RU2563172C1 - METHOD FOR DETERMINING CCR5 delta 32 ALLELE POLYMORPHISM - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from cryopreserved umbilical blood. A polymerase chain reaction (PCR) is conducted with using specific primers. The presence of specific DNA fragments is stated after electrophoretic separation of the PCR amplified mixture and ethydium bromide staining. A normal gene version is accompanied by the PCR fragment length making 224 base pairs. In CCR5 delta 32 allele polymorphism, the PCR fragment length is 192 base pairs.

EFFECT: invention provides the specific detection of CCR5 delta 32 allele polymorphism in the umbilical blood sample and can be used for screening assay.

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Description

The invention relates to medicine, namely genetics, hematology, and can be used to determine the CCR5 delta 32 allelic polymorphism in a screening examination of cord blood samples entering a cord blood bank.

Human Immunodeficiency Virus (HIV) penetrates cells by binding the gp 120 viral envelope glycoprotein to CD4 and CCR5 membrane receptors. Simultaneous expression of CD4 and CCR5 receptors is found on T-lymphocytes, monocytes, macrophages and dendritic cells. The CCR5 protein is encoded by the CCR5 gene located on the short arm of chromosome 3 at position 21 (3p21). There is a CCR5 delta 32 polymorphism, which is a deletion of 32 nucleotide pairs in the coding region of the CCR5 gene. As a result of expression of the mutant gene in a homozygous state, a truncated, functionally inactive CCR5 protein is translated. As a result, homozygous carriers of this polymorphism have almost complete resistance to HIV infection. About 1% of the white population are homozygous carriers of the deletion allele [1, 2]. A successful case of transplantation of hematopoietic stem cells (HSCT) of peripheral blood in 2007 in Germany to an HIV-infected patient with acute myeloid leukemia from a donor, a homozygous carrier of CCR5 delta 32 polymorphism, is known [3, 4]. However, despite the success of the treatment, this was only an isolated case, and subsequently not a single HSCT for the treatment of HIV infection was carried out due to the rare occurrence of CCR5 delta 32 polymorphism and strict conditions for the selection of bone marrow and peripheral blood samples using the HLA system. At the same time, umbilical cord blood hematopoietic stem cells (HSCs) with the genotype CCR5 delta 32 / delta 32 could be much more likely to be used for transplantation during HIV infection due to significantly less stringent requirements for sample selection using the HLA system [5] .

Currently, a small number of test systems for the detection of CCR5 delta 32 mutations are presented in the Russian Federation. Available diagnostic test systems are expensive, expensive equipment is required for research. In addition, certificates of conformity were not received for these diagnostic test systems, which implies the use of these sets only for scientific purposes and does not allow the use of these sets in any types of diagnostic procedures. An example is AmpliSens CCR5del32-screen reagent kit for determining CCR5 delta 32 polymorphism by pyrosequencing.

There are methods for determining allelic polymorphism of CCR5 delta 32 by real-time allele-specific PCR [5, 6].

As a prototype, we can consider a method for determining allelic polymorphism CCR5 delta 32, based on the use of PCR with restriction analysis of amplification products (PCR-RFLP) [7].

The disadvantages of the method that we have chosen as a prototype, as well as the above analogues, include the fact that these methods are characterized by the need to use additional stages of analysis, reagents and equipment for the study, which increases the cost of the method, the complexity and time of the analysis and does not allow the use of this method for screening.

The technical result of the invention is to simplify the method for determining allelic polymorphism CCR5 delta 32, reducing the time it takes and reducing its cost, which will provide the opportunity for screening.

The technical result of the invention is achieved by the fact that the method for determining allelic polymorphism CCR5 delta 32 consists in the isolation of DNA, PCR, detection of the reaction product using electrophoresis, while DNA is isolated from cryopreserved umbilical cord blood, and the following primers are used for PCR:

F: TGTGTTTGCGTCTCTCCCA

R: CTCTTCTTCTCATTTCGACACCG,

PCR is carried out at 94 ° C for 5 min, then 35 cycles are carried out, each of which consists of 3 stages: 94 ° C - 30 s, 56 ° C - 40 s, 72 ° C - 40 s. Then, PCR is carried out at 72 ° C for 10 minutes and 15 ° C for 5 minutes. The polymorphism is detected in an 8% polyacrylamide gel using vertical electrophoresis. The presence of specific DNA fragments is determined after electrophoretic separation of the PCR amplification mixture and staining the gel with ethidium bromide using a transilluminator. The length of the PCR fragments is 224 bp in the normal version of the gene and 192 bp with allelic polymorphism CCR5 delta 32.

The method is as follows:

Prior to the reaction, cryopreserved umbilical cord blood at -70 ° C is thawed in Eppendorf type microtubes at room temperature for 20–40 min. Genomic DNA is isolated from 0.9-1.0 ml of umbilical cord blood using commercial kits Protrans (Germany) or Axygen (USA). Screening of samples for CCR5 delta 32 allele is carried out by PCR. DNA is amplified in a final volume of the reaction mixture of 20 μl. The reaction mixture contains a 10-fold buffer for Taq polymerase, 2 mm MgCl ₂ , 2 mm each deoxynucleoside triphosphate (dNTP), direct and reverse primers (30 ng each), 0.1 Units Taq polymerase, 40-120 ng of the studied genomic DNA The following primers flanking the deletion site are used:

F: TGTGTTTGCGTCTCTCCCA

R: CTCTTCTTCTCATTTCGACACCG.

PCR is carried out in a Biorad My Cycler Version 1.065 thermocycler. Reaction conditions: 94 ° C - 5 min; further 94 ° C - 30 s, 56 ° C - 40 s, 72 ° C - 40 s for 35 cycles; then 72 ° C for 10 minutes and 15 ° C for 5 minutes. The polymorphism is detected in an 8% polyacrylamide gel using vertical electrophoresis. The presence of specific DNA fragments is determined after electrophoretic separation of the PCR amplification mixture and staining the gel with ethidium bromide using a transilluminator. The length of the PCR fragments is 224 bp in the normal version of the gene and 192 bp with allelic polymorphism CCR5 delta 32.

An electrophoregram of amplification products is shown in FIG. one:

1 - heterozygous carriage of polymorphism CCR5 delta 32;

2 - normal type of CCR5 gene;

3 - polymorphism CCR5 delta 32 in the homozygous state.

Salient features of the proposed method are:

- when conducting PCR, the following primers are used:

F: TGTGTTTGCGTCTCTCCCA

R: CTCTTCTTCTCATTTCGACACCG;

- PCR is carried out at 94 ° C for 5 min, then 35 cycles are carried out, each of which consists of 3 stages: 94 ° C - 30 s, 56 ° C -40 s, 72 ° C - 40 s; then carry out PCR at 72 ° C for 10 minutes and 15 ° C for 5 minutes;

- the length of the PCR fragments is 224 bp in the normal version of the gene;

- allelic polymorphism CCR5 delta 32 is determined in the presence of PCR fragments of 192 bp in size.

A causal relationship between the salient features and the result:

The design of specific primers was developed using the NCBI bioinformation database (http://www.ncbi.nlm.). Optimum conditions for PCR were selected — length of primers, annealing temperature, length of DNA amplification, nucleotide ratio. A pair of primers was tested for the possibility of the formation of dimers, duplexes, studs. The results were confirmed by direct sequencing using a CEQ 8800 sequencer (Beckman Coulter, USA).

An example of a specific implementation of the method:

Example 1

Amplification was carried out in a volume of 18 μl. Composition of the reaction mixture: 14.5 μl bidistilled water, 2 μl polymerase buffer (Taq buffer, 10-fold, pH 8.6, without Mg2 +), 1.2 μl 25 mM MgCl _2, 2 μl direct and reverse primer mixture (50 ng each primer), 0.4 μl of a mixture of deoxynucleoside triphosphates (10 mM dNTP), 0.1 μl of Taq polymerase (1 unit of activity), 2 μl of the analyzed genomic DNA (40-120 ng). PCR was performed in a Biorad My Cycler Version 1.065 amplifier. Reaction conditions: 94 ° C - 5 min; further 94 ° C - 30 s, 56 ° C - 40 s, 72 ° C - 40 s for 35 cycles; then 72 ° C for 10 minutes and 15 ° C for 5 minutes. The polymorphism was detected in an 8% polyacrylamide gel using vertical electrophoresis. The presence of specific DNA fragments was determined after electrophoretic separation of the PCR amplification mixture and the gel was stained with ethidium bromide using a transilluminator. The length of the PCR fragments was 224 bp in the normal version of the gene and 192 bp with allelic polymorphism CCR5 delta 32.

Fragment lengths obtained by genotyping of allelic polymorphism:

During the screening study of the detection of CCR5 delta32 allelic polymorphism, 2960 public register cord blood samples were examined. The study revealed 31 samples with the genotype CCR5 delta 32 / delta 32, which amounted to 1.05%. In 534 samples, the allelic polymorphism CCR5 delta 32 was present in the heterozygous state (18.04%).

Thus, the inventive method for determining allelic polymorphism CCR5 delta 32 by PCR compared with the prototype reduces the complexity and the time of the study, reduces the cost of the analysis due to the absence of an additional stage of the study (stage restriction analysis of amplification products), without the need to purchase additional reagents, in particular expensive restrictase. This method can be used for screening for carriage of allelic polymorphism CCR5 delta 32.

Information sources

1. Deng, H. Identification of a major co-receptor for primary isolates of HIV-1 / H. Deng, R. Liu, W. Ellmeier [et al.] // Nature. - 1996. - Vol. 381. - P. 661-666.

2. Dragic, T. HIV-1 entry into CD4 + cells is mediated by the chemokine receptor CC-CKR-5 / T. Dragic, V. Litwin, G.P. Allaway [et al.] // Nature. - 1996. - Vol. 381. - P. 667-673.

3. Hutter, G. Long-Term Control of HIV by CCR5 Delta32 / Delta32 Stem-Cell Transplantation / G. Hutter, D. Nowak, M. Mossner // N. Engl. J. Med. - 2009. - Vol. 360. - P. 692-698.

4. Alters, K. Evidence for the cure of HIV infection by CCR5Δ32 / Δ32 stem cell transplantation / K. Allers, G. Hütter, J. Hofmann // Blood. - 2011 .-- Vol. 117, No. 10. - P. 2791-2799.

5. Petz, L.D. Hematopoietic Cell Transplantation with Cord Blood for Cure of HIV Infections / L.D. Petz, I. Redei, Y. Bryson [et al.] // Biol. Blood Marrow Transplant. - 2013 .-- Vol. 19, No. 3. - P. 393-397.

6. Kofiadi I.A. Distribution of alleles of CCR5, CCR2 and SDF1 genes associated with resistance to HIV infection in Russian populations / I.A. Kofiadi, D.V. Rebrikov, D.Yu. Trofimov [et al.] // Reports of the Academy of Sciences. - 2007. - T. 415, No. 6. - S. 842-845.

7. Patent RU 2249619 C2, 04/23/2003. A method for the simultaneous determination of mutations CCR5del32 and CCR5M303 in the human CCR5 gene, which determine the genetic resistance of the examined to infection with the human immunodeficiency virus of the first type (HIV1). Rakhmanaliev E.R., Apryatin S.A., Nikolaeva I.A., Klimov E.A., Sulimova G.E., Sidorovich I.G.

Claims (1)

The method for determining the allelic polymorphism CCR5 delta 32, which consists in the isolation of DNA, polymerase chain reaction (PCR), detection of the reaction product using electrophoresis, characterized in that the DNA is isolated from cryopreserved umbilical cord blood, and the following primers are used for PCR:
F: TGTGTTTGCGTCTCTCCCA
R: CTCTTCTTCTCATTTCGACACCG
and PCR is carried out at 94 ° C for 5 min, then 35 cycles are carried out, each of which consists of 3 stages: 94 ° C - 30 s, 56 ° C - 40 s, 72 ° C - 40 s, after which PCR is performed at 72 ° C for 10 minutes and 15 ° C for 5 minutes; polymorphism is detected in an 8% polyacrylamide gel using vertical electrophoresis; the presence of specific DNA fragments is determined after electrophoretic separation of the PCR amplification mixture and staining the gel with ethidium bromide using a transilluminator; the length of the PCR fragments is 224 bp in the normal version of the gene and 192 bp with allelic polymorphism CCR5 delta 32.

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