JP4381424B2 - HLA novel gene - Google Patents

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 does not engraft in the patient due to rejection, and conversely, GVHD (Graft-Versus-Host Disease) occurs 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 type of HLA 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 has been a serological procedure performed using antibodies. Due to recent technological innovations, so-called genotyping methods that perform typing based on the sequence of genes encoding HLA molecules have become mainstream. 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 a Sanger method (see, for example, Non-Patent Document 1) in which a sequence is determined by a sequencing reaction. In terms of cost, HLA genotyping uses a method in which a partial sequence is used as a probe or primer, a gene sequence is estimated from its reactivity, and the HLA type is determined (see, for example, Non-Patent Documents 2 and 3). ).

As of January 2006, 66 types of genotypes contained in A24, which is one of the subtypes of HLA-A antigen, have been reported. The most common genotypes except for some regions are A *. 2402 (see, for example, Non-Patent Document 4). However, the present situation is that the existence of allyl has not been sufficiently 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. Allele Frequencies [online], [Search February 1, 2006], Internet <URL: http://www.allelefrequencies.net>

Summary of the Invention

  Accordingly, an object of the present invention is to provide a novel allele contained in A24, which is one of the subtypes of HLA-A antigen.

  The present inventors have now reacted to all probes that are positive for A * 2402 by a method for determining the genotype of the HLA-A gene using a plurality of beads on which an oligonucleotide probe is solid-phased. A gene showing a positive reaction was found. The present invention is based on such knowledge.

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

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

  According to the present invention, detailed typing of the HLA-A antigen becomes possible, which makes it extremely useful not only for determining compatibility at the time of transplantation but also for determining individual susceptibility to diseases.

Detailed description of the invention

The novel allyl according to the present invention is obtained as follows.
Based on the PCR-SSOP (Sequence Specific Oligonucleotide probe) method, which is one of DNA typing methods, using the Luminex xMAP measurement technology (http://www.luminexcorp.com/01_xMAPTechnology/index.html) When the genotype of HLA-A antigen was typed using a WAKFlow HLA typing reagent (manufactured by Yunaga Pharmaceutical Co., Ltd.) that is capable of typing, two types of DNA samples extracted from blood and oral mucosal swabs showed known genotype reactions. Not shown. In these specimens, a positive reaction was observed in 3 probes in addition to a probe set that became positive when the genotype was A * 2402 homozygous.

  When these samples were retyped using the same reagent, all the three types of probes showing reactions other than A * 2402 were near the 114th amino acid (near the 5 ′ end of exon 3) 20 Although it is set to the area | region about a base and it is a part of probe which becomes positive by alleles, such as A * 31, the probe set out of this area did not show a positive reaction.

  The sequence of this region was 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 3, the sequence was confirmed from both sides of the sense strand and antisense strand. As a result, bimodal peaks indicating the presence of two different types of bases in two bases in this region. A sex peak was detected, suggesting that this sample may be a heterozygous allele having an unknown mutation in exon 3 of A * 2402 and A * 2402.

  In order to examine the sequence in detail, a region of about 1 kb from exon 2 to exon 3 of the HLA-A 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-A 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 obtained plasmids, those containing an insert of about 1 kb were selected by restriction enzyme treatment, and the sequences of 13 clones obtained from a blood-derived specimen and 10 clones obtained from a swab-derived specimen were confirmed. .

  As a result, 10 clones of HLA-A * 2402 and 8 clones other than HLA-A were found, and 5 mutations of A * 2402 were found to have mutations in 2 bases suggested by the direct sequencing method. A clone was found.

  As a result of confirming the base sequence of this gene, two mutations were confirmed in exon 3 of the sequence of A * 2402, as shown in FIG. These mutations were non-synonymous substitutions in which the 114th amino acid shown in FIG. 2 was replaced with an amino acid from histidine to glutamine and the 116th amino acid was changed from tyrosine to aspartic acid. These two amino acids are shared among the genotypes contained in A24. In addition, the 114th and 116th amino acids are located inside the region sandwiching the antigenic peptide in the HLA-A molecule.

Thus, these amino acids are also important sites for peptide motifs that bind to HLA-A molecules and are likely to play an important role in the immune system. Therefore, since it is necessary to distinguish between A * 2402 and the genotype having the mutation of the present invention (A * 2402V) in transplantation medicine, the accuracy of typing of HLA-A antigen in the determination of compatibility at the time of organ transplantation, etc. It is extremely useful and important in increasing
Note that the novel allyl found in the present invention is “A * 2462” according to WHO naming. Therefore, in the present specification, the new allele is indicated by either “A * 2402V” or “A * 2462”.

  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 protein having an HLA novel allyl-encoded peptide according to the present invention. The protein having the peptide encoded by the novel allele of HLA according to the present invention is also extremely useful and important in determining compatibility at the time of transplantation as in the case of allyl.

  According to another aspect of the present invention, there is provided a method for typing an HLA-A antigen, wherein the genotype is determined by determining the amino acid sequence of SEQ ID NO: 1, the base sequence of SEQ ID NO: 2 or a complementary sequence thereof, or from them 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 (A * 2402) and the new allele (A * 2402V) 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, in the amino acid sequence of A * 2402, as described above, the 114th amino acid may be replaced from histidine to glutamine, and the 116th amino acid may be replaced from tyrosine to aspartic acid. Can be mentioned.

Comparison of base sequences of known allele (A * 2402) and HLA new allele (A * 2402V). Comparison of amino acid sequences of known alleles (A * 2402) and HLA novel alleles (A * 2402V).

Claims (4)

A novel HLA gene encoding the amino acid sequence of SEQ ID NO: 1. A novel HLA gene having the base sequence of SEQ ID NO: 2 or its complementary sequence. A protein having a peptide encoded by the novel HLA gene according to claim 1 or 2.   A method for typing an HLA-A 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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