Name: investigating the pathogenesis of myh7 mutation gly823glu in familial hypertrophic cardiomyopathy using a mouse model
Uploaded: 2022-08-08T14:00:00
Description: Watch this Scientific Journal Video about Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model at JoVE.com

This work was supported by the Medical Research Fund project of Guangdong Province (A2022363) and the major project of the Guangdong Committee of Science and Technology, China (grant no.2022).
We would like to thank Qingjian Chen of the University of Maryland, College Park for the help during the preparation of this manuscript.

The histories of the families were obtained by interviewing the family members. The study was approved by the Ethics Committee of the Guangdong Provincial Hospital of Chinese Medicine (No. 2019074). Informed written consent was obtained from all the family members. All the animals are treated in accordance with the ethical guidelines of the Guangdong Provincial Hospital of Chinese Medicine (Guangzhou, China).
1. Study subjects
NOTE: The proband III-3 ...

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In this study, we describe one Chinese Han families with HCM. Genetics analysis revealed that a heterozygous MYH6 mutation p.G823E co-segregates with the disease in family members with autosomal dominant inheritance. To validate the pathogenicity of G823E mutation and discuss the underlying mechanisms, we created a C57BL/6N mouse model with G823E at mouse Myh7 locus by CRISPR/Cas9-mediated genome engineering.
Phenotypic characteristics of C57BL/6N-Myh7em1(G823E) knockin mice were ev...

Hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China, with an estimated incidence of 0.2%, affecting 150,000 people1,2.
The pathological anatomical feature that characterizes HCM is asymmetric ventricular hypertrophy, which often involves the ventricular outflow tract and/or interventricular septum3. The clinical manifestation is exertional dyspnea, fatigue, and chest pain. The individual phenotype of HCM has variability ranging from clinically insidious to severe heart failure. Patients with HCM require medical treatment, heart transplantation, life support equipment, and multidisciplinary follow-up4.
In the past century, PCR technology has changed the way we study DNA5. A DNA sequencing method for clinical diagnosis was discovered by Sanger and colleagues6. The Sanger technique was subsequently applied to the Human Genome Project, but this approach was costly and time-consuming7. The advent of whole-genome sequencing (WGS) brought insights into human genetic disease to new heights, but it remained prohibitive in terms of cost. Whole-exome sequencing (WES) technology has long been used to detect germline variants8 and has been successful in identifying somatic driver mutations in the exome of various cancers9. The detection of DNA exons or coding regions by WES can be used to reveal pathogenic variants in most Mendelian diseases. Today, with the decreasing cost of sequencing, WGS is expected to become an important tool in genomics research and can be widely used in the detection of pathogenic variants in the genome.
WES technology has also been used in inherited cardiomyopathy to identify pathogenic variants to further elucidate the etiology. Emerging evidence has implicated that genes coding sarcomere structural protein gene mutations, such as MYH710, MYH611, MYBPC312, MYL213, MYL314, TNNT215, TNNI316, TNNC117, and TPM118 are responsible for the genetic etiology of HCM. Awareness of pathogenic variants in rare disease-causing genes (e.g., obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF (OBSCN, OMIM: 608616)19, acting alpha 2 (ACTN2, OMIM: 102573)20, and cysteine and glycine rich protein 3 (CSRP3, OMIM: 600824)21) has also been associated with HCM. Current genetic studies have identified multiple distinct pathogenic variants in the sarcomeric protein gene in approximately 40%-60% of HCM patients, and genetic testing in HCM patients revealed that most pathogenic variants occur in the myosin heavy chain (MYH7) and myosin-binding protein C (MYBPC3). However,the genetic basis for HCM remains elusive. Exploring the pathogenicity of these variations that underlie the human HCM patients remains a major challenge22.
In this study, we report a pathogenic variant in MYH7 in a Chinese Han family with HCM by WES. In order to verify the pathogenicity of this variant, we established a C57BL/6N-Myh7em1(G823E) knockin mice using the CRISPR/Cas9 system. We also discuss plausible mechanisms of this variant.

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			<td>0.5&#215;TBE</td>
			<td>Shanghai Sangon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2&#215; Taq Master Mix (Dye Plus)</td>
			<td>Nanjing Novizan Biotechnology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Regu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine for small animals</td>
			<td>Reward Life Technology Co., Ltd.</td>
			<td>R500</td>
			<td></td>
		</tr>
		<tr>
			<td>BEDTools</td>
			<td></td>
			<td>2.16.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cas9 in vitro digestion method to detect gRNA target efficiency kit</td>
			<td>Viewsolid Biotechnology Co., Ltd.</td>
			<td>VK007</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Marker</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA stabilizer</td>
			<td>Shanghai Seebio Biotechnology Co., Ltd.</td>
			<td>DNAstable LD</td>
			<td>prevent DNA degradation</td>
		</tr>
		<tr>
			<td>Electric paraffin microtome</td>
			<td>Shenyang Hengsong Technology Co., Ltd.</td>
			<td>HS-S7220-B</td>
			<td></td>
		</tr>
		<tr>
			<td>GATK</td>
			<td></td>
			<td>v3.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentra Puregene blood kit</td>
			<td>Santa Clara</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide, coverslip</td>
			<td>Jiangsu Invotech Biotechnology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin staining solution, Eosin staining solution</td>
			<td>Shanghai Biyuntian Biotechnology Co., Ltd.</td>
			<td>C0107-500ml, C0109</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq X-ten platform</td>
			<td>Illumina</td>
			<td></td>
			<td>perform sequencing on the captured libraries</td>
		</tr>
		<tr>
			<td>Injection of chorionic gonadotropin</td>
			<td>Livzon Pharmaceutical Group Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Injection of pregnant mare serum gonadotropin</td>
			<td>Livzon Pharmaceutical Group Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Local suppliers</td>
			<td></td>
			<td>inhalation anesthesia</td>
		</tr>
		<tr>
			<td>Microinjection microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Embedding Machine</td>
			<td>Shenyang Hengsong Technology Co., Ltd.</td>
			<td>HS-B7126-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Picard</td>
			<td></td>
			<td>(2.2.4) 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Merck KGaA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>samtools</td>
			<td></td>
			<td>1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer</td>
			<td>Applied Biosystems</td>
			<td>ABI 3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>SureSelect Human All Exon V6</td>
			<td>Agilent Technology Co., Ltd.</td>
			<td></td>
			<td>exome probe</td>
		</tr>
		<tr>
			<td>T7 ARCA mRNA Kit</td>
			<td>New England BioLabs, Inc.</td>
			<td>NEB-E2065S</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature box</td>
			<td>BINDER GmbH</td>
			<td>KBF-S Solid.Line</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride Solution</td>
			<td>Sigma, Merck KGaA</td>
			<td>No. T2663</td>
			<td></td>
		</tr>
		<tr>
			<td>Veterinary ultrasound system</td>
			<td>Royal Philips</td>
			<td>CX50</td>
			<td></td>
		</tr>
	</tbody>
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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.

Clinical profile of the families 
The family pedigrees of HCM were obtained and are shown in Figure 2. All the documented family members were diagnosed with HCM at enrollment.
In the family (Figure 2A), the proband was patient III-7, who was diagnosed with HCM and left ventricular outflow tract obstruction (LVOTO) at 46 years old and underwent cardiac surgery. Patient III-3 had minor HCM that did not require surgical treatment. P...

Watch this Scientific Journal Video about Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model at JoVE.com

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

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.

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

This protocol is significant as it can provide a demonstration of ultrasound in small animals. This technique may provide insights into the discovery of pathogenic genes in hereditary cardiomyopathy. Fix the knockin mice horizontally on the platform then tilt the platform 30 degrees coddle to the knockin mouse.Remove hair on the anterior chest wall of the knockin mice using a depilatory cream position the probe vertically with the bump facing the animal's head. Then rotate the probe counterclockwise approximately 45 degrees in the para sternal long axis view. Rotate the probe 90 degrees clockwise to obtain the para sternal short axis view.After rotating the probe, adjust the Y axis displacement to get the correct slice. Observe the long axis image of the heart and then select M mode measurement data. Acquire ultrasound data from para sternal long axis views of mice and measure the heart rate, left ventricular ejection fraction, cardiac output, left ventricular and diastolic dimension, left ventricular and systolic dimension.Left ventricular posterior wall and ventricular septum. The family pedigrees of hypertrophic cardiomyopathy were obtained, and all the documented family members were diagnosed with hypertrophic cardiomyopathy enrollment. Sanger sequencing confirmed the same M Y H 7 P G 8 23 E variant in all patients, but not in the healthy individuals in the families.And 174 controls, the heterozygous M Y H 7 P G 8 23 E completely co segregated in this family. The glycine residue at Codon 8 23 in the neck domain region of M Y H 7 was highly conserved in all the available vertebrate myocin sequences. According to A C M G standards and guidelines the M Y H 7 P G 8 23 E variant was predicted to be a pathogenic variant.The knockin mice developed age-dependent cardiac hypertrophy after birth. Echocardiography revealed that the inter ventricular septum and left ventricular posterior wall developed with increased heart rate in the knockin mice. Also confirmed by the histological analysis.Virus ultrasound data can be obtained by rotating the probe at different angles on different sections of the mouse term.

Research

JoVE Journal

Genetics

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

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

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

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 ...