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

大约每3000名新生儿中就有一人患有最常见的遗传性听力损失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有很大的不同，绝大多数患者患有严重的听力损失，但一小部分患者只表现出轻微的听力损失。有必要开发未来的治疗方法来改善新生儿的DFNB1耳聋，因为这将使这些儿童自然获得口语技能，从长远来看，它可能会使人工耳蜗过时。我的研究目的是确定携带完全相同的GJB2基因(35delG)突变的患者最终患上不同程度听力损失的分子原因。我推测，可能还有其他遗传因素参与调节已知的DFNB1突变对听力的影响。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，这些问题不可能在任何动物模型中得到解决。我将从患者的血液样本开始，通过应用不同的重新编程因子，我将能够产生能够分化为感觉毛发的干细胞群体--并支持内耳的细胞类型。使用这项技术将使我第一次能够将DFNB1表型转移到培养皿中，并将内耳细胞与正常听力个体的细胞进行生理学比较。为了确定影响表型严重程度的因素，我将应用完整的转录组深度测序方法，分析体外产生的细胞类型，以确定转录基因的变异。我建议通过对属于严重和轻微影响组的DFNB1患者进行全基因组关联研究来获得已识别的变异的统计验证。在监测患者来源细胞的生理表现的同时，将通过功能得失实验进一步验证候选方案。深入了解DFNB1的遗传修饰物是为严重影响的儿童开发新的治疗策略的先决条件，这可能在某种程度上帮助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.