De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.
De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.
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DOI：
10.1093/brain/awy092
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发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
Guerrini R
中科院分区：
文献类型：
--
作者：
Fassio A;Esposito A;Kato M;Saitsu H;Mei D;Marini C;Conti V;Nakashima M;Okamoto N;Olmez Turker A;Albuz B;Semerci Gündüz CN;Yanagihara K;Belmonte E;Maragliano L;Ramsey K;Balak C;Siniard A;Narayanan V;C4RCD Research Group;Ohba C;Shiina M;Ogata K;Matsumoto N;Benfenati F;Guerrini R
使用全外显子组测序，Fassio等人在4名发育性脑病和癫痫患者中鉴定了编码v-ATP酶A亚基的ATP 6V1A的新生突变。功能和表达研究表明，受损的溶酶体内稳态，有缺陷的神经突伸长和兴奋性输入的培养神经元的损失。V型质子（H+）ATP酶（v-ATP酶）是一种多亚基质子泵，在所有真核细胞中调节pH稳态;在神经元中，v-ATP酶在突触功能中发挥额外和独特的作用。通过全外显子组测序，我们在4例癫痫性发育性脑病患者中鉴定了编码v-ATP酶A亚基的ATP 6V1A的新生杂合突变（p.Pro27Arg，p.Asp100Tyr，p.Asp349Asn，p.Asp371Gly）。在所有患者中观察到的早期表现为发育迟缓和热性惊厥，2例患者（p.Pro27Arg，p.Asp100Tyr）发展为脑病伴严重延迟、低渗/运动障碍性四肢轻瘫和难治性多种癫痫发作类型，另外2例患者（p.Asp349Asn，p.Asp371Gly）发展为中度延迟伴轻度癫痫发作。对v-ATP酶的原核和真核结构进行的建模预测p.Pro27Arg干扰亚基相互作用，p.Asp100Tyr引起空间位阻并使蛋白质折叠不稳定，p.Asp349Asn影响催化功能，p.Asp371Gly损害质子转运所必需的旋转过程。我们通过分析ATP6V1A过表达的HEK293 T细胞和患者的淋巴母细胞，探讨了p.Asp349Asn和p.Asp100Tyr突变对ATP6V1A表达和功能的影响。p.Asp100Tyr突变体的特征在于由于降解增加而导致的表达降低。相反，未观察到p.Asp349Asn的表达和清除率降低。在过度表达致病性或对照变体的HEK293T细胞中，p.Asp349Asn显著增加LysoTracker®荧光，而对EEA1和LAMP 1表达无影响。相反，p.Asp100Tyr降低LysoTracker®荧光和LAMP 1水平，使EEA1表达不受影响。这两种突变都减少了自噬体的v-ATP酶募集，对自噬没有重大影响。使用LysoSensor ™探针对患者的淋巴母细胞进行的实验显示，p.Asp349Asn的内吞细胞器的pH较低，而p.Asp100Tyr的LAMP 1表达降低，对pH无影响。这些数据表明，p.Asp349Asn功能获得，其特征为细胞内细胞器中质子泵增加，而p.Asp100Tyr功能丧失，ATP 6V1A表达降低，溶酶体标志物水平降低。我们在大鼠海马神经元中表达了p.Asp349Asn和p.Asp100Tyr，并证实了溶酶体标记的显著和相反的作用。然而，这两种突变引起了类似的缺陷，伴随着兴奋性输入的损失，改变溶酶体稳态显着影响神经突的发育和突触连接的神经突伸长。这项研究提供的证据表明，从头杂合ATP6V1A突变引起发育性脑病的病理机制，涉及溶酶体稳态和神经元连接的扰动，揭示了一个新的作用，为v-ATP酶在神经元发育。
Using whole exome sequencing, Fassio et al. identify de novo mutations in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathies and epilepsy. Functional and expression studies demonstrate impaired lysosomal homeostasis, defective neurite elongation and loss of excitatory inputs in cultured neurons. V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients’ lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients’ lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.
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影响因子：
30.8
作者：
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作者：
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