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 ...

<ol>
	<li>Toepfer, C. N., 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=Myosin+sequestration+regulates+sarcomere+function,+cardiomyocyte+energetics,+and+metabolism,+informing+the+pathogenesis+of+hypertrophic+cardiomyopathy.">Myosin sequestration regulates sarcomere function, cardiomyocyte energetics, and metabolism, informing the pathogenesis of hypertrophic cardiomyopathy.</a> Circulation. 141 (10), 828-842 (2020).</li><li>Writing Committee Members 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=2020+AHA/ACC+guideline+for+the+diagnosis+and+treatment+of+patients+with+hypertrophic+cardiomyopathy:+A+report+of+the+American+College+of+Cardiology/American+Heart+Association+Joint+Committee+on+Clinical+Practice+Guidelines.">2020 AHA/ACC guideline for the diagnosis and treatment of patients with hypertrophic cardiomyopathy: A report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines.</a> The Journal of Thoracic and Cardiovascular Surgery. 162 (1), 23-106 (2021).</li><li>Elliott, P., McKenna, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+cardiomyopathy.">Hypertrophic cardiomyopathy.</a> Lancet. 363 (9424), 1881-1891 (2004).</li><li>Maron, B. J., Maron, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+cardiomyopathy.">Hypertrophic cardiomyopathy.</a> Lancet. 381 (9862), 242-255 (2013).</li><li>Inoue, T., Orgel, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonenzymatic+RNA+polymerase+model.">A nonenzymatic RNA polymerase model.</a> Science. 219 (4586), 859-862 (1983).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (12), 5463-5467 (1977).</li><li>Sachidanandam, R., 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+map+of+human+genome+sequence+variation+containing+1.42+million+single+nucleotide+polymorphisms.">A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms.</a> Nature. 409 (6822), 928-933 (2001).</li><li>Ng, S. B., 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=Targeted+capture+and+massively+parallel+sequencing+of+12+human+exomes.">Targeted capture and massively parallel sequencing of 12 human exomes.</a> Nature. 461 (7261), 272-276 (2009).</li><li>Wong, K. M., Hudson, T. J., McPherson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+genetics+of+cancer:+genome+sequencing+and+beyond.">Unraveling the genetics of cancer: genome sequencing and beyond.</a> Annual Review of Genomics and Human Genetics. 12, 407-430 (2011).</li><li>Mattivi, C. 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=Clinical+utility+of+a+phenotype-enhanced+MYH7-specific+variant+classification+framework+in+hypertrophic+cardiomyopathy+genetic+testing.">Clinical utility of a phenotype-enhanced MYH7-specific variant classification framework in hypertrophic cardiomyopathy genetic testing.</a> Circulation. Genomic and Precision Medicine. 13 (5), 453-459 (2020).</li><li>Jiang, J., Wakimoto, H., Seidman, J. G., Seidman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+silencing+of+mutant+Myh6+transcripts+in+mice+suppresses+hypertrophic+cardiomyopathy.">Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy.</a> Science. 342 (6154), 111-114 (2013).</li><li>Hayashi, T., 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=Genetic+background+of+Japanese+patients+with+pediatric+hypertrophic+and+restrictive+cardiomyopathy.">Genetic background of Japanese patients with pediatric hypertrophic and restrictive cardiomyopathy.</a> Journal of Human Genetics. 63 (9), 989-996 (2018).</li><li>Gil, W. S., &#193;vila Vidal, L. A., V&#225;squez Salguero, M. A., Cajiao, M. B., Pe&#241;a, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variant+affecting+the+myosin+light+chain+2+related+to+familial+hypertrophic+cardiomyopathy.">Genetic variant affecting the myosin light chain 2 related to familial hypertrophic cardiomyopathy.</a> Intractable &amp; Rare Diseases Research. 9 (4), 229-232 (2020).</li><li>Berge, K. E., Leren, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+hypertrophic+cardiomyopathy+in+Norway.">Genetics of hypertrophic cardiomyopathy in Norway.</a> Clinical Genetics. 86 (4), 355-360 (2014).</li><li>McNamara, J. W., Schuckman, M., Becker, R. C., Sadayappan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+homozygous+intronic+variant+in+TNNT2+associates+with+feline+cardiomyopathy.">A novel homozygous intronic variant in TNNT2 associates with feline cardiomyopathy.</a> Frontiers in Physiology. 11, 608473 (2020).</li><li>Wang, W., 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=Comparative+transcriptome+analysis+of+atrial+septal+defect+identifies+dysregulated+genes+during+heart+septum+morphogenesis.">Comparative transcriptome analysis of atrial septal defect identifies dysregulated genes during heart septum morphogenesis.</a> Gene. 575, 303-312 (2016).</li><li>Andersen, P. S., 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=Diagnostic+yield,+interpretation,+and+clinical+utility+of+mutation+screening+of+sarcomere+encoding+genes+in+Danish+hypertrophic+cardiomyopathy+patients+and+relatives.">Diagnostic yield, interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives.</a> Human Mutations. 30 (3), 363-370 (2009).</li><li>Nakashima, Y., 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=Lifelong+clinical+impact+of+the+presence+of+sarcomere+gene+mutation+in+Japanese+patients+with+hypertrophic+cardiomyopathy.">Lifelong clinical impact of the presence of sarcomere gene mutation in Japanese patients with hypertrophic cardiomyopathy.</a> Circulation Journal. 84 (10), 1846-1853 (2020).</li><li>Hu, L. R., Kontrogianni-Konstantopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+myocardia+containing+the+Obscurin+R4344Q+mutation+linked+to+hypertrophic+cardiomyopathy.">Proteomic analysis of myocardia containing the Obscurin R4344Q mutation linked to hypertrophic cardiomyopathy.</a> Frontiers in Physiology. 11, 478 (2020).</li><li>Girolami, F., 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=Novel+alpha-actinin+2+variant+associated+with+familial+hypertrophic+cardiomyopathy+and+juvenile+atrial+arrhythmias:+a+massively+parallel+sequencing+study.">Novel alpha-actinin 2 variant associated with familial hypertrophic cardiomyopathy and juvenile atrial arrhythmias: a massively parallel sequencing study.</a> Circulation. Cardiovascular Genetics. 7 (6), 741-750 (2014).</li><li>Salazar-Mendiguchia, J., 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=The+p.(Cys150Tyr)+variant+in+CSRP3+is+associated+with+late-onset+hypertrophic+cardiomyopathy+in+heterozygous+individuals.">The p.(Cys150Tyr) variant in CSRP3 is associated with late-onset hypertrophic cardiomyopathy in heterozygous individuals.</a> European Journal of Medical Genetics. 63 (12), 104079 (2020).</li><li>Teekakirikul, P., Zhu, W., Huang, H. C., Fung, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+cardiomyopathy:+An+overview+of+genetics+and+management.">Hypertrophic cardiomyopathy: An overview of genetics and management.</a> Biomolecules. 9 (12), 878 (2019).</li><li>Crossley, B. 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=Guidelines+for+Sanger+sequencing+and+molecular+assay+monitoring.">Guidelines for Sanger sequencing and molecular assay monitoring.</a> Journal of Veterinary Diagnostic Investigation. 32 (6), 767-775 (2020).</li><li>Song, 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=Mutations+profile+in+Chinese+patients+with+hypertrophic+cardiomyopathy.">Mutations profile in Chinese patients with hypertrophic cardiomyopathy.</a> Clinica Chimica Acta. 351 (1-2), 209-216 (2005).</li><li>Marian, A. J., Braunwald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+cardiomyopathy:+Genetics,+pathogenesis,+clinical+manifestations,+diagnosis,+and+therapy.">Hypertrophic cardiomyopathy: Genetics, pathogenesis, clinical manifestations, diagnosis, and therapy.</a> Circulation Research. 121 (7), 749-770 (2017).</li><li>Cann, F., 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=Phenotype-driven+molecular+autopsy+for+sudden+cardiac+death.">Phenotype-driven molecular autopsy for sudden cardiac death.</a> Clinical Genetics. 91 (1), 22-29 (2017).</li><li>Lafreniere-Roula, 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=Family+screening+for+hypertrophic+cardiomyopathy:+Is+it+time+to+change+practice+guidelines.">Family screening for hypertrophic cardiomyopathy: Is it time to change practice guidelines.</a> European Heart Journal. 40 (45), 3672-3681 (2019).</li><li>Winkelmann, D. A., Forgacs, E., Miller, M. T., Stock, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+drug-induced+allosteric+changes+to+human+beta-cardiac+myosin+motor+activity.">Structural basis for drug-induced allosteric changes to human beta-cardiac myosin motor activity.</a> Nature Communications. 6, 7974 (2015).</li><li>Garc&#237;a-Giustiniani, D., 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=Phenotype+and+prognostic+correlations+of+the+converter+region+mutations+affecting+the+&#946;+myosin+heavy+chain.">Phenotype and prognostic correlations of the converter region mutations affecting the &#946; myosin heavy chain.</a> Heart (British Cardiac Society). 101 (13), 1047-1053 (2015).</li><li>Moore, J. R., Leinwand, L., Warshaw, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+cardiomyopathy+phenotypes+based+on+the+functional+impact+of+mutations+in+the+myosin+motor.">Understanding cardiomyopathy phenotypes based on the functional impact of mutations in the myosin motor.</a> Circulation Research. 111 (3), 375-385 (2012).</li><li>Majewski, J., Schwartzentruber, J., Lalonde, E., Montpetit, A., Jabado, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+exome+sequencing+do+for+you.">What can exome sequencing do for you.</a> Journal of Medical Genetics. 48 (9), 580-589 (2011).</li></ol>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
</table>

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.

investigating-pathogenesis-myh7-mutation-gly823glu-familial

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 microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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 mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

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

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

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

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

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

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

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

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

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

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 diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

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

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

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

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 is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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 each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

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

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 viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

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

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

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 ...

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...