JP2003070498A - Method for detecting abnormality of ubiquitin carboxy- terminal hydrolase l1 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining an abnormality of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene. SOLUTION: The method for determining an abnormality of UCHL1 gene comprises analyzing a polymorphism of UCHL1 gene in a biological sample derived from a testee and detecting an abnormality of UCHL1 gene in the testee based on the analytical result.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to ubiquitin carboxyl terminal hydrolase L1 (Ubiquitin Carboxyl).
-Terminal Hydrolase L1: UCHL1) polymorphism analysis, detecting a UCHL1 gene abnormality in a subject based on the analysis result, a method for detecting a UCHL1 gene abnormality,
The present invention also relates to a method for predicting abnormal UCHL1 protein activity, a method for typing Parkinson's disease, a method for detecting risk factors for Parkinson's disease, and a method for determining susceptibility to Parkinson's disease based on the detection method.

[0002]

BACKGROUND OF THE INVENTION Parkinson's disease was discovered in 1817 by the London surgeon James Parkinson.
It is one of the neurodegenerative diseases first reported by, and usually develops after the age of 40, especially in the 50s and 60s, and has tremor, muscular rigidity, bradykinesia, and postural reflex disorder (prone to fall).
Characterized by symptoms such as. The prevalence of Parkinson's disease is
About 50 people per 100,000 people in Japan, and 2 in white people
It is supposed to be 3.5 times. Pathologically, degenerative atrophy of melanocytes in the substantia nigra of the midbrain and lesions of the basal ganglia (putamen and pallidum) were observed, and the number of nerve cells decreased and An abnormal substance called Lewy bodies is observed in.

Recently, a relationship between Parkinson's disease and an abnormality of ubiquitin carboxyl terminal hydrolase L1 (UCHL1) has been pointed out [Nature, 395 , 451-452 (1998)] and has been drawing attention. However, it remains unclear what mutations in UCHL1 are associated with Parkinson's disease.

[0004] By the way, conventionally, in the diagnosis of Parkinson's disease, it is recognized that the progress is progressive, and subjective symptoms, namely, at least one of shaking at rest, poor movement, and poor walking. , Neurological findings, ie, at least one of resting tremor 4-6 times per second, immobility / perturbation, muscle rigidity with gear phenomenon, and posture / walking disorder, anti-Parkinson's disease It has been carried out based on the diagnostic criteria that treatment with drugs shows clear improvement in subjective symptoms and neurological findings, and differential diagnosis, that is, neither cerebrovascular disorder nor drug-related one. It was However, since the above-mentioned diagnostic method is not only complicated but lacks accuracy, it is difficult to distinguish it from a disease having similar symptoms to Parkinson's disease, and there have been many cases in which a neurologist has difficulty in making a diagnosis. Under such circumstances, there is a demand for a more reliable assay method for Parkinson's disease based on scientific evidence.

[0005]

The present invention analyzes polymorphisms of the ubiquitin carboxyl terminal hydrolase L1 (UCHL1) gene, and based on the analysis results, UCHL1 of a subject is analyzed.
UCHL1 gene abnormality detection method characterized by detecting gene abnormality, and based on the detection method, UCHL1 protein activity abnormality prediction method, Parkinson's disease typing method, Parkinson's disease risk factor detection method and Parkinson's disease susceptibility It is an object of the present invention to provide a determination method of.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found a novel gene polymorphism in the UCHL1 gene that causes a decrease in the activity of the UCHL1 protein, and Has been completed. That is, the present invention is the following (1) to (9).

(1) Polymorphism of ubiquitin carboxyl terminal hydrolase L1 (UCHL1) gene in a biological sample derived from a subject is analyzed, and UCHL of the subject is analyzed based on the analysis result.
A method for detecting a UCHL1 gene abnormality, which comprises detecting a 1-gene abnormality. (2) The method for detecting an abnormal UCHL1 gene according to (1) above, wherein the UCHL1 gene polymorphism is a mutation in codon 9 of exon 6 of the human UCHL1 gene. (3) The mutation in codon 9 of exon 6 is the third in the codon.
U of the above (2), which is a mutation of the th base, thymine to adenine
Method for detecting CHL1 gene abnormality.

(4) The UCHL1 gene polymorphism is a codon mutation in which the 162nd phenylalanine in the human UCHL1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is replaced with leucine. Detection method. (5) The method for detecting an abnormality in the UCHL1 gene according to the above (1), wherein the UCHL1 gene polymorphism is a mutation of the human UCHL1 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 at the 486th base thymine to adenine. (6) Based on the detection result obtained by the detection method of any one of (1) to (5), a method for predicting abnormal activity of UCHL1 protein, which comprises predicting abnormal activity of UCHL1 protein in a subject .

(7) Based on the detection result obtained by the detection method according to any one of (1) to (5) above, the cause of Parkinson's disease development in a subject who has developed Parkinson's disease is due to UCHL1 gene abnormality A method of typing Parkinson's disease, which comprises determining whether or not (8) Based on the detection result obtained by any of the detection methods of (1) to (5) above, detection of a Parkinson's disease risk factor characterized by detecting the presence or absence of a Parkinson's disease risk factor in a subject Method. (9) Based on the detection result obtained by any of the detection methods of (1) to (5), the determination of Parkinson's disease susceptibility, which comprises determining Parkinson's disease susceptibility of a subject. Method. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention analyzes a polymorphism in the UCHL1 gene, and detects a UCHL1 gene abnormality in a subject based on the analysis result, and a method for detecting the UCHL1 gene abnormality. A method for predicting abnormal UCHL1 protein activity, a method for typing Parkinson's disease, a method for detecting risk factors for Parkinson's disease, and a method for determining susceptibility to Parkinson's disease based on the method.

The UCHL1 gene polymorphism includes polymorphisms that affect the activity and / or intracellular behavior of the UCHL1 protein encoded by the UCHL1 gene. For example, UCHL1
Human UC is a polymorphism that affects protein activity.
Codon 9 of exon 6 of the HL1 gene (hereinafter simply referred to as codon 9
(Also referred to as)). The amino acid encoded by this codon is 5 from the active center of UCHL1.
Located within Angstrom, this polymorphism is UCHL1
It changes the activity of. Examples of the mutation of codon 9 include a mutation in which phenylalanine encoded by codon 9 in the normal gene is changed to leucine. More specifically, the mutation of thymine at the third base of codon 9 to adenine Mutation, that is, mutation of codon 9ttt to tta.

The mutation in codon 9 of exon 6 of the human UCHL1 gene corresponds to the mutation in the codon encoding the phenylalanine at the 162nd position in human UCHL1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and exon 6 The mutation of the third base thymine in codon 9 of thymine to adenine is a human UC consisting of the base sequence represented by SEQ ID NO: 1.
It corresponds to the mutation of the 486th base thymine of the HL1 gene to adenine. Here, the nucleotide sequence of the UCHL1 gene having a normal codon 9 is SEQ ID NO: 1, the amino acid sequence of UCHL1 encoded by the gene is SEQ ID NO: 2, and the abnormal codon 9 is
The nucleotide sequence of the UCHL1 gene having SEQ ID NO: 3 is shown in SEQ ID NO: 3, and the amino acid sequence of UCHL1 encoded by the gene is shown in SEQ ID NO: 4.

A UCHL1 gene abnormality in a subject can be detected by examining the presence or absence of the above polymorphism using a biological sample derived from the subject (eg, blood cells, skin mucous membrane cells, etc.). That is, when any one of the above polymorphisms of the UCHL1 gene is detected in the biological sample, the UCHL1 gene of the subject who provided the biological sample can be evaluated to be abnormal.

Further, when a UCHL1 gene abnormality is detected by using a biological sample derived from a subject (eg, blood cells, skin mucous membrane cells, etc.), the biological sample of the subject who provided the biological sample is examined. , UCHL1 protein can be predicted to exhibit abnormal activity in the subject.

Furthermore, when a UCHL1 gene abnormality is detected using a biological sample derived from a subject who has developed Parkinson's disease (for example, blood cells, skin mucous membrane cells, etc.), the biological sample is provided by the supplier. Parkinson's disease in a subject can be determined to be the type that results from the UCHL1 gene abnormality (Parkinson's typing).

Furthermore, when a UCHL1 gene abnormality is detected using a biological sample derived from a subject (for example, blood cells, skin mucous membrane cells, etc.), the subject who provided the biological sample is , It can be determined to carry a risk factor for Parkinson's disease.

Furthermore, when a UCHL1 gene abnormality is detected using a biological sample derived from a subject (eg, blood cells, skin mucous membrane cells, etc.), the subject who provided the biological sample is , Can be determined to be susceptible to Parkinson's disease. Here, the susceptibility of a subject to Parkinson's disease is also referred to as “susceptibility to Parkinson's disease”.

The method for analyzing the UCHL1 gene polymorphism is not particularly limited as long as the above polymorphism can be analyzed. A known method can be used as a method for analyzing a gene polymorphism. For example, from a sample such as peripheral blood collected from a control, a chromosomal DNA is extracted, or a DNA prepared by synthesizing cDNA by reverse transcription to extract mRNA is used as a template, and a portion containing the polymorphism of interest. PCR can be performed by using a primer set so that the PCR product can be amplified and analyzing the base sequence of the obtained PCR product. Further, in the analysis of the base sequence of the PCR product, it is not always necessary to determine the complete base sequence, and it can be analyzed by methods such as PCR-RFLP method and allele-specific PCR method.

The PCR-RFLP method is a gene analysis method based on differences in restriction enzyme sites caused by slight differences in nucleotide sequences. First, a gene fragment containing codon 9 is amplified by a nucleic acid amplification method such as PCR method. The gene that serves as a template may be collected from any cell as long as it is human chromosomal DNA. In the case of amplification by PCR, a sense primer containing a sequence complementary to the upstream intron of exon 6 and an antisense primer containing a sequence complementary to the downstream intron of exon 6 Can be used. In particular,
As the sense primer, 5'-cagcttacactcattttcaa
The aaatttc-3 '(SEQ ID NO: 5) and the antisense primer include 5'-gatatgccaagtgccataaagtcacac-3' (SEQ ID NO: 6), but are not limited thereto. The amplified fragment is then digested with the restriction enzymes MseI or Tru9I. As shown in FIG. 1, in the normal gene, it is thymine, which is the third base of codon 9 of exon 6, but when this is changed to adenine, a restriction enzyme site TTAA recognized and cleaved by restriction enzymes MseI and Tru9I, And restriction enzymes
A restriction enzyme site AATT that is recognized and cleaved by Sse9I and Tsp509I appears. Thus, for example, the amplified fragment is
When digested with MseI or Tru9I, if the gene is normal, it will not be cleaved at codon 9, and if it is abnormal, it will be cleaved. Therefore, by examining the size of the amplified fragment after digestion with the restriction enzyme MseI or Tru9I, it is possible to know whether or not codon 9 of the UCHL1 gene is normal. For example, as shown in FIG. 2, when a 27 bp oligonucleotide primer pair that hybridizes to introns on both sides of exon 6 of the human UCHL1 gene was used to perform amplification by PCR, a 209 bp gene containing exon 6 was obtained. The fragment is amplified. When this is digested with the restriction enzymes MseI or Tru9I, a fragment having the size shown in FIG. 2 is obtained. Therefore, when this is subjected to gel electrophoresis, a band pattern as shown in FIG. 3 is seen. So by this method,
Whether or not codon 9 is normal can be determined. In addition,
Since there are one pair of human chromosomal DNAs derived from parents, when an actual human-derived material is examined by the above method, in the case of normal type or abnormal type homozygosity,
Normal homotype (wt / wt type) and abnormal homotype (mt / mt)
The same band pattern as that shown in FIG. 3 is detected as shown in the lane of FIG. 3). ,
A band pattern in which a normal homotype and an abnormal homotype are mixed is detected.

Allele-specific PCR [Wu, DY, which utilizes the fact that amplification does not occur because a PCR primer does not function as a primer if there is a single base mismatch at the 3'end
et al .: Proc. Natl. Acad. Sci. USA., 86: 2757-2760
(1989)], it is also possible to carry out gene polymorphism analysis of UCHL1. More specifically, this is a method that utilizes the fact that if there is a mismatch in the PCR primer site, annealing of the primer does not occur well, resulting in no amplification by PCR. A major influence on the success of this method is a mismatch at the 3'ends of the primers. Therefore, by designing the primer so that the mutation occurs at the 3'end, whether or not amplification is possible is determined by the type of the allele. Therefore, according to the allele-specific PCR, the type of the allele can be confirmed by the presence or absence of the amplification product. Allele-specific PC
The design of such a primer in R can be performed based on the known base sequence of the UCHL1 gene. Human UC
As an example of a primer for detecting a polymorphism due to mutation of phenylalanine to leucine at codon 9 of exon 6 of HL1 gene, 5'-agatgacaaggtgaatttccatttt-
3 '(SEQ ID NO: 7) and 5'-agatgacaaggtgaatttccattta
-3 '(SEQ ID NO: 8) is included. The primers having the base sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 differ in the base at their 3'ends.
These primers and an appropriately selected reverse primer, for example, 5'-cagacgtactctttgcccagaaagg-3 '(SEQ ID NO: 9) in combination with a primer having a base sequence shown in SEQ ID NO: 7 and SEQ ID NO: 9 A 300 bp product was confirmed only by PCR with the primers, and a normal homotype (wt / wt type) was confirmed, and a 300 bp product was confirmed only by PCR with the primers of SEQ ID NO: 8 and SEQ ID NO: 9. One can be judged as an abnormal homo type (mt / mt type), and if a product can be confirmed in both cases, it can be judged as a normal / abnormal hetero type (wt / mt type).

[0021]

EXAMPLES Examples of the present invention will be specifically described below, but the scope of the present invention is not limited thereto. [Example 1] Search for cSNP on UCHL1 gene Using the bioinformatics method, cSNP on UCHL1 gene was searched for as follows. First, make a query of the amino acid sequence of UCHL1 protein,
From the NCBI database (EST human) containing multiple EST sequences derived from humans, UC using the program tBlastN
The EST sequence of the DNA encoding the HL1 protein was collected.
Then, the collected EST sequences were aligned. As a result, multiple cSNPs with different base sequences
Was found. Table 1 shows the search results for cSNP.

[0022]

[Table 1]

[Example 2] Identification of active center amino acid of UCHL1 protein UCHL3 having 51% amino acid identity with UCHL1 protein
The active center amino acid of the UCHL1 protein was identified by comparison with the protein. That is, in the UCHL3 protein, the amino acids that bind to ubiquitin (active center amino acids) are glutamine at the 89th position from the N-terminal, cysteine at the 95th position, histidine at the 169th position, and aspartic acid at the 184th position. Known [Johnston,
SC et al .: EMBO J. 16 (13): 3787-3796 (1997)]. Therefore, the amino acid sequences of the UCHL3 protein and the UCHL1 protein are aligned, and the amino acids on the UCHL1 protein corresponding to the active center amino acids of the UCHL3 protein, that is, the 84th glutamine from the N-terminal, the 90th cysteine, the 161st The histidine of position No. 1 and the aspartic acid of position No. 176 were identified and these were designated as the active center amino acids of the UCHL1 protein.

[Example 3] Modeling of UCHL1 protein three-dimensional structure Next, modeling of the UCHL1 protein three-dimensional structure was performed. That is, the protein three-dimensional structure prediction program MO based on the UCHL3 protein whose three-dimensional structure is already known.
E (Molecular Operating Environment, CHEMICAL COMPUT
ING GROUP INC.) Was used to predict the three-dimensional structure of the UCHL1 protein. The predicted three-dimensional structure of the UCHL1 protein is shown in Schematic representation in FIG. 5 together with the active center amino acid specified in Example 2.

Example 4 Identification of cSNP Candidate Residues Affecting Activity Based on the three-dimensional structure of the UCHL1 protein obtained in Example 3, substitution of amino acids within 5 angstrom of the active center amino acid was brought about. The resulting cSNP was screened from the cSNP of Example 1. As a result, UCHL
As a cSNP that results in the amino acid substitution of the 162nd phenylalanine within 5 angstroms of any of the four active center amino acids on one protein, the third base thymine in codon 9 of exon 6 of the UCHL1 gene is used. A cSNP to alanine was found.

[0026]

EFFECT OF THE INVENTION According to the present invention, polymorphism of ubiquitin carboxyl terminal hydrolase L1 (UCHL1) gene is analyzed, and detection of UCHL1 gene abnormality in a subject is detected based on the analysis result. Provided are a method, a method for predicting abnormal UCHL1 protein activity, a method for typing Parkinson's disease, a method for detecting a risk factor for Parkinson's disease, and a method for determining susceptibility to Parkinson's disease based on the method.

[0027]

[Sequence list]                                SEQUENCE LISTING           <110> PharmaDesign, Inc.    <120> A method of detection of an abnormal ubiquitin carboxyl-terminal e sterase L1 gene.    <130> PDP-0013    <160> 11    <170> PatentIn Ver. 2.1    <210> 1 <211> 672 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (1) .. (669)    <400> 1 atg cag ctc aag ccg atg gag atc aac ccc gag atg ctg aac aaa gtg 48 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val   1 5 10 15 ctg tcc cgg ctg ggg gtc gcc ggc cag tgg cgc ttc gtg gac gtg ctg 96 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu              20 25 30 ggg ctg gaa gag gag tct ctg ggc tcg gtg cca gcg cct gcc tgc gcg 144 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala          35 40 45 ctg ctg ctg ctg ttt ccc ctc acg gcc cag cat gag aac ttc agg aaa 192 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys      50 55 60 aag cag att gaa gag ctg aag gga caa gaa gtt agt cct aaa gtg tac 240 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr  65 70 75 80 ttc atg aag cag acc att ggg aat tcc tgt ggc aca atc gga ctt att 288 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile                  85 90 95 cac gca gtg gcc aat aat caa gac aaa ctg gga ttt gag gat gga tca 336 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser             100 105 110 gtt ctg aaa cag ttt ctt tct gaa aca gag aaa atg tcc cct gaa gac 384 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp         115 120 125 aga gca aaa tgc ttt gaa aag aat gag gcc ata cag gca gcc cat gat 432 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp     130 135 140 gcc gtg gca cag gaa ggc caa tgt cgg gta gat gac aag gtg aat ttc 480 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 cat ttt att ctg ttt aac aac gtg gat ggc cac ctc tat gaa ctt gat 528 His Phe Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp                 165 170 175 gga cga atg cct ttt ccg gtg aac cat ggc gcc agt tca gag gac acc 576 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr             180 185 190 ctg ctg aag gac gct gcc aag gtg tgc aga gaa ttc acc gag cgt gag 624 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu         195 200 205 caa gga gaa gtc cgc ttc tct gcc gtg gct ctc tgc aag gca gcc taa 672 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala     210 215 220       <210> 2 <211> 223 <212> PRT <213> Homo sapiens    <400> 2 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val   1 5 10 15 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu              20 25 30 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala          35 40 45 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys      50 55 60 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr  65 70 75 80 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile                  85 90 95 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser             100 105 110 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp         115 120 125 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp     130 135 140 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 His Phe Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp                 165 170 175 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr             180 185 190 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu         195 200 205 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala     210 215 220       <210> 3 <211> 672 <212> DNA <213> Homo sapiens    <220> <221> CDS <222> (1) .. (669)    <400> 3 atg cag ctc aag ccg atg gag atc aac ccc gag atg ctg aac aaa gtg 48 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val   1 5 10 15 ctg tcc cgg ctg ggg gtc gcc ggc cag tgg cgc ttc gtg gac gtg ctg 96 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu              20 25 30 ggg ctg gaa gag gag tct ctg ggc tcg gtg cca gcg cct gcc tgc gcg 144 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala          35 40 45 ctg ctg ctg ctg ttt ccc ctc acg gcc cag cat gag aac ttc agg aaa 192 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys      50 55 60 aag cag att gaa gag ctg aag gga caa gaa gtt agt cct aaa gtg tac 240 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr  65 70 75 80 ttc atg aag cag acc att ggg aat tcc tgt ggc aca atc gga ctt att 288 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile                  85 90 95 cac gca gtg gcc aat aat caa gac aaa ctg gga ttt gag gat gga tca 336 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser             100 105 110 gtt ctg aaa cag ttt ctt tct gaa aca gag aaa atg tcc cct gaa gac 384 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp         115 120 125 aga gca aaa tgc ttt gaa aag aat gag gcc ata cag gca gcc cat gat 432 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp     130 135 140 gcc gtg gca cag gaa ggc caa tgt cgg gta gat gac aag gtg aat ttc 480 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 cat tta att ctg ttt aac aac gtg gat ggc cac ctc tat gaa ctt gat 528 His Leu Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp                 165 170 175 gga cga atg cct ttt ccg gtg aac cat ggc gcc agt tca gag gac acc 576 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr             180 185 190 ctg ctg aag gac gct gcc aag gtg tgc aga gaa ttc acc gag cgt gag 624 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu         195 200 205 caa gga gaa gtc cgc ttc tct gcc gtg gct ctc tgc aag gca gcc taa 672 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala     210 215 220       <210> 4 <211> 223 <212> PRT <213> Homo sapiens    <400> 4 Met Gln Leu Lys Pro Met Glu Ile Asn Pro Glu Met Leu Asn Lys Val   1 5 10 15 Leu Ser Arg Leu Gly Val Ala Gly Gln Trp Arg Phe Val Asp Val Leu              20 25 30 Gly Leu Glu Glu Glu Ser Leu Gly Ser Val Pro Ala Pro Ala Cys Ala          35 40 45 Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys      50 55 60 Lys Gln Ile Glu Glu Leu Lys Gly Gln Glu Val Ser Pro Lys Val Tyr  65 70 75 80 Phe Met Lys Gln Thr Ile Gly Asn Ser Cys Gly Thr Ile Gly Leu Ile                  85 90 95 His Ala Val Ala Asn Asn Gln Asp Lys Leu Gly Phe Glu Asp Gly Ser             100 105 110 Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp         115 120 125 Arg Ala Lys Cys Phe Glu Lys Asn Glu Ala Ile Gln Ala Ala His Asp     130 135 140 Ala Val Ala Gln Glu Gly Gln Cys Arg Val Asp Asp Lys Val Asn Phe 145 150 155 160 His Leu Ile Leu Phe Asn Asn Val Asp Gly His Leu Tyr Glu Leu Asp                 165 170 175 Gly Arg Met Pro Phe Pro Val Asn His Gly Ala Ser Ser Glu Asp Thr             180 185 190 Leu Leu Lys Asp Ala Ala Lys Val Cys Arg Glu Phe Thr Glu Arg Glu         195 200 205 Gln Gly Glu Val Arg Phe Ser Ala Val Ala Leu Cys Lys Ala Ala     210 215 220       <210> 5 <211> 27 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 5 cagcttacac tcattttcaa aaatttc 27       <210> 6 <211> 27 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 6 gatatgccaa gtgccataaa gtcacac 27       <210> 7 <211> 25 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 7 agatgacaag gtgaatttcc atttt 25       <210> 8 <211> 25 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 8 agatgacaag gtgaatttcc attta 25       <210> 9 <211> 25 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 9 cagacgtact ctttgcccag aaagg 25       <210> 10 <211> 19 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 10 tttccatttt attctgttt 19       <210> 11 <211> 19 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 11 tttccattta attctgttt 19

[0028]

[Sequence list free text]

SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA SEQ ID NO: 8: Synthetic DNA SEQ ID NO: 9: Synthetic DNA SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: synthetic DNA

[Brief description of drawings]

FIG. 1 is a diagram showing the nucleotide sequences of normal and abnormal genes in the vicinity of codon 9 of human UCHL1 gene exon 6 together with restriction enzyme sites.

FIG. 2 is a diagram showing the sizes and positions of gene fragments detected by the PCR-RFLP method for normal and abnormal genes, respectively.

FIG. 3 is a diagram schematically showing a band pattern observed when the mixture of fragments shown in FIG. 2 is subjected to gel electrophoresis.

FIG. 4 is a diagram schematically showing band patterns of normal homozygous, normal / abnormal heterozygous, and abnormal homozygous subjects.

FIG. 5 is a schematic diagram showing a putative three-dimensional structure of UCHL1 protein.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 C12N 15/00 AF term (reference) 4B024 AA01 AA11 BA08 CA02 CA20 HA11 4B063 QA12 QA13 QA17 QA19 QQ02 QQ22 QQ43 QQ53 QR08 QR14 QR32 QR35 QR40 QR62 QS16 QS25 QS36 QS40 QX01 QX10

Claims (9)

[Claims] 1. A polymorphism of ubiquitin carboxyl terminal hydrolase L1 (UCHL1) gene in a biological sample derived from a subject is analyzed, and UCHL1 of the subject is analyzed based on the analysis result.
A method for detecting a UCHL1 gene abnormality, which comprises detecting a gene abnormality. 2. The UCHL1 gene polymorphism is a mutation in codon 9 of exon 6 of the human UCHL1 gene.
UCHL1 gene abnormality detection method. 3. The method for detecting an abnormality in the UCHL1 gene according to claim 2, wherein the mutation at codon 9 of exon 6 is a mutation at the third base thymine in the codon to adenine. 4. The abnormal UCHL1 gene according to claim 1, wherein the UCHL1 gene polymorphism is a codon mutation that replaces the 162nd phenylalanine with a leucine in the human UCHL1 protein consisting of the amino acid sequence represented by SEQ ID NO: 2. Detection method. 5. The UC according to claim 1, wherein the polymorphism of the UCHL1 gene is a mutation at the 486th base thymine of the human UCHL1 gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 to adenine.
Method for detecting HL1 gene abnormality. 6. A method for predicting UCHL1 protein activity abnormality, which comprises predicting an abnormality in UCHL1 protein activity in a subject based on the detection result obtained by the detection method according to claim 1. 7. Based on the detection result obtained by the detection method according to any one of claims 1 to 5, whether or not the cause of Parkinson's disease development in a subject with Parkinson's disease is due to UCHL1 gene abnormality. A method for typing Parkinson's disease, which comprises: 8. A method for detecting a risk factor for Parkinson's disease, which comprises detecting the presence or absence of a risk factor for Parkinson's disease in a subject based on the detection result obtained by the method for detecting according to claim 1. . 9. A method for determining susceptibility to Parkinson's disease, which comprises determining a subject's susceptibility to Parkinson's disease based on a detection result obtained by the detection method according to claim 1. .