JP2003070485A - Alanine: glyoxylic acid aminotransferase gene and method for diagnosis of type i primary hyperoxaluria using the gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clarify the mutation of alanine: glyoxylic acid aminotransferase (AGT) gene causing type I primary hyperoxaluria (PH1) and provide a method for the diagnosis of PH1 using the gene. SOLUTION: PH1 is diagnosed by using a polynucleotide having a structure of cDNA of AGT gene provided that the 751st thymine is changed to adenine and the 752nd guanine to adenine by missense mutation, a part of the polynucleotide, a polypeptide coded by the polynucleotide or a part of the polypeptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the technical field of gene diagnosis, and in particular, it utilizes an abnormal gene encoding alanine: glyoxylate aminotransferase, which is a causative gene of primary hyperoxaluria type I, and the use thereof. The present invention relates to a method for diagnosing primary hyperoxaluria type I.

[0002]

2. Description of the Related Art Primary hyperoxaluria type I (hereinafter sometimes abbreviated as PH1) is a relatively rare autosomal recessive inherited glyoxylic acid metabolism abnormality, and is associated with oxalic acid and glycolic acid. It is a disease in which insoluble calcium oxalate accumulates throughout the body including the kidneys due to increased production and excretion in urine. Although its clinical manifestations are diverse, many sufferers have a poor prognosis due to renal failure and other factors. The cause is that glyoxylic acid, which is a metabolite of glycolic acid, is not metabolized to glycine due to alanine: glyoxylate transferase (AGT) abnormality of the peroxisome enzyme, and oxalic acid and glycolic acid, which are oxides of glyoxylic acid, are excessively produced.
Due to being discharged. Regarding the abnormality of the AGT gene, several pathogenic mutations are already known. For example, A
A nonsense mutation (Q12fs × 167) in which cytosine is inserted between the 33rd and 34th positions of the GT gene cDNA and a premature stop codon appears has been found.
Since PH1 is a disease that can be treated by liver transplantation before the onset, early diagnosis before the onset is important.
Further investigation is required at the gene level regarding AGT so that it can be surely known whether or not the onset of No. 1 is possible.

[0003]

The present inventor has now found that
We found a new mutation in the AGT gene that causes PH1. That is, as will be described later in detail, when the abnormality of the cDNA of the AGT gene was searched using the gene of the patient definitely diagnosed with PH1 and its parents, the 751th position in the patient's gene was adenine instead of thymine, Also, the 75th
We found that the second position was mutated to adenine instead of guanine, which was derived from the mother, and was compared and searched for healthy subjects 2
Since this mutation was not found in the genes of 00, it was confirmed that these mutations were the etiological factors.

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 alanine: glyoxylate aminotransferase, which is the t at the 751 position in the DNA sequence of SEQ ID NO: 1.
A polynucleotide in which (thymine) is replaced with a (adenine) and g (guanine) at the 752 position is replaced with a (adenine), or a complementary sequence thereof. (2) A part of the polynucleotide of the invention (1), which comprises the 751st substitution base a and the 75th position of SEQ ID NO: 1
An oligonucleotide consisting of 20 to 100 continuous DNA sequences containing a 2-position substitution base a or a complementary sequence thereof. (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 contains 15 to 30 consecutive DNs containing the 751st substitution base a and the 752nd substitution base a of SEQ ID NO: 1.
A primer set which is an oligonucleotide consisting of an A sequence or its complementary sequence. (5) A polypeptide encoded by the polynucleotide of the invention (1) or (3), or obtained by expression of the polynucleotide, wherein the Trp (251) position in the amino acid sequence of SEQ ID NO: 2 is Tryptophan) is a polypeptide in which Lys (lysine) is amino acid substituted. (6) An oligopeptide which is a part of the polypeptide of the invention (5) and comprises 5 to 30 continuous amino acid sequences containing the 251st-substituted amino acid Lys of SEQ ID NO: 2. (7) An antibody produced using the oligopeptide of the invention (6) as an antigen. (8) A method for diagnosing primary hyperoxaluria type I, 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 primary hyperoxaluria type I, comprising:
A method comprising 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 based on AGT.
It is a mutant DNA sequence of the cDNA of the gene (SEQ ID NO: 1), which is the t at position 751 in the DNA sequence of SEQ ID NO: 1.
(Thymine) is a (adenine) and g (guanine) at the 752 position is a (adenine) base-substituted or a complementary sequence thereof. In addition, SEQ ID NO: 1
The DNA sequence of the AGT gene shown by is AccGen in GenBank.
Registered as essin No. NM 000030. As described above, the abnormal gene according to the present invention includes the 751th position and the 752nd position of the cDNA of this normal (wild type) AGT gene.
Missense mutation (missense m
utation).

The polynucleotide of the invention (1) is, for example, a cDNA prepared from the total mRNA of a PH1 patient using the oligonucleotide of the invention (2) described below as a probe.
It can be isolated by screening the library. Further, RT using the primer set of the invention (4) described below and using the mRNA of PH1 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 the wild type (normal) AGT gene 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 above polynucleotide and is an oligonucleotide consisting of 20 to 100 continuous DNA sequences containing the above-mentioned 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. The polynucleotide of the invention (2) can be used in the method for diagnosing PH1 of the invention (8) described below and the like.

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) is screened, for example, by using the oligonucleotide of the invention (2) as a probe to perform a stringent hybridization and washing treatment as described above to screen a genomic library prepared from the chromosomal DNA of a PH1 patient. It can be isolated by
The polynucleotide of the invention (3) is to be detected in the method for diagnosing PH1 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 for the method for diagnosing PH1 of the invention (8) described below and the like.

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 second
The 51st position Trp (tryptophan) is Lys (lysine)
It is a polypeptide in which the amino acid has been replaced. 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 by the two base substitutions as described above. These polypeptides are
A method of isolating from a biological sample of a PH1 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) It can be obtained by a method of producing by recombinant DNA technology using cDNA). These inventions (5)
The polypeptide is, for example, PH1 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 for the method of diagnosing PH1 of the invention (11) described below.

In the invention (8) of this application, the subject is PH1.
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 determined to be high risk for PH1. 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 PH1-related gene mutation, 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. When the subject has a PH1-related gene mutation, 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, "CCANNNN" which is recognized and cleaved by the restriction enzyme Van91I at positions 743 to 753 of the cDNA of normal (wild type) AGT gene (SEQ ID NO: 1).
Although there is a sequence corresponding to "NTGG", this sequence is not present at the corresponding site of the mutated AGT gene as described above.
RFLP (restriction fragme) using such an enzyme
The PCR product can be analyzed by the nt length polymorphism method.

Also in the invention (11) of this application, the subject is PH
1 is a method for diagnosing whether or not there is a possibility of developing 1. When the polypeptide of the invention (5) is present in a biological sample isolated from a subject, the subject is determined to be high risk for PH1. 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. In diagnosing PH1 according to the invention (8) or the invention (11), a conventionally known AGT gene mutation, for example,
By combining the same method as described above based on the mutation in which cytosine is inserted between the 33rd position and the 34th position of the AGT cDNA and the premature stop codon appears, P
A highly reliable diagnosis of H1 can be secured.

The following are the gene mutations on which the present invention is based.
I will explain the research content that was confirmed. Research, clinical picture
Biology of AGT in Japanese sisters with different PH1
Conducted for the purpose of
It wasMethod Peripheral blood of the proband's sister, her sister, her family and healthy controls
The prepared DNA was used as a material for gene analysis. patient,
A part of the liver was biopsied from the parents with a laparoscope, and AGT fermentation was performed.
Used for measuring elementary activity. AGT inheritance in patients and parents
All exons and adjacent introns of the offspring are based on the information of GenBank.
Then, it was amplified by PCR and directly sequenced. Strange
The base substitution that seems to be different is an allele of 100 healthy controls.
Was searched. Analysis provided informed consent
And was approved by the Ethics Committee.

[0019]Case The proband was a younger sister, who was 5 years old at the time of diagnosis, and had no abnormal pregnancy and delivery history.
Born between unrelated, asymptomatic parents. Newborn
Aware that "things like sand" are excreted in Rio diapers
It was, but it was left alone. External urine with vulvar pain at the age of 5
An incarcerated stone in the tunnel and coral-shaped renal stones were found. 7
The old sister had no symptoms including kidney stones.
Oxalic acid and glycol in sister urine by GC / MS analysis
Acid was increased to the same degree, and PH1 was chemically diagnosed.

[0020]Grade First, biopsy liver AGT alanine and glyoxyl
The Michaelis constant (Km) for acid was measured. Understanding
Normal liver obtained from autopsy was used as a control. Against alanine
Km was 7.5 mM in control and parent AGT.
However, that of the daughters was 25 mM. To glyoxylic acid
Km for the controls and parents was 0.3 mM
However, that of the sisters was over 50 mM. Next, Vita
Min B₆Holoenzyme activity against
The liver had 5% holoenzyme, while the parents had almost 50
%, 90% or more of the sisters were holoenzymes. That is,
Vitamin B decreases AGT activity in patients and parents₆Due to deficiency
It is not a thing. Conclusion of analysis of AGT gene
As a result, both sisters insert a single base at exon 1 c. 33-34
insC (cytosine is inserted between the 33rd and 34th positions)
And 2 base substitutions at exon 7 c. 751T> A; c. 7
52G> A (751st is t → a, 752nd is g → a)
It was found to be a composite heterozygote. Previous
Nonsense mutation Q12f that causes premature stop
s × 167 (12 abnormal amino acids starting from glutamine Q
167 amino acids in total)
Yes, the latter is 1,2 of the codons of the 251st amino acid Trp.
Located at the th position and has an amino acid substitution of Trp to Lys
Russ missense mutation (c.751T> A; c.752G
> A; c. 752G> A: W251K). family
Genome analysis showed that the father was Q12fs × 16
7, mother and maternal grandmother using W251K as a heterozygote
Had. Using the resulting restriction enzyme Van91I digestion,
c. 751T> A; c. 752G> A: W251K mutation
Was performed on the DNA of 100 healthy controls.
No mutation was found.

[0021]Conclusion This sister's AGT has a higher affinity for alanine than the control.
3 times lower than that of the affinity for glyoxylic acid
It turned out to be 170 times lower. I.e.
In Bo, it was thought that the sisters' enzymes had little activity
It was Both sisters have Q12f derived from the father in the AGT gene.
sx167 and W251K derived from mother are heterozygous
Had as a body. As already mentioned, c. 33-34
insC: Q12fs 167 has already been reported in PH1
But c. 751T> A; c. 752G> A; c. 752
There is no report of G> A: W251K mutation. However,
The amino acid Trp at position 251 is present in several mammalian AGTs.
Being located in a preserved area,
Since it was not found in the Teru 200 allele, c. 7
51T> A; c. 752G> A; c. 752G> A: W
251K is a novel AGT pathogenic gene that causes PH1
It was judged abnormal. This sister has the same genetic abnormality in AGT,
Urine with similar enzymatic abnormalities and the resulting
Excretion of oxalic acid and glycolic acid in the same amount
, Clinically distinct, with a PH1 phenotype
Diversity was demonstrated molecularly and enzymatically.

[0022]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention provides a novel alanine: glyoxylate aminotransferase gene which is a causative gene of primary hyperoxaluria type I (PH1), and diagnosis of PH1 using the gene. The present invention provides a method and can contribute to the development of early diagnosis and treatment of PH1 at the gene level.

[0023]

[Sequence list]                 SEQUENCE LISTING <100> Japan Science and Technology Corporation <120> A alanine: glyoxylate aminotransferase-endoding gene and a diagn osis of type I primary hyperoxaluria utilizing the same <130> P0476T <160> 2 <210> 1 <211> 1179 <212> DNA <213> Homo sapiens <400> 1 atg gcc tct cac aag ctg ctg gtg acc ccc ccc aag gcc ctg ctc 45 Met Ala Ser His Lys Leu Leu Val Thr Pro Pro Lys Ala Leu Leu                   5 10 15 aag ccc ctc tcc atc ccc aac cag ctc ctg ctg ggg cct ggt cct 90 Lys Pro Leu Ser Ile Pro Asn Gln Leu Leu Leu Gly Pro Gly Pro                 20 25 30 tcc aac ctg cct cct cgc atc atg gca gcc ggg ggg ctg cag atg 135 Ser Asn Leu Pro Pro Arg Ile Met Ala Ala Gly Gly Leu Gln Met                 35 40 45 atc ggg tcc atg agc aag gat atg tac cag atc atg gac gag atc 180 Ile Gly Ser Met Ser Lys Asp Met Tyr Gln Ile Met Asp Glu Ile                 50 55 60 aag gaa ggc atc cag tac gtg ttc cag acc agg aac cca ctc aca 225 Lys Glu Gly Ile Gln Tyr Val Phe Gln Thr Arg Asn Pro Leu Thr                 65 70 75 ctg gtc atc tct ggc tcg gga cac tgt gcc ctg gag gcc gcc ctg 270 Leu Val Ile Ser Gly Ser Gly His Cys Ala Leu Glu Ala Ala Leu                 80 85 90 gtc aat gtg ctg gag cct ggg gac tcc ttc ctg gtt ggg gcc aat 315 Val Asn Val Leu Glu Pro Gly Asp Ser Phe Leu Val Gly Ala Asn                 95 100 105 ggc att tgg ggg cag cga gcc gtg gac atc ggg gag cgc ata gga 360 Gly Ile Trp Gly Gln Arg Ala Val Asp Ile Gly Glu Arg Ile Gly                 110 115 120 gcc cga gtg cac ccg atg acc aag gac cct gga ggc cac tac aca 405 Ala Arg Val His Pro Met Thr Lys Asp Pro Gly Gly His Tyr Thr                 125 130 135 ctg cag gag gtg gag gag ggc ctg gcc cag cac aag cca gtg ctg 450 Leu Gln Glu Val Glu Glu Gly Leu Ala Gln His Lys Pro Val Leu                 140 145 150 ctg ttc tta acc cac ggg gag tcg tcc acc ggc gtg ctg cag ccc 495 Leu Phe Leu Thr His Gly Glu Ser Ser Thr Gly Val Leu Gln Pro                 155 160 165 ctt gat ggc ttc ggg gaa ctc tgc cac agg tac aag tgc ctg ctc 540 Leu Asp Gly Phe Gly Glu Leu Cys His Arg Tyr Lys Cys Leu Leu                 170 175 180 ctg gtg gat tcg gtg gca tcc ctg ggc ggg acc ccc ctt tac atg 585 Leu Val Asp Ser Val Ala Ser Leu Gly Gly Thr Pro Leu Tyr Met                 185 190 195 gac cgg caa ggc atc gac atc ctg tac tcg ggc tcc cag aag gcc 630 Asp Arg Gln Gly Ile Asp Ile Leu Tyr Ser Gly Ser Gln Lys Ala                 200 205 210 ctg aac gcc cct cca ggg acc tcg ctc atc tcc ttc agt gac aag 675 Leu Asn Ala Pro Pro Gly Thr Ser Leu Ile Ser Phe Ser Asp Lys                 215 220 225 gcc aaa aag aag atg tac tcc cgc aag acg aag ccc ttc tcc ttc 720 Ala Lys Lys Lys Met Tyr Ser Arg Lys Thr Lys Pro Phe Ser Phe                 230 235 240 tac ctg gac atc aag tgg ctg gcc aac ttc tgg ggc tgt gac gac 765 Tyr Leu Asp Ile Lys Trp Leu Ala Asn Phe Trp Gly Cys Asp Asp                 245 250 255 cag ccc agg atg tac cat cac aca atc ccc gtc atc agc ctg tac 810 Gln Pro Arg Met Tyr His His Thr Ile Pro Val Ile Ser Leu Tyr                 260 265 270 agc ctg aga gag agc ctg gcc ctc att gcg gaa cag ggc ctg gag 855 Ser Leu Arg Glu Ser Leu Ala Leu Ile Ala Glu Gln Gly Leu Glu                 275 280 285 aac agc tgg cgc cag cac cgc gag gcc gcg gcg tat ctg cat ggg 900 Asn Ser Trp Arg Gln His Arg Glu Ala Ala Ala Tyr Leu His Gly                 290 295 300 cgc ctg cag gca ctg ggg ctg cag ctc ttc gtg aag gac ccg gcg 945 Arg Leu Gln Ala Leu Gly Leu Gln Leu Phe Val Lys Asp Pro Ala                 305 310 315 ctc cgg ctt ccc aca gtc acc act gtg gct gta ccc gct ggc tat 990 Leu Arg Leu Pro Thr Val Thr Thr Val Ala Val Pro Ala Gly Tyr                 320 325 330 gac tgg aga gac atc gtc agc tac gtc ata gac cac ttc gac att 1035 Asp Trp Arg Asp Ile Val Ser Tyr Val Ile Asp His Phe Asp Ile                 335 340 345 gag atc atg ggt ggc ctt ggg ccc tcc acg ggg aag gtg ctg cgg 1080 Glu Ile Met Gly Gly Leu Gly Pro Ser Thr Gly Lys Val Leu Arg                 350 355 360 atc ggc ctg ctg ggc tgc aat gcc acc cgc gag aat gtg gac cgc 1125 Ile Gly Leu Leu Gly Cys Asn Ala Thr Arg Glu Asn Val Asp Arg                 365 370 375 gtg acg gag gcc ctg agg gcg gcc ctg cag cac tgc ccc aag aag 1170 Val Thr Glu Ala Leu Arg Ala Ala Leu Gln His Cys Pro Lys Lys                 380 385 390 aag ctg tga 1179 Lys Leu <210> 2 <211> 392 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ser His Lys Leu Leu Val Thr Pro Pro Lys Ala Leu Leu                   5 10 15 Lys Pro Leu Ser Ile Pro Asn Gln Leu Leu Leu Gly Pro Gly Pro                 20 25 30 Ser Asn Leu Pro Pro Arg Ile Met Ala Ala Gly Gly Leu Gln Met                 35 40 45 Ile Gly Ser Met Ser Lys Asp Met Tyr Gln Ile Met Asp Glu Ile                 50 55 60 Lys Glu Gly Ile Gln Tyr Val Phe Gln Thr Arg Asn Pro Leu Thr                 65 70 75 Leu Val Ile Ser Gly Ser Gly His Cys Ala Leu Glu Ala Ala Leu                 80 85 90 Val Asn Val Leu Glu Pro Gly Asp Ser Phe Leu Val Gly Ala Asn                 95 100 105 Gly Ile Trp Gly Gln Arg Ala Val Asp Ile Gly Glu Arg Ile Gly                 110 115 120 Ala Arg Val His Pro Met Thr Lys Asp Pro Gly Gly His Tyr Thr                 125 130 135 Leu Gln Glu Val Glu Glu Gly Leu Ala Gln His Lys Pro Val Leu                 140 145 150 Leu Phe Leu Thr His Gly Glu Ser Ser Thr Gly Val Leu Gln Pro                 155 160 165 Leu Asp Gly Phe Gly Glu Leu Cys His Arg Tyr Lys Cys Leu Leu                 170 175 180 Leu Val Asp Ser Val Ala Ser Leu Gly Gly Thr Pro Leu Tyr Met                 185 190 195 Asp Arg Gln Gly Ile Asp Ile Leu Tyr Ser Gly Ser Gln Lys Ala                 200 205 210 Leu Asn Ala Pro Pro Gly Thr Ser Leu Ile Ser Phe Ser Asp Lys                 215 220 225 Ala Lys Lys Lys Met Tyr Ser Arg Lys Thr Lys Pro Phe Ser Phe                 230 235 240 Tyr Leu Asp Ile Lys Trp Leu Ala Asn Phe Trp Gly Cys Asp Asp                 245 250 255 Gln Pro Arg Met Tyr His His Thr Ile Pro Val Ile Ser Leu Tyr                 260 265 270 Ser Leu Arg Glu Ser Leu Ala Leu Ile Ala Glu Gln Gly Leu Glu                 275 280 285 Asn Ser Trp Arg Gln His Arg Glu Ala Ala Ala Tyr Leu His Gly                 290 295 300 Arg Leu Gln Ala Leu Gly Leu Gln Leu Phe Val Lys Asp Pro Ala                 305 310 315 Leu Arg Leu Pro Thr Val Thr Thr Val Ala Val Pro Ala Gly Tyr                 320 325 330 Asp Trp Arg Asp Ile Val Ser Tyr Val Ile Asp His Phe Asp Ile                 335 340 345 Glu Ile Met Gly Gly Leu Gly Pro Ser Thr Gly Lys Val Leu Arg                 350 355 360 Ile Gly Leu Leu Gly Cys Asn Ala Thr Arg Glu Asn Val Asp Arg                 365 370 375 Val Thr Glu Ala Leu Arg Ala Ala Leu Gln His Cys Pro Lys Lys                 380 385 390 Lys Leu

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 33/53 M G01N 33/53 33/532 A 33/566 33/532 C12N 15/00 ZNAA 33/566 FF term (reference) 4B024 AA01 AA11 BA10 CA04 CA12 HA11 HA17 4B029 AA07 FA12 4B050 CC03 DD11 LL03 4B063 QA13 QA17 QQ42 QQ52 QR32 QR35 QR55 QR62 QS25 QS34 4H045 AA11 AA74 DA75 FA75

Claims (15)

[Claims] 1. A polynucleotide of a gene encoding alanine: glyoxylate aminotransferase, wherein t (thymine) at the 751st position is a (adenine) and g at the 752nd position in the DNA sequence of SEQ ID NO: 1.
A polynucleotide in which (guanine) has a base substitution with a (adenine), or a complementary sequence thereof. 2. A part of the polynucleotide according to claim 1, wherein the 751st substitution base a and the 75th substitution base a of SEQ ID NO: 1 are used.
An oligonucleotide consisting of 20 to 100 continuous DNA sequences containing a 2-position substitution base a or a complementary sequence thereof. 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 SEQ ID NO.
A primer set which is an oligonucleotide consisting of 15 to 30 continuous DNA sequences containing the 2-position substitution base a or its complementary sequence. 5. A polypeptide encoded by the polynucleotide according to claim 1 or 3 or obtained by expression of said polynucleotide, wherein Trp (tryptophan) at position 251 in the amino acid sequence of SEQ ID NO: 2. Is a polypeptide in which the amino acid is substituted with Lys (lysine). 6. An oligopeptide, which is a part of the polypeptide of claim 5 and comprises 5 to 30 consecutive amino acid sequences including the 251st-substituted amino acid Lys of SEQ ID NO: 2. 7. An antibody produced by using the oligopeptide of claim 6 as an antigen. 8. A method for diagnosing primary hyperoxaluria type I, 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 primary hyperoxaluria type I, 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.