Name: determining the likelihood of variant pathogenicity using amino acid-level signal-to-noise analysis of genetic variation
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Description: Watch this Scientific Journal Video about Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation at JoVE.com

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1. 识别感兴趣的基因和特定的拼接等形式
注: 在这里, 我们演示使用 ensembl15来确定与感兴趣的疾病的发病机制相关的感兴趣的基因的共识序列 (即kcnq1 突变与 lqts 相关)。ensembl 的替代方案包括通过国家生物技术信息中心 (ncbi)16和加州大学圣克鲁斯分校 (ucsc) 人类基因组17 (见材料表)。
<ol><li>在 ensembl 主?...

<ol>
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J., Apweiler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+and+resources+for+identifying+protein+families,+domains+and+motifs.">Tools and resources for identifying protein families, domains and motifs.</a> Genome Biology. 3 (1), (2002).</li><li>Cirino, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+Whole+Genome+Sequencing+to+Multigene+Panel+Testing+in+Hypertrophic+Cardiomyopathy+Patients.">A Comparison of Whole Genome Sequencing to Multigene Panel Testing in Hypertrophic Cardiomyopathy Patients.</a> Circulation Cardiovascular Genetics. 10 (5), (2017).</li><li>Landstrom, A. 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在过去十年中, 高通量基因检测在应用和可用性方面取得了显著进展。然而, 在许多具有成熟遗传基础的疾病中, 如心肌病, 扩大检测未能提高诊断结果21。此外, 许多已确定的变体的诊断效用存在很大的不确定性。这在一定程度上是由于在 wes 和 wgs 上发现的偶然发现的罕见变种越来越多, 这可能导致误诊22。氨基酸级信噪比分析基于公认的预测变异致病性的策略, ...

基因测序平台的迅速改进彻底改变了遗传学在医学中的可获得性和作用。曾经局限于单个基因或少数基因, 下一代基因测序成本的降低和速度的提高导致了基因组整个编码序列 (整个外显子组测序、wes) 和整个基因组 (全基因组测序, wgs) 在临床环境中。wes 和 wgs 经常被用于考虑危重新生儿和关注遗传综合征的儿童, 在这些环境中, wes 和 wgs 是一种经证实的诊断工具, 可以改变临床管理1,2。虽然这导致了对与遗传综合征相关的可能致病性突变的更多识别, 但也大大增加了偶然发现的未知诊断的基因变异或意外阳性结果的数量(vus)。虽然其中一些变异被忽略, 并没有报告, 变异定位与潜在致命或高度病态疾病相关的基因经常报告。目前的指南建议报告在可能对患者有益的特定基因中发现的附带变异, 包括与心脏猝死诱发疾病 (如心肌病和渠道病3。虽然这项建议旨在捕获有可能发生 scd 诱发疾病的个人, 但变种检测的敏感性远远超过特异性。这反映在越来越多的 vus 和偶然识别的变量与不明确的诊断效用, 远远超过频率的各自疾病在给定的人口4。其中一种疾病, 长 qt 综合征 (lqts), 是由突变定位到编码心脏离子通道的基因, 或通道相互作用的蛋白质, 导致心脏复极延迟5引起的典型心脏通道病.这种延迟复极化, 看到一个长期的 qt 间隔在休息心电图, 导致一个潜在的致命室性心律失常的电易感性, 如尖扭转。虽然一些基因与这种疾病的发展有关,但 kcnq1编码的 ik 钾通道 (kcnq1, kv7.1) 的突变是 lqts 1 型的原因, 并作为6型以下的例子.在描述变异解释的复杂性时, 以前所描述的是 lqts 相关基因中罕见的变异, 即所谓的 "背景遗传变异".
除了已知病原变异的大型汇编式数据库外, 还有几种策略可以预测不同变异将产生的影响。一些是基于算法, 如 sift 和 polyphen 2, 它可以过滤大量的新的非同义词变种, 以预测有害性9,10。尽管这些工具被广泛使用, 低特异性限制了其适用性, 当涉及到 "调用" 临床 vus11."信噪比" 分析是一种工具, 它根据已知的病源的已知病理变异频率, 根据种群中罕见的遗传变异, 确定变异与疾病相关的可能性。与基于人群的变异 (高信号到噪声) 相比, 与基于人群的变异相比, 与疾病相关突变的流行率较高的遗传位点的差异更有可能与疾病相关。此外, 与疾病相关频率 (低信噪比频率) 相比, 偶然发现的罕见变异体与罕见种群变异频率较高的基因的定位, 可能不太可能与疾病相关。信号到噪声分析的诊断效用已在最新的心肌病和渠道病基因检测指南中得到说明;然而, 它只被应用于整个基因水平或领域特异性的 12级.最近, 考虑到病理变异 (疾病数据库、文献中的队列研究) 和基于人群的控制变量 (exome 聚合联盟、exac 和基因组聚合数据库, gnomad13) 的可用性增加,这已被应用于蛋白质的原代序列内的单个氨基酸位置。氨基酸水平的信噪比分析已被证明是有用的分类偶然确定的变异的基因与 lqts 相关的可能 "背景" 遗传变异, 而不是疾病相关。在与 lqts 相关的三个主要基因中, 包括kcnq1, 这些偶然识别的变种缺乏显著的信噪比, 这表明这些变种在单个氨基酸位置的频率反映了罕见的种群变异, 而不是与疾病相关的突变。此外, 当蛋白质特异性域拓扑被叠加在高信噪区域时, 病理突变的 "热点" 被定位到蛋白质的关键功能域14。这种方法有希望在确定 1) 变异的可能性是疾病或人口相关和 2) 确定新的关键功能领域的蛋白质与人类疾病。

<table border="0" cellpadding="0" cellspacing="0" style="width:1277px;" width="1277">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:461px;">1000 Genome Project</td>
			<td style="width:109px;">N/A</td>
			<td style="width:359px;">www.internationalgenome.org</td>
			<td style="width:348px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ClinVar</td>
			<td>N/A</td>
			<td>www.ncbi.nlm.nih.gov/clinvar</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ensembl Genome Browser</td>
			<td>N/A</td>
			<td>uswest.ensembl.org/index.html</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Excel</td>
			<td style="width:109px;">Microsoft</td>
			<td style="width:359px;">office.microsoft.com/excel/</td>
			<td>Used for all example formulas and functions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exome Aggregation Consortium&#160;</td>
			<td>N/A</td>
			<td>www.exac.broadinstitute.org</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genome Aggregation Database&#160;</td>
			<td>N/A</td>
			<td>www.gnomad.broadinstitute.org</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">National Center for Biotechnology Information Domain and Structure Database</td>
			<td>N/A</td>
			<td>www.ncbi.nlm.nih.gov/guide/domains-structures/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">National Center for Biotechnology Information Gene Database</td>
			<td>N/A</td>
			<td>www.ncbi.nlm.nih.gov/gene/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">National Center for Biotechnology Information Protein Database</td>
			<td>N/A</td>
			<td>www.ncbi.nlm.nih.gov/protein/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">National Heart, Lung, and Blood Institute GO Exome Sequencing Project</td>
			<td>N/A</td>
			<td>www.evs.gs.washington.edu/EVS/</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:461px;">SnapGene</td>
			<td style="width:109px;">GSL Biotech LCC</td>
			<td style="width:359px;">www.snapgene.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">University of California, Santa Cruz Human Genome Browser</td>
			<td>N/A</td>
			<td>www.genome.ucsc.edu</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the likelihood of variant pathogenicity using amino acid-level signal-to-noise analysis of genetic variation

下一代基因测序的成本和速度的提高产生了临床全外显子组和全基因组检测的爆炸式增长。虽然这导致更多的识别可能与遗传综合征相关的致病性突变, 它也大大增加了偶然发现的未知意义的遗传变异 (vus) 的数量。确定这些变体的临床意义是科学家和临床医生面临的一项重大挑战。一种帮助确定致病性可能性的方法是在蛋白质序列级别进行信号到噪声分析。该协议描述了一种氨基酸级信噪比分析方法, 该方法利用蛋白质已知蛋白质拓扑结构的每个氨基酸位置的变异频率来识别初级序列中具有较高可能性的区域。病理变异 (相对于人口的 "背景" 变异)。该方法可以识别高病理信号的氨基酸残留位 "热点", 可用于细化下一代基因检测发现的 vuss 的诊断权重。

图 6给 kcnq1 的氨基酸级信号噪声分析一个有代表性的结果。在本例中, 描述了在 gnomad 队列 (对照组) 中识别的罕见变体、意外识别的 wes 变体 (实验队列 #1) 和被认为与疾病相关的 lqts 病例相关变体 (实验队列 #2)。此外, 还描述了与 gnomad 变异频率归一化的 wes 和 lqts 队列变量频率的信噪比分析。与 lqts 相关的变种在与通道孔、选择性滤波器和 kcnet 结合?...

Watch this Scientific Journal Video about Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation at JoVE.com

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

氨基酸水平信噪比分析确定了在给定氨基酸位置的遗传变异的流行率归一化为特定种群的背景遗传变异。这样就可以识别蛋白质序列 (信号) 中的变种 "热点", 这些变化高于在人群 (噪声) 中发现的罕见变种的频率。

遗传变异的氨基酸级信噪比分析确定变异致病性的可能性

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

氨基酸级信号-噪声分析，提供基因变异与疾病状态相关的可能性的度量，或是种群内自然遗传变异的一部分。这项技术利用了两种大型遗传资源，即文献中提供的疾病相关突变，或在公共领域进行基于种群的外向体和基因组研究，这些基因资源可以识别罕见的遗传差异。要识别感兴趣的特定基因和拼接异构体，请打开 Ensembl 主页，然后从下拉菜单中选择该物种。输入感兴趣基因的首字母缩略词，然后单击"转到"。从转录表中选择与感兴趣的基因和转录兴趣对应的链接以及感兴趣的 ID。在转录表的参考序列列中注意RNA转录本和RNA转录物识别编号的蛋白质产品，供将来参考。选择与RNA转录带ID号蛋白质产品相关的链接，从国家生物技术信息中心或NCBI蛋白质数据库打开一个新的网页，然后向下滚动到"起源"部分，获取感兴趣的基因转录本的主要蛋白质序列。然后向上滚动到"特征"部分以获取蛋白质特征的列表。要计算每个氨基酸位置的次要等位频率与控制变种，打开一个图形功能电子表格，并创建所有实验变体的位置列。删除变体文本以仅保留变体位置，并在升值中对变体进行排序，以确定哪些位置具有多个关联的变体。通过组合与给定位置关联的每个变体的次要等位基因频率，获取给定位置的所有次要等位基因频率的总和，并计算每个氨基酸位置的次要等位频率与实验变量。接下来，创建一列具有实验变异的氨基酸位置，并计算所有变异位置与该位置关联的所有变异的次要等位频率。若要为实验和控制变体创建次要等位基因的滚动平均值，请创建一个列，其中包含感兴趣的基因中的所有氨基酸位置，并为每个没有控制和实验数据集变体的位置添加一个零的次要等位频率。要为每个实验和控制流行率列创建滚动平均值，请创建一个列，表示控制和实验数据集的次要等位频率的滚动平均值，并在滚动平均值列中将五个变体位置 N 端和 C 端子各自的次要等位频率的平均值放置到每个位置。要计算队列最小频率，请将识别的最低次要等位基因除以 2，并在控制次要等位频率为零的任何单元格中输入此值。在计算信噪比时，这样可以避免除以零。要计算氨基酸级信噪比，将各氨基酸位实验滚动平均值除以各自的控制滚动平均值，并绘制此比率与氨基酸位置的图。要确定功能域和功能的一致氨基酸位置，或感兴趣的蛋白质的转化后修饰区域，确定与蛋白质域和功能相关的氨基酸位置，并打开 NCBI 网页。将感兴趣的蛋白质的RNA转录本的蛋白质产品输入搜索领域，并在特征下识别已知的蛋白质域和特征。识别和记录域名和类型以及氨基酸的位置，并选择与特征对应的链接，以可视化感兴趣的蛋白质主序列上的区域。在信噪柱旁边创建一列，以便引用氨基酸位置列，并识别每个域和功能的 N 或 C 终端方面对应的细胞。然后，在每个单元格中放置一个，在 y 轴上创建具有这些边界的图形，并在 x 轴上创建氨基酸位置的图形，然后用信噪点图覆盖此图形。要映射单个变体位置以覆盖信噪比和蛋白质域拓扑图，请创建域要素列旁边的列，使列中的行与氨基酸位置相对应，并在添加的行的每个单元格中放置一个，对应于包含相应变体的位置。然后，创建一个图形，此列作为 y 轴和 x 轴上的氨基酸位置，并覆盖此图形与信噪比和蛋白质域拓扑图。在这里，对钾电压门控通道亚家庭Q成员一个基因的氨基酸电平信号噪声分析有代表性的结果。在控制队列中识别的罕见差异，以及实验意外识别的有益体外体测序，以及被认为可能与疾病相关的长QT综合征病例相关变种显示。还表现了与控制队列变异频率进行比较的噪声信号分析，以及长QT综合征队列变异频率。在此实验中，长QT综合征相关变异表明，与通道浇注、选择性滤波器和钾电压门控通道亚家庭E成员一个绑定域对应的域中，信噪比很高。相比之下，在健康外向测序组中顺便识别的变异，没有清楚地显示高信号噪声高程的特定区域，这表明这些变异反映了背景遗传变异。这种方法可用于测量临床基因测试期间出现的未知意义变种的诊断重量。

Research

JoVE Journal

Genetics

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...