用两种鲤科鱼类模型解析遗传性脊柱侧凸的致病基因

The hereditary scoliosis, especially the idiopathic scoliosis , is a polygenic complex genetic disease, which affected approximately 4% of population with high incidence in adolescents. Due to the lack of proper animal models for this malformation, its pathogenesis keeps unclear, thus no measures can be take for early prevention or cure, and the treatment options are limited to follow-up and invasive surgical corrections with high health risk and cost. The natural spinal deformities were only found in humans and in teleosts. In our research, two full-sib grass carp famlies were found casually as the wonderful models for the research of hereditary scoliosis. In the project, two cyprind fishes (grass carp and zebrafish) will be used as model animals to investigate the molecular mechanism of hereditary scoliosis. Firstly, four full-sib families will be built from the 2 pairs of parents, and the scoliosis type will be determined by observations of the offspings deformity during somite development and fast growth period. In each full-sib family, malformed samples and controls for resequencing will be selected from those with greater genetic differencesin order to reduce the impact of non-pathogenic linkage sites and to find the predicted pathogenic mutaions and genes. Resulted genome sequeces will applied in genome-wide association studies to screen genes related to hereditary scoliosis. Finally, zebrafish will be used as a validation model to verify key pathogenic mutations through knockout or transgenesis. The study is important to clarify the pathogenic molecular mechanism of spine scoliosis and will provide scientific basis for the study of the hereditary scoliosis pathogenesis in human.

遗传性脊柱侧凸，尤其是特发性脊柱侧凸是一种人类多基因复杂疾病，影响着约4%的人群。由于缺乏合适的动物模型，其致病机制仍不清楚，无法实现早期防治。除人类之外，目前只在硬骨鱼类中有过天然脊柱侧凸群体的报道。我们幸运地发现了两个草鱼天然脊柱侧凸全同胞家系，是目前研究该疾病机制所能找到的最佳实验材料。本项目中，我们将用草鱼和斑马鱼这两种鲤科鱼类作为疾病模型和基因验证模型，探索该疾病的分子机制。首先以2对草鱼亲本构建4个全同胞群体，观测子代胚胎及生长期畸形情况确定侧凸类型，再从每个群体中选择合适的畸形和正常样本进行重测序，通过数据的关联与比较分析，解析与疾病相关的基因及突变型。样本的选择遵循组内遗传差异最大化原则以降低非致病连锁位点的影响。最后通过基因敲除或转基因等方法，以斑马鱼模型，验证部分致病基因。本研究对阐述遗传型脊柱侧凸的分子机制有着重要意义，为人类的脊柱侧凸致病机制研究提供了科学依据。

遗传性脊柱侧凸，尤其是特发性脊柱侧凸是一种人类多基因复杂疾病，高发于青少年时期，影响着约4%的人群。由于缺乏理想的动物模型，其致病基因不易筛选，致病的分子机制也一直不清晰，难以实现早期防治。除人类之外，目前也只在硬骨鱼类中有过天然脊柱侧凸群体的报道。此外，鱼类与人类有着脊柱延伸及运动受力同方向这一共同点，因此有可能成为人类脊柱侧凸研究的合适模型。..本研究从一个存在天然脊柱侧凸个体的草鱼6个全同胞家系的混合群体入手，开发了高通量亲子鉴定分析流程，并利用它获得群体中随机抽样的4083尾个体父母本信息。经统计检验，发现了雌鱼F13的后代以及F004家系具有显著的脊柱侧凸家族聚集性。基于该家系样本的全基因组重测序数据，我们定位到两个QTL区域与脊柱侧凸高度关联，其中qtl1上的fam20ca基因与骨骼发育相关，且在家系中存在一个错义突变。由于该突变所在结构域在脊椎动物中高度保守，我们推测该突变与脊柱侧凸性状相关。于是针对该区域在斑马鱼中进行cas9敲除，结果F1代测交群体及F2代自交群体均在1月龄左右出现了大量脊柱畸形个体，畸形类型分为上/下型侧弯和左/右型侧弯，证实了我们的发现和推测。..在该项目的实施过程中，为了提高群体QTL定位的可读性，本研究升级了草鱼基因组和全发育时期和组织的转录组，并发现了合子激活后期至开口前特异表达的SINE转座子家族；同时为了证明草鱼脊柱侧凸个体的家族聚集性而开发的高通量草鱼亲子鉴定流程，为后期的草鱼遗传育种研究提供了数据和技术两方面的基础支持。