JP6145837B2 - Method for examining bone / joint diseases based on single nucleotide polymorphism in the long arm 24.1 region of chromosome 6 - Google Patents

Description

  The present invention relates to a test method for determining the onset risk of bone and joint diseases such as scoliosis and the presence or absence of the onset, and a reagent used in the test method.

  Scoliosis refers to a condition in which the spine is bent in the left-right direction. Scoliosis whose etiology is not clear is called idiopathic scoliosis and accounts for 80 to 90% of scoliosis.

  Scoliosis occurs more frequently in school-aged children, especially in girls. Adolescent idiopathic scoliosis (Adolescent) shows a curvature of at least 10 ° at the Cobb angle, which is an index of scoliosis, and the etiology is not clear in children from the age of 10 until the skeleton matures. idiopathic scoliosis (AIS). The incidence of AIS is 2% in school-aged children in Japan and 2-3% in the world, and about 10,000 people develop each year in Japan.

  Although scoliosis is diagnosed mainly by X-ray examination, X-ray examination is not effective for diagnosing scoliosis before onset or early onset. Moreover, the treatment of idiopathic scoliosis is performed only by symptomatic therapy, and no causal therapy is performed as long as the etiology is not clear. Therefore, identification of genes and single nucleotide polymorphisms (SNPs) associated with scoliosis is desired in order to enable prediagnosis diagnosis (risk diagnosis) and early diagnosis of scoliosis and to enable causal treatment. .

  Many loci causing AIS have been found by genome-wide linkage analysis, and AIS is considered to be based on a complex genetic predisposition (Non-Patent Documents 1 and 2). . In addition, although AIS susceptibility genes have been reported by candidate gene analysis (Non-Patent Documents 3 to 9), reproducibility of association with AIS is obtained when any race is used as a subject for any gene. (Non-Patent Documents 10 and 11).

  Further, in recent years, candidates for genes related to AIS have been reported by a genome-wide association study (GWAS) based on the Transmission Disequilibrium Test (TDT) method (Non-patent Document 12). . However, they are not reproducible in relation to AIS in case-control association analysis after multiple test correction.

  On the other hand, in recent years, GWAS has seen that SNPs present in the long arm 24.31 region (10q24.31 region) are related to AIS in the Japanese and Chinese populations at a genome-wide level. (Non-Patent Documents 13 and 14).

  The GPR126 gene present in the long arm 24.1 region (6q24.1 region) of chromosome 6 encodes an orphan receptor belonging to the adhesion-GPCR (adhesion class G protein-coupled receptor) family. Rs6570507 present in the GPR126 gene has been found to be associated with trunk length (sitting height) by GWAS meta-analysis in European population (Non-patent Document 15). However, the association between the GPR126 gene and scoliosis is not known.

Wise, C.A. et al., Curr. Genomics 9, 51-59 (2008). Raggio, C. L. et al., J. Orthop. Res. 27, 1366-1372 (2009). Wu, J. et al., Spine 31, 1131-1136 (2006). Zhang, H.Q. et al., Spine 34, 760-764 (2009). Chen, Z. et al., Eur. J. Hum. Genet. 17, 525-532 (2009). Qiu, X. S. et al., Spine 32, 1748-1753 (2007). Wang, H. et al., Spine 33, 2199-2203 (2008). Inoue, M. et al., Stud. Health Technol. Inform. 91, 90-96 (2002). Yeung, H.Y. et al., Stud. Health Technol. Inform. 123, 18-24 (2006). Takahashi, Y. et al., J. Orthop. Res. 29, 834-837 (2011). Takahashi, Y. et al., J. Orthop. Res. 29, 1055-1058 (2011). Sharma, S. et al., Hum Mol Genet. 20, 1456-1466 (2011). Takahashi, Y. et al., Nat. Genet. 43, 1237-1240 (2011). Fan, YH. Et al., J. Hum. Genet. 57, 244-246 (2012). Soranzo, N. et al., PLoS Genet 5, e1000445 (2009).

  An object of the present invention is to provide a method for accurately examining the onset risk of bone and joint diseases such as scoliosis and the presence or absence of onset, and a test reagent used in the method.

  As a result of intensive studies to solve the above problems, the present inventors have found that a single nucleotide polymorphism (SNP) present in the long arm 24.1 region (6q24.1 region) of chromosome 6 is adolescent idiopathic scoliosis ( AIS) was identified. And by investigating these polymorphisms, it discovered that the onset risk of bone / joint diseases, such as scoliosis, and estimation of onset could be implemented correctly, and came to complete this invention.

That is, the present invention includes the following aspects.
[1]
Analyzing single nucleotide polymorphisms present in the long arm 24.1 region of chromosome 6 and examining bone / joint diseases based on the analysis results, and / or risk of developing bone / joint diseases Judgment method of presence or absence.
[2]
The method as described above, wherein the bone / joint disease is scoliosis.
[3]
The method, wherein the scoliosis is adolescent idiopathic scoliosis.
[4]
The method, wherein the single nucleotide polymorphism is a single nucleotide polymorphism existing on the GPR126 gene.
[5]
The method, wherein the single nucleotide polymorphism is a single nucleotide polymorphism in a base corresponding to the 61st base of the base sequence shown in SEQ ID NO: 1 or a base in a linkage disequilibrium relationship with the base. [6]
A probe for bone / joint disease testing, which has a sequence of 10 bases or more including the 61st base in the base sequence shown in SEQ ID NO: 1, or a complementary sequence thereof.
[7]
A primer for bone / joint disease testing, which can amplify a region containing the 61st base in the base sequence shown in SEQ ID NO: 1.

  According to the present invention, it is possible to accurately and simply predict the onset risk (morbidity risk) of bone and joint diseases such as scoliosis, which has been difficult to predict. Moreover, the onset of bone / joint diseases such as scoliosis can be determined accurately and simply. Therefore, the present invention contributes to the prevention and early treatment of bone and joint diseases such as scoliosis.

The figure which shows the outline | summary of GWAS and a series of replication studies (replication studies). The figure which shows the recombination rate (recombination rate) as a result of the analysis of a 6th chromosome long arm 24.1 area | region (6q24.1) and AIS. The result of the association analysis is shown as -log ₁₀ of the P value of the Cochrane Armitage trend test. Diamonds indicate SNPs that showed the strongest association with AIS. The figure and photograph which show the phenotype of the morphant of GPR126 gene of zebrafish. (A) Side view illustrating body length and eyeball diameter. (B)-(E) (B) Body length, (C) Eyeball diameter, (D) Alizarin red stained skeleton, and (E) Number of vertebrae on day 14 after fertilization. *: P <0.05. NS: not significant. ctrl MO: Control. gpr126 MO: A morphant of the GPR126 gene.

<1> Method of the Present Invention The method of the present invention analyzes SNPs present in the long arm 24.1 region (6q24.1 region) of human chromosome 6, and examines bone and joint diseases based on the analysis results. This is a method for determining the risk of developing bone / joint diseases and / or the presence or absence of the onset. That is, in the present invention, “examination” includes examination of the onset risk of bone / joint disease and examination of the presence / absence of development of bone / joint disease. In the method of the present invention, the analysis result of SNP is associated with the risk of developing bone / joint disease and / or the presence or absence of the onset.

  The bone / joint disease is not particularly limited, and examples thereof include spinal diseases, specifically scoliosis. In particular, the method of the present invention is suitably used for testing for idiopathic scoliosis, for which the etiology has not been specified. Scoliosis may occur at any time, such as congenital, juvenile, adolescent, adult, etc., but is preferably adolescent scoliosis, and 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 Asian subjects such as Japanese and Chinese and white subjects, for example. In addition, the method of the present invention can be used for subjects of any gender, but can be suitably used particularly for female subjects.

  Specific examples of the SNP existing in the 6q24.1 region include human rs6570507. Here, the rs number indicates the registration number of the dbSNP database (http // www.ncbi.nlm.nih.gov / projects / SNP /) of National Center for Biotechnology Information. rs6570507 is located in a linkage disequilibrium block containing the GPR126 gene in the 6q24.1 region. Therefore, in particular, bone / joint diseases can be examined by analyzing SNPs present in the linkage disequilibrium block, for example, SNPs present on the GPR126 gene. Specific examples of the GPR126 gene include the region from 142623056 to 142767403 of GenBank Accession No. NC_000006.11.

  rs6570507 is a polymorphism of adenine (A) / guanine (G) at the 142679572th base of GenBank Accession No. NC_000006.11. When this base is A, the possibility or risk of developing bone / joint disease high. Further, when the analysis is performed in consideration of the genotype, the possibility of bone / joint disease or the risk of onset is high in the order of AA> AG> GG.

  Regarding rs6570507, a sequence having a total length of 121 bp including the SNP base and a region of 60 bp before and after it is shown in SEQ ID NO: 1. The 61st base has a polymorphism.

  In the present invention, a base corresponding to the above base is analyzed. The “base corresponding to the above base” means the corresponding base in the above region. That is, “analyzing the base corresponding to the base” includes analyzing the base in the region even if the sequence is slightly changed at a position other than the SNP due to a difference in race. .

In addition, the base to be analyzed in the present invention is not limited to the above, and a polymorphism of a base in linkage disequilibrium with the above base may be analyzed. Here, the “base in linkage disequilibrium with the above-mentioned base” satisfies the relationship of r ² > 0.5, preferably r ² > 0.8, more preferably r ² > 0.9 with the above-mentioned base. Say base. r ² is a linkage disequilibrium coefficient. In addition, a base in linkage disequilibrium with the above base can be identified using, for example, the HapMap database (http://www.hapmap.org/index.html.ja). Alternatively, DNA collected from a plurality of people (usually about 20 to 40 people) can be identified by sequence analysis using a sequencer and searching for SNPs in linkage disequilibrium. Bases that are in linkage disequilibrium with the above bases, when analyzed in consideration of genotype, are homozygotes for risk alleles> heterozygotes for risk and non-risk alleles> homozygotes for non-risk alleles In order, there is a high possibility of bone / joint disease or risk of developing it.

  Specific examples of bases in linkage disequilibrium with rs6570507 include, Is mentioned.

  By examining the type of the SNP base and associating the obtained result with the bone / joint disease based on the above criteria, the bone / joint disease can be examined. 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 SNPs may be analyzed together, or at least one of the SNPs, known SNPs related to bone / joint diseases (Non-patent Document 13 etc.) and linkage disequilibrium with the known SNPs. You may analyze combining a certain SNPs. If a plurality of SNPs related to bone / joint diseases are analyzed together, the accuracy of the examination of bone / joint diseases can be improved. Any SNP may analyze either strand of the double-stranded DNA. For example, the sequence of the GPR126 gene 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 SNP analysis include body fluids such as blood and urine, cells such as oral mucosa, body hair such as hair, and the like. Although these samples can be used directly for the analysis of SNP, it is preferable to isolate chromosomal DNA from these samples by a conventional method and analyze it.

  The analysis of SNP can be performed by a normal gene polymorphism analysis method. Examples include, but are not limited to, sequence analysis, PCR, hybridization, invader method and the like.

Sequence analysis can be performed by an ordinary 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 polymorphism, and the type of base at the corresponding position is determined from the analysis result. can do. In addition, it is preferable to amplify the fragment containing the SNP site in advance by PCR or the like before the sequencing reaction.

  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 showing a polymorphism and having a 3 'end corresponding to each polymorph is prepared. PCR can be performed using each primer, and the type of polymorphism can be determined depending on the presence or absence of the amplification product. Further, the presence or absence of amplification may be examined by the LAMP method (Japanese Patent No. 3313358), NASBA method (Nucleic Acid Sequence-Based Amplification; Japanese Patent No. 2844386), ICAN method (Japanese Patent Laid-Open No. 2002-233379), or the like. it can. In addition, a single-strand amplification method may be used.

  It is also possible to amplify a DNA fragment containing a SNP site and determine which type of polymorphism is based on the mobility of the amplified product in electrophoresis. An example of such a method is a PCR-SSCP (single-strand conformation polymorphism) method (Genomics. 1992 Jan 1; 12 (1): 139-146.). Specifically, first, DNA containing the target SNP is amplified, and the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing 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 polymorphism is included in the restriction enzyme recognition sequence, it can be analyzed by the presence or absence of cleavage by a restriction enzyme (RFLP method). In this case, first, the DNA sample is cleaved with a restriction enzyme. The DNA fragments can then be separated and the type of polymorphism determined by the size of the detected DNA fragment.

  It is also possible to analyze the type of polymorphism by examining the presence or absence of hybridization. That is, a probe corresponding to each base is prepared, and it is also possible to examine which base the SNP is by examining which probe hybridizes.

  By determining which base the SNP is in this way, data for examining bone / joint diseases can be obtained.

<2> Reagent for test | inspection of this invention This invention also provides test reagents, such as a primer and a probe for test | inspecting bone and joint diseases, such as scoliosis. Examples of such a probe include a probe that includes the SNP site and can determine the type of base at the SNP site based on the presence or absence of hybridization. Specifically, a sequence containing the 61st base of the base sequence shown in SEQ ID NO: 1, or a probe having a length of 10 bases or more having its complementary sequence, or a base in a linkage disequilibrium relationship with the base Examples thereof include a probe having a length of 10 bases or more having a sequence or a complementary sequence thereof. The nucleotide sequences of “bases in linkage disequilibrium with the base” and the regions before and after the base are, for example, the dbSNP database (http // www.ncbi.nlm.nih.gov / projects) of National Center for Biotechnology Information. / SNP /). The length of the probe is preferably 15 to 35 bases, more preferably 20 to 35 bases.

Examples of the primer include a primer that can be used for PCR for amplifying the SNP site, or a primer that can be used for sequence analysis (sequencing) of the SNP site. Specifically, a primer capable of amplifying or sequencing a region containing the 61st base of the base sequence shown in SEQ ID NO: 1, or a region containing a base in a linkage disequilibrium relationship with the base Examples include primers that can be amplified or sequenced. The length of such a primer is preferably 10 to 50 bases, more preferably 15 to 35 bases, and even more preferably 20 to 35 bases.

  As a primer for sequencing the SNP site, a primer 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 this region is exemplified. As a primer used to determine polymorphism based on the presence or absence of amplification by PCR, it has a sequence containing the base, a primer containing the base on the 3 ′ side, a complementary sequence of the sequence containing the base, Examples include a primer containing a base complementary to the above base on the 3 ′ side.

  The test reagent of the present invention may contain PCR polymerase, buffer, hybridization reagent, etc. in addition to these primers and probes.

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

(1) Identification of SNPs associated with adolescent idiopathic scoliosis (AIS) Genome-wide association analysis (GWAS) was performed using Japanese subjects to identify genetic mutations that determine AIS sensitivity. . GWAS is a genetic statistical technique for searching for genetic variation related to a phenotype such as a disease. For example, using hundreds of thousands to one million SNPs covering the entire human genome, the frequency of polymorphism between a patient with a certain disease (case) and a subject who does not have the disease (control) By statistically testing for differences, genetic variations associated with the disease can be found.

  An overview of the GWAS and a series of replication studies is shown in FIG. This study was approved by the ethics committee of RIKEN and the ethics committee of participating institutions, and informed consent was obtained from all subjects and parents of some subjects.

(1-1) GWAS
Since AIS often affects women, Japanese women were employed as GWAS subjects. 1,050 AIS subjects (cases) used for GWAS were recruited at 8 hospitals. All AIS subjects underwent clinical and radiological examinations and were diagnosed with AIS by a scoliosis specialist. The diagnostic criteria for AIS are as described in Non-Patent Documents 9 and 10. In all AIS subjects, the Cobb angle, which is an index of scoliosis, exceeded 20 °. The 1,474 control subjects (control) used for GWAS consist of patients with diseases other than AIS registered in BBJ (BioBank Japan) and healthy volunteers recruited at Osaka Midosuji Rotary Club.

The genotypes of 1,050 AIS subjects were analyzed using an Illumina Human610 Genotyping BeadChip (Illumina (USA)). The genotypes of 1,474 control subjects were analyzed using an Illumina HumanHap550v3 Genotyping BeadChip (Illumina (USA)). As quality control, SNPs with a call rate of less than 0.99, SNPs with a Hardy-Weinberg equilibrium test with a cut-off value or less (P ≦ 10 ⁻⁶ ), monomorphic SNPs (monomorphic SNPs) SNPs that are not shared between AIS and control subjects, ambiguous call SNPs, low call rate among closely related pairs with identify-by-state (IBS) ≧ 1.7 Subjects (16 AIS subjects, 1 control subject), and subjects determined by the principal component analysis (PCA) as outliers (1 AIS subject) Was excluded. 465,762 SNPs (including SNPs on the X chromosome) of 1,033 AIS subjects and 1,473 control subjects who passed the quality control were compared with AIS by Cochran-Armitage trend test. The association was evaluated.

  As a result of GWAS, it was suggested that 6 SNPs were associated with AIS in addition to 3 SNPs in the 10q24.31 region (non-patent document 13), which had already been reported to have a significant correlation with AIS at the genome-wide level.

(1-2) Reproduction test by Japanese female subjects Among the above 6 SNPs, SNPs in the same locus (r ² > 0.8) and 10q24.31 region were excluded, and 3 SNPs (rs6570507 , Rs7695327, and rs3123295) were selected for reproducibility testing. In the reproduction test, Japanese female subjects independent of GWAS subjects were used. 680 AIS subjects and 9,672 control subjects were recruited on the same basis as GWAS subjects. The genotypes of 680 AIS subjects were analyzed by multiplex-PCR invader assay (Third Wave Technologies). The genotype of 9,672 control subjects was analyzed using the Illumina HumanHap550v3 Genotyping BeadChip (Illumina (USA)).

As a result of the reproducibility test, among the above three SNPs, rs6570507 present on chromosome 6 is P <1.67 × 10 ⁻² (= 0.05 / 3) which is a threshold value of significance after Bonferroni correction. Satisfied and showed a significant association with AIS ("Replication" column in Table 1).

In addition, as a result of meta-analysis of reproducibility tests by GWAS and Japanese female subjects using the Mantel-Haenszel method, rs6570507 was found to have a P <set as the threshold for genome-wide significance (genome-wide significance) ^Satisfies 5 × 10 ⁻⁸ (P = 4.10 × 10 ⁻¹⁰ ) and showed a significant association with AIS (“Combined” column in Table 1).

RAF: Risk Allele Frequency
CI: Confidence Interval
a: P value of Cochrane Armitage trend test.
b: Allele odds ratio at 95% CI.
c: P value of Breslow-Day test.
d: P value calculated by Mantel-Haenszel method.

(1-3) Meta-analysis of trials by Japanese subjects In addition, 106 male AIS subjects and 14,794 male control subjects were recruited on the same basis as GWAS subjects. A reproduction test was conducted. As a result of a meta-analysis of GWAS and Japanese female and Japanese male subjects using the Mantel-Haenszel method, rs6570507 showed a more significant association with AIS (Table 2). "Japanese Combined" field).

RAF: Risk Allele Frequency
CI: Confidence Interval
a: P value of Cochrane Armitage trend test.
b: Allele odds ratio at 95% CI.
c: P value of Breslow-Day test.
d: P value calculated by Mantel-Haenszel method.

(1-4) Reproducibility study with Chinese and Caucasian subjects In addition, with regard to rs6570507, which was significantly related to AIS in Japanese female subjects, the Chinese (Han Chinese) population and Caucasian population The relationship with AIS was verified.

  743 Chinese AIS subjects were recruited at Nanjing University Hospital. All AIS subjects underwent clinical and radiological examinations and were diagnosed with AIS by a scoliosis specialist. The diagnostic criteria for AIS are as described in Non-Patent Document 11. 1,209 Chinese control subjects consisted of healthy volunteers. The genotype of Chinese subjects was analyzed by multiplex PCR-based invader assay.

  White subjects were recruited according to protocols approved by the Ethics Committee at the University of Texas Southwestern Medical Center. That is, 457 white AIS subjects were recruited from orthopedic patients and their families at the Scottish Light, Texas Children's Hospital (TSRHC). All AIS subjects underwent clinical and radiological examinations and were diagnosed with AIS by a scoliosis specialist. The diagnostic criteria for AIS are as described in Non-Patent Document 12. 744 Caucasian control subjects were recruited from local Texas residents and non-TSRHC orthopedic patients. The genotype of Caucasian subjects was analyzed using Illumina Human OmniExpress 12v1 Chip. Quality control was performed using PLINK v1.07, excluding 10 white AIS subjects and 7 white control subjects.

As a result, the association between rs6570507 and AIS was reproduced in both Chinese and Caucasian populations ("Replication" column in Table 2). In addition, the P value of the Breslow-Day test was less than 0.05 (“P _het ” column in Table 2), and no significant heterogeneity was observed between tests. Moreover, P value by meta-analysis of all the tests was 1.23 × 10 ⁻¹⁴ (“All Combined” column in Table 2).

  That is, this example reports for the first time an area significantly related to AIS regardless of race.

(1-5) Computation
Based on the reference haplotype data of the East Asian population sample from the 1000 Genome Project (http://www.1000genomes.org/), MACH and Minimac software were used to investigate the genotype of the whole genome for further AIS-related region studies. Complementary.

  As a result, it was suggested that 12 loci were associated with AIS. Therefore, among the 12 loci, 786 of the SNPs most strongly associated with AIS (lowest P value) at each of the 8 loci that have not been associated with AIS. Genotype analysis was performed using an independent data set consisting of AIS subjects and 3,422 control subjects. However, none of the SNPs could reproduce the association with AIS after Bonferroni correction.

(1-6) Mapping of chromosome 6 long arm 24.1 region (6q24.1) rs6570507, which was found to have a significant association with AIS, is located in chromosome 6 long arm 24.1 region (6q24.1). Exists. Therefore, the region was mapped using the data of 1000 genome project. The results are shown in FIG. rs7774095 showed the strongest association with AIS, followed by rs6570507. However, in GWAS, the relationship between rs7774095 and AIS (P = 1.23 × 10 ⁻⁵ , OR = 1.29) is the relationship between rs6570507 and AIS (P = 1.37 × 10 ⁻⁶ , OR = 1. It was smaller than 32). This region includes 17 SNPs in strong linkage disequilibrium with rs6570507 (rs9403383, rs6909857, rs972982, rs9403382, rs9389986, rs2050157, rs9496346, rs7741741, rs9389985, rs2039986, rs1891308, rs9403380, rs1418201, rs1040525, rs949509369, rs949509369, rs6937121), and these SNPs are related to AIS, similar to rs6570507. Both rs6570507 and these 17 SNPs were located in the GPR126 gene of the region.

(2) Functional analysis of GPR126
The GPR126 gene encodes an orphan receptor belonging to the adhesion-GPCR (adhesion class G protein-coupled receptor) family.

  In mice, it has been suggested that deletion of the Sox9 gene, which is an important regulator of cartilage formation, reduces the expression of the GPR126 gene in the intervertebral disc. On the other hand, in humans, the expression of GPR126 gene in skeletal tissues associated with scoliosis has not been studied. Therefore, in humans, the expression of GPR126 gene in various tissues including bone, cartilage, and intervertebral disc was examined. As a result, it was revealed that the GPR126 gene was strongly expressed in cartilage. In addition, when the expression of the GPR126 gene in embryonic mice was analyzed by in situ hybridization, it was found that the GPR126 gene was expressed at the growth site of vertebral chondrocytes.

  Next, the function of GPR126 in skeleton formation was examined using zebrafish. GPR126 gene translation inhibition morpholino (gpr126 translation-blocking morpholino; 5′-ACCGACCACTGATGAACGAAATCAT-3 ′; SEQ ID NO: 2) was injected into the 1-cell stage embryo to examine the effects of loss of function of the GPR126 gene. As a result, the morphant of the GPR126 gene (gpr126 morphant) had a shorter body length and delayed ossification of the spine (FIG. 3). In addition, the GPR126 gene morphant had a slower escape response than the control. The same was true when splicing inhibition morpholino (5′-TGCCACTGCAAG CAAACAGGGCACA-3 ′; SEQ ID NO: 3) was used instead of translation inhibition morpholino of the GPR126 gene.

(3) Discussion Scoliosis is widely thought to be caused by abnormal skeletal growth (Kouwenhoven,
JW & Castelein, RM, Spine (Phila Pa 1976) 33, 2898-908 (2008)). Morphological studies have reported that patients with AIS show fast unbalanced vertebral body growth in the longitudinal direction and asymmetry in the length of the skeleton other than the spine (Guo, X., et al ., J Bone Joint Surg Br 85, 1026-31 (2003); Burwell, RG, et al., Stud Health Technol Inform 135, 3-52 (2008); Burwell, RG et al., Scoliosis 4, 24 (2009 )). It has been reported that knockout mice of the GPR126 gene exhibit abnormal limb postures and growth disorders (Monk, KR, et al., Development 138, 2673-80 (2011)). The GPR126 gene has been reported to be associated with body height (Zhao, J. et al., BMC Med Genet 11, 96 (2010); Soranzo, N. et
al., PLoS Genet 5, e1000445 (2009); Sovio, U. et al., PLoS Genet 5, e1000409 (2009); Lettre, G. et al., Nat Genet 40, 584-91 (2008)). Furthermore, rs6570507 present in the GPR126 gene has been found to be associated with trunk length (sitting height) by GWAS meta-analysis in European population (Non-patent Document 15). Thus, the GPR126 gene may affect AIS sensitivity through spinal dysplasia.

  Many studies also suggest that the GPR126 gene is essential for myelination (Monk, KR, et al., Development 138, 2673-80 (2011); Pogoda, HM et al. Dev Biol 298, 118-31 (2006); Monk, KR et al., Science 325, 1402-5 (2009)). The myelin sheath enables fast action potential conduction by axons in the vertebrate nervous system and improves nerve conduction velocity. Scoliosis is also found in various neurological disorders (Kouwenhoven, JW & Castelein, RM, Spine (Phila Pa 1976) 33, 2898-908 (2008); Carter, GT et al., Am J Phys Med Rehabil 74 , S140-9 (1995)). Thus, the GPR126 gene may affect AIS sensitivity through nervous system dysregulation. For example, delayed escape response in morphants of the GPR126 gene may be related to nervous system defects.

  Note that the present invention is not limited to the theory discussed above in any sense.

  As described above, a case-control (Case-Control) -related analysis at the whole genome level has found a region that satisfies the genome-wide level and is related to AIS. SNPs present in this region are useful for examination of bone / joint diseases such as scoliosis and contribute to the prevention and / or treatment of bone / joint diseases such as scoliosis.

Claims (3)

Rs9403383, rs6909857, which is a base corresponding to the 61st base of the base sequence shown in SEQ ID NO: 1, or a base in linkage disequilibrium with the base present in the long arm 24.1 region of chromosome 6 rs972982, rs9403382, rs9389986, rs2050157, rs9496346, rs7741741, rs9389985, rs2039986, rs1891308, rs9403380, rs1418201, rs1040525, rs9496369, rs6570509 or rs6937121 based on the analysis results ,
When the base has a risk allele, the risk of onset of adolescent idiopathic scoliosis is high, or the possibility of adolescent idiopathic scoliosis is determined to be high, so that adolescent idiopathic scoliosis is examined. A method for determining the onset risk and / or presence of adolescent idiopathic scoliosis , characterized by A probe for testing adolescent idiopathic scoliosis having a sequence of 15 bases or more including the 61st base in the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof. A primer for testing adolescent idiopathic scoliosis that can amplify a region containing the base at position 61 in the base sequence shown in SEQ ID NO: 1.