期刊
HUMAN MUTATION
卷 43, 期 1, 页码 56-66出版社
WILEY
DOI: 10.1002/humu.24292
关键词
deep intronic variation; PAH; phenylketonuria; RNA splicing; whole-genome sequencing
资金
- National Key Research and Development Program of China [2016YFC1000307]
- Natural Science Foundation of Gansu Province [18JR3RA036]
The study identified deep intronic variants in the PAH gene that may impact the genetic diagnosis of PKU, with in silico predictions suggesting potential splice site alterations. Furthermore, experimental analysis confirmed the effects of these variants on pseudo-exon inclusion in PAH mRNA, enhancing current understanding of PAH genotypes and the molecular mechanisms underlying PKU.
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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据