WiTNNess: An international natural history study of infantile-onset TNNT1 myopathy.

OBJECTIVE: We created WiTNNess as a hybrid prospective/cross-sectional observational study to simulate a clinical trial for infantile-onset TNNT1 myopathy. Our aims were to identify populations for future trial enrollment, rehearse outcome assessments, specify endpoints, and refine trial logistics. METHODS: Eligible participants had biallelic pathogenic variants of TNNT1 and infantile-onset proximal weakness without confounding conditions. The primary endpoint was ventilator-free survival. "Thriving" was a secondary endpoint defined as the ability to swallow and grow normally without non-oral feeding support. Endpoints of gross motor function included independent sitting and standing as defined by the Word Health Organization, a novel TNNT1 abbreviated motor score, and video mapping of limb movement. We recorded adverse events, concomitant medications, and indices of organ function to serve as comparators of safety in future trials. RESULTS: Sixteen children were enrolled in the aggregate cohort (6 prospective, 10 cross-sectional; median census age 2.3 years, range 0.5-13.8). Median ventilator-free survival was 20.2 months and probability of death or permanent mechanical ventilation was 100% by age 60 months. All six children (100%) in the prospective arm failed to thrive by age 12 months. Only 2 of 16 (13%) children in the aggregate cohort sat independently and none stood alone. Novel exploratory motor assessments also proved informative. Laboratory and imaging data suggest that primary manifestations of TNNT1 deficiency are restricted to skeletal muscle. INTERPRETATION: WiTNNess allowed us to streamline and economize the collection of historical control data without compromising scientific rigor, and thereby establish a sound operational framework for future clinical trials.

Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy.

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.

Clinical characterization of familial hypercholesterolemia due to an amish founder mutation in Apolipoprotein B.

BACKGROUND: Familial hypercholesterolemia (FH) due to a founder variant in Apolipoprotein B (ApoBR3500Q) is reported in 12% of the Pennsylvania Amish community. By studying a cohort of ApoBR3500Q heterozygotes and homozygotes, we aimed to characterize the biochemical and cardiac imaging features in children and young adults with a common genetic background and similar lifestyle. METHODS: We employed advanced lipid profile testing, carotid intima media thickness (CIMT), pulse wave velocity (PWV), and peripheral artery tonometry (PAT) to assess atherosclerosis in a cohort of Amish ApoBR3500Q heterozygotes (n = 13), homozygotes (n = 3), and their unaffected, age-matched siblings (n = 9). ApoBR3500Q homozygotes were not included in statistical comparisons. RESULTS: LDL cholesterol (LDL-C) was significantly elevated among ApoBR3500Q heterozygotes compared to sibling controls, though several ApoBR3500Q heterozygotes had LDL-C levels in the normal range. LDL particles (LDL-P), small, dense LDL particles, and ApoB were also significantly elevated among subjects with ApoBR3500Q. Despite these differences in serum lipids and particles, CIMT and PWV were not significantly different between ApoBR3500Q heterozygotes and controls in age-adjusted analysis. CONCLUSIONS: We provide a detailed description of the serum lipids, atherosclerotic plaque burden, vascular stiffness, and endothelial function among children and young adults with FH due to heterozygous ApoBR3500Q. Fasting LDL-C was lower than what is seen with other forms of FH, and even normal in several ApoBR3500Q heterozygotes, emphasizing the importance of cascade genetic testing among related individuals for diagnosis. We found increased number of LDL particles among ApoBR3500Q heterozygotes but an absence of detectable atherosclerosis.

Nusinersen by subcutaneous intrathecal catheter for symptomatic spinal muscular atrophy patients with complex spine anatomy.

INTRODUCTION/AIMS: Intrathecal administration of nusinersen is challenging in patients with spinal muscular atrophy (SMA) who have spine deformities or fusions. We prospectively studied the safety and efficacy of nusinersen administration via an indwelling subcutaneous intrathecal catheter (SIC) for SMA patients with advanced disease. METHODS: Seventeen participants commenced nusinersen therapy between 2.7 and 31.5 years of age and received 9 to 12 doses via SIC. Safety was assessed in all participants. A separate efficacy analysis comprised 11 nonambulatory, treatment-naive SMA patients (18.1 ± 6.8 years) with three SMN2 copies and complex spine anatomy. RESULTS: In the safety analysis, 14 treatment-related adverse events (AEs) occurred among 12 (71%) participants; all were related to the SIC and not nusinersen. Device-related AEs interfered with 2.5% of nusinersen doses. Four SICs (24%) required surgical revision due to mechanical malfunction with or without cerebrospinal fluid leak (n = 2), and one (6%) was removed due to Staphylococcus epidermidis meningitis. In the efficacy analysis, mean performance on the nine-hole peg test improved in dominant (15.9%, P = 0.012) and nondominant (19.0%, P = 0.008) hands and grip strength increased by 44.9% (P = 0.031). We observed no significant changes in motor scales, muscle force, pulmonary function, or SMA biomarkers. All participants in the efficacy cohort reported one or more subjective improvement(s) in endurance, purposeful hand use, arm strength, head control, and/or speech. DISCUSSION: For SMA patients with complex spine anatomy, the SIC allows for reliable outpatient administration of nusinersen that results in meaningful improvements in upper limb function, but introduces risks of technical malfunction and iatrogenic infection.

APC7 mediates ubiquitin signaling in constitutive heterochromatin in the developing mammalian brain.

Neurodevelopmental cognitive disorders provide insights into mechanisms of human brain development. Here, we report an intellectual disability syndrome caused by the loss of APC7, a core component of the E3 ubiquitin ligase anaphase promoting complex (APC). In mechanistic studies, we uncover a critical role for APC7 during the recruitment and ubiquitination of APC substrates. In proteomics analyses of the brain from mice harboring the patient-specific APC7 mutation, we identify the chromatin-associated protein Ki-67 as an APC7-dependent substrate of the APC in neurons. Conditional knockout of the APC coactivator protein Cdh1, but not Cdc20, leads to the accumulation of Ki-67 protein in neurons in vivo, suggesting that APC7 is required for the function of Cdh1-APC in the brain. Deregulated neuronal Ki-67 upon APC7 loss localizes predominantly to constitutive heterochromatin. Our findings define an essential function for APC7 and Cdh1-APC in neuronal heterochromatin regulation, with implications for understanding human brain development and disease.

Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder.

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

De novo and bi-allelic variants in AP1G1 cause neurodevelopmental disorder with developmental delay, intellectual disability, and epilepsy.

Adaptor protein (AP) complexes mediate selective intracellular vesicular trafficking and polarized localization of somatodendritic proteins in neurons. Disease-causing alleles of various subunits of AP complexes have been implicated in several heritable human disorders, including intellectual disabilities (IDs). Here, we report two bi-allelic (c.737C>A [p.Pro246His] and c.1105A>G [p.Met369Val]) and eight de novo heterozygous variants (c.44G>A [p.Arg15Gln], c.103C>T [p.Arg35Trp], c.104G>A [p.Arg35Gln], c.229delC [p.Gln77Lys∗11], c.399_400del [p.Glu133Aspfs∗37], c.747G>T [p.Gln249His], c.928-2A>C [p.?], and c.2459C>G [p.Pro820Arg]) in AP1G1, encoding gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), associated with a neurodevelopmental disorder (NDD) characterized by mild to severe ID, epilepsy, and developmental delay in eleven families from different ethnicities. The AP1γ1-mediated adaptor complex is essential for the formation of clathrin-coated intracellular vesicles. In silico analysis and 3D protein modeling simulation predicted alteration of AP1γ1 protein folding for missense variants, which was consistent with the observed altered AP1γ1 levels in heterologous cells. Functional studies of the recessively inherited missense variants revealed no apparent impact on the interaction of AP1γ1 with other subunits of the AP-1 complex but rather showed to affect the endosome recycling pathway. Knocking out ap1g1 in zebrafish leads to severe morphological defect and lethality, which was significantly rescued by injection of wild-type AP1G1 mRNA and not by transcripts encoding the missense variants. Furthermore, microinjection of mRNAs with de novo missense variants in wild-type zebrafish resulted in severe developmental abnormalities and increased lethality. We conclude that de novo and bi-allelic variants in AP1G1 are associated with neurodevelopmental disorder in diverse populations.

A recurring NFS1 pathogenic variant causes a mitochondrial disorder with variable intra-familial patient outcomes.

Iron­sulfur clusters (FeSCs) are vital components of a variety of essential proteins, most prominently within mitochondrial respiratory chain complexes I-III; Fe-S assembly and distribution is performed via multi-step pathways. Variants affecting several proteins in these pathways have been described in genetic disorders, including severe mitochondrial disease. Here we describe a Christian Arab kindred with two infants that died due to mitochondrial disorder involving Fe-S containing respiratory chain complexes and a third sibling who survived the initial crisis. A homozygous missense variant in NFS1: c.215G>A; p.Arg72Gln was detected by whole exome sequencing. The NFS1 gene encodes a cysteine desulfurase, which, in complex with ISD11 and ACP, initiates the first step of Fe-S formation. Arginine at position 72 plays a role in NFS1-ISD11 complex formation; therefore, its substitution with glutamine is expected to affect complex stability and function. Interestingly, this is the only pathogenic variant ever reported in the NFS1 gene, previously described once in an Old Order Mennonite family presenting a similar phenotype with intra-familial variability in patient outcomes. Analysis of datasets from both populations did not show a common haplotype, suggesting this variant is a recurrent de novo variant. Our report of the second case of NFS1-related mitochondrial disease corroborates the pathogenicity of this recurring variant and implicates it as a hot-spot variant. While the genetic resolution allows for prenatal diagnosis for the family, it also raises critical clinical questions regarding follow-up and possible treatment options of severely affected and healthy homozygous individuals with mitochondrial co-factor therapy or cysteine supplementation.

Glutaric acidemia type 1: Treatment and outcome of 168 patients over three decades.

Glutaric acidemia type 1 (GA1) is a disorder of cerebral organic acid metabolism resulting from biallelic mutations of GCDH. Without treatment, GA1 causes striatal degeneration in >80% of affected children before two years of age. We analyzed clinical, biochemical, and developmental outcomes for 168 genotypically diverse GA1 patients managed at a single center over 31 years, here separated into three treatment cohorts: children in Cohort I (n = 60; DOB 2006-2019) were identified by newborn screening (NBS) and treated prospectively using a standardized protocol that included a lysine-free, arginine-enriched metabolic formula, enteral l-carnitine (100 mg/kg•day), and emergency intravenous (IV) infusions of dextrose, saline, and l-carnitine during illnesses; children in Cohort II (n = 57; DOB 1989-2018) were identified by NBS and treated with natural protein restriction (1.0-1.3 g/kg•day) and emergency IV infusions; children in Cohort III (n = 51; DOB 1973-2016) did not receive NBS or special diet. The incidence of striatal degeneration in Cohorts I, II, and III was 7%, 47%, and 90%, respectively (p < .0001). No neurologic injuries occurred after 19 months of age. Among uninjured children followed prospectively from birth (Cohort I), measures of growth, nutritional sufficiency, motor development, and cognitive function were normal. Adherence to metabolic formula and l-carnitine supplementation in Cohort I declined to 12% and 32%, respectively, by age 7 years. Cessation of strict dietary therapy altered plasma amino acid and carnitine concentrations but resulted in no serious adverse outcomes. In conclusion, neonatal diagnosis of GA1 coupled to management with lysine-free, arginine-enriched metabolic formula and emergency IV infusions during the first two years of life is safe and effective, preventing more than 90% of striatal injuries while supporting normal growth and psychomotor development. The need for dietary interventions and emergency IV therapies beyond early childhood is uncertain.

De Novo and Inherited Variants in GBF1 are Associated with Axonal Neuropathy Caused by Golgi Fragmentation.

Distal hereditary motor neuropathies (HMNs) and axonal Charcot-Marie-Tooth neuropathy (CMT2) are clinically and genetically heterogeneous diseases characterized primarily by motor neuron degeneration and distal weakness. The genetic cause for about half of the individuals affected by HMN/CMT2 remains unknown. Here, we report the identification of pathogenic variants in GBF1 (Golgi brefeldin A-resistant guanine nucleotide exchange factor 1) in four unrelated families with individuals affected by sporadic or dominant HMN/CMT2. Genomic sequencing analyses in seven affected individuals uncovered four distinct heterozygous GBF1 variants, two of which occurred de novo. Other known HMN/CMT2-implicated genes were excluded. Affected individuals show HMN/CMT2 with slowly progressive distal muscle weakness and musculoskeletal deformities. Electrophysiological studies confirmed axonal damage with chronic neurogenic changes. Three individuals had additional distal sensory loss. GBF1 encodes a guanine-nucleotide exchange factor that facilitates the activation of members of the ARF (ADP-ribosylation factor) family of small GTPases. GBF1 is mainly involved in the formation of coatomer protein complex (COPI) vesicles, maintenance and function of the Golgi apparatus, and mitochondria migration and positioning. We demonstrate that GBF1 is present in mouse spinal cord and muscle tissues and is particularly abundant in neuropathologically relevant sites, such as the motor neuron and the growth cone. Consistent with the described role of GBF1 in Golgi function and maintenance, we observed marked increase in Golgi fragmentation in primary fibroblasts derived from all affected individuals in this study. Our results not only reinforce the existing link between Golgi fragmentation and neurodegeneration but also demonstrate that pathogenic variants in GBF1 are associated with HMN/CMT2.

Branched-chain α-ketoacid dehydrogenase deficiency (maple syrup urine disease): Treatment, biomarkers, and outcomes.

Over the past three decades, we studied 184 individuals with 174 different molecular variants of branched-chain α-ketoacid dehydrogenase activity, and here delineate essential clinical and biochemical aspects of the maple syrup urine disease (MSUD) phenotype. We collected data about treatment, survival, hospitalization, metabolic control, and liver transplantation from patients with classic (i.e., severe; n = 176), intermediate (n = 6) and intermittent (n = 2) forms of MSUD. A total of 13,589 amino acid profiles were used to analyze leucine tolerance, amino acid homeostasis, estimated cerebral amino acid uptake, quantitative responses to anabolic therapy, and metabolic control after liver transplantation. Standard instruments were used to measure neuropsychiatric outcomes. Despite advances in clinical care, classic MSUD remains a morbid and potentially fatal disorder. Stringent dietary therapy maintains metabolic variables within acceptable limits but is challenging to implement, fails to restore appropriate concentration relationships among circulating amino acids, and does not fully prevent cognitive and psychiatric disabilities. Liver transplantation eliminates the need for a prescription diet and safeguards patients from life-threatening metabolic crises, but is associated with predictable morbidities and does not reverse pre-existing neurological sequelae. There is a critical unmet need for safe and effective disease-modifying therapies for MSUD which can be implemented early in life. The biochemistry and physiology of MSUD and its response to liver transplantation afford key insights into the design of new therapies based on gene replacement or editing.

Crigler-Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier.

BACKGROUND AND AIMS: We describe the pathophysiology, treatment, and outcome of Crigler-Najjar type 1 syndrome (CN1) in 28 UGT1A1 c.222C>A homozygotes followed for 520 aggregate patient-years. APPROACH AND RESULTS: Unbound ("free") bilirubin (Bf ) was measured in patient sera to characterize the binding of unconjugated bilirubin (BT ) to albumin (A) and validate their molar concentration ratio (BT /A) as an index of neurological risk. Two custom phototherapy systems were constructed from affordable materials to provide high irradiance in the outpatient setting; light dose was titrated to keep BT /A at least 30% below intravascular BT binding capacity (i.e., BT /A = 1.0). Categorical clinical outcomes were ascertained by chart review, and a measure (Lf ) was used to quantify liver fibrosis. Unbound bilirubin had a nonlinear relationship to BT (R2  = 0.71) and BT /A (R2  = 0.76), and Bf as a percentage of BT correlated inversely to the bilirubin-albumin equilibrium association binding constant (R2  = 0.69), which varied 10-fold among individuals. In newborns with CN1, unconjugated bilirubin increased 4.3 ± 1.1 mg/dL per day. Four (14%) neonates developed kernicterus between days 14 and 45 postnatal days of life; peak BT  ≥ 30 mg/dL and BT /A ≥ 1.0 mol:mol were equally predictive of perinatal brain injury (sensitivity 100%, specificity 93.3%, positive predictive value 88.0%), and starting phototherapy after age 13 days increased this risk 3.5-fold. Consistent phototherapy with 33-103 µW/cm2 •nm for 9.2 ± 1.1 hours/day kept BT and BT /A within safe limits throughout childhood, but BT increased 0.46 mg/dL per year to reach dangerous concentrations by 18 years of age. Liver transplantation (n = 17) normalized BT and eliminated phototherapy dependence. Liver explants showed fibrosis ranging from mild to severe. CONCLUSION: Seven decades after its discovery, CN1 remains a morbid and potentially fatal disorder.

Recessive GM3 synthase deficiency: Natural history, biochemistry, and therapeutic frontier.

GM3 synthase, encoded by ST3GAL5, initiates synthesis of all downstream cerebral gangliosides. Here, we present biochemical, functional, and natural history data from 50 individuals homozygous for a pathogenic ST3GAL5 c.862C>T founder allele (median age 8.1, range 0.7-30.5 years). GM3 and its derivatives were undetectable in plasma. Weight and head circumference were normal at birth and mean Apgar scores were 7.7 ±â€¯2.0 (1 min) and 8.9 ±â€¯0.5 (5 min). Somatic growth failure, progressive microcephaly, global developmental delay, visual inattentiveness, and dyskinetic movements developed within a few months of life. Infantile-onset epileptic encephalopathy was characterized by a slow, disorganized, high-voltage background, poor state transitions, absent posterior rhythm, and spike trains from multiple independent cortical foci; >90% of electrographic seizures were clinically silent. Hearing loss affected cochlea and central auditory pathways and 76% of children tested failed the newborn hearing screen. Development stagnated early in life; only 13 (26%) patients sat independently (median age 30 months), three (6%) learned to crawl, and none achieved reciprocal communication. Incessant irritability, often accompanied by insomnia, began during infancy and contributed to high parental stress. Despite catastrophic neurological dysfunction, neuroimaging showed only subtle or no destructive changes into late childhood and hospitalizations were surprisingly rare (0.2 per patient per year). Median survival was 23.5 years. Our observations corroborate findings from transgenic mice which indicate that gangliosides might have a limited role in embryonic neurodevelopment but become vital for postnatal brain growth and function. These results have critical implications for the design and implementation of ganglioside restitution therapies.

Homozygosity for a mutation affecting the catalytic domain of tyrosyl-tRNA synthetase (YARS) causes multisystem disease.

Aminoacyl-tRNA synthetases (ARSs) are critical for protein translation. Pathogenic variants of ARSs have been previously associated with peripheral neuropathy and multisystem disease in heterozygotes and homozygotes, respectively. We report seven related children homozygous for a novel mutation in tyrosyl-tRNA synthetase (YARS, c.499C > A, p.Pro167Thr) identified by whole exome sequencing. This variant lies within a highly conserved interface required for protein homodimerization, an essential step in YARS catalytic function. Affected children expressed a more severe phenotype than previously reported, including poor growth, developmental delay, brain dysmyelination, sensorineural hearing loss, nystagmus, progressive cholestatic liver disease, pancreatic insufficiency, hypoglycemia, anemia, intermittent proteinuria, recurrent bloodstream infections and chronic pulmonary disease. Related adults heterozygous for YARS p.Pro167Thr showed no evidence of peripheral neuropathy on electromyography, in contrast to previous reports for other YARS variants. Analysis of YARS p.Pro167Thr in yeast complementation assays revealed a loss-of-function, hypomorphic allele that significantly impaired growth. Recombinant YARS p.Pro167Thr demonstrated normal subcellular localization, but greatly diminished ability to homodimerize in human embryonic kidney cells. This work adds to a rapidly growing body of research emphasizing the importance of ARSs in multisystem disease and significantly expands the allelic and clinical heterogeneity of YARS-associated human disease. A deeper understanding of the role of YARS in human disease may inspire innovative therapies and improve care of affected patients.

Bi-allelic CCDC47 Variants Cause a Disorder Characterized by Woolly Hair, Liver Dysfunction, Dysmorphic Features, and Global Developmental Delay.

Ca2+ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, and dysregulation of ER Ca2+ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca2+ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca2+ storage, impaired Ca2+ signaling mediated by the IP3R Ca2+ release channel, and reduced ER Ca2+ refilling via store-operated Ca2+ entry. These results, together with the previously described role of CCDC47 in Ca2+ signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder.

Spinal muscular atrophy within Amish and Mennonite populations: Ancestral haplotypes and natural history.

We correlate chromosome 5 haplotypes and SMN2 copy number with disease expression in 42 Mennonite and 14 Amish patients with spinal muscular atrophy (SMA). A single haplotype (A1) with 1 copy of SMN2 segregated among all Amish patients. SMN1 deletions segregated on four different Mennonite haplotypes that carried 1 (M1a, M1b, M1c) or 2 (M2) copies of SMN2. DNA microsatellite and microarray data revealed structural similarities among A1, M1a, M1b, and M2. Clinical data were parsed according to both SMN1 genotype and SMN2 copy number (2 copies, n = 44; 3 copies, n = 9; or 4 copies, n = 3). No infant with 2 copies of SMN2 sat unassisted. In contrast, all 9 Mennonites with the M1a/M2 genotype (3 copies of SMN2) sat during infancy at a median age of 7 months, and 5 (56%) walked and dressed independently at median ages of 18 and 36 months, respectively. All are alive at a median age of 11 (range 2-31) years without ventilatory support. Among 13 Amish and 26 Mennonite patients with 2 copies of SMN2 who did not receive feeding or ventilatory support, A1/A1 as compared to M1a/M1a genotype was associated with earlier clinical onset (p = 0.0040) and shorter lifespan (median survival 3.9 versus 5.7 months, p = 0.0314). These phenotypic differences were not explained by variation in SMN1 deletion size or SMN2 coding sequence, which were conserved across haplotypes. Distinctive features of SMA within Plain communities provide a population-specific framework to study variations of disease expression and the impact of disease-modifying therapies administered early in life.

Preliminary Safety and Tolerability of a Novel Subcutaneous Intrathecal Catheter System for Repeated Outpatient Dosing of Nusinersen to Children and Adults With Spinal Muscular Atrophy.

BACKGROUND: Many patients with spinal muscular atrophy (SMA) who might benefit from intrathecal antisense oligonucleotide (nusinersen) therapy have scoliosis or spinal fusion that precludes safe drug delivery. To circumvent spinal pathology, we designed a novel subcutaneous intrathecal catheter (SIC) system by connecting an intrathecal catheter to an implantable infusion port. METHODS: Device safety and tolerability were tested in 10 SMA patients (age, 5.4 to 30.5 y; 80% with 3 copies of SMN2); each received 3 sequential doses of nusinersen (n=30 doses). Pretreatment disease burden was evaluated using the Revised Hammersmith Scale, dynamometry, National Institutes of Health pegboard, pulmonary function testing, electromyography, and 2 health-related quality of life tools. RESULTS: Device implantation took ≤2 hours and was well tolerated. All outpatient nusinersen doses were successfully administered via SIC within 20 minutes on the first attempt, and required no regional or systemic analgesia, cognitive distraction, ultrasound guidance, respiratory precautions, or sedation. Cerebrospinal fluid withdrawn from the SIC had normal levels of glucose and protein; cerebrospinal fluid white blood cells were slightly elevated in 2 (22%) of 9 specimens (median, 1 cell/µL; range, 0 to 12 cells/µL) and red blood cells were detected in 7 (78%) specimens (median, 4; range, 0 to 2930 cells/µL). DISCUSSION: Preliminary observations reveal the SIC to be relatively safe and well tolerated in SMA patients with advanced disease and spinal fusion. The SIC warrants further study and, if proven effective in larger trials of longer duration, could double the number of patients able to receive nusinersen worldwide while reducing administration costs 5- to 10-fold.

TNNT1 nemaline myopathy: natural history and therapeutic frontier.

We describe the natural history of 'Amish' nemaline myopathy (ANM), an infantile-onset, lethal disease linked to a pathogenic c.505G>T nonsense mutation of TNNT1, which encodes the slow fiber isoform of troponin T (TNNT1; a.k.a. TnT). The TNNT1 c.505G>T allele has a carrier frequency of 6.5% within Old Order Amish settlements of North America. We collected natural history data for 106 ANM patients born between 1923 and 2017. Over the last two decades, mean age of molecular diagnosis was 16 ± 27 days. TNNT1 c.505G>T homozygotes were normal weight at birth but failed to thrive by age 9 months. Presenting neonatal signs were axial hypotonia, hip and shoulder stiffness, and tremors, followed by progressive muscle weakness, atrophy and contractures. Affected children developed thoracic rigidity, pectus carinatum and restrictive lung disease during infancy, and all succumbed to respiratory failure by 6 years of age (median survival 18 months, range 0.2-66 months). Muscle histology from two affected children showed marked fiber size variation owing to both Type 1 myofiber smallness (hypotrophy) and Type 2 fiber hypertrophy, with evidence of nemaline rods, myofibrillar disarray and vacuolar pathology in both fiber types. The truncated slow TNNT1 (TnT) fragment (p.Glu180Ter) was undetectable in ANM muscle, reflecting its rapid proteolysis and clearance from sarcoplasm. Similar functional and histological phenotypes were observed in other human cohorts and two transgenic murine models (Tnnt1-/- and Tnnt1 c.505G>T). These findings have implications for emerging molecular therapies, including the suitably of TNNT1 gene replacement for newborns with ANM or other TNNT1-associated myopathies.