限于女性的癫痫伴智力低下患儿基因突变分析及父母突变嵌合体研究

In families of epilepsy and mental retardation limited to females (EFMR), PCDH19 gene was the causative gene, which encodes protocadherin 19 and located in the X chromosome. In compared to family cases, sporadic cases were more reported and were identified as de novo mostly. The disease had an unusual X-linked inheritance, females carrying mutation were affected but males carrying mutation were not affected. In our previous study, the Sanger sequencing confirmed that the two sibling sisters had the same PCDH19 mutation but their parent did not carry the mutation, and one of parent was suspected of having a low frequency mutantion. In 2 cases of male epilepsy cases, PCDH19 mosaic mutations were found. 32 sporadic cases of EFMR and 10 EFMR families were collected. The PCDH19 mutation was screened by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). For mutation positive and mosaic mutation male cases, micro-droplet digital PCR were performed to confirm whether their parents carry the same mutation and detect the mutation fraction in genomic DNA of their peripheral blood and multiple tissues. These work could lay a foundation for explore pathogenesis of mosaicism and genetic counseling, and provide guidance for reproduction and prenatal diagnosis.

在限于女性的癫痫伴智力低下(EFMR)家系中，发现致病基因为X染色体上编码原钙粘蛋白19的PCDHl9基因。目前发现该病散发病例较家系更多见，且以新生突变为主。本病遗传方式特殊，携带突变杂合子女性发病，携带突变半合子男性不发病。本课题组前期研究发现，在姐妹同患但父母表型正常的家系，Sanger测序证实姐妹携带相同PCDH19 突变但父母未发现该突变，高度怀疑父或母为低频突变嵌合体；在2例男性癫痫患儿发现PCDH19嵌合突变。本研究拟对已收集的32例EFMR散发病例和10个EFMR家系，采用Sanger测序和MLPA方法筛查PCDH19突变；对突变阳性患儿父母和男性突变嵌合体患儿外周血DNA，采用微滴数字PCR进行突变检测及定量研究；对携带嵌合突变患儿父或母和男性突变嵌合体患儿多种组织样本DNA进行突变定量分析，为探讨嵌合突变致病机理和遗传咨询奠定基础，为再生育产前诊断提供指导。

在限于女性的癫痫伴智力低下(EFMR)家系中，发现致病基因为X染色体上编码原钙粘蛋白19的PCDHl9基因。目前发现该病散发病例较家系更多见，且以新生突变为主。本病遗传方式特殊，携带突变杂合子女性发病，携带突变半合子男性不发病。本课题组至今累计收集到74例PCDH19基因突变相关癫痫病例，包括女性67例和男性7例，62例(83.7%)有局灶性发作，32例(43.2%)病程中出现≥2种发作类型；均有丛集性发作特点(100%)；55例(74.3%)发作有热敏感特点。随访研究发现：发作控制1年以上的患儿33例(44%，末次随访年龄5岁9月～24岁)，63%(38/60)有智力障碍，28%(17/60)有孤独症样表现。74例携带PCDH19基因变异患儿中，共检测到60种不同变异，其中新发现国际上未报道的变异有29种，且热点变异c.1019A>G/p.N340S占8.1%。突变来源分析发现：49例为新生变异，8例为母源变异和4例为父源变异，其中发现2例为男性突变嵌合体无癫痫表型受累。7例PCDH19突变嵌合体的定量分析发现，外周血DNA突变等位基因嵌合比例波动于15.0-89.8%。本项目现有研究结果，对PCDHl9基因相关癫痫的基因型与表型相关性研究提供了大样本的理论基础，对男性嵌合体的嵌合研究，为探索嵌合突变在遗传性癫痫的致病模式提供了研究基础。