Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.
Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.
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DOI：
10.1371/journal.pgen.1005802
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发表时间：
2016-01
期刊：
影响因子：
4.5
通讯作者：
Shukunami C
中科院分区：
文献类型：
--
作者：
Guo L;Yamashita H;Kou I;Takimoto A;Meguro-Horike M;Horike S;Sakuma T;Miura S;Adachi T;Yamamoto T;Ikegawa S;Hiraki Y;Shukunami C
在此之前，我们通过全基因组关联研究，在人类瓢虫同源框1（LBX 1）和FLJ 41350附近发现了一个青少年特发性脊柱侧凸易感基因。在这里，我们描述了相关的非编码变体，并研究了这些基因的功能。染色体构象捕获分析显示，与最显著相关的单核苷酸多态性（rs 11190870）的基因组区域与LBX 1-FLJ 41350的启动子区域物理相互作用。在HEK 293 T细胞中，LBX 1方向的启动子与包括rs 11190870的590-bp区域组合，与风险等位基因相比具有更高的转录活性。在斑马鱼胚胎中，人LBX 1或斑马鱼lbx基因（lbx 1a，lbx 1b和lbx 2）的普遍过表达，而不是FLJ 41350，导致身体弯曲，然后在脊柱形成之前死亡。在lbx 1b或lbx 2的转录激活因子样效应核酸酶介导的敲除斑马鱼中没有观察到这种体轴变形。在斑马鱼中，由GATA 2最小启动子和lbx 1b增强子驱动的lbx 1b嵌合表达显著减轻了胚胎致死表型，从而可以观察到伴有或不伴有椎骨畸形的脊柱弯曲的后期发作。lbx 1b过表达导致的胚胎体轴变形与会聚延伸缺陷有关，会聚延伸是原肠胚中主轴伸长机制的一个组成部分。在过表达lbx 1b的胚胎中，wnt 5 b（非经典Wnt/平面细胞极性（PCP）途径的配体）显著下调。注射Wnt/PCP信号传导的关键下游效应子wnt 5 b或RhoA的mRNA，挽救了有缺陷的会聚延伸表型，并减弱了lbx 1b诱导的体轴弯曲。因此，我们的研究提出了LBX 1及其斑马鱼同源物在斑马鱼胚胎和后续生长的各个阶段体轴变形的新病理特征。脊柱侧凸是最常见的脊柱畸形类型，脊柱侧弯至少10度，影响2-4%的人口。脊柱侧凸主要由脊柱本身的问题引起，分为先天性脊柱侧凸（CS）和特发性脊柱侧凸（IS）。其中，青少年特发性脊柱侧凸（AIS）是脊柱侧凸最常见的形式，被认为是一种常见的多基因疾病。脊柱侧凸的严重脊柱弯曲导致了深刻的心理和社会影响，但由于IS和CS的确切病理机制尚未确定，因此尚未建立基于病因的治疗。在此之前，我们通过全基因组关联研究确定了人类瓢虫同源框1（LBX 1）附近的AIS易感位点。在这里，我们报告的功能特性的最显着相关的单核苷酸多态性（SNP），rs 11190870和LBX 1以及其斑马鱼同源物。LBX 1和斑马鱼lbx基因的过表达导致了与非经典Wnt/平面细胞极性信号传导受损相关的体侧弯曲。因此，我们的研究提出了LBX 1在体轴变形中的一种新的病理特征。
Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish. Scoliosis is the most common type of spinal deformity with a lateral spinal curvature of at least 10 degrees, affecting 2–4% of the population. Scoliosis caused by a primary problem related to the spine itself is classified into congenital scoliosis (CS) and idiopathic scoliosis (IS). Among these, adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is known as a common polygenic disease. Severe curving of the spine in scoliosis leads to profound psychological and social impacts, but etiology-based therapies have not been established since the precise pathological mechanisms of both IS and CS remain undefined. Previously, we identified an AIS susceptibility locus near human ladybird homeobox 1 (LBX1) by a genome-wide association study. Here, we report the functional characterization of the most significantly associated single nucleotide polymorphism (SNP), rs11190870 and LBX1 as well as its zebrafish homologues. Overexpression of LBX1 and zebrafish lbx genes caused lateral body curvature in association with the impairment of non-canonical Wnt/planar cell polarity signaling. Thus, our study presents a novel pathological feature of LBX1 in body axis deformation.