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
1Department of Cardiovascular Surgery, Guangdong Provincial Hospital of Traditional Chinese Medicine,the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, 2The Second Clinical College of Guangzhou University of Chinese Medicine, 3Department of Cardiovascular Surgery, The First Affiliated Hospital,Jinan University, 4Guangdong Provincial Key Laboratory of Laboratory Animals,Guangdong Laboratory Animals Monitoring Institute, 5Department of Anesthesiology, Guangdong Provincial Hospital of Chinese Medicine,the Second Affiliated Hospital of Guangzhou University of Chinese Medicine
Summary
基于我们临床工作中发现的家族性遗传性心肌病家族，我们通过CRISPR / Cas9介导的基因组工程在小鼠MYH7位点创建了一个具有点突变（G823E）的C57BL / 6N小鼠模型来验证该突变。
Abstract
家族性肥厚型心肌病（HCM，OMIM：613690）是中国最常见的心肌病。然而，HCM的潜在遗传病因仍然难以捉摸。
我们之前在一个患有HCM的大型中国汉族家族中发现了肌球蛋白重链7（MYH7）基因杂合变体NM_000257.4：c.G2468A（p.G823E）。在该家族中，变异型 G823E 与常染色体显性遗传病共存。该变体位于MYH7蛋白颈部区域的杠杆臂结构域，并且在同源肌球蛋白和物种中高度保守。为了验证G823E变体的致病性，我们用CRISPR / Cas9介导的基因组工程制作了一个C57BL / 6N小鼠模型，该模型在小鼠MYH7位点具有点突变（G823E）。我们设计了gRNA靶向载体和供体寡核苷酸（靶向序列两侧有134 bp的同源性）。供体寡核苷酸中的p.G823E（GGG至GAG）位点通过同源定向修复引入MYH7的外显子23中。还插入了沉默的p.R819（AGG至CGA），以防止同源定向修复后gRNA结合和序列的重新切割。超声心动图显示 MYH7 G823E/- 小鼠在 2 个月龄时左心室后壁 （LVPW） 肥大伴收缩。这些结果同样通过组织学分析得到验证（图3）。
这些结果表明，G823E变异株在HCM的发病机制中起着重要作用。我们的研究结果丰富了与家族性HCM相关的MYH7变异谱，可为该中国家庭的遗传咨询和产前诊断提供指导。
Introduction
肥厚型心肌病（HCM，OMIM：613690）是中国最常见的心肌病，估计发病率为0.2%，影响15万人1，2。
HCM 的病理解剖学特征是不对称性心室肥厚，通常累及心室流出道和/或室间隔3。临床表现为劳力性呼吸困难、乏力和胸痛。HCM 的个体表型具有从临床隐匿到严重心力衰竭的变异性。HCM 患者需要药物治疗、心脏移植、生命支持设备和多学科随访4.
在过去的一个世纪里，PCR技术改变了我们研究DNA的方式5。Sanger及其同事发现了一种用于临床诊断的DNA测序方法6。桑格技术随后被应用于人类基因组计划，但这种方法既昂贵又耗时7。全基因组测序（WGS）的出现将人类遗传疾病的见解带到了新的高度，但在成本方面仍然令人望而却步。全外显子组测序（WES）技术长期以来一直用于检测种系变异8 ，并已成功鉴定各种癌症外显子组中的体细胞驱动突变9。通过WES检测DNA外显子或编码区可用于揭示大多数孟德尔疾病的致病变异。如今，随着测序成本的降低，WGS有望成为基因组学研究的重要工具，并可广泛应用于基因组中致病变异的检测。
WES 技术也已用于遗传性心肌病，以识别致病变异，以进一步阐明病因。新出现的证据表明，编码肌节结构蛋白基因突变的基因，如MYH7 10、MYH6 11、MYBPC3 12、MYL2 13、MYL3 14、TNNT215、TNNI3 16、TNNC1 17和TPM1 18是HCM遗传病因的原因。对罕见致病基因（例如，暗摜葒素、细胞骨架钙调蛋白和Titin相互作用的RhoGEF（OBSCN，OMIM：608616）19、作用α2（ACTN2，OMIM：102573）20以及半胱氨酸和甘氨酸丰富蛋白3（CSRP3，OMIM：600824）21））中致病变异的认识也与HCM有关。目前的遗传学研究已经在大约40%-60%的HCM患者中确定了肉瘤蛋白基因中多种不同的致病变异，HCM患者的基因检测显示，大多数致病变异发生在肌球蛋白重链（MYH7）和肌球蛋白结合蛋白C（MYBPC3）中。然而，HCM的遗传基础仍然难以捉摸。探索人类HCM患者背后的这些变异的致病性仍然是一个主要挑战22。
在这项研究中，我们报告了WES在中国汉族HCM家族中MYH7的致病变异。为了验证该变体的致病性，我们使用CRISPR / Cas9系统建立了C57BL / 6N-Myh7em1（G823E） 敲入小鼠。我们还讨论了这种变体的合理机制。
Protocol
这些家庭的历史是通过与家庭成员面谈获得的。该研究获得广东省中医院伦理委员会（第2019074号）批准。已获得所有家庭成员的知情书面同意。所有动物均按照广东省中医院（中国广州）的道德准则进行治疗。 1. 研究对象 注：先证者III-3于2019年7月到广东省中医院心血管外科就医。 获取先证者的详细家族病史。通知并呼叫先证者的所…
Representative Results
家庭的临床概况获得了HCM的家族谱系，如图 2所示。所有记录在案的家庭成员在入组时均被诊断为HCM。 在家庭中（图2A），先证者是患者III-7，他在46岁时被诊断出患有HCM和左心室流出道梗阻（LVOTO），并接受了心脏手术。患者III-3患有不需要手术治疗的轻微HCM。患者IV-3也有轻微的HCM，这与他的父亲患者III-3相似。患者II-5患有HCM，…
Discussion
在这项研究中，我们描述了一个患有HCM的中国汉族家庭。遗传学分析显示，杂合子MYH6突变p.G823E与常染色体显性遗传家族成员中的疾病共同分离。为了验证G823E突变的致病性并讨论其潜在机制，我们通过CRISPR / Cas9介导的基因组工程在小鼠Myh7位点上创建了具有G823E的C57BL / 6N小鼠模型。
采用超声心动图评价C57BL/6N-Myh7em1（G823E） 敲入小鼠的表型特征。与对照组相比，在C57BL…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了广东省医学研究基金项目（A2022363）和中国广东省科学技术委员会重大项目（批准号：2022）的支持。
我们要感谢马里兰大学帕克分校的陈青健在准备这份手稿期间提供的帮助。
Materials
| 0.5×TBE | Shanghai Sangon | ||
| 2× Taq Master Mix (Dye Plus) | Nanjing Novizan Biotechnology Co., Ltd. | ||
| Agarose | Regu | ||
| Anesthesia machine for small animals | Reward Life Technology Co., Ltd. | R500 | |
| BEDTools | 2.16.1 | ||
| Cas9 in vitro digestion method to detect gRNA target efficiency kit | Viewsolid Biotechnology Co., Ltd. | VK007 | |
| DNA Marker | Thermo Fisher Scientific | ||
| DNA stabilizer | Shanghai Seebio Biotechnology Co., Ltd. | DNAstable LD | prevent DNA degradation |
| Electric paraffin microtome | Shenyang Hengsong Technology Co., Ltd. | HS-S7220-B | |
| GATK | v3.5 | ||
| Gentra Puregene blood kit | Santa Clara | ||
| Glass slide, coverslip | Jiangsu Invotech Biotechnology Co., Ltd. | ||
| Hematoxylin staining solution, Eosin staining solution | Shanghai Biyuntian Biotechnology Co., Ltd. | C0107-500ml, C0109 | |
| HiSeq X-ten platform | Illumina | perform sequencing on the captured libraries | |
| Injection of chorionic gonadotropin | Livzon Pharmaceutical Group Inc. | ||
| Injection of pregnant mare serum gonadotropin | Livzon Pharmaceutical Group Inc. | ||
| Isoflurane | Local suppliers | inhalation anesthesia | |
| Microinjection microscope | Nikon | ECLIPSE Ts2 | |
| NanoDrop | Thermo Fisher Scientific | 2000 | |
| Paraffin Embedding Machine | Shenyang Hengsong Technology Co., Ltd. | HS-B7126-B | |
| Picard | (2.2.4) 20 | ||
| Proteinase K | Merck KGaA | ||
| samtools | 1.3 | ||
| Sequencer | Applied Biosystems | ABI 3500 | |
| Stereomicroscope | Nikon | SMZ745T | |
| SureSelect Human All Exon V6 | Agilent Technology Co., Ltd. | exome probe | |
| T7 ARCA mRNA Kit | New England BioLabs, Inc. | NEB-E2065S | |
| Temperature box | BINDER GmbH | KBF-S Solid.Line | |
| Trizma Hydrochloride Solution | Sigma, Merck KGaA | No. T2663 | |
| Veterinary ultrasound system | Royal Philips | CX50 |
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Citazione di questo articolo
Xia, Y., Hu, J., Li, X., Zheng, S., Wang, G., Tan, S., Zou, Z., Ling, Q., Yang, F., Fan, X. Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model. J. Vis. Exp. (186), e63949, doi:10.3791/63949 (2022).