SNP site of jk(a+w) blood group causing immune hemolytic transfusion reaction, identification kit, identification method and application

Abstract

This invention provides a SNP site, identification kit, identification method, and application for the Jk(a+w) blood type, which triggers immune hemolytic transfusion reactions. The SNP site is the c.613C>T mutation at position 613, starting from the start codon of the SLC14A1 gene coding region. In the proband, the c.613C>T mutation was detected in one allele, while the other allele carried other known inactivating mutations. The allele carrying the c.613C>T mutation resulted in reduced expression of the Kidd protein in the proband's erythrocytes; the other allele, carrying other inactivating mutations, did not express the Kidd protein. The combined effect of these two mutated alleles resulted in the Jk(a+w) phenotype. This provides a genetic variation basis for the establishment of rare blood type banks and the assurance of transfusion compatibility.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a SNP site, identification kit, identification method, and application of the Jk(a+w) blood type that can trigger immune hemolytic transfusion reactions. Background Technology

[0002] The Kidd blood group system is one of the important blood group systems for human red blood cells (International Society of Blood Transfusion (ISBT) number 009). The Kidd blood group system contains three core antigens: Jk... a (009001), Jk b (009002) and high-frequency antigen Jk3 (009003), wherein Jk a With Jk b As codominant allele products, they combine to form three common phenotypes: Jk(a+b-), Jk(a-b+), and Jk(a+b+). The Jk(a+w) phenotype is characterized by the expression of Jk on the surface of erythrocytes. a The antigen expression intensity is significantly reduced, and a weak phenotype with altered antigenic epitopes may exist. Therefore, its clinical identification requires refined serological methods and is highly prone to false negatives. Its frequency in the population is far lower than the conventional Jk(a+b-) phenotype, making it a weakly expressed subtype requiring close attention. In the field of clinical medicine, the Kidd blood group system has irreplaceable value: on the one hand, Kidd antibodies (especially anti-Jk antibodies)... a 、anti-Jk b Anti-Jk3 antibodies possess a "stealthy" characteristic—their antibody titer decays rapidly, leading to an extremely high false negative rate in routine crossmatching tests. False negatives can trigger severe delayed hemolytic transfusion reactions, and even fatal acute hemolytic events. Individuals with the Jk(a+w) phenotype are more prone to developing low-affinity anti-Jk antibodies due to low red blood cell Jka antigen density and potential epitope alterations. a Antibodies are frequently detected and are often overlooked in routine screening, significantly increasing the risk of transfusion complications. Furthermore, these antibodies are a significant contributing factor to neonatal hemolytic disease. Additionally, Kidd blood type status is associated with kidney transplant rejection. Accurate identification of the Jk(a+w) phenotype can optimize donor-recipient matching strategies and reduce transfusion reactions.

[0003] The genetic basis of this system is regulated by the SLC14A1 gene located on chromosome 18, following Mendelian codominant inheritance. The encoded Kidd glycoprotein is essentially a urea transporter. The molecular mechanisms of the Jk(a+w) phenotype are mostly related to point mutations in the SLC14A1 gene, leading to reduced Jka antigen expression efficiency and alterations in some antigenic epitopes. The mutation frequency of the SLC14A1 gene causing the Jk(a+w) phenotype is extremely low; currently, the International Society of Blood Transfusion (ISBT) has only 12 Jk(a+w) alleles. Accurately elucidating the genetic variation basis of the Jk(a+w) phenotype is of great significance for weak phenotype identification, rare blood type bank establishment, and accurate blood matching in transfusion medicine. It can effectively reduce the incidence of adverse transfusion reactions caused by missed detection of weak antigens and ensure transfusion safety.

[0004] Genotyping technology, through direct analysis of the SLC14A1 gene sequence, can accurately determine allelic composition and clarify blood type status at the molecular level, offering higher accuracy and specificity. Currently, the main Kidd blood typing genotyping technologies include PCR-SSP, RT-qPCR, HRM, PCR-SBT, and next-generation short-read sequencing; however, these technologies have significant technical bottlenecks. Methods such as PCR-SSP and RT-qPCR can only design probes or primers for known mutation sites and cannot detect unknown variations. They are also powerless against structural variations such as large deletions and splice site mutations in the SLC14A1 gene. PCR-SBT requires multiple amplifications and sequencing, which is labor-intensive. Second-generation short-read sequencing has a short read length (usually 100-300 bp), which is difficult to cross repetitive sequences and complex structural regions in the SLC14A1 gene. It requires fragment splicing to construct the full sequence, which is prone to assembly errors and leads to inaccurate haplotype phase determination. The codominant genetic characteristics of the Kidd blood group system require precise differentiation of allele status on homologous chromosomes. Haplotype resolution errors will directly affect the reliability of typing results. The emergence of third-generation long-read sequencing technologies (such as PacBio and Oxford Nanopore technologies) has completely broken through the above technical limitations. Its advantages in full-length sequencing and haplotype typing of the SLC14A1 gene are particularly prominent. However, the existing third-generation detection technologies lack exons 1 and 2 of this gene.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a substance for detecting SNP sites of the SLC14A1 gene in the identification of Jk(a+w) blood types or the preparation of Jk(a+w) blood type identification products, so as to at least solve one of the technical problems existing in the prior art.

[0007] The second objective of this invention is to provide a kit for Jk(a+w) blood typing.

[0008] The third objective of this invention is to provide a method for identifying Jk(a+w) blood type.

[0009] The fourth objective of this invention is to provide the substance containing the SNP site of the SLC14A1 gene, or the kit described above, for use in assessing the risk of immune hemolytic transfusion reactions or in preparing products for assessing the risk of immune hemolytic transfusion reactions.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the application of a substance for detecting SNP sites of the SLC14A1 gene in Jk(a+w) blood typing or in the preparation of Jk(a+w) blood typing products, wherein the SNP site includes the c.613C>T mutation at position 613 starting from the start codon of the coding region of the SLC14A1 gene.

[0011] In a second aspect, the present invention provides a kit for Jk(a+w) blood typing, comprising a substance for detecting SNP sites of the SLC14A1 gene; SNP sites include SNP site a, which is the cause of Jk. a Mutations that reduce antigen expression, leading to Jk a Mutations that reduce antigen expression include the c.613C>T mutation at position 613, starting from the start codon in the coding region of the SLC14A1 gene.

[0012] Furthermore, the SNP site also includes SNP site b, which is the site that leads to Jk. a Mutations that weaken or inactivate antigen expression, with SNP sites a and b located on a pair of alleles on homologous chromosomes.

[0013] Furthermore, the substance for detecting SNP sites of the SLC14A1 gene includes at least one of the following: reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing.

[0014] Furthermore, the reagents for nucleic acid amplification include a first reagent for amplification primer pairs and sequencing primer pairs for amplifying the SLC14A1 gene and / or a second reagent for amplification primer pairs for amplifying the SLC14A1 gene; the amplification fragments of the SLC14A1 gene amplification primer pairs at least cover exons 3 to 10 of the SLC14A1 gene.

[0015] Furthermore, the amplification primer pairs and sequencing primer pairs for amplifying the SLC14A1 gene in the first reagent are independently selected from the nucleotide sequences of the forward and reverse primers, as shown in SEQ ID NO.1~SEQ ID NO.14; Preferably, the amplification primer pairs for amplifying the SLC14A1 gene in the second reagent are independently selected from the forward and reverse primers, and their nucleotide sequences are shown in SEQ ID NO.15~SEQ ID NO.18.

[0016] Thirdly, this invention provides a method for identifying Jk(a+w) blood type, including obtaining the base type of the coding region of the SLC14A1 gene in the sample to be tested, and if the presence of one allele leads to Jk... a SNP sites with weakened antigen expression and the presence of another allele lead to Jk a If the SNP site shows weakened or inactivated antigen expression, the blood type of the sample to be tested is determined to be Jk(a+w). The cause of Jk a The SNP sites with weakened antigen expression include the c.613C>T mutation at position 613, starting from the start codon in the coding region of the SLC14A1 gene.

[0017] Further, this involves amplifying the SLC14A1 gene fragment of the test sample using the first reagent described above, then detecting SNP sites in the amplification product, performing haplotype sequencing, and if a c.613C>T mutation exists in one allele, and a mutation causing Jk is also present in another allele... a If the antigen expression is weakened or inactivated by a mutation, the sample to be tested is determined to be of blood type Jk(a+w); Alternatively, this may involve using primers from the second reagent to perform segmented cascade amplification of the SLC14A1 gene fragment in the test sample, followed by haplotype sequencing. If a c.613C>T mutation is present in one allele, and a mutation causing Jk is present in another allele, then... a If the antigen expression is weakened or inactivated by a mutation, the sample to be tested is determined to be of blood type Jk(a+w).

[0018] Furthermore, inactivating mutations include the c.342-1G>A inactivating mutation; Attenuated mutations include c.613C>T mutations and / or c.130G>A mutations.

[0019] Fourthly, the present invention provides the use of the above-mentioned substance for detecting SNP sites of the SLC14A1 gene, or the above-mentioned kit, in assessing the risk of immune hemolytic transfusion reactions or in preparing products for assessing the risk of immune hemolytic transfusion reactions. Preferably, the reaction comprises Jka or Jk b Delayed hemolytic transfusion reaction caused by antigen-antibody incompatibility.

[0020] This invention reveals the SNP site for the Jk(a+w) blood type in immune hemolytic transfusion reactions. This SNP site is the c.613C>T mutation at position 613 of the start codon in the coding region of the SLC14A1 gene. According to the reference sequence (NCBI accession number NG_011775.4), this SNP site is located in exon 6 of the SLC14A1 gene coding region. In the proband of this invention, the c.613C>T mutation was detected in one allele, which resulted in reduced expression of the Kidd protein in the proband's erythrocytes; while the other allele carried other inactivating mutations and did not express the Kidd protein. The combined effect of these two mutated alleles resulted in the Jk(a+w) phenotype. This mutation provides a genetic basis for the establishment of rare blood type banks and the assurance of transfusion compatibility. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The Sanger sequencing sequence map provided in this embodiment of the invention is shown, wherein the reference sequence number is NG_011775.4; Figure 2 This is a schematic diagram of the full-length segmented cascade amplification of the SLC14A1 gene provided in Example 1 of the present invention, wherein amplified fragment 1 and amplified fragment 2 are represented by light blue lines; exons 1-10 are represented by gray. Figure 3 The electrophoresis results of the full-length PCR amplification products of the SLC14A1 gene provided in Example 1 of this invention are shown below. From left to right, wells 1-3 are the PCR amplification products of fragment 1 of the DNA samples from the proband, random blood donor 1, and blood donor 2, respectively; wells 4-6 are the PCR amplification products of fragment 2 of the DNA samples from the proband, random blood donor 1, and blood donor 2, respectively; and well 7 is the DNA molecular marker DL 15000 bp. Figure 4The haplotype results of c.130G>A from third-generation long sequencing provided in Example 1 of this invention are shown. The sequence of phase 1 is haplotype allele 1, corresponding to the part crossed by the red line; the sequence of phase 2 is haplotype allele 2, corresponding to the part crossed by the blue line. Figure 5 The haplotype results of c.342-1G>A from third-generation long sequencing provided in Example 1 of this invention are shown. The sequence of phase 1 is haplotype allele 1, corresponding to the part crossed by the red line; the sequence of phase 2 is haplotype allele 2, corresponding to the part crossed by the blue line. Figure 6 The haplotype results of c.588A>G from third-generation long sequencing provided in Example 1 of this invention are shown. The sequence of phase 1 is haplotype allele 1, corresponding to the part crossed by the red line; the sequence of phase 2 is haplotype allele 2, corresponding to the part crossed by the blue line. Figure 7 The haplotype results of c.613C>T from third-generation long sequencing provided in Example 1 of this invention are shown. The sequence of phase 1 is haplotype allele 1, corresponding to the part crossed by the red line; the sequence of phase 2 is haplotype allele 2, corresponding to the part crossed by the blue line. Figure 8 The haplotype results of c.838G>A from third-generation long sequencing provided in Example 1 of this invention are shown. The sequence of phase 1 is haplotype allele 1, corresponding to the part crossed by the red line; the sequence of phase 2 is haplotype allele 2, corresponding to the part crossed by the blue line. Figure 9 The serological verification results of the proband provided in Embodiment 2 of the present invention, wherein test tube 1 in A contains the proband's red blood cells and Immuclur's anti-Jk... a Antibody saline agglutination results: Tube 2 contains proband red blood cells and Baorui Sankun anti-Jk antibodies. a Antibody saline agglutination; test tubes 3 and 4 contain red blood cells and anti-Jk antibodies. b All antibodies showed no agglutination; B represents the agglutination microscopy result obtained using the test tube saline method. Figure 10 The above is an APC fluorescence volcano diagram of the empty vector, wild-type JKa gene, and different mutant cell lines expressing the gene in vitro, provided in Example 3 of the present invention. The empty vector cell line is used as a negative control, and the red box represents the expression of positive cells. Figure 11 The empty vector, wild-type JKa gene, and different mutant cell lines for in vitro expression of JKa provided in Example 3 of this invention. a The expression of the antigen is shown in Figure A, where A is a single-parameter fluorescence intensity histogram and B is a statistical graph of average fluorescence intensity. Detailed Implementation

[0023] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0024] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention provides the application of a substance for detecting SNP sites in the SLC14A1 gene in Jk(a+w) blood typing or in the preparation of Jk(a+w) blood typing products, wherein the SNP site includes the c.613C>T mutation at position 613 starting from the start codon in the coding region of the SLC14A1 gene.

[0027] The c.613C>T mutation is a pathogenic SNP site for the Jk(a+w) blood type. This SNP site is the c.613C>T mutation at position 613 of the start codon in the coding region of the SLC14A1 gene. According to the reference sequence (NCBI accession number NG_011775.4), this SNP site is located in exon 6 of the coding region of the SLC14A1 gene. Five mutation sites were detected in the prior witness of this invention. Among them, the c.613C>T mutation is present in one allele, which is located in the same allele haplotype as the detected c.130G>A and c.588A>G mutations; another allele carries an inactivating mutation (c.342-1G>A), which is located in the same haplotype as the detected c.588A>G and c.838G>A. c.130G>A is a reported Jk(a+w) mutation; c.588A>G is a synonymous mutation; c.838G>A is a mutation that distinguishes Jk... a and Jk b The polymorphic sites are unrelated to the Jk(a+w) phenotype. The c.613C>T mutation, present in haplotype 1, combined with the c.130G>A mutation, results in a very weak Jk(a+w) phenotype. Haplotype 2 contains the c.342-1G>A inactivating mutation. The combined effect of these two haplotypes ultimately leads to extremely weakened expression of Kidd protein in the proband's erythrocytes, resulting in a very weak Jk(a+w) phenotype. In routine serological testing, this can easily lead to missed antigen detection and misdiagnosis as the Jk(ab-) phenotype. In vitro validation demonstrates a necessary direct causal relationship between the c.613C>T mutation and the Jk(a+w) phenotype. When individuals with this blood type are recipients, receiving normal Jk blood from ordinary donors... a + or Jk b During red blood cell transfusion, the immune system may recognize it as a "foreign antigen," leading to the production of IgG antibodies. Upon subsequent transfusions, these antibodies rapidly bind to the transfused red blood cells, activating complement or being cleared by the mononuclear phagocytic system, resulting in delayed hemolytic transfusion reaction (DHTR). Furthermore, if a donor is misidentified as having the Jk(ab-) phenotype and transfused to a recipient with the Jk(ab-) phenotype, it will stimulate the recipient to produce antibodies, inducing a hemolytic transfusion reaction. This mutation provides a genetic basis for the establishment of rare blood type banks and the assurance of transfusion compatibility, and can be used for Jk(a+w) blood typing.

[0028] According to another aspect of the present invention, a kit for Jk(a+w) blood typing is also provided, comprising a substance for detecting SNP sites of the SLC14A1 gene; the SNP site includes SNP site a, which is a precursor to Jk(a+w) blood type. a Mutations that reduce antigen expression, leading to Jk aMutations that reduce antigen expression include the c.613C>T mutation at position 613, starting from the start codon in the coding region of the SLC14A1 gene.

[0029] By specifically detecting the first and second SNP sites on a pair of alleles located on homologous chromosomes in the SLC14A1 gene, Jk can be accurately identified at the DNA level. a Weakly expressed alleles avoid the risk of misdiagnosis in serological testing due to low antibody titers, weak antigen expression, or cross-reactivity; on the other hand, it confirms that the individual is Jk. a The combination of weakly expressed alleles and loss-of-function alleles confirms a very weak Jk(a+w) phenotype, improving diagnostic reliability. The kit provides a standardized tool for building rare blood type banks and can be used for large-scale population screening to identify individuals carrying the first and second SNP loci, prevent hemolytic transfusion reactions, and reduce medical risks.

[0030] The kit can be further integrated with other common inactivated SNP sites as second SNP sites as needed. In some specific embodiments, the SNP sites also include SNP site b, which is the site that causes Jk. a Mutations that weaken or inactivate antigen expression, SNP sites a and b are located on a pair of alleles on homologous chromosomes.

[0031] In some specific embodiments, the substance for detecting the SNP site of the SLC14A1 gene includes at least one of the following: reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing.

[0032] Specifically, substances used to detect SNP sites include, but are not limited to, primers, enzymes for nucleic acid amplification reactions, fluorescent labels, buffer reagents, dNTPs, salts, etc. Depending on the specific detection method, those skilled in the art can select the above-mentioned reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing based on the methods described in general and more specific textbooks, references, process manuals, product instructions, and standard documents. This invention does not impose any limitations on these selections.

[0033] In some specific embodiments, the reagents for nucleic acid amplification include a first reagent for amplification primer pairs and sequencing primer pairs for amplifying the SLC14A1 gene and / or a second reagent for amplification primer pairs for amplifying the SLC14A1 gene; the amplification fragments of the SLC14A1 gene amplification primer pairs at least cover exons 3 to 10 of the SLC14A1 gene.

[0034] In some specific embodiments, the amplification primer pair and sequencing primer pair for amplifying the SLC14A1 gene in the first reagent are independently selected from the nucleotide sequences of the forward primer and the reverse primer, as shown in SEQ ID NO.1~SEQ ID NO.14.

[0035] In some specific embodiments, the amplification primer pairs for amplifying the SLC14A1 gene in the second reagent are independently selected from the forward and reverse primers, and their nucleotide sequences are shown in SEQ ID NO.15~SEQ ID NO.18.

[0036] According to another aspect of the present invention, a method for identifying Jk(a+w) blood type is also provided, comprising obtaining the base type of the coding region of the SLC14A1 gene in the sample to be tested, and if the presence of an allele results in Jk... a SNP sites with weakened antigen expression and the presence of another allele lead to Jk a If the SNP site shows weakened or inactivated antigen expression, the blood type of the sample to be tested is determined to be JK(a+w); the aforementioned causes JK a The SNP sites with weakened antigen expression include the c.613C>T mutation at position 613, starting from the start codon in the coding region of the SLC14A1 gene.

[0037] It should be noted that the Jk(a+w) blood type identification method is not for diagnostic or treatment purposes.

[0038] In some specific implementations, the method includes amplifying the SLC14A1 gene fragment of the test sample using the first reagent described above, then detecting SNP sites in the amplification product, performing haplotype sequencing, and if a c.613C>T mutation exists in one allele, and a mutation causing Jk is also present in another allele, the method will determine the specific mutation. a If the antigen expression is weakened or inactivated by a mutation, the sample is determined to be of blood type Jk(a+w); or, this includes using primers from the second reagent to perform segmented cascade amplification of the SLC14A1 gene fragment of the sample, followed by haplotype sequencing. If a c.613C>T mutation exists in one allele, and a mutation causing Jk is present in another allele, then... a If the antigen expression is weakened or inactivated by a mutation, the sample to be tested is determined to be of blood type Jk(a+w).

[0039] In some specific implementations, inactivating mutations include the c.342-1G>A inactivating mutation; attenuated mutations include the c.613C>T mutation and / or the c.130G>A mutation.

[0040] The above kit can accurately identify individuals with the Jk(a+w) phenotype; it indicates that if the individual is given normally expressed Jk...a + or Jk b +blood, which is highly likely to produce IgG-type anti-Jk a Antibodies; upon re-transfusion, exposure to the corresponding antigen will trigger a delayed hemolytic transfusion reaction (DHTR). Furthermore, if a donor is misidentified as having the Jk(ab-) phenotype and transfused to a recipient with the Jk(ab-) phenotype, it will stimulate the recipient to produce antibodies, inducing a hemolytic transfusion reaction. According to another aspect of the invention, the use of the aforementioned substance for detecting SNP sites of the SLC14A1 gene, or the aforementioned kit, in assessing the risk of immune hemolytic transfusion reactions or in preparing products for assessing the risk of immune hemolytic transfusion reactions is also provided.

[0041] In some specific embodiments, the reaction includes the reaction by Jk a or Jk b Delayed hemolytic transfusion reaction caused by antigen-antibody incompatibility.

[0042] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] Example 1 In this embodiment, a blood donor was collected. The urea screening test revealed that the donor's Kidd blood type might be a variant. The specific procedure was as follows: 20µL of 2% red blood cells and 100µL of 2M urea were added to a U-shaped plate, centrifuged at 800rpm for 1min, and then centrifuged again at 2000rpm for 1min. The results showed that the proband's cells underwent partial hemolysis, and it was preliminarily determined to be the Jk variant phenotype.

[0044] Preliminary identification indicates the blood donor is the proband of the Kidd blood type variant. Genotyping was performed using a first-generation PCR-SBT genotyping method, specifically including the following steps: (1) Prepare human genomic DNA as a template for PCR amplification in subsequent steps. With informed consent from the prior witnesses, 200 µL of whole blood was collected for testing. Genomic DNA was extracted according to the instructions of the Pre-Filled Cartridge Reagent kit (catalog number: 101@S4100-22157, RBC Bioscience), and the concentration and purity of the genomic DNA were determined.

[0045] (2) PCR amplification and direct sequencing method to detect the mutation site of the SLC14A1 gene in the proband. The amplification primer sequences are shown in Table 1. The amplification primers were diluted with pure water to 10 μmol / L.

[0046] Table 1

[0047] Prepare LA Taq enzyme (product number: RR02MQ, TaKaRa), RNase-free H2O, and the PCR amplification template prepared in step (1), and prepare the PCR reaction system according to Table 2.

[0048] Table 2 Amplification reaction system

[0049] The amplification conditions were as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 61℃ annealing for 30 s, 72℃ extension for 1 min 10 s, for 35 cycles; 72℃ extension for 10 min, and incubation of the amplified product at 4℃. After PCR amplification, 2 µL of PCR product from each sample was taken for agarose gel electrophoresis to determine the specificity of the amplified fragment.

[0050] The amplified products were digested with 1 µL of shrimp alkaline phosphatase (product number: 55953500, Roche) and 2 µL of exonuclease I (product number: AL21979A, TaKaRa) at 37°C for 30 min, and then inactivated at 80°C for 15 min.

[0051] The purified PCR product was diluted with 20 µL of pure water and mixed well. Two oligonucleotide sequencing primers (nucleotide sequences shown in SEQ ID No. 1~SEQ ID No. 14) were diluted with pure water to a concentration of 3.2 μmol / L. Sequencing was performed using a BigDyeterminator v3.1 sequencing kit (catalog number: 4336699, ABI). Sequencing was performed using the two sequencing primers.

[0052] Five mutations were found in the coding region of the SLC14A1 gene in the proband, such as Figure 1 As shown, the genotyping results are: c.130G>A heterozygous, c.342-1G>A heterozygous mutation, c.588A>G homozygous, c.613C>T heterozygous mutation, and c.838G>A heterozygous. Among these, c.613C>T is a previously unreported mutation leading to an amino acid alteration in p.Pro205Ser. However, due to the lack of haploid sequence information, the genotype cannot be determined, and further haplotype analysis is required.

[0053] Sanger sequencing: The amplification conditions for the seven exons are optimized and adjusted to be uniform, simplifying the operation.

[0054] (3) Establish a third-generation sequencing technology for amplifying the full-length SLC14A1 gene with long fragments and perform haplotype typing of the proband to determine the genotype.

[0055] ① Primer design: like Figure 2 As shown, the full-length SLC14A1 gene was divided into two segments for folded amplification, covering the range from 5'UTR to exon 10. Long fragment amplification primers were designed, with fragment 1 being 19172bp in length and fragment 2 being 16945bp in length. The primer sequences are as follows. The amplification primers were diluted with pure water to 10 μmol / L.

[0056] Amplified fragment 1: Upstream primer Kidd-F1: 5'-TTTCATTCCCAAAGCAGGCC-3' (SEQ ID NO.15).

[0057] Downstream primer Kidd-R1-: 5'-ACTGCRTGGGACCTAAAGCT-3' (SEQ ID NO.16).

[0058] Amplified fragment 2: Upstream primer Kidd-F2: 5'-CCAGTTCTCCGAACCTCCTT-3' (SEQ ID NO.17).

[0059] Downstream primer Kidd-R2-: 5'-TGCTGCTACACCTGGCTAAT-3' (SEQ ID NO.18).

[0060] ②PCR amplification system: The 20μL reaction system contains 1×buffer, 0.5mM dNTPs, 0.2μM upstream and downstream primers, 1U KOD FX Neo DNA polymerase (Toyobo, Japan), about 50ng DNA template, and H2O to make up to 20 µL.

[0061] ③The PCR reaction program is as follows: 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 68℃ annealing and extension for 15 min, 33 cycles, 68℃ extension for 10 min, and then cooling to 12℃.

[0062] Following the above method, DNA samples from the proband were amplified. Simultaneously, DNA samples from randomly selected blood donors 1 and 2 were amplified to verify whether the constructed long-fragment amplification method for the full-length SLC14A1 gene was applicable to the detection of any sample. The electrophoresis results of the full-length SLC14A1 gene PCR amplification products are shown below. Figure 3As shown, the target band was successfully amplified in all three DNA samples, indicating that the method for amplifying the full-length SLC14A1 gene using the constructed long fragment is stable and reliable, and can stably amplify the full-length SLC14A1 gene sequence. The amplification product from the proband was used for ONT third-generation library construction and sequencing.

[0063] ④ ONT third-generation library construction and sequencing The amplification products were purified using the magnetic bead method with Agencourt® AMPure® XP magnetic beads. The ONT library was constructed using the Multiple Samples DNA Library Prep Kit for Ligation Sequencing from Bio-Tech. The third-generation sequencing platform was the ONT sequencing platform, employing the R10 chip. The full-length haplotype 1 sequence of the SLC14A1 gene is shown in SEQ ID NO. 19, and the full-length haplotype 2 sequence of the SLC14A1 gene is shown in SEQ ID NO. 20.

[0064] Using the generated data, we analyzed the full-length haploid sequence of the SLC14A1 gene and performed genotyping analysis for Kidd blood types. The data analysis mainly consisted of steps including basecalling, barcode splitting, reference sequence alignment, variant site identification, homogeneous sequence clustering, alignment with the ISBT-published genotyping standards, and genotyping output. In this step, the SLC14A1 gene (JK*01) (NG_011775.4) served as the standard reference sequence for alignment. Existing analysis methods could be used, or a custom-designed methodology could be developed based on specific needs.

[0065] The results of the analysis are as follows Figures 4-8 As shown: Haploid 1 (phase 1): c.130G>A, c.588A>G, c.613C>T.

[0066] Haploid 2 (phase 2): c.342-1G>A, c.588A>G, c.838G>A.

[0067] Among them, c.588A>G is a synonymous mutation; c.838G>A is a mutation that distinguishes Jk. a and Jk bThe polymorphic sites are not related to the Jk(a+w) phenotype; c.130G>A causes the Jk(a+w) phenotype; the c.613C>T mutation exists in haplotype 1, and together with the c.130G>A mutation, it forms a very weak Jk(a+w) phenotype; haplotype 2 has a c.342-1G>A inactivating mutation. The two haplotypes together cause extremely weakened expression of Kidd protein in the erythrocytes of the proband, forming a very weak Jk(a+w) phenotype. In routine serological testing, this can easily lead to missed antigen detection and misdiagnosis as the Jk(ab-) phenotype.

[0068] The c.613C>T mutation is a novel, unreported mutation that results in an amino acid alteration of p.Pro205Ser. The allele formed by this novel mutation is not yet included in the ISBT allele database published by the International Society of Blood Transfusion (ISBT), indicating that the allele formed by this mutation is a new allele of the SLC14A1 gene.

[0069] Example 2 Serological Verification This embodiment uses serological testing to further verify the proband's blood type, specifically following these steps: Saline tube method for identifying Jk on the surface of red blood cells a and Jk b Antigen: Add 1 drop of each IgM antibody to a test tube. The antibodies are two different anti-Jk antibodies. a (Batch No.: 630102, Immuchurr; Batch No.: 20251218, Baorui Sankun) and 2 types of anti-Jk b (Batch No.: 631122, Immuchurr; Batch No.: 8000461447, Sanquin); Add 1 drop of 2-5% red blood cell suspension, mix well, centrifuge at 1000g for 30s, gently shake the cell container, and observe the cell aggregation.

[0070] The results of the analysis are as follows Figure 9 As shown: Proband erythrocytes and Immuclur's anti-Jk... a Antibody presentation + agglutination, compared with Baorui Sankun anti-Jk a Antibodies exhibit ± agglutination, similar to anti-Jk. b The antibodies did not agglutinate, and serological identification showed the Jk(a+w) phenotype.

[0071] Example 3: In vitro cell expression experiment verification This embodiment uses in vitro cell expression to verify that the SLC14A1 gene carrying the c.613C>T mutation can directly lead to the Jk(a+w) phenotype, and that the combined mutation with the c.130G>A mutation results in a weaker Jk(a+w) phenotype. The specific steps are as follows: (1) Construction of wild-type and mutant expression plasmids Using the wild-type SLC14A1 gene full-length cDNA JK*01 (NM_015865.7) as the wild-type, and based on the haploid sequence characteristics of the c.613C>T mutation, a full-length SLC14A1 cDNA template containing the following mutation was designed. Wild-type plasmid: JK a+ Mutant plasmid: JK 130G>A+588A>G JK 130G>A+588A>G+613C>T JK 588A>G+613C>T These mutant plasmids contain a single mutated JK(a+w) allele and two mutated JK(a+w) alleles, allowing for the verification of both single missense mutations and their combined effects. To enhance expression, a kozak sequence was added to the 5' end, and a 6 bp linker sequence and a 24 bp flag tag sequence were added to the 3' end. Wild-type and mutant cDNAs were synthesized by Nanjing GenScript and cloned into the pcDNA3.1(+)-myc-His A expression vector. The recombinant plasmids were verified using full-sequence sequencing.

[0072] The full-length cDNA sequence (including modified sequences) of the wild-type SLC14A1 gene JK*01 cloned into the expression vector is shown in SEQ ID NO.21. 130G>A+588A>G The mutant clone sequence is shown in SEQ ID NO.22. JK 130G>A+588A>G+613C>T The mutant clone sequence is shown in SEQ ID NO.23. JK 588A>G+613C>T The mutant clone sequence is shown in SEQ ID NO.24.

[0073] (2) Transfection and stabilization screening of CHO cells Inoculate 2×10⁶ cells / well on a 6-well plate the day before transfection. 5 Cells were cultured overnight at 37°C with 5% CO2. Transfection was performed the following day after cells reached approximately 70% confluence. 2.5 µg of plasmid DNA was diluted with 125 µL of Opti-MEM medium to prepare a DNA premix. 4 µL of lipo8000 reagent (batch number: A356260113, Beyotime) was added and thoroughly mixed to prepare the DNA-liposome complex. The liposomes were added to the cells and gently mixed in a figure-eight motion. The cells were incubated at 37°C for 48 h. After 48 hours, the mixture was replaced with 1000 μg / ml G418 (batch number: A356260113, Beyotime) to screen for positive clones.

[0074] (3) Flow cytometry detection of cell surface Jk a Antigen expression The Jk markers on erythrocytes were detected using a flow cytometer via an indirect method. a Antigen expression. Briefly as follows: Prepare 50 µL of cell suspension, with approximately 5 × 10⁶ cells. 5 Add 50µL of primary antibody, an IgM-type anti-Jk antibody. a (Catalog No.: 20251218, Baorui Sankun) Incubate for 1 hour, wash 3 times with PBS, add APC-labeled IgG secondary antibody, mix well, react at room temperature in the dark for 30 minutes, centrifuge and wash once with PBS, then add 500µL PBS and immediately run on the instrument.

[0075] The stained cells were analyzed on a BD FACSuite flow cytometer. Fluorescence was detected in 20,000 cells obtained using BD FACSuite software. The analysis was based on the distribution of positive cells, average fluorescence intensity, and cell surface Jk. a Antigen expression status. The analysis results are as follows: Figure 10 and Figure 11 As shown: Using CHO cells transfected with an empty plasmid as a negative control, comparisons of positive cell distribution and average fluorescence intensity revealed that wild-type SLC14A1 cells successfully expressed a large amount of Jk. a Antigen. And the mutant JK 588A>G+613C>T and JK 130G >A+588A>G Cell surface Jk a All antigens were expressed at a reduced level, while the combined missense mutation JK... 130G>A+588A>G+613C>T Cell surface Jk a Antigen expression compared to a single missense mutation in Jk a The antigen is weaker. This proves that c.613C>T can directly lead to weakened Kidd protein expression, resulting in the Jk(a+w) phenotype. There is a necessary direct causal relationship between the c.613C>T mutation and the Jk(a+w) phenotype. However, in the case of the proband having both c.130G>A and c.613C>T mutations on the same allele, their Jk... a The weakening of antigen expression is the result of the combined effect of two missense mutations.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a substance for detecting SNP sites of the SLC14A1 gene in Jk(a+w) blood typing or in the preparation of Jk(a+w) blood typing products, characterized in that, The SNP site includes the c.613C>T mutation at position 613, starting from the start codon in the coding region of the SLC14A1 gene.

2. A reagent kit for Jk(a+w) blood typing, characterized in that, This includes substances for detecting SNP sites in the SLC14A1 gene; SNP sites include SNP site a, which is the cause of Jk. a Mutations that reduce antigen expression, leading to Jk a The mutation that weakens antigen expression includes the c.613C>T mutation at position 613, starting from the start codon of the SLC14A1 gene coding region as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The SNP sites also include SNP site b, which is the cause of Jk. a Mutations that weaken or inactivate antigen expression, with SNP sites a and b located on a pair of alleles on homologous chromosomes.

4. The kit according to claim 2 or 3, characterized in that, The substance used to detect SNP sites in the SLC14A1 gene includes at least one of the following: reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, reagents for constructing sequencing libraries, and reagents for sequencing.

5. The reagent kit according to claim 4, characterized in that, The reagents used for nucleic acid amplification include a first reagent for amplification primer pairs and sequencing primer pairs for amplifying the SLC14A1 gene and / or a second reagent for amplification primer pairs for amplifying the SLC14A1 gene; the amplification fragments of the SLC14A1 gene amplification primer pairs at least cover exons 3 to 10 of the SLC14A1 gene.

6. The reagent kit according to claim 5, characterized in that, The amplification primer pairs and sequencing primer pairs for amplifying the SLC14A1 gene in the first reagent are independently selected from the nucleotide sequences of the forward and reverse primers, as shown in SEQ ID NO.1~SEQ ID NO.

14. The amplification primer pairs for amplifying the SLC14A1 gene in the second reagent are independently selected from the forward and reverse primers, and their nucleotide sequences are shown in SEQ ID NO.15~SEQ ID NO.

18.

7. A method for identifying Jk(a+w) blood type, characterized in that, This includes obtaining the base type of the coding region of the SLC14A1 gene in the sample to be tested, and if the presence of an allele leads to Jk a SNP sites with weakened antigen expression and the presence of another allele lead to Jk a If the SNP site shows weakened or inactivated antigen expression, the blood type of the sample to be tested is determined to be Jk(a+w). The cause of Jk a The SNP site with weakened antigen expression includes the c.613C>T mutation at position 613, starting from the start codon of the coding region of the SLC14A1 gene as described in claim 1.

8. The identification method according to claim 7, characterized in that, This includes amplifying the SLC14A1 gene fragment of the test sample using the first reagent as described in claim 5 or 6, then detecting SNP sites in the amplification product, performing haplotype cloning and sequencing, and if a c.613C>T mutation exists in one allele, and a mutation causing Jk is also present in another allele... a If the antigen expression is weakened or inactivated by a mutation, the sample to be tested is determined to be of blood type Jk(a+w); Alternatively, it may include segmented cascade amplification of the full-length SLC14A1 gene of the test sample using the second reagent as described in claim 5 or 6, followed by haplotype typing and sequencing. If a c.613C>T mutation is present in one allele, and a mutation causing Jk is present in another allele, then... a If the antigen expression is weakened or inactivated by a mutation, the sample to be tested is determined to be of blood type Jk(a+w).

9. The identification method according to claim 8, characterized in that, Inactivating mutations include the c.342-1G>A mutation; Attenuated mutations include c.613C>T mutations and / or c.130G>A mutations.

10. The substance for detecting SNP sites of the SLC14A1 gene as described in claim 1, or the kit as described in any one of claims 2 to 6, is used in assessing the risk of immune hemolytic transfusion reactions or in preparing products for assessing the risk of immune hemolytic transfusion reactions; The reaction includes Jk a or Jk b Delayed hemolytic transfusion reaction caused by antigen-antibody incompatibility.

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