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.

Fetal skeletal dysplasia is a disease that is difficult to distinguish these types of diseases during the fetal period. Due to the difficulty of fetal ultrasound diagnosis, the severity of fetal skeletal dysplasia is extremely difficult to assess. For this condition, we analyzed 79 fetal samples of skeletal dysplasia from the third affiliated hospital of Zhengzhou University, China from August 2018 to April 2020, which had undergone prenatal whole exome sequencing(WES). By comparing the results of whole-exome sequencing and fetal ultrasound test results, we find that the fetal short limb phenotype found in the range of FL<-4.0SD or HL<-4.0SD through ultrasound test is closely related to FGFR3 gene mutation , and the correlation is stronger when accompanied by macrocephaly. We also find that the fetal limb curved phenotype is closely related to COL1A1 gene mutation. At the same time, we find that nasal dysplasia during fetal period is also a common phenotype of abnormal results detected by whole exome sequencing. Overall, our research shows that WES has different detection rates for various skeletal abnormalities according to the different types of ultrasound detection results, which provides a meaningful guidance for clinical diagnosis of fetal skeletal dysplasia.