Identification of human GJB2 35delG modifying genes using induced pluripotent stem cells

大约每3000个新生儿中就有1个患有最常见的遗传性听力损失，称为DFNB1，这是由GJB2基因突变引起的。听力损失的程度在不同的患者之间差别很大，绝大多数患者有严重的听力损失，但一小部分患者仅表现出轻度的听力损失。有必要开发未来的治疗方法来改善新生儿的DFNB1耳聋，因为它可以让这些孩子自然地获得口语技能，从长远来看，它可以使人工耳蜗植入过时。我的研究目的是找出GJB2基因（35delG）完全相同突变的患者最终出现不同程度听力损失的分子原因。我推测可能有其他遗传因素参与调节已知的DFNB1突变对听觉能力的影响。为了实现这一目标，我将应用基于患者特异性诱导干细胞（iPSC）的技术来帮助我回答我的问题，由于疾病的复杂性，这些问题在任何动物模型中都不可能得到解决。我将从一个病人的血液样本开始，通过应用不同的重编程因子，我将能够产生一个干细胞群，能够分化成感觉毛发和内耳的支持细胞类型。使用这种技术将使我第一次能够将DFNB1表型转移到培养皿中，并与正常听力个体的细胞进行比较，从生理学上表征内耳细胞。为了确定改变表型严重程度的因素，我将采用全转录组深度测序方法分析体外生成的细胞类型，以确定转录基因的变化。我建议通过对属于严重和轻度影响组的DFNB1患者进行全基因组关联研究，获得已识别变异的统计验证。候选药物的进一步验证将通过功能的获得和丧失实验来完成，同时监测患者来源细胞的生理表现。深入了解DFNB1的基因修饰因子是为严重患病儿童开发新的治疗策略的先决条件，这在某种程度上可能有助于他们过上正常的生活。

Approximately 1 in 3,000 babies born suffers from the most common form of hereditary hearing loss called DFNB1, which is caused by mutations in the GJB2 gene. The degree of hearing loss varies substantially among different patients with the vast majority having severe hearing loss but a small subset of patients displays only a mild form of hearing loss. There is a need for developing future treatments that ameliorate DFNB1 deafness in newborn children because it would allow these children to naturally acquire spoken language skills and in the long run, it could make cochlear implantation obsolete.My research aim is to identify the molecular reasons why patients that carry exactly the same mutation in the GJB2 gene (35delG) end up with different levels of hearing loss. I speculate that there might be other genetic factors involved that modulate the impact of the known DFNB1 mutation on the ability to hear sound. To achieve this goal, I will apply patient specific induced stem cell (iPSC)-based technology that helps me to answer my questions, which would not be possible to be addressed in any animal model due to the complexity of the disease. I will start with a blood sample from a patient and by applying different reprogramming factors, I will be able to generate a stem cell population that is capable of differentiating into sensory hair- and supporting cell types from the inner ear. Using this technique will enable me for the first time to transfer the DFNB1 phenotype to the culture dish and to characterize the inner ear cells physiologically in comparison with cells from normal hearing individuals. To identify the factors modifying the severity of the phenotype, I will apply a whole transcriptome deep sequencing approach analyzing the in vitro generated cell types to identify variations in transcribed genes. I propose obtaining statistical validation of identified variations by conducting a genome wide association study with DFNB1 patients that belong to the severely and mildly affected groups. Further validation of the candidates will be done by gain and loss of function experiments while monitoring for physiological performance in patient-derived cells.Insight into genetic modifiers of DFNB1 is a pre-requisite to the development of novel treatment strategies for severely affected children, which at some point might help them to live normal lives.