JP2007014244A - Method for inspecting genetic disposition of intraventricular conduction disturbance or ventricular fibrillation, and reagent therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inspecting genetic disposition of intraventricular conduction disturbance and ventricular fibrillation, or sensitivity to an antiarrhythmic drug. <P>SOLUTION: The method for inspecting the genetic disposition or the like of the intraventricular conduction disturbance, the ventricular fibrillation, and a disease associated with sodium channel disorder of a testee comprises a step for determining the genotype of a transcriptional control region and/or a transcription region of SCN5A possessed by the testee as at least one selected from the group consisting of the kinds of bases at the polymorphic sites of -1418, -1062, -847, -354 and 287 positions, and presence or absence of insertion of a base to the polymorphic site between -835 position and -836 position, and a step for predicting the disposition based on the determined genotype. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for examining a genetic predisposition to a disease associated with ventricular conduction disorder, ventricular fibrillation or sodium channel abnormality or susceptibility to an antiarrhythmic drug or sodium channel blocker, and an oligonucleotide which can be used for the test method , Reagents and kits.

  Heartbeat excitement and conduction are critically dependent on the proper functioning of the cardiac sodium channel. For this reason, a decrease in sodium current due to a drug or a congenital disease such as Brugada syndrome slows conduction and may cause serious arrhythmia (Non-Patent Document 1, Vreede-Swagemakers et al., 1997). . Arrhythmia is a major cause of sudden cardiac death (SCD) and includes ventricular fibrillation (VF), which accounts for approximately 20% of all deaths in adults.

  Sudden cardiac death is an important social problem that kills about 40,000 lives in Japan in an instant, and many of them are thought to be caused by ventricular fibrillation, a fatal ventricular arrhythmia. Brugada syndrome (idiopathic ventricular fibrillation) is a typical disease that causes sudden death due to ventricular fibrillation in patients who do not have obvious organic heart disease. Brugada Syndrome (BS) is characterized by the characteristic ST elevation and ventricular fibrillation of leads 12 to V3 on the 12-lead ECG, and causes sudden nocturnal death in middle-aged and elderly men. It has been suggested to be associated with the reported Pockley disease. Epidemiological studies have reported that Brugada syndrome is common in Asia including Japan, and that ST-elevated electrocardiogram abnormalities characteristic of Brugada syndrome are observed in 1 to 2 of 1,000 Japanese people. Yes. In Brugada syndrome, a mutation of SCN5A, a sodium channel gene related to ventricular muscle excitatory conduction, was first reported in 1998 (Non-Patent Document 2, Chen et al., 1998). Up to now, more than 80 mutations have been reported in the translation region of SCN5A. Any of these mutations causes a decrease in sodium channel function leading to abnormal ventricular muscle excitatory conduction (ventricular conduction disorder), leading to the onset of ventricular fibrillation. It is believed that. Interestingly, this SCN5A is also a common causative gene for other lethal hereditary arrhythmia diseases such as cardiac conduction deficiency, type 3 congenital QT prolongation syndrome, and some familial sinus dysfunction syndromes.

  Thus, mutations in the SCN5A gene encoding the cardiac sodium channel (Non-Patent Document 3, Gellens et al., 1992) cause various early arrhythmia syndromes. Mutations resulting in enhanced channel function cause long QT syndrome, and mutations resulting in loss of channel function result in Brugada syndrome, cardiac conduction deficit (Lenegre disease), familial sinus dysfunction syndrome, atrial resting, familial atrioventricular Blocks and various combinations of these are caused (Non-Patent Document 4, Tan et al., 2003). In addition, it has been preliminarily reported that fiber formation in myocardial tissue can be enhanced by sodium channel mutation (Non-Patent Document 5, Bezzina et al., 2003).

  Here, a region close to the promoter of SCN5A (including the core promoter) has been cloned and functionally analyzed, and a plurality of cis-acting elements that control the expression of the gene have been identified (Non-Patent Document 6, Yang et al.). al, 2004). However, no polymorphism in the transcriptional regulatory region of SCN5A has been reported so far.

  On the other hand, many antiarrhythmic drugs target sodium channels, and exhibit antiarrhythmic action by suppressing them. In this case, for example, if there is a potential sodium channel dysfunction due to a mild dysfunction of SCN5A, that is, an abnormality in ventricular muscle excitatory conduction, administration of an antiarrhythmic drug having a sodium channel inhibitory effect causes unexpected serious Ventricular muscular excitatory conduction abnormalities may be caused and ventricular fibrillation may develop as an arrhythmogenic effect. In addition, it is known that ventricular fibrillation develops due to an increase in ventricular muscle excitatory conduction abnormality during acute myocardial ischemia such as acute myocardial infarction or angina pectoris. In the presence of sodium channel dysfunction, the risk of developing ventricular fibrillation is expected to increase.

Vreede-Swagemakers JJM, Gorgels AP, Duboisarbouw WI, Vanree JW, Daemen MJAP, Houben LGE, Wellens HJJ. Out-Of-Hospital Cardiac Arrest In The 1990s-A Population-Based Study In The Maastricht Area On Incidence, Characteristics And Survival. J Am Coll Cardiol 1997; 30: 1500-1505. Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, Potenza D, Moya A, Borggrefe M, Breithardt G, Ortiz-Lopez R, Wang Z, Antzelevitch C, O'Brien RE, Schulze-Bahr E , Keating MT, Towbin JA, Wang Q: Genetic basis and molecular mechanisms for idiopathic ventricular fibrillation.Nature 392: 293-296, 1998. Gellens, M E .; George, AL, Jr .; Chen, L .; Chahine, M .; Horn, R .; Barchi, RL; Kallen, RG: Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage- dependent sodium channel. Proc. Nat. Acad. Sci. 89: 554-558, 1992. Tan HL, Bezzina CR, Smits JPP, Verkerk AO, Wilde AAM. Genetic control of sodium channel function.Cardiovasc Res 57, 961-973, 2003. Bezzina CR, Rook MB, Groenewegen WA, Herfst LJ, van der Wal AC, Lam J, Jongsma HJ, Wilde AA, Mannens MM. Compound heterozygosity for mutations (W156X and R225W) in SCN5A associated with severe cardiac conduction disturbances and degenerative changes in the conduction system. Circ Res. 2003 Feb 7; 92 (2): 159-68. Yang P, Kupershmidt S, Roden DM.Cloning and initial characterization of the human cardiac sodium channel (SCN5A) promoter.Cardiovasc Res. 2004; 61: 56-65. Wilde AA, Antzelevitch C, Borggrefe M, Brugada J, Brugada R, Brugada P, et al. Proposed diagnostic criteria for the Brugada syndrome: consensus report. Circulation 2002; 106: 2514-2519 Bezzina CR, Verkerk AO, Busjahn A, Jeron A, Erdmann J, Hense HW, Koopmann TT, Bhuiyan ZA, Wilders R, Mannens MMAM, Tan HL, Luft FC, Schunkert H, Wilde AAM.A common polymorphism in KCNH2 (HERG) hastens cardiac repolarization. Cardiovasc Res. 2003b; 59: 27-36. Kupershmidt S, Yang T, Chanthaphaychith S, Wang Z, Towbin JA, Roden DM: Defective human Ether-a-go-go-related gene trafficking linked to an endoplasmic reticulum retention signal in the C terminus.J Biol Chem. 2002; 277 : 27442-27448 Smits JP, Eckardt L, Probst V, Bezzina CR, Schott JJ, Remme CA, Haverkamp W, Breithardt G, Escande D, Schulze-Bahr E, LeMarec H, Wilde AA. Genotype-phenotype relationship in Brugada syndrome: electrocardiographic features differentiated SCN5A -related patients from non-SCN5A-related patients.J Am Coll Cardiol. 2002; 40: 350-6. Excoffier and Slatkin, 1995 Roden DM. Acquired long QT syndrome and complex ion channel genetic disorders. J Clin Invest. In press. Tomaselli GF, Zipes DP.What Causes Sudden Death in Heart Failure? Circ Res 2004; 95: 754-763.

  It is an object of the present invention to provide a method for examining the genetic predisposition of diseases associated with ventricular conduction disturbances, ventricular fibrillation or sodium channel abnormalities, or susceptibility to antiarrhythmic drugs or sodium channel blockers, in particular, The purpose is to predict patients who are likely to develop ventricular conduction disorder and ventricular fibrillation causing death, patients who are likely to have side effects on antiarrhythmic drugs, and patients who are likely to develop ventricular fibrillation during acute ischemia.

According to one aspect of the present invention, a method for testing a subject for a genetic predisposition to a disease associated with impaired ventricular conduction, ventricular fibrillation or sodium channel abnormalities, comprising:
The transcriptional regulatory region and / or genotype of the transcriptional region of SCN5A possessed by the subject is defined as the types of bases of the polymorphic sites at positions -1418, -1062, -847, -354 and 287, and -835. And determining at least one selected from the group consisting of the presence or absence of insertion of a base into the polymorphic site between positions −836;
Predicting the predisposition based on the determined genotype is provided.

According to another aspect of the invention, a method for testing a subject's sensitivity to an antiarrhythmic drug or sodium channel blocker comprising:
The transcriptional regulatory region and / or genotype of the transcriptional region of SCN5A possessed by the subject is defined as the types of bases of the polymorphic sites at positions -1418, -1062, -847, -354 and 287, and -835. And determining at least one selected from the group consisting of the presence or absence of insertion of a base into the polymorphic site between positions −836;
Predicting said susceptibility based on the determined genotype is provided.

  According to another aspect of the present invention, an oligonucleotide capable of hybridizing to a transcriptional regulatory region of SCN5A and / or a region containing a polymorphic site in the transcriptional region, or a region complementary thereto, wherein the polymorphic site comprises: An oligonucleotide is provided which is at least one selected from the group consisting of polymorphic sites at positions −1062, −847 and 287, and polymorphic sites between positions −835 and −836.

  According to another aspect of the present invention, the genotype of the transcriptional regulatory region and / or transcriptional region of SCN5A is defined as the type of base at the polymorphic sites at positions −1062, −847 and 287, and −835 and −836. A reagent for determining at least one selected from the group consisting of the presence or absence of insertion of a base at a polymorphic site between positions is provided. According to another aspect of the present invention, there is provided a kit for determining the genotype of the transcriptional regulatory region and / or transcriptional region of SCN5A, which contains the above reagent.

According to another aspect of the present invention, a method for screening for a drug that modulates the transcription amount of SCN5A, comprising:
Providing a test substance;
Providing a transcriptional regulatory region of SCN5A and / or a plurality of groups of animals having a polynucleotide having the transcriptional region, wherein the transcriptional regulatory region and / or transcriptional region of SCN5A is a transcription amount and / or an expression amount. Is operably linked to a measurable gene, and the transcriptional regulatory region and / or transcription region of SCN5A is a genotype in which the base of the polymorphic site at position -1418 is C, and the polymorphic site at position -1062 The genotype in which the base of C is G, the genotype in which the base of the polymorphic site at position −847 is G, the genotype in which the base of the polymorphic site at position −354 is C, and the base of the polymorphic site at position 287 is And having at least one selected from the group consisting of a genotype that is T and a genotype that has a GC insertion into the polymorphic site between positions −835 and −836;
Contacting the test substance with at least one group of cells or animals;
Measuring the transcription amount and / or the expression level of the gene in a group that has been contacted with the test substance and a group that has not been contacted;
Comparing the amount of transcription and / or the expression level of the group not contacted with the group contacted with the test substance.

  According to the other aspect of this invention, the medicine which adjusts the transcription | transfer amount of SCN5A obtained by the said screening method is provided.

  As described in detail below, the present inventors have found haplotypes involved in ventricular conduction velocity in the transcriptional regulatory region and transcriptional region of SCN5A. By determining the genotype of the polymorphic site in the region, testing for genetic predisposition to diseases associated with ventricular conduction disturbances, ventricular fibrillation or sodium channel abnormalities or susceptibility to antiarrhythmic drugs or sodium channel blockers it can.

  As described above, according to one aspect of the present invention, a method is provided for testing a subject for a genetic predisposition for a disease associated with impaired ventricular conduction, ventricular fibrillation or sodium channel abnormalities.

  Ventricular conduction disorders refer to a state in which the ventricular conduction velocity is faster and a slower state compared to a normal case, and particularly a case in which the ventricular conduction velocity is slow. As a method for non-invasively estimating the ventricular conduction velocity, there is a measurement of QRS time and PQ time of a 12-lead electrocardiogram. The QRS time is an index indicating the conduction time in the ventricle, and the PQ time is an index indicating the total conduction time of the intra-atrial / atrioventricular nodal conduction time and the Hispurkinje system conduction time (corresponding to the HV time). As an invasive method, there is a His-ventricle (HV) time during electrophysiological examination. Generally, the normal range of QRS time is 70 to 100 msec, the normal range of PQ time is 120 to 200 msec, and the normal range of HV time is 30 to 50 msec. A condition exceeding this range may be regarded as ventricular conduction disorder. it can.

  Ventricular fibrillation refers to a state in which the ventricular muscles are excited in a disorderly manner and do not contract to pump out blood. Examples of ventricular fibrillation include ventricular fibrillation that occurs during acute myocardial ischemia and ventricular fibrillation associated with Brugada syndrome. Particularly preferably, the genetic predisposition of ventricular fibrillation occurring during acute myocardial ischemia can be examined by the method according to the present invention. Further, the method according to the present invention can examine the genetic predisposition of ventricular fibrillation associated with Brugada syndrome in a subject.

  Brugada syndrome is a typical disease that may cause sudden death due to ventricular fibrillation. (1) No organic heart disease is found; (2) Right chest lead V1-V3 is type 1 It is characterized in that a coved ST rise is observed and (3) the heart rate corrected QT time is less than 440 msec (Non-Patent Document 7, Wilde et al., 2002).

  Furthermore, according to the present invention, a predisposition for diseases associated with sodium channel abnormality, particularly diseases associated with sodium channel abnormality in the heart, can also be examined. Sodium channel abnormalities include sodium channel quantitative and qualitative (functional) abnormalities. Specifically, examples of diseases associated with sodium channel abnormalities include the above Brugada syndrome, type 3 congenital QT prolongation syndrome, cardiac conduction deficit (Lenegre disease), familial sinus dysfunction syndrome, atrial resting, familial Examples include atrioventricular block and type 3 QT shortening syndrome.

  In addition, according to another aspect of the present invention, a method for examining a subject's sensitivity to an antiarrhythmic drug or sodium channel blocker is provided. Examples of antiarrhythmic drugs include Vaughan-Williams class Ic group pilsicainide, flecainide, propafenone, group Ia disopyramide, procainamide, probenzoin, cibenzoline, (pimenol), quinidine, ajmaline, abrine from group Ib, and bepridil from group IV. Examples of other drugs other than antiarrhythmic drugs having sodium channel blocking action include psychotropic drugs such as tricyclic, tetracyclic antidepressants, phenothiazine derivatives, and selective serotonin reuptake inhibitors. Here, high sensitivity means that the change in the amount of living body when a predetermined amount of drug is administered is large and the change in the amount of living body when a predetermined amount of drug is administered is not different from normal This includes a case where a sufficient change in the amount of the living body can be obtained by the administration of a smaller amount of the medicine because the previous amount of the living body is different (ie, larger or smaller).

  In the method for examining the predisposition or sensitivity according to the present invention, the SCN5A transcription regulatory region and / or the genotype of the transcription region possessed by a subject are determined as follows: −1418, −1062, −847, −354, and 287. And the step of determining at least one selected from the group consisting of the type of base at the polymorphic site and the presence or absence of insertion of the base into the polymorphic site between positions −835 and −836. The present inventors have found a plurality of polymorphic sites in the transcriptional regulatory region and transcription region of SCN5A, and furthermore, genotypes of the polymorphic sites and diseases related to ventricular conduction disorder, ventricular fibrillation or sodium channel abnormality Were found to be associated with a genetic predisposition to or susceptibility to antiarrhythmic drugs or sodium channel blockers.

  SEQ ID NO: 1 shows the nucleotide sequence of the transcriptional regulatory region and transcription region of SCN5A found most widely in Japanese. SEQ ID NOs: 2 and 3 show the transcriptional regulatory region and the nucleotide sequence of the transcriptional region of SCN5A having a polymorphism. In this specification, the position of the transcriptional regulatory region of SCN5A and the base of the transcriptional region are the base sequence of SEQ ID NO: 1, the position of the 2190th base is the +1 position, and the 5 ′ side (upstream) base from the +1 position is The bases on the 3 ′ side (downstream) from the +1 position are described as the 2nd, 3rd, 4th, etc. in order, respectively (Non-Patent Document 6, Yang). et al., 2004). The + 1st position is the major transcription initiation site identified by the RNase protection assay. As shown in FIG. 1, polymorphisms are found at the polymorphic sites at positions −1418, −1062, −847, −354 and 287, and the polymorphic sites between −835 and −836. . In this specification, the polymorphic site at position -1418 refers to the position corresponding to position -1418 of the nucleotide sequence of SEQ ID NO: 1. The same applies to other polymorphic sites.

As shown in SEQ ID NO: 1 and FIGS. 1 and 2, the genotypes most widely found in Japanese are as follows (also referred to as haplotype A or TTT--GC).
The base of the polymorphic site at position -1418 is T;
The base of the polymorphic site at position −1062 is T;
The base of the polymorphic site at position −847 is T,
The base of the polymorphic site at position −354 is G;
The base of the polymorphic site at position 287 is C, and there is no insertion into the polymorphic site between positions −835 and −836.

Further, as shown in SEQ ID NO: 2 and FIGS. 1 and 2, the present inventors have found a genotype having the following polymorphism in the base sequence shown in SEQ ID NO: 1 (also referred to as haplotype B or CCGinsCT). .)
The base of the polymorphic site at position -1418 is C (also referred to as T-1418C),
The base of the polymorphic site at position −1062 is C (also referred to as T-1062C);
The base of the polymorphic site at position −847 is G (also referred to as T-847G),
The base of the polymorphic site at position −354 is C (also referred to as G-354C);
The base of the polymorphic site at position 287 is T (also referred to as C287T), and there is GC insertion at the polymorphic site between positions −835 and −836 (also referred to as −835insGC).

Furthermore, as shown in SEQ ID NO: 3 and FIGS. 1 and 2, the present inventors found a genotype having the following polymorphism in the base sequence shown in SEQ ID NO: 1 (haplotype C or CTT-- -Also referred to as CC.)
The base of the polymorphic site at position -1418 is C;
The base of the polymorphic site at position −1062 is T;
The base of the polymorphic site at position −847 is T,
The base of the polymorphic position at position −354 is C,
The base of the polymorphic site at position 287 is C, and there is no insertion into the polymorphic site between positions −835 and −836.

  Genotypes with haplotype B (AB genotype + BB genotype) were found with a frequency of 21-24% in two independent Japanese groups (n = 182). These six polymorphisms were in almost complete linkage disequilibrium. In addition, haplotype C was found as a rare genotype (2 of 364 cases, 0.5%). Haplotype B may also have a significant correlation with decreased sodium channel expression, increased excitatory conduction time (ie, PQ time and QRS time), and increased QRS time after sodium channel blocker administration. It was found.

  As described above, the transcriptional regulatory region of SCN5A and the six polymorphisms found in the transcriptional region were almost completely in linkage disequilibrium. For this reason, when examining the predisposition or sensitivity, it is not necessary to determine genotypes for all six polymorphic sites. That is, the predisposition or sensitivity can be examined by determining the genotype of at least one of the 6 polymorphisms. Strictly speaking, when the base of the polymorphic site corresponding to position -1418 is C, or the base of the polymorphic site corresponding to position −354 is C, strictly, haplotype B and haplotype C I can't distinguish. However, considering that the frequency of haplotype B is 21 to 24% and the frequency of haplotype C is 0.5%, even in these cases, it can be estimated that haplotype B is present. However, for the sake of more accuracy, the genotype is determined by inserting the base at the polymorphic sites at positions −1062, −847, and 287, and the polymorphic site between positions −835 and −836. It is preferable to determine at least one selected from the group consisting of the presence or absence of. In addition, for further accuracy, genotypes can be determined for more polymorphic sites, particularly for all six polymorphic sites.

  In addition, the method for examining a predisposition or susceptibility according to the present invention includes a step of predicting a predisposition or the susceptibility based on the determined genotype. Specifically, when the subject is a homozygote or heterozygote of the haplotype B allele, it can be predicted that the subject has the above predisposition or is highly sensitive. Predicted that the predisposition is greater or more sensitive when the subject has two haplotype B alleles (homozygotes) than when the subject has one haplotype B allele (heterozygotes) can do.

Determination of the genotype according to the present invention can be performed by those skilled in the art using various methods (see JP 2005-130764 A, JP 2004-313168 A, JP 2004-222503 A, etc.). . Specifically, in addition to the method for determining the nucleotide sequence at the polymorphic site (direct sequencing method), TaqMan PCR method, AcycloPrime method, SNaPshot ^TM method, Pyrosequencing method, MALDI-TOF / MS method, type IIs restriction enzyme SNP-specific labeling method used, genotype determination method at polymorphic sites using magnetic fluorescent beads, Invader method, RCA method, RFLP, PCR-RFLP, PCR-SSCP, denaturing gradient gel method, DNA array And the ASO hybridization method are known. Hereinafter, these techniques will be briefly described. When determining the genotype, it is not always necessary to determine the type of base in the polymorphic site (that is, which is A, G, T, or C) or the type of inserted base. For example, when the type of base at a certain polymorphic site is T or C, it is sufficient to determine that the type of base at that site is “not T” or “not C”.

  As a sample for determining a genotype, any biological sample obtained from a subject can be used. Examples of biological samples include blood, other body fluids, skin, oral mucosa obtained from a subject, Other tissues or cells, etc., or genomic DNA isolated or purified from them can be mentioned.

[Direct sequencing method]
The genotype can be determined by directly determining the base sequence of the DNA containing the polymorphic site according to the present invention. In this method, first, chromosomal DNA is isolated and cloned from a biological sample obtained from a subject, and the base sequence of the cloned DNA is determined. The base sequence can be easily determined using a DNA sequencer or the like.

[TaqMan PCR method]
The genotype can be determined by the TaqMan PCR method (SNP gene polymorphism strategy, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p94-105, Genet Anal. (1999) 14: 143-149). The principle of TaqMan PCR method is as follows. The TaqMan PCR method is an analysis method using a primer set that can amplify a region including a polymorphic site and a TaqMan probe. The TaqMan probe is designed to hybridize to a region containing the target polymorphic site, and is labeled with a reporter dye and a quencher at the 3 ′ end and 5 ′ end.

  When the PCR method is performed in the presence of the TaqMan probe, the extension reaction from the primer eventually reaches the hybridized TaqMan probe. At this time, the TaqMan probe is degraded from its 5 'end by the 5' exonuclease activity of DNA polymerase. The degradation of the TaqMan probe labeled with the reporter dye and quencher can be followed as a change in fluorescence signal. In other words, when the TaqMan probe is decomposed, the reporter dye is released and the distance from the quencher is increased to generate a fluorescent signal. If the target base sequence is hybridized under conditions close to the Tm of the TaqMan probe, the hybridization efficiency of the TaqMan probe is significantly reduced even if there is a difference of one base. If the hybridization of the TaqMan probe decreases due to a difference in one base, the TaqMan probe does not decompose and a fluorescent signal is not generated.

  If a TaqMan probe corresponding to a polymorphism is designed and a different signal is generated by the decomposition of each probe, genotype determination can be performed simultaneously. For example, as a reporter dye, 6-carboxy-fluorescein (FAM) is used for a TaqMan probe corresponding to genotype A at a certain polymorphic site, and VIC is used for a probe corresponding to genotype B. In a state where the probe is not decomposed, generation of a fluorescent signal of the reporter dye is suppressed by the quencher. If each probe hybridizes to the corresponding polymorphic site, a fluorescence signal corresponding to the hybridization is observed. That is, when the signal of either FAM or VIC is stronger than the other, it turns out that the polymorphic site A or polymorphic site B is homozygous. On the other hand, when the polymorphic site is heterozygous, both signals are detected at substantially the same level. By using the TaqMan PCR method, PCR and genotype determination can be performed simultaneously on a genome as an analysis target without a time-consuming step such as separation on a gel. Therefore, the TaqMan PCR method is useful as a method that can determine the genotype of many subjects. Thus, in the present invention, the TaqMan PCR method can be suitably used for determining the genotype of a polymorphic site. Primers and probes used at that time are not particularly limited.

[AcycloPrime method]
The AcycloPrime method has also been put to practical use as a method for determining genotypes using the PCR method (Genome Research (1999) 9: 492-498). In the AcycloPrime method, one primer having a sequence complementary to the base sequence on the 3 ′ side from the base on the 3 ′ side of the base of the polymorphic site is used for polymorphism detection. First, a region containing a polymorphic site in the genome is amplified by PCR. This process is the same as normal genomic PCR. Next, the primer for genome detection is annealed with respect to the obtained PCR product, and extension reaction is performed. The primer for genome detection anneals to a region adjacent to the polymorphic site to be detected. At this time, a nucleotide derivative (terminator) labeled with a fluorescent polarizing dye and blocked with 3′-OH is used as a nucleotide substrate for the extension reaction. As a result, only one base complementary to the base at the position corresponding to the polymorphic site is incorporated, and the extension reaction stops. Incorporation of a nucleotide derivative into a primer can be detected by an increase in fluorescence polarization (FP) due to an increase in molecular weight. If two types of labels having different wavelengths are used for the fluorescent polarizing dye, it is possible to specify which of the two types of bases the specific SNP is. Since the level of fluorescence polarization can be quantified, it is possible to determine whether the polymorphism is homo or hetero in one analysis.

[SNaPshot ^TM method]
In addition, the SNaPshot ^™ method is known as a primer extension method for determining the genotype. In the SNaPshot ^TM method, a primer that is adjacent to a single nucleotide polymorphism site and has a nucleotide that is added to its 3 ′ end by an extension reaction is complementary to the single nucleotide polymorphism site. . Using such a primer, a primer extension reaction is carried out using a nucleic acid sample from a subject as a template, and by using ddNTP (dideoxyNTP) at that time, the extension reaction corresponds to the above polymorphic site. Finish when one nucleotide has been incorporated. The incorporated nucleotide is easily identified by pre-labeling with a fluorescent label or the like, and thus the nucleotide at the polymorphic site is identified. Such methods are known to those skilled in the art, and the procedures for the operation and the specific nucleotide sequences of the primers used can be easily determined by those skilled in the art.

[Pyrosequencing method]
As a primer extension method for determining the genotype, the pyrosequencing method is known (Anal. Biochem. (2000) 10: 103-110). In the pyrosequencing method, four dNTPs are reacted one by one during the primer extension reaction using a nucleic acid sample from a subject as a template. When either dNTP is incorporated, an equal amount of pyrophosphate (PPi) is liberated, and the liberated PPi reacts with sulfurylase to produce ATP, which causes luciferase reaction and luminescence. Therefore, when light emission occurs when a specific dNTP is added, it becomes clear that the nucleotide corresponding to the dNTP has been incorporated, and thereby the nucleotide of the target site in the nucleic acid sample is identified. In this method, since dNTP is used, it is not necessary to use a primer adjacent to the polymorphic site as used in the SNaPshot ^™ method, and a primer separated by several bases may be used. Such methods are known to those skilled in the art, and the procedures for the operation and the specific nucleotide sequences of the primers used can be easily determined by those skilled in the art.

[MALDI-TOF / MS method]
Genotype determination can also be performed by analyzing PCR products with MALDI-TOF / MS. MALDI-TOF / MS can be used in various fields as an analytical method that can clearly identify slight differences in the amino acid sequence of proteins and DNA base sequences because it can know the molecular weight very accurately. Yes. In order to determine a genotype by MALDI-TOF / MS, first, a region containing a polymorphic site to be analyzed is amplified by PCR. The amplification product is then isolated and its molecular weight is measured by MALDI-TOF / MS. Since the base sequence including the polymorphic site is known in advance, the base sequence of the amplification product is uniquely determined based on the molecular weight. In order to determine the genotype using MALDI-TOF / MS, a PCR product separation step is required. However, accurate genotyping can be expected without using labeled primers or labeled probes. It can also be applied to simultaneous detection of polymorphisms at multiple locations.

[SNP-specific labeling method using type IIs restriction enzyme]
A method capable of determining a genotype at higher speed using the PCR method has also been reported. For example, genotypes of polymorphic sites are determined using type IIs restriction enzymes. In this method, a primer having a recognition sequence of type IIs restriction enzyme is used for PCR. A general restriction enzyme (type II) used for gene recombination recognizes a specific base sequence and cleaves a specific site in the base sequence. In contrast, type IIs restriction enzymes recognize specific base sequences and cleave sites away from the recognized base sequences. The number of bases between the recognition sequence and the cleavage site is determined by the enzyme. Therefore, if the primer containing the recognition sequence of the type IIs restriction enzyme is annealed at a position separated by the number of bases, the amplified product can be cleaved at the polymorphic site by the type IIs restriction enzyme. A sticky end containing the SNP base is formed at the end of the amplified product cleaved with the type IIs restriction enzyme. Here, an adapter having a base sequence corresponding to the sticky end of the amplification product is ligated. The adapter has different base sequences including bases corresponding to polymorphic mutations, and can be labeled with different fluorescent dyes. Finally, the amplification product is labeled with a fluorescent dye corresponding to the base at the polymorphic site.

  When PCR is carried out by combining a primer containing the above-mentioned type IIs restriction enzyme recognition sequence with a capture primer, the amplification product is fluorescently labeled and can be immobilized using the capture primer. For example, if a biotin-labeled primer is used as a capture primer, the amplification product can be captured on avidin-bound beads. By tracking the fluorescent dye of the amplification product thus captured, the genotype can be determined.

[Method of determining genotype at polymorphic site using magnetic fluorescent beads]
A technique capable of analyzing a plurality of polymorphic sites in parallel in a single reaction system is also known. Analyzing a plurality of polymorphic sites in parallel is called multiplexing. In general, a typing method using a fluorescent signal requires fluorescent components having different fluorescent wavelengths for multiplexing. However, there are not so many fluorescent components that can be used for actual analysis. On the other hand, when a plurality of types of fluorescent components are mixed in a resin or the like, a variety of fluorescent signals that can be distinguished from each other can be obtained even with limited types of fluorescent components. Furthermore, if a component that is magnetically adsorbed in the resin is added, it is possible to obtain beads that emit fluorescence and can be separated magnetically. Multiplex polymorphic typing using such magnetic fluorescent beads has been devised (Bioscience and Bioindustry, Vol. 60 No. 12, 821-824).

  In multiplexed polymorphic typing using magnetic fluorescent beads, a probe having a base complementary to each polymorphic site at its end is immobilized on the magnetic fluorescent beads. Both are combined so that each polymorphic site corresponds to a magnetic fluorescent bead having a unique fluorescent signal. On the other hand, when the probe fixed to the magnetic fluorescent bead is hybridized to a complementary sequence, a fluorescently labeled oligo DNA having a base sequence complementary to the region adjacent to the polymorphic site is prepared.

  A region containing the polymorphic site is amplified by asymmetric PCR, the above-mentioned magnetic fluorescent bead-immobilized probe and fluorescently labeled oligo DNA are hybridized, and both are further ligated. When the end of the magnetic fluorescent bead-immobilized probe has a base sequence complementary to the base of the polymorphic site, ligation is efficiently performed. On the other hand, if the terminal bases are different due to polymorphism, the ligation efficiency of the two decreases. As a result, the fluorescently labeled oligo DNA is bound to each magnetic fluorescent bead only when the sample has a genotype complementary to the magnetic fluorescent bead.

  The genotype is determined by collecting magnetic fluorescent beads by magnetism and detecting the presence of fluorescently labeled oligo DNA on each magnetic fluorescent bead. Since magnetic fluorescent beads can analyze the fluorescence signal for each bead using a flow cytometer, the signal can be easily separated even if various types of magnetic fluorescent beads are mixed. That is, “multiplexing” in which multiple types of polymorphic sites are analyzed in parallel in a single reaction vessel is achieved.

[Invader method]
As a method for genotyping that does not depend on the PCR method, for example, the Invader method has been put into practical use (SNP gene polymorphism strategy, Kenichi Matsubara, Yoshiyuki Tsuji, Nakayama Shoten, p94-105, Genome Research (2000) 10 : 330-343). In the Invader method, genotype determination is realized using only three types of oligonucleotides, an allele probe, an invader probe, and a FRET probe, and a special nuclease called cleavase. Of these probes, only the FRET probe requires labeling.

  The allele probe is designed to hybridize to a region adjacent to the polymorphic site to be detected. On the 5 'side of the allele probe, a flap consisting of a base sequence unrelated to hybridization is linked. The allele probe has a structure that hybridizes on the 3 'side of the polymorphic site and is linked to a flap on the polymorphic site. On the other hand, the invader probe has a base sequence that hybridizes to the 5 'side of the polymorphic site. The base sequence of the invader probe is designed so that the 3 'end corresponds to the polymorphic site by hybridization. The base at the position corresponding to the polymorphic site in the invader probe may be arbitrary. That is, both base sequences are designed so that the invader probe and the allele probe hybridize adjacently across the polymorphic site.

  If the polymorphic site is a base complementary to the base sequence of the allele probe, when both the invader probe and the allele probe hybridize to the polymorphic site, the invader probe is added to the base corresponding to the polymorphic site of the allele probe. A structure is formed in which an invasion occurs. The cleavase cleaves the invading strand of the oligonucleotide that has formed the invading structure thus formed. Since the cutting occurs on the intrusion structure, the result is that the allele probe flap is cut off. On the other hand, if the base at the polymorphic site is not complementary to the base of the allele probe, there is no competition between the invader probe and the allele probe at the polymorphic site, and no invasion structure is formed. Therefore, the flap is not cut by cleavase.

  The FRET probe is a probe for detecting the flap thus separated. The FRET probe constitutes a hairpin loop having a self-complementary sequence on the 5 'end side and a single-stranded portion arranged on the 3' end side. The single-stranded portion arranged on the 3 'end side of the FRET probe has a base sequence complementary to the flap, and the flap can hybridize there. Both base sequences are designed so that when the flap hybridizes to the FRET probe, a structure is formed in which the 3 'end of the flap enters the 5' end of the self-complementary sequence of the FRET probe. A cleavase recognizes and cleaves an invasion structure. If the FRET probe is cleaved by a cleavase and labeled with a reporter dye and quencher similar to TaqMan PCR, the FRET probe cleavage can be detected as a change in fluorescence signal.

  Theoretically, the flap should hybridize to the FRET probe even in the uncut state. However, in reality, there is a large difference in the binding efficiency to FRET between the cut flap and the flap present in the state of the allele probe. Therefore, the cut flap can be specifically detected using the FRET probe. In order to determine the genotype based on the Invader method, two types of allele probes including a base sequence complementary to each of the polymorphic site A and the polymorphic site B may be prepared. At this time, the base sequences of the flaps are different base sequences. If two types of FRET probes for detecting flaps are prepared and each reporter dye is distinguishable, the genotype can be determined based on the same concept as the TacMan PCR method.

  The advantage of the Invader method is that the FRET probe is the only oligonucleotide that needs to be labeled. The FRET probe can use the same oligonucleotide regardless of the base sequence to be detected. Therefore, mass production is possible. On the other hand, since the allele probe and the invader probe do not need to be labeled, a genotyping reagent can be manufactured at low cost.

[RCA method]
An example of a method for determining a genotype independent of the PCR method is the RCA method (Lizardri PM et al., Nature Genetics 19, 225, 1998). The RCA (Rolling Circle Amplification) method is a DNA amplification method based on a reaction in which a DNA polymerase having a strand displacement action synthesizes a long complementary strand using a circular single-stranded DNA as a template. In the RCA method, an amplification reaction is configured using a primer that anneals to a circular DNA to initiate complementary strand synthesis and a second primer that anneals to a long complementary strand generated by this primer.

  In the RCA method, a DNA polymerase having a strand displacement action is used. For this reason, the portion that has become a double strand by complementary strand synthesis is replaced by a complementary strand synthesis reaction started from another primer annealed to the 5 'side. For this reason, the complementary strand synthesis reaction using circular DNA as a template does not end in one round. Complementary strand synthesis continues while replacing the previously synthesized complementary strand, and a long single-stranded DNA is produced. On the other hand, the second primer anneals to the long single-stranded DNA generated using the circular DNA as a template, and complementary strand synthesis starts. Since single-stranded DNA generated by the RCA method uses circular DNA as a template, the base sequence is a repetition of the same base sequence. Thus, continuous production of long single strands results in continuous annealing of the second primer. As a result, single-stranded portions that can be annealed by the primer are continuously generated without going through a denaturing step. Thus, DNA amplification is achieved.

  If circular single-stranded DNA necessary for the RCA method is generated according to the genotype of the polymorphic site, the genotype can be determined using the RCA method. For this purpose, a linear single-stranded padlock probe is used. The padlock probe has complementary base sequences on both sides of the polymorphic site to be detected at the 5 'end and 3' end. These base sequences are linked at a portion consisting of a special base sequence called a backbone. If the polymorphic site is a base sequence complementary to the end of the padlock probe, the end of the padlock probe hybridized to the polymorphic site can be ligated by DNA ligase. As a result, the linear padlock probe is circularized and the reaction of the RCA method is started. The reaction of the DNA ligase significantly decreases the reaction efficiency when the terminal portion to be ligated is not completely complementary. Therefore, the genotype of the polymorphic site can be determined by confirming the presence or absence of ligation by the RCA method.

  The RCA method can amplify DNA but does not generate a signal as it is. Moreover, if only the presence or absence of amplification is used as an index, the genotype cannot usually be determined unless a reaction is performed for each polymorphism. An improved method for genotyping these points is known. For example, genotypes can be determined in one tube based on the RCA method using molecular beacons. A molecular beacon is a signal generation probe using a fluorescent dye and a quencher, as in the TaqMan method. The 5 'end and 3' end of the molecular beacon are composed of complementary base sequences, and form a hairpin structure alone. If both ends are labeled with a fluorescent dye and a quencher, a fluorescent signal cannot be detected in a state where a hairpin structure is formed. If a part of the molecular beacon is set as a base sequence complementary to the amplification product of the RCA method, the molecular beacon hybridizes to the amplification product of the RCA method. Since the hairpin structure is eliminated by hybridization, a fluorescent signal is generated.

  The advantage of the molecular beacon is that the base sequence of the molecular beacon can be made common regardless of the detection target by using the base sequence of the backbone portion of the padlock probe. The genotype can be determined in one tube by changing the backbone base sequence for each polymorphism and combining two types of molecular beacons with different fluorescence wavelengths. Since the cost of synthesis of fluorescently labeled probes is high, the ability to use a common probe regardless of the measurement target is an economic advantage.

[RFLP, PCR-RFLP]
Genotyping methods that do not use labeled probes have also been performed for a long time. Examples of such a method include a method using restriction fragment length polymorphism (RFLP) and a PCR-RFLP method. RFLP utilizes the fact that a restriction enzyme recognition site mutation or a base insertion or deletion in a DNA fragment caused by restriction enzyme treatment can be detected as a change in the size of the fragment produced after the restriction enzyme treatment. If there is a restriction enzyme that recognizes the base sequence containing the polymorphism to be detected, the base of the polymorphic site can be known by the principle of RFLP.

[PCR-SSCP]
As a method that does not require a labeled probe, a method of detecting a difference in base using a change in secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its nucleotide sequence (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). The PCR-SSCP method is performed by dissociating PCR products into single-stranded DNA and separating them on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.

[Modifier concentration gradient gel method]
Furthermore, examples of methods that do not require a labeled probe include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated depending on the instability of each. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of a partially dissociated DNA fragment becomes very slow and is different from the mobility of a complete double-stranded DNA without a dissociated part, so that both can be separated.

  Specifically, first, a region containing a polymorphic site is amplified by a PCR method or the like. The amplification product is hybridized with a probe DNA whose base sequence is known to make a double strand. This is electrophoresed in a polyacrylamide gel that gradually increases as the concentration of denaturing agents such as urea moves, and is compared with a control. In a DNA fragment in which a mismatch has occurred due to hybridization with the probe DNA, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the moving speed becomes extremely slow. The presence or absence of mismatch can be detected by detecting the difference in mobility generated in this way.

[DNA array method]
In addition, genotypes can also be determined using DNA arrays (cell engineering separate volume "DNA microarray and the latest PCR method", Shujunsha, published 2000.4 / 20, pp97-103 "Analysis of SNPs using oligo DNA chips", Shinichi Kanie). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously.

  In general, a DNA array is composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface layer of the substrate is generally glass, but a porous membrane such as a nitrocellulose membrane can also be used.

  In the present invention, examples of the nucleotide immobilization (array) method include an oligonucleotide-based array developed by Affymetrix. In an oligonucleotide array, oligonucleotides are usually synthesized in vitro. For example, an in-situ synthesis method of an oligonucleotide by a photolithography technique (Affymetrix) and an ink jet (Rosetta Inpharmatics) technique for immobilizing chemical substances is already known.

  The oligonucleotide is composed of a base sequence complementary to a region containing the SNP to be detected. The length of the nucleotide probe to be bound to the substrate is usually 10 to 100 bases, preferably 10 to 50 bases, more preferably 15 to 25 bases when the oligonucleotide is immobilized. Furthermore, in general, mismatch (MM) probes are used in the DNA array method in order to avoid errors due to cross hybridization (non-specific hybridization). The mismatch probe constitutes a pair with an oligonucleotide having a base sequence completely complementary to the target base sequence. An oligonucleotide consisting of a base sequence that is completely complementary to a mismatch probe is called a perfect match (PM) probe. In the process of data analysis, the influence of cross-hybridization can be reduced by eliminating the signal observed with the mismatch probe.

  A sample for genotyping by the DNA array method can be prepared by a method well known to those skilled in the art based on a biological sample collected from a subject. The biological sample is not particularly limited. For example, a DNA sample can be prepared from chromosomal DNA extracted from a subject's blood, tissues or cells such as peripheral blood leukocytes, skin, oral mucosa, tears, saliva, urine, feces, or hair. A specific region of chromosomal DNA is amplified using a primer for amplifying a region containing a polymorphic site to be determined. At this time, a plurality of regions can be simultaneously amplified by the multiplex PCR method. The multiplex PCR method is a PCR method using a plurality of primer sets in the same reaction solution. When analyzing a plurality of polymorphic sites, the multiplex PCR method is useful.

  In general, in the DNA array method, a DNA sample is amplified by the PCR method and the amplification product is labeled. A labeled primer is used for labeling the amplification product. For example, first, genomic DNA is amplified by a PCR method using a primer set specific to a region containing a polymorphic site. Next, DNA labeled with biotin is synthesized by a labeling PCR method using a primer labeled with biotin. The biotin-labeled DNA synthesized in this way is hybridized to the oligonucleotide probe on the chip. The hybridization reaction solution and reaction conditions can be appropriately adjusted according to conditions such as the length of the nucleotide probe immobilized on the substrate and the reaction temperature. One skilled in the art can design appropriate hybridization conditions. In order to detect the hybridized DNA, avidin labeled with a fluorescent dye is added. The array is analyzed with a scanner, and the presence or absence of hybridization is confirmed using fluorescence as an index.

[ASO hybridization method]
In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allele-specific oligonucleotide is composed of a base sequence that hybridizes to a region containing a polymorphic site to be detected. When ASO is hybridized to sample DNA, if a mismatch occurs in the polymorphic site due to polymorphism, the efficiency of hybridization is reduced. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

  Further, according to another aspect of the present invention, there is provided an oligonucleotide capable of hybridizing to a transcriptional regulatory region of SCN5A and / or a region containing a polymorphic site in the transcriptional region, or a region complementary thereto, wherein the polymorphic site Is provided at least one selected from the group consisting of polymorphic sites at positions −1062, −847 and 287, and polymorphic sites between −835 and −836.

  The oligonucleotide according to the present invention specifically hybridizes to a region containing at least one of the polymorphic sites in the transcriptional regulatory region and / or transcription region of SCN5A, or a region complementary thereto. Here, “specifically hybridizes” is preferably performed under stringent hybridization conditions (for example, as described in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition 1989). In (Condition), it means that cross-hybridization with a base sequence different from the target region does not occur significantly.

  Specifically, stringent hybridization conditions are usually about “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent conditions are “0.5 × SSC, 0.1% SDS, 42”. As a more severe condition, a condition of “0.2 × SSC, 0.1% SDS, 65 ° C.” can be exemplified. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will recognize the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.

  Thus, if specific hybridization is possible, the oligonucleotide need not be completely complementary to the transcriptional regulatory region of SCN5A and / or the nucleotide sequence of the transcriptional region. In other words, the oligonucleotide according to the present invention comprises (a) the base sequence (haplotype A) described in SEQ ID NO: 1, and (b) the base sequence described in SEQ ID NO: 1, T-1418C, T-1062C, T- Those having a mutation of 847G, -835insGC, G-354C and C287T (haplotype B), or (c) Those having a mutation of T-1418C and G-354C in the nucleotide sequence described in SEQ ID NO: 1 (haplotype C) Of these, a region containing at least one of the polymorphic sites at positions -1418, -1062, -847, -354 and 287 and the polymorphic sites between positions -835 and -836, or complementary thereto It may have a typical base sequence. In addition, the oligonucleotide according to the present invention can have a base sequence in which one or several nucleotides are deleted, substituted or inserted in the base sequence.

  The oligonucleotide according to the present invention can be used as a probe or primer in the above inspection method. When the oligonucleotide is used as a primer or a probe, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of DNA containing the polymorphic site in the transcriptional regulatory region and / or transcriptional region of SCN5A.

  According to another aspect of the present invention, the genotype of the transcriptional regulatory region and / or transcription region of SCN5A is defined as the types of bases of the polymorphic sites at −1062, −847 and 287, and −835 and A reagent for determining at least one selected from the group consisting of the presence or absence of insertion of a base at the polymorphic site between positions −836 is provided. The reagent can be used to examine a subject's genetic predisposition to diseases associated with ventricular conduction disturbances, ventricular fibrillation or sodium channel abnormalities or susceptibility to antiarrhythmic drugs or sodium channel blockers.

  The reagent can include a primer for amplifying a region containing at least one of the polymorphic sites in the transcriptional regulatory region and / or transcriptional region of SCN5A. The reagent may be a primer set contained in a separate container or a single container for amplifying a region including at least one of the polymorphic sites. In addition, the reagent can include a probe capable of hybridizing to a region containing at least one of the polymorphic sites in the transcription regulatory region and / or transcription region of SCN5A or a region complementary thereto.

  That is, when a primer is used as a reagent for determining the transcriptional regulatory region of SCN5A and / or the genotype of the transcription region, the primer is complementary to the polymorphic site using DNA containing the polymorphic site as a template. Also included are primers capable of initiating strand synthesis. The primer can also be expressed as a primer for providing a replication origin on the 3 'side of the polymorphic site in DNA containing the polymorphic site. The interval between the region where the primer hybridizes and the polymorphic site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the polymorphic site. For example, a primer can be designed so that an amplification product having a length of 20 to 500, usually 50 to 200 bases can be obtained as a region containing a polymorphic site. Based on the base sequence (SEQ ID NO: 1) for the peripheral DNA region containing the polymorphic site, a primer corresponding to the analysis method can be designed. The base sequence constituting the primer can be appropriately modified as well as a base sequence completely complementary to the genomic base sequence.

  In addition to a base sequence complementary to the genomic base sequence, an arbitrary base sequence can be added to the primer. For example, in a primer for a polymorphism analysis method using a type II or type IIs restriction enzyme, a primer to which a type II or type IIs restriction enzyme recognition sequence is added is used. Such a primer having a modified base sequence is included in the primer. Furthermore, the primer can be modified. For example, a fluorescent substance or a primer labeled with a binding affinity substance such as biotin or digoxin is used in various genotyping methods. Primers having these modifications are also included in the present invention.

  On the other hand, when a probe is used as a reagent for determining the transcriptional regulatory region of SCN5A and / or the genotype of the transcriptional region, the above-mentioned oligonucleotide, that is, a polymorphism in the transcriptional regulatory region and / or transcriptional region of SCN5A is used as the probe. An oligonucleotide capable of hybridizing to a region containing a region or a region complementary thereto, wherein the polymorphic sites are polymorphic sites at positions −1062, −847 and 287, and −835 and −836 An oligonucleotide that is at least one selected from the group consisting of polymorphic sites between positions can be used. Alternatively, depending on the genotype determination method, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, a probe containing a base sequence complementary to a region adjacent to the polymorphic site can be shown as a desirable probe reagent in the present invention, although the base sequence of the probe itself does not contain a polymorphic site.

  In other words, a probe that can hybridize to a polymorphic site on genomic DNA or a site adjacent to the polymorphic site is preferable as a probe reagent according to the present invention. The probe is allowed to modify the base sequence, add the base sequence, or modify the same as the primer. For example, a probe used for the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe according to the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe can be designed according to the analysis method based on the base sequence of the surrounding DNA region of the polymorphic site.

  The oligonucleotide, primer, or probe according to the present invention can be synthesized by any method based on the base sequence constituting the oligonucleotide. The length of the base sequence complementary to the genomic DNA of the primer or probe is usually 15 to 100, generally 15 to 50, usually 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  According to another aspect of the present invention, there is provided a kit for determining the genotype of at least one polymorphic site in the transcriptional regulatory region and / or transcriptional region of SCN5A, which contains the above reagent.

  In the kit according to the present invention, various enzymes, enzyme substrates, buffers and the like can be combined according to the genotype determination method. Examples of the enzyme include enzymes necessary for various analysis methods exemplified as the above-described genotyping method, such as DNA polymerase, DNA ligase, or IIs restriction enzyme. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

  Furthermore, the kit according to the present invention can be accompanied by a control in which the base at the polymorphic site is clear. As a control, a genome or a genomic fragment whose polymorphic site genotype is known in advance can be used. The genome may be extracted from cells, or cells or cell fractions may be used. If cells are used as a control, the result of the control can prove that the genomic DNA extraction operation was performed correctly. Alternatively, DNA consisting of a base sequence containing a polymorphic site can be used as a control. Specifically, YAC vectors and BAC vectors containing genomic DNA whose genotype at the polymorphic site has been clarified are useful as controls. Alternatively, a vector in which only several hundred bases corresponding to the polymorphic site are cut out and inserted can be used as a control.

  Furthermore, according to another aspect of the present invention, there is provided a method of screening for a drug that adjusts the transcription amount of SCN5A, particularly a drug that increases the transcription amount of SCN5A. Moreover, according to the other aspect of this invention, the medicine which adjusts the transcription amount of SCN5A obtained by the said screening method is provided. A substance that is positive in the screening method according to the present invention is a candidate substance for a drug that adjusts the transcription amount of SCN5A. In particular, according to the screening method of the present invention, not only a drug that adjusts the transcription amount of SCN5A in a patient having haplotype B, but also a candidate substance that is universally effective as a drug that adjusts the transcription amount of SCN5A in any patient Is obtained.

Specifically, the screening method according to the present invention includes:
Providing a test substance;
Providing a transcriptional regulatory region of SCN5A and / or a plurality of groups of animals having a polynucleotide having the transcriptional region, wherein the transcriptional regulatory region and / or transcriptional region of SCN5A is a transcription amount and / or an expression amount. Is operably linked to a measurable gene, and the transcriptional regulatory region and / or transcription region of SCN5A is a genotype in which the base of the polymorphic site at position -1418 is C, and the polymorphic site at position -1062 The genotype in which the base of C is G, the genotype in which the base of the polymorphic site at position −847 is G, the genotype in which the base of the polymorphic site at position −354 is C, and the base of the polymorphic site at position 287 is And having at least one selected from the group consisting of a genotype that is T and a genotype that has a GC insertion into the polymorphic site between positions −835 and −836;
Contacting the test substance with at least one group of cells or animals;
Measuring the transcription amount and / or the expression level of the gene in a group that has been contacted with the test substance and a group that has not been contacted;
Comparing the amount of transcription and / or the amount of expression of a group not contacted with the test substance.

  Provide a single compound or composition, natural or synthetic compound, organic or inorganic compound, protein, peptide, oligonucleotide, polynucleotide, cell extract, cell culture supernatant, or any other substance as a test substance Can do.

  Screening can be performed both in vitro and in vivo. When performed in vitro, examples of cells include cells derived from humans, monkeys, mice, rats, cows, pigs, dogs and the like. In particular, cardiomyocytes of newborn mice and non-cardiac cells such as Chinese hamster ovary cells can be used. When performed in vivo, examples of animals include monkeys, mice, rats, cows, pigs, dogs and the like.

  The cell or animal used for screening has a polynucleotide. The polynucleotide may be endogenous or exogenous. If exogenous, the polynucleotide may be introduced into the cell or animal by any means. The polynucleotide has a transcriptional regulatory region and / or a transcriptional region of SCN5A, and a gene whose transcription amount and / or expression amount can be measured operably linked thereto.

  The gene that can be measured may be any gene as long as its transcription amount and / or expression level can be measured. As will be apparent to those skilled in the art, the measurable gene can be appropriately selected according to the method for measuring the transcription level and / or the expression level. Examples of the gene that can be measured include genes such as proteins having a predetermined activity such as SCN5A and luciferase, proteins that can bind to known antibodies, and proteins that can specifically bind to known substances such as GST. .

  Further, the transcriptional regulatory region and / or transcription region of SCN5A used for screening are a genotype in which the base of the polymorphic site at position -1418 is C, a genotype in which the base of the polymorphic site at position -1062 is C, A genotype in which the base of the polymorphic site at position 847 is G, a genotype in which the base at the polymorphic site in position -354 is C, a genotype in which the base of the polymorphic site at position 287 is T, and the -835 position And at least one selected from the group consisting of genotypes with GC insertion at the polymorphic site between positions −836. That is, the transcriptional regulatory region and / or transcriptional region of SCN5A used for screening is preferably haplotype B.

  In the present invention, a plurality of groups of cells or animals are provided, and a test substance is brought into contact with only some of the groups. As will be apparent to those skilled in the art, the test substance can be brought into contact with cells or animals by any method. For example, when the cell is a cultured cell, it can be contacted by adding a test substance to the medium. The animal can be contacted by administration by any means. Specifically, the contact can be made by injection from any route.

  By measuring the transcription amount and / or expression level of the gene in a group that has been brought into contact with the test substance and a group that has not been brought into contact, the test substance is compared by comparing the transcription amount and / or the expression level of the gene. Can be determined as a drug candidate substance that adjusts the transcription amount of SCN5A. Specifically, when the amount of transcription and / or expression of the gene is changed in the group in which a certain test substance is contacted, compared to the group in which the test substance is not contacted, Can be a candidate substance.

  A step of measuring the transcription amount and / or expression level of the gene in a group not contacted with the test substance contact group, and a transcription amount of the group not contacted with the test substance contact group And the step of comparing the expression levels can be performed by any method, as will be apparent to those skilled in the art. Specifically, the transcription amount of the gene can be measured by Northern blotting method, DNA array method or the like. In addition, the expression level of the gene can be measured by a luciferase assay method, a CAT method, or the like. Furthermore, when an antibody against the predetermined gene is available, the expression level of the gene can be measured by ELISA, immunoprecipitation, or western blotting. In addition, when a gene of a protein that specifically binds to a predetermined substance such as GST is used as the predetermined gene, the expression level of the gene is determined by precipitating the protein and measuring the amount of the precipitated protein. Can be measured.

[Northern blotting method]
In the Northern blotting method, denatured RNA is separated by agarose electrophoresis under denaturing conditions, transferred to a nitrocellulose filter, and detected with a labeled specific probe. RNA is denatured because RNA has a secondary structure in the molecule, and as such, cannot be accurately separated according to size, and the filter can bind only single-stranded nucleic acids.

[Luciferase assay method, CAT method]
The luciferase assay method and the CAT method are prepared by preparing a plasmid incorporating the LUC (luciferase) or CAT (chloramphenicol acetyltransferase) gene as a reporter downstream of the transcriptional regulatory region of the target gene, and introducing the plasmid into the cell. The enzyme activity is measured. Specifically, in the LUC method, light having a wavelength of 560 nm, which is emitted when luciferase catalyzes a reaction for producing luciferin and AMP from luciferin and ATP in the presence of magnesium, is detected using a luminometer. In the case of the CAT method, the substrate [ ¹⁴ C] chloramphenicol is acetylated by CAT to produce [ ¹⁴ C] acetylchloramphenicol. This sample is extracted with ethyl acetate, developed on a thin layer silica gel plate, and then the radioactivity of spots having different mobilities is measured.

[Target]
The normal control group is a family member who does not have a causative gene mutation in a family with congenital long QT syndrome (LQTS) whose gene mutation has been identified at the National Cardiovascular Center. 102 cases (67 males and 35 females, 9-69 years old, average age 40 ± 14 years old). Only one example was selected from each LQTS family.

  The patient group consists of 80 patients (76 men and 4 women, 1-76 years old, mean age 47 ± 16 years) diagnosed with Brugada syndrome (BS) at the National Cardiovascular Center. In 30 cases, aborted cardiac arrest or ventricular fibrillation was reported, and in 20 cases only fainting experiences were reported. In 30 cases, it was asymptomatic. All patients were subjected to physical examination, chest radiographs, laboratory values and echocardiography with analysis of wall motion and Doppler screening, but no organic heart disease was found . The patient further met the following criteria: 1) typical right type chest lead V1-V3, type 1 coved ST elevation, and 2) heart rate corrected QT time <440 msec. Coved type ST elevation was reported spontaneously in 70 cases and by sodium channel blockers in 10 cases. In 49 of the 80 cases, a response to intravenous administration of a sodium channel blocker (pircicainide, flecainide or disopyramide) was observed. In 9 of the 80 cases, mutations were identified in the region encoding SCN5A.

[Identification of haplotype]
Fragment of 2.8 kbp in total including 2.2 kb upstream of exon 1 of the SCN5A promoter region, exon 1 (173 bp, untranslated region) and 439 bp on the 5 ′ side of intron 1 (from −2190 to +613 based on the main transcription start point) The DNA sequence was determined by PCR. This identified haplotypes with six fully linked polymorphisms in Asians. For PCR, an LA PCR kit (TaKaRa) and the following primers were used.
Sequence number 4: 5'-TAG GAA GTG CCT GTC TCC AGA CAC CTG TTG-3 '(F)
Sequence number 5: 5'-CGC TCT CTG GAA CCA CAT TCA TGG CG-3 '(R)

[Genotyping]
Genomic DNA was prepared from blood leukocytes of Japanese patients clinically diagnosed with Brugada syndrome and normal Japanese control patients. Genotyping was performed on the transcriptional regulatory region and transcriptional region of the SCN5A gene in the National Laboratory for Cardiovascular Disease. For determining the genotype, the direct sequencing method or the PCR-RFLP method was used. All protocols, including molecular screening, were reviewed and approved by the National Cardiovascular Center's Ethics Review Board, and informed consent was obtained from all subjects.

  Genotyping of single nucleotide polymorphisms (SNPs) of T-1418C and T-1062C, as previously described (Non-Patent Document 8, Bezzina et al., 2003b), And performed by primer-induced restriction enzyme digestion.

The following PCR primers were used to amplify a 161 bp fragment containing the T-1418C SNP.
Sequence number 6: 5'-CCCTGATGGCCTGTTTTGTTTA-3 '(sense)
Sequence number 7: 5'-ACTCAGAGACATGGTCACAGGCA-3 '(antisense)

In order to amplify a 123 bp fragment containing the S-1062C SNP, the following PCR primers were used.
Sequence number 8: 5'-ACCTAAGGCGTCCAACGAAGC-3 '(sense)
Sequence number 9: 5'-CCAGGGTCTCAGAGGGCACAG-3 '(antisense)

  Genotyping of the other four polymorphisms (T-847G, -835insGC, G-354C and C287T) was performed by DNA sequencing of both strands by PCR.

The following PCR primers were used to amplify a 638 bp fragment containing the T-847G, -835insGC and G-354C polymorphisms.
Sequence number 10: 5'-AGGCTCTGCATGTGTCAAG-3 '(sense)
Sequence number 11: 5'-GACGCGGACAGGCTCACA-3 '(antisense)

In order to amplify a 599 bp fragment containing the C287T polymorphism, the following PCR primers were used.
Sequence number 12: 5'-GTAGGATGCAGGGATCGCT-3 '(sense)
SEQ ID NO: 13: 5'-CGCTCTCTGGAACCACATTC-3 '(antisense)

  Table 1 shows the number of genotypes and the frequency of minor alleles observed in control and Brugada syndrome patients. As shown in Table 1 and FIG. 1, by screening the promoter region of SCN5A in Asian subjects, 6 nucleotide mutations in this region (T-1418C, T-1062C, T-847G, -835insGC, G -354C and C287T) were revealed. In the two Japanese groups (n = 182), the frequency of genotypes with haplotype B (AB genotype + BB genotype) is 21-24% (24% in 102 control groups, 21% in 80 BS patients) there were. Further, these polymorphisms were not found in Dutch Caucasians or African Americans (each n ≧ 100), and were found to be polymorphisms peculiar to Asians including Japanese. In the tested Japanese (BS patients and controls), the number of all six polymorphic genotypes was in Hardy-Weinberg equilibrium (P> 0.05). No significant difference was found in the number of genotypes between the BS patient group and the control group. These six polymorphisms were in almost complete linkage disequilibrium. In each of the control and BS patient groups, one patient was found each having an allele that did not match them (haplotype C) (364, <1%). Estimated frequencies of haplotype A, haplotype B and haplotype C were 0.755, 0.240 and 0.005 in the control, and 0.782, 0.211 and 0.007 in BS patients without SCN5A mutation, respectively. From these frequencies, heterozygous controls and BS patients are haplotype A carriers with all major alleles and haplotype B with all minor alleles.

[In vitro functional analysis]
To investigate the effect of this haplotype on gene expression, in vitro functional analysis using CHO cells and cardiomyocytes was performed.

(Plasmid)
Using as a template human genomic DNA from a subject who is haplotype A homozygous for all six polymorphic sites, and human genomic DNA from a subject who is haplotype B homozygous for all six polymorphic sites, The above 2.8 kb fragment was amplified. These fragments were cloned into the pGEM-T Easy vector.

  These plasmids were digested with Nco I and Sac I and the insert was subcloned into the pGL3-Basic vector (Promega). The vector has a sequence encoding firefly luciferase, and thus an SCN5A promoter-luciferase fusion construct is obtained. One of these is referred to as pGL-haplotype A, and the other as pGL-haplotype B.

(Transient transfection assay)
As previously described (Non-Patent Document 6, Yang P., 2004), transcription using luciferase activity in neonatal mouse cardiomyocytes and non-cardiac cells (Chinese Hamster Ovary (CHO) cells) Activity was measured. One day old B6D2 mice were sacrificed by dousing in ethanol and decapitation. Neonatal hearts were removed and placed in 1 × PBS solution. The ventricular segment was digested with Lipsin-Wersen (Biofluids, Inc.) and the cells were 15% (v / v) NuSerum (Beckton Dickinson), 2.5 mM in air containing 5% CO ₂ in a humidified manner. The cells were cultured at 37 ° C. in Dulbecco's modified Eagle medium (DMEM) supplemented with thymidine and penicillin-streptomycin (10 units / ml and 10 mg / ml, respectively). Cells were allowed to attach for 48 hours before use. CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured as previously described (9, Kupershmidt et al., 2002).

  SCN5A promoter-luciferase fusion construct (1 μg DNA) was transfected into neonatal mouse heart cells with Fugene 6 (Roche) and CHO cells with pofectamine (Invitrogen). In each experiment, the pRL-TK plasmid (0.05 μg) (Promega) encoding Renilla luciferase was also co-transfected to standardize experimental errors resulting from differences in cell viability and transfection efficiency. . Luminescence was measured 48 hours after transfection by dual luciferase reporter assay system (Promega). In each experiment, the pGL3-Basic (no promoter) plasmid was also tested and based on its activity level.

  As shown in FIG. 3, the transcriptional activity (activity ratio relative to the control) of the SCN5A gene measured using the luciferase activity is as follows: haplotype A: 14.5 ± 2.8 (mean ± SE) vs. haplotype B: 5.5 ± 0.4 in cardiomyocytes Each n = 9, P = 0.006), and in non-cardiomyocytes, haplotype A: 3.6 ± 0.3 vs. haplotype B: 2.7 ± 0.3 (each n = 13, P = 0.04). That is, the transcriptional activity of haplotype B was 62% lower in cardiomyocytes and 25% lower in non-cardiomyocytes than haplotype A. Thus, the transcriptional regulatory activity is significantly decreased in the haplotype B promoter sequence in both cardiomyocytes and non-cardiomyocytes, compared to haplotype A, and the gene expression of the sodium channel is caused by the haplotype B polymorphism. Was shown to decrease.

[Phenotype of BS patients and controls: ECG, electrophysiological parameters and sodium channel blockers]
When sampling blood for genotyping, a baseline 12-lead electrocardiogram (ECG) was recorded for all 102 control subjects and 80 BS patients. One of the inventors blinded to age, gender, genetic information and clinical information, RR time, PQ time in induction II (PQ (II)), QRS time in inductions V1 and V6 (QRS ( V1) and QRS (V6)) (indicative of heart rate, intra-atrial / intra-ventricular nodule / Hispurkinje conduction time, and intra-ventricular conduction, respectively), and ST amplitude at 80 ms after termination of point J and QRS. (ST amplitude) (ST (J) and ST (80)) was measured.

  A standard electrophysiologic study (EPS) was conducted on 47 BS patients. Atrio-His (AH) time and His-ventricle (HV) time were measured. Induction of ventricular fibrillation was evaluated using up to triple extrastimuli from the right ventricular apex (RV apex) and RV outflow tract.

  Forty-nine BS patients were tested for the effect of intravenous sodium channel blocker on ECG parameters. For 37 patients with BS, pilsicainide (maximum 1 mg / kg, rate 0.1 mg / kg / min) was used. For nine BS patients, flecainide (maximum 2 mg / kg, rate 0.2 mg / kg / min) was used. For three BS patients, disopyramide (maximum 2 mg / kg, rate 0.2 mg / kg / min) was used.

  Table 2 shows the clinical characteristics in patients with control and Brugada syndrome (without and with mutations in SCN5A). Control patients were significantly younger than BS patients without SCN5A mutations. Compared to patients without mutations in SCN5A, controls had a significantly higher proportion of women. Compared to controls, patients with Brugada syndrome had significantly longer conduction times (QRS (V1), QRS (V6), PQ (II)) and higher ST segment (ST (J), ST (80)) Had. The RR time (heart rate) was not significantly different between the two groups (925.29 ± 129.98 msec, 913.66 ± 134.27 msec).

  Furthermore, as in previous reports (Non-Patent Document 10, Smiths et al., 2002), BS patients with mutations in SCN5A have significantly longer baseline PQ times and HV than patients without mutations in SCN5A. And had a significantly longer QRS time after class I drug administration. In this example, baseline QRS and RR times were also significantly longer in patients with a mutation in SCN5A compared to patients without a mutation in SCN5A.

[Influence of haplotype pairs]
As described above, it has been observed that haplotype B has a greater negative effect on gene expression compared to haplotype A, so that subjects with haplotype B exhibit a conduction delay detectable by electrocardiogram. It was suggested. To investigate the effect of haplotypes on electrocardiographic parameters of conduction (ie, PQ and QRS times and RR and ST parts, which are indicators of intra-atrial / intra-ventricular nodal / Hispurkinje conduction and intraventricular conduction, respectively) We examined whether haplotypes affect ventricular conduction.

  Specifically, QRS time, PQ time, etc. of 12-lead ECG considered to be an index of excitatory conduction time were examined in 79 Brugada syndrome patients excluding haplotype C and 101 normal control patients. QRS time was measured with V1 and V6 induction, and PQ time was measured with II induction. Among patients with Brugada syndrome, there were 9 cases with mutations in the SCN5A translation region, 5 cases with BB genotype, 20 cases with AB genotype, and 45 cases with AA genotype, and 4 cases with BB genotype in normal control patients. Each index was compared between three groups of 33 cases of AB genotype and 60 cases of AA genotype. In 49 patients with Brugada syndrome, changes in each index before and after intravenous injection of sodium channel blocker were examined.

  Tables 3-5 show the effect of haplotype pairs on electrocardiogram and EPS phenotype in controls, BS patients without mutation in SCN5A and BS patients with mutation in SCN5A.

  As shown in Tables 3 and 4 and FIG. 4, haplotype pairs had a significant correlation with baseline QRS and PQ time in both BS and control groups. That is, in the Brugada syndrome patient, both the index of QRS time and PQ time, which are indicators of sodium channel-dependent ventricular conduction, in the order of SCN5A mutation example, BB genotype example, AB genotype example, AA genotype example, Even in normal control patients, the BB genotype example, the AB genotype example, and the AA genotype example are significantly extended in this order, and in all groups, the excitatory conduction time is significantly extended in patients with haplotype B. (Minor homo> hetero> major homo).

Furthermore, as shown in Tables 4, 5, 6 and FIG. 4, in the Brugada syndrome subset from which the parameters were obtained, haplotype pairs also had a high correlation with conduction time after Na ⁺ channel blockade. That is, in patients with Brugada syndrome, the increase in QRS time by sodium channel blocker intravenous injection increases in the order of BB genotype, AB genotype, AA genotype (P <0.03), and in patients with haplotype B, sodium It has been shown to show excessive prolongation of excitatory conduction time for channel blockers. For all electrocardiographic parameters investigated (RR, QRS (V1, V6), PQ (II), ST (J, 80)), the measured values before and after Na ⁺ channel blocker administration were highly significant (p This was also expected from having <0.0001). The Pearson correlation coefficient r was 0.94, 0.92, 0.92, 0.95, 0.84, and 0.63 for RR, QRS (V1, V6), PQ (II), and ST (J, 80), respectively.

  In both groups (control and BS patient groups) no significant correlation was found between the haplotype pair and baseline RR, ST (J) and ST (80). Also, no significant correlation was found between haplotype pairs and AA, AH, HV, induction and LP in a subset of BS patients studied (Tables 3 and 4).

Tables 3 and 4 show the variance (R ² ) that can be explained by haplotype pairs after adjusting for age and gender. It can be seen that the larger R ² (%), the more affected by the haplotype. As shown in Tables 3 and 4, haplotype pairs can explain significant changes in QRS and PQ before and after administration. That is, a significant proportion of variance in QRS and PQ time could be attributed to this haplotype (for QRS, R ² = 48% in control, 25% in BS group; for PQ, R ² = 12 in control) %, 28% in BS group).

  Table 6 shows the effect of the haplotype pair on the QRS and PQ prolongation (ΔQRS, ΔPQ) after drug administration. As shown in Table 6, in 49 cases where sodium channel blockers were intravenously administered, the QRS prolongation (ΔQRS) after drug administration was genotype dependent and was found to be large in patients with haplotype B. (ΔQRS: AA = 17.8 ± 7.2 ms [mean ± SD], AB = 24.2 ± 7.9 ms, BB = 30 ms; P ≦ 0.03 for AA vs AB and AA vs BB). Note that no significant correlation was found between the haplotype and the RR time.

〔Statistical analysis〕
Statistical analysis was performed as follows. Haplotype frequencies were calculated by counting genotypes. For each polymorphism, the deviation from Hardy-Weinberg equilibrium in the control and BS patients was tested individually with χ ² test with 1 degree of freedom. To compare the genotype frequencies of the six polymorphisms in controls and BS patients, the χ ² test or Fisher's exact test was used as needed. The haplotype frequency was evaluated by the EM algorithm (Non Patent Literature 11, Excoffier and Slatkin, 1995). Using the resulting haplotype frequencies, Bayes' theorem was used to calculate the likelihood of a haplotype pair that matched the genotype combination of multiple heterozygous patients. All quantitative phenotypes showed normal variance. These data are expressed as mean ± standard deviation (SD). By t-test, a quantitative phenotypic comparison was made between BS patients without mutations in SCN5A and BS patients or controls with mutations in SCN5A. Qualitative phenotypes were compared using the χ ² test or Fisher exact test as needed. The impact on quantitative phenotype of haplotype pairs (or genotype combinations) in individual groups was adjusted by age and gender and tested by analysis of variance (ANOVA). Nine patients with mutations in SCN5A and one non-matching patient (with haplotype C) in both groups without mutations in SCN5A were excluded from the analysis. In all analyses, the possible effects of age and gender were adjusted and the percentage of variance (R ² ) that could be attributed to the haplotype pair was calculated. Quantitative phenotypic correlations before and after Na ⁺ channel blockade were expressed as Pearson correlation coefficient (r) (BS patients only). Qualitative phenotypic differences between haplotype pair groups were tested by χ ² test or Fisher exact test as needed. Throughout, it was considered significant when P value <0.05. All statistical analyzes were performed with SAS software (version 9, SAS Institute, Cary, NC).

[Discussion]
As described above, the present inventors have found that a haplotype mutation in the promoter of SCN5A encoding a cardiac sodium channel and that the mutation reduces promoter activity in cardiomyocytes by 62% in vitro. Furthermore, this SCN5A haplotype is associated with up to 28% and 48% PQ and QRS times (intra-atrial / infrared), respectively, in a cohort of 71 Japanese Brugada syndrome subjects without SCN5A mutation and 102 Japanese controls. It is an electrocardiographic marker of atrioventricular intranodal / Hispurkinje conduction velocity and ventricular conduction velocity.

  From the above, a set of 6 polymorphisms within the SCN5A promoter, almost completely in linkage disequilibrium with each other, has a significant effect on the expression level of sodium channels and is large with respect to variations in ECG conduction parameters. It can be seen that it has an effect.

  Changes in conduction through the heart muscle play a major role in reentrant arrhythmias and are associated with sudden death in various animal models and humans. It is thought that the electrocardiogram parameter which is a ventricular conduction, that is, a biometric characteristic, is influenced by a general genetic change (polymorphism). Analyzing genetic determinants of ventricular conduction is important because it provides clues to the etiology of ventricular arrhythmias in common medical conditions where conduction disorders are known, such as ischemia and heart failure.

In the genetic electrical disorder Brugada syndrome, cardiac conduction deficiency (Lenegre disease), familial sinus dysfunction syndrome, atrial resting, familial atrioventricular block, a loss-of-function mutation in the SCN5A gene has occurred An important role of Na ⁺ channels in ventricular conduction is shown. These obstacles have remarkably diverse phenotypes, and common polymorphisms are thought to change the basic diversity.

For example, the relationship between ECG parameters and single nucleotide polymorphisms in the region encoding the ion channel gene that causes amino acid changes (Non-patent Document 12, Roden DM), and Na ⁺ channel, S1102Y polymorphism (generally for Africans) Have been reported to be associated with prolonged QT time and arrhythmia risk in the general population (Splawski et al., 2002). Other mutations that affect ECG parameters include β1 adrenergic receptor mutations (Ranade et al., 2002) for resting heartbeats and delayed rectifier K ⁺ current (KCNH2) for QT time. Mutations in the gene encoding the activating element have been reported (Bezzina et al., 2003). However, the concept that basic electrocardiographic characteristics can be changed by a common functional change in the transcriptional regulatory region has not yet been known.

As described above, haplotype B delayed conduction in normal subjects. Also, the further effect of this genotype in the BS group (the conduction delay in the reference is evident) is also evident. The main determinant of conduction in the ventricular muscle is Na ⁺ current, and severe conduction delay has been shown to be the final common pathway to VF (Non-Patent Document 13, Tomaselli, 2004). In addition, the frequency of haplotype B is relatively high in the Japanese group, haplotype B has a large negative effect on conduction, and the mutation of SCN5A shows the same qualitative phenotype as haplotype B. Various factors such as this suggest a relationship between this haplotype and the high prevalence of BS in Japan. Thus, haplotype B, which has a large effect on ventricular conduction, is considered to be an important factor that alters the susceptibility to arrhythmia, i.e., sudden cardiac death risk, and drug response.

  No mutation of the transcriptional regulatory region of SCN5A has been reported so far, and the present invention is a novel invention. In addition, functional studies have demonstrated that haplotype B reduces sodium channel expression and clinically has a prolonged excitatory conduction time in patients with haplotype B. Reduction of sodium current in the heart delays conduction, which makes it more susceptible to ventricular fibrillation (VF). By screening for these mutations, ventricular conduction disorders and ventricular fibrillation that cause sudden cardiac death in patients who are prone to develop Brugada syndrome, antiarrhythmic drugs with sodium channel inhibitory action, and acute myocardial ischemia It is possible to predict in advance patients who are likely to develop symptoms. Therefore, the haplotype according to the present invention is a pharmacology for predicting ventricular conduction disorder and ventricular fibrillation causing sudden cardiac death, predicting side effects on antiarrhythmic drugs, predicting ventricular fibrillation occurring during acute ischemia, etc. It becomes a genetic marker and realizes customized medicine.

The haplotype found in the cardiac sodium channel gene (SCN5A) promoter is shown. Nucleotide changes are indicated by the position relative to the major transcription start point (+1), as well as the most frequent base before the position and the polymorphic base after the position. * Frequency is in Japanese controls. The transcriptional regulatory region of SCN5A and haplotypes A to C in the transcription region are schematically shown (1-1440). The transcriptional regulatory region of SCN5A and haplotypes A to C in the transcription region are schematically shown (1441 to 2801). The underlined portion indicates exon 1. The double underlined G is the main transfer start point (+ 1st position). Shows reporter activity of two more common haplotypes. Data show mean ± 1 SE (relative to empty vector). FIG. 6 shows the effect of SCN5A promoter genotype on QRS (V6) and PQ (II) time in Brugada syndrome (BS) patients and non-BS controls at baseline and after sodium channel blocker administration. The number of patients is shown in parentheses. QRS (V1) and QRS (V6) have a high correlation (Pearson correlation coefficient r = 0.9), and the genotype effect on QRS (V1) is similar to the effect on QRS (V6).

Claims (13)

A method for examining a subject's genetic predisposition to a disease associated with impaired ventricular conduction, ventricular fibrillation or sodium channel abnormalities, comprising:
The transcriptional regulatory region and / or genotype of the transcriptional region of SCN5A possessed by the subject is defined as the types of bases of the polymorphic sites at positions -1418, -1062, -847, -354 and 287, and -835. And determining at least one selected from the group consisting of the presence or absence of insertion of a base into the polymorphic site between positions −836;
Predicting the predisposition based on the determined genotype. A method for testing a subject's sensitivity to antiarrhythmic drugs or sodium channel blockers, comprising:
The transcriptional regulatory region and / or genotype of the transcriptional region of SCN5A possessed by the subject is defined as the types of bases of the polymorphic sites at positions -1418, -1062, -847, -354 and 287, and -835. And determining at least one selected from the group consisting of the presence or absence of insertion of a base into the polymorphic site between positions −836;
Predicting said susceptibility based on the determined genotype.   In the determining step, the genotype is determined based on the types of bases of the polymorphic sites at positions −1062, −847 and 287, and whether or not a base is inserted into the polymorphic site between positions −835 and −836. The method according to claim 1, wherein the determination is made on at least one selected from the group consisting of: In the predicting step, the subject has the following genotype:
A genotype in which the base of the polymorphic site at position -1418 is C;
A genotype in which the base of the polymorphic site at position −1062 is C;
A genotype in which the base of the polymorphic site at position -847 is G;
A genotype in which the base of the polymorphic site at position −354 is C;
A homozygous or heterozygous allele having at least one of a genotype in which the base of the polymorphic site at position 287 is T, and a genotype having GC insertion into the polymorphic site between positions −835 and −836 The method according to any one of claims 1 to 3, wherein when the subject is a zygote, the subject is predicted to have the predisposition or the sensitivity is high.   An oligonucleotide capable of hybridizing to a region containing a polymorphic site in the transcriptional regulatory region and / or transcription region of SCN5A, or a region complementary thereto, wherein the polymorphic site is at positions −1062, −847 and 287. And / or an oligonucleotide that is at least one selected from the group consisting of polymorphic sites between positions −835 and −836.   The genotype of the transcriptional regulatory region and / or transcriptional region of SCN5A is determined based on the types of bases of the polymorphic sites at positions −1062, −847 and 287, and the bases to the polymorphic sites between −835 and −836. A reagent for determining at least one selected from the group consisting of the presence or absence of insertion.   The reagent according to claim 6, for examining the genetic predisposition of a disease associated with impaired ventricular conduction, ventricular fibrillation or sodium channel abnormality in a subject.   The reagent according to claim 6, for examining a subject's sensitivity to an antiarrhythmic drug or a sodium channel blocker.   The reagent according to any one of claims 6 to 8, comprising a primer for amplifying a region containing at least one of the polymorphic sites in the transcription regulatory region and / or transcription region of SCN5A.   9. The probe according to claim 6, comprising a probe capable of hybridizing to a region containing at least one of the polymorphic sites in the transcription regulatory region and / or transcription region of SCN5A, or a region complementary thereto. reagent.   A kit for determining the genotype of the transcriptional regulatory region and / or transcriptional region of SCN5A, comprising the reagent according to any one of claims 6 to 10. A method of screening for a drug that adjusts the transcription amount of SCN5A, comprising:
Providing a test substance;
Providing a transcriptional regulatory region of SCN5A and / or a plurality of groups of animals having a polynucleotide having the transcriptional region, wherein the transcriptional regulatory region and / or transcriptional region of SCN5A is a transcription amount and / or an expression amount. Is operably linked to a measurable gene, and the transcriptional regulatory region and / or transcription region of SCN5A is a genotype in which the base of the polymorphic site at position -1418 is C, and the polymorphic site at position -1062 The genotype in which the base of C is G, the genotype in which the base of the polymorphic site at position −847 is G, the genotype in which the base of the polymorphic site at position −354 is C, and the base of the polymorphic site at position 287 is And having at least one selected from the group consisting of a genotype that is T and a genotype that has a GC insertion into the polymorphic site between positions −835 and −836;
Contacting the test substance with at least one group of cells or animals;
Measuring the transcription amount and / or the expression level of the gene in a group that has been contacted with the test substance and a group that has not been contacted;
Comparing the amount of transcription and / or the amount of expression of the group not contacted with the group contacted with the test substance.   The medicine which adjusts the transcription | transfer amount of SCN5A obtained by the method of Claim 12.

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