Epithelioid neoplasm of the spinal cord in a child with spinal muscular atrophy treated with onasemnogene abeparvovec.

Spinal muscular atrophy is an autosomal recessive disease resulting in motor neuron degeneration and progressive life-limiting motor deficits when untreated. Onasemnogene abeparvovec is an adeno-associated virus serotype 9-based gene therapy that improves survival, motor function, and motor milestone achievement in symptomatic and presymptomatic patients. Although the adeno-associated virus genome is maintained as an episome, theoretical risk of tumorigenicity persists should genomic insertion occur. We present the case of a 16-month-old male with spinal muscular atrophy who was diagnosed with an epithelioid neoplasm of the spinal cord approximately 14 months after receiving onasemnogene abeparvovec. In situ hybridization analysis detected an onasemnogene abeparvovec nucleic acid signal broadly distributed in many but not all tumor cells. Integration site analysis on patient formalin-fixed, paraffin-embedded tumor samples failed to detect high-confidence integration sites of onasemnogene abeparvovec. The finding was considered inconclusive because of limited remaining tissue/DNA input. The improved life expectancy resulting from innovative spinal muscular atrophy therapies, including onasemnogene abeparvovec, has created an opportunity to analyze the long-term adverse events and durability of these therapies as well as identify potential disease associations that were previously unrecognized because of the premature death of these patients.

GARS-related disease in infantile spinal muscular atrophy: Implications for diagnosis and treatment.

The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.

Clinical and molecular spectrum of thymidine kinase 2-related mtDNA maintenance defect.

Mitochondrial DNA maintenance (mtDNA) defects have a wide range of causes, each with a set of phenotypes that overlap with many other neurological or muscular diseases. Clinicians face the challenge of narrowing down a long list of differential diagnosis when encountered with non-specific neuromuscular symptoms. Biallelic pathogenic variants in the Thymidine Kinase 2 (TK2) gene cause a myopathic form of mitochondrial DNA maintenance defect. Since the first description in 2001, there have been 71 patients reported with 42 unique pathogenic variants. Here we are reporting 11 new cases with 5 novel pathogenic variants. We describe and analyze a total of 82 cases with 47 unique TK2 pathogenic variants in effort to formulate a comprehensive molecular and clinical spectrum of TK2-related mtDNA maintenance disorders.

A comprehensive institutional overview of intrathecal nusinersen injections for spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal-recessive neuromuscular disorder resulting in progressive muscle weakness. In December 2016, the U.S. Food and Drug Administration approved the first treatment for SMA, a drug named nusinersen (Spinraza) that is administered intrathecally. However many children with SMA have neuromuscular scoliosis or spinal instrumentation resulting in challenging intrathecal access. Therefore alternative routes must be considered in these complex patients. OBJECTIVE: To investigate routes of drug access, we reviewed our institutional experience of administering intrathecal nusinersen in all children with spinal muscular atrophy regardless of spinal anatomy or instrumentation. MATERIALS AND METHODS: We reviewed children with SMA who were referred for intrathecal nusinersen injections from March to December 2017 at our institution. In select children with spinal hardware, spinal imaging was requested to facilitate pre-procedure planning. Standard equipment for intrathecal injections was utilized. All children were followed up by their referring neurologist. RESULTS: A total of 104 intrathecal nusinersen injections were performed in 26 children with 100% technical success. Sixty procedures were performed without pre-procedural imaging and via standard interspinous technique. The remaining 44 procedures were performed in 11 complex (i.e. neuromuscular scoliosis or spinal instrumentation) patients requiring pre-procedural imaging for planning purposes. Nineteen of the 44 complex procedures were performed via standard interspinous technique from L2 to S1. Twenty-two of the 44 complex procedures were performed using a neural-foraminal approach from L3 to L5. Three of the 44 complex procedures were performed via cervical puncture technique. There were no immediate or long-term complications but there was one child with short-term complications of meningismus and back pain at the injection site. CONCLUSION: Although we achieved 100% technical success in intrathecal nusinersen administration, our practices evolved during the course of this study. As a result of our early experience we developed an algorithm to assist in promoting safe and effective nusinersen administration in children with spinal muscular atrophy regardless of SMA type, abnormal spinal anatomy and complex spinal instrumentation.

Identification of novel candidate disease genes from de novo exonic copy number variants.

BACKGROUND: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. METHODS: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. RESULTS: In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation of disease association for two recently proposed disease genes (MEIS2 and PTCHD1). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. CONCLUSIONS: Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.

Outcomes of thoracoscopic thymectomy in patients with juvenile myasthenia gravis.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disorder of the postsynaptic neuromuscular junction resulting in fatigability of voluntary muscles. There has been increasing evidence supporting thymectomy for MG in adults, and evidence for the role of surgery in pediatric age groups is increasing. The purpose of this study is to describe the outcomes of our patients with juvenile MG undergoing thoracoscopic thymectomy. MATERIAL AND METHODS: All patients with juvenile MG who underwent thoracoscopic thymectomy at Phoenix Children's Hospital between 1999 and 2014 were included. Patients were diagnosed by their treating neurologist. An Osserman and Genkins criterion was used to classify the severity of the disease and DeFilippi classification was used to assess remission. RESULTS: Twelve patients underwent thoracoscopic thymectomy for juvenile MG during the time frame studied. Nine (75%) patients had an Osserman stage of IIB, with only two patients with ocular disease. There were no conversions to an open procedure. Seven (59%) patients had normal thymic histology, 4 (33%) had evidence of follicular hyperplasia and one (8%) had involutional changes. The median length of hospital stay was 2days (range 1-5days). There was no 30-day postoperative morbidity, reoperations or mortality. The median length of follow-up was 31months (range, 4-91months) and at the time of their last follow-up; all 12 (100%) patients had a DeFilippi Classification of 3 or better. CONCLUSION: Surgery for MG in children is indicated for antibody-receptor-positive patients with moderate to severe disease. Thoracoscopic thymectomy is a safe and acceptable treatment for juvenile MG with good disease control. The low morbidity and shorter hospital duration make it an excellent option for consideration.

Progressive Movement Disorder in Brothers Carrying a GNAO1 Mutation Responsive to Deep Brain Stimulation.

GNAO1, located on chromosome 16q12.2, encodes for 1 of the heterotrimeric guanine binding proteins subunits (G proteins), specifically Gαo, which has been implicated as having an important role in brain function. GNAO1 mutations have been shown to impart oncogene properties as well as cause epileptic encephalopathy. The authors report 2 cases of brothers with a severe movement disorder and hypotonia without epilepsy who have been confirmed by whole exome sequencing to have a novel mutation in GNAO1. Their movement disorder improved significantly with deep brain stimulation.

Novel pathogenic variants and genes for myopathies identified by whole exome sequencing.

Neuromuscular diseases (NMD) account for a significant proportion of infant and childhood mortality and devastating chronic disease. Determining the specific diagnosis of NMD is challenging due to thousands of unique or rare genetic variants that result in overlapping phenotypes. We present four unique childhood myopathy cases characterized by relatively mild muscle weakness, slowly progressing course, mildly elevated creatine phosphokinase (CPK), and contractures. We also present two additional cases characterized by severe prenatal/neonatal myopathy. Prior extensive genetic testing and histology of these cases did not reveal the genetic etiology of disease. Here, we applied whole exome sequencing (WES) and bioinformatics to identify likely causal pathogenic variants in each pedigree. In two cases, we identified novel pathogenic variants in COL6A3. In a third case, we identified novel likely pathogenic variants in COL6A6 and COL6A3. We identified a novel splice variant in EMD in a fourth case. Finally, we classify two cases as calcium channelopathies with identification of novel pathogenic variants in RYR1 and CACNA1S. These are the first cases of myopathies reported to be caused by variants in COL6A6 and CACNA1S. Our results demonstrate the utility and genetic diagnostic value of WES in the broad class of NMD phenotypes.

A De Novo Mutation in TEAD1 Causes Non-X-Linked Aicardi Syndrome.

PURPOSE: Aicardi syndrome (AIC) is a congenital neurodevelopmental disorder characterized by infantile spasms, agenesis of the corpus callosum, and chorioretinal lacunae. Variation in phenotype and disease severity is well documented, but chorioretinal lacunae represent the most constant pathological feature. Aicardi syndrome is believed to be an X-linked-dominant disorder occurring almost exclusively in females, although 46, XY males with AIC have been described. The purpose of this study is to identify genetic factors and pathways involved in AIC. METHODS: We performed exome/genome sequencing of 10 children diagnosed with AIC and their parents and performed RNA sequencing on blood samples from nine cases, their parents, and unrelated controls. RESULTS: We identified a de novo mutation in autosomal gene TEAD1, expressed in the retina and brain, in a patient with AIC. Mutations in TEAD1 have previously been associated with Sveinsson's chorioretinal atrophy, characterized by chorioretinal degeneration. This demonstrates that TEAD1 mutations can lead to different chorioretinal complications. In addition, we found that altered expression of genes associated with synaptic plasticity, neuronal development, retinal development, and cell cycle control/apoptosis is an important underlying potential pathogenic mechanism shared among cases. Last, we found a case with skewed X inactivation, supporting the idea that nonrandom X inactivation might be important in AIC. CONCLUSIONS: We expand the phenotype of TEAD1 mutations, demonstrate its importance in chorioretinal complications, and propose the first putative pathogenic mechanisms underlying AIC. Our data suggest that AIC is a genetically heterogeneous disease and is not restricted to the X chromosome, and that TEAD1 mutations may be present in male patients.

ADCY5-related dyskinesia: Broader spectrum and genotype-phenotype correlations.

OBJECTIVE: To investigate the clinical spectrum and distinguishing features of adenylate cyclase 5 (ADCY5)-related dyskinesia and genotype-phenotype relationship. METHODS: We analyzed ADCY5 in patients with choreiform or dystonic movements by exome or targeted sequencing. Suspected mosaicism was confirmed by allele-specific amplification. We evaluated clinical features in our 50 new and previously reported cases. RESULTS: We identified 3 new families and 12 new sporadic cases with ADCY5 mutations. These mutations cause a mixed hyperkinetic disorder that includes dystonia, chorea, and myoclonus, often with facial involvement. The movements are sometimes painful and show episodic worsening on a fluctuating background. Many patients have axial hypotonia. In 2 unrelated families, a p.A726T mutation in the first cytoplasmic domain (C1) causes a relatively mild disorder of prominent facial and hand dystonia and chorea. Mutations p.R418W or p.R418Q in C1, de novo in 13 individuals and inherited in 1, produce a moderate to severe disorder with axial hypotonia, limb hypertonia, paroxysmal nocturnal or diurnal dyskinesia, chorea, myoclonus, and intermittent facial dyskinesia. Somatic mosaicism is usually associated with a less severe phenotype. In one family, a p.M1029K mutation in the C2 domain causes severe dystonia, hypotonia, and chorea. The progenitor, whose childhood-onset episodic movement disorder almost disappeared in adulthood, was mosaic for the mutation. CONCLUSIONS: ADCY5-related dyskinesia is a childhood-onset disorder with a wide range of hyperkinetic abnormal movements. Genotype-specific correlations and mosaicism play important roles in the phenotypic variability. Recurrent mutations suggest particular functional importance of residues 418 and 726 in disease pathogenesis.

Persistent CNS dysfunction in a boy with CMT1X.

OBJECTIVE: X-linked Charcot Marie Tooth disease (CMT1X) is a hereditary demyelinating neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin 32 (Cx32). Some GJB1 mutations have been reported to cause transient clinical CNS dysfunction. We report a boy with persistent CNS abnormalities possibly caused by CMT1X. METHODS: A five year old boy was evaluated by clinical, electrophysiological, MRI and genetic testing. RESULTS: The patient's early motor milestones were normal to age 5 months. His subsequent course was one of slow improvement punctuated by brief periods of loss of ability to sit between age 5 and 10 months, loss of language between 12 months and 2 years and 1 episode of non-clinically observed resolved left-sided facial weakness. At age 5, he had truncal instability, appendicular ataxia, and dysarthric speech. Cognition was normal. He had mild toe weakness and intrinsic muscle atrophy. MRI evaluation was abnormal. Electrophysiologic testing revealed slowed motor conduction velocities and sensory responses of low amplitude. Genetic workup was normal excepting a novel missense mutation in GJB1, causing a p.54N>H substitution. CONCLUSION: The patient has persistent CNS abnormalities characterized by dysarthria and ataxia. These are similar to transient CNS abnormalities reported in patients with CMT1X. These CNS findings may be the direct result of his novel Cx32 mutation.

Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities.

Chromosome region 1q21.1 contains extensive and complex low-copy repeats, and copy number variants (CNVs) in this region have recently been reported in association with congenital heart defects, developmental delay, schizophrenia and related psychoses. We describe 21 probands with the 1q21.1 microdeletion and 15 probands with the 1q21.1 microduplication. These CNVs were inherited in most of the cases in which parental studies were available. Consistent and statistically significant features of microcephaly and macrocephaly were found in individuals with microdeletion and microduplication, respectively. Notably, a paralog of the HYDIN gene located on 16q22.2 and implicated in autosomal recessive hydrocephalus was inserted into the 1q21.1 region during the evolution of Homo sapiens; we found this locus to be deleted or duplicated in the individuals we studied, making it a probable candidate for the head size abnormalities observed. We propose that recurrent reciprocal microdeletions and microduplications within 1q21.1 represent previously unknown genomic disorders characterized by abnormal head size along with a spectrum of developmental delay, neuropsychiatric abnormalities, dysmorphic features and congenital anomalies. These phenotypes are subject to incomplete penetrance and variable expressivity.