CN115961019B - Reagent for detecting novel pathogenic gene TTN of dilated cardiomyopathy and application thereof - Google Patents
Reagent for detecting novel pathogenic gene TTN of dilated cardiomyopathy and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of gene detection, in particular to a reagent for detecting a novel pathogenic gene TTN of dilated cardiomyopathy, which comprises primers SEQ ID NO. 3 and SEQ ID NO. 4; compared with the wild TTN gene, the novel pathogenic gene TTN has c.32706delAinsTTC heterozygous missense mutation, and the coding nucleotide sequence is SEQ ID NO. 1. The invention also provides application of the reagent in preparing a detection kit. The novel pathogenic gene TTN provided by the invention can be used as a biomarker for clinical auxiliary diagnosis, can distinguish patients carrying c.32706 degrees AinsTTC heterozygous missense mutation from normal people, and has important significance for early diagnosis of dilated cardiomyopathy or auxiliary clinical judgment by detecting a carrier of the mutation; provides a prenatal and postnatal guidance and genetic counseling for the patients with fertility requirements, and can effectively reduce the birth of infants.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a reagent for detecting a novel pathogenic gene TTN of dilated cardiomyopathy and application thereof.
Background
Dilated cardiomyopathy (diated cardiomyopathy, DCM) is one of the common diseases causing heart failure (heart failure), arrhythmia and sudden death, and is clinically manifested as: progressive enlargement of the heart, reduced ventricular contractile function, heart failure, ventricular and supraventricular arrhythmias, abnormal conduction systems, thromboembolism and sudden death. In 2002, the national hierarchy of whole population samples was investigated for 8080 normal populations in 9 regions with a prevalence of 19/10 ten thousand DCM (Wang Zhimin, 2004). One report in china 2004 showed that 767 DCM patients had a mortality rate of 42.24% (Liu X, 2014) at 52 months of follow-up, which places a heavy burden on the home and society.
DCM caused by pathogenic gene mutation is called "familial DCM (familial diated cardiomyopathy, FDCM)", and is expressed as death of more than 2 cases of DCM patients or of first-class relatives under 35 years old in the first-class relatives of the same family, and foreign studies have found that the incidence rate in DCM is 20-50% (Mcnally EM, 2013). The main pathogenic genes of FDCM include TTN, LMNA, MYH, MYH6, TNNT2, ACTC1, BAG3, DSP, MYBPC3, RBM20, SCN5A, TPM1, DES and DMD, and about 40% of familial DCMs can screen clear pathogenic gene mutation, wherein TTN gene truncated mutation accounts for the highest proportion, TTN gene is a coding gene of actin and is expressed in cardiac muscle and skeletal muscle, and the protein has large molecular weight and length of 1/2 of a muscle segment, and is an important structural protein.
With the gradual penetration of FDCM research, the related gene detection is carried out on suspected cases according to the willingness of patients, which is helpful for the early diagnosis and clinical intervention of FDCM, so as to achieve the purpose of delaying the occurrence of diseases or preventing diseases. Although a plurality of TTN mutation sites related to dilated cardiomyopathy are found, based on previous researches, further finding new TTN gene mutation is helpful for further researching dilated cardiomyopathy, and has important significance for early diagnosis of dilated cardiomyopathy or assisting clinical judgment.
Disclosure of Invention
The invention aims at providing a reagent for detecting a novel pathogenic gene TTN of dilated cardiomyopathy and application thereof.
One of the objects of the present invention is to provide: a reagent for detecting a novel pathogenic gene TTN of dilated cardiomyopathy, which comprises primers SEQ ID NO. 3 and SEQ ID NO. 4; compared with the wild TTN gene, the novel pathogenic gene TTN has c.32706delAinsTTC heterozygous missense mutation, the coding nucleotide sequence is SEQ ID NO. 1, and the amino acid sequence of the coding protein is SEQ ID NO. 2.
Specific mutation information is as follows:
the second purpose of the invention is to provide the application of the reagent for detecting the novel pathogenic gene TTN for dilated cardiomyopathy in preparing a detection kit. Preferably, the detection kit comprises a PCR premix, a negative control reagent and a positive control reagent.
The principle and the beneficial effects of the invention are as follows:
the novel pathogenic gene TTN provided by the invention can be used as a biomarker for clinical auxiliary diagnosis, can distinguish patients carrying c.32706 degrees AinsTTC heterozygous missense mutation from normal people, and has important significance for early diagnosis of dilated cardiomyopathy or auxiliary clinical judgment by detecting a carrier of the mutation; provides a prenatal and postnatal guidance and genetic counseling for the patients with fertility requirements, and can effectively reduce the birth of infants.
Drawings
FIG. 1 is a diagram of a prior Sanger sequencing;
FIG. 2 is a family chart of the dilated cardiomyopathy family of example 2.
Detailed Description
The following is a further detailed description of the embodiments:
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. the amplification primers were all synthesized by the division of biological engineering (Shanghai). 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: verification experiment of dilated cardiomyopathy novel pathogenic gene TTN c.32706delAinsTTC
Sample inspection: on the premise that a person with dilated cardiomyopathy forepart (male, 54 years old) and family members thereof voluntarily sign an informed consent form in clinical diagnosis, a 5-10mL whole blood sample (EDTA is added for anticoagulation and preservation at-80 ℃) is sent, a medical record database is established, and information of the disease condition, family condition and the like of the forepart is recorded in detail. The study was approved by the ethics committee of this unit. 500 healthy samples which are irrelevant to the family of the dilated cardiomyopathy forerunner are randomly collected as verification samples, 2-4mL EDTA anticoagulants are collected for each sample, and the samples are stored at-80 ℃.
S1, extracting genome DNA: the whole genome DNA of the human whole blood EDTA anticoagulation sample of the foreigners and the validation sample is extracted by adopting a magnetic bead method whole blood genome DNA extraction kit of Jiangsu Baishino medical science and technology Co., ltd, and the operation steps are carried out 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, SEQ ID NO: 3), 2 mu L of reverse primer (10 mu m, SEQ ID NO: 4), less than 1000ng of template DNA, and RNase-Free H 2 O was added to 50. Mu.L.
Forward primer (SEQ ID NO: 3): 5'AAGAAGACAGAGATGCTCCAAC 3'; reverse primer (SEQ ID NO: 4): 5'CCACCATCACGATCCGGCTT 3'. Length: 739bp.
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, elongation at 72℃for 20s, total 35 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 until the magnetic beads are fully adsorbed (about 1min is needed). (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, 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 until the beads were fully adsorbed (about 1 min). (10) The eluate was transferred to a new 1.5mL centrifuge tube, at which point the beads were discarded.
S6, sanger sequencing the amplified products were subjected to Sanger sequencing using an applied biosystems 3500Dx series gene analyzer.
S7, performing bioinformatics analysis on the sequencing result: sequence alignment was performed in software Chromas between the sequencing results and wild-type TTN gene sequences obtained in NCBI (https:// www.ncbi.nlm.nih.gov /), and it was determined whether the detection sites were mutated.
The precursor carried the TTN gene c.32706delAinsTTC heterozygous missense mutation, and the sequencing result is shown in FIG. 1. This variation changes the 10902 polar positively charged lysine to a polar uncharged asparagine, after which the protein is expressed and the 21 st amino acid has a stop codon, potentially resulting in truncated expression of the protein. Frameshift or nonsense variations downstream of this site were assessed multiple times by the reporter as causative mutations of dilated cardiomyopathy (ClinVar database), and literature searches did not find reports of this variation in relation to disease.
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.
According to the existing evidence: the mutation is rare, it may cause truncated expression of the protein, near and downstream frameshift or nonsense mutation, which is reported multiple times as a pathogenic mutation, frameshift or nonsense mutation as the main pathogenic type of the gene, but lacks family linkage and functional evidence support, so the mutation is a highly suspected pathogenic mutation of dilated cardiomyopathy.
S8, carrying out genetic variation demonstration: none of the 500 healthy control members detected this variation; the dilated cardiomyopathy precursor detected a missense variation in the TTN gene c.32706delainsttc heterozygous.
Example 2: independent sample verification experiment-dilated cardiomyopathy family screening
1 dilated cardiomyopathy family (family chart is shown in fig. 2) was recruited, and laboratory examination, electrocardiographic and dynamic electrocardiographic examination, exercise electrocardiographic examination and imaging examination were performed on all family members to preliminarily confirm that they fit the dilated cardiomyopathy family characteristics. By gene detection, 5 dilated cardiomyopathy patients were examined in this family. As a control, 400 healthy people not suffering from dilated cardiomyopathy were also recruited. TTN gene c.32706delainsttc was amplified for each member of the family and for the control population using the procedure described in example 1, and after amplification, sanger sequencing was performed for analysis. Based on the confidentiality of the sample information, a part of the sample information is now disclosed.
The sample may disclose information: (1) dilated cardiomyopathy family country/region: chinese/beijing; family member male-female ratio: 1:1; family member age distribution: country/region of control population at 10-60 years (2): chinese/beijing; ratio of men and women of control group: 1:1; age distribution of control population: and 10-60 years old.
Sanger sequencing results showed that the ill members of the recruited dilated cardiomyopathy family all carried c.32706delAinsTTC heterozygous mutations; whereas non-diseased members of the family and normal control populations did not see any of the mutations at any of the foregoing sites.
Example 3: dilated cardiomyopathy 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) The sequencing result is analyzed to compare whether the TTN gene c.32706delAinsTTC heterozygous mutation exists.
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 (3)
1. The application of a reagent for detecting a mutant TTN gene in preparing an dilated cardiomyopathy detection kit is characterized in that the coding nucleotide sequence of the mutant TTN gene is SEQ ID NO. 1, the amino acid sequence of the coding protein is SEQ ID NO. 2, and the mutant TTN gene has c.32706delAinsTTC heterozygous missense mutation.
2. The use according to claim 1, wherein the reagents comprise primers SEQ ID NO. 3 and SEQ ID NO. 4.
3. The use according to claim 2, wherein the detection kit comprises a PCR pre-mix, a negative control reagent and a positive control reagent.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028983A (en) * | 2020-05-25 | 2020-12-04 | 百世诺(北京)医学检验实验室有限公司 | Novel mutant protein related to dilated cardiomyopathy, novel mutant gene and application thereof |
| CN113699226A (en) * | 2021-08-16 | 2021-11-26 | 百世诺（北京）医疗科技有限公司 | TTN mutant gene and dilated cardiomyopathy detection kit |
| CN113981066A (en) * | 2021-11-02 | 2022-01-28 | 百世诺（北京）医疗科技有限公司 | Mutant dilated cardiomyopathy pathogenic gene TTN and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9476097B2 (en) * | 2011-04-11 | 2016-10-25 | The Brigham And Women's Hospital, Inc. | Structural mutations in titin cause dilated cardiomyopathy |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028983A (en) * | 2020-05-25 | 2020-12-04 | 百世诺(北京)医学检验实验室有限公司 | Novel mutant protein related to dilated cardiomyopathy, novel mutant gene and application thereof |
| CN113699226A (en) * | 2021-08-16 | 2021-11-26 | 百世诺（北京）医疗科技有限公司 | TTN mutant gene and dilated cardiomyopathy detection kit |
| CN113981066A (en) * | 2021-11-02 | 2022-01-28 | 百世诺（北京）医疗科技有限公司 | Mutant dilated cardiomyopathy pathogenic gene TTN and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| NM_003319.4.Genbank.2022,第1-37页. * |
| Truncations of Titin Causing Dilated Cardiomyopathy;Daniel S. Herman等;N Engl J Med;第366卷(第7期);摘要、补充材料 * |
| 家族性扩张型心肌病一家系基因检测分析;武琼等;山东医药;第62卷(第5期);第48-51页 * |
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