KR20030029201A - Primers for diagnosis of schizophrenia - Google Patents

Abstract

PURPOSE: Provided is a diagnosis primer of schizophrenia particularly by amplifying human endogenous retrovirus-F(HERV-F). Its DNA type and strength are specific to a schizophrenia patient. Therefore, it is useful for the diagnosis of schizophrenia. CONSTITUTION: The diagnosis primer of schizophrenia is characteristically prepared by amplifying human endogenous retrovirus-F(HERV-F). The nucleotide sequence of HERV-F is represented by the SEQ ID NO:1-13. The primer is useful for the diagnosis of schizophrenia early.

Description

Primers for diagnosis of schizophrenia

The present invention relates to a primer for diagnosis of schizophrenia, and more specifically, to specifically amplify human endogenous retrovirus-F (hereinafter referred to as "HERV-F"). It relates to a schizophrenia diagnostic primer, characterized in that.

Schizophrenia is a disorder that causes disorders in emotions, perceptions, and behaviors as the main cause of accidents. It is a complex disease caused by a combination of causes and is called premature dementia. In Korea, it is one of the most common mental illnesses, accounting for more than two-thirds of all inpatients in mental hospitals. It occurs at a rate of 0.13-0.54% of the population. Most of them develop before and after puberty, but can also occur after middle age.

Although studies on genetics, neurobiochemistry, neuroanatomy and sociopsychological aspects have been conducted to clarify the cause of schizophrenia, we have not been able to reach a consistent conclusion. It is becoming. As genetic factors are found to be an important cause of the development of schizophrenia, the possibility of developing schizophrenia is expected through household studies, adoption studies, and twin studies. However, applying these methods to the diagnosis of schizophrenia requires a lot of time and money, and there is a decisive weakness that these studies must be done in advance for each diagnosis target. Therefore, there is a need for a new and easy diagnostic method that can replace it.

Random amplified polymorphic DNA (RAPD) assay, designed by William et al., Amplifies specific target DNA by using a genetic technique called polymerase chain reaction (PCR) of an individual's chromosomal DNA, and agarose gel (agarose). Investigating DNA band patterns on gels can be used to determine the degree of genetic variation between individuals (Kwon, OS and Yoo, M., Kor. J. Microbiol , 1998 , 34 (1-2), 51). -57). PCR amplification using random nucleotides of about 10-mer oligonucleotides as random primers reveals a large number of nonspecific DNA band patterns, which is why RAPD analysis using random primers Use this to compare the overall genetic similarity and difference between individuals. The RAPD assay is based on the classification of eukaryotic organisms with mainly complex genomes, such as the evolutionary genetic studies of bacteria and fungi or the breeding of animals and plants, which require easy handling of many samples in conventional studies, and the simplest genome. It has been used for the identification of bacterial viruses, and recently, it has gone beyond the existing research methods to analyze the DNA of viruses extracted from AIDS patients in connection with human diseases and find mutations or polymorphic gene loci in human cancer tissues. Until the development.

Recently, with the development of molecular biology techniques, research methods for tracking the relationship with diseases based on the DNA base sequence have been introduced, and the research results by these methods appear in various ways. As an example, the candidate genes for schizophrenia are human chromosomes 1, 2q, 3p, 4p, 4q, 5p, 6p, 6q, 7, 8p, 9, 10p, 10q, 11p, 13q, 16p, 18p, 21q, 22q, Xp and Xq have been shown throughout, and the multigeneity of this schizophrenia is the retroviral hypothesis proposed by Professor Timothy J Crow of the Department of Psychiatry, University of Oxford, Oneford Hospital (Crow, TJ, Br. J. Psychiatry , 1984 , 145, 243-). 253). The retroviral hypothesis is that schizophrenia occurs because retroviruses are inserted and moved freely in the human genome, and they are transmitted to the next generation and can be inherited. Since retroviruses cause insertional mutagenesis due to frequent migration, they are thought to be the cause of complex disease syndromes such as schizophrenia due to autoimmune diseases, cancer, and the expression or structural changes of various schizophrenic genes in cells. It is becoming.

The basic structure of the retrovirus consists of the promoter LTR (long terminal repeat), gag encoding core protein, pol encoding reverse transcriptase, and env encoding envelope protein. 5'LTR-gag-pol- env-3'LTR is arrayed. Human endogenous retroviruses (HERVs) are already inserted into germline chromosomes and are normally inherited according to Mendel's law and are known to be present in almost all animals. In humans, they comprise 1-3% of the entire genome.

The term used in the human endogenous retrovirus (HERV) family refers to HERV-K, HERV-H, HERV-W, HERV- by one letter of the amino acid name for each of them due to tRNA complementary to the primer binding site (PBS). It is divided into F etc.

HERV-F is the most recently discovered endogenous retrovirus, first detected in the human chromosome 7q31.1-31.3 region of the XA34cDNA clone isolated from the human glioma cDNA library with ERV-9 as a probe. Widegren, B., et al., J. Gen. Virol ., 1996 , 77, 1631-1641. Gliomas are one of the most common brain tumors, accounting for about 50% of primary intracranial tumors. Sexual incidence is more common in males than in females, with the proportion of males and females being 4.3: 2.7 / 100,000. Positive cases are reported at 1.3: 0.8 / 10 million. Unlike other brain tumors, gliomas invade and destroy normal surrounding brain tissue. These characteristics are important factors in determining the prognosis as well as the malignancy of the tumor cells themselves. Studies of the causes of glioma have revealed changes in various cytogenetic genes, the most common of which are known as structural changes in chromosome 7 and loss of chromosomes 10, 22 and sex chromosomes.

Accordingly, the present inventors have tried to find an easy and convenient method for diagnosing schizophrenia and found that human endogenous retroviruses associated with glioma exist in a large number of genomes in schizophrenic patients. The present invention has been completed by revealing that schizophrenia can be diagnosed early and sensitively using primer sequences that can be amplified automatically.

An object of the present invention is to provide a primer sequence for schizophrenia diagnosis that is easy and simple to diagnose and high in sensitivity.

1 is an electrophoresis picture showing the results of PCR and RAPD analysis using the HERV-F pol sense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

Figure 2 is an electrophoresis picture showing the results of performing PCR and RAPD analysis using the HERV-F pol antisense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

Figure 3 is an electrophoresis picture showing the results of performing PCR and RAPD analysis using the HERV-W pol sense 3 primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

Figure 4 is an electrophoresis picture showing the results of performing PCR and RAPD analysis using the HERV-W pol sense 2 primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

5 is an electrophoresis picture showing the results of PCR and RAPD analysis using the HERV-W LTR sense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

Figure 6 is an electrophoresis picture showing the results of performing PCR and RAPD analysis using the HERV-W LTR antisense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

Figure 7 is an electrophoresis picture showing the results of performing PCR and RAPD analysis using the HERV-W gag sense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

8 is an electrophoresis picture showing the results of PCR and RAPD analysis using the HERV-W env sense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

9 is an electrophoresis picture showing the results of PCR and RAPD analysis using the HERV-K10 env sense primer of the present invention,

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

10 is an electrophoresis picture showing the results of PCR and RAPD analysis using the HERV-K10 env antisense primer of the present invention.

M: size marker

Lane 1-10: group of patients with non-schizophrenia

Lane 11-15: Schizophrenic group

In order to achieve the above object, the present invention provides a primer for diagnosing schizophrenia characterized by specifically amplifying HERV-F.

Hereinafter, the present invention will be described in detail.

The primer for diagnosing schizophrenia of the present invention specifically amplifies in response to a gene of HERV-F, which is one of human endogenous retroviruses, preferably the HERV-F gene described in SEQ ID NO: 1 . To this end, the present invention provides primer sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3 . However, since all primer sequences capable of specifically amplifying HERV-F can also diagnose schizophrenia, it is natural to those skilled in the art that all such sequences also fall within the scope of the present invention.

The present inventors attempted to confirm the relationship between HERV genes and schizophrenia associated with schizophrenia. To this end, blood from patients with schizophrenia, normal people and apes were collected to investigate the relationship between the expression of various HERV genes and schizophrenia. The HERV gene in the above is HERV-F, HERV-K10, HERV-H, HERV-W and the like.

As a result, when amplifying DNA using a primer specific for HERV-F, it was confirmed that a specific band pattern and a large amount of HERV-F were expressed in the schizophrenic patient group (see FIGS . 1 and 2 ). From the above results, it was confirmed that schizophrenia can be easily diagnosed early by amplifying DNA using a primer specific for HERV-F of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples.

Example 1 Preparation of Primer for Diagnosing Schizophrenia

The inventors have prepared primers that can specifically amplify HERV associated with human diseases by the retroviral hypothesis. In addition, 20-mer oligonucleotides were prepared as primers away from the existing 10-mer primers to obtain specific DNA band patterns of schizophrenic patients rather than polymorphisms of the entire human genome ( Table 1 ). The primers were named based on the name of the HERV from which the primers were derived, and the primers produced in the upstream of double-stranded DNA were sense and the primers produced downstream were called antisense. The names and base sequences of the primers of the present invention are shown in Table 1 below.

Primer Name order TM (℃) HERV-F pol sense SEQ ID NO: 2 58 Antisense SEQ ID NO: 3 56 HERV-K10 LTR Sense2 SEQ ID NO: 4 62 Antisense SEQ ID NO: 5 62 HERV-K10 env sense SEQ ID NO: 6 60 Antisense SEQ ID NO: 7 60 HERV-H env sense SEQ ID NO: 8 62 HERV-W pol sense 3 SEQ ID NO: 9 60 Antisense 2 SEQ ID NO: 10 60 HERV-W gag sense SEQ ID NO: 11 62 HERV-W LTR sense SEQ ID NO: 12 62 HERV-W env sense SEQ ID NO: 13 60

Experimental Example 1 Schizophrenia Specific Band Pattern Analysis

<1-1> Sample DNA Collection

A sample of schizophrenic patients was provided with 376 patients (190 women, 186 men) at Suwon Catholic Medical University. A sample of non-psychotic patients received 353 leukocyte samples from a prenatal genetic laboratory at Pusan National University Hospital. Used. Samples of schizophrenic patients included 92 chromosomal abnormalities and 261 chromosomal normals. A sample of leukocytes was used while storing ethanol and acetic acid in a ratio of 9: 1 and stored at -20 ° C.

<1-2> DNA Isolation from White Blood Cell Samples

DNA was isolated from each leukocyte sample in the following manner.

1. Centrifuge each tube of the leukocytes collected at 14000 rpm, 2 minutes, at room temperature (RT), and remove the supernatant except the collected pellets.

2. Add 500 μl of PBS to the tube and mix well.

3. Collect pellets by centrifugation at 14000 rpm, 2 minutes at room temperature (RT) and remove supernatant.

4. Add 200 µl of extraction buffer and 5 µl of proteinase K (20 mg / ml) and mix well.

5. Incubate in a 50-degree water-bath, inverted every 2 hours overnight.

6. Add 200 μl of PCI and mix well to the left and right.

7. Centrifuge at 14000 rpm for 10 minutes at room temperature (RT).

8. Transfer 200 μl of supernatant to a new tube. However, be careful not to climb downstairs.

9. Repeat Step 6 through Step 8 twice.

10. Add 200 μl of isopropanol and mix gently.

11. Centrifuge at 14000 rpm for 10 minutes at room temperature (RT) to completely remove supernatant.

12. Add 500 μl 80% EtOH.

13. Centrifuge at 14000 rpm, 2 minutes at room temperature (RT) and remove the supernatant completely.

14. Dry at room temperature (RT) for about 10 minutes.

In the present invention, among the schizophrenic patients and the schizophrenic patients group divided into chromosomal normal group and chromosomal abnormal group, each of five people from three groups were selected and used in the experiment. In addition, humans and apes (chimpanzees, gorillas, orangutans, gibbons, and Japanese macaques) were selected and compared to each other in two species for accuracy in finding human-specific bands ( Table 2 ).

Genomic DNA Sample Patients with schizophrenia, normal chromosome group One BK99082 (28M, 46XY) 2 BK99096 (29M, 46XY) 3 BK99102 (36M, 46XY) 4 BK99092 (41F, 46XX) 5 BK99106 (28F, 46XX) Patients with schizophrenia, chromosomal abnormalities 6 BK00021 (10M, 45XO, Tuner syn.) 7 BK99169 (34F, 45XO, Tuner syn.) 8 BK99084 (36M, 47XXY, Klienfelter syn.) 9 BK99168 (23M, 47XXY, Klienfelter syn.) 10 BK00003 (30F, 47XXinv (P)) Schizophrenic patients 11 1 19 M 12 3 18 M 13 4 22 M 14 5 33 F 15 10 23 F chimpanzee 16 2856 (lab DNA no.) 17 2857 (〃) Gorilla 18 1531 (〃) 19 1979 (〃) orangutan 20 2847 (〃) 21 2850 (〃) basic 22 2220 (〃) 23 2221 (〃) Japanese monkey 24 2639 & JM from Kyoto 25 2640 (〃)

<1-3> Band pattern analysis of amplified DNA

The inventors investigated the type of amplified DNA in the schizophrenic patient group, the normal group and the apes group using the primers prepared in Example 1 above.

First, PCR reactions were prepared in a total volume of 25 μl with the following composition.

[Reaction solution (25 μl total)]

ddH ₂ O 16.35 μl (autoclaved)

3 μl 2.5M dNTP

10 × Buffer 2.5 μl (promega)

2 μl primer (20 mer / 10 pmol)

0.15 μl Taq polymerase (promega Taq DNA polymerlase: 100 unit M186A)

1 μl DNA (0.6 μg / μl)

PCR was performed for each primer prepared in Example 1 .

The reaction solution was reacted for 3 minutes at 94 ° C, denatured, and then reacted 15 times at 94 ° C for 30 seconds, at 37 ° C for 1 minute, and at 72 ° C for 15 minutes, again at 94 ° C for 30 seconds, and at 40 ° C for 1 minute and 72 ° C. The reaction was carried out for 40 minutes at 1 ° C. and finally at 72 ° C. for 5 minutes. In some cases, the reaction was denatured by reacting at 94 ° C. for 3 minutes, and then reacting 15 times at 30 ° C. at 94 ° C. for 1 minute at 72 ° C. for 1 minute at 72 ° C., and finally at 72 ° C. for 5 minutes. After the reaction, the PCR product was confirmed by electrophoresis (80 V, 1 hour) on a 2% agarose gel. For staining of DNA, 20 mg / ml of ethididum bromide was added at 2 μl per 100 ml of agarose gel, and a band obtained using UV transilluminator (Spectroline) was used for the gel print system. Photographed. At this time, pUC18 treated with restriction enzyme tag I was used as a size marker to confirm the size of the DNA band.

As a result, the DNA band pattern is the same in all groups or is different in some patients, but the specific band pattern is not found in schizophrenic patients. Fig. 3 - Fig. 10).

Experimental Example 2 RAPD Analysis for HERV-F

The present inventors designed the sense primer (HERV-F pol Sense) and antisense primer (HERV-F pol Antisense) in the pol region encoding the reverse transcriptase of the components of HERV-F to perform the experiment according to the method of Experimental Example 1 It was.

As a result, when using the HERV-F pol sense primer as shown in Figure 1 , the DNA band pattern in the schizophrenic patient group (lane 1-10) and the schizophrenic patient group (lanes 11-15) showed a distinct difference. In particular, lanes 14 and 15 show explosively amplified specific band patterns unlike other patients. Therefore, this primer has a good potential as a diagnostic marker for sequencing specific schizophrenia patients.

In addition, as shown in FIG . 2 , the use of the HERV-F pol antisense primer also showed a distinct difference in DNA band patterns between the schizophrenic patients (lanes 1-10) and the schizophrenic patients (lanes 11-15). . Lanes 13, 14, and 15, in particular, are explosively amplified unlike other patients, showing a specific band pattern in schizophrenic patients. Therefore, this primer is also likely to be a diagnostic marker for identifying sequences specific to schizophrenic patients.

As described above, the schizophrenia diagnostic primer of the present invention specifically amplifies HERV-F, and since the type and intensity of the amplified DNA are specific in schizophrenic patients, it is possible to easily and accurately diagnose schizophrenia. have.

Claims (4)

A primer for diagnosing Schizophrenia, which specifically amplifies human endoretrovirus-F (hereinafter referred to as "HERV-F"). The primer for diagnosing schizophrenia according to claim 1, wherein the HERV-F has a nucleotide sequence set forth in SEQ ID NO: 1 . The primer for diagnosing schizophrenia according to claim 1 or 2, which has the nucleotide sequence of SEQ ID NO: 2 . The primer for diagnosing schizophrenia according to claim 1 or 2, which has the nucleotide sequence of SEQ ID NO: 3 .