CN111850141A - Gene polymorphism site marker for detecting Bombay-like blood type - Google Patents
Gene polymorphism site marker for detecting Bombay-like blood type Download PDFInfo
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Abstract
The invention provides a gene polymorphism site marker and a detection primer for detecting Bombay blood type; the gene polymorphism site for detecting the Bombay-like blood group is 803 th base from the start codon in the encoding region of FUT1 gene and G is greater than A. The polymorphic site provided by the invention can be used for detecting the Bombay-like blood type. The invention also provides a product for detecting the Bombay type, wherein the product is used for detecting the polymorphic sites of the genes. The invention provides an effective way for diagnosing the Bombay-like blood type genes by detecting and screening the obtained gene polymorphic sites, and the application effect shows that the gene polymorphic sites and the detection primers provided by the invention can be effectively used for rapid detection of the Bombay-like genes of blood donors and clinical patients.
Description
Technical Field
The invention belongs to the technical field of gene diagnosis products, and particularly relates to a gene polymorphism site marker and a detection primer for detecting a Bombay-like blood group.
Background
The H antigen belongs to the Hh blood group system, is closely related to ABO blood group and Lewis blood group system, and is positioned on the oligosaccharide branched chain molecular structure outside the erythrocyte membrane. The Hh blood group system mainly comprises two rare phenotypes, Bomby and Bomby-like, which are represented by complete or partial deletion of the H antigen on erythrocytes. There are two closely linked gene loci on chromosome 19: h and Se genes. The H gene is also called FUT1 gene, the Se gene is also called FUT2 gene, and each of the two genes codes for an alpha-2-fucosyltransferase. The H (FUT1) gene has 4 exons, and the substrate of the encoded alpha-2-fucosyltransferase is a type 2 sugar chain, which synthesizes H antigenic determinants. The substrates of alpha-2-fucosyltransferase encoded by the Se (FUT2) gene are type 1 and type 2 sugar chains. The H antigen is controlled by alpha 1,2 fucosyltransferase gene (FUT1), and the epitope is composed of L-fucose linked to the terminal lactose of the precursor sugar chain, which is the precursor sugar of galactose and-N acetyl galactose at the terminal of the sugar chain of the blood group A and B antigens. The FUT1 gene has strong polymorphism, and the polymorphism is related to population distribution, and the molecular biological mechanism of the Monsanto-like type shows strong characteristics related to different population distributions. In 1961, Levine found 1 specific blood group in czech, and the red blood cells of this individual had no H antigen, much like the monty type, but unlike the monty type, a small amount of a antigen was present on the red blood cells, and Levine was called the monty-like type (para-bombay phenotype). The existence of the Bombay-like form was subsequently found in Japan, Thailand, Singapore, France, Indiana, USA. In 1979, Zhang Gong beam and the like found the first Bombay blood type in China, and then, tens of Bombay blood types were reported in succession in China. The data show that the Monsanto-like forms in the Japanese population are approximately l-2/300000, hong Kong 1/15620 and Taiwan 1/8000. The lack of H antigen on erythrocytes in Bombay-like individuals, the presence of anti-H antibodies in the serum, which antibodies are hemolytic transfusion reactive with all H antigen positive donor erythrocytes, makes it difficult to find compatible blood for transfusion in clinical transfusions. To further address the clinical transfusion problem of mengamic individuals, extensive molecular mechanistic studies of various H-deficiency phenotypes are required.
Disclosure of Invention
The invention aims to provide a gene polymorphic site marker and a detection primer for detecting Bombay-like blood types; thereby making up for the deficiencies of the prior art.
The invention firstly provides a gene polymorphism site for detecting a Bombay-like blood group, which is 803 th base from an initiation codon in an encoding region of an FUT1 gene, wherein G is more than A;
the invention also provides the application of the polymorphic site, which is the application in detecting the Bombay blood type;
the invention also provides a product for detecting the Bombay blood type, wherein the product is used for detecting the gene polymorphic sites;
the product is a PCR amplification sequencing detection kit;
the invention also provides a method for detecting the Bombay blood type, which is used for detecting whether the gene polymorphic sites exist in individuals;
the detection is to determine whether the gene polymorphic locus exists by PCR amplification sequencing;
the PCR amplification adopts a primer pair, and one sequence information of the primer pair is as follows:
FUT1-4F:5’-ACTTCCTCTTTCCCTGGGACTAA-3’(SEQ ID NO:1)、
FUT1-4R:5’-GTCTCCGCCAGCCAGGTA-3’(SEQ ID NO:2)。
the invention provides an effective way for diagnosing the Bombay-like blood type genes by detecting and screening the obtained gene polymorphic sites, and the application effect shows that the gene polymorphic sites and the detection primers provided by the invention can be effectively used for rapid detection of the Bombay-like genes of blood donors and clinical patients.
Drawings
FIG. 1: example 1 (proband) direct sequencing of the Bombay blood group FUT1, proband FUT1 gene coding region was heterozygous at CT, CT and GA at 35, 424 and 803 from the start codon.
FIG. 2: example 1 (proband) clone sequencing of the Bombay blood group FUT1, proband FUT1 has one allele coding region with 35 th base C > T and 803 th base G > A from the start codon.
FIG. 3: example 1 (proband) clone sequencing of the Bombay blood group FUT1, proband FUT1 has another allele coding region with 424 th bases C > T from the start codon.
Detailed Description
The applicant carried out the FUT1 gene sequencing on a clinical patient suspected of a Bombay-like phenotype in a blood group serological test result, and found that the gene sequencing is a Bombay-like blood group caused by a new gene polymorphic site, thereby contributing to the invention.
The FUT1 gene is located in chromosome 19q13.3 region, can be transcribed into 1095bp mRNA (NCBI accession number NG _007510.1), and finally translated into protein consisting of 365 amino acids. Because of the lack of H antigen on erythrocytes in montmorillonoid-type individuals, resulting in the production of immune anti-H antibodies in the serum, which antibodies are capable of severe hemolytic transfusion reactions with all H antigen positive donor erythrocytes, it is difficult to find compatible blood for transfusion during clinical transfusion. The gene detection system for the Bombay blood type is established by adopting a molecular biological means, is applied to clinical and blood collection work, is favorable for accurately detecting the Bombay blood type individuals, prompts the clinical patients to adopt autologous blood preparation or search suitable blood in a rare blood type bank as soon as possible for the blood transfusion patients, and reduces the occurrence of immune hemolytic transfusion reaction to the maximum extent.
For the allelic polymorphic site markers to which the present invention relates, the applicants explain as follows:
genetic polymorphisms refer to the simultaneous and frequent presence of two or more discrete variants or genotypes (genotypes) or alleles (alleles) in a population of organisms, also known as genetic polymorphisms (genetic polymorphisms).
The nucleotide sequence of the coding sequence is changed by base substitution, deletion and insertion related to gene polymorphism, and the change of genetic code, fragment deletion in protein peptide chain, mRNA splicing abnormality, or mutation of promoter and mutation of non-transcription region are caused in the process of transcribing and translating synthetic protein, so that the transcription abnormality, activity enhancement or reduction of gene and the influence on the arrangement sequence of amino acids in polypeptide chain are caused.
The mutant forms of the FUT1 gene found so far mainly include missense mutations, resulting in one and two amino acid substitutions in the peptide chain of the enzyme protein; nonsense mutations, which convert a codon encoding an amino acid to a stop codon; frame-shifting mutations, resulting in deletions of one or two nucleotides, and the like. Most H-deleted individuals have the genes mutated to homozygous genes, but some are two different mutations, heterozygous genes.
The present invention will be described in detail with reference to examples.
Example 1: screening of Gene polymorphism site marker
1. Extracting peripheral blood genome DNA:
on the basis of meeting the relevant national policy and obtaining the consent of a sampling object, 2-5mL of peripheral venous blood of a suspected Bombay-like blood type patient is extracted and put into an EDTA anticoagulation tube, 500 mu L of the peripheral venous blood is put into a centrifuge tube, an equal volume of erythrocyte lysate (pH8.0) is added, the mixture is evenly mixed for 5 minutes by vortex, the centrifugation is carried out at 12000rpm for 5 minutes, and the supernatant is discarded. The erythrocyte lysate is added repeatedly, 500ul of the erythrocyte lysate is vortexed and mixed for 5 minutes, the centrifugation is carried out for 5 minutes at 12000rpm, and the supernatant is discarded. The precipitate was vortexed for 5 minutes, 50ul of lysis buffer was added, vortexed for 5 minutes, and placed in a 56 ℃ metal bath for 15 minutes. The sample was removed from the water bath and 135ul of protein remover was added. Vortex well and mix, and place at 4 ℃ for 30 min.
Centrifuge at 12000rpm for 5 minutes at room temperature and aspirate the supernatant (approximately 200ul) into a new centrifuge tube. Add 500ul of glacial ethanol, shake the centrifuge tube repeatedly until the white DNA precipitate is separated out. Centrifuge at 12000rpm for 2 minutes at room temperature and discard the supernatant. To the DNA precipitate was added 75% ethanol, rinsed once, centrifuged at 12000rpm for 3 minutes at room temperature, the supernatant was discarded, left to evaporate the remaining ethanol at room temperature, and finally 50. mu.L of TE (pH8.0) was added to dissolve the DNA overnight at 4 ℃.
And (3) performing agarose gel electrophoresis on the extracted DNA, and performing color comparison at 260nm and 280nm by using an ultraviolet spectrophotometer to detect the purity and the concentration of the DNA.
2. Gene sequencing searched for FUT1 gene mutations:
PCR amplification of the fragment of interest: reaction conditions and reaction system:
(1) and (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; 60 seconds at 94 ℃, 90 seconds at 63 ℃, 60 seconds at 72 ℃ and 10 cycles; 60 seconds at 94 ℃, 90 seconds at 61 ℃, 60 seconds at 72 ℃ and 25 cycles; extending for 10min at 72 ℃, and preserving the temperature of the amplification product at 10 ℃.
(2) Reaction system: (TAKARA Ex-Taq polymerase)
The amplification reaction of the genomic DNA template of the proband and the FUT1 primer was performed using this reaction system.
Sequencing a PCR product: the PCR products were sequenced by conventional Sanger sequencing method, and found in exon4 of the proband FUT1 gene, which was heterozygous for CT at position 35, CT at position 424 and GA at position 803, as shown in FIG. 1. Further clone sequencing of exon4 of FUT1 gene revealed that one allele: nt35C > T, nt803G > A; the other allele: nt424C > T, see FIG. 2.
The 35 th base C of one allele is mutated into T, so that a codon is mutated from GCC into GUC, the coded amino acid is converted from alanine (Ala) into valine (Val), the 803 th base G is mutated into A, so that the codon is mutated from UGU into UAU, and the coded amino acid is converted from cysteine (Cys) into tyrosine (Tyr), so that the protein conformation of a-1,2 fucosyltransferase is changed, and the weak expression of H antigen is caused.
The 424 th base C of the other allele is mutated into T, so that the codon is mutated from CGG into UGG, the coded amino acid is converted from arginine (Arg) into tryptophan (Trp), and the conformation of the alpha-1, 2 fucosyltransferase protein is changed, thereby causing the weak expression of H antigen.
The two mutated FUT1 alleles of proband are the molecular basis for the proband to behave as a pygmy blood group.
Multiple sequencing results show that the polymorphic gene is not caused by amplification or sequencing errors, and nt803G > A is a new mutation. This mutation is not present in the following four databases: the single nucleotide polymorphism databases (ftp:// ftp. ncbi. nih. gov/snp/database /), thousand human genome project (ftp:// ftp-trace. ncbi. nih. gov/1000 genes/ftp /), Hapmap8 database (http:// Hapmap. ncbi. nlm. nih. gov /) and Yanhuang database (http:// yh. genomics. org. cn /) indicate that the mutation is rare, and that the mutation causes inactivation of the fucosyltransferase of proband a-1,2, resulting in a blood group-like type.
Through the analysis, the method is proved to be capable of accurately determining the specific Bombay blood type genes, so that the blood type of a person to be determined can be more accurately determined, and the method has very important significance for a patient to formulate a reasonable blood transfusion scheme.
The proband family members collected in the research are extracted to obtain the peripheral blood genome DNA, the DNA is used as a template and FUT1-4F and FUT1-4R primers to amplify PCR target fragments, the conventional Sanger sequencing method is used for directly sequencing PCR products by using the pair of primers, and the gene deletion mutation found in the invention, namely nt35C > T and nt803G > A, exists in the H blood type FUT1 gene Exon4 of one family member of the proband. And (3) continuously carrying out haplotype sequencing on the 4 th exon of the FUT1 gene on the family member with the deletion mutation, carrying out PCR target fragment amplification by using the extracted genomic DNA of the two family members as a template and the primer, cloning the PCR product into a T carrier, selecting a plurality of colonies, carrying out haplotype sequence determination, and determining the haplotype sequence of the sample. Haplotype sequencing finds that the encoding regions nt35C > T and nt803G > A of the family member FUT1 gene, the encoded amino acids are respectively converted into valine (Val) from alanine (Ala) and tyrosine (Tyr) from cysteine (Cys), so that the conformation of the alpha-1, 2 fucosyltransferase protein is changed, the weak expression of H antigen is caused, and the protein is completely consistent with proband.
Sequence listing
<110> Qingdao city central blood station
<120> a gene polymorphic site marker for detecting Bombay-like blood group
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Claims (7)
1. A gene polymorphic site for detecting a Bombay-like blood group is characterized in that the gene polymorphic site is 803 th base from an initiation codon in a coding region of FUT1 gene and is G & gtA.
2. Use of the polymorphic site of the gene according to claim 1 for the preparation of a product for detecting Bombay-like blood group.
3. A product for detecting Bombay type blood, wherein said product is used for detecting the polymorphic site of the gene according to claim 1.
4. The article of claim 3, wherein said article is a PCR amplification sequencing kit.
5. A method for detecting a Bombay-type blood group, which comprises detecting the presence or absence of the polymorphic site in the gene of claim 1 in an individual.
6. The method of claim 5, wherein said detecting is by PCR amplification sequencing to determine the presence of said polymorphic site of the gene of claim 1.
7. The method of claim 6, wherein the PCR amplification sequencing is performed using primer pairs having the sequences of SEQ ID NO. 1 and SEQ ID NO. 2.
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| CN202010841248.6A CN111850141A (en) | 2020-08-20 | 2020-08-20 | Gene polymorphism site marker for detecting Bombay-like blood type |
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| CN202010841248.6A CN111850141A (en) | 2020-08-20 | 2020-08-20 | Gene polymorphism site marker for detecting Bombay-like blood type |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116287321A (en) * | 2023-04-28 | 2023-06-23 | 浙江省血液中心 | SNP site of H blood group system antigen deletion related to immune hemolytic transfusion reaction, application and reagent |
| CN116411059A (en) * | 2023-04-10 | 2023-07-11 | 浙江省血液中心 | SNP locus of simendan-like phenotype for initiating hemolytic transfusion reaction, application and reagent |
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| CN108179185A (en) * | 2018-03-08 | 2018-06-19 | 青岛市中心血站 | A kind of SNP site for being used to detect H blood group anomalies |
| CN109735610A (en) * | 2019-03-07 | 2019-05-10 | 青岛市中心血站 | It is a kind of for detecting the gene polymorphism sites of para-Bombay blood group |
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| CN108179185A (en) * | 2018-03-08 | 2018-06-19 | 青岛市中心血站 | A kind of SNP site for being used to detect H blood group anomalies |
| CN109735610A (en) * | 2019-03-07 | 2019-05-10 | 青岛市中心血站 | It is a kind of for detecting the gene polymorphism sites of para-Bombay blood group |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116411059A (en) * | 2023-04-10 | 2023-07-11 | 浙江省血液中心 | SNP locus of simendan-like phenotype for initiating hemolytic transfusion reaction, application and reagent |
| CN116287321A (en) * | 2023-04-28 | 2023-06-23 | 浙江省血液中心 | SNP site of H blood group system antigen deletion related to immune hemolytic transfusion reaction, application and reagent |
| CN116287321B (en) * | 2023-04-28 | 2023-11-24 | 浙江省血液中心 | SNP site of H blood group system antigen deletion related to immune hemolytic transfusion reaction, application and reagent |
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