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

Summary

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.

Abstract

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.

Introduction

Hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China, with an estimated incidence of 0.2%, affecting 150,000 people^1,2.

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....

Protocol

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 .......

Discussion

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-Myh7^em1(G823E) knockin mice were ev.......

Materials

Name	Company	Catalog Number	Comments
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