JP2007236300A - Method for judging risk of developing integrated dystonia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging the risk of developing integrated dystonia. <P>SOLUTION: The method for judging the risk of developing the integrated dystonia comprises detecting polymorphisms of an fyn gene in the genomic DNA of a subject. A primer and a probe used in the method are provided. A kit for carrying out the method is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for determining the risk of developing schizophrenia, comprising examining polymorphisms of the fyn gene.

  Schizophrenia is a common mental illness that affects approximately 1% of the population. Family, twin, and adoptive studies have shown that heredity plays an important role in the pathogenesis of schizophrenia (1). Linkage analysis reported that several loci on chromosomes 1q, 4q, 5p, 5q, 6p, 6q, 8p, 9q, 10p, 13q, 15q, 22q, and Xp are linked to schizophrenia (Non-patent document 2). In 6q, Cao et al. Showed evidence suggesting linkage in 6q21-22.3 (Non-patent Document 3). This finding is based on the data from the Genetics Initiative (Non-Patent Document 4), multi-center analysis (Non-Patent Document 5), and genome scan meta-analysis (Non-Patent Document 6) of the National Institute of Mental Health (NIMH). It is supported. However, the susceptibility gene for schizophrenia has not yet been identified.

Fyn, a member of the Src family tyrosine kinase, is strongly expressed in the brain, and its gene is located at 6q21. In the mouse brain, Fyn is involved in neural functions such as axon guidance, synapse formation, and neurotransmission (Non-Patent Document 7). In synapses, Fyn localizes to post-synaptic membrane thickening and regulates its activity by phosphorylating NMDA receptor subunits (Non-patent Document 8). Disrupting the fyn gene in mice results in abnormal hippocampal pyramidal neurons (Non-patent Document 9), abnormal formation of cortical layer II / III (Non-patent Document 10), and myelin formation disorder in the forebrain (Non-patent Document 11). ) Causes various brain abnormalities. Interestingly, hippocampal sequence abnormalities (Non-Patent Documents 12-14), upper cortical dysplasia (Non-patent Documents 15-17), and myelinization disorders (Non-patent Documents 18, 19) are associated with schizophrenic patients. It has also been reported in the brain. In terms of behavior, fyn-deficient mice show emotional deficits such as excessive fear and maternal behavioral impairment (Non-Patent Document 7). Recently, the inventors have found that the dopamine receptor blocker haloperidol, a typical antipsychotic, induces Fyn activation and NMDA receptor subunit phosphorylation in the mouse striatum (Non-Patent Literature). 20). Thus, Fyn is also involved in dopamine receptor function. NMDA receptor blockers and dopamine receptor stimulants induce psychiatric symptoms similar to schizophrenia, and dopamine receptor blockers are used to treat schizophrenia. Abnormal body function is assumed. Furthermore, it has been reported that the expression of the fyn gene is increased in the postmortem brain of schizophrenic patients (Non-patent Document 21). For these reasons, the fyn gene is a candidate for a schizophrenia susceptibility gene on 6q21-22. However, no SNP on the fyn gene correlated with schizophrenia has been known so far (Non-patent Documents 22 and 23).
JP-A-2005-55227 TA Ban, Prog Neuropsychopharmacol Biol Psychiatry 28, 753-62 (Aug, 2004). M. Baron, Am J Hum Genet 68, 299-312 (Feb, 2001). Q. Cao et al., Genomics 43, 1-8 (Jul 1, 1997). M. Martinez et al., Am J Med Genet 88, 337-43 (Aug 20, 1999). DF Levinson et al., Am J Hum Genet 67, 652-63 (Sep, 2000). CM Lewis et al., Am J Hum Genet 73, 34-48 (Jul, 2003). T. Yagi, Biochem Pharmacol 57, 845-50 (Apr 15, 1999). MW Salter, LV Kalia, Nat Rev Neurosci 5, 317-28 (Apr, 2004). SG Grant et al., Science 258, 1903-10 (Dec 18, 1992). S. Yuasa, K. Hattori, T. Yagi, Neuroreport 15, 819-22 (Apr 9, 2004). BR Sperber et al., J Neurosci 21, 2039-47 (Mar 15, 2001). AJ Conrad, T. Abebe, R. Austin, S. Forsythe, AB Scheibel, Arch Gen Psychiatry 48, 413-7 (May, 1991). JA Kovelman, AB Scheibel, Acta Neurol Scand 73, 1-32 (Jan, 1986). MF Casanova, B. Rothberg, Schizophr Res 55, 19-24 (May 1, 2002). SE Arnold, BT Hyman, GW Van Hoesen, AR Damasio, Arch Gen Psychiatry 48, 625-32 (Jul, 1991). FM Benes, SL Vincent, M. Todtenkopf, Biol Psychiatry 50, 395-406 (Sep 15, 2001). LD Selemon, G. Rajkowska, PS Goldman-Rakic, J Comp Neurol 392, 402-12 (Mar 16, 1998). KL Davis et al., Arch Gen Psychiatry 60, 443-56 (May, 2003). D. Tkachev et al., Lancet 362, 798-805 (Sep 6, 2003). K. Hattori, S. Uchino, T. Isosaka, M. Maekawa, M. Iyo, T. Sato, S. Kohsaka, T. Yagi, S. Yuasa, J Biol Chem, Epu ahead of print (Jan 2006). T. Ohnuma, H. Kato, H. Arai, PJ McKenna, PC Emson, Brain Res Mol Brain Res 112, 90-4 (Apr 10, 2003). H. Ishiguro, T. Saito, H. Shibuya, M. Toru, T. Arinami, Am J Med Genet 96, 716-20 (Dec 4, 2000). T. Ohnuma et al., Neurosci Lett 343, 70-2 (May 29, 2003).

  An object of the present invention is to provide a method for determining the risk of developing schizophrenia based on the fyn gene.

  The present invention implements a method for determining the risk of developing schizophrenia, comprising detecting a polymorphism of the fyn gene in the genomic DNA of a subject, primers and probes used in the method, and the method Providing a kit for

  The present invention makes it possible to easily determine the risk of developing schizophrenia at the gene level. The method of the present invention is useful for the selection of prevention and treatment of schizophrenia.

  The present invention provides a method for determining the risk of developing schizophrenia, comprising detecting a polymorphism of the fyn gene in a subject's genomic DNA. The base sequence of the fyn gene is disclosed in GenBank HS66H14 and is shown in SEQ ID NO: 1.

  The genomic DNA of the subject can be prepared from a biological sample collected from the subject by a known method. The biological sample is not particularly limited as long as it contains genomic DNA, and examples thereof include various cells, tissues, organs, blood, body fluids, and the like of a subject.

  In a preferred embodiment, the polymorphism is a single nucleotide polymorphism (SNP) at position 5177 (position 5177 in the sequence shown in SEQ ID NO: 1) on the genomic DNA of the fyn gene. When the genotype of the polymorphic site in the subject's genomic DNA is AA, it can be determined that the subject has a high risk of developing schizophrenia.

  In another embodiment, the polymorphism is a deletion at positions 5123-5130 (positions 5123-5130 of the sequence described in SEQ ID NO: 1) on the genomic DNA of the fyn gene. If the deletion is present in the subject's genomic DNA, it can be determined that the subject is at high risk of developing schizophrenia.

Polymorphisms in genomic DNA can be detected by various known methods. Examples of detection methods include, but are not limited to:
TaqMan PCR method (Genet. Anal., 14, 143-149 (1999), J. Clin. Microbiol., 34, 2933-2936 (1996));
Invader method (Science, 5109, 778-783 (1993), J. Biol. Chem., 30, 21387-21394 (1999), Nat. Biotechnol., 17, 292-296 (1999));
Molecular Beacons method (Nat. Biotechnol., 1, p49-53 (1998), Gene Medicine, 4, p46-48 (2000));
DNA microarray or DNA chip method;
Direct sequencing method (Biotechniques, 11, 246-249 (1991));
RFLP (restriction fragment length polymorphism) method;
PCR-SSCP method (Biotechniques, 16,296-297 (1994), Biotechniques, 21, 510-514 (1996));
ASO (Allele Specific Oligonucleotide) hybridization method (Clin. Chim. Acta, 189, 153-157 (1990));
Denaturing Gradient Gel Electrophoresis (DGGE) method ((Biotechniqus, 27, 1016-1018 (1999));
RNaseA cleavage method (DNA Cell. Biol., 14, 87-94 (1995));
MALDI-TOF / MS (Matrix Assisted Laser Desorption-Time of Flight / Mass Spectrometry) method (Genome Res., 7, 378-388 (1997), Eur. J. Clin. Chem. Clin. Biochem., 35, 545- 548 (1997));
RCA method (Nat. Genet., 19, 225-232 (1998)).

  The method of the present invention may detect both polymorphisms of the fyn gene. The method of the present invention may be used in combination with other methods that can predict the risk of developing schizophrenia. For example, when the polymorphism is combined with other gene polymorphisms, more reliable determination is possible.

The present invention also provides a primer for amplifying a region containing at least one of the following positions of the fyn gene:
(A) position 5177 of the sequence set forth in SEQ ID NO: 1;
(B) Positions 5123-5130 of the sequence set forth in SEQ ID NO: 1.
The primer of the present invention is used for detection of a polymorphism at the above-mentioned position in the genomic DNA of a subject. The primer of the present invention is usually an oligonucleotide having 15 to 100 bases, preferably 15 to 30 bases, more preferably 18 to 24 bases. The nucleotides that make up the primer can be natural or non-natural nucleotides. The length of the region amplified by the primer of the present invention and the distance between the position where the primer hybridizes and the position are appropriately changed depending on the detection method used. The region amplified by the primer of the present invention may include both of the above positions. The sequence of the primer can be selected based on the sequence of the fyn gene, but may not be completely complementary to the sequence, and the base sequence may be modified or added depending on the detection method. Further, the primer of the present invention may be modified with a radioisotope, a fluorescent substance, a luminescent substance, an enzyme, biotin or the like for detection. Those skilled in the art can appropriately determine the primer sequence and design depending on the detection method used.

  Examples of primers of the present invention include 5'-GCT ACG TGT GGG TAT AGA TC-3 '(SEQ ID NO: 2) and 5'-CCC AAT TCT GAT GGG CAT GT-3' (SEQ ID NO: 3). These primers can be used in a direct sequencing method in which PCR and sequencing are performed.

The present invention also provides a probe that hybridizes to a region containing at least one of the following positions of the fyn gene:
(A) position 5177 of the sequence set forth in SEQ ID NO: 1;
(B) Positions 5123-5130 of the sequence set forth in SEQ ID NO: 1.
The probe of the present invention is also used for detecting a polymorphism in the base of genomic DNA of a subject. The probe of the present invention is an oligonucleotide having 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases, although it is appropriately changed depending on the detection method. The nucleotides that make up the probe can be natural or non-natural nucleotides. The probe of the present invention may be fixed to a solid phase generally used in the DNA array method, for example, a glass plate, nylon membrane, microbead, capillary or the like. Like the primer, the probe sequence may not be completely complementary to a part of the sequence of the fyn gene, and the base sequence may be modified or added depending on the detection method. The probe may be modified with a suitable substance for detection as described above. Those skilled in the art can appropriately determine the sequence and design of the probe depending on the detection method used.

  The present invention further provides a kit for detecting a polymorphism of the fyn gene in the genomic DNA of a subject comprising the primer and / or probe of the present invention. The kit of the present invention detects (a) a single nucleotide polymorphism at position 5177 of the sequence shown in SEQ ID NO: 1 and / or (b) deletion of positions 5123-5130 of the sequence shown in SEQ ID NO: 1. Yes, used for genetic diagnosis of schizophrenia. The kit of the present invention may further contain an appropriate buffer, standard sample, enzyme, plate and the like, if necessary.

  The present invention also provides a method for determining the risk of developing schizophrenia, comprising examining the expression level of Fyn in a blood sample collected from a subject. Fyn means a protein encoded by the fyn gene. Fyn includes its splice variants FynΔ7 (SEQ ID NO: 4), FynT (SEQ ID NO: 5), and FynB (SEQ ID NO: 6), and amino acid mutations based on naturally occurring individual differences and race differences Those variants containing are also included. In the present specification and claims, the expression level of “Fyn” means the expression level of the entire protein encoded by the fyn gene including each splice variant.

  In this method, when the expression level of Fyn in the subject is low, it can be determined that the risk of developing schizophrenia is high. What is necessary is just to compare an expression level with the average value of the expression level in many (for example, 50 or more) healthy persons, for example.

  The expression level of the protein may be examined by any known method, but is preferably examined using an anti-Fyn antibody. Examples of such methods include Western blotting, ELISA, and dot blot. The anti-Fyn antibody used in this method preferably recognizes an epitope common to each splice variant. Various antibodies that can be used in this method are commercially available: polyclonal anti-Fyn antibodies: GTX13955, GTX74453 and GTX74252 (GeneTex), EB05153 (Everest Biotech), SP1361P, SP1185 and FA1075 (Acris Antibodies), IMG-3040 (IMGENEX ), FA1075 (Fusion Antibodies), AP7709a (ABGENT), H00002534-A01 (Abnova), RB-10325-P (Lab Vision), 01-091-311C (AmProxs), AB1378 (CHEMICON), ab13955 (Novus Biologicals), OBT1252 (OXFORD BIOTECHNOLOGY), SC16, SC30680 and SC28791 (SANTA CRUZ BIOTECHNOLOGY), AHP651, MCA2379 (SEROTEC), 06133 (UPSTATE), AB13955, AB1881 and AB802 (ABCAM), 604102 (BIOLEGEND), AHO065 (BIOSOURCE), 11004 SPRING BIOSCINCE), and AP7709A (ABGENT); Monoclonal anti-Fyn antibodies: 1S (GeneTex, Novus Biologicals, Lab Vision, CHEMICON, Acris Antibodies, Exalpha Biologicals, UPSTATE, ABCAM, BIOSOURCE and ZYMED LABORATORIES), FYN-01 (GeneTex, Bio Vendor Lab oratory Medicine, Novus Biologicals, Acris Antibodies, Exbio Praha and ALEXIS), 15 (SANTA CRUZ BIOTECHNOLOGY), SPM247 (SPRING BIOSCINCE), Y303 (EPITOMICS), and FYN-59 (BIOLEGEND). Further, the anti-Fyn antibody γC3 described in the following literature is preferably used in the method of the present invention: Yasunaga M et al., Involvement of Fyntyrosin kinase in progression of cytokinesis of B lymphocyte progenitor. J Cell Biol. 1996 Jan; 132 (1-2): 91-9; Maekawa M et al., Fyn tyrosine kinase in Sertoli cells is involved in mouse spermatogenesis. Biol Reprod. 2002 Jan; 66 (1): 211-21; Hattori K et al ., Fyn is required for haloperidol-induced catalepsy in mice. J Biol Chem. 2006 Jan 10.

  The present invention also provides a method for determining the risk of developing schizophrenia, comprising examining the expression level of fyn mRNA in a blood sample collected from a subject. In the present specification and claims, “expression level of fyn mRNA” means the expression level of the entire mRNA derived from the fyn gene, including mRNA of each splice variant such as fynΔ7, fynT, and fynB mRNA. . When the expression level of fyn mRNA in a subject is low, it can be determined that the risk of developing schizophrenia is high.

  The expression level of mRNA may be examined by any known method. Examples of such methods include RT-PCR method, competitive PCR method, real-time PCR method, Northern blot method, dot hybridization method, microarray method, RNase protection assay method and the like.

  The present invention also provides a method for determining the risk of developing schizophrenia, comprising examining the expression level of fynΔ7 or fynT mRNA in a blood sample collected from a subject. The cDNA sequences complementary to fynΔ7 and fynT mRNA are shown in SEQ ID NOs: 7 and 8, respectively, and the mRNA sequence of the subject may contain mutations based on individual differences and race differences.

  It can be determined that the risk of developing schizophrenia is high when the expression level of fynΔ7 mRNA in a subject is higher than that of a healthy person, or when the expression level of fynT mRNA is low. The determination is, for example, that the relative expression level of each splice variant mRNA relative to the expression level of the entire transcript (mRNA) of the fyn gene in the subject is significant compared to the average level of a large number (for example, 50 or more) of healthy individuals. By checking whether it is high or low.

  The expression level of mRNA may be examined by any known method as described above. In this method, in order to distinguish individual splice variants, it is preferable to design primers based on regions that differ between the variants.

  The present invention further provides a method for determining the risk of developing schizophrenia, comprising examining the expression level of FynΔ7 or FynT in a blood sample collected from a subject. When the expression level of FynΔ7 in a subject is higher than that of a healthy person, or when the expression level of FynT is low, the subject can be determined to have a high risk of developing schizophrenia. The expression level of the protein can be examined by the method as described above. In this method, it is preferable to use an antibody that specifically recognizes each variant.

  The significant difference test can be performed by one-way analysis of variance (One-way ANOVA), student t-test, F-test, and the like.

  As the blood sample, any sample prepared by any method can be used as long as the protein or mRNA level in the blood can be examined. In the method of the present invention, platelet-rich plasma that can be prepared from a peripheral blood of a subject by a known method (O. Lingjaerde, O. Kildemo, Agents Actions 11, 410-6 (Jul, 1981)) is preferably used. It is done.

  The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.

1. Materials and Methods 1-1. Subjects Peripheral blood samples were collected from the following subjects: 109 patients with schizophrenia diagnosed by DSM-IV (65 men, 44 women, age 36.1 ± 12.6), 75 families within the first degree of the above patients ( 40 men, 35 women, age 54.7 ± 11.4), and 162 healthy people (111 men, 51 women, age 27.0 ± 6.4). None of the subjects showed evidence of acute infection, inflammation, or neurological disease.

1-2. Sample preparation Platelet-rich plasma mainly containing platelets and mononuclear cells was prepared by differential centrifugation (O. Lingjaerde, O. Kildemo, Agents Actions 11, 410-6 (Jul, 1981)). To explain, a sample containing 30 ml of peripheral blood was centrifuged at 50 × g for 15 minutes, and the supernatant was centrifuged at 1,000 × g for 15 minutes. The precipitate was dissolved in 1 mL of PBS, divided into three tubes, and centrifuged at 1,000 xg for 15 minutes. The precipitate was frozen and stored in liquid nitrogen until use. Basically, one of these tubes was used for protein assay, one for RNA analysis and one for genomic analysis. However, due to the limited amount of blood samples collected from subjects and, in some cases, the same assay required repeated tests, not all subjects could perform all three types of assays. It was. The exact number of samples for each analysis is shown in Table 1.

1-3. Western blotting Samples were solubilized with SDS sample buffer (50 mM Tris HCl (pH 6.8), 5% SDS, 10% glycerol, 1 mM sodium orthovanadate), sonicated, and centrifuged at 20,000 xg for 15 minutes . The supernatant was collected and the protein concentration was measured by BCA protein assay (Pierce). They were boiled with or without β-mercaptoethanol (5% of the total amount) and then subjected to SDS-polyacrylamide (10%) gel electrophoresis. The protein was then transferred to a PVDF membrane. The membrane was reacted with a rat monoclonal anti-Fyn antibody (γC3, created by Dr. Masahiro Yasuda (M. Yasunaga et al., J Cell Biol 132, 91-9 (Jan, 1996)), followed by a peroxidase-conjugated secondary antibody. Immunoreactive proteins were visualized with ECL-plus (Amersham) and ATTO Cool Saver (ATTO), followed by stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.7). )) For 30 minutes at 50 ° C. to release the antibody on the membrane, and after washing and blocking, the membrane was reacted with anti-β-tubulin antibody (H-235, Santa Cruz), followed by peroxidase-conjugated antibody. The next antibody was reacted.

1-4. RNA extraction, RT-PCR, and cloning
Total RNA was isolated from the samples with TRIzol reagent (Invitrogen) and reverse transcribed using SuperScript II (Invitrogen) and oligo-dT primers. fynB, fynT, or fynΔ7 cDNA region (38 nucleotides upstream of the start codon to 64 nucleotides downstream of the stop codon), LA-Taq polymerase (Takara) and primer hfyn-30F (5'-AGT TTG TGA TCG TTG GCG- 3 '(SEQ ID NO: 9)) and hfyn + 50R (5'-TGA AAG CTA ATG GGG AGG-3' (SEQ ID NO: 10)). The nucleotide sequences of fynB and fynT were based on GenBank NM_002037 and S74774, respectively. This PCR product was ligated into pBluescript SK-vector (Stratagene).

1-5. Competitive RT-PCR
A method described by Weil et al. (R. Weil et al., J Virol 73, 3709-17 (May, 1999)) was used in a modified manner. To explain, cDNA was amplified using Ex-Taq polymerase (Takara) and primers hfyn390F and FAM-hfyn850R (5′-FAM-GTG TTT CCA TAC CAG GTA CC-3 ′ (SEQ ID NO: 11)). This PCR fragment was purified by Suprec-PCR (Takara) and analyzed by ABI Prism 3100 Genetic Analyzer.

1-6. Real-time RT-PCR
1-4. Real-time PCR was performed on the cDNA product described in 1. using SYBR Green Mix and ABI PRISM 7900 HT (Applied Biosystems). Each PCR reaction contained 10 μl of 2x SYBER Green Mix, 5 pmol of each primer, and 1 μl of cDNA preparation in a total volume of 20 μl. Primers are hGAPDH20F (5'-GTC AAC GGA TTT GGT CGT ATT GG-3 '(SEQ ID NO: 12)) and hGAPDHex3RV (5'-GAC AAG CTT CCC GTT CTC AG-3' (SEQ ID NO: 13)) for GAPDH Hfyn610F (5'-AAA ATT CGC AAA CTT GAC AAT GGT-3 '(SEQ ID NO: 14)) and hfynT719R (5'-AAA CAC AAA CCG TCA GCT TTC TCT-3' (SEQ ID NO: 15)) for fynT Using. Plasmids containing fynT or GAPDH were prepared and serial dilutions of this plasmid were used as standards. Using the linear relationship between the number of gene-containing plasmids in the predetermined calibration curve and the corresponding CT value, the number of molecules of the same gene as the plasmid was determined from the CT (cycle threshold) value of each reaction. The number of each cDNA molecule in a given sample was normalized by the number of GAPDH cDNA molecules in the same sample.

1-7. Sequence of genomic DNA To cover the genomic region from exon 6 to exon 8, two 4 kb regions (0.2 kb overlap) from exon 6 to exon 7B and exon 7A to exon 8 of each sample are LA -Amplified with Taq polymerase. The following primers were used for PCR: 5'-ACA TGT GTG TGT GCA TCC-3 '(SEQ ID NO: 16) and 5'-AGT GTC TTT GTC CCA ACG-3' (SEQ ID NO: 17), 5'- TTT TAA CTT AAC TGT GAT TGC ATC GA-3 ′ (SEQ ID NO: 18) and 5′-TGG GAA TAC TTG ACC CAG-3 ′ (SEQ ID NO: 19). PCR products were purified by PEG precipitation and sequenced directly on an Applied Biosystems 3730 sequencer. Sequence traces were analyzed with SeqScape software (ABI). Genomic nucleotide sequence was based on GenBank HS66H14.
The polymorphism was genotyped by direct sequencing, and two 0.7 kb PCR fragments amplified with two primer pairs were sequenced with the forward and reverse primers used for PCR, respectively. The primers used were: 5'-GCT ACG TGT GGG TAT AGA TC-3 '(SEQ ID NO: 2) and 5'-CCC AAT TCT GAT GGG CAT GT-3' (SEQ ID NO: 3), or 5'- GCA GAA CAG GAA ACC AGT A-3 ′ (SEQ ID NO: 20) and 5′-GCA GCC AGC CTT ATC AAT GA-3 ′ (SEQ ID NO: 21). Repeated sequences were analyzed by RepeatMasker (http://www.repeatmasker.org).

1-8. In vitro expression
The full length cDNAs of fynB, fynT, and fynΔ7 in the pBlueScript SK-vector were transferred to pcDNA 3.1 or pcDNA 3.1 / myc-His (Invitrogen) expression vector. These constructs were transfected into HEK293T cells using LipofectAmine 2000 reagent (Invitrogen). After 24 hours, cell samples were collected with the SDS sample buffer. Lysates were used for Western blotting either directly or after immunoprecipitation with monoclonal anti-Fyn antibody (γC3). In order to evaluate tyrosine phosphorylation, this blot was reacted with anti-phosphotyrosine antibody PY100 (Cell Signaling Technology).

1-9. Kinase assay For immunoprecipitation, HEK293T cells transfected with fynB, fynT, or fynΔ7 were treated with TNE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM Na ₃ VO ₄ , 50 μM Na ₂ Elute with cO ₄ , 1% Nonidet P-40). 2 μg of monoclonal anti-Fyn antibody (γC3) was added to each protein sample (TNE buffer 300 μl). After absorption with Protein G-Sepharose (Amersham), the immunoprecipitate was washed 3 times with TNE buffer and kinase assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MnCl ₂ , and 0.1 mM Na ₃ VO ₄ ) And washed twice. For the kinase assay, 25 μl of kinase assay buffer contained the immunoprecipitate, 2.5 μg of acid-treated enolase, and ³² P-ATP 10 μCu. Phosphorylation was performed at 30 ° C. for 10 minutes, and the phosphorylated protein was separated by SDS-polyacrylamide (10%) gel electrophoresis, followed by autoradiography.

1-10. Statistical analysis The results of Western blotting and relative mRNA expression were analyzed by one-way analysis of variance (One-way ANOVA), or Student's t-test and F-test (StatView-J 5.0). The significance of genomic polymorphism was analyzed by χ ² test (StatView-J 5.0). P levels less than 0.05 were considered significant.

2. Result 2-1. Fyn expression levels in schizotypic patients' serum Fyn (final product of fyn genes) in peripheral blood of schizophrenic patients, their first-degree relatives, and healthy individuals to investigate fyn gene abnormalities in schizophrenic patients Was examined by Western blotting. Platelet rich plasma was used because Fyn is particularly rich in platelets and can be collected in a simple manner.
A strong immunoreactive band of Fyn was observed in the healthy human samples. On the other hand, many of the samples of schizophrenic patients had weak immune responses of Fyn (FIG. 1A). In order to elucidate the relationship between immunoreactivity to Fyn and schizophrenia, the intensity of each band was measured and standardized by comparing it with that of a 4 μL standard sample. There was no significant sex difference or age dependence in the Fyn band intensity (FIGS. 2A and B). The mean intensity of the Fyn band of the patient group was significantly lower than that of the control (healthy person) group, and the family group was also slightly lower than that of the control group (FIG. 1B).

2-2. Alternative splice variants of fyn gene transcripts in schizophrenic patient samples
In order to examine whether the decrease in Fyn was caused by a change in the transcript of the fyn gene, RT-PCR was performed on the mRNA of each sample. As a result, in addition to the full-length fyn cDNA, a fragment about 150 bp shorter than the normal fyn gene transcripts (fynT and fynB) was amplified (FIG. 3A). The intensity of this band was stronger in the schizophrenic patient sample than in the healthy subject sample. As a result of cloning and sequencing this band, this is a splice variant of the fyn gene (JF Goldsmith, CG Hall, TP Atkinson, Biochem Biophys Res Commun 298, 501-4 (Nov 8, 2002)) reported as fynΔ7. It was found (FIG. 3B).

2-3. FynΔ7 mRNA expression level in schizophrenic patient samples Because of the high expression level of fynΔ7 in schizophrenic patient samples, semiquantitative RT-PCR (competitive RT-PCR) was used to determine the relative levels of each fyn isoform. The amount was examined (R. Weil et al., J Virol 73, 3709-17 (May, 1999)).
Using primers that hybridize upstream and downstream of exon 7, mRNA from each sample was amplified (FIG. 3B). From the electropherograms of the PCR products obtained with the automated sequencer, it was found that these PCR products were fynB, fynT, and fynΔ7. fynB was 9 bp different from fynT and fynΔ7 was 156 bp shorter than fynT (FIG. 3C).
Next, the length of each band and the fluorescence intensity were measured and compared. As shown in FIG. 3D, the average ratio of the strength of fynB divided by the sum of the strengths of the three isoforms was comparable in all groups. On the other hand, according to one-way ANOVA, the mean intensity of fynT is significantly lower in the patient group and the family group than in the control group, and the mean intensity of fynΔ7 is significantly higher in the patient group and the family group than in the control group. (FIG. 3D). The decrease in fynT and the increase in fynΔ7 tended to be greater in the family group than in the patient group. Since the fynΔ7 ratio was higher in the female group than in the male group, the male and female samples were examined separately. An increase in the fynΔ7 ratio in the patient group and the family group was observed for all genders (FIG. 4A). On the other hand, no significant age dependency was found in the fynΔ7 ratio (FIG. 4B). Correlation analysis revealed that there was a weak but significant correlation between Fyn band intensity and fynΔ7 expression level (n = 243) (FIG. 5).
Next, quantitative RT-PCR (real-time RT-PCR) was performed on fynT, and analysis was successful for some samples. If samples with less than 1000 GAPDH or less than 10 molecules of fynT in each test tube (cDNA 1 μL) were excluded, the mean expression level (fynT / GAPDH) was 2.41 in the patient group (n = 26) and in the family group It was 2.36 (n = 28) and 3.23 (n = 60) in the control group. In one-way ANOVA, this difference was not significant.

2-4. Kinase activity of FynΔ7
In order to investigate the functional role of the transcript of the fynΔ7 gene, expression analysis in HEK293T cells was performed. Western blotting showed that corresponding protein isoforms were produced from fynB, fynT, and fynΔ7 expression vectors in HEK293T cells. However, when the same membrane was reprobed with an anti-phosphotyrosine antibody, no signal was detected in the lane of fynΔ7 (FIG. 6A).
In addition, an in vitro kinase assay was performed to examine the kinase activity of this protein. As reported by Goldsmith et al. (JF Goldsmith, CG Hall, TP Atkinson, Biochem Biophys Res Commun 298, 501-4 (Nov 8, 2002)), FynΔ7 has no autophosphorylation activity and the reaction system It was confirmed that the enzyme has no ability to phosphorylate enolase added to (Fig. 6B). FynΔ7 protein produced from fynΔ7 vector was less than the corresponding protein produced from fynB and fynT vectors (FIGS. 6C and D). This phenomenon was also observed when equal amounts of fynB and fynΔ7 vectors were mixed and transfected into cells in the same culture plate (data not shown).

2-5. Sequence analysis between exons 6 and 8 of the fyn gene To explore genomic polymorphisms affecting alternative splicing, the genomic sequence between exons 6 and 8 of the fyn gene was examined. First, polymorphisms were searched using 48 samples (42 patients, 6 families) and 11 SNPs and one deletion of two 8 bp repeat sequences were found (FIG. 7). The RepeatMasker program revealed that these polymorphisms are concentrated near the two Alu sequences between exon 7B and exon 8.
Next, pay attention to the polymorphisms observed in more than 10% of these samples, and determine the genotypes of these polymorphisms using more samples (109 patients, 45 families, and 140 healthy people). did. Hardy-Weinberg equilibrium deviations were not statistically significant in each SNP distribution. When χ ² test was performed on the results of patients and healthy individuals, schizophrenia and SNP (rs9481183) (on position 5177 on the genomic DNA of fyn gene (SEQ ID NO: 1)) and 8 bp deletion (genomic DNA of fyn gene) It was found that there was a significant correlation with the above (positions 5123 to 5130 in SEQ ID NO: 1) (Table 2). In some subjects, this SNP was not linked to the 8bp deletion. This was also confirmed by amplification with different primer pairs. In addition, the genotype distribution in these two polymorphic family groups was intermediate between the patient group and the control group. For other polymorphisms, there was no obvious difference in distribution between groups. For SNP (rs9481183), the frequency of wild-type (AA) was higher in the schizophrenic patient group than in the control group. The expression ratio of fynΔ7 was higher when the samples had wild type alleles (+ 17.5%), but this difference was not significant (p = 0.08, student t-test, dominant genetic model).

3. Summary The expression level of Fyn in the subject blood and the expression level of fynΔT or fynT mRNA have been shown to be associated with schizophrenia. In addition, SNPs and 8 bp deletions in the fyn gene that correlate with schizophrenia were identified.

FIG. 1 shows a decrease in Fyn expression level in schizophrenia. (A) Western blotting results. (B) Average intensity of Fyn band for each subject. *: P <0.05, **: p <0.0015, One-way ANOVA. FIG. 2 shows the effect of gender and age on Fyn expression levels. (A) Relationship between Fyn expression level and gender. Male (n = 175), female (n = 116). (B) Relationship between Fyn expression level and age. FIG. 3 shows the fyn gene splicing pattern in schizophrenic patients. (A) Electrophoresis of fyn gene PCR product. The arrow indicates a smaller band (about 1550 bp) than the normal fyn gene transcript (about 1700 bp). (B) Three fyn isoforms by alternative splicing: fynB (using exon 7A), fynT (using exon 7B), and fynΔ7 (exon 7 deleted). (C) Electrophoresis diagram of PCR product by semi-quantitative RT-PCR. (D) Average strength of each isoform. One-way ANOVA, *: p <0.05, **: p <0.01. C: healthy person, F: family, S: patient. FIG. 4 shows the effect of gender and age on fynΔ7 mRNA expression. (A) Relationship between fynΔ7 mRNA expression and gender. * <0.05; ** <0.01; # <0.001, One-way ANOVA. C: healthy person, F: family, S: patient. (B) Relationship between fynΔ7 mRNA expression and age. FIG. 5 shows a correlation analysis between the density of the Fyn band and the expression ratio of fynΔ7 mRNA. FIG. 6 shows the expression of FynB, FynT, and FynΔ7 in HEK293T cells. (A) Autophosphorylation activity of FynB, FynT, and FynΔ7. (B) Self and enolase phosphorylation activity of FynB, FynT, and FynΔ7. (C) Protein produced from fynT, fynB, and fynΔ7 vectors. (D) Comparison of expression levels of FynB, FynT, and FynΔ7. One-way ANOVA, * P <0.01. P-Tyr: phosphotyrosine; WB: Western blot; IP: immunoprecipitation; C: control vector (pcDNA 3.1). FIG. 7 is a schematic diagram showing the genomic region of exon 6 to exon 8 of the fyn gene.

Claims (7)

  A method for determining a risk of developing schizophrenia, comprising detecting a polymorphism of a fyn gene in a genomic DNA of a subject. The method according to claim 1, wherein the polymorphism is at least one selected from the following.
(A) a single nucleotide polymorphism at position 5177 of the sequence shown in SEQ ID NO: 1;
(B) A deletion at positions 5123 to 5130 of the sequence shown in SEQ ID NO: 1. Primer for amplifying a region containing at least one of the following positions of the fyn gene:
(A) position 5177 of the sequence set forth in SEQ ID NO: 1;
(B) Positions 5123-5130 of the sequence set forth in SEQ ID NO: 1.   The primer of Claim 3 which has a sequence of sequence number 2 or 3. Probe that hybridizes to a region containing at least one of the following positions of the fyn gene:
(A) position 5177 of the sequence set forth in SEQ ID NO: 1;
(B) Positions 5123-5130 of the sequence set forth in SEQ ID NO: 1.   A kit for detecting a polymorphism of the fyn gene in genomic DNA of a subject, comprising the primer according to claim 3 and / or the probe according to claim 5. The kit according to claim 6, wherein the polymorphism is at least one selected from the following.
(A) a single nucleotide polymorphism at position 5177 of the sequence shown in SEQ ID NO: 1;
(B) A deletion at positions 5123 to 5130 of the sequence shown in SEQ ID NO: 1.

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