Evaluation of sputum cultures in children with spinal Muscular atrophy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder. Despite increased survival due to novel therapies, morbidity from respiratory complications still persists. We aim to describe these patients' sputum cultures as an expression of chronic infectious airway disease. METHODS: Retrospective review of medical records of all children with SMA followed at the multidisciplinary respiratory neuromuscular clinic at Schneider Childrens' Medical Center of Israel over a 16-year period. Sputum cultures were obtained during routine visits or pulmonary exacerbations. RESULTS: Sixty-one SMA patients, aged 1 month to 21 years, were included in this cohort. Of these, sputum cultures were collected from 41 patients. Overall, 288 sputum cultures were obtained, and 98 (34%) were negative for bacterial growth. For the first culture taken from each patient, 12 out of 41 (29%) were sterile. The most common bacteria were pseudomonas aeruginosa (PSA) (38%) and staphylococcus aureus (19.6%). PSA was found in SMA type I patients more frequently than in type II patients (15/26 = 58% vs 4/13 = 31%, p < 0.001). PSA infection was positively associated with noninvasive ventilation, recurrent atelectasis, recurrent pneumonias, swallowing difficulties, but no significant association was found with cough assist machine usage. The incidence of positive cultures did not differ between those treated with Onasemnogene abeparvovec or Nusinersen compared to those without treatment, but the age of first PSA isolation was slightly older with Nusinersen treatment (p = 0.01). CONCLUSIONS: Airway bacterial colonization is common in SMA type I patients and is not decreased by Onasemnogene abeparvovec or Nusinersen treatment.

Adeno-associated virus serotype 9 antibody titers in patients with SMA pre-screened for treatment with onasemnogene abeparvovec -routine care evidence.

Spinal muscular atrophy (SMA) is characterized by progressive weakness of skeletal and respiratory muscles. This study aimed to evaluate the prevalence of pre-existing anti adeno-associated virus serotype 9 antibody (AAV9-Ab) titers among infantile-onset SMA diagnosed infants pre-screened for treatment with AAV9-based onasemnogene abeparvovec, and to explore whether clinical and/or demographic characteristics are correlated with AAV9 Ab test results. This is a retrospective multicenter study of children diagnosed with 5q SMA younger than two years of age. The obtained data included demographic data, SMA type, SMN2 gene copy number, onset date, and results of AAV9-Ab test and of SMA prior treatments. Thirty-four patients were enrolled; six patients had positive results of AAV9-Ab (titer > 1:50) in the initial screening, 15 patients were re-tested for AAV9-Abs, of whom, three patients had seroreverted [1.5-4.5 months] between the two AAV9-Abs tests. One patient had seroconverted (5.5 months after the first AAV9-Abs test). The remaining 11 patients presented matching titer results in the two tests. No demographic/clinical factors were correlated to high AAV9-Abs titers (P > 0.05).We recommend AAV9-Ab re-tests to be performed until the age of 8 months, or, if 1.5 months or more have passed after the initial AAV9-Abs test.

Targeted transcript analysis in muscles from patients with genetically diverse congenital myopathies.

Congenital myopathies are a group of early onset muscle diseases of variable severity often with characteristic muscle biopsy findings and involvement of specific muscle types. The clinical diagnosis of patients typically relies on histopathological findings and is confirmed by genetic analysis. The most commonly mutated genes encode proteins involved in skeletal muscle excitation-contraction coupling, calcium regulation, sarcomeric proteins and thin-thick filament interaction. However, mutations in genes encoding proteins involved in other physiological functions (for example mutations in SELENON and MTM1, which encode for ubiquitously expressed proteins of low tissue specificity) have also been identified. This intriguing observation indicates that the presence of a genetic mutation impacts the expression of other genes whose product is important for skeletal muscle function. The aim of the present investigation was to verify if there are common changes in transcript and microRNA expression in muscles from patients with genetically heterogeneous congenital myopathies, focusing on genes encoding proteins involved in excitation-contraction coupling and calcium homeostasis, sarcomeric proteins, transcription factors and epigenetic enzymes. Our results identify RYR1, ATPB2B and miRNA-22 as common transcripts whose expression is decreased in muscles from congenital myopathy patients. The resulting protein deficiency may contribute to the muscle weakness observed in these patients. This study also provides information regarding potential biomarkers for monitoring disease progression and response to pharmacological treatments in patients with congenital myopathies.

External lumbar drainage in progressive pediatric idiopathic intracranial hypertension.

OBJECTIVE: Pediatric idiopathic intracranial hypertension (IIH) is characterized by increased intracranial pressure despite normal cerebrospinal fluid and neuroimaging findings. Initial management is typically medical; however, nearly 10% of children will eventually require surgery for persistent headache and/or vision loss. External lumbar drainage, which is a considerably safer treatment option, has not been adequately analyzed in children with medically refractory IIH. METHODS: The authors conducted a single-institution retrospective analysis of children with medically refractory IIH who had undergone external lumbar drain (ELD) placement because of worsening papilledema, reflected as increased retinal nerve fiber layer (RNFL) thickness on optical coherence tomography (OCT) testing. The main outcome measures were effects of external lumbar drainage on papilledema resolution, symptoms, and vision. RESULTS: The authors analyzed the medical records of 13 children with IIH (11 girls, mean age 15.0 ± 2.3 years) whose mean CSF opening pressure was 45.5 ± 6.8 cm H2O. In all children, the average global RNFL thickness in both eyes significantly increased at ELD placement (right eye 371.8 ± 150.2 µm, left eye 400.3 ± 96.9 µm) compared with presentation thickness (right eye 301.6 ± 110.40 µm, left eye 350.2 ± 107.7 µm) despite acetazolamide medical therapy (20-30 mg/kg/day), leading to ELD placement after 9.5 ± 6.9 days (range 3-29 days). After ELD insertion, there was headache resolution, gradual and continuous improvement in optic disc thickness, and preservation of good vision. CONCLUSIONS: ELD placement in children with medically refractory IIH who demonstrated worsening papilledema with increased RNFL thickening on OCT testing typically results in symptom relief and disc edema resolution with good visual outcome, often preventing the need for additional definitive surgeries that carry greater failure and morbidity risks.

Liposomal Bupivacaine (Bupigel) Demonstrates Minimal Local Nerve Toxicity in a Rabbit Functional Model.

We previously reported the development of a novel formulation of an ultra-long-acting local anesthetic based on bupivacaine encapsulated in large multivesicular liposomes (Bupisomes) embedded in hydrogel. This formulation (Bupigel) prolonged bupivacaine release from the formulation in dissolution-like studies in vitro and analgesia in vivo in mouse, rat, and pig models. In this study we assessed Bupigel neurotoxicity on rabbit sciatic nerve using histopathology and electrophysiologic testing. Sciatic nerves of both hind limbs were injected dropwise with different formulations. Nerve conduction studies and needle electromyography two weeks after perineural administration showed signs of neural damage after injection of free lidocaine and bupivacaine, while there was no sign of neural damage after injection with saline, demonstrating the validity of the method. This test also did not show evidence of motor or sensory nerve damage after injection with liposomal bupivacaine at a dose 10-times higher than free bupivacaine. Histologically, signs of neural damage could be observed with lidocaine. Nerves injected with Bupigel showed mild signs of inflammation and small residues of hydrogel in granulomas, indicating a long residence time of the hydrogel at the site of injection, but no histopathological signs of nerve damage. This demonstrated that early signs of neural damage were detected electrophysiologically, showing the usefulness and sensitivity of electrodiagnostic testing in detection of neural damage from new formulations.

Impact of a national population-based carrier-screening program on spinal muscular atrophy births.

Spinal muscular atrophy (SMA) is a genetic neurodegenerative disease. Population carrier screening for SMA was introduced in Israel in 2008 through health-care services' insurance plans and expanded to the entire Israeli population in 2013 by a national health program. The aim of the study was to evaluate the impact of carrier screening on reducing the rate of birth of infants with SMA. All cases of prenatal and postnatal diagnosis of SMA in 2008-2017 were identified from databases of relevant government organizations, genetic laboratories in medical centers, and health care systems in Israel. Since 2013, screening was performed in 309,352 individuals, of whom 5741 were found to be carriers (carrier rate 1:54). Given an average of 180,000 live births annually, the predicted rate of SMA diagnosis was 15 cases per year. Prior to 2013, the average rate of prenatally diagnosed SMA was 4.66 cases per year, compared with 7.75 cases per year following population-wide provision of screening. The annual rate of postnatally diagnosed cases remained steady since 2008, with an average of 7- 7.25 cases per year. Screening has been effective in increasing prenatal detection of SMA but has had no effect on the rate of confirmed postnatal diagnoses. We speculate that screening rates may be affected by social, cultural, and religious factors.

Prognostic Parameters of Acute Transverse Myelitis in Children.

Acute transverse myelitis is a rare and disabling disorder. Data on the imaging features in children are sparse. The aim of this study was to describe the clinical and magnetic resonance imaging findings characteristic of pediatric idiopathic acute transverse myelitis and to identify those with prognostic value. The database of a tertiary pediatric medical center was retrospectively reviewed for patients aged less than 18 years who were diagnosed in 2002-2017 with acute transverse myelitis that was not associated with recurrence of a demyelinating autoimmune event. Data were collected on clinical, laboratory, and imaging findings and outcome. A total of 23 children (11 male, 12 female) met the study criteria. Mean age at disease onset was 10 years, and mean duration of follow-up was 6 years 10 months. Spinal cord and brain magnetic resonance imaging scans were performed on admission or shortly thereafter. The most common finding was cross-sectional involvement, in 16 patients (70%). The mean number of involved spinal segments was 8. The most frequently involved region was the thoracic spine, in 17 patients (74%). Clinical factors predicting good prognosis were cerebrospinal fluid pleocytosis, absence of tetraparesis, and prolonged time to nadir. In conclusion, most children with acute transverse myelitis appear to have a good outcome. Prompt diagnosis and treatment are important. Further research is needed in a larger sample to evaluate the predictive value of imaging features.

Small-fiber neuropathy associated with autoinflammatory syndromes in children and adolescents.

INTRODUCTION: Small-fiber neuropathy is rare in children. It has been associated with several autoimmune disorders, but there are no reports of an autoinflammatory etiology. METHODS: The data of four children/adolescents presenting with erythromelalgia and neuropathic pain from 2014 to 2019 were collected retrospectively from the electronic database of a pediatric medical center. RESULTS: Results of clinical and/or electrophysiological evaluation excluded large nerve fiber involvement. Skin biopsy results confirmed small-fiber neuropathy. According to genetic analysis, two patients were heterozygous and one was homozygous for mutations in the familial Mediterranean fever (MEFV) gene. Behcet disease was diagnosed in the fourth patient. Treatment with anti-interleukin-1 agents, intravenous immunoglobulin, and glucocorticoids was beneficial. DISCUSSION: The diagnosis of small-fiber neuropathy should be considered in children/adolescents presenting with erythromelalgia. A thorough investigation is required to reveal the underlying disorder. Clinicians should be alert to the peripheral neurological manifestations of autoinflammatory syndromes because effective treatments are available.

Pax7, Pax3 and Mamstr genes are involved in skeletal muscle impaired regeneration of dy2J/dy2J mouse model of Lama2-CMD.

Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10-9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin-angiotensin, epithelial-mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

NMO-IgG and AQP4 Peptide Can Induce Aggravation of EAMG and Immune-Mediated Muscle Weakness.

Neuromyelitis optica (NMO) and myasthenia gravis (MG) are autoimmune diseases mediated by autoantibodies against either aquaporin 4 (AQP4) or acetylcholine receptor (AChR), respectively. Recently, we and others have reported an increased prevalence of NMO in patients with MG. To verify whether coexisting autoimmune disease may exacerbate experimental autoimmune MG, we tested whether active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect the severity of EAMG. Injection of either AQP4 peptide or NMO-Ig to EAMG or to naive mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness (but not paralysis), decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, our study shows increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response. This can explain exacerbation or increased susceptibility of patients with one autoimmune disease to develop additional autoimmune syndrome.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.

Variable myopathic presentation in a single family with novel skeletal RYR1 mutation.

We describe an autosomal recessive heterogeneous congenital myopathy in a large consanguineous family. The disease is characterized by variable severity, progressive course in 3 of 4 patients, myopathic face without ophthalmoplegia and proximal muscle weakness. Absence of cores was noted in all patients. Genome wide linkage analysis revealed a single locus on chromosome 19q13 with Zmax = 3.86 at θ = 0.0 and homozygosity of the polymorphic markers at this locus in patients. Direct sequencing of the main candidate gene within the candidate region, RYR1, was performed. A novel homozygous A to G nucleotide substitution (p.Y3016C) within exon 60 of the RYR1 gene was found in patients. ARMS PCR was used to screen for the mutation in all available family members and in an additional 150 healthy individuals. This procedure confirmed sequence analysis and did not reveal the A to G mutation (p.Y3016C) in 300 chromosomes from healthy individuals. Functional analysis on EBV immortalized cell lines showed no effect of the mutation on RyR1 pharmacological activation or the content of intracellular Ca(2+) stores. Western blot analysis demonstrated a significant reduction of the RyR1 protein in the patient's muscle concomitant with a reduction of the DHPRα1.1 protein. This novel mutation resulting in RyR1 protein decrease causes heterogeneous clinical presentation, including slow progression course and absence of centrally localized cores on muscle biopsy. We suggest that RYR1 related myopathy should be considered in a wide variety of clinical and pathological presentation in childhood myopathies.

Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9ß1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9ß1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and ßNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9ß1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of ßNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and ßNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9ß1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9ß1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets.

Improved muscle strength and mobility in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate.

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Electromyography (EMG) accuracy compared to muscle biopsy in childhood.

Reports show wide variability of electromyography (EMG) in detecting pediatric neuromuscular disorders. The study's aim was to determine EMG/nerve conduction study accuracy compared to muscle biopsy and final clinical diagnosis, and sensitivity for myopathic motor unit potential detection in childhood. Of 550 EMG/nerve conduction studies performed by the same examiner from a pediatric neuromuscular service, 27 children (ages 6 days to 16 years [10 boys; M:F, 1:1.7]) with muscle biopsies and final clinical diagnoses were compared retrospectively. Final clinical diagnoses were congenital myopathies (5 of 27,18%), nonspecific myopathies (biopsy myopathic, final diagnosis uncertain; 6 of 27, 22%), congenital myasthenic syndrome (3 of 27, 11%), juvenile myasthenia gravis (1 of 27, 4%), arthrogryposis multiplex congenita (2 of 27, 7%), hereditary motor and sensory neuropathy (1 of 27, 4%), bilateral peroneal neuropathies (1 of 27, 4%), and normal (8 of 27, 30%). There were no muscular dystrophy or spinal muscular atrophy patients. EMG/nerve conduction studies had a 74% agreement with final clinical diagnoses and 100% agreement in neurogenic, neuromuscular junction, and normal categories. Muscle biopsies concurred with final diagnoses in 87%, and 100% in myopathic and normal categories. In congenital myasthenic syndrome, muscle biopsies showed mild variation in fiber size in 2 of 3 children and were normal in 1 of 3. EMG sensitivity for detecting myopathic motor unit potentials in myopathies was 4 of 11 (36%), greater over 2 years of age (3 of 4, 75%), compared to infants less than 2 years (1 of 7, 14%), not statistically significant (P = .0879). EMGs false-negative for myopathy in infants < 2 years of age were frequently neurogenic (3 of 6, 50%). In congenital myopathies EMG detected myopathic motor unit potentials in 40%, with false-negative results neurogenic (20%) or normal (40%). Because our study has no additional tests for active myopathies, for example Duchenne muscular dystrophy genetic testing, our sensitivity for myopathies is lower than if we used a more global view. In conclusion, EMG detection rate of myopathic motor unit potentials at a young age was low, improving in children over 2 years of age. In neurogenic and neuromuscular junction disorders, the EMG has a very high detection rate. In children with mild to moderate neurogenic EMG findings and normal nerve conduction, a myopathy should always be considered.

Hereditary hypotonia, muscle weakness, failure to thrive, and cognitive delay in a large moslem kindred.

Five infants of a Moslem-Arab extended family were evaluated for common and characteristic clinical findings of failure to thrive, extreme muscle weakness, severe motor delay, and moderate to severe cognitive and verbal delay. All children were below the third percentile in weight and height, and three of them had head circumference below the third percentile. Neurologic examination revealed severe hypotonia, muscle weakness, and absent deep tendon reflexes. Two children died at 2 years of age, and none of the children acquired full head control and the motor milestones of rolling and sitting. Laboratory evaluation including muscle biopsies, genetic studies, and metabolic evaluation was nondiagnostic.

Asymptomatic or minimally symptomatic hyperCKemia: histopathologic correlates.

BACKGROUND: Persistent creatine kinase elevation is occasionally encountered in subjects without any clinical manifestation of a neuromuscular disorder or any condition known to be associated with increased serum CK levels. It is still unresolved whether extensive investigations and specifically a muscle biopsy should be performed in clinically normal individuals with elevated CK levels. OBJECTIVE: To study the muscle pathology of patients with asymptomatic or minimally symptomatic hyperCKemia. METHODS: The clinical and laboratory data of patients with persistent hyperCKemia and normal neurologic examination were reviewed and their muscle biopsies evaluated. RESULTS: The study group included 40 patients aged 7-67 years; the male to female ratio was 3:1. Nineteen patients were completely asymptomatic, 20 had mild non-specific myalgia, and 1 had muscle cramps. Electromyography was performed in 27 patients and showed myopathic changes in 7 (26%). Abnormal muscle biopsy findings (e.g., increased variation in fiber size, increased number of central nuclei, and occasional degenerating fibers) were detected in 22 of the 40 patients (55%). No fat or glycogen accumulation was detected. Immunohistochemistry demonstrated abnormal dystrophin staining in 3 patients (8%), resembling the pathologic changes of Becker muscular dystrophy. No abnormal findings were detected on immunohistochemical staining for merosin, dysferlin, caveolin 3, or alpha and gamma sarcoglycans. The EMG findings did not correlate with the pathologic findings. CONCLUSIONS: Abnormal muscle biopsies were found in 55% of patients with asymptomatic or minimally symptomatic hyperCKemia. Specific diagnosis of muscular dystrophy, however, was possible in only 8% of the patients.

Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration.

Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear envelope and Rb and MyoD fail in EDMD at the point of myoblast exit from the cell cycle, leading to poorly coordinated phosphorylation and acetylation steps. Our data is consistent with mutations of nuclear lamina components leading to destabilization of the transcriptome in differentiated cells.

Predominantly upper limb weakness, enlarged cisterna magna, and borderline intelligence in a child with de novo mutation of the skeletal muscle alpha-actin gene.

We present a 10-year-old boy from nonconsanguineous parents of Libyan (Sephardi) Jewish origin. Mild dysmorphism, hypotonia, and clubfoot deformities were noted at birth. On follow-up, he had borderline intelligence and nonprogressive muscle weakness, predominantly in the upper extremities. Physical examination revealed mild facial weakness, a bell-shaped chest cavity, kyphosis, winging of the scapula, and hypotonia of the shoulder girdle. Muscle biopsy demonstrated prominent variation in fiber size and central nuclei and numerous subsarcolemmal particles on modified Gomori trichrome stains. Electron microscopy depicted areas of disrupted sarcomeres with abnormal aggregates. Brain magnetic resonance imaging showed mild widening of the lateral ventricles and an enlarged cisterna magna. Molecular DNA analysis by polymerase chain reaction (PCR) and direct sequencing revealed a de novo heterozygous missense mutation in the skeletal muscle alpha-actin gene (ACTA1) changing codon 348 from TCG serine to TTG leucine.

A new clinical and molecular form of Unverricht-Lundborg disease localized by homozygosity mapping.

Progressive myoclonus epilepsy (PME) has a number of causes, of which Unverricht-Lundborg disease (ULD) is the most common. ULD has previously been mapped to a locus on chromosome 21 (EPM1). Subsequently, mutations in the cystatin B gene have been found in most cases. In the present work we identified an inbred Arab family with a clinical pattern compatible with ULD, but mutations in the cystatin B gene were absent. We sought to characterize the clinical and molecular features of the disorder. The family was studied by multiple field trips to their town to clarify details of the complex consanguineous relationships and to personally examine the family. DNA was collected for subsequent molecular analyses from 21 individuals. A genome-wide screen was performed using 811 microsatellite markers. Homozygosity mapping was used to identify loci of interest. There were eight affected individuals. Clinical onset was at 7.3 +/- 1.5 years with myoclonic or tonic-clonic seizures. All had myoclonus that progressed in severity over time and seven had tonic-clonic seizures. Ataxia, in addition to myoclonus, occurred in all. Detailed cognitive assessment was not possible, but there was no significant progressive dementia. There was intrafamily variation in severity; three required wheelchairs in adult life; the others could walk unaided. MRI, muscle and skin biopsies on one individual were unremarkable. We mapped the family to a 15-megabase region at the pericentromeric region of chromosome 12 with a maximum lod score of 6.32. Although the phenotype of individual subjects was typical of ULD, the mean age of onset (7.3 years versus 11 years for ULD) was younger. The locus on chromosome 12 does not contain genes for any other form of PME, nor does it have genes known to be related to cystatin B. This represents a new form of PME and we have designated the locus as EPM1B.