Physiological role and mechanisms of action for a long noncoding haplotype region.

Most common sequence variants associated with human traits are in noncoding regions of the genome, form haplotypes with other noncoding variants, and exhibit small effect sizes in the general population. Determining the physiological roles and mechanisms of action for these noncoding variants, particularly large haplotypes containing multiple variants, is both critical and challenging. To address this challenge, we developed an approach that integrates physiological studies in genetically engineered and phenotypically permissive animal models, precise editing of large haplotypes in human induced pluripotent stem cells (hiPSCs), and targeted chromatin conformation analysis. We applied this approach to examine the blood pressure associated rs1173771 locus, which includes a haplotype containing 11 single nucleotide polymorphisms (SNPs) spanning 17.4 kbp. Deleting the orthologous noncoding region in the genome of the Dahl salt-sensitive rat attenuated the salt-induced increase in systolic blood pressure by nearly 10 mmHg. This attenuation of hypertension appeared to be mediated by upregulation of the adjacent gene Npr3 (natriuretic peptide receptor 3) in arteries, enhancing vasodilation. The blood pressure-elevating and -lowering haplotypes were precisely reconstituted in hiPSCs using an efficient, two-step genome editing technique. The blood pressure-elevating haplotype decreased NPR3 expression in endothelial cells and vascular smooth muscle cells derived from the edited, isogenic hiPSCs. The influence of the haplotype was partially recapitulated by the sentinel SNP rs1173771. Additionally, the blood pressure-elevating haplotype showed significantly greater chromatin interactions with the NPR3 promoter region. This study illustrates the feasibility of ascertaining the physiological roles and mechanisms of action for large noncoding haplotypes. Our efficient, integrated, and targeted approach can be applied to investigate other noncoding variants.

High mobility group box 1 (HMGB1) is a potential disease biomarker in cell and mouse models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration, regeneration, and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches, including exon skipping and gene replacement therapy, are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo, HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However, HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT, demonstrating a strain-specific difference in DMD-related immunopathology.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.

The nucleotide prodrug CERC-913 improves mtDNA content in primary hepatocytes from DGUOK-deficient rats.

Loss-of-function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK-deficient, patient-derived fibroblasts and myoblasts. CERC-913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC-913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK-deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC-913 increased mtDNA content in DGUOK-deficient hepatocytes up to 2.4-fold after 4 days of treatment in a dose-dependent fashion, which was significantly more effective than dGMP at similar concentrations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC-913 treatment can improve mtDNA content in this model.

Severe Neonatal RYR1 Myopathy With Pathological Features of Congenital Muscular Dystrophy.

The phenotypes associated with pathogenic variants in the ryanodine receptor 1 gene (RYR1, OMIM# 180901) have greatly expanded over the last few decades as genetic testing for RYR1 variants has become more common. Initially described in association with malignant hyperthermia, pathogenic variants in RYR1 are typically associated with core pathology in muscle biopsies (central core disease or multiminicore disease) and symptomatic myopathies with symptoms ranging from mild weakness to perinatal lethality. We describe a 2-week-old male patient with multiple congenital dysmorphisms, severe perinatal weakness, and subsequent demise, whose histopathology on autopsy was consistent with congenital muscular dystrophy. Immunohistochemical analysis of dystrophy-associated proteins was normal. Rapid exome sequencing revealed a novel heterozygous nonsense variant (p.Trp661Ter) in RYR1, as well as a previously described RYR1 pathogenic variant associated with congenital myopathy (p.Phe4976Leu). This highlights the potential for RYR1 pathogenic variants to produce pathological findings most consistent with congenital muscular dystrophy.