Generation of a gene-corrected human isogenic line (UAMi006-A) from propionic acidemia patient iPSC with an homozygous mutation in the PCCB gene using CRISPR/Cas9 technology.

Propionic acidemia (PA) is an inherited metabolic disease caused by mutations in the PCCA and PCCB genes. We have previously generated an induced pluripotent stem cell (iPSC) line (UAMi004-A) from a PA patient with the c.1218_1231del14ins12 (p.Gly407Argfs*14) homozygous mutation in the PCCB gene. Here, we report the generation of the isogenic control in which the mutation was genetically corrected using CRISPR/Cas9 technology. Off-target editing presence was excluded and the iPSCs had typical embryonic stem cell-like morphology and normal karyotype that expressed pluripotency markers and maintained their in vitro differentiation potential.

Pathogenic implications of dysregulated miRNAs in propionic acidemia related cardiomyopathy.

Cardiac alterations (hypertrophic/dilated cardiomyopathy, and rhythm alterations) are one of the major causes of mortality and morbidity in propionic acidemia (PA), caused by the deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC), involved in the catabolism of branched-chain amino acids, cholesterol, and odd-chain fatty acids. Impaired mitochondrial oxidative phosphorylation has been documented in heart biopsies of PA patients, as well as in the hypomorphic Pcca-/-(A138T) mouse model, in the latter correlating with increased oxidative damage and elevated expression of cardiac dysfunction biomarkers atrial and brain natriuretic peptides (ANP and BNP) and beta-myosin heavy chain (ß-MHC). Here we characterize the cardiac phenotype in the PA mouse model by histological and echocardiography studies and identify a series of upregulated cardiac-enriched microRNAs (miRNAs) in the PA mouse heart, some of them also altered as circulating miRNAs in PA patients' plasma samples. In PA mice hearts, we show alterations in signaling pathways regulated by the identified miRNAs, which could be contributing to cardiac remodeling and dysfunction; notably, an activation of the mammalian target of rapamycin (mTOR) pathway and a decrease in autophagy, which are reverted by rapamycin treatment. In vitro studies in HL-1 cardiomyocytes indicate that propionate, the major toxic metabolite accumulating in the disease, triggers the increase in expression levels of miRNAs, BNP, and ß-MHC, concomitant with an increase in reactive oxygen species. Our results highlight miRNAs and signaling alterations in the PCC-deficient heart which may contribute to the development of PA-associated cardiomyopathy and provide a basis to identify new targets for therapeutic intervention.

Dysregulated miRNAs and their pathogenic implications for the neurometabolic disease propionic acidemia.

miRNome expression profiling was performed in a mouse model of propionic acidemia (PA) and in patients' plasma samples to investigate the role of miRNAs in the pathophysiology of the disease and to identify novel biomarkers and therapeutic targets. PA is a potentially lethal neurometabolic disease with patients developing neurological deficits and cardiomyopathy in the long-term, among other complications. In the PA mouse liver we identified 14 significantly dysregulated miRNAs. Three selected miRNAs, miR-34a-5p, miR-338-3p and miR-350, were found upregulated in brain and heart tissues. Predicted targets involved in apoptosis, stress-signaling and mitochondrial function, were inversely found down-regulated. Functional analysis with miRNA mimics in cellular models confirmed these findings. miRNA profiling in plasma samples from neonatal PA patients and age-matched control individuals identified a set of differentially expressed miRNAs, several were coincident with those identified in the PA mouse, among them miR-34a-5p and miR-338-3p. These two miRNAs were also found dysregulated in childhood and adult PA patients' cohorts. Taken together, the results reveal miRNA signatures in PA useful to identify potential biomarkers, to refine the understanding of the molecular mechanisms of this rare disease and, eventually, to improve the management of patients.