UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Impact of missense mutations in recessive Mendelian disease: insight from Meckel syndrome

Supervisors: Dr Dagan Jenkins, Dr Jeshmi Jeyabalan-Srikaran

In genetic disease, variation in clinical presentation can be attributed to genetic and environmental modifiers, as well as allelic variation. While it is often difficult to unambiguously identify modifiers in rare genetic disorders, it is generally accepted that understanding allelic variation could provide patients with important information about the severity of their disease. Advances in this area have previously been stifled by technical limitations preventing systematic modelling of different mutations in experimental systems. However, this has changed in recent years with advances in gene-editing, where it is now possible to engineer exact patient mutations in cell lines and model organisms. Our lab has developed a pipeline which we have used to create >60 cell, zebrafish and mouse lines carrying such mutations, and to characterize a variety of phenotypes in them [e.g. refs. 1,2]. To understand allelic variation, we are particularly interested in modelling missense mutations which may result in a more mild clinical presentation than nonsense or frameshift mutations which result in gene ‘knockout’. How is it that missense mutations retain a significant degree of gene function and can we use this information to understand the underlying mechanisms of disease? An example of this relates to the gene, TCTN3, whereby patients with bilallelic nonsense/frameshift mutations have Meckel syndrome – which is prenatally lethal – whereas a p.Gly314Arg missense mutation in the same gene causes the related but less severe Joubert syndrome, where patients survive after birth albeit with severe retinal, renal and neurological disease [3,4].

Aims/Objectives and Methods:
Aim 1: To generate clonal cell lines carrying frameshift or missense mutations in TCTN3
Methods - We will first produce clonal hTERT RPE-1 and IMCD3 cell lines carrying biallelic frameshift mutations in TCTN3, as previously described [2]. We will use multiple independent guide RNAs (gRNAs) to control for potential off-target mutations. We typically find that 40-70% of genomic DNA is successfully targeted using this system, as assessed by next generation sequence analysis (MiSeq) of clones in a 96 well plate format. We previously demonstrated the utility of this approach [2], where knockout cell lines revealed that IFT80 is absolutely required for initiation of ciliary axonemogenesis. Importantly 100% of cells exhibited an identical phenotype, a result that had never previously been achievable using gene knockdown. Next we will use our optimised pipeline to systematically create multiple clonal cell lines carrying the p.Gly314Arg missense mutation using an homology directed repair oligo with 60bp homology arms. We will characterise cell clones in detail using qPCR to monitor gDNA copy number, transcript stability and splicing of mutation-encoding exons, and we will use Western blotting to assess gene product production and protein (in)stability.

Aim 2: To characterise cellular phenotypes associated with frameshift versus missense mutations
Methods – We will use the OperaTM High Content Screening Platform and Harmony 3.5.2 automated image analysis software to analyse all of the cell clones generated in Aim 1 in parallel in a 96 well plate format. First, we will monitor known ciliary phenotypes, including basal body, IFT, ciliary transition zone, axoneme, membrane and tip markers. We will also monitor DNA damage repair (H2AX staining), as this process has been linked to cilia. Using qPCR for Ptch1 and Gli1, we will also measure hedgehog signalling which is a quantitative readout of ciliary function that is dependent on ciliary trafficking. We will also perform unbiased phenotyping in an attempt to identify novel phenotypes. Fixed cells will be labelled with EdU (to label proliferating cells), CellMask (cytoplasm staining) and DAPI (nuclei). As part of a separate project, we are performing quantitative proteomics to identify all interacting partners of TCTN3 and the effect of the missense mutations on these, and this will guide further hypotheses about cellular and molecular phenotypes that we will analyse using appropriate assays.  

Aim 3: To characterise genetic threshold effects relating to TCTN3 patient mutations in mice
Methods – The current model is that different alleles cause loss of gene function to different degrees such that allelic variation contributes clinical variation according to genetic threshold effects (Figure 1). We have already successfully generated two lines of mice carrying either the p.Gly314Arg mutation or a frameshift mutation in Tctn3. Direct comparison of missense and frameshift mutant mice will allow us to test the hypothesis that missense mutations are hypomorphic, as for cell lines. For all three compound genotypes, we will monitor viability, gross morphology, skeletal development and embryonic tissue patterning using techniques we routinely use in our lab. Crucially, the production of mutant mice will allow us to analyse disease-relevant cell types, including primary osteoblasts and osteoclasts. Isolation of these cell types will allow us to test new hypotheses about disease mechanisms.

Production and molecular characterisation of TCTN3 mutant cell lines (Aim 1) - Year 1
Cellular phenotyping (Aim 2-3) - Year 2-3
Establishment of a Tctn3 breeding colony and analysis of Mendelian ratios/gross phenotyping (Aim 3) – Year 1
Characterisation of detailed mouse phenotypes (Aim 3) – Year 2-3

gene x image

References (*denotes publications from the host lab):
*1) Seda et al. A CRISPR/Cas9-generated zebrafish mutant implicates PPP2R3B loss in idiopathic scoliosis pathogenesis in Turner syndrome. bioRxiv doi: https://doi.org/10.1101/413526.
*2) Taschner et al. Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis. Elife. doi: 10.7554/eLife.33067.
3) Thomas et al. TCTN3 mutations cause Mohr-Majewski syndrome. Am J Hum Genet. 91:372--8.
4) Huppke et al. Tectonic gene mutations in patients with Joubert syndrome. Eur J Hum Genet. 23:616-20.