CN113789376A

CN113789376A - Kit for predicting and screening adolescent idiopathic scoliosis and application

Info

Publication number: CN113789376A
Application number: CN202111359559.XA
Authority: CN
Inventors: 纪志盛; 马彦明; 高育京; 张国威; 林宏生
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-11-17
Filing date: 2021-11-17
Publication date: 2021-12-14
Anticipated expiration: 2041-11-17
Also published as: CN113789376B

Abstract

The invention provides a kit for predicting and screening adolescent idiopathic scoliosis, which comprises a reagent for detecting TTN gene mutation and/or expression level. The AIS disease condition of a human population is predicted and screened by detecting the mutation condition of the TTN gene so as to carry out related prevention in advance, obviously reduce the damage of the disease to the health of the human body, and simultaneously contribute to carrying out prognosis evaluation on a patient with the disease, thereby providing reasonable and effective guidance for treatment and rehabilitation; due to the obvious family genetic correlation of the AIS disease, TTN gene detection results can be utilized to provide bearing and rearing instructions and genetic challenges for a subject and a family thereof, and the birth of related children is reduced; in addition, a new drug treatment target point can be provided for human beings to overcome AIS, so that a new direction is provided for subsequent drug development, clinical treatment and the like.

Description

Kit for predicting and screening adolescent idiopathic scoliosis and application

Technical Field

The invention belongs to the field of biomedicine, and relates to a kit for predicting and screening adolescent idiopathic scoliosis and application thereof.

Background

Adolescent Idiopathic Scoliosis (AIS) refers to a curved spinal deformity formed by one or more segments of the spine curving laterally away from the body midline in the coronal plane, usually accompanied by an increase or decrease in Scoliosis and anterior or posterior processes in the sagittal plane of the spine, occurring during adolescents, with an incidence of about 1% -3% in adolescents 10-18 years, with girls being more common than boys, one of the most common spinal deformities in adolescents.

Adolescent idiopathic scoliosis is the most common type of idiopathic scoliosis, accounting for 0.47-5.2% of scoliosis and accounting for 90% of idiopathic scoliosis. The cause of AIS is not clear and may be related to factors such as family genetics, melatonin levels, platelet structure and function. It is currently believed that the cause of AIS may be due to multiple factors. Currently, epidemiology indicates that AIS is familial aggregating. Andersen research finding on the incidence of twins idiopathic scoliosis: concordance rate of monozygotic twins is 73% -92%, concordance rate of heterozygotic twins is 36% -63%, and genetic inheritance is considered as one of AIS diseases. The development of molecular biology technology makes it possible to use whole exon sequencing to assist clinical diagnosis. Therefore, researchers have utilized methods of gene screening and candidate genes to find disease genes from patients with disease. Kris also found abnormalities in chromosome 19p13 in large samples (202 families 1198) using gene screening, but did not progress substantially.

Current research is not well understood for AIS pathogenesis. Genetic factors play a non-negligible role in the pathogenesis of AIS. Several studies based on twins and AIS families have shown that genetic factors play a significant role in the development and progression of AIS. In 2003, Jussice et al developed an autosomal dominant inheritance pattern for AIS. Although some studies have now demonstrated the effect of genetic mutations on the development and progression of AIS, it is still not possible to fully explain the genetic mechanism of AIS and guide therapy.

Disclosure of Invention

The invention aims to solve the technical problems that in the prior art, the pathogenesis of adolescent idiopathic scoliosis is unclear and advance prevention is difficult to carry out, so that the kit for predicting and screening the adolescent idiopathic scoliosis is provided. TTN is selected as a marker, so that effective prediction can be performed on adolescent idiopathic scoliosis, and high-morbidity population of AIS can be screened out so as to perform reasonable advanced prevention, remarkably reduce the probability and severity of disease occurrence, and reduce the damage to human health.

In order to solve the above technical problems, the present invention is achieved by the following technical solutions.

The invention provides a kit for predicting and/or screening adolescent idiopathic scoliosis in a first aspect, which comprises an agent for detecting TTN gene mutation and/or expression level.

Preferably, the TTN gene is mutated at a site selected from one or more of the following sites: c.G178T, c.T455C, c.G4322C, c.G22498T, c.G31864A, c.G36508A, c.C51482T, c.G76343A, c.C91276T, c.A98164T, c.G100579A, c.G102833T, c.C105782T, c.T107267C. Most preferably, the site of the TTN gene mutation is selected from one or more of c.g4322, c.c91276t, c.g 102833t.

Preferably, the reagent is selected from one or more of gene specific primers, genomic sequencing reagents, antibodies specific for gene expression products.

Preferably, the gene-specific primers are selected from at least one of the following pairs of primers:

(1) primer pair 1: the sequence of the forward primer is shown as SEQ ID NO. 1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;

(2) and (3) primer pair 2: the sequence of the forward primer is shown as SEQ ID NO. 3, and the sequence of the reverse primer is shown as SEQ ID NO. 4;

(3) and (3) primer pair: the sequence of the forward primer is shown as SEQ ID NO. 5, and the sequence of the reverse primer is shown as SEQ ID NO. 6;

(4) and (3) primer pair 4: the sequence of the forward primer is shown as SEQ ID NO. 7, and the sequence of the reverse primer is shown as SEQ ID NO. 8;

(5) and (3) primer pair 5: the sequence of the forward primer is shown as SEQ ID NO. 9, and the sequence of the reverse primer is shown as SEQ ID NO. 10;

(6) and (3) primer pair 6: the sequence of the forward primer is shown as SEQ ID NO. 11, and the sequence of the reverse primer is shown as SEQ ID NO. 12;

(7) and (3) primer pair 7: the sequence of the forward primer is shown as SEQ ID NO. 13, and the sequence of the reverse primer is shown as SEQ ID NO. 14;

(8) and (3) primer pair 8: the sequence of the forward primer is shown as SEQ ID NO. 15, and the sequence of the reverse primer is shown as SEQ ID NO. 16;

(9) and (3) primer pair 9: the sequence of the forward primer is shown as SEQ ID NO. 17, and the sequence of the reverse primer is shown as SEQ ID NO. 18;

(10) a primer pair 10: the sequence of the forward primer is shown as SEQ ID NO. 19, and the sequence of the reverse primer is shown as SEQ ID NO. 20;

(11) a primer pair 11: the sequence of the forward primer is shown as SEQ ID NO. 21, and the sequence of the reverse primer is shown as SEQ ID NO. 22;

(12) primer pair 12: the sequence of the forward primer is shown as SEQ ID NO. 23, and the sequence of the reverse primer is shown as SEQ ID NO. 24;

(13) a primer pair 13: the forward primer sequence is shown as SEQ ID NO. 25, and the reverse primer sequence is shown as SEQ ID NO. 26.

Preferably, the antibody specific for the gene expression product is selected from a TTN monoclonal antibody and/or a TTN polyclonal antibody.

Preferably, the TTN protein content detection reagent is selected from one or more of TTN monoclonal antibody M06 (brand Abnova, product code: H00007273-M06), Anti-tin antibody (brand Abcam, product code: ab 284860).

Preferably, the kit further comprises one or more of PCR enzymes, PCR buffers, dNTPs, fluorescent substrates.

Preferably, the fluorogenic substrate is selected from Syber Green or a fluorescently labeled probe.

In a second aspect, the present invention provides a use of a composition for preparing a kit for predicting and/or screening adolescent idiopathic scoliosis, the composition comprising an agent for detecting a TTN gene mutation and/or expression level.

In a third aspect, the invention provides an application of an agent for detecting TTN gene mutation and/or expression level in preparing a composition for predicting and/or screening adolescent idiopathic scoliosis.

In a fourth aspect, the invention provides an application of an agent for detecting TTN gene mutation and/or expression level in preparing a composition for adolescent idiopathic scoliosis prognosis evaluation.

Without being particularly specified, in the context of the present invention, the primer and/or primer set refers to a PCR primer for synthesizing a cDNA strand of the TTN gene in a PCR, thereby detecting an expression level of the TTN gene.

The protein tinin encoded by the TTN gene is the largest protein known to date and is also the third protein in the muscle of vertebrates, spanning the entire hemisarcomere from the M-line to the Z-plate. Titin plays many important roles in striated muscle, including maintenance of sarcomere integrity, passive generation of force, and assembly of myofibrils. Due to its size and repetitive sequence, the TTN gene exhibits a high degree of variability. Most disease-related TTN gene variations have a large effect on actin expression, including insertion/deletion mutations, splicing mutations, and the like. There are studies that have shown that mutations in the TTN gene are associated with certain muscle diseases, which may include AIS. The different mechanisms of post-transcriptional and post-translational modification of Titin, as well as the diversity and complexity of the signal functions, have led to the unclear study of the mechanisms underlying muscle disease by this protein.

The inventor finds that in many cases of AIS, usually three generations of idiopathic scoliosis cases exist in a family, through a large amount of clinical and laboratory researches, and therefore deduces that the occurrence of the disease can be highly correlated with certain specific genes. Further, through in vitro experiments combined with whole exome sequencing and molecular biology results, different degrees of mutation of TTN were found in AIS patients. Therefore, the TTN gene mutation has obvious correlation with the occurrence of AIS, and can be used as a new biomarker for the occurrence of AIS. The invention discovers that the mutation of the gene TTN is closely related to AIS, provides an index for predicting the genetic tendency of the AIS, and is beneficial to the advanced prevention of the AIS.

Compared with the prior art, the invention has the following technical effects:

(1) the invention carries out deep research on the pathogenesis of adolescent idiopathic scoliosis, and discovers that the mutation of gene TTN is a factor highly related to AIS pathogenesis; therefore, whether the TTN gene of the subject is mutated or not is judged by detecting the TTN gene of the subject, so that adolescent idiopathic scoliosis patients can be effectively detected and screened, namely the variation of the TTN gene can be used as a biomarker for clinically and auxiliarily diagnosing and screening adolescent idiopathic scoliosis diseases.

(2) Whether a subject carries the TTN variant gene or not can be used for screening out the variant carrier at an early stage, so that the advance preparation work is carried out for preventing the disease, the rapid development and deterioration of the disease are prevented from causing impossible health damage to the patient, the prognosis of the patient can be reasonably evaluated, and a reasonable and effective guiding effect is provided for treatment and rehabilitation; meanwhile, TTN gene detection results can be used for providing bearing and child care guidance and genetic inquiry for the subject and family, and reducing the birth of related children.

(3) The invention provides a new drug treatment target point for the human attack AIS by disclosing the relevance of TTN gene mutation on adolescent idiopathic scoliosis, thereby providing a new direction for subsequent drug development, clinical treatment and the like.

Drawings

FIG. 1 is a schematic flow diagram of whole exon sequencing, detection and filtration.

FIG. 2 is a genetic pedigree map of AIS family.

FIG. 3 is a diagram showing the result of the analysis of the single nucleotide polymorphism sites of the AIS family members.

FIG. 4 is a graph of Wein comparing single base variation sites of AIS patients with normal persons.

FIG. 5 is a diagram showing the single-base mutation sites of AIS patients.

FIG. 6 is a schematic diagram of GO classification results for 86 genes of AIS-associated SNPs.

FIG. 7 is a diagram showing the results of AIS shared gene pathway analysis.

FIG. 8 is a schematic representation of the results of ClueGO and CluePedia analyses performed on the same gene.

FIG. 9 is a diagram showing the analysis of the number of base mutations of the AIS-related gene.

FIG. 10 is a schematic diagram of the distribution of 14 non-synonymous mutations of TTN in TTN protein.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Among the reagents used in the context of the present invention, those commercially available are not specifically mentioned. For the use of clinical specimens, informed consent is signed with patients, and related procedures and methods meet medical ethics requirements and quality management specifications of clinical trials of drugs. The experimental methods used in the present invention, such as DNA extraction, whole genome sequencing, primer design, GO classification and channel analysis, are all conventional methods and techniques in the art.

Representative results from selection of biological experimental replicates are presented in the context figure, and data are presented as mean ± SD and mean ± SEM as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 5.0 or SPSS 22.0 software. And comparing the difference of the average values of two or more groups by adopting conventional medical statistical methods such as t test, chi-square test, variance analysis and the like. p < 0.05 was considered a significant difference.

Example 1 AIS pathogenic Gene analysis

Blood samples are collected from other 3 AIS patients in AIS patients and families thereof, and DNA fragments are extracted by the following specific steps:

(1) taking 400 mu L of venous blood, adding 1mL of RCL into the blood sample, reversing and mixing the venous blood and the RCL evenly for several times, and placing the venous blood at room temperature for 5 minutes;

(2) centrifuging at 10000 Xg for 3 min, removing supernatant, and leaving leukocyte precipitate;

(3) adding 200 mu L B3 and 2 mu l protease K, immediately vortex and uniformly mixing, standing at 65 ℃ for 10 minutes, uniformly mixing for several times, and keeping the solution clear;

(4) adding 200 mu L of isopropanol, and fully reversing and mixing, wherein filamentous or clustered genome DNA can appear;

(5) centrifuging at 10000 Xg for 5 min, and removing supernatant;

(6) adding 200 μ l 70% ethanol, vortexing for 5 seconds, centrifuging at 10000 Xg for 5 minutes, removing supernatant, and repeating once;

(7) the DNA precipitate was dried until all the liquid was evaporated. Add 200uEB, vortex at low speed for 5 seconds, and place at 65 ℃ for 10 minutes to 1 hour to dissolve DNA, during which flick several times to help the dissolution.

Subsequently, the DNA obtained by extraction according to the above steps is subjected to whole exon sequencing to screen the pathogenic gene of AIS, and it is detected and filtered.

The results are shown in FIGS. 1 to 3. FIG. 1 is a schematic diagram of the whole exon sequencing, detection and filtration process in this example. The results show that there are usually many cases of AIS in the AIS patient family, and the genetic pedigree is shown in fig. 2, showing that AIS is autosomal dominant. As a result of gene detection, more than 90000 Single base variations (SNV) were detected for each family member (see FIG. 3).

Analysis of AIS patient family members by wien plots revealed a total of 732 overlapping SNVs in all affected individuals (see figure 4). Among these 732 Single Nucleotide Polymorphisms (SNPs), 506 SNPs were annotated as being functionally unknown, 112 SNPs as synonymous mutations, and 114 SNPs as nonsynonymous mutations (see fig. 5).

Example 2 analysis of relationship between AIS-related mutant genes and morphogenesis of muscle tissue

As can be seen from example 1 above, there were 114 nonsynonymous mutant SNPs in AIS patients. Since nonsynonymous mutations may change the expressed amino acids and may affect the function of the protein, 114 nonsynonymous mutations of 86 AIS patients sharing genes were further selected for further analysis.

By GO classification and channel analysis of 86 AIS patients sharing genes, it was found that the most abundant class of these genes in biological processes is cellular processes, cells, cellular parts and organelles represent the major proportion of cellular components, with binding accounting for a large part of the molecular function (see figure 6). Pathway analysis showed that the shared genes were mainly enriched in 10 pathways, including inflammatory signaling pathway, Rho GTPase regulation of cytoskeleton, and integrin signaling pathway (see fig. 7). Meanwhile, it was found that morphogenesis of muscle tissue is the most important biological process, and genes including TTN and the like are involved in this process (see FIG. 8).

Example 3 analysis of the relationship between TTN mutations and AIS pathogenesis

The number of base mutations in the AIS-sharing gene of the patient of example 1 was analyzed, and the results are shown in FIGS. 9 to 10. The results showed that most AIS-associated mutant genes had only 1 SNP, but that TTN had 14 non-synonymous SNPs (see fig. 9). The distribution of these 14 mutants in the TTN protein is shown in fig. 10. Since the TTN gene encodes titin, which is the largest known protein and is closely related to muscle development, it is an excellent candidate gene for AIS.

Subsequently, to confirm whether there was a correlation between the TTN mutation and the onset of AIS, the test sample size was further expanded and 140 AIS patients and 121 normal persons were subjected to TTN sequencing. Firstly, 13 pairs of primers are designed according to the mutation sites of TTN, wherein the first pair of primers comprises two mutation sites, and the rest of primer pairs respectively comprise 1 mutation site. The sequences of the primer pairs and the mutation sites targeted are shown in table 1 below.

TABLE 1 sequences of the primer pairs

Subsequently, DNA in the serum of the subjects was extracted, PCR was performed by designed primers, the obtained PCR products were sequenced, and all 14 variation analyses were tested in the TTN gene by Sanger sequencing for TTN mutation in AIS patients and normal humans. The results are shown in table 2 below:

TABLE 2 TTN mutation in AIS patients and Normal humans

	Is not affected	Disease of the disease	Total of
				TTN wild type	110	44	154
Mutant TTN	11	96	107
				Total of	121	140	261

Among them, a total of 7 mutations were confirmed in AIS patients carrying TTN mutants, which were c.g4322, c.c91276t, c.g102833t, c.g36508a, c.a98164t, c.c105782t, and c.t 107267c. At least one of the 7 site mutations in the TTN gene was found in 96 patients with TTN mutant AIS.

The chi-square test analysis was performed on the above results, which are shown in table 3 below:

TABLE 3 chi fang test results for TTN mutations in AIS patients and normal humans

Note that: a: 0 cells (0.0%) have an expected count of less than 5. The minimum expected count is 49.61. b: only 2 x 2 table calculations.

Further, the calculated scores of PolyPhen-2 and SIFT were used for the prediction of harmfulness of the non-synonymous variants within the above 7 TTNs, where the variants of c.g4322, c.c91276t and c.g102833t meet the conditions (SIFT score less than 0.05, PolyPhen-2 score greater than 0.95, not shown in the figure).

From the above results, it was found that the percentage of variation in the TTN gene was as high as 68.7% and the percentage of the wild-type TTN gene was only 31.4% in AIS confirmed patients. Therefore, it can be clear that the in vivo TTN gene has 7 SNP site mutations of c.G4322, c.C91276T, c.G102833T, c.G36508A, c.A98164T, c.C105782T and the like, especially the AIS incidence rate of the population with three SNP site mutations of c.G4322, c.C91276T and c.G102833T is obviously increased compared with the population with the wild TTN gene, and the difference has the statistical significance of (thepLess than 0.001), namely, the TTN gene mutation has obvious correlation with the AIS. Therefore, the AIS disease condition of a human population can be predicted and screened by detecting the mutation condition of the TTN gene so as to carry out related prevention in advance, obviously reduce the damage of the disease to the health of the human body, and simultaneously contribute to carrying out prognosis evaluation on a patient with the disease, thereby providing reasonable and effective guidance for treatment and rehabilitation; according to the analysis, the AIS disease has obvious family genetic correlation, so that a TTN gene detection result can be used for providing bearing and rearing instructions and genetic inquiry for a subject and a family thereof, and the birth of related children is reduced; in addition, a new drug treatment target point can be provided for human beings to overcome AIS, so that a new direction is provided for subsequent drug development, clinical treatment and the like.

The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

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Claims

1. A kit for predicting and/or screening adolescent idiopathic scoliosis comprising an agent for detecting a TTN gene mutation and/or expression level at a site selected from one or more of the following sites: c.G178T, c.T455C, c.G4322C, c.G22498T, c.G31864A, c.G36508A, c.C51482T, c.G76343A, c.C91276T, c.A98164T, c.G100579A, c.G102833T, c.C105782T, c.T107267C.

2. The kit of claim 1, wherein the reagents are selected from one or more of gene specific primers, genomic sequencing reagents, antibodies specific for gene expression products.

3. The kit according to claim 2, wherein the gene-specific primers are selected from at least one of the following pairs of primers:

4. Use of a composition for the manufacture of a kit for predicting and/or screening adolescent idiopathic scoliosis, said composition comprising an agent for detecting a TTN gene mutation and/or expression level at a site selected from one or more of the following sites: c.G178T, c.T455C, c.G4322C, c.G22498T, c.G31864A, c.G36508A, c.C51482T, c.G76343A, c.C91276T, c.A98164T, c.G100579A, c.G102833T, c.C105782T, c.T107267C.

5. Use of an agent for detecting TTN gene mutation and/or expression level for the preparation of a composition for predicting and/or screening adolescent idiopathic scoliosis, wherein the site of TTN gene mutation is selected from one or more of the following sites: c.G178T, c.T455C, c.G4322C, c.G22498T, c.G31864A, c.G36508A, c.C51482T, c.G76343A, c.C91276T, c.A98164T, c.G100579A, c.G102833T, c.C105782T, c.T107267C.

6. Use of an agent for detecting TTN gene mutation and/or expression level for the preparation of a composition for adolescent idiopathic scoliosis prognosis evaluation, wherein the site of TTN gene mutation is selected from one or more of the following sites: c.G178T, c.T455C, c.G4322C, c.G22498T, c.G31864A, c.G36508A, c.C51482T, c.G76343A, c.C91276T, c.A98164T, c.G100579A, c.G102833T, c.C105782T, c.T107267C.