CN109680055B

CN109680055B - CNV fragment related to congenital tracheomalacia and application thereof

Info

Publication number: CN109680055B
Application number: CN201811511780.0A
Authority: CN
Inventors: 陈俊; 邰隽; 王生才; 李晓丹; 姫婷婷; 杜江南; 房孝莲
Original assignee: Beijing Childrens Hospital
Current assignee: Beijing Childrens Hospital
Priority date: 2018-12-11
Filing date: 2018-12-11
Publication date: 2022-09-02
Anticipated expiration: 2038-12-11
Also published as: CN109680055A

Abstract

The invention discloses a CNV fragment related to congenital tracheomalacia, wherein the CNV fragment is a Chr17:16673072-20374018 interval fragment, and the Chr17:16673072-20374018 fragment has copy number deletion in blood of a patient with congenital tracheomalacia. The kit for detecting congenital tracheomalacia comprises detection of copy number variation of genes Chr17:16673072-20374018 or ALDH3A1 and ATPAF2, and can be used for preliminarily detecting whether a sample to be detected has congenital tracheomalacia diseases. The copy number variation sites of the Chr17:16673072-20374018 or ALDH3A1 and ATPAF2 genes can be used as molecular markers for early screening and diagnosis of congenital tracheomalacia.

Description

CNV fragment related to congenital tracheomalacia and application thereof

Technical Field

The invention relates to the field of molecular biology detection, in particular to a CNV fragment related to congenital tracheomalacia and application thereof.

Background

Tracheomalacia refers to a disease in which the integrity of the tracheal cartilaginous ring is destroyed or part of the longitudinal elastic fibers of the tracheal wall are atrophied or reduced, resulting in collapse and stenosis of the trachea due to hypo-elastic tension. They can be roughly classified into Congenital (primary) tracheitis (CTM) and acquired (secondary) tracheitis according to the cause of the disease. The pathogenesis of CTM is not fully elucidated at present, with an incidence of about 1/2100, accounting for 34% of bronchoscopy diagnoses. CTMs are found in isolation in healthy people, but are more common in premature infants, with an age of onset of mostly within 6 months. CTM is often associated with other diseases such as congenital heart disease, gastroesophageal reflux, TEF, bronchopulmonary dysplasia, impaired neurological function, developmental delay, etc., with an incidence of 1.7% -78%. According to the collapse degree of the trachea, the CTM can be divided into a light level, a medium level and a heavy level, wherein the severe CTM patients mainly show repeated infection, atelectasis and dyspnea, conservative treatment is ineffective, the death rate reaches 80% in the last 80 centuries, even today, the severe CTM patients often have symptoms such as sputum retention, upper respiratory tract obstruction, respiratory arrest and even cardiac arrest, and serious economic and mental burdens are brought to families and society. Therefore, it is important and urgent to search the genetic basis of CTM and to explore its possible pathogenic mechanism.

In recent years, the role of Copy Number Variation (CNV) in complex diseases has received increasing attention. CNV refers to the increase or decrease of copy number of large genomic fragments with a general length of 1kb or more, mainly expressed as deletion and duplication at a sub-microscopic level, caused by genome rearrangement, and is an important component of genome structural variation, and the CNV can cover 29.74% of genome, carries more information than SNP, and has a mutation rate at CNV sites much higher than SNP, which is one of important pathogenic factors for genetic differences and diseases among human individuals.

Currently, microarray-based comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP array) based array technologies are commonly used for genome-wide CNVs. The known CNV detection of genome mainly comprises Real-time fluorescent Quantitative PCR (Real-time qPCR), Ligation-Dependent Multiplex Amplification Probe hybridization (Multiplex Ligation-Dependent Probe Amplification, MLPA and short-fragment Multiplex quantification (Quantitative Multiplex PCR RofShortfluorescence fragments, QMPSF).

Specific CNVs may explain a part of the risk of disease in addition to sequence variation and have been shown to be associated with a variety of complex diseases. There is relatively little current research on the correlation of CNVs with CTMs. The invention aims to discuss the genetic basis of CTM pathogenesis, searches possible biomarkers of CTM from the molecular level, and is beneficial to early diagnosis and treatment of CTM.

Disclosure of Invention

The invention aims to provide a CNV fragment related to congenital tracheomalacia and application of the CNV fragment in preparing products for preventing, early detecting or treating congenital tracheomalacia.

In order to achieve the purpose, the invention firstly provides a CNV fragment related to congenital tracheomalacia, wherein the CNV fragment is a fragment in the interval of Chr17:16673072-20374018, and the fragment of Chr17:16673072-20374018 has copy number loss in blood of a patient with congenital tracheomalacia.

Preferably, the CNV fragment comprises ALDH3a1, ATPAF2, AKAP10, DRG2, FAM106A, FAM83G, LLGL1, RAI1, SLC47a1, SREBF1, TBC1D28 genes.

Preferably, the CNV fragment is used for preparing products for preventing, early detecting or treating congenital tracheomalacia.

Preferably, the product comprises a kit, a reagent or a pharmaceutical product.

Furthermore, the invention provides a kit for detecting congenital tracheomalacia, which comprises a primer for specifically detecting the Chr17:16673072 and 20374018 interval fragment or the gene CNV contained in the interval fragment.

Preferably, the primers for specifically detecting the fragment Chr17:16673072 and 20374018 or the fragment in the interval comprise ALDH3A1 and ATPAF2 gene CNV, the nucleotide sequence of the ALDH3A1 primer is shown in SEQ ID NO.1-4, and the nucleotide sequence of the ATPAF2 primer is shown in SEQ ID NO. 5-8.

Preferably, the kit further comprises 10 XPCR buffer, TaqDNA polymerase, DNA template, dNTP and MgCl ₂ And normal sample genomic DNA.

Preferably, the kit detects the Chr17:16673072-20374018 fragment or the copy number of the gene contained in the fragment in the interval by real-time fluorescent quantitative PCR to preliminarily detect whether the congenital tracheomalacia disease exists.

Preferably, the detecting specifically includes: using genome DNA of sample to be detected as template, using primers shown in SEQ ID NO.1-8 and reference gene primer to make fluorescent quantitative PCR reaction, using

The method calculates the copy number of the CNV fragment of the sample to be detected and compares the copy number with the normal control group, the ratio is within the range of 0.5-1.5, the normal is prompted, and the ratio<0.5 suggests that there is a fragment deletion,ratio of>1.5 suggested fragment repeats.

Preferably, when detecting that the copy number of Chr17:16673072-20374018 or ALDH3A1 or ATPAF2 gene of the sample to be detected is missing, the congenital tracheomalacia disease is preliminarily detected.

Advantageous effects

The invention detects the copy number of the gene of the congenital tracheomalacia patient and verifies the copy number by fluorescence quantitative PCR, and the result shows that the copy number of the gene of the congenital tracheomalacia patient Chr17:16673072 20374018 or ALDH3A1 and ATPAF2 is obviously lower than that of a normal person (P < 0.05). Therefore, the copy number variation sites of the genes Chr17:16673072-20374018 or ALDH3A1 and ATPAF2 can be used as molecular markers for early screening and diagnosis of congenital tracheomalacia.

The kit for detecting congenital tracheomalacia comprises detection of copy number variation of genes Chr17:16673072-20374018 or ALDH3A1 and ATPAF2, and can be used for preliminarily detecting whether a sample to be detected has congenital tracheomalacia diseases.

Drawings

FIG. 1 shows the copy numbers of the congenital tracheomalacia ALDH3A1 and ATPAF2 genes.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The materials, reagents and the like used in the examples are commercially available unless otherwise specified.

EXAMPLE 1 sample Collection

1. Inclusion exclusion criteria for CTM patients

(1) Chinese Han patients, the age of which is not more than 1 year old;

(2) on the basis of combining the medical history, physical examination and fiberbronchoscopy, more than two experienced high-age specialist doctors consistently diagnose the patient to be CTM, and the degree of trachea collapse is severe (namely, circular tube cavities cannot be seen);

(3) eliminating secondary TM and trachea injury caused by other reasons;

(4) excluding other system (e.g., heart, esophagus) malformations;

(5) excluding other known clinical syndromes;

(6) exclusion of patients with combined malignant tumors

(7) Excluding the mother with definite medical history, toxic contact history and high temperature exposure history during pregnancy;

(8) eliminating the incomplete epidemiological data, clinical data and image data;

(9) excluding those who did not obtain informed consent.

2. Inclusion exclusion criteria for normal controls (including parents in the core family)

(1) Normal chinese han children, age not more than 1 year, sex ratio matched with patient group, all samples from health examination children; parents in the core family are Chinese Han nationality people, and no obvious organic or psychogenic pathological changes exist through conventional image and biochemical detection;

(2) all the compared data were evaluated as non-CTM by two high-age specialties blindly;

(3) excluding clear family history of other diseases;

(4) no history of other diseases of the head and the neck, and no medicine is taken at present;

(5) parents in the core family do not smoke, drink, stay up to night, take irritant food and the like within 2 weeks before blood collection.

Sample data are collected strictly according to inclusion and exclusion standards, and 6-8mL of peripheral blood of 8 typical patients and parents (core family) of the patients are collected, wherein the patients are diagnosed in the children respiratory department of Beijing child hospital affiliated to capital medical university from 5 months to 2018 months in 2017. And signing an informed consent.

Example 2 CTM core family genome-wide CNV scanning and data analysis

Selecting 8 typical patients with phenotype and parents (core family) as study objects, carrying out genome-wide CNV scanning, and screening and analyzing pathogenic CNV on the scanning data according to corresponding standards.

1. Whole genome CNV scanning Using aCGH technique

Sample preparation, 6-8mL of venous peripheral blood of the patient was extracted, genomic DNA was extracted using QIAGEN kit, and stored at-20 ℃ for further use. Taking 2.0 mu L of extracted genome DNA, diluting by 100 times, measuring the DNA concentration in a spectrophotometer, reading the numerical value, and calculating the DNA content and the DAN concentration of the genome; simultaneous reading of OD ₂₆₀ /OD ₂₈₀ Ratio of 1.8<OD ₂₆₀ /OD ₂₈₀ <And 2.0 is qualified. The aCGH detection analysis can be carried out on the sample with the DNA concentration not less than 40 ng/mu L and the content not less than 0.5 mu g.

The aCGH detection comprises the following specific steps:

(1) enzyme digestion: the DNA samples of patients and parents select AluI and RsaI enzyme to carry out enzyme digestion reaction (2 h at 37 ℃ to 65 ℃ for 20min to 12 ℃ for 8 min);

(2) and (3) electrophoresis detection: the product after enzyme digestion should be between 200bp and 500 bp;

(3) fluorescence labeling: labeling the digested DNA fragments with Cy3 (red fluorescence) and Cy5 (green fluorescence), respectively;

(4) and (3) purification: purifying the fluorescence-labeled DNA sample by a column;

(5) pre-hybridization: mixing the purified DNA samples with fluorescent labels, and carrying out prehybridization reaction at 37 ℃;

(6) and (3) hybridization reaction: closely combining the washed chip with the probe surface, and putting the chip into a hybridization furnace for hybridization for more than 40 h;

(7) chip washing: fully washing the chip by buffer I and buffer II;

(8) scanning by a scanner: fluorescence signal intensity scans were performed using a scanner provided by Agilent corporation.

2. CTM whole genome CNV data screening and pathogenic gene analysis

(1) For all The discovered CNVs, firstly, The CNVs are compared with a genome variation Database (DGV), a Human Structural variation Database (The Human Structural variant project) and The like to judge whether The CNVs are rare CNVs or common CNVs;

(2) the pathogenicity of CNVs was analyzed by querying the databases, deciper, PubMed and OMIM.

(3) In PubMed data, performing literature retrieval, comparing and analyzing to confirm whether the CNV found in the research is reported in the previous research, and performing more effective CNV screening according to known related genes and signal paths thereof;

(4) according to the principle of 'sharing patients and not carrying patients', site screening is carried out, and intersection is taken from all mutation sets of diseased individuals as much as possible. And finally, predicting the damage degree of the mutation to find out the candidate pathogenic CNV mutation.

The inventor compares the CNV results of 8 patients with a genome variation Database (DGV), a DECIPHER Database, a PubMed Database and an OMIM Database, and finds that 2 patients have the deletion of the same chromosome DNA copy number, namely the deletion of about 3.7Mb in the 17p11.2 interval, are respectively positioned in the Chr17: 16657357364-20374018 and 16673072-20374018 intervals of the 17 chromosome, and finally thinks that the deletion in the common Chr17:16673072-20374018 interval is possibly related to the congenital tracheomalacia disease.

Example 3 identification of chromosome copy number by real-time quantitative PCR (real-time PCR)

1. Primer design

Through analysis of the detection result of aCGH, the inventor obtains 1 CNV interval related to congenital tracheomalacia, namely Chr17:16673072-20374018, and further verifies the CNV interval through a real-time PCR method.

And inquiring the chromosome deletion interval and the gene contained in the chromosome deletion interval in a UCSC database. And selecting key genes ALDH3A1 and ATPAF2 in the CNV interval, and querying a genome sequence by using an Ensembel website. The real-time fluorescent quantitative PCR primers are designed by using Primer 5.0, and two sections of primers are respectively designed to be evenly distributed in key ALDH3A1 and ATPAF2 genes, and 4 pairs of primers are used in total. The GC content of the primer is required to be between 40 and 60 percent; the annealing temperature is between 55 and 65 ℃; the length of the product is controlled between 80bp and 250 bp. The primer sequences, the length of the target fragment, and the annealing temperature are as follows:

TABLE 1 primer sequences

2. Real-time fluorescent quantitative PCR

This study was carried out using ABI Stepone quantitative PCR Instrument manufactured by ABI, USA

Green I chimeric fluorescence real-time PCR.

Real-time reaction system (20. mu.L): the genomic DNA of 2 cases of 17p11.2 interval deletion patients found in the aCGH detection is taken as a pathological sample, a well-amplified normal control is selected as a reference sample, the patients are respectively subjected to specific primer real-time fluorescence quantitative PCR, and a universal primer GAPDH is selected as an internal reference for calibration. The Real-time PCR was performed by TaKaRa, Japan

The Green kit is used for amplification, and the reaction system is as follows:

TABLE 2 reaction System

Real-time PCR reaction conditions:

after denaturation at 94 ℃ for 30s of pre-denaturation, the amplification cycle was started: denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 34s, and elongation at 72 ℃ for 20s, and circulating for 45 times. Drawing a dissolution curve: 95 ℃ for 15S, 60 ℃ for 1min and 95 ℃ for 15S. Whether the primers are suitable for qPCR analysis was determined by plotting amplification curves and dissolution peaks. According to the drawn dissolution curve, the curves of all samples are matched together, the curve trend is smooth, the peak height is sharp, and no primer dimer or hetero-peak caused by non-specific amplification exists, so that the primer specificity in the research is good, and the amplification product is single.

3. Real-time relative quantitative analysis:

using relative quantitative analysis

Method for specificity of copy number of primer specific to 2 patients and normal humanThe primer copy number is quantitatively analyzed, and the universal primer GAPDH is taken as an internal reference point. The Relative quantitative value Relative Quantification (RQ) of amplification of the normal human specific primer and the universal primer was set to 1, 2 cases of amplification of the patient specific primer and the universal primer were compared with the normal control group to obtain RQ values (the experiment was repeated 3 times, the average value was calculated), and the RQ values were plotted on the ordinate, and the results are shown in FIG. 1.

The relative amount of the specific primers of the key genes in the Chr17:16673072-20374018P region of chromosome 17 can be obtained by quantitative analysis, and the ratio of the relative amount of the specific primers to the normal control group is less than 0.5. This result indicates a reduction in the copy number of the key gene in this region, which also reflects a reduction in the copy number of this region, consistent with the aCGH assay.

Example 4 validation of pathogenic CNV at the population level in small-sample CTM patients

The deletion of the Chr17:16673072-20374018 region was verified in 20 small samples of patients who distributed CTM and 8 normal controls by fluorescence real-time quantitative PCR.

PCR amplification was performed on an ABI Stepone platform using

Green I chimeric fluorescence method using GAPDH as internal reference sequence

The method calculates relative copy numbers, and compares the difference in copy numbers of the patient and control pathogenic CNV to assess the relevance of the pathogenic CNV to the disease. Wherein the PCR reaction system, reaction conditions and reaction primers were performed as described in example 3.

The results show that 3 patients in 20 patients with sporadic CTM have reduced copy numbers of pathogenic CNV, and further show that the deletion of chromosome 17 in the Chr17:16673072-20374018 interval is related to congenital tracheomalacia diseases, and can be used as a molecular target for further research on treatment and diagnosis of congenital tracheomalacia diseases.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. Application of a gene copy number variation detection reagent in preparing a product for predicting the risk of congenital tracheomalacia comprises ALDH3A1 and ATPAF2 genes, and the genes are subjected to copy number deletion in blood of a patient suffering from congenital tracheomalacia.

2. The use of claim 1, wherein the product comprises a kit, reagent or pharmaceutical product.

3. The application of the gene copy number variation detection reagent in preparing a kit for predicting the risk of congenital tracheomalacia is characterized in that the kit comprises primers for specifically detecting ALDH3A1 and ATPAF 2.

4. The use of claim 3, wherein the ALDH3A1 primer has a nucleotide sequence shown in SEQ ID NO.1-4 and the ATPAF2 primer has a nucleotide sequence shown in SEQ ID NO. 5-8.

5. The use of claim 4, wherein the kit further comprises 10 XPCR buffer, Taq DNA polymerase, DNA template, dNTP, MgCl ₂ And normal sample genomic DNA.

6. The use of claim 3, wherein the kit is used for predicting whether the risk of congenital tracheomalacia disease is increased by detecting the copy number of genes through real-time fluorescent quantitative PCR.

7. The use according to claim 6, wherein said detecting comprises in particular: taking genome DNA of a sample to be detected as a template, performing fluorescent quantitative PCR reaction by using primers shown as SEQ ID NO.1-8 and primers of an internal reference gene, and performing fluorescent quantitative PCR reaction by using 2 ^-△△ct The method calculates the copy number of the CNV fragment of the sample to be detected, compared with the normal control group, the ratio is in the range of 0.5-1.5, the normal is prompted, and the ratio<0.5 indicates fragment deletion, ratio>1.5 suggested fragment repeats.