CN112410418A

CN112410418A - Dilated cardiomyopathy molecular marker

Info

Publication number: CN112410418A
Application number: CN202011319028.3A
Authority: CN
Inventors: 周年伟; 舒先红; 王小林; 潘翠珍; 唐璐; 李雪洁; 谢惠琳; 蒋英英; 赵维鹏
Original assignee: Zhongshan Hospital Fudan University
Current assignee: Zhongshan Hospital Fudan University
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-02-26

Abstract

The invention discloses an expansion type cardiomyopathy molecular marker, which is a mutant SEQ ID NO. 3 of CHMP4C gene SEQ ID NO. 1 or a polypeptide SEQ ID NO. 4 coded by the mutant SEQ ID NO. 3. The molecular marker is used for preparing a kit for diagnosing dilated cardiomyopathy caused by autophagy dysfunction.

Description

Dilated cardiomyopathy molecular marker

Technical Field

The invention belongs to the field of medicine, and particularly relates to an expanded cardiomyopathy molecular marker and application thereof in preparation of a kit for diagnosing expanded cardiomyopathy.

Background

Dilated Cardiomyopathy (DCM), a primary cardiomyopathy of unknown cause, is characterized by enlargement of the ventricles and accompanying dysfunction of ventricular contraction, with early onset of arrhythmia. The disease becomes progressively worse and death may occur at any stage of the disease. According to clinical research, the incidence rate of people is about 1/2500, gene mutation is the main cause of DCM patients, and gene screening shows that about 35% of patients are mainly caused by gene mutation and relate to myocardial cytoskeletal genes, nuclear membrane genes, ion channel genes and the like, but most of patients do not find clear pathogenic genes, which indicates that potential dilated cardiomyopathy related new genes are to be discovered.

The disease course of the dilated cardiomyopathy patients has obvious heterogeneity, from asymptomatic to arrhythmia, the most serious is heart failure and sudden death, and DCM has intersection with other cardiomyopathies such as hypertrophic cardiomyopathy, right ventricular cardiomyopathy and ion channel disease in clinical manifestation and gene mutation. Since 2015, we screened patients clinically diagnosed with cardiomyopathy for causative genes. Into a total of 411 families, each family including patients and their relatives, we first sequenced the full exons of the proband of cardiomyopathy, and if necessary, the full exons of the relatives, and then performed sanger validation in the families of proband where causative gene mutations were found. In the Chinese invention patent CN201710732660.2, the pathogenic genes and pathogenic mutations related to dilated cardiomyopathy are found by capturing and enriching the whole genome DNA coding sequence through exons by using a second-generation sequencing technology, wherein the pathogenic genes and pathogenic mutations comprise Chmp4C gene c.488A > G mutation, LMNA gene c.961C > T mutation and MYH7 gene c.248G > C mutation.

Based on the findings, the pathogenic genes are screened by continuously utilizing whole-exon sequencing, and for families in which no definite pathogenic genes are found, other members with very definite phenotypes in the families are selected to perform whole-exon sequencing analysis, and the whole families are subjected to cosegregation verification to search possible pathogenic genes. The complete external sequencing of 4 Dilated Cardiomyopathy (DCM) patients carried CHMP4C mutation in the family, and a rare CHMP4C variant (c.488A > G, p.E163G) was found in family 1, indicating that the mutation is possibly related to DCM in the family; further, a new mutation was found in another 3 families: CHMP4C missense variants (c.c611t, p.s204l, gnomAD, 21/282456 and 0.00007435), were subsequently further validated for CHMP 4C-related function in animal models.

Disclosure of Invention

By constructing mouse models, it was found that hybrid CHMP4C mice exhibited increased heart rate, increased left ventricular end-systolic diameter, and decreased heart weight. In zebrafish experiments, CHMP4C overexpressing mutants resulted in cardiac malformations, pericardial edema and accelerated heart rhythm, suggesting that CHMP4C plays a key role in cardiac development and cardiovascular function. Further, the discovery of CHMP4C variants in HEK293 cells resulted in aberrant autophagy and activated apoptosis in HEK293T cells, suggesting a potential pathogenic mechanism for CHMP4C mutations in the heart, i.e., the induction of dilated cardiomyopathy via autophagy-apoptotic signaling pathways. It follows from this that: the CHMP4C gene mutation causes autophagy dysfunction of cardiac muscle cells, and then activates apoptosis signal pathways, resulting in abnormal cardiac muscle cell function, which is manifested as abnormal cardiac function. The above studies form the basis of the present invention. Specifically, the present invention includes the following technical solutions.

An expanded cardiomyopathy molecular marker, which is a CHMP4C gene SEQ ID NO 1 mutant SEQ ID NO 3; or CHMP4C gene mutant SEQ ID NO. 3 coded polypeptide SEQ ID NO. 4.

Wherein, SEQ ID NO. 3 is a (c.A488G, c.C611T) mutant of CHMP4C gene SEQ ID NO. 1. Correspondingly, the polypeptide SEQ ID NO. 4 is a (p.E163G, p.S204L) mutant of the wild-type polypeptide SEQ ID NO. 2 encoded by SEQ ID NO. 1.

atgagcaagttgggcaagttctttaaagggggcggctcttctaagagccgagccgctcccagtccccaggaggccctggtccgacttcgggagactgaggagatgctgggcaagaaacaagagtacctggaaaatcgaatccagagagaaatcgccctggccaagaagcacggcacgcagaataagcgagctgcattacaggcactaaagagaaagaagaggttcgagaaacagctcactcagattgatggcacactttctaccattgagttccagagagaagccctggagaactcacacaccaacactgaggtgttgaggaacatgggctttgcagcaaaagcgatgaaatctgttcatgaaaacatggatctgaacaaaatagatgatttgatgcaagagatcacagagcaacaggatatcgcccaagaaatctcagaagcattttctcaacgggttggctttggtgatgactttgatgaggatgagttgatggcagaacttgaagaattggaacaggaggaattaaataagaagatgacaaatatccgccttccaaatgtgccttcctcttctctcccagcacagccaaatagaaaaccaggcatgtcgtccactgcacgtcgatcccgagcagcatcttcccagagggcagaagaagaggatgatgatatcaaacaattggcagcttgggctacctaa(SEQ ID NO:1)；

MSKLGKFFKGGGSSKSRAAPSPQEALVRLRETEEMLGKKQEYLENRIQREIALAKKHGTQNKRAALQALKRKKRFEKQLTQIDGTLSTIEFQREALENSHTNTEVLRNMGFAAKAMKSVHENMDLNKIDDLMQEITEQQDIAQEISEAFSQRVGFGDDFDEDELMAELEELEQEELNKKMTNIRLPNVPSSSLPAQPNRKPGMSSTARRSRAASSQRAEEEDDDIKQLAAWAT(SEQ ID NO:2)；

atgagcaagttgggcaagttctttaaagggggcggctcttctaagagccgagccgctcccagtccccaggaggccctggtccgacttcgggagactgaggagatgctgggcaagaaacaagagtacctggaaaatcgaatccagagagaaatcgccctggccaagaagcacggcacgcagaataagcgagctgcattacaggcactaaagagaaagaagaggttcgagaaacagctcactcagattgatggcacactttctaccattgagttccagagagaagccctggagaactcacacaccaacactgaggtgttgaggaacatgggctttgcagcaaaagcgatgaaatctgttcatgaaaacatggatctgaacaaaatagatgatttgatgcaagagatcacagagcaacaggatatcgcccaagaaatctcagaagcattttctcaacgggttggctttggtgatgactttgatgaggatgggttgatggcagaacttgaagaattggaacaggaggaattaaataagaagatgacaaatatccgccttccaaatgtgccttcctcttctctcccagcacagccaaatagaaaaccaggcatgttgtccactgcacgtcgatcccgagcagcatcttcccagagggcagaagaagaggatgatgatatcaaacaattggcagcttgggctacctaa(SEQ ID NO:3)；

MSKLGKFFKGGGSSKSRAAPSPQEALVRLRETEEMLGKKQEYLENRIQREIALAKKHGTQNKRAALQALKRKKRFEKQLTQIDGTLSTIEFQREALENSHTNTEVLRNMGFAAKAMKSVHENMDLNKIDDLMQEITEQQDIAQEISEAFSQRVGFGDDFDEDGLMAELEELEQEELNKKMTNIRLPNVPSSSLPAQPNRKPGMLSTARRSRAASSQRAEEEDDDIKQLAAWAT(SEQ ID NO:4)。

Further, the dilated cardiomyopathy particularly refers to dilated cardiomyopathy caused by autophagy dysfunction.

Obviously, the CHMP4C gene mutant SEQ ID NO. 3 and the polypeptide SEQ ID NO. 4 coded by the same can be used as targets for developing drugs for treating or preventing dilated cardiomyopathy, in particular drugs for treating or preventing dilated cardiomyopathy caused by autophagy dysfunction; and can be used for preparing a kit for diagnosing dilated cardiomyopathy, including a gene detection kit for gene sequencing and a protein detection kit such as an ELISA kit.

Accordingly, a second aspect of the present invention provides a kit for diagnosis of dilated cardiomyopathy, which comprises a primer, a DNA/RNA probe, or a microarray chip of DNA/RNA probes for detecting the CHMP4C gene, and determines dilated cardiomyopathy when the determined nucleotide sequence is SEQ ID NO. 3.

For example, the above primers include a forward primer F and a reverse primer R:

F：5’-CGCCCAAGAAATCTCAGAAG-3’(SEQ ID NO:5)；

R：5’-CTGTGCTGGGAGAGAAGAGG-3’(SEQ ID NO:6)。

in another embodiment, there is provided another ELISA kit for diagnosis of dilated cardiomyopathy, comprising: (1) a first antibody capable of specifically binding polypeptide SEQ ID NO 4; (2) a labeled antibody (i.e., a second antibody) capable of binding to the polypeptide SEQ ID NO. 4 when the polypeptide SEQ ID NO. 4 binds to the first antibody as defined in (1); and (3) a polypeptide SEQ ID NO. 4 as a standard.

Preferably, the first antibody does not bind to CHMP4C gene SEQ ID NO. 1 encoded wild-type polypeptide SEQ ID NO. 2, thereby improving the accuracy and precision of diagnosis.

The above ELISA kit further comprises an initiator solution for initiating a chemical reaction between the label on the labeled antibody (i.e., the second antibody) and a chemiluminescent compound or a chromogenic substrate or a fluorescent dye as a reaction target of the label.

The label on the labeled antibody (i.e., the second antibody) is capable of catalyzing or activating a chemiluminescent compound or fluorochrome to rapidly convert a colorless substrate to a colored product, or to cause a change in light, or to convert a non-fluorescent fluorochrome to an intense fluorescent product, which may be peroxidase, phosphatase, or luciferase, preferably horseradish peroxidase.

The sample detected by the kit can be at least one of whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretion, skin and subcutaneous tissue, and peripheral blood is preferred.

The molecular markers SEQ ID NO. 3 and SEQ ID NO. 4 of the invention provide new targets for diagnosing and treating dilated cardiomyopathy, expand the gene spectrum for screening pathogenic genes of dilated cardiomyopathy, contribute to improving DCM management clinically and contribute to developing new targeted therapies. In clinical gene detection, the method can be used for conventional pathogenic gene screening, and the accuracy of pathogenic gene screening is improved.

Drawings

FIG. 1 shows photographs of the differences existing between wild type and Chmp4c heterozygous mice, HE and Masson staining of myocardial tissue. Among them, Chmp4c^+/-Mice exhibited cardiac abnormalities. a, model mouse construction strategy schematic diagram. b, genotype identification map of knockout mice. Separation was performed on a 1% agarose gel by gel electrophoresis. c, wild type and Chmp4c^+/-Heart morphology comparison of mice. Chmp4c compared with wild type^+/-The heart of the mice is small. d, HE and Masson staining showed irregular morphology of the myocardial fibers, with a large amount of collagen analogue deposited in the myocardial tissue. Electron microscopy revealed disorganization of the myofibers of the myocardial tissue. e, wild type and Chmp4c^+/-Representative echocardiograms of mice (taken in B mode). Chmp4c compared with wild type^+/-The mice had increased left ventricular end-systolic diameter and heart rate. f, Chmp4c, in comparison with the wild type^+/-The heart rate of the mice increased significantly. Chmp4c compared with wild type^+/-The heart weight of the mice was significantly reduced. P < 0.05.

Fig. 2 shows functional confirmation of CHMP4C mutation in zebrafish model and stained photographs of myocardial tissue sections. Wherein, a and b are the overall shapes of each group of zebra fish in different development stages. The CHMP4C mutant group showed significant pericardial edema (red arrow) compared to the control group and CHMP 4C-WT. C, f, H & E staining of the mutant zebrafish myocardial tissue showed significant growth retardation and morphological abnormalities compared to the other two groups. d. g, the percentage of pericardial edema was significantly increased in the CHMP4C mutant group compared to the wild-type and control groups. e. h, heart rate was also significantly increased compared to wild type and control groups (n ═ 20 zebrafish/group, heart rate/20 s). i, electron microscopy analysis showed accumulation of large amounts of abnormally black, high-density material in zebrafish cardiomyocytes.

FIG. 3 shows a schematic of the structure of CHMP4C plasmid for transfection of HEK293T cells ordered from Nanjing Kinshire. Wild-type CHMP4C or mutant CHMP4C (E163G) is subjected to PCR, then is cut by BamHI/EcoRI, and is then connected into a FUGW (BamHI/EcoRI) vector to obtain target plasmids FUGW-CHMP4C (wild type) and FUGW-CHMP4C (mutant).

FIG. 4 shows photographs of autophagy fluorescent particles taken by confocal immunofluorescence microscopy after transfection of HEK293 cells with plasmids containing CHMP4C wild-type gene or CHMP4C mutant gene fragments, respectively, and a control plasmid. Wherein, a, heat map of cluster analysis of different expressed genes. b, the top 20 with the highest GO abundance of chmp4 c-responsive genes. c, representative confocal images LC3 (green), blue (DAPI) of four groups of HEK293T cells.

Detailed Description

In the Dilated Cardiomyopathy (DCM) study, the inventors found by whole exome sequencing that 4 patient families carried the CHMP4C mutation. The prover of family 1 is a 58-year-old Han female, and the patient repeatedly suffers syncope and fatigue and goes to our hospital for a doctor. The electrocardiogram shows atrial fibrillation and left bundle branch block. Echocardiography and MRI suggest that the entire heart is enlarged. The contraction function of the left ventricle is reduced comprehensively, and the Left Ventricular Ejection Fraction (LVEF) is 32%. Serum creatine kinase and troponin T are significantly higher than in normal persons. The patient had no prior history of hypertension, coronary heart disease or diabetes. She was diagnosed as DCM. Family history of probands revealed more family members with abnormal cardiac function and malignant cardiovascular events. Some of her family members also suffered from cardiac dysfunction after clinical examination. A rare CHMP4C variant (c.488a > G, p.glu163gly) was found in this family, suggesting that it may be associated with DCM in this family. SIFT and Polyphen2 software predicted it to be detrimental to CHMP4C protein. Furthermore, such mutations are located in conserved regions across multiple species, consistent with inheritance between mendelian family members. Through the genetic analysis of family 1, CHMP4C is presumed to be a new cardiomyopathy causing gene. Based on this, 400 families of hereditary cardiovascular diseases were screened for CHMP4C variants using whole exome sequencing. Two new mutations were found in 3 families: both family 2 and family 3 carry CHMP4C missense variants (c.c611t, p.sg204l, gnomAD, 21/282456 and 0.00007435); family 4 carries frameshift variants (c.73 — 104 delgccctggtccgacttcgggagagactcgaggagat, p.l26gfs 26, gnomAD, 1/213196, 0.000004691). Subsequently, the functions related to CHMP4C are verified on animal model mice, zebrafish and HEK293 cells, and the possible pathogenic mechanism is found: the CHMP4C gene mutation causes autophagy dysfunction of cardiac muscle cells, and then activates apoptosis signal pathways, resulting in cardiac muscle cell dysfunction, which is manifested as cardiac dysfunction. Thus, a molecular marker of DCM, i.e., a CHMP4C variant (mutant), which is SEQ ID NO. 3; the protein sequence is SEQ ID NO. 4.

For ease of writing, CHMP4C is sometimes referred to herein as the english lowercase form CHMP4c, which are used synonymously.

Herein, for the sake of convenience of description, the protein is sometimes used in combination with its encoding gene (DNA) name, and those skilled in the art will understand that they represent different substances in different description occasions. Their meaning will be readily understood by those skilled in the art based on the context and context. For example, for the CHMP4C mutant, when used to describe a mutant CHMP4C protein (p.E163G, p.S204L), reference is made to the protein SEQ ID NO: 4; when described as a gene, it refers to the gene encoding the mutant (c.488A > G, c.C611T) mutant of SEQ ID NO. 3.

In this context, the normal CHMP4C protein SEQ ID NO:2 and its coding gene SEQ ID NO:1 can be referred to as "wild-type" for the purpose of distinguishing them from mutants.

As will be readily understood by those skilled in the art, the molecular markers SEQ ID NO:3 (nucleic acid molecule) and SEQ ID NO:4 (protein molecule) for detecting the morphology of different substances can be determined using detection means customary in the art. For nucleic acid molecules, detection can be performed using a genetic testing device such as an illumina sequencer. Therefore, necessary gene detection kits are required to be provided.

In a preferred embodiment, the kit may further comprise at least one of the following in addition to the various primers or DNA/RNA probes for the specific gene SEQ ID NO. 3: a carrier means, the space of which is divided into defined spaces that can receive one or more containers, such as kits, vials, tubes, and the like, each container containing a separate component for use in the method of the invention; instructions, which may be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, for example paper with a download window for the operation demonstration video APP, such as a two-dimensional code, or in the form of multimedia, such as a CD, a usb-disc, a web-disc, etc.

It is understood that the term "nucleic acid molecule" may be whole genomic DNA extracted directly from a biological sample, or a portion of the whole genome that contains the coding sequence of a disease-causing gene, or total RNA extracted from a biological sample, or mRNA extracted from a biological sample.

For the protein molecule SEQ ID NO 4, ELISA methods can be used for detection. The necessary ELISA kits need to be provided for this purpose. In a preferred embodiment, the kit may further comprise at least one of the following items in addition to the first antibody, the second antibody, the standard of SEQ ID NO:4, and the initiator solution, respectively: a carrier means, the space of which is divided into defined spaces that can receive one or more containers, such as kits, vials, tubes, and the like, each container containing a separate component for use in the method of the invention; instructions, which may be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, for example paper with a download window for the operation demonstration video APP, such as a two-dimensional code, or in the form of multimedia, such as a CD, a usb-disc, a web-disc, etc.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1 mouse experiment

To confirm the observations found in human DCM, we generated a Chmp4c mouse model.

The project adopts CRISPR/Cas9 technology to establish a CHMP4C gene condition knockout flox mouse. The technology is that sgRNA aiming at a target gene is designed to guide Cas9 nuclease to modify a genome at an insertion position, so that homologous recombination efficiency of a gene modification region is increased, and a target fragment is recombined to the target site.

The route for preparing conditional gene knockout flox mice by the technology is as follows:

1. designing a sgRNA target sequence aiming at a target site genome, and carrying out in vitro transcription on the sgRNA target sequence according to the sequence to obtain a sgRNA aiming at the CHMP4C gene;

2. constructing a donor DNA recombinant plasmid for target fragment recombination;

3. injecting fertilized eggs of the sgRNA and the donor DNA recombinant plasmid which are transcribed in vitro to obtain an F0-generation mouse;

4. carrying out genotype identification on the obtained F0 mouse by a PCR (polymerase chain reaction) and sequencing method;

5. mating F0 mouse with wild C57BL/6 mouse to obtain F1 mouse;

6. genotyping F1 mice by PCR and sequencing;

7. the sequencing is verified to be correct, and 6-8 weeks old positive F1 generation mice are taken as a Chmp4c mouse model.

F1 generation of a CHMP4C gene partially knocked-out mouse constructed by earlier research is utilized to breed a homozygous CHMP4C mutant mouse, a c57b/6 mouse is used as a control group, a control, heterozygous, pure and mutant mouse with the age of 4 weeks is taken, pentobarbital sodium with the weight of 40mg/kg is injected into an abdominal cavity, after the mouse is anesthetized, the chest is opened, the heart is taken out, the chest is cut, a fine needle is inserted from the apex of the heart, the left auricle is cut, after PBS perfusion is carried out, the heart is taken out, tissues are reserved for immunohistochemistry and electron microscope observation, and cardiac tissue protein is extracted to prepare the protein related to the autophagy signal pathway detected by western blot. Animal carcasses were treated in a standardized manner according to the animal house management regulations of the subsidiary Zhongshan hospital of the university of Compound Dan.

Chmp4c mice had normal lifespan. Echocardiography analysis was performed at 6 weeks to assess cardiac function in normal wild type mice WT and Chmp4c heterozygous mice. A significant increase in the end-systolic diameter and heart rate of the left ventricle was observed in Chmp4c heterozygous mice compared to wild type. Compared with wild type mice, the heart weight of Chmp4c heterozygous mice was significantly reduced, indicating that abnormal CHMP4C may affect the heart function of the mice.

To test whether there was a difference between the myocardial tissues of wild type and Chmp4c heterozygous mice, we performed HE and Masson staining observations, see fig. 1. In the Chmp4c heterozygous mouse group, the morphology of the myocardial fibers was irregular, and a large number of collagen fiber analogues were deposited in the myocardial tissue. Also, under an electron microscope, disorganization of the myofibers of the myocardial tissue was observed. These findings indicate that cardiac abnormalities in Chmp4c heterozygous mutant mice result in DCM pathogenesis.

Example 2 Zebra fish experiment

Chmp4c heterozygous mice display cardiac abnormalities, while truncated muteins may have a disruptive effect. However, the effect of these variants (E163G and S204L) on protein function is unclear. In this regard, we conducted in vivo and in vitro studies. To investigate whether the CHMP4C mutation may affect cardiac development in vivo, a study was performed using zebrafish. Based on the zebrafish phenotype as a function of the injected concentration, experimental conditions (50 ng/. mu.l and 120hpf) were selected for further analysis.

To verify the effect of the CHMP4C mutation (p.e163g; p.s204l) on cardiac function, wild-type and mutant CHMP4C mrnas were first prepared in a zebrafish model. Collecting AB strain zebra fish fertilized eggs, dividing CHMP4C mRNA (wild type WT and mutant MUT) into six groups according to final concentration of 50, 100 and 200 ng/ul respectively, injecting zebra fish fertilized eggs (at least 200 per group) in a micro-injection amount of 1nl per embryo, and setting a non-injection control group. Embryos were observed with a stereomicroscope (objective 1 x; eyepiece 3 x magnification, brightfield) at 50hpf and 120hpf, respectively, and zebrafish pericardial edema, heart rate and cardiac development were observed and counted. According to the phenotype condition of the zebra fish, the zebra fish with the concentration of 50 ng/. mu.l and the frequency of 120hpf is selected for statistics, and the shape of the myocardial tissue is observed by section staining, and the microstructure of the myocardial tissue is observed under an electron microscope, which is shown in figure 2.

The morphology of the myocardial tissue was observed by section staining and the microstructure of the myocardial tissue was observed by electron microscopy, see fig. 2. Compared with the wild type and the control group, the heart rate of the zebra fish of the CHMP4C mutant group is obviously increased, and the heart malformation and pericardial edema rate are obviously increased. There was no significant difference between wild type and control. Similar changes were also found by staining of myocardial tissue, see fig. 2.

In addition, the microstructure of the myocardial tissue of zebrafish was observed by an electron microscope, and it was found that gap junctions of the mutant group were widened compared to the wild type and the control group. In addition, mitochondrial swelling was evident in the mutant group. We also found that a large number of apoptotic bodies were accumulated in the CHMP4C mutant group, suggesting activation of apoptotic signaling pathways.

Transcriptome sequencing analysis was performed using tissues collected from each group of zebrafish. A hierarchical clustering heatmap is provided, visualizing this differential expression pattern, which strongly suggests the effect of CHMP4C variants (p.s204l and p.e163g) on gene expression. To determine the biological function associated with CHMP4C mutation, Gene Ontology (GO) analysis was performed using the DAVID software package. The GO content of microtubule cytoskeleton and autophagosome membranes was significantly enriched. CHMP4C is part of ESCT-III, an essential internal fractional complex, and previous studies have shown that other proteins are involved in autophagy. Therefore, we speculate that CHMP4C is involved in autophagy regulation.

Example 3 HEK293T cell assay

To assess the effect of CHMP4C variants (p.sz204l and p.e163g) on autophagy, we performed further studies using HEK293T cells.

CHMP4C wild-type plasmid, CHMP4C wild-type, mutant (p.sg204l and p.e163g) plasmid and control plasmid for transfection of HEK293T cells were ordered from the company jinsbury, south kyo.

CHMP4C wild-type plasmid, mutant (p.sg204l and p.e163g) plasmid, and control plasmid were transfected into HEK293T cells, respectively, using Lipofectamine 2000.

Using DMEM medium containing 10% FBS by volume fraction at 37 deg.C and 5% CO by volume fraction₂The cells were cultured in an incubator and then routinely subcultured. 5X 10 before transfection⁵HEK293T cells were inoculated into 12-well plates and cultured, and when the cell density reached more than half, the CHMP4C gene high expression vector was transfected into cells using Lipofectamine 2000. Cells were divided into 3 groups: control group, wild type group, mutant group, each group set with 4 multiple holes. After 48h, the normal group and the serum-free starved group were treated for 2 hours. The groups of cells after treatment were collected for subsequent autophagy-related protein detection.

5% CO at 37 ℃ in DMEM medium₂After 48 hours incubation in the incubator, starved groups were treated with serum-free medium (FBS-free) for 2 hours and then assayed for autophagy-related proteins.

It was found that the autophagy-related protein LC3-I/II was significantly reduced in the mutant group (p.E163G and p.S204L) compared to the control group and the wild-type group. Corresponding changes were also found in proteins upstream of the autophagy pathway (SQSTM1/p62 and ATG12-ATG 5). Under confocal immunofluorescence microscopy, we found a reduction in autophagic fluorescent particles in the mutant group, indicating that autophagic flow was impeded and impaired, as shown in figure 4. These effects may be due to inhibition of autophagy vesicle and lysosomal fusion.

In addition, apoptosis signaling pathways in cells were also evaluated, and it was found that apoptosis-activating protein (Bax/Bcl2) was activated in mutant groups (p.e163g and p.s204l). Apoptosis-related proteins such as caspase 3 and caspase 9 have corresponding changes. Under electron microscopy, a large number of autophagosome-like structures were observed in the mutant group and the autophagosomes were significantly reduced, indicating the presence of autophagosomal pathway abnormalities, see FIG. 4. These effects may be due to inhibition of autophagy vesicle and lysosomal fusion. In addition, a large number of black apoptotic-corpuscle-like high-density substances were observed in the mutant group, indicating activation of apoptotic pathways. It follows that CHMP4C variants (p.e163g and p.s204l) lead to abnormal autophagosomal pathways, which in turn activate apoptotic signals and impair cardiac muscle function.

The embodiments described above are presented to facilitate a person of ordinary skill in the art to understand and use the invention. It will be readily apparent to those skilled in the art that modifications may be made to the embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.

Sequence listing

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<120> an expansive cardiomyopathy molecular marker

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<400> 3

atgagcaagt tgggcaagtt ctttaaaggg ggcggctctt ctaagagccg agccgctccc 60

agtccccagg aggccctggt ccgacttcgg gagactgagg agatgctggg caagaaacaa 120

gagtacctgg aaaatcgaat ccagagagaa atcgccctgg ccaagaagca cggcacgcag 180

aataagcgag ctgcattaca ggcactaaag agaaagaaga ggttcgagaa acagctcact 240

cagattgatg gcacactttc taccattgag ttccagagag aagccctgga gaactcacac 300

accaacactg aggtgttgag gaacatgggc tttgcagcaa aagcgatgaa atctgttcat 360

gaaaacatgg atctgaacaa aatagatgat ttgatgcaag agatcacaga gcaacaggat 420

atcgcccaag aaatctcaga agcattttct caacgggttg gctttggtga tgactttgat 480

gaggatgggt tgatggcaga acttgaagaa ttggaacagg aggaattaaa taagaagatg 540

acaaatatcc gccttccaaa tgtgccttcc tcttctctcc cagcacagcc aaatagaaaa 600

ccaggcatgt tgtccactgc acgtcgatcc cgagcagcat cttcccagag ggcagaagaa 660

gaggatgatg atatcaaaca attggcagct tgggctacct aa 702

<210> 4

<211> 233

<212> PRT

<213> human

<400> 4

Met Ser Lys Leu Gly Lys Phe Phe Lys Gly Gly Gly Ser Ser Lys Ser

1 5 10 15

Arg Ala Ala Pro Ser Pro Gln Glu Ala Leu Val Arg Leu Arg Glu Thr

20 25 30

Glu Glu Met Leu Gly Lys Lys Gln Glu Tyr Leu Glu Asn Arg Ile Gln

35 40 45

Arg Glu Ile Ala Leu Ala Lys Lys His Gly Thr Gln Asn Lys Arg Ala

50 55 60

Ala Leu Gln Ala Leu Lys Arg Lys Lys Arg Phe Glu Lys Gln Leu Thr

65 70 75 80

Gln Ile Asp Gly Thr Leu Ser Thr Ile Glu Phe Gln Arg Glu Ala Leu

85 90 95

Glu Asn Ser His Thr Asn Thr Glu Val Leu Arg Asn Met Gly Phe Ala

100 105 110

Ala Lys Ala Met Lys Ser Val His Glu Asn Met Asp Leu Asn Lys Ile

115 120 125

Asp Asp Leu Met Gln Glu Ile Thr Glu Gln Gln Asp Ile Ala Gln Glu

130 135 140

Ile Ser Glu Ala Phe Ser Gln Arg Val Gly Phe Gly Asp Asp Phe Asp

145 150 155 160

Glu Asp Gly Leu Met Ala Glu Leu Glu Glu Leu Glu Gln Glu Glu Leu

165 170 175

Asn Lys Lys Met Thr Asn Ile Arg Leu Pro Asn Val Pro Ser Ser Ser

180 185 190

Leu Pro Ala Gln Pro Asn Arg Lys Pro Gly Met Leu Ser Thr Ala Arg

195 200 205

Arg Ser Arg Ala Ala Ser Ser Gln Arg Ala Glu Glu Glu Asp Asp Asp

210 215 220

Ile Lys Gln Leu Ala Ala Trp Ala Thr

225 230

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 5

cgcccaagaa atctcagaag 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 6

ctgtgctggg agagaagagg 20

Claims

1. An expanded cardiomyopathy molecular marker, which is a CHMP4C gene SEQ ID NO 1 mutant SEQ ID NO 3; or CHMP4C gene mutant SEQ ID NO. 3 coded polypeptide SEQ ID NO. 4.

2. The dilated cardiomyopathy molecular marker of claim 1, wherein the dilated cardiomyopathy is a dilated cardiomyopathy resulting from autophagy dysfunction.

3. A kit for diagnosing dilated cardiomyopathy is characterized by comprising a primer for detecting CHMP4C gene, a DNA/RNA probe or a microarray chip of the DNA/RNA probe, wherein when the determined nucleotide sequence is SEQ ID NO. 3, the dilated cardiomyopathy is judged.

4. The kit of claim 3, wherein the primers comprise a forward primer F and a reverse primer R:

F：5’-CGCCCAAGAAATCTCAGAAG-3’(SEQ ID NO:5)；

R：5’-CTGTGCTGGGAGAGAAGAGG-3’(SEQ ID NO:6)。

5. a kit for diagnosis of dilated cardiomyopathy, comprising:

(1) a first antibody capable of specifically binding polypeptide SEQ ID NO 4;

(2) a labeled antibody or second antibody capable of binding to the polypeptide SEQ ID NO. 4 when the polypeptide SEQ ID NO. 4 binds to the first antibody as defined in step (1); and

(3) the polypeptide SEQ ID NO. 4 as a standard substance.

6. The kit of claim 5, wherein the first antibody does not bind to the wild-type polypeptide of SEQ ID NO. 2 encoded by the CHMP4C gene of SEQ ID NO. 1.

7. The kit according to claim 5, further comprising an initiator solution for initiating a chemical reaction between the label on the labeled antibody and a chemiluminescent compound or a chromogenic substrate or a fluorescent dye as a reaction target for the label.

8. The kit of claim 7, wherein the label on the labeled antibody is peroxidase, phosphatase, or luciferase.

9. The kit of claim 3 or 5, wherein the sample to be tested is at least one of whole blood, plasma, serum, saliva, oral mucosa, nasopharyngeal secretions, skin, subcutaneous tissue.