Forward genetics of human immunity: the molecular basis of a novel form of combined immunodeficiency

摘要：先天性免疫系统缺陷可导致幼儿严重感染、自身免疫和癌症。这些原发性免疫缺陷是了解人类免疫中个体基因和途径功能的重要模型。下一代测序技术在揭示新基因的致病变异方面具有强大的潜力，但是为了使科学理解和临床护理的益处最大化，需要进行机制研究。这项研究的目的是确定一种新发现的联合免疫缺陷的潜在分子基础。学生将研究选择的候选变体如何在受影响的家庭中与疾病分离，它们对相关蛋白质的表达和功能的影响以及这种变化导致疾病的机制。来自受影响儿童的整个外显子组测序数据的生物信息学分析将通过内部、专有和公开可用的软件工具的组合来实现，这些软件工具已经在宿主实验室内建立使用。将这些筛选到具有预期遗传模式的罕见的、可预测的有害变异后，学生将评估基因和表型之间的拟合度。例如，他们将询问有关基因表达和功能的已发表数据，检查任何突变残基的进化保护程度，将已知的变异映射到蛋白质结构，并与已知的变异（例如小鼠同源物或癌症中的体细胞突变）进行比较。选择的变体将被用于遗传和功能验证。为了进一步确认和筛选候选变异，将使用未受影响和受影响家庭成员的基因组DNA作为模板，通过聚合酶链反应（PCR）扩增对每个变异进行分离评估，然后对PCR扩增子进行Sanger测序（商业执行）。在可能的情况下，将在患者细胞中评估该变异对mRNA和蛋白质丰度的影响（分别通过定量RT-PCR和免疫印迹+/-免疫荧光）。一般来说，我们可以使用患者的成纤维细胞和外周血单个核细胞，通常还有B淋巴母细胞样细胞系。或者，突变将通过哺乳动物表达结构的定点诱变来模拟，并在重组系统中进行研究。对于带有残余蛋白表达的错义突变，氨基酸取代的结构效应将被模拟成已知的分子结构-这可能会深入了解它们对蛋白质功能的影响。进一步的研究将针对感兴趣的蛋白质进行定制，以描述对受影响途径的功能影响，无论是在患者来源的细胞中，还是在基因敲除、基因组编辑甚至是培养细胞中的小分子抑制剂产生的模型系统中。培训将提供核心技术（组织培养，转染，流式细胞术，免疫印迹，成像，ELISA， qPCR等）。我们拥有最先进的设备，包括生物成像、流式细胞术、提供CyTOF和illumina NextSeq的单细胞基因组学设备，用于现场转录组学等。

Keywords: Genetics, immunology, immunodeficiencyAbstract: Inborn errors of the immune system can lead to serious infection, autoimmunity and cancer in young children. These primary immunodeficiencies represent important models through which to understand the function of individual genes and pathways in human immunity. Next generation sequencing techniques have powerful potential to reveal disease-causing variants in novel genes but mechanistic studies are required to maximise the benefits for both scientific understanding and clinical care. The aim of this studentship will be to determine the underlying molecular basis for a newly recognised form of combined immunodeficiency. The student will examine how selected candidate variants segregate with disease in the affected families, their effect on expression and function of the related protein and the mechanism whereby such changes cause disease. Bioinformatic analysis of whole exome sequencing data from affected children will be achieved through a combination of in house, proprietary and publicly available software tools that are already in established use within the host laboratory. Having filtered this down to rare, predicted deleterious variants with the expected pattern of inheritance, the student will assess the goodness of fit between gene and phenotype. For example, they will interrogate published data on gene expression and function, examine the degree of evolutionary conservation of any mutated residue, map variants to protein structure where known and compare with known variants eg in murine homologues or somatic mutations in cancer. Selected variants will be taken forward for genetic and functional validation. To confirm and filter candidate variants further, segregation will be assessed by polymerase chain reaction (PCR) amplification across each variant, using genomic DNA from unaffected and affected family members as template, followed by Sanger sequencing of PCR amplicons (performed commercially). The effects of the variant on mRNA and protein abundance will be assessed in patient cells where possible (by quantitative RT-PCR and immunoblotting +/- immunofluorescence, respectively). Generally we will have access to patient fibroblasts and peripheral blood mononuclear cells, and often B lymphoblastoid cell lines. Alternatively the mutation will be modelled by site-directed mutagenesis of a mammalian expression construct and studied in a recombinant system. For missense mutations with residual protein expression, the structural effect of amino acid substitutions will be modelled into known molecular structures - this may give insight into their effect on protein function. Further studies will be tailored to the protein of interest to delineate functional effects on the affected pathway, whether in patient-derived cells or model systems generated by gene knock down, genome editing or even small molecule inhibitors in cultured cells. Training will be provided in core techniques (tissue culture, transfection, flow cytometry, immunoblotting, imaging, ELISA, qPCR etc). We have access to state of the art facilities including bioimaging, flow cytometry, a new single cell genomics facility offering CyTOF and Illlumina NextSeq for on-site transcriptomics etc.