一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因
1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5Royal Children’s Hospital, 6Murdoch Children’s Research Institute, 7University of Melbourne, 8Plateforme postgenomique INMED, 9University of Pavia, 10Wellcome Trust Centre for Human Genetics, 11Oxford Radcliffe NHS Trust, 12IRCCS Casimiro Mondino Foundation, 13Research Institute of Molecular Pathology, 14IRCCS Stella Maris, 15Columbia University
Summary
脑室结节性异 (PNH) 是成年期皮质发育畸形最常见的形式, 但在大多数偶发病例中, 其遗传基础仍然未知。我们最近制定了一项战略, 以确定新的候选基因的 MCDs, 并直接确认其致病作用在体内。
Abstract
涉及大脑皮层的先天缺陷–也称为皮质发育畸形–是导致智力残疾的重要原因, 也是儿童时期20-40% 的耐药性癫痫的病因。高分辨率脑成像促进了在体内识别一大群的型式。尽管在脑部成像、基因组分析和动物模型的产生方面取得了进展, 但一个简单的工作流程, 系统地确定候选基因的优先顺序, 并测试假定突变的功能效应。为了解决这一问题, 开发并验证了一种能够识别新的致病基因的实验策略。这一策略是基于通过阵列全息或全外测序确定候选基因组区域或基因, 并通过宫内来描述它们的失活或在发展啮齿动物大脑中的特定突变的过度表达的影响。穿孔.这种方法导致识别的C6orf70基因, 编码为一个假定的水泡蛋白, 脑室结节性异的发病机制, 由缺陷的神经元迁移引起的。
Introduction
大脑皮层在认知和智力过程中起着关键的作用, 并且参与情绪控制以及学习和记忆。因此, 许多神经和精神疾病都是由皮质发育畸形引起的, 这并不奇怪。由于后天和遗传因素的影响, 其病因是复杂的。在大多数情况下, 遗传决定的肥胖率的累计患病比例约为 2%, 而且是零星的。例如, 据估计, 在人类人口中, 先天性脑发育的发病率高于 1%, 而且在14% 以上的癫痫患者和40% 的严重或顽固性癫痫中, 出现了某些形式的高血压,1,2。
脑室结节异 (PNH) 是最常见的 MCDs 之一, 是由心室区 (VZ) 神经元异常迁移到发育中的大脑皮层引起的。神经元迁移的失败会导致侧脑室壁的异位神经元聚集, 这通常可以用磁共振成像 (MRI) 进行可视化。PNH 的临床、解剖学和影像学特征均为异质性。结核的范围可以从小的和单边的到双边的和对称的。常见的临床后遗症包括癫痫和智力残疾3。在Filamin A (或FLNA) 基因中的突变, 在 Xq28 中映射, 在100% 的家庭中发现有 X 链接的双边 PNH 和26% 的散发性患者34。一个罕见的, 隐性形式的 PNH 引起的突变在ARFGEF2基因, 其中映射在 20q13, 已报告在两个近亲家庭5。最近, 在包括 PNH6在内的九例受断奶紊乱影响的患者中, 发现了受体配体粘蛋白对DCHS1和FAT4的基因 biallelic 突变。PNH 也已与脆性 X 综合征7, 威廉斯综合征8, 22q11 缺失综合征9, 复制在 5p1510, 删除在 1p3611, 5q14.3-q1512, 6p2513和6q 终端删除综合征14,15,16,17,18,19, 提示其他致病基因分散整个基因组。然而, 大约74% 的散发性 PNH 患者的遗传基础仍有待阐明17。
经典的基因映射方法, 如阵列比较基因组杂交 (数组-全息图) 已被证明是一个强大的工具, 检测亚显微染色体异常, 但是, 使用这种方法确定的基因组区域是通常大并且包含许多基因。
大规模并行测序技术 (即全外测序 (WES) 和全基因组测序 (WGS)) 的出现大大降低了整个人类外或基因组序列所需的成本和时间。然而, 在大多数情况下, 对 WES 和 WGS 数据的解释仍然具有挑战性, 因为对于每位病人来说, 从数据过滤中产生的变种到数百人 (甚至数千名患者) 都有不同的分析方法。
为了加快识别新的致病基因的过程, 设计了一种结合阵列全息、WES 和宫内电穿孔 (井上靖) 筛选候选基因的新系统策略。井上靖允许有选择地灭活 (或过度) 啮齿动物大脑中的特定基因或突变, 使其参与 corticogenesis18,19的快速评估。当基因与疾病的发展、神经元迁移和/或成熟的局部缺陷有关时, rna 介导-击倒或过度表达一个或多个候选基因将导致。在鉴定的基因的失活 (或过度表达) 再现的表型观察到啮齿动物的病人, 它成为一个杰出的候选者筛查的零星患者的。使用这种方法, 我们最近揭示了C6orf70基因 (也称为ERMARD) 在 PNH 发病机制中的重要贡献, 这些患者窝藏6q27 染色体删除16。
Protocol
伦理声明: 大鼠交配, 维护和使用的 INMED 动物设施, 与欧洲联盟和法国立法的协议。 1. 阵列全息和 WES 的 DNA 提取和定量 根据制造商的协议, 从使用自动 dna 隔离机器人的患者或商业上可用的人工 dna 提取试剂盒中提取人血白细胞的基因组 dna (gDNA)。用分光光度计对所有样品进行量化。 2. 阵列全息协议 gDNA 的限制性消化 <…
Representative Results
在图 1中扼要了用于识别新的致病基因的实验策略。 通过在155例发育性脑异常变合并 PNH (图 2A)、胼胝体发育、colpocephaly、小脑发育不良和 polymicrogyria 与癫痫、共济失调和认知障碍, 我们确定了 1.2 Mb 的最小关键删除在6q27 共享的12患者 (图 2B)21。基因组区域包含四已知基因…
Discussion
MCDs 是智力残疾的重要原因, 占20-40% 的耐药性儿童癫癎1,2。在过去十年中, 由于两个主要因素, 对 MCDs 的兴趣显著增加。第一个是大脑成像 (特别是 MRI) 的改善, 这使得医生和科学家能够想象出许多以前未被识别的大脑畸形。另一种是基因工具的进化, 使得许多新奇的致病基因得以鉴定。这大大提高了我们对大脑发育和功能的机制的认识, 并允许更准确的遗…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢 Dr. G. McGillivray, 公关. 美 Dobyns, 公关. Striano, 公关. 夏侯雅伯, 公关. 某地 Roberston 和公关. 顺 Berkovic 为患者提供服务。我们感谢 Dr. ·米歇尔和 d. 梅的技术咨询和帮助。这项工作得到了欧盟七框架方案的资助, 愿望项目, 合同编号: Health-F2-602531-2013, (共,., ar, 个和 cc), INSERM (ar 和 cc), ô勒琼 (R13083AA, 电 P 和 c。Région 普罗旺斯阿尔卑斯科特迪瓦蔚蓝海岸 (APO2014-通俗到 cc 和 d。检察官 K 是一个 EMBO 年轻的调查员, 并得到 FWF 赠款 (I914 和 P24367) 的支持。
Materials
| Picospritzer III | Parker Hannifin Corp | P/N 051-0500-900 | Intracellular Microinjection Dispense Systems |
| Fast Green FCF | Sigma-Aldrich | F7252 | Fast green allow visual monitoring of the injection |
| BTX ECM 830 electroporator | BTX Harvard Apparatus | 45-0002 | The ECM 830 is a Square Wave Pulse generator designed for in vitro and in vivo applications |
| Microtome HM 650 V | Microm | 10076838 | Microtome HM 650 V vibratome 240V 50/60Hz with vibrating blade |
| FluoView 300 | Olympus | The Olympus FluoViewTM 300 is a point-scanning, point-detection, confocal laser scanning microscope designed for biology research application | |
| eCELLence software | Glance Vision Technologies | eCELLence is software designed for the quantitive analysis of cell migration | |
| Agilent Microarray Scanner Bundle | Array slides | ||
| for 1 x 244K, 2 x 105K, 4 x 44K or 8 x 15K | Agilent | Agilent p/n G4900DA, G2565CA or G2565BA | |
| for 1 x 1M, 2 x 400K, 4 x 180K or 8 x 60K | Agilent | Agilent p/n G4900DA or G2565CA | |
| Hybridization Chamber, stainless | Agilent | Agilent p/n G2534A | Chamber for array CGH hybridization |
| Hybridization Chamber gasket slides, 5-pack | Gasket for array CGH hybridization | ||
| for 1-pack microarrays or | Agilent | Agilent p/n G2534-60003 | |
| for 2-pack microarrays or | Agilent | Agilent p/n G2534-60002 | |
| for 4-pack microarrays or | Agilent | Agilent p/n G2534-60011 | |
| for 8-pack microarrays | Agilent | Agilent p/n G2534-60014 | |
| Hybridization oven | Agilent | Agilent p/n G2545A | |
| Hybridization oven rotator for Agilent Microarray Hybridization Chambers | Agilent | Agilent p/n G2530-60029 | |
| Thermal cycler with heated lid | Agilent | Agilent p/n G8800A or equivalent | Termal cycler for incubations |
| 1.5 mL RNase-free Microfuge Tube | Ambion | p/n AM12400 or equivalent | Microcentrifuge |
| Magnetic stir bar | Corning | p/n 401435 or equivalent | Instrument for stirring |
| Qubit Fluorometer | Life Technologies | p/n Q32857 | Instrument for DNA quantification |
| Qubit dsDNA BR Assay Kit, for use with the Qubit fluorometer | Invitrogen | p/n Q32850 | Kit for Qubit fluorometer |
| UV-VIS spectrophotometer | Thermo Scientific | NanoDrop 8000 or 2000, or equivalent | Instrument for DNA quantification |
| P10, P20, P200 and P1000 pipettes | Pipetman or equivalent | DNA dispensation | |
| Vacuum Concentrator | Thermo Scientific | p/n DNA120-115 or equivalent |
Instrument to concentrate DNA |
| SureTag Complete DNA Labeling Kit | Agilent | p/n 5190-4240 | DNA Labeling Kit (for Human Samples) |
| Purification Columns (50 units) | Agilent | p/n 5190-3391 | DNA Labeling Kit (for Human Samples) |
| AutoScreen A, 96-well plates | GE Healthcare | p/n 25-9005-98 | DNA Labeling Kit (for Human Samples) |
| GenElute PCR Clean-Up Kit | Sigma-Aldrich | p/n NA1020 | DNA Labeling Kit (for Human Samples) |
| Human Genomic DNA | p/n G1521 | DNA Labeling Kit (for Human Samples) | |
| For CGH microarrays: | Promega | (female) or p/n G1471 (male) | Array CGH control DNA |
| For CGH+SNP microarrays: | Coriell | p/n NA18507, NA18517, NA12891, NA12878, or NA18579 | Array CGH control DNA |
| Oligo aCGH/ChIP-on-chip Wash Buffer Kit or | Agilent | p/n 5188-5226 | Array CGH hybridization and wash |
| Oligo aCGH/ChIP-on-chip Wash Buffer 1 and | Agilent | p/n 5188-5221 | Array CGH hybridization and wash |
| Oligo aCGH/ChIP-on-chip Wash Buffer 2 | Agilent | p/n 5188-5222 | Array CGH hybridization and wash |
| Stabilization and Drying Solution | Agilent | p/n 5185-5979 | Array CGH hybridization and wash |
| Oligo aCGH/ChIP-on-chip Hybridization Kit | Agilent | p/n 5188-5220 (25) or p/n 5188-5380 (100) | Array CGH hybridization and wash |
| Human Cot-1 DNA | Agilent | p/n 5190-3393 | Array CGH hybridization and wash |
| Agilent C scanner | Agilent | Scanner for array CGH slides | |
| SureSelect XT2 Reagent Kit | Kit for target enrichment | ||
| HiSeq platform (HSQ), 16 Samples | Agilent | p/n G9621A | |
| HiSeq platform (HSQ), 96 Samples | Agilent | p/n G9621B | |
| HiSeq platform (HSQ), 480 Samples | Agilent | p/n G9621C | |
| MiSeq platform (MSQ), 16 Samples | Agilent | p/n G9622A | |
| MiSeq platform (MSQ), 96 Samples | Agilent | p/n G9622B | |
| MiSeq platform (MSQ), 480 Samples | Agilent | p/n G9622C | |
| DNA 1000 Kit | Agilent | p/n 5067-1504 | Kit for the separation, sizing and quantification of dsDNA fragments from 25 to 1000 bp. |
| High Sensitivity DNA Kit | Agilent | p/n 5067-4626 | Kit for analysis of fragmented DNA or DNA libraries. |
| AMPure XP Kit | Kit for automated PCR purification. | ||
| 5 mL | Agencourt | p/n A63880 | |
| 60 mL | Agencourt | p/n A63881 | |
| 450 mL | Agencourt | p/n A63882 | |
| Dynabeads MyOne Streptavidin T1 | Isolation and handling of biotinylated nucleic acids | ||
| 2 mL | Life Technologies | Cat #65601 | |
| 10 mL | Life Technologies | Cat #65602 | |
| Quant-iT dsDNA BR Assay Kit, for the Qubit fluorometer | DNA quantification | ||
| 100 assays, 2-1000 ng | Life Technologies | Cat #Q32850 | |
| 500 assays, 2-1000 ng | Life Technologies | Cat #Q32853 | |
| Qubit assay tubes | Life Technologies | p/n Q32856 | DNA quantification |
| SureSelec tXT2 Capture Libraries | Agilent | depending on the experiment | Kit for libraries capture |
| SureCycler 8800 Thermal Cycler | Agilent | p/n G8800A | DNA amplification |
| 96 well plate module for SureCycler 8800 Thermal Cycler | Agilent | p/n G8810A | DNA amplification |
| SureCycler 8800-compatible 96-well plates | Agilent | p/n 410088 | DNA amplification |
| Optical strip caps | Agilent | p/n 401425 | DNA amplification |
| Tube cap strips, domed | Agilent | p/n 410096 | DNA amplification |
| Compression mats | Agilent | p/n 410187 | DNA amplification |
| 2100 Bioanalyzer Laptop Bundle | Agilent | p/n G2943CA | DNA amplification |
| 2100 Bioanalyzer Electrophoresis Set | Agilent | p/n G2947CA | DNA amplification |
| Covaris Sample Preparation System, E-series or S-series | Covaris | DNA shearing | |
| Covaris sample holders | p/n 520078 | DNA shearing | |
| Nutator plate mixer | BD Diagnostics | p/n 421105 or equivalent | Plate Mixer |
| GaIIx | Illumina | next generation sequencing machine |
Referências
- Meencke, H. J., Veith, G. Migration disturbances in epilepsy. Epilepsy Res. 9, 31-39 (1992).
- Shorvon, S., Stefan, H. Overview of the safety of newer antiepileptic drugs. Epilepsia. 38 (1), 45-51 (1997).
- Parrini, E., et al. Periventricular heterotopia: phenotypic heterogeneity and correlation with Filamin A mutations. Brain. 129 (7), 1892-1906 (2006).
- Fox, J. W., et al. Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia. Neuron. 21 (6), 1315-1325 (1998).
- Sheen, V. L., et al. Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex. Nat Genet. 36 (1), 69-76 (2004).
- Cappello, S., et al. Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development. Nat Genet. 45 (11), 1300-1308 (2013).
- Moro, F., et al. Periventricular heterotopia in fragile X syndrome. Neurology. 67 (4), 713-715 (2006).
- Ferland, R. J., Gaitanis, J. N., Apse, K., Tantravahi, U., Walsh, C. A., Sheen, V. L. Periventricular nodular heterotopia and Williams syndrome. Am J Med Genet A. 140 (12), 1305-1311 (2006).
- van Kogelenberg, M., et al. Periventricular heterotopia in common microdeletion syndromes. Mol Syndromol. 1 (1), 35-41 (2010).
- Sheen, V. L., et al. Periventricular heterotopia associated with chromosome 5p anomalies. Neurology. 60 (6), 1033-1036 (2003).
- Neal, J., Apse, K., Sahin, M., Walsh, C. A., Sheen, V. L. Deletion of chromosome 1p36 is associated with periventricular nodular heterotopia. Am J Med Genet A. 140 (15), 1692-1695 (2006).
- Cardoso, C., et al. Periventricular heterotopia, mental retardation, and epilepsy associated with 5q14.3-q15 deletion. Neurology. 72 (9), 784-792 (2009).
- Cellini, E., et al. Periventricular heterotopia with white matter abnormalities associated with 6p25 deletion. Am J Med Genet A. 158 (7), 1793-1797 (2006).
- Bertini, V., De Vito, G., Costa, R., Simi, P., Valetto, A. Isolated 6q terminal deletions: an emerging new syndrome. Am J Med Genet A. 140 (1), 74-81 (2006).
- Dobyns, W. B., et al. Consistent chromosome abnormalities identify novel polymicrogyria loci in 1p36.3, 2p16.1-p23.1, 4q21.21-q22.1, 6q26-q27, and 21q2. Am J Med Genet A. 146 (13), 1637-1654 (2008).
- Conti, V., et al. Periventricular heterotopia in 6q terminal deletion syndrome: role of the C6orf70 gene. Brain. 136 (11), 3378-3394 (2013).
- Sheen, V. L., et al. Etiological heterogeneity of familial periventricular heterotopia and hydrocephalus. Brain Dev. 26 (5), 326-334 (2004).
- Jaglin, X. H., et al. Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria. Nat. Genet. 41 (6), 746-752 (2009).
- Falace, A., et al. TBC1D24 regulates neuronal migration and maturation through modulation of the ARF6-dependent pathway. Proc Natl Acad Sci U S A. 111 (6), 2337-2342 (2014).
- Lunter, G., Goodson, M. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 21 (6), 936-939 (2011).
- Pagnamenta, A. T., et al. Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies. J Hum Genet. 57 (1), 70-72 (2012).
- Yu, J. Y., DeRuiter, S. L., Turner, D. L. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 99 (9), 6047-6052 (2002).
- Bai, J., et al. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci. 6 (12), 1277-1283 (2003).
- Carabalona, A., et al. A glial origin for periventricular nodular heterotopia caused by impaired expression of Filamin-A. Hum Mol Genet. 21 (5), 1004-1017 (2012).
Citar este artigo
Conti, V., Carabalona, A., Pallesi-Pocachard, E., Leventer, R. J., Schaller, F., Parrini, E., Deparis, A. A., Watrin, F., Buhler, E., Novara, F., Lise, S., Pagnamenta, A. T., Kini, U., Taylor, J. C., Zuffardi, O., Represa, A., Keays, D. A., Guerrini, R., Falace, A., Cardoso, C. A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations. J. Vis. Exp. (130), e53570, doi:10.3791/53570 (2017).