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KR-102960723-B1 - Primers for RHD exome sequencing for detecting RhD variants and use of the same

KR102960723B1KR 102960723 B1KR102960723 B1KR 102960723B1KR-102960723-B1

Abstract

The present invention relates to a method for detecting RHD variants comprising the step of analyzing all 10 exons involved in RhD antigen expression in a sample and adjacent intron regions, a primer set for PCR reaction for RHD exome sequencing analysis consisting of nucleotide sequences described in SEQ ID NOs 1 to 18, a primer set for sequencing for RHD exome sequencing analysis consisting of nucleotide sequences described in SEQ ID NOs 19 to 38, and a kit or composition for detecting RHD variants comprising the primer set. Through the present invention, it was possible to detect gene variants in all cases showing atypical RhD blood type test results.

Inventors

  • 박경운
  • 장호은
  • 오수진

Assignees

  • 주식회사 픽스인스티튜트

Dates

Publication Date
20260507
Application Date
20230703

Claims (9)

  1. The method includes the step of analyzing all 10 exons involved in RhD antigen expression and adjacent intron regions in a sample, and The step of analyzing the intron regions adjacent to all 10 exons comprises performing a PCR reaction and sequencing using a primer set for RHD exome sequencing analysis consisting of the nucleotide sequences described in SEQ ID NOs. 1 to 38. RHD variant detection method.
  2. delete
  3. In claim 1, the PCR reaction detects exons 1, 3, 7, and 8 through conditions of 1 cycle at 94°C for 3 minutes, 32 cycles at 95°C for 30 seconds, 63°C for 30 seconds, and 65°C for 2 minutes, and 1 cycle at 65°C for 10 minutes. Exons 2, 4, 5, 9, and 10 are detected through conditions of 1 cycle of 3 minutes at 95℃, 35 cycles of 30 seconds at 95℃ and 2 minutes 30 seconds at 65℃, and 1 cycle of 10 minutes at 65℃. A method for detecting RHD variants characterized by detecting exon 6 through conditions of 1 cycle of 3 minutes at 95℃, 30 seconds at 95℃, 35 cycles of 30 seconds at 60℃ and 1 minute at 65℃, and 1 cycle of 10 minutes at 65℃.
  4. It consists of the nucleotide sequences described in SEQ ID NOs 1 to 18, and Characterized by being designed to amplify 10 exons of the RHD gene using three sets of PCR reaction conditions, PCR reaction primer set for RHD exome sequencing.
  5. It consists of the nucleotide sequences described in SEQ ID NOs 19 to 38, and Characterized by being used for base sequence analysis of a PCR product obtained by amplifying 10 exons of the RHD gene with three sets of PCR reaction conditions, Sequencing primer set for RHD exome sequencing.
  6. A kit for detecting RHD variants comprising the primer set of claim 4 or 5.
  7. A kit for detecting RHD variants comprising the primer set of claim 4 and the primer set of claim 5.
  8. A composition for detecting RHD variants comprising the primer set of claim 4 or 5.
  9. A composition for detecting RHD variants comprising the primer set of claim 4 and the primer set of claim 5.

Description

Primers for RHD exome sequencing for detecting RhD variants and use of the same The present invention relates to a primer for RHD exome sequencing analysis for RhD variant detection and its use. Blood group antigens and corresponding antibodies are a major area of interest in transfusion medicine. In 2022, the International Society of Blood Transfusion Medicine (ISBT) classified human red blood cell types into 43 groups and officially recognized 349 individual antigens. This is determined by genes that govern the various blood group systems. The most important blood type determining the success or failure of a blood transfusion is the ABO blood type, followed by the Rh blood type in clinical importance. Therefore, ABO and Rh blood type testing are essential tests before a transfusion. There are 57 antigens belonging to the Rh blood group, including D, C, E, c, and e; among these, the D antigen is the most antigenic and clinically important. RhD-positive individuals do not form anti-D even when exposed to the D antigen, whereas RhD-negative individuals can produce anti-D. Due to this principle, Rh blood type must be considered importantly in areas such as blood transfusions, organ transplants, and neonatal hemolytic disease; however, various RhD variants resulting from RHD gene mutations often cause confusion in the medical field. Since there are limitations in identifying RhD variants using commonly performed serological tests, RHD genotyping should be conducted in parallel; however, unlike serological tests, this is not routinely performed in hospital laboratories but is carried out in only a very limited number of laboratories. One reason why RHD genotyping is not commonly performed is that it is not as frequently used as serological testing, but also because it is difficult to conduct without specialized background knowledge due to the specificity of the RHD gene structure. The RHD gene consists of 10 exons and is located on the short arm of chromosome 1. It is situated adjacent to the RHCE gene and is arranged symmetrically to it, and the two have the specific characteristic of having more than 97% identical nucleotide sequences (Fig. 1). Generally, the presence of RhD antigens is determined as RhD positive, while the absence of RhD antigens is determined as RhD negative. The most typical scenario involves the deletion of all 10 exons of the RHD gene, which prevents the production of RhD antigens and results in RhD negative expression. However, various other genetic mutations can cause partial presence or loss of RhD antigens, or single nucleotide mutations can lead to atypical phenotypes, which can cause confusion in determining RhD positive or negative status. Since such atypical phenotypes can trigger immune responses during processes such as blood transfusion, organ transplantation, and pregnancy, genotyping is considered necessary (Figs. 2, 3, 4). The RHD gene consists of a total of 10 exons, and phenotype-related RHD gene variants can be detected by analyzing the 10 exons critical for antigen protein synthesis and some introns surrounding the exons. However, a crucial consideration in this process is that the RHD gene must be analyzed purely while excluding the RHCE gene, which shares more than 97% identity. Due to this difficulty, researchers have used simplified analysis methods that selectively analyze only 3-4 regions of the RHD gene to examine structural variations or analyze only a few high-frequency single nucleotide variations (SNVs); however, in such cases, the detectable variants are extremely limited. [Prior Art] Republic of Korea Patent Publication No. 1020110017387 Figure 1 is a figure showing the structure of the RHD gene and the RHCE gene. Figure 2 shows the structure of the weak D type among RhD variants. Figure 3 shows the structure of partial D type among RhD variants. Figure 4 is a figure showing examples of various RHD variants registered with the International Society of Blood Transfusion Medicine (ISBT). Fig. 5 is Figure showing the selection of PCR and sequencing primers for Exon 1, Fig. 6 is Figure showing the selection of PCR and sequencing primers for Exon 2, Fig. 7 is Figure showing the selection of PCR and sequencing primers for Exon 3, Figure 8 shows the selection of PCR and sequencing primers for Exons 4 and 5. Fig. 9 is Figure showing the selection of PCR and sequencing primers for Exon 6, Fig. 10 is Figure showing the selection of PCR and sequencing primers for Exon 7, Fig. 11 is Figure showing the selection of PCR and sequencing primers for Exon 8, Fig. 12 is Figure showing the selection of PCR and sequencing primers for Exon 9, Fig. 13 is Figure showing the selection of PCR and sequencing primers for Exon 10, Figure 14 shows the PCR results for a total of 10 exons. It is a figure showing the results of an RhD-positive (S1) sample and an RHD gene complete deletion (S2) sample, In the case of RHD exome sequencing, the result is determined by the sequencing result ra