Prenatal phenotype of PNKP-related primary microcephaly associated with variants in the FHA domain

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Detailed pathological examination of a male fetus revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities in a sibling fetus. Prenatal trio exome sequencing identified compound-heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sibling fetus. Through RNA analyses, we characterized skipping of exon 4 affecting the PNKP Forkhead-associated (FHA) and Phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the same splice-donor variant c.1029+2T>C in trans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro) and c.151G>C, p.(Val51Leu), respectively). RNA-seq showed complex splicing events for c.1029+2T>C and c.151G>C. Computational modelling and structural analysis revealed significant clustering of missense variants in the FHA domain, with some variants potentially generating structural damage. Our detailed clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modelling, we highlight the mutational complexity and exemplify a framework for variant characterization in this multi-domain protein.