Screening of MYH7 Gene Mutation Sites in Hypertrophic Cardiomyopathy and Its Significance

Screening of MYH7 Gene Mutation Sites in Hypertrophic Cardiomyopathy and Its Significance

Hypertrophic cardiomyopathy (HCM) is a primary cardiomyopathy characterized by asymmetrical hypertrophy of the ventricular septum, narrowing of the ventricular cavity, thickening of the ventricular wall, or increasing heart weight. It is the most common cause of sudden cardiac death (SCD) in young adults and a major cause of morbidity and mortality in the elderly. HCM is one of the most common autosomal dominant single-gene hereditary diseases in cardiomyopathy, with a prevalence of approximately one in 300 individuals, regardless of race or gender. In China, the prevalence of HCM is 80/100,000 among the adult population, and about 60% of adult patients with HCM exhibit clear disease-causing gene mutations.

The hereditary pattern of HCM involves autosomal dominant inheritance, and the high heritability of the disease often has a profound impact on the entire family of the patient. Therefore, it is of great significance to screen novel gene mutations in HCM. More than 1600 pathogenic mutations have been identified in at least 27 genes related to HCM, among which the most frequent mutations are in the MYH7 gene. MYH7 gene mutations related to HCM may lead to insufficiency of ventricular muscle energy supply, resulting in ventricular or ventricular septal hypertrophy. The pathogenic mutations involved in HCM are divided into two types: familial and sporadic. Familial HCM involves familial aggregation, making it easier to investigate the cause of death. However, the randomization of individuals with sporadic HCM (SHCM) has led to difficulties in determining the cause of death, making SHCM a research hotspot in forensic medicine.

This study aimed to screen the mutation sites in the sarcomeric gene MYH7 in Chinese patients with HCM and analyze the pathogenicity of the mutation sites as well as their significance in clinical and forensic medicine. The study was conducted on 18 cases of sudden death in patients with pathological diagnosis of HCM (non-hypertensive) and 20 cases of violent death autopsies that excluded cardiac hypertrophy and structural changes of the heart. The MYH7 gene status was detected in formalin-fixed paraffin-embedded tissues from the autopsy cases. Common mutation exon fragments of the MYH7 gene were amplified by polymerase chain reaction (PCR), and the end-of-deoxygenation method and gene cloning method were further performed to analyze the mutation sites. Homologous comparison among mutant sites was conducted using the BLAST online database.

The results showed that the 1336th nucleotide of the MYH7 gene at exon 14 was converted from T to G in one HCM case, resulting in the conversion of threonine (Thr) at position 446 to proline (Pro). In another case, the 1402th nucleotide at exon 14 was converted from T to C, resulting in the conversion of phenylalanine (Phe) at position 468 to leucine (Leu). Homologous comparison results showed that the two amino acid residues of Thr446 and Phe468 are highly conserved among different species. These findings indicate that fatal HCM harbored mutations of Thr446Pro and Phe468Leu in the MYH7 gene.

The Thr446Pro and Phe468Leu mutations of the MYH7 gene are located in exon 14, which corresponds to a link between two homocysteines in the adjacent hinge region of the beta myosin heavy chain gene, an important functional area of the spherical head. Mutations in the head region of the MYH7 gene can enhance the adenosine triphosphate enzyme activity of myosin S1, which may prevent changes in the conformation of myosin or alter its interaction with actin and other molecules. This ultimately can cause HCM, which is characterized by high penetrance, rapid progression, heart failure, and other malignant clinical manifestations. The high conservation of Thr446 and Phe468 among different species suggests that once the mutation occurs, it will play an important role in life activities.

The discovery of the Thr446Pro and Phe468Leu mutations in the MYH7 gene is of vital significance for patients at early stages of the disease, affecting risk assessment, prognosis, corresponding preventative measures, improvement of quality of life, and other specific treatments. Furthermore, such precise diagnosis and treatment methods and personalized medicine research on the basis of gene sequencing can prevent medical waste and help clinicians in achieving individual precision treatment. In terms of forensic medicine, continuous development of molecular testing could allow us to clarify the identity of the dead as well as determine specific reasons of death. Thus, the analysis of genetic mutations could play an important role in the identification of unknown individuals.

Among patients with MYH7 mutations, 30% to 40% are sporadic cases. Higher penetration has been reported for MYH7 mutations, which would increase the probability that the disease will manifest in the carriers of each family. Patients with MYH7 mutations can manifest the disease at an earlier age and have a higher degree of hypertrophy, a more malignant phenotype, and a poorer prognosis. The encoding MYH7 gene is located in chromosome 14q12, contains 40 exons, and its overall exon length is 5808 bp. The head of the MYH7 gene is made up of 3 to 21 exons, the neck consists of 21 to 25 exons, also known as the head-rod joints, and 25 to 40 exons form the valve stem. The mutation of the MYH7 gene involves aggregation, which can be found in the head and head-rod joints.

The exons selected in this study are common mutant exons reported in the literature. The exons measured have a high frequency of mutation and most of them are malignant mutations, which has research value and needs to be further studied and improved. Through Sanger sequence analysis of common mutations exons 3, 8, 14, 16, 17, 18, 19, 20, 22, 23, 26, and 27 of the MYH7 gene, we found that exon 14 in the MYH7 gene clearly exhibited the missense mutation Thr446Pro and Phe468Leu. Interestingly, these two mutations were detected previously in China but were not reported in the ESP, Online Mendelian Inheritance in Man, UCSC, and NCBI databases. The death of the Thr446Pro mutation was 48 years old, and the death of the Phe468Leu mutation was 35 years old. Both of the deceased died of unexplained sudden fall to the ground, and the age of sudden death was younger. In these two cases of mutated myocardial tissue, under the microscope, typical changes of HCM can be seen under HE staining, and myocardial fibrosis can be seen under Masson staining. Myocardial fibrosis is a cardiac interstitial remodeling characterized by excessive proliferation, collagen deposition, and abnormal distribution of cardiac interstitial fibroblasts, which is a potential risk factor for SCD. Cardiac myocytes are non-renewable cells. Once fibrous tissue proliferates, the inter-ventricular septum becomes thicker and harder, which limits ventricular filling and decreases diastolic capacity, leading to dysfunction of cardiac blood supply and eventually sudden death.

In addition, there are various reasons that can influence the occurrence and development of HCM, including genetic differences, insertion, and deletion of modified genes, geographical and environmental factors, and ethnicity. Some experiments have studied patients with SHCM and screened the mutation sites of HCM pathogenic genes. Five cases of cardiac myosin binding protein C (MYBPC3) gene mutation, two cases of cardiac troponin I (TNNI3) gene mutation, and one case of MYH7 gene mutation have been found. It shows that familial and SHCM have the same pathogenic genes. Therefore, SHCM patients should be alert to the risk of hereditary offspring. Because the two positive cases in this study were sudden death, no clinical data and no family members were found in autopsy records, it is impossible to do family analysis, but it can provide reference for other cases to make these two positive mutations again, which is still of great significance.

Sometimes the propositus may carry two disease-causing mutations, but genetic screening tests can only find one, thus affecting the results of genetic testing. In this study, only common mutant exons were detected, and the exons that were not detected had the possibility of mutation. However, due to a large amount of literature reading, no mutation sites of other exons were reported. In addition, because of the experimental method, only exons that are prone to mutation are studied, and deletion mutation is possible in undetected exons. These are the reasons for the low detection rate. Therefore, it is necessary to carry out genetic testing analysis and clinical phenotyping of large samples, in order to provide sufficient basis for the formulation of HCM treatments and prevention strategies. Further research studies are needed on the distribution of pathogenic genes in HCM and analysis of its pathological parameters, especially with regards to the relationship between severe clinical phenotypes and mutation genotypes as well as related factors that can affect the phenotype.

In conclusion, this study discovered that the Thr446Pro and Phe468Leu mutations of the MYH7 gene had been found earlier in Chinese patients, and that it could cause HCM. Furthermore, the risk of passing mutations involved in SHCM to the next generation is high, so it will be crucial to conduct family history screenings for patients with the disease. Detailed investigation and analysis of gene mutations related to HCM will not only have value in forensic medicine teaching and research services, but can also lead to medical determination of HCM through genetic testing and prevention of sudden deaths associated with the disease.

doi.org/10.1097/CM9.0000000000000428

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