Understanding the Cellular Consequences of Meier-Gorlin Syndrome

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterised by short stature, microtia and patella aplasia/hypoplasia. Genetic variants, which cause MGS, have previously been found in genes involved in the initiation of DNA replication. This study examined the cellular consequences of novel variants in MGS genes; ORC1, CDC45 and DONSON. The ORC1 gene encodes an essential component of the pre-replication complex and functions during late mitosis/early G1 phase to initiate DNA replication. MGS individuals previously reported to have variants in ORC1 have had at least one variant in the bromo-adjacent homology (BAH) domain at the N-terminus of the protein, a region suggested to be important for protein-protein interactions. In this study we report a patient with a novel homozygous variant (c.1865T>C, p.L622P) in the ATPase Associated with a wide range of cellular Activities (AAA) domain at the C-terminus of ORC1, and attempted to investigate how variants in this region of the protein lead to a MGS phenotype using CRISPR-Cas9 genome editing and a minigene splicing assay. Due to time constraints, results were inconclusive and more work is required to better understand variants in this area of ORC1. CDC45 encodes an essential component of the pre-initiation and CDC45-MCM-GINS (CMG) complex, required during G1 and S-phase. The novel homozygous CDC45 variant (c. 1441-2 A>G) under investigation in this study was found in two siblings presenting with a severe MGS phenotype alongside a range of secondary phenotypes some of which are not typically associated with MGS. It was hypothesised that this novel variant may represent the extreme end of the CDC45 phenotypic spectrum. A splicing assay showed that the variant, located within the canonical splice acceptor site for CDC45 exon 16, caused aberrant splicing and use of an alternative 5′ splice acceptor within exon 16. This resulted in a two amino acid deletion (p.Thr481_Lys482del) and a 72.9% reduction in CDC45 mRNA levels, which was confirmed by RT-qPCR analysis using patient fibroblasts. DONSON is required for stabilising replication forks during S-phase when replication stress is encountered. Biallelic variants in this gene have previously been described in patients presenting with Microcephaly-Micromelia syndrome (MIMIS) and Microcephaly Short Stature, and Limb Abnormalities (MISSLA), both of which are characterized by severe microcephaly and a slight reduction in height. In this study we discovered a novel DONSON variant (c.631C>T, p.R211C), for the first time in clinically diagnosed MGS patients, presenting with a global reduction in size. This project used CRISPR-Cas9 genome editing as well as a range of cellular techniques including subcellular localisation, immunocytochemistry and DNA fibre combing to better understand the cellular consequences of this novel MGS gene. Taken together, results confirmed DONSON as a novel MGS gene and showed that MGS variants led to more subtle changes in subcellular localisation, DNA damage, and replication events than variants seen in non-MGS DONSON patients, reflecting the difference in phenotype. This project aimed to study the cellular consequences of novel MGS variants using CRISPR-Cas9 and a variety of cellular techniques to understand the effect these novel variants had on initiation of DNA replication