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

Dette arbejde blev støttet af Medical Research Fund-projektet i Guangdong-provinsen (A2022363) og det store projekt fra Guangdong-udvalget for videnskab og teknologi, Kina (bevilling nr. 2022).
Vi vil gerne takke Qingjian Chen fra University of Maryland, College Park for hjælpen under udarbejdelsen af dette manuskript.

Familiernes historier blev opnået ved at interviewe familiemedlemmerne. Undersøgelsen blev godkendt af den etiske komité på Guangdong Provincial Hospital of Chinese Medicine (nr. 2019074). Der blev indhentet informeret skriftligt samtykke fra alle familiemedlemmer. Alle dyrene behandles i overensstemmelse med de etiske retningslinjer fra Guangdong Provincial Hospital of Chinese Medicine (Guangzhou, Kina).
1. Studiefag
BEMÆRK: Proband III-3 søgte...

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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. 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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. 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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. 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I denne undersøgelse beskriver vi en kinesisk Han-familie med HCM. Genetikanalyse afslørede, at en heterozygot MYH6-mutation p.G823E co-segregerer med sygdommen hos familiemedlemmer med autosomal dominerende arv. For at validere patogeniciteten af G823E-mutationen og diskutere de underliggende mekanismer skabte vi en C57BL/6N-musemodel med G823E ved mus Myh7-locus ved CRISPR/Cas9-medieret genomteknik.
Fænotypiske egenskaber ved C57BL/6N-Myh7em1(G823E) knockinmus blev evalueret ve...

Hypertrofisk kardiomyopati (HCM, OMIM: 613690) er den mest almindelige kardiomyopati i Kina med en anslået forekomst på 0,2%, der påvirker 150.000 mennesker 1,2.
Det patologiske anatomiske træk, der karakteriserer HCM, er asymmetrisk ventrikulær hypertrofi, som ofte involverer ventrikulær udstrømningskanal og / eller interventrikulær septum3. Den kliniske manifestation er anstrengende dyspnø, træthed og brystsmerter. Den enkelte fænotype af HCM har variabilitet lige fra klinisk snigende til svær hjertesvigt. Patienter med HCM kræver medicinsk behandling, hjertetransplantation, livsstøtteudstyr og tværfaglig opfølgning4.
I det sidste århundrede har PCR-teknologi ændret den måde, vi studerer DNA5 på. En DNA-sekventeringsmetode til klinisk diagnose blev opdaget af Sanger og kolleger6. Sanger-teknikken blev efterfølgende anvendt på Human Genome Project, men denne tilgang var dyr og tidskrævende7. Fremkomsten af helgenomsekventering (WGS) bragte indsigt i menneskelig genetisk sygdom til nye højder, men det forblev uoverkommeligt med hensyn til omkostninger. Whole-exome sekventering (WES) teknologi har længe været brugt til at detektere kimlinje varianter8 og har haft succes med at identificere somatiske drivermutationer i exome af forskellige kræftformer9. Påvisning af DNA-exoner eller kodende regioner af WES kan bruges til at afsløre patogene varianter i de fleste mendelske sygdomme. I dag, med de faldende omkostninger ved sekventering, forventes WGS at blive et vigtigt redskab i genomforskning og kan i vid udstrækning anvendes til påvisning af patogene varianter i genomet.
WES-teknologi er også blevet brugt i arvelig kardiomyopati til at identificere patogene varianter for yderligere at belyse ætiologien. Nye beviser har antydet, at gener, der koder for sarcomere strukturelle proteingenmutationer, såsom MYH7 10, MYH6 11, MYBPC3 12, MYL2 13, MYL3 14, TNNT215, TNNI3 16, TNNC1 17 ogTPM1 18 er ansvarlige for HCM's genetiske ætiologi. Bevidsthed om patogene varianter i sjældne sygdomsfremkaldende gener (f.eks. obscurin, cytoskeletal calmodulin og titin-interagerende RhoGEF (OBSCN, OMIM: 608616)19, virkende alfa 2 (ACTN2, OMIM: 102573)20 og cystein- og glycinrigt protein 3 (CSRP3, OMIM: 600824)21) har også været forbundet med HCM. Nuværende genetiske undersøgelser har identificeret flere forskellige patogene varianter i det sarkomeriske proteingen hos ca. 40% -60% af HCM-patienter, og genetisk test hos HCM-patienter afslørede, at de fleste patogene varianter forekommer i myosin tunge kæde (MYH7) og myosinbindende protein C (MYBPC3). Det genetiske grundlag for HCM er dog fortsat uklart. At undersøge patogeniciteten af disse variationer, der ligger til grund for de menneskelige HCM-patienter, er fortsat en stor udfordring22.
I denne undersøgelse rapporterer vi en patogen variant i MYH7 i en kinesisk Han-familie med HCM af WES. For at verificere patogeniciteten af denne variant etablerede vi en C57BL/6N-Myh7em1(G823E) knockinmus ved hjælp af CRISPR/Cas9-systemet. Vi diskuterer også plausible mekanismer af denne 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

Familiær hypertrofisk kardiomyopati (HCM, OMIM: 613690) er den mest almindelige kardiomyopati i Kina. Den underliggende genetiske ætiologi af HCM forbliver imidlertid undvigende.
Vi har tidligere identificeret en myosin tung kæde 7 (MYH7) gen heterozygot variant, NM_000257.4: c.G2468A (p.G823E), i en stor kinesisk Han-familie med HCM. I denne familie cosegregerer variant G823E med en autosomal dominerende lidelse. Denne variant er placeret i håndtagsarmdomænet i halsområdet af MYH7-proteinet og er stærkt bevaret blandt homologe myosiner og arter. For at verificere patogeniciteten af G823E-varianten producerede vi en C57BL/6N-musemodel med en punktmutation (G823E) ved musens MYH7-locus med CRISPR/Cas9-medieret genomteknik. Vi designede gRNA-målretningsvektorer og donoroligonukleotider (med målretningssekvenser flankeret af 134 bp homologi). P.G823E (GGG til GAG) -stedet i donoroligonukleotid blev introduceret i exon 23 af MYH7 ved homologi-rettet reparation. En tavs p.R819 (AGG til CGA) blev også indsat for at forhindre gRNA-binding og genspaltning af sekvensen efter homologi-rettet reparation. Ekkokardiografi afslørede venstre ventrikulær posterior væg (LVPW) hypertrofi med systole i MYH7 G823E / - mus ved 2 måneders alder. Disse resultater blev ligeledes valideret ved histologisk analyse (figur 3).
Disse resultater viser, at G823E-varianten spiller en vigtig rolle i patogenesen af HCM. Vores resultater beriger spektret af MYH7-varianter forbundet med familiær HCM og kan give vejledning til genetisk rådgivning og prænatal diagnose i denne kinesiske familie.

Familiernes kliniske profil HCM's stamtavler blev opnået og er vist i figur 2. Alle de dokumenterede familiemedlemmer blev diagnosticeret med HCM ved tilmelding.
I familien (figur 2A) var probanden patient III-7, der blev diagnosticeret med HCM og forlod ventrikulær udstrømningskanalobstruktion (LVOTO) ved 46 år og gennemgik hjertekirurgi. Patient III-3 havde mindre HCM, der ikke krævede kirurgisk behandling. Patient IV-3 havd...

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

Baseret på den familiære arvelige kardiomyopatifamilie, der findes i vores kliniske arbejde, skabte vi en C57BL/6N-musemodel med en punktmutation (G823E) ved musen MYH7-locus gennem CRISPR/Cas9-medieret genomteknik for at verificere denne mutation.

Undersøgelse af patogenesen af MYH7-mutationen Gly823Glu i familiær hypertrofisk kardiomyopati ved hjælp af en musemodel

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

Denne protokol er vigtig, da den kan give en demonstration af ultralyd hos små dyr. Denne teknik kan give indsigt i opdagelsen af patogene gener i arvelig kardiomyopati. Fastgør knockinmusene vandret på platformen, og vip derefter platformen 30 grader til knockinmusen.Fjern hår på knockinmusenes forreste brystvæg ved hjælp af en hårfjerningscreme, der placerer sonden lodret med bulen vendt mod dyrets hoved. Drej derefter sonden mod uret ca. 45 grader i para sternal lang aksevisning. Drej sonden 90 grader med uret for at opnå den para sternale korte aksevisning.Når du har drejet sonden, skal du justere Y-aksens forskydning for at få den korrekte skive. Overhold hjertets lange aksebillede, og vælg derefter måledata for M-tilstand. Få ultralydsdata fra para sternal langakse visninger af mus og mål puls, venstre ventrikulær udstødningsfraktion, hjerteudgang, venstre ventrikulær og diastolisk dimension, venstre ventrikulær og systolisk dimension.Venstre ventrikulær bagvæg og ventrikulær septum. Familiens stamtavler af hypertrofisk kardiomyopati blev opnået, og alle de dokumenterede familiemedlemmer blev diagnosticeret med hypertrofisk kardiomyopati tilmelding. Sanger-sekventering bekræftede den samme M Y H 7 P G 8 23 E-variant hos alle patienter, men ikke hos de raske individer i familierne.Og 174 kontroller, den heterozygote M Y H 7 P G 8 23 E helt adskilt i denne familie. Glycinresten ved Codon 8 23 i halsdomæneområdet i M Y H 7 var stærkt konserveret i alle tilgængelige hvirveldyrs myocinsekvenser. I henhold til A C M G standarder og retningslinjer blev M Y H 7 P G 8 23 E-varianten forudsagt at være en patogen variant.Knockinmusene udviklede aldersafhængig hjertehypertrofi efter fødslen. Ekkokardiografi afslørede, at interventrikulær septum og venstre ventrikulær posterior væg udviklede sig med øget hjertefrekvens i knockinmusene. Også bekræftet af den histologiske analyse.Virus ultralydsdata kan opnås ved at rotere sonden i forskellige vinkler på forskellige sektioner af museperioden.

Research

JoVE Journal

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