Identification of novel deep intronic PAH gene variants in patients diagnosed with phenylketonuria

Hum Mutat. 2022 Jan;43(1):56-66. doi: 10.1002/humu.24292. Epub 2021 Nov 16.


Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previously, 94.21% of variants were identified using Sanger sequencing and multiplex ligation-dependent probe amplification. To investigate the remaining variants, we performed whole-genome sequencing for four patients with PKU and unknown genotypes to identify deep intronic or structural variants. We identified three novel heterozygous variants (c.706+368T>C, c.1065+241C>A, and c.1199+502A>T) in a deep PAH gene intron. We detected a c.1199+502A>T variant in 60% (6/10) of PKU patients with genetically undetermined PKU. In silico predictions indicated that the three deep variants may impact splice site selection and result in the inclusion of a pseudo-exon. A c.1199+502A>T PAH minigene and reverse transcription PCR (RT-PCR) on blood RNA from a PKU patient with biallelic variants c.1199+502A>T and c.1199G>A confirmed that the c.1199+502A>T variant may strengthen the predicted branch point and leads to the inclusion of a 25-nt pseudo-exon in the PAH mRNA. Reverse transcription polymerase chain reaction (RT-PCR) on the minigene revealed that c.706+368T>C may create an SRSF2 (SC35) binding site via a 313-nt pseudo-exon, whereas c.1065+241C>A may produce an 81-nt pseudo-exon that strengthens the predicted SRSF1 (SF2/ASF) binding site. These results augment current knowledge of PAH genotypes and show that deep intronic analysis of PAH can genetically diagnose PKU.

Keywords: PAH; RNA splicing; deep intronic variation; phenylketonuria; whole-genome sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Exons
  • Humans
  • Introns
  • Mutation
  • Phenylalanine Hydroxylase* / genetics
  • Phenylketonurias* / diagnosis
  • Phenylketonurias* / genetics
  • Serine-Arginine Splicing Factors


  • SRSF1 protein, human
  • Serine-Arginine Splicing Factors
  • Phenylalanine Hydroxylase