SNP (Single Nucleotide Polymorphism) site associated with moso-like blood type and detection preparation

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) site associated with a Bengal-like blood type, and the site generates a new allele of an H blood group system and can lead to generation of a Bengal-like phenotype. The new SNP mutation site is the 520th nucleotide from the initiation codon of the FUT1 gene coding region, and is varied from T to C. According to the SNP locus marker obtained through detection and screening, an effective way for performing Bengal-to-buy-like gene diagnosis is provided, and the application effect shows that the polymorphic locus marker of the gene and the detection primer provided by the invention can be effectively used for quickly detecting the FUT1 gene for clinical patients and blood station blood donors.

Description

Technical Field

[0001] The invention belongs to the technical field of gene diagnosis products, and in particular relates to a SNP site associated with Bombay-like blood type and a detection preparation. Background Art

[0002] Blood type is a genetic characteristic of the surface of red blood cells detected by specific alloantibodies. Correct blood type identification is the key to ensuring safe clinical use of blood. In clinical transfusion therapy, hemolytic transfusion reactions may occur due to red blood cell blood type incompatibility. Acute hemolytic reactions are accompanied by some typical clinical manifestations: chills, fever, cyanosis of the lips, and coldness of the waist and back. In severe cases, progressive renal failure, disseminated intravascular coagulation, and even death may occur. The most common is the wrong transfusion of ABO blood types, such as transfusing type A blood to type B patients; transfusing type A, type B, and type AB blood to type O patients, etc.

[0003] As the precursor of A and B blood group antigens, H antigen is closely related to the ABO blood group system. The H blood group system has been named as an independent blood group system by the International Committee on Nomenclature of Red Cell Blood Groups, namely the ISBT (International Society of Blood Transfusion) 018-Hh blood group system, with the system symbol H and only one antigen, namely the H antigen. The H antigen is encoded by the highly homologous FUT1 gene (H gene) and FUT2 gene (Se gene). Mutations in the FUT1 and FUT2 genes can cause rare blood types, such as the Bombay type and the Bombay-like type. The Bombay-like type is a secretory type lacking H antigen, in which red blood cells completely lack H antigen or only a small amount of H antigen exists, and ABH blood type substances are also present in the secretory cells, and anti-HI is usually present in the serum. Compared with other countries, the Bombay-like type has a higher distribution frequency in the Chinese population. Usually, anti-H / anti-HI antibodies are present in the plasma of Bombay-like individuals, which can lead to a decrease in the survival rate of red blood cells after transfusion and a hemolytic transfusion reaction. Therefore, missed detection of Bombay-like blood types can cause hemolytic transfusion reactions, and the detection of this type of Bombay blood type is of great significance for transfusion safety. Bombay-like blood types are easily missed in routine testing, especially when using microcolumn gel cards for ABO blood typing. Since microcolumn gel cards do not have anti-stereotyping O cells, Bombay-like blood types with extremely weak or missing A / B antigens are easily missed.

[0004] The genotyping method can accurately determine the red blood cell type, accurately detect what alleles the specimen carries and whether new alleles exist, etc. It has important application value in the fields of clinical blood transfusion and stem cell transplantation. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP site associated with Bombay-like blood group, as well as a molecular preparation for detecting the SNP site and its application.

[0006] The applicant sequenced the FUT1 gene of the H blood type system, which is dominantly inherited on autosome 19q13.3, and found that the FUT1 gene of the proband and a family member had genetic mutations, which caused changes in the quantity and quality of H antigens (extremely weak expression or absence). After searching databases and literature, this mutation site was discovered for the first time in the world. Since the H antigen is extremely weakly expressed or absent, the human body's natural immunity can produce anti-H antibodies without additional immune stimulation.

[0007] The present invention provides a SNP site that causes mutation of the FUT1 gene. The provided SNP site is located at the 520th base from the start codon of the FUT1 gene coding region, and the base mutates from T to C (c.520T>C).

[0008] That is, it is located at position 520 of the nucleic acid fragment with the sequence of SEQ ID NO:1.

[0009]

[0010]

[0011] In yet another aspect, the present invention provides a use of the SNP site as a detection target in the preparation of a product for detecting Bombay blood type.

[0012] The product is a PCR amplification sequencing detection kit;

[0013] The present invention also provides a PCR amplification sequencing detection kit for detecting Bombay-like blood types, wherein the kit comprises a primer pair for amplifying a nucleic acid fragment containing the above-mentioned SNP site;

[0014] As a specific record of the embodiment, the sequence information of the primer pair is as follows:

[0015] FUT1-4F:5'-CCCGGTATTCCGCATCAC-3'(SEQ ID NO:2),

[0016] FUT1-4R: 5'-CGGTGTAGCCTCCTGTCCAT-3' (SEQ ID NO: 3).

[0017] The present invention provides a new application of detecting variant genes of H antigen FUT1 encoded by the H blood type system, thereby providing an effective approach to genetic diagnosis and genetic counseling for avoiding hemolytic transfusion reactions. The application results show that the SNP sites and detection primers of the gene provided by the present invention can be effectively used for rapid detection of FUT1 gene mutation sites of H blood type Bombay phenotype in blood donors and clinical patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Direct sequencing of the proband showed that the proband was CT heterozygous at the 520th base from the start codon in the FUT1 gene coding region.

[0019] Figure 2 : The proband's clone sequencing map showed that the 520th base from the start codon in the FUT1 gene coding region of the proband mutated from T to C (c.520T>C). DETAILED DESCRIPTION

[0020] The applicant discovered the mutation site of the FUT1 gene in a proband with H blood type-like Bombay phenotype and family members, and confirmed that the mutation of this gene was the pathogenic mutation that caused the proband to produce allogeneic anti-HI antibodies and thus induce hemolytic transfusion reactions.

[0021] The FUT1 gene of the H blood type system is 7380bp in length and is located on the long arm of chromosome 19. It has 4 exons. The protein-coding sequence is located in the 4th exon and consists of 1098 bases. The 365 amino acids encoded by the FUT1 gene (NCBI accession number is NG_007510.1) constitute α(1,2) fucosyltransferase. The function of this enzyme is to connect the fucosyl to the galactose at the end of the precursor core sugar chain branch to form the H antigen.

[0022] The FUT1 gene has 4 exons, the protein coding sequence is located in the 4th exon, and the glycosyltransferase molecule consists of 365 amino acids. FUT1 gene mutation can lead to H antigen deficiency, which in turn leads to A and / or B antigen deficiency. The present invention screened and obtained a new H blood type variant gene SNP mutation site, sequenced the H antigen coding sequence of the proband's H blood type system, and found that the exon sequence of the FUT1 gene encoding the H antigen of this blood type system in the proband had 520T>C. After database and literature search, this mutation site was discovered for the first time internationally. Due to the presence of this mutation site, c.520T>C causes the 174th amino acid to change from phenylalanine to leucine. The change in amino acids may cause changes in the activity of α-1,2-fucosyltransferase, thereby affecting the expression of H antigen. Due to the lack of H antigen, anti-HI antibodies were produced in the serum of the proband. By analyzing the FUT1 gene sequence of the proband's parents, the 520T>C mutation was detected in their mother.

[0023] The present invention will be described in detail below with reference to the embodiments.

[0024] Example 1: Screening of gene polymorphism site markers

[0025] 1. Extraction of peripheral blood genomic DNA:

[0026] In accordance with relevant national policies and regulations, and based on the consent of the sampling subjects, 2-5 ml of peripheral venous blood was drawn from the mutant gene family members, placed in EDTA-Na2 anticoagulant tubes, and frozen at -80°C for future use; DNA extraction was performed using the DNA extraction kit of Invitrogen. The specific operation process is as follows:

[0027] After the frozen EDTA anticoagulated blood is thawed at room temperature, take 500ul and put it in a centrifuge tube, add an equal volume of red blood cell lysis solution (pH8.0), vortex mix for 5 minutes, centrifuge at 12000rpm for 5 minutes, and discard the supernatant. Repeat adding 500ul of red blood cell lysis solution, vortex mix for 5 minutes, centrifuge at 12000rpm for 5 minutes, and discard the supernatant.

[0028] The precipitate was vortexed for 5 minutes, 50 μL of nuclear lysis solution was added, vortexed for 5 minutes, and placed in a 56°C metal bath for 15 minutes. The sample was removed from the water bath, and 135 μL of protein clearing solution was added. Vortexed thoroughly and incubated at 4°C for 30 minutes.

[0029] Centrifuge at 12000rpm for 5 minutes at room temperature, and pipette the supernatant (about 200μL) into a new centrifuge tube. Add 500μL of ice ethanol and shake the centrifuge tube repeatedly until the white DNA precipitate is precipitated. Centrifuge at 12000rpm for 2 minutes at room temperature and discard the supernatant. Add 75% ethanol to the DNA precipitate, rinse once, centrifuge at 12000rpm for 3 minutes at room temperature, discard the supernatant, and place at room temperature to evaporate the remaining ethanol. Finally, add 50μL TE (pH8.0) and dissolve the DNA at 4℃ overnight.

[0030] The extracted DNA was subjected to agarose gel electrophoresis, and the purity and concentration of the DNA were detected by ultraviolet spectrophotometer at 260nm and 280nm.

[0031] 2. Gene sequencing to find the mutation site of FUT1 gene

[0032] PCR amplification of target fragments: reaction conditions and reaction system:

[0033] (1) PCR reaction conditions: pre-denaturation at 95°C for 10 min; 10 cycles of 94°C for 60 s, 63°C for 90 s, and 72°C for 60 s; 25 cycles of 94°C for 60 s, 61°C for 90 s, and 72°C for 60 s; extension at 72°C for 10 min, and incubation of the amplified product at 10°C.

[0034] (2) Reaction system: (TAKARA Ex-Taq polymerase)

[0035]

[0036]

[0037] This reaction system was used to perform an amplification reaction between the proband's genomic DNA template and the above-mentioned FUT1 primers.

[0038] PCR product sequencing: The PCR product was sequenced using the conventional Sanger sequencing method. Multiple sequencing results showed that this polymorphic gene was not caused by amplification or sequencing errors, and nt520 T>C was a new mutation site. This mutation did not exist in the following four databases: Single Nucleotide Polymorphism Database (ftp: / / ftp.ncbi.nih.gov / snp / database / ), 1000 Genomes Project (ftp: / / ftp-tracncbi.nihg ov / 1000genomes / ftp / ), Hapmap 8 Database (http: / /

[0039] / hapmap.ncbi.nlm.nih.gov / ) and Yanhuang database (http: / / yh.genomics.org.cn / ), indicating that the variant is very rare and causes a decrease in the activity of α-1,2-fucosyltransferase in the proband, leading to the Bombay-like type.

[0040] In this study, the family members of the proband were collected, and their peripheral blood genomic DNA was extracted. The above DNA was used as a template and FUT1-4F and FUT1-4R primers were used to amplify the PCR target fragment. The conventional Sanger sequencing method used the above primers to directly sequence the PCR product, and it was found that the gene heterozygosity described in the present invention, namely nt520CT, existed in the 4th exon of the FUT1 gene of the H blood type system of the mother of the proband. The haplotype sequencing of the 4th exon of the FUT1 gene was continued for the mutation, and it was found that the FUT1 gene coding region c.520T>C, and the encoded amino acids were changed from phenylalanine (Phe) to leucine (Leu), resulting in changes in the activity of α-1,2 fucosyltransferase and weak expression of H antigen, which was completely consistent with the proband.

[0041] Through the above analysis, it is proved that the primers designed by the inventors can be used to detect whether the FUT1 gene of the individual H blood type system has a Bombay-like type caused by the mutation of the 520th base from the start codon from T to C. The specific primers and detection method can achieve specific, sensitive and rapid detection of the Bombay-like type, which is of great significance for improving the safety of clinical blood transfusion.

Claims

1. A SNP site causing mutation in the FUT1 gene, characterized in that: The SNP site is located at the 520th position of the nucleic acid fragment with the sequence of SEQ ID NO: 1, and its base changes from T to C.

2. Use of the SNP site described in claim 1 as a detection target in the preparation of a product for detecting Bombay blood group.

3. The use according to claim 2, characterized in that The product is a PCR amplification sequencing detection kit.

4. A PCR amplification sequencing detection kit for detecting Bombay-like blood group, characterized in that: The kit comprises a primer pair for amplifying a nucleic acid fragment comprising the SNP site of claim 1.

5. The kit according to claim 4, characterized in that The upstream primer of the primer pair has a sequence of SEQ ID NO: 2, and the downstream primer has a sequence of SEQ ID NO: 3.

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