JP2003070476A - Gene of electron transfer flavin protein dehydrogenase and method for diagnosing glutaric aciduria type ii - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate the mutation of the gene of an electron transfer flavin protein dehydrogenase (ETF-QO) becoming an etiology of glutaric aciduria type II (GAII), and to provide a method for diagnosing the GAII by utilizing the mutation. SOLUTION: The GAII is diagnosed by utilizing a polynucleotide having a cDNA of a ETF-QO gene of which thymine at the 1,519-position is converted to guanine by missense mutation, a part thereof, a polypeptide encoded thereby, a part thereof or the like.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the technical field of gene diagnosis, and in particular, an abnormal gene encoding an electron transfer flavoprotein dehydrogenase, which is a causative gene for glutamic aciduria type II, and glutamic acid urine utilizing the abnormal gene. A method for diagnosing illness type II

[0002]

2. Description of the Related Art Glutamic aciduria type II (hereinafter sometimes referred to as GAII) is a multiple acyl Co
-Also called A dehydrogenase deficiency, clinical symptoms include severe hypoglycemia immediately after birth, sudden infant death syndrome or syndrome-like symptoms in infancy, and almost no symptoms up to adulthood. Its severity varies. The cause of GAII is the mitochondrial electron transfer flavoprotein (electron transfer).
flavoprotein: ETF) or electron transfer flavoprotein dehydrogenase (ETF-QO: EC 1. 5.5.1). ETF is a dimer composed of α (α-ETF) and β subunit (β-ETF). α, β-
ETF and ETF-QO are encoded by independent genes. One ETF-QO mutation has already been reported. However, ETF-QO genomic structure and GA
The relationship with II was not clear. Although GAII is a rare disease, early diagnosis is difficult due to the variety of clinical symptoms, and development of an early diagnosis method at the gene level is desired.

[0003]

The present inventor has now found that
A new ETF-QO gene mutation leading to GAII was found. That is, as described later in detail, when the ETF-QO gene of patients diagnosed with GAII and their families was examined, position 1519 of the ETF-QO gene cDNA was mutated to guanine instead of thymine, and It was confirmed that the above-mentioned mutation is one of the etiological factors of GAII since it is a zygote, that it is derived from parents, and that this mutation is not found in the ETF-QO gene cDNA of healthy subjects. did.

The present invention is based on such knowledge, and this application provides the following inventions (1) to (15) as a solution to the above-mentioned problems. (1) A polynucleotide of a gene encoding an electron transfer flavoprotein dehydrogenase, which comprises DN of SEQ ID NO: 1.
A polynucleotide in which t (thymine) at the 1519th position in the A sequence is replaced with g (guanine), or a complementary sequence thereof. (2) A part of the polynucleotide of the invention (1), which contains the 1519th substitution base g of SEQ ID NO: 20
An oligonucleotide consisting of ~ 100 contiguous DNA sequences or its complementary sequence. (3) A polynucleotide derived from human chromosomal DNA which hybridizes with the polynucleotide of the invention (1) or the oligonucleotide of the invention (2), or a complementary sequence thereof under stringent conditions. (4) A double-stranded polynucleotide consisting of the polynucleotide of the invention (3) and its complementary sequence, or the mRNA transcribed from the polynucleotide of the invention (3) is added to P
A primer set for CR amplification, wherein one primer is an oligonucleotide consisting of 15 to 30 continuous DNA sequences containing the 1519th substitution base g of SEQ ID NO: 1 or its complementary sequence. (5) A polypeptide which is encoded by the polynucleotide of the invention (1) or (3) or is obtained by expression of the polynucleotide, wherein the Tyr (r) at position 507 in the amino acid sequence of SEQ ID NO: 2 ( A polypeptide in which (tyrosine) is amino acid substituted with Asp (aspartic acid). (6) An oligopeptide which is a part of the polypeptide of the invention (5) and comprises 5 to 30 consecutive amino acid sequences containing the 507th substitution amino acid Asp of SEQ ID NO: 2. (7) An antibody produced using the oligopeptide of the invention (6) as an antigen. (8) A method for diagnosing glutaric aciduria type II, which comprises detecting whether or not the polynucleotide of the invention (3) is present in chromosomal DNA isolated from a subject. (9) Chromosomal DNA isolated from a subject or m thereof
The method of the above-mentioned invention (8), which detects whether RNA is hybridized with the polynucleotide of the above-mentioned invention (1) or the oligonucleotide of the above-mentioned invention (2), or a complementary sequence thereof under stringent conditions. (10) Chromosomal DNA or mR isolated from a subject
The method according to the invention (8), wherein the presence or absence of a PCR product is detected when PCR is performed using NA as a template and the primer set according to the invention (4). (11) A method for diagnosing glutaric aciduria type II, which comprises detecting whether or not the polypeptide of the invention (5) is present in a biological sample isolated from a subject. (12) The method of the above-mentioned invention (11), which detects whether or not a polypeptide that reacts with the antibody of the above-mentioned invention (7) is present in a biological sample isolated from a subject. (13) A DNA probe obtained by labeling the oligonucleotide of the invention (2). (14) A DNA chip comprising the oligonucleotide of the invention (2). (15) A labeled antibody, which is obtained by labeling the antibody of the invention (7).

The embodiments of each of these inventions will be described in detail below. In the present specification, a continuous sequence of 101 or more nucleotides is a polynucleotide,
An oligonucleotide is defined as a sequence of ~ 100 contiguous nucleotides. Further, a continuous sequence of 31 or more amino acids is defined as a polypeptide, and a continuous amino acid sequence of 2 to 30 is defined as an oligopeptide.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The invention (1) of this application is the ETF.
-A mutant DNA sequence of the cDNA (SEQ ID NO: 1) of the QO gene, which is 1519th in the DNA sequence of SEQ ID NO: 1.
Position t (thymine) is a polynucleotide in which g (guanine) is base-substituted or its complementary sequence. The DNA sequence of the ETF-QO gene represented by SEQ ID NO: 1 is registered in GenBank as Accessin No. S69232. As described above, the abnormal gene according to the present invention is the cDNA of this normal (wild type) ETF-QO gene.
Missense mutatio with a point mutation at position 19
n).

The polynucleotide of the invention (1) is, for example, a cDNA prepared from the total mRNA of a GAII patient using the oligonucleotide of the invention (2) described below as a probe.
It can be isolated by screening the library. In addition, using the primer set of the invention (4) described below, RT using the mRNA of GAII patient as a template
-It can also be isolated by PCR. Alternatively, it can also be obtained by introducing the above base substitution into the cDNA of wild type (normal) ETF-QO using a commercially available mutation kit or the like.

The invention (2) of this application is the above invention (1).
Which is a part of the polynucleotide, and is an oligonucleotide consisting of 20 to 100 continuous DNA sequences containing the respective substituted bases or a complementary sequence thereof. The oligonucleotide of the invention (2) can be produced by chemically synthesizing it by a known method. Alternatively, the polynucleotide of the invention (1) can be prepared by a method such as cutting with a suitable restriction enzyme. It can be used for the polynucleotide of the invention (2) and the method for diagnosing GAII of the invention (8) described later.

The invention (3) of this application relates to the above invention (1).
Or a polynucleotide of the invention (2) or a polynucleotide derived from human chromosomal DNA (genomic DNA) which hybridizes with the complementary sequence thereof under stringent conditions. here,
Stringent conditions are conditions that allow selective and detectable specific binding of those polynucleotides or oligonucleotides to genomic DNA derived from a chromosome. As is well known,
Stringent conditions are determined by salt concentration, temperature, and other conditions. For example, the lower the salt concentration and the higher the temperature, the higher the stringency and the less likely it is to hybridize. The salt concentration is generally adjusted by adjusting the concentration of the SSC solution (NaCl + trisodium citrate) and the stringent salt concentration is, for example, about 250 mM NaCl or less and about 25 mM trisodium citrate or less. The stringent temperature is generally 15 to 25 ° C. lower than the melting temperature (Tm) of the perfect hybrid, for example, about 30 ° C. or higher. The temperature can be lowered by adding an organic solvent (eg formamide) to the solution. Other conditions include hybridization time, detergent (eg, SDS) concentration, presence or absence of carrier DNA, and the like, and various stringencies can be set by combining these conditions. As one preferred example, 250 mM
NaCl, 25 mM trisodium citrate, 1% SD
Hybridization is carried out at a temperature of 42 ° C. under the conditions of S, 50% formamide and 200 μg / ml denatured salmon sperm DNA. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, with decreasing salt concentration and increasing temperature increasing wash stringency. As one preferred example, 15 mM NaC
1, 1.5 mM trisodium citrate and 0.1% S
Washing is performed at a temperature of 68 ° C. under the condition of DS. For the stringent conditions, see, for example, J. Sambrook et al., “Molecular Cloning, A Laboratory Mannual, S.
econd Edition, Cold Spring Harbor Laboratory Press
(1989), especially Section 11.45 “Conditions for Hybridiz
ation of Oligonucleotide Probes "", and appropriate conditions can be easily used by referring to those descriptions. The polynucleotide of the invention (3) screens a genomic library prepared from the chromosomal DNA of a GAII patient by, for example, the above-mentioned stringent hybridization and washing treatment using the oligonucleotide of the invention (2) as a probe. It can be isolated by
The polynucleotide of the invention (3) is to be detected in the diagnostic method of the invention (8) described below.

The invention (4) of this application is the above invention (3).
Double-stranded polynucleotide (genomic DNA) comprising the polynucleotide of claim 1 and its complementary sequence, or the invention (3)
Is a set of primers for PCR amplification of mRNA transcribed from the polynucleotide. In these primer sets, one of the oligonucleotide primers is composed of 15 to 30 continuous DNA sequences containing the substitution base of SEQ ID NO: 1 or its complementary sequence. The other primer can be any continuous DNA sequence 5'or 3'to the substituted base of SEQ ID NO: 1 or its complementary sequence. These primer sets are known DN based on SEQ ID NO: 1 containing the respective substituted bases.
It can be produced by the A synthesis method. Further, a linker sequence or the like can be added to the end of the primer.
The primer set of the invention (4) can be used in the method for diagnosing GAII of the invention (8) described below.

The invention (5) of this application is a polypeptide encoded by the polynucleotide of the invention (1) or (3) or obtained by the expression of the polynucleotide, wherein the amino acid sequence of SEQ ID NO: 2 is present. In the fifth
It is a polypeptide in which Tyr (tyrosine) at position 07 is amino acid substituted with Asp (aspartic acid). That is, as described above, the base substitution in the polynucleotide of the invention (1) is a missense mutation, and the amino acid of the normal (wild type) polypeptide is mutated as described above by one base substitution. These polypeptides are
A method of isolating a biological sample of a GAII patient according to a known method, a method of preparing a peptide by chemical synthesis based on the amino acid sequence of SEQ ID NO: 2 containing each substituted amino acid residue, or the polynucleotide (mutation of the invention (1) (mutation It can be obtained by a method of producing by recombinant DNA technology using cDNA). These inventions (5)
The polypeptide is, for example, GAII of the invention (11) described below.
It can be the inspection target of the diagnostic method.

The invention (6) of this application is a part of the polypeptide of the invention (5), which is an oligopeptide having a continuous amino acid sequence of 5 to 30 including a substituted amino acid. These oligopeptides can be produced by a method of chemically synthesizing based on a predetermined amino acid sequence, a method of digesting the polypeptide of the invention (5) with an appropriate protease, or the like. These oligopeptides can be used, for example, as an antigen for producing the antibody of the invention (7) described below.

The antibody of the invention (7) is a polyclonal antibody or a monoclonal antibody prepared by using the oligopolypeptide of the invention (6) as an antigen. These antibodies can be produced by known antibody production methods. Further, this antibody can specifically recognize the polypeptide of the invention (5), and can be used in the diagnostic method of the invention (11) described below.

In the invention (8) of this application, the subject is GAII.
It is a method of diagnosing whether there is a possibility of developing.
That is, chromosomal DNA is isolated from a biological sample of a subject, and when the polynucleotide of the invention (3) is present in this DNA, this subject is judged to be high risk for GAII. The polynucleotide can be detected by various known methods, but the method of the invention (9) or (10) is preferable.

In the method of the invention (9), the chromosomal DNA or mRNA thereof isolated from the subject and the above-mentioned invention (1) are used.
It is detected whether the polynucleotide of 1) or the oligonucleotide of the invention (2) hybridizes under stringent conditions. If the subject has a genetic mutation associated with GAII, chromosomal DNA or its mR
NA and the polynucleotide or oligonucleotide are
It hybridizes even under stringent conditions. Hybridization can be detected by a known method. For example, the DNA probe of the invention (13),
Using the DNA chip of the invention (14), for example, ASO
By performing hybridization by the (allele specific oligomer) method, it can be performed easily and with high accuracy.

In the method of the invention (10), PCR (including RT-PCR) is performed according to a known method using the chromosomal DNA or mRNA isolated from the subject as a template and the primer set of the invention (4). P when performing
The presence or absence of CR products is detected. If the subject has a genetic mutation associated with GAII, a PCR product of the polynucleotide defined by the primer set is obtained. The analysis of PCR products can also be performed according to known methods. For example, G found by the inventor
The abnormal gene of ETF-QO, which is the etiological gene of AII, has a nucleotide sequence from t to g at position 1519 in SEQ ID NO: 1, resulting in the sequence "gatc" at positions 1519 to 1522. It is cleaved by DpnI, and since the "ggatcc" sequence is generated at positions 1518 to 1523, it can be cleaved by the restriction enzyme BamHI. Therefore, PFLP (restriction
The presence or absence of the polynucleotide having the gene mutation can be detected by the fragment length polymorphism) method.

Also in the invention (11) of this application, the subject is GA
It is a method of diagnosing whether or not there is a possibility of developing II, and in the case where the polypeptide of the invention (5) is present in the biological sample isolated from the subject, the subject is judged to be at high risk for GAII. To do. The presence of the polypeptide can be achieved by various known methods, but the invention (12)
Is preferred. The method of the invention (12) is the invention (7).
This is a method using an antibody, and particularly, by using the labeled antibody of the invention (15), simple and highly accurate detection becomes possible. As the label, various known labels such as enzymes, isotopes, and fluorescent dyes can be used.

The following are the gene mutations on which the present invention is based.
I will explain the research content that was confirmed. Study Japanese
The molecular biology of GAII in Escherichia coli
To clarify the genomic structure of the F-QO gene
It wasMethod Lymphoblasts established from patient B lymphocytes and patient skin fibers
The blast cells were used as the material. [<SUP> 3 </ SUP> H]-
Using labeled myristic acid, palmitic acid and oleic acid
The fatty acid β-oxidation capacity of the cells was compared with that of control cells. Fine
Of α- and β-ETF in cell lysate
The patient method and control cells were compared by the Gott method. α and β-E
GenBank all exons and flanking introns of TF gene
Amplify by PCR based on the information of
went. Regarding ETF-QO, RT that sandwiches the entire translation region
-After performing PCR, direct sequencing was performed. simultaneous
Of ETF-QO from PAC human genomic library
ETF-QO gene isolated from a clone containing a translation region
Patients, families and healthy subjects after elucidation of the genomic structure of
Performed ETF-QO gene analysis using the control's genome
It was Analysis is done with informed consent
Approved by the Science Committee.

[0019]Case The case was a boy who was 2 months old at the time of diagnosis, and had no abnormality in histories of pregnancy and delivery.
Cousin was born between married parents. 2 of 7 brothers
He presented with psychomotor retardation and died in early childhood. This child
Significant fat caused by injection nutrition
I had a liver problem and required intensive care. Psychomotor development after that
A delay was noted, but he died suddenly at the age of three. 2 in patient's urine
-Hydroxyglutarate, ethylmalonic acid and glutarate are not
The number of patients was increasing, and GAII was chemically diagnosed based on the time of onset.
(Hirose S. et al., Acta Paediatr89: 887-887, 200
0).

[0020]Grade Fatty acid β-oxidation ability of patients' lymphoblasts is lower than normal
And supported the chemical diagnosis of GAII. Western block
Method, qualitatively and quantitatively for α and β-ETF in patient cells
I didn't find any abnormalities. Similarly for α and β-ETF
There was no abnormality in the gene. Obtained by RT-PCR
There was no difference in the size and expression of ETF-QO mRNA.
Although it was not found, the
A mutation of min → guanine was found. This mutation is the 507th
Located at the first codon of amino acid Tyr of
Patients are homozygous for missense mutations that result in amino acid mutations
It seemed to have as a united body. Family and patient
A nom analysis revealed that 3 of the parents and siblings had this change.
Have heterozygosity and the patient is homozygous
Was confirmed. ETF-Q within the analyzed range
There were no other mutations. Restriction enzyme Ba generated by this mutation
Genome from 100 healthy controls using mHI cleavage
This mutation was not found in the analysis.

[0021]Conclusion The patient had no abnormalities in α and β-ETF, and ETF-QO
T → g base mutation at the 1519th position in the gene (507th position)
Tyr → Asp amino acid mutation) as a homozygote
Was. In addition, Tyr at the 507th crossed the seeds and ET
Conserved in F-QO family, healthy controls
The absence of the same mutation in the 200 allele derived from
T → g base mutation at position 519 (Tyr → Asp at position 507)
Amino acid mutation), a novel ETF that causes delayed GAII
-Determined to be QO gene abnormality.

[0022]

As described above in detail, the present invention is based on GAII.
Novel ETF-QO mutant gene, which is the etiological gene of
The present invention provides a method for diagnosing GAII using the method, and can contribute to the development of early diagnosis of GAII at the gene level.

[0023]

[Sequence list]                 SEQUENCE LISTING <100> Japan Science and Technology Corporation <120> An electron transfer flavoprotein ubiquinone oxidoreductase-encod ing gene and a diagnosis of GAII utilizing the same <130> P0466T <160> 2 <210> 1 <211> 1854 <212> DNA <213> Homo sapiens <400> 1 atg ctg gtg ccg cta gcc aag ctg tcc tgc ctg gca tat cag tgc 45 Met Leu Val Pro Leu Ala Lys Leu Ser Cys Leu Ala Tyr Gln Cys                 5 10 15 ttt cat gcc tta aaa att aag aaa aat tat cta cct cta tgt gct 90 Phe His Ala Leu Lys Ile Lys Lys Asn Tyr Leu Pro Leu Cys Ala                 20 25 30 ata aga tgg tct tca act tct act gtg cct cga att act acc cat 135 Ile Arg Trp Ser Ser Thr Ser Thr Val Pro Arg Ile Thr Thr His                 35 40 45 tat act att tat ccc cgg gat aag gac aag aga tgg gaa gga gtg 180 Tyr Thr Ile Tyr Pro Arg Asp Lys Asp Lys Arg Trp Glu Gly Val                 50 55 60 aac atg gaa agg ttt gca gaa gaa gca gat gtt gta ata gtt ggt 225 Asn Met Glu Arg Phe Ala Glu Glu Ala Asp Val Val Ile Val Gly                 65 70 75 gca ggc cct gca ggg ctc tct gca gct gtt cgt cta aaa cag ttg 270 Ala Gly Pro Ala Gly Leu Ser Ala Ala Val Arg Leu Lys Gln Leu                 80 85 90 gct gtg gca cat gaa aag gac atc cgt gtg tgt cta gtg gag aaa 315 Ala Val Ala His Glu Lys Asp Ile Arg Val Cys Leu Val Glu Lys                 95 100 105 gct gcc cag ata gga gct cat act ctc tca ggg gct tgc ctt gat 360 Ala Ala Gln Ile Gly Ala His Thr Leu Ser Gly Ala Cys Leu Asp                 110 115 120 cca ggt gct ttt aaa gaa ctc ttc cca gac tgg aaa gag aag ggg 405 Pro Gly Ala Phe Lys Glu Leu Phe Pro Asp Trp Lys Glu Lys Gly                 125 130 135 gct cca ctt aac act cct gta aca gaa gac aga ttt gga att tta 450 Ala Pro Leu Asn Thr Pro Val Thr Glu Asp Arg Phe Gly Ile Leu                 140 145 150 aca gag aaa tac aga att cct gtg cca att ctt cca ggg ctt cca 495 Thr Glu Lys Tyr Arg Ile Pro Val Pro Ile Leu Pro Gly Leu Pro                 155 160 165 atg aat aat cat ggc aat tac att gta cgc ttg gga cat tta gtg 540 Met Asn Asn His Gly Asn Tyr Ile Val Arg Leu Gly His Leu Val                 170 175 180 agc tgg atg ggc gaa caa gca gaa gcc ctt ggt gtt gaa gta tac 585 Ser Trp Met Gly Glu Gln Ala Glu Ala Leu Gly Val Glu Val Tyr                 185 190 195 cct ggt tat gca gct gct gag gtc ctt ttt cat gat gat ggt agt 630 Pro Gly Tyr Ala Ala Ala Glu Val Leu Phe His Asp Asp Gly Ser                 200 205 210 gta aaa gga att gcc act aac gat gta ggg ata caa aag gat ggt 675 Val Lys Gly Ile Ala Thr Asn Asp Val Gly Ile Gln Lys Asp Gly                 215 220 225 gca cca aag gca aca ttt gag aga gga ctg gaa cta cat gct aaa 720 Ala Pro Lys Ala Thr Phe Glu Arg Gly Leu Glu Leu His Ala Lys                 230 235 240 gtc aca att ttt gca gaa ggt tgc cat gga cat cta gcc aag caa 765 Val Thr Ile Phe Ala Glu Gly Cys His Gly His Leu Ala Lys Gln                 245 250 255 cta tat aag aag ttt gat ttg aga gca aat tgt gaa cct caa acc 810 Leu Tyr Lys Lys Phe Asp Leu Arg Ala Asn Cys Glu Pro Gln Thr                 260 265 270 tac ggg att gga ctg aag gag tta tgg gtt att gat gaa aag aac 855 Tyr Gly Ile Gly Leu Lys Glu Leu Trp Val Ile Asp Glu Lys Asn                 275 280 285 tgg aaa cct ggg aga gta gat cac act gtt ggt tgg ccc ttg gac 900 Trp Lys Pro Gly Arg Val Asp His Thr Val Gly Trp Pro Leu Asp                 290 295 300 aga cat acc tat gga gga tct ttc ctc tat cat ttg aat gaa ggt 945 Arg His Thr Tyr Gly Gly Ser Phe Leu Tyr His Leu Asn Glu Gly                 305 310 315 gaa ccc cta gta gct ctt ggt ctt gtg gtt ggt cta gac tat cag 990 Glu Pro Leu Val Ala Leu Gly Leu Val Val Gly Leu Asp Tyr Gln                 320 325 330 aat cca tac ctg agt cca ttt aga gag ttc caa agg tgg aaa cac 1035 Asn Pro Tyr Leu Ser Pro Phe Arg Glu Phe Gln Arg Trp Lys His                 335 340 345 cat cct agc att cgg cca acc ttg gaa ggt gga aaa agg att gca 1080 His Pro Ser Ile Arg Pro Thr Leu Glu Gly Gly Lys Arg Ile Ala                 350 355 360 tac gga gcc aga gct ctc aat gaa ggt ggc ttt cag tct ata cca 1125 Tyr Gly Ala Arg Ala Leu Asn Glu Gly Gly Phe Gln Ser Ile Pro                 365 370 375 aaa ctc acc ttt cct ggt ggt tta cta att ggt tgt agt cct ggt 1170 Lys Leu Thr Phe Pro Gly Gly Leu Leu Ile Gly Cys Ser Pro Gly                 380 385 390 ttt atg aat gtt ccc aag atc aaa ggt act cac aca gca atg aaa 1215 Phe Met Asn Val Pro Lys Ile Lys Gly Thr His Thr Ala Met Lys                 395 400 405 agt gga att tta gca gca gaa tct att ttt aat caa cta act agt 1260 Ser Gly Ile Leu Ala Ala Glu Ser Ile Phe Asn Gln Leu Thr Ser                 410 415 420 gaa aat ctc caa tca aag aca ata gga ctc cat gta act gaa tat 1305 Glu Asn Leu Gln Ser Lys Thr Ile Gly Leu His Val Thr Glu Tyr                 425 430 435 gag gac aat ttg aag aac tca tgg gta tgg aaa gag cta tat tct 1350 Glu Asp Asn Leu Lys Asn Ser Trp Val Trp Lys Glu Leu Tyr Ser                 440 445 450 gtt aga aat ata aga ccg tcc tgc cac gga gta ctg ggt gta tat 1395 Val Arg Asn Ile Arg Pro Ser Cys His Gly Val Leu Gly Val Tyr                 455 460 465 gga ggg atg att tac act gga atc ttt tac tgg ata ttg aga gga 1440 Gly Gly Met Ile Tyr Thr Gly Ile Phe Tyr Trp Ile Leu Arg Gly                 470 475 480 atg gag ccg tgg act ctg aaa cat aaa ggt tct gac ttt gaa cgg 1485 Met Glu Pro Trp Thr Leu Lys His Lys Gly Ser Asp Phe Glu Arg                 485 490 495 ctc aag cca gcc aag gat tgc aca cct att gag tat cca aaa ccc 1530 Leu Lys Pro Ala Lys Asp Cys Thr Pro Ile Glu Tyr Pro Lys Pro                 500 505 510 gat gga cag atc agt ttt gac ctc ttg tca tct gtg gct ctg agt 1575 Asp Gly Gln Ile Ser Phe Asp Leu Leu Ser Ser Val Ala Leu Ser                 515 520 525 ggt act aat cat gaa cat gac cag ccg gca cac tta acc tta agg 1620 Gly Thr Asn His Glu His Asp Gln Pro Ala His Leu Thr Leu Arg                 530 535 540 gat gac agt ata cct gta aat aga aat ctg tcg ata tat gat ggg 1665 Asp Asp Ser Ile Pro Val Asn Arg Asn Leu Ser Ile Tyr Asp Gly                 545 550 555 ccc gag cag cga ttc tgt cct gca gga gtt tat gaa ttt gta cct 1710 Pro Glu Gln Arg Phe Cys Pro Ala Gly Val Tyr Glu Phe Val Pro                 560 565 570 gtg gaa caa ggt gat gga ttt cgg tta cag ata aat gct cag aac 1755 Val Glu Gln Gly Asp Gly Phe Arg Leu Gln Ile Asn Ala Gln Asn                 575 580 585 tgt gta cat tgt aaa aca tgt gat att aaa gat cca agt cag aat 1800 Cys Val His Cys Lys Thr Cys Asp Ile Lys Asp Pro Ser Gln Asn                 590 595 600 att aac tgg gtg gta cct gaa ggt gga gga gga cct gct tac aat 1845 Ile Asn Trp Val Val Glu Gly Gly Gly Gly Pro Ala Tyr Asn                 605 610 615 gga atg taa 1854 Gly Met <210> 2 <211> 617 <212> PRT <213> Homo sapiens <400> 2 Met Leu Val Pro Leu Ala Lys Leu Ser Cys Leu Ala Tyr Gln Cys                 5 10 15 Phe His Ala Leu Lys Ile Lys Lys Asn Tyr Leu Pro Leu Cys Ala                 20 25 30 Ile Arg Trp Ser Ser Thr Ser Thr Val Pro Arg Ile Thr Thr His                 35 40 45 Tyr Thr Ile Tyr Pro Arg Asp Lys Asp Lys Arg Trp Glu Gly Val                 50 55 60 Asn Met Glu Arg Phe Ala Glu Glu Ala Asp Val Val Ile Val Gly                 65 70 75 Ala Gly Pro Ala Gly Leu Ser Ala Ala Val Arg Leu Lys Gln Leu                 80 85 90 Ala Val Ala His Glu Lys Asp Ile Arg Val Cys Leu Val Glu Lys                 95 100 105 Ala Ala Gln Ile Gly Ala His Thr Leu Ser Gly Ala Cys Leu Asp                 110 115 120 Pro Gly Ala Phe Lys Glu Leu Phe Pro Asp Trp Lys Glu Lys Gly                 125 130 135 Ala Pro Leu Asn Thr Pro Val Thr Glu Asp Arg Phe Gly Ile Leu                 140 145 150 Thr Glu Lys Tyr Arg Ile Pro Val Pro Ile Leu Pro Gly Leu Pro                 155 160 165 Met Asn Asn His Gly Asn Tyr Ile Val Arg Leu Gly His Leu Val                 170 175 180 Ser Trp Met Gly Glu Gln Ala Glu Ala Leu Gly Val Glu Val Tyr                 185 190 195 Pro Gly Tyr Ala Ala Ala Glu Val Leu Phe His Asp Asp Gly Ser                 200 205 210 Val Lys Gly Ile Ala Thr Asn Asp Val Gly Ile Gln Lys Asp Gly                 215 220 225 Ala Pro Lys Ala Thr Phe Glu Arg Gly Leu Glu Leu His Ala Lys                 230 235 240 Val Thr Ile Phe Ala Glu Gly Cys His Gly His Leu Ala Lys Gln                 245 250 255 Leu Tyr Lys Lys Phe Asp Leu Arg Ala Asn Cys Glu Pro Gln Thr                 260 265 270 Tyr Gly Ile Gly Leu Lys Glu Leu Trp Val Ile Asp Glu Lys Asn                 275 280 285 Trp Lys Pro Gly Arg Val Asp His Thr Val Gly Trp Pro Leu Asp                 290 295 300 Arg His Thr Tyr Gly Gly Ser Phe Leu Tyr His Leu Asn Glu Gly                 305 310 315 Glu Pro Leu Val Ala Leu Gly Leu Val Val Gly Leu Asp Tyr Gln                 320 325 330 Asn Pro Tyr Leu Ser Pro Phe Arg Glu Phe Gln Arg Trp Lys His                 335 340 345 His Pro Ser Ile Arg Pro Thr Leu Glu Gly Gly Lys Arg Ile Ala                 350 355 360 Tyr Gly Ala Arg Ala Leu Asn Glu Gly Gly Phe Gln Ser Ile Pro                 365 370 375 Lys Leu Thr Phe Pro Gly Gly Leu Leu Ile Gly Cys Ser Pro Gly                 380 385 390 Phe Met Asn Val Pro Lys Ile Lys Gly Thr His Thr Ala Met Lys                 395 400 405 Ser Gly Ile Leu Ala Ala Glu Ser Ile Phe Asn Gln Leu Thr Ser                 410 415 420 Glu Asn Leu Gln Ser Lys Thr Ile Gly Leu His Val Thr Glu Tyr                 425 430 435 Glu Asp Asn Leu Lys Asn Ser Trp Val Trp Lys Glu Leu Tyr Ser                 440 445 450 Val Arg Asn Ile Arg Pro Ser Cys His Gly Val Leu Gly Val Tyr                 455 460 465 Gly Gly Met Ile Tyr Thr Gly Ile Phe Tyr Trp Ile Leu Arg Gly                 470 475 480 Met Glu Pro Trp Thr Leu Lys His Lys Gly Ser Asp Phe Glu Arg                 485 490 495 Leu Lys Pro Ala Lys Asp Cys Thr Pro Ile Glu Tyr Pro Lys Pro                 500 505 510 Asp Gly Gln Ile Ser Phe Asp Leu Leu Ser Ser Val Ala Leu Ser                 515 520 525 Gly Thr Asn His Glu His Asp Gln Pro Ala His Leu Thr Leu Arg                 530 535 540 Asp Asp Ser Ile Pro Val Asn Arg Asn Leu Ser Ile Tyr Asp Gly                 545 550 555 Pro Glu Gln Arg Phe Cys Pro Ala Gly Val Tyr Glu Phe Val Pro                 560 565 570 Val Glu Gln Gly Asp Gly Phe Arg Leu Gln Ile Asn Ala Gln Asn                 575 580 585 Cys Val His Cys Lys Thr Cys Asp Ile Lys Asp Pro Ser Gln Asn                 590 595 600 Ile Asn Trp Val Val Glu Gly Gly Gly Gly Pro Ala Tyr Asn                 605 610 615 Gly Met

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) G01N 33/53 G01N 33/566 33/58 A 33/566 Z 33/58 37/00 102 C12N 15/00 ZNAA 37/00 102 F F term (reference) 2G045 AA25 DA12 DA13 DA14 DA36 DA77 FB02 FB03 FB07 4B024 AA11 BA80 CA01 CA09 CA11 HA12 4B050 CC01 CC03 CC04 DD11 LL03 4B063 QA01 QA18 QA19 QQ01 QQ42 QQ52 QR08 QR42 QR55 QR62 QR82 QS25 QS34 QS36 QX02 4H045 AA11 AA30 CA40 DA76 EA50 FA72 FA74

Claims (15)

[Claims] 1. A polynucleotide of a gene encoding an electron transfer flavoprotein dehydrogenase, which is SEQ ID NO: 1.
In the DNA sequence of, the t (thymine) at position 1519 is g
A polynucleotide having a base substitution of (guanine) or its complementary sequence. 2. A part of the polynucleotide according to claim 1, which comprises the substitution base g at the 1519th position of SEQ ID NO: 1.
An oligonucleotide consisting of ~ 100 contiguous DNA sequences or its complementary sequence. 3. A polynucleotide derived from human chromosomal DNA which hybridizes with the polynucleotide according to claim 1 or the oligonucleotide according to claim 2 or a complementary sequence thereof under stringent conditions. 4. A double-stranded polynucleotide comprising the polynucleotide of claim 3 and its complementary sequence, or claim 3.
Is a primer set for PCR-amplifying mRNA transcribed from the polynucleotide of No. 1, wherein one primer contains the 1519th substitution base g of SEQ ID NO: 1.
A primer set which is an oligonucleotide consisting of ~ 30 continuous DNA sequences or their complementary sequences. 5. A polypeptide encoded by the polynucleotide according to claim 1 or 3 or obtained by expression of said polynucleotide, wherein Tyr (tyrosine) at position 507 in the amino acid sequence of SEQ ID NO: 2. Is a polypeptide in which the amino acid has been replaced by Asp (aspartic acid). 6. An oligopeptide which is a part of the polypeptide of claim 5 and which comprises a continuous amino acid sequence of 5 to 30 including the 507th substitution amino acid Asp of SEQ ID NO: 2. 7. An antibody produced by using the oligopeptide of claim 6 as an antigen. 8. A method for diagnosing glutaric aciduria type II, which comprises detecting whether or not the polynucleotide of claim 3 is present in chromosomal DNA isolated from a subject. 9. It is determined whether the chromosomal DNA isolated from a subject or its mRNA is hybridized with the polynucleotide of claim 1 or the oligonucleotide of claim 2 or a complementary sequence thereof under stringent conditions. 9. The method of claim 8 to detect. 10. The method according to claim 8, wherein presence or absence of a PCR product is detected when PCR is performed using the chromosomal DNA or mRNA isolated from a subject as a template and using the primer set according to claim 4. 11. A method for diagnosing glutaric aciduria type II, which comprises detecting whether or not the polypeptide of claim 5 is present in a biological sample isolated from a subject. 12. The method according to claim 11, which comprises detecting whether or not a polypeptide reactive with the antibody according to claim 7 is present in a biological sample isolated from a subject. 13. A DNA probe obtained by labeling the oligonucleotide of claim 2. 14. A DNA chip comprising the oligonucleotide according to claim 2. 15. A labeled antibody obtained by labeling the antibody of claim 7.