Altered gene transcription profiles in fibroblasts harboring either TK2 or DGUOK mutations indicate compensatory mechanisms

Abstract Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder characterized by a reduced amount of mtDNA, which impairs synthesis of respiratory chain complexes. MDS has been classified into two main groups, the hepatocerebral form affecting liver and the central nervous system, and the myopathic form targeting the skeletal muscle. We have compared the molecular genetic characteristics of fibroblasts derived from two patients harboring TK2 mutations with two harboring mutations in DGUOK gene. Real-time PCR revealed mtDNA depletion in dGK-deficient fibroblasts (dGK − ) but not in TK2-deficient cells (TK2 − ). Real-time RT-PCR and western blotting demonstrated significant differences in the expression of the human equilibrative nucleoside transporter 1 (hENT1) at the mRNA and protein levels. hENT1 transcript and protein were increased in quiescent control and TK2 − fibroblasts relative to cycling cells. In contrast, hENT1 was stable in quiescent and cycling dGK − cells. Moreover, siRNA down-regulation of hENT1, but not of TK1, induced mtDNA depletion in TK2 − fibroblasts indicating that hENT1 contributes to the maintenance of normal mtDNA levels in cells lacking TK2. Transcripts for thymidine phosphorylase, the mitochondrial transcription factor A (TFAM), and the polymerase gamma (Pol γ), were reduced in dGK − , but not in TK2 − cells while the mRNA expression of thymidylate synthase (TS) increased. Our results suggested differential gene expression in TK2 and dGK-deficient fibroblasts, and highlighted the importance of hENT1 as a compensatory factor in MDS disorder.