Delineating genetic underpinnings and disease pathogenesis of primary microcephaly and Filippi syndrome

The genetic underpinnings of autosomal recessive genetic disorders have been extensively scrutinized for decades. Nevertheless, the requisites for dissecting the role of causative gene variants are indispensable for elucidation of underlying disease pathogenesis. Since thoroughgoing research has been conducted to decipher complexities of brain development, congenital microcephaly (CM) stands prominent as an immaculate model of the in utero development of the brain. The isolated form of CM — primary microcephaly (MCPH) — is a rare disorder portrayed by reduced brain size and intellectual disability. Contrarily, the syndromic form of CM has a shared genetic architecture and expounds additional roles of CM proteins in other organ systems. This study aimed to comprehend the underlying pathology of 17 families afflicted with different inherited conditions. A consanguineous Pakistani MCPH family was characterized to harbour a mutation in NUP37. The mutational consequences of this gene, along with NUP107 — mutated in syndromic microcephaly — were studied in patient derived cells to delineate the pathomechanisms. Both genes encode crucial components of the outer rings of the nuclear pore complex (NPC). Mutant proteins were discerned to be unstable and dislocated from the nuclear envelope in patient cells. Comparable mutational effects were observed on NUP160 and NUP133 — further components of the NPC. Consequently, abnormalities of the nuclear shape, chromatin organization, nucleoli as well as reduction of nuclear pore density were revealed in primary fibroblasts carrying a mutation in NUP37. My observations accentuate that the components of the NPC play a pivotal role in embryonic brain development and that a defective function of nuclear pore proteins leads to congenital microcephaly with or without associated anomalies. CM is also a prominent feature of an extremely rare disorder, Filippi syndrome (FLPIS) typically characterized by craniodigital features (syndactyly of fingers and/or toes), short stature, intellectual disability and peculiar facial gestalt. A slight variability in clinical presentation causes an allelic disorder called Filippi like syndrome (FLS). Hitherto, only CKAP2L has been biochemically characterized for unraveling disease pathogenesis of FS. In this study, mutational consequences of CSNK2B — a novel candidate gene based on two missense and a nonsense mutation identified in three unrelated cases of FS and FLS — were explored. CSNK2B encodes the β subunit of CK2, which is the master regulator of all kinases. Mutant CSNK2B showed abnormal expression at transcript as well as 2 protein level. One of the missense mutations resulted in an impaired cross talk between α and β subunits and both missense mutations compromised the kinase activity of CK2. Delving deeper into the mutational effects, two crucial pathways were mainly investigated; canonical Wnt signaling (CWS) and DNA damage response (DDR). In CWS, a compromised interaction of CK2β with two key regulators of Wnt signaling, DVL3 and β-Catenin (along with its dysregulated expression) was observed. The likely effects of β-Catenin dysregulation on Wnt and DDR related genes were traced by transcriptional profiling. Furthermore, cell cycle arrest and elevated apoptosis were observed in mutant LCLs due to delayed DDR. Additionally, whole proteome profiling demonstrated disturbed interaction of 38 proteins crucial for pivotal cellular pathways, specifically CWS. Intriguingly, whole phosphoproteome profiling revealed impaired phosphorylation of more than 300 CK2 substrates in patient LCLs, thus suggesting global mutational effects. In general, it was noticed that the impact of both missense mutations slightly differed from each other on protein level. Data suggest that mutations in CSNK2B exert pleiotropic mutational effects, thereby causing FS and FSL. In the remaining families of FS and MCPH, whole-exome sequencing and subsequent bioinformatics analyses revealed strong novel candidate genes for each of them. Taken together, this study provides novel insights into the genetic and functional bases of MCPH and FS. It lays the foundation for future genomic and therapeutic investigations for these and related disorders.