JP7088519B2 - How to test for scoliosis - Google Patents

Description

The present invention relates to a test method for determining the risk of developing scoliosis and the risk of progression, and a reagent used in the test method. It also relates to a method for screening a prophylactic or therapeutic drug for scoliosis.

Scoliosis is a condition in which the spine is curved in the left-right direction. Among scoliosis, those whose etiology is not clear are called idiopathic scoliosis, which accounts for 80 to 90% of scoliosis.
Scoliosis often occurs in children of school age, and in children from the age of 10 to the maturity of the skeleton, it shows a curve of at least 10 ° at the Cobb angle, which is an index of scoliosis, and its etiology. Is defined as Adolescent idiopathic scoliosis (AIS). The incidence of AIS is 2% in Japan and 2 to 3% in the world among children of school age, and about 10,000 people in Japan develop AIS every year.

The diagnosis of scoliosis is mainly performed by X-ray examination, but the X-ray examination is not effective for the diagnosis of scoliosis before the onset or in the early stage of the onset. Moreover, the treatment of idiopathic scoliosis is performed only by symptomatic treatment, and as long as the etiology is not clear, no causative treatment is performed. Therefore, in order to enable presymptomatic diagnosis (risk diagnosis) and early diagnosis of scoliosis, and to enable treatment of the cause, identification of genes and single nucleotide polymorphisms (SNPs) related to scoliosis is desired. ..

Many loci that contribute to the pathogenesis of AIS have been found by genome-wide linkage analysis, and AIS is thought to be based on a complex genetic predisposition. In addition, AIS susceptibility genes have been reported by candidate gene analysis, but there are problems in terms of reproducibility and the like.
Furthermore, in recent years, a candidate for a gene related to AIS has been reported by a genome-wide association study (GWAS) based on the Transmission Disequilibrium Test (TDT) method (Non-Patent Document 1). .. However, they have not been reproducible in relation to AIS in case-control association studies after multiple test correction.

On the other hand, in recent years, GWAS has found that SNPs present in the 24.31 region (10q24.31 region) of the long arm of chromosome 10 satisfy genome-wide associations in the Japanese and Chinese populations and are associated with AIS. (Patent Document 1). Similarly, GWAS suggested that SNPs present in the long arm 24.1 region (6q24.1 region) of chromosome 6 are associated with AIS in the Japanese population (Patent Document 2).
In addition, the relationship between SNP of the BNC2 (basonuclin-2) gene present in the short arm 22.2 region (9p22.2 region) of chromosome 9 and scoliosis has also been reported (Patent Document 3). However, little is known about female-specific SNPs and SNPs associated with the progression of scoliosis.

Japanese Unexamined Patent Publication No. 2013-042673 Japanese Unexamined Patent Publication No. 2014-132875 Japanese Unexamined Patent Publication No. 2016-214091

Sharma, S. et al., Hum Mol Genet. 20, 1456-1466 (2011).

An object of the present invention is to provide a method for accurately inspecting the risk of developing scoliosis and the risk of progression, and a test reagent used in the method. It is also an object of the present invention to provide a method for screening a prophylactic or therapeutic agent for scoliosis.

As a result of diligent studies to solve the above problems, the present inventors have found that single nucleotide polymorphisms (SNPs) selected from the group consisting of rs59404763, rs10196262, rs1475712 and rs1828853 are associated with adolescent idiopathic scoliosis (AIS). Identified to do. Then, by investigating these polymorphisms, it was found that the risk of developing and progressing scoliosis can be accurately predicted. Furthermore, they have found that a substance that increases the expression of the Obscurin (OBSCN) gene and a substance that suppresses the expression of the EML4 gene are useful as a prophylactic or therapeutic agent for scoliosis, and have completed the present invention.

That is, the present invention includes the following aspects.
[1] Single nucleotide polymorphisms selected from the group consisting of rs59404763, rs10196262, rs1475712 and rs1828853, or single nucleotide polymorphisms that are in linkage disequilibrium with the single nucleotide polymorphisms are analyzed, and scoliosis is detected based on the analysis results. A method for testing scoliosis, which is characterized by testing.
[2] The method according to [1], wherein the scoliosis is idiopathic scoliosis in adolescence.
[3] The method according to [1], wherein the scoliosis is scoliosis in a woman, and the single nucleotide polymorphism is rs59404763, rs10196262 or rs1475712.
[4] The method according to [1], wherein the scoliosis test is a test for the degree of scoliosis progression, and the single nucleotide polymorphism is rs1828853.
[5] A probe for scoliosis examination having a sequence of 15 or more bases including the 26th base of the base number in the base sequence shown in any of SEQ ID NOs: 1 to 9 or a complementary sequence thereof.
[6] A primer for scoliosis examination capable of amplifying a region containing the 26th base in the base sequence shown in any of SEQ ID NOs: 1 to 9.
[7] A step of adding a drug candidate to cells expressing a reporter gene linked to the Obscurin gene or the promoter of the Obscurin gene, a step of measuring the expression level of the Obscurin gene or the reporter gene, and a substance for increasing the expression level. A method of screening for a prophylactic or therapeutic agent for scoliosis, including the step of selecting.
[8] A method for examining scoliosis, which comprises a step of measuring the expression level of mRNA or protein of the Obscurin gene.
[9] A step of adding a drug candidate substance to a cell expressing a reporter gene linked to the EML4 gene or the promoter of the EML4 gene, a step of measuring the expression level of the EML4 gene or the reporter gene, and a substance for reducing the expression level. A method of screening for a prophylactic or therapeutic agent for scoliosis, including the step of selecting.
[10] A method for examining scoliosis, which comprises a step of measuring the expression level of mRNA or protein of the EML4 gene.

According to the present invention, it is possible to accurately and easily predict the risk of developing scoliosis and the risk of progression of scoliosis, which has been difficult to predict. In particular, by examining single nucleotide polymorphisms of rs59404763, rs10196262, and rs1475712, the risk of developing scoliosis in women can be investigated. In addition, by examining the single nucleotide polymorphism of rs1828853, the risk of progression of scoliosis can be investigated. Further, according to the screening method of the present invention, a therapeutic agent or a preventive agent for scoliosis can be obtained. Therefore, the present invention contributes to the prevention and early treatment of scoliosis.

Figure showing intra-regional association plots of two sites associated with female AIS ((a) 1q42.13. (B) 2p21.). The value of -log10 (P) showing the association between AIS and SNP by logistic regression analysis is plotted at the position on the chromosome where SNP is present, along with the orientation and position of the gene. The estimated recombination rate is shown by the red line. The SNPs with the highest association signals (rs59404763, rs10196262) are indicated by red triangles, and the other SNPs are colored according to linkage disequilibrium (r ² ) with the SNP. The figure which shows the result of having investigated the influence on the transcriptional activity of the allele of rs58194408. (a) is the result of the electrophoretic mobility shift assay using the nuclear extract of HEK293 cells (photo). An unlabeled probe was used in the competitive assay. The band intensity of the complex (arrow) was lower in the rs58194408 risk allele probe than in the rs58194408 non-risk allele probe (NR). (b) shows the evaluation results of the transcriptional activation ability of the rs58194408 allele in HEK293 cells. Cells were transfected with a plasmid containing the luciferase gene linked to the OBSCN promoter containing the risk allele (R) or non-risk allele (NR) of rs58194408 and examined for luciferase activity. The relative activity of luciferase is shown by the mean ± S.D. of the two experiments. * Indicates P <0.01 (Student's t test). The figure which shows the result of having investigated the influence on the transcriptional activity of the allele of rs144297067. (a) is the result of an electrophoretic mobility shift assay using a nuclear extract of A172 cells (photo). An unlabeled probe was used in the competitive assay. The band intensity of the complex (arrow) was higher in the rs144297067 risk allele probe than in the rs144297067 non-risk allele probe (NR). (b) shows the evaluation results of the transcriptional activation ability of the rs144297067 allele in A172 cells. Cells were transformed with a plasmid containing the luciferase gene linked to the EML4 promoter containing rs144297067 risk allele (R) or non-risk allele (NR) and examined for luciferase activity. The relative activity of luciferase is shown by the mean ± S.D. of the two experiments. * Indicates P <0.01 (Student's t test). Diagram showing the topologically related domains (TADs) of rs10196262 and rs1475712. The TAD of rs10196262 in (a) contains the three genes LOC102723824, EML4 and COX7A2L, and the TAD of rs1475712 in (b) contains EYA2. The figure which shows the analysis result of SNP which is related to the progress of AIS. a) shows a Manhattan plot showing the P value of GWAS. The P-value of each SNP was plotted against its position on the chromosome. The blue line represents the threshold value (P = 1.0 × ^10-5 ) for verification analysis. b) shows an association plot for the sitting position of 11q. SNPs were plotted by position on the chromosome (x-axis) and GWAS association (-log ₁₀ P-value; y-axis). The degree of linkage disequilibrium (r ² ) of the SNP with the SNP (rs1828853: purple circle) having the strongest associated signal is indicated by color. c) shows a local association plot around rs1828853. A significantly related SNP is present in Intron 1 of MIR4300HG. The SNPs indicated by the black circles overlap with the enhancer sequences epigenomically annotated with Chrom HMM.

<1> Inspection method of the present invention The method of the present invention analyzes a single nucleotide polymorphism (SNP) selected from rs59404763, rs10196262, rs1475712 and rs1828853 or SNP that is in linkage disequilibrium with the single nucleotide polymorphism, and the present invention is described. It is characterized by examining scoliosis based on the analysis result. In the present invention, the "test" includes a test for the risk of developing scoliosis, a test for the presence or absence of the onset of scoliosis, and a test and prediction of the degree of progression of scoliosis. In the method of the present invention, the analysis result of SNP is associated with the risk of developing scoliosis, the presence or absence of onset, and the risk of progression.

The scoliosis is not particularly limited, but is suitably used, for example, for the examination of idiopathic scoliosis for which the etiology has not been conventionally specified. Scoliosis may occur at any time such as congenital, juvenile, adolescent, adult, etc., but adolescent scoliosis is preferable, and adolescent idiopathic scoliosis (adolescent idiopathic scoliosis) AIS) is more preferred.

The method of the present invention can be used for subjects of any race. The method of the present invention can be suitably used for, for example, Asian subjects such as Japanese and Chinese, and Caucasian subjects. In addition, the method of the present invention can be used for subjects of any gender, and can be particularly preferably used for female subjects.

SNPs useful for testing female scoliosis include rs59404763, rs10196262 and rs1475712. Here, the rs number indicates the registration number of the dbSNP database (http //www.ncbi.nlm.nih.gov/projects/SNP/) of the National Center for Biotechnology Information.

rs59404763 is an SNP located in the linkage disequilibrium block containing the OBSCN gene in the 1q42.13 region, meaning a cytosine (C) / thymine (T) polymorphism at base 228511629 of GenBank Accession No. NC_000001.10. However, if this base is C, there is a high possibility or risk of developing scoliosis in women (Table 2). In addition, when analyzed in consideration of genotype, the possibility of scoliosis or the risk of developing scoliosis is high in the order of CC> TC> TT. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is C.

For rs59404763, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 1. If the 26th base in SEQ ID NO: 1 has a polymorphism and this base is T (risk allele), it is determined that the possibility of scoliosis or the risk of developing scoliosis is high.

In the present invention, the base corresponding to the above base is analyzed. The "base corresponding to the above base" means the corresponding base in the above region on the human chromosome. That is, "analyzing a base corresponding to the above base" includes analyzing the corresponding base in the above region even if the above sequence changes slightly at a position other than SNP due to a difference in race or the like. ..

Further, the base to be analyzed in the present invention is not limited to the above-mentioned ones, and polymorphisms of bases having a linkage disequilibrium with the above-mentioned bases may be analyzed. Here, the "base in linkage disequilibrium with the above base" means a base satisfying the relationship of preferably r ² > 0.8, more preferably r ² > 0.9 with the above base. r ² is the linkage disequilibrium coefficient. Bases in linkage disequilibrium can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja).

Specific examples of the base having a linkage disequilibrium with rs59404763 include, for example, rs58194408 (Table 3).
rs58194408 means a polymorphism of guanine (G) / adenine (A) at the 228401707th base of GenBank Accession No. NC_000001.10, and if this base is G, the possibility of scoliosis or the risk of developing scoliosis is high ( Table 3). In addition, when analyzed in consideration of genotype, the possibility of scoliosis or the risk of developing scoliosis is high in the order of GG>AG> AA. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is G.
For rs58194408, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 2. If the 26th base in SEQ ID NO: 2 has a polymorphism and this base is A (risk allele), it is determined that the possibility of scoliosis or the risk of developing scoliosis is high.
Other bases that are in linkage disequilibrium with rs59404763 include rs117147433 and rs75785474.

rs10196262 is an SNP located near the LOC102723824 gene in the human chromosome 2p21 region, and is guanine (G) at the 42362087th base of GenBank Accession No. NC_000002.11.
/ Means a polymorphism of adenine (A), and if this base is A, the possibility of scoliosis or the risk of developing it is high (Table 2). In addition, when analyzed in consideration of genotype, the possibility of scoliosis or the risk of developing scoliosis is high in the order of AA>AG> GG. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is A.

For rs10196262, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 3. If the 26th base in SEQ ID NO: 3 has a polymorphism and this base is A (risk allele), it is determined that the possibility of scoliosis or the risk of developing scoliosis is high.

Specific examples of the base having a linkage disequilibrium with rs10196262 include, for example, rs144297067 (Table 3).
rs144297067 means a polymorphism of- (no base) / TGTTTTGTTT (insertion) at base 42362286 of GenBank Accession No. NC_000002.11, and if TGTTTTGTTT is inserted in this base, the possibility or onset of scoliosis The risk is high (Table 3). In addition, when analyzed in consideration of genotype, the possibility of scoliosis or the risk of developing kyphosis is high in the order of homozygous insertion mutation> heterozygous without insertion and homozygous without insertion. Therefore, it is conceivable to prevent or treat scoliosis in subjects whose base is an insertion mutagenesis.
For rs144297067, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 4. If the 26th base in SEQ ID NO: 4 has a polymorphism and there is an insertion (risk allele) of TGTTTTGTTT here, it is judged that the possibility of scoliosis or the risk of developing scoliosis is high.
Other bases that are in linkage disequilibrium with rs10196262 include rs11676073, rs13392988, rs13386561, rs11684533, rs17669917, and rs13410780.

rs1475712 is an SNP located in the intergenic region between SLC2A10 and EYA2 in the human chromosome 20q13.12 region, and is a polymorphism of adenine (A) / guanine (G) at the 45492024th base of GenBank Accession No. NC_ 000020.10. If this base is A, the possibility of scoliosis or the risk of developing it is high (Table 2). In addition, when analyzed in consideration of genotype, the possibility of scoliosis or the risk of developing scoliosis is high in the order of AA> GA> GG. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is A.

For rs1475712, a sequence (complementary strand) having a total length of 51 bp including the SNP base and a region of 25 bp before and after it is shown in SEQ ID NO: 5. If the 26th base in SEQ ID NO: 5 has a polymorphism and this base is T (a complementary base of risk allele A of rs1475712), it is determined that the possibility of scoliosis or the risk of developing scoliosis is high.
Examples of bases that are in linkage disequilibrium with rs1475712 include rs144779633.

On the other hand, rs1828853 is mentioned as an SNP related to the degree of progression of scoliosis.
rs1828853 is an SNP located in the MIR4300HG gene in the 11q14.1 region, which means a guanine (G) / adenine (A) polymorphism at the 82371520th base of GenBank Accession No. NC_000011.10. If this is the case, the risk of developing and progressing scoliosis is high (Table 4). In addition, when analyzed in consideration of genotype, the risk of developing scoliosis and the risk of progression are higher in the order of AA>AG> GG. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is A.

For rs1828853, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 6. When the 26th base in SEQ ID NO: 6 has a polymorphism and this base is A (risk allele), it is determined that the risk of developing scoliosis and the risk of progression are high.

Specific examples of bases that are in linkage disequilibrium with rs1828853 include rs971548, rs35333564 and rs7949305.

rs971548 means a guanine (G) / thymine (T) polymorphism at the 82379683th base of GenBank Accession No. NC_ 000011.10. When this base is T, the risk of developing and progressing scoliosis is high. In addition, when analyzed in consideration of genotype, the risk of developing scoliosis and the risk of progression are higher in the order of TT>TG> GG. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is T.
For rs971548, a sequence (complementary strand) having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 7. When the 26th base in SEQ ID NO: 7 has a polymorphism and this base is A (a complementary base of the risk allele of rs971548), it is determined that the risk of developing scoliosis and the risk of progression are high.

rs35333564 means a polymorphism of- (single nucleotide deletion) / guanine (G) at the 82398145th base of GenBank Accession No. NC_000011.10. When this base is G, the risk of developing and progressing scoliosis Is high. In addition, when analyzed in consideration of genotype, the risk of developing scoliosis and the risk of progression are higher in the order of->G-> GG. Therefore, it is conceivable to prevent or treat scoliosis in subjects whose base is-.
For rs35333564, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 8. When the 26th base in SEQ ID NO: 8 has a polymorphism and this base is- (risk allele), it is determined that the risk of developing scoliosis and the risk of progression are high.

rs7949305 means a polymorphism of thymine (T) / cytosine (C) at the 82399142th base of GenBank Accession No. NC_000011.10, and when this base is C, the risk of developing scoliosis and the risk of progression are high. In addition, when analyzed in consideration of genotype, the risk of developing scoliosis and the risk of progression are higher in the order of CC>CT> TT. Therefore, it is conceivable to prevent or treat scoliosis in a subject whose base is C.
For rs7949305, a sequence having a total length of 51 bp including the SNP base and a region 25 bp before and after the SNP base is shown in SEQ ID NO: 9. When the 26th base in SEQ ID NO: 9 has a polymorphism and this base is C (risk allele), it is determined that the risk of developing scoliosis and the risk of progression are high.

Scoliosis can be examined by examining the types of SNP bases and associating the obtained results with scoliosis based on the criteria as described above. The SNP may be analyzed alone, or a plurality of SNPs including at least one of the SNPs may be collectively analyzed (haplotype analysis). For example, a plurality of the above SNPs may be analyzed collectively, or at least one of the above SNPs may be analyzed in combination with a known SNP associated with scoliosis or SNPs in linkage disequilibrium with the known SNP. May be good. Collective analysis of multiple SNPs associated with scoliosis improves the accuracy of scoliosis testing. For any SNP, either strand of double-stranded DNA may be analyzed. For example, each sequence may be analyzed for the sense strand or the antisense strand.

The sample used for SNP analysis is not particularly limited as long as it is a sample containing chromosomal DNA. Examples of the sample used for the analysis of SNP include body fluids such as blood, urine and cerebrospinal fluid, cells such as the cervix and oral mucosa, and body hair such as hair. Although these samples can be used directly for SNP analysis, it is preferable to isolate chromosomal DNA from these samples by a conventional method and use it for analysis.

The analysis of SNP can be performed by a usual gene polymorphism analysis method. For example, sequence analysis, PCR, hybridization, invader method and the like can be mentioned, but the present invention is not limited thereto.

Sequence analysis can be performed by a conventional method. Specifically, a sequence reaction is performed using a primer set at a position of several tens of bases on the 5'side of a base showing a polymorphism, and from the analysis result, it is determined which kind of base the corresponding position is. can do. Before the sequence reaction, it is preferable to amplify the fragment containing the SNP site by PCR or the like in advance.

In addition, SNP analysis can be performed by examining the presence or absence of amplification by PCR. For example, a primer having a sequence corresponding to a region containing a base indicating a polymorphism and having a 3'end corresponding to each polymorphism is prepared. PCR can be performed using each primer to determine which type of polymorphism is present depending on the presence or absence of amplification products. In addition, the LAMP method (Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Patent No. 2843586), ICAN method (Japanese Patent Laid-Open No. 2002-233379), SmartAmp method (Patent No. 3897805). It is also possible to check the presence or absence of amplification by means of a book). In addition, a single chain amplification method may be used.

It is also possible to amplify DNA fragments containing SNP sites and determine which type of polymorphism is due to the difference in mobility of the amplified product in electrophoresis. Such a method includes, for example, the PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1;
12 (1): 139-146.). Specifically, first, the DNA containing the target SNP is amplified, and the amplified DNA is dissociated into a single-stranded DNA. The dissociated single-stranded DNA can then be separated on a non-denatured gel and the type of polymorphism can be determined by the difference in mobility of the separated single-stranded DNA on the gel.

Furthermore, when a base showing a polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by the restriction enzyme (RFLP method). In this case, first, the DNA sample is cleaved with a restriction enzyme. The DNA fragment can then be isolated and the size of the detected DNA fragment can be used to determine what type of polymorphism it is.

It is also possible to analyze the types of polymorphisms by examining the presence or absence of hybridization. That is, it is also possible to investigate which base the SNP is by preparing a probe corresponding to each base and examining which probe it hybridizes to.

By determining which base the SNP is in this way, data for examining scoliosis can be obtained.
The results determined by the method of the present invention are provided to doctors and the like as necessary. The doctor or the like who provided the results can diagnose the risk of onset and the risk of progression after performing necessary tests such as physical examination, X-ray examination, and CT examination. If a doctor or the like diagnoses that the risk of developing or progressing AIS is high, treatment such as wearing a device or cast or traction can be performed. In addition, if the risk is diagnosed as low, these treatments, which are burdensome for the patient, can be avoided.

<2> Reagent for scoliosis test of the present invention The present invention also provides a test reagent such as a primer or a probe for testing scoliosis. Examples of such a probe include a probe that includes the above SNP site and can determine the type of base of the SNP site depending on the presence or absence of hybridization. Specifically, a probe having a length of 15 bases or more having a sequence containing the 26th base of the base sequence shown in any of SEQ ID NOs: 1 to 9 or a complementary sequence thereof, and a chain imbalance relationship with the base. Examples thereof include a probe containing a base in the above, or a probe having a length of 15 bases or more having a complementary sequence thereof. For the base sequences of "bases having a linkage disequilibrium relationship with the relevant base" and the regions before and after it, for example, the dbSNP database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects) It can be obtained from / SNP /). The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases.
If the 26th base is a deletion or insertion of several bases, the deletion or insertion of several bases is regarded as the 26th base.

Examples of the primer include a primer that can be used for PCR for amplifying the SNP site, and a primer that can be used for sequence analysis (sequencing) of the SNP site. Specifically, it is a primer capable of amplifying or sequencing the region containing the 26th base of the base sequence shown in any of SEQ ID NOs: 1 to 9, and has a linkage disequilibrium relationship with the base. Examples thereof include primers capable of amplifying or sequencing a region containing a base. The length of such a primer is preferably 15 to 50 bases, more preferably 15 to 35 bases, still more preferably 20 to 35 bases.

As a primer for sequencing the SNP site, a primer having a sequence having a sequence 5'side of the base, preferably 30 to 100 bases upstream, or a 3'side region of the base, preferably 30 to 100 bases downstream. A primer having a sequence complementary to the region of is exemplified. The primer used for determining the polymorphism by the presence or absence of amplification by PCR has a sequence containing the above base, a primer containing the base on the 3'side, and a complementary sequence of the sequence containing the base. Examples thereof include a primer containing a complementary base of the above base on the 3'side.

In addition to these primers and probes, the test reagent of the present invention may include a polymerase and buffer for PCR, a reagent for hybridization and the like.

<3> Screening method for preventive or therapeutic agents for scoliosis In the present invention, as described later, the risk allele of rs58194408 causes a decrease in Obscurin (OBSCN) expression due to a decrease in transcription factor binding and a decrease in transcription activity, resulting in a decrease in Obscurin (OBSCN) expression. It was suggested that reduced expression may affect the onset of AIS in women. It was also suggested that the risk allele of rs10196262 causes an increase in EML4 expression by increasing transcription factor binding and transcription activity, and that the increase in EML4 expression may affect the onset of AIS in women. Therefore, by screening drugs using the activity of increasing the expression of OBSCN or the activity of decreasing the expression of EML4 as an index, a substance that can be a prophylactic or therapeutic drug for scoliosis can be obtained.

OBSCN is a Rho guanine nucleotide exchange factor involved in sarcomere assembly (J Cell Biol 154, 123-36 (2001)) and is registered as an OBSCN gene, specifically, for example, in GenBank Accession No. NM_052843.3. Genes that are listed.

EML4 (EMAP (echinoderm microtubule-associated protein) -like protein 4) is a protein belonging to the EMAP family involved in microtubule formation (Exp Cell Res 312, 3241-51 (2006)), and is specifically as an EML4 gene. For example, a gene registered in GenBank Accession No. NM_019063.4 can be mentioned.

That is, the screening method of the present invention includes a step of adding a drug candidate to cells expressing a reporter gene linked to the promoter of the OBSCN or EML4 gene or the OBSCN or EML4 gene, and the expression level of the OBSCN or EML4 gene or the reporter gene. A method for screening a prophylactic or therapeutic agent for scoliosis, which comprises a step of measuring the amount of the gene and a step of selecting a substance that changes the expression level thereof can be mentioned.

For example, when the expression level of the OBSCN gene or the reporter gene is increased in the presence of a drug candidate substance as compared with the absence of the drug candidate substance, the drug candidate substance is used as a candidate substance for a prophylactic or therapeutic drug for scoliosis. You can choose.

For example, when the expression level of the EML4 gene or the reporter gene is suppressed in the presence of a drug candidate substance as compared with the absence of the drug candidate substance, the drug candidate substance is used as a candidate substance for a prophylactic or therapeutic drug for scoliosis. Can be selected as.

Examples of cells expressing the OBSCN or EML4 gene include mammalian cells such as mice and humans, and for example, cells such as ovary, uterus, skeletal muscle, liver, skin, and brain can be used.

When a reporter gene linked to the promoter of the OBSCN or EML4 gene is used, the promoter of the OBSCN or EML4 gene is preferably a region containing about 2 kbp upstream of the transcription initiation site, and more preferably a region containing about 5 kbp upstream. Promoter sequence information is available from GenBank Accession No. NC_000001.10 and NC_000002.11 respectively.

Examples of the reporter gene include a luciferase gene, a GFP gene, and a chloramphenicol acetyltransferase gene. These reporter genes are ligated to the promoter of the OBSCN or EML4 gene, integrated into a plasmid used to introduce the gene into mammalian cells, and transfected into the cells by conventional methods such as lipofection.

A drug candidate is added to the cells expressing the OBSCN or EML4 gene as described above, or the cells into which the reporter gene has been introduced, and the expression level of the OBSCN or EML4 gene or the reporter gene is measured.

The drug candidate substance is not particularly limited, and may be, for example, a small molecule synthetic compound or a compound contained in a natural product. It may also be a peptide or nucleic acid. Individual candidate substances may be used for screening, or a compound library containing these substances may be used. By selecting a candidate substance that changes the expression level of the OBSCN or EML4 gene or reporter gene (compared to when it is not added) (increase in the case of OBSCN, decrease in the case of EML4), Substances that can be prophylactic or therapeutic agents can be obtained.

The expression level of the OBSCN or EML4 gene can be measured by methods such as RT-PCR, quantitative PCR, Northern blot, ELISA, Western blotting, in situ hybridization, and immune tissue staining. The expression level of the reporter gene depends on the type of the reporter gene, but can be measured by the fluorescence intensity, the luminescence intensity, the radioactivity intensity, and the like.

<4> Test method based on the expression level of the OBSCN or EML4 gene The present invention also provides a test method for scoliosis (method for obtaining test data) including a step of measuring the expression level of mRNA or protein of the OBSCN or EML4 gene. offer. If the expression level of the mRNA or protein of the OBSCN gene is decreased as compared with a control such as a healthy person, it can be determined that the patient has scoliosis or is at high risk of scoliosis. If the expression level of the EML4 gene mRNA or protein is increased as compared with a control such as a healthy person, it can be determined that the patient has scoliosis or is at high risk of scoliosis.

The expression level of the OBSCN or EML4 gene can be examined by RT-PCR, Northern blot, microarray method, or the like. The expression level of the OBSCN or EML4 gene product can be examined by ELISA, Western blotting, or the like. As the antibody, a commercially available antibody can be used, or an antibody against OBSCN or EML4 can be used. Examples of the sample used for the examination include body fluids such as blood, urine, and cerebrospinal fluid, cells such as the cervix and oral mucosa, and body hair such as hair.

Further, in the present invention, the expression level of the OBSCN or EML4 gene can be compared between the presence and absence of the therapeutic agent for scoliosis to determine the effect of the therapeutic agent for scoliosis. For example, if a therapeutic agent for scoliosis is added to cells expressing the OBSCN gene and the induction of expression of the OBSCN gene is increased as compared with the case where the therapeutic agent for scoliosis is not added, it can be determined that the therapeutic agent is effective. Further, it can be determined that the higher the suppression rate, the higher the effect of the therapeutic agent. In addition, if a therapeutic agent for scoliosis is added to cells that overexpress the EML4 gene and the induction of EML4 gene expression is suppressed as compared with the case where the therapeutic agent for scoliosis is not added, it is judged that the therapeutic agent is effective. It can be determined that the higher the suppression rate, the higher the effect of the therapeutic agent.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following aspects.

<Search for SNPs that specifically correlate with scoliosis in women>
[AIS Gender Hierarchized Genome-Wide Association Study]
First, genome-wide association studies were performed using GWAS subjects (2104 cases and 10736 controls) and the latest 1000 Genomes Project reference haplotypes. Informed consent was obtained from all the subjects and the guardians of a small number of subjects. This study was approved by the testing institution and the institutional review board of RIKEN. The analysis was performed using a multiplex PCR-based invader assay (Third Wave Technologies).

Males and females were analyzed separately to identify clusters of nominally significant variants within each dataset.
Two previously identified AIS-sensitive loci on chromosome 10q24.31 and chromosome 6q24.1 within the female dataset (2007 cases and 4751 controls) have genome-wide significance (P < It reached 5 × 10 ^-8 ), and 21 sitting positions showed a suggestive association (P <1 × 10 ^-5 ).

[Reproduction analysis of association in women]
Six previously identified or investigated loci were excluded from the 23 female-related loci. For each of the remaining 17 loci, we selected the SNP with the lowest P-value for reproducibility analysis. Of them, rs144779633, rs35518445 and rs3045832 were replaced with rs1475712, rs10208206 and rs12537799 in high linkage disequilibrium (R ² > 0.98) with each SNP, respectively.
An independent set of Japanese female subjects (941 cases and 1387 controls) was used to type representative SNP genotypes for each locus and have a significant association (P <2.94 × 10 ^-3 ). We identified two SNPs (rs59404763 and rs10196262) with Bonferroni-corrected P-value thresholds and one SNP (rs1475712) showing a promising association (P = 6.09 × ^10-3 , OR = 1.19). Combining the results of GWAS and reproducibility analysis, rs59404763 and rs10196262 reached the genome-wide significance threshold of P <5 × ^10-8 , and rs1475712 showed a suggestive association (Table 1). These novel susceptibility variants showed a significant association in females but not in males (Table 2).

[Lotus coition feature analysis and candidate causal variants and gene identification]
To analyze the characteristics of the related loci, typed and imputated SNPs around rs59404763 and rs10196262 were plotted (Fig. 1). The most significantly associated SNP, rs59404763 (composite P-value = 2.51 × 10 ^-10 , OR = 1.53, 95% CI = 1.34 to 1.75), is located within the OBSCN intron on chromosome 1q42.13 (region). Figure 1a), the other associated SNP, rs10196262 (complex P-value = 3.16 × 10 ^-9 , OR = 1.33, 95% CI = 1.21 to 1.46), is a gene of unknown character located in the chromosome 2p21 region, LOC102723824. It was located in the vicinity of (Fig. 1b).
Variants that were strongly correlated with rs59404763 and rs10196262, respectively (R ² > 0.8) were selected to identify the causal SNP for each locus, and Haploreg was used to assess the accommodation capacity of those variants (Table 3).

[Identification of causative SNPs and candidate susceptibility genes in the locus 1q42.13]
The most significant association of SNPs within the locus 1q42.13 was in the OBSCN gene (Fig. 1a). Among them, rs58194408 has strong evidence of accommodation ability including changes in motifs for DNase I hypersensitive sites, histone marks and some transcription factors, so that SNP is used as a candidate causative SNP. Selected.
Electrophoretic mobility shift assay (EMSA) was performed to discover allergen differences in transcription factor binding that the binding strength of risk alleles to nuclear proteins was lower than that of non-risk alleles (Fig. 2a). did. Next, we investigated whether rs58194408 has an allele-specific effect on the expression of OBSCN. Luciferase reporter vectors containing the OBSCN promoter and the genomic segments surrounding its SNP were constructed and the transcriptional activity of those reporter vectors was analyzed. As a result, risk alleles showed significantly lower transcriptional activity than non-risk alleles (Fig. 2b). These results suggest that decreased transcription factor binding due to risk alleles and decreased OBSCN expression due to decreased transcriptional activity may affect the pathogenesis of AIS in women.

[Identification of causative SNPs and candidate susceptibility genes in the 2p21 locus]
The most significant association of SNPs within the chromosome 2p21 locus was between genes. Therefore, to identify candidate susceptibility genes, the Hi-C interaction browser was used to search for topologically relevant domains (TADs) that overlap rs10196262 and other SNPs that were significantly associated, and of LOC102723824, EML4 and COX7A2L. A TAD containing three genes was discovered (Fig. 4a). Based on the expression quantitative trait position (eQTL) data of those genes from the genotype-tissue expression (GTEx) project, rs10196262 is significantly associated with EML4 expression and risk alleles are more highly expressed than non-risk alleles. It became clear to show.

EMSA was performed to identify allergen differences in transcription factor binding (Fig. 3a). The variant rs144297067 is in complete linkage disequilibrium (LD) with rs10196262, suggesting that rs144297067 is significantly associated with EML4 expression.
Next, we investigated whether rs144297067 has an allele-specific effect on EML4 expression. Luciferase reporter vectors containing the EML4 promoter and the genomic segments surrounding its variants were constructed and the changes in transcriptional activity of those reporter vectors were analyzed. As a result, it was found that the risk variant rs144297067 showed significantly higher transcriptional activity than the non-risk variant (Fig. 3b). These observations were consistent with the eQTL data from the GTEx portal.

The reporter assay and gel shift assay were performed according to the following procedure.
Cell culture and luciferase reporter assay In order to construct a luciferase reporter vector for each allele of the candidate cause variant, a genomic fragment adjacent to each variant was synthesized as a double-stranded oligonucleotide (Invitrogen) containing an XhoI cleavage site at both ends. Their genomic fragments were cloned upstream of the SV40 promoter in the pGL3 promoter vector. We also clone the OBSCN promoter region and the EML4 promoter region (-987 to +53 and -765 to +274, respectively) into the pGL4.10 vector (Promega) to obtain the OBSCN promoter-luciferase fusion vector and the EML4 promoter-luciferase fusion vector. It was constructed. The genomic fragment surrounding rs58194408 was then cloned into the OBSCN promoter luciferase fusion vector, and the genomic fragment surrounding rs144297067 was cloned into the EML4 promoter luciferase fusion vector. All inserts in those constructs were confirmed by sequencing. Transit-LT1 (Mirus Bio) was used to transfect HEK293 cells and A172 cells with these constructs and the luciferase activity was measured 24 hours after transfection according to the manufacturer's instructions. In these experiments, the phRL-TK vector or phRL-SV40 vector (Promega) was used as an internal control for normalization. All assays were performed in triplicate at least twice.

Electrophoretic mobility shift assay (EMSA)
Each probe was prepared by annealing complementary oligonucleotides and labeled with digoxigenin (DIG) -11-dd UTP using the DIG gel shift kit (Roche). Nuclear extracts were prepared as previously described. Competitive experiments were performed by pre-incubating an excess of unlabeled oligonucleotide and nuclear extract prior to adding the labeled oligonucleotide. The reaction mixture was allowed to stand on ice for 20 minutes at a constant temperature. The DNA protein complex was separated on a 6% TBE gel (Invitrogen) and the signal was detected using a chemiluminescence detection system (Roche) according to the manufacturer's instructions.

[Suggestive sitting position]
In addition to the two genome-wide associations mentioned above, we have identified suggestive loci on the chromosome 20q13.12 region. The associated SNP, rs1475712, was located in the intergenic region between SLC2A10 and EYA2, and the TAD of rs1475712 contained only one gene, EYA2 (Fig. 4b). EYA2 is a human homologue of the eyes absent gene of the fruit fly (Drosophila). Eya2 physically interacts with Dach2 or Six1 to synergistically regulate myogenic gene expression.

<Search for SNPs related to progress>
We mobilized 2142 AIS patients previously described (Am J Hum Genet 97, 337-342 (2015).). All patients underwent medical examination and radiographic evaluation by a surgeon specializing in scoliosis. Informed consent was obtained from all patients or their parents. This study was approved by the Institute of Physical and Chemical Research and the Institutional Review Board of the testing organization. A curve with a bump angle of 40 ° or more was defined as progression of the curve regardless of the maturity of the skeleton, and a curve with a bump angle of 30 ° or less in a patient with a mature skeleton was defined as non-progression of the curve. This criterion is based on the natural course of AIS that curves with hump angles greater than 40 ° are progressive even after skeletal maturation, while curves with hump angles less than 30 ° are non-progressive. Is based. This time, we placed a gap of 10 ° between the two groups mentioned above for a clear classification. The Cobb method has an intra-observer error or an inter-observer error of 5-10 °. Skeletal maturation was defined by a Risser grade of 4 or higher (reference number 24). For unoperated cases, the patient's latest radiographs are used to assess the hump angle, and for operated cases, the hump angle is assessed using information from the patient's last visit before surgery. did.

The 2142 AIS patients mentioned above were divided into an advanced group and a non-advanced group, 1105 patients were assigned to the advanced group, and 832 patients were assigned to the non-advanced group. The genotyping data of those patients determined in previous GWAS for AIS susceptibility genes using two Illumina Genotyping BeadChips (Human610 and OmniExpress) were used. After quality control filtering, we investigated the association between 502857 SNPs in Human 610 and 592392 SNPs in OmniExpress. Quantile-quantile (QQ) plots were used to check for possible population structuring effects and genomic inflation coefficients of 1.03 and 1.05 were obtained for each platform. These mean that the likelihood of false positive associations due to population structure or cryptorelatedness of the population is low. Since the tag SNPs contained in these two types of genotyping chips are different, we performed whole-genome implantation and meta-analysis as previously described. A total of 7521072 SNPs were analyzed. We identified 10 candidate loci with suggestive relevant evidence (P <1 × 10 ^-5 ; Figure 5a).

Reproducibility analysis An independent Japanese AIS cohort (323 advanced patients and 283 non-advanced patients) was used to investigate the reproducibility of the association of the 10 candidate loci. We find evidence of an association with one locus represented by the SNP, rs1828853, on chromosome 11q14.1 with a P-value of 3.73 × 10 ^-5 (Bonferroni-corrected P-value less than 5.00 × 10 ^-3 ). rice field. Combining the results of GWAS and reproducibility analysis, rs1828853 reached the genome-wide significance threshold of P <5 × 10 ^-8 (composite P value = 1.98 × 10 ^-9 ; odds ratio = 1.56; 95% confidence interval (CI). ) = 1.35 to 1.80; Table 4).

Precision Mapping To analyze the features of loci on chromosome 11q14.1, we plotted SNPs typed around rs1828853 and complemented by imputation. A local association plot was created using Locus Zoom. The landmark SNP rs1828853 and the SNP in the strong LD were extracted and plotted (Fig. 5c). These SNPs were in the same LD block containing only a single gene, MIR4300HG. Their most significantly related SNPs were located within Intron 1 of MIR4300HG (FIGS. 5b and 5c).

Specific Functional SNPs in the Lous Coition To search for functional SNPs, we used HaploReg version 4.1 to extract SNPs that strongly correlate with rs1828853 (r ² ≧ 0.9). Three SNPs, rs971548, rs35333564 and rs7949305, were located within the enhancer histone mark. Therefore, the genomic region containing the SNP may act as a cis sequence for some enhancers.

Claims (9)

Single nucleotide polymorphism selected from the group consisting of rs59404763, rs10196262 and rs1475712 or rs58194408, rs117147433, rs75785474, rs144297067, rs11676073, rs13392988, rs13386561, rs11684533, rs17669917, rs13410780 and rs144779633 that are in linkage disequilibrium with the single nucleotide polymorphism. A method for testing scoliosis in women , which comprises analyzing a single nucleotide polymorphism selected from the group consisting of two and testing for scoliosis based on the analysis result. The method according to claim 1, wherein the scoliosis is idiopathic scoliosis in adolescence. In addition, the single nucleotide polymorphism rs1828853 or rs971548 in linkage disequilibrium with the single nucleotide polymorphism.
, Rs35333564 and rs7949305, specializing in the analysis of single nucleotide polymorphisms selected from the group.
The method according to claim 1, which is a symptom. A probe for scoliosis examination having a sequence of 15 or more bases containing the 26th base of the base number or a complementary sequence thereof in the base sequence shown in any of SEQ ID NOs: 1 to 5 . A primer for scoliosis examination capable of amplifying a region containing the 26th base in the base sequence shown in any of SEQ ID NOs: 1 to 5 . A step of adding a drug candidate substance in vitro to a cell expressing a reporter gene linked to the Obscurin gene or the promoter of the Obscurin gene, a step of measuring the expression level of the Obscurin gene or the reporter gene, and a substance for increasing the expression level. A method of screening for a prophylactic or therapeutic agent for scoliosis, including the step of selecting. The expression level of the mRNA or protein of the Obscurin gene is measured , and if the expression level of the mRNA or protein is reduced as compared with the control, it is determined that the patient has scoliosis or is at high risk of scoliosis. A method for testing for scoliosis, including steps. A step of adding a drug candidate substance in vitro to a cell expressing a reporter gene linked to the EML4 gene or the promoter of the EML4 gene, a step of measuring the expression level of the EML4 gene or the reporter gene, and a substance for reducing the expression level. A method of screening for a prophylactic or therapeutic agent for scoliosis, including the step of selecting. The expression level of the mRNA or protein of the EML4 gene is measured , and if the expression level of the mRNA or protein is increased as compared with the control, it is determined that the patient has scoliosis or is at high risk of scoliosis. A method for testing for scoliosis, including steps.

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