Differential inclusion of NEB exons 143 and 144 provides insight into NEB-related myopathy variant interpretation and disease manifestation.

Biallelic pathogenic variants in the gene encoding nebulin (NEB) are a known cause of congenital myopathy. We present two brothers with congenital myopathy and compound heterozygous variants (NC_000002.12:g.151692086G>T; NM_001271208.2: c.2079C>A; p.(Cys693Ter) and NC_000002.12:g.151533439T>C; NM_001271208.2:c.21522+3A>G) in NEB. Transcriptomic sequencing on affected individual muscles revealed that the extended splice variant c.21522+3A>G causes exon 144 skipping. Nebulin isoforms containing exon 144 are known to be mutually exclusive with isoforms containing exon 143, and these isoforms are differentially expressed during development and in adult skeletal muscles. Affected individuals' MRI patterns of muscle involvement were compared with the known pattern of relative abundance of these two isoforms in muscle. We propose that the pattern of muscle involvement in these affected individuals better fits the distribution of exon 144-containing isoforms in muscle than with previously published MRI findings in NEB-related disease due to other variants. Our report introduces disease pathogenesis and manifestation as a result of alteration of isoform distributions in muscle.

CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes.

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Differential inclusion of NEB exons 143 and 144 provides insight into NEB-related myopathy variant interpretation and disease manifestation.

Biallelic pathogenic variants in the gene encoding nebulin (NEB) are a known cause of congenital myopathy. We present two individuals with congenital myopathy and compound heterozygous variants (NM_001271208.2: c.2079C>A; p.(Cys693Ter) and c.21522+3A>G ) in NEB. Transcriptomic sequencing on patient muscle revealed that the extended splice variant c.21522+3A>G causes exon 144 skipping. Nebulin isoforms containing exon 144 are known to be mutually exclusive with isoforms containing exon 143, and these isoforms are differentially expressed during development and in adult skeletal muscles. Patients MRIs were compared to the known pattern of relative abundance of these two isoforms in muscle. We propose that the pattern of muscle involvement in these patients better fits the distribution of exon 144-containing isoforms in muscle than with previously published MRI findings in NEB-related disease due to other variants. To our knowledge this is the first report hypothesizing disease pathogenesis through the alteration of isoform distributions in muscle.

A humanized knock-in Col6a1 mouse recapitulates a deep-intronic splice-activating variant.

Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

Clinical, immunohistochemical, and genetic characterization of splice-altering biallelic DES variants: Therapeutic implications.

Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory insufficiency, and are collectively known as desminopathies. While most DES pathogenic variants act via a dominant mechanism, recessively acting variants have also been reported. Currently, there are no effective therapeutic interventions for desminopathies of any type. Here, we report an affected individual with rapidly progressive dilated cardiomyopathy, requiring heart transplantation at age 13 years, in the setting of childhood-onset skeletal muscle weakness. We identified biallelic DES variants (c.640-13 T>A and c.1288+1 G>A) and show aberrant DES gene splicing in the affected individual's muscle. Through the generation of an inducible lentiviral system, we transdifferentiated fibroblast cultures derived from the affected individual into myoblasts and validated this system using RNA sequencing. We tested rationally designed, custom antisense oligonucleotides to screen for splice correction in these transdifferentiated cells and a functional minigene splicing assay. However, rather than correctly redirecting splicing, we found them to induce undesired exon skipping. Our results indicate that, while an individual precision-based molecular therapeutic approach to splice-altering pathogenic variants is promising, careful preclinical testing is imperative for each novel variant to test the feasibility of this type of approach for translation.

Collagen type VI regulates TGFß bioavailability in skeletal muscle.

Collagen VI-related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalizatoion of collagen VI in the ECM underlies the non-cell autonomous dysfunction and dystrophic changes in skeletal muscle with an as of yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conduct a comprehensive natural history and outcome study in a novel mouse model of COL6-RDs (Col6a2-/- mice) using standardized (Treat-NMD) functional, histological, and physiologic parameter. Notably, we identify a conspicuous dysregulation of the TGFß pathway early in the disease process and propose that the collagen VI deficient matrix is not capable of regulating the dynamic TGFß bioavailability at baseline and also in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGFß with downstream skeletal muscle abnormalities, paving the way for developing and validating therapeutics that target this pathway.

Loss of Function of the Cytoplasmic Fe-S Assembly Protein CIAO1 Causes a Neuromuscular Disorder with Compromise of Nucleocytoplasmic Fe-S Enzymes.

Cytoplasmic and nuclear iron-sulfur enzymes that are essential for genome maintenance and replication depend on the cytoplasmic iron-sulfur assembly (CIA) machinery for cluster acquisition. Here we report that patients with biallelic loss of function in CIAO1 , a key CIA component, develop proximal and axial muscle weakness, fluctuating creatine kinase elevation and respiratory insufficiency. In addition, they present with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, macrocytic anemia and gastrointestinal symptoms. Mutational analysis and functional assays revealed reduced stability of the variants compared to wild-type CIAO1. Loss of CIAO1 impaired DNA helicases, polymerases and repair enzymes which rely on the CIA complex to acquire their Fe-S cofactors, with lentiviral restoration reversing all patient-derived cellular abnormalities. Our study identifies CIAO1 as a novel human disease gene and provides insights into the broader implications of the iron-sulfur assembly pathway in human health and disease.

BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy.

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin-5 for fusion of endoplasmic reticulum-derived vesicles with the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER-to-Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild-type, among them ERGIC-53. The BET1/ERGIC-53 interaction was validated by endogenous co-immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC-53 was observed in P1 and P2's derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC-53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD.

Stress and coping strategies in pediatric dental residents.

PURPOSE/OBJECTIVES: This study examines the amount and sources of stress, as well as coping strategies, exercise, and alcohol use, among pediatric dental residents in the United States. METHODS: One hundred fifty pediatric dental residents (n = 76 postgraduate year [PGY] 1; n = 74 PGY2) in 2-year residency programs responded to an anonymous survey that included demographic questions, the Perceived Stress Scale (PSS), Graduate Dental Environment Stress Scale (GDES), Tactics For Coping With Stress Inventory, and questions about alcohol consumption and exercise. RESULTS: Stress scores were moderate (mean PSS = 16.7 ± 7.1; GDES = 61.7 ± 16.0). The largest sources of stress were research requirements, program/clinic issues, and finances. There were no significant differences in amount and sources of stress between PGY1 and PGY2 residents (P ≥ 0.10). Residents from western programs (based on AAPD districts) reported less stress than those in other areas (PSS, P = 0.04; GDES, P = 0.09). Number of negative coping tactics used was positively correlated (PPS, P < 0.0001; GDES, P = 0.0004), while number of positive coping tactics was negatively correlated (PSS, P < 0.0001; GDES, P = 0.0008) with stress scores. Younger residents (< 30yrs) used more coping tactics than older residents (P = 0.0002). Hospital-based residents used more negative coping tactics than those in university-based and combined programs (P = 0.05). Residents exercising > 150 min/wk had lower PSS (P = 0.03) and GDES (P = 0.09) scores. Alcohol consumption was unrelated to stress scores. CONCLUSION(S): Amount and sources of stress do not differ by residency year. Residents utilizing positive coping strategies and exercising had lower stress than those using negative coping strategies. Pediatric dental residency programs should educate and encourage residents to use positive coping strategies and exercise.

A novel intronic homozygous mutation in the AMT gene of a patient with nonketotic hyperglycinemia and hyperammonemia.

Nonketotic Hyperglycinemia is an autosomal recessive disorder characterized by defects in the mitochondrial glycine cleavage system. Most patients present soon after birth with seizures and hypotonia, and infants that survive the newborn period often have profound intellectual disability and intractable seizures. Here we present a case report of a 4-year-old girl with NKH as well as hyperammonemia, an uncommon finding in NKH. Genetic analysis found a previously unreported homozygous mutation (c.878-1 G > A) in the AMT gene. Maximum Entropy Principle modeling predicted that this mutation most likely breaks the splice site at the border of intron 7 and exon 8 of the AMT gene. Treatment with L-Arginine significantly reduced both the proband's glycine and ammonia levels, in turn aiding in control of seizures and mental status. This is the first time the use of L-Arginine is reported to successfully treat elevated glycine levels.

α2* Nicotinic acetylcholine receptors influence hippocampus-dependent learning and memory in adolescent mice.

The absence of α2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of α2* nAChRs (Chrna2L9'S/L9'S and Chrna2KO) influence hippocampus-dependent learning and memory and CA1 synaptic plasticity. We substituted a serine for a leucine (L9'S) in the α2 subunit (encoded by the Chrna2 gene) to make a hypersensitive nAChR. Using a dorsal hippocampus-dependent task of preexposure-dependent contextual fear conditioning, adolescent hypersensitive Chrna2L9'S/L9'S male mice exhibited impaired learning and memory. The deficit was rescued by low-dose nicotine exposure. Electrophysiological studies demonstrated that hypersensitive α2 nAChRs potentiate acetylcholine-induced ion channel flux in oocytes and acute nicotine-induced facilitation of dorsal/intermediate CA1 hippocampal long-term potentiation in Chrna2L9'S/L9'S mice. Adolescent male mice null for the α2 nAChR subunit exhibited a baseline deficit in learning that was not reversed by an acute dose of nicotine. These effects were not influenced by locomotor, sensory or anxiety-related measures. Our results demonstrated that α2* nAChRs influenced hippocampus-dependent learning and memory, as well as nicotine-facilitated CA1 hippocampal synaptic plasticity.

A longitudinal study of stress in first-year dental students.

This study examines the association of stress with performance and health in first-year dental students and changes in the amount and sources of stress over one year. Students at four U.S. dental schools completed the Dental Environment Stress (DES) scale, Perceived Stress Scale (PSS), stress rating, and demographic questions at the start of their first year of school (baseline), 11.7 weeks, and one year later when first-year GPA, illnesses, health ratings, and symptom frequency were also assessed. Overall, 296 (186 men, 110 women) responded at baseline and after one year; 205 responded all three times. Stress scores were negatively correlated with GPA (DES, p=.006; PSS, p=.04; stress rating, p=.002) and with physical and emotional health ratings (p's< or =.002), but positively associated with illness (p<.05), symptoms (p<.0001), and symptom frequency (p's<.05). Stress was higher after one year (p's<.001) and varied by school (p<.0001). Women (p<.01), younger (p<.003), and single students (p<.03) had higher stress at baseline, but after one year, there were no differences by gender, age, or marital status. Ratings for items on the Dental Environment Stress scale related to schoolwork were high at baseline and increased further by one year (p's< or =.0001); items related to school atmosphere had low ratings initially with large increases over time (p's<.0001). In conclusion, stress increases over time in first-year dental students and is related to detrimental effects on performance and health. Variation between schools may reflect different teaching methods. Changes in sources of stress may reflect the different contributions of anticipatory and situational stress over time. First-year dental students may benefit from stress reduction programs.

The effect of condyle fossa relationships on head posture.

Although it is commonly accepted that there is an interrelationship between the temporomandibular joint (TMJ) and head posture, few, if any, previous studies have quantified this effect. The purpose of this study is to quantify the effect of a change in the condyle fossa relationship of symptomatic temporomandibular joints on head posture. Charts of 51 patients (N=10 men and N=41 women) with symptomatic TMJ pathology were reviewed. The condyle fossa relationships were measured pre- and posttreatment using sagittal corrected hypocycloidal tomography. The amount of slant between the shoulder and external auditory meatus (EAM) was measured in pre- and posttreatment photographs as an indicator of forward head posture; less slant indicates better posture. Subjects ranged in age from 13-74 years (mean=43.1) and had been treated for an average of 5 months. Comparisons with pre-treatment measures showed that after treatment, the amount of retrodiskal space was significantly increased by an average of 1.67 mm on the left side (t=-10.11, p<0.0001) and 1.92 mm on the right (t=-9.62, p<0.0001). Comparisons also showed that after treatment, the amount of slant between the shoulder and EAM decreased by 4.43 inches on average which was also significant (t=13.08, p<0.0001). Improvement in the condyle fossa relationship was related to decreased forward head posture. This suggests that optimizing mandibular condyle position should be considered in the management of forward head posture (adaptive posture).