WO2020111169A1 - Genetic testing method for multifactorial genetic disease and testing kit - Google Patents

Abstract

The present invention can be used as a method for testing for the risk of morbidity or for a disease from a functional polymorphism (functional SNP) of a gene of a functional protein which is assumed to be abnormal or responsible for the disease. Since the risk of morbidity for a multifunctional genetic disease can be tested by analyzing a functional SNP, the present invention can be used to assist diagnosis and prevent the disease. Provided are a method, testing chip, and testing kit for testing functional SNPs.

Description

Genetic test method and test kit for multifactorial genetic diseases

The present invention relates to a method for testing a morbidity risk of a multifactorial genetic disease using a plurality of Single Nucleotide Polymorphisms (single nucleotide polymorphism, hereinafter referred to as SNP), an auxiliary diagnostic method, a test chip and a test kit.

∙ Diversity in which one base in the base sequence of the genome of a certain biological population is mutated is called SNP. In many cases, SNPs are present outside the gene region and their regulatory regions in genomic DNA, and do not cause changes in genetic characteristics. However, there are many studies that analyze SNPs and compare their frequencies because some of them can serve as genetic markers.

For example, studies have been conducted to compare the frequency of SNPs used as markers, which are related to the susceptibility to illness and the side effects of drugs. In Japan, the SNP frequency for each disease in the Japanese patient population of 35 diseases including malignant tumors such as lung cancer and breast cancer, and cardiovascular diseases such as heart failure and myocardial infarction, and standard SNP frequency information of the Japanese are available. Comparative research has been published (JSNP database, URL: http://snp.ims.u-tokyo.ac.jp/index_ja.html).

-SNPs that are clearly associated with these diseases have been proposed for application as markers that identify disease-related genes by linkage analysis and association analysis. Many SNPs associated with these diseases do not cause changes such as gene expression, but the expression and properties of disease-related genes such as gene expression levels, qualitative changes in proteins, and changes in non-coding RNA. It is known that there is a SNP called a functional SNP (functional SNP) that influences (non-patent document 1).

Currently, although the relationship between SNPs and diseases has been clarified, there is no functional SNP that has been put to practical use as a marker for diagnosing diseases and analyzing risk of morbidity. Moreover, in multifactorial genetic diseases, it is very difficult to judge the risk of morbidity by a single functional SNP, because there are multiple genes themselves that are considered to be involved.

Multifactorial genetic disease is a disease that is caused by multiple factors such as environmental factors in addition to multiple genetic predispositions. It is considered difficult to predict morbidity risk because environmental factors are involved in onset in addition to multiple genetic predispositions. However, if the morbidity risk can be predicted in advance, the onset can be suppressed by various methods such as removing the environmental factors of the disease. As a result, it is possible to reduce the number of affected persons.

Multifactorial genetic diseases include various diseases such as diabetes, hypertension, ischemic heart disease, NASH (non-alcoholic steatohepatitis), rheumatoid arthritis, gout, and malignant tumors. In neuropsychiatric disorders, schizophrenia, mood disorders (bipolar disorder, major depressive disorder, etc.), addictive diseases, autism spectrum disorders, attention deficit/hyperactivity disorder, Alzheimer's disease, narcolepsy , Panic disorder, social anxiety disorder, obsessive-compulsive disorder, generalized anxiety disorder, eating disorder, personality tendency and disorder are considered to be multifactorial diseases. Although the genetic background of these diseases remains largely unknown, it is believed that there are multiple genetic predispositions. If the morbidity risk of these multifactorial diseases can be known in advance, the morbidity risk can be reduced by reducing the environmental factors.

Furthermore, diseases in the field of neuropsychiatry are considered to be extremely useful if an auxiliary diagnosis can be made by examination using SNP. In the field of psychiatry and neurology, diagnosis is based on an inquiry. Unlike in the case of visceral diseases such as digestive organs, the diseases of the neuropsychiatric region cannot collect the brain tissue of the patient for examination and cannot be diagnosed by tissue biopsy. Although one depressive symptom may be a different disease, most of the diseases have been diagnosed by a doctor's inquiry, and establishment of an objective test method is desired.

For example, schizophrenia is said to affect 1 in 100 people, and the number of patients is high following anxiety and depression, and it is estimated that there are 79.5 million patients in Japan ( Ministry of Health, Labor and Welfare, 2008 patient survey). Schizophrenia is a psychotic disorder that often develops from late adolescence to adolescence and has a chronic course. The symptoms of schizophrenia are diverse, and many diseases exhibit similar symptoms such as mood disorders, and differential diagnosis is difficult especially for early symptoms. Schizophrenia is a disease that can be expected to have a favorable prognosis if it is diagnosed and treated as accurately as possible at the earliest stage, and early diagnosis is desired. If there is a means for objectively making an auxiliary diagnosis in addition to the inquiry, it is possible to make a reliable diagnosis early.

It has also been reported that antipsychotics, omega-3 fatty acid administration, cognitive behavioral therapy, and family therapy significantly suppress the transition to schizophrenia in the high-risk group who are prone to schizophrenia (Non-patent Reference). Reference 2), early identification of high-risk groups has a great merit in preventing schizophrenia. For other multifactorial genetic diseases, if the high-risk group can be identified at an early stage, many diseases can be taken preventive measures, and it is considered that the onset of the diseases can be suppressed.

JP 2004-135667 A

Regarding schizophrenia whose analysis examples are shown below as examples, test kits and auxiliary diagnosis methods have already been disclosed (Patent Document 1, Non-Patent Document 3). Patent Document 1 is a test kit in which gene expression levels are comprehensively examined by a DNA microarray, and those having a change compared with healthy subjects are selected. Although the analysis was performed using samples from 5 acute patients, 12 chronic patients, and 9 healthy subjects, the number of samples in each group used in the analysis was small, and the gene expression level was the subject's health condition and environmental factors. Since it is highly likely to be affected by such factors, it is considered that the diagnostic accuracy is low. Non-Patent Document 3 is an attempt to use a plurality of gene polymorphisms obtained by comprehensive gene analysis (Genome-Wide Association Study, GWAS) as a polymorphism risk for auxiliary diagnosis of schizophrenia and bipolar disorder. However, each of the candidate genes has a very low effective dose, and there is a big problem in its validity. None of the results have been put to practical use, and new test methods and test kits are required. Regarding the above-mentioned multifactorial genetic diseases, various markers for examining morbidity risk have been proposed, but none of them has been put into practical use.

As described above, in a multifactorial genetic disease, not only the genetic predisposition but also environmental factors have an important influence on the onset, so when the morbidity risk is high, avoid the environmental factors highly associated with the onset risk, Can be used to prevent the onset of symptoms such as taking prophylactic drugs. Further, if it is known in advance that the disease risk is high, it is possible to perform regular examinations and receive appropriate treatment early. However, effective markers for predicting morbidity risk have not been developed.

Until now, when SNPs were used as test markers, studies were conducted on markers that existed in a single gene. In addition, there is no example in which the risk of morbidity was analyzed by examining the relationship with a disease only for functional SNPs. The present invention provides a new test method using a combination of a plurality of SNPs that cause a quantitative or qualitative change in gene expression as a disease marker. An object of the present invention is to provide a method, a test chip, and a test kit for testing the risk of morbidity of a multifactorial genetic disease, which has been difficult to predict the risk of morbidity. It is another object of the present invention to provide a test method and a test kit for assisting diagnosis in diseases in the neuropsychiatric field for which there is no objective diagnostic criterion.

The present invention relates to an inspection method and an inspection kit for the following diseases.
(1) A test method for assisting a morbidity risk or diagnosis of a multifactorial genetic disease by using a functional SNP of at least two or more genes known to be involved in the multifactorial genetic disease.
(2) The multifactorial genetic diseases include schizophrenia, bipolar disorder, mood disorders, addictive disorders, autism spectrum disorders, attention deficit/hyperactivity disorder, Alzheimer's disease, as neuropsychiatric disorders. Narcolepsy, panic disorder, social anxiety disorder, obsessive-compulsive disorder, general anxiety disorder, eating disorder, personality tendency and disorder, and other areas of disease include diabetes, hypertension, ischemic heart disease, and NASH (non-alcoholic steatohepatitis). ), Rheumatoid arthritis, gout, and malignant tumor.
(3) The test method according to (1) or (2), wherein the multifactorial genetic disease is schizophrenia or bipolar disorder.
(4) The test method according to (3), wherein the SNP is a functional polymorphism involved in dopamine fluctuation.
(5) The functional polymorphisms involved in the fluctuation of dopamine are gene polymorphisms of tyrosine hydroxylase (TH) gene, catechol-O-methyltransferase (COMT) gene, and dopamine D2 receptor (DRD2) gene. (4) The inspection method according to (4).
(6) The inspection method according to (4) or (5), wherein the functional SNPs are rs10770141, rs4680, rs1799732, and rs1800497.
(7) In addition to the functional SNP according to (6), rs2070762, rs6356, rs921451, rs3837091, rs1079597, rs1076560, rs6277, rs1799836, rs1040399, DAT1 Promoter -67A / T, DAT1 40-bp variable number of tandem repeats An inspection method comprising at least one of the above.
(8) A test chip for use in a test for assisting the morbidity risk or diagnosis of the multifactorial genetic disease according to any one of (1) to (7), which is known to be involved in the multifactorial genetic disease. A test chip in which functional SNPs of a plurality of genes are retained in a detectable manner.
(9) A test kit used for a test for assisting a morbidity risk of a multifactorial genetic disease or a diagnosis, wherein the primer used for the test according to any one of (1) to (7) and a reagent necessary for the test, Alternatively, an inspection kit including the inspection chip of (8) and a reagent necessary for the inspection.
(10) A DNA of interest is analyzed for rs10770141, rs4680, and rs1800497, which are functional SNPs, and at least two of T(+) for rs10770141, Met(−) for rs4680, and A1(+) for rs1800497 are combined. If you have schizophrenia, or you are at high risk of morbidity, if you have all three, you have bipolar disorder or you are at high risk of morbidity To test for schizophrenia and bipolar disorder.
(11) A method for examining a patient suspected of schizophrenia, which comprises analyzing the DNA of a target for functional SNPs rs10770141, rs4680, and rs1800497. Is a T(+), or rs4680 has a combination of Met(−) and rs1800497 has a combination of A1(+), a method of determining that the possibility of schizophrenia is high.
(12) A method for assisting the dose determination of an antipsychotic drug in a schizophrenic patient by analyzing functional SNPs rs10770141, rs4680, and rs1800497.
(13) It is recommended that patients with T(+) rs10770141 and Met(−) rs4680 study high-dose dopamine receptor blockers. (12) Assist the dose determination of antipsychotic drugs. Method.
(14) A method for diagnosing a disease in a neuropsychiatric region, which comprises examining functional SNPs of at least two or more genes known to be involved in a multifactorial genetic disease and sickness of the disease due to the functional SNP. A diagnostic method that refers to and diagnoses risks.
(15) The diagnostic method according to (14), wherein the disease in the neuropsychiatric region is schizophrenia or bipolar disorder, and the functional SNP is a functional polymorphism involved in dopamine fluctuation.
(16) If the test method described in any one of (1) to (7) determines that the risk of developing a multifactorial genetic disease is high, administration of a prophylactic drug or non-drug therapy Preventive methods to suppress the onset.
(17) A treatment method for differentially diagnosing a multifactorial genetic disease by the test method according to any one of (1) to (7), and administering a therapeutic drug or non-pharmacotherapy.

The figure which shows typically dopamine synthesis and decomposition. The figure which shows the relationship between functional SNPs and antipsychotic drug dose.

Previously, a method of comprehensively analyzing genes to find SNPs associated with a disease was used. On the other hand, the present inventors have found that the “function that is assumed to be responsible (or abnormal)” in a disease is affected by the combination of functional polymorphisms of the constituent protein genes required for the expression of the function. Assuming that they received it, we performed functional polymorphism analysis. Since this method is a method of analyzing the risk of morbidity based on the functional polymorphism of a gene associated with a disease, it will be associated with an innate characteristic or abnormality of a function that is supposed to be involved in the disease. Therefore, it becomes possible to explain the pathology and pathological condition of the disease, and there is a high possibility that an appropriate preventive method or therapeutic method can be selected for each patient.

As used herein, the term “functional SNP” refers to an SNP existing in a gene region or its regulatory region, which causes some change such as increase/decrease in gene expression or qualitative change in translated protein. .. The functional SNP in a certain disease includes not only a functional SNP directly involved in the disease but also a functional SNP of a gene group involved in the disease such as a signal transduction system or an enzyme cascade that is clearly involved in the disease. In the case of schizophrenia shown below as an example, it exists in the gene region involved in dopamine synthetic ability, resolution, and the density of its receptor, dopamine D2 receptor (Dopamine D2 Receptor, DRD2), and its regulatory region. SNP that causes a change in gene expression level or protein. Furthermore, not only dopamine nervous system but also serotonin nervous system, noradrenaline nervous system, and other proteins related to other neurotransmission systems that have been reported to be involved in schizophrenia, and functional SNPs of proteins that will be found in the future. Can be included.

In the test chip of the present invention, oligonucleotides complementary to a plurality of SNPs to be detected may be directly immobilized on a substrate, or may be immobilized on a carrier such as beads and then retained on the substrate. Nucleic acid may be extracted from the blood of a patient and hybridized by a conventional method to detect the double-stranded nucleic acid. Alternatively, a plurality of SNPs to be detected may be simultaneously detected by a known inspection method using PCR such as the invader method and the real-time PCR method. Further, a functional SNP capable of testing other diseases may also be fixed to one test chip so that a plurality of diseases can be tested at the same time. Any known method can be used as long as it is a known method that can specify the sequence of functional SNPs. Further, the test kit can include a primer for detecting functional SNPs, a reagent for use in PCR, or the above-mentioned test chip and necessary reagents.

Until now, the present inventors have reported morbidity risk for dopamine hypersensitivity psychosis (DSP) whose morbidity and mechanism of action are clear (Non-patent document 4). However, it is considered that many mechanisms other than the dopamine neurotransmission system are involved in the morbidity risk of schizophrenia itself, and it has been considered that SNP analysis of these genes alone cannot be performed. However, as a result of the analysis by the present inventors, it is possible to diagnose a morbidity risk even with respect to a multifactorial genetic disease having a wide variety of pathogenic mechanisms, by analyzing SNPs of genes considered to be involved in combination. It became clear. It has been suggested that the mechanism involved in the pathogenesis is not limited to the dopaminergic nervous system, and construction of a system capable of judging the morbidity risk of nearly half of the population is considered to be extremely meaningful.

Hereinafter, the present invention will be described focusing on schizophrenia, but the analysis method using a combination of “functional SNPs” is effective for other diseases, particularly multifactorial genetic diseases. In particular, schizophrenia, mood disorders, addictive diseases, diabetes, hypertension, and hyperlipidemia are diseases that have many patients all over the world, and early diagnosis and prevention are desired. The combination of functional polymorphisms of genes is inherent and can be predicted before the onset, and of course, the prognosis and progress of the disease can be predicted without being affected by the condition and stage of the disease. In particular, being able to predict the risk of morbidity before onset is a very important test from the viewpoint of prevention. In addition, in the case of neuropsychiatric diseases, in which inquiry is an important factor for diagnosis, it is a useful tool as an auxiliary diagnosis method that provides objective judgment material for diagnosis. In particular, in diseases where differential diagnosis is difficult in the early stage of onset, such as schizophrenia and bipolar disorder, the objective judgment material of functional SNP is very useful for diagnosis.

It is said that schizophrenia is highly heterogeneous, but about 75% is effective with antipsychotic drugs, which are dopamine D2 receptor blockers. It has been reported that the dopamine synthesizing ability is enhanced in patients for which a DRD2 blocker is effective (Non-patent Document 5). Furthermore, it has been reported that DRD2 density may be low in schizophrenia patients (Non-Patent Document 6). The proteins that compose the dopamine neurotransmission system and their functional SNPs are schematically shown in FIG.

Dopamine is synthesized by tyrosine hydroxylase (Tyrosine Hydroxylase, TH) and released into the synaptic cleft. The dopamine released in the synaptic cleft is decomposed by catechol-O-methyltransferase (COMT) or reintroduced into the cell by the dopamine transporter (DAT). Also, dopamine is released and suppressed by the dopamine D2 receptor (DRD2).

In schizophrenia, it has been suggested that dopamine release is enhanced at nerve endings in limbic dopaminergic nerves and dopamine concentration in synaptic cleft is increased (Non-patent Documents 7 and 8). On the other hand, it has been reported that the group in which the positive symptoms are improved by the antipsychotic drug has an increased dopamine synthetic ability, and in the group that does not improve the synthetic ability is equivalent to that of a healthy person (non-patent document 9), and dopamine. It has been suggested that synthetic ability and dopamine concentration in the synaptic cleft are involved in symptoms and treatment responsiveness to antipsychotic drugs.

The action of dopamine synthesized from tyrosine in cells is regulated by these enzymes and receptors, and it is known to be associated with the onset of schizophrenia. Among the proteins schematically shown in FIG. 1, TH, COMT, and DRD2 are proteins known to have functional polymorphisms.

Not only in the dopamine neurotransmission system, but also in schizophrenia, which is thought to involve more genes, if the dopamine neurotransmission function is involved in the onset risk, a functional SNP involved in the onset risk of DSP May increase the risk of developing schizophrenia. Schizophrenia is formed by the repetitive hyperactivity and elimination of dopaminergic function. It is believed that this is a mechanism similar to psychosis that occurs with repeated administration of dopamine antagonists. Therefore, the risk of developing schizophrenia was examined using a gene set having a high correlation with the risk of developing DSP.

The SNP used is a polymorphism of the TH gene promoter region, COMT gene, DRD2 gene (Table 1). Specifically, C-824T gene polymorphism in the TH gene promoter region, polymorphism in which Val at position 158 of COMT gene is replaced with Met, -141C Ins/Del polymorphism in DRD2 gene, Taq1A polymorphism in ANKK1 gene is there. The ANKK1 (the ankyrin repeat and kinase domain maintaining 1) gene is a gene close to the DRD2 gene, and the Taq1A polymorphism of ANKK1 is a polymorphism suggested to be associated with the DRD2 density. These polymorphisms are single nucleotide polymorphisms registered as rs10770141, rs4680, rs1799732, and rs1800497 in the SNP database of the National Center for Biotechnology Information (Non-Patent Documents 10 to 13). We analyzed whether polymorphisms of these three genes are involved in schizophrenia risk.

This example was approved by the ethics review committee of Chiba University School of Medicine, and in accordance with the Declaration of Helsinki by the World Medical Association and the ethical rules of the Japanese Society of Psychiatry and Neurology, with sufficient informed consent to obtain privacy. Observing confidentiality obligations, and giving due consideration to maintaining anonymity.

Patients with schizophrenia meet the criteria for diagnosis of mental disorders published by American Psychiatric Association and a revised text manual 4th edition (DSM-4-TR; diagnostic and statistical manual of mental disorders-IV-text revision). I chose one. The combination of the above functional polymorphisms was analyzed in 349 patients with schizophrenia and 273 healthy subjects.

Blood-derived DNA collected from schizophrenia patients and healthy subjects was extracted and analyzed using QIAamp DNA Blood Minikit (Qinagen). For the analysis, the genotypes of each patient and healthy subjects were identified for the above four SNPs by a real-time PCR method (TaqMan SNP Genotyping Assay (Thermo Fisher Scientific)). That is, the TH gene C-824T gene polymorphism (rs10770141, T allele), the COMT gene Val158Met polymorphism (rs4680, Met allele), the DRD2 gene Taq1A polymorphism (rs1800497, A1 allele), and ANKK1 located in the upstream vicinity thereof. The -141CIns/Del polymorphism (rs1799732, Del allele) was analyzed (Table 2).

In the C-824T polymorphism (rs10770141) of the TH gene, the one in which C is replaced by T is T(+), and in the COMT gene Val158Met polymorphism (rs4680), the one in which it is replaced by Met is Met(+). ), the A1 allele carrier in the DRD2 gene Taq1A polymorphism (rs1800497) is indicated as A1(+), and the deletion in the -141CIns/Del polymorphism (rs17979932) is indicated as Del(+). The indication of (-) in each polymorphism indicates that the polymorphism is not possessed.

Persons who carry the polymorphism T(+) in the TH gene have high activity of tyrosine hydroxylase, that is, high dopamine-producing ability. In the COMT gene, a person who possesses Met(−) substituting for Val has a high activity of the dopamine-degrading enzyme COMT and has a high rate of dopamine degradation. Those who carry the A1(+) or Del(+) gene polymorphism have low expression of the dopamine D2 receptor.

From the combinations of each polymorphism, we summarized dopamine synthesis, degradation, and receptor expression (Table 3). Activity indicates dopamine neurotransmitter activity, from the left, high and low levels of dopamine synthesis, high and low levels of dopamine degradation, high and low receptor expression, specific combinations of polymorphisms, schizophrenia, number of healthy individuals, The appearance frequency and the appearance frequency ratio are summarized.

As a result, when TH is highly expressed (T(+)), COMT is highly expressed (Met(−)), and DRD2 is lowly expressed (A1(+) or Del(+)), other It became clear that the incidence of schizophrenia was much higher than that of the combination. Table 4 shows the results of calculating the odds ratio by comparing the case of T(+), Met(−), which is A1(+), or Del(+), with other combinations. In the case of T(+), Met(-), and the combination of A1(+) or Del(+) gene polymorphisms, the odds ratio was 6.5 (p<0.029), resulting in a high rate. The result was that they were prone to schizophrenia. As a result of the Fisher's exact test, the P value was 0.00357 in the one-sided test, 0.00296 in the two-sided test, and the P-value was corrected by Bonferroni's method when the gene polymorphism was present. The results were 0.02856 and 0.02368, which indicates that schizophrenia was significantly more likely to occur. In the case of the combination of the above SNPs, the risk of developing DSP increases (Non-Patent Document 4). However, the DSP onset risk does not completely match the onset risk of schizophrenia because the onset risk increases not only in the combination of the above SNPs but also in other combinations.

Due to the nature of functional polymorphism, it is considered that the person with this combination has the following dopamine neurotransmission function. A person with a functional SNP of this combination has a high ability to synthesize dopamine by the TH gene, and thus a large amount of dopamine is synthesized due to stress or the like. Moreover, since DRD2 is low-expressed, release inhibition is less likely to occur, and a large amount of dopamine is released in the synaptic cleft. However, since the dopamine decomposing ability by COMT is high, there is a possibility that the dopamine has a dopamine transmitting function of being immediately decomposed. The person with this combination has a dopamine neurotransmission function in which the dopaminergic nerve activity is rapidly enhanced and disappears naturally, and it is considered that repeated stress causes sensitization and manifests schizophrenia. This is consistent with the conventional pathological hypothesis of schizophrenia, and it is possible to select an appropriate treatment method, and it is highly likely to lead to the development of a prevention method.

This combination accounted for 4.6% of the analyzed schizophrenia patients, but as shown in Table 5, there are other functional polymorphisms related to dopamine fluctuations in the synaptic cleft, which also show similar dopamine fluctuations. It is believed that there are combinations of functional SNPs shown. Furthermore, by combining functional polymorphisms related to the neurotransmission system, which are said to be associated with schizophrenia, such as the serotonin neurotransmission system and the noradrenaline neurotransmission system, the risk of schizophrenia is predicted more widely and accurately. be able to.

Most patients with schizophrenia are ameliorated by antipsychotic drugs, which are DRD2 blockers. In antipsychotic treatment, it is considered that there is an optimal DRD2 occupancy rate by the antipsychotic and that there is an optimal dose for each antipsychotic. However, there are large individual differences in the therapeutic dose in actual clinical practice. If the function of the gene is changed by the functional SNP, it is considered that the effect of the antipsychotic drug is also affected. Therefore, the correlation between functional SNP and antipsychotic dose was analyzed. In the case of schizophrenia, the chlorpromazine equivalent, which is the value obtained by converting the prescribed amount of the prescribed oral drug into chlorpromazine, is used as a standard, so it was decided to analyze the correlation between the chlorpromazine equivalent and SNP.

The relationship between a single SNP (Single SNP) or a combination of two SNPs (Double SNP) and the chlorpromazine equivalent (CP equivalent equivalent) for the samples of schizophrenia patients collected at Chiba University shown above. Analyzed. As a result of detailed analysis, it was revealed that TaqIA among the DRD2 gene polymorphisms had a high correlation with the onset of disease. Therefore, among the functional polymorphisms of genes of the dopamine neurotransmission system, 3 of TaqIA, TH, and COMT were identified. The functional SNPs of one gene were analyzed (Fig. 2).

▽ For individual genes, the CP equivalent conversion tended to be high for T(+) for TH and Met(-) for COMT. As for the combination of genes, the result that the CP equivalent conversion amount was significantly high was obtained in the combination of T(+) of TH and Met(−) of COMT (p<0.05). In addition, the CP equivalent conversion amount also tended to be high in the combination of T(+) of TH and A1(-) of TaqIA.

Based on these results, we analyzed the frequency of occurrence of TH, COMT, and TaqIA functional polymorphisms in individual schizophrenia patients and healthy individuals (Table 6). The combination of T(+) of TH and Met(−) of COMT, which had significantly high CP equivalent conversion values, had an odds ratio of 2.59 (p<0.05), which was clearly high.

　Antipsychotics do not exert antipsychotic effects at low doses. On the other hand, overdose of antipsychotic drugs causes side effects such as extrapyramidal symptoms, depression, and discomfort, resulting in decreased medication adherence and recurrence, leading to a poor long-term prognosis. In addition, overdose causes a compensatory increase in DRD2, induces dopamine hypersensitivity psychosis, and develops treatment resistance. While a biological index is needed to provide the optimal dose for each individual, it is also possible to use a combination of functional SNPs as a guide for determining the optimal dose.

Next, we conducted analysis using a large number of RIKEN samples. 2012 Japanese schizophrenia patients (1111 men, average age 47.2±14.1 years, 901 females, average age 49.2±14.7 years) and 2170 healthy people ( From 889 men, average age 39.2±13.8 years, 1281 women, average age 44.6±14.1 years), use “Greedy 5-To-1 Digit-Matching” algorithm for age and gender By using the tendency score matching (property score matching), an analysis was performed for each of 1272 individuals with schizophrenia and healthy individuals. The number of schizophrenia patients is 574 men, 698 women, average age 47.4±13.9 years, average onset age 24.9±13.4 years, healthy persons 603 men, 669 women, The average age was 46.5±13.9 years. In addition, in order to verify the preliminary results of the pilot study described above, the subjects used in the analysis exclude the patients employed at Chiba University and related hospitals.

Since rs10770141 (TH), rs4680 (COMT), and rs1800497 (TaqA1) were considered to have a high correlation with the fluctuation of dopamine, these three functional SNPs were analyzed. The statistical analysis method is based on the method described above.

All ages: schizophrenia (SZ) n=1272, healthy people (CNT) n=1272; 20-30 years old: SZ n=182, CNT n=170, Fisher's exact test (two-sided test) #p< 0.05, *p<0.01, **p<0.001
All Double Combination means a combination of two SNPs, and also includes a combination of all three SNPs (Met(-)*A1(+)*T(+)).

First, the genetic risk in schizophrenia patients at all ages was 48.1% for rs4680(Met(-)), 61.1% for rs1800497(A1(+)), and rs10770141(T(+)). ) Was 11.5%, those with all these risk factors were 2.9%, and those without all were 18.1%. On the other hand, in healthy subjects, rs4680(Met(−)) was 42.7%, rs1800497(A1(+)) was 47.6%, and rs10770141(T(+)) was 6.8%. 2.4% had all risk factors, and 25.3% did not. As shown in Table 7, the odds ratio of each SNP calculated by correlating with schizophrenia for age and sex is 1.24 for rs4680 (Met(-)) and 1 for rs1800497(A1(+)). 0.73 and rs10770141(T(+)) were 1.79, which were both significant.

Also, the odds ratios of two genetic risk carriers, rs4680 (Met(−)) and rs1800497(A1(+)), and rs1800497(A1(+)) and rs10770141(T(+)) were 2.01 respectively. A significant difference was observed with 1.87. In addition, rs4680 (Met(−)) and rs10770141(T(+)) tended to correlate with schizophrenia (p=0.068). The ratio of possessing either of the above two risk factors is 35.8% in the schizophrenia patient group, and the odds ratio is 2.22, which is a very high value.

Furthermore, a more prominent tendency was recognized when analyzed by dividing into the prevalence of schizophrenia onset (30 years or younger) (Table 7, 20-30 years). rs4680(Met(-)) has 53.0%, rs1800497(A1(+)) has 62.1%, rs10770141(T(+)) has 13.7%, all of these risk factors Was 3.3%. On the other hand, in healthy subjects, rs4680(Met(−)) was 38.8%, rs1800497(A1(+)) was 48.8%, and rs10770141(T(+)) was 1.2%. 1.2% had all risk factors. As shown in Table 7, the odds ratio of each SNP calculated for correlation with schizophrenia was 1.78 for rs4680(Met(−)), 1.72 for rs1800497(A1(+)), and rs10770141(T (+)) was 13.38, showing a higher correlation than the analysis at all ages.

In addition, in the analysis of the two risk factors, significant differences were found in all combinations, and rs4680(Met(−)) and rs1800497(A1(+)), rs4680(Met(−)) and rs10770141(T(+) )), the odds ratios of rs1800497(A1(+)) and rs10770141(T(+)) were 8.97, 5.96, and 6.46, respectively. Furthermore, those who possess either of the above two risk factors are very high, 42.9% of the schizophrenia patient group (5.9% of the healthy person group) and the odds ratio of 12.00. There is.

Higher odds ratios at prevalence suggest that genetic factors are more involved in the development of schizophrenia at prevalence. If preventive intervention can be performed at a prevalence age of schizophrenia in a group with a high genetic risk factor, the onset of schizophrenia can be suppressed. For example, the presence or absence of a genetic factor can be grasped in advance by a test chip or the like, and preventive intervention such as medication, cognitive behavior therapy, or family therapy can be performed for a group with a high risk of onset.

It is known that schizophrenia has a pre-symptomatic stage (at risk mental state, ARMS) or a pre-symptomatic phase called attenuated psychosis syndrome in the American Psychiatric Association diagnostic criteria DSM-5 before the onset of schizophrenia. There is. ARMS may develop not only schizophrenia but also bipolar disorder, and it is said that diagnosis of both diseases is often wrong even after the onset. ARMS has been reported to be effective for some antipsychotic drugs, family intervention, and omega-3 fatty acids, and there is a high risk of developing schizophrenia by testing SNPs at the ARMS stage, or If it is possible to judge whether the risk of developing bipolar disorder is high, more appropriate measures can be taken.

Bipolar disorder known to have genetic factors other than schizophrenia, autism and ADHD, social anxiety disorder, dementia, etc. if functional SNPs can be used for testing, medication, family It is possible to take appropriate measures early or before the onset, such as intervention of.

Also, it is known that psychiatric diseases become less responsive to antipsychotic drugs when the time from the onset of symptoms to the start of treatment becomes longer. However, it has been pointed out that diagnosis is difficult in the early stage of onset, for example, it is difficult to distinguish bipolar disorder from schizophrenia. The test using the functional SNP can obtain information that assists the diagnosis, and thus an appropriate treatment method can be selected earlier.

Next, as a result of analyzing the bipolar disorder using a sample of Chiba University, it was revealed that the dopaminergic nervous system gene polymorphism was also associated with the bipolar disorder. It is necessary to wait for verification in a large number of cases planned in the future, but in the case of having two risk factors of rs4680 (Met(-)) and rs10770141 (T(+)), an odds ratio of 5.86 and three When there was a risk factor, a significant difference was observed between the odds ratio of 12.0 and healthy subjects (Table 8).

Bipolar disorder (BP) n=172, healthy person (CNT) n=273, #p<0.05, *p<0.01, **p<0.001 by Fisher's exact test (two-sided test)

 In schizophrenia, there was no significant difference in the possession of the three risk factors from the healthy subjects, whereas in bipolar disorder, it was revealed that the possession of the three risk factors correlates with the disease. Based on these results, it is clear that the characteristics of the dopaminergic nervous system differ depending on the disease. In schizophrenia, a combination of a highly active allele of COMT and a low expression allele of DDR2 density of presynapse (Met(-)*A1(+)) is characteristic, and in bipolar disorder, a highly active allele of COMT. A combination of TH highly expressing alleles (Met(−)*T(+)) is characteristic. It has been suggested that there is a difference in executive function, working memory, and attention function depending on the presence or absence of Met in COMT. Therefore, Met(-) is involved in responsiveness to environmental factors involved in the onset of both diseases. Is inferred. Furthermore, it is speculated that A1(+) of TaqIA and T(+) of TH may cause schizophrenia or bipolar disorder.

Also, as mentioned above, there are other functional SNPs involved in dopamine fluctuations. Furthermore, there are other functional SNPs that are presumed to be involved in mental disorders such as schizophrenia, such as the serotonin nervous system and the noradrenaline nervous system. If it becomes possible to differentiate diseases in the psychiatric region with high heterogeneity by advancing these analyses, it becomes possible to make a differential diagnosis earlier and select an appropriate treatment method.

Here, schizophrenia and bipolar disorder are shown as examples, but as a functional SNP for assaying the risk of diabetes, for example, the functional SNP of the insulin resistance-related gene adiponectin (G-276T, T94G) is used. , CD14 functional SNP (T-159C), amylin gene SNP (Ser20Gly), hepatocyte nuclear factor gene HNF4α (P1-HNF4α, P2-HNF4α), PPARγ (Pro12Ala), insulin receptor gene IRS-1 (Gly972Arg) , IRS-2 (Gly1057Asp), gene polymorphism of β3 adrenergic receptor (Trp64Arg) which is a functional SNP of gene polymorphism associated with obesity, β2 adrenergic receptor gene polymorphism (Arg16Gly), UCP1 (Uncoupling Protein 1) ) Gene polymorphism (A-3826G), FTO (A/T) and the like (Non-patent Documents 14 to 17). In mood disorders, serotonin transporter gene 5HTLTPR (l/s), serotonin 5HT _1A receptor gene 5HTR1 (C-1019G, Gly272Asp), serotonin 5HT _2A receptor gene 5HTR2A (T102C, A-1438G), tryptophan synthase TPH1. In addition, functional dependent SNPs such as opioid receptor gene OPRM1 (A11G) are known in addition to gene polymorphisms of dopamine neurotransmission system in dependent diseases such as gene polymorphism of dopamine neurotransmission system (non-patent document 18 ~19), by combining these SNPs, morbidity risk can be similarly examined.

As described above, the analysis method shown here is a method capable of analyzing the morbidity risk of a multifactorial genetic disease by combining and analyzing the functional SNPs reported so far. , It is a very versatile analysis method.

Claims (13)

1. A test method that assists the morbidity risk or diagnosis of a multifactorial genetic disease by using functional SNPs of at least two genes known to be involved in the multifactorial genetic disease.
2. The multifactorial disease is
   Diseases in the field of neuropsychiatry include:
   Schizophrenia, bipolar disorder, mood disorder, addictive disease, autism spectrum disorder, attention deficit/hyperactivity disorder, Alzheimer's disease, narcolepsy, panic disorder, social anxiety disorder, obsessive-compulsive disorder, generalized anxiety disorder, intake Eating disorders, personality trends and disabilities,
   Diseases in other areas include
   The test method according to claim 1, which is diabetes, hypertension, ischemic heart disease, NASH (non-alcoholic steatohepatitis), rheumatoid arthritis, gout, or malignant tumor.
3. The multifactorial disease is
   The test method according to claim 1 or 2, wherein the test method is schizophrenia or bipolar disorder.
4. The test method according to claim 3, wherein the SNP is a functional polymorphism involved in fluctuation of dopamine.
5. The functional polymorphisms involved in the fluctuation of dopamine are gene polymorphisms of tyrosine hydroxylase (TH) gene, catechol-O-methyltransferase (COMT) gene, and dopamine D2 receptor (DRD2) gene. The inspection method according to claim 4, which is characterized in that:
6. The inspection method according to claim 4 or 5, wherein the functional SNPs are rs10770141, rs4680, rs1799732, and rs1800497.
7. In addition to the functional SNP according to claim 6, rs2070762, rs6356, rs9221451, rs3837091, rs1079597, rs1076560, rs6277, rs1799836, rs1040399, DAT1 Promoter-67a/T, DAT1 40-bp alien varieval-varia vari. An inspection method characterized by including one of them.
8. A test chip for use in a test for assisting diagnosis of morbidity of a multifactorial genetic disease according to any one of claims 1 to 7, or diagnosis,
   A test chip in which functional SNPs of a plurality of genes known to be involved in a multifactorial genetic disease are detectably retained.
9. A test kit for use in a test for assisting diagnosis of a multifactorial genetic disease risk, or
   A primer used in the test according to any one of claims 1 to 7 and a reagent necessary for the test,
   Alternatively, an inspection kit containing the inspection chip of claim 8 and a reagent necessary for the inspection.
10. DNA of interest was analyzed for the functional SNPs rs10770141, rs4680, and rs1800497, and rs10770141 has T(+), rs4680 has Met(−), and rs1800497 has at least one of A1(+) in combination. If you have schizophrenia, or you are at high risk of morbidity,
    A method for testing schizophrenia and bipolar disorder, which is determined to be bipolar disorder or has a high risk of morbidity if all three are present.
11. A method for testing a patient suspected of having schizophrenia,
    DNA of interest was analyzed for functional SNPs rs10770141, rs4680, and rs1800497,
    If the target age is 30 years or younger, rs10770141 is T(+),
    A test method that determines that schizophrenia is likely when rs4680 has a combination of Met(-) and rs1800497 has a combination of A1(+).
12. A method of assisting in determining the dose of an antipsychotic drug in schizophrenia patients by analyzing functional SNPs rs10770141, rs4680, and rs1800497.
13. The method for assisting in determining the dose of an antipsychotic drug according to claim 12, which is recommended for studying the administration of a high dose of a dopamine receptor blocker to a patient in which rs10770141 is T(+) and rs4680 is Met(−).
    

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