Biallelic variants in IGHMBP2 are a known cause of spinal muscular atrophy with respiratory distress type I (SMARD1). Here we present a series of patients in whom there was a high clinical suspicion for SMARD1-related disease with one pathogenic missense or truncating variant in IGHMBP2 but no second variant identified on standard genetic testing. Genome sequencing identified variants of uncertain significance in intron 8 of IGHMBP2 for four patients in compound heterozygosity with a pathogenic coding variant: c.1235 +1076G>A/ c.1241_1254dup14; p.Ser419ArgfsX9; c.1235 + 894 C>A/ c.1730T>C; p.Leu577Pro; c.1235 +450 G>A / c.455T>C;p.Leu152Pro and c.1235 + 450 G>A / c.660A>T; p.Lys220Asn. To determine pathogenicity of these deep intronic variants, patient-derived iPSC motor neurons were generated to obtain short-read and long-read RNA sequencing. Subsequent analysis identified novel pseudoexon inclusions at the site of each deep intronic variant, leading to a frameshift and premature truncating codon. While each variant utilizes a unique splice acceptor site, they all make use of the same intronic cryptic donor. Thus, this hotspot of aberrant splice induction is an excellent candidate for therapeutic splice modulation through antisense oligonucleotide (ASO) administration. Work to determine a single efficacious ASO to treat all hotspot variants utilizing the common cryptic donor is ongoing. Additional SMARD1 patients with a single variant in IGHMBP2 are undergoing pseudoexon investigation and will likely identify more novel variants in this region. This series highlights the importance of phenotype-driven genomic re-analysis and full characterization of deep intronic variants, which have important variant-specific therapeutic implications.

PIEZO2 is a large stretch-gated ion channel highly expressed in specific sensory neurons which mediates human mechanosensation. Individuals with biallelic loss of function (LOF) variants in the PIEZO2 gene highlight the impact of the congenital absence of specific mechanotransduction-dependent senses, including proprioception aspects of touch and interception. We report on twelve unrelated individuals (except one sibling pair), ranging in age from 4 years to 42 years old with biallelic PIEZO2 LOF, and report on detailed neurological examination, neurophysiology, and evaluations of pulmonary, gastrointestinal and urinary function. We identified common early neonatal and developmental consequences of PIEZO2 LOF including: reduced fetal movements (n=6), hypotonia (n=12), neonatal respiratory distress (n=9), and feeding difficulty (n=11). The age at achievement of independent ambulation (n=8) was markedly delayed (5-16 years old). Pseudoathetosis was frequently clinically observed. Orthopedic and systemic findings included: congenital hip dysplasia (n=11), arthrogryposis (n=12), early progressive scoliosis (n=12), constipation (n=11), reduced voiding frequency (n=10). The findings in these participants with LOF in PIEZO2 recapitulates what is observed in preclinical models, and highlights the developmental, motor functional, systemic, and orthopedic consequences of absent mechanotransduction and proprioception. Initially slow but improving functional gains over time suggest that these maturational processes may rely on emerging executive and motor planning skills combined with visual compensatory input. This expanded series represents the largest cohort of individuals with PIEZO2 LOF across a wide age range. Recognizing and elucidating this PIEZO2-related phenotype improves our understanding of human sensory perception and allows for development of rehabilitative measures aimed at compensatory recruitment of intact sensory modalities to optimize function.

Loss of function of FDX2, encoding the essential iron-sulfur (Fe-S) cluster biogenesis component ferredoxin 2, has been reported to cause optic atrophy, myopathy, partially reversible leukoencephalopathy, and sensorineural axonal neuropathy. We describe the first patient with FDX2 loss of function presenting with blindness (no light perception) at age 17 months. Our evaluation revealed clinical and muscle imaging evidence of a myopathy. The patient was found to harbor novel FDX2 variants: c.271C>T, p.L91F; c.344A>G, p.H115R. In vitro studies in the patient's fibroblasts revealed defective interactions of the FDX2 variants with the Fe-S assembly components, resulting in dysfunction of the mitochondrial oxidative phosphorylation system. We obtained a Single Patient Expanded Access IND for Idebenone, a Co-enzyme Q10 analog with increased solubility, which the patient started at age 3 years. Titration of Idebenone was limited by neutropenia, subsequently recognized to be independent of Idebenone. Treatment of patient fibroblasts with mitoquinol mesylate (MitoQ), a Co-enzyme Q10 analog targeted to the mitochondrial matrix, increased: ATP-linked and maximal respiration, basal oxygen consumption rate, and spare respiratory capacity. A second Single Patient Expanded Access IND was obtained for MitoQ, which the patient started at age 5 years and has tolerated at a dose of 10mg twice daily. Anecdotally, the patient's stamina has increased, and her walking ability is stable-to-improved. While the patient's roving eye movements have decreased following exposure to Idebenone and MitoQ, formal ophthalmology examinations demonstrate continued no light perception, and optical coherence tomography (OCT) reveals retinal and ganglion cell layer thinning while photoreceptors remain intact centrally. L-carnitine, biotin, alpha-lipoic acid, riboflavin and vitamin E have been added to the regimen of MitoQ, and in vitro testing in the patient's fibroblasts of these supplements is ongoing. Loss of function of FDX2, encoding the essential iron-sulfur (Fe-S) cluster biogenesis component ferredoxin 2, has been reported to cause optic atrophy, myopathy, partially reversible leukoencephalopathy, and sensorineural axonal neuropathy. We describe the first patient with FDX2 loss of function presenting with blindness (no light perception) at age 17 months. Our evaluation revealed clinical and muscle imaging evidence of a myopathy. The patient was found to harbor novel FDX2 variants: c.271C>T, p.L91F; c.344A>G, p.H115R. In vitro studies in the patient's fibroblasts revealed defective interactions of the FDX2 variants with the Fe-S assembly components, resulting in dysfunction of the mitochondrial oxidative phosphorylation system. We obtained a Single Patient Expanded Access IND for Idebenone, a Co-enzyme Q10 analog with increased solubility, which the patient started at age 3 years. Titration of Idebenone was limited by neutropenia, subsequently recognized to be independent of Idebenone. Treatment of patient fibroblasts with mitoquinol mesylate (MitoQ), a Co-enzyme Q10 analog targeted to the mitochondrial matrix, increased: ATP-linked and maximal respiration, basal oxygen consumption rate, and spare respiratory capacity. A second Single Patient Expanded Access IND was obtained for MitoQ, which the patient started at age 5 years and has tolerated at a dose of 10mg twice daily. Anecdotally, the patient's stamina has increased, and her walking ability is stable-to-improved. While the patient's roving eye movements have decreased following exposure to Idebenone and MitoQ, formal ophthalmology examinations demonstrate continued no light perception, and optical coherence tomography (OCT) reveals retinal and ganglion cell layer thinning while photoreceptors remain intact centrally. L-carnitine, biotin, alpha-lipoic acid, riboflavin and vitamin E have been added to the regimen of MitoQ, and in vitro testing in the patient's fibroblasts of these supplements is ongoing.

Trendelenberg gait; no contractures were noted.A neuromuscular gene panel test reported as a variant of uncertain significance a novel CFL2 variant (c.449G > A, p.Gluy150Glu, inherited homozygously.Muscle biopsy was performed and revealed the presence of scattered nemaline rods, confirming the mutation as pathogenic.Recent examination (age 10 yr) showed progression of weakness throughout.Neck flexion strength was minimal, and she maintains a posture of next extension.In the legs, hip flexion weakness was pronounced (2/5), with relative preservation of knee flexion (5/5) and extension (4 + /5) but significant ankle dorsiflexion weakness (4-/5).Significant contractures were noted at elbows and knees, and markedly at the ankles.Ultrasound of affected muscles showed a uniform ground-glass appearance.Immunofluorescent analysis showed abnormal CFL-2 distribution, with accumulation within scattered fibers associated with disruption of sarcomeric organization as judged by alpha-actinin staining.CFL-2 expression by western blot results showed a level of 5.6% of normal of a 17 kDa band, but the presence of a 50 kDa band not seen in control muscle; peptide sequencing of this band by mass spectrometry is underway.These results confirm the importance of the C-terminal region to actin organization and suggest the potential of altered CFL-2 self-assembly, providing hypotheses for future studies of CFL-2 structure and function.

Skeletal muscle ultrasound (MUS) is a non-invasive ancillary tool for evaluation of neuromuscular conditions that can enhance the physical exam and complement electrodiagnostic studies. While MUS is more extensively studied in primary myopathies, it can also enhance the diagnostic evaluation of patients with neuropathies. Neurogenic disease typically results in heterogenous "streaky" echogenicity in skeletal muscle while myopathic disease typically appears as homogenously increased echogenicity in a granular pattern. In addition, the superiority of MUS in detecting fasciculations compared to EMG is reported in amyotrophic lateral sclerosis, which is likely due to the higher volume of sampled muscle with MUS. Here, we describe MUS findings and EMG/NCS correlates in a cohort of 17 individuals with genetically confirmed childhood onset neuropathies and neuronopathies evaluated at the National Institutes of Health. MUS was graded using the modified Heckmatt scale (MHS) and a description of increased echogenicity as "streaky" or "granular" by three independent reviewers. In 17 patients evaluated, there was a length-dependent pattern of increased echogenicity. Tibialis anterior and gastrocnemius were most affected, and deltoid, triceps and biceps were most spared. "Streaky" appearance was seen in most muscles but was more difficult to appreciate in muscles with low echogenicity scores (grade 0 MHS) or in "end-stage" muscle (grade 3 MHS). MUS was more sensitive at detecting fasciculations than EMG in this setting. No clear pattern emerged to differentiate specific genetic etiologies of neuropathy/neuronopathy from one another via MUS. This study demonstrates the utility of muscle ultrasound in detecting neuropathic disease.

Background/Purpose: Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is caused by mutations in the β-sarcoglycan gene (SGCB), resulting in loss of SGCB protein and other components of the dystrophin-associated protein complex (DAPC). LGMD2E/R4 manifests as progressive hip/shoulder muscle weakness. This first-in-human, phase 1/2 trial (NCT03652259) evaluated SRP-9003, a self-complementary rAAVrh74.MHCK7.hSGCB construct restoring SGCB.

Bi-allelic loss-of-function mutations in the collagen XII-encoding gene, COL12A1, are known to cause congenital hypotonia, respiratory insufficiency, joint hyperlaxity and joint contractures. Monoallelic, dominantly acting mutations in this gene cause a similar but milder phenotype and have been reported only in a handful of patients. We report detailed clinical characteristics of 6 patients (age 3-62 years) from 4 unrelated families with novel dominant mutations in COL12A1. All patients developed weakness at birth or during early childhood. Adult patients reported improvement of weakness and had little-to-no limitation of motor activities in young adulthood and mild weakness reemerged during 4th decade of life or later. Three pediatric patients had generalized weakness, while three adult patients had predominant distal upper and lower limb weakness. Joint contractures or hyperlaxity was observed in 5 patients. Three families carried dominant missense mutations affecting glycine residues, one in a laminin G-like domain and two in the Gly-X-Y repeats of the triple helix domain, while one family carried heterozygous in-frame deletion of exon 52. All of the mutations resulted in increased intracellular retention of collagen XII and loss of fibrillar pattern of staining in patients' fibroblasts. Since haploinsufficiency is not known to cause disease, we designed and screened small interfering RNAs (siRNAs) that specifically target the exon 52 mutant allele in patient-derived skin fibroblasts, thus converting the genetic abnormality to a haploinsufficient state. Immunostaining of the patient's fibroblasts treated with targeting siRNA corrected the intracellular retention and restored the fibrillar pattern of collagen XII staining. This study characterizes a distal myopathy phenotype in adults with dominant COL12A1 mutations, further defines the phenotypic spectrum and natural history of COL12A1-related myopathies, and provides proof of concept of a precision medicine treatment approach by proposing and validating allele-specific knockdown using siRNAs specifically designed to target a patient's COL12A1 mutation.

UNC-45B (unc45 myosin chaperone B) encodes a myosin-specific co-chaperone essential for the folding, stability and maintenance of sarcomeric myosins, thus facilitating proper assembly and function of myosins in skeletal and cardiac muscle. However, mutations in UNC-45B have not yet been established as a cause for muscle disease. We report 3 independent patients with a phenotype of childhood-onset progressive proximal and axial muscle weakness and respiratory insufficiency whose muscle biopsy findings include "eccentric" and unstructured cores with accumulation of Z-disc material. Whole exome sequencing identified rare, predicted to be damaging, bi-allic mutations in the C-terminal UCS domain of UNC-45B which is critical for myosin binding. The recurring c. 2261G>A; p.Arg754Gln UNC-45B mutation was identified in homozygosity in two patients of Hispanic and Turkish descent respectively, while the third patient was found to have a rare bi-allic missense mutation c.2332C>T; p.Arg778Trp in compound heterozygosity with a splice site mutation c.2261+5G>C resulting in aberrant splicing. Western blot revealed a severe reduction of UNC-45B protein in patient muscle compared to control, and immunofluorescence localization studies demonstrated abnormal relocalization of the residual UNC-45B protein within the sarcomere as well as focal disruption of the myofibrillar apparatus. Functional analyses of the effect of these UNC-45B mutations on muscle fiber mechanics, through force generation and myosin binding in patient's muscle, in addition to in vivo studies in C.elegans of the effect of these mutations on UNC45b function will provide further insights into this novel disease mechanism. Our series establishes recessive mutations in UNC-45B as a cause of a novel form of progressive myopathy in humans, which we propose to be classified as a secondary myosinopathy, manifesting histologically and ultrastructurally with eccentric and unstructured cores.

Collagen XII-related disorders are comprised of overlapping symptoms involving both connective tissue and muscle. Both homozygous recessive loss-of-function (in 2 siblings) as well as dominant mutations have been recognized in our initial description of this group. While additional dominant cases and families have now been documented, presenting with hyperlaxity, contractures, and milder weakness, no additional cases of the much more severe recessive loss of function phenotype have been reported to define the range of this presentation. Here, we report two additional cases of unrelated patients with recessive loss-of-function mutations in collagen XII (COL12A1). Patient 1 is a female of European descent, evaluated at one year of age, carrying compound heterozygous mutations: c.5794+2A>T and c.5269C>T/p.R1757X. Patient 2 was evaluated at 13 years of age and is a female, of consanguineous Pakistani descent carrying a homozygous truncating mutation: c.8464C>T/p.Arg2822X. Both patients had reduced fetal movements, congenital hypotonia and severe dysphagia with feeding difficulties. Physical findings included a narrow, elongated face, a high-arched palate, micrognathia and mild webbing of the neck. Contractures were present at the elbows, knees, hips and long finger flexors. Both patients had notable equinovalgus foot deformities with a prominent calcaneus and foot hyperlaxity. Both patients had early-onset kyphoscoliosis, with Patient 2 requiring surgical repair. Both had widespread, significant joint hyperlaxity along with proximal and distal weakness precluding independent ambulation. Of note, Patient 2 had significant hypertrophic cardiomyopathy with left ventricular outflow tract obstruction and mitral valve prolapse diagnosed before 6 months of age, which has improved over time. These additional patients confirm the initial observation of recessive loss of function of collagen XII as a considerably more severe phenotype compared to dominant mutations in COL12A1, while highlighting the striking clinical involvement of both connective tissue and muscle and expanding the phenotype to potentially include cardiomyopathy.

Omigapil is an inhibitor of the GAPDH-Siah1-mediated apoptosis pathway and has been shown to improve weight and locomotor activity in the LAMA2-related dystrophy (LAMA2-RD) mouse model (dyw/dyw mouse). Studies of Omigapil in the COL6-related dystrophy (COL6-RD) mouse model (Col6a1−/− mouse) demonstrated decreased apoptosis, in particular of the diaphragm muscle. The NIH was the single site for a phase I PK study of omigapil in patients with LAMA2-RD or COL6-RD. A phase I open-label, sequential group, cohort study of Omigapil for patients 5-16 years with LAMA2-RD or COL6-RD: 1. to establish the PK profile of Omigapil at a range of doses, using novel adaptive algorithms (CRM and SAVOR) 2. to evaluate the safety and tolerability of Omigapil at a range of doses, and 3. to establish the feasibility of conducting disease-relevant clinical assessments. The study met recruitment goals and study endpoints. Twenty patients were randomly assigned to 1 of 3 dosing cohorts, with each patient receiving 4 weeks of vehicle run-in and 12 weeks on study drug. Slightly greater than dose proportional increases in systemic exposure to Omigapil were seen at doses 0.02 - 0.08 mg/kg/day. The dose which achieved patient exposure within the pre-established target of AUC0-24h range was 0.06 mg/kg/day. In general, Omigapil was safe and well tolerated. No consistent changes were seen in the disease-relevant clinical assessments. This was the first clinical trial of a therapeutic compound for LAMA2-RD and COL6-RD, completed with an adaptive trial design which supported dose adjustments. While the results of the disease-relevant clinical assessments did not demonstrate significant changes, their interpretation is limited due to the short duration of the study. Nevertheless, they provide essential information on the feasibility of their use as potential outcome measures and will aid in the design of future clinical trials for LAMA2-RD and COL6-RD patients.