WO2015037681A1 - 抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法及び判定用キット - Google Patents
抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法及び判定用キット Download PDFInfo
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Definitions
- the present invention relates to a test method and a determination kit for determining an antithyroid drug-induced agranulocytosis risk. More specifically, anti-thyroid drug-induced agranulocytosis risk, including testing susceptibility polymorphisms for anti-thyroid drug-induced agranulocytosis and determining the risk of anti-thyroid drug-induced agranulocytosis.
- the present invention relates to a test method for determination and a kit for determining an antithyroid drug-induced agranulocytosis risk, comprising a polynucleotide capable of detecting a susceptibility polymorphism to antithyroid drug-induced agranulocytosis.
- Hyperthyroidism is a disease in which a large amount of thyroid hormone is secreted from the thyroid gland, and the cause is mostly Graves' disease. Graves' disease is an autoimmune disease caused by unlimited stimulation of the thyroid by autoantibodies to thyroid-stimulating hormone receptors (TSHceptReceptor Antibody: TRAb), and is characterized by a large number of women.
- TSHceptReceptor Antibody TSHceptReceptor Antibody
- treatment methods for hyperthyroidism are roughly divided into three types: pharmacotherapy, radioisotope therapy, and surgical therapy, and treatment is generally started by drug therapy using an antithyroid drug.
- Antithyroid drugs are drugs that suppress the synthesis of thyroid hormones, and usually the blood concentration of thyroid hormones returns to normal within a few months after taking the drug.
- antithyroid drugs must continue to be taken as long as the causative TRAb is positive, and treatment is often prolonged. Across.
- Agranulocytosis is known as one of the serious side effects of antithyroid drugs.
- Agranulocytosis refers to a state in which the number of granulocytes showing non-specific bactericidal action among leukocytes responsible for immune responses to foreign antigens such as bacteria and viruses is significantly reduced (500 / ⁇ L or less or 100,200 or less) It is estimated that about 70% of agranulocytosis patients develop agranulocytosis due to drugs such as antithyroid drugs (eg, methimazole).
- antithyroid drugs eg, methimazole
- the incidence of antithyroid drug-induced agranulocytosis is about 1 in 300 and is not high.
- the number of granulocytes in the blood is significantly reduced, resulting in an easily infectious state and a fatal pathology caused by a serious infection can occur.
- a doctor may be able to manage the patient's white blood cell count and the amount of leukocyte disease as long as the drug continues to be administered due to its severe side effects despite the low incidence of agranulocytosis.
- the granulocyte count must be monitored regularly and in detail, which is an overburden for both the physician and the patient, especially when the treatment is long term.
- agranulocytosis usually has few subjective symptoms when granulocytes begin to decrease. Therefore, in many cases, the infection has already progressed at the time when the disease is found, and in that case, the administration of the antithyroid drug is stopped and the treatment of hyperthyroidism is interrupted, Appropriate treatment for infectious diseases must be performed, and the patient suffers from double suffering.
- HLA Human Leukocyte Antigen
- MHC human major histocompatibility complex
- HLA genes show the highest degree of polymorphism among functional genes, for example, to test for the presence of disease susceptibility genes in the HLA region by comparing HLA allele frequencies between patient populations and healthy control populations Can do. If the association analysis shows a significant association between a particular HLA allele and the disease, the HLA allele itself determines disease susceptibility, and within an HLA region that is in linkage disequilibrium with the HLA allele There may be other genes that determine this. To date, case-control studies have shown that specific HLA alleles are significantly increased or decreased in patient populations with specific diseases, especially for immune-related diseases Many reports have been made.
- HLA-DRB1 * 08: 03: 02 is an antithyroid drug-induced methimazole It has been reported that it is related to atopic agranulocytosis (Non-patent Document 1). However, since there are only 24 patient samples used in the study and the target genes are limited to HLA class II genes, HLA-DRB1 * 08: 03: 02 is a disease for methimazole-induced agranulocytosis Whether it is a susceptibility gene is uncertain, results need to be revalidated, and in fact have not reached clinical application.
- GWAS Gene Wide Association Study
- SNP Single Nucleotide Polymorphism
- An object of the present invention is to provide a test method and a determination kit for simply and highly accurately determining an anti-thyroid drug-induced agranulocytosis risk.
- the present inventors collected 115 specimens of antithyroid drug-induced agranulocytosis patient specimens having strict and precise clinical information in cooperation with two thyroid hospitals representing Japan.
- a case-control study with GWAS using a human SNP array we attempted to search for genetic factors associated with antithyroid drug-induced agranulocytosis.
- 115 patient specimens were typed using 2,635,435 SNPs on the genome as markers, and as a control group, general Japanese DNA specimens (1,798 specimens) collected in a community-based genome cohort
- HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, and HLA-DRB1 * 08: 02 HLA alleles are antithyroid.
- HLA-B * 39: 01 and HLA-B * 38: 02 were in a linkage disequilibrium relationship with rs41560220
- HLA-DRB1 * 08: 03 and HLA-DRB1 * 08: 02 were in a linkage disequilibrium relationship.
- HLA-B and HLA-DRB1 genes correlated with agranulocytosis, it was possible that these sensitive HLA alleles may have important amino acid residues. Therefore, set-up multiple logistic regression analysis was performed using alignment of HLA amino acid sequences. As a result, the phenylalanine residue at position 116 (B-Phe116) of HLA-B protein and the leucine residue at position 74 (DRB1-Leu74) of HLA-DRB1 protein are strongly associated with antithyroid drug-induced agranulocytosis. Correlated.
- the present invention relates to the following.
- [1] (1) Using a sample derived from a subject, a polymorphism existing in the HLA region, A) a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 1 (G> T * ), B) Polymorphism at the 201st base in the base sequence represented by SEQ ID NO: 2 (C> T * ), C) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 3 (C> T * ), D) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 4 (T * > G), E) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 5 (C> T * ), (However, the parentheses indicate the reference allele> variant allele, * indicates the risk allele, and G, A, T, and C indicate guanine, adenine, thymine, and cyto
- Test method to determine the risk of drug-induced agranulocytosis [2] Polymorph of the above A) or a polymorphism in which the polymorphism and the linkage disequilibrium coefficient D ′ are in a linkage disequilibrium state of D ′ of 0.8 or more, and / or a polymorphism of B) or the polymorphism and linkage disequilibrium coefficient
- the polymorphism in the linkage disequilibrium state with the polymorphism of A) and linkage disequilibrium D 'of 0.8 or more is the amino acid at position 74 of HLA-DRB1 * 08: 03 or HLA-DRB1 * 08: 02 Is a polymorphism at the position that encodes the polymorphism in the linkage disequilibrium state with
- the risk of antithyroid drug-induced agranulocytosis can be determined easily and with high accuracy. Therefore, for patients who are determined to be at high risk for antithyroid drug-induced agranulocytosis, the treatment of hyperthyroidism can result in the development of extremely serious side effects. Can be avoided, and invasive tests such as excessive blood sampling can be avoided for patients who are determined to be at low risk, resulting in safe, secure and accurate hyperthyroidism Treatment of the disease is possible.
- FIG. 1 shows a Manhattan plot showing which SNPs associated with antithyroid drug-induced agranulocytosis are present on the chromosome.
- the P value (-log 10 (P)) of each SNP obtained by GWAS is shown on the vertical axis, and the position on the chromosome (Chromosome) is shown on the horizontal axis.
- FIG. 2 shows an enlarged view of the result of the HLA region in FIG.
- FIG. 3 shows a linkage disequilibrium (LD) map of the human chromosome 6 short arm 6p21.3 region.
- FIG. 4 shows a linkage disequilibrium (LD) map of the human chromosome 6 short arm 6p21.3 region.
- FIG. 5 shows a linkage disequilibrium (LD) map of the human chromosome 6 short arm 6p21.3 region.
- FIG. 6 shows the agranulocytosis odds ratio for the number of risk alleles of the four SNP markers (rs6457580, rs41560220, rs1736959 and rs3135387).
- FIG. 7 shows the association between agranulocytosis and amino acids at positions 74 and 116 in HLA-DRB1 and HLA-B proteins.
- the present invention provides: (1) Using a subject-derived sample, a polymorphism present in the HLA region, A) a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 1 (G> T * ), B) Polymorphism at the 201st base in the base sequence represented by SEQ ID NO: 2 (C> T * ), C) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 3 (C> T * ), D) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 4 (T * > G), E) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 5 (C> T * ), (However, the parentheses indicate the reference allele> variant allele, * indicates the risk allele, and G, A, T, and C indicate guanine, adenine, thymine, and cytosine, respectively) and F) A
- the term “antithyroid drug” is used for the treatment of hyperthyroidism, and is not particularly limited as long as it can suppress the synthesis of thyroid stimulating hormone, but preferably a thionamide-based drug such as carbimazole, methimazole, It means propylthiouracil and the like, more preferably methimazole or propylthiouracil, most preferably methimazole.
- a thionamide-based drug such as carbimazole, methimazole, It means propylthiouracil and the like, more preferably methimazole or propylthiouracil, most preferably methimazole.
- hypothyroidism means a disease in which a large amount of thyroid hormone is secreted from the thyroid gland, and examples include, but are not limited to, Graves' disease, inflammation due to poisons and radiation, and the like.
- the “subject” to be measured is a hyperthyroid patient, preferably a Graves' disease patient, who is taking or is taking an antithyroid drug for the treatment or prevention of the disease A person who has a plan to do.
- the race of the subject is not particularly limited, but is preferably East Asian, and more preferably Japanese.
- sample derived from the subject to be measured in the test method of the present invention is preferably a biological sample containing the genomic DNA of the subject, but the polymorphism to be detected is a non-transcribed region such as a promoter or an intron.
- a biological sample containing mRNA or total RNA may be used instead of genomic DNA.
- the sample is directly used, for example, a living tissue of a subject, specifically, for example, excrement such as feces, urine, sputum, saliva, body fluid such as blood, cells such as oral mucosa and skin, body hair, and the like.
- genomic DNA may be isolated from a biological tissue of a subject by a method well known to those skilled in the art and used.
- genomic DNA can be isolated from a specimen such as blood, saliva, skin, etc. collected from a human by a phenol extraction method or the like.
- a commercially available genomic DNA extraction kit or apparatus may be used.
- anti-thyroid drug-induced agranulocytosis risk means the risk of developing or aggravating agranulocytosis by the administration of anti-thyroid drugs
- anti-thyroid drug-induced “High risk of granulocytosis” means that there is a high possibility of developing agranulocytosis in the future, or a high possibility of becoming severe. Therefore, the test method of the present invention may be performed, for example, to determine the risk of developing agranulocytosis before administration of the antithyroid drug, or agranulocytosis becomes severe during the administration of the antithyroid drug. It may be done to determine the risk of doing.
- the ⁇ HLA region '' means the entire genomic DNA region spanning about 3.6 Mb from the telomere side HCP5P15 gene to the centromere side KIFC1 gene present in the human chromosome 6 short arm 6p21.3 region, It does not mean only a specific gene part.
- polymorphism is a change in one or more bases (substitution, deletion, insertion, transposition, inversion, etc.) on genomic DNA, for example, one base is another base.
- SNP substitution, deletion, insertion, transposition, inversion, etc.
- DIP 1 to several tens of bases deleted or inserted
- VNTR variable number of tandem repeats
- reference allele in polymorphisms such as SNP represents the same base or base sequence as the human genome standard sequence (referred to as RefSeq), and “variant allele” appears as a polymorphism but is identical to RefSeq. Represents something that is not.
- risk allele in polymorphisms such as SNP refers to an allele that increases the risk of antithyroid drug-induced agranulocytosis
- non-risk allele refers to an allele of the risk allele
- single nucleotide polymorphism is SNP database dbSNP [http: //www.ncbi.nlm.nih.] Provided by NCBI [http://www.ncbi.nlm.nih.gov/]. In gov / SNP /], it is indicated by rs number which is reference SNP ID number, and the base position is based on NCBI Genome Reference Consortium Human Build 37 (GRCh37).
- the polymorphism of A) is a gene polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 1, and is represented by ID number: rs6457580 in the SNP database dbSNP provided by NCBI SNP.
- the polymorphism is an SNP in which the base of 32393141 in the genome sequence NC_000006.11 is G> T (reference allele> variant allele; the same applies hereinafter).
- the base sequence represented by SEQ ID NO: 1 represents the genomic DNA sequence of human chromosome 6 of 500 bp before and after the SNP. Case and control studies with GWAS using human SNP arrays showed that the allele frequency of the T allele was significantly higher in the case group than in the control group.
- T allele is a risk allele of antithyroid drug-induced agranulocytosis.
- the polymorphism of B) is a polymorphism at the 201st base in the base sequence represented by SEQ ID NO: 2, and is an SNP represented by ID number rs41560220 in the SNP database dbSNP.
- the polymorphism is an SNP in which the base of 31323875 in the genome sequence NC_000006.11 is C> T.
- the base sequence represented by SEQ ID NO: 2 represents the genomic DNA sequence of human chromosome 6 of 200 bp before and after the SNP. Case and control studies with GWAS using human SNP arrays showed that the allele frequency of the T allele was significantly higher in the case group than in the control group. Thus, the T allele is a risk allele of antithyroid drug-induced agranulocytosis.
- the polymorphism of C) is a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 3, and is an SNP represented by ID number rs1736959 in the SNP database dbSNP.
- the polymorphism is an SNP in which the base of 29782470 in the genome sequence NC — 000006.11 is C> T.
- the base sequence represented by SEQ ID NO: 3 represents a genomic DNA sequence of human chromosome 6 of 500 bp before and after the SNP. Case and control studies with GWAS using human SNP arrays showed that the allele frequency of the T allele was significantly higher in the case group than in the control group. Thus, the T allele is a risk allele of antithyroid drug-induced agranulocytosis.
- the polymorphism of the above D) is a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 4, and is an SNP represented by ID number: rs3135387 in the SNP database dbSNP.
- the polymorphism is an SNP in which the base of 32415109 of the genome sequence NC_000006.11 is T> G.
- the base sequence represented by SEQ ID NO: 4 shows the genomic DNA sequence of human chromosome 6 of 500 bp before and after the SNP. Case and control studies with GWAS using human SNP arrays showed that the allele frequency of the T allele was significantly higher in the case group than in the control group. Thus, the T allele is a risk allele of antithyroid drug-induced agranulocytosis.
- the polymorphism of E) is a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 5, and is an SNP represented by ID number rs17576984 in the SNP database dbSNP.
- the polymorphism is an SNP in which the base of 32212985 in the genome sequence NC_000006.11 is C> T.
- the base sequence represented by SEQ ID NO: 5 represents the genomic DNA sequence of human chromosome 6 of 500 bp before and after the SNP.
- Case and control studies with GWAS using human SNP arrays showed that the allele frequency of the T allele was significantly higher in the case group than in the control group.
- the T allele is a risk allele of antithyroid drug-induced agranulocytosis.
- the polymorphism F) is a polymorphism in any linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more with any of the polymorphisms A) to E).
- the “linkage disequilibrium coefficient D ′” is defined as (A, a) for each of the first polymorphism and (B, b) for each allele of the second polymorphism. If the frequencies of the two haplotypes (AB, Ab, aB, ab) are P AB , P Ab , P aB , P ab , the following formula is obtained.
- D ′ (P AB P ab -P Ab P aB ) / Min [(P AB + P aB ) (P aB + P ab ), (P AB + P Ab ) (P Ab + P ab )] [ Wherein Min [(P AB + P aB ) (P aB + P ab ), (P AB + P Ab ) (P Ab + P ab )] is (P AB + P aB ) (P aB + P ab ) and (P AB + P Ab ) (P Ab + P ab ) means taking the smaller value. ]
- linkage disequilibrium coefficient r 2 may be used as an index representing the linkage disequilibrium state.
- the polymorphism of F) in linkage disequilibrium with any of the polymorphisms of A) to E) can be identified by a method known per se, such as the HapMap database (http://www.hapmap.org/ index.html.ja), etc., or a sequencer can be used to analyze the sequence of multiple sample DNAs to search for SNPs in linkage disequilibrium.
- the F) polymorphism can be easily identified by preparing an LD block by a method known per se using Haploview software (FIGS. 3 to 5).
- the linkage disequilibrium coefficient D ′ of any of the polymorphs of A) to E) above is 0.8 or more, preferably 0.9 or more, more preferably 0.95 or more, still more preferably 0.99 or more, most Preferably, it is in a chain disequilibrium state of 1 (complete chain), or the chain disequilibrium coefficient r 2 is 0.6 or more, preferably 0.8 or more, more preferably 0.9 or more, further preferably 0.95 or more, most preferably 1 (complete chain) ) In the linkage disequilibrium state.
- polymorphisms in linkage disequilibrium state where the linkage disequilibrium coefficient D ′ is 1 (complete linkage) with the polymorphism in A) are rs28362683, rs10947262, rs4959028, rs6930615, rs732162, rs9501626, rs3135392, rs8084, rs2239806, rs7192, rs3129888, rs7195, rs1051336, rs1041885, rs2213586, rs2213585, rs9268832, rs6903608, rs9268877, rs9268880, rs9268979, rs9269110, rs1964995, rs4713555, rs7744001 etc.
- rs2596487 and the like are exemplified as the polymorphism in the linkage disequilibrium state in which the polymorphism in B) and the linkage disequilibrium coefficient D ′ are 1 (complete linkage).
- polymorphisms in the linkage disequilibrium state in which the linkage disequilibrium coefficient D ′ is 1 (complete linkage) with the polymorphism in C) are rs1633041, rs1737041, rs1002046, rs1610644, rs1633011, Examples include rs1630969, rs1632988, rs1632987, rs1736971, rs1736969, rs1610663, rs1610699, rs11753629, rs1736957, rs1620173, rs1619379, rs2735028, rs1049033, and the like.
- rs2395148, rs12524661 and the like are exemplified as polymorphisms in the linkage disequilibrium state in which the polymorphism in D) and the linkage disequilibrium coefficient D ′ are 1 (complete linkage).
- the inspection method of the present invention includes a step of testing at least one polymorphism selected from the group consisting of the above A) to F) (hereinafter also referred to as polymorphism of the present invention).
- testing the polymorphisms of the present invention includes detecting the presence or absence of risk alleles for the polymorphisms of the present invention.
- the step of testing the polymorphism of the present invention includes a step of detecting the presence or absence of a risk allele for the polymorphism of the present invention and a step of detecting the presence or absence of a non-risk allele. .
- Whether the polymorphism of the present invention has a risk allele and / or a non-risk allele can be determined by any polymorphism analysis method known in the art. For example, genomic DNA extracted from a subject's cells or the like is used as a sample, and a nucleic acid containing a continuous base sequence of about 15 to about 500 bases including any of the polymorphic site bases described above is used as a probe. According to the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)), hybridization is carried out while accurately controlling stringency, and only sequences that are completely complementary to the probe are detected.
- the reaction temperature is gradually increased from the denaturation temperature using a method or a mixed probe in which one of the nucleic acid and the nucleic acid in which the polymorphic site in the nucleic acid is replaced with another base is labeled and the other is unlabeled.
- the labeling agent examples include radioisotopes (eg, 32 P), enzymes (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, Fluorescamine, fluorescein isothiocyanate, Cy3, Cy5, etc.), examples of luminescent substances, luminol, luminol derivatives, luciferin, lucigenin, etc.) are used.
- radioisotopes eg, 32 P
- enzymes eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.
- fluorescent substances eg, Fluorescamine, fluorescein isothiocyanate, Cy3, Cy5, etc.
- the detection of the polymorphism is performed by various methods described in, for example, WO 03/023063, such as RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturant concentration gradient gel electrophoresis. Electrophoresis, RNase A cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method , UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method, etc. (see WO 03/023063, page 17, line 5 to page 28, line 20).
- the TaqMan PCR method and the invader method will be described in more detail as representative methods.
- the TaqMan PCR method is a method using PCR using fluorescently labeled allele-specific oligonucleotides (TaqMan probes) and Taq DNA polymerase.
- TaqMan probe an oligonucleotide having a continuous base sequence of about 15 to about 30 bases including any of the above polymorphic site bases is used.
- the probe is labeled with a fluorescent dye such as FAM or VIC at the 5 'end and labeled with a quencher (quenching substance) such as TAMRA at the 3' end, and the quencher absorbs the fluorescence energy as it is. Therefore, fluorescence is not detected.
- Probes are preferably prepared for both alleles and labeled with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is VIC) for batch detection.
- the 3 ′ end is phosphorylated so that a PCR extension reaction from the TaqMan probe does not occur.
- PCR is performed with a primer designed to amplify a partial sequence of genomic DNA containing a region that hybridizes with the TaqMan probe and Taq DNA polymerase, the TaqMan probe hybridizes with the template DNA and simultaneously extends from the PCR primer.
- the hybridized TaqMan probe is cleaved by the 5 ′ nuclease activity of Taq DNA polymerase, the fluorescent dye is released and is not affected by the quencher, and fluorescence is detected.
- the fluorescence intensity increases exponentially by amplification of the template.
- an allele-specific oligonucleotide containing a base at the polymorphic site about 15 to about 30 bases in length; the G allele is FAM, the T allele is VIC and is labeled at the 5 ′ end
- the subject genotype is GG or TT, strong fluorescence intensity of FAM or VIC is observed, and the other fluorescence is almost observed. I can't.
- the subject's genotype is GT, both FAM and VIC fluorescence are detected.
- the invader method does not label the allele-specific oligonucleotide (allele probe) itself, and a sequence that does not complement the template DNA (flap) on the 5 'side of the base of the polymorphic site. And a complementary sequence specific to the template on the 3 ′ side.
- an oligonucleotide having a specific complementary sequence on the 3 ′ side of the polymorphic site of the template (invader probe; the base corresponding to the polymorphic site at the 5 ′ end of the probe is arbitrary)
- the 5 'side has a sequence that can take a hairpin structure, and when the hairpin structure is formed, the sequence that is continuous 3' from the base paired with the 5 'end base is a sequence complementary to the allele probe flap.
- FRET fluorescence resonance energy transfer
- the 5 ′ end of the FRET probe is fluorescently labeled (for example, FAM, VIC, etc.), and a quencher (for example, TAMRA, etc.) is bound in the vicinity thereof, and fluorescence is not detected as it is (hairpin structure).
- a quencher for example, TAMRA, etc.
- the 3 ′ end of the invader probe enters the polymorphic site when the three members complementarily bind.
- the enzyme that recognizes the structure of this polymorphic site cleavase
- the single-stranded part of the allele probe ie, the 5'-side flap from the base of the polymorphic site
- the flap is complementary to the FRET probe.
- the polymorphic site of the flap enters the hairpin structure of the FRET probe.
- the end-labeled fluorescent dye of the FRET probe is released, and the fluorescence is detected without being affected by the quencher.
- An allele probe whose base at the polymorphic site does not match the template is not cleaved by cleavase, but an allele probe that is not cleaved can also hybridize with the FRET probe, and thus fluorescence is detected in the same manner.
- the allele probe that matches the base of the polymorphic site has significantly higher fluorescence intensity than the allele probe that does not match.
- the template DNA is preferably amplified by PCR using a primer capable of amplifying a region containing a portion to which the allele probe and invader probe hybridize.
- the test method of the present invention includes a step of determining an antithyroid drug-induced agranulocytosis risk based on the test result of the polymorphism of the present invention. Accordingly, in one embodiment, the step of determining the antithyroid drug-induced agranulocytosis risk of the present invention comprises the step of having the risk allele when the subject has a risk allele for the polymorphism of the present invention. A step of determining that the risk of antithyroid drug-induced agranulocytosis is higher than those who do not.
- the step of determining the risk of antithyroid drug-induced agranulocytosis of the present invention comprises the step of determining whether the polymorphic genotype of the present invention is a homozygote of risk allele, non-risk allele or non-risk allele. The step of determining that the subject has a high risk of antithyroid drug-induced agranulocytosis in the order of a heterozygote with a risk allele and a homozygote with a non-risk allele is included.
- the determination accuracy is improved if the number of polymorphs to be tested is large. Therefore, it is preferable to test two or more polymorphisms selected from the polymorphisms of the present invention to determine the risk of antithyroid drug-induced agranulocytosis. For example, if two polymorphisms of A) and B) above are tested, and both polymorphic genotypes of A) and B) are non-risk allele homozygotes, then antithyroid drug-induced agranulocytosis It can be judged that the risk is extremely low. Conversely, if the polymorphic genotypes of A) and B) are both homozygotes of risk alleles, it can be determined that the risk of antithyroid drug-induced agranulocytosis is extremely high.
- the polymorphism of B) and the polymorphism (rs2596487) in a complete linkage relationship of D ′ 1, that is, the polymorphism of F) and the polymorphism described above E)
- the risk allele homozygote is score 2
- the risk allele heterozygote heterozygote is score 1
- the non-risk allele homozygote is score 0
- the scores of rs2596487 / rs17576984 are in the order of genotypes: 2/2, 2/1, 1/2, 1/1, 2/0, 0/2, 1/0, 0/1, 0/0. It can be determined that the risk of antithyroid drug-induced agranulocytosis is high.
- the polymorphism A) or B it is preferable to test the polymorphism A) or B), and it is most preferable to test two polymorphisms A) and B).
- the step of testing the polymorphism of the present invention only two or more polymorphisms F) may be tested. When more than one polymorphism is detected, it is desirable that they are not fully linked, most preferably not in linkage disequilibrium.
- the polymorphism of the above A) and the polymorphism in the linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more and / or the polymorphism of the above B) are linked.
- Polymorphs in linkage disequilibrium with an unbalance coefficient D ′ of 0.8 or more are tested.
- Examples of the polymorphism in the linkage disequilibrium state in which the polymorphism in A) and the linkage disequilibrium coefficient D ′ are 0.8 or more include, for example, a polymorphism completely linked to the polymorphism in A) described above. It is particularly preferred to test the polymorphism at the position encoding the 74th amino acid (ie Leu) of -DRB1 * 08: 03 and HLA-DRB1 * 08: 02.
- examples of the polymorphism in the linkage disequilibrium state in which the polymorphism of B) and the linkage disequilibrium coefficient D ′ are 0.8 or more include, for example, a polymorphism completely linked to the polymorphism of B) described above. Testing for polymorphisms at positions encoding amino acid at position 116 (ie Phe) or amino acid at position 158 (ie not Ala) of HLA-B * 39: 01 or HLA-B * 38: 02 Particularly preferred.
- HLA-B * 39: 01, HLA-DRB1 * 14: 03, and HLA-DRB1 * 08: 02 are strongly associated with antithyroid agranulopathies. Furthermore, HLA-B * 38: 02 and HLA-DRB1 * 08: 03 also correlate with antithyroid drug-induced agranulation.
- the subject is one or more alleles selected from HLA-B * 39: 01, HLA-DRB1 * 14: 03, HLA-DRB1 * 08: 02, HLA-B * 38: 02 and HLA-DRB1 * 08: 03
- HLA-B * 39: 01 and / or HLA-DRB1 * 14: 03 and / or HLA-DRB1 * 08: 02 alleles the risk of antithyroid drug-induced agranulocytosis is It can be determined to be high.
- the subject is HLA-B * 39: 01, HLA-DRB1 * 14: 03, HLA-DRB1 * 08: 02, HLA-B * 38: 02 and HLA-DRB1 * Test for having one or more alleles selected from 08:03, preferably HLA-B * 39: 01 and / or HLA-DRB1 * 14: 03 and / or HLA-DRB1 * 08: 02 May determine the risk of antithyroid drug-induced agranulocytosis.
- HLA-B * 39: 01, HLA-B * 38: 02, HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, or HLA-DRB1 * 08: 02 HLA-B * 39: 01, HLA-B * 38: 02, HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, or HLA-DRB1 * 08: 02 alleles and other HLA alleles Is not particularly limited as long as it can be distinguished from each other.
- the above-described polymorphism detection method can be used.
- the entire corresponding gene sequence may be analyzed, or only a part of the gene sequence may be analyzed.
- the sample used for the analysis of the HLA allele the same sample as the “sample derived from the subject” described above can be preferably used.
- HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, and HLA-DRB1 * 08: 02 are common in that the amino acid at position 74 of the HLA-DRB1 protein is a polymorph with an amino acid substitution that is Leu. To do.
- HLA-B * 39: 01 and HLA-B * 38: 02 are common in that they are polymorphisms with an amino acid substitution in which the amino acid at position 116 of the HLA-B protein is Phe.
- the present invention also provides (1) Using a sample derived from the subject, the following (a) and / or (b): (A) Whether the amino acid at position 74 of the HLA-DRB1 protein is Leu (b) Whether the amino acid at position 116 of the HLA-B protein is Phe, or whether the amino acid at position 158 is Ala And determining the risk of antithyroid drug-induced agranulocytosis, including the step of determining the risk of antithyroid drug-induced agranulocytosis based on the test results of (2) and (1) Inspection method for; (Hereinafter, it may be referred to as the inspection method (2) of the present invention).
- the test subject of the test method (2) of the present invention is a hyperthyroid patient, preferably a patient with Graves' disease, who is taking or taking an antithyroid drug for the treatment or prevention of the disease A person who has a plan.
- the race of the subject is not particularly limited, and may be, for example, any of Mongoloid, Caucasian, and Niggloid.
- (A) Alleles whose amino acid at position 74 of the HLA-DRB1 protein is Leu are HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, and HLA-DRB1 * 08: 02
- HLA-DRB1 * 08: 09 and the like can be mentioned.
- alleles in which the amino acid at position 116 of the HLA-B protein is Phe include HLA-B * 39: 01 and HLA-B * 38: 02, for example, , HLA-B * 37: 01, HLA-B * 39: 04, HLA-B * 67: 01 and the like. Since the frequency of these risk alleles is not negligible not only in Mongoloid but also in Caucasian and Niggloid, the inspection method (2) of the present invention can be widely used regardless of race.
- the “subject-derived sample” to be measured is a subject in addition to the biological sample containing the genomic DNA, mRNA, and total RNA of the test subject described in the “test method of the present invention”.
- a sample containing HLA-DRB1 and / or HLA-B protein collected from the sample may be used.
- an antibody or aptamer that specifically binds to a partial peptide containing Leu at position 74 of the HLA-DRB1 protein and / or a partial peptide containing Phe at position 116 of the HLA-B protein or Ala at position 158 By detecting a complex of HLA-DRB1 or HLA-B protein and the antibody or aptamer by an immunological or equivalent technique using an antibody or aptamer that specifically binds to a partial peptide ( a) whether the amino acid at position 74 of the HLA-DRB1 protein is Leu, and / or (b) whether the amino acid at position 116 of the HLA-B protein is Phe, or the amino acid at position 158 is Ala You can test for it.
- the subject can be determined to be at high risk for antithyroid drug-induced agranulocytosis.
- the invention is a polymorphism present in the HLA region, A) a polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 1 (G> T * ), B) Polymorphism at the 201st base in the base sequence represented by SEQ ID NO: 2 (C> T * ), C) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 3 (C> T * ), D) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 4 (T * > G), E) Polymorphism at the 501st base in the base sequence represented by SEQ ID NO: 5 (C> T * ), (However, the parentheses indicate the reference allele> variant allele, * indicates the risk allele, and G, A, T, and C indicate guanine, adenine, thymine, and cytosine, respectively) and F) A polymorphism in any of the polymorphism
- the determination kit of the present invention contains a polynucleotide capable of detecting a risk allele in the polymorphism of the present invention.
- a polynucleotide capable of detecting a risk allele in the polymorphism of the present invention has a sequence in which the 501st base in the base sequence represented by SEQ ID NO: 1 is T (A for a complementary strand sequence).
- the 201st base in the base sequence represented by SEQ ID NO: 2 is T (A in the case of a complementary strand sequence). The presence can be detected.
- the determination kit of the present invention preferably contains a polynucleotide capable of detecting a risk allele in the polymorphism of A) and / or B).
- a polynucleotide capable of detecting a risk allele in the polymorphism of A) and / or B in a preferred embodiment of the determination kit of the present invention, the polymorphism in the above A), the polymorphism in the linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more, and / or the polymorphism in the above B)
- Polynucleotides capable of detecting a risk allele in a polymorphism in a linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more are included.
- Examples of the polymorphism in the linkage disequilibrium state in which the polymorphism in A) and the linkage disequilibrium coefficient D ′ are 0.8 or more include, for example, a polymorphism completely linked to the polymorphism in A) described above. It is particularly preferable to include a polynucleotide capable of detecting a polymorphism at a position encoding the 74th amino acid (ie, Leu) of -DRB1 * 08: 03 or HLA-DRB1 * 08: 02.
- examples of the polymorphism in the linkage disequilibrium state in which the polymorphism of B) and the linkage disequilibrium coefficient D ′ are 0.8 or more include, for example, a polymorphism completely linked to the polymorphism of B) described above.
- the determination kit of the present invention preferably further contains a polynucleotide capable of detecting a non-risk allele in the polymorphism of the present invention.
- a polynucleotide capable of detecting a non-risk allele in the polymorphism of the present invention.
- such a polynucleotide has a sequence in which the 501st base is G (C in the case of a complementary strand sequence) in the base sequence represented by SEQ ID NO: 1.
- the 201st base in the base sequence represented by SEQ ID NO: 2 is C (G for complementary strand sequences) The presence can be detected.
- polynucleotide capable of detecting the risk allele and the polynucleotide capable of detecting the non-risk allele are collectively referred to as “polynucleotide capable of detecting the polymorphism of the present invention”.
- the polynucleotide capable of detecting the polymorphism of the present invention is the polymorphism detection method described above, for example, RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturant concentration gradient.
- the primer is designed to specifically amplify a region of genomic DNA (or mRNA) containing the base of the polymorphic site of the present invention. Any thing can be used.
- the polynucleotide capable of detecting the polymorphism of the present invention is a probe
- the probe can hybridize with a region of genomic DNA (or mRNA) containing the base of the polymorphic site of the present invention under stringent conditions. Anything designed as described above may be used.
- stringent conditions refers to a homology of about 90% or more, preferably about 95% or more, particularly preferably about 96, 97, 98, 99% or more, most preferably 100% in the nucleotide sequence. This refers to the conditions under which polynucleotides having can hybridize. The stringency can be adjusted by appropriately changing the salt concentration and temperature during the hybridization reaction and washing, and those skilled in the art can easily set suitable conditions.
- the length of the polynucleotide capable of detecting the polymorphism of the present invention is not particularly limited as long as it can detect a DNA fragment in the HLA region having a continuous base sequence of 10 to 200 including the polymorphic site.
- those skilled in the art can appropriately select the length according to the use of the polynucleotide.
- those having a base length of 10 to 200 bp, preferably 15 to 100 bp, more preferably 15 to 35 bp can be exemplified.
- the length of the DNA that can be amplified by the primer is, for example, 15 to 1000 bp, preferably 20 to 500 bp, more preferably 20 to 200 bp.
- those having a base length of 10 to 200 bp, preferably 15 to 100 bp, more preferably 15 to 35 bp can be exemplified.
- the primer contains an additional sequence suitable for detection of the polymorphism (sequence not complementary to genomic DNA (or mRNA)), for example, a linker sequence. May be included.
- the primer may be an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase).
- the probe may contain an additional sequence suitable for detection of the polymorphism (a sequence not complementary to genomic DNA (or mRNA)).
- the probe used in the Invader probe method may have an additional sequence called a flap at the 5 ′ end of the base at the polymorphic site.
- the probe may be an appropriate labeling agent such as a radioisotope (eg, 125 I, 131 I, 3 H, 14 C, etc.), an enzyme (eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, Cy3, Cy5, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) Also good.
- a radioisotope eg, 125 I, 131 I, 3 H, 14 C, etc.
- an enzyme eg, ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase. , Malate dehydrogenase, etc.
- fluorescent substances eg
- a quencher quenching substance that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM, VIC, etc.).
- the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.
- the probe and / or primer are each separately (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer) at an appropriate concentration (eg, 2 to 20 ⁇ ). 1-50 ⁇ M) and can be stored at about -20 ° C.
- the determination kit of the present invention contains at least one polynucleotide capable of detecting the polymorphism of the present invention (at least one capable of detecting a risk allele). If the number of detected polymorphisms is large, the determination accuracy is high. Therefore, it is preferable to include two or more kinds of polynucleotides capable of detecting the polymorphism of the present invention (at least those capable of detecting risk alleles).
- the polynucleotide capable of detecting the polymorphism of the present invention may be DNA or RNA, and may be single-stranded or double-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA / RNA hybrid. Therefore, when describing a nucleic acid having a certain base sequence in the present specification, unless otherwise specified, a single-stranded nucleic acid having the base sequence, a single-stranded nucleic acid having a sequence complementary to the base sequence, and a hybrid thereof It should be understood that the term is used to encompass all double-stranded nucleic acids.
- the polynucleotide can be synthesized according to a conventional method using an automatic DNA / RNA synthesizer based on, for example, information on each nucleotide sequence represented by SEQ ID NOs: 1 to 5.
- the subject “subject” is a patient with hyperthyroidism, preferably a patient with Graves' disease, who is taking or taking an antithyroid drug for the treatment or prevention of the disease.
- the race of the subject is not particularly limited, but is preferably East Asian, and more preferably Japanese.
- the determination kit of the present invention may further include other components necessary for carrying out the method, depending on the polymorphism detection method.
- the kit when the kit is for polymorphism detection by TaqMan PCR method, the kit includes 10 ⁇ PCR reaction buffer, 10 ⁇ MgCl 2 aqueous solution, 10 ⁇ dNTPs aqueous solution, Taq DNA polymerase (5 U / ⁇ L), It may further include, but is not limited to, a criterion table for risk of antithyroid drug-induced agranulocytosis, instructions describing the kit operation method, and the like.
- the present invention also provides the following (a) and / or (b): (A) The amino acid at position 74 of the HLA-DRB1 protein is Leu (b) The substance capable of identifying that the amino acid at position 116 of the HLA-B protein is Phe, or that the amino acid at position 158 is Ala An antithyroid drug-induced agranulocytosis risk determination kit (determination kit (2) of the present invention) is provided.
- the amino acid at position 74 of the HLA-DRB1 protein is Leu
- the amino acid at position 116 of the HLA-B protein is Phe
- the amino acid at position 158 is Ala
- a polynucleotide that can detect a partial nucleotide sequence including a codon encoding the amino acid at position 158 can be exemplified.
- polynucleotides can be designed and prepared by the same method as described in detail in the above “determination kit of the present invention”.
- Other substances that can identify that the 74th amino acid of the HLA-DRB1 protein is Leu, the 116th amino acid of the HLA-B protein is Phe, or the 158th amino acid is Ala for example, An antibody or aptamer that specifically binds to a partial peptide containing Leu at position 74 of the HLA-DRB1 protein, a partial peptide containing Phe at position 116 or a partial peptide containing Ala at position 158 of the HLA-B protein Antibody or aptamer that specifically binds. Such an antibody or aptamer can be obtained by a known method.
- the subject of the determination kit (2) of the present invention is a hyperthyroid patient, preferably a Graves' disease patient, who is taking or is taking an antithyroid drug for the treatment or prevention of the disease A person who has a plan to do.
- the race of the subject is not particularly limited, and may be, for example, any of Mongoloid, Caucasian, and Niggloid.
- the determination kit (2) of the present invention may further include other components necessary for carrying out the method according to the polymorphism detection method.
- the kit when the kit is for detecting a polymorphism by the TaqMan PCR method, the same components as those in the “determination kit of the present invention” can be included.
- the kit when the kit is for detection of polymorphism with an antibody or an aptamer, the kit may be a reagent, a container or the like usually used in an immunological assay, such as a reaction buffer, a secondary antibody, a labeling substance, Plates and the like can be included as additional components.
- Example 1 Search for SNPs correlated with anti-thyroid drug-induced agranulocytosis (1) Subjects DNA samples of patients diagnosed with anti-thyroid drug-induced agranulocytosis (500 / ⁇ L or less) in Sakai Hospital (Kobe, Japan) A total of 115 cases were collected from 63 cases and 52 cases from Ito Hospital (Tokyo, Japan). Of the 115 cases, 113 were diagnosed with Graves' disease and the other two were painless thyroiditis and thyroid poisoning and were treated with antithyroid drugs. Antithyroid drugs used for treatment were methimazole (95 cases) and propylthiouracil (hereinafter also referred to as PTU, 20 cases).
- DNA samples from 89 Graves' disease patients were obtained from Kyoto University. In this study, approval was obtained from the ethics committee of each research institution, and informed consent was obtained from all subjects.
- GWAS Genome-wide association analysis
- DNA samples from Ito Hospital were used as the case group and 375 DNA samples from the Nagahama Cohort were used as the control group, both of which were Illumina infinium HumanOmni 2.5-8 v1.0 Genotyping was performed using a DNA analysis kit (Illumina). After genotyping using SNP array, DNA sample with call success rate of less than 95% (12 cases), DNA sample with high relative relationship with other specimens (PI_HAT ⁇ 0.35 by PLINK software) (122 cases), principal components DNA samples (5 cases) that were not included in the Asian cluster were excluded from the analysis. As a result, the first set of DNA samples after quality control consisted of 63 case groups and 1,445 control groups, and the second set of DNA samples consisted of 52 case groups and 353 control groups.
- SNP markers we focused on 2,635,435 SNPs common to the two arrays. Of these, call success rate was 95% or more, minor allele frequency in case group or control group was 0.01 or more, Hardy-Weinberg equilibrium test p-value> SNP markers that were 1.0 ⁇ 10 ⁇ 7 were selected. As a result, a total of 1,223,017 SNPs (first set) and 1,246,969 SNPs (second set) were used in the analysis. DNA samples from Graves' disease patients were used to genotype SNPs associated with agranulocytosis by capillary sequencing using a 3730xl DNA analyzer (Life Technologies).
- SNPs which are strongly associated with antithyroid-induced agranulocytosis, are 2 regions in the Class I region (upstream of the HLA-A region and around the HLA-B region) and 2 regions in the Class II region (HLA -2 areas around the DR area) (4 areas in total) (Figure 2).
- HLA genotyping Since all of the SNP markers obtained above are in the HLA region, antithyroid drug-induced agranulocytosis is an HLA gene. It may be related to itself. Therefore, targeting around the HLA-DR region and HLA-B region that were found to be very strongly related, using the patient sample HLA-B, HLA-C, HLA-DRB1, HLA-DPB1 and HLA-DQB1 genes Genotyping was conducted.
- HLA-B, HLA-C, HLA-DRB1, HLA-DPB1 and HLA-DQB1 genes were determined.
- HLA-B, HLA-C, HLA-DRB1, HLA-DPB1, and HLA-DQB1 allele frequency information of 1,000 general Japanese populations were obtained from the NPO HLA Research Institute (Kyoto, Japan).
- HLA alleles with an allele frequency greater than 1% were used for association analysis in either case or control groups.
- HLA-B * 39: 01, HLA-B * 38: 02, HLA-C * 07: 02, HLA-DRB1 * 08: 03, HLA-DRB1 * 14: 03, HLA-DRB1 * 08: 02, HLA-DRB1 * 09: 01 and HLA-DQB1 * 06: 01 showed significant association with antithyroid drug-induced agranulocytosis (Table 10).
- Example 3 Search for amino acids related to sensitive HLA alleles (1) Logistic regression analysis of HLA amino acids The amino acid sequences corresponding to either HLA alleles that were genotyped or obtained from HLA laboratories (http: // www. ebi.ac.uk/ipd/imgt/hla/) and aligned for each HLA gene. A total of 462 amino acid variants were identified in 278 locations. Three-dimensional structural analysis of HLA-DRB1 and HLA-B proteins was performed with UCSF chimera software.
- AIC Akaike Information Criterion
- HLA-B * 39: 01 and HLA-B * 38: 02 have B-Phe116
- HLA-DRB1 * 14: 03 and HLA-DRB1 * 08 : 02 had DRB1-Leu74 (Table 11).
- Rs41560220 in HLA-B and rs6457580 and rs3135387 in HLA-DRB1 showed the same AIC improvement compared to B-Phe116 and DRB1-Leu74, respectively.
- the ratio of the haplotype involved in the onset was analyzed from the frequency of haplotypes in healthy individuals and the above OR. The results are shown in Table 12 below.
- the risk allele homozygote is represented as score 2, the risk allele and non-risk allele heterozygote as score 1, and the non-risk allele homozygote as score 0.
- the case where neither SNP has a risk allele was set as a control.
- geno10 and later genotypes ie geno10, 02, 20, 11, 12, 21, 22
- 10% of patients will receive antithyroid drugs. Will be discontinued, which can reduce the incidence of agranulocytosis by 50%.
- To determine which genotype a patient should stop taking antithyroid drugs is the severity of the disease or complications (especially whether it is causing kidney function decline), the patient's age (whether they are elderly), Appropriate judgment can be made at the discretion of the doctor in consideration of various factors such as gender.
- the risk of antithyroid drug-induced agranulocytosis can be determined easily and with high accuracy. Therefore, for patients who are determined to be at high risk for antithyroid drug-induced agranulocytosis, the treatment of hyperthyroidism can result in the development of extremely serious side effects. Can be avoided, and invasive tests such as excessive blood sampling can be avoided for patients who are determined to be at low risk, resulting in safe, secure and accurate hyperthyroidism Treatment of the disease is possible.
- This application is based on Japanese Patent Application No. 2013-188806 filed in Japan (filing date: September 11, 2013), the contents of which are incorporated in full herein.
Abstract
Description
HLA領域は、染色体のテロメア側からセントロメア側に向かって、主に、(1)HLA-A、B、C抗原系などを支配するクラスI領域、(2)補体成分などを支配するクラスIII領域、及び(3)HLA-DP、DQ、DR抗原系などを支配するクラスII領域に分類される。
現在までに、症例・対照研究(case-control study)によって、特定のHLAアレルが、特定の疾患の患者集団で有意に増加或いは減少していることが明らかにされており、とりわけ免疫関連疾患に関する報告が多くなされている。
具体的には、ゲノム上の2,635,435箇所のSNPをマーカーとして患者検体115例をタイピングし、また対照群としては、地域ベースのゲノムコホートで収集された一般的な日本人のDNA検体(1,798検体)のタイピング情報を用い、各SNPについてジェノタイプ頻度を比較する症例・対照研究を実施した。
その結果、ヒト第6染色体短腕部6p21.3上のヒトHLA領域に、抗甲状腺薬誘発性無顆粒球症と極めて強い関連を示す191個のSNPを同定した。それらは、ClassI領域にある2領域(HLA-A領域上流側及びHLA-B領域周辺)とClassII領域にある2領域(HLA-DR領域周辺の2領域)の合計4領域に大きく分類された。そのうち最も強い関連はHLA-DR領域周辺で得られ(最も強い関連多型:rs6457580)、続いて、HLA-B領域周辺(最も強い関連多型:rs41560220)であった。各領域内において有意差を示すSNPの多くは強い連鎖不平衡の関係にあり、また4領域間では互いに連鎖不平衡は見られず、独立したものであることがわかった。
上記で得られた多型マーカーは全てHLA遺伝子領域に存在するため、抗甲状腺薬誘発性無顆粒球症がHLA遺伝子そのものと関係する可能性がある。そのため、極めて強い関連の見られたHLA-DR領域及びHLA-B領域周辺にターゲットを絞って、患者検体を用いてHLA-B、HLA-C、HLA-DRB1、HLA-DPB1及びHLA-DQB1遺伝子のジェノタイピングを実施した。その結果、HLA-B*39:01及びHLA-B*38:02、並びにHLA-DRB1*08:03、HLA-DRB1*14:03及びHLA-DRB1*08:02のHLAアレルが、抗甲状腺薬誘発性無顆粒球症と相関していた。また、HLA-B*39:01及びHLA-B*38:02はrs41560220と、HLA-DRB1*08:03及びHLA-DRB1*08:02はrs6457580と、それぞれ連鎖不平衡の関係にあった。
HLA-B及びHLA-DRB1遺伝子の両方において複数のアレルが無顆粒球症と相関があったので、これらの感受性HLAアレルには重要なアミノ酸残基が存在する可能性が考えられた。そこで、HLAアミノ酸配列のアライメントを使って、set up多重ロジスティック回帰分析を実施した。その結果、HLA-Bタンパク質の116位のフェニルアラニン残基(B-Phe116)、並びにHLA-DRB1タンパク質の74位のロイシン残基(DRB1-Leu74)が、抗甲状腺薬誘発性無顆粒球症と強く相関した。また、HLA-Bタンパク質における158位におけるアラニン残基の不存在は、B-Phe116の存在と連鎖不平衡(r2=0.92)であることが見出された。
本発明者らは、これらの知見に基づき更に検討を進め、これら多型について試験することにより、抗甲状腺薬誘発性無顆粒球症リスクを判定できることを確認し、本発明を完成するに至った。
[1](1)被験者由来のサンプルを使用し、HLA領域に存在する多型であって、
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法;
[2]上記A)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又はB)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型を試験する工程を含む、上記[1]記載の検査方法;
[3]上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-DRB1*08:03又はHLA-DRB1*08:02の74位のアミノ酸をコードする位置における多型であり、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-B*39:01又はHLA-B*38:02の116位又は158位のアミノ酸をコードする位置における多型である、[2]記載の検査方法;
[4]被験者由来のサンプルがゲノムDNAを含む、上記[1]~[3]のいずれかに記載の検査方法;
[5]被験者が東アジア人である、上記[1]~[4]のいずれかに記載の検査方法;
[6]HLA領域に存在する多型であって、
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型において、リスクアレルを検出し得るポリヌクレオチドを含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット;
[7]上記A)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又はB)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型において、リスクアレルを検出し得るポリヌクレオチドを含んでなる、上記[6]記載のキット;
[8]上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-DRB1*08:03又はHLA-DRB1*08:02の74位のアミノ酸をコードする位置における多型であり、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-B*39:01又はHLA-B*38:02の116位又は158位のアミノ酸をコードする位置における多型である、[7]記載のキット;
[9]非リスクアレルを検出し得るポリヌクレオチドをさらに含む、上記[6]~[8]記載のキット;
[10]上記アレルを検出し得るポリヌクレオチドが、該アレルを含む10~200の連続した塩基配列若しくはその相補鎖配列からなる断片と、ストリンジェントな条件下でハイブリダイズし得るプローブ、及び/又は該断片を増幅し得るプライマーである、上記[6]~[9]のいずれかに記載のキット;
[11]東アジア人に対して、抗甲状腺薬誘発性無顆粒球症リスクを判定するために用いられる、上記[6]~[10]のいずれかに記載のキット;
[12](1)被験者由来のサンプルを使用し、以下の(a)及び/又は(b):
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであるか否か
(b)HLA-Bタンパク質の116位のアミノ酸がPheであるか否か、又は158位のアミノ酸がAlaであるか否か
を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法;
[13]以下の(a)及び/又は(b):
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであること
(b)HLA-Bタンパク質の116位のアミノ酸がPheであること、又は158位のアミノ酸がAlaであること
を同定し得る物質を含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット;
に関する。
(1)被験者由来のサンプルを使用し、HLA領域に存在する多型であって、
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法(以下、本発明の検査方法とも記す。)を提供する。
該サンプルは、例えば、被験者の生体組織、具体的には、例えば、糞便・尿・喀痰・唾液などの排泄物、血液などの体液、口腔粘膜・皮膚などの細胞、体毛などを直接使用してもよく、或いは当業者に周知の方法により被験者の生体組織からゲノムDNAを単離し、これを用いてもよい。例えば、ヒトから採取した血液、唾液、皮膚などの検体からフェノール抽出法などによりゲノムDNAを単離することができる。その際、市販のゲノムDNA抽出キットや装置を用いてもよい。
ヒトSNPアレイを用いたGWASによる症例・対照研究の結果、Tアレルのアレル頻度は、対照群よりも症例群において有意に高いことが示された。ここで、「有意」とは、カイ二乗検定又はフィッシャーの正確確率検定におけるp値が5.0×10-8未満であることを意味する。従って、Tアレルは、抗甲状腺薬誘発性無顆粒球症のリスクアレルである。
ヒトSNPアレイを用いたGWASによる症例・対照研究の結果、Tアレルのアレル頻度は、対照群よりも症例群において有意に高いことが示された。従って、Tアレルは、抗甲状腺薬誘発性無顆粒球症のリスクアレルである。
ヒトSNPアレイを用いたGWASによる症例・対照研究の結果、Tアレルのアレル頻度は、対照群よりも症例群において有意に高いことが示された。従って、Tアレルは、抗甲状腺薬誘発性無顆粒球症のリスクアレルである。
ヒトSNPアレイを用いたGWASによる症例・対照研究の結果、Tアレルのアレル頻度は、対照群よりも症例群において有意に高いことが示された。従って、Tアレルは、抗甲状腺薬誘発性無顆粒球症のリスクアレルである。
ヒトSNPアレイを用いたGWASによる症例・対照研究の結果、Tアレルのアレル頻度は、対照群よりも症例群において有意に高いことが示された。従って、Tアレルは、抗甲状腺薬誘発性無顆粒球症のリスクアレルである。
D'=(PABPab-PAbPaB)/Min[(PAB+PaB)(PaB+Pab),(PAB+PAb)(PAb+Pab)]
[式中、Min[(PAB+PaB)(PaB+Pab),(PAB+PAb)(PAb+Pab)]は、(PAB+PaB)(PaB+Pab)と(PAB+PAb)(PAb+Pab)とのうち、値の小さい方をとることを意味する。]
r2=(PABPab-PAbPaB)2/(PAB+PaB)(PaB+Pab)(PAB+PAb)(PAb+Pab)
TaqMan PCR法は、蛍光標識したアレル特異的オリゴヌクレオチド(TaqManプローブ)とTaq DNAポリメラーゼによるPCRとを利用した方法である。TaqManプローブとしては、上記したいずれかの多型部位の塩基を含む約15~約30塩基の連続した塩基配列からなるオリゴヌクレオチドが用いられる。該プローブは、その5’末端がFAMやVICなどの蛍光色素で、3’末端がTAMRAなどのクエンチャー(消光物質)でそれぞれ標識されており、そのままの状態ではクエンチャーが蛍光エネルギーを吸収するため蛍光は検出されない。プローブは双方のアレルについて調製し、一括検出のために互いに蛍光波長の異なる蛍光色素(例えば、一方のアレルをFAM、他方をVIC)で標識することが好ましい。また、TaqManプローブからのPCR伸長反応が起こらないように3’末端はリン酸化されている。TaqManプローブとハイブリダイズする領域を含むゲノムDNAの部分配列を増幅するように設計されたプライマー及びTaq DNAポリメラーゼとともにPCRを行うと、TaqManプローブが鋳型DNAとハイブリダイズし、同時にPCRプライマーからの伸長反応が起こるが、伸長反応が進むとTaq DNAポリメラーゼの5’ヌクレア-ゼ活性によりハイブリダイズしたTaqManプローブが切断され、蛍光色素が遊離してクエンチャーの影響を受けなくなり、蛍光が検出される。鋳型の増幅により蛍光強度は指数関数的に増大する。
例えば、上記1)の多型の検出においては、多型部位の塩基を含むアレル特異的オリゴヌクレオチド(約15~約30塩基長;GアレルはFAMで、TアレルはVICでそれぞれ5’末端標識し、3’末端はいずれもTAMRAで標識)をTaqManプローブとして用いた場合、被験者のジェノタイプがGG、あるいはTTであれば、それぞれFAMあるいはVICの強い蛍光強度を認め、他方の蛍光はほとんど認められない。一方、被験者のジェノタイプがGTであれば、FAM及びVIC両方の蛍光が検出される。
インベーダー法では、TaqMan PCR法と異なり、アレル特異的オリゴヌクレオチド(アレルプローブ)自体は標識されず、多型部位の塩基の5’側に鋳型DNAと相補性のない配列(フラップ)を有し、3’側には鋳型に特異的な相補配列を有する。インベーダー法では、さらに鋳型の多型部位の3’側に特異的な相補配列を有するオリゴヌクレオチド(インベーダープローブ;該プローブの5’末端である多型部位に相当する塩基は任意である)と、5’側がヘアピン構造をとり得る配列を有し、ヘアピン構造を形成した際に5’末端の塩基と対をなす塩基から3’側に連続する配列がアレルプローブのフラップと相補的な配列であることを特徴とするFRET(fluorescence resonance energy transfer)プローブとが用いられる。FRETプローブの5’末端は蛍光標識(例えば、FAMやVICなど)され、その近傍にはクエンチャー(例えば、TAMRAなど)が結合しており、そのままの状態(ヘアピン構造)では蛍光は検出されない。
鋳型であるゲノムDNAにアレルプローブ及びインベーダープローブを反応させると、三者が相補結合した際に多型部位にインベーダープローブの3’末端が侵入する。この多型部位の構造を認識する酵素(cleavase)を用いてアレルプローブの一本鎖部分(即ち、多型部位の塩基から5’側のフラップ部分)を切り出すと、フラップはFRETプローブと相補的に結合し、フラップの多型部位がFRETプローブのヘアピン構造に侵入する。この構造をcleavaseが認識して切断することにより、FRETプローブの末端標識された蛍光色素が遊離してクエンチャーの影響を受けなくなって蛍光が検出される。多型部位の塩基が鋳型とマッチしないアレルプローブはcleavaseによって切断されないが、切断されないアレルプローブもFRETプローブとハイブリダイズすることができるので、同様に蛍光が検出される。但し、反応効率が異なるため、多型部位の塩基がマッチするアレルプローブでは、マッチしないアレルプローブに比べて蛍光強度が顕著に強い。
通常、3種のプローブ及びcleavaseと反応させる前に、鋳型DNAはアレルプローブ及びインベーダープローブがハイブリダイズする部分を含む領域を増幅し得るプライマーを用いてPCRにより増幅しておくことが好ましい。
それゆえ、別の実施態様において、本発明の抗甲状腺薬誘発性無顆粒球症リスクを判定する工程には、被験者における本発明の多型ジェノタイプがリスクアレルのホモ接合体、リスクアレルと非リスクアレルとのヘテロ接合体、非リスクアレルのホモ接合体の順に、該被験者は抗甲状腺薬誘発性無顆粒球症リスクが高いと判定する工程が含まれる。
本発明の検査方法の好ましい一実施態様においては、上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又は上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が試験される。上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型としては、例えば、上述のA)の多型と完全連鎖した多型等が挙げられるが、HLA-DRB1*08:03やHLA-DRB1*08:02の74位のアミノ酸(即ち、Leu)をコードする位置における多型を試験することが特に好ましい。一方、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型としては、例えば、上述のB)の多型と完全連鎖した多型等が挙げられるが、HLA-B*39:01やHLA-B*38:02の116位のアミノ酸(即ち、Phe)又は158位のアミノ酸(即ち、Alaではない)をコードする位置における多型を試験することが特に好ましい。
被験者がHLA-B*39:01、HLA-B*38:02、HLA-DRB1*08:03、HLA-DRB1*14:03、又はHLA-DRB1*08:02を有するか否かを試験する方法は、HLA-B*39:01、HLA-B*38:02、HLA-DRB1*08:03、HLA-DRB1*14:03、又はHLA-DRB1*08:02アレルとそれ以外のHLAアレルとを区別できる限り特に限定されず、例えば、上記した多型検出方法を使用することができる。
HLAアレルの解析においては、該当する遺伝子配列全体を解析しても、遺伝子配列の一部のみを解析してもよい。また、HLAアレルの解析に用いる試料は、上記した「被験者由来のサンプル」と同様のものを好適に使用することができる。
従って、本発明はまた、
(1)被験者由来のサンプルを使用し、以下の(a)及び/又は(b):
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであるか否か
(b)HLA-Bタンパク質の116位のアミノ酸がPheであるか否か、又は158位のアミノ酸がAlaであるか否か
を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法;
(以下、本発明の検査方法(2)という場合もある)を提供する。
検出対象となる(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであるアレルとしては、上述のHLA-DRB1*08:03、HLA-DRB1*14:03、及びHLA-DRB1*08:02の他、例えば、HLA-DRB1*08:09等が挙げられる。また、(b) HLA-Bタンパク質の116位のアミノ酸がPheである(158位のアミノ酸がAlaでない)アレルとしては、HLA-B*39:01及びHLA-B*38:02の他、例えば、HLA-B*37:01、HLA-B*39:04、HLA-B*67:01等が挙げられる。これらのリスクアレルの頻度はモンゴロイドのみならず、コーカソイドやニグロイドにおいても無視できない程度であるので、本発明の検査方法(2)は、人種を問わず広く使用可能である。
試験の結果、(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであった場合、及び/又は(b)HLA-Bタンパク質の116位のアミノ酸がPheであるか、もしくは158位のアミノ酸がAlaでなかった場合、被験者は抗甲状腺薬誘発性無顆粒球症リスクが高いと判定することができる。
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型(すなわち、本発明の多型)において、リスクアレルを検出し得るポリヌクレオチドを含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット(以下、本発明の判定用キットとも記す。)を提供する。
本発明の判定用キットの好ましい一実施態様においては、上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又は上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型において、リスクアレルを検出し得るポリヌクレオチドが含まれる。上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型としては、例えば、上述のA)の多型と完全連鎖した多型等が挙げられるが、HLA-DRB1*08:03やHLA-DRB1*08:02の74位のアミノ酸(即ち、Leu)をコードする位置における多型を検出し得るポリヌクレオチドを含むことが特に好ましい。一方、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型としては、例えば、上述のB)の多型と完全連鎖した多型等が挙げられるが、HLA-B*39:01やHLA-B*38:02の116位のアミノ酸(即ち、Phe)又は158位のアミノ酸(即ち、Alaではない)をコードする位置における多型を検出し得るポリヌクレオチドを含むことが特に好ましい。
本発明の多型を検出し得るポリヌクレオチドがプライマーである場合、当該プライマーは、本発明の多型部位の塩基を含むゲノムDNA(もしくはmRNA)の領域を特異的に増幅し得るよう設計されたものであればいかなるものであってもよい。本発明の多型を検出し得るポリヌクレオチドがプローブである場合、当該プローブは、本発明の多型部位の塩基を含むゲノムDNA(もしくはmRNA)の領域とストリンジェントな条件下でハイブリダイズし得るよう設計されたものであればいかなるものであってもよい。
本発明のポリヌクレオチドをプライマーとして用いる場合には、10~200 bp、好ましくは15~100 bp、より好ましくは15~35 bpの塩基長を有するものが例示できる。プライマーが増幅することができるDNAの長さは、例えば15~1000 bp、好ましくは20~500 bp、より好ましくは20~200 bpである。
本発明のポリヌクレオチドをプローブとして用いる場合には、10~200 bp、好ましくは15~100 bp、より好ましくは15~35 bpの塩基長を有するものが例示できる。
また、該プライマーは、適当な標識剤、例えば、放射性同位元素(例、125I、131I、3H、14Cなど)、酵素(例、β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素など)、蛍光物質(例、フルオレスカミン、フルオレッセンイソチオシアネート、Cy3、Cy5など)、発光物質(例、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなど)などで標識されていてもよい。
本発明の多型を検出し得るポリヌクレオチドがプローブとして用いられる場合、該プローブは多型の検出に適した付加的配列(ゲノムDNA(若しくはmRNA)と相補的でない配列)を含んでいてもよい。例えば、Invaderプローブ法に用いられるプローブは、多型部位の塩基の5’末端にフラップと呼ばれる付加的配列を有し得る。
また、該プローブは、適当な標識剤、例えば、放射性同位元素(例、125I、131I、3H、14Cなど)、酵素(例、β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素など)、蛍光物質(例、フルオレスカミン、フルオレッセンイソチオシアネート、Cy3、Cy5など)、発光物質(例、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなど)などで標識されていてもよい。或いは、蛍光物質(例、FAM、VICなど)の近傍に該蛍光物質の発する蛍光エネルギーを吸収するクエンチャー(消光物質)がさらに結合されていてもよい。かかる実施態様においては、検出反応の際に蛍光物質とクエンチャーとが分離して蛍光が検出される。
上記プローブ及び/又はプライマーは、各々別個に(或いは可能であれば混合した状態で)水若しくは適当な緩衝液(例、TEバッファーなど)中に適当な濃度(例、2~20×の濃度で1~50μMなど)となるように溶解し、約-20℃で保存することができる。
上記ポリヌクレオチドは、例えば、配列番号:1~5で表される各塩基配列の情報に基づいて、DNA/RNA自動合成機を用いて常法に従って合成することができる。
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであること
(b)HLA-Bタンパク質の116位のアミノ酸がPheであること、又は158位のアミノ酸がAlaであること
を同定し得る物質を含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット(本発明の判定用キット(2))を提供する。
HLA-DRB1タンパク質の74位のアミノ酸がLeuであること、HLA-Bタンパク質の116位のアミノ酸がPheであること、又は158位のアミノ酸がAlaであることを同定し得る物質としては、例えば、HLA-DRB1遺伝子又はmRNA中の、HLA-DRB1タンパク質の74位のアミノ酸をコードするコドンを含む部分ヌクレオチド配列を検出し得るポリヌクレオチド、HLA-B遺伝子又はmRNA中の、HLA-Bタンパク質の116位又は158位のアミノ酸をコードするコドンを含む部分ヌクレオチド配列を検出し得るポリヌクレオチド等を挙げることができる。これらのポリヌクレオチドは、上記「本発明の判定用キット」において詳述したのと同様の方法により、デザインし、調製することができる。
HLA-DRB1タンパク質の74位のアミノ酸がLeuであること、HLA-Bタンパク質の116位のアミノ酸がPheであること、又は158位のアミノ酸がAlaであることを同定し得る他の物質として、例えば、HLA-DRB1タンパク質の74位のLeuを含む部分ペプチドに対して特異的に結合する抗体もしくはアプタマー、HLA-Bタンパク質の116位のPheを含む部分ペプチドもしくは158位のAlaを含む部分ペプチドに対して特異的に結合する抗体もしくはアプタマー等を挙げることができる。そのような抗体もしくはアプタマーは、周知の方法によりそれぞれ取得することができる。
抗甲状腺薬誘発性無顆粒球症と相関するSNPの探索
(1)被験者
抗甲状腺薬誘発性無顆粒球症(500/μL以下)と診断された患者のDNAサンプルを隈病院(神戸、日本)から63例、伊藤病院(東京、日本)から52例の計115例収集した。115例のうち113例はバセドウ病と、残り2例は無痛性甲状腺炎及び甲状腺中毒症と診断され、抗甲状腺薬による治療を受けていた。治療に使用された抗甲状腺薬は、メチマゾール(95例)及びプロピルチオウラシル(以下、PTUとも記す。20例)であった。年齢、性別、顆粒球数などの患者臨床情報は、各機関における甲状腺専門医により収集された。
対照群としては、滋賀県長浜市のゲノムコホート研究(以下、長浜コホートとも記す)で収集された1,937例のDNAサンプルを使用した。本試験に採用した患者の特徴を以下の表1に示す。
GWASは、2つのセットで実施した。第一セットでは、症例群として隈病院から得た63例のDNAサンプルを使用し、Illumina infinium HumanOmni 2.5-8 v1.0 DNA分析キット(Illumina社)を用いてジェノタイピングした。対照群としては、長浜コホートにより得た1,562例のDNAサンプルをIllumina infinium HumanOmni 2.5-4 v1.0 DNA分析キット(Illumina社)によりジェノタイピングした。
第二セットでは、症例群として伊藤病院から得た52例のDNAサンプルを使用し、対照群として長浜コホートより得た375例のDNAサンプルを使用し、いずれもIllumina infinium HumanOmni 2.5-8 v1.0 DNA分析キット(Illumina社)を用いてジェノタイピングした。
SNPアレイを用いたジェノタイピングの後、コール成功率95%未満のDNAサンプル(12例)、他の検体と高い血縁関係(PLINKソフトウェアによるPI_HAT≧0.35)にあるDNAサンプル(122例)、主成分分析によりアジア人クラスターから外れているDNAサンプル(5例)を除外した。結果として、クオリティコントロール後の第一セットのDNAサンプルは、症例群:63例と対照群:1,445例からなり、第二セットのDNAサンプルは、症例群:52例と対照群:353例からなった。
SNPマーカーとしては、2つのアレイに共通する2,635,435個のSNPに着目し、これらのうち、コール成功率95%以上、症例群又は対照群におけるマイナーアレル頻度0.01以上、Hardy-Weinberg平衡検定p値>1.0×10-7であるSNPマーカーを選択した。結果として、計1,223,017個のSNP(第一セット)及び1,246,969個のSNP(第二セット)を解析に使用した。
バセドウ病患者のDNAサンプルは、3730xl DNA分析装置(Life Technologies社)を用いたキャピラリーシーケンシングにより無顆粒球症と関連するSNPをジェノタイピングするために使用した。
2セットのGWAS及び統合評価の統計解析は、PLINKソフトウェア又はRソフトウェアを用いた、カイ二乗検定又はフィッシャーの正確確率検定(Fisher’s exact test)により実施した。試験間の不均一性テストは、ブレスロー・デイ検定(Breslow-Day test)を用いて行った。フィッシャーの正確確率検定は、2×2分割表の期待値に5以下のものがある場合に使用した。HLA領域におけるSNPの独立性を調べるために多重ロジスティック回帰分析を実施した。多重検定におけるボンフェローニ補正(Bonferroni’s correction)後のGWAS有意水準は5.0×10-8に設定した。関連するSNP間の相互作用は、従来公知の方法(Andersson et al., European journal of epidemiology 2005; 20: 575-9)により評価した。SNP間のLD値の解析及び連鎖不平衡地図の作成には、Haploview version 4.1ソフトウェアを用いた。
第一セット(症例群:63例及び対照群:1,445例)及び第二セット(症例群:52例及び対照群:353例)について、症例・対照研究を行ったところ、ヒト第6染色体短腕部6p21.3上のヒトHLA領域に、抗甲状腺薬誘発性無顆粒球症と極めて強い関連を示すSNPが191個同定された(図1)。集団の構造化は観察されなかった。同定した191個のSNPを2つのセットの統合解析により得られたp値、オッズ比(OR)、95%信頼区間(CI)とともに以下の表2~6に示す。
抗甲状腺薬誘発性無顆粒球症と関連するHLAアレルの探索
(1)HLAジェノタイピング
上記で得られたSNPマーカーは全てHLA領域に存在するため、抗甲状腺薬誘発性無顆粒球症がHLA遺伝子そのものと関係する可能性がある。そのため、極めて強い関連の見られたHLA-DR領域及びHLA-B領域周辺にターゲットを絞って、患者検体を用いてHLA-B、HLA-C、HLA-DRB1、HLA-DPB1及びHLA-DQB1遺伝子のジェノタイピングを実施した。
具体的には、WAKFlowシステム(湧永製薬株式会社、大阪、日本)を用いた高解像度(4-digit)ジェノタイピングにより、実施例1記載の抗甲状腺薬誘発性無顆粒球症患者115例についてHLA-B、HLA-C、HLA-DRB1、HLA-DPB1及びHLA-DQB1遺伝子のジェノタイプを決定した。一般的な日本人集団1,000人のHLA-B、HLA-C、HLA-DRB1、HLA-DPB1及びHLA-DQB1アレル頻度情報は、NPO法人HLA研究所(京都、日本)から得た。HLAジェノタイピングでは、症例群又は対照群のいずれかにおいて1%超のアレル頻度を有するHLAアレルを関連解析に使用した。
統計解析では、カイ二乗検定又はフィッシャーの正確確率検定により、症例群と対照群におけるHLAアレルの頻度を比較した。多重ロジスティック回帰分析を実施して、HLAアレルの独立性を検証した。多重検定におけるボンフェローニ補正後の有意水準は、6.9×10-4に設定した。
多重検定におけるボンフェローニ補正後、HLA-B*39:01、HLA-B*38:02、HLA-C*07:02、HLA-DRB1*08:03、HLA-DRB1*14:03、HLA-DRB1*08:02、HLA-DRB1*09:01及びHLA-DQB1*06:01が、抗甲状腺薬誘発性無顆粒球症と有意な関連を示した(表10)。
感受性HLAアレルと関連するアミノ酸の探索
(1)HLAアミノ酸のロジスティック回帰分析
ジェノタイピングした又はHLA研究所から得た、どちらか一方のHLAアレルと対応するアミノ酸配列をIMGTデータベース(http://www.ebi.ac.uk/ipd/imgt/hla/)から検索し、各HLA遺伝子ごとにアライメントした。合計462のアミノ酸のバリアントが278箇所で同定された。HLA-DRB1及びHLA-Bタンパク質の3次元構造解析をUCSF chimeraソフトウェアにより行った。
(2)set up多重ロジスティック回帰分析
HLA-B及びHLA-DRB1遺伝子の両方において、複数のアレルが無顆粒球症と関連があったので、感受性HLAアレルにおける重要なアミノ酸残基が存在すると思われた。そこで、HLAアミノ酸配列のアライメントを使って、set up多重ロジスティック回帰分析を実施した。
赤池情報量基準(AIC)をアミノ酸分析のために算出した。アミノ酸変異は、変異を含むロジスティック回帰モデルが最少のAICを示す場合、他のアミノ酸変異を含むそれらと比較して7より大きいAIC(ΔAIC>7)で、他の変異に対して有意とみなした。並べ替え検定を1,000回実施し、アミノ酸変異によるAICの向上が偶然にもたらされたのかどうかと、有意なアミノ酸変異によるAICの向上は、有意なSNPマーカーによる向上と同等であるかどうかとを評価した。
HLA-DRB1タンパク質の74位のロイシン残基(DRB1-Leu74)は、他のアミノ酸変異に比べて21.0のΔAICと最も強い関連を示した(p=7.9x10-26、並べ替え p=0.001、図7A)。DRB1-Leu74のコンディショニングに続き、116位のフェニルアラニン(B-Phe116)は、14.1のΔAICと2番目に強い関連を示した(p<8.2x10-12、並べ替え p=0.001、図7B)。HLA-Bタンパク質における158位のアラニン残基の不存在は、B-Phe116の存在と連鎖不平衡(r2=0.92)であることが見出された。DRB1-Leu74及びB-Phe116におけるコンディショニング後、他のアミノ酸は有意な関連を示さなかった(p>0.00024、図7C)。重要なことには、HLA-B*39:01及びHLA-B*38:02はB-Phe116を有し、HLA-DRB1*08:03、HLA-DRB1*14:03及びHLA-DRB1*08:02はDRB1-Leu74を有した(表11)。HLA-Bにおけるrs41560220と、HLA-DRB1におけるrs6457580及びrs3135387は、それぞれB-Phe116と、DRB1-Leu74と比較して同等のAIC向上を示した。
本発明のSNPマーカーについてのハプロタイプ別オッズ比とシミュレーション
115人の抗甲状腺薬による無顆粒球症患者と1937人の健常人のサンプルを用いて、本発明のSNPマーカーである、rs41560220とD'=1の完全連鎖の関係にあるrs2596487と、rs17576984のハプロタイプ別オッズ比(OR)をSNPごとの関連解析により解析した。両方のSNPにつきリスクアレルを有する場合には多重ロジスティック回帰分析のORを適用した。1937人の健常人サンプル結果によるSNPのアレル頻度を基に、ハーディワインバーグ平衡が成立するとして、一般健常人集団における各ハプロタイプ頻度の割合を計算した。また、健常人におけるハプロタイプ頻度と上記のORから、当該ハプロタイプが発症に関与する割合を解析した。結果を以下の表12に示す。表12では、リスクアレルのホモ接合体をスコア2、リスクアレルと非リスクアレルとのヘテロ接合体をスコア1、非リスクアレルのホモ接合体をスコア0として表す。また、どちらのSNPについてもリスクアレルを有さない場合を対照として設定した。
本出願は、日本で出願された特願2013-188806(出願日:平成25年9月11日)を基礎としており、その内容はすべて本明細書に包含されるものとする。
Claims (13)
- (1)被験者由来のサンプルを使用し、HLA領域に存在する多型であって、
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法。 - 上記A)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又はB)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型を試験する工程を含む、請求項1記載の検査方法。
- 上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-DRB1*08:03又はHLA-DRB1*08:02の74位のアミノ酸をコードする位置における多型であり、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-B*39:01又はHLA-B*38:02の116位又は158位のアミノ酸をコードする位置における多型である、請求項2記載の検査方法。
- 被験者由来のサンプルがゲノムDNAを含む、請求項1~3のいずれか一項記載の検査方法。
- 被験者が東アジア人である、請求項1~4のいずれか一項記載の検査方法。
- HLA領域に存在する多型であって、
A)配列番号:1で表わされる塩基配列中第501番目の塩基における多型(G>T*)、
B)配列番号:2で表わされる塩基配列中第201番目の塩基における多型(C>T*)、
C)配列番号:3で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
D)配列番号:4で表わされる塩基配列中第501番目の塩基における多型(T*>G)、
E)配列番号:5で表わされる塩基配列中第501番目の塩基における多型(C>T*)、
(但し、カッコ内はリファレンスアレル>バリアントアレルを示し、*はリスクアレルを示し、G、A、T及びCは、それぞれ、グアニン、アデニン、チミン及びシトシンを示す。)及び
F)上記A)~E)のいずれかの多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、
からなる群から選択される少なくとも1つの多型において、リスクアレルを検出し得るポリヌクレオチドを含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット。 - 上記A)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型、及び/又はB)の多型もしくは該多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型において、リスクアレルを検出し得るポリヌクレオチドを含んでなる、請求項6記載のキット。
- 上記A)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-DRB1*08:03又はHLA-DRB1*08:02の74位のアミノ酸をコードする位置における多型であり、上記B)の多型と連鎖不平衡係数D'が0.8以上の連鎖不平衡状態にある多型が、HLA-B*39:01又はHLA-B*38:02の116位又は158位のアミノ酸をコードする位置における多型である、請求項7記載のキット。
- 非リスクアレルを検出し得るポリヌクレオチドをさらに含む、請求項6~8のいずれか一項記載のキット。
- 上記アレルを検出し得るポリヌクレオチドが、該アレルを含む10~200の連続した塩基配列若しくはその相補鎖配列からなる断片と、ストリンジェントな条件下でハイブリダイズし得るプローブ、及び/又は該断片を増幅し得るプライマーである、請求項6~9のいずれか一項記載のキット。
- 東アジア人に対して、抗甲状腺薬誘発性無顆粒球症リスクを判定するために用いられる、請求項6~10のいずれか一項記載のキット。
- (1)被験者由来のサンプルを使用し、以下の(a)及び/又は(b):
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであるか否か
(b)HLA-Bタンパク質の116位のアミノ酸がPheであるか否か、又は158位のアミノ酸がAlaであるか否か
を試験する工程、及び
(2)(1)の試験結果に基づいて、抗甲状腺薬誘発性無顆粒球症リスクを判定する工程
を含む、抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法。 - 以下の(a)及び/又は(b):
(a)HLA-DRB1タンパク質の74位のアミノ酸がLeuであること
(b)HLA-Bタンパク質の116位のアミノ酸がPheであること、又は158位のアミノ酸がAlaであること
を同定し得る物質を含んでなる、抗甲状腺薬誘発性無顆粒球症リスク判定用キット。
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US15/021,401 US20160222453A1 (en) | 2013-09-11 | 2014-09-11 | Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit |
JP2015536634A JP6516128B2 (ja) | 2013-09-11 | 2014-09-11 | 抗甲状腺薬誘発性無顆粒球症リスクを判定するための検査方法及び判定用キット |
EP14843658.7A EP3045541A4 (en) | 2013-09-11 | 2014-09-11 | Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit |
CN201480050278.8A CN105765077B (zh) | 2013-09-11 | 2014-09-11 | 测定抗甲状腺药物诱导的粒细胞缺乏症风险的检测方法以及测定用试剂盒 |
US16/207,650 US20190316199A1 (en) | 2013-09-11 | 2018-12-03 | Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit |
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US16/207,650 Division US20190316199A1 (en) | 2013-09-11 | 2018-12-03 | Test method for evaluating the risk of anti-thyroid drug-induced agranulocytosis, and evaluation kit |
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WO2003023063A1 (fr) | 2001-09-07 | 2003-03-20 | Sankyo Company, Limited | Methode d'estimation du risque d'apparition de diabetes |
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Cited By (2)
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CN105886609A (zh) * | 2015-02-17 | 2016-08-24 | 陈沛隆 | 药物不良反应风险评估方法及其装置 |
CN105886609B (zh) * | 2015-02-17 | 2020-02-21 | 陈沛隆 | 药物不良反应风险评估方法及其装置 |
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EP3045541A4 (en) | 2017-05-10 |
EP3045541A1 (en) | 2016-07-20 |
JPWO2015037681A1 (ja) | 2017-03-02 |
CN105765077B (zh) | 2021-09-28 |
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US20190316199A1 (en) | 2019-10-17 |
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