JP2011097933A - Method for predicting risk of polypoidal choroidal vasculopathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for predicting risk of polypoidal choroidal vasculopathy by detecting a single nucleotide polymorphism associated with the polypoidal choroidal vasculopathy, and a kit used for the detection method. <P>SOLUTION: The method for predicting risk of polypoidal choroidal vasculopathy comprises a process for detecting in vitro alleles of the single nucleotide polymorphism located at position-17 of a nucleotide sequence in at least one nucleotide sequence selected from the group consisting of specific nucleotide sequences or a complementary sequence thereof, in nucleic acid molecules in a sample derived from a subject to be inspected (detection process), and a process for determining if at least one of the detected alleles is a risk allele or not (determination process). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for predicting the risk of polypoidal choroidal vasculopathy. More specifically, the present invention relates to a method for predicting the risk of polypoidal choroidal vasculopathy by detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy, and a kit used for the predicting method.

  Polypoidal choroidal vasculopathy (hereinafter also referred to as PCV) is a disease characterized by the formation of an abnormal vascular network in the choroid and a polypoid lesion at the end. Polypoidal choroidal vasculopathy has been confused for a long time with exudative age-related macular degeneration (hereinafter also referred to as AMD) accompanied by abnormal angiogenesis in the choroid. In recent years, it has been established as a new disease concept.

  In the diagnosis of polypoidal choroidal vasculopathy, by indocyanine green (ICG) fluorescence contrast examination, characteristic vascular abnormalities such as dendritic vascular network and polypoid lesions are observed, and / or by fundus examination, It is required that an orange-red raised lesion be observed consistent with the polypoid lesion. However, these findings are rarely found together, and there may be a problem of differentiation from wet age-related macular degeneration. These findings are found near the optic disc or in the macular region. In addition to the above findings, bleeding or serous retinal pigment epithelium detachment or retinal pigment epithelium atrophy may occur. On the other hand, the frequency of formation of fibrous scars and drusen, which is characteristic of wet age-related macular degeneration, is low. Furthermore, polypoidal choroidal vasculopathy often remits and recurs frequently, but the visual acuity is maintained for a relatively long time (see Non-Patent Documents 1 and 2 above).

  The cause of polypoidal choroidal vasculopathy has not yet been elucidated, but its association with hypertension has been suggested. Although sufficient knowledge about the treatment method has not been obtained, treatment by photodynamic therapy has been attempted. However, even with photodynamic therapy, the abnormal vascular network does not disappear and the risk of recurrence remains.

  On the other hand, a single nucleotide polymorphism is a substitution mutation in which one base changes to another base in the base sequence of an individual's genome. It is a polymorphism that exists at a frequency of 1% or more. Single nucleotide polymorphisms exist in introns, exons, or other genomic regions on genes.

Japan Polypoidal Choroidal Angiopathy Research Association, Journal of the Japanese Ophthalmological Society, 2005, 109, 417-427 Mitsuko Yuzawa et al., Bulletin of Japanese Ophthalmology, 2006, 57, 321-335

  In general, as a test for polypoidal choroidal vasculopathy (PCV), a fundus test, for example, a method of inspecting the fundus after intravenous injection of a fluorescent dye such as ICG is used. The burden on the patient is not necessarily light, such as the mydriatic state persisting for several hours. On the other hand, as described above, photodynamic therapy is performed for the treatment of polypoidal choroidal vasculopathy. However, patients who have progressed in the treatment also have a risk of recurrence, and it is necessary to repeat the treatment. From the viewpoint of reducing the physical, mental and economic burdens on patients, it is extremely important to obtain early diagnosis and early diagnosis in the treatment of polypoidal choroidal vasculopathy. That is, it is an important unresolved need to receive a diagnosis of polypoidal choroidal vasculopathy at an earlier stage or to know the risk of developing polypoidal choroidal vasculopathy in the future.

  On the other hand, the dominant causative gene of polypoidal choroidal vasculopathy has not yet been identified. In addition, even though it cannot explain the involvement of a single gene mutation or polymorphism in the disease, there are many gene mutations or polymorphisms that are relatively mildly involved in polypoidal choroidal vasculopathy, and these are combined. It may be possible to explain the involvement of genetic factors in the development of polypoidal choroidal vasculopathy.

  The present inventors focused on polymorphisms on the genome, particularly single nucleotide polymorphisms, in order to find genes associated with polypoidal choroidal vasculopathy.

  By finding a polymorphism related to polypoidal choroidal vasculopathy, it becomes possible to determine the risk of polypoidal choroidal vasculopathy based on whether or not the polymorphic choroidal vasculopathy is present. In the future, it is possible to know that there is a risk of polypoidal choroidal vasculopathy in the future, or it can be used to assist in the diagnosis of early polypoidal choroidal vasculopathy. That is, by knowing the risk of polypoidal choroidal vasculopathy, the sample provider can take preventive measures for the onset of polypoidal choroidal vasculopathy, and early treatment can be started with an early confirmed diagnosis. Therefore, it is significant to find polymorphisms involved in polypoidal choroidal vasculopathy.

  An object of the present invention is to provide a method for predicting the risk of polypoidal choroidal vasculopathy by detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy, and a kit used for the detection method.

  The present inventors cover known polymorphic sites present on the genome (in particular, autosome) of polypoidal choroidal vasculopathy patients (PCV patients) and non-polypoidal choroidal vasculopathy patients (non-PCV patients). Analysis, a single nucleotide polymorphism related to polypoidal choroidal vasculopathy was found, and furthermore, a risk allele and a non-risk allele that was the opposite allele in the single nucleotide polymorphism were found, and the present invention was completed.

That is, the present invention
[1] A nucleic acid molecule in a sample derived from a subject, which is present in the 17th position of the base sequence in at least one base sequence selected from the group consisting of base sequences represented by SEQ ID NOs: 1 to 70 or a complementary sequence thereof A step of detecting in vitro a single nucleotide polymorphism allele to be detected (detection step), and a step of determining whether at least one of the detected alleles is a risk allele (determination step)
A method for predicting the risk of polypoidal choroidal vasculopathy, comprising:
And [2] the nucleic acid molecule in the subject-derived sample, in at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 or a complementary sequence thereof, in the 17th position of the base sequence The present invention relates to a kit for predicting the risk of polypoidal choroidal vasculopathy, comprising a nucleic acid molecule for detecting an existing single nucleotide polymorphism allele in vitro.

  By analyzing the single nucleotide polymorphism allele of the present invention contained in a genome-derived nucleic acid molecule present in a sample by the method of the present invention, the presence / absence and / or risk of polypoidal choroidal vasculopathy of the sample provider Can be predicted. Based on this risk, the sample provider can take an early diagnosis by taking preventive measures for polypoidal choroidal vasculopathy or regularly seeing it, and can start appropriate treatment at an early stage when the disease does not progress. In addition, according to the method of the present invention, a sample provider suspected of having polypoidal choroidal vasculopathy can be determined to have a high probability of being diagnosed with polypoidal choroidal vasculopathy, and treatment can be started at an early stage. it can.

  The present invention relates to a method for predicting the risk of polypoidal choroidal vasculopathy based on a single nucleotide polymorphism of a gene (hereinafter sometimes referred to as SNP). A step of detecting in vitro a single nucleotide polymorphism allele present in the 17th position of the base sequence in at least one base sequence selected from the group consisting of base sequences or a complementary sequence thereof (detection step); and A method for predicting the risk of polypoidal choroidal vasculopathy, comprising a step of determining whether or not a detected allele is a risk allele (determination step). The present invention finds single nucleotide polymorphisms related to polypoidal choroidal vasculopathy (also referred to as single nucleotide polymorphisms of the present invention), further finds risk alleles and their alleles in the single nucleotide polymorphisms, and uses them In particular, it has a great feature. Therefore, using the risk allele and / or using the odds ratio calculated based on the frequency of the risk allele and its allele as an index, predicting the onset of such polypoidal choroidal vasculopathy It is also possible to test the susceptibility to polypoidal choroidal vasculopathy, and the method can also be used as a method for measuring or determining a polypoidal choroidal vasculopathy onset factor. In the present specification, the polymorphism means that there is diversity in the sequence at a specific position of the genome in a certain biological species, and the site where the polymorphism exists (hereinafter also referred to as polymorphic site) The site on the genome where a single nucleotide polymorphism exists.

  Further, in the present specification, an allele refers to each type having different bases that can be taken at a certain polymorphic site. In this specification, the genotype refers to an arbitrary combination of a specific allele (also referred to as one allele) and an allele opposite to the allele (also referred to as the other allele) at a certain polymorphic site. Furthermore, at a certain polymorphic site, there are three types of genotypes that are the above combinations, and the same allele combination is called a homotype and the different allele combination is called a heterotype.

  In the present specification, an allele that conflicts (also referred to as an allele) refers to another allele that corresponds to the specified allele that constitutes a single nucleotide polymorphism.

  In the present specification, a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy is defined as a single nucleotide polymorphism between a polypoidal choroidal vasculopathy patient (PCV patient) and a non-polypoidal choroidal vasculopathy patient (non-PCV patient). A single nucleotide polymorphism in which the ratio of the frequencies of alleles in a nucleotide polymorphism is statistically significantly different by a certain p value.

  In the present invention, the risk of polypoidal choroidal vasculopathy refers to the risk of polypoidal choroidal vasculopathy. The risk of polypoidal choroidal vasculopathy in the present invention includes the possibility of developing polypoidal choroidal vasculopathy in the future and the likelihood of developing polypoidal choroidal vasculopathy. In the present invention, risk prediction means that the presence or absence of a future risk is determined at the present time, or the magnitude of the future risk is determined at the present time.

  Although details will be described later, in the present invention, the risk allele is such that the odds ratio obtained for each allele of the single nucleotide polymorphism alleles related to the polypoidal choroidal vasculopathy is greater than 1. Say allele. A non-risk allele is an allele whose risk ratio is 1 or less because it is a confrontation allele of a risk allele. Here, the odds ratio obtained for each allele, that is, the odds ratio obtained for one allele is the ratio of the frequency of one allele to the frequency of the other allele (opposite allele) in a PCV patient group. Divided by the ratio of the frequency of the one allele to the frequency of the other allele in the group. That is, the odds ratio obtained for one allele is the reciprocal of the odds ratio obtained for the other allele. Details of the odds ratio will be described later. In this specification, the allele frequency (allele frequency) means the ratio of a specific allele to the whole in a certain group.For example, the allele of each individual belonging to a certain group is determined by the method described in the examples. Can be obtained. Other matters not described in this specification include an introduction to genetic statistics (Naoya Kamatani; Iwanami Shoten, 2007) or bioinformatics 2nd edition (translated by Mount DW, Koji Okazaki et al .; Medical Science International, 2005), etc. You can refer to the book.

  Below, the identification method of the single nucleotide polymorphism relevant to polypoidal choroidal vasculopathy is demonstrated.

  Single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy extract total DNA from the blood of PCV patients diagnosed with polypoidal choroidal vasculopathy and non-PCV patients diagnosed with polypoidal choroidal vasculopathy In addition, using about 500,000 known single nucleotide polymorphisms on the human genome as an index, individual single nucleotide polymorphism alleles are specific to PCV patients or non-PCV patients, more specifically, statistically significant. It was found by analyzing whether or not it exists. Here, the non-PCV patient as a control may be a person who is not polypoidal choroidal vasculopathy. Patients diagnosed as not having polypoidal choroidal vasculopathy and diagnosed with other diseases such as glaucoma and / or cataract may be used as controls. In this case, in order to find a single nucleotide polymorphism specifically associated with polypoidal choroidal vasculopathy, a comparison between patients (group) with a disease used as a control and patients (group) with polypoidal choroidal vasculopathy It is preferable to use a single nucleotide polymorphism that is performed on a disease and is commonly determined to have a significant difference at a certain p value. By using such a single nucleotide polymorphism allele, the risk of polypoidal choroidal vasculopathy can be predicted. Although details will be described in the Examples section, single nucleotide polymorphisms associated with the polypoidal choroidal vasculopathy disclosed in the present invention can be identified by the following method.

(Identification of single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy)
First, total DNA is extracted from the blood of PCV patients diagnosed with polypoidal choroidal vasculopathy and non-PCV patients diagnosed with no polypoidal choroidal vasculopathy. Total DNA in blood can be extracted by any known method, but can be extracted by a known method such as a phenol extraction method (Methods in Enzymology, 152, 181 pages, 1987).

  The type of base in the single nucleotide polymorphism in the extracted DNA sample, that is, the identification of the allele at each polymorphic site is performed by, for example, a method using a solidified probe (microarray) described later, PCR such as TaqMan (registered trademark) method, etc. It can be performed by an arbitrary method such as an applied method. In that case, the probe used for detection can be designed based on the target single nucleotide polymorphism and its peripheral sequence information. In designing, for example, a database of known single nucleotide polymorphisms such as dbSNP provided by the National Center for Biotechnology Information (NCBI) in the United States or SNP information provided by the HapMap project, or Genbank provided by NCBI, etc. It is also possible to refer to information such as sequences obtained from these databases using known database of sequence information. The probe used for detecting a single nucleotide polymorphism can be detected by either a probe complementary to the genomic sense strand or a probe complementary to the antisense strand. There are also commercially available kits that can detect a large number of single nucleotide polymorphism alleles in the same procedure by solidifying a large number of probes capable of detecting single nucleotide polymorphisms existing on the human genome. It is also possible to detect alleles in a sample efficiently. Many of such kits are configured to detect each opposing allele contained in one sample by the same operation, and can determine the genotype. As a suitable example of such a commercially available kit, a microarray type single nucleotide polymorphism analysis kit (Affymetrix, Genechip Human Mapping 250K Nsp / Sty Array (GeneChip (registered trademark) Human Mapping 250K Nsp / Sty Array)) ].

  For single nucleotide polymorphisms related to polypoidal choroidal vasculopathy, alleles present in DNA derived from PCV patients and non-PCV patients are identified by the method described above, and the frequency of each allele is determined between PCV patients and non-PCV patients. And the allele frequency can be determined based on whether or not there is a significant difference in the p value described later.

  For statistical analysis, for example, Fisher's exact test, chi-square test, or Cochran-Armitage trend test can be used. In addition, when it is necessary to correct the increase in risk of occurrence of the first type of error due to repeated comparisons, it can be corrected by a known method such as Bonferroni, Holm, or Benjamini and Hofberg. it can.

Illustrating the case based on Bonferroni's correction, it is related to polypoidal choroidal vasculopathy by dividing the desired significance level, eg 5 × 10 ^-2 significance level, by the number of iterations, ie the number of polymorphic sites used in the analysis Determine the single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy with a risk factor of at most 5 × 10 ^-2 even if the first type of error is considered at the maximum by using the significance level used for SNP selection Can do. A single nucleotide polymorphism lower than the significance level set in this way can be selected as a more preferable single nucleotide polymorphism. However, in the analysis of single nucleotide polymorphisms across the entire genome as in the present invention, Bonferroni correction is overcorrection, and the p value is significantly reduced, so there is a possibility of overlooking single nucleotide polymorphisms related to diseases. It has also been reported to increase (Schymick JC et al., Lancet Neurology. 2007: 6: 322-8, Van Steen K et al., Nature Genetics. 2005: 37: 683-691).

  On the other hand, the method by Benjamini and Hoffberg is a method of suppressing the false discovery rate (FDR) to a desired level in the multiple analysis as a whole even though it is not actually significant (False Discovery Rate; FDR). Benjamini Y, Hochberg T: J. Royal Stat Soc B85 pp289-300, 1995). That is, when statistical analysis is performed on n single nucleotide polymorphisms, p values of single nucleotide polymorphisms not related to a disease are considered to be uniformly distributed in the range of 0 to 1. On the other hand, it is considered that the p value of single nucleotide polymorphisms related to the disease is concentrated around 0. Assume that the ratio of single nucleotide polymorphisms that are truly related to a disease is σ, and the ratio of single nucleotide polymorphisms that are not related to a disease is 1−σ. When a certain significance level α is considered, the expected value of the number of single nucleotide polymorphisms having a p value of not more than the significance level α and not related to a disease is nα (1-σ). If the number of single nucleotide polymorphisms that became significant by statistical analysis is R, the ratio of single nucleotide polymorphisms incorrectly determined, that is, FDR is nα (1-σ) / R. Here, even if the actual value of σ is unknown, it is always nα (1-σ) / R ≦ nα / R, so by setting the value of nα / R to a desired ratio, the actual FDR is It can be suppressed below the desired value. On the other hand, since n and R are values determined by analysis, the value of nα / R can be set to a desired value by varying α. Single nucleotide polymorphisms are arranged in ascending order of p value, and each p value is pi (i is a positive integer). When α = pi, there are i single nucleotide polymorphisms having a p value of pi or less. Among these, the expected value of the ratio of single nucleotide polymorphisms unrelated to the disease is npi (1-σ) / i. Since this value decreases as i decreases, it can be considered that r single nucleotide polymorphisms are associated with the desired disease in order from the smallest p value, where r is the desired FDR.

In addition, academically desirable multiplicity correction methods have not yet been established, but when multiplicity correction is performed using these methods, excessive correction will not be below the significance level adjusted by the correction method. Significance levels can be set at any suitable level in the range. In the case of setting any appropriate level, for example, the significance level when repeatedly analyzing 500,000 single nucleotide polymorphisms is preferably, for example, 1 × 10 ⁻³ , more preferably 1 × 10 ⁻⁴ , and more preferably 1 × 10 ^{− 5} is more preferable, 3 × 10 ⁻⁶ is more preferable, 1 × 10 ⁻⁶ is more preferable, and 3 × 10 ⁻⁷ is more preferable. In general, it is known that the first type of error and the statistical power are inversely proportional. One method of maintaining power while reducing the first type of error is to perform single nucleotide polymorphism analysis in two stages (Skol AD et al., Nature Genetics. 2006: 38: 209-213). For example, first, a large number of single nucleotide polymorphism analyzes over the entire genome are performed as a primary analysis, and then an analysis is performed using a single nucleotide polymorphism narrowed down to some extent in the primary analysis as a secondary analysis. In this case, a single nucleotide polymorphism that has a relatively low p value in any analysis may be selected. Preferably, a single nucleotide polymorphism that is a candidate in the first analysis is selected as a desired p value, for example, 0.001 to What is less than 0.05 is selected, and the selected single nucleotide polymorphism may be further analyzed in consideration of multiplicity.

  In addition, in a certain region on the genome, there may be a region where SNPs that are associated with diseases are continuously present with a relatively low p value. SNPs belonging to such areas are highly related to diseases. On the other hand, a region in which single nucleotide polymorphisms related to a disease are continuously present in this manner may have a so-called linkage disequilibrium state in which the recombination rate of genomic DNA is 0 or extremely low. High nature. A region on the genome in linkage disequilibrium is called an LD block. Whether or not the region is in a linkage disequilibrium state is determined by a known method based on the analysis result of the region or a database of linkage disequilibrium, for example, a database of known LD block structures provided by the HapMap project. This can be confirmed. Since single nucleotide polymorphisms in the linkage disequilibrium state are very likely to show the same behavior, any single nucleotide polymorphism in the linkage disequilibrium state, preferably one single nucleotide polymorphism, preferably the pairwise tagging method A single nucleotide polymorphism determined by narrowing down by the so-called tag SNP, a plurality of single nucleotide polymorphisms in the linkage disequilibrium state can be represented. Further, by reanalyzing the vicinity of the single nucleotide polymorphism related to the polypoidal choroidal vasculopathy of the present invention, for example, using TaqMan (registered trademark) SNP Genotyping Assays (Applied Biosystems), the single nucleotide polymorphism is analyzed. New disease-related single nucleotide polymorphisms may be found that are in linkage disequilibrium with the type.

In the present invention, in the analysis of single nucleotide polymorphisms related to polypoidal choroidal vasculopathy on autosomes, as a primary analysis, as a combination of genotypes in individual single nucleotide polymorphisms, that is, the homotype of one allele A genotype that is a heterotype and a homotype of the other allele, a homotype of one allele + a heterotype of the other allele, a homotype of one allele vs. a heterotype of the other allele + a homotype of the other allele, And three kinds of statistical models of homotypes of one allele versus heterotypes of the other and the homotype of the other allele (these are also referred to as dominant model, recessive model, and genotype model when the one allele is a risk allele) The Fisher's exact test is applied to each of the PCV patient group versus glaucoma patient group (hereinafter referred to as Comparative A) and the PCV patient group versus cataract patient group (hereinafter referred to as Comparative B). Thus, the p value when three kinds of statistical models were applied was obtained. Next, in order to exclude single nucleotide polymorphisms specifically related to diseases other than polypoidal choroidal vasculopathy and to select single nucleotide polymorphisms specifically related to polypoidal choroidal vasculopathy, In both cases, 370 single nucleotide polymorphisms having a minimum p value of less than 0.01 in the comparison A and the comparison B in the three statistical models obtained as described above in polypoidal choroidal vasculopathy Selected as a candidate for related single nucleotide polymorphisms. Further, as a secondary analysis, cataract patients and glaucoma patients are collectively set as a control group, and the single nucleotide polymorphism candidate related to the polypoidal choroidal vasculopathy described above is again set to the same p value for the PCV patient group and the control group. The smallest p value obtained by applying three statistical models was used as the p value representing the single nucleotide polymorphism. A single nucleotide polymorphism used for detection of an allele or genotype associated with polypoidal choroidal vasculopathy is a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy described above when the multiplicity is adjusted by Bonferroni correction. Bonferroni correction is performed on the number of single nucleotide polymorphisms that are considered as type candidates, and a significance level for selecting a suitable single nucleotide polymorphism can be set. Specifically, the desired significance level of 5 × 10 ⁻² is divided by the number of repetitions of 370 times, and a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy is converted to a single nucleotide polymorphism of p value of 1.35 × 10 ⁻⁴ or less. Among them, a more preferable single nucleotide polymorphism was used. However, as described above, since Bonferroni correction is known to be overcorrection, a single nucleotide polymorphism whose p value is less than 0.01 is generally polyp-like, for example, a level approximately 1 to 2 orders of magnitude higher than the above significant level. It can be considered related to choroidal vasculopathy. In the present invention, all the 363 single nucleotide polymorphisms having the p value determined as described above below 0.01 are within the range where the FDR calculated for 370 candidate single nucleotide polymorphisms is less than 5%. It was included. That is, all of these 363 single nucleotide polymorphisms can be suitably used in the present invention.

  In order to obtain a highly reliable result in the analysis, the quality of the detection result is confirmed in advance for the single nucleotide polymorphism used in the analysis, and the single nucleotide polymorphism detected at a reasonable quality is analyzed. It is desirable to provide. Elements used for such quality confirmation include call rate, Hardy-Weinberg equilibrium and minor allele frequency.

  The determination rate of each single nucleotide polymorphism with respect to all samples, that is, the polymorphic site having a low call rate has a high rate of occurrence of typing errors and is not highly reliable. For this reason, it is desirable to analyze using a single nucleotide polymorphic site having a sufficiently high call rate. As a call rate as a criterion for accepting or rejecting a single nucleotide polymorphism, for example, a single nucleotide polymorphism showing a call rate of 70%, preferably 75%, more preferably 80%, still more preferably 85%, more preferably 90% or more. It is desirable to adopt a mold.

  Hardy-Weinberg equilibrium means that in a genetically uniform population with no sufficient variation or selection pressure and a sufficient number of individuals formed by random mating, the distribution frequency of alleles at a given locus Say that it is constant. Whether or not the Hardy-Weinberg equilibrium is established can be statistically tested by several known methods such as the Chi-square test and Fisher's exact test. In general, Hardy-Weinberg equilibria are established in a sufficient number of human populations, so generally genotyping errors are detected on the assumption that Hardy-Weinberg equilibria are established in the population. The study of Hardy-Weinberg equilibrium is used for this purpose. When statistically testing the establishment of the Hardy-Weinberg equilibrium, 0.0001 is used as the significance level, for example.

  In the case of single nucleotide polymorphisms contained in two alleles, the minor allele frequency refers to the frequency of the lower allele of the two alleles. The threshold value can be set arbitrarily. As described above, since the concept of single nucleotide polymorphism has a minor allele frequency exceeding about 1%, it is desirable to reject single nucleotide polymorphisms having a minor allele frequency of less than 1%. In addition, in the search for polymorphisms that cause multifactor diseases such as the present invention, it is considered that a plurality of polymorphisms having relatively low relative involvement in the disease are involved. It is desirable to reject single nucleotide polymorphisms with a certain frequency, for example, less than 5%.

  The single nucleotide polymorphism related to polypoidal choroidal vasculopathy thus obtained is a known sequence database such as Genbank provided by NCBI or a database of dbSNP or HapMap project provided by NCBI. By referring to the nucleotide polymorphism database, the SNP ID in the database (especially dbSNP ID), the physical position on the genome where the single nucleotide polymorphism exists, the sequence information around the single nucleotide polymorphism, the single nucleotide A gene having a polymorphism or a gene present in the vicinity of a single nucleotide polymorphism, and if present on a gene, distinguishing whether the position of the single nucleotide polymorphism is an intron or an exon, the function of the gene, Information such as homologous genes can be obtained, nucleic acid molecules used in the present invention can be obtained based on these information, and probes used in the present invention can be designed. As information for specifying a single nucleotide polymorphism when searching for such information in a database, one or a plurality of information recorded in these databases can be used.

  In the single nucleotide polymorphism associated with polypoidal choroidal vasculopathy determined in this way, odds are used as an index of how strong association exists between one allele and the presence or absence of polypoidal choroidal vasculopathy. The ratio can be determined. Odds is the value of the probability of an event occurring in a group divided by the probability that the event does not occur. The odds ratio is the ratio of odds in two different groups. When determining the odds ratio for an allele, the odds ratio that is a disease when having an allele, that is, the ratio of the frequency of one allele to the frequency of the other allele in the PCV patient group. Is divided by the ratio of the frequency of one allele to the other frequency in the non-PCV patient group. In this case, the obtained odds ratio is referred to as the obtained odds ratio for the “one allele”.

  When finding the odds ratio of an allele, if a certain frequency is 0, and therefore the odds ratio is infinite, the odds ratio can be approximated using an appropriate number instead of 0. . As an appropriate number used in place of 0, for example, a real number exceeding 0 such as 0.5 or 0.1 can be used.

  Furthermore, in the present invention, the risk allele is determined using the odds ratio obtained for the allele. As is clear from the above definition, the odds ratio obtained for one allele is the reciprocal of the odds ratio obtained for the other allele. In the present invention, an allele whose odds ratio obtained for an allele is greater than 1 is called a risk allele, and an allele that opposes the risk allele is called a non-risk allele. In the present invention, the term “allele odds ratio” refers to the odds ratio obtained for the risk allele, that is, the odds ratio of polypoidal choroidal vasculopathy when the risk allele is present.

  In the present invention, the risk of polypoidal choroidal vasculopathy can be predicted by these indices. A certain single nucleotide polymorphism risk allele is an allele observed more frequently in the PCV patient group than in the non-PCV patient group, that is, an allele involved in the risk of polypoidal choroidal vasculopathy. When predicting the risk of polypoidal choroidal vasculopathy using an allele, it is predicted that there is a risk of polypoidal choroidal vasculopathy by having at least one risk allele of the single nucleotide polymorphism. In this case, the larger the odds ratio, the higher the risk of polypoidal choroidal vasculopathy of the sample provider having the risk allele. On the other hand, as is clear from the definition of the odds ratio, in the present invention, by using the reciprocal of the odds ratio of the allele, it is possible to predict the disease risk of the non-risk allele. That is, as the reciprocal of the odds ratio of the allele is smaller than 1, it can be used for predicting that the disease risk of the non-risk allele is low.

  In the present invention, the risk of polypoidal choroidal vasculopathy can be predicted using a genotype in addition to or instead of an allele, that is, a genotype that is a combination of one allele and its allele. . In this case as well, the risk allele may be used as a reference as in the case of the allele, and the genotype considered to be at risk of polypoidal choroidal vasculopathy can be determined in consideration of the odds ratio.

  When determining the risk of polypoidal choroidal vasculopathy by obtaining the genotype odds ratio, first, two odds ratios of a dominant type odd ratio and a recessive type odds ratio are obtained based on the risk allele.

  In the present invention, the dominant type odds ratio is the odds ratio of polypoidal choroidal vasculopathy when there is at least one risk allele (ie, when the risk allele is homozygous or heterozygous), ie, the PCV patient group The ratio of the risk allele homozygous frequency and the heterozygous frequency to the frequency of the non-risk allele homozygote in the non-PCV patient group Recessive odds ratio is the odds ratio of polypoidal choroidal vasculopathy when the risk allele is homozygous, that is, the frequency of the risk allele homotype in the PCV patient group and the non-risk The ratio of the allele homozygous frequency to the sum of the heterozygous frequencies is calculated by comparing the risk allele homozygous frequency, the non-risk allele homotypic frequency, and the heterozygous frequency in the non-PCV patient group. Divided by the ratio of the sum of degrees.

  Next, when determining a genotype considered to be a risk of polypoidal choroidal vasculopathy, it is determined as follows in consideration of the dominant-type odds ratio and the recessive-type odds ratio obtained as described above.

  In each single nucleotide polymorphism associated with polypoidal choroidal vasculopathy, if the dominant odds ratio is greater than the recessive odds ratio, the risk allele of the single nucleotide polymorphism is associated with the dominant odds ratio. On the other hand, when the recessive odds ratio is larger than the dominant odds ratio, the single nucleotide polymorphism risk allele is related to the recessive odds ratio. When the risk allele is related to the dominant odds ratio, the genotype that is considered to be at risk for polypoidal choroidal vasculopathy in the single nucleotide polymorphism is a homozygous or heterozygous risk allele, and the risk allele is a recessive type When related to the odds ratio, the genotype considered to be at risk for polypoidal choroidal vasculopathy in the single nucleotide polymorphism is a homozygous risk allele.

  When obtaining the odds ratio of a genotype, the frequency of any genotype becomes 0, the odds ratio becomes infinite, and therefore it may be impossible to calculate. In such a case, as in the case of the allele odds ratio, the odds ratio can be approximately calculated using an appropriate number instead of the zero. As an appropriate number used in place of 0, for example, a real number exceeding 0 such as 0.5 or 0.1 can be used.

  If a genotype that is determined to be at risk for polypoidal choroidal vasculopathy determined in this way is detected in the sample, the sample provider may be predicted to be at risk for polypoidal choroidal vasculopathy. it can. In either case, the greater the odds ratio, the higher the risk of polypoidal choroidal vasculopathy.

  Furthermore, also when predicting the risk of polypoidal choroidal vasculopathy by the combination of single nucleotide polymorphisms of the present invention, the prediction accuracy can be improved by using the magnitude of the odds ratio. That is, there are many genotypes that are considered to be at risk for risk alleles or polypoidal choroidal vasculopathy and / or few genotypes that are not at risk for non-risk alleles or polypoidal choroidal vasculopathy The risk of polypoidal choroidal vasculopathy in a sample provider who only has higher risk than that of a non-sampled provider, in which case the polypoidal choroidal vessel is more likely if it has more alleles or genotypes with higher odds ratios The risk of illness is expected to be higher.

  Preferably, the odds ratio or the odds ratio used for predicting the risk of polypoidal choroidal vasculopathy as an index indicating the relationship between the allele or genotype and the degree of disease is a single nucleotide polymorphism determined to be statistically significant. The odds ratio calculated for the mold.

  As described later, the single nucleotide polymorphism of the present invention may exist in any gene region such as an intron or exon on the genome, or may exist in any region that is not a gene on the genome. When present in introns or exons, the gene may be one of the causative genes for polypoidal choroidal vasculopathy. In addition, when the single nucleotide polymorphism of the present invention is present in a region that is not a gene on the genome, the gene in the vicinity of the single nucleotide polymorphism and the single nucleotide polymorphism are linked, and the gene in the vicinity May be one of the causative genes of polypoidal choroidal vasculopathy, or the region containing the single nucleotide polymorphism or its vicinity is transcribed to mRNA, and other genes via phenomena such as RNA interference May be involved in the expression of. That is, a gene to which the single nucleotide polymorphism of the present invention belongs on the genome, or a gene present in the vicinity of the single nucleotide polymorphism of the present invention on the genome is included in the scope of the present invention.

  In the present invention, intron type single nucleotide polymorphism (iSNP) means that an intron has a single nucleotide polymorphism. Translational region type single nucleotide polymorphism (cSNP) is a region where single nucleotide polymorphism exists in the region translated into protein, and the codon containing the single nucleotide polymorphism is a codon encoding another amino acid or a stop codon. It means a change in the amino acid sequence such as mutation. Silent single nucleotide polymorphism (sSNP) refers to one having a single nucleotide polymorphism in the translation region and not accompanied by a change in amino acid sequence. Genomic single nucleotide polymorphism (gSNP) means that a single nucleotide polymorphism exists in a region that does not encode a gene on the genome. Transcriptional regulatory polymorphism (rSNP) refers to a single nucleotide polymorphism present at a site considered to be involved in transcriptional regulation on an exon.

  Thus, a single nucleotide polymorphism may exist at any position on the genome, and in any case, it may have an association with a disease. The presence of single nucleotide polymorphisms in introns or untranslated regions may affect gene expression control, splicing that occurs after gene transcription, and mRNA stability. If there is a single nucleotide polymorphism in the translation region, the substitution of the base changes the codon corresponding to one amino acid to a codon corresponding to another amino acid, or changes to a stop codon. Changes may occur in the structure of the protein encoded by. These changes cause changes in the expression level and function of the gene, and in turn the expression level or function of the protein encoded by the gene, which can cause various diseases. When a genomic single nucleotide polymorphism is associated with a disease, the region containing the polymorphic site is actually translated and may have some influence on the expression of other genes. When a silent single nucleotide polymorphism is associated with a disease, there is another polymorphism associated with the disease around the single nucleotide polymorphism, and the polymorphism and the silent single nucleotide polymorphism are in a linkage disequilibrium state. In some cases, an association with a disease may be observed. Similarly, even a single nucleotide polymorphism other than the silent single nucleotide polymorphism, the single nucleotide polymorphism itself is not a direct cause of polypoidal choroidal angiopathy, but a polypoidal choroidal angiopathy present in the vicinity. An association between these single nucleotide polymorphisms and polypoidal choroidal vasculopathy may also be observed when in linkage disequilibrium with the true polymorphism.

(Nucleic acid molecules containing alleles associated with polypoidal choroidal vasculopathy)
In another aspect of the present invention, a nucleic acid molecule comprising a single nucleotide polymorphism associated with polypoidal choroidal angiopathy and a sequence complementary to a nucleic acid molecule comprising a single nucleotide polymorphism associated with polypoidal choroidal angiopathy Nucleic acid molecules are provided.

  A nucleic acid molecule containing a nucleic acid sequence containing a single nucleotide polymorphism described in Tables 1 to 10 and / or a nucleic acid molecule containing a complementary sequence of the sequence is a nucleic acid molecule used for allele detection in the present invention. . The nucleic acid molecule used for allele detection in the present invention is preferably a nucleic acid molecule containing a single nucleotide polymorphism described in Table 1 or Table 2 or a fragment thereof, and at least one of the nucleic acid molecules or the nucleic acid concerned A nucleic acid molecule complementary to the molecule or a fragment thereof, more preferably a nucleic acid molecule comprising a single nucleotide polymorphism described in Table 1, or a fragment thereof, wherein at least any one of said nucleic acid molecules or said nucleic acid molecule Or a fragment thereof. Further, among nucleic acid molecules containing single nucleotide polymorphisms described in Tables 1 to 10 or nucleic acid molecules complementary to the nucleic acid molecules, nucleic acid molecules containing risk alleles or nucleic acid molecules complementary to the nucleic acid molecules It is desirable to be.

  Nucleic acid molecules containing single nucleotide polymorphisms associated with these polypoidal choroidal vasculopathy, or nucleic acid molecules having a sequence complementary to the nucleic acid molecule can be used as a marker for determining the level of polypoidal choroidal vasculopathy risk. it can. Furthermore, these nucleic acid molecules can be used as a probe for detecting a risk allele or its allele in the single nucleotide polymorphism or determining a genotype. Further, when the single nucleotide polymorphism is present on an exon, these nucleic acid molecules can be used for detection of a gene transcript.

  In the present invention, a more preferable nucleic acid molecule can be selected by the same method as described in the section for identifying a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy.

  Nucleic acid molecules constituting a eukaryotic genome are composed of a sense strand and a double strand of an antisense strand complementary to the sense strand. That is, a single nucleotide polymorphism is also composed of a sense strand and an antisense strand, and detecting any single nucleotide polymorphism has the same meaning, so the nucleic acid molecule of the present invention includes any of these. .

  Regardless of whether the nucleic acid molecule in the present invention is deoxyribonucleic acid or ribonucleic acid, a nucleic acid molecule having both of them in the same nucleic acid molecule is also included in the present invention. When a nucleic acid molecule containing ribonucleic acid is used, the nucleoside defined by thymidine (T) in the sequence of the present invention can be read as uridine '(U). The same applies to the case where the nucleoside of the complementary strand becomes thymidine. In addition, these nucleic acid molecules can be chemically modified as necessary within the range not impairing the function for use in the present invention. The function in this case refers to the function of achieving the purpose of using the nucleic acid molecule.

  The nucleic acid molecule in the present invention is based on the sequence information disclosed in the present specification or the sequence information obtained by searching information such as the database ID disclosed in the present specification in a database such as dbSNP, HapMap or Genbank. Can be synthesized using a known method such as the phosphoramidite method. It is also possible to synthesize using a commercially available DNA synthesizer. In addition, the nucleic acid molecule in the present invention is obtained from a sample containing human-derived DNA, by a known method such as PCR method, or for some nucleic acid molecules from a sample containing human-derived RNA, such as RT-PCR method. It can be obtained by a known method. Primers necessary for acquisition can be designed by those skilled in the art based on sequence information disclosed in the present specification or sequence information that can be searched from information such as database IDs disclosed in the present specification. For example, when using the PCR method, a primer of about 10 to 30 bases having a sequence homologous to a partial sequence of the target nucleic acid molecule can be used, and when using the RT-PCR method, A reverse transcription reaction is performed using an oligo dT primer, random hexamer or the like to prepare a complementary DNA (cDNA), and the target sequence in the cDNA can be amplified by the PCR method described above.

  The nucleic acid molecule preferably has a length of 16 to 50 bases, more preferably 23 to 27 bases, and even more preferably 25 bases, and is complementary to the above-described polymorphic site and its surrounding sequence, or a complementary sequence thereto. Desirably, the nucleic acid molecule comprises a sequence. The nucleic acid molecule can be subjected to any chemical modification as long as its function is not impaired. The function in this case refers to the function of achieving the purpose of using the nucleic acid molecule.

In the method for detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy and the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention, the nucleic acid molecule used for allele detection is:
Preferably, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 (each corresponding to one single nucleotide polymorphism in a pair of odd-numbered and even-numbered base sequences) or A nucleic acid molecule comprising a sequence having an allele corresponding to the 17th of the base sequence represented by SEQ ID NOs: 1 to 70 in the complementary sequence or a partial sequence thereof,
More preferably, in at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 12 and SEQ ID NOs: 69 to 70, or a complementary sequence thereof, or a partial sequence thereof, the sequence numbers 1 to 12 or a nucleic acid molecule comprising a sequence having an allele corresponding to the 17th position of the base sequence represented by SEQ ID NOs: 69 to 70,
More preferably, in the following at least one base sequence selected from the group consisting of a base sequence comprising a single nucleotide polymorphism of a to g or a complementary sequence thereof, or a partial sequence thereof: A nucleic acid molecule containing a sequence having an allele corresponding to the 17th of the base sequence included in the pair. (Here, as described above, in the set of SEQ ID NOs indicated by a to g, each set of base sequences is a base sequence containing one allomorphic allele of each single nucleotide polymorphism in the 17th base. )
a: SEQ ID NO: 1 and / or SEQ ID NO: 2,
b: SEQ ID NO: 3 and / or SEQ ID NO: 4,
c: SEQ ID NO: 5 and / or SEQ ID NO: 6,
d: SEQ ID NO: 7 and / or SEQ ID NO: 8,
e: SEQ ID NO: 9 and / or SEQ ID NO: 10,
f: SEQ ID NO: 11 and / or SEQ ID NO: 12, and g: SEQ ID NO: 69 and / or SEQ ID NO: 70
In the present specification, the partial sequence means a fragment of a nucleic acid sequence, and the sequence length is not limited as long as it has an allele corresponding to the 17th of the sequence. A fragment having a length of 16 to 50 bases, more preferably 23 to 27 bases, and even more preferably 25 bases. A fragment of a nucleic acid sequence containing a single nucleotide polymorphism of the present invention obtained by searching a single nucleotide polymorphism of the present invention in a database of known single nucleotide polymorphisms such as dbSNP or a database of known sequence information is also a partial sequence of the present invention. include.

  In the case of using a nucleic acid molecule containing any one of the above single nucleotide polymorphisms, among them, the sequence represented by the odd-numbered sequence number of the set of sequences containing each single nucleotide polymorphism, or a complementary sequence thereof, or these It is preferable to use a nucleic acid molecule comprising a single nucleotide polymorphism allele present at the 17th position of the selected nucleotide sequence in the nucleotide sequence consisting of the partial sequence of The nucleic acid molecule is preferably composed of a sequence containing the allele. These are base sequences containing risk alleles at each polymorphic site.

(Probe capable of detecting alleles associated with polypoidal choroidal vasculopathy)
In another aspect of the invention, a probe capable of detecting an allele associated with polypoidal choroidal vasculopathy is provided. The probe in the present invention can be prepared in the same manner as the nucleic acid molecule of the present invention described above.

  The probe is capable of hybridizing with a nucleic acid sequence containing each single nucleotide polymorphism allele described in Tables 1 to 10 below or a complementary sequence thereof under stringent conditions (also referred to as high stringency conditions). Anything can be used. Preferably, the probe of the present invention is a probe capable of detecting a risk allele and / or a non-risk allele included in at least one single nucleotide polymorphism described in Table 1 or Table 2, more preferably 1 is a probe capable of detecting a risk allele and / or a non-risk allele contained in at least one single nucleotide polymorphism described in 1. In addition, in a probe capable of detecting a risk allele and / or a non-risk allele included in any one single nucleotide polymorphism described in Tables 1 to 10, a risk allele can be detected as one of preferred embodiments. A probe. On the other hand, by using both a probe capable of detecting a risk allele and a probe capable of detecting a non-risk allele, the genotype of the single nucleotide polymorphism can be determined.

  In this specification, the term “hybridization” means that a nucleic acid molecule having a certain sequence and a nucleic acid molecule complementary to at least a part of the nucleic acid molecule associate with each other through hydrogen bonds based on complementary base sequences. To do. The types of complementary nucleic acid molecules associated with the original nucleic acid molecule may be the same or different, and the types of nucleic acid molecules constituting these nucleic acid molecules may be deoxyribonucleic acid, ribonucleic acid, or the same nucleic acid It may have both in the molecule.

  In the present specification, stringent conditions or high stringency conditions mean conditions under which a nucleic acid molecule having a sequence complementary to a partial sequence of the nucleic acid molecule specifically hybridizes to a nucleic acid molecule having a certain sequence. (Fred M. Ausuble et al., Current Protocols in Molecular Biology p.2.10.1-2.10.16; John Wiley and Sons, Inc.). Specific examples of using a solid-phase probe consisting of a 25-base long DNA molecule include, for example, 0.056M 2- (N-Morpholino) ethanesulfonic acid, 5% DMSO, 2.5 × Denhardt's solution, 5.77 mM EDTA, 0.0115% Examples of the conditions include hybridization at 49 ° C after denaturation at 95 ° C for 10 minutes in the presence of Tween-20, 2.69M Tetrametyl Ammonium Chloride. It is known that such conditions may vary depending on the sequence length of nucleic acid molecules used as probes and the degree of chemical modification. Optimizing hybridization and / or washing conditions that allow specific hybridization This is possible with the ordinary creative ability of those skilled in the art.

  In the present invention, allele-specific (or allele-specific) means that the allele is contained in a nucleic acid molecule containing the polymorphic site, or that a certain nucleic acid molecule contains the allele in the polymorphic site. Hybridization specifically to stringent nucleic acid molecules under stringent conditions, that is, in such a manner that alleles that oppose the alleles can be identified.

  Since the polymorphic site allele can be determined by detecting any polymorphic site in the sense strand or antisense strand of the genome, the probe in the present invention has a sequence specific to the allele of the sense strand. Any nucleic acid molecule having a complementary sequence, ie a sequence specific to the antisense strand allele, and a sequence complementary to the sequence specific to the antisense strand allele, ie a sequence specific to the sense strand allele included. The probe of the present invention can also be used for detection of cDNA or mRNA containing the single nucleotide polymorphism of the present invention. When used for detection of cDNA or mRNA, the single nucleotide polymorphism that is present in the exon or in the vicinity thereof is preferably used.

  The probe in the present invention consists of a nucleic acid molecule containing an allele-specific sequence or a complementary strand thereof or a partial sequence thereof described in Tables 1 to 10 below.

  The probe in the present invention has any number of bases not derived from the sequence for the purpose of providing a spacer or stabilization at the end, as long as it can hybridize to the allele-specific sequence or its complementary strand under stringent conditions. Sequences can be added. Preferably, the sequence contributing to allele-specific hybridization in the probe of the present invention comprises only an allele-specific sequence or its complementary strand.

  The probe can be given any label for use in detection. Any label used for the probe can be used, but in general, fluorescent labels such as FITC and Cy3, biotin, or enzyme labels such as alkaline phosphatase and horseradish peroxidase are generally used. When a biotin label is used, streptavidin or avidin that specifically binds to biotin is further subjected to a detectable label, and the labeled streptavidin or the like is used as a secondary label. A labeled anti-biotin antibody can be used instead of labeled streptavidin or the like. The detection sensitivity can be improved by using a labeled anti-streptavidin antibody as a tertiary label for streptavidin bound to biotin. Any known method can be used for labeling the probe, and this method is well known to those skilled in the art. Arbitrary sequences serving as the spacers described above can be added to the probe, and the spacers can be labeled. Reagents for labeling probes, labeled streptavidin, labeled anti-biotin antibodies, and the like are commercially available as reagents and can be purchased and used.

  As long as the probe in the present invention can specifically hybridize to a nucleic acid molecule having the target allele, it is a nucleic acid having both of them in the same nucleic acid molecule regardless of whether it is deoxyribonucleic acid or ribonucleic acid. Molecular probes are also included in the present invention. When a nucleic acid molecule containing ribonucleic acid is used as a probe, the nucleoside defined by thymidine (T) in the sequence of the present invention can be read as uridine '(U). The same applies to the case where the nucleoside of the complementary strand of the sequence of the present invention is thymidine, or the case where the probe of the present invention is used for detection of mRNA. In addition, the probe in the present invention can be chemically modified as necessary as long as it can specifically hybridize to a nucleic acid molecule having the target allele under stringent conditions. Any known method can be used for chemical labeling.

  The probe for detection can be detected by a known method by reacting with a sample in a solution state, or can be solidified on a carrier in advance. The probe corresponding to each allele of several to several hundred thousand different single nucleotide polymorphisms is solidified at a predetermined position on a solid support in advance of one to ten or more probes for each single nucleotide polymorphism. In addition, it is also possible to take the form of a so-called microarray, which is a solidified probe in which a sample is reacted with this, a signal generated from the hybridized probe is scanned and analyzed using a computer. When taking the form of a solidified probe, the maximum number of probes to be solidified is limited by the solidification density of the probe and the area of the solidified site.

  When taking the form of such a solidified probe, the nucleic acid molecule in the sample should be labeled by a known method and bound to the solidified unlabeled probe of the present invention or detected. On the solid layer derived from the labeled nucleic acid molecule having the allele to be detected by binding the nucleic acid molecule having the allele to the unlabeled probe of the present invention which has been solidified and then labeling by a known method. Signal can be detected.

  The solidification can be performed by any known method, and for example, methods such as synthetic oligo print and spotting photolithograph can be used. Moreover, there is no restriction | limiting in the material of a support | carrier, Generally used materials, for example, polymers, such as a polycarbonate and a polystyrene, glass, a silicon crystal, etc. are used. In addition, in order to increase the adhesion of nucleic acid, a coating such as cationization may be applied to the carrier in advance before solidification. Further, in order to prevent adsorption of non-specific nucleic acid to the carrier, blocking can be performed with a known blocking agent after solidification. Any blocking agent can be used as long as it can suppress adsorption of non-specific nucleic acid to the carrier. Examples include salmon sperm DNA, Denhardt's solution, Cot-I DNA extracted from placenta, sodium dodecyl sulfate. Anionic surfactants such as nonionic surfactants such as polyoxyethylene sorbitan monolaurate can be used.

  In addition, in the case of solidifying a probe, by consolidating a probe specific to each allele that opposes on the same carrier, each opposing allele contained in one sample can be operated by the same operation. It can also be set as the structure to detect. In such a configuration, not only the allele of the sample but also the genotype can be determined by the same operation.

  Although the suitable sequence length of the detection probe varies depending on the detection method, it may be any one that can specifically detect the single nucleotide polymorphism allele or genotype associated with the polypoidal choroidal vasculopathy of the present invention. For example, the probe in the case of using a solidified probe for the detection of an allele preferably has a length of 16 to 50 bases, more preferably 23 to 27 bases, still more preferably 25 bases, and the polymorphism described above A probe comprising a nucleic acid molecule comprising a site and its peripheral sequence, or a sequence complementary thereto, and the probe for detecting an allele in a solution state is preferably 16 to 50 bases, more preferably 23 to 27. A probe comprising a nucleic acid molecule having a base, more preferably a length of 25 bases, and comprising the aforementioned polymorphic site and its peripheral sequence, or a sequence complementary thereto.

In the method for detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy and the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention, the probe used for allele detection is:
At least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 (each of which corresponds to one single nucleotide polymorphism in a pair of odd-numbered and even-numbered base sequences) or its complementary A probe comprising a nucleotide sequence having an allele corresponding to the 17th of the nucleotide sequence represented by SEQ ID NOs: 1 to 70 in the nucleotide sequence consisting of these sequences or partial sequences thereof;
Preferably, in the base sequence consisting of at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 12 and SEQ ID NOs: 69 to 70, a complementary sequence thereof, or a partial sequence thereof, A probe containing a base sequence having an allele corresponding to the 17th position of the base sequence represented by Nos. 1 to 12 or SEQ ID Nos. 69 to 70,
More preferably, in the following base sequence consisting of at least one base sequence selected from the group consisting of base sequences including single nucleotide polymorphisms a to g or a complementary sequence thereof, or a partial sequence thereof: It is a probe containing a base sequence having an allele corresponding to the 17th base sequence included in the set of a to g. (Here, as described above, in the set of SEQ ID NOs indicated by a to g, each set of base sequences is a base sequence containing one allomorphic allele of each single nucleotide polymorphism in the 17th base. )
a: SEQ ID NO: 1 and / or SEQ ID NO: 2,
b: SEQ ID NO: 3 and / or SEQ ID NO: 4,
c: SEQ ID NO: 5 and / or SEQ ID NO: 6,
d: SEQ ID NO: 7 and / or SEQ ID NO: 8,
e: SEQ ID NO: 9 and / or SEQ ID NO: 10
f: SEQ ID NO: 11 and / or SEQ ID NO: 12, and
g: SEQ ID NO: 69 and / or SEQ ID NO: 70

  In the case of using a probe containing any one of the above-mentioned single nucleotide polymorphisms, among them, a sequence represented by an odd-numbered sequence number or a complementary sequence thereof among a set of sequences containing each single nucleotide polymorphism, or these It is preferable to use a probe containing a single nucleotide polymorphism allele present at the 17th position of the selected base sequence in the base sequence consisting of the partial sequence. These are base sequences containing risk alleles at each polymorphic site.

(Method for detecting single nucleotide polymorphism associated with polypoidal choroidal vasculopathy in sample and predicting risk of polypoidal choroidal vasculopathy)
In one aspect of the present invention, a method is provided for detecting whether a sample containing nucleic acid molecules has an allele or genotype that is frequent in PCV patients. Samples include nucleic acid molecules, that is, those capable of extracting genome-derived nucleic acid molecules (DNA and / or RNA), or mRNA or cDNA containing a gene present in the vicinity of the single nucleotide polymorphism of the present invention Any substance can be used as long as it can be extracted, for example, blood, white blood cells, hair roots, hair, saliva, oral mucosal cells, nasal mucosal cells, skin, or tissues such as muscles or organs obtained by biopsy are used. .

  As described above, the nucleic acid molecules constituting the eukaryotic genome are composed of complementary sense strands and antisense strands, and the single nucleotide polymorphism allele of the present invention is detected in the nucleic acid molecules contained in the sample. Can be performed by detecting either the sense strand or the antisense strand of the polymorphic site.

  The 33-base nucleic acid sequence containing a single nucleotide polymorphism related to polypoidal choroidal vascular disease disclosed in the present invention is a base sequence consisting of two base sequences that differ only in the center (i.e., the 17th base). (That is, a pair with an odd number and an even number) and the 17th base is a polymorphic site. The base sequence including the risk allele in the polymorphic site is the previous (that is, odd-numbered) sequence in the set of base sequences. Moreover, the risk allele is described in Tables 1 to 10 described later. By detecting a risk allele in a nucleic acid molecule contained in a sample for at least one of these single nucleotide polymorphisms, the risk of polypoidal choroidal vasculopathy of the sample provider can be predicted. That is, when at least one risk allele in the single nucleotide polymorphism is present in the nucleic acid molecule in the sample, it may be predicted that there is a risk of polypoidal choroidal vasculopathy.

  Moreover, about the single nucleotide polymorphism relevant to the polypoidal choroidal vasculopathy identified above, each allele which opposes the specific allele contained in one sample can be detected, and a genotype can be determined. That is, when only a certain allele is detected, it is a homotype having the allele as a homozygote, and when two alleles are detected, it is a heterotype having the two alleles as heterogeneous. For at least one of these single nucleotide polymorphisms, the genotype of the single nucleotide polymorphism contained in the nucleic acid molecule in the sample is determined, and the genes of the PCV patient group and the control group described in Tables 11 to 19 described below Based on the type frequency data, a genotype that is considered to be at risk of polypoidal choroidal vasculopathy is determined by the above-described method, and risk prediction may be similarly performed by comparing these genotypes. Note that the determination of the genotype of a single nucleotide polymorphism contained in a nucleic acid molecule in a sample does not necessarily require identification of the risk allele. Can be performed either before or after determining whether at least one risk allele is present, and there is a risk of polypoidal choroidal vasculopathy determined based on the determined genotype and the risk allele And the risk of polypoidal choroidal vasculopathy can be predicted.

  Any method can be used to detect the presence of these alleles in a sample containing nucleic acid molecules, but each can be detected by hybridizing using a probe specific for each of the aforementioned alleles and detecting the signal. Can detect alleles. By comparing the detected allele with the risk allele in the single nucleotide polymorphism disclosed in the present invention, it is possible to determine whether or not the risk allele in the single nucleotide polymorphism is present in the nucleic acid molecule in the sample. For example, the above-described nucleic acid molecule of the present invention and / or the above-described probe of the present invention is a risk in the method for detecting a single nucleotide polymorphism related to polypoidal choroidal vasculopathy and the risk predicting method for polypoidal choroidal vasculopathy of the present invention. It can be suitably used to determine genotypes that are considered to be at risk of allele or polypoidal choroidal vasculopathy.

  In addition, a microarray in which probes corresponding to each allele to be opposed, that is, a risk allele and a non-risk allele included in a single nucleotide polymorphism, are respectively labeled, or each opposing allele is solidified By using the solidified probe, it is possible to detect each of the opposing alleles contained in the same sample. In such a configuration, not only the allele of the sample but also the genotype can be determined. In addition, when using a solid-phase probe such as a microarray in which each opposing allele is solid-layered on the same carrier, it can be configured such that hybridization is performed by the same operation and detected by the same operation.

  As a method for detecting a single nucleotide polymorphism of the present invention, any known method other than the above-described methods can be used. For example, the following methods can be used. Examples of the method of hybridizing using a probe include the TaqMan (registered trademark) method and the Invader (registered trademark) method, and any of these can be used. Even in the case of probes for detecting the same allele, a more preferable probe may differ depending on the method used for detection. The determination of the single nucleotide polymorphism allele or genotype of the present invention does not depend on the detection method, but it is desirable to use a probe suitable for the detection method. Examples of the method that does not perform hybridization with a probe include a direct sequencing method for directly determining a base sequence.

  As described above, in the present specification, “allele-specific” or “allele-specific” means that a nucleic acid molecule or base sequence containing the polymorphic site contains the allele, or that a certain nucleic acid molecule is In the polymorphic site, it is possible to hybridize specifically to a nucleic acid molecule having a sequence containing the allele under stringent conditions, i.e., so that alleles that are opposite to the allele can be identified. To tell.

  If the sample is analyzed in this way and the sample contains a genotype that is considered to be at risk for a risk allele or polypoidal choroidal vasculopathy, the person who provided the sample is at risk for polypoidal choroidal vasculopathy. A person who is predicted to be high and suspected of having polypoidal choroidal vasculopathy that provided the sample is likely to be diagnosed with polypoidal choroidal vasculopathy. The magnitude of the risk of polypoidal choroidal vasculopathy can be predicted using the magnitude of the odds ratio.

  Furthermore, by using a single sample to detect two or more of the single nucleotide polymorphisms, risk alleles or genotypes associated with the polypoidal choroidal vasculopathy of the present invention, It is also possible to improve the accuracy of risk determination. In this case, the magnitude of the risk can be predicted using as an index the number of genotypes considered to be at risk of risk allele or polypoidal choroidal vasculopathy detected in the sample or the odds ratio thereof. The number of genotypes that are considered to be at risk for the risk allele or polypoidal choroidal vasculopathy or the odds ratio of these is greater, or if these values exceed a certain threshold, the risk is determined to be higher can do.

  As described above, in the method for detecting a single nucleotide polymorphism related to polypoidal choroidal vasculopathy and the risk predicting method for polypoidal choroidal vasculopathy of the present invention, the nucleic acid molecules in the sample are described in Tables 1 to 10 described later. Determination of any single nucleotide polymorphism allele and / or genotype determined, the single nucleotide polymorphism allele and / or genotype in the sample and the single nucleotide polymorphism disclosed in the present invention Anything that includes at least one of risk alleles and / or genotype comparisons considered to be at risk for polypoidal choroidal vasculopathy disclosed in the present invention is included.

The method for predicting the risk of polypoidal choroidal vasculopathy of the present invention,
Determining at least one single nucleotide polymorphism allele and / or genotype described in Tables 1 to 10 for nucleic acid molecules in the sample, single nucleotide polymorphism allele and / or genotype in the sample And comparing the genotype considered to be at risk of polypoidal choroidal vasculopathy disclosed in the present invention and / or the risk allele of the single nucleotide polymorphism disclosed in the present invention Including
Preferably, in the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention, at least one single nucleotide polymorphism allele and / or genotype described in Table 1 or Table 2 is determined in the nucleic acid molecule in the sample. The allele and / or genotype of the single nucleotide polymorphism in the sample and the risk allele of the single nucleotide polymorphism disclosed in the present invention and / or the risk of polypoidal choroidal vascular disease disclosed in the present invention Including at least one of comparing possible genotypes,
More preferably, the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention comprises determining the allele and / or genotype of at least one single nucleotide polymorphism described in Table 1 in the nucleic acid molecule in the sample, It is considered that there is a risk of the single nucleotide polymorphism allele and / or genotype in the sample and the risk allele of the single nucleotide polymorphism disclosed in the present invention and / or the polypoidal choroidal vascular disease disclosed in the present invention. Performing at least one of genotype comparisons.

In the method for detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy and the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention, the single nucleotide polymorphism used for detection is:
Preferably, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 (each corresponding to one single nucleotide polymorphism in a pair of odd-numbered and even-numbered base sequences) or In its complementary sequence, it is a single nucleotide polymorphism present at the 17th position of each base sequence,
More preferably, in at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 12 and SEQ ID NOs: 69 to 70, or a complementary sequence thereof, a single base present at the 17th position of each base sequence Polymorphic,
More preferably, in the following at least one base sequence selected from the group consisting of base sequences including single nucleotide polymorphisms a to g, or a complementary sequence thereof, one present at the 17th position of each base sequence It is a base polymorphism. (Here, as described above, in the set of SEQ ID NOs indicated by a to g, each set of base sequences is a base sequence containing one allomorphic allele of each single nucleotide polymorphism in the 17th base. )
a: SEQ ID NO: 1 and / or SEQ ID NO: 2,
b: SEQ ID NO: 3 and / or SEQ ID NO: 4,
c: SEQ ID NO: 5 and / or SEQ ID NO: 6,
d: SEQ ID NO: 7 and / or SEQ ID NO: 8,
e: SEQ ID NO: 9 and / or SEQ ID NO: 10
f: SEQ ID NO: 11 and / or SEQ ID NO: 12, and
g: SEQ ID NO: 69 and / or SEQ ID NO: 70

  When using any one of the above-mentioned single nucleotide polymorphisms, in the sequence set including each single nucleotide polymorphism, in the sequence indicated by the odd-numbered sequence number or its complementary sequence, 17 of each nucleotide sequence It is preferable to use the second single nucleotide polymorphism allele. These are base sequences containing risk alleles at each polymorphic site.

  In detecting these single nucleotide polymorphisms in the sample, in addition to any one of the single nucleotide polymorphisms, in addition to the single nucleotide polymorphism, at least one of the single nucleotide polymorphisms described in Tables 1 to 10 is used. Combining determination of single nucleotide polymorphism alleles or genotypes can improve the accuracy of disease determination. The single nucleotide polymorphism used in the combination is preferably the single nucleotide polymorphism described in Table 1 or Table 2, and more preferably the single nucleotide polymorphism described in Table 1.

(Kit for detecting alleles associated with polypoidal choroidal vasculopathy)
In another aspect of the invention, a kit for detecting single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy is provided.

  The kit of the present invention includes all nucleic acid molecules in the sample as long as they can detect any single nucleotide polymorphism risk allele described in Tables 1 to 10 described later. Preferably, the kit of the present invention is a kit capable of detecting at least one single nucleotide polymorphism risk allele and / or non-risk allele described in Table 1 or Table 2, more preferably Table 1 A kit capable of detecting a risk allele and / or a non-risk allele of at least one single nucleotide polymorphism described in 1. By using a kit capable of detecting both a risk allele and a non-risk allele included in one single nucleotide polymorphism, the genotype of the single nucleotide polymorphism can be determined.

  In the kit of the present invention, in addition to any one single nucleotide polymorphism described above, in addition to the single nucleotide polymorphism, at least one single nucleotide polymorphism risk allele described in Tables 1 to 10 and / or Alternatively, a kit capable of detecting a non-risk allele or determining a genotype is also one aspect of the present invention. In the case of a kit for determining an allele or genotype of a plurality of single nucleotide polymorphisms, in addition to any one of the above single nucleotide polymorphisms, preferably the risk of single nucleotide polymorphisms described in Table 1 or Table 2 It is a kit capable of detecting alleles and / or non-risk alleles or determining genotypes, and more preferably detecting single nucleotide polymorphism risk alleles and / or non-risk alleles or genotypes described in Table 1. It is a kit that can be determined.

  Among the kits capable of detecting a risk allele and / or non-risk allele of at least one single nucleotide polymorphism in Tables 1 to 10 or determining a genotype, one of the preferred embodiments includes the single nucleotide polymorphism. Kits that can detect risk alleles.

  As described in the section of the probe, the kit of the present invention may detect either the single-stranded polymorphic sense strand or the antisense strand, or may detect both of them. good. There is no limitation on the method for detecting a single nucleotide polymorphism, and for example, the detection method exemplified in the section of the probe is used.

  A kit for detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy using the nucleic acid molecule of the present invention and / or the probe of the present invention is included in the present invention. That is, the kit of the present invention contains the nucleic acid molecule of the present invention and / or the probe of the present invention so that the allele or genotype of a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy can be determined. is there.

  A kit for detecting both the aforementioned risk alleles and non-risk alleles is also a preferred embodiment of the present invention. Using such a kit, it is also possible to determine the genotype of each allele as already described.

  By using the kit of the present invention to detect the presence of a risk allele or a genotype that is considered to be at risk of polypoidal choroidal vasculopathy in a sample, future polypoidal choroidal vasculopathy in non-PCV patients Can be diagnosed or diagnosed or aided in diagnosis of polypoidal choroidal vasculopathy in a person suspected of having polypoidal choroidal vasculopathy.

  In addition, as described above, by using a probe specific to each of the opposing alleles, and by making the label of the probe different, or by adopting the form of the above-mentioned solidified probe (microarray), It can also be set as the kit which measures the opposing allele by the same operation.

  By using a single sample to detect a plurality of these alleles or loci, it is possible to improve the accuracy of risk prediction and diagnosis of future polypoidal choroidal vasculopathy. Even in such a configuration, it is possible to adopt a configuration in which detection is performed by the same operation by adopting a form of a probe with a different label or the above-described microarray.

In the method for detecting a single nucleotide polymorphism associated with polypoidal choroidal vasculopathy and the method for predicting the risk of polypoidal choroidal vasculopathy of the present invention, a kit used for detection or risk prediction,
At least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 (each of which corresponds to one single nucleotide polymorphism in a pair of odd-numbered and even-numbered base sequences) or its complementary In the nucleotide sequence consisting of these sequences or partial sequences thereof, a kit capable of determining an allele or genotype of a single nucleotide polymorphism corresponding to the 17th of each nucleotide sequence represented by SEQ ID NOs: 1 to 70,
Preferably, in the base sequence consisting of at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 12 and SEQ ID NOs: 69 to 70, a complementary sequence thereof, or a partial sequence thereof, It is a kit capable of determining an allele or genotype of a single nucleotide polymorphism corresponding to the 17th position of each base sequence represented by Nos. 1 to 12 or SEQ ID Nos. 69 to 70,
More preferably, in the following base sequence consisting of at least one base sequence selected from the group consisting of base sequences including single nucleotide polymorphisms a to g or a complementary sequence thereof, or a partial sequence thereof: The kit is capable of determining an allele or genotype of a single nucleotide polymorphism corresponding to the 17th of each base sequence included in the set of a to g. (Here, as described above, in the set of SEQ ID NOs indicated by a to g, each set of base sequences is a base sequence containing one allomorphic allele of each single nucleotide polymorphism in the 17th base. )
a: SEQ ID NO: 1 and / or SEQ ID NO: 2,
b: SEQ ID NO: 3 and / or SEQ ID NO: 4,
c: SEQ ID NO: 5 and / or SEQ ID NO: 6,
d: SEQ ID NO: 7 and / or SEQ ID NO: 8,
e: SEQ ID NO: 9 and / or SEQ ID NO: 10
f: SEQ ID NO: 11 and / or SEQ ID NO: 12, and
g: SEQ ID NO: 69 and / or SEQ ID NO: 70

  In the kit capable of determining any single nucleotide polymorphism allele or genotype of any of the above, among them, a sequence represented by an odd-numbered sequence number or a complement thereof among a set of sequences containing each single nucleotide polymorphism It is preferable that the kit is capable of determining an allele of a single nucleotide polymorphism corresponding to the 17th of each base sequence included in the set of a to g in a target sequence or a base sequence composed of these partial sequences. These are base sequences containing risk alleles at each polymorphic site.

(Preferred race)
The single nucleotide polymorphism associated with polypoidal choroidal vasculopathy used in the present invention is used in any race as long as it has the polymorphism, but the preferred race is Mongoloid, more preferably East Asian. More preferably, it is a Japanese or a person who has Japanese as an ancestor.

  According to the present invention, a person who has a genotype on the genome which is considered to be at risk for the risk allele or polypoidal choroidal vasculopathy disclosed in the present invention has a high risk of developing polypoidal choroidal vasculopathy in the future. Those who do not have a risk allele or a genotype that is considered to be at risk of polypoidal choroidal vasculopathy can be determined to have a low risk of developing polypoidal choroidal vasculopathy in the future.

  In addition, those who have a risk allele disclosed in the present invention or a genotype that is considered to be at risk of polypoidal choroidal vasculopathy on the genome and who are suspected of having polypoidal choroidal vasculopathy at an early stage are difficult to detect. Since there is a possibility of developing polypoidal choroidal vasculopathy, detection of the single nucleotide polymorphism of the present invention can be used for diagnosis of polypoidal choroidal vasculopathy or for assisting diagnosis.

EXAMPLES The present invention will be described below with reference to examples. However, the examples are provided for better understanding of the present invention, and the scope of the present invention is intended to be limited to these examples. It is not a thing. In the following examples, for general molecular biology methods that are not described in detail, molecular cloning (Joseph Sambrook et al., Molexular Cloning-A Laboratory Manual, 3 ^rd Edition, Cold Spring Harbor Laboratory Press , 2001), etc., and molecular biology grade reagents are used.

  In the present invention, total DNA is extracted from the blood of patients diagnosed with polypoidal choroidal vasculopathy, patients diagnosed with glaucoma, and patients diagnosed with glaucoma, and a known single nucleotide sequence on the human genome is extracted. By analyzing about 500,000 types of loci, the gene loci related to the disease were analyzed, and single nucleotide polymorphisms considered to be specifically related to polypoidal choroidal vasculopathy were examined.

Example 1 Extraction of DNA from a sample Extraction of DNA from a sample was performed according to the phenol extraction method (Methods in Enzymology, vol. 152, page 181 (1987)). Leukocytes were purified by adding a sufficient amount of hypotonic hemolysis buffer containing ammonium salt and EDTA to the blood sample to which citrate had been added. After several washes, the cells were lysed with a lysis buffer containing Proteinase K and SDS. After confirming that the suspended cells were lysed and clear, phenol was added and shaken at room temperature for 1-3 hours. The phenol layer was discarded and only the aqueous layer was centrifuged to further separate impurities. The aqueous layer was dialyzed against TE buffer (10 mM Tris pH 8.0, 0.1 mM EDTA, Teknova), and then DNA was collected by ethanol precipitation and dissolved in TE buffer. To 20 μL of the genomic DNA solution thus obtained, 7.5 μM NH ₄ OAc 10 μL, 20 mg / mL Glycogen 0.5 μL and 100% ice-cold ethanol 50 μL were added and left at -20 ° C. for 1 hour. After centrifugation at 12,000 × g for 20 minutes, the supernatant was removed. After adding 500 μL of 80% ice-cold ethanol and centrifuging at 12,000 × g for 5 minutes, the supernatant was removed and the precipitate was dried. The precipitate was dissolved in TE buffer and adjusted to a concentration of 50 ng / μL. The DNA concentration was measured by absorbance at 260 nm using a spectrophotometer (Biophotometer, Eppendorf). For some samples, DNA was extracted using an automatic nucleic acid extraction apparatus (Magtration System 8Lx, Precision System Science Co., Ltd.) according to the instructions of the apparatus.

Example 2 Analysis of Single Nucleotide Polymorphism Single nucleotide polymorphism analysis is performed by using a commercially available microarray single nucleotide polymorphism analysis kit (Affymetrix) capable of analyzing about 500,000 known single nucleotide polymorphisms on the human genome. GeneChip Human Mapping 250K Nsp / Sty Array (GeneChip (registered trademark) Human Mapping 250K Nsp / Sty Array) (hereinafter referred to as microarray) was used.

  The total DNA extracted in Example 1 (genomic DNA) is processed according to the instructions of the kit and instrument, and applied to the microarray to analyze single nucleotide polymorphisms present in the DNA extracted from the specimen. Specifically, the following operation is performed.

(Restriction enzyme treatment)
Each 250 ng of genomic DNA is cut with Sty I (New England Biolabs) or Nsp I (New England Biolabs) restriction enzymes, respectively, according to the following procedure. That is, 5 μL of genomic DNA solution, 11.5 μL of commercially available molecular biology grade ultrapure water (AccuGENE® molecular biology-grade water, Cambrex), 2 μL of buffer solution attached to each restriction enzyme, 10 mg / mL Restriction enzyme treatment was performed by adding 0.2 μL of BSA and 1 μL of 10 U / mL restriction enzyme. The reaction was carried out at 37 ° C. for 2 hours and then incubated at 65 ° C. for 20 minutes to inactivate the restriction enzyme.

(Addition of adapter sequence)
The adapter sequence is added to the sample after the restriction enzyme treatment by the following procedure. For the restriction enzyme-treated sample, 50 μM Adaptor Nsp I or Adaptor Sty I (New England Biolabs) 0.75 μL corresponding to the cleaved restriction enzyme, T4 DNA ligase buffer (New England Biolabs) 2.5 μL and T4 DNA ligase (New England Biolabs) 2 μL was added and reacted at 16 ° C. for 3 hours to add an adapter sequence, and then reacted at 70 ° C. for 20 minutes to inactivate T4 DNA ligase. The sample after the addition of the adapter was diluted by adding 75 μL of cooled AccuGENE water.

(Amplification and purification of gene fragments)
The gene to which the adapter sequence is added is amplified by PCR using the TITANIUM ^™ (registered trademark) DNA Amplification kit (Clontech) according to the following procedure. That is, AccuGENE water 39.5 μL, TITANIUM ^TM (registered trademark) taq PCR buffer 10 μL, 5 M GC-Melt (Clonetech) 20 μL, 2.5 mM dNTP (Takara Bio) 4.5 μL, 100 μM PCR primer002 (Affymetrix, GeneChip ( (Registered Trademark) 4.5 μL (included in Mapping 250K Nsp / Sty Assay Kit) and TITANIUM ^™ (registered trademark) taq DNA polymerase 2 μL were added and reacted. The reaction temperature and time are as follows. First, after standing at 94 ° C. for 3 minutes, the reaction was carried out for 30 cycles of 94 ° C. for 30 seconds → 60 ° C. for 30 seconds → 68 ° C. for 15 seconds and left at 68 ° C. for 7 minutes. With this as a series of reaction operations, the same PCR reaction operation was performed three times for each sample. The PCR product was subjected to 2% agarose gel electrophoresis, and it was confirmed that a gene fragment of 200 bp to 1000 bp was amplified. Three PCR products were combined into one and purified using a DNA amplification Clean-Up kit (Clontech). The purified PCR product was dissolved in 45 μL RB buffer (Clontech) and recovered. The sequence of PCR primer 002 used as a primer for PCR reaction is as follows: 5′-attatgagcacgacagacgcctgatct-3 ′ (SEQ ID NO: 71).

(Fragmentation of amplified gene)
The amplified gene is fragmented by the following procedure using GeneChip (registered trademark) Mapping 250K Nsp / Sty Assay Kit (Affymetrix) reagent. That is, the absorbance of the purified PCR product at 260 nm was measured using a spectrophotometer (Biophotometer, Eppendorf), and 90 μg of the PCR product was diluted with RB buffer (Clontech) to a total volume of 45 μL. Fragmentation buffer 5 μL and 0.05 U / μL Fragmentation Reagent 5 μL included in the kit were added to the diluted sample, reacted at 37 ° C. for 35 minutes, and allowed to stand at 95 ° C. for 15 minutes to inactivate Fragmentation Reagent. The Fragmentation Reagent was diluted with 10 × fragmentation buffer and AccuGENE water so that the final concentration was 0.05 U / μL Fragmentation Reagent, 1 × fragmentation buffer. The fragmented product was subjected to 4% agarose gel electrophoresis to confirm that the gene was fragmented.

(Add biotin label to fragmented gene)
A biotin label is added to the fragmented gene by GeneChip (registered trademark) Mapping 250K Assay Kit (Affymetrix) according to the following procedure. Add 5 μL of the fragmented sample to the terminal deoxynucleotidyl transferase buffer 14 μL, 30 mM GeneChip DNA Labeling Reagent 2 μL and 30 U / μL Terminal Deoxynucleotidyl Transferase enzyme 3.5 μL included in the kit, react at 37 ° C. for 4 hours, and then react at 95 ° C. for 15 minutes. The enzyme was inactivated by leaving to stand as a sample for hybridization.

(Hybridization)
Hybridization cocktail master mix in 70 μL sample with biotin label (composition is as follows; 0.056M 2- (N-Morpholino) ethanesulfonic acid, 5% DMSO, 2.5 × Denhard'ts solution (Sigma), 5.77 mM EDTA pH 8. 0, 0.115 mg / mL Herring Sperm DNA (Promega), 1 × Oligo Control Reagent 0100 (Affymetrix), 11.5 μg / mL Human Cot-1 DNA (Invitrogen), 0.0115% Tween-20, 2.69 M Tetrametyl Ammonium Chloride (Sigma) ) 190 μL was added, denatured at 95 ° C. for 10 minutes, and maintained at 49 ° C. Hybrid oven (GeneChip Hybridization Oven 640, Affymetrix) with 200 μL sample applied to GeneChip (registered trademark) Human Mapping 250K Nsp / Sty Array (hereinafter also referred to as microarray), temperature set to 49 ° C, rotation speed 60 r / min For 16 to 18 hours. The sample was removed from the microarray, and Array Holding Buffer (1 mM MES, 1M NaCl, 0.01% Tween-20) was filled inside the microarray.

(Staining and detection)
Staining of the microarray after hybridization was performed using GeneChip Fluidics Station 450 of Affymetrix, Inc., GeneChip (registered trademark) Operating Software 1.4 (hereinafter GCOS), protocol: MAP500Kv1_450. Primary and tertiary staining were each performed with 10 μg / mL Streptavidin-Phycoerythrin (Invitrogen) prepared with Stain Buffer, and secondary staining was performed with 5 μg / mL biotinylated Anti-Streptavidin Antibody (Vector Lab.). Detection was performed with GeneChip (registered trademark) Scanner 3000 7G using GCOS. The specific protocol for the staining / washing process and the composition of the washing solution are as follows.
(Dyeing and washing process protocol (FS-450 Fluidics Protocol-Mapping 500Kv1_450))
-Post Hyb Wash # 1: 6 cycles of 5 mixes / cycle with Wash Buffer A at 25 ℃
-Post Hyb Wash # 2: 24 cycles of 5 mixes / cycle with Wash Buffer B at 45 ℃
-Stain: Stain the probe array for 10 minutes in SAPE solution at 25 ℃
-Post Stain: Wash 6 cycles of 5 mixes / cycle with Wash Buffer A at 25 ℃
-2nd Stain: Stain the probe array for 10 minutes in Antibody Stain Solution at 25 ℃
-3rd Stain: Stain the probe array for 10 minutes in SAPE solution at 25 ℃
-Final Wash: 10 cycles of 6 mixes / cycle with Wash Buffer A at 30 ℃
The final holding temperature is 25 ℃
[Solution composition]
・ Wash A: Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween 20)
For 1000 mL:
300 mL of 20X SSPE, 1.0 mL of 10% Tween-20, 699 mL of water
Filter through a 0.2 μm filter.Store at room temperature.
・ Wash B: Stringent Wash Buffer (0.6X SSPE, 0.01% Tween 20)
For 1000 mL:
30 mL of 20X SSPE, 1.0 mL of 10% Tween-20, 969 mL of water
Filter through a 0.2 μm filter.Store at room temperature.
The pH should be 8.
・ Stain Buffer: (6X SSPE, 0.01% Tween 20, 1X Denhardt's solution)
For 1188 μL:
360 μL of 20X SSPE, 3.6 μL of 3% Tween-20, 24 μL 50X Denhardt's solution, 800.04 μL of water

(Genotyping analysis)
SNP Genotyping analysis was performed using Affymerix Power Tools ver.1.10.0 from Affymetrix. The included BRLMM genotype calling algorithm was used for analysis.

Example 3 Comparison of single nucleotide polymorphisms of PCV patients and non-PCV patients on autosomes Comparison of disease-related single nucleotide polymorphisms was performed according to the method of Klein et al. (Science 308, 385, 2005) as follows. Went so.

(Test group setting)
Patients who satisfy the following criteria were set as PCV patients. That is, at least one eye has a clear orange-red raised lesion confirmed by fundus examination, and indocyanine green fluorescence fundus contrast examination confirms a characteristic polyp-like choroidal lesion. Set to. On the other hand, patients diagnosed with glaucoma, i.e., optic discs showed glaucoma-specific findings (such as enlarged optic disc depression) and retinal nerve fiber layer defects in fundus examination. One hundred patients with primary open-angle glaucoma with consistent visual field abnormalities were assigned to the glaucoma patient group. In addition, 100 patients who were diagnosed with cataract and did not recognize eye diseases other than cataract and refractive error were included in the cataract patient group.

(Single nucleotide polymorphism analysis)
Using the blood provided with the consent obtained by the participants' voluntary will as a sample, total DNA was extracted by the method described in Example 1, and single nucleotide polymorphisms were analyzed by the method described in Example 2. It was. The following analysis was performed using statistical analysis language R ver.2.7.0. That is, a single nucleotide polymorphism with a call rate of less than 90%, a single nucleotide polymorphism with a minor allele frequency of less than 5%, and a single nucleotide polymorphism that is judged to have no Hardy-Weinberg equilibrium at a significance level of 0.0001. A single nucleotide polymorphism that was considered to be highly experimentally reliable was selected by rejecting all types and confirming that they were correctly called by visual inspection. The test for the establishment of the Hardy-Weinberg equilibrium was performed using the chi-square test.

(Primary analysis of single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy)
In order to extract candidates for single nucleotide polymorphisms related to polypoidal choroidal vasculopathy with respect to the single nucleotide polymorphism thus selected, (1) PCV patient group vs. glaucoma patient group, (2) PCV patient group pair Two comparative analyzes of cataract patient groups were performed. In order to extract candidates for single nucleotide polymorphisms specifically related to polypoidal choroidal vasculopathy, in common with (1) and (2), three genotypes (homo, hetero, one allele, 370 single nucleotide polymorphisms having a minimum p value of less than 0.01 when compared for each homozygote of alleles were extracted and used as candidates for single nucleotide polymorphisms related to polypoidal choroidal vasculopathy. Note that Fisher's exact test was used to calculate the p-value of the genotype. Here, in order to confirm the validity of the selected 370 disease-related single nucleotide polymorphism candidates, the False discovery rate for the 370 SNPs to be analyzed was determined by the method of Benjamini and Hochberg (Benjamini Y, Hochberg T: J Royal Stat Soc B85 pp289-300, 1995). All 370 single nucleotide polymorphisms that were candidates were included in the range where the false discovery rate was below 5%. That is, these single nucleotide polymorphisms are considered to have an extremely high probability of being related to a disease.

(Secondary analysis of single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy)
The single nucleotide polymorphism candidates related to polypoidal choroidal vasculopathy selected in this way were analyzed in more detail. That is, the patients employed in the glaucoma patient group and the patients employed in the cataract patient group were collectively used as a control group, and a comparative analysis of the PCV patient group and the control group was performed as follows.

(Allele odds ratio)
The odds ratio of the allele was calculated by the following formula as an index of how strong a relationship exists between the presence or absence of the risk allele and the presence or absence of polypoidal choroidal vasculopathy.

  Here, the risk allele was set so that the odds ratio was greater than 1 when the odds ratio of the allele was calculated by substituting the number of detected alleles constituting the single nucleotide polymorphism into the above formula. . When the calculation is performed by exchanging one allele and the other allele, the odds ratio is a reciprocal relationship with each other, that is, if one odds ratio is smaller than 1, the other odds ratio is larger than 1. It is clear from the definition formula.

(Calculation of genotype p-value)
The genotype p value of each SNP was calculated using Fisher's exact test for the PCV patient group versus the control group. In that case, genotype model (risk allele homotype vs heterotype vs non-risk allele homotype), dominant model (risk allele homotype + heterotype vs non-risk allele homotype), recessive model (risk allele homotype vs non-risk allele) The p value was calculated for three types of statistical models (homo type + hetero type), and the one with the lowest p value was used as the p value representing the single nucleotide polymorphism.

Analysis was conducted as described above, and 363 single nucleotide polymorphisms having a p value of less than 0.01 were selected as single nucleotide polymorphisms related to polypoidal choroidal vasculopathy. Furthermore, Bonferroni correction, that is, the p value (the single nucleotide polymorphism) below the corrected significance level (1.35 × 10 ⁻⁴ ) or less obtained by setting the overall significance level to 0.05 and dividing the significance level 0.05 by 370 iterations. A single nucleotide polymorphism whose genotype is related to polypoidal choroidal vasculopathy was extracted.

(Odds ratio of genotype)
Further, for these single nucleotide polymorphisms, the dominant-type odds ratio and the recessive-type odds ratio were determined by the following formula based on the risk allele determined as described above. Here, for any single nucleotide polymorphism, if any genotype frequency is 0, and thus any odds ratio becomes infinite, approximate calculation is performed with the genotype frequency set to 0.5. It was. As described above, in the present invention, the dominant odds ratio is the odds ratio of polypoidal choroidal vasculopathy when at least one risk allele is present (that is, when the risk allele is homozygous or heterozygous). Yes, the recessive odds ratio is the odds ratio of polypoidal choroidal vasculopathy when the risk allele is homozygous.

(result)
Among the single nucleotide polymorphisms related to polypoidal choroidal vasculopathy thus obtained, Table 1 shows single nucleotide polymorphisms with lower p-values, that is, single nucleotide polymorphisms with p <1.0 × 10 ^−5. Table 2 shows the single nucleotide polymorphisms of 1.0 × 10 ⁻⁵ ≦ p ≦ 1.35 × 10 ⁻⁴ for each SNP ID, chromosome number and physical position where the single nucleotide polymorphism exists, and the single nucleotide polymorphism. The name of the gene to which it belongs or the name of the gene present in the vicinity of the single nucleotide polymorphism, the relative position (if the single nucleotide polymorphism is present on the intron of the gene indicated by the gene name, this is indicated. If the type does not exist on the intron of the gene, it indicates the distance between the gene and the single nucleotide polymorphism), used to calculate the risk allele base, the non-risk allele base, and the genotype p-value. Statistical model, p-value of genotype, odds ratio of allele, and risk A sequence number corresponding to a 33-base sequence consisting of a allele and 16 bases before and after it (sequence including a risk allele) and a sequence corresponding to a 33-base sequence composed of a non-risk allele and 16 bases before and after that Enter the number (sequence containing the non-risk allele). The SNP ID is the dbSNP database ID. Among these information, chromosome number and physical position, gene name and relative position, and sequence information including each allele are available from technical data provided by Affymetrix and public databases such as NCBI SNP and NCBI Nucleotide using dbSNP ID etc. as a key Extracted. In the p value column, each p value is displayed in an exponential format with 10 as the base. Here, the numerical values described before and after the symbol E indicate the mantissa part and the exponent part when the p-value is displayed in the exponential format (the same applies to the tables in the examples below).

Furthermore, single nucleotide polymorphisms with 1.35 × 10 ⁻⁴ <p <1.0 × 10 ⁻² are shown in Tables 3-10.

  All of the 363 single nucleotide polymorphisms listed in Tables 1 to 10 are 370 single nucleotide polymorphisms selected in the primary analysis (that is, the FDR calculated for all of the 370 single nucleotide polymorphisms is 5). And a preferred single nucleotide polymorphism is selected by secondary analysis using a population as a population) and can be used in the present invention with sufficient reliability.

  Furthermore, for these single nucleotide polymorphisms, the risk allele frequency, the non-risk allele frequency, the risk allele homotype frequency, the hetero frequency, the non-risk allele homotype frequency are shown in Tables 11 to 19 for each of the PCV patient group and the control group. Describe. Tables 20 to 28 list the genotype odds ratio of each single nucleotide polymorphism, the related genotype odds ratio, and the genotype at risk. Here, the genotype odds ratio is the dominant type odds ratio and the recessive type odds ratio determined for the single nucleotide polymorphism (that is, the genotype odds ratio of the genotype related to the single nucleotide polymorphism). Odds ratio) value is described, and the related odds ratio indicates whether the odds ratio of the genotype related to the single nucleotide polymorphism is a dominant type odd ratio or a recessive type odds ratio.

Example 4 Comparison of single nucleotide polymorphisms related to wet age-related macular degeneration and single nucleotide polymorphisms related to polypoidal choroidal angiopathy Genes reported to have single nucleotide polymorphisms related to age-related macular degeneration A part of single nucleotide polymorphisms present in the locus was compared with whether it was associated with each of the diseases of wet age-related macular degeneration and polypoidal choroidal vasculopathy. Staging of patients with wet age-related macular degeneration was performed according to a report by Sedon et al. (Ophthalmology 113, 260, 2006). 100 Japanese patients with at least one of the left and right eyes diagnosed with sedon staging system grade 5b, that is, wet age-related macular degeneration with choroidal neovascularization or scar formation, and who are not polypoidal choroidal angiopathy 100 An example was employed in a wet age-related macular degeneration patient group (hereinafter, wAMD patient group). DNA was extracted in accordance with Example 1 from blood provided from 100 wAM patient groups with the consent obtained by the participants' voluntary intention, and single nucleotide polymorphisms were analyzed according to Example 2. The control group was 190 patients with glaucoma and cataract.

  The p-value was calculated using Fisher's exact test and Cochran-Armitage trend test for the age-related macular degeneration patient group versus the control group. In Ficher's exact test, the genotype model (risk allele homotype vs. heterotype vs. non-risk allele homotype), dominant model (risk allele homotype + heterotype vs. non-risk allele homotype), recessive model (risk allele homology) The p value was calculated for three types of statistical models (type vs. non-risk allele homotype + heterotype). The minimum value of these four p values (that is, three by Fishcer's exact test and one by trend test) was used as the p value of the single nucleotide polymorphism. On the other hand, for PCV patients, the same patient group as in Example 3 was used, and in the same manner as in the case of wet age-related macular degeneration, compared to the 190 control group used in the analysis of wet age-related macular degeneration. Analysis was performed. The odds ratio was calculated in the same manner as in Example 3.

(result)
The single nucleotide polymorphism rs2241394 located at the C3 locus was significantly associated with polypoidal choroidal vasculopathy by the method of Benjamini and Hochberg in this study (FDR <0.05). On the other hand, the single nucleotide polymorphism did not show an association with wet age-related macular degeneration. The examination result in the polypoidal choroidal angiopathy of the said single nucleotide polymorphism is described in Tables 29-31. Table 29 shows the SNP ID (dbSNP ID), the chromosome number and physical position where the single nucleotide polymorphism exists, the gene to which the single nucleotide polymorphism belongs or the name of the gene present in the vicinity of the single nucleotide polymorphism, relative Position (when the single nucleotide polymorphism is present on the intron of the gene indicated by the gene name, this is indicated, and when the single nucleotide polymorphism is not present on the intron of the gene, the gene and the single nucleotide (Indicates the distance between polymorphisms), risk allele bases, non-risk allele bases, statistical model used to calculate genotype p-value, genotype p-value, allele odds ratio, dominant odds Ratio, Recessive odds ratio, and a sequence number corresponding to a 33-base sequence consisting of a risk allele and 16 bases each before and after it (non-risk allele and 16 bases each before and after it) 33 base length The sequence number corresponding to the column (sequence including non-risk allele) is described. Table 30 lists the risk allele frequency, non-risk allele frequency, risk allele homozygote frequency, heterozygous frequency, and non-risk allele homozygous frequency of the single nucleotide polymorphism. Furthermore, in Table 31, the genotype odds ratio of the single nucleotide polymorphism, the related genotype odds ratio, and the genotype at risk are listed. Here, the genotype odds ratio describes the value of the Dominant odds ratio and Recessive odds ratio obtained for the single nucleotide polymorphism that are large, and the related odds ratio is the single nucleotide polymorphism. Indicates whether the odds ratio of the genotype related to the type is a Dominant odds ratio or a Recessive odds ratio.

Example 5 Estimation of LD block structure and extraction of tag SNP The LD block structure to which the single nucleotide polymorphism of the present invention belongs can be estimated as follows.

Using the genotyping data of the single nucleotide polymorphism determined by the HapMap project, the LD block containing the single nucleotide polymorphism related to the polypoidal choroidal vasculopathy determined in Example 3 or Example 4 is estimated. For single nucleotide polymorphisms that have been clarified to exist in the LD block, for example, narrowing is performed by the pairwise tagging method that rejects R ² <0.8 and MAF <0.2, and the single nucleotide polymorphism that represents this LD block is represented. Extract the type, ie tag SNP. R ² is a measure of linkage disequilibrium. Assuming that the two single nucleotide polymorphisms S1 and S2 are each composed of two alleles (1, 2), there are four possible haplotypes. When the frequency of each of the four haplotypes is (h ₁₁ , h ₁₂ , h ₂₁ , h ₂₂ ) (the first subscript represents the S1 allele, the second subscript represents the S2 allele), R ² Is defined by the following equation.

Example 6 Reanalysis of single nucleotide polymorphism on LD block Reanalyze the tag SNP estimated in Example 5 and any single nucleotide polymorphism belonging to the LD block containing the tag SNP as follows. Can do. This example illustrates a reanalysis method using TaqMan (registered trademark) SNP Genotyping Assays (Applied Biosystems).

  The patient group and the control group may be the same as or different from the patient group and the control group of Example 3 or Example 4. Using the blood provided by these participants as a sample, for example, total DNA was extracted by the method described in Example 1, and the single nucleotide polymorphism contained in the LD block was compared with the patient group according to the protocol of Applied Biosystems. Perform comparative analysis of groups. The reagents, thermal cycler, and detector for the real-time PCR reaction can be those specified by Applied Biosystems. When the StepOne Plus real-time PCR system is used for the thermal cycler and detector, StepOne Software V2.0.1 can be used to detect the single nucleotide polymorphism allele in the PCR product.

  Using the obtained single nucleotide polymorphism detection result, the allele odds ratio is calculated in the same manner as in Example 3 to determine the risk allele, and the genotype p-value is calculated.

  In this way, it is possible to confirm an association with a disease for any single nucleotide polymorphism in the LD block, and to determine a risk allele and a genotype that is a risk of the disease.

Example 7 Analysis of single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy using a sample containing genomic DNA In the single nucleotide polymorphisms associated with polypoidal choroidal vasculopathy disclosed in the present invention, Tables 1 to 10 or 29 The frequency of alleles described in 1 is statistically different between the PCV patient group and the control group. By determining the allele of at least one of these single nucleotide polymorphisms contained in a sample containing genomic DNA, it can be determined whether or not a risk allele exists. When a risk allele is detected for at least one of the single nucleotide polymorphisms of the present invention in the genomic DNA of the sample, it is determined that the single nucleotide polymorphism risk allele associated with polypoidal choroidal vasculopathy is included in the sample Is done. Instead of an allele, it is also possible to determine the genotype considered to be at risk for the aforementioned polypoidal choroidal vasculopathy. Furthermore, using one sample, it is also possible to detect two or more alleles or genotypes of single nucleotide polymorphisms associated with the polypoidal choroidal vasculopathy of the present invention, which are frequently observed in diseases.

Example 8 Precise determination of risk of polypoidal choroidal vasculopathy in non-PCV patients and diagnostic assistance for persons suspected of polypoidal choroidal vasculopathy In the same manner as in the previous examples, genomic DNA provided by non-PCV patients was obtained. Using the containing sample, the risk of polypoidal choroidal vasculopathy of the sample provider can be accurately determined. Specifically, for a single nucleotide polymorphism associated with the disease disclosed in the present invention, when at least one genotype that is considered to be at risk of a risk allele or polypoidal choroidal vasculopathy is detected in the sample, Determined to be at risk for polypoidal choroidal vasculopathy. It is possible to improve the accuracy of predicting the risk of polypoidal choroidal vasculopathy by detecting a combination of two or more of the single nucleotide polymorphisms of the present invention by combining a plurality of probes for detecting single nucleotide polymorphisms related to a disease. it can. In this case, the risk is predicted using the sum of the number of genotypes considered to be at risk of the risk allele or polypoidal choroidal vasculopathy detected in the sample or the odds ratio thereof as an index. Depending on the magnitude of these numbers, the risk of polypoidal choroidal vasculopathy is predicted to be large or small, and when a certain threshold value is exceeded, it is determined that the risk of polypoidal choroidal vasculopathy is high.

  Further, similarly, diagnosis assistance for a person suspected of having polypoidal choroidal vasculopathy can be performed. If a sample provided by a person suspected of having polypoidal choroidal vasculopathy and at least one of the risk alleles or a genotype that is considered to be at risk of polypoidal choroidal vasculopathy is detected in the sample, polypoidal choroidal vessels There is a high probability that it should be judged as a disease. Further, a risk allele or polypoidal choroidal blood vessel detected in a sample by combining a plurality of probes for detecting a single nucleotide polymorphism associated with a disease and detecting two or more single nucleotide polymorphisms of the present invention in combination. The degree to which the probability to be judged as polypoidal choroidal vasculopathy is evaluated using the sum of the number of genotypes considered to be at risk of the disease or the odds ratio thereof as an index. When a certain threshold value is exceeded, it can be diagnosed as polypoidal choroidal vasculopathy.

  By analyzing the allele or genotype of the single nucleotide polymorphism of the present invention in a sample by the method of the present invention, the presence / absence and / or level of risk of future polypoidal choroidal vasculopathy of the sample provider is determined. be able to. Based on this risk, the sample provider will take precautionary measures for polypoidal choroidal vasculopathy and / or periodically undergo examination of the fundus, eg, ICG fluorescence imaging, etc. to diagnose polypoidal choroidal vasculopathy at an early stage Can start treatment. A sample provider suspected to have polypoidal choroidal angiopathy having a genotype considered to be at risk of a single nucleotide polymorphism risk allele of the present invention or polypoidal choroidal angiopathy is polypoidal choroidal angiopathy and This is useful because there is a high probability of being diagnosed and treatment can be started early.

Claims (12)

About the nucleic acid molecule in the sample derived from the subject, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 or a complementary sequence thereof, a single base present at the 17th position of the base sequence A step of detecting a polymorphic allele in vitro (detection step), and a step of determining whether at least one of the detected alleles is a risk allele (determination step)
A method for predicting the risk of polypoidal choroidal vasculopathy.   The method for predicting the risk of polypoidal choroidal vasculopathy according to claim 1, further comprising a step of determining a genotype based on the allele detected in the detection step.   In at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70, or a complementary sequence thereof, or a partial sequence thereof, the 17th of the base sequences represented by SEQ ID NOs: 1 to 70 The method for predicting the risk of polypoidal choroidal vasculopathy according to claim 1 or 2, wherein the detection step is performed using a nucleic acid molecule containing a sequence having a corresponding allele.   The method for predicting the risk of polypoidal choroidal vasculopathy according to claim 3, wherein the nucleic acid molecule is used as a probe.   The method for predicting the risk of polypoidal choroidal vasculopathy according to claim 4, wherein the probe is 23 to 27 bases in length.   The method for predicting the risk of polypoidal choroidal vasculopathy according to claim 4 or 5, wherein the probe is solidified.   About the nucleic acid molecule in the sample derived from the subject, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 or a complementary sequence thereof, a single base present at the 17th position of the base sequence A kit for predicting the risk of polypoidal choroidal vascular disease, comprising a nucleic acid molecule for detecting a polymorphic allele in vitro.   In the nucleic acid molecule, at least one base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 1 to 70 or a complementary sequence thereof, or a partial sequence thereof, the base sequences represented by the above-mentioned SEQ ID NOs: 1 to 70 The kit for predicting the risk of polypoidal choroidal vasculopathy according to claim 7, which is a nucleic acid molecule comprising a sequence having an allele corresponding to the 17 th of the above.   The polypoidal choroidal vasculopathy according to claim 8, wherein the nucleic acid molecule is at least one base sequence selected from the group consisting of base sequences represented by SEQ ID NOs: 1 to 70, a complementary sequence thereof, or a partial sequence thereof. Risk prediction kit.   The prediction kit for the risk of polypoidal choroidal vasculopathy according to any one of claims 7 to 9, wherein the nucleic acid molecule is used as a probe.   The kit for predicting the risk of polypoidal choroidal vasculopathy according to claim 10, wherein the probe has a length of 23 to 27 bases.   12. The kit for predicting the risk of polypoidal choroidal vasculopathy according to claim 10 or 11, wherein the probe is solidified.