Patent Application
20100196895
The present invention relates to a method of detecting the presence of a single nucleotide polymorphism associated with the onset of glaucoma, or a single nucleotide polymorphism with a high onset risk of glaucoma, and a kit used in the detection method.
Glaucoma is a disease which causes a characteristic optic nerve cupping and an impairment in a visual field by retinal ganglion cell death.
An elevation in an intraocular pressure is considered to be a major cause for the nerve cupping and the impairment in the visual field in glaucoma. On the other hand, there is also glaucoma in which an intraocular pressure is held within a normal range in statistical calculation, and even in this case, it is considered that glaucoma develops because the intraocular pressure is at a sufficiently high level for causing the impairment in a visual field for an individual.
The basic treatment for glaucoma is to maintain an intraocular pressure at a low level, and it is necessary to consider the causes for a high intraocular pressure in order to maintain a low intraocular pressure. Therefore, in the diagnosis of glaucoma, it is important to classify the types of glaucoma in accordance with the levels of intraocular pressures and causes therefor. As the causes for an elevation in an intraocular pressure, the presence or absence of closure of angle which is a major drainage pathway for an aqueous humor filling an eye is important. From these viewpoints, the primary glaucoma is roughly classified into the two groups of closed-angle glaucoma accompanying angle closure and open-angle glaucoma without accompanying angle closure. Among them, the open-angle glaucoma is classified into open-angle glaucoma, in a narrow sense, accompanying an elevation in an intraocular pressure, i.e. primary open-angle glaucoma, and normal tension glaucoma in which an intraocular pressure is held within a normal range.
It is known from old times that glaucoma is associated with inheritance. It is reported that 5 to 50% of individuals with open-angle glaucoma have a family history, and it is generally understood that 20 to 25% of individuals have hereditary causes. Based on these reports, studies on a search for a gene responsible for glaucoma are performed. As a result, it is reported that a mutation in myocilin (MYOC) gene is associated with open-angle glaucoma (See Patent Publication: 1), and that a mutation in optineurin gene (OPTN) is associated with normal tension glaucoma (See
Non-Patent Publication: 1). However, all the genetic causes of glaucoma cannot be explained only by these genes, and the presence of unknown glaucoma-related genes is expected.
On the other hand, a single nucleotide polymorphism means that a substitution mutation in which a single base is changed into another base is found in base sequences of the genome of an individual, and the mutation exists in a certain frequency, generally a frequency of about 1% or more, in the population of an organism species. A single nucleotide polymorphism exists at intron or exon on genes, or any of the regions of the genome other than these.
Patent Publication 1: Japanese Patent-Laid Open No. 2000-306165 Non Patent Publication 1: Rezaie T and eleven others, Science, 2002, 295(5557), 1077-1079.
Generally, an intraocular pressure or an ocular fundus photograph is used as a simple examination for glaucoma; however, these examinations do not necessarily lead to a definite diagnosis for glaucoma. Usually, in addition to these, visual field examinations are performed; however, there are some disadvantages that the examination is carried out for a long period of time, causing burdens on patients, and that one must be accustomed to the examination, so that initial examination results have low reliability.
On the other hand, as mentioned above, the involvement of hereditary causes is strongly suspected in the onset of glaucoma, but critical responsible genes are not identified. On the other hand, even if the involvement of a single gene to the disease cannot be explained by a mutation or polymorphism, it is considered that there are numerous mutations or polymorphisms of a gene of which involvement to glaucoma is relatively moderate, and the involvement of hereditary causes to the onset of glaucoma can be explained by a combined action of each of these mutations or polymorphisms.
The inventors have remarked on a polymorphism on the genome, especially a single nucleotide polymorphism, in order to find a gene associated with glaucoma.
By finding polymorphisms involved in the onset of glaucoma, a person having the polymorphisms which are found in a high frequency in glaucoma patients is predicted to have a high onset risk of glaucoma in future even before the onset thereof. Also, the polymorphisms can be applied to screening of whether or not a visual field examination is required, in an early stage of glaucoma which is difficult to be detected by the simple determination of glaucoma, i.e. a method such as a measurement of intraocular pressure or an ocular fundus photograph, which is available to be diagnosed only by carrying out the visual field examination. In other words, a sample donor can take a preventive measure for the onset of glaucoma by knowing the onset risk of glaucoma, and in addition, a necessary measure for preventing visual constriction such as a definite diagnosis and an initiation of treatment at an early stage according to a precision examination can be taken; therefore, it is important to find a polymorphism involved in the onset of glaucoma.
An object of the present invention is to provide a method of detecting a single nucleotide polymorphism involved in the onset of glaucoma, thereby predicting an onset risk of glaucoma, and a kit used in the detection method.
The present inventors have found a single nucleotide polymorphism associated with the onset of glaucoma by a comprehensive analysis of known polymorphic sites existing on the genome (autosome) in glaucoma patients and non-patients without a family history of glaucoma, and further found an allele identified in a high frequency in glaucoma patients and an opposite allele thereof, and a genotype identified in a high frequency in glaucoma patients, which is a combination of each of the alleles, in the single nucleotide polymorphism. Furthermore, the present inventors have found that a determination on whether or not a sample donor is a person who is more likely to suffer from the onset of glaucoma can be made at an even higher precision by performing the determination in a combination of these plural single nucleotide polymorphisms associated with the onset of glaucoma. Thus, the present invention has been perfected thereby.
Concretely, the present invention relates to:
[1] a method of determining the presence or the absence of a glaucoma risk, including the steps of:
detecting in vitro an allele and/or a genotype of a single nucleotide polymorphism which is located on a 31st base of a base sequence, in a sample from a subject, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto (step A), and
comparing the allele and/or the genotype detected in the step A with at least one of an allele and/or a genotype, containing a high-risk allele, in the base sequences shown in SEQ ID NOs: 203 to 514 (step B),
detecting in vitro, in a sample from a subject, an allele and/or a genotype of a single nucleotide polymorphism which is located on a 31st base of a base sequence in a nucleic acid molecule, wherein the nucleic acid molecule comprises at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto (step C1), or
detecting in vitro, in a sample from a subject, an allele and/or a genotype of a single nucleotide polymorphism, using a nucleic acid molecule comprising a base sequence containing at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 515 to 694 or a complementary sequence thereto (step C2), and
comparing the allele and/or the genotype detected in the step C1 or C2 with at least one nucleic acid molecule comprising an allele and/or a genotype, containing a high-risk allele, in the base sequences shown in the SEQ ID NOs: 203 to 514 (step D),
a nucleic acid molecule comprising at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto, or a partial sequence thereof, wherein the nucleic acid molecule comprises a single nucleotide polymorphism which is located on a 31st base of a base sequence, and/or
a nucleic acid molecule comprising a base sequence containing at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 515 to 694 or a complementary sequence thereto,
detecting in vitro an allele and/or a genotype of a single nucleotide polymorphism which is located on a 31st base of a base sequence, in a sample from a subject, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto (step E), and
comparing the allele and/or the genotype detected in the step E with at least one of an allele and/or a genotype, containing a high-risk allele, in the base sequences shown in SEQ ID NOs: 203 to 514 (step F), wherein the subject is diagnosed as glaucoma in a case where the allele detected in the step E is the high-risk allele, or
According to the method of the present invention, the presence or the absence of the onset risk of glaucoma in a sample donor can be determined, and further the level of the risk can be predicted, by analyzing an allele or a genotype of a single nucleotide polymorphism in the present invention contained in a nucleic acid molecule derived from the genome existing in a sample. A sample donor can be provided with a preventive measure for glaucoma, or can receive appropriate treatments, on the basis of this risk. In addition, according to the method of the present invention, a sample donor who is suspected of glaucoma, having an allele or a genotype containing a single nucleotide polymorphism in the genome that is identified in a high frequency in glaucoma patients, can be given a detailed examination on whether or not the donor is with early glaucoma, which is difficult to be determined sufficiently by an intraocular pressure or an ocular fundus photograph, and can be started with a treatment at an early stage in a case where the donor is diagnosed as glaucoma.
The present invention is a method of determining the presence or the absence of a glaucoma risk, including the step of detecting in vitro an allele and/or a genotype having at least one single nucleotide polymorphism using at least one single nucleotide polymorphism (hereinafter may be referred to as SNP) contained in a base sequence selected from the group consisting of specified base sequences or a complementary sequence thereto, wherein the method of determining the presence or the absence of a glaucoma risk further includes the step of:
comparing the allele and/or the genotype detected in the step with at least one of an allele and/or a genotype, containing a high-risk allele, in the specified base sequences, in a sample from a subject,
A great feature of the present invention resides in that a single nucleotide polymorphism associated with the onset of glaucoma is found, further that in the single nucleotide polymorphism, an allele identified in a high frequency in glaucoma patients and an opposite allele thereof, and a genotype, which is a combination of each of the alleles identified in a high frequency in glaucoma patients are found, and used. The polymorphism as used herein refers to a matter that a diversity is found in sequences of a specified location on the genome in a certain organism species, and a site at which the polymorphism exists (hereinafter also referred to as polymorphic site) refers to a site on the genome that a single nucleotide polymorphism is found.
In addition, the allele as used herein refers to each of types having a different base from each other that can be taken in a certain polymorphic site. The genotype as used herein refers to a combination of opposite alleles in a certain polymorphic site. Further, in a certain polymorphic site, there are three types for a genotype which is a combination of opposite alleles, wherein a combination of the same alleles is referred to as a homozygote, and a combination of different alleles is referred to as a heterozygote.
The opposite allele as used herein refers to another allele corresponding to a specified allele among the alleles constituting a certain single nucleotide polymorphism.
In the present invention, the single nucleotide polymorphism associated with glaucoma refers to a single nucleotide polymorphism associated with the onset of glaucoma or a single nucleotide polymorphism associated with the progression of glaucoma. In other words, the single nucleotide polymorphism associated with the onset of glaucoma refers to a single nucleotide polymorphism in which each allele or each genotype frequency in the single nucleotide polymorphism significantly differs in a statistical analysis at a given p-value between glaucoma patients and non-patients; and the single nucleotide polymorphism associated with the progression of glaucoma refers to a single nucleotide polymorphism in which each allele or each genotype frequency in the single nucleotide polymorphism significantly differs in a statistical analysis at a given p-value between the progressive glaucoma cases and the nonprogressive glaucoma cases.
In the present invention, the high-risk allele refers to an allele having a higher frequency in a glaucoma patient group than that of a non-patient group among each of the alleles of the single nucleotide polymorphism associated with glaucoma. On the other hand, in the present invention, the low-risk allele refers to an allele opposite to the high-risk allele in a certain polymorphic site.
In addition, the homozygote and the heterozygote of a genotype are defined in the same manner as in the high-risk allele and the low-risk allele.
In other words, in certain polymorphic sites, a combination of high-risk alleles or low-risk alleles themselves is referred to a homozygote, and a combination of a high-risk allele and a low-risk allele is referred to as a heterozygote.
An embodiment where allele frequencies of the glaucoma patient group and the non-patient group are statistically compared is referred to as an allele model, and an embodiment where genotype frequencies thereof are compared is referred to as a genotype model. There are a dominant genetic model and a recessive genetic model in the genotype models, wherein the former means an embodiment where both a homozygote of high-risk alleles and a heterozygote are involved with the onset risk, and the latter means an embodiment where a homozygote of a high-risk allele is involved with the onset risk.
In the present invention, the glaucoma risk refers to a risk concerning glaucoma. The onset risk of glaucoma refers to a possibility of the future onset of glaucoma determined by susceptibility to a disease. In the present invention, the prediction of a risk refers to a determination of the presence or the absence of a future risk at the present stage, or determining the level of a future risk at the present stage.
Also, the glaucoma as used herein means preferably open-angle glaucoma (OAG) or normal tension glaucoma (NTG), and the open-angle glaucoma, when used without specifying otherwise, means primary open-angle glaucoma (POAG) in a narrow sense, without embracing normal tension glaucoma.
A method of identifying a single nucleotide polymorphism associated with glaucoma will be explained hereinbelow.
In the present invention, in selecting the single nucleotide polymorphism associated with glaucoma, in particular, a candidate single nucleotide polymorphism is selected by the steps including extracting a total DNA from blood of each of glaucoma patients diagnosed as primary open-angle glaucoma or normal tension glaucoma and non-patients diagnosed as not being with glaucoma and determined to have no family history of glaucoma according to a medical interview (also referred to as control individuals), and comparing allele or genotype frequencies of individual single nucleotide polymorphisms in the glaucoma patients and the non-patients using known single nucleotide polymorphisms of about 500,000 on the human genome as an index. Further, the allele or genotype frequencies of individual single nucleotide polymorphisms for the single nucleotide polymorphisms that are selected as candidates are obtained for glaucoma patients and non-patients that are different from the sample groups mentioned above. By combining these results, a single nucleotide polymorphism of which difference in frequencies is recognized with high statistical significance is found. Here, a group composed of the glaucoma patients is referred to as a glaucoma patient group, and a group composed of the non-patients is referred to as a non-patient group. By using the alleles or genotypes having a single nucleotide polymorphism associated with the onset of glaucoma found according to these analyses, the determination of the presence or the absence of the onset risk of glaucoma, and the prediction of the level of an onset risk can be enabled. Although the details will be explained in the section of Examples, a single nucleotide polymorphism associated with glaucoma disclosed in the present invention can be identified according to a method given below.
(Identification of Single Nucleotide Polymorphism Associated with Glaucoma)
First, a total DNA is extracted from blood of each of patients diagnosed as glaucoma and non-patients determined to have no family history of glaucoma. The total DNA in blood can be extracted by any known methods; for example, a DNA can be extracted by binding a DNA eluted by lysing cells to surfaces of magnetic beads coated with silica, and separating and collecting the DNA utilizing a magnetic force.
The kind of a base in a single nucleotide polymorphism in the extracted DNA sample, i.e. an allele having a single nucleotide polymorphism can be identified by any methods, including, for example, a method using an immobilized probe described later, or the like. Upon the identification, a probe used in the detection can be designed on the basis of the sequence information of a single nucleotide polymorphism of interest and surrounding sequences thereof When the probe is designed, the sequence information obtained from the database for known single nucleotide polymorphisms such as dbSNP can be used as a reference. As to a probe used in the detection of a single nucleotide polymorphism, the detection can be made with either a probe complementary to a sense strand of the genome, or a probe complementary to an antisense strand. Although the details will be described later, a kit in which probes capable of detecting single nucleotide polymorphisms existing on the human genome are immobilized in large amounts, thereby making it possible to determine alleles of numerous single nucleotide polymorphisms in a single operation is commercially available, and whereby an allele in a sample can be efficiently determined using the kit. Many of the kits also have the constitution that the alleles that are opposite to each other contained in one sample are detected in a single operation, so that a genotype can be determined.
The single nucleotide polymorphism associated with glaucoma can be determined by previously identifying an allele existing on DNA from glaucoma patients and non-patients according to the method as mentioned above, statistically comparing each of the allele frequencies and the genotype frequencies in a glaucoma patient group against a non-patient group, and determining whether or not a difference that a p-value is below the significance level as defined by a given standard is caused in at least one of the allele frequencies and the genotype frequencies. In a case where the difference is caused, the allele frequencies or genotype frequencies for these factors in the glaucoma patient group and the non-patient group are compared to determine whether any of the alleles or genotypes are identified in a high frequency in the glaucoma patient group.
In the statistical analysis, for example, a chi-square test can be used. Type I error caused by multiple comparisons can be corrected by a known correction method, for example, Bonferroni method. In a case where a correction is based on Bonferroni correction, for example, a significance level can be obtained by dividing a p-value of 5×10−2 by the number of multiple comparisons, i.e. the number of polymorphisms to be compared in the chi-square test. A single nucleotide polymorphism below the significance level determined in the manner described above can be selected as a more preferred single nucleotide polymorphism, and a method used in other known multiple corrections, for example, an FDR method or a permutation method may also be used in the selection of a preferred single nucleotide polymorphism. However, a known multiple correction method such as Bonferroni correction is a method presupposing that the phenomenon of carrying out multiple analyses is completely independent; on the other hand, there are some cases where the phenomenon is not completely independent because linkage disequilibrium is found in a single nucleotide polymorphism as described later. In other words, in the case as mentioned above, it is considered that overcorrection takes place when correction is carried out according to Bonferroni method. Especially, in the analysis of a single nucleotide polymorphism over the whole genome as in the present invention, factors to be statistically compared are highly enormous in number; therefore, a p-value serving as a standard is drastically lowered when multiple corrections are performed, so that a possibility of an oversight of a single nucleotide polymorphism associated with a disease becomes high (Schymick J C et al., Lancet Neurology. 2007: 6: 322-8; Van Steen K et al., Nature Genetics. 2005: 37: 683-691). An academically preferred multiple correction method is not yet established, and as other correction methods, correction by another known correction method can be carried out, or a significance level can be set at any appropriate levels within the range that would not be below the significance level according to the Bonferroni correction. When any appropriate level is set, for example, the significance level in a case where about 500,000 single nucleotide polymorphisms are analyzed repeatedly of 5×10−2 is used, more preferably 1×10−2, even more preferably 1×10−3, even more preferably 1×10−4, even more preferably 3×10−5, and even more preferably 1×10−5. As described later, the adjustment of the significance level as described above is useful from the fact that it is confirmed that a single nucleotide polymorphism identified to be associated with glaucoma in the present invention exists continuously in a certain region on the genome.
In addition, in general, it is known that type I error and the statistical power are inversely proportional. A method of maintaining the statistical power while lowering type I error includes a method of performing a single nucleotide polymorphism analysis in two divided steps (Skol A. D. et al., Nature Genetics. 2006: 38: 209-213). For example, in a case where a single nucleotide polymorphism analysis is carried out for a fixed number of samples, firstly, analysis of enormous single nucleotide polymorphisms over the whole genome for a part of samples thereof is carried out as primary analysis, and secondly, analysis of single nucleotide polymorphisms narrowed down in the first analysis to some degree is carried out for the remainder samples as secondary analysis. In this case, in both of the analyses, a single nucleotide polymorphism may be selected so as to have a relatively low p-value, for example, 0.05; preferably, a single nucleotide polymorphism serving as a candidate in the first analysis may be selected at a given significance level, and the selected single nucleotide polymorphism may be further analyzed using another sample. On the other hand, it is more preferable that the results of the first analysis and the secondary analysis are not individually statistically analyzed but these results are combined and analyzed. In the case as mentioned above, the two analytical results can be combined by a known method of meta-analysis, for example, Mantel-Haenszel method (Mantel N et al., Journal of the National Cancer Institute 1959: 22: 719-748). When the analytical results are combined according to a meta-analysis method such as Mantel-Haenszel method, the significance level for the selection of a single nucleotide polymorphism in individual analysis is not needed to be at the level of Bonferroni correction, and the significance level may be set by taking narrowing-down efficiency or the like into consideration. On the other hand, upon determination of whether or not a single nucleotide polymorphism is significant by a p-value combined by a meta-analysis method such as Mantel-Haenszel method, it is preferable to use a significance level with considering multiple comparisons. Here, the Mantel-Haenszel method refers to a method of combining analytical results by weighting the results obtained by multiple analyses when a chi-square test or the like is carried out. A statistical parameter combined by Mantel-Haenszel method includes, in addition to the p-value, an odds ratio described later or the like.
A single nucleotide polymorphism for the detection of the allele or genotype associated with glaucoma is preferably a single nucleotide polymorphism having a p-value of 1×10−3 or less, more preferably a single nucleotide polymorphism having a p-value of 3×10−4 or less, even more preferably a single nucleotide polymorphism having a p-value of 1×10−4 or less, and even more preferably a single nucleotide polymorphism having a p-value of 3×10−5 or less, when the single nucleotide polymorphism for the detection is based on the results obtained in a single analysis using, for example, a microarray in which about 500,000 single nucleotide polymorphisms are detected in a single operation. When the results are obtained by combining multiple analytical results according to a meta-analysis method such as Mantel-Haenszel method, the single nucleotide polymorphism for the detection is preferably a single nucleotide polymorphism having a p-value of 1×10−2 or less, more preferably a single nucleotide polymorphism having a p-value of 3×10−3 or less, even more preferably a single nucleotide polymorphism having a p-value of 1×10−4 or less, and even more preferably a single nucleotide polymorphism having a p-value of 3×10−4 or less.
It is preferable that a sufficient number of single nucleotide polymorphisms are analyzed, in order to obtain highly reliable results upon analysis. For example, a polymorphic site having a low determination rate of each single nucleotide polymorphism to the whole sample, i.e. a low call rate, is likely to have a high rate of typing errors, so that the reliability is not high. Therefore, it is preferable that the analysis is performed using a single nucleotide polymorphism having a sufficiently high call rate. As to the call rate that serves as a standard of accepting or rejecting a single nucleotide polymorphism, for example, it is preferable that a single nucleotide polymorphism showing a call rate of preferably 70%, more preferably 75%, even more preferably 80%, even more preferably 85%, and even more preferably 90% or more is employed.
Besides them, factors that can be considered upon analysis are Hardy-Weinberg's equilibrium and minor allele frequency.
The Hardy-Weinberg's equilibrium means that a distribution frequency of the opposite alleles in a certain gene locus is constant even after generations, in a genetically homogeneous population having a sufficient number of individuals formed by panmixia without a mutation or selection pressure. Whether or not the Hardy-Weinberg's equilibrium is established can be confirmed by some known methods, for example, a chi-square test and a direct probability calculation method of Fischer. In a population of a sufficient number, it is considered that the Hardy-Weinberg's equilibrium is established by a single panmixia, i.e. the Hardy-Weinberg's equilibrium is established as long as inbreeding does not exist. Therefore, generally, under the assumption that the
Hardy-Weinberg's equilibrium is established in the general population, analysis of the Hardy-Weinberg's equilibrium is used for the purpose of detecting errors of genotype determination of a sample. However, even if the Hardy-Weinberg's equilibrium is established as a whole, when a certain genotype is unevenly distributed in a disease group or a control group in a certain gene locus, for example, there are some cases where a certain genotype has a predominant influence on a disease, or the like; therefore, said analysis can be omitted, in a case where a search for disease-associated genes is carried out.
The minor allele frequency refers to an allele frequency with a lower frequency of the frequencies of two alleles in a case where single nucleotide polymorphisms are contained in two alleles. It is possible that a threshold thereof is arbitrarily set. As mentioned above, it is preferable that a single nucleotide polymorphism having a minor allele frequency of below 1% is rejected, because the concept of a single nucleotide polymorphism is in that the single nucleotide polymorphism has a minor allele frequency exceeding about 1%. On the other hand, there is a possibility that an allele having a very high or very low allele frequency in a disease group has a predominant influence on a disease. It is considered that polymorphisms of which relative involvement to a disease is relatively low are multiply involved in search of polymorphisms causative of multi-factorial diseases; therefore, for the purpose of searching the polymorphisms as mentioned above, an analysis excluding a frequency of a certain level or lower, for example, a minor allele of less than 5% can be a preferred means. On the other hand, in order to search polymorphisms that have predominant influences on a disease, it is effective not to reject the polymorphisms of the minor allele frequency.
From the allele or genotype associated with glaucoma thus obtained, the information such as a location on the genome at which a single nucleotide polymorphism exists, the sequence information, a gene in which a single nucleotide polymorphism exists or a gene existing in the neighborhood, discrimination of intron or exon or a function thereof in a case where the single nucleotide polymorphism exists on the gene, and a homologous gene in other organism species can be obtained, by referring to the database of known sequences such as GenBank, or the database of known single nucleotide polymorphisms such as dbSNP, whereby a nucleic acid molecule used in the present invention is obtained, on the basis of the information, and a probe or the like used in the present invention can be designed.
As the criteria for determining the presence or the absence of a risk in a single nucleotide polymorphism associated with glaucoma determined as mentioned above, a high-risk allele is defined. As mentioned above, in the present invention, the high-risk allele refers to an allele having a higher frequency in a glaucoma patient group than that of a non-patient group among each of the alleles of single nucleotide polymorphisms associated with glaucoma, and in the present invention, the low-risk allele refers to an allele opposite to a high-risk allele in a certain polymorphic site.
The determination of the presence or the absence of an onset risk can be carried out according to an allele or a genotype.
In a case where the determination is carried out according to an allele, the presence of the onset risk is determined for the single nucleotide polymorphism because of having a high-risk allele.
In a case where the determination is carried out according to a genotype, the onset risk is determined by taking into consideration whether the high-risk allele complies with a dominant genetic model, or with a recessive genetic model. In a certain polymorphic site, when the frequency of a homozygote of the high-risk allele and a heterozygote is significantly high in a glaucoma patient group as compared to that of a non-patient group, it is said that these genotypes comply with a dominant genetic model. The presence of an onset risk is determined for the single nucleotide polymorphism in a case where the genotype is a homozygote of the high-risk allele or a heterozygote, when the high-risk allele complies with a dominant genetic model. On the other hand, when the frequency of a homozygote of the high-risk allele is significantly high in a glaucoma patient group as compared to that of a non-patient group, it is said that these genotypes comply with a recessive genetic model. The presence of an onset risk is determined for the single nucleotide polymorphism in a case where the genotype is a homozygote of the high-risk allele, when the high-risk allele complies with a recessive genetic model.
The determination of the presence or the absence of an onset risk can be also carried out according to a low-risk allele. As mentioned above, the low-risk allele is an allele opposite to a high-risk allele, i.e. an allele identified in a high frequency in a non-patient group. In a case where the determination is carried out according to an allele, the presence of an onset risk is determined for the single nucleotide polymorphism because of not having a low-risk allele.
The same applies to a case of a genotype as well. When the determination is carried out according to a genotype, an onset risk is determined by taking into consideration whether the low-risk allele complies with a dominant genetic model, or with a recessive genetic model. In a certain polymorphic site, when the frequency of a homozygote of the low-risk allele and a heterozygote is significantly high in a non-patient group as compared to that of a glaucoma patient group, it is said that these genotypes comply with a dominant genetic model. The presence of an onset risk is determined for the single nucleotide polymorphism in a case where the genotype is not a homozygote of the low-risk allele or a heterozygote, when the low-risk allele complies with a dominant genetic model. On the other hand, when the frequency of a homozygote of the low-risk allele is significantly high in a non-patient group as compared to that of a glaucoma patient group, it is said that these genotypes comply with a recessive genetic model. The presence of an onset risk is determined for the single nucleotide polymorphism in a case where the genotype is not a homozygote of the low-risk allele, when the low-risk allele complies with a recessive genetic model.
As to whether the determination is carried out using a method for any of an allele, a dominant genetic model, and a recessive genetic model, the same method as in a method where a p-value judged to be significant is obtained can be used. In a case where the methods where a p-value judged to be significant is obtained exist in a plurality for one single nucleotide polymorphism, any of these methods may be used, and preferably, the same method as in a method where the lowest p-value is calculated is used.
Generally, in a single nucleotide polymorphism associated with a disease, a relative risk or an odds ratio is used as an index of an extent of the strength of the association that exists between one allele or genotype and the presence or the absence of a disease.
Generally, the relative risk refers to a ratio of an incidence rate in a group with a risk factor to an incidence rate in a group without a risk factor. On the other hand, the odds ratio generally refers to a ratio obtained by dividing odds, which is a ratio of a proportion of individuals with a risk factor to a proportion of individuals without a risk factor in a patient group, by odds obtained in a non-patient group in the same manner, which is in many cases used in a case-control study as in the present invention. The odds ratio in the present invention is determined on the basis of the allele frequency or the genotype frequency. In other words, the odds ratio of a single nucleotide polymorphism associated with the onset refers to a value obtained by calculating a quotient obtained in a ratio of an allele or genotype frequency to another allele or genotype frequency in a glaucoma patient group, over a ratio of frequencies obtained in the same manner in a non-patient group. In the present invention, an extent to which an onset risk of glaucoma increases can be predicted by comparing a case of having a certain allele or genotype to a case of having an allele or genotype other than the above, using these indices. For example, when an odds ratio of a certain allele in a certain single nucleotide polymorphism is greater than 1, the allele is an allele found in a high frequency in a glaucoma patient group, in which the larger the odds ratio, the higher the onset risk of glaucoma for a sample donor having the allele. On the other hand, when an odds ratio of an allele is less than 1, the allele is an opposite allele of the allele that is identified in a high frequency in a disease, in which the smaller the odds ratio, the lower the onset risk of glaucoma for a sample donor having the allele. The risk of a disease can also be predicted in the same manner for a genotype.
In the present invention, the value of the odds ratio would be always greater than 1 by obtaining an odds ratio based on the high-risk allele. The risk prediction in a combination of plural single nucleotide polymorphisms is facilitated by defining so that the odds ratio is greater than 1 when having the high-risk allele as mentioned above.
Although the details are shown by the numerical formulas in the section of Examples, in a case where an odds ratio is obtained for an allele, the odds ratio may be a value obtained by calculating a quotient obtained in a ratio of the high-risk allele frequency to the low-risk allele frequency in a glaucoma patient group, over a ratio of the high-risk allele frequency to the low-risk allele frequency in a non-patient group. In order to obtain an odds ratio in a genotype, the odds ratio is obtained by taking into consideration whether the high-risk allele complies with a dominant genetic model, or with a recessive genetic model. In other words, a homozygote of the high-risk allele and a heterozygote becomes a risk factor when the high-risk allele complies with a dominant genetic model, and a homozygote of the high-risk allele becomes a risk factor when the high-risk allele complies with a recessive genetic model. Therefore, when the high-risk allele complies with a dominant genetic model, the odds ratio may be obtained by obtaining the sum of the homozygote frequency of the high-risk allele and the heterozygote frequency in a glaucoma patient group, and calculating a quotient obtained in a ratio of the above sum to the homozygote frequency of the low-risk allele, over a ratio of frequencies obtained in the same manner in a non-patient group. When the high-risk allele complies with a recessive genetic model, the odds ratio may be obtained by obtaining the sum of the homozygote frequency of the low-risk allele and the heterozygote frequency in a glaucoma patient group, and calculating a quotient obtained in a ratio of the homozygote frequency of the high-risk allele to the above sum, over a ratio of frequencies obtained in the same manner in a non-patient group.
Further, the reliability of a single nucleotide polymorphism used in the prediction of a risk can be confirmed with an odds ratio. As mentioned above, the meaning for the prediction of a risk reverses in a case where the odds ratio is 1 or more and a case where the odds ratio is 1 or less. Therefore, in a case where a calculated 95% confidence interval of the odds ratio includes 1, it cannot be said that the reliability for the prediction of a risk for the odds ratio as mentioned above would be high.
In addition, in a case where an onset risk of glaucoma is predicted by a combination of single nucleotide polymorphisms of the present invention, the level of the risk can be predicted by using the level of the odds ratio.
In the odds ratio according to an allele, the odds ratio of combined two or more single nucleotide polymorphisms can be calculated according to the following formula:
(RA1combRA2comb)/(RA3combRA4comb)
wherein
RA4comb: an allele frequency in a case where all the alleles are low-risk alleles in the non-patient group.
For example, in a case where two single nucleotide polymorphisms associated with the onset risk of glaucoma are combined, an odds is determined by dividing the frequencies in a glaucoma patient group all having high-risk alleles of a single nucleotide polymorphism, or having any one of high-risk alleles, by the frequency in the glaucoma patient group not having any one of high-risk alleles. The odds ratio in a case of a combination of the single nucleotide polymorphisms can be determined by calculating a ratio of said odds to the odds of that in a non-patient group obtained in the same manner.
In order to obtain an odds ratio according to a combination in cases of genotypes, the odds ratio is obtained by taking into consideration whether the high-risk allele complies with a dominant genetic model, or with a recessive genetic model, in the same manner as that alone.
In the odds ratio according to a dominant genetic model, the odds ratio of combined two or more single nucleotide polymorphisms can be calculated according to the following formula:
(RGd1combRGd2comb)/(RGd3combRGd4comb)
wherein
RGd1comb: a frequency at which at least one genotype is a homozygote of a high-risk allele or a heterozygote, in a glaucoma patient group;
For example, in a case where both the high-risk alleles of the two single nucleotide polymorphisms comply with a dominant genetic model, the odds ratio may be obtained by calculating a quotient obtained in a ratio of the frequency at which any of the two single nucleotide polymorphisms are a homozygote of a high-risk allele or a heterozygote in a glaucoma patient group to the frequency at which both the two single nucleotide polymorphisms are a homozygote of a low-risk allele in the glaucoma patient group, over a ratio of frequencies of those obtained in the same manner in a non-patient group.
In the odds ratio according to a recessive genetic model, the odds ratio of combined two or more single nucleotide polymorphisms can be calculated according to the following formula:
(RGr1combRGr2comb)/(RGr3combRGr4comb)
wherein
For example, in a case where both the high-risk alleles of the two single nucleotide polymorphisms comply with a recessive genetic model, the odds ratio may be obtained by calculating a quotient obtained in a ratio of the frequency at which any of the two single nucleotide polymorphisms are a homozygote of a high-risk allele in a glaucoma patient group to the frequency at which both the two single nucleotide polymorphisms are a homozygote of a low-risk allele in the glaucoma patient group, over a ratio of frequencies of those obtained in the same manner in a non-patient group. Here, the odds ratio for a combination of single nucleotide polymorphisms can also be calculated by combining single nucleotide polymorphisms having different genetic forms.
Generally, the odds ratio increases by combining two or more single nucleotide polymorphisms, as compared to a case where these single nucleotide polymorphisms are used alone. Therefore, by a combination of two or more single nucleotide polymorphisms, a sample donor with a higher onset risk of glaucoma would be identified, whereby the improvement in the precision of the prediction can be made possible, as compared to the case where a single nucleotide polymorphism is used alone.
In order to confirm the improvement of the precision of the prediction of an onset risk of glaucoma according to a combination of single nucleotide polymorphisms in the present invention, a multivariate analysis can be employed. As the multivariate analysis method, a method well known to one of ordinary skill in the art such as logistic regression analysis method, discriminant analysis method, multiple linear regression analysis method, or proportional hazard analysis method can be employed, among which the logistic regression analysis method is effective in a case where a dichotomous variable such as the presence or the absence of an onset risk of glaucoma is handled.
The logistic regression analysis method refers to a method of analyzing a degree to which multiple independent variables (H) contribute in order to describe a single dependent variable (Φ) (Wakariyasui Igaku Tokeigaku (Easy Medical Statistics), pp. 148-179, Toshio MORIZANE, Medical Tribune). By performing the logistic regression analysis, a regression coefficient (λ) for each independent variable can be obtained, and this regression coefficient can be utilized as an index showing a degree to which each independent variable describes a dependent variable. In addition, a dependent variable on each obtained independent variable can be calculated by substituting this regression coefficient into the following formula:
Φ=1/{1+exp[−(λ0+λ1Π1+λ2Π2+λ3Π3+ . . . )]}
Here, when the logistic regression analysis is performed, the independent variables π used in analysis can be previously narrowed down using a stepwise method or the like. The stepwise method refers to a method for selecting independent variables Π so as to maximize the regression coefficients by adding an optional independent variable Π. In other words, it means that after the regression coefficient is maximized by adding an arbitrary independent variable Π, the same outcome is obtained even if another independent variable Π is further added.
In the present invention, by combining any two or more single nucleotide polymorphisms determined to be involved in the onset of glaucoma, the extent to which the precision of the prediction of an onset risk is improved can be known, as compared to that where each of the single nucleotide polymorphisms is used alone. Concretely, the above formula is obtained according to logistic regression analysis by using each of any two or more single nucleotide polymorphisms as an independent variable Π (homozygote of one allele=0, heterozygote=1, homozygote of an opposite allele=2). In each sample, a dependent variable Φ is calculated by substituting a variable for each single nucleotide polymorphism into this formula. When a dependent variable Φ is greater than a given threshold (for example, 0.5), this sample donor is determined to be a glaucoma patient. The determination results are collated with the matter of whether the sample donor having a single nucleotide polymorphism was actually the glaucoma patient. According to the combination of the two or more single nucleotide polymorphisms in the present invention, an improvement in a concordance proportion is confirmed, whereby the precision improvement by the combination can be confirmed.
In addition, the single nucleotide polymorphisms which exist in genetically sufficiently close locations to each other are inherited in linkage, not inherited independently, in some cases. In a certain population, a state in which a linkage as described above is held regardless of occurrence of a recombination by mating is referred to as a linkage disequilibrium, and a unit holding the linkage is referred to a haplotype block or an LD block.
In the experiment results by the present inventors, it is found that a single nucleotide polymorphism associated with glaucoma actually may exist in clusters in a relatively closely on the genome in some cases. It is considered that these regions belong to an LD block associated with glaucoma. In order to determine an LD block associated with glaucoma, the LD block can be determined by analyzing single nucleotide polymorphisms which exist in the region as many as possible by the method mentioned above, and applying an algorithm to determine an LD block, for example, an EM algorithm. In addition, when the single nucleotide polymorphism associated with glaucoma in the present invention belongs to a known LD block, the LD block can be considered as an LD block associated with glaucoma. Genome Browser provided on the internet web sites by California University at Santa Cruz, or the like can be consulted for a known LD block.
Because a single nucleotide polymorphism that belongs to an LD block associated with glaucoma is linked to a single nucleotide polymorphism associated with glaucoma identified according to the experiments of the present inventors, it can be considered that the single nucleotide polymorphism that belongs to an LD block associated with glaucoma also associates with glaucoma in the same manner; therefore, the single nucleotide polymorphism is used in the prediction of an onset risk or progressive risk of glaucoma. In addition, by re-determining a sequence within the LD block associated with glaucoma, or a sequence surrounding the single nucleotide polymorphism associated with glaucoma that is identified according to the experiments by the present inventors, there is a possibility that an unknown single nucleotide polymorphism which is linked with the single nucleotide polymorphism, in other words, which is associated with the onset of glaucoma or the progression thereof, is found. Whether or not the found single nucleotide polymorphism is actually associated with the onset of glaucoma or the progress thereof can be determined by comparing an allele or genotype frequency of a disease group with that of a control group in the same manner as explained above.
In the present invention, an intronic single nucleotide polymorphism (iSNP) refers to one in which a single nucleotide polymorphism is identified in intron. A coding single nucleotide polymorphism (cSNP) refers to one that is accompanied by a change in an amino acid sequence, such as a codon in which the single nucleotide polymorphism is mutated to a codon encoding other amino acids or a termination codon, among those in which single nucleotide polymorphisms exist in regions translated in a protein. A silent single nucleotide polymorphism (sSNP) refers to one without accompanying a change in an amino acid sequence, among those in which a single nucleotide polymorphism is identified in a coding region. A genomic single nucleotide polymorphism (gSNP) refers to one in which a single nucleotide polymorphism exists in a region not encoding the gene on the genome. A regulatory polymorphism (rSNP) refers to a single nucleotide polymorphism existing in a site that is thought to be involved in the transcriptional regulation.
As described above, a single nucleotide polymorphism may exist in any location on the genome, any cases of which can be associated with a disease. In a case where a single nucleotide polymorphism exists in the intron or a non-coding region, there may be some cases where the single nucleotide polymorphism may influence a gene expression control, or splicing that takes place after the gene transcription or stability of mRNA. In a case where a single nucleotide polymorphism exists in the coding region, by substitution of its base, a codon corresponding to a certain amino acid may be changed to a codon corresponding to a different amino acid, or may undergo a change, for example, a change to a termination codon, or the like, which may lead to a change in the structure of a protein encoded thereby. Changes in expression levels or functions of genes by these changes consequently lead to changes in expression levels or functions of proteins encoded by the genes, which can be causes for various diseases. In a case where the genomic single nucleotide polymorphism is associated with a disease, there is a possibility that a region including the polymorphic site is actually translated, and influences in some way to other gene expressions. In a case where a silent single nucleotide polymorphism is associated with a disease, it is considered that a different polymorphism associating with the disease exists in the surrounding of the single nucleotide polymorphism, and the polymorphism and the silent single nucleotide polymorphism are linked, so that the association with the disease is found. Similarly, in a single nucleotide polymorphism other than the silent single nucleotide polymorphism, even when the single nucleotide polymorphism itself is not a direct cause for glaucoma but links to a polymorphism which is the true cause for glaucoma existing in the surrounding, the association of these single nucleotide polymorphisms and glaucoma may be found in some cases. In the case as described above, as described later, a polymorphism which is causative of glaucoma can be found by re-sequencing the surrounding of the single nucleotide polymorphism in the present invention. However, in any case, these single nucleotide polymorphisms can be also used for the purpose of predicting an onset risk of glaucoma, regardless of whether or not these would be the true causes for the disease.
(Nucleic Acid Molecule Comprising Allele Associated with Glaucoma)
In an embodiment of the present invention, there are provided a nucleic acid molecule comprising a single nucleotide polymorphism associated with glaucoma, and a nucleic acid molecule having a sequence complementary to the nucleic acid molecule comprising a single nucleotide polymorphism associated with glaucoma.
The nucleic acid molecule comprising a single nucleotide polymorphism associated with glaucoma or the nucleic acid molecule having a sequence complementary to the nucleic acid molecule can be used as a marker for determining the level of the onset risk of glaucoma. Further, these nucleic acid molecules can be used as a probe for detecting an allele or an opposite allele thereof identified in a high frequency in glaucoma patients, or determining a genotype, in the single nucleotide polymorphism. In addition, in a case where the single nucleotide polymorphism exists on exon or in the neighborhood thereof, these nucleic acid molecules can be used in the detection of transcripts of genes.
The nucleic acid molecule constituting the genome of an eukaryote is constituted by double strands of a sense strand and an antisense strand complementary to the sense strand. In other words, the single nucleotide polymorphism also exists on the sense strand and the antisense strand, and the nucleic acid molecule of the present invention embraces both of these strands because the detection of a single nucleotide polymorphism of both the strands is equally significant.
Nucleic acid molecules comprising any one of single nucleotide polymorphisms listed in Tables 1 and 2, Tables 5 to 25, Tables 26 to 28 and Tables 29 to 51 shown later, nucleic acid molecules comprising any single nucleotide polymorphisms existing in a region or on a gene determined by the linkage disequilibrium data or the like listed in Tables 3 and 4 shown later, and nucleic acid molecules complementary to these nucleic acid molecules are all embraced in the nucleic acid molecule of the present invention.
In an embodiment of the present invention, the nucleic acid molecule of the present invention is preferably nucleic acid molecules comprising a single nucleotide polymorphism listed in Tables 1 and 2, Tables 26 to 28 or Tables 52 to 62 shown later, or nucleic acid molecules complementary thereto, wherein
in a case where the single nucleotide polymorphism is gSNP, the nucleic acid molecule is a nucleic acid molecule comprising a sequence from a next base of a known single nucleotide polymorphism on an upstream side of the sense strand to a base before a known single nucleotide polymorphism on a downstream side, or a nucleic acid molecule comprising a sequence complementary thereto,
in a case where the single nucleotide polymorphism is iSNP, sSNP or cSNP, the nucleic acid molecule is a nucleic acid molecule comprising a full length of the gene on the genome including the single nucleotide polymorphism, a nucleic acid molecule comprising a sequence complementary thereto, and a nucleic acid molecule containing a complementary DNA (cDNA) molecule comprising the single nucleotide polymorphism or a sequence complementary thereto,
in a case where the single nucleotide polymorphism is rSNP, the nucleic acid molecule is a nucleic acid molecule comprising a sequence from a next base of a known single nucleotide polymorphism on an upstream side of the sense strand to a full length of the gene existing downstream of a promoter region in which the single nucleotide polymorphism exists, or a nucleic acid molecule comprising a sequence complementary thereto.
The nucleic acid molecule in the present invention is not limited whether it is a deoxyribonucleic acid, a ribonucleic acid, or a peptide nucleic acid, and a nucleic acid molecule comprising a mixed sequence thereof is also embraced in the present invention. In a case where a ribonucleic acid is used in the nucleic acid molecule in the present invention, in the sequence of the nucleic acid molecule in the present invention (including a sequence complementary thereto), thymine may be read as uracil. In addition, these nucleic acid molecules may be subjected to chemical modifications as occasion demands, within the range that would not impair a function to be used in the present invention. In this case, the function refers to a function of accomplishing the purpose of using the nucleic acid molecule.
The nucleic acid molecule in the present invention can be synthesized by a known method, for example, a phosphoramidite method, on the basis of the sequence information disclosed herein, or the sequence information obtained by searching the information disclosed herein with the database. The nucleic acid molecule can be synthesized using a commercially available DNA synthesizer. In addition, the nucleic acid molecule in the present invention can be obtained from a sample comprising DNA from human according to a known method such as a PCR method, or in some nucleic acid molecules, can be obtained from a sample containing RNA from human according to a known method such as an RT-PCR method. As to primers that are necessary for the obtainment, one of ordinary skill in the art can design the primers on the basis of the sequence information disclosed herein, or the sequence information that can be searched from ID of the database disclosed herein. For example, in a case where a PCR method is used, primers having about 10 to about 30 bases that have sequences homologous to a part of the sequences of the nucleic acid molecule of interest can be used, and in a case where an RT-PCR method is used, the nucleic acid molecule can be obtained by carrying out reverse transcription reaction using an oligo dT primer, or a random hexamer, or the like to prepare cDNA, and amplifying a sequence of interest in the cDNA by the PCR method mentioned above.
The nucleic acid molecule has a length of preferably from 16 to 55 bases, and more preferably from 23 to 27 bases or 47 to 53 bases. It is preferable that the nucleic acid molecule is a nucleic acid molecule containing the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto.
When a nucleic acid molecule comprising a single nucleotide polymorphism associated with the onset of glaucoma is selected, in a case where the nucleic acid molecule is selected based on the results obtained in a single analysis using a microarray in which, for example, 500,000 nucleic acid molecules are detected in a single operation, the nucleic acid molecule in the present invention is preferably a nucleic acid molecule having a p-value of 1×10−3 or less, more preferably a nucleic acid molecule having a p-value of 3×10−4 or less, even more preferably a nucleic acid molecule having a p-value of 1×10−4 or less, and even more preferably a nucleic acid molecule having a p-value of 3×10−5 or less. In a case where plural analytic results are combined and obtained according to a method of meta-analysis, such as Mantel-Haenszel method, the nucleic acid molecule is preferably a nucleic acid molecule having a p-value of 1×10−2 or less, more preferably a nucleic acid molecule having a p-value of 3×10−3 or less, even more preferably a nucleic acid molecule having a p-value of 1×10−3 or less, even more preferably a nucleic acid molecule having a p-value of 3×10−4 or less, and even more preferably a nucleic acid molecule having a p-value of 1×10−4 or less.
As a different means of selecting a preferred nucleic acid molecule, a significance level is set according to a known multiple correction method, whereby a preferred nucleic acid molecule can be selected. In a case where a correction is based on Bonferroni correction, for example, a significance level can be obtained by dividing a p-value of 5×10−2 by the number of multiple comparisons, i.e. the number of polymorphisms to be compared in the chi-square test. A nucleic acid molecule having a single nucleotide polymorphism below the significance level thus obtained may be selected as a more preferred nucleic acid molecule. Upon the selection, Bonferroni correction may be performed using a p-value that is combined according to a method of meta-analysis, such as Mantel-Haenszel method, and the number of single nucleotide polymorphisms to be subject for the meta-analysis. Other known methods used in multiple corrections, for example, an FDR method or a permutation method may be used in the selection of a preferred nucleic acid molecule.
(Method of Detecting Single Nucleotide Polymorphism Associated with Glaucoma and Method of Predicting Onset Risk of Glaucoma)
Another embodiment of the present invention provides a method of detecting the presence or absence of an allele or genotype having a high frequency in glaucoma patients in a sample containing a nucleic acid molecule from the genome. The samples may be any ones so long as the nucleic acid molecules from the genome can be extracted, and for example, blood, white blood cells, hair root, hair, saliva, oral mucosa cells, skin, tissues such as muscles or organs obtained by biopsy, or the like can be used.
As mentioned above, the nucleic acid molecule constituting the genome of an eukaryote is constituted by a sense strand and an antisense strand that are complementary to each other, and the determination of the allele of the single nucleotide polymorphism in the present invention can also be performed by detecting any one of the bases of the sense strand and the antisense strand of the polymorphic site.
As mentioned above, in the method of determining the presence or the absence of the allele or genotype in a sample containing a nucleic acid molecule, any means can be used. For example, hybridization is carried out using a probe specific to each of the alleles, preferably a probe in the present invention described later, which is designed based on the sequence information disclosed in the present invention, and each of the alleles can be detected by detecting signals therefor. In addition, each of the alleles opposite to each other, in other words, an allele having a high association to a disease for a certain single nucleotide polymorphism and an allele having a low association thereto are each provided with different labels, and a probe capable of hybridizing these alleles to a polymorphic site, or an immobilized probe such as a microarray in which each of alleles opposite to each other is immobilized is used, whereby each of the alleles opposite to each other contained in the same sample can be detected. In the constitution as described above, not only the alleles of the sample, but also the genotypes can be determined. In addition, in a case where an immobilized probe such as a microarray in which each of the alleles opposite to each other is immobilized on the same carrier is used, a constitution that the hybridization is carried out in a single operation, and that the detection is carried out in a single operation can be also taken.
As another method of detecting a single nucleotide polymorphism in the present invention, the following method can be utilized. Examples of a method of hybridization using a probe are Taqman method, Invader (registered trademark) method, LightCycler method, cyclin probe method, MPSS method, beads-array method, and the like, and any of these methods can be employed. As to the probe for detecting the same allele, a more preferred probe may differ in some cases depending upon a method used in the detection. The determination of the allele or genotype of the single nucleotide polymorphism in the present invention does not depend upon the detection method, and it is preferable to use a suitable probe depending upon the detection method.
The Taqman method is a method of detecting a genetic polymorphism using an oligoprobe having a given length in which a fluorescent substance is bound to a 5′-side, and a quencher is bound to a 3′-side. The presence or absence of the polymorphism is determined by hybridizing a probe to a nucleic acid molecule having a polymorphism of interest, cutting off a part of the probe on the 5′-side by a PCR reaction, and measuring a fluorescent amount emitted by a fluorescent substance.
The Invader method is a method of detecting a genetic polymorphism using a probe (reporter) which has a sequence common to a 3′-side of a nucleic acid molecule having a polymorphism, but the sequence on a 5′-side being completely different therefrom, and a probe (invader) having only a sequence common to a 5′-side. The nucleic acid molecules of interest and these two probes are hybridized, a product is then treated with a nuclease, a part of the cut-out reporter probe is hybridized with a probe for detection having a fluorescent substance and a quencher, a hybridization product is treated with a nuclease, and the fluorescent substance is released, whereby the presence or absence of the polymorphism is determined by a fluorescent amount thereof.
The LightCycler method is a method of detecting a polymorphism including the step of hybridizing a polymorphic detection probe having a fluorescent substance and an anchor probe having a quencher, to a nucleic acid molecule having a polymorphism previously amplified by PCR. If the hybridized DNA is gradually heated, the polymorphic detection probe is released when a given temperature is reached, and the presence or absence of the polymorphism is determined by measuring this fluorescent amount.
The cyclin probe method is a polymorphic analysis method utilizing a probe having a fluorescent substance or a quencher on each end of a DNA (DRD probe), wherein DNA sequences are bound in a manner that both ends of an RNA sequence having a sequence complementary to a polymorphic site of a nucleic acid molecule of interest are sandwiched. A DRD probe is hybridized to a nucleic acid molecule of interest previously amplified by PCR or the like, RNase is allowed to act on this complex, and a fluorescent dye is released, whereby the presence or absence of the polymorphism is determined by measuring this fluorescent amount.
The MPSS method is a method of performing polymorphic analysis using an encoded adaptor probe and a decoder probe. The encoded adaptor probe is an oligo DNA having a 4-bases long protruding end on a 5′-side, subsequently a recognition sequence for a restriction enzyme BbvI, and a single-stranded sequence bound to a decoder probe on a 3′-side. On the other hand, the decoder probe is a single-stranded oligo DNA having a fluorescent substance on a 3′-side, and the decoder probe containing 4 different sequences, each sequence specifically hybridizing to a single encoded adaptor probe. The nucleic acid molecule having a polymorphism is previously immobilized on beads, and an initiation adaptor containing a recognition sequence for BbvI is bound thereto, to digest with BbvI to form a 4-bases long protruding end. The ligation with the encoded adaptor probe is carried out sequentially from a 3′-side of the protruding 4 bases, and the sequence of the bound encoded adaptor is detected with a specified decoder probe.
The beads array method is a method of performing the determination of a genotype including the step of combining beads to which a probe for allele detection and an oligonucleotide (address sequence) specifying the location information on the array of signals detected by the probe for allele detection are bound. For example, there are Golden Gate Assay using beads immobilized with only an address sequence (23 bases) of Illumina, and Infinium (registered trademark) Assay using beads in which probes (50 bases) for allele detection are bound to an address sequence (30 bases). In both the methods, which location on an array the probes for allele detection are bound can be known for each of the beads arranged arbitrarily on the array, on the basis of the address sequence.
The method of the Golden Gate Assay will be shown hereinbelow. In the detection of a single nucleotide polymorphism, two kinds of probes (allele-specific probes) specifically hybridizing to each allele, and a probe capable of specifically hybridizing to a sequence located 1 to 20 bases downstream on the 3′-side of the single nucleotide polymorphism (downstream sequence recognition probe) are used. In the downstream sequence recognition probe, an address sequence for specifying the location on the array is provided. In addition, these three probes contain a sequence to which universal primers described later are bound. The three probes are annealed with a genomic DNA, and a DNA polymerase and a ligase are added thereto. By carrying out an extension reaction and a ligation reaction, an allele-specific product ligating a gap between the allele-specific probe and the downstream sequence recognition probe is formed. A reaction for PCR is carried out with this allele-specific product as a template using two kinds of fluorescent-labeled universal primers, each being specific to each allele, and a universal primer bound to the downstream sequence recognition probe. A labeled PCR product is hybridized to an oligonucleotide immobilized on beads via an address sequence. The fluorescence on the beads is detected with a confocal laser scanner, thereby determining an allele and a genotype.
The method of the Infinium Assay will be shown hereinbelow. An array by Illumina [Illumina, iSelect™ Genotyping BeadChip] described later is in accordance with this method. There are two methods in the detection of an allele by this array. In one method, two kinds of probes (probes for allele detection of 50 bases long, Infinium I type) only differing by a base at a 3′-end, wherein the 3′-end is a site for detecting a single nucleotide polymorphism, are used. Whole genome amplification for a genomic DNA is previously carried out, and fragmentation with an enzyme is carried out. The probe and the fragmented genomic DNA are hybridized, and thereafter an allele-specific extension reaction takes place, whereby a base on the downstream (3′-side) by a single base of a polymorphic site labeled with a single kind of a fluorescent dye is incorporated corresponding to the probe. In another method, one kind of probe without having an allele-specific sequence of a single nucleotide polymorphism in the probe is used (probe for allele detection of 50 bases, Infinium II type). A 3′-end of this probe has a sequence up to a single base upstream (5′-side) from a polymorphic site. The probe and the fragmented genomic DNA are hybridized, and according to a single base extension reaction, a base labeled with either one of two kinds of fluorescent dyes is incorporated corresponding to a single nucleotide polymorphic site of interest. In both the methods, the fluorescence is detected by a confocal laser scanner, thereby determining an allele and a genotype.
Here, the details of properties for length, modification and the like of probes used in the hybridization method mentioned above will be described later.
In addition, a method without carrying out hybridization with a probe includes PCR-RFLP method, SSCP method, mass spectrometry and direct sequencing method.
The PCR-RFLP method is a method including the steps of forming different DNA fragments according to enzymatic digestion of a nucleic acid molecule having a polymorphism due to the existence of a polymorphism in a cleavage site of the restriction enzyme in the nucleic acid molecule, and determining the presence or absence of a polymorphism from a difference in electrophoretic patterns thereof. A nucleic acid molecule of interest is amplified by PCR, this amplified fragment is cleaved with a restriction enzyme, and a fragment formed electrophoretically is analyzed. The length of the nucleic acid molecule comprising an amplified polymorphism is usually from 50 to 10,000 base pairs, and more preferably from 100 to 1,000 base pairs.
The SSCP method is a method including the steps of amplifying a nucleic acid molecule having a polymorphism by PCR, forming a single-stranded DNA, electrophoresing the product, and determining the presence or absence of a polymorphism from a difference in the electrophoretic patterns thereof. The nucleic acid molecule of interest is amplified by PCR, and a single-stranded DNA is formed by subjecting this amplified fragment to heat or an alkali treatment. This single-stranded DNA forms a base sequence-specific higher-order structure; therefore, if these amplified fragments are electrophoresed, a difference in the electrophoretic mobility is found due to the difference in its structure. The primer used in PCR is labeled with a radioisotope or fluorescent substance.
In addition, the length of the nucleic acid molecule comprising an amplified polymorphism is usually from 50 to 10,000 base pairs, and more preferably from 100 to 1,000 base pairs.
The mass spectrometry is a method including the steps of ionizing a polymer with a matrix and a laser or the like, accelerating the ionized polymer in a high electric field to allow a flight to a detector, and identifying mass from a difference in the flight time, or the like. This mass spectrometry is combined with the above primer extension method or the like to detect a polymorphism. Concretely, a single base extension reaction is carried out with a primer complementary to a sequence up to a single base upstream of a polymorphic site of a nucleic acid molecule having a polymorphism, any one of 4 kinds of dideoxyribonucleotides, and deoxyribonucleic acids other than those corresponding the above, and a difference in mass of nucleic acid products having different sequences incorporated in a 3′-end is determined, whereby a polymorphism can be identified.
The direct sequencing method is a method of directly reading off a base sequence of a nucleic acid molecule having a polymorphism. Representative methods are called Sanger method (dideoxy method). A primer that is unlabeled or labeled with a radioisotope or a fluorescent substance is bound to a nucleic acid molecule of interest, an extension reaction with Klenow enzyme or the like is stopped with four kinds of dideoxyribonucleotides that are unlabeled or labeled with a radioisotope or a fluorescent substance, the product is digested with a restriction enzyme, and a DNA fragment generated is separated by electrophoresis. The base sequence of a 3′-end is read off in the order of fragments having a lower molecular weight on the basis of an electrophoretic image, thereby a base sequence containing a few bases before and after a polymorphism is determined. As a modified method thereof, there is a method called a primer extension method. This is a method including the steps of carrying out a single base extension reaction using a primer complementary to a sequence up to a single base upstream of a polymorphic site of a nucleic acid molecule having a polymorphism, and reading off any one of the sequences of the 4 kinds of dideoxyribonucleotides incorporated in the 3-end. There are various methods in the identification of this dideoxyribonucleotides; for example, 4 kinds of nucleotides are labeled with different fluorescent substances, and separated and identified electrophoretically. In addition, a method of converting pyrophosphoric acid formed during an extension reaction to ATP, and identifying its ATP from luminescence of luciferase is also employed. The length of the primer used in the extension reaction is usually from 10 to 300 base pairs, and preferably from 15 to 25 base pairs.
In the present invention, the hybridization means that a nucleic acid molecule having a certain sequence is associated with a nucleic acid molecule complementary to at least a part of the nucleic acid molecule via a hydrogen bond on the basis of base sequences that are complementary to each other. The kind of the complementary nucleic acid molecule associated with the original nucleic acid molecule may be identical or different, and a nucleic acid constituting these nucleic acid molecules can be a deoxyribonucleic acid, a ribonucleic acid, or a peptide nucleic acid. In these nucleic acid molecules, when referred to the ribonucleic acid, in the sequence of the nucleic acid molecule (including a complementary sequence), thymine may be read as uracil.
The stringent conditions in the present invention mean conditions in which a nucleic acid molecule having a sequence complementary to a partial sequence of a nucleic acid molecule having a certain sequence is specifically hybridized to the nucleic acid molecule (Fred M. Ausuble et al., Current Protocols in Molecular Biology, 2.10.1-2.10.16, John Wiley and Sons, Inc). Concrete examples of the conditions as described above include conditions such as a temperature lower than a melting temperature (Tm) of a complex formed between a nucleic acid molecule having a certain sequence and a complementary nucleic acid molecule hybridized to the nucleic acid molecule by preferably from about 5° to about 30° C., and by more preferably about 10° to about 25° C., a reaction solution for hybridization, such as SSC (mixed solution of sodium chloride and sodium citrate) in a concentration of 0.01 to 6-folds, SSPE (mixed solution of sodium chloride, sodium dihydrogenphosphate, and EDTA) or MES (a mixed solution of 2-(N-morpholino)ethanesulfonic acid and tetramethylammonium chloride) buffer, and hydrogen ion concentrations of a pH of from 6 to 8. For example, the stringent conditions in a case where an immobilized probe is prepared by immobilizing a 25 by DNA probe include conditions of hybridization at 49° C. in the MES buffer (hydrogen ion concentrations being from 6.5 to 6.7) in a 1-fold concentration, and sequentially washing with SSC (hydrogen ion concentrations being 8.0) in a 6-fold concentration at 25° C., and thereafter SSC (hydrogen ion concentrations being 8.0) in a 0.6-fold concentration at 45° C.
In the present invention, the term allele-specific (or specific to allele) means that the allele is contained in a sequence from the genome including the polymorphic site or in a prepared nucleic acid molecule including the polymorphic site, or a certain nucleic acid molecule is capable of specifically hybridizing under stringent conditions to a nucleic acid molecule having a sequence containing the allele in the polymorphic site, in other words, in the manner of being capable of discriminating the allele and the opposite allele.
Base sequences of 61 bases in length including a single nucleotide polymorphism associated with the onset of glaucoma, disclosed in the present invention, are composed of two pairs of base sequences which differ only by a base in the center (i.e. 31st base) (i.e. those pairs are consisting of a sequence having odd number of SEQ ID No. and a sequence having even number of SEQ ID No.), and the 31st base is a polymorphic site. The high-risk alleles in the polymorphic sites are listed in Tables 1 and 2 or Tables 52 to 63 given later. In any of these single nucleotide polymorphisms, in a case where the existence of an allele that exists in a high frequency in glaucoma patients is determined, a high-risk allele in a sample is detected, whereby the existence of the allele that exists in a high frequency in glaucoma patients can be determined.
In addition, as to any single nucleotide polymorphisms associated with the onset of glaucoma identified above, the genotype can be determined by detecting the presence or the absence of each of the alleles opposite to each other contained in one sample. In detail, in a case where only a certain allele is detected, the genotype is a homozygote of the allele, and in a case where two alleles are detected, the genotype is a heterozygote having the two alleles. In at least one of these single nucleotide polymorphisms, by detecting a genotype, it is determined whether or not the genotype that is identified in a higher frequency in a glaucoma patient group than that of a non-patient group exists in a sample. In other words, in the single nucleotide polymorphism mentioned above, when the high-risk allele complies with a dominant genetic model, a homozygote of the high-risk allele or a heterozygote is a genotype that is identified in a higher frequency in a glaucoma patient group than that of a non-patient group, and when the high-risk allele complies with a recessive genetic model, a homozygote of the high-risk allele is a genotype that is identified in a higher frequency in a glaucoma patient group than that of a non-patient group. It is preferable that each of the opposite alleles is measured in a single operation, from the viewpoint of reducing judgmental error.
The sample is analyzed in the manner described above, and in a case where the allele or genotype that is identified in a higher frequency in a glaucoma patient group than that of a non-patient group exists in the sample, there are some high probabilities that an individual donating the sample not having glaucoma at the present point is predicted to have a high onset risk of glaucoma, or is determined that a precision examination for glaucoma such as visual field examination is necessary, and that an individual donating the sample who is suspected of having glaucoma should be diagnosed as glaucoma.
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, the single nucleotide polymorphism used in the detection is a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto, more preferably a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in
SEQ ID NOs: 203 to 238 or a complementary sequence thereto, even more preferably a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of pairs of base sequences containing a single nucleotide polymorphism listed below or a complementary sequence thereto, wherein, as mentioned above, in the pairs of SEQ ID NOs: shown in a to r, each of the pairs of sequences corresponds to one single nucleotide polymorphism, and each of the base sequences is a base sequence containing an allele opposite to each other of the single nucleotide polymorphism in a 31st base:
In a case where any one of the single nucleotide polymorphisms is used, especially, it is preferable that an allele of a single nucleotide polymorphism located on a 31st base of a base sequence is used, wherein the base sequence is at least one base sequence selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
SEQ ID NO: 203, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 234, SEQ ID NO: 235, and SEQ ID NO: 238,
or a complementary sequence thereto.
Here, these sequences are sequences containing a high-risk allele in each of polymorphic sites.
Further, the precision of the determination of a future onset risk of glaucoma can be improved by detecting a combination of two or more of alleles or genotypes associated with glaucoma in the present invention, using one sample.
For the single nucleotide polymorphisms to be combined, any ones can be used so long as they are a single nucleotide polymorphism in the present invention, preferably a single nucleotide polymorphism having a low p-value, and more preferably a single nucleotide polymorphism of which p-value obtained by combining the results obtained in two analyses by a meta-analysis method, such as Mantel-Haenszel method, is determined to be significant even below the level of Bonferroni correction. In addition, from a different viewpoint, it is preferable to use a single nucleotide polymorphism that is confirmed to contribute to the improvement in the precision of the risk prediction by a combination according to the logistic regression analysis described later. On the other hand, since the single nucleotide polymorphisms in a state of linkage disequilibrium mentioned above show the same behavior, in a case where plural single nucleotide polymorphisms in a state of linkage disequilibrium are combined, risks of glaucoma based on the same region may be evaluated unnecessarily seriously in some cases. In a case where a risk of a disease is predicted by combining the single nucleotide polymorphisms in the present invention, when it is intended to evaluate all the risks in even weighting, it is preferable that the prediction is carried out employing only one of the single nucleotide polymorphisms in the state of linkage disequilibrium, in a case that the plural single nucleotide polymorphisms that are in the state of linkage disequilibrium mentioned above are contained.
In a case where a risk is predicted according to a combination of any two or more single nucleotide polymorphisms in the present invention, an onset risk of glaucoma can be predicted using the regression formula obtained by the logistic regression analysis. Concretely, the regression formula according to the logistic regression analysis is obtained by respectively using each of the any two or more single nucleotide polymorphisms as an independent variable Π (homozygote of one allele=0, heterozygote=1, homozygote of an opposite allele=2). In each sample, a dependent variable Φ is calculated by substituting a value corresponding to each single nucleotide polymorphism into this formula. When a dependent variable Φ is greater than a given threshold (for example, 0.5), the determination can be made that this sample donor has an onset risk.
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, in a case where any two or more single nucleotide polymorphisms are combined, the single nucleotide polymorphisms used in the detection are preferably single nucleotide polymorphisms which are located on 31st bases of base sequences, wherein the base sequences are base sequences containing two or more different single nucleotide polymorphisms, selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto,
more preferably single nucleotide polymorphisms which are located on 31st bases of base sequences, wherein the base sequences are base sequences containing two or more different single nucleotide polymorphisms, selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 238 or a complementary sequence thereto,
even more preferably single nucleotide polymorphisms which are located on 31st bases of base sequences, wherein the base sequences are base sequences containing two or more different single nucleotide polymorphisms, selected from the group consisting of pairs of base sequences containing a single nucleotide polymorphism listed below or a complementary sequence thereto,
wherein, as mentioned above, in the pairs of SEQ ID NOs: shown in a to r, each of the pairs of sequences corresponds to one single nucleotide polymorphism, and each of the base sequences is a base sequence containing an allele opposite to each other of the single nucleotide polymorphism in a 31st base:
In addition, it is preferable that the single nucleotide polymorphisms to be used in combination are those that are not in the state of linkage disequilibrium, and from this viewpoint, in all the embodiments of the combinations mentioned above, supposing that
Further, in all the embodiments of the combinations mentioned above, it is preferable that an allele of a single nucleotide polymorphism located on a 31st base of a base sequence is used, wherein the base sequence is a base sequence containing two or more different single nucleotide polymorphisms, selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
(Probe Capable of Detecting Allele Associated with Glaucoma)
In another embodiment of the present invention, an allele-specific nucleic acid molecule or probe (hereinafter referred to as probe) capable of detecting an allele associated with glaucoma, and a method of detecting an allele or a genotype associated with glaucoma using the probe are provided.
Any probes may be used so long as the probe is capable of hybridizing under the stringent conditions to an allele-specific sequence, in a polymorphic site of the single nucleotide polymorphism associated with glaucoma in the present invention. The determination of the allele in a polymorphic site can be made by detecting any one of polymorphic sites of the sense strand and the antisense strand on the genome; therefore, the probe in the present invention embraces any one of sequences complementary to a sequence specific to an allele of the sense strand and sequences complementary to a sequence specific to an allele of the antisense strand, in other words, sequences specific to an allele of the sense strand. The probe in the present invention can also be used in the detection of cDNA or mRNA, containing a single nucleotide polymorphism in the present invention. In a case where the probe is used in the detection of cDNA or mRNA, a probe in which the single nucleotide polymorphism exists in exon or neighborhood thereof is used.
The probes capable of detecting each of alleles of the single nucleotide polymorphisms listed in Tables 1 and 2, Tables 5 to 25, Tables 26 to 28, Tables 29 to 51, or Tables 52 to 62 given later or a complementary strand thereto, and the probes capable of specifically detecting each of alleles of any single nucleotide polymorphisms that exist in a region associated with glaucoma listed in Tables 3 and 4 or Tables 63 to 70 given later or a complementary strand thereto are all embraced in the probe in the present invention. In a case where, for example, the obtained results are based on a single analysis using a microarray in which a probe capable of specifically detecting each of alleles of 500,000 single nucleotide polymorphisms, or a complementary strand thereto, is detected in a single operation, the probe of the present invention is preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 1×10−3 or less, more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 3×10−4 or less, even more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 1×10−4 or less, and even more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 3×10−5 or less. In a case where plural analytical results are combined and obtained according to a method of meta-analysis, such as Mantel-Haenszel method, the probe is preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 1×10−2 or less, more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 3×10−3 or less, even more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 1×10−3 or less, even more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 3×10−4 or less, and even more preferably a probe capable of specifically detecting each of alleles of a single nucleotide polymorphism or a complementary strand thereto, of which p-value is 1×10−4 or less.
The probe in the present invention preferably contains an allele-specific sequence or a complementary strand thereto, and even more preferably in the probe in the present invention, a sequence contributing to an allele-specific hybridization consists only of an allele-specific sequence or a complementary strand thereto. To the probe in the present invention, a spacer or any sequences of several bases that are not from an allele-specific sequence for the purpose of providing stabilization or the like can be added in an end, within the range that the probe is capable of hybridizing to the sequence under the stringent conditions. The added sequence is preferably a sequence that does not take a three-dimensional structure, such as a hairpin structure.
The probe can be provided with any labels for use in the detection. Any labels to be provided to the probe that are ordinarily used can be used, and in general, a fluorescent label such as FITC or Cy3, biotin, an enzyme label such as an alkaline phosphatase and horseradish peroxidase, or the like is usable. In a case where a biotin label is used, streptavidin capable of specifically binding to biotin is previously provided with a further detectable label, and the labeled streptavidin is used as a secondary label. A labeled anti-biotin antibody can also be used in place of the labeled streptavidin. As a method of providing a label to a probe, any known methods may be used, and the methods are well known to one of ordinary skill in the art. An arbitrary sequence which serves as a spacer as mentioned above may be added to the probe, and the spacer may be provided with a label. A reagent for labeling a probe, a labeled streptavidin, a labeled anti-biotin antibody or the like is commercially available as a reagent, and can also be purchased.
The probe in the present invention is not limited whether it is a deoxyribonucleic acid, a ribonucleic acid, or a peptide nucleic acid, and a probe containing a mixed sequence thereof is also embraced in the present invention, so long as the probe is capable of specifically hybridizing to a nucleic acid molecule having an allele of interest. In a case where a probe containing a ribonucleic acid is used as the probe in the present invention, in the sequence of the probe in the present invention (including a sequence complementary thereto), thymine may read as uracil. In addition, the probe in the present invention may be subjected to chemical modifications as needed, so long as the probe is capable of specifically hybridizing under stringent conditions to a nucleic acid molecule having an allele of interest. As the method of providing a chemical label, any known methods may be used.
The probe for the detection can be reacted with the sample in the state of solution and then detected by a known method, or previously immobilized to a carrier. The probe can take the form of an immobilized probe obtained by previously immobilizing a probe corresponding to each of the alleles of several to several hundred-thousand different single nucleotide polymorphisms to a location defined on a solid carrier in the number of from one to dozen probes per one single nucleotide polymorphism, reacting a sample to the immobilized probes, scanning a signal generated from a hybridized probe, and analyzing the scanned data with a computer, which is a so-called microarray. In a case where the probe takes the form of an immobilized probe, the largest number of the immobilized probes are limited by immobilization density and area of immobilized sites for the probes.
In a case where the probe takes the form of an immobilized probe as described above, signals on the solid phase from the nucleic acid molecule having a labeled target allele can be detected by previously labeling a nucleic acid molecule in a sample by a known method, and binding the labeled nucleic acid molecule with an immobilized unlabeled probe in the present invention, or by binding a nucleic acid molecule having an allele to be detected to an immobilized unlabeled probe in the present invention, and thereafter labeling the product according to a known method.
The immobilization can be carried out by any of known method, and for example, a method such as synthetic oligoprint or spotting photolithograph can be used. Also, the material for the carrier is not limited, and a generally used material, for example, a polymer such as a polycarbonate or a polystyrene, glass, silicon crystal or the like can be used. In addition, in order to enhance adhesive strength of the nucleic acids, a carrier may be provided with a coating such as cationization before the immobilization. In addition, in order to prevent nonspecific nucleic acids from being adsorbed to a carrier, blocking can be carried out with a known blocking agent after the immobilization. The blocking agent as mentioned above may be any ones so long as the blocking agent is capable of controlling the nonspecific nucleic acids from being adsorbed to the carrier, and for example, salmon sperm DNA, Denhardt's solution, Cot-I DNA extracted from human placenta, an anionic surfactant such as sodium dodecyl sulfate, a nonionic surfactant such as polyoxyethylene sorbitan monolaurate, or the like can be used.
In addition, in a case where the probe is immobilized, it is possible to construct that each of the opposite alleles contained in one sample is detected under the same operation by immobilizing a probe specific to each of the alleles opposite to each other on the same carrier. In the construction as described above, not only the alleles but also the genotypes in the samples can be determined.
It is preferable that the probe used in the detection of the allele is a probe having a length of preferably from 16 to 55 bases, more preferably from 23 to 27 bases or 47 to 53 bases, and even more preferably 25 bases in total of a length of the polymorphic site and some bases before and after the polymorphic site, the probe containing the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, that the probe is a probe containing the polymorphic site mentioned above and a 5′-upstream side thereof, preferably a sequence of 49 bases (i.e. a sequence of 50 bases), the probe containing the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, or that the probe is a probe containing a sequence of 50 bases on a 5′-upstream side of the polymorphic site mentioned above, the probe having a sequence adjoining the polymorphic site mentioned above, or a sequence complementary thereto.
An even more preferred probe used in the detection of the allele is:
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, the probe usable in the detection is a probe containing a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto, or a partial sequence thereof, and/or a probe having a base sequence containing at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 515 to 694 or a complementary sequence thereto, more preferably a probe containing a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 238 or a complementary sequence thereto, or a partial sequence thereof, and/or a probe having a base sequence containing at least one base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 515 to 535 or a complementary sequence thereto, and
In a case where any one of the single nucleotide polymorphisms is used, especially, it is preferable that in Group A, a probe containing an allele of a single nucleotide polymorphism located on a 31st base of a base sequence is used, wherein the base sequence is at least one base sequence selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, in a case where any two or more single nucleotide polymorphisms are combined, the probes usable in the detection are preferably probes containing a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is a base sequence containing a single nucleotide polymorphism, selected from the group consisting of base sequences shown in SEQ ID NOs: 203 to 514 or a complementary sequence thereto, or a partial sequence thereof, and/or probes having a base sequence containing a base sequence selected from the group consisting of base sequences shown in SEQ ID NOs: 515 to 694 or a complementary sequence thereto, wherein the probes are probes corresponding to two or more different single nucleotide polymorphisms thereof,
In addition, it is preferable that the single nucleotide polymorphisms to be used in combination are those that are not in the state of linkage disequilibrium, and from this viewpoint, in all the embodiments of the combinations mentioned above, supposing that, in Group A, a group composed of a base sequence containing a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is a base sequence belonging to the group consisting of:
Further, in all the embodiment of the combinations mentioned above, in Group A, a probe containing an allele of a single nucleotide polymorphism located on a 31st base of a base sequence, wherein the base sequence is a base sequence containing a single nucleotide polymorphism selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
The probe in a case where a Taqman method is used in the detection of an allele usually has a length of preferably from 10 to 300 bases, and contains the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, and the probe also contains a fluorescent substance and a quencher. More preferably, the probe has a length of 20 to 60 bases, and contains the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, and the probe contains a fluorescent substance and a quencher.
The probes in a case where an Invader method is used in the detection of an allele comprise a probe (reporter) which have a common sequence to a 3′-side of the polymorphic site mentioned above and a sequence on a 5′-side being completely different therefrom, and a probe (invader) only composed of the common sequence to a 5′-side. These probes usually have a length of preferably from 10 to 300 bases, and more preferably a length of from 20 to 60 bases.
The probe in a case where a LightCycler method is used in the detection of an allele, usually has a length of preferably from 10 to 300 bases, and contains the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, and the probe contains a fluorescent substance and a quencher. More preferably, the probe has a length of 20 to 60 bases, and contains the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, and the probe contains a fluorescent substance and a quencher.
The probe in a case where a cyclin probe method is used in the detection of an allele is a probe in which DNA sequences are bound in a manner that both ends of an RNA sequence having the polymorphic site and a surrounding sequence thereof, or a sequence complementary thereto, are sandwiched, and each of DNA ends has a fluorescent substance or a quencher. These probes usually have a length of preferably from 10 to 300 bases, and contain the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto. More preferably, the probe has a length of 20 to 60 bases, and contains the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto.
The probes in a case where an MPSS method is used in the detection of an allele comprise an oligo DNA (encoded adaptor probe) having a protruding end of 4 bases on a 5′-side, subsequently a recognition sequence for a restriction enzyme BbvI, and a single-stranded sequence to which a decoder probe is bound on a 3′-side, and a single strand oligo DNA (decoder probe) which has fluorescent substance on a 3′-side, and containing 4 different sequences, each sequence specifically hybridizing to one of the encoded adaptor probes. Here, a DNA sequence is bound in a manner that both ends of an RNA sequence having the polymorphic site mentioned above and a surrounding sequence thereof, or a sequence complementary thereto, are sandwiched, and each of DNA ends has a fluorescent substance or a quencher. The encoded adaptor probe usually has a length of preferably from 10 to 300 base pairs, and more preferably from 15 to 40 base pairs. On the other hand, the decoder probe usually has a length of preferably from 10 to 300 base pairs, and more preferably from 5 to 30 base pairs.
(Kit of Detecting Allele Associated with Glaucoma)
In another embodiment of the present invention, a kit of detecting a single nucleotide polymorphism associated with glaucoma is provided.
The kit of the present invention (or a composition for predicting a risk) embraces all those kits so long as the allele or genotype of any one of single nucleotide polymorphisms associated with glaucoma disclosed in the present invention can be detected in a nucleic acid molecule in a sample. As mentioned above, the kit of the present invention may be those that detect a base of either the sense strand or the antisense strand of the single nucleotide polymorphism, or those that detect bases of both the strands. In a case where the kit of the present invention is based on the results obtained in a single analysis using a microarray for a kit of detecting an allele or genotype associated with glaucoma for detecting, for example, 500,000 single nucleotide polymorphisms in a single operation, the kit is preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 1×10−4 or less listed in Tables 1 and 2 set forth below, more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 3×10−4 or less, even more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 1×10−4 or less, and even more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 3×10−5 or less. In a case where the plural analytic results are combined and obtained according to a method of meta-analysis, such as Mantel-Haenszel method, the kit is preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value listed in Tables 52 to B set forth below of 1×10−2 or less, more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 3−10−3 or less, even more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 1×10−3 or less, even more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 3×10−4 or less, and even more preferably a kit of detecting an allele or genotype associated with glaucoma for single nucleotide polymorphisms having a p-value of 1×10−4 or less.
A kit of detecting both an allele identified in a high frequency in the glaucoma patient group mentioned above and an allele opposite to the allele is also one embodiment of the present invention. In a case where a kit as described above is used, as already explained, a genotype of each of the alleles can also be determined.
By detecting the presence of an allele or a genotype that is identified in a high frequency in glaucoma patients in the sample using the kit of the present invention, a future onset risk of glaucoma of an individual not having glaucoma at the present stage can be predicted, whether or not precise visual field examinations for glaucoma are required can be determined, or the diagnosis of an individual who is suspected of glaucoma can be made for glaucoma.
In addition, as mentioned above, a kit for determining alleles that are opposite to each other in a single operation can be prepared by using a probe specific to each of the alleles that are opposite to each other, and providing different labels to the probes, or providing in the form of a microarray or beads array as mentioned above.
The precision for the prediction of the onset risk of glaucoma or the determination of whether or not precise visual field examinations are required can also be improved by providing a kit having the constitution of detecting these plural alleles or genotypes using one sample. Even in the constitution as described above, a constitution can be taken that the detection is carried out in a single operation by having the form of probes provided with different labels, or the form of the microarray or beads array mentioned above.
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, the kit usable in detecting or predicting a risk is
In a case where any one of the single nucleotide polymorphisms is used, especially, in Group A, preferred is a kit of detecting a single nucleotide polymorphism associated with the onset of glaucoma or predicting an onset risk of glaucoma, using a nucleic acid molecule containing an allele of a single nucleotide polymorphism located on a 31st base of a base sequence, wherein the base sequence is at least one base sequence selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
In the method of detecting a single nucleotide polymorphism associated with glaucoma and the method of predicting an onset risk of glaucoma in the present invention, in a case where any two or more single nucleotide polymorphisms are combined, the kit usable in detecting or predicting a risk is
In addition, it is preferable that the single nucleotide polymorphisms to be used in combination are those that are not in the state of linkage disequilibrium, and from this viewpoint, in all the embodiments of the combinations mentioned above, supposing that, in Group A, a group composed of a base sequence containing a single nucleotide polymorphism which is located on a 31st base of a base sequence, wherein the base sequence is a base sequence belonging to the group consisting of:
In all the combinations mentioned above, in Group A, preferred is a kit of detecting a single nucleotide polymorphism associated with the onset of glaucoma or a kit of predicting an onset risk of glaucoma, using a nucleic acid molecule comprising an allele of a single nucleotide polymorphism located on a 31st base of a base sequence, wherein the base sequence is a base sequence containing a single nucleotide polymorphism, selected from the group consisting of the following base sequences containing a single nucleotide polymorphism:
(Method of Predicting Onset Risk of Glaucoma, Including Performing the Predicting Risk in Two-Steps or Multi-Steps)
When a prediction of an onset risk of glaucoma using a single nucleotide polymorphism in the present invention is carried out, it can be performed in two or more steps as follows; candidates who are considered that precise prediction of an onset risk of glaucoma is necessary are selected, and the candidates are subjected to detailed prediction of a risk.
In a case where prediction of a risk is performed in two or more multi-steps, first, prediction of an onset risk of glaucoma mentioned above is preformed on at least one single nucleotide polymorphism in the present invention, preferably any one or several single nucleotide polymorphisms, and subsequently, prediction of detailed risks may be performed using a combination of the single nucleotide polymorphisms of the present invention mentioned above. The number of combinations may be further increased as occasion demands, whereby precision of the prediction of a risk can also be improved. As described above, by performing prediction of a risk in two or more multi-steps, the reduction in costs for performing the prediction of a risk and the prediction of a risk in a high precision can be both accomplished.
The prediction of a risk in an initial step may be a convenient method of predicting a risk. For example, a method of predicting a risk so that an immobilized probe capable of detecting at least one of the single nucleotide polymorphisms, preferably any one or several single nucleotide polymorphisms, is immobilized in a manner that at least one of the single nucleotide polymorphisms in the present invention is detectable is a convenient method, and can be realized at a low cost. Here, as to a method for nucleic acid extraction in this case, a kit that can be realized according to a known technique, or a commercially available simple kit for nucleic acid extraction can be used. It is convenient to use a method including the steps of using, for example, an enzyme-labeled probe as the immobilized probe used in the prediction of a risk as described above, and detecting the probe according to a colorimetric method. As to the samples used in the detection, those that are obtained in a relatively low penetration, such as saliva, oral mucosa cells, urine, hair root, blood or white blood cells are preferred.
The prediction of a risk in a next step may be a method of predicting a risk with an emphasis on precision. For example, the detection of a single nucleotide polymorphism associated with the onset of glaucoma is carried out by combining two or more single nucleotide polymorphisms in the present invention mentioned above, whereby prediction of a risk may be performed in a high precision.
By performing prediction of a risk in two or more multi-steps, the precision for prediction of a risk can be improved, while reducing the costs or lowering a burden on a subject at an initial step to a minimum level.
According to the method disclosed in the present invention, the determination can be made that an individual who has an allele or genotype on the genome that is identified in a high frequency in glaucoma patients disclosed in the present invention has a high risk of the onset of glaucoma in future, and that an individual who does not have an allele or genotype that is identified in a high frequency in the glaucoma patients has a low risk of the onset of glaucoma in future.
In addition, an individual having an allele or genotype on the genome that is identified in a high frequency in glaucoma patients disclosed in the present invention has a possibility of being in an early stage of glaucoma that is difficult to be diagnosed according to a simple method of determination of glaucoma, such as measurement of intraocular pressure or examination of ocular fundus, and that is diagnosed for the first time after performing visual field examination. Therefore, a single nucleotide polymorphism in the present invention is detected, whereby whether or not the visual field examination is required can be screened. On the other hand, in a case where an individual who is suspected of being glaucoma has an allele or genotype associated with glaucoma in the present invention on the genome, there is a high probability that the individual who is suspected of being glaucoma is to be diagnosed as glaucoma.
The present invention will be specifically described hereinbelow by Examples, and Examples are given for illustration purposes for a better comprehension of the present invention, without intending to limit the scope of the present invention thereto. Here, in the following Examples, as to generally used molecular biological methods that are not specifically described in detail, methods and conditions described in a textbook such as Molecular Cloning (Joseph Sambrook et al., Molexular Cloning—A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001) or the like are used.
In the present invention, a total DNA was extracted from blood of each of patients diagnosed as glaucoma, and non-patients diagnosed as being not with glaucoma and determined not to have any family history in glaucoma according to a medical interview, and gene loci associated with the disease were analyzed based on about 500,000 known single nucleotide polymorphisms on the human genome as an index to determine an association of a single nucleotide polymorphism and the disease. In addition, patients with fast progression of glaucoma, i.e. progressive glaucoma cases, and patients with slow progression of glaucoma, i.e. nonprogressive glaucoma cases were subjected to the identification of a single nucleotide polymorphism and the association of the single nucleotide polymorphism with the progression in the same manner as above.
In DNA extraction from specimens, a commercially available automated nucleic acid extraction apparatus (QUIAGEN, BIOROBOT (registered trademark) EZ1), and a kit for extraction of a nucleic acid (EZ1 DNA Blood 350 μl Kit) compatible to the extraction apparatus and in which nucleic acids absorbed to magnetic beads were collected by a magnetic force were used. A total DNA was extracted in accordance with the instruction manuals of the apparatus and kit. According to the present method, a total DNA of about 5 μg was obtained from 350 μL of a blood specimen.
In the analysis of single nucleotide polymorphisms, a commercially available microarray type single nucleotide polymorphism analysis kit (Affimetrix (GeneChip(registered trademark) Human Mapping 500K) (hereinafter also referred to as microarray) capable of analyzing about 500,000 known single nucleotide polymorphisms on the human genome was used. In the detection of single nucleotide polymorphisms, a scanner (Affimetrix (GeneChip(registered trademark) Scanner 3000)) compatible to the kit was used. In the analysis of single nucleotide polymorphisms, a specialized analysis software (Affimetrix (GTYPE(registered trademark))) was used.
The total DNA extracted in Example 1 was treated in accordance with the instruction manuals of the kit and apparatus, and applied to a microarray, and a single nucleotide polymorphism existing on the DNA extracted from the specimen was analyzed. Briefly explaining, a sample obtained by treating 250 ng of a total DNA with a restriction enzyme NspI and a sample obtained by treating 250 ng of a total DNA with a restriction enzyme StyI were prepared, and amplified by a PCR method with adaptors bound to the protruding ends of each of the samples. A PCR product was collected, and fragmented with DNaseI, and the ends of the fragmented PCR products were biotin-labeled using the labeling reagent contained in the kit. A buffer for hybridization was added to the PCR products that were already fragmented at both ends and labeled, the mixture was heat-treated at 99° C. for 10 minutes, and incubated at 49° C. for 1 minute, and the resulting mixture was injected to a microarray for NspI-treated sample or a microarray for StyI-treated sample depending on a firstly treated restriction enzyme, and hybridized at 49° C. for 16 to 18 hours. After the termination of hybridization, the microarray was stained with streptavidin-phycoerythrin. A fluorescence from phycoerythrin bound via biotin and streptavidin to DNA ends of samples hybridized with an immobilized allele-specific probe was read using the scanner mentioned above, and analyzed with the software mentioned above. Probes corresponding to about 250,000 single nucleotide polymorphisms each are previously immobilized to the microarray for NspI-treated sample and the microarray for StyI-treated sample, respectively, and analytical results for about 500,000 single nucleotide polymorphisms per one sample were obtained by combination of both the results. According to the present method, opposite alleles of each of the single nucleotide polymorphisms were read with a single operation, and consequently, a genotype was determined. In this case, it was determined that the genotype was a heterozygote in a case where both signals from each of the alleles constituting a single nucleotide polymorphism were detected, and that the genotype was a homozygote of the detected allele in a case where only either one of the signals was detected.
Here, in accordance with the instruction manual of the kit, as the probe immobilized to the kit, a probe for a sense strand or a probe for an antisense strand of the genome is used. In addition, according to the datasheet of the kit, the determination results for the present kit using 270 samples and those in HapMap are compared for single nucleotide polymorphisms overlapping between single nucleotide polymorphisms reported in the HapMap project and single nucleotide polymorphisms in the kit. As a result, a concordance rate of the single nucleotide polymorphisms shows 99% or more.
The comparison on single nucleotide polymorphisms associated with a disease was made in accordance with the method used in the studies on genes responsible for age-related macular degeneration by Klein et al (Science, 308, 385, 2005).
Primary open-angle glaucoma patients and normal tension glaucoma patients that were diagnosed on the basis of Guidelines offered by Japan Glaucoma Society were assigned to a glaucoma patient group, and healthy individuals that were confirmed to have no family history of glaucoma according to a medical interview were assigned to a non-patient group. Blood donated under the consent on free will of the participants after having sufficiently explained the contents of studies from 418 cases of the glaucoma patient group and 300 controls of the non-patient group was used as specimens, a total DNA was extracted from the specimens according to the method described in Example 1, and the analysis of single nucleotide polymorphisms was performed according to the method described in Example 2. The analytical results of a single nucleotide polymorphism obtained in each of the patients were stored in the Laboratory Information Management System (World Fusion, LaboServer) adopting a relational database. A specialized analysis program for a single nucleotide polymorphism was created and loaded within the system, and the analysis was performed as follows: A single nucleotide polymorphism considered to have a high experimental reliability was extracted by rejecting a single nucleotide polymorphism having a call rate of less than 90% in both the glaucoma patient group and the non-patient group, a single nucleotide polymorphism having a difference in call rates between the glaucoma patient group and the non-patient group by 5% or more, a single nucleotide polymorphism having a minor allele frequency of less than 5%, and a single nucleotide polymorphism that is determined to deviate from the Hardy-Weinberg's equilibrium under conditions of a p-value of 1×10−4 or less according to a chi-square test, and allele frequencies and genotype frequencies of the single nucleotide polymorphisms were compared between the groups. The allele frequencies and the genotype frequencies were statistically compared according to the chi-square test. As to single nucleotide polymorphisms showing a p-value of 1×10−3 or less, cluster images serving as a basis for the determination of a genotype were confirmed. In a case where the determination of a genotype was made regardless of unclearness of the separation among clusters, the single nucleotide polymorphism was considered to be a non-subject of the analysis. In other words, the errors in the determination of a genotype were excluded by this step. The evaluation of the cluster was performed without informing the names of single nucleotide polymorphisms and the critical rates. Single nucleotide polymorphisms of which allele or genotype shows association with glaucoma at a p-value of 1×10−4 or less, i.e. −log P of 4 or more are listed in Tables 1 to 2. Here, the odds ratio for association of an allele with a disease, and the odds ratio for association of a genotype with a disease in each of the tables, respectively, were calculated on the basis of the following formulas (1) to (5).
Allele Frequency=Number of Detection of an Allele in Group/Total Number of Detection of Alleles in Group formula (1)
Genotype Frequency=Number of Detection of a Genotype in Group/Total Number of Detection of Genotypes in Group formula (2)
Odds Ratio for Allele=[(Number of Detection of an Allele Identified in High Frequency in Glaucoma Patient Group, in Glaucoma Patient Group)/(Number of Detection of an Allele Opposite to the Allele Identified in High Frequency in Glaucoma Patient Group, in Glaucoma Patient Group)]/[(Number of Detection of the Allele Identified in High Frequency in Glaucoma Patient Group, in Non-Patient Group)/(Number of Detection of the Allele Opposite to the Allele Identified in High Frequency in Glaucoma Patient Group, in Non-Patient Group)] formula (3)
Odds Ratio for Genotype of Homozygote=[(Number of Detection of a Genotype Having Homozygote of an Allele Identified in High Frequency in Glaucoma Patient Group, in Glaucoma Patient Group)/(Number of Detection of a Genotype Having Homozygote of an Allele Identified in High Frequency in Non-Patient Group, in Glaucoma Patient Group)]/[(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Glaucoma Patient Group, in Non-Patient Group)/(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Non-Patient Group, in Non-Patient Group)] formula (4)
Odds Ratio for Genotype of Heterozygote=[(Number of Detection of a Genotype of Heterozygote in Glaucoma Patient Group)/(Number of Detection of a Genotype Having Homozygote of an Allele Identified in High Frequency in Non-Patient Group, in Glaucoma Patient Group)]/[(Number of Detection of the Genotype Having Homozygote in Non-Patient Group)/(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Non-Patient Group, in Non-Patient Group)] formula (5)
Tables 1 and 2 list dbSNP ID number or Affimetrix Array ID number specifying known single nucleotide polymorphisms obtained, each of bases constituting Allele 1 and Allele 2, the exon, intron information (in a case where a single nucleotide polymorphism exists on a gene, the gene name and the exon or intron in which SNP exists are shown, and in a case where a single nucleotide polymorphism does not exist on a gene, neighboring genes and a distance between the gene and the single nucleotide polymorphism are shown), the chromosome number at which a single nucleotide polymorphism exists, the physical location of a single nucleotide polymorphism, the p-value for an allele according to a chi-square test (−log P), the high-risk allele frequencies in the glaucoma patient group and the non-patient group, the type of the high-risk allele (indicating whether the high-risk allele is Allele 1 or Allele 2), the odds ratio for an allele, the p-value for a genotype according to a chi-square test (−log P), the odds ratio for a genotype of a homozygote and the odds ratio for a genotype of a heterozygote, and SEQ ID NO of the sequence containing Allele 1 and Allele 2 in each of the polymorphic sites. Here, one of ordinary skill in the art can obtain the information for sequences or alleles of the single nucleotide polymorphisms from dbSNP ID number or Affimetrix array ID number mentioned above.
When the allele or genotype frequencies listed in Tables 1 to 2 were compared between the non-patients without family history and the glaucoma patients, a statistical difference was found. By determining an allele of any one of these single nucleotide polymorphisms, whether or not an allele that is identified in a higher frequency in the glaucoma patient group than that of the non-patient group exists in the sample can be determined.
Specifically, when a first single nucleotide polymorphism listed in Tables 1 and 2 is explained as an example, one polymorphic site exists in a nucleic acid molecule shown in SEQ ID NO: 1 or 2 occupying a gene locus homologous to each other. In detail, a single nucleotide polymorphism is associated with the onset of glaucoma, of which 31st base is either A (Allele 1) or G (Allele 2), wherein Allele 1 indicated as a high-risk allele, that is, an allele of being A in the single nucleotide polymorphism is identified in a high frequency in the glaucoma patient group. Further, using the odds ratio for an allele, or the odds ratio for a genotype of a homozygote and the odds ratio for a genotype of a heterozygote, the degree of which the risk of a disease increases can be predicted in a case of having the allele or genotype. Similarly, all the sequences disclosed in Tables 1 and 2 have a polymorphic site associated with glaucoma in the sequence, and one allele or at least one genotype in the polymorphic site is identified in a high frequency in the glaucoma patient group.
According to the above studies, 40 single nucleotide polymorphisms of which alleles or genotypes were associated with glaucoma at a p-value of 1×10−4 or less existing in clusters in relatively adjacent regions on the genome were found in 21 regions.
The allele or genotype identified in a high frequency in the glaucoma patient group of a single nucleotide polymorphism listed in Tables 1 and 2 can be used as a marker showing that an onset risk of glaucoma is high. On the other hand, an allele that is opposite to the allele or a genotype other than the genotype can be used as a marker showing that an onset risk of glaucoma is low.
Next, the surrounding regions and/or genes of the single nucleotide polymorphisms listed in Tables 1 and 2 were determined on the basis of the database provided by the HapMap project. In detail, regions in which single nucleotide polymorphisms that were considered to be in a linkage disequilibrium with the single nucleotide polymorphisms listed in Tables 1 and 2 exist were determined, on the basis of the linkage disequilibrium data in combination of the Japanese and the Chinese in the HapMap project.
Also, in a case where the single nucleotide polymorphism listed in Tables 1 and 2 exists in the linkage disequilibrium region containing the gene, the physical location and the gene name of the region were determined. On the other hand, in a case where the single nucleotide polymorphism listed in Tables 1 and 2 exists in the linkage disequilibrium region without containing the gene, only the physical location of the region was determined. In addition, in a case where the single nucleotide polymorphism listed in Tables 1 and 2 exists on one gene beyond the linkage disequilibrium region, only the gene name was determined.
A single nucleotide polymorphism of which p-value is lowest in each region is considered to be a single nucleotide polymorphism representing the region. Tables 3 and 4 list a single nucleotide polymorphism representing the region, the chromosome number at which the region exists, the physical location of the region (start point and end point) and the gene name contained in the region.
The region listed in Tables 3 and 4 is a region or gene considered to be linked with a single nucleotide polymorphism listed in Tables 3 and 4 which is associated with glaucoma in the present invention, and a single nucleotide polymorphism which exists in these regions or genes is considered to be linked with a single nucleotide polymorphism in the present invention. In other words, any single nucleotide polymorphisms which exist in these regions are linked with the single nucleotide polymorphism which exists in the region as listed in Tables 3 and 4, and any of these single nucleotide polymorphisms can be used in the prediction of a risk of glaucoma in the same manner.
Also, a single nucleotide polymorphism of which allele or genotype shows association with glaucoma at a p-value of 1×10−3 or less, i.e. −log P of 3 or more, is also listed in Tables 5 to 25.
Tables 5 to 25 list dbSNP ID number or Affimetrix Array ID number for specifying known single nucleotide polymorphisms obtained, the exon, intron information (in a case where a single nucleotide polymorphism exists on a gene, the gene name and the exon or intron in which SNP exists are shown, and in a case where a single nucleotide polymorphism does not exist on a gene, neighboring genes and a distance between the gene and the single nucleotide polymorphism are shown), the chromosome number at which the single nucleotide polymorphism exists, the physical location of the single nucleotide polymorphism, the p-value for an allele according to a chi-square test (−log P), the high-risk allele frequencies in the glaucoma patient group and the non-patient group, the odds ratio for an allele, the p-value for a genotype according to a chi-square test (−log P), the odds ratio for a genotype of a homozygote, and the odds ratio for a genotype of a heterozygote. Here, in the tables, a portion of which odds ratio is indicated as ND shows a case where any one of the number of detection in the denominator is 0, so that the odds ratio could not be calculated.
According to the above studies, 413 single nucleotide polymorphisms of which alleles or genotypes were associated with glaucoma at a p-value of 1×10−3 or less were found.
When the allele or genotype frequencies listed in Tables 5 to 25 were compared between the non-patients without having family history and the glaucoma patients, a statistical difference was found. By determining an allele of any one of these single nucleotide polymorphisms, whether or not an allele that is identified in a higher frequency in the glaucoma patient group than that of the non-patient group exists in the sample can be determined.
The comparison on single nucleotide polymorphisms was made for progressive glaucoma cases and nonprogressive glaucoma cases in the same manner as in Example 3.
Concretely, blood donated under the consent on free will of the participants after having sufficiently explained the contents of studies from 210 cases of patients with progressive visual loss within a given time period, despite the treatments for lowering an intraocular pressure such as a drug for lowering an intraocular pressure or a surgical operation (progressive glaucoma cases), and 175 cases of patients without the progression (nonprogressive glaucoma cases), among the primary open-angle glaucoma patients and the normal tension glaucoma patients diagnosed on the basis of Guidelines offered by Japan Glaucoma Society, was used as a specimen, and alleles frequencies and genotypes frequencies between the groups were also compared by performing the analysis in the same manner as in Example 3. Alleles frequencies and genotype frequencies were statistically compared according to the chi-square test in the same manner. Single nucleotide polymorphisms of which alleles or genotypes show association with the progression of glaucoma at a p-value of 1×10−4 or less, i.e. −log P of 4 or more are listed in Tables 26 to 28. Here, the odds ratio for association of an allele with the progression of glaucoma, and the odds ratio for association of a genotype with the progression of glaucoma in each of the tables, respectively were calculated on the basis of the following formulas (6) to (8).
Odds Ratio for Allele=[(Number of Detection of an Allele Identified in High Frequency in Progressive Glaucoma Group, in Progressive Glaucoma Group)/(Number of Detection of an Allele Opposite to the Allele Identified in High Frequency in Progressive Glaucoma Group, in Progressive Glaucoma Group)]/[(Number of Detection of the Allele Identified in High Frequency in Progressive Glaucoma Group, in Nonprogressive Glaucoma Group)/(Number of Detection of the Allele Opposite to the Allele Identified in High Frequency in Progressive Glaucoma Group, in Nonprogressive Glaucoma Group)] formula (6)
Odds Ratio for Genotype of Homozygote=[(Number of Detection of a Genotype Having Homozygote of an Allele Identified in High Frequency in Progressive Glaucoma Group, in Progressive Glaucoma Group)/(Number of Detection of a Genotypes Having Homozygote of an Allele Identified in High Frequency in Nonprogressive Glaucoma Group, in Progressive Glaucoma Group)]/[(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Progressive Glaucoma Group, in Nonprogressive Glaucoma Group)/(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Nonprogressive Glaucoma Group, in Nonprogressive Glaucoma Group)] formula (7)
Odds Ratio for Genotype of Heterozygote=[(Number of Detection of a Genotype of Heterozygote in Progressive Glaucoma Group)/(Number of Detection of a Genotype Having Homozygote of an Allele Identified in High Frequency in Nonprogressive Glaucoma Group, in Progressive Glaucoma Group)]/[(Number of Detection of the Genotype of Heterozygote in Nonprogressive Glaucoma Group)/(Number of Detection of the Genotype Having Homozygote of the Allele Identified in High Frequency in Nonprogressive Glaucoma Group, in Nonprogressive Glaucoma Group)] formula (8)
Tables 26 to 28 list dbSNP ID number or Affimetrix Array ID number specifying known single nucleotide polymorphisms obtained, each of bases constituting Allele 1 and Allele 2, the exon, intron information (in a case where a single nucleotide polymorphism exists on a gene, the gene name and the exon or intron in which SNP exists are shown, and in a case where a single nucleotide polymorphism does not exist on a gene, neighboring genes and a distance between the gene and the single nucleotide polymorphism are shown), the chromosome number at which the single nucleotide polymorphism exists, the physical location of the single nucleotide polymorphism, the p-value for an allele according to a chi-square test (−log P), the high-risk allele frequencies in the progressive glaucoma group and the nonprogressive glaucoma group, the type of the high-risk allele (indicating whether the high-risk allele is Allele 1 or Allele 2), the odds ratio for an allele, the p-value for genotype according to a chi-square test (−log P), the odds ratio for a genotype of a homozygote, the odds ratio for a genotype of a heterozygote, and SEQ ID NO of the sequence containing Allele 1 and SEQ ID NO of the sequence containing Allele 2 in each of the polymorphic sites. Here, in the tables, a portion of which odds ratio is indicated as ND shows a case where any one of the number of detection in the denominator is 0, so that the odds ratio could not be calculated.
According to the above studies, 61 single nucleotide polymorphisms of which alleles or genotypes were associated with the progression of glaucoma at a p-value of 1×10−4 or less were found.
When the allele or genotype frequencies listed in Tables 26 to 28 were compared between the progressive glaucoma cases and the nonprogressive glaucoma cases, a statistical difference was found. By determining an allele of any one of these single nucleotide polymorphisms, whether or not an allele that is identified in a higher frequency in the progressive glaucoma group than that of the nonprogressive glaucoma group exists in the sample can be determined.
The allele or genotype identified in a high frequency in the progressive glaucoma group for a single nucleotide polymorphism listed in Tables 26 to 28 can be used as a marker showing that a progressive risk of glaucoma is high. On the other hand, an allele that is opposite to the allele or a genotype other than the genotype can be used as a marker showing that a progressive risk of glaucoma is low.
Also, a single nucleotide polymorphism of which allele or genotype shows association with the progression of glaucoma at a p-value of 1×10−3 or less, i.e. −log P of 3 or more, is listed in Tables 29 to 51.
Tables 29 to 51 list dbSNP ID number or Affimetrix Array ID number specifying known single nucleotide polymorphisms obtained, the exon, intron information (in a case where a single nucleotide polymorphism exists on a gene, the gene name and the exon or intron in which SNP exists are shown, and in a case where a single nucleotide polymorphism does not exist on a gene, neighboring genes and a distance between the gene and the single nucleotide polymorphism are shown), the chromosome number at which the single nucleotide polymorphism exists, the physical location of the single nucleotide polymorphism, the p-value for an allele according to a chi-square test (−log P), the high-risk allele frequencies in the progressive glaucoma group and the nonprogressive glaucoma group, the odds ratio for an allele, the p-value for a genotype according to a chi-square test (−log P), the odds ratio for a genotype of a homozygote, and the odds ratio for a genotype of a heterozygote. Here, in the tables, a portion of which odds ratio is indicated as ND shows a case where any one of the number of detection in the denominator is 0, so that the odds ratio could not be calculated.
According to the above studies, 480 single nucleotide polymorphisms of which alleles or genotypes were associated with the progression of glaucoma at a p-value of 1×10−3 or less were found.
When the allele or genotype frequencies listed in Tables 29 to 51 were compared between the progressive glaucoma cases and the nonprogressive glaucoma cases, a statistical difference was found. By determining an allele of any one of these single nucleotide polymorphisms, whether or not an allele that is identified in a higher frequency in the progressive glaucoma group than that of the nonprogressive glaucoma group exists in the sample can be determined.
Surrounding sequences of single nucleotide polymorphisms described in Tables 1 to 2 or Tables 26 to 28 are subjected to re-sequencing, so that the detection of a single nucleotide polymorphism can be confirmed, and that an unknown single nucleotide polymorphism that possibly exists can be identified. The re-sequencing can be performed according to any known methods, and for example, the re-sequencing can be performed by a direct sequencing method.
In order to determine the single nucleotide polymorphisms associated with glaucoma identified in Example 3 or 4, or the alleles and genotypes of known single nucleotide polymorphisms existing in the surrounding sequences of the single nucleotide polymorphisms listed in Tables 1 to 51, an immobilized probe can be prepared. A known single nucleotide polymorphism can be referred to, for example, the database of dbSNP or J SNP. In the immobilized probe, for example, an oligonucleotide probe designed so as to maximize its sensitivity, specificity or reproducibility for several probes to several hundred-thousand probes can be loaded. The immobilized probe can be produced according to a method such as a method of synthesizing an oligonucleotide on a solid carrier or a method including the steps of previously synthesizing an oligonucleotide and immobilizing the oligonucleotide in a high density on a solid carrier.
The presence or the absence of the onset of glaucoma can be determined at a more accurate level using the immobilized probe produced in Example 6. A probe for detecting a single nucleotide polymorphism associated with a disease is plurally combined, so that the level of which the onset risk of glaucoma increases is evaluated. In a case where a value exceeds a threshold, it is determined that the onset of glaucoma takes place.
In addition, using the immobilized probe produced in Example 6, the single nucleotide polymorphism existing on the genome of the glaucoma patients and that of the non-glaucoma patients can be compared. There is a possibility that single nucleotide polymorphisms existing in locations with an adjacent distance to each other are linked and inherited by linkage disequilibrium. There is a possibility that single nucleotide polymorphisms linked with the single nucleotide polymorphisms listed in Tables 1 and 2 or Tables 26 to 28 can be identified by the immobilized probe, so that it can be expected that a single nucleotide polymorphism having an even stronger association with glaucoma is found.
In order to maintain a statistical power while lowering type I error, candidate single nucleotide polymorphisms associated with the onset of glaucoma identified in the primary analysis of Example 3 were subjected to a secondary analysis of a single nucleotide polymorphism in separately collected samples using an array for analyzing a single nucleotide polymorphism designed in an original style (hereinafter, referred to as a custom array).
For the custom array, a kit for analyzing a single nucleotide polymorphism commercially available from Illumina [Illumina, iSelect™ Genotyping BeadChip] was used. For 446 single nucleotide polymorphisms associated with the onset of glaucoma showing a p-value of 1×10−3 or less in Example 3, the designing of a probe for specifically detecting these single nucleotide polymorphisms was tried. Since these probes are randomly immobilized to the substrate via beads, the step of specifying a location of the beads (decoding) is needed. A probe for detecting a single nucleotide polymorphism of which location was unable to be specified in a process of decoding was excluded from the subject for analysis. As a result, the preparation of a custom array capable of typing 412 single nucleotide polymorphisms out of 446 single nucleotide polymorphisms is made possible, and the custom array was used in the analysis of a single nucleotide polymorphism described later. Here, as described in the section of Infinium (registered trademark) assay in a beads-array method, in these assay methods, there are two methods, i.e. a method using one kind of a probe and a method using two kinds of probes. Basically, in the detection of one single nucleotide polymorphism, one kind of the probe was used, and two probes were used for some single nucleotide polymorphisms.
The experiment was performed in accordance with the instruction manuals of the custom array kit and the analyzing instrument of Illumina, using specialized reagents contained in the kit. Briefly, the experimental procedures will be explained as follows. A reagent specialized in the treatment of the genome and a sodium hydroxide solution were added to 150 to 300 ng of the total DNA extracted in Example 1. Next, an enzyme for amplifying a whole genome was added thereto, and the mixture was incubated at 37° C. for 20 to 24 hours, and a whole genome was amplified. Further, an enzyme for fragmentation was added thereto, and the mixture was incubated at 37° C. for one hour. After the DNA was precipitated with isopropanol, a reagent for solubilization was added to the precipitates, and the mixture was suspended at 48° C. for one hour. A mixture was heat-denatured at 95° C. for 20 minutes, and this solution was injected into the custom array, and hybridization was carried out at 48° C. for 16 to 24 hours.
After the hybridization, an allele-specific extension reaction or a single base extension reaction was performed for each probe, and the fluorescent signals were amplified. The signals were read with a scanner (Illumina, BeadArray Reader) compatible to the kit. In addition, a specialized software (Illumina, BeadStudio 3.1) was used in the analysis of the single nucleotide polymorphisms. According to the present analytical method, the opposite alleles of a single nucleotide polymorphism can be determined simultaneously, and the genotypes were determined on the basis of the analytical results. The genotype was determined to be a heterozygote when both the signals of each of the alleles constituting a single nucleotide polymorphism were detected, and the genotype was determined to be a homozygote of the detected allele when only one of the signals of the alleles was detected.
The precision of the determination of a genotype was confirmed for all the single nucleotide polymorphisms to be analyzed on the basis of a cluster image showing a distribution of fluorescent signals, in accordance with Infinium (registered trademark) Genotyping Data Analysis, an analyzing manual of Illumina. The genotypes of the single nucleotide polymorphisms that are determined accurately are indicated on the image as three clusters of fluorescent signals that are completely separated from each other (two kinds of homozygotes and a heterozygote).
On the other hand, boundary lines of the three clusters become unclear for the single nucleotide polymorphisms that are not determined accurately. In a case where a degree of separation of the clusters is determined not to be high according to analysis software, the cluster image of the single nucleotide polymorphism was reconfirmed. In a case where a genotype was determined regardless of unclearness of the clusters, the sample was excluded from the subsequent analytical operations. Here, the confirmation of the cluster image was carried out under masking, in other words, in a state that the names of single nucleotide polymorphisms and p-values could not be compared with the single nucleotide polymorphisms. Here, the single nucleotide polymorphisms overlapping between the custom array used in the secondary analysis and GeneChip Human Mapping 500K of Affimetrix used in the primary analysis showed a concordance rate of 99% or more, when the concordance rates of the determination of genotypes were compared using 104 samples.
Primary open-angle glaucoma patients and normal tension glaucoma patients diagnosed on the basis of Guidelines offered by Japan Glaucoma Society were assigned to a glaucoma patient group, and healthy individuals confirmed to have no family history of glaucoma according to a medical interview were assigned to a non-patient group. For the present analysis, the same samples used in Example 3 for performing the primary analysis were not used, and new samples were collected. Blood donated under the consent on free will of the participants after having sufficiently explained the contents of studies from 409 cases of the glaucoma patient group and 448 controls of the non-patient group, each group being different from those of Example 3 was used as a specimen, a total DNA was extracted according to the method described in Example 1, and the analysis of single nucleotide polymorphisms was performed according to the method described in Example 9. The analytical results of a single nucleotide polymorphism obtained in each of the patients were stored in the Laboratory Information Management System (World Fusion, LaboServer) adopting a relational database. A specialized analysis program for a single nucleotide polymorphism was created and loaded within the system, and the analysis described as follows was performed. In detail, a single nucleotide polymorphism considered to have a high experimental reliability was extracted by rejecting a single nucleotide polymorphism having a call rate of less than 90% in both the glaucoma patient group and the non-patient group, and a single nucleotide polymorphism having a minor allele frequency of less than 5%.
In a meta-analysis, the Mantel-Haenszel method was used (Wakariyasui Igaku Tokeigaku (Easy Medical Statistics), pp. 48-80, Toshio
MORIZANE, Medical Tribune). In detail, 402 single nucleotide polymorphisms considered to have a high experimental reliability in both of the methods described in Example 3 and Example 10 were subjected to statistical comparisons of the allele frequency and two genotype frequencies (a dominant genetic model and a recessive genetic model) using Mantel-Haenszel chi-square test. Single nucleotide polymorphisms of which any one of an allele model, a dominant genetic model, and a recessive genetic model shows association with the onset of glaucoma at a p-value of 1.2×10−4 or less (the level of Bonferroni correction corresponding to p<5×10−2 when 402 times of multiple comparisons were performed), that is, −log P of 3.91 or more, are listed in Table 52.
The calculations of the Mantel-Haenszel chi-square test, and the odds ratio in the Mantel-Haenszel method for these single nucleotide polymorphisms, and a 95% confidence interval were performed according to the following procedures.
A Mantel-Haenszel chi-square value was determined for the allele model, the dominant genetic model, and the recessive genetic model, and a p-value was calculated by comparing the value with the chi-square distribution of a degree of freedom of 1.
The Mantel-Haenszel chi-square value (λAMH2) of the allele model was calculated according to the following formulas.
The Mantel-Haenszel chi-square value (χDMH2) of the dominant genetic model was calculated according to the following formulas.
The Mantel-Haenszel chi-square value (χRMH2) of the recessive genetic model was calculated according to the following formulas.
The odds ratio in the Mantel-Haenszel test was calculated for the allele model, the dominant genetic model, and the recessive genetic model.
The odds ratio in the Mantel-Haenszel test (ORaaH) for the allele model was calculated according to the following formula.
The odds ratio in the Mantel-Haenszel test (ORdMH) for the dominant genetic model was calculated according to the following formula.
The odds ratio in the Mantel-Haenszel test (ORrMH) for the recessive genetic model was calculated according to the following formula.
A 95% confidence interval of the odds ratio in the Mantel-Haenszel test was calculated for the allele model, the dominant genetic model, and the recessive genetic model.
The 95% confidence interval (95% CIA) for the allele model was calculated according to the following formulas.
A 95% confidence interval (95% CId) for the dominant genetic model was calculated according to the following formulas.
A 95% confidence interval (95% CIr) for the recessive genetic model was calculated according to the following formulas.
Table 52 lists dbSNP ID number specifying known single nucleotide polymorphisms obtained, the chromosome number at which a single nucleotide polymorphism exists, the physical location of a single nucleotide polymorphism, the exon, intron information (in a case where a single nucleotide polymorphism exists on a gene, the gene name and the exon or intron in which SNP exists are shown, and in a case where a single nucleotide polymorphism does not exist on a gene, neighboring genes and a distance between the gene and the single nucleotide polymorphism are shown), the information on the linkage disequilibrium state (the numbers of LD1 to LD4 were assigned to single nucleotide polymorphisms which exist in the same linkage disequilibrium region), each of bases constituting Allele 1 and Allele 2, the base of a high-risk allele, high-risk allele frequencies of the glaucoma patient group and the non-patient group, the p-value in a test method having the lowest p-value among three Mantel-Haenszel tests (allele frequency, dominant genetic model, and recessive genetic model), the kinds of the tests thereof, the odds ratio thereof, the 95% confidence interval thereof, SEQ ID NO: of the sequence containing Allele 1 and SEQ ID NO: of the sequence containing Allele 2 in each of the polymorphic sites, and SEQ ID NO: showing a base sequence of a probe used in a secondary analysis (basically, both the alleles are detected by the same probe, and in a case where the alleles are discriminated using two kinds of probes, both the sequences are listed together.). Here, one of ordinary skill in the art can obtain the information for sequences or alleles of the single nucleotide polymorphisms from dbSNP ID number listed above.
When the allele or genotype frequencies of the single nucleotide polymorphisms listed in Table 52 were compared between the non-patients and the glaucoma patients, a statistical difference was found according to Mantel-Haenszel chi-square test. By determining an allele of any one of these single nucleotide polymorphisms in the same manner as that in Example 3, whether or not an allele that is identified in a higher frequency in the glaucoma patient group than that of the non-patient group exists in the sample can be determined.
According to the above studies, 18 single nucleotide polymorphisms of which alleles or genotypes were associated with glaucoma at a p-value of 1.2×10−4 or less existing in clusters in relatively adjacent regions on the genome were found in 11 regions.
An allele identified in a high frequency in the glaucoma patient group for single nucleotide polymorphisms listed in Table 52 (in other words, a high-risk allele) or a genotype (in other words,
Similarly, a single nucleotide polymorphism of which allele or genotype shows association with the onset of glaucoma at a p-value of 1×10−2 or less, i.e. −log P of 2 or more is listed in Tables 53 to 62.
The single nucleotide polymorphisms listed in Tables 53 to 62 can be also used as a marker for predicting an onset risk of glaucoma in the same manner.
Next, regions and/or genes of the surrounding of single nucleotide polymorphism listed in Table 52 were determined by making reference to the database provided by the HapMap project. In detail, regions in which the single nucleotide polymorphism considered to be in a linkage disequilibrium with the single nucleotide polymorphisms listed in Table 52 exists were determined, on the basis of the linkage disequilibrium data in combination of the Japanese and the Chinese in the HapMap project.
Also, in a case where the single nucleotide polymorphism listed in
Table 52 exists in the linkage disequilibrium region containing genes, the physical location of the region and the gene name were determined. On the other hand, in a case where the single nucleotide polymorphism listed in Table 52 exists in the linkage disequilibrium region without containing the genes, only the physical location of the region was determined. In addition, in a case where the single nucleotide polymorphism listed in Table 52 exists on one gene beyond the linkage disequilibrium region, the gene name and the physical location of the gene were determined.
A single nucleotide polymorphism of which p-value is the lowest for each region is considered to be a single nucleotide polymorphism representing the region, and Tables 63 to 70 list a single nucleotide polymorphism representing the region, the chromosome number at which the region exists, the physical location of the region (start point and end point) and the gene name contained in the region.
The region listed in Tables 63 to 70 is a region or gene considered to be linked with a single nucleotide polymorphism associated with the onset of glaucoma in the present invention listed in Tables 53 to 62, and a single nucleotide polymorphism which exists in these regions or genes is considered to be linked with a single nucleotide polymorphism in the present invention. In other words, any single nucleotide polymorphisms which exist in these regions are linked with the single nucleotide polymorphism which exists in the region listed in Tables 53 to 62, and any of these single nucleotide polymorphisms can be used in the prediction of an onset risk of glaucoma in the same manner.
In the present invention, by combining any two or more single nucleotide polymorphisms determined to be involved in the onset of glaucoma, an extent to which the precision of the prediction of a risk of a disease improves is examined with logistic regression analysis, as compared to that where each of the single nucleotide polymorphisms is used alone. In the present analysis, any combinations of the single nucleotide polymorphisms determined to be significantly associated with the onset of glaucoma by statistically comparing allele or genotype frequencies can be used. In one example, 17 single nucleotide polymorphisms that showed a significant difference under the Bonferroni correction were subjected to the logistic regression analysis.
Out of 17 single nucleotide polymorphisms that had a significance under the Bonferroni correction, single nucleotide polymorphisms for use in the logistic regression analysis were further narrowed down according to a stepwise method. The value of 0.01 was adopted as criteria of variable incorporation and variable exclusion in the stepwise method. Upon the application of a stepwise method, a single nucleotide polymorphism belonging to the same LD block (ones having the same description in the column of linkage disequilibrium in Table 52) is represented by any one of single nucleotide polymorphisms belonging to each of the LD blocks, and it is set so that any one of the single nucleotide polymorphisms is to be a subject to be incorporated. Each of the narrowed-down single nucleotide polymorphisms is defined as an independent variable (Π) (homozygote of one allele=0, heterozygote=1, homozygote of opposite allele=2), and each regression coefficient (λ) can be determined according to the logistic regression analysis, and the following formula (18) was obtained formula (18)
Φ=1/{1+exp[−(λ0+λ1Π1+λ2Π2+λ3Π3+ . . . )]}
Next, in each sample, a value for risk prediction (Φ) was calculated by substituting a variable for each single nucleotide polymorphisms into this formula. When Φ is greater than 0.5, this sample donor was determined to be with an onset risk. A concordance rate was calculated by comparing the determination results with the matter of whether the sample donor having a single nucleotide polymorphism was actually a glaucoma patient. Further, the concordance rate was determined as mentioned above for each of the incorporated single nucleotide polymorphisms alone, and all the combinations of any two or more single nucleotide polymorphisms, and means and standard deviations of the concordance rate were obtained for each of the number of single nucleotide polymorphisms used in combination. Table 71 lists the number of single nucleotide polymorphisms, alone or in a combination of arbitrary number, the number of combinations when arbitrary number of single nucleotide polymorphism is combined, and the relationship between the mean and the standard deviation of the concordance rate. Here, all the calculations were performed using SAS 9.1.3, Windows (registered trademark) Edition, SAS Institute Japan Corporation.
As listed in Table 71, according to a stepwise method, out of the 17 single nucleotide polymorphisms, assuming that a pair of single nucleotide polymorphisms belonging to the same LD block were each counted as one, all ten single nucleotide polymorphisms were selected (rs7081455, rs693421, rs9358578, rs7961953, rs16935718, rs11123034, rs13110551, rs7559118, rs10517556, and rs6451268). A value for risk prediction (Φ) of individual cases was calculated using a logistic regression formula, alone or in a combination of any two or more of these 10 single nucleotide polymorphisms. When a cut-off value for a value for risk prediction is defined as 0.5, mean±standard deviation of the concordance rate was 54.7±1.4% in a case that each of the single nucleotide polymorphisms was used alone. This concordance rate was elevated as an increase in the number of single nucleotide polymorphisms used in combination, and reached the maximum of 59.9% in a case that all the ten were combined.
As described above, it was evident that in the determination of an onset risk of glaucoma by a single nucleotide polymorphism, an excellent concordance rate can be obtained even in a case that each of the single nucleotide polymorphisms are used alone, and the diagnostic precision can be further enhanced by combining these single nucleotide polymorphisms.
As described above, an individual who has an allele or a genotype that is identified in a high frequency in the glaucoma patients disclosed in the present invention on the genome has a high onset risk of glaucoma in future, and an individual who does not have an allele or a genotype that is identified in a high frequency in the glaucoma patients has a low onset risk of glaucoma in future.
According to the method of the present invention, the level of an onset risk of glaucoma of a sample donor can be determined by analyzing an allele or a genotype of a single nucleotide polymorphism in the present invention in a sample. A sample donor can take a preventive measure of glaucoma, or can receive appropriate treatments, on the basis of this risk. In addition, it is useful because, a sample donor who has an allele or a genotype that is identified in a high frequency in the glaucoma patients of a single nucleotide polymorphism in the present invention on the genome can be given a precision examination in whether or not the donor is with an early glaucoma which is difficult to be determined sufficiently by an intraocular pressure or an ocular fundus photograph, and can be started with a treatment at an early stage in a case where the donor is diagnosed as glaucoma.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-108688 | Apr 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/057527 | 4/17/2008 | WO | 00 | 10/16/2009 |
