Each mutation predits truncation of the ε subunit at the level of the long cytoplasmic loop, between the third (M3) and fourth (M4) membrane spanning domains. The propositus' asymptomatic son carries ε1293G, indicating that the two mutations are heteroallelic. Expression of AChR harboring either mutation in human embryonic kidney (HEK) fibroblasts was markedly reduced. Single-channel activit recorded from HEK cells cells expressin ε 1101insT-AChR was infrequent but resembled activty of wild-tpe AChR channels in amplitude and open duration. No channel activity could be recorded from HEK cells expressing ε 1293insG-AChR. Expression of γAChR at the EPs may serve as the means of phenotypic rescue from potentially fatal nonsense mutations in the ε-subunit gene.     

Congenital myasthenic syndromes: progress over the past decade

Congenital myasthenic syndromes (CMS) stem from defects in presynaptic, synaptic basal lamina, and postsynaptic proteins. The presynaptic CMS are associated with defects that curtail the evoked release of acetylcholine (ACh) quanta or ACh resynthesis. Defects in ACh resynthesis have now been traced to mutations in choline acetyltransferase. A basal lamina CMS is caused by mutations in the collagenic tail subunit (ColQ) of the endplate species of acetylcholinesterase that prevent the tail subunit from associating with catalytic subunits or from becoming inserted into the synaptic basal lamina. Most postsynaptic CMS are caused by mutations in subunits of the acetylcholine receptor (AChR) that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh and result in slow- and fast-channel syndromes, respectively. Most low-expressor mutations reside in the AChR epsilon subunit and are partially compensated by residual expression of the fetal type gamma subunit. In a subset of CMS patients, endplate AChR deficiency is caused by mutations in rapsyn, a molecule that plays a critical role in concentrating AChR in the postsynaptic membrane.     

Beneficial effect of albuterol in congenital myasthenic syndrome with epsilon-subunit mutations

Mutations in the epsilon subunit of the acetylcholine receptor (AChR) are a common cause of congenital myasthenic syndrome (CMS). Patients are usually treated with acetylcholinesterase inhibitors and 3,4-diaminopyridine with modest clinical benefit. We report 2 patients with CMS due to mutations in the AChR epsilon subunit. The first patient carries two heterozygous frameshift mutations, ε127ins5 and ε1293insG. The second patient is homozygous for the εC142Y mutation that curtails AChR expression to 22% of wild-type in HEK cells. Treatment with pyridostigmine and 3,4-diaminopyridine had a limited beneficial effect in the first patient, and the second patient became wheelchair-bound during therapy. The additional use of albuterol produced dramatic improvement in strength and in activities of daily living in both patients. The efficacy and safety of albuterol in patients who harbor identified low-expressor or null mutations in the epsilon or other subunits of AChR merits a well-designed clinical trial.     

Congenital myasthenic syndrome (CMS) in three European kinships due to a novel splice mutation (IVS7 - 2 A/G) in the epsilon acetylcholine receptor (AChR) subunit gene

Mutations in the epsilon-acetylcholine receptor (AChR epsilon) subunit gene cause congenital myasthenic syndromes (CMS) with postsynaptic neural transmission defects. We present 3 male and 2 female patients from three unrelated Croatian, Hungarian, and Russian families with autosomal recessive CMS. All patients manifested with variable degrees of ophthalmoparesis and generalized, fatiguable muscle weakness since birth or early infancy. Electrophysiological studies showed a decremental response in all patients indicating a neuromuscular transmission defect. Pyridostigmine treatment improved the proximal muscle weakness whereas the ophthalmoparesis remained unchanged in all patients. Analysis of the AChR epsilon subunit gene showed homozygosity for a novel splice site mutation of intron 7 epsilon(IVS7-2A/G) in the two Croatian siblings. epsilon-mRNA analysis by RT-PCR and direct sequencing revealed that exon 7 was spliced directly to exon 9 with skipping of exon 8. The Hungarian and Russian patients were heteroallelic carriers of the same mutation epsilon(IVS7-2A/G) and of a frameshifting mutation epsilon 70insG and epsilon 1293insG, respectively. We hypothesize that altered splice products may not be expressed as functional receptors at the cell surface. A haplotype analysis with polymorphic markers revealed a high degree of similarity for the epsilon(IVS7-2A/G) carrying allele in all families and may therefore indicate a common origin of the mutation.     

A common mutation (epsilon1267delG) in congenital myasthenic patients of Gypsy ethnic origin.

OBJECTIVE: Mutation analysis of the acetylcholine receptor (AChR) epsilon subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). BACKGROUND: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR epsilon subunit (epsilon1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR epsilon subunit. METHODS: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for epsilon1267delG by PCR amplification and sequence analysis. RESULTS: The authors report the complete genomic sequence and organization of the gene coding for the epsilon subunit of the human AChR (accession number AF105999). Homozygous epsilon1267delG was identified in 13 CMS patients from 11 independent families. All epsilon1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. CONCLUSIONS: The mutation epsilon1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (epsilon1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease.     

A newly identified chromosomal microdeletion of the rapsyn gene causes a congenital myasthenic syndrome

The objective is mutation analysis of the RAPSN gene in a patient with sporadic congenital myasthenic syndrome (CMS). Mutations in various genes encoding proteins expressed at the neuromuscular junction may cause CMS. Most mutations affect the epsilon subunit gene of the acetylcholine receptor (AChR) leading to endplate AChR deficiency. Recently, mutations in the RAPSN gene have been identified in several CMS patients with AChR deficiency. In most patients, RAPSN N88K was identified, either homozygously or heteroallelic to a second missense mutation. A sporadic CMS patient from Germany was analyzed for RAPSN mutations by RFLP, long-range PCR and sequence analysis. Clinically, the patient presents with an early onset CMS, associated with arthrogryposis multiplex congenita, recurrent episodes of respiratory insufficiency provoked by infections, and a moderate general weakness, responsive to anticholinesterase treatment. The mutation RAPSN N88K was found heterozygously to a large deletion of about 4.5 kb disrupting the RAPSN gene. Interestingly, an Alu-mediated unequal homologous recombination may have caused the deletion. We hypothesize that numerous interspersed Alu elements may predispose the RAPSN locus for genetic rearrangements.     

Phenotypic comparison of two Scottish families with mutations in different genes causing autosomal dominant nocturnal frontal lobe epilepsy

PURPOSE: Mutations in genes coding for the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor receptor (CHRN) are known to cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Here we examined the phenotypes in two families, from the same ethnic and geographic backgrounds, with ADNFLE as a result of mutations in these two different subunits of CHRN. METHODS: All affected family members underwent a detailed clinical evaluation and review of available EEG, neuroimaging, and videotapes of seizures. The molecular study of family D is reported here; family S has a previously reported mutation in the beta 2 subunit of CHRN. RESULTS: A total of 16 individuals with ADNFLE were identified in the two families. In both families, seizure semiology, age at seizure onset, and the natural history of the seizure disorder was similar. Intrafamilial variation in terms of severity of epilepsy syndrome was present in both families. A significant number of individuals from each family had a history of psychological problems. The molecular study of family D revealed a Ser248Phe mutation in the alpha 4 subunit of CHRN. CONCLUSIONS: The epilepsy phenotype is not distinguishable in the two families who have ADNFLE as a result of mutations in genes coding for different CHRN subunits. This is likely to be due to the similar functional consequences of each mutation on the CHRN receptor.     

Congenital myasthenic syndromes

Congenital myasthenic syndromes (CMS) constitute a heterogenous group of inherited disorders in which neuromuscular transmission is compromised by one or more specific mechanisms. Clinical evidence for the diagnosis of a CMS includes a history of increased fatigable weakness since infancy or early childhood, a decremental EMG response, and the absence of acetylcholine receptor (AChR) antibodies. There has been rapid progress in understanding of the molecular basis of CMS. Mutation analysis of the AChR subunits has revealed numerous disease-associated mutations. These mutations alter the response to acetylcholine. It is decreased in the fast-channel syndromes and in primary AChR deficiency; and it is increased in the slow-channel syndrome due to prolonged open-time of the AChR. Acetylcholinesterase deficiency is associated with mutations in the gene encoding the collagenic tail subunit of the enzyme. Mutations in the gene encoding for choline acetyltransferase causes the CMS associated with episodic apnea.     

Electrophysiological and morphological characterization of a case of autosomal recessive congenital myasthenic syndrome with acetylcholine receptor deficiency due to a N88K rapsyn homozygous mutation

Congenital myasthenic syndromes are rare heterogeneous hereditary disorders, which lead to defective neuromuscular transmission resulting in fatigable muscle weakness. Post-synaptic congenital myasthenic syndromes are caused by acetylcholine receptor kinetic abnormalities or by acetylcholine receptor deficiency. Most of the congenital myasthenic syndromes with acetylcholine receptor deficiency are due to mutations in acetylcholine receptor subunit genes. Some have recently been attributed to mutations in the rapsyn gene. Here, we report the case of a 28-year-old French congenital myasthenic syndrome patient who had mild diplopia and fatigability from the age of 5 years. His muscle biopsy revealed a marked reduction in rapsyn and acetylcholine receptor at neuromuscular junctions together with a simplification of the subneural apparatus structure. In this patient, we excluded mutations in the acetylcholine receptor subunit genes and identified the homozygous N88K rapsyn mutation, which has already been shown by cell expression to impair rapsyn and acetylcholine receptor aggregation at the neuromuscular junction. The detection of the N88K mutation at the heterozygous state in five of 300 unrelated control subjects shows that this mutation is not infrequent in the healthy population. Electrophysiological measurements on biopsied intercostal muscle from this patient showed that his rapsyn mutation-induced fatigable weakness is expressed not only in a diminution in acetylcholine receptor membrane density but also in a decline of endplate potentials evoked at low frequency.     

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.     

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes

BACKGROUND: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. METHODS: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. RESULTS: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation. CONCLUSION: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.     

Congenital myasthenic syndromes: recent advances

Congenital myasthenic syndromes (CMS) can arise from presynaptic, synaptic, or postsynaptic defects. Mutations of the acetylcholine receptor (AChR) that increase or decrease the synaptic response to acetylcholine (ACh) are a common cause of the postsynaptic CMS. An increased response occurs in the slow-channel syndromes. Here, dominant mutations in different AChR subunits and in different domains of the subunits prolong the activation episodes of AChR by either delaying channel closure or increasing the affinity of AChR for ACh. A decreased synaptic response to ACh occurs with recessive, loss-of-function mutations. Missense mutations in the low-affinity, fast-channel syndrome and in a disorder associated with mode-switching kinetics of AChR result in brief activation episodes and reduce the probability of channel opening. Mutations causing premature termination of the translational chain or missense mutations preventing the assembly or glycosylation of AChR curtail the expression of AChR. These mutations are concentrated in the epsilon subunit, probably because substitution of the fetal gamma for the adult epsilon subunit can rescue humans from fatal null mutations in epsilon. Recent molecular genetic studies have also elucidated the pathogenesis of the CMS caused by absence of the asymmetric form of acetylcholinesterase from the synaptic basal lamina. Endplate acetylcholinesterase deficiency is now known to be caused by mutations in the collagenic tail subunit of the asymmetric enzyme that prevents the association of the collagenic tail subunit with the catalytic subunit or its insertion into the basal lamina.     

Congenital myasthenic syndromes: phenotypic expression and pathophysiological characterisation

Congenital Myasthenic Syndromes (CMS) are a heterogeneous group of diseases caused by genetic defects affecting neuromuscular transmission. The twenty five past Years saw major advances in identifying different types of CMS due to abnormal presynaptic, synaptic, and postsynaptic proteins. CMS diagnosis requires two steps: 1) positive diagnosis supported by myasthenic signs beginning in neonatal period, efficacy of anticholinesterase medications, positive family history, negative tests for anti-acetylcholine receptor (AChR) antibodies, electromyographic studies (decremental response at low frequency, repetitive CMAP after one single stimulation); 2) pathophysiological characterisation of CMS implying specific studies: light and electron microscopic analysis of endplate (EP) morphology, estimation of the number of AChR per EP, acetylcholinesterase (AChE) expression, molecular genetic analysis. Most CMS are postsynaptic due to mutations in the AChR subunits genes that alter the kinetic properties or decrease the expression of AChR. The kinetic mutations increase or decrease the synaptic response to ACh resulting respectively in Slow Channel Syndrome (characterized by a autosomal dominant transmission, repetitive CMAP, refractoriness to anticholinesterase medication) and fast channel, recessively transmitted. AChR deficiency without kinetic abnormalities is caused by recessive mutations in AChR genes (mostly epsilon subunit) or by primary rapsyn deficiency, a post synaptic protein involved in AChR concentration. Recently, mutations in SCN4A sodium channel have been reported in one patient. AChE deficiency is identified on the following data: recessive transmission, presence of repetitive CMAP, refractoriness to cholinesterase inhibitors, slow pupillary response to light and absent expression of the enzyme at EP. This synaptic CMS is caused by mutations in the collagenic tail subunit (ColQ) that anchors the catalytic subunits in the synaptic basal lamina. The most frequent presynaptic CMS is caused by mutations of choline acetyltransferase. Several CMS are still not characterized. Many EP molecules are potential etiological candidates. In these unidentified cases, other methods of investigations are required: linkage analysis, when sufficient number of informative relatives are available, microelectrophysiological studies performed in intercostal or anconeus muscles. Prognosis of CMS, depending on severity and evolution of symptoms, is difficult to assess, and it cannot not be simply derived from mutation identification. Most patients respond favourably to anticholinesterase medications or to 3,4 DAP which is effective not only in presynaptic but also in postsynaptic CMS. Specific therapies for slow channel CMS are quinidine and fluoxetine that normalize the prolonged opening episodes. Clinical benefits derived from the full characterisation of each case include genetic counselling and specific therapy.     

Evidence for S284L mutation of the CHRNA4 in a white family with autosomal dominant nocturnal frontal lobe epilepsy

PURPOSE: To identify mutations of the neuronal nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) in a group of white patients. METHODS: A group of 47 patients from 21 unrelated families with ADNFLE were screened for mutations in CHRNA4. Clinical features and EEG findings in the patients were consistent with those reported in the literature for other affected families. The entire gene was amplified from genomic DNA by polymerase chain reaction (PCR) followed by multitemperature single-strand conformation polymorphism analysis (MSSCP) and sequencing. RESULTS: A c.851C>T transition in exon 5 of CHRNA4 was identified in three affected individuals from two generations of the same family, but not in the remaining patients or in 100 healthy volunteers. This mutation caused an S284L substitution in the transmembrane domain M2 segment of the alpha4 subunit of the neuronal nicotinic acetylcholine receptor. The same mutation had previously been detected in a single Japanese family with ADNFLE, and in an Australian woman with a sporadic form of NFLE. CONCLUSIONS: This is the first report of an occurrence of c.851C>T transition in a white family with ADNFLE.     

Synaptic congenital myasthenic syndrome in three patients due to a novel missense mutation (T441A) of the COLQ gene

Congenital myasthenic syndromes (CMS) with deficiency of endplate acetylcholinesterase (AChE) are caused by mutations in the synapse specific collagenic tail subunit gene (COLQ) of AChE. We identified a novel missense mutation (T441A) homozygously in three CMS patients from two unrelated German families. The mutation is located in the C-terminal region of the ColQ protein, which initiates assembly of the triple helix, and is essential for insertion of the tail subunit into the basal lamina. Density gradient analysis of AChE extracted from muscle of one of the patients revealed the absence of asymmetric AChE. All patients were characterized by an onset of disease in childhood, exercise-induced proximal weakness, absence of ptosis and ophthalmoparesis, a decremental EMG response, and deterioration in response to anticholinesterase drugs. However, age at onset, disease progression, disease severity, and functional impairment varied considerably among the three patients. As adults, two siblings from one family experience only mild impairment, while the third patient requires a wheelchair for most of the day and assisted ventilation at night.