Molecular characterization and therapy of acyl-CoA dehydrogenase deficiencies

Acyl-CoA dehydrogenase (ACAD) deficiencies are autosomal recessive inborn errors of metabolism together affecting 1/50,000 babies born in the United States. Isovaleric academia (IVA) is caused by genetic defects in isovaleryl-CoA dehydrogenase (IVDH) catalyzing the third step in the leucine (Leu) degradation pathway resulting from mutations in the isovaleryl-CoA dehydrogenase (IVD). Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of β-oxidation of fatty acids with carbon length chain of 14 to 22 and its deficiency results from biallelic mutations in ACADVL. Both deficiencies are detected through tandem mass spectrometry as part of newborn screening (NBS) and confirmed via DNA sequencing of IVD or ACADVL. However, the identification of variants of unknown significance (VUS) leads to uncertainty in patient diagnosis and treatment. Therapy for both disorders is dietary based, but most patients still experience episodes of metabolic decompensation triggered by physiological stress with significant morbidity and mortality. Using CRISPR/Cas9 technology, I generated IVD and ACADVL null HEK293T cell lines to examine VUS pathogenicity. Cell lines contained no IVDH or VLCAD protein or enzyme activity, respectively. Expression plasmids containing VUSs were synthesized and transfected null cells assayed with a high through-put system with results comparing favorably to those obtained with patient fibroblasts. I examined peroxisome proliferator activated delta receptor (PPAR) activators as a treatment for VLCADD, predicted to improve the expression of ACADVL. Functional studies in patient cells indeed demonstrated an improvement in bioenergetics in some cell lines, confirming a potential for therapy, but indicating the need for a personalized medicine approach based on patient genotype. NBS has been designated as one of the top public health successes of the 20th century, and has continued to expand in the past two decades to include more than 50 disorders. However, techniques to confirm the diagnosis in screen positive babies has lagged. VUS pathogenicity confirmation provides a major public health benefit to the newborn screening community. In addition, targeting therapeutics to specific genetic disease variants will maximize public health outcomes of NBS. The technologies utilized here are generalizable to many IEMs and will improve the testing and treatment of affected babies identified by NBS