JP6782412B2 - Pain genes and their uses - Google Patents

Description

The present invention relates to a gene involved in pain, a mutation in the gene that causes young periodic limb pain, and a method for screening a pain suppressant using the gene.

Pain (hereinafter, also referred to as "pain") is roughly classified into acute pain and chronic pain. Acute pain acts as a defense system that protects the body from further injuries, causing temporary pain due to injuries, burns, etc., but disappears as the pain stimulus disappears or heals. On the other hand, chronic pain is when the pain continues for a long period of time and the pain continues even after the injury etc. recovers, or the musculoskeletal system such as degenerative spondylosis and low back pain including diabetes and cancer. It may accompany various diseases such as connective tissue diseases, neurological diseases, rheumatic diseases, and diseases of unknown cause such as fibromyalgia.

Pain affects as much as 10% of the world's population. Chronic pain, in which pain continues for a long period of time, significantly reduces the quality of life of patients and causes difficulty in working, resulting in great social loss. Therefore, appropriate measures for pain are required. However, the underlying pain-causing substance associated with chronic pain has not been identified. Non-inflammatory limb joint pain is one of the most common chronic pains in the elderly, but the mechanism is still unknown.
On the other hand, there are families in which limb and / or limb joint pain occurs periodically in childhood and the mechanism is unknown. Patients in this family do not have cardiac conduction disorders or epilepsy, except that periodic limb / limb joint pain begins in infancy, remits in adulthood, and sometimes leaves migraine. Moreover, the cause of the pain (hereinafter, may be abbreviated as "juvenile periodic limb pain" in the present specification) has been unknown for a long time, such as no inflammatory findings in clinical examination.

Studies of hereditary pain disorders with a single gene have enhanced the molecular understanding of pain and provided new drug discovery targets for analgesics.
Voltage-gated sodium channels (Nav) are essential to generate action potentials in excitatory cells. Ten genes encoding the α-subunit of sodium channels have been identified in mammals. sodium channel voltage gated type XI alpha (SCN11A) is a potential-dependent sodium channel (Nav1.9) with a gate property that is resistant to tetrodotoxin, and is a small-diameter sensory sensation present in the dorsal root ganglion and trigeminal ganglion. It encodes an injury-acceptor localized in a small-diameter sensory neuron. Since knockout mice of this gene show a painless phenotype and various inflammatory mediators relieve hyperalgesia (Non-Patent Document 1), Nav1.9 is a major peripheral inflammatory hyperalgesia. It is considered to be an effector.
SCN9A and SCN10A are known as paralog genes for SCN11A. SCN9A encodes Nav1.7, which has a tetrodotoxin-sensitive gate characteristic, such as inherited erythromelalgia, paroxysmal extreme pain, and congenital insensitivity to Pain. Is the causative gene of. On the other hand, SCN10A, like SCN11A, has a gate property resistant to tetrodotoxin and encodes Nav1.8, which is presumed to be functionally similar. SCN10A is known to be the causative gene for small fiber neuropathy and Brugada syndrome. However, for SCN11A, the human symptoms caused by the mutation are unknown, and the elucidation of its function has been delayed.

As described above, the pain and organ localization associated with each gene of SCN9A and SCN10A have been elucidated, and inhibitors specific to each gene product have been developed. However, since SCN9A and SCN10A exist in addition to nerves, their effects on the autonomic nervous system and the conduction system of the heart are recognized as complications. Therefore, inhibitors of SCN11A localized in the dorsal root ganglion and trigeminal ganglion are promising as headache agents, and a wide range of sodium channel inhibitors including the gene product (Nav1.9) have been reported so far. (See, for example, Patent Document 1).
However, since there were no cases in humans of what kind of pain the mutation of SCN11A causes, the effect of treatment could not be predicted, and a specific inhibitor of SCN11A has not been developed.

Japanese Unexamined Patent Publication No. 2013-100374

Proc Natl Acad Sci USA Vol.102 No.26 Page.9382-9387 (2005)

An object of the present invention is to provide a means for identifying a novel causative gene associated with pain and utilizing the gene or a product thereof to screen a pain suppressant (analgesic) based on a novel mechanism. ..
Another object of the present invention is to clarify the relationship between SCN11A and pain, thereby providing a new and useful drug discovery target for pain.

The present inventors focused on juvenile periodic limb pain as a hereditary pain disorder of unknown cause, and described three patient families who complained of limb joint pain and limb pain periodically in childhood for four generations. Linkage analysis and whole-genome exome analysis were performed to investigate the causative gene. As a result, only those with juvenile periodic limb pain in the family concerned had a missense mutation (C A T (c.665G> A)) or A GT (c) at the 222nd codon (CGT) of the SCN11A gene. It was found that .664C> A)) was present and that the mutation replaced arginine at position 222 of the SCN11A translation product with histidine (p.R222H) or serine (p.R222S). As a result of mutation search in the general population in the residential area of these patients, the above mutation was not found. Therefore, SCN11A is the causative gene of pain, and the cause of juvenile periodic limb pain in 3 families is the gene. It was identified as a p.R222H or p.R222S mutation in.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows:
[1] Any of the following proteins (a) to (d).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids were deleted and / or substituted and / or inserted and / or added. A protein having an amino acid sequence (excluding the substitution at position 222 with arginine) and having a pain-inducing effect (c) A nucleic acid consisting of a complementary strand sequence of the base sequence shown in SEQ ID NO: 1 and a stringent. A protein encoded by a nucleic acid that hybridizes under conditions and having a pain-inducing effect (d) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, which is an ortholog in another mammal of the amino acid sequence 222. A nucleic acid encoding the protein according to the proteins [2] and [1], wherein the amino acid residue corresponding to the position is an amino acid other than arginine and has a pain-inducing action.
[3] A vector containing the nucleic acid according to [2].
[4] A host cell transformed with the vector according to [3].
[5] The cell according to [4], wherein the host cell is a mammalian cell, and the nucleic acid encoding endogenous SCN11A is replaced with the nucleic acid according to [2].
[6] A method for producing the protein, which comprises culturing the cell according to [4] and recovering the protein according to [1] from the culture.
[7] A transgenic non-human mammal or a part of a living body thereof that exogenously expresses the nucleic acid according to [2] and exhibits a pain phenotype.
[8] The animal according to [7] or a part of a living body thereof, wherein the nucleic acid encoding the endogenous SCN11A of a non-human mammal is replaced with the nucleic acid according to [2].
[9] The animal according to [7] or [8] or a part of a living body thereof, wherein the non-human mammal is a mouse or rat.
[10] A test substance in which the test substance is brought into contact with the cell according to [5] or a part of the animal or a living body thereof according to any one of [7]-[9] to improve the pain phenotype. A method of screening for substances that suppress pain, characterized by the selection of.
[11] A method for screening a substance that suppresses pain.
(A) A step of bringing the test substance into contact with the cell according to [5] or the non-human mammal according to any one of [7]-[9] or a part of the living body thereof.
(B) The steps of measuring the expression level of the protein according to [1] or the nucleic acid according to [2] in each of the cases where the test substance is brought into contact with the test substance and the case where the test substance is not brought into contact with the test substance, and (c) (b) A method comprising the step of selecting a test substance having a reduced expression level as a substance that suppresses pain.
[12] An antibody or fragment thereof that specifically binds to the protein according to [1].
[13] A test for diagnosing a pain-related disease in a subject, which comprises detecting the presence of the protein according to [1] or the nucleic acid according to [2] in a biological sample collected from the subject. Method.
[14] An agent for treating pain sensation in mammals having a pain-inducing mutation or a hypoalgesia-inducing mutation in the SCN11A gene, which comprises an ion channel regulator composed of the gene product.
[15] The SCN11A gene having the pain-inducing mutation is
An ortholog of (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in other mammals, corresponding to position 222 of the amino acid sequence. The agent according to [14], wherein the amino acid residue is an amino acid other than arginine, and encodes a protein having a pain-inducing action.

The present invention reveals that SCN11A is a pain-related gene and its p.R222H or p.R222S mutations cause juvenile periodic limb pain. Since the expression of SCN11A is localized in the dorsal root ganglion and trigeminal ganglion as compared with the paralog genes SCN9A and SCN10A, the expression of SCN11A or the function of its gene product Nav1.9 is selectively used. By inhibiting, pain can be suppressed.

FIG. 1 shows a family tree (top) of the family tree (family tree 1) analyzed in the present invention, and an exome analysis result (bottom). ■ and ● indicate pain onset, □ and ○ indicate non-onset, ■ and □ indicate male, ● and ○ indicate female. The arrow indicates the proband. FIG. 2 shows a family tree of the family tree (family tree 2) analyzed in the present invention. Squares indicate men and circles indicate women. For each mark, the left half fill indicates the person with limb pain, and the right half fill indicates the person with headache. The arrow indicates the proband. Specimens were collected from the circled individuals and analyzed. FIG. 3 shows a family tree of the family tree (family tree 3) analyzed in the present invention. ■ and ● indicate pain onset, □ and ○ indicate non-onset, ■ and □ indicate male, ● and ○ indicate female. The diagonal lines indicate that the symptoms are unknown because they have already died. P indicates the proband. The number + y indicates age. Specimens were collected from the circled individuals and analyzed. FIG. 4 shows the sequencing results of the nucleotide sequence of codon 222 of the SCN11A gene in the proband of family 3 and their parents.

The details of the present invention will be described below.
In the present invention, "pain" or "pain" is defined by the International Association for the Study of Pain (IASP, 1981) as "leading to or using a term that describes a substantial or potential injury to a tissue. It is used to mean "expressed, unpleasant sensory or emotional experience" and includes any of nociceptive pain (somatic pain, visceral pain), neuropathic pain, and psychogenic pain. In addition, it may include both acute pain and chronic pain, but mainly refers to the case of chronic pain.
In the present invention, "having a pain-inducing action" means having a pain-inducing action on mammals such as humans regardless of the degree of pain such as dull pain and severe pain and the type of pain. For example, the degree of pain expressed by VAS (visual analogue pain scale) is statistically significant on the side of 1 mm or more, preferably 10 mm or more, more preferably 20 mm or more, and 100 mm (that is, the maximum pain that can be imagined). It means that it has an action to change the pain, and more preferably, it means to increase the degree of pain expressed by VAS to 70 mm or more, more preferably 80 mm or less.
On the other hand, "suppressing pain" means reducing or eliminating pain regardless of the degree and type of pain. For example, it means to statistically significantly change the degree of pain expressed by VAS to 1 mm or more, preferably 10 mm or more, more preferably 20 mm or more, and 0 mm (that is, a state where there is no pain at all). More preferably, it means reducing the degree of pain expressed by VAS to 30 mm or less, more preferably 20 mm or less.
In the present invention, the "pain-inducing mutation" means a gene mutation such that the mutant protein generated by the mutation has the above-mentioned "pain-inducing action". On the other hand, the "hypoalgesia-induced mutation" means a gene mutation in which the mutant protein produced by the mutation causes the sensation of pain to be dull or lost (painlessness).

Potential-dependent sodium channels (Navs) are ion channels that are essential for excitatory cells to induce action potentials, and the gate opens and closes due to changes in membrane potential to allow Na ions to pass through according to the difference in concentration inside and outside the cell. , It is a protein that changes the charged state of the cell membrane. Nav is composed of α subunit and several β subunits, which are responsible for most of its functions. To date, 10 subunits (Nav1.1-1.9 and Nax) have been cloned as α subunits. Of these, Nav1.8 and Nav1.9 are specifically expressed in small nociceptive neurons that are present in peripheral sensory ganglia such as the dorsal root ganglion (DRG) and trigeminal ganglion and are thought to transmit noxious stimulus information. Therefore, it may be involved in the onset of pain. In fact, there are many reports suggesting a link between Nav1.8 and inflammatory hyperalgesia and neuropathic pain. On the other hand, there are very few reports on the association between Nav1.9 and the development of pathological pain.

The sodium channel voltage gated type XI alpha (SCN11A) gene is a gene encoding Nav1.9, and the human SCN11A gene encodes a protein consisting of 1791 amino acids. The nucleotide sequence of the human SCN11A cDNA and the amino acid sequence of the translation product are registered in the National Center for Biotechnology Information (NCBI) database as NCBI RefSeq numbers NM_001287223 and NP_001274152, respectively.

The present invention provides a novel SCN11A gene having a pain-inducing mutation (hereinafter, also referred to as “mutant SCN11A gene of the present invention”), and a mutant Nav1.9 protein having a pain-inducing action, which is a translation product thereof. The mutant Nav1.9 protein of the present invention is one of the following (a) to (d).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids were deleted and / or substituted and / or inserted and / or added. A protein having an amino acid sequence (excluding the substitution at position 222 with arginine) and having a pain-inducing effect (c) A nucleic acid consisting of a complementary strand sequence of the base sequence shown in SEQ ID NO: 1 and a stringent. A protein encoded by a nucleic acid that hybridizes under conditions and having a pain-inducing effect (d) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, which is an ortholog in another mammal of the amino acid sequence 222. A protein in which the amino acid residue corresponding to the position is an amino acid other than arginine and has a pain-inducing effect In addition, "and / or" is used in the present specification in the sense of including either one or both. ..
Here, the "amino acid sequence shown in SEQ ID NO: 2" is an amino acid sequence in which arginine at position 222 is replaced with histidine (p.R222H) or serine (p.R222S) in the amino acid sequence of a known human Nav1.9 protein. is there. In the "base sequence shown in SEQ ID NO: 1", in the known human SCN11A cDNA sequence, the 665th G of the coding sequence (CDS) is A (corresponding to the p.R222H mutation) or the 664th C is A. It is a base sequence substituted with (corresponding to p.R222S). That is, in the present invention, the causative gene of familial juvenile periodic limb pain having the following characteristics (i) to (vi) is the SCN11A gene, and the mutation that induces the pain is p.R222H (c.665G). > A) or p.R222S (c.664C> A) based on the discovery.
(i) It is autosomal dominant.
(ii) Pain begins in infancy, resolves in adulthood, and is not accompanied by cardiac conduction damage or epilepsy, except for occasional migraines.
(iii) The nature of the pain is strong spontaneous limb and limb joint pain that touches the bones.
(iv) No inflammatory findings.
(v) Pain is periodic and is induced by weather and cold exposure and ameliorated by warming.
(vi) Non-steroidal anti-inflammatory drugs relieve pain mildly.

With respect to (b) above, more specifically, (i) 1 to 50, preferably 1 to 20, and more preferably 1 to number (5, 4, 3 or) in the amino acid sequence shown in SEQ ID NO: 2. 2) Amino acid sequence lacking 1 amino acid, (ii) 1 to 50, preferably 1 to 20, and more preferably 1 to number (5, 4, 3 or 2) in the amino acid sequence shown in SEQ ID NO: 2. ) Amino acid sequence with added amino acids, (iii) 1 to 50, preferably 1 to 20, and more preferably 1 to number (5, 4, 3 or 2) to the amino acid sequence shown in SEQ ID NO: 2. 1 to 50, preferably 1 to 20, and more preferably 1 to number (5, 4, 3 or 2) in the amino acid sequence shown in SEQ ID NO: 2 (iv). Examples thereof include a protein containing an amino acid sequence in which one of the amino acids is replaced with another amino acid, or (v) an amino acid sequence in which they are combined. However, this excludes the case where position 222 is replaced with arginine in SEQ ID NO: 2.
For example, naturally found polymorphisms of human SCN11A that result in amino acid substitutions include glycine at position 481 replaced with glutamate (p.G481E; registered as rs12059805 in the NCBI dbSNP database), position 777. Methionin replaced with arginine (p.M777R; registered as rs4302324 in NCBI dbSNP database), valine at position 909 replaced with isoleucine (p.V909I; registered as rs33985936 in NCBI dbSNP database) ), The tyrosine at position 1198 is replaced with histidine (p.Y1198H; registered as rs12638601 in the NCBI dbSNP database).

Further, substitution of amino acids having similar properties (for example, glycine and alanine, valine and leucine and isoleucine, serine and threonine, aspartic acid and glutamine, aspartic acid and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) If so, there may be a larger number of substitutions and the like. When an amino acid is deleted, substituted or inserted as described above, the position of the deletion, substitution or insertion is not particularly limited as long as the pain-inducing action is maintained. Then, the mutagenesis can be carried out by a method known per se, such as site-specific mutagenesis, alanine scanning, or mutagenesis by PCR.

In the above (c), the "stringent condition" refers to nucleotide sequences having high identity, such as 95% or more, preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and particularly preferably. Conditions under which nucleotide sequences having 99.5% or more identity hybridize with each other and nucleotide sequences with lower identity do not hybridize, specifically, Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1 -Matters described in 6.3.6, 1999, eg, hybridization at 6 x SSC (sodium chloride / sodium citrate) / 45 ° C, followed by one or more times at 0.2 x SSC / 0.1% SDS / 50-65 ° C. However, those skilled in the art can appropriately select hybridization conditions that give stringency equivalent to this.
Here, "identity of nucleotide sequence" uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; gap cost = Linear; filtering = ON; It can be calculated with match score = 1; mismatch score = -2).
The protein of (c) above also includes an isoform of Nav1.9 encoded by a splicing variant of the SCN11A gene consisting of the nucleotide sequence shown in SEQ ID NO: 1. For example, as an isoform of human Nav1.9, in the amino acid sequence shown in SEQ ID NO: 2, the isoform in which threonine at position 1444 is replaced with lysine and the amino acid at positions 1445-1791 is deleted (UniprotKB identifier: Q9UI33-2). In the amino acid sequence shown in SEQ ID NO: 2, an isoform in which the amino acid at positions 946 to 983 is deleted (see UniprotKB identifier: Q9UI33-3) can be mentioned.

In the above (d), as the SCN11A gene ortholog derived from a mammalian species other than human, arginine at position 222 is preferably another amino acid in the amino acid sequence of the mouse Nav1.9 protein registered as RefSeq number NP_036017 in the NCBI database. Has an amino acid sequence substituted with histidine or serine, an amino acid sequence in which arginine at position 219 is substituted with another amino acid, preferably histidine or serine, in the amino acid sequence of rat Nav1.9 protein registered as RefSeq No. NP_062138. In the amino acid sequence of bovine Nav1.9 protein registered as RefSeq No. XP_002696965, the amino acid sequence in which arginine at position 225 is replaced with another amino acid, preferably histidine or serine, has an amino acid sequence, and the accession number is given to Uniprot KB. Examples of the amino acid sequence of the chimpanzee Nav1.9 protein registered as H2QMB6 include those having an amino acid sequence in which arginine at position 222 is replaced with another amino acid, preferably histidine or serine.

In addition to the above sequences, the SCN11A gene or its gene products in other mammals are queried from the nucleotide sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2, and the genomes of non-human mammals and / Alternatively, the nucleotide sequence and amino acid sequence of SCN11A derived from a desired species can be obtained by searching the cDNA database or protein database using BLAST or FASTA.

The mutant Nav1.9 protein having a pain-inducing action can be isolated from small nociceptive neurons of peripheral sensory ganglia of mammals having the mutation by using a method for separating and purifying a membrane protein known per se. Alternatively, the mutant SCN11A cDNA is cloned by extracting mRNA from the cell, designing a primer based on the cDNA sequence information of human SCN11A shown in SEQ ID NO: 1, and performing RT-PCR using the mRNA as a template. It can also be obtained by inserting the cDNA into an appropriate expression vector, introducing and expressing it in a host cell, and recovering the mutant Nav1.9 protein from the culture of the host cell.
However, more simply, normal SCN11A cDNA is cloned from a mammal having a normal SCN11A gene having no pain-inducing mutation by the same method as described above, and SEQ ID NO: 2 is performed by a site-specific mutagenesis method known per se. It can be obtained by introducing a mutation such that the codon encoding the arginine residue corresponding to position 222 of the amino acid sequence shown in is replaced with another amino acid, preferably a histidine or serine residue.

The mutant SCN11A gene of the present invention is not particularly limited as long as it is a nucleic acid containing a base sequence encoding the above-mentioned mutant Nav1.9 protein having a pain-inducing action. The nucleic acid may be any of double-stranded DNA, single-stranded DNA, single-stranded RNA, double-stranded RNA, and DNA / RNA hybrid, but is preferably double-stranded DNA or single-stranded RNA. , More preferably double-stranded DNA. The mutant SCN11A gene of the present invention can be used by linking to a vector (cloning vector or expression vector). Further, depending on the purpose (for example, expression of protein), an appropriate vector suitable for the host cell into which the vector is introduced can be appropriately selected, and the vector can be transformed into the host cell. The vector can also include suitable promoters, terminator regions, selectable marker genes for selecting transformants (drug resistance genes, genes that complement auxotrophic mutations, etc.) and the like.

In addition, the vector may contain a sequence encoding a tag sequence useful for protein separation / purification (for example, His tag, GST tag, FLAG ™ tag, etc.), and the vector may contain a target host cell. It may be integrated into the genome of.

The type of vector is not particularly limited, but pBTrp2, pBTac1, pBTac2, pBluescript II SK (+), pBluescript II SK (-), pSTV28, pUC118, pUC19, pKK233-2, pSE280, pSupex, pUB110, pTP5, pC194. , PET vector, pAGE vector, pcDNA vector, pGEX vector, pMC1neo, λgt11, pkk223-3, pBlue BacIII, pVL1392, pVL1393 and the like.

Examples of the host cell include any cell capable of expressing the vector, for example, a bacterial cell, a fungal cell, an insect cell, an animal cell and the like.
Bacterial cells include, but are not limited to, Escherichia coli, Bacillus, Brevibacillus, Corynebacterium, Streptomyces and the like. The fungal cells are not particularly limited, and examples thereof include Saccharomyces cerevisiae and Aspergillus fungi. The insect cells are not particularly limited, and examples thereof include silkworms (Bombyx mori) and cabbage loopers (Trichoplusia ni). The animal cells are not particularly limited, but are cell lines such as CHO cells, HeLa cells, COS-1 cells, COS-7 cells, HEK293 cells, BALL-1 cells, HCT-15 cells, PC12 cells, and dorsal root ganglion. (DRG) Primary culture of neurons, ES cells, iPS cells, etc. can be mentioned.

For the introduction of the vector into the host cell, a transformation method known per se, for example, a competent cell method, a protoplast method, a calcium phosphate co-precipitation method, an electroporation method, a microinjection method, a lipofection method, etc. Can be done using.

When transformed cells are used for the purpose of purifying mutant Nav1.9 protein, it is preferable to use cells that do not express the endogenous SCN11A gene as host cells in order to facilitate purification, but even when animal cells are used. Since there are some differences in the amino acid sequence of SCN11A protein between humans and mice, rats, etc., for example, when recombinantly producing a human-derived mutant Nav1.9 protein, primates such as mice or rats Other mammalian cells can also be used.

On the other hand, when the transformed cell is intended to be used, for example, as a screening system for a pain-suppressing substance described later, it is preferable to use a mammalian cell, preferably a nerve cell, particularly a DRG neuron or the like as a host cell, and further, an intrinsic cause. It is desirable to replace (knock in) the coding region of the sex SCN11A gene with a nucleic acid encoding the mutant Nav1.9 protein loaded on the vector. In order to easily obtain mutant SCN11A knock-in cells, ES cells or iPS cells can be used as host cells, knock-in ES or iPS cells can be produced by homologous recombination, and then differentiated into nerve cells by a differentiation induction method known per se. it can.

The above-mentioned transformed cells can be cultured by a method known per se. The medium used for culturing can be appropriately selected for each desired cell. For example, LB medium or the like can be used for Escherichia coli culture, SD medium or the like can be used for yeast culture, and DMEM medium, EMEM medium or the like can be used for mammalian culture. In addition, the medium (semi-solid medium, liquid medium, powder medium) contains various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, etc.) as required. Amino acids (including natural amino acids or synthetic amino acids), nucleic acid bases (purines, pyrimidines), inorganic salts (DDL4, MnSO4, FeSO4, NaCl, etc.), antibiotics (ampicillin, canamycin, hyglomycin) and the like can be added.

Those skilled in the art can appropriately determine the culturing time, culturing temperature, and the like according to the type of cells. For example, the culture temperature can be set to 10 to 45 ° C., preferably 20 to 40 ° C., and the culture time can be set to 1 hour to 14 days, preferably 10 hours to 7 days, or the like. The pH can also be adjusted as appropriate.

The mutant Nav1.9 protein obtained by the above method can be purified by a method known per se. For example, cells collected by centrifugation or the like are ground by ultrasound or glass beads, and then solid substances such as cell fragments are removed by centrifugation or the like to prepare a crude enzyme solution, and further, sulfate and sulfuric acid are prepared. A chromatographic method such as salting out method using sodium, ion exchange chromatography, gel filtration chromatography, affinity chromatography, gel electrophoresis or the like can be used.

Since the mutant Nav1.9 protein encoded by the mutant SCN11A gene of the present invention has a pain-inducing effect in mammals, a nucleic acid encoding the mutant Nav1.9 protein is introduced into non-human mammalian cells and transgenic animals. By producing the above, a pain model animal can be provided. Accordingly, the present invention also provides transgenic (hereinafter also referred to as Tg) non-human mammals that exogenously express the mutant SCN11A gene of the present invention and exhibit a pain phenotype.

The Tg animal of the present invention includes fertilized eggs of non-human mammals, unfertilized eggs, sperms and their precursor cells (primitive germ cells, egg protozoa, egg mother cells, egg cells, sperm cells, sperm cells, sperm cells). Etc.), preferably in the early stages of embryonic development of fertilized eggs (more preferably before the 8-cell stage), calcium phosphate co-precipitation method, electroporation method, lipofection method, aggregation method, microinjection (microinjection), etc. It is produced by introducing the mutant SCN11A gene of the present invention by a gene transfer method such as an injection method, a gene gun (particle gun) method, or a DEAE-dextran method.
One embodiment of the case where a virus is used as an expression vector is a method of infecting an early embryo or ES cell of a non-human mammal with a virus containing a DNA encoding a mutant Nav1.9 protein (eg, Proc. Natl). . Acad. Sci. USA, 99 (4): 2140-2145, 2002). For example, when using retrovirus or lentivirus, cells are sown in an appropriate incubator such as a dish, a viral vector is added to the culture medium, and after culturing for 1 to 2 days, if it is an early embryo, it is pseudo-pregnant. If it is an ES cell, it is transplanted into the oviduct or uterus of a female non-human mammal for embryo, and the culture is continued by adding a selective agent such as G418 or hygromycin, and the cell in which the vector is incorporated is selected.
In addition, as described in Proc. Natl. Acad. Sci. USA, 98: 13090-13095 (2001), a viral vector during co-culture of spermatogonia collected from male non-human mammals with STO feeder cells. Efficiently obtain exogenous mutant SCN11A gene hetero Tg (+/-) offspring by injecting into the seminiferous tubules of male infertile non-human mammals and mating with female non-human mammals. Can be done.

Further, by the gene transfer method, DNA containing the mutant SCN11A gene of the present invention can be introduced into somatic cells, tissues, organs, etc. of non-human mammals and used for cell culture, tissue culture, etc. Tg animals can also be produced by fusing cells with the embryonic (or germ) cells described above using known cell fusion methods. Alternatively, as in the case of producing a knockout animal, the embryonic stem cell (ES cell) or induced pluripotent stem cell (iPS cell) of a non-human mammal contains the nucleic acid of the present invention using the above gene transfer method. After introducing the DNA and selecting a clone in which the DNA is stably integrated, the ES cells are injected into the blastocyst or the ES cell mass and the 8-cell stage embryo are aggregated to prepare a chimeric mouse. It is also possible to obtain Tg animals by selecting those in which the introduced DNA has been transmitted to the germ line.

Furthermore, the Tg animal is more preferably a knock-in (KI) animal in which the endogenous SCN11A gene is replaced with a sequence encoding a mutant human Nav1.9 protein. Therefore, in the Tg animal of the present invention, the target DNA is introduced into ES or iPS cells using an appropriate targeting vector, and the endogenous SCN11A gene is obtained by homologous recombination with a sequence encoding the mutant human Nav1.9 protein. It can also be made by substitution.

The present invention relates to a part of a living body of a Tg animal (preferably a KI animal) prepared as described above (for example, (1) cells stably holding the chimeric SCN11A gene (for example, small nociceptive neurons), tissues. (Example, dorsal root ganglion, trigeminal ganglion), etc. (2) cells or tissues derived from these are cultured and passaged as necessary, etc.), and the mutant human Nav1.9 protein of the present invention is also used. Retaining a capable chimeric SCN11A gene in an expressible state As a part of the living body of a transgenic non-human mammal, in a state in which the chimeric SCN11A gene capable of producing the human-type mutant Nav1.9 protein of the present invention can be expressed. It can be used for the same purposes as non-human mammals to be retained.
The Tg non-human mammal obtained by the above method or a part of the living body thereof can be used as a pain-inducing model animal or a model cell.

The non-human mammal (recipient animal) that can be targeted in the present invention is not particularly limited as long as it is a non-human mammal for which a transgenic system (knockout system in the case of a KI animal) has been established. Examples include rats, cows, monkeys, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters and the like. Preferably, rodents, which have a relatively short ontogeny and biological cycle and are easy to reproduce, are more preferable from the viewpoint of producing disease model animals, and in particular, mice (for example, C57BL / 6 strains, BALB / c strains, and C3H strains as pure strains) are preferable. , FVB line, DBA2 line, etc., and as hybrid lines, B6C3F1 line, BDF1 line, B6D2F1 line, ICR line, etc.) and rats (for example, Wistar, SD, etc.) are preferable.
In addition to mammals, birds such as chickens can be used for the same purposes as non-human mammals targeted in the present invention.

Furthermore, the present invention also includes a method of screening for substances acting on the mutant Nav1.9 protein of the present invention. The substance that acts includes a substance that binds to the mutant Nav1.9 protein, a substance that is related to the pain-inducing action of the mutant Nav1.9 protein, and the like, and for example, inhibits or inhibits the sodium channel function of the mutant Nav1.9 protein. The screening can be performed for the purpose of searching for a substance to be promoted.

The method is not particularly limited, but for cells, tissues or transgenic non-human mammals expressing the above-mentioned mutant Nav1.9 protein or fragments thereof, the cells and the like in the presence and absence of the test substance, etc. Sodium ion current, action potential, etc. in the cells are measured by a method for evaluating and comparing the changes in the cells, for example, a method known per se, such as the patch clamp method, and the sodium current in the presence and absence of the test substance can be measured. Examples thereof include a method of examining fluctuations in action potentials and the like.

Furthermore, the present invention also includes a method of screening for substances that suppress pain. The method is not particularly limited, but for example, the test substance is brought into contact with a cell, tissue or transgenic non-human mammal expressing the above-mentioned mutant Nav1.9 protein or a fragment thereof, and the test substance is not brought into contact with the test substance. Examples thereof include a method of measuring and comparing changes in the pain phenotype in the cells, tissues or animals, and selecting a test substance having an improved pain phenotype as a candidate for a pain suppressant.

In the case of cells or tissues, the test substance is contacted by adding the test substance to a medium or buffer solution, and in the case of transgenic non-human mammals, the test substance is orally or parenterally. It can be done by administering to. The amount added to the medium and the like and the amount to be administered to the animal can be appropriately selected according to the test substance.

The pain-suppressing action can be evaluated as an index of pain phenotype, for example, change of sodium ion current in cells / tissues, and relief of pain symptoms in animals.

Alternatively, as another method for screening a substance that suppresses pain, the following steps (a) to (c):
(A) A step of contacting a test substance with a cell, tissue or transgenic non-human mammal expressing the above-mentioned mutant Nav1.9 protein.
(B) The expression level was determined in the steps of measuring the expression level of the mutant Nav1.9 protein or the nucleic acid encoding the mutant Nav1.9 protein or the nucleic acid encoding the same in the case where the test substance was brought into contact with the test substance and the case where the test substance was not brought into contact with the test substance, respectively. Examples thereof include a method including a step of selecting a reduced test substance as a candidate for a pain suppressant.

The contact of the test substance in the step (a) can be performed in the same manner as described above. The expression level in the step (b) can be measured by using a method for detecting a protein or RNA known per se. The cells, tissues, and Tg animals used here are preferably those that do not express the endogenous SCN11A gene, and the expression of the mutant SCN11A gene is preferably regulated by the expression regulatory region of the endogenous SCN11A gene. Therefore, in one preferred embodiment, knock-in cells / tissues or animals in which the coding region of the endogenous SCN11A gene is replaced with a nucleic acid encoding a mutant Nav1.9 protein are used. However, even when transformed cells or Tg animals expressing the endogenous SCN11A gene are used, anti-mutant Nav1.9 protein-specific antibodies (ie, that can be used in clinical tests for the diagnosis of pain-related diseases described below). An antibody that does not cross-react with the endogenous Nav1.9 protein) or a primer and / or probe that specifically detects only the mRNA encoding the mutant Nav1.9 protein is used to encode the mutant Nav1.9 protein or it. Only the mRNA that can be measured can be measured.

The test substance that can be tested in any of the above screening methods is not particularly limited, and is, for example, a low molecular weight compound, a high molecular weight compound, a protein (cytogen, chemokine, antibody, etc.), a nucleic acid (DNA, RNA, etc.), a virus, Examples thereof include a compound library prepared by using combinatorial chemistry technology, a random peptide library prepared by solid phase synthesis or a phage display method, and natural components derived from microorganisms, animals, plants, marine organisms, and the like. Such substances can be appropriately determined by those skilled in the art.

The substance that suppresses pain obtained by the above method contains the substance as an active ingredient, is formulated by adding excipients, binders, disintegrants, lubricants, etc., and is used as a pain therapeutic agent or a preventive agent. You can also do it.

In a further aspect of the present invention, the p.R222H (c.665G> A) or p.R222S (c.664C> A) mutation of the subject's SCN11A gene (human if the subject is a non-human mammal). Diagnosis of pain-related disorders in the subject, comprising the step of detecting a mutation in which the arginine residue corresponding to arginine at position 222 of the Nav1.9 protein is replaced by another amino acid, preferably histidine or serine). It also provides an inspection method for.

In one embodiment, the mutation can be detected using any known SNP detection method. For example, a genomic DNA extracted from a subject's cell or the like is used as a sample, and a nucleic acid containing a continuous base sequence of about 15 to about 500 bases containing the base of any of the above polymorphism sites is used as a probe. Hybridization is performed according to the method of Wallace et al. (Proc. Natl. Acad. Sci. USA, 80, 278-282 (1983)) with precise control of stringency to detect only sequences that are completely complementary to the probe. Using a mixed probe in which one of the above-mentioned nucleic acid and the above-mentioned nucleic acid in which the base of the polymorphism site is replaced with another base is labeled and the other is unlabeled, the reaction temperature is gradually increased from the modification temperature. Examples thereof include a method in which hybridization is performed while reducing the amount, and a sequence that is completely complementary to one of the probes is first hybridized to prevent cross-reaction with a probe having a mismatch. Here, examples of the labeling agent include radioisotopes (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malic acid dehydrogenation. Enzymes, etc.), fluorescent substances (eg, fluorescamine, fluoressen isothiocyanate, Cy3, Cy5, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) are used.

Preferably, the detection of the polymorphism is carried out by, for example, various methods described in WO 03/023063, such as RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturing agent concentration gradient gel electricity. Migration method, RNase A cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, Invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allergic hybridization method, padlock probe method , UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method, etc. (see WO 03/023063, page 17, line 5 to page 28, line 20).

As a kit suitable for carrying out the above-mentioned test method, for example, from the base sequence of the nucleic acid encoding the above-mentioned mutant Nav1.9 protein, for example, p.R222H (c.665G> A) or p.R222S (p.R222H (c.665G> A)) or p.R222S ( Examples include kits containing primers capable of amplifying regions containing c.664C> A). The primer can be produced by a method known per se. For example, in the case of humans, it has 15 or more consecutive nucleic acids of the nucleotide sequence shown in SEQ ID NO: 1 and has a mutant SCN11A gene p.R222H (c.665G> A) or p.R222S (c.664C> A). It can be a primer capable of amplifying a region containing.

Another aspect of detecting the p.R222H (c.665G> A) or p.R222S (c.664C> A) mutation of the SCN11A gene is a method of detecting the expression of the mutant Nav1.9 protein in a subject. Be done. For example, the mutant Nav1.9 protein in a DRG biopsy sample is detected using, for example, immunological techniques such as Western blotting, radioimmunoassay (RIA), fluorescent antibody, and ELISA. be able to.

For the detection of the above-mentioned mutant Nav1.9 protein, an antibody or a fragment thereof that specifically binds only to the mutant Nav1.9 protein and does not bind to the Nav1.9 protein that does not have the p.R222H or p.R222S mutation is used. To. The method for producing the antibody is well known, and the antibody can be produced by a method known per se. As the immunogen, all of the above-mentioned mutant Nav1.9 protein may be used, or a fragment thereof containing histidine or serine at position 222 may be used. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. The antibody fragment is not particularly limited, and examples thereof include scFv, Fab, Fab', (Fab') ₂ , Fv, Fd, scFv, and sdFv.

Pain-related diseases include, but are not limited to, limb arthralgia, neuropathy due to trauma or pain due to metabolic nerve inflammation, diabetic peripheral nerve injury, and cancer pain. It is preferably familial juvenile periodic limb pain, and particularly preferably familial juvenile periodic limb pain having the following characteristics (i) to (vi).
(i) It is autosomal dominant.
(ii) Pain begins in infancy, resolves in adulthood, and is not accompanied by cardiac conduction damage or epilepsy, except for occasional migraines.
(iii) The nature of the pain is strong spontaneous limb and limb joint pain that touches the bones.
(iv) No inflammatory findings.
(v) Pain is periodic and is induced by weather and cold exposure and ameliorated by warming.
(vi) Non-steroidal anti-inflammatory drugs relieve pain mildly.
Among the characteristics of these pains are (ii) beginning in infancy, remission in adulthood, without cardiac or autonomic abnormalities, and (v) being induced by cold exposure and ameliorating by warming. The two points are significantly different from the pain of hereditary pain caused by mutations in the SCN11A paralog genes SCN9A and SCN10A, so they are peculiar to the SCN11A gene regardless of familial or sporadic (due to de novo mutation). The presence of pain based on this mechanism was shown.

The present invention is based on the first finding that SCN11A is the causative gene of pain and that a particular gene mutation induces familial juvenile periodic limb pain. That is, it was demonstrated for the first time by the present invention that dysfunction of Nav1.9 sodium channel, which is an SCN11A gene product, can cause the onset of pain or hypoalgesia. This means that in mammals with pain-inducing mutations or hypoalgesia-inducing mutations in the SCN11A gene, the pain-sensing abnormalities are caused by regulating the function of the Nav1.9 sodium channel, which is the SCN11A gene product. It shows that it can be treated.
Therefore, the present invention is also a therapeutic agent for hyperalgesia in mammals having a pain-inducing mutation or a hypoalgesia-inducing mutation in the SCN11A gene, and contains an ion channel regulator composed of the gene product. Providing an agent.

Mammalian animals (eg, humans, monkeys, cows, pigs, dogs, cats, mice, rats, etc.) that have a pain-inducing or blunting mutation in the SCN11A gene to be treated develop pain or blunting. The indicated mammal can be estimated, for example, by isolating genomic DNA or mRNA derived from DRG neurons and examining whether or not there is a mutation in the coding region of the SCN11A gene that causes a change in amino acids. Pain-inducing mutations p.R222H or p.R222S (in non-human mammals, substitution of other amino acids of the arginine residue corresponding to arginine at position 222 of the human Nav1.9 protein, preferably histidine or serine) If this is the case, the mutation can be detected by using the same method as the above-mentioned test method for diagnosing a pain-related disease.

DRG neurons derived from mammals with the identified mutant SCN11A gene to characterize a sodium channel composed of a mutant Nav1.9 protein encoded by the SCN11A gene with a pain-inducing or blunting-induced mutation. In the mutant Nav1.9 protein-expressing cells obtained by introducing into cells or the like, it is possible to carry out an electrophysiological assay known per se, such as the patch clamp method, to compare the characteristics of sodium channels with wild-type Nav1.9. ..

The action of the mutant Nav1.9 protein encoded by the identified mutant SCN11A gene to bring channel characteristics different from wild-type Nav1.9 (eg, sodium current magnitude, persistence, etc.) closer to those of wild-type Nav1.9. By administering a Nav1.9 sodium channel regulator having the above to a mammal having the mutant SCN11A gene, it is possible to treat pain and pain sensation abnormalities such as blunted pain in the mammal. For example, if the hyperalgesia is due to the generation of sodium currents due to mutation Nav1.9, which is excessive compared to wild-type Nav1.9, the symptoms can be alleviated by administering a Nav1.9 sodium channel blocker. .. Examples of such channel blockers include compounds described in JP2013-100374, JP2012-149084, JP2012-126751, JP2012-126745, JP2012-126744 and the like. However, but not limited to these, other known channel blockers and novel agents selected by the screening method of the present invention described above can also be preferably used. Alternatively, siRNA, shRNA, miRNA, antisense oligonucleic acid, and ribozyme targeting SCN11A mRNA can be used as an expression inhibitor of the mutant Nav1.9 protein for the purpose of selectively inhibiting the Nav1.9 sodium channel.

The present invention will be described in more detail with reference to the following examples, but the examples are merely examples of the present invention and do not limit the scope of the present invention in any way.

Samples In the examples below, three patient families (family 1-3) who periodically complain of limb pain during childhood were selected for four generations. Informed consent was obtained from all subjects. This study was approved by the Ethics Committee of Kyoto University and Akita University.

Example 1 Linkage analysis and whole-genome exome analysis of family 1 In this family, sudden limb pain occurs from infancy. Limb pain lasts for 30 to 40 minutes when a low pressure system arrives until puberty, after which there is a pain-free time of about one and a half hours, and then it becomes painful again. Although it changes, it gradually improves after that, and in adulthood, the frequency is about 1 to 2 times / year. Although the pain is limited to the limbs, the pain site is indefinite, and varies from the case of only one limb to the case of all limbs, and may be only the upper limb. There is no swelling or redness of the painful area, no pulsatile pain, and dull pain with a feeling of coldness, which is slightly improved by warming. No bone / muscle changes were observed, and no abnormal neurological findings were observed. No abnormalities in physique after adulthood are observed. The family tree of the main family (generations II to IV) is shown in Fig. 1 (upper panel).

Linkage analysis was performed in the original family, and a linkage region with an LOD score of 1.20 was found in the chromosome 3 region. Figure 1 (lower panel) shows the results of the exomes of all eight participants of the original family. The five novel genetic polymorphisms that all patients have and no non-symptomatic individuals have are ANKRD23, SCN11A, STAB1, RPAP1, and ITGA11, and their functions predicted that SCN11A was the causative gene. The mutation in the SCN11A gene was p.R222H. Details of Quality Control (QC) and Quality Assurance (QA) for exome analysis are shown below.

Whole-genome exome analysis was performed using the next-generation sequencer, Illumina HiSeq 2000. The SureSelect Human All Exon V4 + UTRs Kit from Agilent was used to capture the genomic DNA, and the library was prepared according to the Illumina protocol. Genomic DNA prepared from the subjects was fragmented, and the 150-200 bp (without adapter) genomic fragment was ligated with the capture to form a library. Multiplex sequencing was performed using a flow cell specified by Illumina. The Read length was set to 100 bps. The UCSC Genome Browser hg19 was used as the RefSeq database, and the analysis was performed using Burrows-Wheeler Aligner 0.6.2 (BWA). The Genome Analysis Toolkit 1.6-13-g91f02df (GATK) was used for SNP and In / del analysis. The number of effective reads and bases was about 84.86 million read pairs and about 5.3 Gbps on average for a total of 11 samples including 3 samples from family 2 described later. The analysis results are summarized in Table 1.

Mutations in each sample, including MS / NS / Indel / SS / FS / RT (MS, Missense; NS, Nonsense; Indel, Insertion or deletion; SS, Splice Site; FS, Fragile Sites; RT, Read through) Approximately 10,000 were found, and there were 61 heterozygous mutations that were common to affected individuals and were not present in non-affected individuals. Among them, in the 1k genome, there were 23 mutations with a minor allele frequency (MAF) of 1% or less, and 11 mutations with a MAF of 1% or less in Japanese. Among them, 5 new mutations were not registered in dbSNP135.

Example 2 Linkage analysis of family tree 2 and whole-genome exome analysis Family tree 2 (family tree is shown in Fig. 2) has already been reported as a paper (Brain Res., 2006, 28 (10): 660-662). , Shows almost the same symptoms as Family 1. Genomic DNA was collected from the proband (IV-1) brother (IV-2), younger brother (IV-3), mother (III-1) and father (spouse of III-1), of which 3 samples (IV) -3, III-1 and III-1 spouses) were subjected to exome analysis in the same manner as in Example 1, and the results are summarized in Table 2.

Approximately 10,000 mutations were found in each sample, including MS / NS / Indel / SS / FS / RT, and there were 1332 heterozygous mutations common to affected individuals and not present in non-affected individuals. .. Of these, in the 1k genome, there were 238 mutations with MAF of 1% or less, and 165 mutations with MAF of 1% or less in Japanese. Among them, 59 new mutations were not registered in dbSNP135. Of these, the only ion channel gene was SCN11A, which is common to family 1, and the mutation was p.R222S. In this case, elevation of substance P (9.3-12.6 pg / ml; control 4.8 + 2.4 pg / ml) and elevation of CGRP (20.9), which are known to be released from nerve endings present in the trigeminal ganglion at the time of onset. -30.7 pg / ml; control 13.4 + 4.4 pg / ml) was observed.

Example 3 Analysis of Family 3 The IV-1 proband (IV-1) of the original family was a 2-year-old boy, 41 weeks, 2954 g, without asphyxia. He has a history of febrile convulsions 5 times, but no EEG abnormalities are observed. From around 1 year and 11 months, an episode that suddenly started crying appeared. I noticed that I started complaining about my lower limbs not being able to touch the floor. The pain area is the joints of the limbs, such as the elbows, knees, ankles, and wrists. I have a lot of knees. One pain is usually about 5 minutes, but it occurs many times a day. There are no other concomitant symptoms during a pain attack (fever, redness, swelling, etc.). More often at night than during the day, when the weather is bad, when the air pressure drops. I take bufferin when I have pain. My father (III-5) also had the same symptoms from an early age, but he belonged to the soccer club when he was a student, and the frequency of pain decreased overwhelmingly as an adult. In the original family, at least II-4, III-5, IV-1 have no headache, and III-3, III-4 also have no headache according to III-5 (III-1, III-). Details of 2 are unknown). III-5 and III-6 are not consanguineous marriages. I-2 has already died and no symptoms have been confirmed. II-2 has no symptoms as far as III-5 knows.
In the original family, the gene sequences of all exons of SCN11A were examined for the genomes of proband IV-1 and its parents (III-5 and III-6), and as a result, IV-1 and father (III-5) p. R222H mutation was observed (Fig. 4) This mutation was not observed in non-onset mothers (III-6).

Example 4 Allele frequency of SCN11A gene mutation in the general population of the patient's residential area The frequencies of the mutant alleles p.R222H and p.R222S were analyzed using restriction enzymes in the general population of 150 in the patient's residential area. In the general population of 150, no two mutations were found, with a 95% CI of 0-0.0122.

Although the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that preferred embodiments can be modified.

The contents of all publications, including the patents and patent application specifications mentioned herein, are incorporated herein by reference in their entirety to the same extent as expressly stated. ..

To date, the pain associated with SCN11A has been unknown. The present invention reveals that SCN11A is the causative gene and that the amino acid site involved in pain development is a substitution of arginine at position 222 in three families that develop extremely characteristic juvenile periodic limb pain. did. This indicates the possibility of discovering analgesics with few side effects by a new mechanism that is effective for pain in limb joints that frequently occur in elderly people, pain due to chronic inflammation, and bone pain in cancer-bearing patients. ..
Since the expression of the SCN11A gene is localized to nerve cells, especially small nociceptive neurons of peripheral sensory ganglia, it is possible to develop highly selective pain therapeutic agents having very little effect on other organs. Therefore, the present invention is extremely useful industrially.

Claims (13)

Any of the following proteins (a) to ( c ).
(A) Protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) In the amino acid sequence shown in SEQ ID NO: 2, 1 to 50 amino acids were deleted and / or substituted and / or inserted and / or added. amino acid sequence (except for substitution of position 222) has, and protein having a pain-inducing activity
(C ) An ortholog of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in other mammals, in which the amino acid residue corresponding to position 222 of the amino acid sequence is histidine or serine, and has a pain-inducing effect. Protein A nucleic acid encoding the protein according to claim 1. A vector containing the nucleic acid according to claim 2. A host cell transformed with the vector according to claim 3. The cell according to claim 4, wherein the host cell is a mammalian cell, and the nucleic acid encoding endogenous SCN11A is replaced with the nucleic acid according to claim 2. A method for producing a protein, which comprises culturing the cell according to claim 4 and recovering the protein according to claim 1 from the culture. A transgenic non-human mammal or a part of a living body thereof that exogenously expresses the nucleic acid according to claim 2 and exhibits a pain phenotype. The animal or part of a living body thereof according to claim 7, wherein the nucleic acid encoding the endogenous SCN11A of a non-human mammal is replaced with the nucleic acid according to claim 2. The animal or part of a living body thereof according to claim 7 or 8, wherein the non-human mammal is a mouse or rat. A test substance is selected by bringing the test substance into contact with the cell according to claim 5 or a part of the animal or a living body thereof according to any one of claims 7-9 to improve the pain phenotype. A method of screening for substances that suppress pain. A method of screening for substances that suppress pain
(A) A step of bringing a test substance into contact with the cell according to claim 5 or a part of the non-human mammal according to any one of claims 7-9 or a living body thereof.
(B) The steps of measuring the expression level of the protein according to claim 1 or the nucleic acid according to claim 2 in the case where the test substance is brought into contact with the test substance and in the case where the test substance is not brought into contact with each other, and (c) (b). A method comprising the step of selecting a test substance having a reduced expression level as a substance that suppresses pain. A test method for diagnosing a pain-related disease in a subject, which comprises detecting the presence of the protein according to claim 1 or the nucleic acid according to claim 2 in a biological sample collected from a subject. A therapeutic agent for pain sensation abnormalities in mammals having a pain-inducing mutation in the SCN11A gene, which is a blocker of an ion channel composed of the gene product, or siRNA, shRNA, miRNA, anti, which targets the mRNA of the gene. The SCN11A gene, which contains an expression inhibitor of the gene selected from the group consisting of sense oligonucleic acid and ribozyme, and has the pain-inducing mutation,
An ortholog of (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in other mammals, corresponding to position 222 of the amino acid sequence. An agent that encodes a protein whose amino acid residue is histidine or serine and has a pain-inducing effect.