Biallelic pathogenic variants in PNKP are associated with microcephaly and early-onset seizures (MCSZ), ataxia with oculomotor apraxia type 4 and Charcot-Marie-Tooth disease type 2B2. We describe the clinical and neuroimaging features of 27 new patients with PNKP variants. All patients presented with early-onset seizures, congenital microcephaly and intellectual disability. In addition, we compared our results with data in the literature. Twenty-five patients presented with the classic MCSZ phenotype, while two showed a more severe clinical phenotype. The brain imaging features of the 25 patients varied significantly, but widening of the frontal lobe gyri with frontal hypoplasia and prominent cerebellar folia (consistent with atrophy) could point to PNKP-related microcephaly . In contrast, the two patients with severe phenotype showed additional brain MRI features of white matter loss and pontocerebellar hypoplasia fulfilling the criteria of microlissencephaly. Exome sequencing identified seven different PNKP variants, including two novel ones. The c.1253_1269dup p.(Thr424GlyfsTer49) and c.1381_1383dup p.(Asn461dup) variants, each was recurrent in 10 patients (37%), while the c.1381_1383del p.(Asn461del) variant was recurrent in four patients (14.8%). Haplotype analysis confirmed that the p.Asn461dup variant has a founder effect in our population. No genotype-phenotype correlation was observed in our cohort. Our results provide 'microlissencephaly' as an emerging distinct phenotype linked to PNKP variants. As such, PNKP variants could be associated with four overlapping subgroups that lie along a unifying phenotypic continuum.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSCs) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives an unprecedented opportunity to explore human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e. Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy syndrome, tuberous sclerosis syndrome) and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems in the realm of personalized medicine.

Microlissencephaly is a subtype of congenital microcephaly characterized by extreme microcephaly with simplified gyral pattern. Other brain malformations may accompany it. WDR81 encodes a multi-domain transmembrane protein that is predominantly expressed in the brain and is thought to play a role in endolysosomal trafficking and autophagy. We reported two siblings with microlissencephaly born to consanguineous Turkish parents and reviewed all previously reported patients with WDR81 variants presenting with severe microcephaly accompanied by congenital brain malformations. Whole-exome sequencing was performed on both siblings. Sanger DNA sequencing was performed on the patients' parents. We also examined LC3, p62, and Beclin 1 mRNA and protein expression levels from blood samples of both siblings using real-time PCR and Western blotting, respectively. Whole-exome sequencing revealed a novel biallelic c.4157+5G>A splice variant in the WDR81 gene in both siblings. In our study, LC3, p62, and Beclin 1 mRNA expression levels were high, LC3 protein expression level was also high and p62 and Beclin 1 protein expression levels were low. High LC3 and low p62 protein expression levels supported studies concluding that WDR81 inhibits autophagy through PI3KC3 inhibition, while low Beclin 1 protein expression level supported studies concluding that autophagy is suppressed in case of loss of function variants in the WDR81 gene. We suggest that due to its role in endolysosomal trafficking, WDR81-related diseases should be included in vesicular trafficking disorders.

The development of the cerebral cortex involves a series of dynamic events, including cell proliferation and migration, which rely on the motor protein dynein and its regulators NDE1 and NDEL1. While the loss of function in NDE1 leads to microcephaly-related malformations of cortical development (MCDs), NDEL1 variants have not been detected in MCD patients. Here, we identified two patients with pachygyria, with or without subcortical band heterotopia (SBH), carrying the same de novo somatic mosaic NDEL1 variant, p.Arg105Pro (p.R105P). Through single-cell RNA sequencing and spatial transcriptomic analysis, we observed complementary expression of Nde1/NDE1 and Ndel1/NDEL1 in neural progenitors and post-mitotic neurons, respectively. Ndel1 knockdown by in utero electroporation resulted in impaired neuronal migration, a phenotype that could not be rescued by p.R105P. Remarkably, p.R105P expression alone strongly disrupted neuronal migration, increased the length of the leading process, and impaired nucleus-centrosome coupling, suggesting a failure in nucleokinesis. Mechanistically, p.R105P disrupted NDEL1 binding to the dynein regulator LIS1. This study identifies the first lissencephaly-associated NDEL1 variant and sheds light on the distinct roles of NDE1 and NDEL1 in nucleokinesis and MCD pathogenesis.

Biallelic variants in PPIL1 have been recently found to cause a very rare type of pontocerebellar hypoplasia and congenital microcephaly in which simplified gyral pattern was not observed in all of the patients. Here, we describe a series of nine patients from eight unrelated Egyptian families in whom whole exome sequencing detected a previously reported homozygous missense variant (c.295G>A, p.Ala99Thr) in PPIL1. Haplotype analysis confirmed that this variant has a founder effect in our population. All our patients displayed early onset drug-resistant epilepsy, profound developmental delay, and visual impairment. Remarkably, they presented with recognizable imaging findings showing profound microcephaly, hypoplastic frontal lobe and posteriorly predominant pachygyria, agenesis of corpus callosum with colpocephaly, and pontocerebellar hypoplasia. In addition, Dandy-Walker malformation was evident in three patients. Interestingly, four of our patients exhibited hematopoietic disorder (44% of cases). We compared the phenotype of our patients with other previously reported PPIL1 patients. Our results reinforce the hypothesis that the alterative splicing of PPIL1 causes a heterogeneous phenotype. Further, we affirm that hematopoietic disorder is a common feature of the condition and underscore the role of major spliceosomes in brain development.