阅读说明：本技术 Shank3片段序列甲基化检测试剂在制备精神分裂症诊断试剂盒中的用途 (Application of SHANK3 fragment sequence methylation detection reagent in preparation of schizophrenia diagnostic kit ) 是由 李涛 倪培艳 于 2021-07-16 设计创作，主要内容包括：本发明提供了SHANK3片段序列甲基化检测试剂在制备精神分裂症诊断试剂盒中的用途,属于体外诊断试剂技术领域。本发明研究发现SHANK3基因启动子长度为144bp的片段序列可以作为诊断精神分裂症的生物标记物。与健康人群相比,精神分裂症患者该片段序列第83、84位的CG和第109、110位的CG甲基化水平显著升高。因此,SHANK3基因启动子长度为144bp的片段序列甲基化检测试剂可制备精神分裂症诊断试剂盒,用于精神分裂症早期诊断,具有良好的应用前景。(The invention provides an application of a SHANK3 fragment sequence methylation detection reagent in preparation of a schizophrenia diagnostic kit, belonging to the technical field of in-vitro diagnostic reagents. The research of the invention finds that the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used as a biomarker for diagnosing schizophrenia. Compared with healthy people, the methylation level of CG at 83 th and 84 th positions and CG at 109 th and 110 th positions of the fragment sequence of the schizophrenia patient is obviously increased. Therefore, the methylation detection reagent of the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used for preparing a schizophrenia diagnosis kit, is used for early diagnosis of schizophrenia, and has good application prospect.)

1. The application of a reagent for detecting the methylation of the promoter fragment sequence of the SHANK3 gene in the preparation of a schizophrenia diagnostic kit; the sequence of the fragment is as follows:

TCAGCTGCACCACTCAGGCCAGGCCAGTGGCCTTGGGAGGGGCCTGTGATGCTGGGACCACAGTTCCTGGGCAGGGAGCAACCGTCTAGGCGTGGGGAGAACGCAGGACGTGACCCACACACCGCACTGGAGGCTCCGCTCTGC。

2. use according to claim 1, characterized in that: the sites of methylation of the fragment sequence are the CG at positions 83 and 84 and the CG at positions 109 and 110 of the fragment sequence.

3. Use according to claim 1, characterized in that: the reagent for detecting methylation of the fragment sequences is a sequencing reagent;

and/or the reagent for detecting the methylation of the fragment sequences is a PCR reagent.

4. Use according to claim 3, characterized in that: the reagent for detecting methylation of the fragment sequence is a reagent for pyrosequencing.

5. Use according to any one of claims 1 to 4, characterized in that: the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human cortical interneurons;

and/or the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human peripheral mononuclear cells.

6. Use according to claim 5, characterized in that: the human cortical interneuron is a cortical interneuron directionally induced by the human induced pluripotent stem cells.

7. Use according to claim 6, characterized in that: the human induced pluripotent stem cells are reprogrammed by human skin cells.

8. A diagnostic kit for schizophrenia characterized by comprising: it comprises a reagent for detecting methylation of a promoter fragment sequence of a SHANK3 gene; the sequence of the fragment is as follows:

TCAGCTGCACCACTCAGGCCAGGCCAGTGGCCTTGGGAGGGGCCTGTGATGCTGGGACCACAGTTCCTGGGCAGGGAGCAACCGTCTAGGCGTGGGGAGAACGCAGGACGTGACCCACACACCGCACTGGAGGCTCCGCTCTGC；

preferably, the sites of methylation of the fragment sequence are the CG at positions 83 and 84 and the CG at positions 109 and 110 of the fragment sequence.

9. The diagnostic kit for schizophrenia according to claim 8, wherein: the reagent for detecting methylation of the fragment sequences is a sequencing reagent;

and/or, the reagent for detecting the methylation of the fragment sequence is a PCR reagent;

preferably, the reagent for detecting methylation of the fragment sequence is a reagent for pyrosequencing.

10. The diagnostic kit for schizophrenia according to claim 8 or 9, characterized in that: the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human cortical interneurons;

and/or the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human peripheral mononuclear cells;

preferably, the human cortical interneurons are cortical interneurons directionally induced by human induced pluripotent stem cells;

more preferably, the human induced pluripotent stem cells are reprogrammed from human skin cells.

Technical Field

The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to an application of a SHANK3 fragment sequence methylation detection reagent in preparation of a schizophrenia diagnostic kit.

Background

Schizophrenia is a chronic disabling encephalopathy (SCZ) characterized by various damages of thinking, emotion, will behaviors and the like, and the chronic disabling encephalopathy mostly occurs in the late stage or early adult stage of teenagers, has a lifetime prevalence rate of about 1%, and can seriously affect the life, work, learning and social functions of patients. However, the pathophysiological mechanism of schizophrenia has not been fully elucidated so far, and the diagnosis and classification criteria of schizophrenia are still based on clinical symptoms and behavioral descriptions of patients, and lack of reliable biological basis for guiding clinical diagnosis and treatment, which greatly limits objective diagnosis, early intervention and effective treatment of schizophrenia in clinic, so that there is a need for developing an objective diagnostic reagent for schizophrenia.

The diagnostic reagent is prepared by adopting the principles or methods of immunology, microbiology, molecular biology and the like, and is used for diagnosing and detecting human diseases, investigating epidemiology and the like in vitro. The diagnostic reagent can be used for early diagnosis of diseases and has important significance for early intervention and effective treatment of the diseases. Therefore, the development of effective diagnostic agents for schizophrenia is important for the diagnosis and treatment of schizophrenia.

However, the lack of markers for diagnosing schizophrenia is a significant cause of the current difficulty in diagnosing schizophrenia. Due to the high complexity of the brain, it is difficult to accurately find biomarkers for the diagnosis of schizophrenia.

Therefore, there is a high necessity to find a biomarker for diagnosing schizophrenia.

Disclosure of Invention

The invention aims to provide application of a SHANK3 fragment sequence methylation detection reagent in preparation of a schizophrenia diagnostic kit.

The invention provides an application of a reagent for detecting the methylation of a promoter fragment sequence of a SHANK3 gene in preparing a schizophrenia diagnostic kit; the sequence of the fragment is as follows:

TCAGCTGCACCACTCAGGCCAGGCCAGTGGCCTTGGGAGGGGCCTGTGATGCTGGGACCACAGTTCCTGGGCAGGGAGCAACCGTCTAGGCGTGGGGAGAACGCAGGACGTGACCCACACACCGCACTGGAGGCTCCGCTCTGC。

further, the sites of methylation of the fragment sequence are CG at positions 83 and 84 and CG at positions 109 and 110 of the fragment sequence.

Further, the reagent for detecting the methylation of the fragment sequences is a reagent for sequencing;

and/or the reagent for detecting the methylation of the fragment sequences is a PCR reagent.

Furthermore, the reagent for detecting methylation of the fragment sequence is a reagent for pyrosequencing.

Further, the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human cortical interneurons;

and/or the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human peripheral mononuclear cells.

Further, the human cortical interneurons are cortical interneurons directionally induced by the human induced pluripotent stem cells.

Further, the human induced pluripotent stem cells are reprogrammed by human skin cells.

The invention also provides a schizophrenia diagnostic kit, which comprises a reagent for detecting the methylation of the promoter fragment sequence of the SHANK3 gene; the sequence of the fragment is as follows:

TCAGCTGCACCACTCAGGCCAGGCCAGTGGCCTTGGGAGGGGCCTGTGATGCTGGGACCACAGTTCCTGGGCAGGGAGCAACCGTCTAGGCGTGGGGAGAACGCAGGACGTGACCCACACACCGCACTGGAGGCTCCGCTCTGC；

preferably, the sites of methylation of the fragment sequence are the CG at positions 83 and 84 and the CG at positions 109 and 110 of the fragment sequence.

Further, the reagent for detecting the methylation of the fragment sequences is a reagent for sequencing;

and/or, the reagent for detecting the methylation of the fragment sequence is a PCR reagent;

preferably, the reagent for detecting methylation of the fragment sequence is a reagent for pyrosequencing.

Further, the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human cortical interneurons;

and/or the reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene is a reagent for detecting the methylation of the sequence of the promoter fragment of the SHANK3 gene in human peripheral mononuclear cells;

preferably, the human cortical interneurons are cortical interneurons directionally induced by human induced pluripotent stem cells;

more preferably, the human induced pluripotent stem cells are reprogrammed from human skin cells.

The research of the invention finds that the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used as a biomarker for diagnosing schizophrenia. Compared with healthy people, the methylation level of CG at 83 th and 84 th positions and CG at 109 th and 110 th positions of the fragment sequence of the schizophrenia patient is obviously increased. Therefore, the methylation detection reagent of the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used for preparing a schizophrenia diagnosis kit, is used for early diagnosis of schizophrenia, and has good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a result of diagnosis of schizophrenia using the biomarkers of the present invention; wherein, a is the process of directional differentiation of cortical interneurons, b is the result of immunofluorescence staining of cortical interneurons of patients with Schizophrenia (SCZ) and normal control groups (HC), and c is the comparison of the methylation levels of CG (CG1) at positions 83 and 84 and CG (CG4) at positions 109 and 110 in promoter fragment sequences of genes of patients with Schizophrenia (SCZ) and normal control groups (HC) SHANK 3.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1 diagnosis of schizophrenia Using biomarkers

1. The gene name: SHANK3

2. Fragment sequence (length 144bp, underlined two CGs are hypermethylation sites):

TCAGCTGCACCACTCAGGCCAGGCCAGTGGCCTTGGGAGGGGCCTGTGATGCTGGGACCACAGTTCCTGGGCAGGGAGCAACCGTCTAGGCGTGGGGAGAACGCAGGACGTGACCCACACACCGCACTGGAGGCTCCGCTCTGC(SEQ ID NO.1)

the hypermethylation sites are CG at positions 83 and 84 and CG at positions 109 and 110 of the fragment sequence respectively.

3. Experimental methods

Induced pluripotent stem cells (iPSCs) from 5 schizophrenic patients and 4 normal controls were prepared and directed to induce cortical interneurons using reprogramming techniques. The significant increase of the methylation level of the gene fragment sequence in the schizophrenia patients compared with the normal control is verified in the neurons, and the increase of the methylation level of the fragment is proved to be a biological marker for schizophrenia diagnosis.

The specific method comprises the following steps:

a. cortical interneuron differentiation method: reference is made to Peiyan Ni, Haneul Noh, Zhuiching Shao, Qian Zhu, Youxin Guan, Joshua J.park, Fatima Arif, James M.park, Chiderah Abani, Cameron Beaudreault, Joy S.park, Elizabeth Berry, Alexander Moghadam, Patric Stanton, John N N.Hutchon, Biandrews, Clare Faux, John Parnevelas, Leonard M.Eisenberg, Kyunjook Park, Vadim Y.Bolshakov, Sangmi 201ung, Large-Scale Generation and Characterification of Hormogeneous powders of cell filtration, moisture of cell, gradient 430, Pluronic Stem City crack, moisture filtration of moisture, device of cell, device family of moisture, device family of cell, device family of moisture, moisture of cell, moisture of cell of the family of the genus of the family of the species of the family of the genus of the species of the family of the species of the family of the species of the genus of the family of the genus of the family of the genus of the family of the genus of the family.

i. Preparation of Induced pluripotent Stem cells (iPSCs)

Reprogramming is accomplished by conventional techniques from skin cells derived from the subject. The subjects (schizophrenic patients and normal controls) were all from Mclean Hospital, Harvard medical school, and the detailed information was referred to (Mol Psychiatry,2020,25(11): 2873-2888.) as part of the samples in the articles, namely ID 292, L5, L7 and L9 of HC group (normal control group) in article Figure 3a, and ID 58, 285, 483, 1442 and L8 of SCZ group (schizophrenic patients).

ii directional differentiation of cortical interneurons

Cortical interneurons are committed to differentiate as shown in FIG. 1 a.

(1) The prepared iPSCs were cultured in KSR medium (20% serum replacement, 2mM L-glutamine and 10. mu.M. beta. -mercaptoethanol in DMEM medium) at week 1, and then cultured with L (LDN193189,100nM), S (SB431542, 10. mu.M), Sg (SHH signaling pathway agonist SAG, 0.1. mu.M) and W (Wnt signaling pathway inhibitor IWP2, 5. mu.M), and 10. mu.M of ROCK inhibitor (Y-27632) was added on the day of differentiation. And obtaining the neurosphere after differentiation.

(2) KSR medium was supplemented with L (ldn193189,100nm) and Sg (SHH signaling pathway agonist SAG,0.1 μ M) at week 2; and the neurospheres are transferred to spinner conditions for culture (80rpm), and the obtained 3D embryoid bodies are uniform in size and stable in cell viability.

(3) Sg (0.1. mu.M) and FGF8(100ng/ml) were added at week 3 using N2 medium (2% N2 and 200. mu.M ascorbic acid in DF12 medium); after three weeks, neural precursor cells are obtained, and after optimized pancreatin (0.5% pancreatin, 2U/ml Turbo DNase, 1mM Trehalose) digestion, the neural precursor cells are inoculated on a glass slide, and after fixation, immunofluorescence staining is carried out to identify the proportion of Nkx2.1 and Nestin. Simultaneously, a one-week synchronization of Curtureone (1 ‰), DAPT (10. mu.M) and PD0332991 (2. mu.M) was initiated.

(4) After 4 weeks, the medium was changed to B27 medium (2% B27 in DF12 medium) and 10ng/ml GDNF and 10ng/ml BDNF were added; cortical interneurons (cINs) were obtained after 8 weeks, digested with optimized pancreatin (0.5% pancreatin, 2U/ml Turbo DNAse, 1mM Trehalose), inoculated on slides, and fixed for immunofluorescence staining to identify the ratios of GAD, SOX6 and β -Tublin.

b. Phenotypic identification of cortical interneurons:

and (3) performing phenotype identification on the differentiated cortical interneurons of each subject by adopting an immunofluorescence staining method, wherein fig. 1b shows a typical staining result, and table 1 shows the specific expression amount of each marker of the differentiated cortical interneurons of the ipscs from each tested source. The conclusion was that there was no difference in phenotype between the schizophrenic patients and the normal controls, suggesting that the subsequent differences in methylation levels were not due to abnormal cellular phenotype.

TABLE 1 specific expression levels of each marker for iPSC differentiated cortical interneurons from each test source

c. The SHANK3 gene promoter has high methylation level in cortical interneuron

Extracting genome DNA of cortical interneurons of the subjects, and carrying out methylation sequencing on the fragment sequence (SEQ ID NO.1, 144bp) of the SHANK3 gene promoter by adopting a pyrosequencing method. As a result, the methylation level of two CG sites (CG at 83 th and 84 th positions and CG at 109 th and 110 th positions of the fragment sequence, respectively, underlined in the fragment sequence) in the fragment sequence of the schizophrenia patient is remarkably increased compared with that of the normal control, and the result is displayed as a histogram (FIG. 1c, CG at 83 th and 84 th positions is CG1, and CG at 109 th and 110 th positions is CG4 in FIG. 1 c).

Example 2 diagnosis of schizophrenia Using biomarkers

Except that the methylation level of the above-mentioned fragment sequence of the promoter of the SHANK3 gene was detected in the cortical interneuron of example 1; schizophrenia can also be diagnosed by detecting the methylation level of the sequence of the fragment of the SHANK3 gene promoter in peripheral mononuclear cells.

In conclusion, the research of the invention finds that the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used as a biomarker for diagnosing schizophrenia. Compared with healthy people, the methylation level of CG at 83 th and 84 th positions and CG at 109 th and 110 th positions of the fragment sequence of the schizophrenia patient is obviously increased. Therefore, the methylation detection reagent of the segment sequence with the length of 144bp of the SHANK3 gene promoter can be used for preparing a schizophrenia diagnosis kit, is used for early diagnosis of schizophrenia, and has good application prospect.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

Application of <120> SHANK3 fragment sequence methylation detection reagent in preparation of schizophrenia diagnosis kit

<130> GYKH1970-2021P0113328CCR4

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 144

<212> DNA

<213> Artificial sequence

<400> 1

tcagctgcac cactcaggcc aggccagtgg ccttgggagg ggcctgtgat gctgggacca 60

cagttcctgg gcagggagca accgtctagg cgtggggaga acgcaggacg tgacccacac 120

accgcactgg aggctccgct ctgc 144