Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Brain. 2022 Apr 29;145(3):925-938. doi: 10.1093/brain/awab376.
Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.
局灶性皮质发育不良包括局灶性皮质发育不良、偏侧巨脑畸形和巨脑畸形，是一组与脑过度生长、细胞和结构发育不良、难治性癫痫、自闭症和智力残疾相关的神经发育障碍。重要的是，局灶性皮质发育不良是小儿局灶性难治性癫痫最常见的原因。在该谱系中已经发现了 PI3K-AKT-MTOR 通路的功能获得和功能丧失变异，其镶嵌率和组织分布存在差异。在这项研究中，我们使用液滴数字聚合酶链反应 (ddPCR) 对手术切除组织中的常见 PI3K-AKT-MTOR 通路变异进行了深度分子分析，并结合关键表型数据进行了分析。总共收集了 58 名局灶性皮质发育不良儿童的 159 个样本，包括 124 个脑组织样本。我们使用 ddPCR 针对三个 PI3K-AKT-MTOR 通路基因中的六个突变热点设计了一个超灵敏且高度靶向的分子诊断面板，即 PIK3CA(p.E542K，p.E545K，p.H1047R)、AKT3(p.E17K)和 MTOR(p.S2215F，p.S2215Y)。我们量化了所有样本的镶嵌率，并将基因型与关键的临床、神经影像学和组织病理学数据相关联。在 17 名个体中鉴定出致病性变异体，整体分子解决率为 29.31%。所有突变阳性样本的变异等位基因分数范围为 0.14%至 22.67%。我们的数据表明，致病性 MTOR 变异体主要与局灶性皮质发育不良相关，而致病性 PIK3CA 变异体在偏侧巨脑畸形中更为常见。此外，这些热点突变之一的存在与癫痫发作的早期发作相关。然而，镶嵌率与神经影像学或组织病理学上皮质畸形的严重程度无关。重要的是，我们在其他类型的手术切除癫痫病变（如多微小脑回或内侧颞叶硬化）中无法识别这些突变热点，这表明 PI3K-AKT-MTOR 突变在局灶性皮质发育不良-偏侧巨脑畸形谱中是特异性的致病原因。最后，我们的数据表明，通过靶向测序液滴数字聚合酶链反应对最常见的 PI3K-AKT-MTOR 突变进行超灵敏分子分析是一种有效的分子方法，当与神经影像学和组织病理学相结合时，具有良好的诊断效果。
