Uncovering new mechanisms of craniosynostosis associated with structural and copy number variation, using mouse modelling and human neural crest cells

Growth of the skull to make space for the growing brain is possible because new bone is added to narrow gaps between the skull bones, termed cranial sutures, as the head grows. When one of these gaps fuses, this prevents further growth at the fused suture, a disorder termed craniosynostosis (CRS). This serious condition affects about 350 babies annually in the UK.
CRS has many causes, but we know that in about 25% it occurs because of an altered genetic instruction regulating the complex signalling processes in the cranial sutures. Finding a genetic cause is important for many reasons. For parents, it ends the diagnostic odyssey; for clinical geneticists, it enables correct risks to be given and tests to be recommended; for the medical team, there may be specific complications to screen for. In biochemical disorders, the information can be critical for correct treatment. We can also learn more about how cranial sutures normally function.
Despite this progress, there are many children with CRS in whom a genetic cause is suspected, but cannot be identified. For example in the 100,000 Genomes whole genome sequencing (WGS) Project (100kGP), 119 carefully selected patients/families with CRS have been analysed, but a diagnosis has only been achieved in ~18%. How might we be missing some genetic causes?
Although DNA sequencing technology has achieved remarkable advances, there is still "dark matter" in the genome that is poorly characterised. For example 70% of our genome is made up of repetitive DNA that is hard to sequence and often gets scrambled when it is being copied. Consequently sections of DNA may be present in too many or too few copies (copy number variants - CNVs), or pieces may be altered in their position or orientation (structural variants - SVs). These CNVs and SVs can cause changes to the way nearby genes are expressed (RNA is made); as a result, a protein might be made in a tissue where the gene should normally be silent.
In this project we want to look in more detail for these SVs and CNVs in children with complex CRS who remain without a diagnosis, as we suspect these could cause misexpression in cranial sutures leading to CRS. The 100kGP resource is ideal to study, because it represents the largest resource of WGS of these children in the world.
Our proposed work comprises four broad elements:
First, we want to take another look at the WGS data, using combinations of computational methods to find SV or CNV that might be causative. We will also use two relatively new technologies, nanopore sequencing and optical mapping, which look at genomes in different ways to the previous WGS; we expect these will reveal previously unknown SV and CNV.
Second, for carefully chosen SV or CNV that we suspect may cause CRS, we will make the equivalent rearrangement in mice by genome engineering. If the mice show abnormal skull growth, this provides strong support that the rearrangement is causative of the human condition too. We plan to construct 4 different mouse mutants during the project.
Third, we will test how well a different technology, that avoids the use of mice, might work. This involves inducing human cells to change into a type of cell termed neural crest, starting from a patient blood sample. This cell type is one of the major constituents of cranial sutures. We plan to construct 6 different neural crest lines during the project.
Fourth, we will perform detailed tests comparing mouse tissues and neural crest cells, to see if cell function is disturbed. We will measure expression of the genes around the SV/CNV; assess how the DNA is folded inside the cell; and analyse the chemical modifications of proteins wrapping around the DNA.
Our overall goals will be to extend understanding of the ways by which SV/CNV cause CRS and other diseases; obtain specific comparative data on methods of genome mapping and functional assessment to improve future analyses; and find new diagnoses for patients and families.

Despite adoption of whole genome sequencing in large clinical samples such as the 100,000 Genomes Project (100kGP), finding causative mutations outside the exome remains difficult. For the disorder craniosynostosis (CRS; premature fusion of cranial sutures), the diagnostic rate in 100kGP is only ~18%, despite careful case selection and intensive scrutiny for pathogenic single nucleotide variants (SNV).
Structural and copy number variants (SV/CNV) are potent sources of genetic disease, for example SVs >20 kb are ~50-fold more likely than SNVs to alter gene expression. However, SV/CNV are difficult to identify in short read sequence such as in 100kGP, moreover the clinical pipeline in current use is suboptimal.
We present several examples of SV/CNV segregating with CRS, and propose that the cranial suture acts as a sensor for aberrant gene expression caused by occult SV/CNV, with CRS providing an abnormal phenotypic readout. We will seek further SV/CNV in 100kGP data from cause-unknown CRS, by systematically seeking unique segregating/de novo rearrangements and intersecting the findings with clinical features suggesting a genetic cause. For the strongest candidate SV/CNV, we will construct mouse models and/or patient-derived neural crest cell (NCC) lines, phenotype mice for CRS, and measure functional parameters (RNA expression, including single cell transcriptomics in sutures; topologically-associating domain [TAD] structure; and chromatin landscape in NCC), providing evidence to assist pathogenic interpretation.
Outcomes will include 1) Technology comparison of nanopore sequencing and optical mapping for identification of SV/CNV; 2) Single cell transcriptomic and TAD data will evaluate the cranial suture as a mis-expression sensor, and address how well disturbed TAD structure explains how SV/CNV cause CRS and other diseases; 3) Comparison of results in mouse models and NCC for the same SV/CNV, to determine feasibility of NCC to replace animal use.

Given that this work addresses the problem of maximizing the utility of whole genome sequence (WGS) data generated in the flagship 100,000 Genomes Project (100kGP)-to which the MRC has also contributed £24M- its impact is predicted to be broad. Direct beneficiaries will include applied and basic biomedical scientists, clinicians, patients and policymakers (such as NHS commissioners) wishing to ensure that resources allocated to diagnostic WGS are optimally exploited.
The path ahead is signposted by our preliminary analysis of the WGS data for craniosynostosis (CRS), which showed that a small expert research team can double the diagnostic rate compared to the combined efforts of Genomics England (GE) and the NHS Laboratories. The current proposal seeks to provide an end-to-end solution to identify functionally significant structural and copy number variation (SV/CNV) from WGS data, using CRS as a demonstration project. Scientists at varied points along the applied/pure spectrum will be impacted including bioinformaticians (SV/CNV calling, predicting topologically-associating domain [TAD] structures), genome sequencing technologists (comparison of nanopore sequencing and optical mapping), epigenomicists (integration of RNA/ChIP sequencing and experimentally deduced TAD structures), and developmental biologists (misexpression in cranial sutures and CRS). In particular we expect that bioinformatics prediction of TAD structures and associated experimental analysis will provide evidence on the strengths and limitations of using a TAD-centric approach to dissecting the functional consequences of SV/CNV.
Analogous to identifying why GE/NHS Genetics Laboratories had not prioritized single nucleotide variants in the 100kGP dataset that we consider likely causative, we will find multiple SV/CNV that have been missed by the clinical pipelines. By pointing out these issues, and suggesting solutions, this work will contribute to improved clinical SV/CNV calling across a broad spectrum of rare diseases. We will work closely with GE to improve pathways to access stored samples to undertake confirmatory analyses, and where fresh samples or additional clinical information are required, to recontact patients within an ethically rigorous framework.
We are interested in finding methods to replace mice to provide functional evidence of pathogenicity. Analysis of readily available patient cells often fails because they do not represent a cell type similar to that affected in a complex developmental phenotype such as CRS. Our exploration of reprogrammed neural crest cells will be of interest to all those concerned with new applications of 3Rs principles.
Both the doctors who have referred cases in whom we make new diagnoses, and the patients/families will benefit directly. For the doctor, a genetic diagnosis enables definitive management and reproductive risk information to be given and appropriate genetic testing to be offered. For the patient/family, it ends the diagnostic odyssey and often facilitates provision of additional social and educational support.
Clinical geneticists and other doctors submit samples to WGS programmes such as 100kGP with the notion that this represents a "definitive" genetic test. Doctors need to know that this is not yet the case, but that efforts are under way to improve the diagnostic dividend. Patient groups, specifically those concerned with CRS (such as Headlines), but also many other engaged support groups, need to be informed of potential benefit for themselves if they consent to additional research.
Overall this work will impact the effectiveness of public services and policy by increasing the diagnostic dividend of WGS and by increasing confidence that it is an effective test with high sensitivity and specificity. Quality of life of patients and families will be improved through the empowerment provided by greater autonomy to take decisions about their life, based on best available genetic information.