JoVE Journal > Genetics
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Genética

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Published: August 20, 2019
doi:
10.3791/59658
, , , ,
1Department of Molecular and Human Genetics,Baylor College of Medicine, 2Program in Developmental Biology,Baylor College of Medicine, 3Department of Pediatrics, Section of Neurology and Developmental Neuroscience,Baylor College of Medicine, 4Jan and Dan Duncan Neurological Research Institute,Texas Children’s Hospital, 5Department of Neuroscience,Baylor College of Medicine

Summary

该协议的目的是概述在果蝇黑色素体内实验的设计和性能，以评估与人类疾病相关的罕见基因变异的功能后果。

Abstract

测序技术的进步使全基因组和全外体数据集更易于用于临床诊断和尖端人类遗传学研究。尽管已经开发了许多silico算法来预测这些数据集中识别的变异的致病性，但功能研究对于确定特定基因组变异如何影响蛋白质功能至关重要，尤其是对于感知错误变异。在未诊断疾病网络（UDN）和其他罕见疾病研究联盟中，模型生物（MO），包括果蝇、甲鱼、斑马鱼和小鼠，被积极用于评估假定的人类疾病引起功能变异。该协议描述了一种用于UDN的模拟生物筛选中心果蝇核心的罕见人类变异的功能评估方法。工作流从从多个公共数据库收集人工和 MO 信息开始，使用 MARRVEL Web 资源评估变体是否可能导致患者病情，并根据可用情况设计有效的实验知识和资源。接下来，生成遗传工具（例如T2A-GAL4和UAS-人cDNA线），以评估果蝇感兴趣的变异的功能。在开发这些试剂后，可以进行基于抢救和过度表达实验的双管齐下的功能测定，以评估变异功能。在抢救分支中，内源性苍蝇基因通过用参考或变异人类转基因取代正交果蝇基因而”人性化”。在过度表达分支中，参考和变异的人类蛋白质在各种组织中被外向驱动。在这两种情况下，任何可感染的表型（例如，杀伤性、眼睛形态学、电生理学）都可以用作读出，而不管感兴趣的疾病如何。参考和变异等位基因之间的差异表明存在变异特异性效应，因此可能具有致病性。该协议允许快速，在体内评估具有已知和未知功能的基因的假定人类致病变异。

Introduction

患有罕见疾病的患者往往经历一段被称为”诊断奥德赛”的艰难旅程，以获得准确的诊断1。大多数罕见疾病被认为有很强的遗传来源，使得遗传/基因组分析成为临床工作的关键要素。除了基于染色体微阵列的候选基因面板测序和拷贝数变异分析外，全外显体 （WES） 和全基因组测序 （WGS） 技术在过去十年中已成为越来越有价值的工具2 3.目前，在WES和WGS中识别已知致病变异的诊断率为+25%（在儿科病例中较高）4，5。对于大多数在临床WES/WGS后仍未确诊的病例，一个常见的问题是有许多候选基因和变异。下一代测序通常能够识别许多基因中的新奇或超罕见变异，而解释这些变异是否导致疾病表型是具有挑战性的。例如，尽管基因中的大多数无意义突变或帧移突变被认为是功能丧失 （LOF） 等位基因，因为编码转录本的无意义介导衰减，但截断最后一个外源区中发现的突变会逃脱此过程，并可能充当良性或函数增益 （GOF） 等位各位数6.

此外，预测一个误感等位基因的影响是一项艰巨的任务，因为它可能导致许多不同的遗传场景，正如赫尔曼·穆勒在20世纪30年代首次描述的那样（即异构、低态、超态、反变形、新变形或异构）7.在silico计划和方法已经开发了许多预测误感变异的致病性基于进化保护，氨基酸变化的类型，在功能域的位置，等位基因频率在一般人群中，和其他参数8。然而，这些程序不是解决变体解释这一复杂问题的全面解决方案。有趣的是，最近的一项研究表明，五种广泛使用的变异致病性预测算法（Polyphen9，SIFT 10，CADD11，PROVEAN12，突变味觉）同意致病性 +80% 的时间 80%8.值得注意的是，即使所有算法都同意，它们也会在 11% 的时间内返回对致病性的错误预测。这不仅导致有缺陷的临床解释，而且还可能劝阻研究人员跟进新的变种，错误地列出他们为良性。补充目前硅酮建模限制的一个方法是提供实验数据，证明体外、体外（如培养细胞、有机体）或体内变异功能的效果。

对MO中罕见疾病相关变异的体内功能研究具有独特的优势13，已被世界各地的许多罕见疾病研究倡议所采用，包括美国的未诊断疾病网络（UDN）和稀有疾病模型和机制 （RDMM） 网络在加拿大，日本， 欧洲和澳大利亚14.除了这些协调的努力，将MO研究人员纳入全国范围的罕见疾病诊断和机械研究工作流程，临床和MO研究人员之间的一些单独合作研究已导致发现和许多新的人类致病基因和变异的表征82，83，84。

在UDN中，一个集中的模型生物筛选中心（MOSC）接收候选基因和变异的提交，并描述患者的病情，并评估该变异是否可能是病原菌使用信息学工具和体内实验。在UDN的第一阶段（2015-2018年），MOSC由果蝇核心[贝勒医学院（BCM）]和斑马鱼核心（俄勒冈大学）组成，他们合作评估病例。MOSC利用信息学分析和在果蝇和斑马鱼中的若干不同实验策略，迄今已为132名患者的诊断、31种新综合征的识别、发现几种新人类做出了贡献。疾病基因（例如，EBF315、ATP5F1D 16、TBX2 17、IRF2BPL 18、COG4 19、WDR37 20）和已知疾病的型皮扩张基因（例如，CACNA1A21，ACOX122）。

除了UDN内的项目外，MOSC果蝇核心研究人员还与孟德尔基因组学中心和其他倡议（例如，ANKLE223，TM2D3）合作，为新的疾病基因发现做出了贡献。 24， NRD125， OGDHL25， ATAD3A26， ARIH127， MARK328， DNMBP29） 使用相同的信息学和遗传学集为UDN制定的战略。鉴于MO研究对罕见疾病诊断的重要性，MOSC已扩大到包括UDN第二阶段（2018-2022年）的C.elegans核心和第二斑马鱼核心（均位于圣路易斯华盛顿大学）。

本手稿描述了在UDN MOSC果蝇核中积极使用的体内功能研究方案，以确定误感变异是否对使用表达人类的转基因苍蝇感兴趣的蛋白质产生功能影响蛋白质。该协议的目的是帮助MO研究人员与临床研究组合作，提供实验证据，证明感兴趣的基因中的候选变异具有功能性后果，从而促进临床诊断。在果蝇研究人员与一名临床研究人员接触的情况下，此协议最有用，该患者有一个罕见疾病患者，其基因中具有特定的候选变体。

该协议可以分为三个要素：（1） 收集信息以评估兴趣变异对患者表型负责的可能性，以及在果蝇中进行功能性研究的可行性，（2） 收集现有的遗传工具和建立新的，和（3）在体内进行功能研究。第三个要素可以根据如何评估利益变式的功能（救援实验或基于过度表达的策略）进一步细分为两个子元素。需要注意的是，该协议可以针对罕见单源疾病研究以外的许多情况进行调整和优化（例如，常见疾病、基因环境相互作用和药理学/基因筛选，以确定治疗靶点）。确定变异的功能和致病性的能力不仅通过提供准确的分子诊断使感兴趣的患者受益，而且还将对转化和基础科学研究产生更广泛的影响。

Protocol

1. 收集人类和MO信息进行评估：利益变异对疾病表型负责的可能性和果蝇功能研究的可行性 执行广泛的数据库和文献搜索，以确定感兴趣的特定基因和变体是否是解释感兴趣的患者表型的好候选者。具体而言，收集以下信息。 评估感兴趣的基因以前是否涉及其他遗传疾病（已知疾病基因的表型扩张），或者这是一个全新的疾病候选基因[具有不确定意义的基因变?…

Representative Results

EBF3中与神经发育表型相关的新感变异的功能研究国家卫生研究院未确诊疾病项目（UDP）的医生和人类遗传学家在7岁男性患有神经发育表型，包括肌张力障碍、失语症、全球发育迟缓和言语障碍。在EBF3（早期B细胞因子3）15中识别出一个错误感变异（p.R163Q），这是一种编码COE（Collier/Olfactory-1/早期B细胞因子）家族转录因子的基因。该案于2016年3…

Discussion

使用果蝇黑色素的实验性研究提供了一个强大的测定系统，以评估与疾病相关的人类变异的后果。这是由于在过去的一个世纪里，许多在飞行领域的研究人员产生了大量的知识和多样化的基因工具。然而，就像任何其他实验系统一样，承认存在的警告和限制是很重要的。

与数据挖掘相关的注意事项
虽然该协议的第一步是挖掘数据库，?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢何塞·萨拉萨尔、王朱莉娅和卡伦·舒尔兹博士对手稿的批判性阅读。我们感谢刘宁博士和罗希博士对这里讨论的TBX2变异的功能特性。未诊断疾病网络模型生物筛选中心通过国家卫生研究院（NIH）共同基金（U54 NS093793）提供支持。H.T.C. 得到了NIH[CNCDP-K12和NINDS（1K12 NS098482）]、美国神经学学会（神经科学研究资助）、伯罗斯·惠康基金（医学科学家职业奖）、儿童神经学学会和儿童神经学基金会（） 的进一步支持。PERF Elterman 奖）和 NIH 主任早期独立奖 （DP5 OD026426）。M.F.W.得到了西蒙斯基金会（SFARI奖：368479）的进一步支持。S. Y. 得到了NIH（R01 DC014932）、西蒙斯基金会（SFARI奖：368479）、阿尔茨海默氏症协会（新调查员研究补助金：15-364099）、纳曼家庭基础研究基金和卡罗琳·威斯法律研究基金的支持。分子医学。BCM 的共聚焦显微镜部分由 NIH Grant U54HD083092 支持给智力和发育障碍研究中心 （IDDRC） 神经可视化核心。

Materials

Drosophila Stocks for UAS-human cDNA transgenesis
Injection strains for transgenesis (D. melanogaster) BDSC #24871 Specific Reagent: VK33 (3rd chromosome) Injection line
Injection strains for transgenesis (D. melanogaster) BDSC #24872 Specific Reagent: VK37 (2nd chromosome) Injection line
Plasmid DNA
Cloning vector Thermo Fisher #12536-017 Specific Reagent: pDONR221
Drosophila transgenesis vector Gift from Drs. Johannes Bischof and Konrad Basler (Bischof et al., 2013 PNAS) Specific Reagent: pGW-HA.attB
Molecular biology kits and reagents
Agarose Sigma-Aldrich #A2790 Specific Reagent: Agarose (molecular biology grade)
Chemically Competent Cells (E. coli) Thermo Fisher #18265017 Specific Reagent: DH5α
DNA Gel Extraction kit Thermo Fisher #K210012 Specific Reagent: PureLink Gel Extraction Kit
DNA Isolation and purification kit Qiagen #27104 Specific Reagent: QIAprep Spin Miniprep Kit
High Fidelity Polymerase NEB #M0491 Specific Reagent: Q5 Polymerase kit
Recombinase mediated cloning system Thermo Fisher #11789020 Specific Reagent: Gateway BP Clonase kit
Recombinase mediated cloning system Thermo Fisher #11791100 Specific Reagent: Gateway LR Clonase II Enzyme kit
Site Directed Mutagenesis kit Agilent #200523 Specific Reagent: Quick Change II Mutagenesis kit
Electroretinogram Rig related equipment
ERG Analysis Molecular Devices N/A Specific Reagent: Axon pCLAMP 10 Data Software Package
ERG Data Collection LabX #R150358 Specific Reagent: ISO-DAM Isolated Biologic Amplifier
ERG Stimulator Astro-Med #S48 Specific Reagent: Square Pulse Stimulator
DOWNLOAD MATERIALS LIST

Referências

  1. Boycott, K. M., et al. International Cooperation to Enable the Diagnosis of All Rare Genetic Diseases. The American Journal of Human Genetics. 100 (5), 695-705 (2017).
  2. Lupski, J. R., et al. Whole-Genome Sequencing in a Patient with Charcot-Marie-Tooth Neuropathy. New England Journal of Medicine. 362 (13), 1181-1191 (2010).
  3. Boycott, K. M., Vanstone, M. R., Bulman, D. E., MacKenzie, A. E. Rare-disease genetics in the era of next-generation sequencing: discovery to translation. Nature Reviews Genetics. 14 (10), 681-691 (2013).
  4. Yang, Y., et al. Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing. JAMA. 312 (18), 1870 (2014).
  5. Lee, H., et al. Clinical Exome Sequencing for Genetic Identification of Rare Mendelian Disorders. JAMA. 312 (18), 1880 (2014).
  6. Coban-Akdemir, Z., et al. Identifying Genes Whose Mutant Transcripts Cause Dominant Disease Traits by Potential Gain-of-Function Alleles. The American Journal of Human Genetics. 103 (2), 171-187 (2018).
  7. Muller, H. J. Further studies on the nature and causes of gene mutations. Proceedings of the Sixth International Congress of Genetics. , 213-255 (1932).
  8. Ghosh, R., Oak, N., Plon, S. E. Evaluation of in silico algorithms for use with ACMG/AMP clinical variant interpretation guidelines. Genome Biology. 18 (1), 225 (2017).
  9. Adzhubei, I. A., et al. A method and server for predicting damaging missense mutations. Nature Methods. 7 (4), 248-249 (2010).
  10. Vaser, R., Adusumalli, S., Leng, S. N., Sikic, M., Ng, P. C. SIFT missense predictions for genomes. Nature Protocols. 11 (1), 1-9 (2016).
  11. Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J. l., Kircher, M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Research. , (2018).
  12. Choi, Y., Sims, G. E., Murphy, S., Miller, J. R., Chan, A. P. Predicting the functional effect of amino acid substitutions and indels. PloS ONE. 7 (10), 46688 (2012).
  13. Wangler, M. F., et al. Model Organisms Facilitate Rare Disease Diagnosis and Therapeutic Research. Genética. 207 (1), 9-27 (2017).
  14. Oriel, C., Lasko, P. Recent Developments in Using Drosophila as a Model for Human Genetic Disease. International Journal of Molecular Sciences. 19 (7), 2041 (2018).
  15. Chao, H. T., et al. A Syndromic Neurodevelopmental Disorder Caused by De Novo Variants in EBF3. American Journal of Human Genetics. 100 (1), 128-137 (2017).
  16. Oláhová, M., et al. Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder. American Journal of Human Genetics. 102 (3), 494-504 (2018).
  17. Liu, N., et al. Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder. Human Molecular Genetics. 27 (14), 2454-2465 (2018).
  18. Marcogliese, P. C., et al. IRF2BPL Is Associated with Neurological Phenotypes. American Journal of Human Genetics. 103 (2), 245-260 (2018).
  19. Ferreira, C. R., et al. A Recurrent De Novo Heterozygous COG4 Substitution Leads to Saul-Wilson Syndrome, Disrupted Vesicular Trafficking, and Altered Proteoglycan Glycosylation. The American Journal of Human Genetics. 103 (4), 553-567 (2018).
  20. Kanca, O., et al. De novo variants in WDR37 are associated with epilepsy, colobomas and cerebellar hypoplasia. Americal Journal of Human Genetics. , (2019).
  21. Luo, X., et al. Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially. PLOS Genetics. 13 (7), 1006905 (2017).
  22. Chung, H., et al. ACOX1 induces autoimmunity whereas a de novo gain of function variant induces elevated ROS and glial loss in humans and flies. Cell Metabolism. , (2019).
  23. Yamamoto, S., et al. A Drosophila Genetic Resource of Mutants to Study Mechanisms Underlying Human Genetic Diseases. Cell. 159 (1), 200-214 (2014).
  24. Jakobsdottir, J., et al. Rare Functional Variant in TM2D3 is Associated with Late-Onset Alzheimer’s Disease. PLoS Genetics. 12 (10), 1006327 (2016).
  25. Yoon, W. H., et al. Loss of Nardilysin, a Mitochondrial Co-chaperone for α-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration. Neuron. 93 (1), 115-131 (2017).
  26. Harel, T., et al. Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes. American Journal of Human Genetics. 99 (4), 831-845 (2016).
  27. Tan, K. L., et al. Ari-1 Regulates Myonuclear Organization Together with Parkin and Is Associated with Aortic Aneurysms. Developmental Cell. 45 (2), 226-244 (2018).
  28. Ansar, M., et al. Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3. Human Molecular Genetics. 27 (15), 2703-2711 (2018).
  29. Ansar, M., et al. Bi-allelic Loss-of-Function Variants in DNMBP Cause Infantile Cataracts. The American Journal of Human Genetics. 103 (4), 568-578 (2018).
  30. Wang, J., et al. MARRVEL: Integration of Human and Model Organism Genetic Resources to Facilitate Functional Annotation of the Human Genome. The American Journal of Human Genetics. 100 (6), 843-853 (2017).
  31. Wang, J., Liu, Z., Bellen, H., Yamamoto, S. MARRVEL, a web-based tool that integrates human and model organism genomics information. Journal of Visualized Experiments. , (2019).
  32. Mungall, C. J., et al. The Monarch Initiative: an integrative data and analytic platform connecting phenotypes to genotypes across species. Nucleic Acids Research. 45, 712-722 (2017).
  33. Hu, Y., Comjean, A., Mohr, S. E., Perrimon, N., Perrimon, N. Gene2Function: An Integrated Online Resource for Gene Function Discovery. Genes|Genomes|Genetics. 7 (8), 2855-2858 (2017).
  34. Ioannidis, N. M., et al. An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. The American Journal of Human Genetics. 99 (4), 877-885 (2016).
  35. Szklarczyk, D., et al. The STRING database in 2017: quality-controlled protein–protein association networks, made broadly accessible. Nucleic Acids Research. 45 (1), 362-368 (2017).
  36. Hu, Y., et al. Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data. Nucleic Acids Research. 46 (1), 567-574 (2018).
  37. Lawson, C. L., et al. EMDataBank unified data resource for 3DEM. Nucleic Acids Research. 44 (1), 396-403 (2016).
  38. Bienert, S., et al. The SWISS-MODEL Repository-new features and functionality. Nucleic Acids Research. 45 (1), 313-319 (2017).
  39. Webb, B., Sali, A. Comparative Protein Structure Modeling Using MODELLER. Current Protocols in Bioinformatics. 54, 1-37 (2016).
  40. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols. 10 (6), 845-858 (2015).
  41. Bamshad, M. J., et al. The Centers for Mendelian Genomics: A new large-scale initiative to identify the genes underlying rare Mendelian conditions. American Journal of Medical Genetics Part A. 158 (7), 1523-1525 (2012).
  42. Sobreira, N. L. M., et al. Matchmaker Exchange. Current Protocols in Human Genetics. 95, 1-15 (2017).
  43. Temple, G., et al. The completion of the Mammalian Gene Collection (MGC). Genome Research. 19 (12), 2324-2333 (2009).
  44. Katzen, F. Gateway ®Recombinational cloning: a biological operating system. Expert Opinion on Drug Discovery. 2 (4), 571-589 (2007).
  45. Venken, K. J. T., He, Y., Hoskins, R. A., Bellen, H. J. P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster. Science. 314 (5806), 1747-1751 (2006).
  46. Bischof, J., et al. A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila. Development. 140 (11), 2434-2442 (2013).
  47. Bischof, J., Sheils, E. M., Björklund, M., Basler, K. Generation of a transgenic ORFeome library in Drosophila. Nature Protocols. 9 (7), 1607-1620 (2014).
  48. Laible, M., Boonrod, K. Homemade site directed mutagenesis of whole plasmids. Journal of Visualized Experiments. (27), e1135 (2009).
  49. Balana, B., Taylor, N., Slesinger, P. A. Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells. Journal of Visualized Experiments. (44), e2189 (2010).
  50. Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific C31 integrases. Proceedings of the National Academy of Sciences. 104 (9), 3312-3317 (2007).
  51. Ringrose, L. Transgenesis in Drosophila melanogaster. Methods in Molecular Biology. 561, 3-19 (2009).
  52. Venken, K. J. T., He, Y., Hoskins, R. A., Bellen, H. J. P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster. Science. 314 (5806), 1747-1751 (2006).
  53. Groth, A. C., Fish, M., Nusse, R., Calos, M. P. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genética. 166 (4), 1775-1782 (2004).
  54. Greenspan, R. . Fly Pushing: The Thory and Practice of Drosophila Genetics. , (2004).
  55. Ashburner, M., Golic, K., Hawley, R. S. . Drosophila: A Laboratory Handbook. , (2005).
  56. Diao, F., White, B. H. A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion. Genética. 190 (3), 1139-1144 (2012).
  57. Diao, F., et al. Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes. Cell Reports. 10 (8), 1410-1421 (2015).
  58. Bellen, H. J., et al. The Drosophila Gene Disruption Project: Progress Using Transposons With Distinctive Site Specificities. Genética. 188 (3), 731-743 (2011).
  59. Lee, P. -. T., et al. A gene-specific T2A-GAL4 library for Drosophila. eLife. 7, (2018).
  60. Venken, K. J. T., et al. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nature Methods. 8 (9), 737-743 (2011).
  61. Li-Kroeger, D., et al. An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila. eLife. 7, (2018).
  62. Salazar, J. L., Yamamoto, S. Integration of Drosophila and Human Genetics to Understand Notch Signaling Related Diseases. Advances in Experimental Medicine and Biology. 1066, 141-185 (2018).
  63. Wangler, M. F., Yamamoto, S., Bellen, H. J. Fruit Flies in Biomedical Research. Genética. 199 (3), 639-653 (2015).
  64. Duffy, J. B. GAL4 system indrosophila: A fly geneticist’s swiss army knife. Genesis. 34 (1-2), 1-15 (2002).
  65. Nagarkar-Jaiswal, S., et al. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila. eLife. 4, (2015).
  66. Dolph, P., Nair, A., Raghu, P. . Electroretinogram Recordings of Drosophila. (1), (2011).
  67. Lauwers, E., Verstreken, P. Assaying Mutants of Clathrin-Mediated Endocytosis in the Fly Eye. Methods in Molecular Biology. 1847, 109-119 (2018).
  68. Rhodes-Mordov, E., Samra, H., Minke, B. Electroretinogram (ERG) Recordings from Drosophila. Bio-Protocol. 5 (21), (2015).
  69. Deal, S., Yamamoto, S. Unraveling novel mechanisms of neurodegeneration through a large-scale forward genetic screen in Drosophila. Frontiers in Genetics. , (2019).
  70. Chouhan, A. K., et al. Uncoupling neuronal death and dysfunction in Drosophila models of neurodegenerative disease. Acta Neuropathologica Communications. 4 (1), 62 (2016).
  71. Lek, M., et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 536 (7616), 285-291 (2016).
  72. Liberg, D., Sigvardsson, M., Akerblad, P. The EBF/Olf/Collier family of transcription factors: regulators of differentiation in cells originating from all three embryonal germ layers. Molecular and Cellular Biology. 22 (24), 8389-8397 (2002).
  73. Prasad, B. C., et al. Unc-3, a gene required for axonal guidance in Caenorhabditis elegans, encodes a member of the O/E family of transcription factors. Development. 125 (8), 1561-1568 (1998).
  74. Jinushi-Nakao, S., et al. Knot/Collier and Cut Control Different Aspects of Dendrite Cytoskeleton and Synergize to Define Final Arbor Shape. Neuron. 56 (6), 963-978 (2007).
  75. Pozzoli, O., Bosetti, A., Croci, L., Consalez, G. G., Vetter, M. L. Xebf3 is a regulator of neuronal differentiation during primary neurogenesis in Xenopus. Biologia do Desenvolvimento. 233 (2), 495-512 (2001).
  76. Wang, S. S., Lewcock, J. W., Feinstein, P., Mombaerts, P., Reed, R. R. Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection. Development. 131 (6), 1377-1388 (2004).
  77. Fulp, C. T., et al. Identification of Arx transcriptional targets in the developing basal forebrain. Human Molecular Genetics. 17 (23), 3740-3760 (2008).
  78. Gécz, J., Cloosterman, D., Partington, M. ARX: a gene for all seasons. Current Opinion in Genetics & Development. 16 (3), 308-316 (2006).
  79. Dubois, L., Vincent, A. The COE–Collier/Olf1/EBF–transcription factors: structural conservation and diversity of developmental functions. Mechanisms of Development. 108 (1-2), 3-12 (2001).
  80. Cook, R. K., et al. The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome. Genome Biology. 13 (3), 21 (2012).
  81. Chao, H. -. T., et al. A Syndromic Neurodevelopmental Disorder Caused by De Novo Variants in EBF3. The American Journal of Human Genetics. 100 (1), 128-137 (2017).
  82. Sleven, H., et al. De Novo Mutations in EBF3 Cause a Neurodevelopmental Syndrome. The American Journal of Human Genetics. 100 (1), 138-150 (2017).
  83. Harms, F. L., et al. Mutations in EBF3 Disturb Transcriptional Profiles and Cause Intellectual Disability, Ataxia, and Facial Dysmorphism. The American Journal of Human Genetics. 100 (1), 117-127 (2017).
  84. Tanaka, A. J., et al. De novo variants in EBF3 are associated with hypotonia, developmental delay, intellectual disability, and autism. Molecular Case Studies. 3 (6), 002097 (2017).
  85. Blackburn, P. R., et al. Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases. Molecular Case Studies. 3 (3), 001743 (2017).
  86. Lopes, F., Soares, G., Gonçalves-Rocha, M., Pinto-Basto, J., Maciel, P. Whole Gene Deletion of EBF3 Supporting Haploinsufficiency of This Gene as a Mechanism of Neurodevelopmental Disease. Frontiers in Genetics. 8, 143 (2017).
  87. Liu, N., et al. Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder. Human Molecular Genetics. 27 (14), 2454-2465 (2018).
  88. Mesbah, K., et al. Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and arterial pole morphogenesis. Human Molecular Genetics. 21 (6), 1217-1229 (2012).
  89. Bellen, H. J., Yamamoto, S. Morgan’s legacy: fruit flies and the functional annotation of conserved genes. Cell. 163 (1), 12-14 (2015).
  90. Richards, S., et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. 17 (5), 405-423 (2015).
  91. McGary, K. L., Park, T. J., Woods, J. O., Cha, H. J., Wallingford, J. B., Marcotte, E. M. Systematic discovery of nonobvious human disease models through orthologous phenotypes. Proceedings of the National Academy of Sciences of the United States of America. 107 (14), 6544-6549 (2010).
  92. Yamamoto, S., et al. A Drosophila Genetic Resource of Mutants to Study Mechanisms Underlying Human Genetic Diseases. Cell. 159, 200-214 (2014).
  93. Ausubel, F. M. . Current Protocols in Molecular Biology. , (1989).
  94. Rubin, G., Spradling, A. Genetic transformation of Drosophila with transposable element vectors. Science. 218 (4570), 348-353 (1982).
  95. Sun, Y., Sriramajayam, K., Luo, D., Liao, D. J. A Quick, Cost-Free Method of Purification of DNA Fragments from Agarose Gel. Journal of Cancer. 3, 93-95 (2012).
  96. Ronaghi, M. DNA Sequencing :A Sequencing Method Based on Real-Time Pyrophosphate. Science. 281 (5375), 363-365 (1998).
  97. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 77 (1), 51-59 (1989).

Tags

DrosophilaIn Vivo Functional StudyRare Human VariantsDisease-associated VariantsProtein FunctionRescue ExperimentsOverexpression StudiesGal4-UAS SystemVisual SystemPhotoreceptors
check_url/pt/59658?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Harnish, J. M., Deal, S. L., Chao, H., Wangler, M. F., Yamamoto, S. In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila. J. Vis. Exp. (150), e59658, doi:10.3791/59658 (2019).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image