JP2009132696A - Hla-b54 new allele - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new alleles included in B54 as one of HLA-B antigen subtypes. <P>SOLUTION: The HLA-B54 new alleles which encodes a specific amino acid sequence and the HLA-B54 new alleles which has a specific base sequence or a sequence complementary thereto are provided, each exhibiting a positive reaction entirely different from those in known alleles through analyzing the genotype of HLA-B gene using a plurality of beads solid-phased with an oligonucleotide probe. For these new alleles, it has been found to exhibit a positive reaction entirely different from those in known alleles when the genotype of HLA-B gene is analyzed using a plurality of beads solid-phased with an oligonucleotide probe. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel allele of human leukocyte antigen (hereinafter abbreviated as HLA).

  The importance of HLA typing has been attracting attention not only in determining suitability at the time of transplantation but also in determining individual susceptibility to diseases.

  When organ transplantation is performed, how much the HLA type matches between the donor and the patient greatly affects the success rate of transplantation. If the HLA types do not match, the organ will not engraft the patient due to rejection, and conversely, GVHD (Graft-Versus-Host Disease) will occur due to donor-derived immune cells, The patient's life is at risk. The relationship between the incidence of specific diseases such as diabetes and the HLA type has also been pointed out. It can be said that HLA typing has become more important as medical technology becomes more advanced.

  Conventional HLA typing is a serological method performed using antibodies, but so-called genotyping methods in which typing is performed based on the sequence of genes encoding HLA molecules have become mainstream due to recent technological innovation. Genotype matching in bone marrow transplantation has been shown to correlate with transplantation results, and HLA genotyping has become increasingly important in the medical field.

  As a method for confirming the base sequence, there is the Sanger method (for example, see Non-Patent Document 1) for determining the sequence by sequencing reaction. However, HLA genotyping uses a partial sequence as a probe or primer for cost reasons. A method is used in which the gene sequence is estimated from the reactivity and the HLA type is determined (see, for example, Non-Patent Documents 2 and 3).

  HLA-B54, which is one of the subtypes of HLA-B antigen, has a very low frequency of appearance in Europe and the United States, and is a characteristic antigen of various ethnic groups in East Asia such as Japanese. Association with diseases such as AIDS sensitivity of immunodeficiency virus (for example, see Non-Patent Document 4), silicosis (for example, see Non-Patent Document 5), and diffuse panbronchiolitis (for example, see Non-Patent Document 6) Has been suggested.

As of October 2007, 13 types of genotypes contained in HLA-B54 have been reported (see, for example, Non-Patent Document 7), but the existence of allyl has not been fully studied. .
Santamaria P. et al. HLA class I sequence-based typing. Hum Immunol. 37 (1): 39-50, 1993. Bunce M. et al. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens. 46 (5): 355-67, 1995. Kawai S. et al. Routine low and high resolution typing of the HLA-DRB gene using the PCR-MPH (microtitre plate hybridization) method.Eur J Immunogenet. 23 (6): 471-86, 1996. Association analysis of HIV infection resistance and HLA-B in Japanese hemophilia patients.Journal of the Japanese Society for Histocompatibility. 12 (2): 124,2005. A case of HLA-B54 positive rheumatoid arthritis and silicosis associated with lung cancer. Journal of the Japanese Respiratory Society, 44 (12): 993-996, 2006. Diffuse panbronchitis. Journal of Toho Medical Society, 51 (5): 257-264, 2004. Allele Frequencies [online], [October 22, 2007 search], Internet <URL: http://www.ebi.ac.uk/imgt/hla/>

Summary of the Invention

  Accordingly, an object of the present invention is to provide a novel allele contained in B54, which is one of the subtypes of HLA-B antigen.

  The present inventors have now found a gene that shows a positive reaction different from that of a known allele by a method for determining the genotype of the HLA-B gene using a plurality of beads on which an oligonucleotide probe is immobilized. The present invention is based on such knowledge.

  That is, the present invention provides a novel allele of HLA-B54 encoding the amino acid sequence set forth in SEQ ID NO: 1.

  The present invention also provides a novel allele of HLA-B54 having the base sequence set forth in SEQ ID NO: 2 or its complementary sequence.

  According to the present invention, detailed typing of HLA-B antigen becomes possible, so not only the suitability at the time of transplantation but also the determination of individual susceptibility to diseases such as AIDS, silicosis, diffuse panbronchitis, etc. Is extremely useful.

Detailed description of the invention

  Based on PCR-SSOP (Sequence Specific Oligonucleotide probe) method, which is one of DNA typing methods, Luminex's xMAP measurement technology (http://www.luminexcorp.com/01_xMAPTechnology/index.html) When the genotype of the HLA-B antigen was typed using a WAKFlow HLA typing reagent (manufactured by Yugenaga Pharmaceutical Co., Ltd.) that was capable of typing, the reactivity of the known genotype was not shown in the DNA sample extracted from blood.

  The sequences of exon 2 and exon 3 were confirmed by the direct sequencing method (Wong C. et al. Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature. 330: 384-6, 1987.). Using the primers set upstream and downstream of exon 2 and exon 3, respectively, the sequences were confirmed from both sides of the sense strand antisense strand. Exon 2 is a sequence of B * 5201 and B * 5401, but exon 3 Detected a sequence of B * 5201 and a sequence not previously reported. Therefore, there was a possibility that this specimen was a heterojunction of B * 5201 and an unknown allyl.

  In order to examine the sequence in detail, a region of about 1 Kb from exon 2 to exon 3 of the HLA-B antigen gene was isolated by subcloning. This region is amplified by PCR reaction using primers set upstream of exon 2 and downstream of exon 3 of the HLA-B antigen, and a fragment of the desired length is extracted by electrophoresis and incorporated into a plasmid vector by TA cloning. It was. E. coli transformed with this plasmid was cultured in a small amount of medium, and the plasmid was extracted from E. coli recovered from the medium. Among the resulting plasmids, those containing an insert of about 1 Kb were selected by restriction enzyme treatment, and the sequences of the resulting 5 clone plasmids were confirmed.

  As a result, 2 clones of HLA-B * 5201 and 3 clones having a novel sequence confirmed by the direct sequencing method were obtained.

  As a result of confirming the base sequence of the gene for 3 mutant clones having a novel sequence, 3 mutations were confirmed in exon 3 of the sequence B * 5401 as shown in FIG. These mutations were non-synonymous substitutions in which the 114th amino acid shown in FIG. 2 was replaced from asparagine to aspartic acid and the 116th amino acid was replaced from leucine to serine. The 114th and 116th amino acids are located inside the region sandwiching the antigenic peptide in the HLA-B molecule.

Therefore, these amino acids are also important sites for peptide motifs that bind to HLA-B molecules and are likely to play an important role in the immune system. Therefore, it is necessary to distinguish between B * 5401 and the genotype (B * 54V) having the mutation of the present invention in transplantation medical treatment, and increase the typing accuracy of HLA-B antigen in suitability determination at the time of transplantation. Above, it is extremely useful and important. It is also extremely useful and important in determining an individual's sensitivity to diseases such as AIDS, silicosis, and diffuse panbronchitis.
Note that the novel allyl found in the present invention is “B * 5414” according to WHO naming. Therefore, in the present specification, the new allele is indicated by either “B * 54V” or “B * 5414”.

  Therefore, as described above, the novel allele according to the present invention encodes the amino acid sequence described in SEQ ID NO: 1, and has the base sequence described in SEQ ID NO: 2 or a complementary sequence thereof.

  According to another aspect of the present invention, there is provided a method for typing an HLA-B54 antigen, wherein the amino acid sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2 or a complementary sequence thereof, A method is provided that uses the resulting sequence variation information. Here, the sequence variation information obtained from them is the base sequence or amino acid sequence of the known allele (B * 5401) and the new allele (B * 54V) as shown in FIG. 1 and FIG. It is the variation information on the sequence obtained by comparing. For example, in the case of an amino acid sequence, the case where the 114th amino acid is changed from asparagine to aspartic acid and the 116th amino acid is replaced from leucine to serine on the amino acid sequence of B * 5401, as described above. It is done.

Comparison of nucleotide sequences of known allele (B * 5401) and HLA-B54 new allele (B * 54V). Comparison of amino acid sequences of known alleles (B * 5401) and HLA-B54 novel alleles (B * 54V).

Claims (4)

  A novel allele of HLA-B54 encoding the amino acid sequence of SEQ ID NO: 1. A novel allele of HLA-B54 having the base sequence of SEQ ID NO: 2 or its complementary sequence.   A protein having a peptide encoded by the novel allele of HLA-B according to claim 1 or 2. A method for typing an HLA-B54 antigen, which comprises using the amino acid sequence of SEQ ID NO: 1, the base sequence of SEQ ID NO: 2 or a complementary sequence thereof, or sequence variation information obtained therefrom when determining a genotype.

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