DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria

Mol Genet Metab. 2016 Sep;119(1-2):1-7. doi: 10.1016/j.ymgme.2016.01.001. Epub 2016 Jan 14.


Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients.

Keywords: DNA methylation; PAH(enu2); Phenylketonuria; Toxicity.

MeSH terms

  • Animals
  • Brain / metabolism
  • Brain / pathology
  • Calcium-Binding Proteins
  • DNA Methylation / genetics*
  • Disease Models, Animal
  • Gene Expression Regulation
  • Genomic Imprinting
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Iodide Peroxidase / genetics*
  • Liver / metabolism
  • Liver / pathology
  • Mice
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Phenylalanine Hydroxylase / deficiency
  • Phenylalanine Hydroxylase / genetics*
  • Phenylketonurias / genetics*
  • Promoter Regions, Genetic
  • RNA, Untranslated / genetics


  • Calcium-Binding Proteins
  • Dlk1 protein, mouse
  • Intercellular Signaling Peptides and Proteins
  • RNA, Untranslated
  • Phenylalanine
  • iodothyronine deiodinase type III
  • Iodide Peroxidase
  • Phenylalanine Hydroxylase