JP4314094B2 - Benign familial neonatal seizure-related gene mutation and benign familial neonatal seizure diagnosis method - Google Patents

Description

  The invention of this application relates to a gene mutation associated with benign familial neonatal convulsions and a diagnostic method thereof. More particularly, a polynucleotide derived from a benign familial neonatal seizure-related gene characterized by point mutations and / or deletion mutations, a polypeptide that is a gene product thereof, and a benign subject to these polynucleotides or polypeptides. The present invention relates to a method for diagnosing familial neonatal seizures.

  Benign familial neonatal convulsions (BFNC) are characterized by early seizures in neonates and generally resolve spontaneously in months. However, there are many acute diseases with a poor prognosis among diseases that cause seizures in newborns. For this reason, newborns with seizures should be promptly diagnosed as to whether it is BFNC or due to an acute disease with a poor prognosis, and if it is the latter, early treatment is necessary. It is said. The diagnosis is currently based on guidelines submitted by the Commission on Classification and Terminology of the International League against Epilepsy (Non-Patent Document 1).

  However, BFNC and other acute diseases are very similar in the state of seizures, and it is very difficult to diagnose them by observing the physical symptoms at the first onset. Therefore, great expectations are placed on the diagnosis at the gene level.

On the other hand, some relations between mutations in neuronal voltage-dependent K ⁺ channel genes (KCNQ2: cDNA is GenBank / AF033348) and neonatal seizures have been reported (Patent Document 1, Non-Patent Documents 5-8). The inventors of this application have also reported a base substitution mutation of the KCNQ2 gene related to BFNC (Non-patent Document 9).
Special table 2001-521041 Commission on Classification and Terminology of the ILAE (1989) Epilepsia, 30, 389-399. Biervert, C. Et al. Science 279: 403-406, 1998 Singh, NA et al. Nat. Genet. 18 (1): 25-29, 1998 Biervert, C. and Steinlein, OK Hum. Genet. 104 (3): 234-240, 1999 Lerche H. et al. Ann Neurol. 46 (3): 305-312, 1999 Steinlein OK et al. Neuroreport. 26; 10 (6): 1163-1166, 1999 Lee WL et al. Neuropediatrics. 31 (1): 9-12, 2000 Miraglia del Giudice E. et al. Eur J Hum Genet. 8 (12): 994-7, 2000 Hirose S. et al. Ann Neurol. 47 (6): 822-6, 2000

  The identification of gene mutations offers new possibilities for the diagnosis of BFNC and other poor prognosis diseases, or for the rapid development of prevention and treatment for poor prognosis diseases. . For example, by examining the presence of a mutated gene or the presence of a mutated protein expressed by a mutated gene in a neonate who has a convulsive seizure, whether the seizure is due to a BFNC or a poor prognosis disease Can be diagnosed with a high probability in advance. In addition, such a pre-diagnosis makes it possible to prescribe a treatment for prevention or treatment from an early stage if the disease has a poor prognosis.

  However, because the BFNC phenotype has incomplete penetrance and frequent family mutations, it is necessary to identify as many genetic mutations as possible and make a diagnosis by comprehensive judgment of multiple mutations. .

  The invention of this application was made in view of the circumstances as described above, and provides a novel gene mutation related to BFNC existing in the KCNQ2 gene and a BFNC diagnosis method using this gene mutation. It is an issue.

This application provides the following inventions (1) to (13) to solve the above-mentioned problems.
(1) A polynucleotide derived from a gene related to benign familial neonatal seizure, comprising the nucleotide sequence of SEQ ID NO: 1,
(a) deletion of positions 910-912 ttc; and
(b) 967 c replaced with t
A polynucleotide having at least one of the mutations.
(2) An oligonucleotide which is a part of the polynucleotide of the invention (1) and contains the mutation site.
(3) A polynucleotide derived from human chromosomal DNA that hybridizes with the polynucleotide of the invention (1) or the oligonucleotide of claim 2 under stringent conditions.
(4) A primer set for PCR amplification of the polynucleotide of the invention (1) or the invention (3), wherein one primer is an oligonucleotide containing a mutation site or a complementary sequence thereof.
(5) A microarray comprising the polynucleotide of the invention (1) or the oligonucleotide of the invention (2).
(6) An expression product of the polynucleotide of the invention (1) or (3) derived from a gene related to benign familial neonatal seizure, wherein the amino acid sequence of SEQ ID NO: 2
(A) deletion of position 304 Phe; and
(B) No. 323 Gln or less deleted,
A polypeptide having at least one of the mutations.
(7) An oligopeptide which is a part of the polypeptide of the invention (6) and contains a mutation site.
(8) An antibody that is prepared using the oligopeptide of the invention (7) as an antigen and recognizes an oligopeptide containing a mutation.
(9) A method for diagnosing benign familial neonatal convulsion, which comprises detecting whether or not the polynucleotide of the invention (3) is present in chromosomal DNA isolated from a subject.
(11) The invention for detecting 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. Method (9).
(11) The method according to the invention (9), wherein the presence or absence of a PCR product is detected when PCR is performed using a chromosomal DNA or mRNA isolated from a subject as a template and the primer set according to the invention (4).
(12) A method for diagnosing benign familial neonatal convulsions, comprising detecting whether or not the polypeptide of the invention (6) is present in a biological sample isolated from a subject.
(13) The method of the invention (12), wherein it is detected whether or not a polypeptide that reacts with the antibody of the invention (8) is present in a biological sample isolated from a subject.

Specifically, the inventors of this application searched for 61 mutations of KCNQ2 in 61 Japanese patients diagnosed with BFNC based on clinical course and EEG findings, and directly searched for two novel KCNQ2 genes shown in the invention (1). Nucleotide mutations were found. Further, it was found that the two amino acid mutations shown in the invention (6) occur in the protein (polypeptide) which is an expression product of these nucleotide mutations in the KCNQ2 gene. That is, these mutations are described as follows according to the literature (Dunnen, J. and Antonarakis, S. (2001) Hum. Genet., 109, 121-124).
(i) c.910-912delTTC or TTT, F304del
(ii) c.967C> T, Q323X
The human KCNQ2 gene transcript (mRNA: GenBank / AF033348) has four types of splicing variants (1: GenBank / NM_172107, 2: GenBank / NM_172106, 3: GenBank / NM_004518, 4: GenBank / NM_172108). However, the above mutations (i) and (ii) also apply to all these transcripts. In this application, the cDNA nucleotide sequence of the splicing variant 2 of the KCNQ2 gene is exemplified as SEQ ID NO: 1, and the amino acid sequence of its expression product (polypeptide) is exemplified as SEQ ID NO: 2.

  The inventions (1) to (13) have been completed based on the novel findings as described above.

  In the present invention, “polynucleotide” refers to a nucleoside phosphate ester (nucleotide ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP, dTTP) in which purine or pyrimidine is β-N-glycoside-linked to a sugar. A molecule in which 100 or more molecules are bound is referred to, and an “oligonucleotide” is a molecule in which 2-99 molecules are linked. The polynucleotide and oligonucleotide in the present invention are single-stranded or double-stranded, and in the case of single-stranded, it is either the sense strand or the antisense strand.

  “Polypeptide” means a molecule composed of 34 or more amino acid residues linked to each other by an amide bond (peptide bond), and “oligonucleotide” refers to a molecule composed of 2-33 amino acid residues. means.

  Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents and the like, except for a technique that clearly indicates the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc.

Invention (1) of this application is a mutated polynucleotide derived from the human KCNQ2 gene, and the following nucleotide mutation in the DNA sequence of SEQ ID NO: 1:
(a) Positions 910-912 ttc deleted
(b) 967 c replaced with t
A polynucleotide having at least one of the following.

  The polynucleotide of this invention (1) can be isolated, for example, by screening a cDNA library prepared from the total mRNA of BFNC patients using the oligonucleotide of the following invention (2) as a probe. It can also be isolated by RT-PCR using the mRNA of a BFNC patient as a template using the primer set of invention (4) described later. Alternatively, it can also be obtained by introducing the above-mentioned base substitution into wild-type KCNQ2 cDNA using a commercially available mutation kit or the like. The obtained cDNA can be obtained by a conventional gene amplification method such as PCR (Polymerase Chain Reaction) method, NASBN (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method and SDA (Strand Displacement Amplification) method. Can be amplified.

  The polynucleotide of this invention (1) can be used in the BFNC diagnostic method of invention (10) described later. It can also be used as a material for preparing the polypeptide of invention (6) described later by genetic engineering.

  The invention (2) of this application is an oligonucleotide which is a part of the polynucleotide of the invention (1) and comprises 20 to 99 consecutive DNA sequences including each mutation site. The oligonucleotide of this invention (2) can be obtained by known methods (for example, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661 Belousov (1997) Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; US Pat. No. 4,458,066). It can be synthesized in vitro. The polynucleotide of the invention (1) can also be prepared by a method such as cleaving with an appropriate restriction enzyme.

Such an oligonucleotide can be labeled with a labeling substance, particularly when used as a probe. The labeling can be performed by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescence labeling method, a biotin labeling method, a chemiluminescence method, and the like, but it is preferable to use a fluorescence labeling method. As the fluorescent substance, those capable of binding to the base moiety of the oligonucleotide can be appropriately selected and used. However, cyanine dyes (eg, Cy Dye ^TM series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy- N ² -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used.

  The oligonucleotide of the invention (2) can also be used in the BFNC diagnostic method of the invention (10) described later. Alternatively, the oligopeptide of the invention (7) described later can also be used as a material for genetic engineering.

  Invention (3) of this application is a polynucleotide (genomic DNA) derived from human chromosomal DNA that hybridizes with the polynucleotide of the invention (1) or the oligonucleotide of the invention (2) under stringent conditions. Here, the stringent conditions are conditions that enable selective and detectable specific binding between the polynucleotide or oligonucleotide and genomic DNA derived from a chromosome. Stringent conditions are defined by salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency increases depending on whether the salt concentration is reduced, the organic solvent concentration is increased, or the hybridization temperature is increased. For example, stringent salt concentrations are generally about 750 mM or less NaCl and about 75 mM or less trisodium citrate, more preferably about 500 mM or less NaCl and about 50 mM or less trisodium citrate, most preferably about 250 or less NaCl. less than mM and trisodium citrate less than about 25 mM. The stringent organic solvent concentration is about 35% or more, most preferably about 50% or more of formamide. The stringent temperature condition is about 30 ° C. or higher, more preferably about 37 ° C. or higher, and most preferably about 42 ° C. or higher. Other conditions include hybridization time, detergent (eg, SDS) concentration, presence / absence of carrier DNA, and various stringencies can be set by combining these conditions. In one preferred embodiment, hybridization is performed at a temperature of 30 ° C. under conditions of 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. In a more preferred embodiment, hybridization is performed at a temperature of 37 ° C. under conditions of 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA. In the most preferred embodiment, hybridization is performed at a temperature of 42 ° C. under conditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 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, and the stringency of the wash increases with decreasing salt concentration and increasing temperature. For example, stringent salt conditions for washing are preferably no more than about 30 mM NaCl and no more than about 3 mM trisodium citrate, most preferably no more than about 15 mM NaCl and no more than about 1.5 mM trisodium citrate. Stringent temperature conditions for washing are about 25 ° C. or higher, more preferably about 42 ° C. or higher, and most preferably about 68 ° C. or higher. In one preferred embodiment, washing is performed at a temperature of 25 ° C. under conditions of 30 mM NaCl, 3 mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, washing is performed at a temperature of 42 ° C. under conditions of 15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS. In the most preferred embodiment, 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 the polynucleotide of the invention (3), for example, using the oligonucleotide of the invention (2) as a probe, the stringent as described above is screened for a genomic library prepared from chromosomal DNA of BFNC patients by hybridization and washing treatment Can be isolated.

  The polynucleotide of this invention (3) becomes a detection target in the diagnostic method of invention (9) described later.

  Invention (4) of this application PCR amplifies a 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) It is a primer set for. In these primer sets, one oligonucleotide primer is composed of a 15-30 nt continuous DNA sequence including each mutation (substitution or deletion) site of SEQ ID NO: 1 (KCNQ2) or a complementary sequence thereof. The other primer can be any continuous DNA sequence 5 ′ or 3 ′ of each mutation site 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 including each mutation site. A linker sequence or the like can also be added to the end of the primer. Furthermore, commercially available software such as Oligo ™ [manufactured by National Bioscience Inc. (USA)], GENETYX [manufactured by Software Development Co., Ltd. (Japan)] and the like can also be used for sequence design.

  The primer set of the invention (4) can be used for the BFNC diagnosis method of the invention (12) described later.

  Invention (5) of this application is a microarray (DNA chip) provided with the polynucleotide and / or oligonucleotide as a capture probe. As a method for producing a microarray, a method of directly synthesizing an oligonucleotide on the surface of a solid phase carrier (on-chip method) and a method of fixing a previously prepared oligonucleotide on the surface of a solid phase carrier are known. The microarray of the present invention can be produced by any of these methods. As an on-chip method, a combination of the use of a protective group that is selectively removed by light irradiation and the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing can be combined in a predetermined area of a minute matrix. It can be performed by a method of performing selective synthesis (masking technique: for example, Fodor, SPA Science 251: 767, 1991). On the other hand, when immobilizing an oligonucleotide prepared in advance on the surface of a solid phase carrier, an oligonucleotide into which a functional group has been introduced is synthesized, and the oligonucleotide is spotted on the surface of the surface of the solid phase carrier and covalently bonded (for example Lamture, JB et al. Nucl. Acids Res. 22: 2121-2125, 1994; Guo, Z. et al. Nucl. Acids Res. 22: 5456-5465, 1994). Oligonucleotides are generally covalently bound to a surface-treated solid support via a spacer or a crosslinker. A method is also known in which polyacrylamide gel fragments are aligned on a glass surface and a synthetic oligonucleotide is covalently bound thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996). ). In addition, an array of microelectrodes was prepared on a silica microarray, an agarose permeation layer containing streptavidin was provided on the electrode as a reaction site, and this site was charged positively to immobilize the biotinylated oligonucleotide. There is also known a method that enables precise hybridization at high speed by controlling the site charge (Sosnowski, RG et al. Proc. Natl. Acad. Sci. USA 94: 1119-1123, 1997). The microarray of the present invention can be produced by any of the above methods.

Invention (6) of this application is an expression product of the polynucleotide of invention (1) or invention (3), wherein the amino acid sequence of SEQ ID NO: 2 has the following amino acid mutation:
(A) No. 304 Phe deleted
(B) A polypeptide having at least one of deletions at position 323 and below Gln.

  That is, the amino acid mutation in these polypeptides may be caused by a missense mutation or deletion mutation in the polynucleotide of the invention (1), whereby the amino acid of a normal (wild-type) polypeptide (SEQ ID NO: 2) is mutated, or a stop codon. It becomes a short-chain polypeptide.

  These polypeptides can be isolated from a biological sample of a BFNC 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 mutated amino acid residue, or the invention ( It can be obtained by the method of producing by recombinant DNA technology using the polynucleotide (mutant cDNA) of 1). The polypeptide of this invention (6) can be used as a test object of the BFNC diagnostic method of the following invention (23), for example.

  The invention (7) of this application is an oligopeptide which is a part of the polypeptide of the invention (6) and has a 5-30 continuous amino acid sequence containing each amino acid mutation. 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 (6) with an appropriate protease. These oligopeptides can be used, for example, as antigens for antibody production of invention (8) described later.

The antibody of the invention (8) is a polyclonal antibody or a monoclonal antibody prepared using the oligopeptide of the invention (7) as an antigen, a whole molecule capable of binding to the epitope of the polypeptide of the invention (6), and a Fab , F (ab ′) ₂ , Fv fragment, etc. are all included. For example, in the case of a polyclonal antibody, such an antibody can be obtained from serum after immunizing an animal using the oligopeptide as an immunogen. Alternatively, it can be prepared by introducing the expression vector for eukaryotic cells into animal muscle or skin by injection or gene gun, and then collecting serum. As the animal, mouse, rat, rabbit, goat, chicken and the like are used. Monoclonal antibodies are known monoclonal antibody production methods ("monoclonal antibodies", Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996) Can be created according to. These antibodies can specifically recognize the polypeptide of the invention (6) and can be used in the diagnostic method of the invention (12) described later.

  Invention (9) of this application is a method for diagnosing whether or not a convulsive seizure in a subject (newborn) is BFNC. 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 a BFNC patient. The polynucleotide can also be detected by a method of directly sequencing it, but the method of the invention (10) or (11) is preferred.

  In the method of the invention (10), it is determined whether or not the chromosomal DNA or mRNA thereof isolated from the 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 gene mutation associated with BFNC, the chromosomal DNA or mRNA thereof and the polynucleotide or oligonucleotide hybridize even under stringent conditions. Hybridization can be performed easily and with high accuracy using, for example, the above-described labeled probe or microarray. Specifically, as a hybridization method using a labeled DNA probe, for example, a known method such as the Allele-specific Oligonucleotide Probe method, Oligonucleotide Ligation Assay method, Invader method or the like can be employed. Diagnosis using a microarray can be performed, for example, as follows. That is, cDNA is synthesized and amplified by PCR using mRNA isolated from a biological sample (eg, blood) of a subject as a template. At that time, labeled dNTP is incorporated into labeled cDNA. The labeled cDNA is brought into contact with the macroarray, and the cDNA hybridized with the capture probe (oligonucleotide) of the microarray is detected. Hybridization can be performed by dispensing a 96- or 384-well plastic plate and spotting a labeled cDNA aqueous solution on the microarray. The amount of spotting can be about 1 to 100 nl. Hybridization is preferably performed in the temperature range of room temperature to 70 ° C. for 6 to 20 hours. After completion of hybridization, washing is performed using a mixed solution of a surfactant and a buffer to remove unreacted labeled cDNA. As the surfactant, sodium dodecyl sulfate (SDS) is preferably used. As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good buffer solution, or the like can be used, and a citrate buffer solution is preferably used.

  In the method of the invention (11), the presence or absence of a PCR product when PCR is carried out using the chromosomal DNA or mRNA isolated from the subject as a template and the primer set of the invention (4) is detected. When the subject has a gene mutation associated with BFNC, a polynucleotide PCR product defined by the primer set is obtained. PCR or RT-PCR can be performed by a known method. In addition to the method of directly sequencing PCR products, for example, PCR-SSCP method, PCR-CFLP method, PCR-PHFA method and the like may be used for detection of nucleotide mutation. Moreover, the well-known methods of Rolling Circle Amplification method and Primer Oligo Base Extension method can also be adopted.

Invention (12) of this application is also a method for diagnosing whether or not a subject is a BFNC patient, and when the polypeptide of the invention (6) is present in a biological sample isolated from the subject, The subject is determined to be a BFNC patient. The presence of the polypeptide can be carried out by various known methods, but the method of the invention (13) is preferred. The method of the invention (13) is a method using the antibody of the invention (8), and in particular, by using a labeled antibody, simple and highly accurate detection is possible. The label can be an enzyme, a radioisotope or a fluorescent dye. The enzyme is not particularly limited as long as it satisfies the conditions such as a large turnover number, stability even when bound to an antibody, and specific coloring of a substrate, and is used for normal EIA. Enzymes such as peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase and the like can also be used. Moreover, an enzyme inhibitor, a coenzyme, etc. can also be used. These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used according to the type of enzyme used. For example, when peroxidase is used as an enzyme, 3,3 ′, 5,5′-tetramethylbenzidine can be used, and when alkaline phosphatase is used as an enzyme, paranitrophenol or the like can be used. As the radioisotope, those used in usual RIA such as ¹²⁵ I and ³ H can be used. As the fluorescent dye, those used in usual fluorescent antibody methods such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used. When using an enzyme, add a substrate that decomposes due to enzymatic action and develops color, optically measures the amount of degradation of the substrate, obtains the enzyme activity, converts this to the amount of bound antibody, and compares it with the standard value. From this, the antibody amount is calculated. When using a radioactive isotope, the radiation dose emitted by the radioactive isotope is measured with a scintillation counter or the like. In addition, when a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope. Furthermore, a sandwich method using a primary antibody and a labeled secondary antibody (“ELISA method” when an enzyme is used as a label) can be preferably used.

  The above BFNC diagnosis can also be performed in combination with the detection of the mutation of the KCNQ2 gene or its expression product known so far.

Hereinafter, screening procedures and results for confirming the gene mutation that is the basis of the invention will be described.
1． Method
1-1. patient
Diagnosis of BFNC was performed by a pediatric neurologist according to the criteria in the International Anti-Epileptic Federation classification and terminology committee guidelines (Non-Patent Document 1) and the like. The survey was conducted on 61 BFNC patients and 100 healthy volunteers.
1-2. Genetic analysis
Genomic DNA was prepared from an EDTA-treated whole blood sample using a QIAamp DNA Blood kit (Qiagen, Germany). Screening for KCNQ2 and KCNQ3 gene abnormalities was performed using a direct sequencing method with an automated sequencer. The primers and PCR reaction conditions for PCR amplification of all exons of the gene were those described in Non-Patent Document 9. Human KCNQ2: GenBank / NM_172106 was used as a reference sequence for each mRNA.
1-3. Statistical analysis Differences in the incidence of abnormalities were determined based on statistical significance using the χ ² test. Statistical significance was obtained when the P value was less than 0.05.
2． result
As a result of genetic analysis of BFNC patients, the following novel mutations in the KCNQ2 gene were found.
(i) c.910-912delTTC or TTT, F304del
(ii) c.967C> T, Q323X
Mutation (i) is a frameshift that results in a stop codon, resulting in a short peptide lacking positions 304 and below in SEQ ID NO: 2. Mutation (ii) also results in a mutant peptide lacking the Phe residue at position 304 of SEQ ID NO: 2. This Phe residue is an amino acid residue highly conserved across species in the KCNQ2 protein, and deletion of this Phe residue greatly affects the function of the KCNQ2 protein.

  None of these mutations were observed in 100 healthy subjects.

  From the above results, it was confirmed that the aforementioned KCNQ2 gene mutations (i) and (ii) were mutations closely related to BFNC.

  As described above in detail, the invention of this application provides a novel gene mutation associated with benign familial neonatal seizures present in the KCNQ2 gene and a method for diagnosing benign familial neonatal seizures using this gene mutation. By these inventions, it is possible to reliably perform a diagnosis for distinguishing benign familial neonatal seizure from a disease having a poor prognosis.

Claims (6)

A polynucleotide derived from a gene related to benign familial neonatal seizure, wherein the ttc positions 910 to 912 are deleted in the nucleotide sequence of SEQ ID NO: 1. An oligonucleotide comprising a part of the polynucleotide of claim 1 and comprising a base sequence before and after positions 910 to 912 in SEQ ID NO: 1 and comprising 20 to 99 consecutive DNA sequences not containing said ttc . The polynucleotide according to claim 1 a primer set for PCR amplification, one primer is made from the front and back of the base sequence of the 910-912 of ttc in SEQ ID NO: 1, and does not include the ttc 15 to 30 A primer set which is an oligonucleotide consisting of a continuous DNA sequence or a complementary sequence thereof.   A microarray comprising the polynucleotide of claim 1 or the oligonucleotide of claim 2. A method for testing benign familial neonatal convulsions, wherein chromosomal DNA or mRNA thereof isolated from a subject and whether or not the polynucleotide of claim 1 or the oligonucleotide of claim 2 hybridizes under stringent conditions A method characterized by detecting. A method for testing benign familial neonatal seizures , characterized by detecting the presence or absence of a PCR product when PCR is performed using a chromosomal DNA or mRNA isolated from a subject as a template and using the primer set of claim 3. And how to.