# Patent

## Title
Method for assessment of potential for development of dravet syndrome and use thereof

## Abstract

Provided is a method of assessing a potential for development of Dravet syndrome with high accuracy, and use thereof. The method according to the present invention of assessing a potential for development of Dravet syndrome includes, with use of a sample taken from a subject, detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1, and detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/574,977, filed Oct. 10, 2012, which is a national stage of International (PCT) Patent Application Serial No. PCT/JP2011/051636, filed Jan. 27, 2011, which claims the benefit of and priority to JP Patent Application No. 2010-018705 filed Jan. 29, 2010, the contents of each of which are hereby incorporated by reference for all purposes.


TECHNICAL FIELD
The present invention relates to a method for assessing a potential for development of Dravet syndrome, and use thereof.
BACKGROUND ART
Febrile seizure is a disease that has a high incidence rate of approximately 8% in infants. A main symptom of febrile seizure is known as a continuation of generalized convulsions for 1 to 5 minutes while suffering a fever at or over 38° C. caused by a viral or bacterial infection such as a cold, or microbism. Most cases of febrile seizure that have an onset of between 6 months after birth and around 5 years old cure by the time when the patient turns 6 years old. In many cases, febrile seizure does not require active treatment. Therefore, febrile seizure is considered, in principle, as a benign disease.
However, among patients whose onset of febrile seizure was under the age of one, other than the patients of the benign disease which cease as a regular febrile seizure, there are some patients who suffer from convulsions continuously even after turning 6 years old, and there are some patients who are patients of Dravet syndrome (previously called “Severe Myoclonic Epilepsy in Infancy; SMEI”), which are patients of an intractable epilepsy disease.
The patients of Dravet syndrome are triggered in the onset of convulsions under the age of one. An average age of the onset of convulsions for patients of Dravet syndrome is 4 months to 6 months after birth. An incipient seizure of convulsion for a patient of Dravet syndrome is generally a systemic or a unilateral tonic-clonic or clonic convulsion, and during infancy, may lead to status epilepticus. Moreover, this convulsion seizure is easily induced by fever or bathing.
Conventionally, febrile seizure was diagnosed and treated by a general pediatrician or a family doctor, and Dravet syndrome is also diagnosed based on clinical symptoms characteristic of Dravet syndrome such as convulsion seizure or the like. However, by the time the patients of Dravet syndrome turn two to three years old, that is around when the clinical symptoms of Dravet syndrome have all appeared, these patients would have suffered repetitive convulsions many times and would often have had experienced critical conditions such as status epilepticus or the like. Hence, it is necessary to develop a diagnosis method that enables detection of Dravet syndrome in its possible earliest stage by a general pediatrician or family doctor, who is engaged in primary medical care. Detection of Dravet syndrome at an earlier stage would allow for the patent to see an epilepsy specialist in advance, which would allow for preventing the patient from reaching a critical condition.
Recently, it has been reported that 30% to 80% of Dravet syndrome patients find missense mutation (mutation causing a substitution of an amino acid) and nonsense mutation (mutation causing protein synthesis to stop in an incomplete state) on a SCN1A gene that encodes a voltage-gated sodium ion channel NaV1.1 α-subunit type 1 (see Non Patent Literature 1 and 2). From such a point in view, attempts have been made to examine abnormalities in the SCN1A gene to diagnose Dravet syndrome on the basis of genes.
For example, Patent Literatures 1 to 4 disclose that mutation of the SCN1A gene is related to SMEI. Moreover, Patent Literatures 1 to 4 disclose that SMEI can be diagnosed by use of the mutation of the SCN1A gene as an indicator.
More specifically, Patent Literature 1 discloses the diagnosis of SMEI by assessing a plurality of mutations on the SCN1A gene that relate to SMEI, as a whole.
Patent Literature 2 discloses the diagnosis of SMEI performed by detecting a presence of a mutation that frequently occurs on the SCN1A gene of a nerve that is affected by SMEI.
Patent Literatures 3 and 4 disclose a method of diagnosing epilepsy syndromes including SMEI and syndromes associated with SMEI, by detecting a change in the SCN1A gene and confirming whether that change is known as being related to SMEI or a syndrome associated with SMEI or is known as not being related to SMEI or a syndrome associated with SMEI.
CITATION LIST
Patent Literature
Patent Literature 1

    Japanese Patent Application Publication, Tokukai, No. 2004-329153 A (Publication Date: Nov. 25, 2004)


Patent Literature 2

    Japanese Patent Application Publication, Tokukai, No. 2004-73058 A (Publication Date: Mar. 11, 2004)


Patent Literature 3

    Published Japanese Translations of PCT International Publication, Tokuhyo, No. 2008-546376 A (Publication Date: Dec. 25, 2008)


Patent Literature 4

    Published Japanese Translations of PCT International Publication, Tokuhyo, No. 2006-524490 A (Publication Date: Nov. 2, 2006)


Non Patent Literature
Non Patent Literature 1

    Sugawara T, Mazaki-Miyazaki E, Fukushima K, Shimomura J, Fujiwara T, Hamano S, Inoue Y, Yamakawa K. 2002. Frequent mutations of SCN1A in severe myoclonic epilepsy in infancy. Neurology 58: 1122-1124.


Non Patent Literature 2

    Ohmori I, Ouchida M, Ohtsuka Y, Oka E, Shimizu K. 2002. Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy. Biochem Biophys Res Commun 295: 17-23.


Non Patent Literature 3

    Escayg A, Heils A, MacDonald B T, Haug K, Sander T, and Meisler M H. 2001. A novel SCN1A mutation associated with generalized epilepsy with febrile seizures plus- and prevalence of variants in patients with epilepsy. Am J Hum Genet. 68: 866-873.


SUMMARY OF INVENTION
Technical Problem
As described above, the mutation on the SCN1A gene is found in an extremely large number of Dravet syndrome patients (30% to 80%). However, it is becoming revealed that the presence of a mutation on the SCN1A gene does not necessarily mean that the symptoms of Dravet syndrome would appear.
For example, Non Patent Literature 3 reports that not just the patients of the intractable Dravet syndrome, but also patients of febrile seizure and patients with a certain kind of benign epilepsy (e.g. GEFS+ (Generalized epilepsy with febrile seizure plus)) have a mutation on the SCN1A gene.
As such, the mutation on the SCN1A gene is not a phenomenon specific to Dravet syndrome. Hence, the conventional methods of examining just the abnormalities on the SCN1A gene as described in Patent Literatures 1 to 4 can be said as insufficient for specifically diagnosing Dravet syndrome.
Therefore, in order to distinguish between the patients with benign febrile seizure and the patients with Dravet syndrome and to allow for the patients with Dravet syndrome to receive appropriate treatment by a specialist, further development is required in techniques for more accurately diagnosing Dravet syndrome.
The present invention is accomplished in view of the foregoing problems, and an object thereof is to provide a method of (specifically) assessing with high accuracy a potential for development of Dravet syndrome.
Solution to Problem
Patients of GEFS+ and the patients of Dravet syndrome are common in a point that the SCN1A gene has a mutation. Meanwhile, the inventors performed diligent study based on their unique point of view of focusing on the difference in malignancy between the diseases; they considered that the development of Dravet syndrome is related to not just the mutation on the SCN1A gene but also another factor, and that another cause is related to the worsening and intractableness of Dravet syndrome. As a result, the inventors uniquely found out that many Dravet syndrome patients have a mutation on the SCN1A gene and further a mutation on the CACNA1A gene that encodes a P/Q type voltage-gated calcium ion channel CaV2.1 α1 subunit.
Furthermore, based on this finding, the inventors produced a rat having both the mutations on the SCN1A gene and the CACNA1A gene, and demonstrated that the rat having both the mutations on the SCN1A gene and the CACNA1A gene experienced more serious convulsion seizures as compared to rats having just the mutation on the SCN1A gene.
Based on these results of analyzing genes and animal testing results, it was found that the potential for development of Dravet syndrome can be assessed with high accuracy by detecting mutations for both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, and accomplished the present invention.
Namely, the present invention includes the following inventions.
An assessment method according to the present invention is a method of assessing a potential for development of Dravet syndrome, the method including:
with use of a sample taken from a subject,
detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and
detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. It is preferable that the assessment method according to the present invention is a method of obtaining data for assessing potential for development of Dravet syndrome.
A kit according to the present invention is a kit for assessing a potential for development of Dravet syndrome, the kit comprising:
a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and
a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. The kit according to the present invention may be a kit for obtaining data for assessing a potential for development of Dravet syndrome.
A model animal of Dravet syndrome according to the present invention has a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
A production method according to the present invention of a model animal of Dravet syndrome is a method of producing the model animal of Dravet syndrome described above, which method includes:
introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and
introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
A cell according to the present invention has a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
A method of producing a cell according to the present invention is a method of producing the cell described above, which method includes:
introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1; and
introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
A screening method according to the present invention of a drug for treating Dravet syndrome includes:
administering a candidate agent to the model animal of Dravet syndrome according to the present invention; and
assessing whether or not the administering of the candidate agent has made Dravet syndrome improve or cure in the model animal of Dravet syndrome.
A screening method according to the present invention of a drug for treating Dravet syndrome includes:
administering a candidate agent to the cell according to the present invention; and
assessing whether or not the administering of the candidate agent has made activity of the voltage-gated sodium ion channel NaV1.1 and/or activity of the voltage-gated calcium ion channel CaV2.1 change in the cell.
For a fuller understanding of the nature and advantages of the invention, reference should be made to the ensuing detailed description taken in conjunction with the accompanying drawings.
Advantageous Effects of Invention
The method according to the present invention of assessing a potential for development of Dravet syndrome allows for obtaining data for assessing the potential for development of Dravet syndrome, by detecting mutations for both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
Patients of GEFS+, being a benign epilepsy, inherit the mutation of the SCN1A gene within the family. In comparison, in patients of Dravet syndrome, approximately 90% of the mutations on SCN1A gene are de novo mutation, i.e. are anew mutations in which a mutation arises even though their parents have no mutation. As such, although the GEFS+ patients and the Dravet syndrome patients are common in that a mutation is on the SCN1A gene, the cause for the difference in malignancy of the disease was unknown. However, it was clarified by the present inventors for the first time, that the presence of mutations on both the SCN1A gene and the CACNA1A gene is related to the worsening and intractableness of Dravet syndrome.
As described above, reports have already been made that a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, referred to as “sodium ion channel α1 subunit”) is related to the development of Dravet syndrome. However, no reports have been made whatsoever that Dravet syndrome is related to a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 (hereinafter, referred to as “calcium ion channel α1 subunit”).
Reports have been made that a mutation on α subunit other than the α1 subunit of voltage-gated calcium ion channel CaV2.1 is associated with Dravet syndrome (see Iori Ohmori et. Al., Neurobiology of Disease 32 (2008) 349-354). More specifically, this literature (Iori Ohmori et. Al.) discloses that a mutation on β4 subunit of voltage-gated calcium ion channel CaV2.1 (hereinafter, simply referred to as “calcium ion channel β4 subunit”) is associated with Dravet syndrome.
However, the foregoing literature strongly teaches regarding Dravet syndrome that a mutation on the “calcium ion channel β4 subunit” is important together with the mutation on the “α-subunit of sodium ion channel NaV1.1”. This description in the literature hinders a motivation to arrive at a point that a mutation suitable for detecting Dravet syndrome is present in the calcium ion channel α1 subunit.
In the first place, a skilled person would not arrive at considering, just because a relationship of a mutation on a specific subunit with a disease is known for a specific channel, that other subunits would also have a mutation related to that disease. At least, the finding that the voltage-gated sodium ion channel NaV1.1 is related to Dravet syndrome is only known regarding the mutation on the “α1 subunit”; this does not give motivation for analyzing mutations on other subunits.
As to a mutation on the calcium ion channel α1 subunit, reports have been made stating a relationship with (1) epixodic ataxia type 2 (characterized in paroxysmal cerebellar ataxia), (2) familial hemiplegic migraine type 1 (e.g. hemiplegia, hemianopsia, dysphagia, throbbing headache), and (3) spinocerebellar ataxia type 6 (e.g. ataxic gait, limb ataxia, cerebellar dysarthria, nystagmus) (see Keiji IMOTO et al., “Igaku no Ayumi” (Development in Medical Science), Vol. 201, No. 13 (Issued Jun. 29, 2002); Taiji TSUNEMI et al., “Igaku no Ayumi” (Development in Medical Science), Vol. 201, No. 13 (Issued Jun. 29, 2002)). However, the diseases of (1) to (3) all show no symptoms of epilepsy, and neither are diseases related to Dravet syndrome. At least, although the finding regarding the mutation on the calcium ion channel α1 subunit is known as related to the diseases of (1) to (3), it is not one that gives motivation for analyzing a mutation on the calcium ion channel α1 subunit in Dravet syndrome, which disease is completely unrelated to the diseases of (1) to (3).
The assessment method according to the present invention detects a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1. Hence, it is possible to detect Dravet syndrome with high accuracy. Consequently, the assessment method of the present invention brings about an effect that it is possible to improve reliability of a potential for detecting Dravet syndrome as compared to the conventional method by detecting a mutation on the SCN1A gene. Furthermore, detection of a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 is possible even with an infant under the age of one. Hence, according to the assessment method of the present invention, an effect is brought about that data for assessing the potential for development in Dravet syndrome can be obtained from a patient in an early stage of development or in a stage prior to the onset of the intractable disease, in particular of an infant under the age of one.
Moreover, as shown in Examples later described, an effect is brought about that by detecting a mutation on both α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1, the detection sensitivity of Dravet syndrome patients dramatically improve.
Furthermore, with use of the kit according to the present invention, it is possible to easily detect the mutation on both α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1. Hence, the kit according to the present invention is useful for a general pediatrician to screen, at an early stage of disease of under the age of one, a patient of Dravet syndrome that requires treatment by a specialist, among benign febrile epilepsy.
By using the assessment method and kit according to the present invention, it is possible to detect the patients of Dravet syndrome with high accuracy at the point in time of an age under one, which is an age difficult to detect until now. Moreover, by sending a blood sample to an examination center and examining its abnormal genes, it is possible to detect a Dravet syndrome patient with high accuracy even in a private hospital at a remote location or the like.
Moreover, the Dravet syndrome model animal and cell according to the present invention can be usefully used for resolving a development mechanism of the intractable Dravet syndrome, and for development and the like of medicament for Dravet syndrome.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view illustrating an amino acid sequence of a protein encoded by a human SCN1A gene and an amino acid sequence of a protein encoded by a rat SCN1A gene.
FIG. 2 is a view illustrating a result of performing function analysis of sodium ion channel, by use of patch clamping. Illustrated in (a) is a typical example of a sodium current effected by a change in potential of a normal sodium ion channel and a mutant sodium ion channel. Illustrated in (b) is a result of examining a time constant (i) at inactivation.
FIG. 3 is a view illustrating a result of performing function analysis of a sodium ion channel, by use of patch clamping. Illustrated in (a) is a current-voltage relationship, illustrated in (b) is an activation curve of the sodium ion channel, illustrated in (c) is an inactivation curve of the sodium ion channel, and illustrated in (d) is a recovery curve from the inactivation of the sodium ion channel.
FIG. 4 is a view illustrating a result of performing function analysis of a sodium ion channel, by use of patch clamping. Illustrated in (a) is a sodium current flowing in the sodium ion channel, and illustrated in (b) is a relative value (%) of a persistent sodium current amount flowing into the sodium ion channel.
FIG. 5 is a view illustrating genotypes of parent rats (P), first filial generation (F1) rats, and second filial generation (F2) rats. Illustrated in (a) is a view showing genotypes of the parent rats (P) and the F1 rats. Illustrated in (b) are genotypes of the F1 rats and the F2 rats.
FIG. 6 is a view illustrating a method of identifying genotypes of the Scn1a gene and the Cacna1a gene of the F2 rat, by sequencing.
FIG. 7 is a view illustrating a method of identifying a genotype of the Scn1a gene of the F2 rat, by restriction enzyme digestion. Illustrated in (a) is a nucleotide sequence of where mutation is on a mutant Scn1a gene (N1417H), and a nucleotide sequence of a wild-type Scn1a gene corresponding to that nucleotide sequence of the mutant Scn1a gene. Illustrated in (b) is a size of a DNA fragment expected by the restriction enzyme digestion. Illustrated in (c) is a result of electrophoresis.
FIG. 8 is a view illustrating a method of identifying a genotype of the Cacna1a gene in a F2 rat, by restriction enzyme digestion. Illustrated in (a) is a nucleotide sequence of where a mutation is on a mutant Cacna1a gene (M251K), and a nucleotide sequence of a wild-type Cacna1a gene corresponding to that nucleotide sequence of the mutant Cacna1a gene. Illustrated in (b) is a size of a DNA fragment expected by the restriction enzyme digestion. Illustrated in (c) is a result of electrophoresis.
FIG. 9 is a view illustrating a result of examining an effect of a mutation on the Cacna1a gene, in a rat having a mutation on Scn1a gene. Illustrated in (a) is a body temperature at a time of convulsion onset (convulsion threshold), illustrated in (b) is a severity score, and illustrated in (c) is duration of the convulsion.
FIG. 10 is a view illustrating a part of an electroencephalogram at a time of seizure of a rat in group (3) (Scn1a mutant (homo)+Cacna1a mutant (hetero)).
FIG. 11 is a view illustrating an amino acid sequence of a protein encoded by a human CACNA1A gene and an amino acid sequence of a protein encoded by a rat Cacna1a gene.
FIG. 12 is a view illustrating a result of detecting a mutation on voltage-gated calcium ion channel CaV2.1 α1 subunit. Illustrated in (a) is a result of a mutation analysis of the CACNA1A gene, and schematically illustrated in (b) is a part where a mutation was detected in the calcium ion channel α1 subunit.
FIG. 13 is a view illustrating a result of performing function analysis of the calcium ion channel, by use of patch clamping. Illustrated in (a) is a barium current record effected by a change in potential of a normal calcium ion channel and a mutant calcium ion channel. Illustrated in (b) is a current-voltage relationship, and illustrated in (c) is peak current value (pA), a total charge (pF) and a peak current density (pA/pF).
FIG. 14 is a view illustrating a result of performing function analysis of a calcium ion channel, by use of patch clamping. Illustrated in (a) is an activation curve of the calcium ion channel. Illustrated in (b) is a time constant of voltage-gated activation of the calcium ion channel. Illustrated in (c) is a time constant of voltage-gated activation at 20 mV. Illustrated in (d) is a voltage-gated inactivation curve of the calcium ion channel. Illustrated in (e) is a result of examining fast and slow inactivation time constants (i).



DESCRIPTION OF EMBODIMENTS
Described below is an embodiment of the present invention in detail. The present invention is not limited to this embodiment however, and may be carried out in modes of various modifications that are made within the described scope. Moreover, all academic literature and patent literature disclosed in the present specification are incorporated as reference. Unless mentioned otherwise, numerical ranges expressed as “A to B” denote “not less than A but not more than B”.
1. Assessment Method According to the Present Invention
A method of assessing a potential for development of Dravet syndrome according to the present invention (also referred to as “assessment method according to the present invention”) is a method of assessing a potential for development of Dravet syndrome in a subject, by use of a sample taken from the subject. In the present specification, the “potential for development of Dravet syndrome” includes a potential that the Dravet syndrome is already developed and a potential that the Dravet syndrome may develop in the future.
The subject is not particularly limited, and may be an individual in which Dravet syndrome has developed (individual having potential for development) or may be an individual in which the Dravet syndrome is not developed (individual having no potential for development). Out of such individuals, it is preferable that the subject is of either infants or children.
The assessment method according to the present invention, more specifically, may be of any method as long as it includes, with use of a sample taken from the subject: detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. Any other specific configurations are not limited in particular.
In the embodiment, the voltage-gated sodium ion channel NaV1.1 is made up of α-subunit type 1, β1 subunit, and β2 subunit. The β1 subunit and the β2 subunit are auxiliary subunits.
The α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, referred to as “sodium ion channel α1 subunit”) is for example a polypeptide that is registered as GenBank accession No. AB093548 (i.e. amino acid sequence represented by SEQ ID NO. 1). Moreover, an example of a gene that encodes the α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (hereinafter, called “sodium ion channel α1 subunit gene”) is, as a SCN1A gene, a polynucleotide made up of a nucleotide sequence registered as GenBank accession No. AB093548 (i.e. nucleotide sequence represented by SEQ ID NO. 2).
The voltage-gated calcium ion channel CaV2.1 is made up of α-subunit type 1, β subunit, γ subunit, and α2δ subunit.
The voltage-gated calcium ion channel CaV2.1 α-subunit type 1 (hereinafter, referred to as “calcium ion channel α1 subunit”) is for example a polypeptide registered as GenBank accession No. NM 023035 (i.e. amino acid sequence represented by SEQ ID NO. 3). Moreover, an example of a gene that codes the α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 (hereinafter, referred to as “calcium ion channel α1 subunit gene”) is, as a CACNA1A gene, a polynucleotide made up of a nucleotide sequence registered as GenBank accession No. NM 023035 (i.e. nucleotide sequence represented by SEQ ID NO. 4).
In the present specification, for example, the term “α-subunit type 1 of voltage-gated sodium ion channel NaV1.1” denotes “α-subunit type 1 protein of voltage-gated sodium ion channel NaV1.1”. Namely, in the present specification, unless it is clearly described as indicating a gene as like “gene encoding α-subunit type 1 of voltage-gated sodium ion channel NaV1.1” or “α-subunit type 1 gene of voltage-gated sodium ion channel NaV1.1”, a protein is denoted. This way of description is not limited to the “α-subunit type 1 of voltage-gated sodium ion channel NaV1.1”, and “α-subunit type 1 of voltage-gated calcium ion channel CaV2.1” is denoted similarly thereto.
It is preferable that the assessment method according to the present invention further includes, in addition to the detecting the presence of a mutation: detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
The assessment method according to the present invention may include, for detecting the mutation, a step such as preprocessing of a sample that is taken from the living organism. The “preprocessing” indicates, for example, a process of extracting DNA from the sample taken from the living organism, a process of extracting RNA from the sample taken from the living organism, a process of extracting protein from the sample taken from the living organism, or like process. These preprocessing can be carried out by use of conventionally known methods.
The assessment method according to the present invention may be a method of obtaining data for assessing a potential for development of Dravet syndrome. In this case, the present invention does not include the step of determining by a doctor.
(1-1. Detecting Presence of Mutation)
In the present specification, the “detecting presence of a mutation” denotes detecting a presence of a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and detecting a presence of a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1.
In the assessment method according to the present invention, the detecting of the presence of a mutation on the α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 may be performed prior to the detecting of the presence of a mutation on the α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 or vice versa, or may be performed simultaneously.
By detecting the presence of a mutation in both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, it is possible to obtain the data that enables accurate assessment of the potential for development of Dravet syndrome.
The mutation detected by the assessment method according to the present invention may be a mutation on a nucleotide sequence of a gene, or may be a mutation on an amino acid of a protein. The “mutation on a nucleotide sequence of a gene” is not limited in particular by a specific kind of mutation as long as it is a mutation that causes a change in an amino acid sequence of a protein encoded by a gene having a mutation on its nucleotide sequence as compared to an amino acid sequence of a protein encoded by a wild-type gene. Mutations on the nucleotide sequence as described above are, for example, missense mutation (substitution of an amino acid), nonsense mutation (synthesis of an amino acid stops in an incomplete state), frameshift (a frame of an amino acid codon shifts caused by insertion or deletion of a nucleotide, which causes an amino acid sequence downstream of the mutation position to change, thereby losing its original function), splicing defect (e.g. deletion of its exon region), minority nucleotide insertion or deletion (a part of amino acids is newly added or lost however its downstream is synthesized as normal amino acid), and minor deletion of an exon region (loss of one or a plurality of exon). Variations on the nucleotide sequence as such are not limited to mutations, and may also include gene polymorphism.
Moreover, in the assessment method according to the present invention, the detection of mutation may be performed to mRNA, cDNA, and proteins obtained from these genes.
In the present specification, “gene” can be replaced by “polynucleotide”, “nucleic acid” or “nucleic acid molecule”.
The “polynucleotide” means a polymer of a nucleotide. Hence, the term “gene” in the present specification includes not only the double stranded DNA but also a single stranded DNA and RNA (mRNA, etc.) such as a sense strand and an antisense strand that construct the double stranded DNA.
The term “DNA” encompasses cDNA, genomic DNA and the like that can be obtained by cloning, a chemically synthesized technique or a combination of these. Namely, DNA may be a “genome” type DNA, which includes a noncoding sequence such as intron or the like that is a form included in an animal genome, or may be a cDNA obtained from mRNA with use of reverse transcriptase or polymerase, i.e. “transcription” type DNA that does not include a noncoding sequence such as intron.
Examples of the mutation on sodium ion channel α1 subunit is, more specifically, a mutation of asparagine (N) at position 1417 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, and is preferably a mutation of asparagine (N) at position 1417 to histidine (H) (“N1417H” in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4249 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4249 with cytosine (C) (A4249C).
Moreover, another embodiment is a mutation of lysine (K) at position 1027 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of lysine (K) at position 1027 to a stop codon (“K1027X” in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 3079 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 3079 with thymine (T) (A3079T).
Yet another embodiment is a mutation of glutamine (Q) at position 1450 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glutamine (Q) at position 1450 to arginine (R) (“Q1450R” in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4349 of a nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4349 with guanine (G) (A4349G).
Yet another embodiment is a mutation of threonine (T) at position 1082 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1086 by frameshift (“T1082fsX1086” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3245 of a nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3245 (C3245del).
Yet another embodiment is a mutation of lysine (K) at position 547 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 570 by frameshift (“K547fsX570” in Table 1). This mutation is caused by, for example, a mutation at position 1641 of the nucleotide sequence of the sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably an insertion of adenine (A) into position 1641 (1641insA).
Yet another embodiment is a mutation of proline (P) at position 707 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 714 by frameshift (“P707fsX714” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2120 in the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 2120 (C2120del).
Yet another embodiment is a mutation of arginine (R) at position 712 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of arginine (R) at position 712 to a stop codon (“R712X” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2134 of the nucleotide sequence of the sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2134 with thymine (T) (C2134T).
Yet another embodiment is a mutation of leucine (L) at position 1265 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of leucine (L) at position 1265 to proline (P) (“L1265P” in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 3794 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 3794 with cytosine (C) (T3794C).
Yet another embodiment is a deletion of amino acid of positions 460 to 554 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1 (“Exon10” in Table 1). This mutation is caused by, for example, a deletion of nucleotide at positions 1378 to 1662 (exon 10) of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2.
Yet another embodiment is a mutation of arginine (R) at position 865 of the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of arginine (R) at position 865 to a stop codon (“R865X” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2593 of the nucleotide sequence of the sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2593 with thymine (T) (C2593T).
Yet another embodiment is a mutation of arginine (R) at position 1648 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 1648 with cysteine (C) (“R1648C” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4942 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4942 with thymine (T) (C4942T).
Yet another embodiment is a mutation of arginine (R) at position 931 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 931 with cysteine (C) (“R931C” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 2791 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 2791 with thymine (T) (C2791T).
Yet another embodiment is a mutation of arginine (R) at position 501 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 543 by frameshift (“R501fsX543” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 1502 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of guanine (G) at position 1502 (G1502del).
Yet another embodiment is a mutation of alanine (A) at position 1002 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1009 by frameshift (“A1002fsX1009” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3006 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3006.
Yet another embodiment is a mutation of phenylalanine (F) at position 902 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of phenylalanine (F) at position 902 to cysteine (C) (“F902C” in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 2705 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of thymine (T) at position 2705 with guanine (G) (T2705G).
Yet another embodiment is a mutation of glycine (G) at position 1674 of the amino acid sequence of aodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of glycine (G) at position 1674 with arginine (R) (“G1674R” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 5020 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 5020 with cytosine (C) (G5020C).
Yet another embodiment is a mutation of valine (V) at position 1390 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of valine (V) at position 1390 to methionine (M) (“V1390M” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 4168 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 4168 with adenine (A) (G4168A).
Yet another embodiment is a mutation of serine (S) at position 607 in the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 622 by frameshift (“S607fsX622” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 1820 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 1820 (C1820del).
Yet another embodiment is a mutation of tryptophan (W) at position 1434 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of tryptophan (W) at position 1434 with arginine (R) (“W1434R” in Table 1). This mutation is caused by a mutation of thymine (T) at position 4300 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 4300 with cytosine (C) (T4300C).
Yet another embodiment is a mutation of threonine (T) at position 1909 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of threonine (T) at position 1909 with isoleucine (I) (“T1909I” in Table 1). This mutation is caused by, for example, the mutation of cytosine (C) at position 5726 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 5726 with thymine (T) (C5726T).
Yet another embodiment is a mutation of phenylalanine (F) at position 1289 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a deletion of phenylalanine (F) at position 1289 (“F1289del” in Table 1). This mutation is caused by, for example, mutations of cytosine (C) at position 3867, thymine (T) at position 3868, and thymine (T) at position 3869, each in the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C) at position 3867, thymine (T) at position 3868, and thymine (T) at position 3869.
Yet another embodiment is a mutation of tryptophan (W) at position 1271 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of tryptophan (W) at position 1271 to a stop codon (“W1271X” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 3812 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 3812 with adenine (A) (G3812A).
Yet another embodiment is a mutation of alanine (A) at position 1429 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1443 by frameshift (“A1429fsX1443” in Table 1). This mutation is caused by, for example, a mutation of five-nucleotide CCACA between positions 4286 to 4290 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of CCACA at positions 4286 to 4290, with ATGTCC.
Moreover, another embodiment is a mutation of glycine (G) at position 1880 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1881 by frameshift (“G1880fsX1881” in Table 1). This mutation is caused by mutation of six-nucleotide AGAGAT between positions 5640 to 5645 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of six-nucleotide AGAGAT between positions 5640 to 5645 with CTAGAGTA.
Yet another embodiment is a mutation of alanine (A) at position 1685 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of alanine (A) at position 1685 with aspartic acid (D) (“A1685D” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 5054 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 5054 with adenine (A) (C5054A).
Yet another embodiment is a mutation of arginine (R) at position 377 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of arginine (R) at position 377 with leucine (L) (“R377L” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 1130 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by substitution of guanine (G) at position 1130 with thymine (T) (G1130T).
Yet another embodiment is a mutation of serine (S) at position 1574 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to a stop codon (“S1574X” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4721 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4721 with guanine (G) (C4721G).
Yet another embodiment is a mutation of glutamine (Q) at position 1277 in the amino acid sequence of the sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glutamine (Q) at position 1277 to a stop codon (“Q1277X” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 3829 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 3829 with thymine (T) (C3829T).
Yet another embodiment is a mutation of glycine (G) at position 177 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glycine (G) at position 177 to arginine (R) (“G177R” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 529 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 529 with adenine (A) (G529A).
Yet another embodiment is a mutation of glutamic acid (E) at position 788 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of glutamic acid (E) at position 788 with lysine (K) (“E788K” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2362 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of guanine (G) at position 2362 with adenine (A) (G2362A).
Yet another embodiment is splicing defects at positions 1429 and subsequent positions of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a deletion of positions at and subsequent to 1429 (“intron 21” in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at a second last position (position −2), preferably a mutation in which adenine (A) at a second last position (position −2) of the intron 21 is substituted with guanine (G) (intron 21 ag(−2)gg), out of the intron 21 present in a genomic DNA between positions 4284 and 4285 of the nucleotide sequence of sodium ion channel al subunit gene represented by SEQ ID NO. 2. Namely, the second last nucleotide sequence of the intron 21 present in the genomic DNA between positions 4284 (exon 21) and 4285 (exon 22) of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2 is ag, and is connected to the beginning of the exon 22. Generally, since the ag of the intron 21 is a recognition sequence that is spliced, in a case in which an abnormality exists at that position, the intron is determined as still continuing, which thus causes the exon immediately after (or in its downstream) to be abnormally spliced. This makes it impossible to generate a full-length protein.
Yet another embodiment is a mutation of serine (S) at position 1574 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to a stop codon (“S1574X” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4721 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of cytosine (C) at position 4721 with guanine (G).
Yet another embodiment is a mutation of valine (V) at position 212 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a substitution of valine (V) at position 212 with alanine (A) (“V212A” in Table 1). This mutation is caused by, for example, a mutation of thymine (T) at position 635 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of thymine (T) at position 635 with cytosine (C) (T635C).
Yet another embodiment is a mutation of threonine (T) at position 1539 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of threonine (T) at position 1539 to proline (P) (“T1539P” in Table 1). This mutation is caused by, for example, a mutation of adenine (A) at position 4615 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of adenine (A) at position 4615 with cytosine (C) (A4615C).
Yet another embodiment is a mutation of tryptophan (W) at position 738 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably by mutation causing generation of a stop codon at position 746 by frameshift (“W738fsX746” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2213 in the nucleotide sequence of the sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a deletion of guanine (G) at position 2213 (G2213del).
Yet another embodiment is a mutation of leucine (L) at position 990 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably by a mutation of leucine (L) at position 990 to phenylalanine (F) (“L990F” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 2970 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 2970 with thymine (T) (G2970T).
Yet another embodiment is a mutation of glycine (G) at position 163 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of glycine (G) at position 163 to glutamic acid (E) (“G163E” in Table 1). This mutation is caused by, for example, a mutation of guanine (G) at position 488 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of guanine (G) at position 488 with adenine (A) (G488A).
Yet another embodiment is a mutation of alanine (A) at position 1662 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation of alanine (A) at position 1662 to valine (V) (“A1662V” in Table 1). This mutation is caused by, for example, a mutation of cytosine (C) at position 4985 in the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably by a substitution of cytosine (C) at position 4985 with thymine (T) (C4985T).
Yet another embodiment is a mutation of lysine (K) at position 1057 of the amino acid sequence of sodium ion channel α1 subunit represented by SEQ ID NO. 1, preferably a mutation causing generation of a stop codon at position 1073 by frameshift (“K1057fsX1073” in Table 1). This mutation is caused by, for example, a mutation of 14 nucleotides (AGAAAGACAGTTGT) between positions 3170 to 3183 of the nucleotide sequence of sodium ion channel α1 subunit gene represented by SEQ ID NO. 2, preferably a substitution of the 14 nucleotides between the positions 3170 to 3183 with TCATTCTGTATG.
It is needless to say that the mutation on the α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 is not limited to the mutations exemplified above.
Examples of mutations on a calcium ion channel α1 subunit encompass, more specifically, a mutation on methionine (M) at position 249 of an amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on methionine (M) at position 249 to lysine (K) (“M249K” in Table 2). This mutation is caused by, for example, a mutation on thymidine (T) at position 746 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a mutation on thymidine (T) at position 746 substituted with adenine (A) (T746A).
Moreover, another embodiment is a mutation on glutamic acid (E) at position 921 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on glutamic acid (E) at position 921 to aspartic acid (D) (“E921D” in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 2762 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of adenine (A) at position 2762 with cytosine (C) (A2762C).
Yet another embodiment is a mutation on glutamic acid (E) at position 996 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on glutamic acid (E) at position 996 to valine (V) (“E996V” in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 2987 of the nucleotide sequence of the calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of adenine (A) at position 2987 with thymine (T) (A2987T).
Yet another embodiment is a mutation on arginine (R) at position 1126 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on arginine (R) at position 1126 to histidine (H) (“R1126H” in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 3377 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of guanine (G) at position 3377 with adenine (A) (G3377A).
Yet another embodiment is a mutation on arginine (R) at position 2201 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on arginine (R) at position 2201 to glutamine (Q) (“R2201Q” in Table 2). This mutation is, for example, caused by mutation on guanine (G) at position 6602 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of guanine (G) at position 6602 with adenine (A) (G6602A).
Yet another embodiment is a mutation on glycine (G) at position 1108 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on glycine (G) at position 1108 to serine (S) (“G1108S” in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 3322 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of guanine (G) at position 3322 with adenine (A) (G3322A).
Yet another embodiment is a mutation on alanine (A) at position 924 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation of alanine (A) at position 924 to glycine (G) (“A924G” in Table 2). This mutation is, for example, caused by a mutation on cytosine (C) at position 2771 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a substitution of cytosine (C) at position 2771 with guanine (G) (C2771G).
Yet another embodiment is a mutation on glycine (G) at position 266 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on glycine (G) at position 266 to serine (S) (“G266S” in Table 2). This mutation is, for example, caused by a mutation on guanine (G) at position 796 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of guanine (G) at position 796 with adenine (A) (G796A).
Yet another embodiment is a mutation on lysine (K) at position 472 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3, preferably a mutation on lysine (K) at position 472 to arginine (R) (“K472R” in Table 2). This mutation is, for example, caused by a mutation on adenine (A) at position 1415 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably by a substitution of adenine (A) at position 1415 with guanine (G) (A1415G).
Yet another embodiment is a deletion of an amino acid at positions 2202 to 2205 of the amino acid sequence of calcium ion channel α1 subunit represented by SEQ ID NO. 3 (“del2202-2205” in Table 2). This mutation is, for example, caused by a mutation on ACCAGGAGCGGG of positions 6605 to 6616 of the nucleotide sequence of calcium ion channel α1 subunit gene represented by SEQ ID NO. 4, preferably a deletion of ACCAGGAGCGGG at positions 6605 to 6616 (del6605-6616).
It is needless to say that the mutations related to the function abnormality of voltage-gated calcium ion channel CaV2.1 is not limited to the mutations exemplified above.
The mutations on the foregoing sodium ion channel α1 subunit and the mutations on the foregoing calcium ion channel α1 subunit are organized into Table 1 and Table 2.







TABLE 1





Mutations on sodium ion channel a1 subunit


















1289delF,
G177R,
Q1450R,
T1539P,


A1002fsX1009,
G1880fsX1881,
R1648C,
T1909I,


A1429fsX1443,
intron 21,
R377L,
V1390M,


A1662V,
K1027X,
R501fsX543,
V212A,


A1685D,
K1057fsX1073,
R712X,
W1271X,


E788K,
K547fsX570,
R865X,
W1434R,


Exon10*,
L1265P,
R931C,
W738fsX746,


F902C,
L990F,
S1574X,
N1417H,


G163E,
P707fsX714,
S607fsX622,


G1674R,
Q1277X,
T1082fsX1086,





Exon10* exon deletion detected by MLPA













TABLE 2





Mutations on calcium ion channel a1 subunit



















A924G,
E996V,
K472R,



del 2202-2205,
G1108S,
R1126H,



E921D,
G266S,
R2201Q,



M249K










In the assessment method according to the present invention, it is preferable that the mutation on sodium ion channel α1 subunit is, more specifically, at least one mutation shown in Table 1, and the mutation on calcium ion channel α1 subunit is, more specifically, at least one mutation shown in Table 2.
The assessment method according to the present invention is not limited in particular of how the presence of a mutation is detected for both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, and any method conventionally known may be used.
Examples of methods for detecting the presence of the mutation for both the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene encompass mutation detecting methods such as DNA sequencing method using PCR, SSCP method (Single strand conformation polymorphism), DHPLC method (denaturing high performance liquid chromatography); polymorphism detecting methods using real-time PCR or DNA chip; method of detecting micro-deletion of exons of a gene; and Northern blotting, RT-PCR, Real-time PCR, and cDNA array, each of which detect an increase and decrease of mRNA. Moreover, when the presence of mutation is to be detected for both of sodium ion channel α1 subunit protein and calcium ion channel α1 subunit protein, a method such as Western blotting, immunostaining, protein array or the like may be used.
The following provides more specific descriptions, by separating into the following embodiments: (A) an embodiment detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, (B) an embodiment detecting a gene mutation with use of mRNA (cDNA) included in a sample taken from a subject, and (C) an embodiment detecting a protein mutation with use of a protein included in a sample taken from a subject.
(A) Embodiment Using Genomic DNA
In the embodiment detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, first, a genomic DNA is extracted from the sample taken from the subject, by a conventionally known method.
The “sample taken from the subject” is not limited in particular, and any sample from which a genomic DNA is extractable can be used. More specifically, a sample of blood, oral mucosa cells, bone marrow fluid, hair, various organs, peripheral lymphocytes, synovial cells or the like can be used. Moreover, cells taken from the subject may be cultured and a genomic DNA may be extracted from its proliferated cells.
Moreover, the extracted genomic DNA may be used upon amplification by a gene amplification method generally performed, for example, PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification), SDA (Strand Displacement Amplification), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids).
The method of detecting the presence of mutation for both the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene with use of a sample including a genomic DNA prepared as such is not limited in particular, and examples encompass allele-specific oligonucleotide probe method, Oligonucleotide Ligation Assay, PCR-SSCP, PCR-CFLP, PCR-PHFA, invader method, RCA (Rolling Circle Amplification), Primer Oligo Base Extension, and like methods.
More specifically, a polynucleotide for detecting a mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a polynucleotide for detecting a mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 are used to detect, from the genomic DNA, the presence of a mutation for both the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene.
The “polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1” is indicative of a polynucleotide having a nucleotide sequence complementary to a set region in a sodium ion channel α1 subunit gene (e.g. a region including an exon, or boundary region between an exon and an intron). The “polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1” is indicative of a polynucleotide having a nucleotide sequence complementary to a set region in the calcium ion channel α1 subunit gene (e.g. a region including an exon, or a boundary region between an exon and an intron).
The “polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1” is, more specifically, a polynucleotide having a nucleotide sequence represented by any one of SEQ ID NOs.: 5, 6, and 9 to 62, for example. Moreover, the “polynucleotide for detecting a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1” is, more specifically, a polynucleotide having a nucleotide sequence represented by any one of SEQ ID NOs.: 7, 8, and 63 to 143.
Two kinds of the polynucleotides may be used in combination as a primer pair, or one kind may be used as a probe. When the two kinds are used in combination as a primer pair, the polynucleotides may be used in combinations as exemplified in Examples described later.
When two kinds of the polynucleotides are used in combination as a primer pair, it is possible, for example, to amplify a set region in the gene by PCR with use of a corresponding primer pair, and thereafter, directly sequence the obtained PCR product, to detect the presence of the mutation in the gene.
Moreover, two kinds of fluorescence-labeled polynucleotides may be used as a primer pair, to amplify a set region of the gene by PCR, perform gel electrophoresis or capillary electrophoresis with the obtained PCR product, and study a strength of the signals, so as to detect the presence of a mutation in the gene.
Moreover, when one kind of the polynucleotides is to be solely used as a probe, the presence of the mutation on the gene can be detected by, for example, digesting the genomic DNA with an appropriate restriction enzyme and detecting a difference in size of the digested genomic DNA fragment by Southern blotting or the like.
As such, by detecting the presence of mutations for both the sodium ion channel α1 subunit gene and calcium ion channel α1 subunit gene with use of the genomic DNA included in the sample taken from the subject, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found on both the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene in the obtained data, it can be assessed that the subject has a high potential for development of Dravet syndrome.
The primer pair and probe used in the method of detecting the mutation may be prepared by a DNA synthesizer or the like, as in law of the art.
(B) Embodiment Using mRNA (cDNA)
In the embodiment of detecting a mutation with use of mRNA included in a sample taken from the subject, first, mRNA is extracted from a sample taken from the subject, with use of a conventionally known method.
The “sample taken from the subject” is not limited in particular, and any sample can be used as long as mRNA can be extracted therefrom and a gene that can be subjected to the detection of a mutation is expressed or is possibly expressed. The “sample taken from the subject” is preferably, for example, a peripheral blood leukemic cell, dermal fibroblast, oral mucosa cell, neuron, or muscle cell, each of a patient.
Subsequently, cDNA is prepared from the extracted mRNA by reverse transcription reaction. Furthermore, if necessary, the obtained cDNA may be amplified by a gene amplification method generally performed, for example PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification), SDA (Strand Displacement Amplification), LAMP (Loop-Mediated Isothermal Amplification), and ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids).
The method of detecting the presence of the mutation for both the sodium ion channel α1 subunit gene and calcium ion channel α1 subunit gene with use of a sample including cDNA prepared as such is not limited in particular; whether or not a gene mutation is present in a subject that is subjected to mutation detection may be detected with use of a similar method as with a case in which a gene mutation is detected with use of a genomic DNA, as described in the foregoing “(A) Embodiment using genomic DNA”.
By detecting the presence of the mutation for both the sodium ion channel α1 subunit gene and calcium ion channel α1 subunit gene with use of mRNA included in the sample that is taken from the subject, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found in both the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene in the obtained data, it can be assessed that the subject has a high potential for the development of Dravet syndrome.
(C) Embodiment Using Protein
In the embodiment of detecting a mutation using protein included in the sample taken from a subject, first, protein is extracted from the sample taken from the subject with use of a conventionally known method.
The sample taken from the subject is not limited in particular, and may be any sample from which protein is extractable and in which both of sodium ion channel α1 subunit protein and calcium ion channel α1 subunit protein are expressed or is possibly expressed.
The method of detecting the presence of mutation for both the sodium ion channel α1 subunit protein and the calcium ion channel α1 subunit protein with use of the sample including the protein prepared as described above is not limited in particular, and for example an antibody which specifically recognizes just a protein having a set mutation may be prepared, to detect the mutation by ELISA or Western blotting using that antibody. In the present specification, the term “protein” may be used replaceable with “polypeptide” or “peptide”.
Moreover, mutation may be detected by isolating a protein to be subjected to the mutation detection from the sample including the foregoing protein, and digesting the isolated protein with an enzyme or the like directly or if necessary, with use of a protein sequencer or a mass spectrometer. Alternatively, the mutation may be detected on the basis of an isoelectric point of the isolated protein.
As such, by detecting the presence of a mutation for both of the sodium ion channel α1 subunit protein and the calcium ion channel α1 subunit protein with use of a protein included in the sample taken from the subject, it is possible to obtain data for assessing potential for development of Dravet syndrome in the subject. More specifically, when a mutation is found on both the sodium ion channel α1 subunit protein and the calcium ion channel α1 subunit protein in the obtained data, it is possible to assess that the subject has a high potential for development of Dravet syndrome.
(1-2. Step of Detecting Change in Activity)
In the present specification, the “step of detecting change in activity” is indicative of a step of detecting whether activity of the voltage-gated sodium ion channel NaV1.1 has changed and a step of detecting whether activity of the voltage-gated calcium ion channel CaV2.1 has changed.
As described in Examples later described, it is considered that the change in activity in both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1, caused by the mutations on the sodium ion channel α1 subunit and on the calcium ion channel α1 subunit, is related to the development of Dravet syndrome. Hence, although the mutation on the sodium ion channel α1 subunit is not particularly limited in its position, it is preferable that the mutation is on a position that causes a change in the activity of the voltage-gated sodium ion channel NaV1.1. Moreover, although the mutation on the calcium ion channel α1 subunit is not particularly limited in its position, it is preferable that the mutation is on a position that causes a change in the activity of the voltage-gated calcium ion channel CaV2.1.
Here, the activity of the voltage-gated sodium ion channel NaV1.1 is, more specifically, an activity to allow transmission of sodium ion (Na+) into the cell by depending on membrane potential. The change in activity of the voltage-gated sodium ion channel NaV1.1 is not limited in particular, and may be an increase of activity or may be a decrease in activity. Namely, the change is sufficiently one that shows an abnormality in the activity of the voltage-gated sodium ion channel NaV1.1.
In the present specification, “the activity of the voltage-gated sodium ion channel NaV1.1 is changed” indicates that an activity of a mutant voltage-gated sodium ion channel NaV1.1 including the sodium ion channel α1 subunit on which the mutation is present is of a value having a statistically significant difference based on a significant test as compared to an activity of a wild-type voltage-gated sodium ion channel NaV1.1, and preferably indicates that p is equal to or smaller than 0.05 by Student's t-test.
Moreover, the activity of the voltage-gated calcium ion channel CaV2.1 is, more specifically, an activity that causes transmission of calcium ion (Ca2+) into the cell to be membrane voltage-gated. The change in function of the voltage-gated calcium ion channel CaV2.1 is not particularly limited, and may be the increase of activity or the decrease in activity. Namely, the change is sufficiently one that shows abnormality of the activity of the voltage-gated calcium ion channel CaV2.1.
In the present specification, “the activity of the voltage-gated calcium ion channel CaV2.1 is changed” indicates that the activity of a mutant voltage-gated calcium ion channel CaV2.1 including the calcium ion channel α1 subunit on which a mutation is present is of a value having a statistically significant difference based on a significant test as compared to an activity of a wild-type voltage-gated calcium ion channel CaV2.1, and preferably indicates that p is equal to or smaller than 0.05 by Student's t-test.
An example of a method of detecting that the activity of the voltage-gated sodium ion channel NaV1.1 is changed by the mutation is, for example, (i) coexpressing, in a culture cell with use of a expression vector or the like, a sodium ion channel α1 subunit gene on which a mutation is present with a wild-type gene (β1 subunit gene and β2 subunit gene) that encodes a subunit (β1 subunit and β2 subunit) other than the α1 subunit, which wild-type gene makes up the voltage-gated sodium ion channel NaV1.1, (ii) measuring an activity of the voltage-gated sodium ion channel NaV1.1 on which a mutation is present with use of the obtained cultured cell, and (iii) comparing the activity with an activity of the wild-type voltage-gated sodium ion channel NaV1.1, to confirm whether the activity of the voltage-gated sodium ion channel NaV1.1 is changed. The method of measuring the activity of the voltage-gated sodium ion channel NaV1.1 is not particularly limited, however it is possible to use the conventionally known patch clamping, imaging with use of a fluorescence probe, or like method.
An example of a method of detecting that the activity of the voltage-gated calcium ion channel CaV2.1 is changed by mutation is by (i) coexpressing, in a culture cell with use of an expression vector or the like, a calcium ion channel α1 subunit gene on which a mutation is present with a wild-type gene (β subunit gene, γ subunit gene, and α2δ subunit gene) that encodes a subunit (β subunit, γ subunit, and α2δ subunit) other than the α1 subunit, which wild-type gene makes up the voltage-gated calcium ion channel CaV2.1, (ii) measuring, with the obtained cultured cell, an activity of the voltage-gated calcium ion channel CaV2.1 on which the mutation is present, and (iii) comparing the activity with an activity of the wild-type voltage-gated calcium ion channel CaV2.1, to confirm whether the activity of the voltage-gated calcium ion channel CaV2.1 is changed. The method of measuring the activity of the voltage-gated calcium ion channel CaV2.1 is not limited in particular, however it is possible to use the conventionally known patch clamping, imaging using an optical probe, a calcium indicator, or a caged compound, for example.
The assessment method according to the present invention, since it includes the foregoing configuration, it is possible to obtain data for assessing a potential for development of Dravet syndrome in the subject. Hence, with the assessment method according to the present invention, it is possible to find out, with high accuracy and at an early stage, Dravet syndrome having the unfavorable prognosis, which thus allows for preparing a treatment management system by an epilepsy specialist from an earlier stage for a Dravet syndrome patient. As a result, it is possible to improve treatment intervention of the patient, reduce the mental burden on their families, and reduce the economical burden. Furthermore, it is possible to provide appropriate treatment for the patient of Dravet syndrome; this hence reduces medical fees.
2. Kit According to the Present Invention
The present invention also encompasses a kit for assessing the potential for development of Dravet syndrome, with use of the assessment method according to the present invention (hereinafter, also referred simply as “kit according to the present invention”).
The kit according to the present invention is not limited in its specific configuration in particular as long as it includes at least a reagent for detecting the presence of mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and a reagent for detecting the presence of mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1.
As described in “1. Assessment method according to the present invention”, ways considered to detect the presence of mutation for both of α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 are (A) detecting a gene mutation with use of a genomic DNA included in a sample taken from a subject, or (B) detecting a gene mutation with use of mRNA (cDNA) included in a sample taken from the subject.
Hence, in order to detect a mutation using a genomic DNA included in the sample taken from the subject or mRNA (cDNA) included in the sample taken from the subject, the kit according to the present invention includes a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated sodium ion channel NaV1.1; and a polynucleotide being used for determining a mutation on α-subunit type 1 of voltage-gated calcium ion channel CaV2.1. Such polynucleotides can be used as, for example, a primer pair or a probe. These polynucleotides may be included solely or may be included as a combination of a plurality thereof.
The kit according to the present invention encompasses (A) a kit for detecting a mutation with use of a genomic DNA included in a sample taken from a subject and (B) a kit for detecting a mutation with use of a mRNA (cDNA) included in a sample taken from a subject. The following specifically describes the reagents included in the embodiments of the kits in (A) or (B).
(A) Kit for Detecting Mutation with Use of Genomic DNA Included in Sample Taken from Subject
For example, a configuration of the sodium ion channel α1 subunit and the calcium ion channel α1 subunit may include a primer pair designed so as to allow amplification of the genomic DNA of each of the genes or a part of its region, or may include a probe designed so that one of genomic DNA of its mutant type or wild-type can be specifically detected. These polynucleotides are as described in the foregoing (A) Embodiment using genomic DNA in “1. Assessment method according to the present invention”, so hence its description has been omitted here.
Furthermore, such a kit may be configured to include, in addition to the primer pair or probe, a combination of one or more reagent necessary for detecting the presence of the mutation on the gene, such as a reagent used in PCR, Southern blotting, and nucleic acid sequencing.
The reagent is selected and employed as appropriate in accordance with the detection method of the present invention, and examples thereof are dATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, the kit according to the present invention may include a suitable buffer solution and a washing solution that can be used in the PCR, Southern blotting, and nucleic acid sequencing.
(B) Kit Detecting Mutation with Use of mRNA (cDNA) Included in Sample Taken from Subject
For example, a configuration of the sodium ion channel α1 subunit and the calcium ion channel α1 subunit may include a primer pair designed so as to allow amplification of the cDNA of each of the genes or a part of its region, or include a probe designed so that one of mRNA of its mutant type or wild-type can be specifically detected. These polynucleotides are as described in (B) Embodiment using mRNA (cDNA) in “1. Assessment method according to the present invention”, so hence its description has been omitted here.
Furthermore, such a kit may be configured to include, in addition to the primer pair or probe, a combination of one or more reagent necessary for detecting the presence of a mutation on the gene, such as a reagent used in RT-PCR, Northern blotting, nucleic acid sequencing or the like.
The reagent is selected and employed as appropriate in accordance with the detection method of the present invention, and examples thereof are dATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, the kit according to the present invention may include a suitable buffer solution and a washing solution that can be used in RT-PCR, Northern blotting, and nucleic acid sequencing.
The kit according to the present invention may include the exemplified configuration in any combination. Furthermore, the kit may include other reagents other than the reagents exemplified above.
As described in the item “1. Assessment method according to the present invention”, in order to detect the presence of mutation for both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, it is further considerable to (C) detect the mutation with use of a protein included in the sample taken from a subject.
Therefore, the kit according to the present invention may include, for example, an antibody that specifically bonds to just the wild-type or mutant protein among the proteins of the sodium ion channel α1 subunit and the calcium ion channel α1 subunit. Furthermore, the configuration may be one which, in addition to the antibody, includes one or more reagent in combination, which reagent is used for ELISA or Western blotting.
Furthermore, the kit according to the present invention may include a reagent used for measuring activity of the voltage-gated sodium ion channel NaV1.1, a reagent used for measuring activity of the voltage-gated calcium ion channel CaV2.1, or the like.
With use of the kit according to the present invention as described above, it is possible to easily obtain data for assessing the potential for development of Dravet syndrome in the subject. A subject to which the kit may be applied is not particularly limited, however is preferably applied to infants or children.
3. Model Animal of Dravet Syndrome According to the Present Invention and its Production Method
The present invention encompasses a model animal of Dravet syndrome, and its production method.
(3-1. Model Animal of Dravet Syndrome According to the Present Invention)
The model animal of Dravet syndrome according to the present invention has a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit. The mutation on the sodium ion channel α1 subunit and the mutation on the calcium ion channel α1 subunit are as described in the item “1. Assessment method according to the present invention” described above, so therefore specific descriptions thereof are omitted here.
It is preferable in the model animal of the Dravet syndrome that both the activity of the voltage-gated sodium ion channel NaV1.1 and the activity of the voltage-gated calcium ion channel CaV2.1 are changed as compared to a wild-type animal. This change in activity is not particularly limited, and may be an increase of activity or may be a decrease in activity. The method of confirming whether or not an activity of the voltage-gated sodium ion channel NaV1.1 of the model animal of Dravet syndrome according to the present invention is changed from that of a wild-type, and the method of confirming whether or not an activity of the voltage-gated calcium ion channel CaV2.1 of the model animal of Dravet syndrome according to the present invention is changed from that of a wild-type, are both not particularly limited. For example, with an individual of a model animal of Dravet syndrome according to the present invention or cells collected from the model animal of Dravet syndrome according to the present invention, confirmation may be made by measuring the activity by use of the conventionally known patch clamping, slice patching, imaging with use of fluorescence probe and like method.
The model animal of Dravet syndrome according to the present invention has the mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, so therefore develops Dravet syndrome. Such a model animal of Dravet syndrome can be used advantageously for clarification of the development mechanism of the intractable Dravet syndrome, and for development of medicament for Dravet syndrome.
In the present specification, “model animal” denotes an experiment animal used for developing a prevention method or treatment against human diseases, and more specifically is a non-human mammal such as a mouse, rat, rabbit, monkey, goat, pig, sheep, cow, or dog, and other vertebrates.
(3-2. Production Method of Model Animal of Dravet Syndrome According to the Present Invention)
A method of producing a model animal of Dravet syndrome, according to the present invention, includes: introducing a mutation on sodium ion channel α1 subunit and introducing a mutation on calcium ion channel α1 subunit.
More specifically, a mutation can be introduced on each of the genes by manipulating the gene of the model animal. Here, the “manipulating the gene of the model animal” intends to mean manipulation of a gene of a model animal by use of a conventionally known gene manipulation technique. More specifically, this encompasses all of destruction of a gene of the model animal, an introduction of a mutation to that gene, a substitution of that gene with a mutant gene, and furthermore, introduction of a foreign gene into the model animal, and crossing of model animals.
The production method according to the present invention of the model animal of Dravet syndrome may include steps other than those described above. Specific steps, materials, conditions, used devices, used equipment and the like are not limited in particular.
With the production method according to the present invention of a model animal of Dravet syndrome, it is possible to produce a model animal developed in Dravet syndrome by manipulating genes of a model animal so that a mutation is introduced into the genes of the sodium ion channel α1 subunit and the calcium ion channel α1 subunit.
4. Cells According to the Present Invention and its Production Method
The present invention also encompasses cells having a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, and its production method.
(4-1. Cell According to the Present Invention)
The cell according to the present invention is a cell having a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit. The mutation on the sodium ion channel α1 subunit and the mutation on the calcium ion channel α1 subunit are as described in the item “1. Assessment method according to the present invention” described above, so therefore specific description thereof have been omitted here.
The cell according to the present invention intends to mean experimental culture cells having a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit. More specifically, the cell is an experimental culture cell derived from a mammal such as a human, mouse, rat, hamster, rabbit, monkey and the like, and other vertebrates.
It is preferable that with such a cell, both of activity of the voltage-gated sodium ion channel NaV1.1 and activity of the voltage-gated calcium ion channel CaV2.1 are changed. This change in activity is not particularly limited, and may be an increase of activity or a decrease in activity. The method of confirming whether or not the activity of the voltage-gated sodium ion channel NaV1.1 of the cell according to the present invention is changed from that of a wild-type, and a method of confirming whether or not the activity of both of the voltage-gated calcium ion channel CaV2.1 of the cell according to the present invention is changed from that of the wild-type are as described in “1. Assessment method according to the present invention” described above, so hence specific description thereof have been omitted here.
Such a cell can be used for clarification of a development mechanism of the intractable Dravet syndrome, and for the development in medicament for Dravet syndrome. For example, it is possible to suitably use this for screening of a drug for treating Dravet syndrome. Namely, this cell can also be said as a screening cell for a drug for treating Dravet syndrome. Accordingly, the present invention also encompasses a screening cell of a drug for treating Dravet syndrome (hereinafter, simply called “screening cell”), and its production method.
(4-2. Production Method of Cell According to Present Invention)
A method of producing a cell according to the present invention is a method of producing a cell that has the foregoing properties, and includes: introducing a mutation on a sodium ion channel α1 subunit; and introducing a mutation on a calcium ion channel α1 subunit. More specifically, the following three embodiments can be raised. The following three embodiments are described specifically below, however the present invention is not limited to these.
(1) Method of Using Expression Vector Etc.
This method produces a cell that expresses a mutant voltage-gated sodium ion channel NaV1.1 and mutant voltage-gated calcium ion channel CaV2.1, with use of an expression vector or the like. More specifically described, in order to make a cell express the mutant voltage-gated sodium ion channel NaV1.1, for example, a sodium ion channel α1 subunit gene having a mutation that causes a change in an amino acid is coexpressed, in a culture cell that serves as a host, with a wild-type gene (β1 subunit gene and β2 subunit gene) making up the voltage-gated sodium ion channel NaV1.1, which wild-type gene encodes a subunit other than the α1 subunit (β1 subunit and β2 subunit), with use of an expression vector or the like. This enables the cell to express the mutant voltage-gated sodium ion channel NaV1.1 that includes the mutant sodium ion channel α1 subunit.
Similarly, in order to make the cell express the mutant voltage-gated calcium ion channel CaV2.1, for example, a calcium ion channel α1 subunit gene having a mutation that causes a change in an amino acid is coexpressed, in a culture cell that serves as a host, with a wild-type gene (β subunit gene, γ subunit gene, and α2δ subunit gene) making up a voltage-gated calcium ion channel CaV2.1, which wild-type gene encodes a subunit other than the α1 subunit (β subunit, γ subunit, and α2δ subunit), with the expression vector or the like. This hence enables the cell to express a mutant voltage-gated calcium ion channel CaV2.1 that includes the mutant calcium ion channel α1 subunit.
At this time, it is preferable that the culture cell serving as a host is a cell from which no voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1 is expressed. With use of such a cell, no effect is caused by the residing voltage-gated sodium ion channel NaV1.1 and residing voltage-gated calcium ion channel CaV2.1.
(2) Method of Using Artificial Mutation Introduction
This method introduces mutation for both of the sodium ion channel α1 subunit and the calcium ion channel α1 in a culture cell expressing both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1.
The method of introducing the mutation on the culture cell is not particularly limited, and a conventionally known gene manipulation technique is used in combination as appropriate.
(3) Method of Using Model Animal of Dravet Syndrome According to the Present Invention
This method extracts a tissue from the model animal of Dravet syndrome according to the present invention as described above, and prepares a culture cell from that tissue. The model animal of Dravet syndrome according to the present invention is as described in “3. Model animal of Dravet syndrome according to the present invention and its production method”, and so therefore specific description thereof has been omitted here. Of course, the “tissue” that is extracted is intended to mean a tissue in which both the sodium ion channel α1 subunit on which a mutation is introduced and the calcium ion channel α1 subunit on which a mutation is introduced are expressed.
This hence allows for easy production of a cell that has a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit. The kinds of tissues extracted from the model animal of Dravet syndrome is not limited in particular, and may be selected as appropriate depending on its purpose.
The method according to the present invention of producing a cell may include steps other than the steps described above. Specific steps, materials, conditions, used devices, used equipment and the like are not limited in particular.
5. Screening Method of Drug for Treating Dravet Syndrome
The model animal of Dravet syndrome according to the present invention and the cell according to the present invention can be used in development of a new treatment method and drug for treating Dravet syndrome. Hence, the present invention encompasses a screening method of a drug for treating Dravet syndrome, which screens a drug for treating Dravet syndrome (hereinafter, also called “screening method according to the present invention”).
In the specification, an embodiment using a model animal of Dravet syndrome according to the present invention and an embodiment using a screening cell have been explained as embodiments of the screening method according to the present application. However, the present invention is not limited to these embodiments.
Namely, for example, the embodiment may use another model animal of Dravet syndrome instead of the model animal of Dravet syndrome according to the present invention.
(1) Case of Using Model Animal of Dravet Syndrome According to the Present Invention
The method is sufficient as long as it includes administering a candidate agent to the model animal of Dravet syndrome according to the present invention, and assessing whether or not Dravet syndrome shows improvement or is cured in the model animal of Dravet syndrome to which the candidate agent is administered.
Namely, according to the screening method of the drug for treating Dravet syndrome according to the present invention, a candidate agent is administered to the model animal of Dravet syndrome, to assess whether or not that candidate agent can serve as a drug for treating Dravet syndrome in the model animal of Dravet syndrome to which the candidate agent is administered, by having the improvement or curing of Dravet syndrome serve as an indicator.
The method of assessing whether or not Dravet syndrome is improved or cured in the model animal of Dravet syndrome to which the candidate agent is administered is not limited in particular, and is sufficiently assessed by use of characteristic symptoms of Dravet syndrome as indicators. For example, it is possible to determine whether Dravet syndrome is improved or cured by comparing a control animal not having a mutation that causes an amino acid change on the sodium ion channel α1 subunit gene and the calcium ion channel α1 subunit gene (i.e. an animal not having a mutation on both of α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1) with the model animal of Dravet syndrome according to the present invention, in terms of “body temperature at convulsion onset (convulsion threshold)”, “severity score”, “duration of convulsion”, and the like each shown in the Examples later described.
The candidate agent is not limited in particular, however it is preferable that it is a compound expectable of giving effect on the expression of voltage-gated sodium ion channel NaV1.1 and/or expression of voltage-gated calcium ion channel CaV2.1, or a compound expectable of giving effect on the activity of the voltage-gated sodium ion channel NaV1.1 and/or the activity of voltage-gated calcium ion channel CaV2.1 (e.g. an inhibitor or candidate substance of an inhibitor, or an agonist or a candidate substance of an agonist, each of which has effect on both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1).
Moreover, the candidate agent may be an expression plasmid vector or a virus vector that includes a polynucleotide made of a sodium ion channel α1 subunit gene or a part of its nucleotide sequence. Moreover, the candidate agent may be an expression plasmid vector or a virus vector that includes a polynucleotide made of the calcium ion channel α1 subunit gene or a part of its nucleotide sequence.
The method of administering such a candidate agent to the Dravet syndrome model animal according to the present invention is not limited in particular, and a suitable method is sufficiently selected from conventionally known methods in accordance with physical properties of that candidate agent.
(2) Case of Using Screening Cell According to the Present Invention
The method at least includes administering a candidate agent to a screening cell according to the present invention, and assessing whether or not activity of voltage-gated sodium ion channel NaV1.1 and/or activity of voltage-gated calcium ion channel CaV2.1 in the screening cell of a drug for treating Dravet syndrome to which the candidate agent was administered, is changed.
Namely, with the screening method according to the present embodiment, it is possible to assess whether a candidate agent can serve as a drug for treating Dravet syndrome, by administering the candidate agent to the screening cell according to the present invention, based on an indicator of whether the activity of the voltage-gated sodium ion channel NaV1.1 and/or the activity of the voltage-gated calcium ion channel CaV2.1 in the screening cell to which the candidate agent is administered, is changed.
Moreover, the method of assessing, in the screening cell to which the candidate agent is administered, whether or not the activity of the voltage-gated sodium ion channel NaV1.1 is changed and whether or not the activity of the voltage-gated calcium ion channel CaV2.1 is changed are not limited in particular, and the assessments are sufficiently carried out by use of an electrophysiologic measurement device, fluorescence observation device, or the like.
The candidate agent is not limited in particular, and similar substances as those described in the foregoing “(1) Case of using model animal of Dravet syndrome according to the present invention” may be used.
The method of administering such a candidate agent to a cell according to the present invention is not limited in particular, and a suitable method based on the physical properties and the like of that candidate agent is selected and used from conventionally known methods.
It is preferable in the assessment method according to the present invention that the mutation on α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 is at least one of a mutation shown in Table 1, and
the mutation on α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 is at least one of a mutation shown in Table 2.
It is preferable in the assessment method according to the present invention to further include:
detecting a change in activity of the voltage-gated sodium ion channel NaV1.1; and
detecting a change in activity of the voltage-gated calcium ion channel CaV2.1.
The present invention is not limited to the description of the embodiments above, but may be altered by a skilled person within the scope of the claims. An embodiment based on a proper combination of technical means disclosed in different embodiments is encompassed in the technical scope of the present invention.
EXAMPLES
The following describes more specifically of the present invention with use of Examples, however the present invention is not limited to the Examples.
Example 1
Identification of Risk Factors for Predicting Development of Dravet Syndrome
DNA were extracted from peripheral blood of 47 Dravet syndrome patients who visited Okayama University Hospital and/or its related hospitals, and mutations on various genes were analyzed. This study was performed upon receiving approval from Okayama University, Institutional Review Board of Human Genome and Gene Analysis Research.
More specifically, a genomic DNA was extracted from peripheral blood of a patient with use of a DNA extraction kit (WB kit; Nippon gene, Tokyo, Japan), and all exons were amplified by PCR. In PCR, a reaction solution of 25 μl was used, which includes 50 ng of human genomic DNA, 20 μmol of various primers, 0.8 mM of dNTPs, 1 reaction buffer, 1.5 mM of MgCl2, and 0.7 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, Calif., USA). As to the nucleotide sequence (SEQ ID NOs.: 9-62) of the primer pair used, see “Sequence of primers” described later.
An obtained PCR product was purified with use of PCR products pre-sequencing kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Subsequently, with use of Big Dye Terminator FS ready-reaction kit (Applied Biosystems), a sequence reaction was performed, and with use of a fluorescence sequencer (ABI PRISM3100 sequencer; Applied Biosystems), a nucleotide sequence of the obtained PCR product was determined.
First, mutation analysis was performed of SCN1A gene that encodes α-subunit type 1 (also called “α1 subunit”) making up the voltage-gated sodium ion channel NaV1.1, for the 47 Dravet syndrome patients. As a result, a mutation in the SCN1A gene was found in 38 patients out of the 47 Dravet syndrome patients. For the 9 patients in which no mutation was detected, a further analysis was performed on the number of gene copies of the SCN1A gene, with use of Multiplex Ligation-dependent Probe Amplification (MLPA; MRC-Holland; SALSA MLPA kit P137). As a result, a deletion of exon 10 was detected in 1 patient. The number of patients in which no mutation of the SCN1A gene was found was 8. The mutation detected in the SCN1A gene is as shown in Table 1.
Next, with use of the DNA of the 47 patients, gene analysis was performed for GABRG2 gene, CACNA1A gene, CACNB4 gene, SCN1B gene, and SCN3A gene. These genes encode proteins as follows:
GABRG2: GABAA receptor γ2 subunit gene
CACNA1A: α1 subunit of voltage-gated calcium ion channel CaV2.1
CACNB4: β4 subunit of voltage-gated calcium ion channel
SCN1B: β1 subunit of voltage-gated sodium ion channel
SCN3A: α3 subunit of voltage-gated sodium ion channel NaV1.3
The nucleotide sequence (SEQ ID NOs.: 63-143) of the primer pair used for the gene analysis of the CACNA1A gene is shown in “Sequence of primers” described later.
As a result, various kinds of gene mutations were found in the CACNA1A gene that encodes α-subunit type 1 (also called “α1 subunit”) making up the voltage-gated calcium ion channel CaV2.1 (see Table 2 and FIG. 12).
Table 3 shows the gene mutations of SCN1A and CACNA1A that were detected in the Dravet syndrome patients.







TABLE 3







SCN1A and CACNA1A gene mutations


detected in Dravet syndrome patients









P.




No.
SCN1A gene
CACNA1A gene















1
G177R
G266S





2
W738fsX746
K472R


3
V1390M
A924G


4
V212A
E921D
E996V


5
R377L
E921D
E996V


6
Deletion of exon 10
E921D
E996V



(Exon10*)


7
P707fsX714
E921D
E996V


8
R865X
E921D
E996V


9
F902C
E921D
E996V


10
T1082fsX1086
E921D
E996V


11
Q1277X
E921D
E996V


12
Q1450R
E921D
E996V


13
A1685D
E921D
E996V


14
T1909I
E921D
E996V
R1126H
R2201Q


15
G163E
R1126H
R2201Q


16
K547fsX570
R1126H
R2201Q


17
S1574X
R1126H
R2201Q


18
R712X
G1108S


19
R1648C
G1108S


20
negative
G1108S


21
negative
Del2202-2205


22
R501fsX543
negative


23
S607fsX622
negative


24
E788K
negative


25
R931C
negative


26
R931C
negative


27
L990F
negative


28
A1002fsX1009
negative


29
K1027X
negative


30
K1057fsX1073
negative


31
L1265P
negative


32
W1271X
negative


33
1289delF
negative


34
Intron 21 splicing
negative



error


35
A1429fsX1443
negative


36
W1434R
negative


37
T1539R
negative


38
S1574X
negative


39
G1674R
negative


40
A1662V
negative


41
G1880fsX1881
negative


42
negative
negative


43
negative
negative


44
negative
negative


45
negative
negative


46
negative
negative


47
negative
negative





P. No. Patient Number


Exon10* exon deletion detected by MPLA






The following mutations are mutations of the CACNA1A gene detected this time. These mutations were mutations that cause an amino acid substitution, mutations that cause no amino acid substitution, and intron mutations.
(1) Missense Mutations
G266S 1 case
K472R 1 case
E921D 11 cases
A924G 1 case
E996V 11 cases
G1108S 3 cases
R1126H 4 cases
R2201Q 4 cases
(2) Deletion of Amino Acids
4 amino acid deletions (deletion 2202-2205) 1 case
(3) Gene Mutation Causing No Amino Acid Change in Exon
E292E (rs16006), E394E (rs2248069), 15251 (rs16010), T698T (rs16016), R1023R (rs16025), F1291F (rs16030), T1458T (new SNP or mutation), S1472S (new SNP or mutation), V1890V (rs17846921), H2225H (rs16051)
(4) Gene Mutation in Intron
exon 1 upstream (rs16000), intron 1 (rs16003), intron 3 (rs17846942), intron 8 (rs2306348), intron 11 (rs10407951), intron 17 (rs16018), intron 39 (rs3816027), intron 40 (rs17846925), intron 42 (new SNP or mutation).
The missense mutations and deletion mutations detected in coding regions of the CACNA1A gene shown in the foregoing (1), and (2) are shown in Table 4.







TABLE 4







Summary of mutations detected in coding


region of CACNA1A gene


Coding Region














Mutation
SNP Reg.



Exon No.
Amino acid
type
No.





1
Exon 6
G266S
Missense
—


2
Exon 11
K472R
Missense
—


3
Exon 19
E921D
Missense
rs16022


4
Exon 19
A924G
Missense
—


5
Exon 19
E996V
Missense
rs16023


6
Exon 20
G1108S
Missense
rs16027


7
Exon 20
R1126H
Missense
—


8
Exon 47
R2201Q
Missense
—


9
Exon 47
Del 2202-2205
Deletion
—





SNP Reg. No.: Single Nucleotide Polymorphism Registration Number






These mutations were compared and studied with a gene polymorphism (Single Nucleotide Polymorphism; SNP) database of NCBI (National Center for Biotechnology Information). As a result, it was found that 3 kinds of the mutations out of the 9 kinds of mutations were registered in the SNP database as gene polymorphism (Single Nucleotide Polymorphism; SNP).
The gene mutation shown in (3) and (4) were either a gene polymorphism registered in the SNP database, or a new gene polymorphism or mutation. The registered number in the SNP database is shown in the brackets.
Out of the SNP already reported, the mutations which caused a change in the amino acid were considered probably that although no seizure occurs just by that individual case having the CACNA1A gene SNP, but when an abnormality of SCN1A gene is simultaneously present, this is somewhat involved in the worsening of the symptom.
A comparison of patients having a mutation in either of the SCN1A gene and the CACNA1A gene or both of the SCN1A gene and CACNA1A gene, out of the 47 Dravet syndrome patients, resulted as follows.
Patients having a mutation on both SCN1A and CACNA1A: 19 cases
Patients having a mutation on just SCN1A: 20 cases
Patients having a mutation on just CACNA1A: 2 cases
Patients having no mutation on either of SCN1A or CACNA1A: 6 cases.
No reports whatsoever have been made regarding abnormalities in the CACNA1A gene of the patients of Dravet syndrome, until now. The result of the present study shows that Dravet syndrome patients highly frequently has a mutation in SCN1A, i.e. a α1 subunit gene of the voltage-gated sodium ion channel NaV1.1, and in CACNA1A, i.e. a α1 subunit gene of the voltage-gated calcium ion channel CaV2.1.
A literature disclosing that a mutation on a β4 subunit of the voltage-gated calcium ion channel CaV2.1 (hereinafter, simply referred to as “calcium ion channel β4 subunit”) is involved with Dravet syndrome (Iori Ohmori et al., Neurobiology of Disease 32 (2008) 349-354) describes that out of 38 patients in which a mutation was detected in the sodium ion channel α1 subunit, 1 Dravet syndrome patient had a mutation on both the sodium ion channel α1 subunit and the calcium ion channel β4 subunit.
In comparison, out of 39 patients in which a mutation was detected on the sodium ion channel α1 subunit, the patients of Dravet syndrome having a mutation on both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit were 19 patients (6 patients when excluding patients having registered SNP that cause a change in an amino acid in an exon). This result shows that by detecting the mutation for both the sodium ion channel α1 subunit and the calcium ion channel α1 subunit, the detection sensitivity of Dravet syndrome patients dramatically increase as compared to detecting the mutation for both the sodium ion channel α1 subunit and the calcium ion channel β4 subunit.
In the present specification, a nucleotide number in mRNA of the SCN1A gene and an amino acid number in a protein of SCN1A were made to be in line with GenBank accession No. AB093548; methionine, encoded by the initiation codon (ATG), was numbered as the first amino acid, and the initial A of the initiation codon was numbered as the first nucleotide.
Moreover, a genome sequence of the CACNA1A gene was in line with the GenBank accession number NC—000019. The number of the nucleotide in mRNA of CACNA1A gene and the number of the amino acid in CACNA1A protein was made to be in line with the GenBank accession number NM 023035; methionine, encoded by the initiation codon (ATG), was numbered as the primacy amino acid, and the initial A of the initiation codon was numbered as the primacy nucleotide.
Example 2
Study of Gene Mutation in Benign Febrile Seizure Patient
A study was performed of a SCN1A gene and CACNA1A gene abnormality in a benign febrile seizure patient. DNA was extracted from peripheral blood of 50 patients of benign generalized epilepsy with febrile seizure plus (GEFS+), who visited Okayama University Hospital and/or its related hospitals, and mutations on various genes were analyzed. The DNA extraction, PCR amplification of the gene, and sequencing reactions were performed by the methods described above.
First, mutation analysis of voltage-gated sodium ion channel SCN1A gene was performed, which resulted in detecting gene mutation that caused amino acid changes in 6 patients. Next, mutation analysis was performed for 9 kinds of mutations of missense mutations and deletion mutations that were detected in the coding region of the CACNA1A gene, which resulted in detecting a mutation in 16 patients. Each of the mutations are shown in Table 5.







TABLE 5







SCN1A and CACNA1A gene mutations


detected in benign febrile seizure










Patient No.
SCN1A
CACNA1A














1





2
M1856T


3

del 2202-2205


4


5

del 2202-2205


6
R1575C


7

E921D
E996V


8

E921D
E996V


9

E921D
E996V


10


11


12
I1616T


13


14


15


16


17


18

E921D
E996V


19


20


21


22

E921D
E996V


23

E921D
E996V


24


25


26

E921D
E996V


27


28

E921D
E996V


29

A924G


30

E921D
E996V


31


32


33

E921D
E996V


34

G1108S


35


36
I1616T


37
I1616T


38


39
Y1769H


40

E921D
E996V


41


42


43


44


45


46


47


48

E921D
E996V


49


50









Out of the 50 benign epilepsy patients, it was confirmed that no patient had mutations simultaneously on both SCN1A gene and CACNA1A gene.
The following shows a result of gene mutation analysis of a total of 97 patients, of 47 malignant Dravet syndrome cases and 50 benign febrile seizure patient cases.
(1) As a result of screening patients having a mutation on the SCN1A gene among the 97 patients, 39 Dravet syndrome patients (39 cases out of 47 cases) and 6 benign epilepsy patients (6 cases out of 50 cases) were detected.
(2) As a result of screening patients having a mutation on both the SCN1A gene and CACNA1A gene out of the 97 patients, 19 Dravet syndrome patients (19 cases out of 47) were detected, and no (0) benign epilepsy patients were detected.
These results suggest that by examining both the SCN1A gene mutation and the CACNA1A gene mutation, it is possible to eliminate the false positive (benign febrile seizure patients) better than examining just the SCN1A gene mutation, and suggest a possibility of detecting the Dravet syndrome patients with higher accuracy.
Example 3
Study of Gene Mutation in a Healthy Person
To investigate whether the remaining 6 kinds of gene mutations excluding the registered 3 kinds out of the 9 kinds of missense mutations and deletion mutations detected in the coding region of the CACNA1A gene are of the gene polymorphism (SNP), gene mutation of the CACNA1A gene was similarly analyzed for DNA extracted from blood of 190 healthy persons. Results of the 9 kinds of the missense mutations and deletion mutations detected in the coding region of the CACNA1A gene are shown in Table 6. As a result, one kind of the CACNA1A gene mutation (G266S) was not detected from the healthy persons. From this result, it was found that the CACNA1A gene mutation of G266S is not an SNP, and is a novel gene mutation (gene abnormality) not found in the 190 healthy persons, which neither is in the NCBI SNP database.







TABLE 6







CACNA1A gene mutation detected in healthy persons and Dravet syndrome













Nucleotide
Amino Acid

Control



Exon
Substitution
Substitution
Dravet (n = 47)
(n = 188-190)
p-value













Frequency of variants















6
A876G
G266S
1/47
2.1%
0/188
  0%
0.20


11
A1415G
K472R
1/47
2.1%
1/188
0.53%
0.36


19
A2762C
E921D
11/47 
23.4% 
49/188 
26.06% 
0.71


19
C2771G
A924G
1/47
2.1%
7/190
3.68%
1.00


19
A2987T
E996V
11/47 
23.4% 
49/188 
26.06% 
0.71


20
G3322A
G1108S
3/47
6.4%
16/189 
8.46%
0.77


20
G3377A
R1126H
 4/47*
8.5%
1/188
0.53%
0.0061


47
G6602A
R2201Q
4/47
8.5%
4/189
2.12%
0.052


47
6605-6616del
DQER2202-
1/47
2.1%
3/190
1.58%
1.00




2205del










Frequency of




combined mutations














19

E921D + E996V
11/47 
23.4% 
49/188 
26.06% 
0.71


20 + 47

R1126H + R2201Q
 4/47*
8.50% 
0/188
  0%
0.0014









As a result of studying the comparison of frequencies in which mutations occur in healthy persons and Dravet syndrome patients, it was shown that the CACNA1A gene mutation R1126H was of a larger number with Dravet syndrome in terms of statistical significance (p=0.0061), and it was found that the CACNA1A gene mutation R2201Q also had a trend having a larger number with Dravet syndrome patients (p=0.052). The patients simultaneously having both mutations of R1126H and R2201Q on the CACNA1A gene were detected significantly in just the Dravet syndrome patients (4 cases out of 47 cases), and no healthy persons were detected (p=0.0014). Examination of DNA of the parents of these four patients revealed that the two mutations of R1126H and R2201Q were simultaneously present on one chromosome, i.e. within the same CACNA1A protein molecule, and that this double mutation was inherited from the parents.
Example 4
Study of Relation Between Genotype and Symptoms
A study was performed on how the 9 kinds of missense mutations and deletion mutations detected in the coding region of CACNA1A gene give effect on the worsening of symptoms of the disease. Out of Dravet syndrome patients whose seizure symptom data is managed in detail, the seizure symptoms under the age of 1 were compared between 20 patients who have just the SCN1A gene mutation and 19 patients who have a mutation on both the SCN1A gene and the CACNA1A gene. A result thereof is shown in Table 7. Note that “GTC” in Table 7 is an abbreviation of a generalized tonic-clonic seizure, and “CPS” is an abbreviation of a complex partial seizure.







TABLE 7







Relation of symptoms under the age of 1 with genotype










Total no.




of
Type of Seizures

















Seizure
Total no.
prolonged


Hemi-
Myoclonic




onset
of
(>10 min)
GTC
CPS
convulsion
seizure


Genotype
N
(months)
seizures
seizures
(%)
(%)
(%)
(%)





SCN1A
20
5.6 ± 0.3 
10.2 ± 1.2
2.4 ± 0.4 
95
45
50 
15


mutation +


No CACNA1A


variants


SCN1A
19
4.6 ± 0.4*
10.7 ± 1.3
4.4 ± 0.7*
95
26
84*
11


mutation +


CACNA1A


variants





GTC: generalized tonic-clonc seizure,


CPS: complex partial seizure


*p < 0.05






It was found that the patients having a CACNA1A variant, as compared to the patients having no CACNA1A variant, are (i) significantly quicker in seizure onset (p=0.049), (ii) significantly greater in the number of times prolonged seizures occur, which prolonged seizure is a convulsion seizure that continues for 10 or more minutes (p=0.019), and (iii) significantly higher in the frequency that a hemiconvulsion occurs (p=0.041). This indicates that when there is a variation of the CACNA1A gene including the polymorphism in addition to a SCN1A gene abnormality, there is a possibility that the symptom may worsen.
Example 5
Analysis on Functions of Mutant Voltage-Gated Calcium Ion Channel
An analysis was performed on functions of a mutant calcium ion channel and a normal (wild-type) calcium ion channel, with use of culture cells. First, cDNA of a human CACNA1A gene (SEQ ID NO.: 4) was used to prepare an expression vector having a mutant CACNA1A (double mutation of G266S; R1126H; R2201Q; deletion 2202-2205; double mutation of R1126H and R2201Q) gene. After obtaining DNA fragments including the mutated parts by PCR, regions of a normal cDNA corresponding to those fragments were substituted with those fragments, to prepare the mutant cDNA. As a control, an expression vector (pMO14X2-CACNA1A) having a normal (wild-type) CACNA1A gene was used.
Analysis was performed on functions of the mutant calcium ion channel and the normal calcium ion channel, with use of the culture cells. A α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1, which is a CACNA1A gene product, had been subjected to function adjustment by the α2δ subunit and β4 subunit that similarly configure the voltage-gated calcium ion channel CaV2.1. Hence, an expression vector having a CACNA1A gene that encodes a α-subunit type 1, and an expression vector having a human CACNB4 gene (GenBank accession No. U95020) (SEQ ID NO.: 151) encoding a β4 subunit and a rabbit α2δ gene (GenBank accession No. NM—001082276) (SEQ ID NO.: 152) encoding a α2δ subunit. were coexpressed on a human renal cell HEK293 with use of a transfection reagent. Electrophysiologic properties were studied by patch clamping of a whole cell record.
More specifically, recording of a calcium ion channel current was carried out at room temperature of 22° C. to 24° C., 72 hours after transfection. With use of a multistage P-97 Flaming-Brown micropipette puller, a patch electrode was prepared from borosilicate glass.
The composition of intracellular fluid was 110 mM CsOH, 20 mM CsCl, 5 mM MgCl2, 10 mM EGTA, 5 mM MgATP, 5 mM creatine-phosphate, and 10 mM HEPES. On the other hand, the composition of the used extracellular fluid was 5 mM BaCl, 150 mM TEA-Cl, 10 mM glucose, and 10 mM HEPES. The amplifier used was Axopatch200B (Axon Instruments).
Electrophysiologic properties of the mutation channel were compared with those of a normal channel, by studying voltage-gated channel activation, inactivation, recovery from inactivation, and duration current. The activation curve and the inactivation curve were analyzed by Boltzmann function, to find a half-maximal activation/inactivation (V1/2) and a slope factor (k). The recovery curve from the inactivation was analyzed by a two exponential function. Statistics used the unpaired Student's t test. Clampfit 8.2 software and OriginPro 7.0 (OriginLab) were used for data analysis.
FIG. 13 and FIG. 14 are views illustrating results of performing function analysis of the calcium ion channel, by patch clamping. In the graphs in FIG. 13 and FIG. 14, the normal calcium ion channel is shown as “WT”, and the mutant calcium ion channels are shown as “R266S”, “R1126H”, “R2201Q”, “Del2202”, and “RH+RQ”. The mutation “Del2202” means the mutation “Deletion 2202-2205”, and the mutation “RH+RQ” means the mutation “R1126H+R2201Q”.
Illustrated in (a) of FIG. 13 is a barium current record in accordance with a change in potential of the normal calcium ion channel and the mutant calcium ion channel. Illustrated in (b) is a current-voltage relationship, and illustrated in (c) are a peak current value (pA), a total charge (pF), and a peak current density (pA/pF).
More specifically, (a) of FIG. 13 illustrates a current record of measuring barium current that is depolarized by changing a depolarizing stimulus by 10 mV each from −40 mV to +60 mV and is flowed therein. The current-voltage relationship illustrated in (b) of FIG. 13 is a graph obtained by (i) measuring a flowing barium current for every membrane potential while having a holding potential, being deeper than a resting membrane potential, as −100 mV, and a depolarizing stimulus being changed by 10 mV each from −40 mV to +60 mV, and (ii) plotting the membrane potential on a horizontal axis and a current value on a vertical axis. The view illustrated on the lower right of the graph in (b) of FIG. 13 shows that in this experiment, “the depolarizing stimulus was changed by 10 mV each from −40 mV to +60 mV for 30 ms (milliseconds), with the holding potential being −100 mV, which holding potential is deeper than the resting membrane potential”.
As a result, it was found that the mutant calcium ion channel “Deletion2202-2205” and “R1126H+R2201Q” significantly increased in its flowed current amount, peak current value, and peak current density, as compared to the normal calcium ion channel.
Next, in order to specifically study the electrophysiologic properties of the calcium ion channel, a voltage-gated activity of the calcium ion channel ((a) of FIG. 14), a time constant (τ) at activation ((b) and (c) of FIG. 14), inactivation of the calcium ion channel ((d) of FIG. 14), and a time constant (τ) at inactivation ((e) FIG. 14) were measured.
The activation curve illustrated in (a) of FIG. 14 shows a barium current value flowing per membrane potential as a relative value, by having a maximum sodium current value obtained from the graph of (b) of FIG. 13 be 1, and an obtained curve was analyzed by Boltzmann function to find a half-maximal activation (V1/2) and a slope factor (k). The view provided on the lower right of the graph in (a) of FIG. 14 represents that, in this experiment, “the depolarizing stimulus was changed by 10 mV each from −40 mV to +60 mV for 30 ms (milliseconds), with the holding potential being −100 mV, which holding potential is deeper than the resting membrane potential”.
As a result of analyzing the voltage-gated activity of the calcium ion channel, it was found that (i) the mutant calcium ion channel “G266S” and “R1126H” show a significant hyperpolarization shift as compared to the normal channel, and that (ii) the mutant calcium ion channel “R1126H” and “Deletion2202-2205” significantly increased in the voltage-gated property as compared to the normal channel, by comparing the slope factor (k) (see (a) of FIG. 14 and Table 8). This means that the mutant calcium ion channel “G266S”, “R1126H” and “Deletion2202-2205” are easily activated even in a low membrane potential, thereby tending to cause excess hyperexcitability of nerve cells.
Table 8 shows electrophysiologic properties of the calcium ion channel. Statistical comparison of the normal CACNA1A and the mutant CACNA1A were performed by the Student's t test. The asterisk (*) in Table 8 indicates that there is a significant difference between the normal CACNA1A and the mutant CACNA1A when a critical rate is under 5%, and the double asterisk (**) indicates that there is a significant difference between the normal CACNA1A and the mutant CACNA1A when the critical rate is under 1%.







TABLE 8







Electrophysiologic properties of calcium ion channel










Activation
Inactivation














V1/2 (mV)
k (mV)
n
V1/2 (mV)
k (mV)
n

















WT-CACNA1A
6.3 ± 1.3
4.3 ± 0.2
16
−16.9 ± 1.5
−4.5 ± 0.6
10


G266S
 1.0 ± 1.2**
4.3 ± 0.4
11
−13.8 ± 1.6
−5.5 ± 0.3
10


R1126H
 0.4 ± 1.6**
 3.3 ± 0.3*
10
−18.9 ± 0.6
−6.1 ± 0.7
8


R2201Q
6.4 ± 1.5
4.1 ± 0.2
8
−13.4 ± 1.7
−5.7 ± 0.4
10


Deletion2202-2205
1.3 ± 1.4
 3.4 ± 0.2*
8
−13.3 ± 1.2
−4.7 ± 0.6
9


R1126H + R2201Q
2.6 ± 1.1
3.5 ± 0.2
10
−15.2 ± 0.9
−5.4 ± 0.1
10





V1/2, half-maximal voltage activation and inactivation;


k, slope factor.


Statistical coparison between WT-CACNA1A and mutant channels was performed by Student's t test


(*P < 0.05 and **P < 0.01 versus WT-CACNA1A).






Illustrated in (b) of FIG. 14 is a time constant of channel voltage-gated activation, that is to say, a time required for each current to reach 66.7%. Moreover, (c) of FIG. 14 illustrates a time constant of voltage-gated activation at 20 mV. From (b) and (c) of FIG. 14, it was demonstrated that the mutant calcium ion channel “G266S” was significantly small in the time constant of voltage-gated activation at 20 mV, as compared to a normal channel. Since this point is considered as that the mutant calcium ion channel “G266S” is made so as to flow a lot of current within a short depolarization, this means that there is a trend of causing hyperexcitement in the nerve cells.
Illustrated in (d) of FIG. 14 is a voltage-gated inactivation curve of the calcium ion channel, which was measured upon changing a membrane potential to activate the calcium ion channel and thereafter providing a depolarizing stimulus to measure how much barium current was flown. Note that the view illustrated on the lower left of the graph illustrated in (d) of FIG. 14 shows that, in this experiment, “the depolarizing stimulus was changed by 20 mV each from −120 mV to +60 mV for 2 s (seconds), and subsequently be changed to 20 mV, with the holding potential being −100 mV, which holding potential is deeper than the resting membrane potential”.
The voltage-gated inactivation curve of the calcium ion channel showed no recognizable significant difference, in either of the mutant channel or the normal channel.
Illustrated in (e) of FIG. 14 is a result of studying an inactivation time constant (i). There are two kinds of inactivation: inactivation of a fast component and inactivation of a slow component. The “τfast” in the left graph of (e) of FIG. 14 is a constant representing a time required until the inactivation of the fast component reaches 33.3%, and the “τslow” in the right graph is a constant representing a time required until the inactivation of the slow component reaches 33.3%. These inactivation time constants were, more specifically, calculated by analyzing the inactivation curve with use of Clampfit 8.2 software.
As a result, there was no significant difference in the inactivation time constant between that of the normal calcium ion channel and that of the mutant calcium ion channel. Table 9 shows physiological properties of the mutant calcium ion channel. The arrow pointing upwards ↑T) in Table 9 indicates that an increase in channel activity was recognized, and the hyphen “−” indicates that no change was recognized in the channel activity.







TABLE 9







Summary of electrophysiological properties


of mutant calcium ion channel









CACNA1A












Biophysical



Del 2202-
R1126H +


property
G266S
R1126H
R2201Q
2205
R2201Q





Peak current density
—
—
—
↑
↑ ↑


Activation V1/2
↑
↑
—
—
—


Activation slop
—
↑
—
↑
—


factor


Activation time
↑
—
—
—
—


constants


Inactivation V1/2
—
—
—
—
—


Inactivation slope
—
—
—
—
—


factor





↑, predicted gain of channel activity,


—, no predicted change in channel activity.






It was found that the mutations other than “R2201Q” in the calcium ion channel were mutations of a gain of function kind, and tends to cause excitement of the nerve cells.
Example 6
Production of Dravet Syndrome Model Rat
From the foregoing findings, it was considered that having some kind of mutation on both of SCN1A and CACNA1A is important in the development of Dravet syndrome.
Accordingly, a rat was produced which has both of the mutation on α1-subunit gene Scn1a of the voltage-gated sodium ion channel NaV1.1 and the mutation on α1-subunit gene Cacna1a of the voltage-gated calcium ion channel CaV2.1, to study the worsening of symptoms (human genes are represented as SCN1A and CACNA1A, and rat genes are represented as Scn1a and Cacna1a).
More specifically, a rat having a mutation on the Scn1a gene (F344-Scn1aKyo811) and a rat having a mutation on the Cacna1a gene (GRY (groggy rat, Cacna1agry)) were used as parent rats. Each of these mice is described below.
<F344-Scn1aKyo811>
A rat produced by ENU mutagenesis, having a missense mutation on a α1 subunit gene (Scn1a) of the voltage-gated sodium channel NaV1.1. Asparagine (N), which is an amino acid at position 1417, was mutated to histidine (H) (represented as “N1417H”). This rat served as a model animal of human generalized epilepsy febrile seizure plus (GEFS+). Background genealogy is F344/NS1c rat. This rat was provided from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University.
<GRY (Groggy Rat, Cacna1agry)>
A mutant rat produced by administering methyl nitrosourea to Sc1:Wistar, whose main symptoms are ataxia and absence-like seizure. This rat has an autosomal recessive mode of inheritance, and has a missense mutation on the α1-subunit of the voltage-gated calcium ion channel CaV2.1. Methionine (M), which is an amino acid at position 251, is mutated to lysine (K) (M251K). This rat was provided from the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University.
FIG. 11 is a view showing an amino acid sequence of a protein encoded by a human CACNA1A gene and an amino acid sequence of a protein encoded by a rat Cacna1a gene. The upper line of the amino acid sequence shown in FIG. 11 represents an amino acid sequence of the protein encoded by the rat Cacna1a gene (GenBank accession No. NM—012918) (SEQ ID NO.: 147), and the lower line is the amino acid sequence of the protein encoded by the human CACNA1A gene (GenBank accession No. NM—023035) (SEQ ID NO.: 3). Moreover, the squared amino acid “M” in FIG. 11 is an amino acid that is mutated from the amino acid “M” to an amino acid “K” in the human mutant CACNA1A (M249K) protein (SEQ ID NO.: 148) and the rat mutant Cacna1a (M251K) protein (SEQ ID NO.: 149).
As illustrated in FIG. 11, the mutation (M251K) on the α1 subunit of the rat voltage-gated calcium ion channel CaV2.1 corresponds to the mutation (M249K) on the α1 subunit of the human voltage-gated calcium ion channel CaV2.1.
The F344-Scn1aKyo811 and GRY (groggy rat, Cacna1agry) as described above were mated to produce a rat having each of the gene mutations.
(1. Analysis on Functions of Mutant Voltage-Gated Sodium Ion Channel)
An analysis was performed with use of culture cells, on functions of a mutant sodium ion channel and normal sodium ion channel, before tests using the rats were performed. The rat having a mutation on the Scn1a gene (F344-Scn1aKyo811) has asparagine (AAT), which is an amino acid at position 1417 of a protein encoded by the Scn1a gene, was changed to histidine (CAT) (N1417H). The asparagine at position 1417 is located in a pore formation region that is related to ionic permeation of sodium ion channel third domain. On this account, first, the function analysis of the mutant voltage-gated sodium ion channel included in F344-Scn1aKyo811 was performed.
More specifically, an expression vector having a mutant SCN1A (N1417H) gene (SEQ ID NO.: 150) including a missense mutation was prepared with use of cDNA of human SCN1A gene. As control, an expression vector having a normal (wild-type) SCN1A gene (SEQ ID NO.: 2) was prepared.
FIG. 1 is a view showing an amino acid sequence of a protein encoded by the human SCN1A gene and an amino acid sequence of a protein encoded by the rat Scn1a gene. The upper line in the amino acid sequence shown in FIG. 1 represents an amino acid sequence of a protein that is encoded by the human SCN1A gene (SEQ ID NO.: 1), and the lower line represents an amino acid sequence of a protein that is encoded by the rat Scn1a gene (SEQ ID NO.: 144). Moreover, the squared amino acid “N” in FIG. 1 is an amino acid on which a mutation from an amino acid “N” to an amino acid “H” occurs, of the human mutant SCN1A (N1417H) protein (SEQ ID NO.: 145) and the rat mutant SCN1A (N1417H) protein (SEQ ID NO.: 146).
An analysis was performed with use of culture cells, on functions of the mutant sodium ion channel and the normal sodium ion channel. The α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1, which is a SCN1A gene product, was adjusted in its function by β1 subunit and β2 subunit that similarly make up the voltage-gated sodium ion channel NaV1.1. Hence, an expression vector having the SCN1A gene that encodes the α-subunit type 1 was coexpressed with an expression vector having the SCN1B gene that encodes the β1 subunit and the SCN2B gene that encodes the β2 subunit in a human renal cell HEK293, with use of a transfection reagent. The electrophysiologic properties were studied by patch clamping based on whole cell recording.
More specifically, recording of the sodium ion channel current was carried out at room temperature of 22° C. to 24° C., 24 hours to 48 hours after transfection. A patch electrode was prepared from borosilicate glass by use of multistage P-97 Flaming-Brown micropipette puller.
Composition of intracellular fluid was 110 mM CsF, 10 mM NaF, 20 mM CsCl, 2 mM EGTA, and 10 mM HEPES. On the other hand, the composition of extracellular fluid was 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. Axopatch200B (Axon Instruments) was used as the amplifier.
Electrophysiologic properties of the mutation channel were compared with those of a normal channel, by studying voltage-gated channel activation, inactivation, recovery from inactivation, and duration current. The activation curve and the inactivation curve were analyzed by Boltzmann function, to find a half-maximal activation/inactivation (V1/2) and a slope factor (k). The recovery curve from the inactivation was analyzed by a two exponential function. Durable Na current was found by a difference in the duration current when depolarized at −10 mV for 100 ms, before and after addition of 10 μM of tetrodotoxin (TTX). Statistics used were unpaired Student's t test. Clampfit 8.2 software and OriginPro 7.0 (OriginLab) were used for data analysis.
FIGS. 2 to 4 are views illustrating results of performing function analysis of the sodium ion channel by patch clamping. The graphs of FIGS. 2 to 4 show the normal sodium ion channel as “WT” or “WT-SCN1A”, and show the mutant sodium ion channel as “N1417H”.
Illustrated in (a) of FIG. 2 is a typical example of a sodium current in response to a change in potential of the normal sodium ion channel and the mutant sodium ion channel. More specifically, a depolarizing stimulus was changed 10 mV each from −80 mV to +60 mV for depolarization, and sodium current that flowed in was measured. As a result, both of the normal sodium ion channel and the mutant sodium ion channel function as a channel, and there was no significant difference between the two.
Illustrated in (b) of FIG. 2 is a result of studying the inactivation time constant (τ). There are two types of inactivation; an inactivation of a fast component and an inactivation of a slow component. The “τ1” in (b) of FIG. 2 is indicative of a constant indicative of a time required for the inactivation of the fast component to reach 33.3%, and the “τ2” is indicative of a constant indicative of a time required for the inactivation of the slow component to reach 33.3%. These inactivation time constants, more specifically, were calculated by analyzing the inactive curve with use of the Clampfit 8.2 software. As a result, there was no significant difference in the inactivation time constant between that of the normal sodium ion channel and that of the mutant sodium ion channel.
Next, in order to specifically study the electrophysiologic properties of the sodium ion channel, a current-voltage relationship ((a) of FIG. 3), an activation of the sodium ion channel ((b) of FIG. 3), an inactivation of the sodium ion channel ((c) of FIG. 3), and recovery from the inactivation of the sodium ion channel ((d) of FIG. 3) were measured.
More specifically, the current-voltage relationship illustrated in (a) of FIG. 3 was obtained by (i) measuring a flowing sodium current for every membrane potential while having a holding potential, being deeper than a resting membrane potential, as −120 mV, and a depolarizing stimulus being changed by 10 mV each from −80 mV to +60 mV, and (ii) plotting the membrane potential on a horizontal axis and a current value on a vertical axis. The view illustrated on the lower left of the graph in (a) of FIG. 3 shows that in this experiment, “the depolarizing stimulus was changed by 10 mV each from −80 mV to +60 mV for 20 ms (milliseconds), with the holding potential being −120 mV, which holding potential is deeper than the resting membrane potential”.
The activation curve illustrated in (b) of FIG. 3 shows a sodium current value flowing per membrane potential as a relative value, by having a maximum sodium current value obtained from the graph of (a) of FIG. 3 be 1, and an obtained curve was analyzed by Boltzmann function to find a half-maximal activation (V1/2) and a slope factor (k). The view provided on the lower right of the graph in (b) of FIG. 3 represents that in this experiment, “the depolarizing stimulus was changed by 10 mV each from −80 mV to +60 mV, for 20 ms (milliseconds), with the holding potential being −120 mV, which holding potential is deeper than the resting membrane potential”.
The inactive curve illustrated in (c) of FIG. 3 was obtained by similarly changing the membrane potential to activate the channel and thereafter providing depolarizing stimulus and measuring how much the sodium current flows, to find the half-maximal inactivation (V1/2) and the slope factor (k). Note that the view provided on the lower left of the graph of (c) of FIG. 3 represents that in this experiment, “the depolarizing stimulus was changed by 10 mV each from −140 mV to +0 mV for 100 ms (milliseconds) and subsequently changed to −10 mV, with the holding potential being −120 mV”.
The recovery curve from the inactivation illustrated in (d) of FIG. 3 was obtained as follows. When a depolarizing stimulus was provided with pulse 1 (P1), the channel became inactive upon opening. When the depolarizing stimulus was returned to the original −120 mV, the sodium ion channel returned to its resting state, and upon stimulation of pulse 2 (P2), the channel opened again. The recovery time of this pulse 1 and pulse 2 were changed to obtain the recovery curve from the inactivation. This curve was analyzed by a two exponential function. It was determined whether the function of the channel was made easily excited or in the opposite was made difficult to be excited, depending on whether the recovery was quicker or slower as compared to the normal channel. The view provided on the lower right of the graph of (d) of FIG. 3 indicates that in this experiment, “a holding potential was −120 mV, −10 mV was provided for 100 ms (milliseconds) as the depolarizing stimulus and thereafter was returned to −120 mV, and after elapse of each of the times (milliseconds) shown on the x-axis, −10 mV was provided for 20 ms (milliseconds)”.
As a result, no significant difference was recognized in the current-voltage relationship and the channel activation, between the normal sodium ion channel and the mutant sodium ion channel (see (a) and (b) of FIG. 3). Meanwhile, a significant test was performed regarding the channel inactivation, on a point that the normal sodium ion channel and the mutant sodium ion channel are inactivated by 50%, whereby resulted in finding that the mutant sodium ion channel had shifted significantly to the depolarization side (p<0.05) ((c) of FIG. 3).
As to the recovery from the channel inactivation, it was found that the recovery was significantly slow in the mutant sodium ion channel ((d) of FIG. 3). In (d) of FIG. 3, a part in which a period of recovery (Recovery period (ms)) from the inactivation was 1 ms to 8 ms corresponds to a “fast component”, and a part in which the period of recovery from the inactivation was 10 ms to 100 ms corresponds to a “slow component”.
More specifically, upon comparison between the normal sodium ion channel and an abnormal sodium ion channel based on a time required for the fast component in recovering from the inactivation to recover from the inactivation to 33.3%, it was found that the recovery was significantly slow for the mutant sodium ion channel (normal: τf=1.7±0.1 ms, n=14; mutant: τf=2.5±0.2 ms (P<0.01), n=12).
Similarly, upon comparison of the normal sodium ion channel with the abnormal sodium ion channel based on the time required for the slow component in recovering from the inactivation to recover from the inactivation to 33.3%, it was found that the mutant sodium ion channel was significantly slow in recovering (normal: τs=40.3±5.3 ms, n=14; mutant: τs=60.9±7.9 ms (P<0.05), n=12).
FIG. 4 shows that, even if the sodium ion channel was made inactivated after the potential was changed to activate the sodium ion channel, the baseline of the mutant sodium channel does not return back in the whole cell record, which indicates clearly that the sodium current was persistently flowing into the mutant sodium ion channel. The persistent sodium current is considered as an obstruction of an inactivation gate. From the view of (a) of FIG. 4, it was confirmed that even after the elapse of time, the inactivation was insufficient in the mutant sodium ion channel as compared to that of the normal sodium ion channel.
So as to find the persistent sodium current shown in (a) of FIG. 4, a relative value (%) was found by dividing, with a maximum current amount, a final current amount that flowed between 80 milliseconds to 100 milliseconds when a depolarizing stimulus of 100 milliseconds was given. Results thereof are shown in (b) of FIG. 4. From these results, it was found that the mutant sodium ion channel had properties that the persistent sodium current increases.
This data show that the function of the voltage-gated sodium ion channel NaV1.1 became abnormal by the mutation. Namely, this means that by having the mutation, the nerve cells are easily excessively excited, that is to say, more easily causes the occurrence of a convulsion.
Literature (Satoko Tokuda et. al., BRAINRESEARCH 1133 (2007) 168-177; Kenta Tanaka et. al., Neuroscience Letters 426 (2007) 75-80) discloses that the function of the voltage-gated calcium ion channel CaV2.1 of a rat becomes abnormal due to a mutation (M251K) on the al subunit of the voltage-gated calcium ion channel CaV2.1 of the rat.
Therefore, with a rat having the mutation on both the Scn1a gene and Cacna1a gene described later, it can be considered that the functions of both the voltage-gated sodium ion channel NaV1.1 and the voltage-gated calcium ion channel CaV2.1 are abnormal.
(2. Confirmation of Gene Mutation in Dravet Syndrome Model Rat)
The foregoing F344-Scn1aKyo811 and the GRY (groggy rat, Cacna1agry) were mated as parent rats (P) to produce F1 (first filial generation) rats, and these F1 rats were mated to produce F2 (second filial generation) rats. FIG. 5 is a view showing genotypes of the parent rats (P), the F1 rats and the F2 rats. As illustrated in (a) of FIG. 5, the F1 rats have the heterozygous mutation on both the Scn1a gene and the Cacna1a gene (referred to as “Scn1a mutant (hetero)+Cacna1a mutant (hetero)”). Moreover, as illustrated in (b) of FIG. 5, rats showing 9 types of genotypes were born from the F2 rats. The genotypes of each of the rats were identified by extracting a tip tissue of the tail of the rats and extracting its DNA, to perform DNA sequencing with the extracted DNA and detect its gene mutation, or by detecting a digested pattern with use of a restriction enzyme.
(Method of Confirming Gene Mutation by DNA Sequencing)
Confirmation of gene mutation by DNA sequencing was performed as follows. First, a genomic DNA was amplified with use of a primer pair that sandwiches a mutation point (a nucleotide sequence of a Scn1a amplification primer pair is represented by SEQ ID NO.: 5 and SEQ ID NO.: 6, and a nucleotide sequence of a Cacna1a amplification primer pair is represented by SEQ ID NO.: 7 and SEQ ID NO.: 8), and thereafter, an obtained PCR product was purified with use of a PCR products pre-sequencing kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, England). See the item “Sequence of primers” later described for the nucleotide sequence of the used primer pairs.
Next, sequence reaction was performed with use of a Big Dye Terminator FS ready-reaction kit (Applied Biosystems), to determine a nucleotide sequence with a fluorescence sequencer (ABI PRISM3100 sequencer; Applied Biosystems).
FIG. 6 is a view illustrating a method of identifying a genotype of the Scn1a gene and the Cacna1a gene of the F2 rats, by sequencing. As illustrated in FIG. 6, a wild-type Scn1a gene has a nucleotide at position 4249 be “A”. In comparison, a mutant Scn1a gene (N1417H) has a nucleotide at position 4249 that is mutated from “A” to “C”. As a result, a codon “AAT” that designates asparagine (N) being an amino acid at position 1417 in the wild-type Scn1a gene, is mutated to a codon “CAT” which designates histidine (H), in the mutant Scn1a gene (N1417H).
Moreover, the wild-type Cacna1a gene has a nucleotide at position 752 be “T”. In comparison, the mutant Cacna1a gene (M251K) has a nucleotide at position 752 that is mutated from “T” to “A”. As a result, a codon “ATG” that designates methionine, which is an amino acid at position 251, is mutated to a codon “AAG” that designates lysine.
(Method of Confirming Gene Mutation by Restriction Enzyme Digestion)
The method of confirming gene mutation by the restriction enzyme digestion was performed as follows. When detecting mutation in the Scn1a gene, a genomic DNA was amplified with use of a primer pair (SEQ ID NOs.: 5 and 6) that sandwich a mutation point in the Scn1a gene, and thereafter an obtained PCR product was reacted for three hours at 50° C., with use of a restriction enzyme Bc1I. Thereafter, the PCR product reacted with the restriction enzyme was subjected to electrophoresis with use of 4% agarose gel, and the size of the band was detected. FIG. 7 is a view illustrating a method of identifying the genotype of the Scn1a gene of the F2 rats, by restriction enzyme digestion.
As shown in (a) and (b) of FIG. 7, the wild-type Scn1a gene was not digested with Bc1I, so the size of the band remained as the size of the PCR product (nucleotide of 380 bp). On the other hand, the mutant Scn1a gene (N1417H) was digested with Bc1I, so two fragments (nucleotides of 276 bp and 104 bp) were detected. In a case of a heterozygous rat of the wild-type Scn1a gene and the mutant Scn1a gene (N1417H), three fragments (nucleotides of 380 bp, 276 bp, and 104 bp) were detected. Illustrated in (c) of FIG. 7 shows a result of electrophoresis.
In a case of detecting the mutation on the Cacna1a gene, a genomic DNA was amplified with use of a primer pair (SEQ ID NOs.: 7 and 8) that sandwich a mutation point of the Cacna1a gene, and thereafter, an obtained PCR product was reacted for 1 hour at 37° C. with use of a restriction enzyme PciI. Thereafter, the PCR product reacted with the restriction enzyme was subjected to electrophoresis with use of 4% agarose gel, to detect the size of a band.
FIG. 8 is a view illustrating a method of identifying a genotype of the Cacna1a gene of the F2 rats, by restriction enzyme digestion. As illustrated in (a) and (b) of FIG. 8, a wild-type Cacna1a gene was not digested with PciI, so hence the size of the band remained as the size of the PCR product (nucleotide of 352 bp). On the other hand, the mutant Cacna1a gene (M251K) was digested with PciI, and thus two fragments (nucleotides of 219 bp and 133 bp) were detected. With a heterozygous rat of the wild-type Cacna1a gene and an abnormal Cacna1a gene (M251K), three fragments (nucleotides of 352 bp, 219 bp, and 133 bp) were detected. Illustrated in (c) of FIG. 8 is a result of electrophoresis.
Example 7
Analysis of Dravet Syndrome Model Rat
A study was performed on what kind of (worsening) effect was given on the seizure when a mutation on the Cacna1a gene was added to a mutation on the Scn1a gene, with use of a Dravet syndrome model rat. More specifically, comparison was made regarding symptoms when a convulsion seizure was induced by heat load, between a rat having a homozygous mutation on the Scn1a gene (referred to as “Scn1a mutant (homo)+Cacna1a wild-type (homo)”) and a rat having a homozygous mutation on the Scn1a gene and a heterozygous mutation on the Cacna1a gene (referred to as “Scn1a mutant (homo)+Cacna1a mutant (hetero)”).
The Scn1a mutant (homo)+Cacna1a wild-type (homo) and the Scn1a mutant (homo)+Cacna1a mutant (hetero) both have a homozygous mutation on the Scn1a gene (N1417H). Hence, comparison is made between the wild-type Cacna1a gene and the mutant Cacna1a gene (M251K), under the condition of the homozygous mutation of the Scn1a gene.
Moreover, a rat having a wild-type Scn1a gene and a wild-type Cacna1a gene (referred to as “Scn1a wild-type (homo)+Cacna1a wild-type (homo)”) and a rat having a wild-type homozygous mutation on the Scn1a gene and a heterozygous mutation on the Cacna1a gene (referred to as “Scn1a wild-type (homo)+Cacna1a mutation (hetero)”) were used as control. The following lists the genotypes of the rats used in the experiment. The following numbers (1) to (4) correspond to the numbers in (b) of FIG. 5.
(1) Scn1awt/wtCacna1awt/wt (Scn1a wild-type (homo)+Cacna1a wild-type (homo)) 14 males
(2) Scn1amut/mutCacna1awt/wt (Scn1a mutant (homo)+Cacna1a wild-type (homo)) 7 males
(3) Scn1amut/mutCacna1awt/mut (Scn1a mutant (homo)+Cacna1a mutant (hetero)) 17 males
(4) Scn1awt/wtCacna1awt/mut (Scn1a wild-type (homo)+Cacna1a mutant (hetero)) 12 males.
Hot bath load (45° C.) were given on male rats of 5 weeks old of the groups (1) to (4) described above, to compare their body temperatures at a time when a convulsion is induced, their duration of the convulsion, and their severity score of the convulsion. A rectal temperature at the time when the seizure started was measured, to serve as the body temperature at the time when the convulsion was induced. The seizure severity score of the convulsion were evaluated as follows: 0=no seizure, 1=facial convulsion, 2=clonic convulsion of both arms while maintaining posture, 3=sprint or jump, 4=generalized convulsion unable to maintain posture, and 5=death caused by persistent convulsion.
The results were as shown in FIG. 9. FIG. 9 is a view showing a result of the effect caused by the mutation on the Cacna1a gene in the Scn1a gene-mutated rat. In the graphs of (a) to (c) in FIG. 9, Scn1amut/mutCacna1awt/wt (the foregoing rat (2)) is shown as “Scn1a mutant (homo)”. Scn1amut/mutCacna1awt/mut (the foregoing rat (3)) is shown as “Scn1a mutant (homo)+Cacna1a mutant (hetero)”. Moreover, control Scn1awt/wtCacna1awt/wt (foregoing rat (1)) is shown as “WT”, and control Scn1awt/wtCacna1awt/mut (foregoing rat (4)) is shown as “Cacna1a mutant (hetero)”.
As a result of analysis, the group (3) rats (Scn1a mutant (homo)+Cacna1a mutant (hetero)) had no large difference in the body temperatures at the time of convulsion onset (convulsion threshold) ((a) of FIG. 9) and severity scores ((b) of FIG. 9), from those of the group (2) rats (Scn1a mutant (homo)+Cacna1a wild-type (homo)). However, it was found that the duration of the convulsion ((c) of FIG. 9) became significantly long. This result demonstrates that the mutation of the Cacna1a gene relates to the worsening of the symptoms of convulsion.
Furthermore, FIG. 10 shows a part of an electroencephalogram during a seizure of a group (3) rat (Scn1a mutant (homo)+Cacna1a mutant (hetero)). It was considered from this result that a rat having a mutation on the Scn1a gene and the Cacna1a gene could serve as a model rat of the intractable Dravet syndrome. The model rat is expected to be usefully used in the future for clarification of the onset mechanism of the intractable Dravet syndrome, development of medicament for Dravet syndrome, and like uses.
Moreover, these results are considered as supporting the gene analysis data of Example 1, that a variation of the CACNA1A gene was detected in addition to a mutation on the SCN1A gene, in a patient of Dravet syndrome which is an intractable epilepsy. Namely, the method according to the present invention of obtaining data for assessing the potential for development of Dravet syndrome can be said as a technique supported by the gene analysis results of the Examples, a mutant channel function analysis result, and animal experiment results.
CONCLUSION
The present invention was developed based on a molecular foundation of development of the intractable Dravet syndrome; the assessment method according to the present invention can be said as useful as an early detection method of Dravet syndrome patients. By use of the assessment method according to the present invention, it is possible to find Dravet syndrome, which has an unfavorable prognosis, in high accuracy and at an early stage. This allows for an epilepsy specialist to prepare a treatment management system for the patient of Dravet syndrome from an early stage. As a result, this leads to improvement in therapeutic intervention of the patient, reduction of mental load on the family, and reduction of economical burden. Moreover, it is possible to carry out appropriate treatment to the Dravet syndrome patient, so therefore is considered as contributive to the reduction of medical fees.
Furthermore, with use of the kit according to the present invention, it is possible to easily detect the mutation for both the SCN1A gene and CACNA1A gene. Consequently, the kit according to the present invention is useful for a general pediatrician to distinguish a patient of Dravet syndrome who requires treatment by a specialist out of the benign febrile epilepsies, during the initial stage of the disease under the age of one.
By use of the assessment method and the kit according to the present invention, it is possible to detect with high accuracy a patient of Dravet syndrome at the point in time of under the age of one, which was difficult to detect until now. Moreover, by examining gene abnormalities upon sending the blood taken to an examination center, it is possible to detect Dravet syndrome patients in high accuracy even for a remote personal hospital or the like.
Moreover, the model animal and cell according to the present invention may be usefully used in the clarification of an onset mechanism of the intractable Dravet syndrome, the development of medicament for Dravet syndrome, and like uses.
<Primer Sequences>
Table 10 shows a nucleotide sequence of a primer pair used for amplifying the Scn1a gene and amplifying the Cacna1a gene.









TABLE 10







Scn1a
Sense primer:
SEQ ID


amplifi-
5′-TGA CTT TTC TTT CTC TCC GTT
NO.: 5


cation
TG-3′




Antisense primer:
SEQ ID



5′-TGG CTG CAA TAA TCA CTT TGT
NO.: 6



T-3′



 


Cacna1a
Sense primer:
SEQ ID


amplifi-
5′- TCT CTG TCT CCC CAG GTT TAC-
NO: 7


cation
3′




Antisense primer:
SEQ ID



5′-GTG GCT AAC ACA CAG CTT TGC-3′
NO.: 8









Tables 11 and 12 show nucleotide sequences of primer pairs used for detecting SCN1A gene genomes.









TABLE 11







Exon 1
Sense primer:
SEQ ID


amplifi-
5′-tcatggcacagttcctgtatc-3′
NO.: 9


cation
Antisense primer:
SEQ ID



5′-gcagtaggcaattagcagcaa-3′
NO.: 10


 


Exon 2
Sense primer:
SEQ ID


amplifi-
5′-tggggcactttagaaattgtg-3′
NO.: 11


cation
Antisense primer:
SEQ ID



5′-tgacaaagatgcaaaatgagag-3′
NO.: 12


 


Exon 3
Sense primer:
SEQ ID


amplifi-
5′-gcagtttgggcttttcaatg-3′
NO.: 13


cation
Antisense primer:
SEQ ID



5′-tgagcattgtcctcttgctg-3′
NO.: 14


 


Exon 4
Sense primer:
SEQ ID


amplifi-
5′-agggctacgtttcatttgtatg-3′
NO.: 15


cation
Antisense primer:
SEQ ID



5′-tgtgctaaattgaaatccagag-3′
NO.: 16


 


Exon 5
Sense primer:
SEQ ID


amplifi-
5′-CAGCTCTTCGCACTTTCAGA-3′
NO.: 17


cation
Antisense primer:
SEQ ID



5′-TCAAGCAGAGAAGGATGCTGA-3′
NO.: 18


 


Exon 6
Sense primer:
SEQ ID


amplifi- 
5′-agcgttgcaaacattcttgg-3′
NO.: 19


cation
Antisense primer:
SEQ ID



5′-gggatatccagcccctcaag-3′
NO.: 20


 


Exon 7
Sense primer:
SEQ ID


amplifi- 
5′-gacaaatacttgtgcctttgaatg-3′
NO.: 21


cation
Antisense primer:
SEQ ID



5′-acataatctcatactttatcaaaaacc-3′ 
NO.: 22


 


Exon 8
Sense primer:
SEQ ID


amplifi-
5′-gaaatggaggtgttgaaaatgc-3′
NO.: 23


cation
Antisense primer:
SEQ ID



5′-aatccttggcatcactctgc-3′
NO.: 24


 


Exon 9
Sense primer:
SEQ ID


amplifi-
5′-agtacagggtgctatgaccaac-3′
NO.: 25


cation
Antisense primer:
SEQ ID



5′-tcctcatacaaccacctgctc-3′
NO.: 26


 


Exon 10
Sense primer:
SEQ ID


amplifi- 
5′-tctccaaaagccttcattagg-3′
NO.: 27


cation
Antisense primer:
SEQ ID



5′-ttctaattctccccctctctcc-3′
NO.: 28


 


Exon 11
Sense primer:
SEQ ID


amplifi-
5′-tcctcattctttaatcccaagg-3′
NO.: 29


cation
Antisense primer:
SEQ ID



5′-gccgttctgtagaaacactgg-3′
NO.: 30


 


Exon 12
Sense primer:
SEQ ID


amplifi-
5′-gtcagaaatatctgccatcacc-3′
NO.: 31


cation
Antisense primer:
SEQID



5′-gaatgcactattcccaactcac-3′
NO.: 32


 


Exon 13
Sense primer:
SEQ ID


amplifi-
5′-tgggctctatgtgtgtgtctg-3′
NO.: 33


cation
Antisense primer:
SEQ ID



5′-ggaagcatgaaggatggttg-3′
NO.: 34


 


Exon 14
Sense primer:
SEQ ID


amplifi-
5′-tacttcgcgtttccacaagg-3′
NO.: 35


cation
Antisense primer:
SEQ ID



5′-gctatgcaagaaccctgattg-3′
NO.: 36


















TABLE 12







Exon 15
Sense primer:
SEQ ID


amplifi-
5′-atgagcctgagcggttagg-3′
NO.: 37


cation
Antisense primer:
SEQ ID



5′-atacatgtgccatgctggtg-3′
NO.: 38


 


Exon 16
Sense primer:
SEQ ID


amplifi-
5′-tgctgtggtgtttccttctc-3′
NO.: 39


cation
Antisense primer:
SEQ ID



5′-tgtattcataccttcccacacc-3′
NO.: 40


 


Exon 17
Sense primer:
SEQ ID


amplifi-
5′-aaaagggttagcacagacaatg-3′
NO.: 41


cation
Antisense primer:
SEQ ID



5′-attgggcagatataatcaaagc-3′
NO.: 42


 


Exon 18
Sense primer:
SEQ ID


amplifi-
5′-cacacagctgatgaatgtgc-3′
NO.: 43


cation
Antisense primer:
SEQ ID



5′-tgaagggctacactttctgg-3′
NO.: 44


 


Exon 19
Sense primer:
SEQ ID


amplifi-
5′-tctgccctcctattccaatg-3′
NO.: 45


cation
Antisense primer:
SEQ ID



5′-gcccttgtcttccagaaatg-3′
NO.: 46


 


Exon 20
Sense primer:
SEQ ID


amplifi- 
5′-aaaaattacatcctttacatcaaactg-3′
NO.: 47


cation
Antisense primer:
SEQ ID



5′-ttttgcatgcatagattttcc-3′
NO.: 48


 


Exon 21
Sense primer:
SEQ ID


amplifi- 
5′-tgaaccttgcttttacatatcc-3′
NO.: 49


cation
Antisense primer:
SEQ ID



5′-acccatctgggctcataaac-3′ 
NO.: 50


 


Exon 22
Sense primer:
SEQ ID


amplifi-
5′-tgtcttggtccaaaatctgtg-3′
NO.: 51


cation
Antisense primer:
SEQ ID



5′-ttggtcgtttatgctttattcg-3′
NO.: 52


 


Exon 23
Sense primer:
SEQ ID


amplifi-
5′-ccctaaaggccaatttcagg-3′
NO.: 53


cation
Antisense primer:
SEQ ID



5′-atttggcagagaaaacactcc-3′
NO.: 54


 


Exon 24
Sense primer:
SEQ ID


amplifi- 
5′-gagatttgggggtgtttgtc-3′
NO.: 55


cation
Antisense primer:
SEQ ID



5′-ggattgtaatggggtgcttc-3′
NO.: 56


 


Exon 25
Sense primer:
SEQ ID


amplifi-
5′-caaaaatcagggccaatgac-3′
NO.: 57


cation
Antisense primer:
SEQ ID



5′-tgattgctgggatgatcttg-3′
NO.: 58


 


Exon 26(1)
Sense primer:
SEQ ID


amplifi-
5′-aggactctgaaccttaccttgg-3′
NO.: 59


cation
Antisense primer:
SEQ ID



5′-ccatgaatcgctcttccatc-3′
NO.: 60


 


Exon 26(2)
Sense primer:
SEQ ID


amplifi-
5′-tgtgggaacccatctgttg-3′
NO.: 61


cation
Antisense primer:
SEQ ID



5′-gtttgctgacaaggggtcac-3′
NO.: 62









Tables 13 and 14 show nucleotide sequences of primer pairs used for detecting the CACNA1A gene genome. In Tables 13 and 14, for example, E1F indicates an Exon 1 amplification sense primer, and E1Rv indicates an Exon 1 amplification antisense primer.









TABLE 13







Exon 1
CACNA1A-E1F:
SEQ ID


amplifi-
5′-tctccgcagtcgtagctccag-3′
NO.: 63


cation
CACNA1A-E1Rv:
SEQ ID



5′-agagattctttcacactcctcc-3′
NO.: 64


 


Exon 2
CACNA1A-E2F:
SEQ ID


amplifi-
5′-tttagaagtcacctgatctggg-3′
NO.: 65


cation
CACNA1A-E2Rv:
SEQ ID



5′-gacagagcgagactctggttca-3′ 
NO.: 66


 


Exon 3
CACNA1A-E3F:
SEQ ID


amplifi-
5′-gacaagagaactctgcaagagg-3′
NO.: 67


cation
CACNA1A-E3Rv:
SEQ ID



5′-atacagctgagacatggaggtg-3′ 
NO.: 68


 


Exon 4
CACNA1A-E4F:
SEQ ID


amplifi-
5′-tttatcccgtgaggcaggtactg-3′
NO.: 69


cation
CACNA1A-E4Rv:
SEQ ID



5′-cctcctgagatgctctgcatag-3′ 
NO.: 70


 


Exon 5
CACNA1A-E5F:
SEQ ID


amplifi-
5′-tgtggtgcttccttcaccattg-3′
NO.: 71


cation
CACNA1A-E5Rv:
SEQ ID



5′-cagaggctatttcactcactgc-3′ 
NO.: 72


 


Exon 6
CACNA1A-E6F:
SEQ ID


amplifi-
5′-ccccaaagccaaacattgatctc-3′
NO.: 73


cation
CACNA1A-E6Rv:
SEQ ID



5′-actctgattgtccacacacactg-3′
NO.: 74


 


Exon 7
CACNA1A-E7F:
SEQ ID


amplifi-
5′-cagaaaacgttcctccatttccc-3′
NO.: 75


cation
CACNA1A-E7Rv:
SEQ ID



5′-aagcttcaatggcctctacttgg-3′
NO.: 76


 


Exon 8
CACNA1A-E8F:
SEQ ID


amplifi-
5′-gccatactctggcttttctatgc-3′
NO.: 77


cation
CACNA1A-E8Rv:
SEQ ID



5′-cgtgatgtcagatcctggcttc-3′ 
NO.: 78


 


Exon 9
CACNA1A-E9F:
SEQ ID


amplifi-
5′-gttggctattgctactgttgcg-3′
NO.: 79


cation
CACNA1A-E9Rv:
SEQ ID



5′-gatccttagaaccagtcacctg-3′ 
NO.: 80


 


Exon 10
CACNA1A-E10F:
SEQ ID


amplifi-
5′-tgatagtgccaccttgaacctc-3′
NO.: 81


cation
CACNA1A-E10Rv:
SEQ ID



5′-tgatgtaatctgcccaggacac-3′ 
NO.: 82


 


Exon 11
CACNA1A-E11F:
SEQ ID


amplifi-
5′-ctgcaacagagaactatcagcc-3′
NO.: 83


cation
CACNA1A-E11Rv:
SEQ ID



5′-aagagaagtggaaaaagggtgtg-3′
NO.: 84


 


Exon 12
CACNA1A-E12F:
SEQ ID


amplifi-
5′-gtagttctagcatgttggaggc-3′
NO.: 85


cation
CACNA1A-E12Rv:
SEQ ID



5′-atctgtcattccaggcaagagc-3′ 
NO.: 86


 


Exon 13~15
CACNA1A-E13F:
SEQ ID


amplifi-
5′-atggatgaatgagggggtcaag-3′
NO.: 87


cation
CACNA1A-E15Rv:
SEQ ID



5′-agcaggcactttcatctgtgac-3′ 
NO.: 88


 


Exon 13~15
CACNA1A-E13F2:
SEQ ID


amplifi-
5′-tccatttggagggaggagtttg-3′
NO.: 89


cation
CACNA1A-E15Rv:
SEQ ID



5′-agcaggcactttcatctgtgac-3′ 
NO.: 88


 


Exon 14~15
CACNA1A-E14F:
SEQ ID


amplifi-
5′-cctccagaaagttgggaaagtg-3′
NO.: 90


cation
CACNA1A-E15Rv:
SEQ ID



5′-agcaggcactttcatctgtgac-3′ 
NO.: 88


 


Exon 16~17
CACNA1A-E16F:
SEQ ID


amplifi-
5′-aaggagaagccaacacggagtc-3′
NO.: 91


cation
CACNA1A-E17Rv:
SEQ ID



5′-ggtggtaactttgccagagaaac-3′
NO.: 92


 


Exon 18
CACNA1A-E18F:
SEQ ID


amplifi-
5′-agcaggtacccattccaattgg-3′
NO.: 93


cation
CACNA1A-E18Rv:
SEQ ID



5′-aatctgtgcctgggatagtgtg-3′ 
NO.: 94


 


Exon 19
CACNA1A-E19F:
SEQ ID


amplifi-
5′-cctgactcagatgctcacagac-3′
NO.: 95


cation (1)
CACNA1A-E19Rv:
SEQ ID



5′-acacagcacgtgctactttggc-3′
NO.: 96


 


Exon 19
CACNA1A-E19F2:
SEQ ID


amplifi-
5′-gaggacttcctcaggaaacag-3′
NO.: 97


cation (2)
CACNA1A-E19Rv: 
SEQ ID



5′-acacagcacgtgctactttggc-3′
NO.: 96


 


Exon 20
CACNA1A-E20F:
SEQ ID


amplifi-
5′-agatggaatcttagctaggatcc-3′
NO.: 98


cation
CACNA1A-E20Rv:
SEQ ID



5′-aattatctcactgaaccctccac-3′
NO.: 99


 


Exon 21
CACNA1A-E21F:
SEQ ID


amplifi-
5′-agaaatgtcagccgcttcttgc-3′
NO.: 100


cation
CACNA1A-E21Rv:
SEQ ID



5′-ggtggtcaacactcactcattg-3′
NO.: 101


 


Exon 22
CACNA1A-E22F:
SEQ ID


amplifi-
5′-tttgttgtgtaggaggccttgg-3′
NO.: 102


cation
CACNA1A-E22Rv:
SEQ ID



5′-aacatcccaccctacctatgag-3′
NO.: 103


















TABLE 14







Exon 23
CACNA1A-E23F:
SEQ ID


amplifi- 
5′-cctgcgcaactgtatatagcag-3′
NO.: 104


cation
CACNA1A-E23Rv:
SEQ ID



5′-ctcaacctcctgatctcaagtg-3′
NO.: 105


 


Exon 24
CACNA1A-E24F:
SEQ ID


amplifi- 
5′-cccaaagtttggatctaagagcc-3′
NO.: 106


cation
CACNA1A-E24Rv:
SEQ ID



5′-aaagccatcgaagctcttcctg-3′ 
NO.: 107


 


Exon 25
CACNA1A-E25F:
SEQ ID


amplifi- 
5′-caggtgaaatggaccactcttc-3′
NO.: 108


cation
CACNA1A-E25Rv:
SEQ ID



5′-tccttgagcagtgtacaacctg-3′ 
NO.: 109


 


Exon 26
CACNA1A-E26F:
SEQ ID


amplifi- 
5′-gaatgccaggattgagtccaac-3′
NO.: 110


cation
CACNA1A-E26Rv:
SEQ ID



5′-gaatgtgctggaaagtggagac-3′ 
NO.: 111


 


Exon 27
CACNA1A-E27F:
SEQ ID


amplifi- 
5′-cactgcttcccaagcagtctag-3′
NO.: 112


cation
CACNA1A-E27Rv:
SEQ ID



5′-attacaggcgtgagccaccatg-3′ 
NO.: 113


 


Exon 28
CACNA1A-E28F:
SEQ ID


amplifi- 
5′-tttccctctgttcctgttctgc-3′
NO.: 114


cation
CACNA1A-E28Rv: 
SEQ ID 



5′-ttcggttgggacaatgcttctg-3'
NO.: 115


 


Exon 29
CACNA1A-E29F:
SEQ ID


amplifi- 
5′-ctcaagcaactgtagctgttgg-3′
NO.: 116


cation
CACNA1A-E29Rv:
SEQ ID



5′-ttatcagggtagaggcaggaac-3′ 
NO.: 117


 


Exon 30
CACNA1A-E30F:
SEQ ID


amplifi- 
5′-gtgaaaagaagagcctagtccg-3′
NO.: 118


cation
CACNA1A-E30Rv:
SEQ ID



5′-atggtaacactcacaggttgg-3′ 
NO.: 119


 


Exon 31
CACNA1A-E31F:
SEQ ID


amplifi- 
5′-gcccttcgaacaaccataactg-3′
NO.: 120


cation
CACNA1A-E31Rv:
SEQ ID



5′-cctacagccaagctttggttac-3′ 
NO.: 121


 


Exon 32
CACNA1A-E32F:
SEQ ID 


amplifi-
5′-cccattggttttttggcactgg-3′
NO.: 122


cation
CACNA1A-E32Rv:
SEQ ID


 
5′-ggacagacagacagaggagag-3′
NO.: 123


 


Exon 33~35
CACNA1A-E33F:
SEQ ID


amplifi- 
5′-tgttggttggcttcatgtaggg-3'
NO.: 124


cation
CACNA1A-E35Rv:
SEQ ID



5′-cagaattatcagagcaggtccc-3′ 
NO.: 125


 


Exon 36
CACNA1A-E36F:
SEQ ID


amplifi- 
5′-tctcagctcccagtaaaaggag-3′
NO.: 126


cation
CACNA1A-E36Rv:
SEQ ID



5′-caacagtgctgagtttgagacg-3′ 
NO.: 127


 


Exon 37
CACNA1A-E37F:
SEQ ID


amplifi- 
5′-ggcctctgtgtacatgtctttg-3′
NO.: 128


cation
CACNA1A-E37Rv:
SEQ ID



5′-gggtatgcaagggtgatgattc-3′ 
NO.: 129


 


Exon 38
CACNA1A-E38F:
SEQ ID


amplifi- 
5′-tgtttctccccacctctcttc-3'
NO.: 130


cation
CACNA1A-E38Rv:
SEQ ID



5′-aaaaaaacccagtgcctggacg-3′ 
NO.: 131


 


Exon 39
CACNA1A-E39F:
SEQ ID


amplifi- 
5′-agaaactgagtactgggacagg-3′
NO.: 132


cation
CACNA1A-E39Rv:
SEQ ID



5′-ggaagagtgaatgaagatccgg-3′ 
NO.: 133


 


Exon 40~41
CACNA1A-E40F:
SEQ ID


amplifi- 
5′-aaagattggggtctcgttctcg-3′
NO.: 134


cation
CACNA1A-E41Rv:
SEQ ID



5′-ccctcatattccagttggttcc-3′ 
NO.: 135


 


Exon 42~44
CACNA1A-E42F:
SEQ ID


amplifi- 
5′-gtgtgtgtgtgtgtatactggg-3′
NO.: 136


cation
CACNA1A-E44Rv:
SEQ ID



5′-cagactgcttcagagactgaag-3′ 
NO.: 137


 


Exon 45
CACNA1A-E45F:
SEQ ID


amplifi- 
5′-ccgatttctcttgatgccagtg-3′
NO.: 138


cation
CACNA1A-E45Rv:
SEQ ID



5′-agggtgcgattgccaaagaaag-3′ 
NO.: 139


 


Exon 46~47
CACNA1A-E46F:
SEQ ID


amplifi- 
5′-acccagagccctgattgatcag-3′
NO.: 140


cation
CACNA1A-E47Rv:
SEQ ID



5′-ttggatggggtatccccttctc-3′ 
NO.: 141


 


Exon 48
CACNA1A-E48F:
SEQ ID


amplifi- 
5′-tctcttcctcccaatcccgtg-3′
NO.: 142


cation
CACNA1A-E48Rv:
SEQ ID



5′-tgcccaggagggtctcttttg-3′
NO.: 143









INDUSTRIAL APPLICABILITY
As described above, by detecting the presence of a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, it is possible to obtain data for assessing a potential for development of Dravet syndrome of a subject who has not yet been subjected to onset of Dravet syndrome, with high accuracy. Hence, it is possible to distinguish a patient of Dravet syndrome that requires treatment by a specialist, out of benign febrile seizure patents, at an initial stage of disease under the age of one. Hence, it is possible to use not only in the field of diagnosis medical treatment such as medical devices, diagnosis kits and the like, but broadly in the health science and medical field industry.
Moreover, in the present invention, by introducing a mutation on both of α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1, it is possible to produce a model animal of Dravet syndrome. Such a model animal of Dravet syndrome can be used for development of medicament and treatment methods of Dravet syndrome. Hence, the present invention can be widely used in the industry of life science fields including the pharmaceutical field.

## Claims

1. A model animal of Dravet syndrome, having a mutation on both α-subunit type 1 of voltage-gated sodium ion channel NaV1.1 (Nav1.1α1) and α-subunit type 1 of voltage-gated calcium ion channel CaV2.1 (Cav2.1α1), wherein
the mutation on the Nav1.1α1 is selected from the group consisting of A1685D, F902C, deletion of Exon 10, G163E, G177R, K547fsX570, P707fsX714, Q1277X, Q1450R, R1648C, R377L, R712X, R865X, S1574X, T1082fsX1086, T1909I, V1390M, V212A, and W738fsX746, and
the mutation on the Cav2.1α1 is selected from the group consisting of A924G, E921D, E996V, G1108S, G266S, K472R, R1126H and R2201Q,
wherein
in the case that the mutation on the Nav1.1α1 is G177R, the mutation on the Cav2.1α1 is G266S,
in the case that the mutation on the Nav1.1α1 is W738fsX746, the mutation on the Cav2.1α1 is K472R,
in the case that the mutation on the Nav1.1α1 is V1390M, the mutation on the Cav2.1α1 is A924G,
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of A1685D, F902C, deletion of Exon 10, P707fsX714, Q1277X, Q1450R, R377L, R865X, T1082fsX1086 and V212A, the mutation on the Cav2.1α1 is at least one of E921D and E996V,
in the case that the mutation on the Nav1.1α1 is T1909I, the mutation of Cav2.1α1 is at least one of E921D, E996V, R1126H and R2201Q,
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of G163E, K547fsX570 and S1574X, the mutation on the Cav2.1α1 is at least one of R1126H and R2201Q, and
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of R712X and R1648C, the mutation on the Cav2.1α1 is G1108S.

2. The model animal according to claim 1,
wherein the mutation on the Nav1.1α1 is selected from the group consisting of V212A, R377L, deletion of exon 10, P707fsX714, R865X, F902C, T1082fsX1086, Q1277X, Q1450R, A1685D, and
wherein the mutation on the Cav2.1α1 is either E921D or E996V.

3. The model animal according to claim 1,
wherein the mutation on the Nav1.1α1 is T1909I, and
wherein the mutation on the Cav2.1α1 is selected form the group consisting of E921D, E996V, R1126H, and R2201Q.

4. The method according to claim 1,
wherein the mutation on the Nav1.1α1 is selected from the group consisting of G163E, K547fsX570, and S1574X, and

wherein the mutation on the Cav2.1α1 is either R1126H or R2201Q.

5. A method of producing a model animal of Dravet syndrome as set forth in claim 1, the method comprising:
introducing a mutation on a α-subunit type 1 of the voltage-gated sodium ion channel NaV1.1 (Nav1.1α1); and
introducing a mutation on a α-subunit type 1 of the voltage-gated calcium ion channel CaV2.1 (Cav2.1α1), wherein
the mutation on the Nav1.1α1 is selected from the group consisting of A1685D, F902C, deletion of Exon 10, G163E, G177R, K547fsX570, P707fsX714, Q1277X, Q1450R, R1648C, R377L, R712X, R865X, S1574X, T1082fsX1086, T1909I, V1390M, V212A, and W738fsX746, and
the mutation on the Nav1.1α1 is selected from the group consisting of A924G, E921D, E996V, G1108S, G266S, K472R, R1126H and R2201Q,
wherein
in the case that the mutation on the Nav1.1α1 is G177R, the mutation on the Cav2.1α1 is G266S,
in the case that the mutation on the Nav1.1α1 is W738fsX746, the mutation on the Cav2.1α1 is K472R,
in the case that the mutation on the Nav1.1α1 is V1390M, the mutation on the Cav2.1α1 is A924G,
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of A1685D, F902C, deletion of Exon 10, P707fsX714, Q1277X, Q1450R, R377L, R865X, T1082fsX1086 and V212A, the mutation on the Cav2.1α1 is at least one of E921D and E996V,
in the case that the mutation on the Nav1.1α1 is T1909I, the mutation of Cav2.1α1 is at least one of E921D, E996V, R1126H and R2201Q,
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of G163E, K547fsX570 and S1574X, the mutation on the Cav2.1α1 is at least one of R1126H and R2201Q, and
in the case that the mutation on the Nav1.1α1 is selected from the group consisting of R712X and R1648C, the mutation on the Cav2.1α1 is G1108S.

6. The method according to claim 5,
wherein the mutation on the Nav1.1α1 is selected from the group consisting of V212A, R377L, deletion of exon 10, P707fsX714, R865X, F902C, T1082fsX1086, Q1277X, Q1450R, A1685D, and
wherein the mutation on the Cav2.1α1 is either E921D or E996V.

7. The method according to claim 5,
wherein the mutation on the Nav1.1α1 is T1909I, and
wherein the mutation on the Cav2.1α1 is selected form the group consisting of E921D, E996V, R1126H, and R2201Q.

8. The method according to claim 5,
wherein the mutation on the Nav1.1α1 is selected from the group consisting of G163E, K547fsX570, and S1574X, and

wherein the mutation on the Cav2.1α1 is either R1126H or R2201Q.

9. A screening method of a drug for treating Dravet syndrome, the method comprising:
administering a candidate agent to the model animal of Dravet syndrome as set forth in claim 1; and
assessing whether or not the administering of the candidate agent has made Dravet syndrome improve or cure in the model animal of Dravet syndrome.