Identification of novel deep intronic PAH gene variants in patients with
phenylketonuria
Abstract
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, whole-genome sequencing (WGS) was
performed in four patients with PKU with unknown genotype to identify
deep intronic or structural variants. Three novel heterozygous variants
(c.706+368T>C; c.1065+241C>A; and
c.1199+502A>T) were identified in a deep PAH gene intron.
The c.1199+502A>T variant was detected in 60% (6/10) PKU
patients. In silico prediction showed that the three deep variants may
impact splice site selection and result in inclusion of a pseudo-exon.
The c.1199+502A>T PAH minigene and reverse transcription
PCR of blood RNA in a patient with PKU and compound heterozygous
variants (c.1199+502A>T/ c.1199G>A) confirmed
that the c.1199+502A>T variant creates a novel branch point
and leads to the inclusion of a 25 bp in PAH mRNA
(r.1199_2000ins1199+538_1199+562). Furthermore, the
c.1199G>A mutation leads to the retention of an additional
17 nt in the PAH mRNA transcript (r.1199_2000ins1199+1_1199+17). These
results expand the PAH genotypic spectrum and highlight that deep
intronic analysis of PAH can improve genetic diagnosis in undiagnostic
patients.