Titel: Prenatal phenotype of PNKP-related primary microcephaly and unexpected complex variant mechanisms in RNA-analysis
ID: P-ClinG-087
Art: Postertalk
Redezeit: 2 min
Session: Poster Session

Referent: Sonja Neuser (Leipzig/DE)

Abstract - Text


The polynucleotide kinase 3"-phosphatase (PNKP) is involved in the repair of single- and double-strand DNA breaks. Biallelic pathogenic variants in PNKP cause four formally distinct neurodevelopmental and neurological diseases ranging from syndromic childhood manifestations as well as developmental and epileptic encephalopathy to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions with a broad symptomatic variability exist.

By prenatal trio-exome sequencing, we identified two compound heterozygous PNKP variants (c.302C>T, p.(Pro101Leu) and c.498G>A, p.[(=),0?]) in a male fetus with micro- and brachycephaly, brain malformations and microretrognathia diagnosed at 13th gestational week. Segregation analysis confirmed both variants in a previous affected sister fetus. Both pregnancies were terminated and fetopathological examination of the index fetus revealed micrencephaly with pronounced hypoplastic frontal lobes, shortened occipital lobes, missing temporo-parietal lobulation and hypoplastic cerebellum. These findings suggested a recessive primary microcephaly, especially in the view of a similar phenotype in the previous pregnancy.

We performed RT-PCR analysis on RNA from fetal muscle and a paternal PAXgene sample to characterize the silent variant c.498G>A, which affects the last base of exon 4. This showed an in-frame deletion of parts of the FHA- and phosphatase-domains by exon skipping. We retrospectively investigated two unrelated individuals with the same splice-donor variant c.1029+2T>C and a second missense variant. Interestingly, our RNAseq revealed unexpected complex splicing effects. Additionally, all identified missense variants were located in the FHA-domain and computational modelling approved significant clustering of the affected amino acids. The distinct variant effects were analyzed through the 3D protein model.

We conclude that the range of PNKP associated manifestations extends to severe prenatal presentation, indicating a continuous phenotypical spectrum. Syndrome-oriented autopsy and knowledge of distinct fetal phenotypes are crucial for validation and weighing of unknown genetic variants. In silico tools implicate a splice effect for 35 % of all reported PNKP variants. RNA analysis regarding hidden and complex splicing events will support variant interpretation - also for novel identified variants. Genotype-phenotype correlation in PNKP may be related with affection of different domains, but a precise prediction – especially in setting of prenatal counselling – is still not possible.