利用模式动物探索DFNX1型耳聋发病机制及临床药物干预策略

Hearing Loss is a common disease causes human disability. The identification of the genes underlying hereditary hearing loss and to decipher its mechanism will be important to prevent the recurrence of hearing loss. This research field has always had originally innovative achievements.In the previous study, our research group found that mutations in PRPS1 causing DFNX1 nonsyndromic hearing impairment. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients.These results were published in Am.J.Hum.Genet.,2010 (IF=11.68, the applicant was the joint first anthors).In this proposal, we are going to establish a knock-in mouse model mimicing human DFNX1 hearing loss. With these mouse model, we are going to decipher the deep mechanism of DFNX1 hearing loss. This study will evaluate the effectiveness and the security of utilizing SAM supplementation in the diet alleviate some of the symptoms of patients with PRPS1 spectrum diseases by replenishing purine nucleotides and attenuate the progression of hearing loss in DFNX1 individuals. This project will provide the evidence to evaluate the clinical feasibility of presymptomatic prevention of DFNX1 hearing loss.

耳聋是严重高发的致残性疾病，新致聋基因的发现及其致聋机制的诠释在耳聋防治研究领域一直占据着重要的源头创新地位。2010年本课题组在Am J Hum Genet杂志（影响因子11.68，申请人为并列第一作者）首次报道了DFNX1型耳聋的致病基因PRPS1，发现PRPS1基因突变后PRS-1酶活性的部分下降导致了内耳能量代谢障碍，从而引发了DFNX1型耳聋的发生和进行性加重。在此工作基础上，本申请课题拟模拟我们发现的人类DFNX1型耳聋基因突变构建knock-in小鼠模型，利用模式动物深入研究PRPS1基因突变致聋机制，验证S-腺苷甲硫氨酸（SAM）对PRS-1经典代谢途径的代偿作用，在实体动物模型上评估SAM对DFNX1型耳聋症状前预防作用的有效性和安全性，为DFNX1型耳聋药物干预的临床可行性提供可靠的依据。

本课题基于前期研究基础，首次成功鉴定出DFNX1的致病基因——PRPS1，为了进一步揭示了其致聋机制，本研究利用ES细胞基因打靶技术，通过构建PRPS1基因敲入载体，在ES细胞基因组内置入与人类PRPS1突变同源的c.193G>A（D65N）突变，经过筛选、鉴定、和传代培养后，进行ES细胞囊胚注射以及胚胎移植，将突变位点整合进入小鼠胚胎，初步得到了携带有prps1基因c.193G>A突变的嵌合体小鼠。成功建立knock-in小鼠模型后，利用PCR扩增测序法以及Genotyping鉴定小鼠的基因型。将得到的突变体小鼠与野生型小鼠进行配种繁育，得到五种基因型的小鼠：雄鼠突变型（prps1D65N/null）、雄鼠野生型(prps1+/null)、雌鼠纯合型（prps1D65N/D65N）、雌鼠杂合型（prps1D65N/+）、雌鼠野生型（prps1+/+）。从小鼠出生后4周持续观察至25周以上，每间隔一月左右，对上述五种基因型的小鼠进行ABR测听，观察听力变化。此外，取突变型与野生型小鼠分别进行形态学观察，包括应用苏木精伊红火棉胶切片染色法在光镜下观察，以及应用免疫荧光染色法在共聚焦显微镜下观察。本研究成功建立了PRPS1基因敲入小鼠模型，并对其进行了初步听力学和形态学观察。