|Microcephaly describes a reduced brain size in patients that manifests in an evidently reduced head circumference compared to healthy individuals of same age, sex and ethnicity. Even though microcephaly is still considered a rare clinical feature in Europe, hundreds of syndromes manifest with microcephaly indicating that the identification and characterization of microcephaly-associated genes can help to shed light on cellular processes important for brain development, but also beyond.
Since a substantial number of microcephaly patients remain without a genetic diagnosis, I investigated in the first part of my thesis whole-exome sequencing (WES) data of three patients from non-consanguineous families. In the first family of Pakistani descent, different family members were presenting with varying severities of syndromic microcephaly pointing towards an autosomal dominant or X-linked mode of inheritance. Using a combination of WES and deep amplicon sequencing, I identified the c.848delA mutation in the novel and highly convincing microcephaly-associated candidate gene DLG3 and confirmed a low-level somatic mosaicism in one affected family member. In the second family, I applied WES and identified two novel nonsense mutations (c.442C>T; p.Gln148* and c.2557C>T; p.Arg879*) in the DDX11 gene, associated with Warsaw breakage syndrome. With this, I expanded the spectrum of mutations and phenotypes associated with the DDX11 gene. In the last family, I identified two novel mutations affecting splicing in the FANCI gene (c.2890-2A>G and c.2108A>G; p.Asp703Gly), associated with Fanconi anemia.
In the second part of my thesis, I characterized the novel microcephaly-associated gene KMT2B and the previously identified de novo c.5462insC (p.Leu1822Thrfs*12) mutation in more detail. KMT2B encodes a H3K4 methyltransferase and exerts essential functions especially during early embryonic developmental stages. Ectopic overexpression of wild-type and p.Leu1822Thrfs*12 KMT2B in COS-7 cells confirmed stability of the mutant protein, albeit the nuclear import was impaired. Further characterization of endogenous RNA and protein levels in patient-derived cells demonstrated a reduction of KMT2B RNA and protein. I applied deep amplicon sequencing and verified stability of the c.5462insC transcript. However, I did not detect the p.Leu1822Thrfs*12 protein, neither by using Western Blot nor by using mass spectrometry, pointing towards protein instability and favoring haploinsufficiency as disease underlying effect. The bulk levels of H3K4me3 were not affected in patient-derived fibroblasts, and cells showed no increased apoptosis activation. Patient-derived fibroblasts were successfully reprogrammed into induced pluripotent stem cells (iPSC), and subsequent RNA sequencing (RNAseq) uncovered differential expression of several WNT-signaling-associated genes. Differentiation of iPSC into early neural progenitor cells (NPC) confirmed a normal course in the expression of pluripotency marker OCT4 and the NPC markers SOX1 as well as PAX6 indicating successful initiation of differentiation. In order to investigate the pathomechanism in vivo, I used a CRISPR/Cas9-based strategy and generated a knock out (KO) model of KMT2B in the zebrafish Danio rerio. Targeting the KMT2B homologs kmt2ba and kmt2bb, I showed that homozygous kmt2ba as well as kmt2bb KO animals were viable. KO of kmt2bb, but not kmt2ba, resulted in growth retardation and microcephaly at 14 days post fertilization (dpf), thereby mimicking the phenotype of our patient. Whole-mount immunostainings for activated Caspase 3 illustrated increased activation of apoptosis in affected embryos at 32 hours post fertilization (hpf). In order to characterize early corticogenesis prior to the onset of apoptosis, I investigated a murine conditional heterozygous Kmt2b KO model (heterozygous Kmt2b cKO) at embryonic day 13.5 (E13.5). Immunohistochemistry did not reveal any differences between control and heterozygous Kmt2b cKO embryos. Based on this, only a very mild phenotype was to be expected and the sensitive next-generation sequencing (NGS)-based approach of single-cell RNA sequencing (scRNAseq) was applied for gene expression analysis. Subsequently, analysis of scRNAseq data pointed towards a mild imbalance in the number of proliferating vs. non-proliferating radial glial cells (RGCs) in developing forebrains of heterozygous Kmt2b cKO embryos.
In summary, via WES analysis, the data of my PhD thesis end the long odyssey of an unknown diagnosis for three families and provide important information about the underlying genetic cause and open perspectives in the field of therapeutic approaches. The characterization of KMT2B and the c.5462insC mutation provides novel insights into the pathomechanism of microcephaly and also into cellular processes beyond.