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Completed
Uncovering new mechanisms of craniosynostosis associated with structural and copy number variation, using mouse modelling and human neural crest cells
FunderUK Research and Innovation
Funder Contribution £789,024.00
Duration01 Nov 2020 - 29 Sep 2024
Funded underMRC
Project codeMR/T031670/1
Open Access mandatePublications: NoResearch data: No
Identifiers
Project timeline100%
Description
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.
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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.
