CN113416776B

CN113416776B - Biomarker for detecting ventricular septal defect and application thereof

Info

Publication number: CN113416776B
Application number: CN202110687661.6A
Authority: CN
Inventors: 李博宁; 刘琮; 贾红玲; 范骏
Original assignee: Shenzhen Childrens Hospital
Current assignee: Shenzhen Childrens Hospital
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2022-04-01
Anticipated expiration: 2041-06-21
Also published as: CN113416776A

Abstract

The invention relates to a molecular marker for rapidly and accurately detecting ventricular septal defect, which is DDIT4 gene, and the inventor researches to find that the expression level of DDIT4 is obviously increased only in VSD children, and the VSD can be detected by quantitatively analyzing the content of DDIT4 gene in peripheral blood. The method has the advantages of convenient material acquisition, simple operation, strong specificity, rapidness, accuracy, stable result and the like, can diagnose the VSD in time, rapidly, objectively and accurately, can separate the VSD from the ASD and the PDA through one experiment, and has the advantages which are not possessed by the traditional diagnosis method. Therefore, the method has great clinical application value for quick diagnosis of the VSD infant, and provides directionality for further developing a VSD quick diagnosis kit.

Description

Biomarker for detecting ventricular septal defect and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a biomarker for detecting ventricular septal defect and application thereof.

Background

Septal defects are the most common congenital heart disease and can cause communication between the left and right sides (atria/ventricles) of the heart. This can be classified as Ventricular Septal Defect (VSD) and Atrial Septal Defect (ASD), accounting for 40% and 10% of the incidence of congenital heart disease, respectively. Nearly 50% of these defects heal spontaneously during a period of time after birth, and if the defects are small and free of hemodynamic changes, they can be treated with regular follow-up visits, while the other half requires treatment by medical intervention or surgical repair. Patients with septal defects often have a clinical manifestation of a susceptibility to growth retardation, decreased endurance, and respiratory disorders, and may be accompanied by cardiac hypertrophy, increased pulmonary circulatory pressure, arrhythmia, and heart failure.

VSD is one of the most common congenital malformations of the heart, accounting for 40% of all cardiac abnormalities. VSDs are not only common isolated cardiac malformations, but may also be a component of complex cardiac malformations, including french tetrad or single ventricular atrioventricular connections, and the like. Since VSD tends to heal itself after birth as children grow, the prevalence of this defect varies with age, and the exact statistics of its incidence depend on the sensitivity of the examination technique. Neonatal VSD screening by high-sensitivity color doppler echocardiography is reported to have up to 5% incidence and mostly minor muscle defects that heal spontaneously the first year after birth. Since many patients do not have clinical symptoms and the majority of small defects heal with age, the exact prevalence of VSD among the population varies from study to study, depending on the mode of diagnosis and the age of the population. Just as after applying echocardiography to VSD diagnosis, the prevalence was recorded as high as 3.94/1000, which is much higher than previously diagnosed by clinical physical examination or autopsy.

Several key factors determine the pathophysiological process and clinical presentation of VSDs. The main factors are the ventricular horizontal shunt volume, the direction of the shunt, and the degree of heart chamber volume loading. Secondary effects include aortic valve prolapse and obstruction of the pulmonary vessels or outflow tract. Ventricular shunt is determined by the size of the defect, the relative resistance of the pulmonary vessels and the systemic vascular bed. The small defects themselves, so-called restrictive defects, provide an inherent resistance to flow, and the split flow is often small. While the amount of blood flow through a large non-limiting defect depends on the relative resistance of the lungs and the systemic vascular bed. Although various non-limiting defect definition values are proposed based on the ratio of the cross-sectional area of the defect to the area of the aortic orifice, the ratio of the cross-sectional area of the defect to the diameter of the body surface area or the flow rate through the periosteum, there is currently no precise standard for defining non-limiting defects. When the defect is non-limiting, the primary determinant of ultimate shunt and clinical symptoms is the relative resistance of the lung and systemic vascular bed. Importantly, the amount of resistance between the two may be constantly variable, depending in particular on the age of the patient. The left-to-right flow distribution of the infant may be initially small, and the defect may actually be quite large due to the early pulmonary vascular resistance characteristic of the newborn, but as pulmonary vascular resistance decreases, left and right ventricular shunts increase, and the patient's symptoms become more pronounced due to excessive pulmonary blood flow. In some VSD patients, pulmonary vascular disease may occur in advance. In a small number of people, the typical decline in postnatal pulmonary vascular resistance in the presence of VSD may be delayed or halted. Thus, they may never develop symptoms due to excessive shunting from left to right, with signs of pulmonary vascular disease only at a later stage. If a large lesion is not corrected, the number of left and right ventricular shunts may decrease over time and eventually their direction may reverse, resulting in cyanosis and Eisenmener's syndrome. In large VSD patients without pulmonary vascular disease, an increase in the volume loading of the left atrium and ventricle (due to increased pulmonary blood flow and thus increased pulmonary venous return) can result in left cardiac dilation throughout the cardiac cycle. Eccentric left ventricular hypertrophy may occur due to increased end diastolic volume loading. The long-term presence of a ventricular septal defect relative to pulmonary hypertension may eventually lead to right ventricular hypertrophy and dilation. These features will dominate when patients enter the end stage of the severe Eisenmenger syndrome, which is often characterized by right heart failure, and these patients often lose the opportunity for surgical treatment, with poor prognosis, and therefore early diagnosis of VSD is particularly important.

The diagnosis of septal defects mainly depends on echocardiography combined with clinical symptoms, and other examinations include chest X-ray, CT and Magnetic Resonance Imaging (MRI), and a small part of the examinations are feasible for cardiac catheter examination diagnosis. Echocardiography can determine the presence, location, size, and hemodynamic characteristics of a defect. These imaging methods provide direct clinical evidence for diagnosing septal defects, especially VSD of children, but have some disadvantages, such as long examination time, high examination accuracy affected by the experience of technicians, difficulty in matching the patients to cause misdiagnosis, poor radiation for examining the VSD to be beneficial to the growth and development of the children, and the like. Therefore, it is very important to find a molecular marker capable of quickly and accurately diagnosing VSD, which can indicate the direction for clinical treatment, diagnose the occurrence of lesion at early stage, improve prognosis and improve the quality of life of children patients.

It has been proved that DDIT4, DDIT4, DNA injury-inducing transcription factor 4, is involved in various intracellular stress reactions induced by various stress conditions such as oxidative stress, endoplasmic reticulum stress, hypoxia and starvation. DDIT4 is a proven mTORC1 upstream regulatory molecule, and DDIT4 can induce TSC1 and TSC2 to form heterodimer, participate in the regulation of mTORC1, and play an important role in cell proliferation and autophagy. But is it associated with ventricular septal defect as a biomarker? No report of applying DDIT4 gene to ventricular septal defect detection is found at present.

Disclosure of Invention

Based on this, one of the objectives of the present invention is to provide a molecular marker for rapid and accurate detection of ventricular septal defects.

The specific technical scheme is as follows:

a biomarker for ventricular septal defect detection, wherein the biomarker is DDIT4 gene.

The invention also aims to provide application of the biomarker in preparing a ventricular septal defect detection reagent.

The invention also aims to provide a kit for detecting the ventricular septal defect.

The technical scheme for realizing the purpose is as follows:

a kit for detecting ventricular septal defect comprises a reagent for detecting the biomarker.

In some embodiments, the kit is prepared by using polymerase chain reaction technology, in situ hybridization technology, enzymatic mutation detection technology, chemical shear mismatch technology, mass spectrometry technology, gene chip technology or gene sequencing technology, or a combination thereof.

In some embodiments, the above-described kits preferably employ techniques that include polymerase chain reaction techniques, which are any of RT-PCR, immuno-PCR, nested PCR, fluorescence PCR, in situ PCR, membrane-bound PCR, anchored PCR, anchorage PCR, in situ PCR, asymmetric PCR, long-range PCR, parachute PCR, gradient PCR.

In some of these embodiments, the polymerase chain reaction is RT-PCR and the kit comprises upstream and downstream primers for DDIT4, the sequences of the upstream and downstream primers being shown in SEQ ID NO.17-SEQ ID NO.18, respectively.

In some embodiments, the test sample of the kit is blood.

The invention also aims to provide the application of the ventricular septal defect detection kit in the detection of ventricular septal defects.

It is also an object of the present invention to provide a method of detecting a ventricular septal defect.

The technical scheme for realizing the purpose is as follows:

a method of detecting a ventricular septal defect in a child comprising the steps of:

(1) obtaining genome DNA of a biological sample to be detected;

(2) and detecting the expression level of the DDIT4 gene.

In some embodiments, the biological sample to be tested is blood, preferably peripheral blood.

Compared with the prior art, the invention has the following beneficial effects:

the inventor finds a molecular marker which can be used for quickly and accurately detecting the Ventricular Septal Defect (VSD), and the molecular marker is DDIT4 gene. The inventor researches and discovers that the expression level of DDIT4 gene is remarkably increased only in VSD children, and VSD can be detected by quantitatively analyzing the content of DDIT4 gene in peripheral blood.

The method has the advantages of convenient material acquisition, simple operation, strong specificity, rapidness, accuracy, stable result and the like, can diagnose the VSD in time, rapidly, objectively and accurately, can separate the VSD from the ASD and the PDA through one experiment, and has the advantages which are not possessed by the traditional diagnosis method. Therefore, the method has great clinical application value for quick diagnosis of the VSD infant, and provides directionality for further developing a VSD quick diagnosis kit.

Drawings

FIG. 1 is a plot of Principal Component Analysis (PCA) of blood samples from a clinical child of VSD and before and after ASD treatment in example 1, where the 1APCA plot shows the changes in the VSD child's blood samples before and after surgery, each point representing a clinical sample; the 1B PCA plot shows the changes in blood samples from ASD children before and after surgery, with each dot representing a clinical specimen.

Figure 2 is a Venn plot of the differential genes identified before and after treatment with VSD and ASD in example 1.

FIG. 3 is a profile of differentially expressed genes as shown by mRNA sequencing analysis in example 1, where the blue dots represent down-regulated genes and the red dots represent up-regulated genes; wherein FIG. 3A shows 377 down-regulated genes and 668 up-regulated genes before and after VSD treatment; fig. 3B shows 737 down-regulated genes and 786 up-regulated genes before and after treatment of an ASD infant.

FIG. 4 is a heat map of the expression levels of the first 50 differential genes from the ASD and VSD groups in example 1, with red showing up-regulation of gene expression and fold difference and green showing down-regulation of gene expression and fold difference; wherein figure 4A is the relative expression levels of differential genes before, after VSD treatment and in control groups (healthy volunteers); FIG. 4B is the relative expression levels of differential genes before, after and in control (healthy volunteers) of ASD treatment; figure 4C is a heat map of the relative expression levels of overlapping differential genes between the ASD and VSD groups.

Figure 5 is a GO analysis of the differential genes in VSD and ASD infants in example 1. Wherein FIG. 5A is a molecular functional analysis of differential genes in the VSD group; fig. 5B is a biological process analysis of the differential genes in the ASD group.

FIG. 6 is a statistical plot of the differential gene qRT-PCR detection results of VSD and ASD samples in example 1, wherein FIG. 6A is the validation of the differential genes of the VSD pre-treatment group and the VSD post-treatment group; figure 6B is a validation of differential genes before and after ASD treatment.

FIG. 7 is a graph showing the results of qRT-PCR detection of DDIT4 expression in VSD, ASD and PDA samples in example 2.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification the meaning of "a", "an" and "the" include plural referents. The meaning of "in.

The present invention will be described in further detail with reference to specific examples.

Example 1 screening for Rapid diagnostic VSD molecular markers

1. Transcriptome sequencing

(1) Collecting Shenzhen children hospital cardiology samples, wherein 3 samples from children before ASD treatment (< 18 years old), 4 samples from children after ASD treatment, 4 samples from children before VSD treatment, 2 samples from children after VSD treatment and 4 samples from healthy volunteers, and 1ml of peripheral blood of healthy children, ASD and VSD children is collected respectively;

(2) RNA was extracted from peripheral blood samples according to the Trizol reagent manual (Invitrogen Life Technologies, Carlsbad, CA) and concentration and purity were measured;

(4) extracted mRNA was used for sequencing;

(5) preliminary screening of nucleic acid markers: performing principal component analysis on the results of sequencing of blood samples from VSD and clinical children before and after ASD treatment to obtain a PCA plot as shown in figure 1, wherein a 1APCA plot shows the changes in the blood samples from VSD children before and after surgery, each point representing a clinical sample; the 1B PCA plot shows the changes in blood samples from ASD children before and after surgery, with each dot representing a clinical specimen.

Further, the ASD group, VSD group and healthy group were subjected to RNA-Seq analysis, and mRNA was primarily screened according to the criteria of log FC (Fold Change) > 2 Fold and P <0.05 for up-or down-regulated expression difference, and genes with significant up-or down-regulated expression difference were obtained by mRNA omics analysis, including 1408 difference genes in ASD, 930 difference genes in VSD, and 115 difference genes overlapping them (see fig. 2). That is, in the child VSD, 1045 genes were differentially expressed, and in the child ASD, 1523 genes were differentially expressed, 115 genes among which were differentially expressed in common between the child ASD and the child VSD.

As shown in the volcano plot of the distribution of differentially expressed genes shown in the mRNA sequencing analysis of fig. 3, genes with p-values less than 0.05 and absolute fold changes of 2 or more were considered significantly changed genes; in VSD children, 337 genes were down-regulated and 668 genes were up-regulated; in ASD infants, 737 genes were down-regulated and 786 genes were up-regulated. Figure 4 shows that DDIT4, both in ASD and VSD children, was elevated in expression and that DDIT4 expression was significantly reduced after treatment, no different from the normal group.

GO analysis is carried out to difference Gene among VSD and the ASD infant, GO database, is called Gene Ontology entirely, wherein the function of Gene has been divided into three parts and is respectively: cellular Components (CC), Molecular Functions (MF), Biological Processes (BP). Wherein FIG. 5A is a molecular functional analysis of the differential genes in the VSD group, which was found to be associated with immune response modulating signal pathways, immune response modulating cell surface receptors, leukocyte migration, immune response activating signaling and immune response activating cell surface receptors; FIG. 5B is a biological process analysis of the differential genes in the ASD group, which was found to be associated with membrane localization of proteins, mRNA catabolic processes of nuclear transcription, protein targeting to ER processes, and protein localization to ER. The biological processes identified in this analysis may contribute to the pathobiology of the treatment of cardiac septal defects. This result indicates that the development of septal defects and remodeling mechanisms may be different in VSD or ASD treatment.

2. Verification of differential genes

After the differential genes were determined, we further validated with qRT-PCR and detected the screened differential genes.

(1) RNA was extracted from peripheral blood samples according to the Trizol reagent manual (Invitrogen Life Technologies, Carlsbad, CA) and concentration and purity were measured;

(2) synthesizing cDNA using a cDNA synthesis kit (reverse transcription kit: TOYOBO FSQ-301); the method comprises the following steps:

first step (genome removal reaction)

Reaction system: (16ul)

4X DN MasterMix 4ul

RNAtemplate 1ug

Sterilized water supplement of 16ul

Reaction procedure: 37 ℃ for 5min

The second step is that: cDNA Synthesis reaction

System (20ul):

16ul of first-step reaction solution

5X RT Master Mix II 4ul

Reaction procedure: hold at 37 deg.C, 15min, 98 deg.C, 5min, 4 deg.C

(3) The cDNA levels were measured in real time by SYBR green in a Lightcycler. Normalization was performed in each individual sample using the housekeeping gene GAPDH, and 2 was used^-ΔΔCtThe method quantifies relative expression changes. The specific reaction system preparation and reaction procedures are as follows:

RT-QPCR reaction System 20 ul: (detection reagent: TOYOBO QPS-201)

Reaction procedure: 95 deg.C 2min 95 deg.C 15S 60 deg.C 1min

40cycles Melt Curve Stage

TABLE 1 primer sequences for differential genes

The results of qRT-PCR are shown in figure 6, with mean downregulation of the expression levels of DDIT4, IRS2 and TOB1 after ASD and VSD treatment, and significant reduction in expression of these genes in the DESeq2 differential expression assay. In particular, the expression of DDIT4 was significantly reduced after treatment, which was not different from the normal group.

In conclusion, the qRT-PCR results show that the expression of DDIT4 is increased before treatment and that the expression of DDIT4 is significantly reduced after treatment, regardless of whether it is in children with ASD or VSD, and is not different from that in the normal group.

Example 2 validation of Rapid diagnostic VSD molecular markers

To further confirm whether DDIT4 was elevated only in VSD patients or in other patients with congenital heart disease, we examined the expression levels of DDIT4 in peripheral blood of Patent of Ductus Atriosus (PDA) patients and ASD patients.

(1) Blood was drawn from 30 healthy groups (control group), 30 PDA, 30 ASD, 30 VSD patients according to Trizol reagent manual (Invitrogen Life Technologies, Carlsbad, CA), RNA was extracted from peripheral blood samples, and concentration and purity were determined;

(2) synthesizing cDNA using a cDNA synthesis kit;

(3) DDIT4 in these samples was detected using qRT-PCR, normalized in each individual sample using housekeeping gene GAPDH, and using 2^-ΔΔCtThe method quantifies relative expression changes.

The data show that, as shown in fig. 7, the expression levels of DDIT4 in peripheral blood of PDA, VSD, and ASD children were all increased compared to the control group, but the expression level of DDIT4 was significantly increased only in VSD children, and was statistically significant. Indicating that DDIT4 can be used as a specific biomarker for VSD infants. Based on the above data, we can conclude that DDIT4 can be used as a biomarker for VSD children, which is of great significance to VSD children.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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Claims

1. Application of reagent for detecting DDIT4 gene expression level in peripheral blood sample in preparing ventricular septal defect detection kit.

2. The use of claim 1, wherein the kit is prepared using polymerase chain reaction technology, in situ hybridization technology, gene chip technology, or gene sequencing technology, or a combination thereof.

3. Use according to claim 2, wherein the technology used in the kit comprises polymerase chain reaction technology, wherein the polymerase chain reaction is any one of qRT-PCR, immuno PCR, nested PCR, in situ PCR, membrane-bound PCR, anchored PCR, anchor PCR, asymmetric PCR, long-range PCR, parachute PCR, gradient PCR.

4. The use of claim 3, wherein the polymerase chain reaction is qRT-PCR and the kit comprises upstream and downstream primers for DDIT4, the sequences of the upstream and downstream primers are shown as SEQ ID number 17-SEQ ID number 18, respectively.