Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.
Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.
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DOI：
10.1371/journal.pgen.1006905
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发表时间：
2017-07
期刊：
影响因子：
4.5
通讯作者：
Members of the UDN
中科院分区：
文献类型：
--
作者：
Luo X;Rosenfeld JA;Yamamoto S;Harel T;Zuo Z;Hall M;Wierenga KJ;Pastore MT;Bartholomew D;Delgado MR;Rotenberg J;Lewis RA;Emrick L;Bacino CA;Eldomery MK;Coban Akdemir Z;Xia F;Yang Y;Lalani SR;Lotze T;Lupski JR;Lee B;Bellen HJ;Wangler MF;Members of the UDN
CACNA1A 编码神经元 P/Q 型电压依赖性 Ca2+ 通道的 α-1A 亚基，其显性突变可导致不同的神经表型。罕见的明显严重的早发性发育迟缓和先天性共济失调可能是由新的 CACNA1A 错义等位基因引起的，其中的变异影响通道的 S4 跨膜段，据报道其中一些会丧失功能。对五名患有严重早发性共济失调的个体进行外显子组测序，在一名患有整体发育迟缓和进行性小脑萎缩的女孩中发现了一种新的变异（p.R1673P），在四名患有整体发育迟缓、肌张力低下和眼科异常的个体中发现了一种复发性的从头 p.R1664Q 变异。鉴于这些表型的严重性，我们探讨了它们对果蝇的功能影响。我们之前生成了 cac 的无效和部分功能丧失等位基因，cac 是果蝇中 CACNA1A 的同源物。在这里，我们创建了转基因野生型和突变基因组救援构建体，具有两个著名的保守点突变。 p.R1673P突变体未能挽救cac致死性，在突变体克隆记录的视网膜电图（ERG）中显示出功能获得表型，并根据ERG和透射电子显微镜在衰老果蝇中进化出神经退行性表型。相比之下，p.R1664Q 变体表现出功能丧失，并且未能形成神经退行性表型。因此，新的 R1673P 等位基因在果蝇和人类中产生神经退行性表型，可能是由于功能的毒性获得。钙通道控制细胞内钙的水平，对人类健康很重要。事实上，已经发现患有共济失调的平衡障碍的患者群体的人类基因组中名为 CACNA1A 的钙通道基因发生突变。在患有特定形式偏头痛的患者中也观察到了 CACNA1A 突变，导致身体一侧暂时瘫痪（偏瘫）。 CACNA1A 突变越来越多地出现在儿童时期更严重的大脑表型中。这项研究的重点是 5 名患有特别严重的 CACNA1A 相关疾病的患者。其中一名患者的 CACNA1A 存在特定的遗传拼写错误，而其他四名患者则存在附近的拼写错误。我们使用果蝇（Drosophila melanogaster）在缺乏果蝇钙通道的遗传背景下产生了具有相同拼写错误的果蝇。有趣的是，通过研究这些果蝇，我们发现了 1 号患者和其他四名患者的突变之间的差异。这些差异表明其中一种突变会产生更多的神经退行性变，实际上我们在该患者身上看到了更多的退行性变。果蝇研究使我们能够了解这些患者的突变功能，并有助于指导治疗决策。
Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function. Calcium channels control the levels of calcium within cells and are important in human health. Indeed, groups of patients with disorders of balance known as ataxia have been found to have mutations in a calcium channel gene in the human genome called CACNA1A. CACNA1A mutations have also been observed in patients with particular forms of migraine leading to temporary paralysis on one side of the body (hemiplegia). Mutations in CACNA1A are increasingly found in even more severe brain phenotypes in childhood. This research focused on a group of 5 patients with that particularly severe CACNA1A-related disease. One of the patients had a particular genetic misspelling in CACNA1A while the other four had nearby misspellings. We used the fruitfly, Drosophila melanogaster, to generate flies with these same misspellings in a genetic background that lacked the fly version of the calcium channel. Interestingly, by studying these flies we saw differences between the mutation in Patient 1 and the other four patients. These differences suggest one of the mutations produces more neurodegeneration, and indeed we see more degeneration in that patient. The fly studies allowed us to understand the function of the mutations in these patients, and were helpful in guiding treatment decisions.
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