Name: genetic analysis of hereditary transthyretin ala97ser related amyloidosis
Uploaded: 2018-06-09T15:00:00.000+00:00
Duration: 6 min 33 s
Description: Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

我们要感谢新趣吴小姐在实验中的帮助。这项研究得到了长功夫医学研究计划 (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) 和 100-4470A3, 104-2462A3, 台湾的资助。

在实验室进行的测试是根据1988年《临床实验室改善修订》 (CLIA) 的要求而进行的, 由昌热心纪念医院机构评审委员会批准的规例及大学 (执照100-4470A3 和 104-2462A3)。所有患者均获得知情同意。
1. 采血标本收集
<ol><li>将全血收集到商用的 EDTA 处理管中。轻轻混合, 将血液样本储存在4摄氏度, 直到加工。</li>
</ol>2. 从外周血中提取 DNA
使用 dna 提取试剂盒进行基因组 dna 提取 (见材料表)。
<ol><li>在4.0 毫升血液样本中加入10毫升溶液1。在冰上孵化样品10分钟。</li>
	<li>离心样品在 2000 x g 5 分钟在4°c。小心去除和丢弃上清液。并用重悬3毫升溶液1中的颗粒。</li>
	<li>离心样品在 2000 x g 5 分钟在4°c 再和放弃上清。并用重悬3毫升溶液2中的颗粒。</li>
	<li>添加10µL 的链霉蛋白酶库存溶液 (225 毫克/毫升), 以获得最终浓度100µg/毫升和混合良好。孵化在37°c 过夜。</li>
	<li>在冰上把管子冷却10分钟. 将0.8 毫升溶液3添加到样品中, 反转 3-5 次。将样品放在冰上5分钟。</li>
	<li>离心样品在 3000 x g 15 分钟在4°c。小心地将上清转移到15毫升无菌锥形离心管。</li>
	<li>添加6µL 的 RNase 库存溶液 (10 毫克/毫升), 以获得最终浓度20µg/毫升。孵育37°c 15 分钟。</li>
	<li>加入2.5 毫升的异丙醇, 并通过轻轻反转几次, 以沉淀 DNA (白色的股, 絮凝材料将形成) 彻底混合。使用大口径吸管, 将 DNA 沉淀转移到一个新的1.5 毫升离心管。</li>
	<li>离心机在 1.2万 x g 3 分钟在4摄氏度。小心丢弃上清液。通过添加500µL 70% 乙醇来清洗 DNA 颗粒。</li>
	<li>离心机在 1.2万 x g 1 分钟, 并丢弃上清。室温下的空气干 DNA 颗粒。添加100µL ddH2O 和孵化65°c 15 分钟并用重悬 DNA。</li>
</ol>3. 基因组 DNA 量化
使用分光光度计 (见材料表) 来检测基因组 DNA 的数量和质量。
<ol><li>登录到连接到分光光度计机器的计算机上。打开软件。</li>
	<li>初始化分光光度机:<ol>
<li>在分光光度计软件上单击 "核酸"。</li>
		<li>用一次性纸擦拭和纯净水清洁底座。</li>
	</ol>
</li>
	<li>空白分光光度计:<ol>
<li>在基座上装载2µL 纯净水。</li>
		<li>降低分光光度计的上臂, 点击 "空白" 校准。</li>
		<li>完成后, 抬起上臂, 用擦拭擦干底座。</li>
	</ol>
</li>
	<li>测量样品:<ol>
<li>在示例类型上单击 "DNA-50"。</li>
		<li>在基座上装载2µL 的 DNA 样本。</li>
		<li>下上臂, 点击 "测量" 测量 A260/A280 比和样品浓度。</li>
		<li>清洁的基座之间的每一个运行与擦拭和纯净水。</li>
	</ol>
</li>
	<li>单击 "打印屏幕" 以收集数据。</li>
</ol>4. 突变的遗传分析
用 PCR4放大靶 DNA。使用 DNA 聚合酶试剂盒在25µL 反应混合物中进行 PCR (见材料表)。表 1显示了 TTR 基因内含子底漆。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>基因</td>
			<td>底漆序列</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5 '-TCAGATTGGCAGGGATAAGC-3 ' R: 5 '-GCAAAGCTGGAAGGAGTCAC-3 '</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5 '-TCTTGTTTCGCTCCAGATTTC-3 ' R: 5 '-TCTACCAAGTGAGGGGCAAA-3 '</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5 '-GTGTTAGTTGGTGGGGGTGT-3 ' R: 5 '-TGAGTAAAACTGTGCATTTCCTG-3 '</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5 '-GACTTCCGGTGGTCAGTCAT-3 ' R: 5 '-GCGTTCTGCCCAGATACTTT-3 '</td>
		</tr>
</tbody></table>表1。TTR 基因内含子底漆.(NCBI 参考序列: NC_000018.10)
<ol><li>将表 2中的试剂添加到0.2 毫升 PCR 管中。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>组件</td>
			<td>容积 (µL)</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>10x 缓冲器</td>
			<td>2。5</td>
			<td>1x</td>
		</tr>
<tr>
<td>氯化镁2 (25 毫米)</td>
			<td>1。5</td>
			<td>1.5 毫米</td>
		</tr>
<tr>
<td>360 GC 增强器</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360脱氧核糖核酸聚合酶</td>
			<td>0。2</td>
			<td>2 U 或反应</td>
		</tr>
<tr>
<td>dNTP 混合 (每25毫米)</td>
			<td>2</td>
			<td>每2毫米</td>
		</tr>
<tr>
<td>底漆 F (10 µM)</td>
			<td>1</td>
			<td>0.4 µM</td>
		</tr>
<tr>
<td>底漆 R (10 µM)</td>
			<td>1</td>
			<td>0.4 µM</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>PCR 级水</td>
			<td>13。8</td>
			<td>-</td>
		</tr>
<tr>
<td>总容积</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>表2。PCR 反应条件。
<ol start="2"><li>在台式离心机上轻轻地混合离心机, 将所有部件收集到管子的底部。把管子放在 thermocycler。</li>
	<li>进行30个周期的 PCR。每个周期包括变性步骤三十年代在94°c, 底漆退火为三十年代在58°c, 并且聚合酶引伸为四十五年代在72°c (参见表 3)。</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>步</td>
			<td>温度 (°c)</td>
			<td>时间</td>
			<td>周期</td>
		</tr>
<tr>
<td>初始变性</td>
			<td>95</td>
			<td>5分钟</td>
			<td>1</td>
		</tr>
<tr>
<td>DNA 变性步骤</td>
			<td>94</td>
			<td>三十年代</td>
			<td>30个周期</td>
		</tr>
<tr>
<td>底漆退火步骤</td>
			<td>58</td>
			<td>三十年代</td>
			<td>30个周期</td>
		</tr>
<tr>
<td>聚合酶扩展步骤</td>
			<td>72</td>
			<td>四十五年代</td>
			<td>30个周期</td>
		</tr>
<tr>
<td>后伸长步骤</td>
			<td>72</td>
			<td>10分钟</td>
			<td>1</td>
		</tr>
<tr>
<td>PCR 循环结束</td>
			<td>4</td>
			<td>不定</td>
			<td></td>
		</tr>
</tbody></table>表3。用于扩增 PCR 产品的循环条件。
<ol start="4"><li>用凝胶电泳可视化技术验证 PCR 产品:<ol>
<li>准备1.2% 琼脂糖凝胶:<ol>
<li>将1.2 克琼脂糖粉与100毫升的0.5x 微波瓶混合在一起。微波2分钟, 直到琼脂糖完全溶解。将琼脂糖混合物冷却到55摄氏度。</li>
			<li>加入2µL 的溴溴溶液 (10 毫克/毫升) 的琼脂糖混合物, 并轻轻搅拌。将琼脂糖混合物倒入凝胶托盘中约 5-7 毫米, 然后添加梳 (s) 到位。允许它固化 (至少30分钟), 并小心地移除梳子 (s)。</li>
		</ol>
</li>
		<li>载入样品并运行琼脂糖凝胶:<ol>
<li>将凝胶和托盘放入电泳室。用0.5x 填充电泳细胞, 直到凝胶覆盖。</li>
			<li>仔细地装载2µL 100 bp 脱氧核糖核酸梯子 (参见材料表) 入胶凝体的一条车道作为分子大小标记。负载染料/样品混合物 (5 µL PCR 产品混合1µL 6x 负载染料) 进入额外的车道。</li>
			<li>打开电源。在 100 v 的时候用25分钟的凝胶。</li>
			<li>从电泳细胞中取出凝胶。在成像系统中, 在紫外线照射下对 DNA 片段进行可视化。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. DNA 测序
<ol><li>使用商业工具包和序列进行商业或自动排序器 (参见材料表) 来纯化 PCR 产品。</li>
	<li>提交核苷酸序列到 NCBI 爆炸服务器, 以比较核苷酸查询到核苷酸数据库10。显示结果后, 执行序列对齐并识别预期的突变。</li>
</ol>

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B., Lundgren, E., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mutation,+two+distinct+disease+variants:+Unravelling+the+impact+of+transthyretin+amyloid+fibril+composition.">One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition.</a> Journal of Internal Medicine. 281 (4), 337-347 (2017).</li><li>Vil&#224;-Rico, M., 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=Quantitative+analysis+of+post-translational+modifications+in+human+serum+transthyretin+associated+with+familial+amyloidotic+polyneuropathy+by+targeted+LC-MS+and+intact+protein+MS.">Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.</a> Journal of Proteomics. 127, 234-246 (2015).</li></ol>

作者没有什么可透露的。

协议中有两个关键步骤。首先, 为了有足够数量的白细胞, 应避免 hemodiluted 标本11。其次, 使用合适的 PCR 引物是取得可靠结果12的根本。我们使用的底漆爆破 web 工具设计引物4,13;在四 TTR 外显子的每一侧至少有40基对。我们还在 NCBI 上进行爆炸检查底漆的特异性。
在 DNA 提取步骤中进行了一些修改。首先, 使用的三解决方案的数量减少。这种修改还会产生足够数量的 DNA 并保存解决方案。第二, 异丙醇或100% 乙醇是在 DNA14,15的沉淀中使用的两种替代品。与乙醇相比, 异丙醇在室温下沉淀 DNA, 降低了盐15的共沉淀。此外, 在冷冻温度下, 可能需要更长的降水时间以最大限度地提高15的 DNA 产量。三是用 TE 缓冲液代替双蒸馏水 (ddH2O) 并用重悬 DNA。该协议中的 DNA 制备用于后续 PCR, 因此对存储的保护并不令人关注。此外, EDTA 将抑制酶活性, 当 DNA 用于 PCR15。
为了降低成本和更少的等待时间, PCR 产品纯化和排序是由生物技术公司进行的。该协议的局限性应该是手动方法, 包括重复的离心步骤。开发了一种自动化的核酸提取系统, 有利于缩短工作时间, 提高结果的重现性和质量16。
在所有淀粉样蛋白沉积的患者中, 应进行系统性 amyloidoses 的鉴别诊断。质谱 (MS) 可用于确定蛋白亚基, 并将该病归类为免疫球蛋白轻链淀粉样变性 (AL, 中位存活率低于 ATTR)17或 transthyretin 相关的淀粉样变性18,19, 直接下游基因测试不可能做, 特别是为那些遗传性 ATTR 最初不怀疑19的患者。
以质谱为基础的蛋白质组学分析已用于 TTR 淀粉样变性19、20、21的筛选和分型。然而, 单 ms 并不足以排除遗传性 ATTR22患者的致病性突变, 因为一些非典型或罕见的 TTR 变种可能不会被分离的淀粉样体沉积标本或血清样品的 MS 分析临床实验室19,23。此外, 可能的取样误差, 淀粉样纤维的分布不均匀可能导致假阴性组织活检5,21,24, 使下游蛋白质组分析困难。因此, DNA 检测是最可靠的 ATTR 测试, 应在大多数情况下进行 ATTR5,22, 以及任何特发性进展轴突周围神经病变或远端对称性疼痛小纤维神经病变25,26。
其他可能与 ATTR 表型变异相关的因素和指导未来方向包括在血清和 ATTR 纤维成分27,28的转化后修饰 (PTMs) 变体。尽管遗传性 ATTR 是一种单疾病, 但在相同突变或同一家族内的变异人中, 也有相当大的变异性27。单基因异质性无法阐明 ATTR28的不同发病和病理。因此, 当考虑到这些因素时, 应利用遗传和蛋白质组学方法。

Transthyretin (TTR) 淀粉样变性 (ATTR) 是遗传性系统性淀粉样变性1的最常见形式, 可由 Transthyretin (TTR) 基因2中的常染色体显性遗传突变引起。TTR 突变破坏 tetrameric 蛋白结构的稳定, 并导致其离解成单体, 重组成淀粉样纤维2。超过 100 amyloidogenic TTR 突变已被报告全球1。用聚合酶链反应 (PCR) 方法进行遗传分析, 证实了 TTR 点突变的存在, 具有避免放射性标记探针处理与南方印迹3相比较的优点。PCR 是一种快速、简便、廉价、可靠的技术, 已应用于现代科学的众多领域4。
由于其表型异质性, 早期诊断这一进步和致命的疾病是有挑战性的。随着对 ATTR 早期表现的关注和广泛的认识, 以及新出现的治疗方法5, 适当的诊断研究, 包括 TTR 基因检测, 对于改善预后至关重要。此外, 不同的突变与不同显性的特点, 发病年龄, 进展模式, 疾病严重性, 中位生存率, 肝移植的疗效, 或 TTR 稳定剂2,6, 和不同程度的神经和心脏参与, 这对基因咨询有很大的影响7,8,9。此外, 高度准确的基因检测是唯一的工具, 区分两种不同类型的 ATTR: 遗传性 (突变) 和野生类型 (非突变体, 老年系统性淀粉样变性, SSA)7。必须确认 ATTR 的类型, 因为治疗方法相差很大2。因此, TTR 遗传试验的逐步性协议的描述越来越有必要。
以台湾最常见的地方性突变 Ala97Ser 为例, 说明了检测突变的分子方法。dna 提取步骤的修改减少了三种溶液的用量, 并产生了足够数量的 dna。在该协议中, 对所有四 TTR 外显子进行了分析, 而包括 5 ' 上游、3 ' 下游、启动子、内含子和未翻译区域 (UTR) 的区域未进行排序。

<table><tbody><tr><td>EDTA-treated tubes</td><td>BD</td><td></td><td></td></tr><tr><td>DNA Extraction Kit</td><td>Stratagen</td><td>200600</td><td></td></tr><tr><td>NanoDrop ND2000 spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Delicate Task Wipers</td><td>Kimberly-Clark</td><td>Kimtech Science Kimwipes</td><td></td></tr><tr><td>AmpliTaq Gold 360 DNA Polymerase kit</td><td>Applied Biosystems</td><td>4398823</td><td></td></tr><tr><td>TTR gene intronic primers&amp;nbsp;</td><td>Exon1</td><td>F: 5&amp;rsquo;-TCAGATTGGCAGGGATAAGC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon1</td><td>R: 5&amp;rsquo;-GCAAAGCTGGAAGGAGTCAC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>F: 5&amp;rsquo;-TCTTGTTTCGCTCCAGATTTC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>R: 5&amp;rsquo;-TCTACCAAGTGAGGGGCAAA-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>F: 5&amp;rsquo;-GTGTTAGTTGGTGGGGGTGT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>R: 5&amp;rsquo;-TGAGTAAAACTGTGCATTTCCTG-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>F: 5&amp;rsquo;-GACTTCCGGTGGTCAGTCAT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>R: 5&amp;rsquo;-GCGTTCTGCCCAGATACTTT-3&amp;rsquo;</td><td></td></tr><tr><td>thermocycler</td><td>Applied Biosystems</td><td>GeneAmp PCR System 9700</td><td></td></tr><tr><td>electrophoresis cell&amp;nbsp;</td><td>ADVANCE</td><td>Mupid-2plus</td><td></td></tr><tr><td>DNA ladder</td><td>Protech</td><td>PT-M1-100</td><td></td></tr><tr><td>dye</td><td>BioLabs</td><td>B7021</td><td></td></tr><tr><td>AlphaImager EC</td><td>Alpha Innotech</td><td>AlphaImager EC</td><td></td></tr><tr><td>automatic sequencer&amp;nbsp;</td><td>Applied Biosystems</td><td>3730xl DNA Analyzer</td><td></td></tr></tbody></table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

遗传检测是遗传性 transthyretin 相关淀粉样变性的最可靠的试验, 应在 transthyretin 淀粉样变性 (ATTR) 的大多数病例中进行。ATTR 是一种罕见的, 但致命的疾病与异构表型;因此, 诊断有时会延迟。因此, 随着对 ATTR 早期表现的关注和广泛的认识, 以及新的治疗方法, 适当的诊断研究, 包括 transthyretin (TTR) 基因检测, 以确认 ATTR 的类型和变种, 因此改善预后的基础。利用聚合酶链反应 (PCR) 方法进行遗传分析, 证实 TTR 点突变比传统方法 (如南方印迹) 的存在更为迅速和安全。在此, 我们展示了基因确认的 ATTR Ala97Ser 突变, 最常见的地方性突变在台湾。该协议包括四个主要步骤: 收集全血标本, dna 提取, 所有四 TTR 外显子的基因分析与 PCR, 和 DNA 测序。

琼脂糖凝胶电泳两个病人和一个健康的个人揭示了预期大小的带, 包括 454 bp PCR 产品的外显子4的 TTR 基因 (图 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> 图 1.凝胶电泳描述 PCR 扩增 TTR 基因.正常: 健康的个体。车道 M: 100 bp DNA 梯子作为分子大小标记。车道 1: 246 bp (外显子 1)。车道 2: 292 bp (外显子 2)。车道 3: 299 bp (外显子 3)。车道 4: 454 bp (外显子 4)。<a href="/files/ftp_upload/57743/57743fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
直接测序揭示了 TTR 基因外显子4中 G 的核苷酸 T 取代。这无义突变导致在两个病人的氨基酸位置97的丙氨酸到丝酸的替代。图 2显示了这两名患者和一个健康个体的序列图谱。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57743/57743fig2v2.jpg"> 图 2.TTR 基因 (患者1和 2) 外显子4的健康个体 (野生型) 和异型 G > T 突变的测序.箭头在患者1和患者2图谱表明两个重叠的高峰不同的颜色。两个峰值大约是序列其余部分高度的一半。这个异型替代, (克/T) 是确认的反向序列, (C/A)。这个数字的部分已经从我们以前的出版物5中的一个数字中修改过。<a href="/files/ftp_upload/57743/57743fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

在这里, 我们提出了一个协议, 以确认存在的点突变诊断遗传性 transthyretin 淀粉样变性, 使用 Ala97Ser, 最常见的地方性突变在台湾, 作为一个例子。

遗传性 Transthyretin Ala97Ser 相关淀粉样变性的遗传分析

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

Nanyang Technological University

Research

JoVE Journal

Medicine

7.6K 次观看.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. 在这里, 我们提出了一个协议, 以确认存在的点突变诊断遗传性 transthyretin 淀粉样变性, 使用 Ala97Ser, 最常见的地方性突变在台湾, 作为一个例子。

视频: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

高分辨率融合PCR用于补体受体1长度多态性基因分型:阿尔茨海默病基因易感性评估的创新工具

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

科研

遗传学

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

用高光谱共聚焦显微镜和荧光寿命成像对发光共轭吩染色的淀粉样组织的成像

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

生物化学

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

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

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. ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

利用染色质配置的计算分析，将阿尔茨海默氏病变异体映射到其目标基因

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

生物工程

Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM remains elusive.
We previously identified a myosin heavy chain 7 (MYH7) gene heterozygous variant, NM_000257.4: c.G2468A (p.G823E), in a large Chinese Han family with HCM. In this family, variant G823E cosegregates with an autosomal dominant disorder. This variant is located in the lever arm domain of the neck region of the MYH7 protein and is highly conserved among homologous myosins and species. To verify the pathogenicity of the G823E variant, we produced a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus with CRISPR/Cas9-mediated genome engineering. We designed gRNA targeting vectors and donor oligonucleotides (with targeting sequences flanked by 134 bp of homology). The p.G823E (GGG to GAG) site in the donor oligonucleotide was introduced into exon 23 of MYH7 by homology-directed repair. A silenced p.R819 (AGG to CGA) was also inserted to prevent gRNA binding and re-cleavage of the sequence after homology-directed repair. Echocardiography revealed left ventricular posterior wall (LVPW) hypertrophy with systole in MYH7 G823E/- mice at 2 months of age. These results were likewise validated by histological analysis (Figure 3).
These results demonstrate that the G823E variant plays an important role in the pathogenesis of HCM. Our findings enrich the spectrum of MYH7 variants linked to familial HCM and may provide guidance for genetic counseling and prenatal diagnosis in this Chinese family.

Based on the familial hereditary cardiomyopathy family found in our clinical work, we created a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus through CRISPR/Cas9-mediated genome engineering to verify this mutation.

利用小鼠模型研究MYH7突变Gly823Glu在家族性肥厚型心肌病中的发病机制

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM ...

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics.
Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS&#39; strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Here, we present a protocol to utilize the latest version of the US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. This protocol demonstrates the application of the online tool to rapidly analyze protein conservation and provide customizable and easily interpretable predictions of chemical susceptibility across species.

演示序列比对以预测跨物种敏感性的工具，用于快速评估蛋白质保存

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

一个表型养生为用于研究衰老的人类疾病基因牵连转基因小鼠

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...