JP4461263B2 - Method for obtaining data for enabling early diagnosis of Dravet syndrome and use thereof - Google Patents

Description

  The present invention relates to a method for acquiring data for determining the possibility of onset of Dravet syndrome and its use, and in particular, enables early diagnosis of Dravet syndrome applicable to infants under 1 year of age. The present invention relates to a method for acquiring data for use and its use.

  Febrile convulsions are a common disease that occurs in about 8% of children. Thermal convulsions are generalized convulsions that last for 1 to 5 minutes with fever of 38 ° C. or higher mainly due to viral infection such as the common cold or bacterial infection. It develops from 6 months to about 5 years of age, and the majority of cases are cured by the age of 6. For this reason, active treatment is often unnecessary, and as a rule, it is a benign disease. However, among patients with febrile convulsions that develop before the age of 1 year, in addition to patients with benign diseases that begin with febrile convulsions, those with persistent convulsions after 6 years of age, Dravet syndrome (also known as infantile severe myoclonic epilepsy (Severe Myoclonic Epilepsy in Infancy); also referred to as SMEI).

  Dravet syndrome convulsions occur before the age of 1 year, with an average age of onset of 4 to 6 months of age. In general, the first seizure is generalized or unilateral tonic clonic or clonic convulsions, and often causes a state of convulsions during infancy. In addition, seizures are easily induced by fever and bathing. After infancy, unilateral convulsions decrease and convulsion status is less likely to occur.

  Myoclonic seizures appear in addition to seizures from around 1 year old to around 4 years old. Atypical absence seizures also appear in about 40-90% of patients at the same time, often with fine myoclonies in the eyelids, neck, and upper limbs. Non-convulsive status epilepticus with atypical absence seizures appearing for several hours to several days, with asynchrony in various parts of the body, irregular myoclonies, and increased tonus of the extremities There is. Also, myoclonies without epilepsy on EEG are observed, which are mainly asynchronous irregular myoclonies in the distal extremities and facial muscles. Complex partial seizures appear at various times during the course.

  The development before the onset of Dravet syndrome is in principle normal. Walking may be slightly delayed, with significant words but rarely speaking. Gait instability may persist or progress. Also, it is often hyperactive during infancy.

  The electroencephalogram shows normal findings at the onset of Dravet syndrome. Slow waves became apparent from about 1 to 2 years of age, and diffuse θ activity was often observed despite the superiority of the central parietal region. Epilepsy launches will be detected until after the age of one. It is mainly composed of 2.5 to 4 c / s wide spikes and slow waves, but multifocal spikes are often observed. Moreover, photosensitivity is recognized by 42 to 65%, and figure hypersensitivity is recognized by 11 to 35%.

  In recent years, attempts have been made to diagnose Dravet syndrome at the gene level. For example, Patent Documents 1 and 2 disclose that a voltage-dependent sodium channel α-subunit type I gene (SCN1A) mutation in the nervous system is involved in SMEI. It is also disclosed that SMEI can be diagnosed using SCN1A mutation as an index.

  Patent Document 3 discloses that a mutation of a voltage-type sodium channel type II gene (SCN2A) in the nervous system is involved in intractable pediatric epilepsy accompanied by severe regression of intellectual ability. Further, it is disclosed that it is possible to diagnose whether or not there is a predisposition to refractory childhood epilepsy accompanied by severe intellectual ability regression using the mutation of SCN2A as an index.

Patent Document 4 discloses that a mutation in the GABA _A receptor γ ² subunit gene (GABRG2) is involved in SMEI. Further, it is disclosed that SMEI can be diagnosed using GABRG2 mutation as an index.
Japanese Patent Laying-Open No. 2004-73058 (published on March 11, 2004) JP 2004-329153 A (published on November 25, 2004) Japanese Patent Laying-Open No. 2004-275115 (released on October 7, 2004) JP 2005-192411 A (published July 21, 2005)

  Traditionally, febrile convulsions have been diagnosed and treated mainly by general pediatricians and family doctors. However, early diagnosis is difficult because diagnosis based on the clinical symptoms described above requires specialized knowledge. By the age of 2-3 years, when clinical symptoms appear, patients with Dravet syndrome often experience convulsions many times and experience dangerous conditions such as status epilepticus. Therefore, it is necessary to detect Dravet syndrome as early as possible, and there is a need for the development of an early diagnosis method that can be used by general pediatricians and family doctors who are involved in primary care.

  As an example of such a technique, the techniques disclosed in Patent Documents 1 to 4 are listed. However, since the causal relationship between the mutation of each gene, which is an index of the onset of Dravet syndrome, and Dravet syndrome has not been sufficiently elucidated by these methods, there is a problem that the accuracy is not sufficient. For example, regarding the technique of Patent Document 4, the mutation of GABRG2 gene is about 0.5% (1 case / about 200 cases) of Dravet syndrome, and the detection rate is low. In addition, benign thermal convulsions are detected in about 1% (2 cases / about 200 cases) and thus have no specificity. Further, in the techniques of Patent Documents 1 and 2, a specific mutation site of the SCN1A gene is analyzed, but the specific mutation site is not a multiple mutation and has a problem that the detection frequency is considerably low.

  Therefore, there is a need for further development of technology that can early diagnose the onset of Dravet syndrome even for infants under 1 year of age.

  The present invention has been made in view of the above problems, and its purpose is to obtain data for determining the possibility of the onset of Dravet syndrome that can be applied to infants younger than 1 year, and its To provide usage.

As a result of intensive investigations in view of the above problems, the present inventors subdivide the results of mutation analysis of specific genes and clinical symptoms, and detect patients with Dravet syndrome from patients who develop febrile convulsions under the age of 1 year. Several risk factors were uniquely discovered. According to the diagnostic prediction criteria in which the risk factors are scored based on the risk rate, it has been found that the possibility of the onset of Dravet syndrome can be determined with high accuracy at the time of less than 1 year old, which has been difficult to detect conventionally. The invention has been completed.
That is, the present invention includes the following inventions.

(1) A method for acquiring data for determining the possibility of the onset of Dravet syndrome, wherein at least one risk factor among the risk factors shown in the following (a) to (i) is included in a document: Determining whether or not it is included.
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience (2) Above (a The method according to (1), wherein the number of risk factors included in the material among the risk factors indicated in (i) to (i) is calculated.

  (3) A risk score is set for each of the risk factors shown in (a) to (i) above, and among the risk factors shown in (a) to (i) above, the risk included in the above document The method according to (1), wherein a sum of risk scores set for each factor is calculated as a judgment prediction score.

  (4) The relative relationship of the risk score set for each of the risk factors shown in (a) to (i) above is the risk score set for the risk factor shown in (a) above. The risk scores set for the risk factors shown in (b) to (i) above are 3, 3, 2, 2, 2, 1, 1, 1, respectively. The method according to (3).

(5) A recording medium used for determining the possibility of onset of Dravet syndrome, and whether or not at least one risk factor is possessed among the risk factors shown in (a) to (i) below: A recording medium on which inquiry items to be asked are recorded.
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience (6) Above (a The risk score set for each of the risk factors shown in (i) to (i) is recorded in association with each of the risk factors shown in (a) to (i) above. The recording medium according to (5).

  (7) The relative relationship of the risk score set for each of the risk factors shown in (a) to (i) above is the risk score set for the risk factor shown in (a) above. The risk scores set for the risk factors shown in (b) to (i) above are 3, 3, 2, 2, 2, 1, 1, 1, respectively. The recording medium according to (6).

  (8) A kit used for determining the possibility of onset of Dravet syndrome, comprising the recording medium according to any one of (5) to (7).

  (9) The method according to (8), further comprising a primer for amplifying all or a part of at least one of the exon region and exon / intron boundary region of the human SCN1A gene. The described kit.

(10) A system for acquiring data for determining the possibility of the onset of Dravet syndrome, comprising an inquiry means and a determination means. The inquiry means includes the following (a) to (i) Among the risk factors shown, at least one risk factor is interrogated to determine whether or not the subject possesses the risk factor, and an interrogation result file in which the interrogation result is recorded is generated. (A) to (i), wherein at least one risk factor is determined to be included in the interview result recorded in the interview result file.
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience (11) The determination means Calculates the number of risk factors included in the interrogation results among the risk factors shown in (a) to (i) above, (10).

  (12) Storage means for storing a risk score set for each of the risk factors shown in the above (a) to (i) is further provided, and the determination means uses the risk score stored in the storage means. Using the risk factors shown in (a) to (i) above, the sum of the risk scores set for each of the risk factors included in the interview result is calculated as a prediction prediction score. The system according to (10).

  (13) The relative relationship of the risk score set for each of the risk factors shown in (a) to (i) above is the risk score set for the risk factor shown in (a) above. The risk scores set for the risk factors shown in (b) to (i) above are 3, 3, 2, 2, 2, 1, 1, 1, respectively. The system according to (12).

  (14) A program for operating the system according to any one of (10) to (13), which causes a computer to function as the inquiry unit and the determination unit.

  (15) A computer-readable recording medium on which the program according to (14) is recorded.

  The data acquisition method for determining the possibility of onset of Dravet syndrome according to the present invention determines the possibility of onset of Dravet syndrome by determining the presence or absence of risk factors that are highly relevant to the onset of Dravet syndrome. Get data to do. The risk factors can also be detected in infants younger than 1 year. Therefore, there is an effect that data for determining the possibility of the onset of Dravet syndrome can be easily obtained at low cost and at an early stage of the onset or before the intractable course.

  Embodiments according to the present invention will be described below with reference to FIGS. 1 to 6, but the present invention is not limited thereto.

<I. Data acquisition method for determining the possibility of Dravet syndrome>
The data acquisition method for determining the possibility of development of Dravet syndrome according to the present invention (hereinafter simply referred to as “data acquisition method according to the present invention”) This is a method of acquiring data for determination. In this specification, “possibility of developing Dravet syndrome” includes both the possibility of developing Dravet syndrome and the possibility of developing Dravet syndrome in the future. It is.

Specifically, the data acquisition method according to the present invention determines whether a risk factor is included in a document. As the risk factor, a symptom highly correlated with Dravet syndrome is used for persons who have developed febrile convulsions under the age of one year. Specific examples of such risk factors include the risk factors shown in the following (a) to (i).
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) There is a truncation mutation in the SCN1A gene (g) There is experience in partial seizures (h) There is a missense mutation in the SCN1A gene (i) There is experience in myoclonic seizures.

  That is, the data acquisition method according to the present invention determines whether or not at least one risk factor among the risk factors shown in the above (a) to (i) is included in the material, and develops Dravet syndrome. It is a method of acquiring data for determining the possibility of being. In the present specification, “prolonged convulsions” means that convulsions or disturbances of consciousness are prolonged than normal convulsive seizures, and specifically means convulsions of 10 minutes or longer. In the present specification, the “missense mutation” means a mutation in which a mutation occurs in a codon of an amino acid encoded by DNA, resulting in an amino acid substitution. Here, a single amino acid deletion abnormality is also included in the missense mutation. `` Truncation mutation '' means a mutation in which a codon encoding an amino acid is changed to a stop codon that does not encode an amino acid, a mutation in which the codon is shifted due to a frame shift, resulting in a stop codon, and It means a mutation in which a downstream amino acid is different from a normal amino acid due to a splicing abnormality resulting from a mutation in an exon / intron region.

  The above-mentioned material means a material in which information related to the health of the subject such as past illness, treatment history, current disease, and treatment status thereof is described. Specifically, for example, the subject's medical record, test results, answer results to the questionnaire, and the like can be mentioned.

  Where the material is a test result, it preferably includes a test result for diagnosing the onset of Dravet syndrome. For example, the analysis result of SCN1A gene can be mentioned. In particular, it is preferable to include the results of analyzing the presence or absence of truncation mutations and missense mutations in the SCN1A gene. In the present invention, when the analysis result of the SCN1A gene is used, the analysis method is not particularly limited. Specifically, it can be performed using a conventionally known gene mutation detection method.

  For example, first, tissues such as hair and oral mucosal epithelium collected from a subject, and specimens such as blood and body fluid are prepared. When blood or body fluid is used as a specimen, it is not limited to fresh blood or body fluid, but blood, coagulated blood, or body fluid soaked in blood that has been subjected to anticoagulation treatment or filter paper may be used. Alternatively, blood, coagulated blood, or body fluid may be soaked in filter paper and then dried.

  Next, genomic DNA is isolated from the specimen. For isolation of genomic DNA, a conventionally known method may be used. For example, a method using a conventional SDS / Proteinase treatment can be used. Commercially available white blood cell separation reagents and DNA extraction reagents may also be used.

  Using the genomic DNA as a template, the full length or specific region of the SCN1A gene is amplified by a conventionally known method. Examples of DNA amplification methods include PCR, LAMP (Loop-mediated Isothermal Amplification) (Eiken Chemical Co., Ltd.), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids) (Takara Shuzo Co., Ltd.). Can do. Examples of the specific region include exons and exon / intron boundary regions of the SCN1A gene.

  Further, instead of purified genomic DNA, DNA may be amplified by directly using hair, oral epithelium, trace blood and the like. Furthermore, instead of genomic DNA, cDNA synthesized from mRNA isolated from a subject using reverse transcriptase may be used. In that case, examples of the specific region to be amplified include a 5 'untranslated region, a 3' untranslated region, and a region within an exon.

  Next, the base sequence of the amplified gene fragment is determined. For the base sequence determination method, a conventionally known method such as a method using a fluorescent label or a method using a radioisotope can be used. The presence or absence of mutation can be determined by comparing the determined base sequence with the genomic sequence or cDNA sequence of the SCN1A gene of a healthy person.

  In addition, mutation analysis of the SCN1A gene includes SSCP (Single Strand Conformation Polymorphism) method, DHPLC (Denaturing High Performance Liquid Chromatography) method, a method of searching for mutations using differences in melting temperature, and mutation using microarray and real-time PCR. Any conventionally known mutation detection method such as a detection method and a polymorphism detection method can be used.

  The SCN1A gene in the present invention is a human-derived voltage-dependent sodium channel α-subunit type I gene in the nervous system. As the genomic sequence, for example, the base sequence shown in SEQ ID NO: 1 can be mentioned. In addition, examples of the base sequence of the cDNA include the base sequence shown in SEQ ID NO: 2 (base sequence registered as GenBank accession number AB093548).

  In the present invention, the positions of missense mutations and truncation mutations in the SCN1A gene described in the risk factors shown in (f) and (h) above are not particularly limited. Although details will be described in the examples described later, for example, in the base sequence shown in SEQ ID NO: 1, mutations shown in Table 1 can be mentioned, but the present invention is not limited thereto.

  Moreover, when the said document is an answer result with respect to an inquiry table, it is preferable that the inquiry result performed when diagnosing Dravet syndrome is included.

More specifically, what is necessary is just to be able to determine whether a test subject includes the risk factors shown in the above (a) to (i). For example, the interrogation in the above-mentioned interrogation chart includes interrogation of the number of seizures, seizure type, seizure-inducing factor, age at the first occurrence of seizures, and the presence or absence of abnormality of the SCN1A gene. These interviews are performed using, for example, an interview table in which the following questions Q1 to Q9 are described, such as the interview table shown in FIG. 1 (a), and the risk factors shown in (a) to (i) above. This can be done by directly asking whether or not
(Q1) Is febrile convulsions occurring less than 8 months after birth? (Q2) Is the number of seizures 5 or more? (Q3) Is convulsions continued for more than 10 minutes? (Q4) Is there any experience of convulsions (Q5)? ) Do you have partial seizures? (Q6) Do you have myoclonic seizures? (Q7) Have you experienced convulsions during or after bathing? (Q8) Are there missense mutations in the SCN1A gene? (Q9) SCN1A Is there a truncation mutation in the gene? Also, using the questionnaire shown in Fig. 1 (b), the number of seizures, seizure type, seizure-inducing factors, and the age at which seizures occurred for the first time You may ask. In addition, it cannot be overemphasized that the questionnaire which can be used in this invention is not limited to FIG. 1 (a) and (b).

  In the data acquisition method according to the present invention, first, it is determined whether or not the material includes the risk factors shown in (a) to (i) above. In the present invention, it is only necessary to examine whether at least one risk factor among the risk factors shown in (a) to (i) above is included in the above document, but it is highly possible to develop Dravet syndrome. From the viewpoint of acquiring data for accurate determination, it is preferable to determine whether or not the risk factor is included in the above-mentioned material for a plurality of risk factors, and it is more preferable to determine all the risk factors. .

  Moreover, in embodiment which determines whether only the one part risk factor shown to said (a)-(i) is included in the said data, said (a)-(e), (g), and ( It is preferable to determine whether or not the seven risk factors shown in i) are included in the material. According to such a configuration, since there is no need to examine the presence or absence of a mutation in the SCN1A gene, the possibility of developing Dravet syndrome can be easily determined at low cost without being restricted by physical and physical limitations. Data can be obtained.

  Furthermore, in another embodiment for determining whether or not some of the risk factors shown in the above (a) to (i) are included in the material, there are 8 risks excluding the risk factors shown in (e) above. It is preferable to determine whether or not a factor is included in the material. According to such a configuration, even for a subject who does not have a bathing habit, data for determining the possibility of developing Dravet syndrome can be easily acquired.

  In addition, among the risk factors shown in (a) to (i) above, whether the six risk factors shown in (a) to (d), (g), and (i) are included in the above document It can be set as the embodiment which determines.

  Furthermore, in another embodiment for determining whether or not some of the risk factors shown in (a) to (i) are included in the material, the four risks shown in (b) to (e) above are used. It is preferable to determine whether or not a total of five risk factors including the factor and the risk factor indicated in either one of (f) and (h) are included in the material.

  In addition, among the risk factors shown in (a) to (i) above, an embodiment for determining whether or not the four risk factors shown in (b) to (e) above are included in the material is used. Can do.

  Furthermore, in another embodiment for determining whether or not some of the risk factors shown in (a) to (i) are included in the material, the three risks shown in (b) to (d) above are used. It is preferable to determine whether the factor is included in the material.

  In addition, among the risk factors shown in (a) to (i) above, an embodiment for determining whether or not the two risk factors shown in (b) and (d) above are included in the material is used. Can do.

  According to the configuration not including the test item for the SCN1A gene mutation, it is not necessary to test for the presence or absence of the mutation in the SCN1A gene. Data for determining the possibility of the onset of can be acquired. According to the configuration not including the three risk factors shown in the above (a), (g), and (i), since no special classification knowledge of seizure type is required, the medical condition of the subject's family etc. This makes it easy to understand the data, and can easily acquire data for determining the possibility of the onset of Dravet syndrome. According to the configuration that does not include the risk factor shown in (c) above, there is no need to wait until the number of seizures reaches 5, so data for determining the possibility of developing Dravet syndrome earlier Can be obtained.

  Furthermore, in the data acquisition method according to the present invention, in addition to the risk factors shown in (a) to (i), additional risk factors may be added as risk factors, or (a) to (i) Of the risk factors shown, some risk factors may be deleted and further risk factors added.

  As a result of the determination, when data indicating that the risk factor is included in the material is acquired, it can be determined that the subject is highly likely to develop Dravet syndrome using the data. .

  In the data acquisition method according to the present invention, the number of risk factors included in the material may be calculated. According to this, data for determining the possibility of the onset of Dravet syndrome with higher accuracy can be acquired. According to the data acquired by the data acquisition method according to this embodiment, the greater the number of risk factors included in the material, the higher the possibility that the subject has developed Dravet syndrome.

  Furthermore, in the data acquisition method according to the present invention, for each of the risk factors shown in the above (a) to (i), a corresponding risk score (numerical value that is an index of the probability of the onset of Dravet syndrome) is obtained. It may be set, and the sum of the risk scores corresponding to each of the risk factors included in the material may be calculated as the judgment prediction score.

  Specifically, for example, among risk factors shown in the above (a) to (i), a higher value is set for a risk factor having a higher relationship with the onset of Dravet syndrome, and the onset of Dravet syndrome For risk factors that have a relatively low relationship with, set a smaller number. That is, instead of considering all the risk factors shown in (a) to (i) above uniformly as to the high possibility of the onset of Dravet syndrome, the risk factors shown in (a) to (i) above. Give superiority or inferiority. With such a configuration, it is possible to acquire data for determining the possibility of the onset of Dravet syndrome with higher accuracy. In addition, according to the data acquired by the data acquisition method of the present embodiment, the greater the determination prediction score, the higher the likelihood of developing Dravet syndrome, and the lower the determination prediction score, the lower the likelihood of developing Dravet syndrome. Can be determined.

  Needless to say, among the risk factors shown in (a) to (i) above, a smaller risk score is set for a risk factor having a higher association with the onset of Dravet syndrome, and the onset of Dravet syndrome. It is also possible to adopt an embodiment in which a larger risk score is set for a risk factor having a relatively low relationship with. According to the data acquired by the data acquisition method according to such an embodiment, the smaller the determination prediction score, the higher the possibility of developing Dravet syndrome, and the higher the determination prediction score, the more likely the development of Dravet syndrome. Can be determined to be low.

  Here, the superiority or inferiority of the causal relationship between the onset of Dravet syndrome and the risk factors shown in the above (a) to (i) will be described. That is, a risk score setting method corresponding to each of the risk factors shown in the above (a) to (i) will be described.

  The present inventors independently found out that the risk factors shown in the above (a) to (i) are symptoms highly related to the onset of Dravet syndrome, as shown in the Examples described later. Furthermore, the relationship between the possession of risk factors shown in (a) to (i) and the onset of Dravet syndrome is not all the same, and the magnitude of the possibility of the onset of Dravet syndrome depends on the retained risk factors. Found out that there is superiority or inferiority. Specifically, the risk factors shown in the above (a) to (i) are classified into a group having the highest association with the onset of Dravet syndrome (hereinafter also referred to as “first group”), and then Dravet syndrome. A group with moderately high relevance to the onset (hereinafter also referred to as “second group”) and a group with the lightest relevance to the onset of Dravet syndrome (hereinafter also referred to as “third group”) ), The first group includes the risk factors shown in the above (a) to (c), and the second group contains the risk factors shown in the above (d) to (f). It was found that the third group includes the risk factors shown in (g) to (i) above.

  In one embodiment of the data acquisition method according to the present invention, when a corresponding risk score is set for each of the risk factors shown in (a) to (i) above, it belongs to the first group based on the above knowledge. A high risk score is set for the risk factor, a lower risk score is set for the risk factor belonging to the second group, and a lower risk score is set for the risk factor belonging to the third group. Is preferably set. Moreover, it is preferable to set the same risk score for risk factors belonging to the same group.

  The method for setting the corresponding risk score for each of the risk factors shown in (a) to (i) above is not particularly limited. For example, each risk shown in (a) to (i) above is used. As for the association between factors and Dravet syndrome, a significant difference test between a patient group of Dravet syndrome and a patient group of non-Dravet syndrome by Chi-square or Fisher test, and a method of performing an odds ratio test can be mentioned. Furthermore, sensitivity (sensitivity) for examining the ability to detect a disease and specificity (specificity) for examining the ability to not misdiagnose a non-sick person as a disease may be used. Other statistical processing may be used.

  Here, an embodiment of a method for setting a risk score corresponding to each risk factor using a Chi-square or Fisher test and an odds ratio test will be described, but the present invention is not limited to this. Absent.

  In the present embodiment, if the p value is less than 0.01, it is determined that it is statistically significant. Also, a test based on the odds ratio is performed, and if the value of the odds ratio (hereinafter also referred to as “odds value”) is greater than 1, it is determined to be statistically significant. A risk score corresponding to each risk factor may be set based on the odds value and the p value in the Chi-square or Fisher test.

  For example, when the odds value ≦ 1 or p> 0.01, the risk score is 0 (not a risk factor), and when 1 <odds value <20 or 0.01 ≧ p> 1.0E-5, If the risk score is 1 and 20 ≦ odds value <40 or 1.0E−5 ≧ p> 1.0E−10, the risk score is 2 and 40 ≦ odds value or 1.0E−10 ≧ p In this case, the risk score is 3. According to the above criteria, the risk scores set for the risk factors belonging to the first to third groups are 3, 2, and 1, respectively. The specific risk score value corresponding to each risk factor is not limited to the above, but the relative risk score values set for the risk factors belonging to the first to third groups. It is sufficient that the above relationship is the above relationship. The standard for setting the risk score is not limited to the above.

  In the embodiment in which the risk score is set for each risk factor, as described above, a judgment prediction score is calculated by summing up the risk scores of the risk factors included in the material. According to the determination prediction score calculated in this manner, the higher the determination prediction score, the higher the possibility that the subject has developed Drevet syndrome. In the data acquisition method according to the present invention, it is preferable to set a reference value as a determination criterion for the possibility of the onset of Dravet syndrome for the determination prediction score. According to such a configuration, it is possible to easily determine the possibility of the onset of Dravet syndrome in the subject simply by comparing the determination prediction score of the subject with the reference value. In this case, the reference value that is a criterion for determining the possibility of the onset of the Dravet syndrome is the number and type of risk factors for determining whether or not they are included in the material, and the risk score set for each risk factor. It is changed according to the value of.

  For example, the case where the risk score set for each risk factor shown in the above (a) to (i) is 3, 3, 3, 2, 2, 2, 1, 1, 1 will be described. To do. In the embodiment for determining whether or not all the risk factors shown in the above (a) to (i) are included in the material, the reference value for determining that there is a high possibility of developing Dravet syndrome in the subject is described above. The determination prediction score is preferably 7 or more. In addition, the embodiment for determining whether the seven risk factors shown in (a) to (e), (g), and (i) are included in the above-mentioned material is the possibility of developing Dravet syndrome in a subject. It is preferable that the determination prediction score is 7 or more as a reference value for determining that the property is high. Among the risk factors shown in (a) to (i) above, in the embodiment for determining whether or not eight risk factors other than the risk factor shown in (e) above are included in the material, The reference value for determining that the possibility of developing the syndrome is high is preferably such that the determination prediction score is 6 or more. Furthermore, in the embodiment for determining whether or not the six risk factors shown in the above (a) to (d), (g), and (i) are included in the above-mentioned material, the onset of Dravet syndrome in the subject is possible. It is preferable that the determination prediction score is 6 or more as a reference value for determining that the property is high. In addition, whether or not the above-mentioned materials include a total of five risk factors including the four risk factors shown in (b) to (e) above and the risk factor shown in either (f) or (h). In the embodiment for determining whether or not the reference value for determining that the Dravet syndrome is highly likely to develop in the subject, the determination prediction score is preferably 6 or more. In the embodiment for determining whether or not four risk factors shown in the above (b) to (e) among the risk factors shown in the above (a) to (i) are included in the material, The reference value for determining that there is a high possibility of the onset of Dravet syndrome is preferably such that the determination prediction score is 5 or more. Further, in the embodiment for determining whether or not the three risk factors shown in (b) to (d) are included in the material, the reference value for determining that there is a high possibility of developing Dravet syndrome in the subject is The determination prediction score is preferably 5 or more. The embodiment for determining whether or not two risk factors shown in (b) and (d) among the risk factors shown in (a) to (i) are included in the material The reference value for determining that there is a high possibility of the onset of Dravet syndrome is preferably such that the determination prediction score is 3 or more.

  As another embodiment, the risk factor belonging to the first group is set to a low risk score, the risk factor belonging to the second group is set to a higher risk score, and the third group It is also possible to adopt an embodiment in which a risk score higher than that is set for the risk factors belonging to. If the data acquired by the data acquisition method according to such an embodiment is used, it can be determined that the lower the determination prediction score, the higher the possibility of developing Dravet syndrome.

  As described above, in the data acquisition method according to the present invention, the risk factors shown in the above (a) to (i) are included in the material using the medical record, the examination result, the answer result of the questionnaire, and the like. By determining whether or not there is, data for determining the possibility of the onset of Dravet syndrome is acquired. According to the data acquired by the data acquisition method according to the present invention, it is possible to determine the possibility of developing Dravet syndrome easily and at low cost without requiring specialized knowledge.

  Moreover, any of the risk factors shown in the above (a) to (i) can be detected in a subject under 1 year old. Therefore, according to the data acquisition method of the present invention, it is possible to acquire data for determining the possibility of the onset of Dravet syndrome in a subject under the age of 1, which was difficult in the past. Therefore, in the data acquisition method according to the present invention, the subject is preferably an infant under 1 and more preferably an infant under 1 year with thermal convulsions. These subjects are highly likely to develop Dravet syndrome, determine whether Dravet syndrome has occurred as early as possible, and refer to an epilepsy specialist before the symptoms become serious, and prepare a treatment management system. is required. According to the data acquisition method of the present invention, data for determining the possibility of the onset of Dravet syndrome can be acquired at an early stage of less than one year old, which can be made possible.

  In addition, according to the data acquisition method of the present invention, it is possible to improve the patient's treatment results and reduce the mental burden and economic burden of the family. Furthermore, since appropriate treatment can be performed early in the onset of Dravet syndrome, it can contribute to the reduction of medical costs.

  The present invention also includes a method of determining the possibility of developing Dravet syndrome in a subject using data acquired using the data acquisition method according to the present invention. In the present embodiment, truncation mutation and missense mutation are used as risk factors for the SCN1A gene, but the present invention is not limited to this, and mutations such as SCN1A gene deletion and rearrangement are dangerous. It can also be a factor. Specifically, an abnormality in the promoter / enhancer region of the SCN1A gene can be used as one of risk factors. In this case, it is preferable to use a primer capable of amplifying the promoter / enhancer region.

<II. Recording medium and kit for determining the possibility of Dravet syndrome>
(II-1) Recording medium for determining possibility of onset of Dravet syndrome Recording medium for determining possibility of onset of Dravet syndrome according to the present invention (hereinafter also simply referred to as “recording medium for determination according to the present invention”) <I. It is a recording medium that can be suitably used to implement the data acquisition method according to the present invention described in the section> Data acquisition method for determining the possibility of Dravet syndrome. Specifically, it is a recording medium on which an inquiry item asking whether or not there is a symptom highly associated with the onset of Dravet syndrome is recorded. For example, among the risk factors shown in the following (a) to (i) And a recording medium in which an inquiry item for checking whether or not at least one risk factor is possessed is recorded. It is preferable that all the risk factors shown in (a) to (i) are recorded on the determination recording medium according to the present invention, but only a part of the risk factors may be recorded. Further, in addition to the risk factors shown in (a) to (i), further risk factors may be recorded, or some of the risk factors shown in (a) to (i) are deleted, and further Risk factors may be recorded.
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience In the embodiment in which only a part of the risk factors shown in the above (a) to (i) is recorded in the determination recording medium, the above (a) to (e), (g), and (i) ) In which seven risk factors are recorded, in which eight risk factors other than (e) above are recorded, (a) to (d), (g), and (i) above Configurations that record the six risk factors shown A configuration in which five risk factors including the four risk factors shown in the above (b) to (e) and one risk factor shown in either (f) or (h) are recorded, The configuration in which the four risk factors shown in (b) to (e) are recorded, the configuration in which the three risk factors shown in (b) to (d) are recorded, (b) and ( A configuration in which two risk factors shown in d) are recorded is preferable.

  The form of the determination recording medium according to the present invention is not particularly limited. For example, paper, floppy (registered trademark) disk, CD-R, CD-ROM, CD-R / RW, DVD-R, DVD -R / RW, DVD-ROM, etc. can be mentioned. That is, the inquiry table shown in FIGS. 1A and 1B can be said to be an embodiment of the determination recording medium according to the present invention.

  According to the recording medium for determination according to the present invention, the user simply answers the inquiry item recorded on the recording medium for determination and determines whether or not the subject possesses the risk factor. Data for easily determining the possibility of onset of Dravet syndrome in a subject can be acquired.

  In addition, the determination recording medium according to the present invention may record a determination criterion as to whether or not the subject has developed Dravet syndrome. Specifically, a criterion for determining whether or not the subject develops Dravet syndrome may be recorded based on the number of risk factors held by the subject. With such a configuration, the possibility of developing Dravet syndrome in the subject can be determined with higher determination accuracy by comparing the number of the risk factors possessed by the subject with the reference.

  It is preferable that a risk score associated with each risk factor is further recorded on the determination recording medium according to the present invention. Since the risk score has been described above, the description thereof is omitted here. According to such a configuration, the user determines whether or not the subject possesses the risk factor recorded on the determination recording medium, and the risk score associated with the risk factor possessed by the subject. Can be summed to calculate a judgment prediction score. According to such a configuration, the possibility of the onset of Dravet syndrome in the subject can be determined based on the magnitude of the determination prediction score. Therefore, it is possible to further improve the determination accuracy of the possibility of the onset of Dravet syndrome.

  In the embodiment in which the risk score is recorded in the determination recording medium according to the present invention, a reference value for determining the possibility that the subject has developed Dravet syndrome is further recorded from the determination prediction score. It is preferable. Since the reference value setting method and the specific example of the reference value have been described above, the description thereof is omitted here.

  In this way, the recording medium for determination according to the present invention can be used as a check sheet when acquiring data for determining the possibility of onset of Dravet syndrome, in other words, the present invention. It can utilize suitably for implementation of the data acquisition method concerning.

  The present invention also includes a kit for determining the possibility of developing Dravet syndrome (hereinafter, also simply referred to as “determination kit according to the present invention”) including the above-described recording medium for determination. Hereinafter, the determination kit according to the present invention will be described.

(II-2) Kit for determining the possibility of developing Dravet syndrome The kit for determining the possibility of developing Dravet syndrome according to the present invention (hereinafter, also simply referred to as “the kit for determining according to the present invention”) The recording medium for determination according to the invention may be included, and the other components are not particularly limited.

  For example, the structure containing the reagent, instrument, etc. for analyzing SCN1A gene can be mentioned. Specific examples include leukocyte cell separation reagents and instruments, DNA extraction reagents and instruments, SCN1A gene DNA amplification reagents and instruments, and SCN1A gene mutation detection reagents and instruments.

  The reagent for amplifying the SCN1A gene DNA may include a primer for amplifying the DNA of the human SCN1A gene. The primer only needs to be located in a region capable of amplifying all or a part of at least one of the exon region and exon / intron boundary region of the human SCN1A gene. It is not limited to the sequence and its position. For example, oligonucleotides having the base sequences shown in SEQ ID NOs: 3 to 56 used in the examples described later can be used in the combinations described in the examples. Further, as described above, in the data acquisition method according to the present invention, deletion and rearrangement of the SCN1A gene can be used as one of risk factors. Therefore, as a primer for amplifying the DNA of the human SCN1A gene, a primer capable of detecting an abnormality in the promoter / enhancer region of the human SCN1A gene may be used.

  Further, the DNA to be amplified is not limited to genomic DNA, but may be cDNA synthesized by reverse transcriptase using mRNA. In that case, the primer for amplifying cDNA may be capable of amplifying all or a part of at least one of the 5 ′ untranslated region, 3 ′ untranslated region and exon region. .

  As another embodiment, the determination kit according to the present invention may include a component that enables transport of a sample from which genomic DNA or mRNA can be extracted. Specifically, for example, containers for transporting samples capable of extracting DNA, such as liquid blood and body fluids, coagulated blood, blood and body fluids adsorbed and dried on filter paper, hair, and oral epithelium and / or A configuration including an instrument and a transporting means for transporting the container containing the sample to a medical facility, a hospital, an inspection organization, or the like can be given. Examples of the conveying means include mail envelopes and stamps. Moreover, it is preferable that the determination kit according to the present embodiment includes an agreement for obtaining consent from a subject for genetic DNA analysis, or a guardian or a parental authority of the subject. According to the said structure, the data for determining the possibility of onset of Dravet syndrome in a test subject can be acquired, without being influenced by a user's residence etc.

<III. Data acquisition system for determining the possibility of Dravet syndrome>
A data acquisition system 1 (system) according to the present embodiment is configured by a computer device, and includes a CPU, memory (RAM, ROM, etc.), external storage device (hard disk drive, MO drive, etc.), display device, input device ( Keyboard, mouse, etc.) and an output device (printer, etc.). In addition, the data acquisition system 1 obtains data for determining the possibility of the onset of Dravet syndrome, as shown in FIG. 2, a personal information file generation unit 10, an inquiry unit 20 (interrogation means), determination A unit 30 (determination unit) and a storage unit 40 (storage unit) are provided.

  The personal information file generation unit 10, the inquiry unit 20, and the determination unit 30 include a CPU (Central Processing Unit), a RAM (Random Access Memory), a ROM (Read Only Memory), and the like. The storage unit 40 includes an information storage device (memory such as RAM and ROM, an external storage device such as a hard disk drive and an MO drive), an information recording medium (CD-ROM, DVD, etc.) and the like.

  Here, operation | movement of the data acquisition system 1 concerning embodiment is demonstrated using the flowchart shown in FIG.

  When the data acquisition system 1 according to the present embodiment is activated, the personal information file generation unit 10 displays a personal data input screen on the display screen of the display device (Step 1; (Omitted). The personal data input screen assists the user in inputting the personal information of the subject into the data acquisition system 1. Specifically, for example, as shown in FIG. 4, a name 17 capable of inputting character data of a name, a radio button 18 capable of selecting and inputting a male or female gender, and four ABO blood types An example of the screen includes a radio button 22 that can be selected and input, and a moon age 19 that can input numeric data of the age.

  When the personal data input screen is displayed, the user uses the input device to support the personal data input screen to the personal information file generation unit 10 such as name, gender, age, blood type, etc. Enter personal data to identify an individual.

  More specifically, using the personal data input screen shown in FIG. 4, the name is input as character data in the name 17 on the personal data input screen. The sex and blood type are input by clicking the radio button 18 for male or female, respectively, and the blood type is selected by clicking one of the four radio buttons 22 for ABO blood type. Further, the age 19 is input as numerical data. If it is desired to correct the input data while inputting these data, clicking the correction button 26 deletes the input data and allows the input data to be input again. When all the data has been input, the “Next” button 25 is clicked using the input device.

  When the “Next” button 25 is clicked, a signal is transmitted from the input device to the personal information file generation unit 10, and the personal information file generation unit 10 generates a personal information file with the signal as a cue (S2). . After S2, the personal information file generation unit 10 transmits a signal to the inquiry unit 20.

  When receiving the signal from the personal information file generating unit 10, the inquiry unit 20 displays the inquiry screen on the display screen of the display device with the signal as a cue (S3). The inquiry screen assists the user in inputting the answer result to the subject's inquiry to the data acquisition system 1. Specifically, for example, as shown in FIG. 5, a screen including a radio button 28 capable of selecting and inputting an answer as to whether or not the subject has the risk factors shown in (a) to (i) above. Can be mentioned.

  When the personal data input screen is displayed, the user can use the input device to assist the examinee 20 in the interview section 20 with the assistance of the interview screen, and the risk that the subject is shown in the above (a) to (i). Enter the result of answering whether or not you own the factor. The answer may be input while the user is actually interviewing the subject, or may be input based on material obtained from the subject in advance. The material referred to here is the above <I. Refers to the data described in Data acquisition method for determining the possibility of Dravet syndrome.

To explain in more detail using the inquiry screen shown in FIG. 5, an answer is input to the inquiry item 27 on the inquiry screen by clicking the radio button 28 of “Yes” or “No”. The inquiry item 27 may be an inquiry asking whether or not the subject possesses the risk factors shown in the above (a) to (i). For example, the interviews shown in the following (A) to (I) are mentioned.
(A) Do you have any experience of convulsions?
(B) Have you ever experienced convulsions for more than 10 minutes?
(C) Do you have more than 5 seizures?
(D) Is the onset of febrile convulsions less than 8 months old?
(E) Have you ever experienced convulsions by bathing?
(F) Is there a truncation mutation in the SCN1A gene?
(G) Do you have experience with partial seizures?
(H) Is there a missense mutation in the SCN1A gene?
(I) Do you have experience with myoclonic attacks?
According to such an inquiry format, since the answer can be handled as binary data, the processing in the determination unit 30 described later can be simplified.

  If it is desired to correct the input answer while inputting the answer, the input answer can be deleted by clicking the correction button 26 and input again. When the input of answers to all the interviews on the interview screen is completed, the “Next” button 25 is clicked using the input device.

  When the “Next” button 25 is clicked, a signal is transmitted from the input device to the interrogation unit 20, and the interrogation unit 20 generates an interrogation result file in which the interrogation results are recorded (S 4). . After S4, the inquiry unit 20 transmits a signal to the determination unit 30.

  The determination unit 30 receives a signal from the interrogation unit 20 and uses the interrogation result file as a signal to determine the possibility of the onset of Dravet syndrome in the subject. Specifically, the determination unit 30 first determines whether or not the interview result recorded in the interview result file includes the risk factor (S5). More specifically, taking the generated inquiry result file as an example in accordance with the inquiry screen shown in FIG. 5, it is determined whether or not there is an item for which “Yes” is selected among the inquiry items 27. When the determination unit 30 determines that the inquiry result in the inquiry result file does not include the risk factor, that is, the inquiry item 27 does not include an item for which “Yes” is selected, the display screen of the display device displays A determination result screen indicating that the test subject is less likely to develop Dravet syndrome is displayed on the display screen of the display device (S6).

  On the other hand, when the determination unit 30 determines that the inquiry result of the inquiry result file includes the risk factor, that is, among the inquiry items 27, there is an item for which “Yes” is selected, the determination prediction score is calculated. (S7). The determination unit 30 calculates the determination prediction score using the risk score for each risk factor stored in the storage unit 40. Regarding the judgment prediction score and the risk score, the above <I. Since the explanation has been given in the section “Data acquisition method for determining the possibility of the onset of Dravet syndrome”, the description thereof is omitted here.

  Next, the determination unit 30 compares the determination prediction score calculated in S <b> 7 with a reference value that can be determined to cause Drevet syndrome. Specifically, it is determined whether or not the determination prediction score is equal to or greater than the reference value (S8). As a result, if the determination prediction score is equal to or higher than the reference value, the determination unit 30 displays a determination result screen indicating that the subject is highly likely to develop Dravet syndrome on the display screen of the display device. (S9). On the other hand, if the determination prediction score is smaller than the reference value, the determination unit 30 displays a determination result screen indicating that the test subject is less likely to develop Dravet syndrome on the display screen of the display device (S6). For the reference value, <I. Since the explanation has been given in the section “Data acquisition method for determining the possibility of the onset of Dravet syndrome”, the description thereof is omitted here.

  The screen displayed by the determination unit 30 only needs to allow the user to confirm that the subject has non-Dravet syndrome or Dravet syndrome, but may include other information. For example, as shown in FIG. 6, the determination prediction score may be displayed. In addition, an end button 32 is provided, and the user can end the operation by clicking the end button 32 using an input device.

  Furthermore, a “treatment method” button 34 is provided, and by clicking the “treatment method” button 34 using an input device, treatment methods and coping methods stored in a memory or an external storage device, nationwide An information file of a specialist or the like can be displayed on the display screen of the display device.

  By operating as described above, the data acquisition system 1 according to the present embodiment performs the data acquisition method for determining the possibility of the onset of Dravet syndrome according to the present invention, thereby Data for determining the possibility of onset can be acquired. According to the data acquired using the data acquisition system 1 according to the present embodiment, the possibility of the onset of Dravet syndrome can be determined easily and with high accuracy.

  Since the data acquisition system 1 according to the present embodiment includes the personal information file generation unit 10, the inquiry result file can be associated with the personal information file and stored in an external storage device or the like. However, when there is no need to save the inquiry result file or the determination result, the personal information file generation unit 10 may be omitted.

  Moreover, although embodiment which the determination part 30 determines possibility of onset of Dravet syndrome based on the determination prediction score was demonstrated, this invention is not limited to this, <I. As described in Data Acquisition Method for Determining the Possibility of Onset of Dravet Syndrome>, whether the answer result recorded in the interview result file includes the risk factors shown in (a) to (i) above No may be used as a criterion, or the number of risk factors included in the answer result may be used as a criterion.

Furthermore, the data acquisition system according to the present invention is configured to variously modify and modify the data acquisition system 1 described above and output a display screen using an output device, or to generate a determination result file in which the determination result is recorded The determination result, the personal information file, and the inquiry result file are associated with each other and stored in an external storage device, or the input device and the display device, the personal information file generation unit 10, the inquiry unit 20, and the determination unit 30 are stored. And a system configuration in which the two are connected via a network. That is, the data acquisition system according to the present invention includes systems having any configuration as long as the possibility of the onset of Dravet syndrome is determined by executing the data acquisition method according to the present invention.

Finally, each block of the data acquisition system 1, particularly the personal information file generation unit 10, the inquiry unit 20, and the determination unit 30 may be configured by hardware logic, or by software using a CPU as follows. It may be realized.

  That is, the data acquisition system 1 includes a CPU (central processing unit) that executes instructions of a control program that realizes each function, a ROM (read only memory) that stores the program, and a RAM (random access memory) that expands the program. And a storage device (recording medium) such as a memory for storing the program and various data. An object of the present invention is a recording medium on which a program code (execution format program, intermediate code program, source program) of a control program of the data acquisition system 1 which is software for realizing the functions described above is recorded in a computer-readable manner. This can also be achieved by supplying the data acquisition system 1 and reading and executing the program code recorded on the recording medium by the computer (or CPU or MPU).

  Examples of the recording medium include tapes such as magnetic tapes and cassette tapes, magnetic disks such as floppy (registered trademark) disks / hard disks, and disks including optical disks such as CD-ROM / MO / MD / DVD / CD-R. Card system such as IC card, IC card (including memory card) / optical card, or semiconductor memory system such as mask ROM / EPROM / EEPROM / flash ROM.

  The data acquisition system 1 may be configured to be connectable to a communication network, and the program code may be supplied via the communication network. The communication network is not particularly limited. For example, the Internet, intranet, extranet, LAN, ISDN, VAN, CATV communication network, virtual private network, telephone line network, mobile communication network, satellite communication. A net or the like is available. Further, the transmission medium constituting the communication network is not particularly limited. For example, even in the case of wired such as IEEE 1394, USB, power line carrier, cable TV line, telephone line, ADSL line, etc., infrared rays such as IrDA and remote control, Bluetooth ( (Registered trademark), 802.11 wireless, HDR, mobile phone network, satellite line, terrestrial digital network, and the like can also be used. The present invention can also be realized in the form of a computer data signal embedded in a carrier wave in which the program code is embodied by electronic transmission.

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and a comparative example and FIGS. 7-10, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[Example 1: Identification of risk factors for predicting onset of Dravet syndrome]
(1) Selection of Dravet syndrome patients and non-Dravet syndrome patients First, he had experience of developing febrile convulsions under 1 year of age. At the time of the implementation of this example, the condition and name of each disease became clear. 112 Japanese patients were selected. Next, 96 patients who satisfy all of the following conditions (i) to (v) were selected using a medical chart or the like for each symptom at the time of less than 1 year of age.
(I) febrile convulsions before the age of 1 (ii) normal development at onset (iii) a follow-up period of 3 years or more (iv) not convulsions caused by central nervous system infections (v) basics Next, the 96 patients were classified into two groups of Dravet syndrome patients (46 persons) and non-Dravet syndrome patients (50 persons) according to their medical records. In this classification, Dravet's syndrome is defined as normal development at the onset, general convulsions, half-body convulsions, or partial seizures, most often causing convulsions during fever, followed by myoclonic seizures. A case in which developmental delay gradually appears after 2 years of age, in which epilepsy firing appears and resistance to treatment occurs. In addition, for cases with recurrent seizures, head CT and MRI were performed to confirm that there were no brain lesions. Furthermore, the developmental classification was divided into three groups: normal, mild lag, and moderate to severe lag.

  In addition, the 50 patients classified as non-Dravet syndrome were further classified into a group that started with febrile convulsions and a group that later combined epilepsy.

  Table 2 shows the clinical characteristics of the 96 patients at the time of implementation of this example.

(2) Retrospective analysis of clinical data of Dravet syndrome patients and non-Dravet syndrome patients Retrospective analysis of clinical data at the age of 1 year for the above Dravet syndrome patients (46) and non-Dravet syndrome patients (50) Was analyzed. As clinical factors, the age at onset, family history of convulsive disease, total number of seizures up to 1 year, the presence of prolonged seizures of 10 minutes or more, seizure type, provoking factors, and EEG findings were examined.

(2-1) Age at onset (onset age) and total number of seizures up to 1 year (total number of seizures) First, examine age at onset (onset age) and total number of seizures up to 1 year (total number of seizures) It was. In the above Dravet syndrome patients (46) and non-Dravet syndrome patients (50), the total was taken. FIGS. 7A and 7B are graphs showing the distribution.

Next, in these distributions, we decided to find a threshold to divide Dravet syndrome from non-Dravet syndrome. First, when the age of onset is 5 months or less (less than 6 months) is divided as a threshold that is prone to Dravet syndrome, 6 months or less is divided as a threshold, 7 months or less is divided as a threshold, and so on. The number of patients was counted. Next, the sensitivity for examining the ability to detect a disease (sensitivity) and the specificity for examining the ability not to misdiagnose a non-sick person as a disease were calculated by the following equations.
Sensitivity = a / (a + c)
Specificity = d / (b + d)
In the formula, a to d are as shown in Table 3 below.

  The calculated “sensitivity” and “1-specificity” were continuously plotted on a graph, and ROC curve analysis (Receiver Operating Characteristic Curve Analysis) was performed. The result is shown in FIG. In FIG. 7C, the threshold value was obtained by searching for a point where both “sensitivity” and “specificity” had high values. Similarly, the cases in which the number of seizures was divided as threshold values were totaled, ROC curve analysis was performed, and points with good “sensitivity” and “specificity” were determined to obtain threshold values. As a result, the onset age was 7 months or less (less than 8 months), and the total number of seizures was 5 or more as risk factors for Dravet syndrome.

(2-2) Other clinical factors About the above-mentioned Dravet syndrome patients (46) and non-Dravet syndrome patients (50), the medical history at the age of 1 and the family history of convulsive diseases up to the age of 1 The total number of seizures, the presence of prolonged seizures of 10 minutes or more, seizure type, provoking factors, and EEG findings were examined. The results are shown in Table 4.

(2-3) Mutation of SCN1A gene in Dravet syndrome patient and non-Dravet syndrome patient The SCN1A gene of the above Dravet syndrome patients (46) and non-Dravet syndrome patients (50) was analyzed. Genomic DNA was extracted from the patient's peripheral blood using a DNA extraction kit (WB kit; Nippon gene, Tokyo, Japan), and all exons were amplified by PCR. The PCR was 25 μl containing 50 ng human genomic DNA, 20 pmol each primer, 0.8 mM dNTPs, 1 × reaction buffer, 1.5 mM MgCl ₂ , 0.7 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA). Performed in the reaction. The resulting PCR product was purified using PCR products pre-sequencing kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Subsequently, a sequence reaction was performed using Big Dye Terminator FS ready-reaction kit (Applied Biosystems), and the nucleotide sequence was determined using a fluorescent sequencer (ABI PRISM3100 sequencer; Applied Biosystems). The results of SCN1A gene analysis thus obtained are shown in Table 5. As shown in Table 5, there were 19 missense mutations (41%) in Dravet syndrome, 19 cases (41%) in truncation mutation, and 6 missense mutations (12%) in non-Dravet syndrome.

  The genomic sequence and cDNA sequence of the SCN1A gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The primer sequences for detecting the SCN1A gene genome used are as follows.

Exon 1 amplification
Sense primer: 5'-tcatggcacagttcctgtatc-3 '(SEQ ID NO: 3)
Antisense primer: 5'-gcagtaggcaattagcagcaa-3 '(SEQ ID NO: 30)
Exon 2 amplification
Sense primer: 5'-tggggcactttagaaattgtg -3 '(SEQ ID NO: 4)
Antisense primer: 5'- tgacaaagatgcaaaatgagag -3 '(SEQ ID NO: 31)
Exon 3 amplification
Sense primer: 5'-gcagtttgggcttttcaatg-3 '(SEQ ID NO: 5)
Antisense primer: 5'-tgagcattgtcctcttgctg-3 '(SEQ ID NO: 32)
Exon 4 amplification
Sense primer: 5'-agggctacgtttcatttgtatg-3 '(SEQ ID NO: 6)
Antisense primer: 5'- tgtgctaaattgaaatccagag -3 '(SEQ ID NO: 33)
Exon 5 amplification
Sense primer: 5'-CAGCTCTTCGCACTTTCAGA-3 '(SEQ ID NO: 7)
Antisense primer: 5'-TCAAGCAGAGAAGGATGCTGA-3 '(SEQ ID NO: 34)
Exon 6 amplification
Sense primer: 5'-agcgttgcaaacattcttgg-3 '(SEQ ID NO: 8)
Antisense primer: 5'-gggatatccagcccctcaag-3 '(SEQ ID NO: 35)
Exon 7 amplification
Sense primer: 5'- gacaaatacttgtgcctttgaatg -3 '(SEQ ID NO: 9)
Antisense primer: 5'-acataatctcatactttatcaaaaacc-3 '(SEQ ID NO: 36)
Exon 8 amplification
Sense primer: 5'-gaaatggaggtgttgaaaatgc-3 '(SEQ ID NO: 10)
Antisense primer: 5'-aatccttggcatcactctgc-3 '(SEQ ID NO: 37)
Exon 9 amplification
Sense primer: 5'- agtacagggtgctatgaccaac -3 '(SEQ ID NO: 11)
Antisense primer: 5'- tcctcatacaaccacctgctc -3 '(SEQ ID NO: 38)
Exon 10 amplification
Sense primer: 5'-tctccaaaagccttcattagg -3 '(SEQ ID NO: 12)
Antisense primer: 5'-ttctaattctccccctctctcc -3 '(SEQ ID NO: 39)
Exon 11 amplification
Sense primer: 5'- tcctcattctttaatcccaagg -3 '(SEQ ID NO: 13)
Antisense primer: 5'- gccgttctgtagaaacactgg -3 '(SEQ ID NO: 40)
Exon 12 amplification
Sense primer: 5'-gtcagaaatatctgccatcacc-3 '(SEQ ID NO: 14)
Antisense primer: 5'-gaatgcactattcccaactcac-3 '(SEQ ID NO: 41)
Exon 13 amplification
Sense primer: 5'-tgggctctatgtgtgtgtctg-3 '(SEQ ID NO: 15)
Antisense primer: 5'-ggaagcatgaaggatggttg-3 '(SEQ ID NO: 42)
Exon 14 amplification
Sense primer: 5'-tacttcgcgtttccacaagg -3 '(SEQ ID NO: 16)
Antisense primer: 5'-gctatgcaagaaccctgattg-3 '(SEQ ID NO: 43)
Exon 15 amplification
Sense primer: 5'- atgagcctgagacggttagg -3 '(SEQ ID NO: 17)
Antisense primer: 5'-atacatgtgccatgctggtg-3 '(SEQ ID NO: 44)
Exon 16 amplification
Sense primer: 5'-tgctgtggtgtttccttctc-3 '(SEQ ID NO: 18)
Antisense primer: 5'-tgtattcataccttcccacacc -3 '(SEQ ID NO: 45)
Exon 17 amplification
Sense primer: 5'-aaaagggttagcacagacaatg-3 '(SEQ ID NO: 19)
Antisense primer: 5'- attgggcagatataatcaaagc -3 '(SEQ ID NO: 46)
Exon 18 amplification
Sense primer: 5'- cacacagctgatgaatgtgc -3 '(SEQ ID NO: 20)
Antisense primer: 5'-tgaagggctacactttctgg-3 '(SEQ ID NO: 47)
Exon 19 amplification
Sense primer: 5'-tctgccctcctattccaatg-3 '(SEQ ID NO: 21)
Antisense primer: 5'- gcccttgtcttccagaaatg -3 '(SEQ ID NO: 48)
Exon 20 amplification
Sense primer: 5'- aaaaattacatcctttacatcaaactg -3 '(SEQ ID NO: 22)
Antisense primer: 5'-ttttgcatgcatagattttcc -3 '(SEQ ID NO: 49)
Exon 21 amplification
Sense primer: 5'- tgaaccttgcttttacatatcc -3 '(SEQ ID NO: 23)
Antisense primer: 5'-acccatctgggctcataaac-3 '(SEQ ID NO: 50)
Exon 22 amplification
Sense primer: 5'-tgtcttggtccaaaatctgtg-3 '(SEQ ID NO: 24)
Antisense primer: 5'-ttggtcgtttatgctttattcg-3 '(SEQ ID NO: 51)
Exon 23 amplification
Sense primer: 5'- ccctaaaggccaatttcagg -3 '(SEQ ID NO: 25)
Antisense primer: 5'- atttggcagagaaaacactcc -3 '(SEQ ID NO: 52)
Exon 24 amplification
Sense primer: 5'-gagatttgggggtgtttgtc-3 '(SEQ ID NO: 26)
Antisense primer: 5'-ggattgtaatggggtgcttc-3 '(SEQ ID NO: 53)
Exon 25 amplification
Sense primer: 5'-caaaaatcagggccaatgac-3 '(SEQ ID NO: 27)
Antisense primer: 5'-tgattgctgggatgatcttg-3 '(SEQ ID NO: 54)
Exon 26 (1) amplification
Sense primer: 5'- aggactctgaaccttaccttgg -3 '(SEQ ID NO: 28)
Antisense primer: 5'-ccatgaatcgctcttccatc-3 '(SEQ ID NO: 55)
Exon 26 (2) amplification
Sense primer: 5'-tgtgggaacccatctgttg-3 '(SEQ ID NO: 29)
Antisense primer: 5'-gtttgctgacaaggggtcac-3 '(SEQ ID NO: 56)
(3) Identification of risk factors In order to investigate the relationship between each clinical factor and Dravet syndrome, Chi-square test or Fisher test is performed on the data obtained in (2-1) to (2-3) above. Group significance tests were performed. If the p value was less than 0.01, it was determined to be statistically significant. In addition, a test based on the odds ratio was also performed. The results are shown in Table 4.

The following 9 clinical factors were considered to be risk factors for Dravet syndrome.
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) There is a truncation mutation in the SCN1A gene (g) There is experience in partial seizures (h) There is a missense mutation in the SCN1A gene (i) There is experience in myoclonic seizures.

(4) Risk factor scoring For the risk factor identified in (3) above, the odds ratio value (odds value) calculated by the statistical processing and the p-value in the Chi-square test or Fisher test are calculated. Based on each risk factor, a risk score (risk score) of 1 to 3 was assigned (Table 4). Specifically, 1 <odds value <20 or 0.01 ≧ p> 1.0E-5 is 1 point, 20 ≦ odds value <40 or 1.0E-5 ≧ p> 1.0E-10 is 2 points. 40 ≦ Odds value or 1.0E−10 ≧ p was set to 3 points. The results are shown in Table 4. In Table 4, as a result of the statistical calculation in the above (3), items that are not considered as risk factors, specifically, items that have an odds value of 1 or less or p> 0.01 are described as 0 points. did.

[Example 2: Detection efficiency of Dravet syndrome using the data acquisition method according to the present invention]
The detection efficiency of Dravet syndrome was examined using the data acquisition method according to the present invention. The risk score of each risk factor determined in Example 1 was applied to each patient, each symptom of the patient was quantified, and a determination prediction score was obtained. The distribution of judgment prediction scores was compared between Dravet syndrome (46 people) and non-Dravet syndrome (50 people). The result is shown in FIG.

  As shown in FIG. 8, when the determination prediction score is 7 points or more, it is determined that the risk of becoming a Dravet syndrome increases. Thus, although there was a slight overlap of scores, it was shown that both groups can be distinguished with high accuracy by adapting the score table in this way.

[Example 3: Comparative study of seizure prognosis using the data acquisition method according to the present invention]
It was examined whether the seizure prognosis can be compared using the data acquired by the data acquisition method according to the present invention. First, after combining Dravet syndrome (46 people) and non-Dravet syndrome (50 people) without distinguishing, the prediction prediction score was divided into a group of 6 points or less and a group of 7 points or more, and Kaplan-Meier method was used. Used to compare the suppression course of seizures up to 96 months after birth. The result is shown in FIG.

  As shown in FIG. 9, the number of seizures attenuated with time in the group with a judgment prediction score of 6 or less. On the other hand, it became clear that seizures continued in the group with a predicted prediction score of 7 or more until 96 months after birth. This indicates that the data acquisition method according to the present invention selects patients with intractable epilepsy with high accuracy.

[Example 4: Detection efficiency of Dravet syndrome using data acquisition method according to the present invention (2)]
In the data acquisition method according to the present invention, among the nine risk factors shown in (a) to (i) of Example 1, the combination of risk factors to be used was changed, and the detection efficiency of Dravet syndrome was examined. Specifically, (1) when all nine risk factors are used, (2) when the seven risk factors shown in the above (a) to (e), (g), and (i) are used, ( 3) When using eight risk factors excluding the risk factors shown in (e) above, (4) using the six risk factors shown in (a) to (d), (g), and (i) above. (5) When using a total of five risk factors including the four risk factors shown in (b) to (e) above and the risk factor shown in either (f) or (h), ( 6) When using the four risk factors shown in (b) to (e) above, (7) When using the three risk factors shown in (b) to (d) above, (8) (b) and (b) above In the case of using the two risk factors shown in (d), the detection efficiency of Dravet syndrome is increased using the same method as in Example 2. It was 討. The result is shown to Fig.10 (a)-(h).

  As shown in FIG. 10 (a), (1) when all nine risk factors were used, it was determined that the risk of becoming a Dravet syndrome increased when the determination prediction score was 7 points or more. (2) When the seven risk factors shown in the above (a) to (e), (g), and (i) are used for the determination, as shown in FIG. Above, it was determined that the risk of developing Dravet syndrome increased. (3) When using 8 risk factors excluding the risk factors shown in (e) above, as shown in FIG. 10 (c), if the judgment prediction score is 6 points or more, the risk of becoming Dravet syndrome is high. It was determined to be. (4) When using the six risk factors shown in the above (a) to (d), (g), and (i), as shown in FIG. The risk of developing Dravet syndrome was determined to be high. (5) When using a total of five risk factors including the four risk factors shown in (b) to (e) above and the risk factor shown in either (f) or (h), FIG. As shown in e), when the judgment prediction score is 6 points or more, it is judged that the risk of becoming Dravet syndrome increases. (6) When using the four risk factors shown in the above (b) to (e), as shown in FIG. 10 (f), when the judgment prediction score is 5 points or more, the risk of becoming a Dravet syndrome increases. It was judged. (7) When the three risk factors shown in the above (b) to (d) are used, as shown in FIG. 10 (g), when the judgment prediction score is 5 points or more, the risk of becoming a Dravet syndrome increases. It was judged. (8) When the two risk factors shown in the above (b) and (d) are used, as shown in FIG. 10 (h), when the judgment prediction score is 3 or more, the risk of becoming a Dravet syndrome increases. It was judged.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  As described above, the present invention obtains data for determining the possibility of developing Dravet syndrome by determining the presence or absence of a specific risk factor that can be detected even in infants younger than one year. Therefore, it is possible to select patients with intractable epilepsy who need treatment by a specialist from benign febrile convulsions in the early stage of the disease under 1 year old. Therefore, the present invention can be applied to industries in the medical field such as medical devices and diagnostic kits.

FIGS. 1A and 1B are diagrams showing an inquiry table according to an embodiment of the present invention. FIG. 2 is a functional block diagram of a data acquisition system for determining the possibility of development of Dravet syndrome according to an embodiment of the present invention. FIG. 3 is a flowchart showing the operation of the data acquisition system for determining the possibility of the onset of Dravet syndrome according to an embodiment of the present invention. FIG. 4 is a diagram showing a personal data input screen displayed by the personal information file generation unit of the data acquisition system for determining the possibility of the onset of Dravet syndrome according to an embodiment of the present invention. FIG. 5 is a diagram showing an inquiry screen displayed by the inquiry unit of the data acquisition system for determining the possibility of the onset of Dravet syndrome according to an embodiment of the present invention. FIG. 6 is a diagram showing a determination result screen displayed by the determination unit of the data acquisition system for determining the possibility of the onset of Dravet syndrome according to an embodiment of the present invention. 7 (a) and 7 (b) are diagrams showing the distribution of the number of seizures up to the age of onset (onset age) and 1 year of age, respectively, in Dravet syndrome patients and non-Dravet syndrome patients (50), 7 (c) is a graph showing the results of ROC curve analysis by continuously plotting “sensitivity” and “1-specificity” calculated based on FIGS. 7 (a) and (b) on a graph. is there. FIG. 8 is a diagram showing the results of measuring the detection efficiency of Dravet syndrome in the example according to the present invention. FIG. 9 is a diagram showing the results of observing the course of seizure suppression up to 96 months after birth using the Kaplan-Meier method in the examples according to the present invention. FIGS. 10A to 10H are diagrams showing the results of measuring the detection efficiency of Dravet syndrome in another example according to the present invention.

Explanation of symbols

1 Data acquisition system (system)
10 Personal information file generator 20 Interview department (interview means)
30 determination unit (determination means)
40 Storage unit (storage means)

Claims (6)

A method for acquiring data for determining the possibility of the onset of Dravet syndrome,
Based on the interview result or test result for the presence of holdings of risk factors shown in the following (a) ~ (i), set for each of the risk factors shown in the following (a) ~ (i) Including a step of calculating a sum of the obtained risk scores as a judgment prediction score,
When the risk score set for the risk factor shown in (a) below is 3,
The risk score set with respect to the risk factor shown in the following (b)-(i) is 3, 3, 2, 2, 2, 1, 1, 1, respectively .
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience The method according to claim 1, further comprising a step of calculating the number of risk factors included in the inquiry result or the test result among the risk factors indicated in (a) to (i) above. A system for acquiring data for determining the possibility of the onset of Dravet syndrome,
Interview means,
Storage means;
Determination means,
The interrogation means conducts an interrogation or examination in which at least one risk factor among the risk factors shown in the following (a) to (i) is asked whether the subject possesses the risk factor, and the interrogation result Or generate an interview result file that records the test results ,
The storage means stores a risk score set for each of the risk factors shown in the following (a) to (i):
The determination means determines whether at least one risk factor among the risk factors shown in the following (a) to (i) is included in the interview result or the test result recorded in the interview result file: And among the risk factors shown in the following (a) to (i) using the risk score stored in the storage means, the risk factors included in the interview result or test result are respectively Calculated as the judgment prediction score,
The relative relationship of the risk score set for each of the risk factors shown in (a) to (i) below is:
When the risk score set for the risk factor shown in (a) below is 3,
The risk score set with respect to the risk factors shown in the following (b) to (i) is 3, 3, 2, 2, 2, 1, 1, 1, respectively .
(A) Has experience of convulsions of the body (b) Has experience of convulsions (c) Has 5 or more seizures (d) Onset of thermal convulsions is less than 8 months old (e) Induction of convulsions by bathing (F) SCN1A gene truncation mutation exists (g) Partial seizure experience (h) SCN1A gene missense mutation exists (i) Myoclonic seizure experience The system according to claim 3 , wherein the determination means calculates the number of risk factors included in the inquiry result or the test result among the risk factors indicated in (a) to (i). A program for operating the system according to claim 3 or 4 for causing a computer to function as the inquiry means and the determination means. A computer-readable recording medium on which the program according to claim 5 is recorded.

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