JP2005073626A - Variation of electron transfer flavin protein dehydrogenase gene and method for diagnosis of glutaric aciduria type ii using the variation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for the early and sure diagnosis of glutaric aciduria type II (GAII) which is a rare disease but difficult to perform early diagnosis. <P>SOLUTION: The diagnosis of GAII is carried out by using a polynucleotide formed by the missense mutation of the 1096th cytosine to thymine and the 1208th cytosine to thymine in a cDNA of a gene encoding an electron transfer flavin protein dehydrogenase (ETF-QO) causing GAII, a part of the polynucleotide, a polypeptide encoded by the gene or a part of the polypeptide. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention belongs to the technical field of genetic diagnosis, and in particular, an abnormal gene encoding an electron transfer flavin protein dehydrogenase that is a pathogenic gene of glutaric aciduria type II and diagnosis of glutaric aciduria type II using the same Regarding the method.

  Glutaric aciduria type II (hereinafter sometimes referred to as GAII) is also called multiple acyl-Co-A dehydrogenase deficiency, and clinical symptoms are severe hypoglycemia immediately after birth, in infancy Severe infant death syndrome or syndrome-like symptoms, completely asymptomatic up to near adults, and their severity vary. The cause of GAII is due to an abnormality in mitochondrial 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. However, the relationship between the genomic structure of ETF-QO and GAII has hardly been clarified.

The present inventors have previously found one mutation in the ETF-QO gene that brings about GAII, and have proposed a diagnostic method for GAII that uses this mutation (Patent Document 1). This discovery is extremely useful for the diagnosis of GAII, but further progress is required to continue research at the genetic level.
JP 2003-70476 A

  An object of the present invention is to provide a means that contributes to early and reliable diagnosis of GAII, which is a rare disease but is difficult to diagnose early due to the diversity of clinical symptoms.

  The present inventor has now found a new mutation in the ETF-QO gene that causes GAII. That is, as will be described in detail later, when the ETF-QO gene of patients diagnosed with GAII and their families was examined, position 1096 of the ETF-QO gene cDNA was mutated to thymine instead of cytosine. Since the position is mutated to thymine instead of cytosine, it is a homozygote, it is derived from parents, and this mutation is not found in the ETF-QO gene cDNA of healthy subjects. It was confirmed that the mutation was one of the pathogenesis of GAII.

The present invention has been derived on the basis of such knowledge, and this application provides the following inventions (1) to (15) to solve the above-mentioned problems.
(1) A polynucleotide of a gene encoding an electron transfer flavin protein dehydrogenase, wherein c (cytosine) at position 1096 is changed to t (thymine) in the DNA sequence of SEQ ID NO: 1, and c ( A polynucleotide in which cytosine) is substituted with t (thymine) or a complementary sequence thereof.
(2) an oligonucleotide comprising a part of the polynucleotide of the invention (1) and comprising 20 to 100 consecutive DNA sequences containing the substitution base t at position 1096 of SEQ ID NO: 1 or the substitution base t at position 1208 Or its complementary sequence.
(3) A polynucleotide derived from human chromosomal DNA that hybridizes with the polynucleotide of the invention (1), the oligonucleotide of the invention (2), or a complementary sequence thereof under stringent conditions.
(4) A primer set for PCR amplification of a double-stranded polynucleotide comprising the polynucleotide of the invention (3) and its complementary sequence, or mRNA transcribed from the polynucleotide of the invention (3), A primer set which is an oligonucleotide consisting of 15 to 30 consecutive DNA sequences containing the 1096th substituted base t of SEQ ID NO: 1, or the 1208th substituted base t of SEQ ID NO: 1 or a complementary sequence thereof.
(5) A polypeptide encoded by the polynucleotide of the invention (1) or (3) or obtained by expression of the polynucleotide, wherein the Leu at position 366 in the amino acid sequence of SEQ ID NO: 2 A polypeptide in which leucine is amino acid substituted for Phe (phenylalanine) and Ala (alanine) at position 403 is Val (valine).
(6) A part of the polypeptide of the invention (5), comprising 5 to 30 consecutive amino acid sequences including the 366th substituted amino acid residue Phe in SEQ ID NO: 2 or the 403th substituted amino acid residue An oligopeptide.
(7) An antibody produced using the oligopeptide of the invention (6) as an antigen.
(8) A method for diagnosing glutaric aciduria type II, comprising detecting whether or not the polynucleotide of the invention (3) is present in chromosomal DNA isolated from a subject.
(9) Whether or not the chromosomal DNA or mRNA thereof isolated from the subject and the polynucleotide of the invention (1), the oligonucleotide of the invention (2), or their complementary sequences hybridize under stringent conditions The method of the invention (8) for detecting the above.
(10) The method according to the invention (8), wherein the 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 the primer set of the invention (4).
(11) A method for diagnosing glutaric aciduria type II, the 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 invention (11), wherein it is detected whether or not a polypeptide that reacts with the antibody of the invention (7) is present in a biological sample isolated from a subject.
(13) A DNA probe characterized by labeling the oligonucleotide of the invention (2).
(14) A DNA chip comprising the oligonucleotide of the invention (2).
(15) A labeled antibody, wherein the antibody of the invention (7) is labeled.

  According to the present invention, by confirming a mutation in the ETF-QO gene, which is a pathogenic gene for GAII, early diagnosis of GAII can be accurately performed without establishing cultured lymphoblasts.

  Hereinafter, embodiments of each of these inventions will be described in detail. In the present specification, a continuous sequence of 101 or more nucleotides is defined as a polynucleotide, and a continuous nucleotide sequence of 2 to 100 nucleotides is defined as an oligonucleotide. A continuous amino acid sequence of 31 or more is defined as a polypeptide, and a continuous amino acid sequence of 2 to 30 is defined as an oligopeptide.

Invention (1) of this application is a mutated DNA sequence of cDNA of the ETF-QO gene (SEQ ID NO: 1), and c (cytosine) at position 1096 in the DNA sequence of SEQ ID NO: 1 is changed to t (thymine), and This is a polynucleotide in which c (cytosine) at position 1208 is substituted with t (thymine) or a complementary sequence thereof.
The DNA sequence of the ETF-QO gene represented by SEQ ID NO: 1 is stored in GenBank as Accessin No. S.
It is registered as 69232. As described above, the abnormal gene according to the present invention is caused by a missense mutation in which the points 1096 and 1208 of this normal (wild-type) ETF-QO gene cDNA are point-mutated.

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

Invention (2) of this application is a part of the polynucleotide of the invention (1), which is an oligonucleotide consisting of 20 to 100 consecutive DNA sequences each containing a substituted base, or a complementary sequence thereof.
The oligonucleotide of the invention (2) can be produced by chemically synthesizing by a known method. In addition, the polynucleotide of the invention (1) can also be prepared by a method such as cleaving with an appropriate restriction enzyme.
It can be used in the polynucleotide of the invention (2), the GAII diagnostic method of the invention (8) described later, and the like.

Invention (3) of this application is a polynucleotide derived from human chromosomal DNA that hybridizes under stringent conditions with the polynucleotide of the invention (1) or the oligonucleotide of the invention (2), or a complementary sequence thereof ( Genomic DNA).
Here, the stringent condition is a condition that enables selective and detectable specific binding between the polynucleotide or oligonucleotide and genomic DNA derived from a chromosome. As is well known, stringent conditions depend on salt concentration, temperature, and other conditions; for example, the lower the salt concentration, the higher the temperature, the higher the stringency and the more hybridized. It becomes difficult. 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 or less NaCl and about 25 mM or less trisodium citrate. The stringent temperature is generally 15 to 25 ° C. lower than the melting temperature (Tm) of the complete hybrid, for example, about 30 ° C. or more. 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 various stringencies can be set by combining these conditions.

As one preferred example, hybridization is performed at a temperature of 42 ° C. under conditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, 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, and the stringency of the wash increases with decreasing salt concentration and increasing temperature. As one preferred example, washing is performed at a temperature of 68 ° C. under conditions of 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. For stringent conditions, see, for example, “Molecular Cloning, A Laboratory Mannual,” by J. Sambrook et al.
Second Edition, Cold Spring Harbor Laboratory Press (1989), especially detailed in section 11.45 “Conditions for Hybridization of Oligonucleotide Probes” and should be used easily by referring to those descriptions. Can do.

For the polynucleotide of the invention (3), for example, a genomic library prepared from the chromosomal DNA of a GAII patient is screened by the stringent hybridization and washing treatment as described above using the oligonucleotide of the invention (2) as a probe. Can be isolated.
These polynucleotides of the invention (3) are to be detected in the diagnostic method of the invention (8) described later.

Invention (4) of this application is a PCR amplification of double-stranded polynucleotide (genomic DNA) comprising the polynucleotide of the invention (3) and its complementary sequence, or mRNA transcribed from the polynucleotide of the invention (3). This is a primer set. In these primer sets, one oligonucleotide primer is composed of 15 to 30 continuous DNA sequences containing the substituted base of SEQ ID NO: 1 or a complementary sequence thereof. The other primer can be any continuous DNA sequence 5 ′ or 3 ′ side of the substituted base of SEQ ID NO: 1 or a complementary sequence thereof.
These primer sets can be prepared by a known DNA synthesis method based on SEQ ID NO: 1 containing each substituted base. A linker sequence or the like can also be added to the end of the primer.
The primer set of the invention (4) can be used for the GAII diagnosis method of the invention (8) described later.

Invention (5) of this application is a polypeptide encoded by the polynucleotide of invention (1) or (3) or obtained by expression of the polynucleotide, wherein the amino acid sequence of SEQ ID NO: 2 This is a polypeptide in which Leu (leucine) at position 366 is substituted with Phe (phenylalanine) and Ala (alanine) at position 403 is substituted with Val (valine).
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 the base substitution.
These polypeptides can be isolated from a biological sample of 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 invention (1 ) Polynucleotide (mutant cDNA) and the like can be obtained by a method of producing by recombinant DNA technology. These invention (5) polypeptides can be used, for example, as a test object in the GAII diagnostic method of the following invention (11).

  The invention (6) of this application is an oligopeptide which is a part of the polypeptide of the invention (5) and has a continuous amino acid sequence of 5 to 30 including substituted amino acids. These oligopeptides can be prepared by a method of chemically synthesizing based on a predetermined amino acid sequence or a method of digesting the polypeptide of the invention (5) with an appropriate protease. These oligopeptides can be used, for example, as antigens for antibody production of invention (7) described later.

  The antibody of the invention (7) is a polyclonal antibody or a monoclonal antibody produced using the oligopolypeptide of the invention (6) as an antigen. These antibodies can be produced by a known antibody production method. 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 later.

  Invention (8) of this application is a method for diagnosing whether or not a subject has a possibility of developing GAII. 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 with respect to GAII. The polynucleotide can be detected by various known methods, but the method of the invention (9) or (10) is preferred.

  In the method of the invention (9), it is determined whether or not the chromosomal DNA or mRNA thereof isolated from a subject and the polynucleotide of the invention (1) or the oligonucleotide of the invention (2) hybridize under stringent conditions. To detect. When the subject has a genetic mutation associated with GAII, the chromosomal DNA or mRNA thereof and the polynucleotide or oligonucleotide hybridize even under stringent conditions. Hybridization can be detected by a known method. For example, hybridization using the DNA probe of the invention (13) or the DNA chip of the invention (14) is performed, for example, by the ASO (allele specific oligomer) method. Therefore, it can be performed simply and with high accuracy.

  In the method of the invention (10), PCR (including RT-PCR) was 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). The presence or absence of a PCR product is detected. If the subject has a genetic mutation associated with GAII, a polynucleotide PCR product defined by the primer set is obtained. Analysis of the PCR product can also be performed according to a known method, for example, RFLP (restriction fragment length polymorphism).

  The invention (11) of this application is also a method for diagnosing whether or not a subject is likely to develop GAII. When the polypeptide of the invention (5) is present in a biological sample isolated from the subject In addition, the subject is determined to be high risk for GAII. The presence of the polypeptide can be carried out by various known methods, but the method of the invention (12) is preferred. The method of the invention (12) is a method using the antibody of the invention (7). In particular, by using the labeled antibody of the invention (15), simple and highly accurate detection is possible. Various known labels such as enzymes, isotopes and fluorescent dyes can be used as the label.

  In the following, the contents of the study confirming the gene mutation that is the basis of the present invention will be described. The study was conducted for the purpose of identifying molecular biological abnormalities in Japanese GAII and clarifying the genomic structure of the ETF-QO gene.

Methods Lymphoblasts established from patient B lymphocytes and patient skin fibroblasts were used as materials. [<SUP> 3 </ SUP> H] -labeled myristic acid, palmitic acid and oleic acid were used to compare the fatty acid β-oxidation ability of cells with that of control cells. The α and β-ETF in the cell lysate were compared between patients and control cells by Western blotting. All exons and adjacent introns of the α and β-ETF genes were amplified by PCR based on GenBank information and directly sequenced. For ETF-QO, RT-PCR was performed across the entire translation region, followed by direct sequencing. At the same time, the genome structure of the ETF-QO gene was isolated from the PAC human genomic library and the clone containing the entire translation region of ETF-QO was clarified, and then the ETF-QO gene analysis using the genomes of patients, families and healthy controls Went. The analysis was performed with informed consent and was approved by the Ethics Committee.

The case was a boy who was 2 months old at the time of diagnosis. Two brothers out of seven siblings suffered psychomotor development and died in early childhood. The infant developed due to poor feeding and weak muscles, and due to infused nutrition, he had significant fatty liver and required intensive care. After that, psychomotor development was delayed, but suddenly died at the age of three. Urinary 2-hydroxyglutaric acid, ethylmalonic acid, and glutaric acid in the patient's urine increased markedly, and GAII was chemically diagnosed from the time of onset (Hirose).
S. et al., Acta Paediatr 89: 887-887, 2000).

Results The fatty acid β-oxidation ability of lymphoblasts was lower than normal, supporting the chemical diagnosis of GAII. Western blotting found no qualitative or quantitative abnormalities in α and β-ETF in patient cells. Similarly, there was no abnormality in the genes of α and β-ETF. Although no difference was observed in the size and expression of mRNA of ETF-QO obtained by RT-PCR, mutations of cytosine → thymine at position 1096 and cytosine → thymine at position 1208 of the patient's cDNA were found. The former mutation is located at the first codon of the 366th amino acid Leu and is an amino acid mutation to Phe, and the latter mutation is located at the second of the codon of the 403rd amino acid Ala and results in an amino acid mutation to Val. The mutation seemed to have the patient as a homozygote. Genomic analysis of family and patients revealed that 3 of their parents and siblings had this mutation as a heterozygote, and the patient was homozygous. There were no other mutations in ETF-Q within the analyzed range. Genomic analysis from 96 healthy controls using the restriction enzyme BamHI cleavage produced by this mutation did not find this mutation.

Conclusion The child has no abnormality in α and β-ETF, and the c → t base mutation at position 1096 (Leu → Phe amino acid mutation at position 366) and the c → t base mutation at position 1208 (Ala at position 403) in the ETF-QO gene. → Val amino acid mutation) as a homozygote. In addition, Leu at position 366 and Ala at position 403 are cross-species and are conserved in the ETF-QO family (see FIG. 1), and the 200 allele from healthy controls did not have the same mutation, resulting in position 1096. C → t base mutation (Leu → Phe amino acid mutation at position 366) and c → t base mutation at position 1208 (Ala → Val amino acid mutation at position 403) and a novel ETF-QO gene abnormality that causes delayed GAII It was judged.

  The present invention provides tools and techniques that enable early diagnosis of GAII and can be used in related fields.

It is a comparison table | surface which shows that the 366 leucine and 403 alanine of electron transfer flavitan protein dehydrogenase protein are damaged beyond a seed | species. In the figure, (A) shows 366 leucine and (B) shows 403 alanine.

Claims (15)

  A polynucleotide of a gene encoding an electron transfer flavin protein dehydrogenase, wherein c (cytosine) at position 1096 is t (thymine) and c (cytosine) at position 1208 in the DNA sequence of SEQ ID NO: 1. A polynucleotide having a base substitution to t (thymine) or a complementary sequence thereof.   A part of the polynucleotide of claim 1, which is an oligonucleotide consisting of 20 to 100 consecutive DNA sequences containing the substitution base t at position 1096 or the substitution base t at position 1208 of SEQ ID NO: 1, or a complementary sequence thereof.   A polynucleotide derived from human chromosomal DNA that hybridizes under stringent conditions with the polynucleotide of claim 1 or the oligonucleotide of claim 2, or a complementary sequence thereof.   A primer set for PCR amplification of a double-stranded polynucleotide comprising the polynucleotide of claim 3 and its complementary sequence, or mRNA transcribed from the polynucleotide of claim 3, wherein one primer is of SEQ ID NO: 1. A primer set, which is an oligonucleotide consisting of 15 to 30 consecutive DNA sequences containing the 1096th substitution base t, or the 1208th substitution base t, or a complementary sequence thereof.   A polypeptide encoded by the polynucleotide of claim 1 or 3 or obtained by expression of said polynucleotide, wherein Leu (leucine) at position 366 in the amino acid sequence of SEQ ID NO: 2 is Phe (phenylalanine) A polypeptide in which Ala (alanine) at position 403 is substituted with Val (valine).   An oligopeptide which is a part of the polypeptide of claim 5 and consists of 5 to 30 consecutive amino acid sequences containing the 366th substituted amino acid residue Phe or the 403th substituted amino acid residue of SEQ ID NO: 2.   An antibody produced using the oligopeptide of claim 6 as an antigen.   A method for diagnosing glutaric aciduria type II, comprising detecting whether or not the polynucleotide of claim 3 is present in chromosomal DNA isolated from a subject.   9. Detecting whether chromosomal DNA or mRNA thereof isolated from a subject and the polynucleotide of claim 1 or the oligonucleotide of claim 2 or a complementary sequence thereof hybridize under stringent conditions. the method of.   The method according to claim 8, wherein the presence or absence of a PCR product is detected when PCR is carried out using the chromosomal DNA or mRNA isolated from a subject as a template and the primer set of claim 4.   A method for diagnosing glutaric aciduria type II, comprising detecting whether the polypeptide of claim 5 is present in a biological sample isolated from a subject.   The method according to claim 11, wherein it is detected whether a polypeptide that reacts with the antibody according to claim 7 is present in a biological sample isolated from a subject.   A DNA probe, wherein the oligonucleotide of claim 2 is labeled.   A DNA chip comprising the oligonucleotide of claim 2. A labeled antibody, wherein the antibody of claim 7 is labeled.

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