高原藏族新生儿持续肺动脉高压CPS1基因突变分析与机制研究

Persistent pulmonary hypertension of the newborn (PPHN) is a common critical disease in neonatal intensive care unit (NICU), and is the main cause of neonatal hypoxic respiratory failure with high mortality and morbidity. Low-dose of inhaled NO (inhaled nitric oxide, iNO) can effectively treat neonates with PPHN. Endogenous NO comes from L-arginine, and CPS1 is the rate limiting enzymes of L-arginine synthesis. Previous study showed that the polymorphism of CPS1 is associated with PPHN. By using whole-exome sequencing, we recently found a de novo heterozygous missense mutation (N410H) in a Tibetan neonatal baby death caused by PPHN.The present study is proposed to go on further confirmation of CPS1 gene variations with larger sample size in Highland-Tibetan and Plain-Han PPHN neonates. Then, newborn umbilical endothelial cells will be cultured in vitro for those CPS1 gene mutation cases, the mRNA and protein expression, and the impact on the downstream NO synthesis will be observed. Third, CRISPR/Cas9 site-directed mutant mice model will be constructed. We will detection gene expression, protein and enzyme activity in NO pathway, to confirm CPS1 gene mutation can lead to PPHN, and try to treat by using NO treatment.In the conclusion, through this study, we try to confirm CPS1 gene mutation can lead PPHN and try to clarify the molecular mechanisms of gene action in the pathogenesis of PPHN. Our study can provide new ideas for early intervention of PPHN.

新生儿持续肺动脉高压（PPHN）是导致新生儿严重呼吸衰竭的主要原因之一，伴高病死率和致残率。已有证据显示，遗传因素参与了严重PPHN的发生。CPS1是内源性一氧化氮（NO）生产原料L-精氨酸合成的限速酶，本课题组在1例PPHN死亡藏族新生儿中发现了CPS1基因新发错义突变（N410H）。本课题拟：1.扩大样本量检测高原藏族和平原汉族PPHN新生儿CPS1基因突变情况；2. 培养新生儿脐带内皮细胞，体外观察CPS1基因突变对该基因mRNA和蛋白表达影响，以及对下游NO合成的影响；3.构建CRISPR/Cas9基因定点突变小鼠模型，检测CPS1基因突变对NO通路基因表达、蛋白定量及酶活性的影响，证实CPS1基因突变可以导致PPHN，并观察对外源性吸入NO的治疗反应。通过本研究明确CPS1基因在严重呼吸衰竭/PPHN发病中的作用和分子机制，阐明PPHN的遗传学机制，并为早期干预提供新思路。

新生儿持续肺动脉高压（PPHN）是导致新生儿严重呼吸衰竭的主要原因之一，伴高病死率和致残率。氨基甲酰磷酸合成酶I(CPS1)是内源性一氧化氮生产原料L-精氨酸合成的限速酶。本研究对一个高原藏族散发PPHN核心家系，进行全外显子组测序，发现CPS1基因新发错义突变（c.A1228C，p.N410H），继续共收集了30例高原藏族和50例平原汉族PPHN患儿；对PPHN患儿进行测序，CPS1基因编码区及其侧翼进行序列分析，获得了CPS1基因变异谱，提示CPS1是PPHN的致病突变基因之一。进一步通过体外培养肺微血管内皮细胞，探讨了CPS1基因突变对该基因mRNA和蛋白表达影响，以及对下游NO合成的影响，证实CPS1基因突变通过对NO通路的影响，是导致PPHN发生的可能机制。本研究结果有助于阐明PPHN的遗传学机制，为早期干预提供新思路。