SNP (Single Nucleotide Polymorphism) marker for detecting RHD blood group mutation site and application

Abstract

The invention discloses an SNP marker for detecting an RHD blood group mutation site and application, and relates to the technical field of gene diagnosis products, the SNP marker is located on a fourth exon of an RHD gene coding region for coding a D antigen on the surface of red blood cells, a 519th base from an initiation codon is mutated, and C is mutated into G; the 173rd amino acid is converted into a termination codon from tyrosine. An effective RHD blood group gene diagnosis approach is provided, and the application effect shows that the SNP site and the detection primer provided by the invention can be effectively used for rapid detection of RHD variant gene mutation sites of blood transfusion patients and blood station blood donors.

Description

Technical Field

[0001] The present invention relates to the technical field of gene diagnosis products, and in particular to a SNP marker for detecting RHD blood type mutation sites and its application. Background Art

[0002] The RHD blood group system, also known as the Rh blood group system, is a blood group system used to classify human red blood cells. The most famous antigen in the Rh blood group system is the D antigen, so the Rh blood group commonly referred to refers to the RHD blood group. The Rh blood group system was discovered by scientists Landsteiner and Wiener in 1940. Rh is the abbreviation of Rhesus (rhesus monkey) because this blood group system was first discovered in rhesus monkeys. The Rh blood group system contains more than 50 different antigens, among which the D antigen is the most important. Depending on the presence or absence of the D antigen, the Rh blood type is divided into Rh positive (RHD positive) and Rh negative (RHD negative). Inheritance of Rh blood type: Rh blood type is inherited from parents to children. If both parents are Rh positive, the children may also be Rh negative, but this is rare. If one parent is Rh negative, the children may also be Rh negative.

[0003] Rh-negative blood type is relatively rare in my country. Because its preciousness is similar to that of pandas, it is called "panda blood". In my country, the proportion of Rh-negative blood type is about 0.2%-0.5%, which is much lower than that of Rh-positive blood type. During blood transfusion, if the recipient is Rh-negative and Rh-positive blood is transfused, hemolytic reaction may occur. Therefore, for patients with Rh-negative blood type, Rh-negative blood is preferred for transfusion. If the pregnant woman is Rh-negative and the fetus is Rh-positive, the immune system of the pregnant woman may produce antibodies to attack the red blood cells of the fetus, leading to hemolytic disease of the newborn. Therefore, Rh-negative pregnant women need to pay special attention during pregnancy and, if necessary, receive anti-D immunoglobulin injections to prevent hemolytic disease of the newborn.

[0004] In summary, the RHD blood type system is a classification method for human red blood cells. Panda blood (Rh-negative blood type) has attracted much attention due to its rarity and particularity during blood transfusion. During medical treatment and pregnancy, special measures need to be taken for patients and pregnant women with Rh-negative blood type. Based on this, the present invention proposes a SNP marker for detecting RHD blood type mutation sites and its application. Summary of the invention

[0005] The present invention provides a SNP marker for detecting RHD blood type mutation sites and its application, which can accurately confirm the RHD mutation blood type gene of red blood cells, thereby making up for the deficiencies of the prior art.

[0006] According to one aspect of the present disclosure, there is provided an SNP marker for detecting RHD blood group mutation sites. The SNP marker is located in the fourth exon of the coding region of the RHD gene encoding the D antigen on the red blood cell surface. The base at the 519th position from the start codon has mutated from C to G; the 173rd amino acid has changed from tyrosine to a stop codon.

[0007] A primer pair for performing a PCR reaction in RHD blood group gene detection to detect the presence of the SNP marker. The sequence information of the primers is as follows: Primer RHD-4F: 5’-CTATCAGGGCTTGCCCCG-3’, Primer RHD-4R: 5’-CTTCAGACACCCAGGGGAACT-3’.

[0008] A primer for gene sequencing. The sequence information of the primer is as follows: RHD-4F: 5’-CTATCAGGGCTTGCCCCG-3’. Primer RHD-4F is the primer used for forward sequencing of the SNP marker.

[0009] An application of a primer pair in the preparation of a product for detecting RHD variant blood groups of red blood cells.

[0010] In a possible implementation, the product is a PCR amplification reaction kit.

[0011] A kit for detecting RHD blood group, which contains the primer pair.

[0012] An application of an SNP marker, which is the application of the SNP marker in detecting RHD blood group mutations of red blood cells, including: Performing PCR amplification on the blood sample DNA to be detected using the primer pair to obtain an amplification product; Sequencing the amplification product to obtain a sequencing result; Performing genotype analysis according to the sequencing result to determine whether the SNP marker exists in the sample to be detected.

[0013] Compared with the prior art, the beneficial effects of the present invention are: An SNP marker for detecting RHD blood group mutation sites and its application in embodiments of the present disclosure, an SNP marker for detecting silent genes caused by RHD gene mutations. The SNP marker has a C>G mutation at the 519th base from the start codon in the coding region of RHD gene Exon4, resulting in the 173rd amino acid being changed from tyrosine to a stop codon; thus affecting the expression of the variant RHD gene and becoming a silent gene without protein transcription function. The present invention provides a new use for the RHD variant gene, thus providing an effective way for rapid gene diagnosis, gene screening and genetic counseling for hemolytic transfusion reactions and severe hemolytic disease of the newborn caused by blood transfusion in the RHD blood group system. The application results show that the SNP locus and detection primers of the gene provided by the present invention can be effectively used for rapid detection of new RHD variant gene mutation sites in the peripheral blood of clinical blood transfusion patients and blood donors.

[0014] By means of molecular biology, an RHD gene detection system is established and applied to clinical blood transfusion work, which helps to accurately detect RHD-negative individuals, helps to reduce the production of irregular antibodies in blood recipients, helps to prevent and monitor prenatal irregular antibodies, and helps to reduce the occurrence of hemolytic disease of the newborn. Brief Description of the Drawings

[0015] Figure 1 Shows the direct sequencing map of a normal RHD individual in an embodiment of the present disclosure.

[0016] Figure 2 Shows the direct sequencing map of a proband in an embodiment of the present disclosure, carrying one RHD mutant gene and one RHD gene with complete deletion. Detailed Embodiments

[0017] The following will describe in detail various exemplary embodiments, features and aspects of the present disclosure with reference to the accompanying drawings. The same reference numerals in the drawings denote elements having the same or similar functions. Although various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0018] The special term "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0019] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present disclosure can also be implemented without some specific details. In some instances, methods, means, elements and circuits well-known to those skilled in the art are not described in detail so as to highlight the gist of the present disclosure.

[0020] Figure 1 Direct sequencing map of a normal RHD individual showing an embodiment of the present disclosure. Figure 2 Direct sequencing map of a proband showing an embodiment of the present disclosure, carrying one RHD mutant gene and one RHD gene with complete deletion.

[0021] The applicant sequenced the RHD gene from 309 individuals with serological tests showing RHD negative or variant phenotypes, and found a new SNP mutation resulting in an RHD negative gene. Although this individual has one RHD gene, its phenotype is RHD negative, thus contributing to the present invention.

[0022] The RH gene is located in the region of chromosome 1p34.3 - 1p36.1 and can be transcribed into an mRNA of 2837 bp (NCBI accession number: NM_016124.4), which is ultimately translated into a protein composed of 417 amino acids. It is composed of the RHD gene (encoding the RHD antigen) and the RHCE gene (encoding the RHC, c, E, and e antigens) arranged in tandem. The RHD gene and the RHCE gene have a high degree of structural homology, both consisting of 10 exons and 9 introns. Among them, the 8th exon of both is exactly the same, and the exons 3, 4, 5, 7, 9 and the intron 4 have relatively large differences. Therefore, by using molecular biology methods to establish an RHD gene detection system and applying it to clinical blood transfusion work, it helps to accurately detect RHD negative individuals, helps to reduce the production of irregular antibodies in blood recipients, helps to prevent and monitor irregular antibodies during pregnancy, and helps to reduce the occurrence of hemolytic disease of the newborn.

[0023] Regarding the SNP markers involved in the present invention, the applicant explains as follows: SNP (single nucleotide polymorphism, SNP, that is, single nucleotide polymorphism) refers to DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. The polymorphism exhibited by SNPs only involves the variation of a single base, and the manifestations include transition, transversion, insertion, and deletion, etc.

[0024] The present invention will be described in detail below with reference to embodiments.

[0025] Screening of SNP markers: 1. Extract genomic DNA from peripheral blood: On the basis of compliance with relevant national policies and with the consent of the sampling subjects, 2-5 mL of RHD-negative or variant peripheral venous blood is drawn and placed into an EDTA anticoagulation tube for storage at -80 °C for future use; after the frozen EDTA anticoagulated blood is melted at room temperature, 500 μL is taken and placed in a centrifuge tube, and an equal volume of TE (a commonly used buffer solution composed of Tris (a tris(hydroxymethyl)aminomethane) and EDTA (an ethylenediaminetetraacetic acid), where Tris acts as a pH buffer and EDTA acts as a metal ion chelating agent) is added. The pH of TE is 8.0. Mix well, centrifuge at 10,000 revolutions per minute at 4 °C for 10 minutes, and discard the supernatant.

[0026] Add 180 μL of TE, 20 μL of SDS (10%) (SDS is the abbreviation of sodium dodecyl sulfate in biology. SDS is an anionic detergent. As a denaturing agent and solubilizing reagent, it can break the hydrogen bonds within and between molecules, unfold the molecules, and thus destroy the secondary and tertiary structures of protein molecules), and 8 μL of proteinase K (10 mg / ml), mix well, and place in a 37 °C water bath overnight. Take out the sample from the water bath and centrifuge briefly to precipitate the sample. Add an equal volume of Tris-saturated phenol (about 300 μL) to the reaction tube, mix well, centrifuge at 10,000 revolutions per minute at room temperature for 10 minutes, and aspirate the supernatant (about 300 μL) into a new centrifuge tube. Repeat the phenol extraction once and aspirate the supernatant into a new centrifuge tube.

[0027] Add an equal volume of Tris-saturated phenol:chloroform mixture (150 μL each of phenol and chloroform), mix well, centrifuge at 10,000 revolutions per minute at room temperature for 10 minutes, and transfer the supernatant to a new centrifuge tube.

[0028] Add an equal volume of Tris-saturated phenol:chloroform:isoamyl alcohol mixture (100 μL each of phenol, chloroform, and isoamyl alcohol), mix well, centrifuge at 10,000 revolutions per minute at room temperature for 10 minutes, and transfer the supernatant to a new centrifuge tube.

[0029] Add 1 / 10 volume of 3 mol / L, pH 5.2 sodium acetate (about 30 μL) and 2 volumes of pre-cooled 100% ethanol, mix gently, and white flocculent precipitates can be seen. Centrifuge at 10,000 revolutions per minute at room temperature for 10 minutes to precipitate the DNA at the bottom of the tube, and discard the supernatant.

[0030] Add 70% ethanol to the DNA precipitate, rinse once, centrifuge at 7,000 revolutions per minute at room temperature for 5 minutes, discard the supernatant, place at room temperature to volatilize the remaining ethanol, and finally add 50 μL of TE (pH 8.0) to dissolve the DNA at 4 °C overnight.

[0031] Perform agarose gel electrophoresis on the extracted DNA and use an ultraviolet spectrophotometer to measure the absorbance at 260 nm and 280 nm to detect the purity and concentration of the DNA.

[0032] 2. Searching for mutations in the RHD gene of the proband by direct sequencing PCR amplification of the target fragment: Reaction conditions and reaction system: (1) PCR reaction conditions: 95°C for 5 minutes; 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 60 seconds, 35 cycles; 72°C for 5 minutes.

[0033] (2) Reaction system: (Fast start Taq polymerase from Onelambda company) 2×mix buffer 10µl RHD-4F primer (2.5µM) 2µl RHD-4R primer (2.5µM) 2µl Fast start Taq polymerase 1µl (1.25U) Genomic DNA template (50ng / ul) 1µl dH 2 O 4 µl Total 20µl Among them, 2×mix buffer is the reaction solution premixed in the PCR reaction, and Fast start Taq polymerase is the fast start polymerase, dH 2 O is single-distilled water.

[0034] Apply this reaction system to perform amplification reactions of the genomic DNA template of each RHD-negative or variant individual with this RHD primer.

[0035] Direct sequencing of the PCR product: Use the conventional Sanger sequencing method to sequence the above PCR product. The primer used is RHD-4F: 5’-CTATCAGGGCTTGCCCCG-3’. This proband carries one RHD mutant gene and one RHD full deletion gene. A mutation was found at the fourth exon of the RHD gene in this RHD variant individual, with a C>G mutation at the 519th base starting from the start codon (such as Figure 2As shown in Figure 1 ), the 173rd amino acid was converted from tyrosine to a stop codon, which affected the expression of the RHD gene and formed a silent gene. Although the proband expressed a complete RHD gene, the serological phenotype was RHD-negative. Multiple sequencing results showed that this mutation site was not introduced due to amplification or sequencing errors, but should be a new mutation. This mutation does not exist in the following four databases: single nucleotide polymorphism database (ftp: / / ftp.ncbi.nih.gov / snp / database / ), 1000 Genomes Project (ftp: / / ftp-trace.ncbi.nih.gov / 1000genomes / ftp / ), Hapmap8 database (http: / / hapmap.ncbi.nlm.nih.gov / ), and Yanhuang database (http: / / yh.genomics.org.cn / ), indicating that this mutation is very rare. The 519th base from the start codon of Exon4 mutated from C to G, resulting in the 173rd amino acid being replaced by tyrosine to form a stop codon (p.Tyr173STOP). With the appearance of the stop codon, although the proband's RHD gene is fully expressed, the stop codon caused by the mutation appears, and the proband's RHD phenotype is negative (D antigen is not expressed on the surface of red blood cells).

[0036] This mutation causes a conformational change in the D antigen protein, leading to the RHD variant. However, this mutation was not found in the peripheral blood genomic DNA samples of 309 RHD-positive individuals. The above analysis proves that this method can accurately determine the RHD gene, thereby more accurately determining the RHD blood type of the person being tested, which is of great significance for the formulation of blood transfusion policies for patients.

[0037] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A SNP marker for detecting RHD blood type mutation sites, characterized in that: The SNP marker is located in the fourth exon of the RHD gene coding region encoding the D antigen on the red blood cell surface. The 519th base from the start codon mutated from C to G; the 173rd amino acid changed from tyrosine to a stop codon.

2. A primer pair, characterized in that: The primer pair is used for PCR reaction in RHD blood type gene detection to detect whether the SNP marker in claim 1 exists. The sequence information of the primers is as follows: Primer RHD-4F: 5'-CTATCAGGGCTTGCCCCG-3', Primer RHD-4R: 5'-CTTCAGACACCCAGGGGAACT-3'.

3. A primer for gene sequencing, characterized in that: The primer sequence information is as follows: RHD-4F: 5'-CTATCAGGGCTTGCCCCG-3', and the primer RHD-4F is a primer used in forward sequencing of the SNP marker described in claim 1.

4. An application of a primer pair, characterized in that: The primer pair is the primer pair in claim 2, and the primer pair is used in the preparation of a product for detecting RHD variant blood type of red blood cells.

5. The use of a primer pair according to claim 4, characterized in that: The product is a PCR amplification reaction kit.

6. A kit for detecting RHD blood type, characterized in that: The kit comprises the primer pair according to claim 2.

7. An application of a SNP marker, characterized in that: The SNP marker is the SNP marker in claim 1, and the application is the application of the SNP marker in detecting red blood cell RHD blood type mutation, including: Using the primers described in claim 2 to perform PCR amplification on the blood sample DNA to be tested to obtain an amplification product; Sequencing the amplified product to obtain a sequencing result; Perform genotype analysis based on the sequencing results to determine whether the sample to be tested contains the SNP marker described in claim 1.

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