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Genetic testing of patient constitutional DNA (i.e., their genome) is increasingly performed in medical practice. Sequencing an entire human genome (about 3.2 billion nucleotides) is now possible to complete in days to weeks, and at a similar cost to some advanced imaging tests or to a brief admission to hospital. Genome sequencing is being integrated into health care systems internationally, most notably in the UK. Starting in 2021, genome sequencing is being performed as a clinical genetic test in Ontario, Canada. Genome sequencing is broader in scope than other commonly used genetic tests, and data can be analyzed in both hypothesis-driven and hypothesis-generating ways. For these reasons, genome sequencing will likely eventually supplant exome sequencing, large next-generation sequencing gene panel tests and chromosomal microarray analysis. Genome sequencing is a consideration for children and adults with suspected genetic disorders for whom a targeted genetic testing approach is unlikely to succeed or has already failed.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1 , which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease 1 . However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy 2j /dy 2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy 2j /dy 2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR–dCas9-mediated upregulation of Lama1 , which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases. When Lama1 was upregulated using CRISPR and a catalytically inactive Cas9 in a mouse model of congenital muscular dystrophy type 1A, apparent hindlimb paralysis, muscle fibrosis and nerve myelination defects were ameliorated in symptomatic mice.

Cerebral palsy (CP) represents a group of non-progressive clinically heterogeneous disorders that are characterized by motor impairment and early age of onset, frequently accompanied by co-morbidities. The cause of CP has historically been attributed to environmental stressors resulting in brain damage. While genetic risk factors are also implicated, guidelines for diagnostic assessment of CP do not recommend for routine genetic testing. Given numerous reports of aetiologic copy number variations (CNVs) in other neurodevelopmental disorders, we used microarrays to genotype a population-based prospective cohort of children with CP and their parents. Here we identify de novo CNVs in 8/115 (7.0%) CP patients (∼1% rate in controls). In four children, large chromosomal abnormalities deemed likely pathogenic were found, and they were significantly more likely to have severe neuromotor impairments than those CP subjects without such alterations. Overall, the CNV data would have impacted our diagnosis or classification of CP in 11/115 (9.6%) families. Cerebral palsy (CP) is a heterogeneous disorder that has been historically attributed to environmental factors with genetic contributions being discovered more recently. Here the authors perform microarray-based analysis of copy number variations in a cohort of children with CP and their parents and find chromosomal abnormalities linked to the disease.

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Humans with gain-of-function (GOF) mutations in STAT1 (Signal Transducer and Activator of Transcription 1), a potent immune regulator, experience frequent infections. About one-third, especially those with DNA-binding domain (DBD) mutations such as T385M, also develop autoimmunity, sometimes accompanied by increases in T-helper 1 (Th1) and T-follicular helper (Tfh) CD4 effector T cells, resembling those that differentiate following infection-induced STAT1 signaling. However, environmental and molecular mechanisms contributing to autoimmunity in STAT1 GOF patients are not defined. We generated Stat1T385M/+ mutant mice to model the immune impacts of STAT1 DBD GOF under specific-pathogen free (SPF) conditions. Stat1T385M/+ lymphocytes had more total Stat1 at baseline and also higher amounts of IFNg-induced pStat1. Young mutants exhibited expansion of Tfh-like cells, while older mutants developed autoimmunity accompanied by increased Tfh-like cells, B cell activation and germinal center (GC) formation. Mutant females exhibited these immune changes sooner and more robustly than males, identifying significant sex effects of Stat1T385M-induced immune dysregulation. Single cell RNA-Seq (scRNA-Seq) analysis revealed that Stat1T385M activated transcription of GC-associated programs in both B and T cells. However, it had the strongest transcriptional impact on T cells, promoting aberrant CD4 T cell activation and imparting both Tfh-like and Th1-like effector programs. Collectively, these data demonstrate that in the absence of overt infection, Stat1T385M disrupted naïve CD4 T cell homeostasis and promoted expansion and differentiation of abnormal Tfh/Th1-like helper and GC-like B cells, eventually leading to sex-biased autoimmunity, suggesting a model for STAT1 GOF-induced immune dysregulation and autoimmune sequelae in humans.

An HDR-independent therapeutic genome-editing approach corrected the splice-site mutation in Lama2 in a mouse model of congenital muscular dystrophy type 1A, and may be applied more broadly to correct splice-site mutations associated with other diseases. Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases 1 . Prevalence is higher in neuromuscular disorders (NMDs) 2 , owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway 3 , 4 , 5 , which is extremely inefficient in postmitotic tissues such as skeletal muscle 6 . Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis 7 . Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy 2J /dy 2J mouse model of MDC1A 8 . Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy 2J /dy 2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.

Tubulinopathies and neurodevelopmental ciliopathies are two groups of genetic disorders that cause structural brain malformations. Tubulinopathies result from mutations in tubulins, the building blocks of microtubules, most of which are dominant. Neurodevelopmental ciliopathies are mostly recessive disorders caused by defects in the primary cilium, an organelle that regulates key signaling pathways during brain development. Although more than 40 genes have been associated with neurodevelopmental ciliopathies, many patients still lack a known genetic cause. Here, we present a de novo heterozygous missense variant (p.G308S) in Tubulin Beta Class I ( TUBB ) identified in a patient showing features of both ciliopathy and tubulinopathy. While microtubules are essential for primary cilia formation and function, an association between tubulin mutations and neurodevelopmental ciliopathies has not been established. Using patient-derived cells and gene-edited isogenic cell lines, we show that the identified variant impairs the early stages of cilia formation by altering microtubule dynamics and structure. Consistent with this, mice carrying the variant display decreased ciliation in the cerebellum and kidney. Furthermore, we demonstrate that the disease mechanism is not haploinsufficiency and that other patient mutations in TUBB affect cilia formation, putting forward defective ciliogenesis as a contributing pathogenic factor in a subset of tubulinopathy patients. Tubulin mutations cause a group of disorders named tubulinopathies. Here, the authors show that specific variants in the tubulin TUBB impair primary cilia formation, putting forward primary cilia defects as a pathogenic factor in some tubulinopathy cases.

Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor-{szligbeta} (TGF-{szligbeta}) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-{szligbeta} signaling and can be prevented by TGF-{szligbeta} antagonists such as TGF-{szligbeta}-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.

Becker muscular dystrophy (BMD) is a rare X-linked recessive neuromuscular disorder, frequently caused by in-frame deletions in the DMD gene that result in the production of a truncated, yet functional, dystrophin protein. The consequences of BMD-causing in-frame deletions on the organism are difficult to predict, especially in regard to long-term prognosis. Here, we used CRISPR-Cas9 to generate a new Dmd Δ52-55 mouse model by deleting exons 52-55 in the Dmd gene, resulting in a BMD-like in-frame deletion. To delineate the long-term effects of this deletion, we studied these mice over 52 weeks by performing histology and echocardiography analyses and assessing motor functions. Our results suggest that truncated dystrophin is sufficient to maintain wildtype-like muscle and heart histology and functions in young mice. However, the truncated protein appeared to be insufficient to maintain normal muscle homeostasis and protect against exercise-induced damage at 52 weeks. To further delineate the effects of this exon 52-55 in-frame deletion, we performed RNA sequencing pre- and post-exercise and identified several differentially expressed pathways that reflect the abnormal muscle phenotype observed at 52 weeks in the BMD model.