JP5416962B2 - How to determine the risk of developing photosensitivity to drugs - Google Patents

Description

  The present invention relates to a method for determining the risk of developing photosensitivity caused by a drug comprising detecting a specific gene polymorphism, a method for selecting a patient to be administered with a drug associated with photosensitivity as a side effect, and the method described above. The present invention relates to a kit for determining the risk of developing photosensitivity caused by a drug containing the oligonucleotide.

  5-Methyl-1-phenyl-2- (1H) -pyridone (generic name: pirfenidone) is a drug intended for pulmonary fibrosis. Various effects have been reported so far for pirfenidone. For example, 1) it shows a therapeutic effect on fibrosis in the lungs, arteriosclerotic lesions, etc., and JP-A-2-215719 shows that 2) its analogs have the same action. 3) U.S. Pat. No. 3,974,281, U.S. Pat. No. 4,042,699, and U.S. Pat. No. 4,052,509 are useful for treating inflammatory conditions in the respiratory tract and skin. No. 11-512699 discloses that 4) suppresses the synthesis and release of TNF-α. In addition, International Publication WO02 / 060446 discloses a tablet containing 5-methyl-1-phenyl-2- (1H) -pyridone as a main drug, masking odor and bitterness, and improving light stability. Has been.

  As one of the side effects of pirfenidone, photosensitivity has been reported in some patients (Arata Azuma et al., American Jounal of Respiratory and Critical Care Medicine, Vol 171, pp1040-1057, 2005). In photosensitivity, eczema and hives develop when the skin is exposed to sunlight. However, the presence or absence of photosensitivity varies from patient to patient. If the risk of developing photosensitivity can be determined before administration of pirfenidone, patients to be administered pirfenidone can be selected, but such a determination method has not been reported so far.

Arata Azuma et al., American Jounal of Respiratory and Critical Care Medicine, Vol 171, pp1040-1057, 2005 JP-A-2-215719 JP-T 8-510251 US Pat. No. 3,974,281 U.S. Pat. No. 4,042,699 US Pat. No. 4,052,509 Japanese National Patent Publication No. 11-512699 International Publication WO02 / 060446

  An object of the present invention is to identify a genetic polymorphism associated with the risk of developing photosensitivity due to a drug, and to use that genetic polymorphism to develop the risk of developing photosensitivity due to a drug such as pirfenidone. It is to provide a method for determining Still another object of the present invention is to provide a method for selecting a patient who administers a drug associated with photosensitivity as a side effect, an oligonucleotide that can be used in a method for determining the risk of developing photosensitivity caused by the drug, and It is to provide a kit for determining the risk of developing photosensitivity caused by a drug.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have succeeded in identifying a genetic polymorphism associated with the risk of developing photosensitivity caused by pirfenidone and completing the present invention. It came.

  That is, according to the present invention, CRISP2 (cystein-rich secretpory protein 2) gene, CRISP3 (cystein-rich secretpory protein 3) gene, GOLSYN (golgi-localized protein) gene, MN1 (meningioma 1) gene, and KCNIP4 (Kv There is provided a method for determining the risk of developing photosensitivity caused by a drug, comprising detecting at least one gene polymorphism present in a gene selected from a channel interacting protein 4) gene.

  Preferably, according to the present invention, CRISP2 (cystein-rich secretpory protein 2) gene, CRISP3 (cystein-rich secretpory protein 3) gene, GOLSYN (golgi-localized protein) gene, MN1 (meningioma 1) gene, and KCNIP4 (Kv A method for determining the risk of developing photosensitivity caused by a drug, comprising detecting at least one single nucleotide polymorphism present in a gene selected from channel interacting protein 4) gene is provided.

Preferably, according to the present invention, the risk of developing photosensitivity caused by a drug, comprising detecting at least one single nucleotide polymorphism selected from the group consisting of (1) to (5) below is preferably determined. A method is provided.
(1) T / C polymorphism at the −161th base (161st position from the 3 ′ end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) T / C polymorphism at the + 14184th base (14184th from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) T / C polymorphism at the −8320th base (the 8320th from the 3 ′ end) of the first intron of the MN1 (meningioma 1) gene;
(4) A / G polymorphism at the base of the first intron of the KCNIP4 (Kv channel interacting protein 4) +8504 position (8504 position from the 5 ′ end): and (5) From (1) to (4 ) And a polymorphism in linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more.

  Preferably, the drug is pirfenidone.

  According to the present invention, there is further provided a method for selecting a patient to administer a drug, comprising selecting a patient to administer the drug based on the risk of developing photosensitivity caused by the drug obtained by the method of the present invention. Is provided.

According to the present invention, it can further hybridize to a sequence of at least 10 bases including at least one site selected from the group consisting of the following (1) to (5), or a complementary sequence thereof, as described above. Oligonucleotides are provided for use as probes in the methods of the invention.
(1) -161th position (161th position from the 3 'end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) the + 14184th position (the 14184th position from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) the -8320th position of the first intron of the MN1 (meningioma 1) gene (the 8320th position from the 3 'end);
(4) the + 8504th position (8504th counted from the 5 ′ end) of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene: and (5) the above described in any one of (1) to (4) A polymorphic site in a linkage disequilibrium state where the polymorphism and linkage disequilibrium coefficient D 'is 0.8 or more.

According to the present invention, it is possible to further amplify a sequence of at least 10 bases including at least one site selected from the group consisting of the following (1) to (6) and / or a complementary sequence thereof, An oligonucleotide used as a primer in the method of the present invention is provided.
(1) -161th position (161th position from the 3 'end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) the + 14184th position (the 14184th position from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) the -8320th position of the first intron of the MN1 (meningioma 1) gene (the 8320th position from the 3 'end);
(4) the + 8504th position (8504th counted from the 5 ′ end) of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene: and (5) the above described in any one of (1) to (4) A polymorphic site in a linkage disequilibrium state where the polymorphism and linkage disequilibrium coefficient D 'is 0.8 or more.

  Preferably, the primer is a forward primer and / or a reverse primer.

According to the present invention, there is further provided a kit for determining the risk of developing photosensitivity caused by a drug, comprising one or more of the oligonucleotides described above.
Preferably, the drug is pirfenidone.

  According to the method of the present invention, the risk of developing photosensitivity caused by a drug such as pirfenidone can be determined accurately and quickly.

[1] Method for Determining Risk of Photosensitivity Due to Drug The method of the present invention is a gene polymorphism present in a specific gene that is related to the onset of drug photosensitivity, particularly a single nucleotide polymorphism. This is a method for determining the risk of developing photosensitivity caused by a drug by detecting (SNPs).

  The above-mentioned specific genes are CRISP2 (cystein-rich secretpory protein 2) gene, CRISP3 (cystein-rich secretpory protein 3) gene, GOLSYN (golgi-localized protein) gene, MN1 (meningioma 1) gene, and KCNIP4 (Kv channel interacting protein 4) A gene selected from genes, and gene polymorphisms exist in exons or introns of genomic DNA containing this gene.

  In the present invention, “detecting at least one gene polymorphism (such as a single nucleotide polymorphism) present in a gene” means (i) directly detecting the gene polymorphism (referred to as gene-side polymorphism), and (ii) It refers to both detecting a gene polymorphism (referred to as a complementary polymorphism) present on the complementary sequence side of the gene and estimating the gene polymorphism from the detection result. However, it is more preferable to directly detect the gene polymorphism because the base on the gene side and the base on the complementary sequence side are not necessarily in a completely complementary relationship.

The single nucleotide polymorphisms to be detected in the present invention include CRISP2 (cystein-rich secretpory protein 2) gene, CRISP3 (cystein-rich secretpory protein 3) gene, GOLSYN (golgi-localized protein) gene, MN1 (meningioma 1) Genes and gene polymorphisms present in genes selected from the KCNIP4 (Kv channel interacting protein 4) gene, and more specifically, selected from the group consisting of (1) to (5) below Examples include at least one single nucleotide polymorphism.
(1) T / C polymorphism at the −161th base (161st position from the 3 ′ end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) T / C polymorphism at the + 14184th base (14184th from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) T / C polymorphism at the −8320th base (the 8320th from the 3 ′ end) of the first intron of the MN1 (meningioma 1) gene;
(4) A / G polymorphism at the base of the first intron of the KCNIP4 (Kv channel interacting protein 4) +8504 position (8504 position from the 5 ′ end): and (5) From (1) to (4 ) And a polymorphism in linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more.

  The base sequence of CRISP2 (cystein-rich secretpory protein 2) gene is known and is registered, for example, with GeneID: 7180 in The National Center for Biotechnology Information (NCBI). The base sequence of CRISP3 (cystein-rich secretpory protein 3) gene is known, and is registered, for example, with NCBI as GeneID: 10321. The base sequence of the GOLSYN (golgi-localized protein) gene is known, and is registered, for example, with NCBI as GeneID: 55638. The base sequence of the MN1 (meningioma 1) gene is known, and is registered, for example, with NCBI as GeneID: 4330. The base sequence of the KCNIP4 (Kv channel interacting protein 4) gene is known, and is registered, for example, with NCBI as GeneID: 80333.

  The position of the T / C polymorphism in the −161th base of the 6th intron of the CRISP2 (cystein-rich secretpory protein 2) gene is the 1136th base (from the 3 ′ end side) of the base sequence shown in SEQ ID NO: 1. Corresponds to the 161st base). The base sequence described in SEQ ID NO: 1 is the base sequence of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene.

  The position of the T / C polymorphism at the + 14184th base of the second intron of the GOLSYN (golgi-localized protein) gene corresponds to the 4185th base of the base sequence described in SEQ ID NO: 2. The base sequence described in SEQ ID NO: 2 is the base sequence of the 10,000th to 15000th bases of the second intron of the GOLSYN (golgi-localized protein) gene.

  The position of the T / C polymorphism in the −8320th base of the first intron of the MN1 (meningioma 1) gene corresponds to the 1348th base of the base sequence described in SEQ ID NO: 3. The base sequence described in SEQ ID NO: 3 is the base sequence from the 36000th base of the first intron of the MN1 (meningioma 1) gene to the 3 'terminal base. The -8320th base (the 8320th counting from the 3 'end) of the first intron of the MN1 (meningioma 1) gene corresponds to the 37347th base of the first intron of the MN1 (meningioma 1) gene.

  The position of the A / G polymorphism at the + 8504th base of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene corresponds to the 3505th base of the base sequence described in SEQ ID NO: 4. The base sequence described in SEQ ID NO: 4 is the base sequence from the 5000th position to the 10,000th position of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene.

  In the present invention, when the −161th base (161th counted from the 3 ′ end) of the sixth intron of the CRISP2 gene is T, the + 14184th (14184 counted from the 5 ′ end) of the second intron of the GOLSYN gene. ) Base is C, the −8320th base of the first intron of the MN1 gene (the 8320th base from the 3 ′ end) is T, and the + 8504th of the first intron of the KCNIP4 gene (5 If the base (the 8504th from the end) is A, it can be determined that the risk of developing photosensitivity due to the drug is high.

  On the other hand, when the base of the 161st intron of the 6th intron of the CRISP2 gene is C (161st counted from the 3 'end), the + 14184th (the 14184th counted from the 5' end) of the 2nd intron of the GOLSYN gene ) Is T, when the −8320th base of the first intron of the MN1 gene is 8320 (8320th from the 3 ′ end), and when the base is +8504 (5 ′ of the first intron of the KCNIP4 gene) When the 8504th base from the end is G, it can be determined that the risk of developing photosensitivity due to the drug is low.

Furthermore, in the present invention, the polymorphism described in any of (1) to (4) above and a polymorphism in a linkage disequilibrium state having a linkage disequilibrium coefficient D ′ of 0.8 or more can be used. Linkage disequilibrium refers to inheriting linked to each other at a higher frequency than when two alleles are inherited independently. The SNP markers are maintained in linkage disequilibrium within an average of 22 kb in Asians including Japanese and Europeans. It has also been reported that linkage disequilibrium is maintained within an average of 11 kb in Africans. Furthermore, a group of alleles exhibiting such linkage disequilibrium is called a haplotype. If there are multiple SNPs at a given locus, the combination of polymorphisms varies from individual to individual. This combination is a so-called haplotype marker and represents individual diversity. Such haplotype markers can be used to correlate genetic information of a subject with a predisposition to the development of photosensitivity caused by a drug. The linkage disequilibrium coefficient D ′ is defined as four haplotypes (AB, Ab, aB), with each allele of the first SNP (A, a) and each allele of the second SNP (B, b) for two SNPs. , Ab), where P _AB , P _Ab , P _aB , P _ab are obtained by the following equation.
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 to take the smaller value. ]

  In the present invention, preferably, a polymorphism having a linkage disequilibrium coefficient D ′ of 0.8 or more, more preferably 0.95 or more, further preferably 0.99 or more, and most preferably 1 can be used.

  In this specification, “determination” of the risk of developing photosensitivity caused by a drug means determination of the presence or absence of the development of photosensitivity, determination of the possibility of development of photosensitivity, genetic factors of photosensitivity It means elucidation.

  In addition, the “determination” of the risk of developing photosensitivity due to a drug may be determined by the results of the single nucleotide polymorphism detection method described above and, if desired, other polymorphism analysis (VNTR or RFLP) and / or other test results. Can also be performed.

  In the present specification, the “drug” is not particularly limited as long as it is a drug that may cause photosensitivity due to a side effect, but pirfenidone is particularly preferable.

(Detection target)
The target of detection of a genetic polymorphism is preferably genomic DNA, but in some cases (ie, when the polymorphic site and its adjacent region are identical or completely complementary to the genome), cDNA or mRNA is used. You can also. Examples of the sample from which the subject is collected include arbitrary biological samples such as blood, bone marrow fluid, semen, peritoneal fluid, urine and other body fluids; tissue cells such as liver; hair such as hair. Genomic DNA and the like can be extracted and purified from these samples according to a conventional method.

(amplification)
In detecting a gene polymorphism, first, a portion containing the gene polymorphism is amplified. Amplification is performed by, for example, the PCR method, but may be performed by other known amplification methods such as the NASBA method, the LCR method, the SDA method, and the LAMP method.
Selection of the primer is, for example, a sequence of at least 10 bases or more, preferably 10 to 100 bases, more preferably 10 to 50 bases including the single nucleotide polymorphism site in the base sequences shown in SEQ ID NOs: 1 to 4. And / or amplifying its complementary sequence.

  The primer may contain one or more substitutions, deletions and additions in the sequence as long as it can function as a primer for amplifying a sequence having a predetermined number of bases including the single nucleotide polymorphism site.

  The primer used for amplification may be selected so that one of the forward primer or the reverse primer hybridizes to the single nucleotide polymorphism site so that it is amplified only when the sample is of one allelic type. The primer can be labeled with a fluorescent substance, a radioactive substance, or the like as necessary.

(Detection of genetic polymorphism)
Detection of a gene polymorphism can be performed by hybridization with a probe specific to one allele type. The probe may be labeled with an appropriate means such as a fluorescent substance or a radioactive substance, if necessary. The probe is not particularly limited as long as it contains the above-mentioned single nucleotide polymorphic site, hybridizes with a test sample, and gives a specificity that can be detected under the detection conditions employed. As the probe, for example, in the sequence shown in SEQ ID NOs: 1 to 4, at least 10 bases or more, preferably a sequence of 10 to 100 bases, more preferably a sequence of 10 to 50 bases including the single nucleotide polymorphism site, Alternatively, oligonucleotides that can hybridize to their complementary sequences can be used. In addition, it is preferable to select the oligonucleotide so that the single nucleotide polymorphism site is present at substantially the center of the probe. As long as the oligonucleotide can function as a probe, i.e. hybridizes with a sequence of interest allelic but does not hybridize with other allelic sequences, One or more substitutions, deletions, additions may be included. In addition, probes such as a single-stranded probe (padlock probe) used for amplification by the RCA (rolling circle amplification) method are annealed with genomic DNA and become circular, thereby satisfying the above probe conditions. included.

  Hybridization conditions used in the present invention are conditions sufficient to distinguish allelic types. For example, the conditions are such that the sample hybridizes if it is one allelic type but does not hybridize if it is another allelic type, eg, stringent conditions. Here, examples of the “stringent conditions” include conditions described in, for example, Molecular Cloning a Laboratory Manual Second Edition (Sambrook et al., 1989). Specifically, for example, 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhart and 100 mg / ml herring sperm DNA For example, a condition of incubating overnight at 65 ° C. with a probe in a solution containing.

  The probe can also be used as a DNA chip with one end fixed to a substrate. In this case, only a probe corresponding to one allele type may be fixed to the DNA chip, or probes corresponding to both allele types may be fixed.

  The detection of gene polymorphism can also be performed by restriction fragment length polymorphism (RFLP). In this method, the sample nucleic acid is digested with a restriction enzyme that is cleaved by a restriction enzyme depending on which genotype the single nucleotide polymorphic site takes, and the size of the digest fragment is examined. To determine whether the sample nucleic acid has been cleaved by the restriction enzyme, and thereby analyze the polymorphism of the sample.

  Genetic polymorphism may be detected by directly sequencing the amplification product (direct sequencing method). The sequencing can be performed by a known method such as dideoxy method or Maxam-Gilbert method.

  Genetic polymorphism may be detected by an invader assay. In this method, an invader oligo having a complementary sequence to a DNA target fragment to be tested for the presence of SNP and a 5 ′ flap structure and a complementary oligo (signal probe) for detecting SNP are used. First, an invader oligo and a signal probe are hybridized to the target DNA. At this time, the invader oligo and the probe have an invasive structure in which one base overlaps. Cleavase (a flap endonuclease isolated from Archaeoglobus fulgidus) acts on this part. When the base of the signal probe at the SNP site and the target base are complementary (no SNP), the 5 'flip of the signal probe is cleaved. Is done. The cut 5 'flip hybridizes to a FRET Probe (Fluorescence resonance energy transfer probe). A fluorescent dye and a quencher are close to each other on the FRET probe, and the fluorescence is suppressed. However, when the 5 ′ flip DNA is bound, the fluorescent dye part is cleaved by Cleavase, and the fluorescent signal can be detected. .

  The detection of gene polymorphism also includes denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP), allele-specific PCR (allele-specific PCR). ), Hybridization method using ASO (allele-specific oligonucleotide), chemical cleavage of mismatches (CCM), heteroduplex method (HET) method, primer extension (PEX) method, rolling circle amplification (RCA) method Etc. can be used.

[2] Kit for determining the risk of developing photosensitivity caused by a drug The oligonucleotide as the primer or probe described above is used as a kit for determining the risk of developing photosensitivity caused by a drug containing the drug. The kit that can be provided may contain a restriction enzyme, a polymerase, a nucleoside triphosphate, a label, a buffer, and the like used in the above-described method for analyzing a gene polymorphism.

  The present invention will be described with reference to the following examples, but the present invention is not limited to such examples. Unless otherwise specified, the following examples are described in Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience). The method used was used.

Example 1 Search for related SNPs “Pirfenidone (S-7701) for idiopathic pulmonary fibrosis” Phase III clinical study and documented consent for blood sampling for genetic analysis In pulmonary fibrosis patients, 54 patients who developed photosensitivity due to pirfenidone (S-7701) and 42 non-developed patients were studied.

First, DNA extraction was performed at a contract facility using peripheral blood obtained from the above-mentioned patients with and without photosensitivity. Using this DNA sample, genotype determination was performed by using an array for SNP analysis Sentrix ^™ HumanHap300 Genotyping BeadChip by Illumina's BeadArray method. The SNP to be analyzed was selected by Illumina based on data from the international HapMap project.

  Genotyping was performed on 307570 SNP loci in each of 54 patients with photosensitivity caused by pirfenidone (S-7701) and 42 patients without disease. Next, a genetic statistical analysis was performed using a 2 × 2 contingency table, and SNPs that showed a significant difference in genotype frequency or allele frequency between the photosensitivity patient group and the non-onset patient group were selected. (Relevant analysis). The related analysis was performed according to reports of Devlin B et al. Genomics, 29, 311-322 (1995) and Nielsen DM et al. Am J Hum Genet, 63, 1531-1540 (1998).

  The results are shown in Table 1. The distribution of genotypes at the 19 SNP locus was strongly associated with the development of photosensitivity due to S-7701. In the table, 11 indicates a homozygote of a major allele, 12 indicates a heterozygote, and 22 indicates a homozygote of a minor allele.

  Of the 19 SNPs, those showing an Odds ratio of 20 or more were selected as SNPs highly related to the disease. Of these, rs515257, rs3133926, rs2267113, and rs6858452, excluding those judged to be linked from the comparison of the case and control values, were further analyzed. rs515257 is the SNP locus (T / C) at the −161st base of the 6th intron of CRISP2 gene, rs3133926 is the SNP locus (T / C) at the + 14184th base of the 2nd intron of the GOLSYN gene, rs2267113 is the MN1 gene The SNP locus (T / C) at the -8320th base of the first intron and rs6858452 were the SNP locus (A / G) at the + 8504th base of the first intron of the KCNIP4 gene. (The position information of the base is counted from the direction in which the numerical value decreases, and + is added when counting from the 5 'side, and-is added when counting from the 3' side). Moreover, the pattern of the allele in each SNP is as follows (Table 2).

Example 2: Construction of Photosensitivity Prediction System Combining 4 SNPs In order to improve the photosensitivity prediction accuracy with pirfenidone (S-7701), the above 4 SNPs, which are considered to be strongly related to diseases, were combined. Analysis was performed. Patients with a risk type genotype for the development of photosensitivity were given 1 point, and patients with a non-risk type genotype were given 0 points. (See Scoring in Figure 1). Next, the scores were summed for each patient, a patient with a total score of 0 was determined as a non-risk type, and a patient with a score of 1 or more was determined as a risk type, and statistical analysis using a 2 × 2 contingency table was performed. As a result, P value was found to be significantly related to 3.9 × 10 ^-15, and it was shown that the onset of photosensitivity caused by pirfenidone (S-7701) can be predicted by combining genotypes in 4 SNPs. It was done.

In addition, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the photosensitivity prediction system for pirfenidone (S-7701) were examined. Specifically, in Table 3 of the prediction results as follows,
Sensitivity = a / (a + b)
Specificity = d / (c + d)
Positive predictive value = sensitivity x incidence / (sensitivity x incidence + (1-specificity) x (1-incidence))
Negative predictive value = specificity x (1-incidence) / (specificity x (1-incidence) + (1-sensitivity) x incidence)
The diagnostic characteristics of the prediction system can be evaluated by the following formula.

  As a result, the sensitivity was 0.81, the specificity was 0.95, the positive predictive value (PPV) was 0.94, and the negative predictive value (NPV) was 0.84 (FIG. 2). . In other words, when this prediction system is used, both the false positive and false negative ratios are expected to be low, and a highly reliable diagnosis can be expected.

Example 3: Search for candidate genes related to the development of photosensitivity
The region around the photosensitivity-related SNPs (rs6935473, rs515257, rs699983, rs699986) in the CRISP2, CRISP3, and PGK2 genes was searched for a more detailed genetic polymorphism by the direct sequence method. Target sites were amplified using PCR primers designed based on GenBank information (accession numbers: NM_003296, NM_006061 and NM_138733) related to the genomic sequence including CRISP2, CRISP3 and PGK2. In designing PCR primers, the REPEATMASKER program (supplied by the University of Washington, http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker, according to the report of Bedell et al., Bioinformatics, 16, 1040-1041 (2000) The repetitive element is excluded from the search target. Genotyping was performed on 54 photosensitivity patients and 42 non-onset patients for SNPs identified by direct sequencing of the search area. Genetic statistical analysis was performed on the difference in genotype frequency or allele frequency between the photosensitivity patient group and the non-onset patient group using a 2 × 2 contingency table. The negative common logarithm (-log (P-value)) of the obtained P value was calculated and plotted against the position of each SNP on the chromosome, thereby obtaining FIG.

  As a result of the analysis, it was shown that an additional photosensitivity-related SNP exists in the region from about 60 kb upstream to about 30 kb downstream of rs6935473 (FIG. 3). This area was almost consistent with the range of linkage disequilibrium blocks based on data from the international HapMap project.

  Therefore, the region important for the development of photosensitivity due to pirfenidone (S-7701) is considered to exist within this 90 kb region including rs6935473. In addition, CRISP2, CRISP3, and PGK2 present in this linkage disequilibrium block are considered to be candidate genes for the onset of photosensitivity caused by pirfenidone (S-7701).

Figure 1 gives 4 points for patients with genotypes of risk type for developing photosensitivity for 4 SNPs, 0 for patients with non-risk type genotypes, and then scores for each patient The results are shown as a result of statistical analysis using a 2 × 2 contingency table in which patients with a total score of 0 are determined as non-risk type and patients with 1 or more points as risk type. FIG. 2 shows the results of examining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the photosensitivity prediction system for pirfenidone according to the present invention. FIG. 3 shows the results of searching for candidate genes associated with the development of photosensitivity.

Claims (6)

A method for determining the risk of developing photosensitivity caused by pirfenidone , comprising detecting at least one single nucleotide polymorphism selected from the group consisting of (1) to (4) below.
(1) T / C polymorphism at the −161th base (161st position from the 3 ′ end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) T / C polymorphism at the + 14184th base (14184th from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) T / C polymorphism at the −8320th base of the first intron of the MN1 (meningioma 1) gene (8320th from the 3 ′ end); and (4) KCNIP4 (Kv channel interacting protein 4) gene. A / G polymorphism at base +8504 of the first intron (8504 from the 5 'end). The screening method of the patient who administers the pirfenidone including selecting the patient who administers the said pirfenidone based on the risk of the onset of photosensitivity by the pirfenidone acquired by the method of Claim 1. The method according to claim 1 or 2 , which can hybridize to a sequence of at least 10 bases including at least one site selected from the group consisting of the following (1) to (4) or a complementary sequence thereof: Used as a probe in.
(1) -161th position (161th position from the 3 'end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) the + 14184th position (the 14184th position from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) -8320th position of the first intron of the MN1 (meningioma 1) gene (8320th position counted from the 3 'end); and (4) + 8504th position of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene. (8504th from the 5 'end). The sequence of at least 10 bases including at least one site selected from the group consisting of the following (1) to (4) and / or a complementary sequence thereof can be amplified, and according to claim 1 or 2 An oligonucleotide used as a primer in the method.
(1) -161th position (161th position from the 3 'end) of the sixth intron of the CRISP2 (cystein-rich secretpory protein 2) gene;
(2) the + 14184th position (the 14184th position from the 5 'end) of the second intron of the GOLSYN (golgi-localized protein) gene;
(3) -8320th position of the first intron of the MN1 (meningioma 1) gene (8320th position counted from the 3 'end); and (4) + 8504th position of the first intron of the KCNIP4 (Kv channel interacting protein 4) gene. (8504th from the 5 'end). The oligonucleotide according to claim 4 , wherein the primer is a forward primer and / or a reverse primer. A kit for determining the risk of developing photosensitivity caused by pirfenidone , comprising one or more of the oligonucleotides according to any one of claims 3 to 5 .