智力发育迟缓新候选基因POSTN对脑损伤神经元体外保护作用研究

目的 通过对1例智力发育迟缓患儿全外显子组测序（WES），旨在为该患儿寻找潜在的致病原因。方法 纳入1例在复旦大学附属儿科医院（我院）住院期间诊断不明患儿，采用SureSelct Human All Exon捕获试剂盒和Illumina HiSeq2000测序平台，行WES检测；数据分析采用我院分子诊断中心建立的高通量测序数据分析流程；结果采用Sanger直接测序法进行验证。建立体外大鼠神经元原代培养和糖氧剥夺（OGD）模型，分为空白对照组（Sham+PBS亚组、Sham+POSTN亚组）、OGD组（OGD+PBS亚组和OGD+POSTN亚组），采用免疫荧光、LDH毒性检测、BrdU和TUNEL方法检测POSTN对2组及其亚组神经元生长增殖和凋亡的影响。结果 患儿WES共检测到532 151个变异，筛选后检测到POSTN基因（NM_006475）外显子23:c.A2475T:p.G825G和外显子19:c.G2251A:p.E751K。Sanger测序显示p.G825G来自母亲，p.E751K来自父亲，符合复合杂合遗传模式。培养的大鼠原代神经元（MAP-2或NeuN阳性细胞）中有内源性POSTN的表达。LDH活性Sham+POSTN亚组与Sham+PBS亚组差异无统计学意义，OGD+POSTN亚组与OGD+PBS亚组差异有统计学意义。OGD+PBS亚组致神经元细胞缺氧2、6、12 d后比Sham+PBS亚组BrdU阳性的神经元数量增高，TUNEL阳性神经元凋亡也增高；OGD+POSTN亚组各时间点神经元增殖较OGD+PBS亚组均明显增高，但凋亡减少。结论 POSTN可以减少脑损伤后LDH对于神经元的细胞毒性，促进损伤后神经元的增殖，抑制凋亡。

Study on the pretective effect of a novel cerebral dysplasia gene,POSTN on cultured neurons from OGD injury in vitro

Objective In this study,whole-exome sequencing was used to identify pathogenic mutations in a newborn with cerebral dysplasia.Methods The exome targets of the patient's DNA was captured with the SureSelct Human All Exon kit followed by sequencing with the Illumina HiSeq2000 platform.The data analysis pipeline of Children's Hospital of Fudan University was used for the annotation and variant calling.The detected variant was confirmed with Sanger direct sequencing.Neurons were isolated from neonatal rats and the oxygen-glucose deprivation (OGD) model was established.The cultured neurons were divided into 2 control group (Sham + PBS and Sham + POSTN subgroups) and OGD group (OGD + PBS and OGD + POSTN subgroups).Immunofluorescence,LDH cytotoxicity detection,BrdU staining and TUNEL staining were used to detect the proliferation and apoptosis of cultured neurons.Results Whole-exome sequencing identified total of 532 151 variants in the proband,and revealed two novel compound heterozygous variants in exon 23:c.A2475T:p.G825G and exon 19:c.G2251A:p.E751K in POSTN (NM_ 006475) gene.Sanger direct sequencing confirmed these two variants.POSTN was expressed in the cultured rat neurons (MAP-2 and NeuN positive cells).LDH cytotoxicity in Sham + POSTN subgroup was compromised with Sham + PBS subgroup.However,POSTN protected neurons from LDH cytotoxicity in OGD groups.The neurons were treated with OGD for 2,6,12 days,compared with Sham + PBS subgroups,the proliferation of neurons was increased in OGD + PBS subgroup as well as apoptosis of neurons.After POSTN treatment,the number of proliferative neurons was markedly increased in OGD group,however,the apoptotic neuron number was significantly decreased.Conclusion POSTN treatment decreased LDH cytotoxicity,increased the proliferation and decreased apoptosis of OGD-treated neurons.