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 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.
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.
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 sought medical advice in the Department of Cardiovascular Surgery of the Guangdong Provincial Hospital of Chinese Medicine in July 2019.
2. DNA extraction
NOTE: DNA is extracted with a commercial blood kit according to the manufacturer's instructions.
3. Whole exome sequencing and variant analysis
NOTE: To systematically search for disease-causing gene mutations, exome sequencing in affected individuals (II-5, II-7, III-3, III-7, III-8, III-9, and IV-3) and unaffected individuals (III-2, III-5, IV-4) was performed.
4. Sanger sequencing
5. Generation of C57BL/6N-MYH7em1(G823E) knockin mice
6. Evaluation of the cardiac morphology and function
NOTE: Apply M-mode echocardiography to assess heart morphology and function of C57BL/6N-Myh7em1(G823E) knockin mice.
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. Patient IV-3 also had minor HCM, which was similar to his father, Patient III-3. Patient II-5 had HCM and underwent surgery to repair the defect at the age of 51 due to LVOTO. Patient I-1 and patient II-2 died due to cardiac accidents at 57 and 46 years old, respectively. Patient I-1's medical history was unavailable. Patient II-7 and patient III-9 presented with a shortness of breath and were diagnosed with HCM.
Exome sequence analysis and segregation of variants
In this family, exome sequencing of the five individuals generated a mean of a total of 19,978,731 pairs of sequenced reads with an average read length of 125 bp. In total, 98.72% of sequenced reads passed the quality assessment and were mapped to 98.66% of the human reference genome. Even after filtering, more than 42 variants (including single nucleotide substitutions and indels) were shared by these four patients. Of these, 27 were missense SNVs, 15 were predicted to alter splicing. Finally, according to ACMG rating guidelines, a heterozygous c.G2468A; p.G823E variant of MYH7 (NM_0002571) was observed in proband III-7 as well as the other three patients.
Identification of a pathogenic mutation
Sanger sequencing confirmed the same MYH7 p.G823E variant in all patients but not in the healthy individuals in the families and 174 controls (Figure 2B). The heterozygous MYH7 p.G823E completely co-segregated in this family. In this family, patients IV-3 who harbored this variant inherited it from patient III-3. Patients III-7 and III-8 carried the same variant, which was inherited from their father. The information for patient III-9 was unavailable.
This variant, previously described in HCM by a sporadic patient24, has not been reported in the 1000 G, ESP6500, ExAC, HGMD, ClinVar databases or control subjects. The glycine residue at codon 823 in the neck domain region of MYH7 is highly conserved in all the available vertebrate myosin sequences (Figure 2C). MYH7 is a known HCM-causative gene and plays an important role in cardiac development or structure/function. Based on ACMG standards and guidelines, the MYH7 p.G823E variant was predicted to be a pathogenic variant (PVS1 + PS3 + PS4 + PM2). Taken together, these results support that this variant is detrimental and contributes to the pathogenesis of HCM in these families.
C57BL/6N-Myh7em1(G823E) knockin mice developed severe cardiac hypertrophy
To further verify the pathogenesis of MYH7 p.G823E, we generate C57BL/6N-Myh7em1(G823E) knockin mice. C57BL/6N-MYH7em1(G823E) knockin mice developed an age-dependent cardiac hypertrophy after birth (Figure 2A,B). Echocardiography revealed that IVS and LVPW developed with increased HR in C57BL/6N-Myh7em1(G823E) knockin mice (Table 1). These results were likewise validated by histological analysis (Figure 3). There were no obvious differences in LVDd, LVDs, EF, or CO between wild-type and heterozygous mice (Table 1).
Figure 1: Ultrasound data acquisition. (A) The probe is located on the long axis of the sternum. (B) The probe is located on the short axis of the sternum. (C) M-mode ultrasound image of the long-axis view of the sternum. The yellow arrow indicates the location of the interventricular septum. The red arrow indicates the floating valve. (D) M-mode ultrasound image of the short-axis view of the sternum. The yellow arrow indicates the location of the anterior papillary muscle, and the bulge below is the posterior papillary muscle. Please click here to view a larger version of this figure.
Figure 2: The large family carrying the heterozygous MYH7 G823E variant. (A) The proband is marked by an arrow. Full and open circles and squares indicate affected and normal individuals, respectively. (B) The G823E variant in MYH7 confirmed by Sanger sequencing. (C) Conservation of the MYH7 G823E site in different species. The yellow arrow represents the site of G823E in the amino acid sequence of different species, which is highly conserved. Please click here to view a larger version of this figure.
Figure 3: Pathological changes of myocardial tissue. (A) Myocardial fibers are arranged in an orderly manner, and there is no significant difference between each part. (B) Ventricular muscle fiber hypertrophy, disordered arrangement, and lesions are mainly concentrated in the posterior wall of the left ventricle and interventricular septum. Please click here to view a larger version of this figure.
C57BL/6N-Myh7em1(G823E) knockin mice group | Control group | ||
(n=4) | (n=6) | ||
HR (/min) | 451.25 ± 25.786 | 413.83 ± 12.77 | P = 0.015 |
LVDd (mm) | 4.12 ± 0.33 | 3.95 ± 0.20 | P = 0.330 |
LVDs (mm) | 2.95 ± 0.44 | 2.85 ± 0.20 | P = 0.626 |
EF(%) | 55.02 ± 9.52 | 54.31 ± 5.11 | P = 0.881 |
CO (mL) | 18.46 ± 3.05 | 15.30 ± 2.39 | P = 0.102 |
IVS (mm) | 1.13 ± 0.20 | 0.67 ± 0.07 | P = 0.001 |
LVPW (mm) | 1.40 ± 0.60 | 0.70 ± 0.06 | P = 0.000 |
Table 1: Morphology and function analysis for p.G823E and wild type. Data were analyzed using SPSS. Continuous variables are expressed as mean ± standard deviation (SD). The Student's t or Wilcoxon rank sum tests for continuous variables. P-values below 0.05 were considered statistically significant.
Supplemental File 1. Please click here to download this File.
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 evaluated by echocardiography. Multiple trabeculation and a higher ratio of non-compacted to compacted myocardial layer were found in the C57BL/6N-Myh7em1(G823E) knockin mice compared to the controls. The transgenic mice also showed LVPW and IVS hypertrophy and an increase in HR. However, the function of the heart showed no significant change between the two groups. These results suggested that an increased HR may exhibit a compensatory effect on maintenance ventricle function during cardiac remodeling. Further studies are needed to observe and analyze the morphology and function of the heart.
Myosin-7 (MYH7) belongs to the family of myocardial sarcomeric structural proteins. MYH7 encoded by the MYH7 gene (14q11.2; OMIM: 160760), which is found in the first pathogenic gene associated with HCM in 1990. To date, more than 300 variants in the MYH7 gene have been associated with HCM25,26. However, the pathogenicity of great majority of variation remains elusive. Using this protocol, we created the model mice successfully. When planning and designing the model mouse, there are three main issues under consideration. First, the amino acid sequence of the mouse MYH7 protein is highly homologous with human. Second, MYH7 is highly expressed in ventricles, as well as MYH7 in humans. The glycine residue at codon 823 in the MYH7 is highly conserved in all available myosin sequences.
MYH7 contains a conserved head motor domain at the N-terminus, which binds actin and has actin-activated Mg·ATPase activity and a neck region, as well as a myosin tail, which contains a coiled-coil (CC) region at the C-terminus. The glycine residue at codon 823 in the neck domain region of MYH7 suggests that this variation may impact on lever arm rotation in contraction27,28,29,30.
WES mainly focuses on the exons of genes, which only account for 1.5%8 of the total genome DNA. However, most of the human genetic diseases that have been identified are related to exons31. This makes WES widely recognized and applied in clinical practice. There are certain ethical issues with the widespread application of WES. The study of this kind of human genetic disease often involves cross-border discussions. Although researchers try to remove personally identifiable information as much as possible, the huge information contained in DNA can still re-identify the research individuals, and even their families. Only standardized information exchange measures can ensure the rational application of WES. In the future, WES is of great significance for medical strategies for specific gene mutations and individualized diagnosis and treatment measures for multiple diseases.
In summary, we identified a MYH7 heterozygous variant, p.G823E, in a large Chinese Han family with HCM using WES. The C57BL/6N-Myh7em1(G823E) knockin mice were found to have thicker IVS and LVPW, and faster HR than wild-type mice. The p.G823E variant may impair lever arm rotation, suggesting that this mutation plays an important role in familial HCM. Our work broadens the information on the mutation spectrum of the MYH7 gene associated with HCM and provides better clarity into the appropriate diagnosis and genetic counseling of affected families.
The authors have nothing to disclose.
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.
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 |