Effects of HMGCR deficiency on skeletal muscle development.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Cognitive Decline and Other Late-Stage Neurologic Complications in Cockayne Syndrome.

Background and Objectives: Cockayne syndrome (CS) is an ultra-rare, autosomal recessive, premature aging disorder characterized by impaired growth, neurodevelopmental delays, neurodegeneration, polyneuropathy, and other multiorgan system complications. The anatomic aspects of CS neurodegeneration have long been known from postmortem examinations and MRI studies, but the clinical features of this neurodegeneration are not well characterized, especially at later stages of the disease. Methods: This was a retrospective observational study in which individuals with CS who survived beyond 18 years were ascertained at 3 centers in the United States, France, and the United Kingdom. Medical records were examined to determine the frequencies and features of the following neurologic complications: neurocognitive/neuropsychiatric decline (8 symptoms), tremors, neuropathy, seizures, and strokes. Results: Among 18 individuals who met inclusion criteria, all but one (94.4%) experienced at least one symptom of neurocognitive/neuropsychiatric decline, with most individuals experiencing at least half of those symptoms. Most participants experienced tremors and peripheral neuropathy, with a few experiencing seizures and strokes. For individuals with available data, 100.0% were reported to have gait ataxia and neuroimaging showed that 85.7% had generalized cerebral atrophy on MRI while 78.6% had white matter changes. Discussion: Symptoms of neurocognitive/neuropsychiatric decline are nearly universal in our cohort of adults with CS, suggesting that these individuals are at risk of developing neurocognitive/neuropsychiatric decline, with symptoms related to but not specific to dementia. Considering the prominent role of DNA repair defects in CS disease mechanisms and emerging evidence for increased DNA damage in neurodegenerative disease, impaired genome maintenance may be a shared pathway underlying multiple forms of neurocognitive/neuropsychiatric decline. Components of the DNA damage response mechanism may bear further study as potential therapeutic targets that could alleviate neurocognitive/neuropsychiatric symptoms in CS and other neurodegenerative disorders.

Detecting missed diagnoses of spinal muscular atrophy in genome, exome, and panel sequencing datasets.

Spinal muscular atrophy (SMA) is a genetic disorder that causes progressive degeneration of lower motor neurons and the subsequent loss of muscle function throughout the body. It is the second most common recessive disorder in individuals of European descent and is present in all populations. Accurate tools exist for diagnosing SMA from short read and long read genome sequencing data. However, there are no publicly available tools for GRCh38-aligned data from panel or exome sequencing assays which continue to be used as first line tests for neuromuscular disorders. We therefore developed and extensively validated a new tool - SMA Finder - that can diagnose SMA not only in genome, but also exome and targeted sequencing samples aligned to GRCh37, GRCh38, or T2T-CHM13. It works by evaluating aligned reads that overlap the c.840 position of SMN1 and SMN2 in order to detect the most common molecular causes of SMA. We applied SMA Finder to 16,626 exomes and 3,911 genomes from heterogeneous rare disease cohorts sequenced at the Broad Institute Center for Mendelian Genomics as well as 1,157 exomes and 8,762 targeted sequencing samples from Tartu University Hospital. SMA Finder correctly identified all 16 known SMA cases and reported nine novel diagnoses which have since been confirmed by clinical testing, with another four novel diagnoses undergoing validation. Notably, out of the 29 total SMA positive cases, 21 had an initial clinical diagnosis of muscular dystrophy, congenital myasthenic syndrome, or congenital myopathy. This underscored the frequency with which SMA can be misdiagnosed as other neuromuscular disorders and confirmed the utility of using SMA Finder to reanalyze phenotypically diverse neuromuscular disease cohorts. Finally, we evaluated SMA Finder on 198,868 individuals that had both exome and genome sequencing data within the UK Biobank (UKBB) and found that SMA Finder's overall false positive rate was less than 1 / 200,000 exome samples, and its positive predictive value (PPV) was 96%. We also observed 100% concordance between UKBB exome and genome calls. This analysis showed that, even though it is located within a segmental duplication, the most common causal variant for SMA can be detected with comparable accuracy to monogenic disease variants in non-repetitive regions. Additionally, the high PPV demonstrated by SMA Finder, the existence of treatment options for SMA in which early diagnosis is imperative for therapeutic benefit, as well as widespread availability of clinical confirmatory testing for SMA, may warrant the addition of SMN1 to the ACMG list of genes with reportable secondary findings after genome and exome sequencing.

One episode of low intensity aerobic exercise prior to systemic AAV9 administration augments transgene delivery to the heart and skeletal muscle.

INTRODUCTION: The promising potential of adeno-associated virus (AAV) gene delivery strategies to treat genetic disorders continues to grow with an additional three AAV-based therapies recently approved by the Food and Drug Administration and dozens of others currently under evaluation in clinical trials. With these developments, it has become increasingly apparent that the high doses currently needed for efficacy carry risks of toxicity and entail enormous manufacturing costs, especially for clinical grade products. Strategies to increase the therapeutic efficacy of AAV-mediated gene delivery and reduce the minimal effective dose would have a substantial impact on this field. We hypothesized that an exercise-induced redistribution of tissue perfusion in the body to favor specific target organs via acute aerobic exercise prior to systemic intravenous (IV) AAV administration could increase efficacy. BACKGROUND: Aerobic exercise triggers an array of downstream physiological effects including increased perfusion of heart and skeletal muscle, which we expected could enhance AAV transduction. Prior preclinical studies have shown promising results for a gene therapy approach to treat Barth syndrome (BTHS), a rare monogenic cardioskeletal myopathy, and clinical studies have shown the benefit of low intensity exercise in these patients, making this a suitable disease in which to test the ability of aerobic exercise to enhance AAV transduction. METHODS: Wild-type (WT) and BTHS mice were either systemically administered AAV9 or completed one episode of low intensity treadmill exercise immediately prior to systemic administration of AAV9. RESULTS: We demonstrate that a single episode of acute low intensity aerobic exercise immediately prior to IV AAV9 administration improves marker transgene delivery in WT mice as compared to mice injected without the exercise pre-treatment. In BTHS mice, prior exercise improved transgene delivery and additionally increased improvement in mitochondrial gene transcription levels and mitochondrial function in the heart and gastrocnemius muscles as compared to mice treated without exercise. CONCLUSIONS: Our findings suggest that one episode of acute low intensity aerobic exercise improves AAV9 transduction of heart and skeletal muscle. This low-risk, cost effective intervention could be implemented in clinical trials of individuals with inherited cardioskeletal disease as a potential means of improving patient safety for human gene therapy.

Diagnostic capabilities of nanopore long-read sequencing in muscular dystrophy.

Many individuals with muscular dystrophies remain genetically undiagnosed despite clinical diagnostic testing, including exome sequencing. Some may harbor previously undetected structural variants (SVs) or cryptic splice sites. We enrolled 10 unrelated families: nine had muscular dystrophy but lacked complete genetic diagnoses and one had an asymptomatic DMD duplication. Nanopore genomic long-read sequencing identified previously undetected pathogenic variants in four individuals: an SV in DMD, an SV in LAMA2, and two single nucleotide variants in DMD that alter splicing. The DMD duplication in the asymptomatic individual was in tandem. Nanopore sequencing may help streamline genetic diagnostic approaches for muscular dystrophy.

TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis.

Proteomic technologies are powerful methodologies that can aid our understanding of mechanisms of action in biological systems by providing a global view of the impact of a disease, treatment, or other condition on the proteome as a whole. This report provides a detailed protocol for the extraction, quantification, precipitation, digestion, labeling, and subsequent data analysis of protein samples. Our optimized TMT labeling protocol requires a lower tag-label concentration and achieves consistently reliable data. We have used this protocol to evaluate protein expression profiles in a variety of mouse tissues (i.e., heart, skeletal muscle, and brain) as well as cells cultured in vitro. In addition, we demonstrate how to evaluate thousands of proteins from the resulting dataset.