Report of new variants in PPIL1 underlying type 14 pontocerebellar hypoplasia and their associated phenotypic manifestations in two fetuses

Mutations in the PPIL1 gene have been linked to type 14 pontocerebellar hypoplasia (PCH14); however, prenatal clinical characteristics of PCH14 caused by mutations in the PPIL1 gene have not been reported. This study reports the first prenatal case of PCH14 diagnosed by whole‐exome sequencing (WES). Two fetuses with severe microcephaly and cerebral dysplasia, along with their parents, underwent WES. The effects of the discovered PPIL1 variants on PPIL1 protein function were investigated using bioinformatics tools. WES revealed two compound heterozygous missense mutations in PPIL1, c.376C > G (p.His126Asp) and c.392G > T (p.Arg131Leu), inherited from the mother and father, respectively. The co‐segregation of PPIL1 mutations in this family was confirmed using Sanger sequencing, identifying two PCH14‐affected fetuses. Bioinformatics analysis revealed that these mutations could disrupt the formation of hydrogen bonds, altering the structural stability of the PPIL1 protein. This study is the first to define the clinical characteristics of PCH14 during pregnancy and reports a novel heterozygous missense variant, expanding the PCH14‐related mutational spectrum of PPIL1.

It is characterized by congenital progressive microcephaly and significantly delayed psychomotor development after birth, including intellectual disability, absent motor skills, and language impairment.
Additionally, axial hypotonia, spastic quadriplegia with brisk tendon reflexes, and early onset intractable epilepsy may occur. Most patients exhibit abnormalities in the posterior fossa, including pontocerebellar hypoplasia, agenesis or partial agenesis of the corpus callosum, and simplification of gyrus on magnetic resonance imaging (MRI) of the brain. Using whole-exome sequencing (WES), Chai et al. (2021) identified homozygous or compound heterozygous variants in the PPIL1 gene that affect splicing integrity as the cause of PCH14. Up-to-date, only 10 pathogenic PPIL1 variants related to PCH14 have been reported.
This study reports the first prenatal case of PCH14 diagnosed by WES. Two pregnancies in a non-consanguineous Chinese family were terminated due to severe fetal microcephaly (MC) with dysgenesis of the corpus callosum and delayed gyrification. We identified novel compound heterozygous variants in the PPIL1 gene, p.His126Asp and p.Arg131Leu, inherited from their parents in the affected fetuses. This report highlights the prenatal clinical phenotype of PCH14.

| METHODS
A healthy woman had three consecutive pregnancies, and an ultrasound revealed abnormalities in two of them. Therefore, we sequenced the whole-exome of the two abnormal fetuses and the couple to establish the genetic cause of the abnormal pregnancies.
This study was conducted in accordance with the Declaration of Helsinki and approved by the Ningbo Women and Children's Hospital Ethics Committee (NO.EC2020-014). The couple provided written informed consent to conduct this study and publish their data.
Genomic DNA was isolated from skin tissue of two aborted fetuses and the peripheral whole blood of the parents using a QIAamp DNA Kit (Qiagen, Hilden, Germany). Sequencing libraries were generated by probe hybridization and targeting exons of approximately 20,000 genes, adjacent splicing regions ($20 bp), and the entire mitochondrial genome. Prior to sequencing on an Illumina HiSeq2500 platform, the enriched libraries were subjected to quality control evaluation. Sequencing reads with a quality score below 20 were filtered out. The BWA program was used to map the filtered sequencing data to the GRCh37 (hg19) human genome reference sequence. Single nucleotide variations (SNVs) and insertion-deletion (indel) variations were identified using GATK Haplotype Caller and subsequently annotated using Annovar with variants having a minor allele frequency (MAF) of 1% in public databases such as 1000 Genomes Project (1000 GP), Genome Aggregation Database (gnomAD), and Exome Sequencing Project (ESP). Variants were analyzed for potential pathogenicity using various tools including SIFT, Polyphen2, Mutation Taster, Mutation Assessor, CADD, and REVEL. We followed the pathogenicity interpretation criteria recommended by the American College of Medical Genetics (ACMG) (Richards et al., 2015).
Sanger sequencing was performed on all family members, including the unaffected daughter, using an ABI 3500X DNA sequencer to validate the variants (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). Primers were designed using the tool Primer 5 (forward, 5 0 -AACTCACCCAAGATGCCAGG-3 0 ; reverse, 5 0 -GCCTTTGTACAGG GCAGAG A-3 0 ) and polymerase chain reaction (PCR) was carried out under the following conditions: pre-denaturation at 94 C for 5 min; denaturation at 94 C for 30 s, annealing at 60 C for 30 s, and extension at 72 C for 45 s for a total of 30 cycles; followed by extension at 72 C for 10 min and storage at 4 C. The PCR products were then purified using ExoSAP-IT PCR Product Cleanup (Applied Biosystems) and sequenced with BigDye Terminator v3.1 (Applied Biosystems). The sequencing data were analyzed using Chromas software.
PPIL1 amino acid sequences were obtained from the NCBI database. Homology modeling available in the Swiss model online server was used to simulate the 3D structure of PPIL1 protein sequences with and without mutations. PyMOL was used to view protein models that met the criteria of VERFY-3D (https://servicesn.mbi.ucla.edu/ Verify3D/) and ProCheck (https://www.ebi.ac.uk/thornton-srv/ software/PROCHECK/).

| Clinical report
At 26 + 6 weeks of gestation, a 32-year-old healthy pregnant woman with a fetus displaying various brain abnormalities was referred to the prenatal diagnostic center at Ningbo Women and Children's hospital following a routine prenatal ultrasound. The woman was in a nonconsanguineous union and had three consecutive pregnancies, two of which were terminated due to fetal brain abnormalities. The first fetus, at 26 + 6 weeks of gestation, presented with a decreased head circum-  The OMIM classifies PCHM as a PCH14 subtype. Clinical features of PCH14 include hypotonia, seizures, psychomotor retardation, intellectual disability, and progressive microcephaly. Currently, 10 mutations in PPIL1 have been linked to PCH14, including 8 missense variations, a splicing mutation, and a duplication (Chai et al., 2021).
In this study, we performed WES on two aborted fetuses with brain abnormalities and their parents. During the second trimester, both fetuses exhibited reduced HC and abnormal brain development, including ACC and simplification of gyrus. While the inferior vermis of the cerebellum was absent in the first fetus, it was present in the second fetus. The WES analysis revealed two heterozygous missense mutations in PPIL1 which were inherited from both parents. Additionally, Sanger sequencing of the parents and the sibling indicated that PPIL1 mutations segregate into phenotypes. Clinical features of PCH14, including progressive microcephaly after birth and cortical alterations with gyrus simplification and changes in the posterior fossa, have previously been noted (Chai et al., 2021). Furthermore, PCH was previously classified as a neurodegenerative condition with a prenatal onset that primarily affects, but was not limited to, the cerebellum and pons (Barth, 1993). Based on the typical clinical signs of abnormally decreased HC and brain dysplasia, the two fetuses displayed sufficient evidence of an inherited neurodevelopmental disorder. Therefore, we diagnosed the two fetuses with PCH14 owing to compound heterozygous mutations in PPIL1.
PPIL1 is a protein that interacts with the pre-mRNA splicing factors PRP17, SKIP, and RBM22 (Bessonov et al., 2008). During the B act stage of spliceosome formation, PPIL1 is recruited to the major spliceosome complex by binding to SKIP (Rajiv & Davis, 2018). Chai et al. reported that all PPIL1 variants impact the stability of the interaction with SKIP, and found that PPIL1 mutations result in a greater number of significant differential splicing events, such as mutually exclusive exons, skipped exons, alternative 5 0 and 3 0 splice sites, and retained introns, mostly influencing the splicing of short and high-GC-content introns (Chai et al., 2021). PPIL1 variants also alter the splicing of genes that are susceptible to splicing errors in brain neurons. The two  variants identified in this study were predicted to influence protein function, as the Arg131 residue of PPIL1 is known to be critical for its interaction with SKIP (Davis et al., 2010). The PPIL1-SKIP interaction causes the disorder-to-order transition of SKIP, which plays a critical role in coordinating dynamics across the spliceosome (Wang et al., 2010). Specifically, PPIL1 residues Arg131 and Ile97 create a hydrophobic groove with the SKIP Pro65 linker (Stegmann et al., 2010;Wang et al., 2010). Additionally, the side chain of Arg131 in PPIL1 forms a hydrogen bond with that of Glu66 in SKIP (Stegmann et al., 2010). The variant Arg131Gln disrupts the structure of PPIL1 and prevents its binding to SKIP (Chai et al., 2021). Consequently, we hypothesized that the Arg131Leu variant impairs the connection between SKIP and PPIL1, thereby altering the splicing processes.
His126 has been identified as the active site residue of PPIL1 (Davis et al., 2010). PPIL1 and PRP17 interact at the active site to form an active PPIase-substrate pair. PPIL1 can accelerate the isomerization of PRP17 Gly94-Pro95 (Chai et al., 2021). However, these enzymatic interactions are not essential for protein activity. Mutations in the enzymatic region (Ala99Thr, Gly109Cys, and Ala101 Asp106dup) alter the protein folding and stability of PPIL1, leading to brain-specific PCH14 (Chai et al., 2021). In this study, we found that the residue His126 is strictly conserved across all animal species and that the His126Asp substitution affects the formation of hydrogen bonds between His126 and Asn102 residues in PPIL1. Therefore, we hypothesized that the His126Asp variant affects PPIL1 protein stability. However, further functional investigations are necessary to understand the effect of PPIL1 variations and investigate their underlying molecular processes.
PPIL1 mutations have been identified as the source of impaired brain development causing PCH14, whose mechanism is disruption of splicing integrity and significant spliceosomopathies (Chai et al., 2021). The present study broadened the mutation spectrum of PPIL1 and identified the first prenatal instances of PCH14. In addition, we show that prenatal genetic testing can identify PCH14, which has important consequences for prognostic and reproductive counseling.
The implications of the recently discovered PPIL1 mutations in neurodevelopment require further research.

AUTHOR CONTRIBUTIONS
Haibo Li and Yuxin Zhang conceived the study, analyzed the molecular results, and drafted the manuscript. Lulu Yan and Min Xie interpreted the data on the variants. Jiangyang Xue helped revise the manuscript.
Xumian Yang, Yongming Xue, and Liyun Tian collected clinical data. All authors reviewed and approved the final version of the manuscript.