CN106048059A - SNP sites of A variation blood type for triggering acute hemolytic transfusion reaction - Google Patents

Abstract

The invention aims to provide SNP sites used for detecting acute hemolytic transfusion reaction triggered by A variation type of ABO blood type system. The SNP sites are the 467th and the 689th bases starting from initiation codon from the new A variation gene coding region. The invention provides the new application of the A variation gene so as to provide an effective approach to fast gene diagnosis, gene screening and genetic counseling for acute intravascular hemolytic transfusion reaction. Through application effects, it shows that the SNP sites and detection primers of the gene can be effectively applied to clinical patients' peripheral blood and fetal villi or amniotic fluid for fast detection of new A variation gene mutation sites.

Description

A SNP site of A variant blood type triggering acute hemolytic transfusion reaction

technical field

The invention belongs to the technical field of gene diagnosis products, and in particular relates to a SNP site for detecting acute hemolytic transfusion reaction caused by ABO blood group system type A variant blood group gene.

Background of the invention

Acute Hemolytic Transfusion Reaction (AHTR) usually occurs within 24 hours after blood transfusion, more often than immediately after blood transfusion. As long as 5-10ml of ABO-incompatible blood is transfused, obvious symptoms will appear; blood transfusion exceeding 200ml will cause serious consequences. Clinically, it often manifests as intravascular hemolysis. Patients with fever, chills, nausea, and back pain, followed by hemoglobinuria, hemoglobinemia, and hypotensive shock, should be considered as acute intravascular hemolytic transfusion reactions, and the blood transfusion should be stopped immediately and administered immediately. Carry out various checks. At the same time, this process will cause complement activation, start the coagulation system, release bradykinin and catecholamines, and induce DIC; even circulatory and renal failure; therefore, hypotensive shock, DIC, and acute renal failure are usually called acute intravascular hemolysis. disease.

Whether at home or abroad, the wrong infusion of ABO blood type is the most common factor causing acute hemolytic transfusion reactions. According to the US FDA report, 139 of the 159 deaths from acute hemolytic reactions were caused by ABO incompatible blood transfusions. Secondly, it is more common that patients with A variant (A2 variant is the most common) produce anti-A1 antibodies due to immune factors (blood transfusion, pregnancy or miscarriage). If A1 blood is transfused again, it will cause acute or delayed immune hemolysis. In view of this situation, the importance of prevention must be emphasized. Therefore, when identifying the ABO blood type of clinical patients, it is necessary to carry out positive and negative typing. , only 2-5% of the A2 type and 25% of the A2B can produce anti-A1 antibodies in the anti-typing experiment. immune hemolytic reaction.

Contents of the invention

The purpose of the present invention is to provide a SNP site of acute hemolytic transfusion reaction caused by ABO blood group system A variant blood group gene, so as to make up for the deficiencies in the prior art.

The applicant sequenced a family member with autosomal 9q34.1-9q34.2 dominantly inherited type A variant blood type gene, and found a genetic pathogenic mutation in the type A variant gene of the family member, causing The changes in the quantity and quality of the A antigen, and the anti-A1 antibody can be produced through immune stimulation, in order to avoid the acute hemolytic transfusion reaction caused by blood transfusion, thus contributing to the invention.

The invention provides a SNP site for detecting the acute hemolytic transfusion reaction caused by the A-type variant blood type gene of the ABO blood type system, which is the 467th and 689th bases from the start codon in the coding region of the ABO blood type gene.

The direct amplification and sequencing primer information used to detect the above SNP sites is as follows:

ABO-E7F:5'-ATCCGCCTGCCTTGCAGATA-3'SEQ ID NO:1

ABO-E7R:5'-AGCCTAGGCTTCAGTTACTCACAACA-3'SEQ ID NO:2

The primers used to detect the haplotype amplification and sequencing of the above SNP sites are as follows:

ABO-E6mo: 5'-CGGGATCCATGTGGGTGGCACCCTGCCA-3'SEQ ID NO:3

ABO-E7mo: 5'-GGGCCTAGGCTTCAGTTACTC-3' SEQ ID NO: 4

The invention provides a new application of ABO blood type A variant gene detection, thereby providing an effective way to avoid the occurrence of acute hemolytic transfusion reactions in gene diagnosis, prenatal genetic screening and genetic counseling, and the application effect shows that the invention The provided gene SNP sites and detection primers can be effectively used in the rapid detection of ABO blood type A variant gene mutation sites in the peripheral blood of clinical patients.

Description of drawings

Figure 1: Sequencing map of the ABO gene mutation site in a proband with ABO blood type A variant gene;

Figure 2: The direct sequencing map of the proband;

Figure 3: The sequence map of the clone of the proband. The coding region of the ABO gene of the proband has mutations at the 467th and 689th bases from the start codon (nt467C>T; nt689G>T).

detailed description

The applicant found the mutation site of the type A variant gene in an ABO blood type A variant gene family, and confirmed that the mutation of the gene caused the variation of the A blood type to produce the same anti-A1 antibody, which triggered acute hemolytic blood transfusion Responsive pathogenic genes, thus contributing to the present invention.

A variant is located on chromosome 9q34.1-9q34.2, which can be transcribed into mRNA of about 1065bp (NCBI accession number is NM_020469.2), and directly translated into a protein consisting of 355 amino acids.

The present invention will be described in detail below in conjunction with the examples.

Example 1: Confirmation of the mutation site of type A variant from the gene detection of the proband of ABO blood type A variant gene.

1. Extraction of peripheral blood genomic DNA:

In compliance with the relevant national policies and regulations, and on the basis of the consent of the sampling subjects, 2-5ml of peripheral venous blood from members of the ABO blood type A variant gene family was drawn, put into EDTA-Na ₂ anticoagulant tubes, and stored at -80°C for later use; DNA extraction uses the DNA extraction kit of LIFE Company, the specific operation process is as follows:

After thawing the frozen EDTA anticoagulated blood at room temperature, take 500ul and put it in a centrifuge tube, add an equal volume of erythrocyte lysate (pH8.0), vortex and mix for 5 minutes, centrifuge at 12000rpm for 5 minutes, and discard the supernatant. Add 500ul of red blood cell lysate repeatedly, vortex and mix for 5 minutes, centrifuge at 12000rpm for 5 minutes, and discard the supernatant.

The precipitate was vortex-mixed for 5 minutes, 50ul of nuclear lysate was added, vortex-mixed for 5 minutes, and placed in a 56°C metal bath for 15 minutes. Take out the sample from the water bath and add 135ul of protein clearing solution. Vortex well to mix, and place at 4 °C for 30 min.

Centrifuge at 12000 rpm for 5 minutes at room temperature, and pipette the supernatant (about 200ul) into a new centrifuge tube. Add 500ul of ice ethanol, and shake the centrifuge tube repeatedly until the white DNA precipitate is precipitated. Centrifuge at 12,000 rpm for 2 minutes at room temperature, and discard the supernatant. Add 75% ethanol to the DNA pellet, rinse once, centrifuge at 12,000 rpm at room temperature for 3 minutes, discard the supernatant, place at room temperature to evaporate the remaining ethanol, and finally add 50 μL TE (pH 8.0) to dissolve the DNA overnight at 4°C.

Perform agarose gel electrophoresis on the extracted DNA, and use a UV spectrophotometer to compare colors at 260nm and 280nm to detect the purity and concentration of DNA.

2. Find the mutation site of the A variant gene in the proband by direct sequencing

PCR amplification target fragment: reaction conditions and reaction system:

(1) PCR reaction conditions: 95°C pre-denaturation for 10 minutes; 94°C for 60 seconds, 63°C for 90 seconds, 72°C for 60 seconds, 10 cycles; 94°C for 60 seconds, 61°C for 90 seconds, 72°C for 60 seconds, 25 cycles Cycle; extend at 72°C for 10 minutes, and incubate the amplified product at 10°C.

(2) Reaction system: (TAKARA Ex-Taq polymerase)

The reaction system was used to carry out the amplification reaction of the proband's genomic DNA template and the above-mentioned ABO-E7F and ABO-E7R primers.

PCR product sequencing: The above PCR products were sequenced by conventional Sanger sequencing, and the primer pair ABO-E7F:5'-ATCCGCCTGCCTTGCAGATA-3'; ABO-E7R:5'-AGCCTAGGCTTCAGTTACTCACAACA-3' was used to find the ABO blood type in the proband The 467th and 689th bases of the coding region of gene A were mutated from the start codon. Among them, a CT hybrid sequence appeared at the 467th base; a GT hybrid sequence appeared at the 689th base. Multiple sequencing results showed that the mutation site was not introduced due to amplification or sequencing errors (Figure: 1).

3. Perform haplotype sequencing on exons 6, 7 and intron 6 of the ABO gene to determine the mutation site of the A variant gene in the proband

Primers for PCR amplification target fragment: ABO-E6mo: 5'-CGGGATCCATGTGGGTGGCAC CCTGCCA-3'; ABO-E7mo: 5'-GGGCCTAGGCTTCAGTTACTC-3'.

The PCR amplification primers may also select other primer pairs capable of amplifying the above-mentioned SNP sites.

Reaction conditions and reaction system for PCR amplification of target fragments:

(1) PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 50 seconds; extension at 72°C for 5 minutes, and incubation of the amplified product at 10°C.

(2) Reaction system: (TAKARA LA-Taq polymerase)

The reaction system was used to carry out the amplification reaction between the genomic DNA template of each family member and the primers designed by the inventor for cloning sequencing. The PCR product was cloned into the T vector, and multiple colonies were picked for haplotype sequencing. The haplotype sequencing primers used ABO-E6mo: 5'-CGGGATCCATGTGGGT GGCACCCTGCCA-3'; ABO-E7mo: 5'-GGGCCTAGGCTTCAGTTACTC-3' (Sequencing primers can also be selected from other primers designed from the type A variant gene and can amplify SNP sites) to determine the haplotype sequence of the sample (Figure 3). This mutation is a newly discovered mutation. This mutation was not present in the following three databases: Single Nucleotide Polymorphism Database (ftp://ftp.ncbi.nih.gov/snp/database/), Blood Type Gene Mutation Database (http://www. ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo), indicating that the mutation is very rare, and the mutation leads to a change in the nucleotide composition of the protein composition of the A blood group, In the coding region of A gene, the 467th position from the start codon is changed from C>T, resulting in the 156th amino acid being changed from proline to leucine; the 689th base is changed from G>T, resulting in the 230th amino acid Changed from glycine to valine; the mutation of the amino acid caused the deletion of the A epitope in the individual of the A variant, resulting in the ability of the individual to produce the same anti-A1 antibody in the plasma without immune stimulation ( Natural immunity), when the type A blood of a normal person is transfused, an acute hemolytic transfusion reaction will occur, which will endanger the life of the patient. However, the mutation screening of this site was carried out in the peripheral blood genomic DNA samples of nearly 300 cases of normal local population, and the mutation was not found.

Genomic DNA from the peripheral blood of the family members of the proband collected by the Institute of Blood Transfusion Medicine at the Central Blood Bank in Qingdao was used as a template to amplify the PCR target fragment using the above-mentioned DNA as a template and ABO-E7F and ABO-E7R primers. Conventional Sanger The sequencing method used the above pair of primers to directly sequence the PCR product, and found that the ABO blood type A gene of the two family members of the proband had the SNP mutation found in the present invention, that is, the coding region was at position 467 from the start codon A CT heterozygous mutation occurred at the base, and a GT heterozygous mutation occurred at the 689th base. Continue to perform haplotype sequencing of exons 6 and 7 and intron 6 of the ABO gene for the two family members with SNP mutations, and use the genomic DNA of the two family members extracted above as a template to combine with ABO-E6mo, ABO-E7mo primers are used to amplify the PCR target fragment, clone the PCR product into T vector, pick multiple colonies for haplotype sequence determination, use the above primers for haplotype sequencing primers, and determine the haplotype sequence of the sample. Haplotype sequencing found that the 467th position of the A gene coding region of the two family members was changed from C>T from the start codon, resulting in the 156th amino acid being changed from proline to leucine; the 689th base was changed from G>T, resulting in the change of the 230th amino acid from glycine to valine, which is completely consistent with the proband, and the same anti-A1 antibody exists in the plasma of the two families.

Through the above analysis, it is proved that the primers designed by the inventor can be used to detect whether the individual ABO blood group gene has A gene mutation, so there is a potential risk of acute transfusion-type hemolytic reaction after blood transfusion.

Claims (8)

1. A SNP site for detecting the acute hemolytic transfusion reaction caused by ABO blood group A variant, characterized in that, the SNP site is the A type variant gene coding region composed of the 467th and 467th start codons and 689 bases. 2. The application of the SNP site according to claim 1 in the preparation of A variant gene-related products for detecting ABO blood type. 3. application as claimed in claim 2, is characterized in that, described product is the primer pair that is used for PCR purpose fragment amplification and direct sequencing or is used for haplotype PCR purpose fragment amplification and haplotype sequencing. primer pair. 4. A pair of primers for detecting the SNP site of the acute hemolytic transfusion reaction caused by ABO blood group A variant, characterized in that the pair of primers is used for detecting the SNP site of claim 1. 5. The pair of primers according to claim 4, wherein the sequences of the pair of primers are SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. 6. A product for detecting the SNP site according to claim 1, characterized in that the product comprises the primer pair according to claim 4. 7. The product according to claim 6, wherein the sequences of the primer pair are SEQ ID NO:1 and SEQ ID NO:2. 8. The product according to claim 6, wherein the sequences of the primer pair are SEQ ID NO:3 and SEQ ID NO:4.