分析腮耳肾综合征致病基因Eya1等的转录调控进而解析新的致病因素的功能研究

Branchio-oto-renal syndrome (BOR) is an autosomal dominant rare genetic disorder involving the kidneys, ears, and neck, which contribute to 2% deafness. Etiological and genetic study revealed causes of half BOR are mutation related to coding region of Eya1, Six1, and Six5, while causes of other half remain in mist. There are two possible causes, one is mutation of new genes, the other is mutation involved on cis-regulatory elements or trans-acting factors in non-coding region of known genes, especial Eya1 and Six1. In previous study, applicant has found that there are several ear-specific enhancers near BOR related genes, Eya1 and Six1. According to that, it can be inferred that ear-specific enhancers and transcription factors binding to them, are important to expression of Eya1/Six1. If mutation happened either in enhancers or on transcription factors, the expression pattern of Eya1/Six1 will present abnormally, consequently, function of ear and hearing ability will present abnormally. Therefore, in this study, applicant proposed to focus on non-coding region of BOR related genes Eya1 and Six1, 1) to validate functional regulatory elements and to identify disease related mutation in those elements from BOR patients; 2) to identify and study on transcription factors binding to DNA elements or miRNA binding to RNA sites. In order to fulfill them, first of all, bioinformatics screening will be performed on 250kb genomic sequences upstream or downstream of target gene to locate candidate elements. Secondly, a systematic experimental study will be performed to study on activity of each element and factors interact to that. Thirdly, BOR patients’ samples will be collected for genetic analysis. Coding region of Eya1/Six1, functional non-coding element of same gene, region of associated trans-acting factors will be sequenced and analyzed. It can be expected, future achievements of this project will benefit BOR patients. New disease cause will be identified, mechanism of genes involved in disease will be elucidated, so as to promote diagnose and prevention of BOR syndrome.

腮耳肾综合征是发病率1/40000的罕见遗传病,影响多个器官,2%耳聋患儿源于此病。50%病因与Eya1等的编码区突变相关,另50%病因不明,可能是新基因,也可能源于Eya1等已知致病基因的非编码区突变。申请人前期发现,相关致病基因附近有可决定基因在内耳特异表达的增强子。推测,该增强子变异可导致致病基因在耳中表达异常,引发耳功能异常；或,与该增强子结合作用的转录因子异常,而导致表达异常和耳功能异常。因此本研究拟以Eya1等的非编码区为切入点,通过序列分析筛选出的潜在调控区和在细胞及斑马鱼中的功能研究,鉴定新的病因,即1)可影响Eya1等致病基因表达水平、区域变化的功能调控区,2)与这些调控区功能相关的转录因子和miRNA。由此,为诊断病因不明腮耳肾综合征患者提供新的研究依据。对这些患者潜在功能调控区的分析,也可反过来精确指导实验研究,明确靶点与疾病的相关性,促进该疾病的发病机制和防治。

腮耳肾综合征是发病率1/40000的罕见遗传病,影响多个器官，2%耳聋患儿源于此病。50%病因与Eya1等的编码区突变相关，另50%病因不明。本研究针对这50%病因不明的情况，设定目标，通过对腮耳肾综合征致病基因的调控区进行系统的功能分析以解析新的致病因素，从而为该疾病的分子诊断提供依据，并为该疾病的治疗提供线索和靶点。申请人在细胞和斑马鱼水平对腮耳肾综合征致病基因eya1和six1b上下游约250kb的区域进行生物信息学分析和功能实验研究以鉴定潜在的顺式调控元件和反式作用因子。研究发现eya1和six1b上下游分别包含多个具有活性功能的调控元件，尤其重要的是2个内耳特异性的增强子顺式调控元件和miR-124-3p反式作用因子，同时也发现了可调控eya1和six1b基因表达的多个相关的沉默子、启动子及转录因子。进一步的功能研究发现，内耳特异性增强子可调控eya1或six1在内耳局部异常过表达，最终导致听力功能异常；也可通过改善恢复已敲除目标基因eya1-/-或six1-/-转基因斑马鱼内耳中的eya1或six1表达水平对听力功能异常进行挽救。研究还表明miR-124-3p可通过下调eya1表达而引发听力功能异常。这些研究表明增强子、沉默子、启动子以及转录因子和miRNA这些调控区和相关调控因子可以影响eya1和six1基因的表达引起eya1或six1相关的听力障碍，而且增强子还能够通过调控机制来挽救eya1或six1基因缺陷引起的听力损害。因此，对腮耳肾致病基因eya1和six1b相关的非编码调控元件进行的研究拓展了我们对腮耳肾综合征发病机制的认识，鉴定出新的致病因素，为腮耳肾综合征的诊断和治疗提供了有力的线索和促进。

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