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

颅骨的生长为大脑的生长腾出空间是可能的，因为随着头部的生长，新的骨头被添加到颅骨之间狭窄的缝隙中，称为颅缝。当其中一个缝隙融合时，这阻止了融合缝合线的进一步生长，这种疾病称为颅缝闭塞（CRS）。在英国，这种严重的疾病每年影响大约350名婴儿。CRS有许多原因，但我们知道，大约25%的CRS是由于调节颅骨缝合线复杂信号过程的遗传指令改变而发生的。从很多方面来说，找到遗传原因很重要。对父母来说，它结束了漫长的诊断过程；对于临床遗传学家来说，它能够给出正确的风险并推荐测试；对于医疗团队来说，可能会有特定的并发症需要筛查。在生化疾病中，这些信息对于正确治疗至关重要。我们还可以了解更多关于颅缝正常功能的信息。尽管取得了这一进展，但仍有许多患有CRS的儿童被怀疑有遗传原因，但无法确定。例如，在100,000个基因组全基因组测序（WGS）项目（100kGP）中，对119个精心挑选的CRS患者/家庭进行了分析，但只有约18%的患者/家庭得到了诊断。我们怎么可能遗漏了一些遗传因素呢？尽管DNA测序技术已经取得了显著的进步，但基因组中仍有“暗物质”没有得到很好的表征。例如，我们70%的基因组是由重复的DNA组成的，这些DNA很难测序，而且在复制时经常会被打乱。因此，DNA片段可能存在过多或过少的拷贝（拷贝数变异- CNVs），或者片段可能在其位置或方向上发生改变（结构变异- SVs）。这些CNVs和SVs可以改变附近基因的表达方式（RNA的产生）；因此，在正常情况下基因沉默的组织中可能会产生一种蛋白质。在这个项目中，我们想更详细地研究这些SVs和cnv在没有诊断的复杂CRS儿童中的情况，因为我们怀疑这些可能导致颅缝错误表达导致CRS。100kGP资源是理想的研究对象，因为它代表了世界上这些儿童WGS的最大资源。我们提出的工作包括四个主要内容：首先，我们想要重新审视WGS数据，使用计算方法的组合来找到可能导致SV或CNV的原因。我们还将使用两种相对较新的技术，纳米孔测序和光学图谱，它们以不同于之前WGS的方式来观察基因组；我们期望这些将揭示以前未知的SV和CNV。其次，对于我们怀疑可能导致CRS的精心选择的SV或CNV，我们将通过基因组工程在小鼠中进行等效的重排。如果小鼠表现出异常的颅骨生长，这就有力地证明了这种重排也是人类疾病的病因。我们计划在项目中构建4种不同的小鼠突变体。第三，我们将测试另一种避免使用老鼠的技术的效果。这包括从患者血液样本开始，诱导人类细胞转变为一种称为神经嵴的细胞。这种细胞是颅骨缝合线的主要成分之一。我们计划在项目中构建6条不同的神经嵴线。第四，我们将进行详细的测试，比较小鼠组织和神经嵴细胞，看看细胞功能是否受到干扰。我们将测量SV/CNV周围基因的表达；评估DNA如何在细胞内折叠；并分析包裹DNA的蛋白质的化学修饰。我们的总体目标是扩大对SV/CNV引起CRS和其他疾病的途径的理解；获得基因组定位和功能评估方法的具体比较数据，以改进未来的分析；为病人和家属找到新的诊断方法。

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.