Validated assays for the quantification of C9orf72 human pathology.
Validated assays for the quantification of C9orf72 human pathology.
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DOI：
10.1038/s41598-023-50667-3
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发表时间：
2024-01-08
影响因子：
4.6
通讯作者：
中科院分区：
文献类型：
--
作者：
C9orf72基因的重复扩增突变是FTD和ALS的主要已知遗传原因。C9orf72-ALS/FTD领域一直受到缺乏可靠工具来监测该基因组座位及其RNA和蛋白质产物的困扰。我们以敲除的人IPSC系为对照，验证了在DNA、RNA和蛋白质水平上量化C9orf72病理生物学的分析。在这里，我们展示了单分子测序可以准确地测量重复序列的扩展，并忠实地报告在传统上难以测序的基因组区域中C9orf72基因座的变化。这对于确定重复扩展的大小和阶段，以及确定基因编辑后基因座的变化具有特别的价值。我们开发了ddPCR方法来定量两个主要的C9orf72转录变体，我们通过选择性地切除它们不同的转录起始点来验证这两个变体。使用有效的敲除的人IPSC株，我们验证了4个商用抗体(测试的9个)对C9orf72蛋白的免疫印迹定量是特异的，但没有一个是免疫细胞化学特异的。我们使用MSD免疫分析技术测试了66种浓度的15种二肽重复蛋白(Dprs)抗体组合，发现其中两种(针对PolyGA和PolyGP)在患者IPSC运动神经元中产生的信号比基因敲除对照增加1.5倍或更多，并在人类尸检和转基因小鼠脑组织中进行了验证。我们经过验证的DNA、RNA和蛋白质检测方法适用于发现研究和临床试验。
A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.
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影响因子：
16.2
作者：
Ash PE;Bieniek KF;Gendron TF;Caulfield T;Lin WL;Dejesus-Hernandez M;van Blitterswijk MM;Jansen-West K;Paul JW 3rd;Rademakers R;Boylan KB;Dickson DW;Petrucelli L
通讯作者：
Petrucelli L
DOI：
10.1016/s1474-4422(12)70043-1
发表时间：
2012-04
期刊：
The Lancet. Neurology
影响因子：
--
作者：
Majounie E;Renton AE;Mok K;Dopper EG;Waite A;Rollinson S;Chiò A;Restagno G;Nicolaou N;Simon-Sanchez J;van Swieten JC;Abramzon Y;Johnson JO;Sendtner M;Pamphlett R;Orrell RW;Mead S;Sidle KC;Houlden H;Rohrer JD;Morrison KE;Pall H;Talbot K;Ansorge O;Chromosome 9-ALS/FTD Consortium;French research network on FTLD/FTLD/ALS;ITALSGEN Consortium;Hernandez DG;Arepalli S;Sabatelli M;Mora G;Corbo M;Giannini F;Calvo A;Englund E;Borghero G;Floris GL;Remes AM;Laaksovirta H;McCluskey L;Trojanowski JQ;Van Deerlin VM;Schellenberg GD;Nalls MA;Drory VE;Lu CS;Yeh TH;Ishiura H;Takahashi Y;Tsuji S;Le Ber I;Brice A;Drepper C;Williams N;Kirby J;Shaw P;Hardy J;Tienari PJ;Heutink P;Morris HR;Pickering-Brown S;Traynor BJ
通讯作者：
Traynor BJ
影响因子：
5.5
作者：
Feliciano CM;Wu K;Watry HL;Marley CBE;Ramadoss GN;Ghanim HY;Liu AZ;Zholudeva LV;McDevitt TC;Saporta MA;Conklin BR;Judge LM
通讯作者：
Judge LM
影响因子：
7.1
作者：
Frick P;Sellier C;Mackenzie IRA;Cheng CY;Tahraoui-Bories J;Martinat C;Pasterkamp RJ;Prudlo J;Edbauer D;Oulad-Abdelghani M;Feederle R;Charlet-Berguerand N;Neumann M
通讯作者：
Neumann M
影响因子：
12.7
作者：
Mackenzie, Ian R.;Arzberger, Thomas;Neumann, Manuela
通讯作者：
Neumann, Manuela