Characterization of Novel Intron Splice Mutations in Patients with Propionic Acidemia

Abstract

Propionic Acidemia (PA) is a rare genetic metabolic disorder characterized by a deficiency in propionyl CoA carboxylase (PCC), an enzyme responsible for the proper breakdown of certain proteins and lipids. As a result, patients exhibit an abnormal accumulation of toxic organic acids, which can cause detrimental health problems. PA derives from mutations in PCCA and PCCB genes, which are responsible for expressing the vital subunits of PCC. Mutations in intronic sequences may cause errors in RNA processing, specifically with RNA splicing. If intronic segments are improperly included in the final mRNA form, the resultant products become nonfunctional. The goal of this project was to determine whether novel intron splice site mutations induce improper mRNA processing and, thus, nonfunctional proteins. To achieve this, we collaborated with genetics testing company Prevention Genetics to construct minigene plasmids containing intron mutations from patients. The minigene system was transfected into mouse fibroblast cells to allow mRNA processing to occur. RNA was isolated, reverse‐transcribed into cDNA, and sequenced to determine whether the mutated introns are splicing differently from the wildtype. Initial results indicate several identified intronic mutations contribute to an altered splicing pattern in patients with PA. Knowledge of the effect of these mutations on gene expression will strengthen the genetic database to help doctors diagnose PA in their patients, as well as help develop preventive and personalized treatments.Support or Funding InformationUWSP OSCAR Travel Grant (Up to $3,000 for three students)