Test method for predicting prognosis of ALS patients, test kit, and drug screening method

Abstract

To elucidate the pathology and pathogenesis of patients with ALS, in particular sporadic ALS, thereby developing therapeutic agents and methods to extent the survival time.SOLUTION: Genome Wide Association Study was performed on the survival time of sporadic ALS patients to discover that seven genetic polymorphisms within FGF1 and THSD7A affect a decrease in the survival time. It was also found out that these SNPs are associated with FGF1 and THSD7A gene expression. These SNPs, FGF1 expression, and THSD7A expression can be used as markers for the survival time of sporadic ALS, and these markers can be used to develop methods for preventing and treating the pathological conditions.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a testing method, a testing kit, and a pharmaceutical screening method using gene polymorphisms that affect the survival time of amyotrophic lateral sclerosis (hereinafter sometimes referred to as ALS). In addition, since analysis of gene polymorphisms revealed that patients with short survival time have reduced expression of fibroblast growth factor 1 (FGF1), a neurotrophic factor, and thrombospondin type 1 domain containing 7A (THSD7A), a neuroangiogenic factor, in motor neurons, the present invention also provides a testing method, a testing kit, and a pharmaceutical screening method using FGF1 expression and / or THSD7A expression as an indicator. [Background technology]

[0002] ALS is a progressive neurodegenerative disease that causes severe muscle atrophy and muscle weakness, and is a type of motor neuron disease. ALS progresses extremely quickly, causing respiratory paralysis, swallowing disorders, and quadriplegia, and approximately one in five patients die or become respirator-dependent within three to five years of onset. A recent report from Japan suggests that approximately 2.2 people per 100,000 people develop the disease annually, with an estimated prevalence of 9.9 per 100,000 people. Although it is not a rare disease, no effective treatment for a cure has yet been established.

[0003] It is said that 5-10% of ALS cases are familial, and most are sporadic. Although new genes have been discovered one after another in recent years for familial ALS, which develops due to a mutation in a single gene, elucidation of the pathology and cause of sporadic ALS has been delayed (Non-Patent Document 1). Although some sporadic ALS cases have gene mutations found in familial ALS, it is believed that multiple factors are involved in most sporadic ALS cases, and the cause has not been clarified.

[0004] Genome-wide association studies (GWAS) using single nucleotide polymorphisms (hereinafter sometimes referred to as SNPs) as markers have been conducted to study genes involved in sporadic ALS (Non-Patent Document 2). However, the reported associated gene polymorphisms have low odds ratios of about 1.5, which are insufficient for constructing a disease model of sporadic ALS, and are also considered to lack reproducibility (Non-Patent Document 3).

[0005] In addition, many clinical trials have been attempted for the treatment of ALS, but there is still no effective treatment, and there is no satisfactory disease-modifying therapy that suppresses the progression. The average survival time of ALS patients is said to be 3 to 5 years after onset, but it has been reported that this varies greatly from individual to individual, ranging from patients who require a ventilator within about 6 months to patients who are able to walk without the need for a ventilator even 10 years after onset (Non-Patent Document 4). The diversity of disease progression makes it difficult to analyze the results of clinical trials for ALS, and is the reason why effective treatments cannot be found. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2014-526042 [Non-patent literature]

[0007] [Non-Patent Document 1] Finsters, J. & Burgunder, JM, 2014, Eur. J. Med. Genet .,Vol.57(2-3), pp.103-112 [Non-Patent Document 2] Renton, AE, et al., 2014, Nature Neuroscience, Vol.17 (1), pp.17-23 [Non-Patent Document 3] Iida, A., et al., 2011, Neurobiology of aging, Vol.32(4), 757e13-14 [Non-Patent Document 4] Chio A. et al., 2009, Amyotroph. Lateral Scler., Vol.10(5-6),pp.310-23 [Non-Patent Document 5] Atsuta, N., et al., 2011, Brain and Nerve=Shinkei kenky uno shinpo, Vol.63(5), pp.491-496 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide an indicator that is highly useful for predicting or estimating the prognosis of ALS patients and its use (e.g., a method for predicting risks related to survival time).Another objective of the present invention is to provide a means for screening drugs (ALS therapeutic drugs or candidates thereof) that are useful for improving the prognosis of ALS patients. [Means for solving the problem]

[0009] Under the above-mentioned problem, the present inventors performed detailed and comprehensive analysis. As a result, they succeeded in identifying seven SNPs that correlate with the survival time of ALS patients. Of the seven, four are gene polymorphisms of the FGF1 gene, and three are gene polymorphisms of the THSD7A gene. On the other hand, as a result of gene expression analysis in immortalized lymphocytes derived from ALS patients and in cells in which iPS cells created from patient lymphocytes were differentiated into motor neurons, it was found that for the SNPs successfully identified, gene expression (expression of the FGF1 gene, expression of the THSD7A gene) was reduced when the SNPs had effect alleles (i.e., alleles that bring about poor prognosis). According to this result, it can be said that increasing the expression of the FGF1 gene or the THSD7A gene is useful for improving the prognosis. Therefore, the effectiveness of a test substance in improving the prognosis of ALS patients can be evaluated using the increased expression of these genes as an index. This evaluation system is useful as a means for searching (screening) drug candidates that are effective in improving the prognosis of ALS patients. Based on the above results and considerations, the following invention is provided. [1] A testing method for predicting the survival time of an ALS patient, comprising: detecting at least one single nucleotide polymorphism among ALS patient reference SNP ID numbers: rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950, and rs2354951; and determining that there is a high risk of a shortened survival time when the detected single nucleotide polymorphism is an effect allele. [2] A testing method for predicting the survival time of an ALS patient, comprising measuring the expression levels of FGF1 and / or THSD7A in an ALS patient, and determining that there is a high risk of a shortened survival time if the expression levels are lower than a threshold value. [3] The method for testing described in [2], characterized in that the expression level is measured as an mRNA expression level and / or a protein expression level. [4] The testing method described in [3], wherein the mRNA expression level is measured by quantitative PCR. [5] The method according to [3], wherein the protein expression level is measured using an anti-FGF1 antibody and an anti-THSD7A antibody. [6] The method for testing described in any one of [1] to [5], characterized in that the test is performed using lymphocytes obtained from a patient. [7] A test kit for predicting the survival time of an ALS patient, comprising: A test kit comprising at least one of a PCR primer for detecting at least one single nucleotide polymorphism among reference SNP ID numbers: rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950, and rs2354951, a quantitative PCR primer for analyzing FGF1 expression levels, a quantitative PCR primer for analyzing THSD7A expression levels, an antibody for quantifying FGF1 expression levels, and an antibody for quantifying THSD7A expression levels. [8] A method for screening for a drug that extends the survival time of ALS patients, comprising contacting a test substance with cultured cells and selecting an effective substance using an increase in FGF1 expression level and / or THSD7A expression level as an indicator. [9] The screening method according to [8], wherein the reference SNP ID numbers rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950 and rs2354951 are effect alleles in the cultured cells.

[10] The screening method according to [8] or [9], wherein the cultured cells are a lymphocyte cell line established from peripheral blood of an ALS patient.

[11] The screening method according to [8] or [9], wherein the cultured cells are motor neurons derived from iPS cells established from cells of an ALS patient.

[12] The screening method according to any one of [8] to

[11] , wherein the expression levels of FGF1 and THSD7A are determined by measuring mRNA and / or protein. [Brief description of the drawings]

[0010] [Figure 1] A Manhattan plot showing the results of genome-wide association studies of sporadic ALS survival and SNPs. [Figure 2-1] A diagram showing linkage disequilibrium of SNPs that were found to be correlated with survival time in sporadic ALS. [Figure 2-2] Continued from Figure 2. [Figure 3-1] Survival curves (rs11738209) created by classifying the survival time of sporadic ALS patients by SNP. [Figure 3-2] Continued from Figure 3 (survival curves for rs2354952). [Figure 4] Figure showing gene expression levels of FGF1 (A, C) and THSD7A (B, D) in each genotype. Note that A and B are gene expression levels in peripheral blood-derived lymphocyte cell lines, and B and D are gene expression levels in iPS cell-derived motor neurons. [Diagram 5] A graph showing the in vitro survival time of iPS cell-derived motor neurons for each FGF1 (A) and THSD7A (B) genotype (FGF1 rs11738209 genotype, THSD7A rs2354952 genotype). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present inventors conducted a cohort study and performed genome-wide association analysis of the survival time of sporadic ALS using the Cox proportional hazards model, and found seven SNPs (rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950, rs2354951) that were significantly correlated with survival time. Furthermore, the analysis revealed that four of the seven SNPs (rs11738209, rs4912870, rs35215281, rs6878506) are in the FGF1 gene, and three (rs2354952, rs2354950, rs2354951) are in the THSD7A gene, and are in linkage disequilibrium. Therefore, by detecting any one or more of the SNPs of the FGF1 gene and the THSD7A gene, the risk of shortened survival time can be determined. In the present invention, the term "detecting a single nucleotide polymorphism (SNP)" can be replaced with the term "analyzing a single nucleotide polymorphism (SNP)". The detection of SNP reveals the state of the polymorphic position (i.e., the type of base).

[0012] Since linkage disequilibrium is shown, the risk of shortened survival time of sporadic ALS patients can be examined by analyzing any one or more of the seven SNPs and examining whether they are effect alleles or non-effect alleles. The effect allele (risk allele) of rs11738209 is A, the effect allele (risk allele) of rs4912870 is A, the effect allele (risk allele) of rs35215281 is C, the effect allele (risk allele) of rs6878506 is C, the effect allele (risk allele) of rs2354952 is G, the effect allele (risk allele) of rs2354950 is A, and the effect allele (risk allele) of rs2354951 is C. If an effect allele is detected, it is determined that there is a high risk of shortened survival time. As is clear from the criteria, the judgment here can be made automatically / mechanically without relying on the judgment of a doctor, technician, or other person with specialized knowledge.

[0013] As a method for detecting SNPs, known methods such as the Invader method, TaqMan PCR method, single base extension method, pyrosequencing method, and exonuclease cycling assay method can be used. Primers required for detecting the above seven SNPs can be prepared by known methods. Specifically, sequence information of a gene region containing the SNP to be detected is obtained from a database, and a PCR primer set is designed using the software Primer3, keeping in mind that mutations in a single base sequence can be analyzed.

[0014] The scope of application of the present invention is not limited to Japanese people. That is, the present invention can also be applied to Mongoloids other than Japanese people and other races (Caucasoids, etc.). However, considering the fact that the polymorphisms to be detected were identified by comprehensive analysis using a Japanese cohort and that the types and frequencies of polymorphisms often show similar trends in genetically similar populations (for example, Chinese and Korean populations are similar to Japanese populations), the subjects in the present invention are preferably Mongoloids (Japanese, Chinese, Korean, etc.), and more preferably Japanese people.

[0015] On the other hand, analysis using patient-derived lymphocyte cell lines revealed that effect alleles of four SNPs in the FGF1 gene led to decreased FGF1 gene expression, and effect alleles of three SNPs in the THSD7A gene led to decreased THSD7A gene expression.

[0016] Furthermore, when patient-derived iPS cells were differentiated into motor neurons, FGF1 gene expression was lower in motor neurons of patients with effect alleles of four SNPs in the FGF1 gene than in motor neurons obtained from patients with non-effect allele homozygotes, and THSD7A gene expression in motor neurons of patients with effect alleles of three SNPs in the THSD7A gene was significantly lower than in motor neurons obtained from patients with non-effect allele homozygotes. Therefore, in ALS patients, the risk of shortened survival can be determined by typing the seven SNPs or measuring the FGF1 expression level and / or THSD7A expression level. The FGF1 expression level and THSD7A expression level may be measured by measuring either mRNA or protein.

[0017] When measuring FGF1 mRNA and THSD7A mRNA, for example, quantitative PCR may be used. Quantitative PCR may be performed using known methods such as SYBR Green, TaqMan probe, and RT-PCR. In addition, FGF1 protein and THSD7A protein may be quantified using anti-FGF1 antibody and anti-THSD7A antibody by known methods such as ELISA, RIA, and Western blot. By measuring mRNA and protein, FGF1 expression and THSD7A expression can be quantified with high sensitivity.

[0018] If the expression levels of FGF1 and THSD7A are significantly lower than those of the control, it is possible to determine that there is a high risk of shortening survival time. As the control, a sample obtained from a healthy individual or a group in which the above seven SNPs are homozygous non-effect alleles can be used. The threshold value can be determined appropriately depending on the measurement method. The threshold value may be determined by a preliminary experiment using a large number of samples. For example, the average value of the control group may be used as the threshold value to determine the risk.

[0019] In the present invention, ALS patients are subjects. As samples from ALS patients, any sample, such as blood, tissue, etc., may be used as long as it contains cells, but peripheral blood is preferred because it places less physical strain on the patient. Nucleic acid samples used for detecting SNPs can be prepared from the subject's blood, saliva, lymph, urine, sweat, skin cells, mucosal cells, hair, etc. using known extraction and purification methods. Any genomic DNA of any length can be used as a nucleic acid sample as long as it contains the polymorphic site to be detected.

[0020] The present invention also provides a test kit for predicting the survival time of an ALS patient. The test kit includes at least one of the following reagents (1) to (5): (1) PCR primers to detect at least one of the seven SNPs listed above (2) Quantitative PCR primers for analyzing FGF1 expression levels (3) Quantitative PCR primers for analyzing THSD7A expression levels (4) Antibody for quantifying FGF1 expression level (anti-FGF1 antibody) (5) Antibody for quantifying THSD7A expression level (anti-THSD7A antibody)

[0021] The present inventors have shown that the survival time of sporadic ALS correlates with the gene polymorphism and expression level of FGF1 and THSD7A. Therefore, it is possible to screen for therapeutic drugs that extend the survival time of ALS using the expression of FGF1 and / or THSD7A as an index. In addition, since FGF1 is a trophic factor in the nervous system and THSD7A is involved in angiogenesis of motor nerves, the medicine obtained by screening may be applicable not only to sporadic ALS but also to familial ALS and other neurodegenerative diseases that cause muscle atrophy.

[0022] As indicators of FGF1 expression and THSD7A expression, mRNA and protein can be measured by the known methods described above, and a substance that increases their expression can be selected, thereby screening for drugs that extend the survival time of ALS patients.

[0023] Specifically, a library compound is added to cells with low FGF1 and / or THSD7A expression levels, and the changes in FGF1 and / or THSD7A expression levels before and after the addition of the compound are analyzed. Compounds that can increase FGF1 and / or THSD7A expression levels can be candidates for medicines that extend the survival time of ALS. Any cells with low FGF1 and / or THSD7A expression levels may be used, but since cell lines with genotypes in which the SNPs have effect alleles have low FGF1 and / or THSD7A expression levels, these cell lines may be used, and the library compound may be added to the culture solution to analyze the FGF1 and THSD7A expression levels.

[0024] As a specific cell line, it is preferable to use a lymphocyte cell line established from a sporadic ALS patient, in which the above seven SNPs have effect alleles. Among the established cell lines, by using a cell line with particularly low FGF1 expression and / or THSD7A expression, it is possible to capture the increase in FGF1 expression and / or THSD7A expression with high sensitivity. In addition, iPS cells established from cells of a sporadic ALS patient (for example, those established from the patient's immortalized lymphocytes (LCL)) can be used. The present inventors have already established iPS cells derived from 62 cases of patients and induced differentiation into motor neurons. Using these lymphocyte cell lines and iPS cells derived from patient cells with specific genotypes, it is possible to screen for drugs that increase the expression levels of FGF1 and THSD7A and verify their effects.

[0025] The substance to be screened (test substance) may be any substance that is a candidate for a drug, such as a low molecular weight compound, a natural product, or an antibody. Existing drugs (for example, a library of drugs approved by the U.S. Food and Drug Administration (FDA)) may be used as the test substance. In addition, various compound libraries (for example, Ligand Box) are provided (for example, available from Asinex Co., Ltd. and Namiki Shoji Co., Ltd.), and such compound libraries may be used. The test substance may be derived from a natural product or may be synthetic. In the latter case, an efficient screening system can be constructed by using, for example, a combinatorial synthesis technique. In addition, two or more types of test substances may be added simultaneously to examine the interaction, synergistic effect, etc. between the test substances. In addition, the expression levels of FGF1 and THSD7A can be measured by quantifying mRNA or protein. EXAMPLES

[0026] In the present invention, in order to search for gene mutations related to the survival time of sporadic ALS, genome-wide association analysis was performed on sporadic ALS patients. As a result, we succeeded in finding gene polymorphisms related to the survival time of sporadic ALS, and confirmed the effect of the polymorphisms by functional analysis using lymphocytes derived from patients and motor neurons differentiated from iPS cells. The present invention will be described in detail below with reference to the data.

[0027] <Multicenter ALS Patient Cohort Study (JaCALS)> ALS is a typical neurological disease with no curative treatment. Clarifying its pathology and developing a treatment are urgent issues, and a multi-center ALS patient cohort has been established, with 32 facilities nationwide participating. As of November 2019, 1,766 ALS patients and 451 control cases have been registered, and research and studies are being conducted with the aim of clarifying the pathology, developing methods to suppress and prevent the pathology, and improving medical care and patient support. In the multi-center ALS patient cohort study (Japanese Consortium for ALS research, hereinafter referred to as JaCALS), we grasp the prospective clinical profile of registered ALS patients, and accumulate biological resources such as patient genes and immortalized cells, and are proceeding with research to clarify the pathology and develop therapeutic drugs.

[0028] At the 32 facilities participating in JaCALS, ALS patients diagnosed according to the revised El Escorial diagnostic criteria are registered in an anonymized database with their consent. Registered patients are assigned a unique patient number, and clinical data is kept anonymous at each participating facility. The data is sent to the Clinical Data Center at the Nagoya University School of Medicine and stored in the JaCALS database. When ALS patients are registered, they are examined by a neurologist at each medical facility and their blood is drawn, and their DNA and lymphocytes immortalized by the EB virus are stored.

[0029] The time of onset of the disease is defined as the time when each patient first noticed symptoms such as muscle weakness, swallowing disorders, speech disorders, and breathing difficulties. The progression of the disease in registered patients will be assessed every three months through telephone surveys by clinical research coordinators and diagnosis by a doctor. The ALS Functional Rating Scale-Revised (hereinafter sometimes referred to as ALSFRS-R), which is used to evaluate the overall severity and progression of the disease in ALS patients, is used to grasp the patient's condition and the degree of progression.

[0030] The ALSFRS-R is an assessment scale for understanding the daily life of ALS patients, and evaluates the function of ALS patients by rating 12 items, such as speech, swallowing, daily activities, and breathing, on a 5-point scale from 4 to 0. The lower the score, the more advanced the disease and the more impaired the function.

[0031] The fact that accumulating ALSFRS-R data by telephone surveys is a reliable method has already been confirmed by the high correlation (intraclass correlation coefficient 0.97, 95% CI 0.94-0.98) between the ALSFRS-R and a physician's examination (Non-Patent Document 5). In addition, in order to standardize the survey methods at each facility participating in the study, three physicians visited each facility to confirm the evaluation methods.

[0032] The endpoints were the introduction of tracheostomy positive pressure ventilation (TPPV) or patient death, and patients who survived without the introduction of TPPV were defined as "TPPV-free survival."

[0033] <Selection of sporadic ALS patients> One thousand two hundred seventy-two patients with sporadic ALS were genotyped at the RIKEN Institute using the HumanOmniExpressExome BeadChip array (Illumina). Four patients showed discrepancies between genetic sex and database sex. Whole-genome pedigree identity analysis was performed with PLINK 1.9 software, and seven sample pairs with sample duplication or consanguinity (pi-hat > ​​0.1875) were detected, and one sample from each pair was excluded. Principal component analysis (PCA) using the 1000 Genomes Project reference panel (Phase 3) detected seven individuals who appeared to be of a non-Japanese race. These seven individuals were also excluded from the analysis. In addition, PCA based only on samples was performed to identify population outliers. Based on the first 10 principal components, 20 population outliers were identified and excluded from further analysis. Non-chromosomal SNPs and SNPs with a genotype call rate < 0.98 or a Hardy-Weinberg equilibrium exact test p-value < 1 × 10 in control subjects were excluded. -6 After excluding SNPs with minor allele frequency (MAF) <0.01 or deviating from allele frequencies calculated from the 1000 Genomes Project Phase 3 East Asian ethnic samples, 1009 patients enrolled in JaCALS and 232 patients enrolled in other cohorts and 546,755 SNPs were selected.

[0034] Based on the 1000 Genomes Project reference panel (phase 3), we generated a whole-genome reference panel using SHAPEIT2 and Minimac3 software. We excluded variants with r2 < 0.5 and MAF < 0.01, resulting in 8,128,443 variants in JaCALS and 8,102,791 variants in the other cohorts. We excluded cases with insufficient survival or covariate data. Finally, a total of 887 patients enrolled in JaCALS and 189 patients enrolled in the other cohorts were included in the analysis.

[0035] Associations between TPPV-free survival and SNPs were analyzed by Cox proportional hazards regression analysis using ProbABEL. Independent variables included SNPs and imputed dose genotypes as covariates. Covariates included age at onset, sex, use of riluzole, and two principal components calculated from genotype data. Survival curves for SNP genotypes that reached genome-wide significance were estimated based on the Kaplan-Meier method and compared using the log-rank test.

[0036] [Table 1]

[0037] <Example 1> Meta-analysis of the association of SNPs with TPPV-free survival was performed using the inverse variance weighting method with METAL software. Genetic heterogeneity was assessed by Cochran's Q test. The meta-analysis included 7,908,526 SNPs and the results of both studies. p-value <5 × 10 -8 was set as the genome-wide significance level.

[0038] A bioinformatics approach was undertaken to prioritize associated SNPs at novel loci: ANNOVAR was used to add functional annotation, including the genetic location of sentinel SNPs.

[0039] Immortalized lymphocytes were generated from peripheral blood B cells of ALS patients using standard Epstein-Barr virus (EBV) transformation techniques. Recently, we successfully generated iPSC models from LCLs of 32 sporadic ALS patients from JaCALS and differentiated them into motor neurons. We analyzed the in vitro survival of iPSC-derived motor neurons from 32 ALS patients. The in vitro survival of iPSC-derived motor neurons from 32 patients was compared for each genotype of SNPs that reached genome-wide significance levels. Kaplan-Meier curves for each genotype were compared using the log-rank test.

[0040] Quantitative PCR RNA was extracted from immortalized lymphocytes and iPSC-derived motor neurons using the PureLink RNA Mini Kit (Thermo Fisher Scientific). Total RNA was transcribed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and the CFX96 system (BioRad, Hercules) according to the manufacturer's instructions. The reaction conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The expression levels of β2-microglobulin or GAPDH were quantified simultaneously as standard controls.

[0041] <Example 2: Identification of SNPs associated with ALS survival time> Genes associated with survival in sporadic ALS were analyzed using the Cox proportional hazards model.

[0042] Among the 1,076 patients analyzed, an additive model was used to analyze whether there were any SNPs associated with survival time.

[0043] The genome-wide significance level was 5.0 × 10 -8 This value was used as the threshold. In the correlation model, seven SNPs in Table 2 that were associated with survival time were identified (p = 4.85 × 10 -9 ~4.60×10 -8 ).

[0044] [Table 2] In Table 2, Chr stands for chromosome.

[0045] The results of the association analysis are shown in Figure 1. Figure 1 is a Manhattan plot in which the smallest p-value calculated by the additive model for each SNP is plotted on the vertical axis and the position on the chromosome is plotted on the horizontal axis.

[0046] Figure 2 shows that each gene polymorphism is in linkage disequilibrium with the surrounding SNPs.

[0047] Figure 3 shows how each gene polymorphism (rs11738209, rs2354952) actually affects survival time.

[0048] <Example 3> To confirm that the SNPs of FGF1 and THSD7A affect expression in ALS patients, we measured FGF1 expression in lymphocytes and motor neurons differentiated from iPS cells for rs11738209 and THSD7A expression in lymphocytes and motor neurons differentiated from iPS cells for rs2354952.

[0049] Immortalized cell lines (immortalized lymphocytes) were established by infecting peripheral blood B lymphocytes obtained from all ALS patients at the time of registration with EB virus and stored in a liquid nitrogen tank. For rs11738209, which is thought to be involved in ALS survival, 20 samples of immortalized lymphocytes from patients with heterozygosity for the effect allele, 20 samples of non-effect allele homozygosity, 2 samples of iPS cell-derived motor neurons with heterozygosity for the effect allele, and 18 samples of non-effect allele homozygosity were subjected to FGF1 gene expression analysis. For rs2354952, 20 samples of immortalized lymphocytes from patients with homozygosity for the effect allele, 20 samples of non-effect allele homozygosity, and 7 samples of homozygosity for the effect allele and 8 samples of homozygosity for the non-effect allele were subjected to THSD7A gene expression analysis (Figure 4).

[0050] The expression levels of FGF1 mRNA and THSD7A mRNA were measured by quantitative RT-PCR as follows. RNA was extracted from immortalized lymphocytes and iPS cell-derived motor neurons using the PureLink RNA Mini Kit (Thermo Fisher Scientific). Total RNA was transcribed using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and the CFX96 system (BioRad, Hercules) according to the manufacturer's instructions. The reaction conditions were 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The expression levels of β2 microglobulin or GAPDH were quantified simultaneously as standard controls.

[0051] As shown in Figure 4, the expression levels of the FGF1 gene and the THSD7A gene were significantly lower in lymphocytes of patients with an effect allele than in patients with a homozygous non-effect allele genotype (Figure 4A, B). The expression level of the THSD7A gene was significantly lower in motor neurons of patients with a heterozygous effect allele than in patients with a homozygous non-effect allele genotype (Figure 4D). The expression level of the FGF1 gene tended to be lower in motor neurons of patients with a heterozygous effect allele than in patients with a homozygous non-effect allele genotype, although the number of cases was small and there was no significant difference (Figure 4C). From these results, the risk of shortened survival can be evaluated by analyzing the expression levels of FGF1 and THSD7A using peripheral blood and iPS cell-derived motor neurons of ALS patients.

[0052] As shown in Figure 5, the in vitro survival time of iPS cell-derived motor neurons from patients with an effect allele genotype of the THSD7A gene was significantly shorter than that of patients with a homozygous non-effect allele genotype (Figure 5B). The in vitro survival time of iPS cell-derived motor neurons from patients with an effect allele genotype of the FGF1 gene tended to be shorter than that of patients with a homozygous non-effect allele genotype, although the number of cases was small and there was no significant difference (Figure 5A). These results suggest that the risk of shortened survival time can be evaluated by analyzing the in vitro survival time of iPS cell-derived motor neurons from ALS patients. [Industrial Applicability]

[0053] Not only will this provide a basis for elucidating the pathology of sporadic ALS, but it will also be possible to develop therapeutic drugs that extend the survival time of ALS patients, whose survival time is short, by using FGF1 and THSD7A expression as indicators. In addition, the SNPs discovered this time can be used as allocation factors when conducting clinical trials, making it possible to more accurately evaluate investigational drugs. In addition, since it is possible to predict the course of the disease and the underlying pathology, it will be possible to provide more appropriate care when creating nursing care and care support plans.

[0054] The present invention is not limited to the above-mentioned embodiment and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent publications, patent publications, etc. specified in this specification are all incorporated by reference.

Claims

1. A test method for predicting the survival time of an ALS patient, comprising: detecting at least one single nucleotide polymorphism among reference SNP ID numbers of ALS patients: rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950, and rs2354951; and determining that there is a high risk of a shortened survival time when the detected single nucleotide polymorphism is an effect allele; The effect allele of rs11738209 is A, the effect allele of rs4912870 is A, the effect allele of rs35215281 is C, the effect allele of rs6878506 is C, the effect allele of rs2354952 is G, the effect allele of rs2354950 is A, and the effect allele of rs2354951 is C. Testing method.

2. A testing method for predicting the survival time of an ALS patient, characterized in that the expression levels of FGF1 and / or THSD7A in an ALS patient are measured, and if the expression levels are lower than a predetermined threshold, it is determined that there is a high risk of a shortened survival time.

3. The method for testing according to claim 2 , wherein the expression level is determined by measuring an mRNA expression level and / or a protein expression level.

4. The method for testing according to claim 3 , wherein the mRNA expression level is measured by quantitative PCR.

5. The method according to claim 3 , wherein the protein expression level is measured using an anti-FGF1 antibody and an anti-THSD7A antibody.

6. The method according to any one of claims 1 to 5, wherein the test is performed using lymphocytes obtained from a patient.

7. A test kit for use in the test method according to any one of claims 1 to 6, comprising: A test kit comprising at least one of a PCR primer for detecting at least one single nucleotide polymorphism among reference SNP ID numbers: rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950, and rs2354951, a quantitative PCR primer for analyzing FGF1 expression levels, a quantitative PCR primer for analyzing THSD7A expression levels, an antibody for quantifying FGF1 expression levels, and an antibody for quantifying THSD7A expression levels.

8. A method for screening for pharmaceuticals that extend the survival time of ALS patients, comprising contacting a test substance with cultured cells and selecting a test substance that increases the expression level of FGF1 and / or THSD7A as an effective substance.

9. The screening method according to claim 8, wherein the reference SNP ID numbers rs11738209, rs4912870, rs35215281, rs6878506, rs2354952, rs2354950 and rs2354951 are effect alleles for the cultured cells.

10. The method of claim 8 or 9, wherein the cultured cells are a lymphocyte cell line established from peripheral blood of an ALS patient.

11. The method of claim 8 or 9, wherein the cultured cells are motor neurons derived from iPS cells established from cells of an ALS patient.

12. The screening method according to any one of claims 8 to 11, wherein the expression levels of FGF1 and THSD7A are determined by measuring mRNA and / or protein.

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