CN116024224A - Arrhythmia right ventricular dysplasia cardiomyopathy variant gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of gene detection, in particular to a variant gene of arrhythmia-caused right ventricular dysplasia cardiomyopathy, which is PKP2, and compared with a reference sequence SEQ ID NO. 5 of a wild PKP2 gene, the 2018 th site of the variant gene lacks a base C, and the nucleotide sequence is SEQ ID NO. 1. The invention also relates to application of the arrhythmia right ventricular dysplasia cardiomyopathy variation gene in preparation of a detection kit. The arrhythmia right ventricular dysplasia cardiomyopathy variation gene provided by the invention can be used as a biomarker for clinical auxiliary diagnosis; the detection of the variant carrier provides the instruction of prenatal and postnatal care and genetic consultation for the subject, reduces the birth of the infant, and has important significance for early diagnosis of arrhythmia right ventricular dysplasia cardiomyopathy or auxiliary clinical judgment.

Description

Arrhythmia right ventricular dysplasia cardiomyopathy variant gene and application thereof

Technical Field

The invention relates to the technical field of gene detection, in particular to a variant gene of arrhythmia right ventricular dysplasia cardiomyopathy and application thereof.

Background

The arrhythmia right ventricular dysplasia cardiomyopathy (ARVC) is a common type of arrhythmia cardiomyopathy, also called right ventricular cardiomyopathy, and the prevalence is about 1:2000-5000. Clinically, ventricular arrhythmias, progressive heart failure, and even sudden cardiac death are often manifested, especially in young people and athletes, one of the most common causes of sudden death. About 20-30% of patients have a family history of ARVC or sudden death. The disease is one of hereditary cardiomyopathy, mostly autosomal dominant inheritance, accompanied by incomplete exon and differential expression, and also rarely autosomal recessive inheritance, such as Naxos disease. If the disease can be found and diagnosed early, the incidence rate of malignant events can be effectively reduced by intervening in life style, reducing strenuous exercise, preventing early medication and other measures.

The pathogenic gene PKP2 related to arrhythmia right ventricular dysplasia cardiomyopathy codes for plakophilin2, which is involved in connecting cytoplasmic cadherin tail and intermediate silk protein in the cytoskeleton and possibly regulating the signal activity of beta-catenin.

The related gene detection of the suspected cases is helpful to early diagnosis and clinical intervention of the right ventricular dysplasia cardiomyopathy caused by arrhythmia according to the willingness of patients, so as to achieve the purposes of delaying the occurrence of the diseases or preventing the diseases. Revealing the pathogenesis of the arrhythmia right ventricular dysplasia cardiomyopathy related to the PKP2 gene from the molecular level, providing theoretical basis for clinical treatment of the arrhythmia right ventricular dysplasia cardiomyopathy, but currently, a large number of unknown PKP2 gene mutation sites still exist, further finding out new PKP2 gene mutation sites is helpful for further researching the arrhythmia right ventricular dysplasia cardiomyopathy, and has important significance for early diagnosis of the arrhythmia right ventricular dysplasia cardiomyopathy or assisting clinical judgment.

Disclosure of Invention

The invention aims at providing a variant gene of arrhythmia right ventricular dysplasia cardiomyopathy and application thereof aiming at the defects.

The invention aims to provide that: a variant gene of arrhythmia right ventricular dysplasia cardiomyopathy, wherein the variant gene is PKP2, and compared with a reference sequence SEQ ID NO. 5 of a wild PKP2 gene, the 2018 th deletion base C of the variant gene has a nucleotide sequence SEQ ID NO. 1; compared with the amino acid sequence SEQ ID NO. 6 of the wild PKP2 gene encoding protein, the amino acid sequence of the variant gene encoding protein is SEQ ID NO. 2.

The invention successfully screens out variant genes through a large number of experiments, researches and analyses, and utilizes the variant genes to develop a detection kit capable of being used for rapidly, sensitively and effectively detecting the variant genes. The specific information of the variant genes is shown in the following table:

TABLE 1 variant genes

Gene	Genomic location	Transcription number	Base change	Amino acid changes	Reference genome version
						PKP2	chr12:32955486	NM_001005242	c.2018delC	p.Pro673ArgfsTer29	GRCh37/hg19

The invention also provides application of the arrhythmia right ventricular dysplasia cardiomyopathy variation gene in preparing a detection kit, wherein the detection kit comprises primers for amplifying the variation gene, and the sequences of the primers are SEQ ID NO. 3 and SEQ ID NO. 4.

Preferably, the kit for treating the right ventricular dysplasia cardiomyopathy comprises PCR premix, a negative control reagent and a positive control reagent.

The invention has the beneficial effects that: the variant gene disclosed by the invention can be used as a biomarker for clinically assisting in diagnosing the arrhythmia-caused right ventricular dysplasia cardiomyopathy, and has important significance for early diagnosis of the arrhythmia-caused right ventricular dysplasia cardiomyopathy or assisting in clinical judgment; the kit based on the reagent development of variant genes can distinguish patients carrying PKP2c.2018delC heterozygous missense mutation from normal people, provides instruction of prenatal and postnatal care and genetic consultation for subjects, and reduces birth of infants.

Drawings

FIG. 1 is a diagram of Sanger sequencing of example 1 with PKP2c.2018delC patients;

FIG. 2 is a family chart of a person with a pro-arrhythmogenic right ventricular dysplasia cardiomyopathy in example 2.

Detailed Description

The following is a further detailed description of the embodiments, but is not intended to limit the invention thereto.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Reagent source: PCR premix: 2 XTaq Master mix (Dye), available from Jiangsu kang as century Biotech Co., ltd., product number: l01037/70335; comprises the following components: taq DNA Polymerase PCR Buffer, mg ²⁺ Components required for conventional PCR such as dNTPs, PCR stabilizers and enhancers. Agencourt AMPure XP magnetic beads: purchased from beckmann coulter trade (china), product number: 311303. amplification primers were synthesized by the company limited of biological engineering (Beijing). RNase-Free H ₂ O: purchased from beijing solebao technologies limited. Whole blood genome DNA extraction kit by magnetic bead method: purchased from Jiangsu Baishino medical science and technology Co., ltd., lot number: 20031886-01C.

Example 1: variant gene c.2018delC validation experiment

On the premise that the patient with arrhythmia right ventricular dysplasia cardiomyopathy and family members thereof are voluntarily signed with informed consent, 5-10mL of human whole blood EDTA anticoagulation sample is sent, a medical record database is established, and the patient condition, family condition and other data are recorded in detail. The study was approved by the ethics committee of this unit.

S1, extracting genome DNA: the method comprises the steps of extracting whole genome DNA from an EDTA anticoagulated sample of human whole blood of a patient by using a magnetic bead method whole blood genome DNA extraction kit of Jiangsu Baishino medical science and technology Co., ltd, and performing the operation steps according to the product specification. The concentration and purity of the DNA were examined and used as template DNA for PCR amplification.

S2, preparing a PCR reaction system: the PCR reaction system is used for amplifying a section of DNA sequence containing target gene loci, and comprises the following components: 25. Mu.L of PCR premix, 2. Mu.L of forward primer (10. Mu.M), 2. Mu.L of reverse primer (10. Mu.M), less than 1000ng of template DNA, and RNase-Free H2O was added to make up to 50. Mu.L. The forward and reverse primer information used is as follows:

forward primer (SEQ ID NO: 3): 5'CTGTAATCCCAGCTAGTCT 3'; reverse primer (SEQ ID NO: 4): 5'CCGGTTTATCACCTACTCC 3'. Length: 529bp.

S3, amplifying target fragments: mixing the reaction systems, and carrying out amplification reaction of target gene fragments on a PCR instrument, wherein the amplification procedure is as follows: pre-denaturation at 95℃for 2min; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 30s, and a total of 33 cycles. Final extension at 72℃for 2min.

S4, detecting PCR products: 2 mu L of PCR product is taken, 1.5% agarose gel electrophoresis is used for detecting the PCR product, 1000bp Marker is selected as a reference, and detection is performed to verify that the amplified product is in the expected size.

S5, purifying a PCR product: after PCR product detection, the PCR product is purified by using Agencourt AMPure XP magnetic beads, and the purification steps are carried out according to the product specification, and specifically include the following steps: (1) Vortex the beads for 30s to thoroughly mix them into a homogeneous solution. (2) To a 1.5mL centrifuge tube, the PCR product to be purified was added, followed by 2 sample volumes of the magnetic bead solution. After vortexing and mixing, vortexing was performed for 5min at room temperature on a thermo mixer at 1400 rpm. (3) The centrifuge tube of the last step is placed on a magnetic rack for about 1min until the magnetic beads are completely adsorbed. (4) The centrifuge tube was kept fixed on a magnetic rack, and the solution was discarded while avoiding contact with the magnetic beads. (5) After 500 mu L Buffer PW is added into the centrifuge tube in the last step, the centrifuge tube is taken down from the magnetic rack, the centrifuge tube is put back into the magnetic rack again after vortex oscillation for 10s, and the centrifuge tube is kept stand for 1min, and the rinsing liquid is thoroughly discarded after the magnetic beads are completely adsorbed on the side wall of the centrifuge tube. (6) repeating the step (5). (7) Keeping the centrifuge tube fixed on the magnetic rack for standing for 10min, and completely volatilizing the ethanol. (8) Taking the centrifuge tube off the magnetic frame, adding 20-100 mu L Buffer EB, suspending the magnetic beads in the eluent by vortex oscillation, and then placing the centrifuge tube on a thermo mixer at 65 ℃ and 1400rpm for oscillation elution for 5min. (9) The centrifuge tube was placed on a magnetic rack for about 1min until the beads were fully adsorbed. (10) The eluate was transferred to a new 1.5mL centrifuge tube, at which point the beads were discarded.

S6, sanger sequencing was performed on the amplified products using an applied biosystems 3500Dx series gene analyzer.

S7, performing bioinformatics analysis on the sequencing result: the sequencing results were aligned with the wild-type PKP2 gene sequences (SEQ ID NO:5 and SEQ ID NO: 6) obtained in NCBI (https:// www.ncbi.nlm.nih.gov /) in software Chromas to determine whether a mutation occurred in the detection site.

S8, carrying out genetic variation demonstration: the patient detects PKP2c.2018delC heterozygous missense mutation, namely, compared with a reference sequence SEQ ID NO. 5 of a wild PKP2 gene, the 2018 th site of the mutation gene lacks a base C, and the nucleotide sequence is SEQ ID NO. 1; compared with the amino acid sequence SEQ ID NO. 6 of the wild PKP2 gene encoding protein, the amino acid sequence of the variant gene encoding protein is SEQ ID NO. 2, and the Sanger sequencing diagram is shown in FIG. 1.

Thousands of genomes were searched (https:// www.ncbi.nlm.nih.gov/variation/tools/1000genome /): and no. ClinVar (https:// www.snpedia.com/index. Php/ClinVar): and no. ESP6500 (https:// ESP. Gs. Washington. Edu/drive /): and no. ExAC (http:// exac.hms.harvard.edu /): and no. HGMD (http:// www.hgmd.c.ac.uk/ac/index. Php): and no. Neither the cardiomyopathy patients in the century Nuo local population database nor the control population carried the variation.

According to the existing evidence: the variation is rare and the variation is a highly suspected pathogenic mutation of arrhythmogenic right ventricular dysplastic cardiomyopathy.

Example 2: sample validation experiment

2500 arrhythmogenic right ventricular dysplasia cardiomyopathy patients and 1000 healthy people not suffering from arrhythmogenic right ventricular dysplasia cardiomyopathy were recruited. PKP2c.2018delc was amplified for each member of the family and for healthy individuals using the method of example 1, and analyzed after Sanger sequencing was performed after amplification was completed.

Based on the confidentiality of the sample information, a part of the sample information is now disclosed. The sample may disclose information: (1) arrhythmogenic right ventricular dysplasia cardiomyopathy family; country/region: chinese/beijing; family member male-female ratio: 1:1; family member age distribution: 10-60 years old; (2) country/region of healthy people: chinese/beijing; healthy population male-female ratio: 1:1; age distribution of healthy population: 12-60 years old.

Only in the recruited arrhythmogenic right ventricular dysplastic cardiomyopathy family (family diagram shown in fig. 2) the diseased members all carried pkp2c.2018delc heterozygous missense variations; the healthy population did not see any of the mutations at any of the above sites.

Example 3: detection kit

1. The composition is as follows:

TABLE 2 composition

2. The using method comprises the following steps: (1) genomic DNA extraction: the genomic DNA of the peripheral blood sample was extracted using a DNA extraction kit. (2) PCR amplification: PCR amplification was performed using the above-described kit, and the reaction system and reaction conditions were as described in example 1. (3) purifying the PCR amplification product. (4) Sanger sequencing of the purified PCR amplification product. (5) Sequencing results were analyzed to compare if there was a PKP2c.2018delc heterozygous missense variation.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (4)

1. The variant gene of the arrhythmia right ventricular dysplasia cardiomyopathy is characterized in that the variant gene is PKP2, and compared with a reference sequence SEQ ID NO. 5 of a wild PKP2 gene, the 2018 th site of the variant gene lacks a base C, and the nucleotide sequence is SEQ ID NO. 1.

2. The arrhythmogenic right ventricular dysplasia cardiomyopathy variant gene of claim 1, wherein the amino acid sequence of the variant gene encoded protein is SEQ ID No. 2, as compared to the amino acid sequence of the wild-type PKP2 gene encoded protein of SEQ ID No. 6.

3. The use of the arrhythmogenic right ventricular dysplasia cardiomyopathy variant gene according to claim 2 in the preparation of a detection kit, wherein the detection kit comprises primers for amplifying the variant gene, the sequences of the primers are SEQ ID NO. 3 and SEQ ID NO. 4.

4. The test kit of claim 3, further comprising a PCR premix, a negative control reagent, and a positive control reagent.

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