儿童交替性偏瘫患儿基因突变分析及父母突变嵌合体研究

Alternating hemiplegia of childhood (AHC) is a severe neurological disorder and it occurred mostly in sporadic cases. It is characterized by paroxysmal oculomotor abnormalities, alternating hemiplegia, dystonia and cognitive impairment. Some patients has epileptic seizures. In 2012, it is reported that mutations of sodium-potassium ATPase α3 subunit gene ATP1A3 was the major cause of AHC, and almost all AHC-linked mutations were identified as de novo. The causative gene of ATP1A3 negative patients with AHC was still not clear. Our study group has collected 86 AHC cases in the past decade and four cases had family history. The aim of our study was to screen ATP1A3 mutations in Chinese AHC patients by PCR-DNA sequencing and multiplex ligation-dependent probe amplification(MLPA), and further to investigate the genotype-phenotype correlations, which would be valuable in clinical diagnosis and confirming the atypical AHC cases. For ATP1A3 mutation positive cases, we will perform Sanger sequencing and Raindrop digital PCR to confirm whether their parents carry the same mutation and detect the mutation fraction. For patients with de novo ATP1A3 mutation, we will analyze the parental origin of the de novo mutations by allele specific polymerase chain reaction (PCR), and further perform ATP1A3 mutation analysis and quantitative study by Raindrop digital PCR in paternal or maternal genomic DNA of multiple tisstues to detect the mutant allelic fraction. These work are helpful to gene diagnosis and genetic counseling in Chinese AHC patients.

儿童交替性偏瘫（AHC）是一种严重的神经系统发作性疾病，多数为散发病例，主要表现为发作性眼球运动异常、交替性偏瘫、肌张力不全和智力损害，少数可合并癫痫发作。2012年发现其主要致病基因为ATP1A3，且多数为新生突变，少数患儿致病基因尚不明确。本课题组已收集AHC 86例，其中4例有家族史，拟采用PCR-Sanger测序和MLPA方法筛查ATP1A3突变，分析AHC患儿基因突变特点及基因型-表型相关性，协助临床早期诊断和确诊不典型病例；对突变阳性的患儿父母采用Sanger测序和Raindrop数字PCR进行突变检测及定量分析；对新生突变的患儿采用等位基因特异性PCR进行突变亲源性分析，对确定突变位于父或母等位基因的一方多组织样本DNA采用Raindrop数字PCR进行低频突变定量分析；对ATP1A3突变阴性患儿，采用全外显子组测序寻找新的致病基因，为AHC患儿的基因诊断和遗传咨询奠定基础。

儿童交替性偏瘫（AHC）是一种少见的严重的神经系统发作性疾病，多数为散发病例。主要表现为发作性眼球运动异常、交替性偏瘫、肌张力不全和智力损害，少数可合并癫痫发作。 2012年发现其主要致病基因为ATP1A3，且多数为新生突变。本项目按计划共收集到162例AHC患儿，采用Sanger测序或靶向捕获基因测序方法筛查ATP1A3基因突变，发现其中95.7%（155/162）患儿携带ATP1A3基因突变，多数为错义突变，其中最常见的突变类型为D801N、E815K和G947R突变。对突变阳性的148例患儿父母采用Sanger测序方法进行突变来源验证，发现98.5%（146/148）为新生突变。对怀疑可疑嵌合突变及Sanger测序证实为ATP1A3“新生突变”的80例AHC家系，采用Raindrop数字微滴PCR进行嵌合突变鉴定和定量分析，发现约7.5%（6/80）“新生突变”为来自父母一方的嵌合突变。对携带嵌合突变者的多组织样本进行突变定量分析，发现突变等位基因频率波动于0.03%至33.03%，差异性较大。对ATP1A3突变阴性7例患儿，计划下一步采用全外显子组测序寻找新的致病基因。本研究对患儿基因型与表型进行相关性分析，发现AHC患儿起病年龄D801N组与E815K组均早于G947R组，首次偏瘫发作年龄DS01N组与E815K组也均早于 G947R组，合并癫痫比例及智力、运动落 后严重程度，E815K组患儿均高于D801N组和G947R组，ATPIA3基因突变也是中国AHC患儿的主要致病基因，3种热点突变（D801N、E815K和G947R）与国际报道一致；热点突变E815K具有更严重的临床表型，表现为起病年龄及偏瘫出现年龄早，易合并癫痫发作，智力运动发育落后更严重。对其中96例AHC患儿随访口服氟桂利嗪治疗研究发现，氟桂利嗪对于多数AHC患儿有效，可降低偏瘫发作频率，缩短偏瘫持续时间，减轻偏瘫严重程度。起病年龄、治疗年龄、服药剂量、是否携带E815K、D801N或G947R突变，均不是影响疗效的因素。本项目研究成果对AHC患儿的基因诊断和遗传咨询提供了更多科学依据。