JPH11313682A - Fukuyama type congenital muscular dystrophy causing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new protein comprising a Fukuyama type congenital muscular dystrophy(FCMD) containing a specific amino acid sequence, useful for elucidating the crisis and condition of FCMD, diagnosing, preventing and treating FCMD, etc., with its gene. SOLUTION: This new protein comprises a Fukuyama type congenital muscular dystrophy(FCMD) causing protein containing an amino acid sequence of the formula or a protein containing an amino acid sequence in which one or plural amino acids are deleted, substituted or added in the amino acid sequence of the formula and having the same biological function as that of FCMD causing protein. The crisis of FCMD is caused by the function insufficiency of the protein. Useful information for elucidation of the crisis and condition of FCMD, its diagnosis, prevention and treatment, etc., can be provided by the gene of the protein. The protein is readily obtained by screening a human adult brain cDNA library and using the obtained FCMD causing protein gene by an ordinary genetic engineering method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein causing causative disease of Fukuyama type muscular dystrophy (hereinafter also referred to as a protein causing FCMD), and more specifically, a novel protein which causes the onset of congenital muscular dystrophy of Fukuyama type due to its dysfunction. About. The present invention also relates to a novel gene encoding such a protein (hereinafter, also referred to as an FCMD-causing gene). Further, the present invention relates to a genetic abnormality of the FCMD-causing gene,
In particular, it relates to a mutant that causes the development of Fukuyama-type congenital muscular dystrophy.

[0002]

2. Description of the Related Art Muscular dystrophy is a hereditary degenerative disease mainly occurring in skeletal muscle.
These are classified into the above types.

[0003] The most frequently occurring muscular dystrophy, sex-linked recessive Duchenne muscular dystrophy (hereinafter also referred to as DMD), is obtained by a positional cloning method as a defective gene product causing the disease. Has been identified, and intensive research has been conducted on its pathological mechanism. In addition, many studies on other muscular dystrophy have been made since the discovery of dystrophin to elucidate its etiological gene and pathophysiology and to develop therapeutic methods.

One of the autosomal recessive inherited muscular dystrophy is Fukuyama-type congenital muscular dystrophy characterized as severe congenital muscular dystrophy accompanied by impaired brain formation or intelligence.
cular dystrophy (hereinafter also referred to as FCMD).
This was first reported about 40 years ago (Fukuyama, Y., Pae
diatria Universitatis Tokyo, 4, 5-8 (1960)), McKu
The sick catalog describes "MIM253800".

[0005] The FCMD is one of the most common autosomal recessive diseases in Japan, and is the second most common form of childhood muscular dystrophy. The incidence of FCMD in Japan is 0.7-1.2 per 10,000 (Fukuyama,
Y., et al., Brain Dev., 6,373-390 (1984)), which corresponds to almost half the incidence of DMD (Fukuyama, Y., et. Al.).
al., Brain Dev., 3, 1-30 (1981)).

[0006] Patients with FCMD usually show reduced facial and limb muscular strength and generalized muscle tone by nine months of age, and generally develop joint contracture earlier than DMD patients. FC
Motor dysfunction levels in MD patients are more severe than in DMD patients, and most patients cannot even walk. In general, F
CMD patients become bedridden before age 10 due to systemic advanced muscle atrophy and joint contractures, and many have died by age 20 years. Other concomitant symptoms include severe intellectual decline (IQ 30-50) and seizures with EEG abnormalities in about half (Fukuyama, Y., et al., B
rain Dev., 3,1-30 (1981)).

The histopathological changes observed in skeletal muscle are similar to those in DMD patients.

[0008] The most common and characteristic changes in the central nervous system are brain malformations, presumably due to impaired migration of nerve cells, including cerebellar and cerebellar cerebellar gyrus, thick gyrus and cerebral gyrus. . In addition, hydrocephalus, fibrous thickening of the buffy coat, local adhesion of the left and right cerebral hemispheres and hypoplasia of the corticospinal tract are often observed (Fukuyama, Y., et al., Brain Dev.,
3, 1-30 (1981)). Therefore, analysis and identification of the pathogenic gene of FCMD is, of course, useful for understanding the pathophysiology of muscular dystrophy in general,
In addition, the information is believed to provide important insights into brain formation and growth.

[0009] Under such circumstances, the present inventors have found that the locus of the causative gene of FCMD exists in the long arm region of chromosome 9 by means of a combination of homozygosity mapping and linkage analysis. (Toda, T., eta al., Nat. Gen.
et., 5, 283-286 (1993)). In addition, the present inventors have developed homozygous mapping and recombination mapping (recombination mapping).
mapping), the FCMD locus is D9S
127 and the marker CA246 (D9S2111) in the 5cM region.
A linkage disequilibrium was found between the D locus and a microsatellite marker (D9S306) within this 5cM candidate region, and the FCMD causative gene was assumed to be within 1 Mb of chromosome 9q31 around D9S306 ( Toda, T., et al., Am. J. Hum. G
enet., 55, 946-950 (1994); Toda, T., et al., Jpn.
J. Hum. Genet., 40, 333-334 (1995)).

[0010] Furthermore, by comparing the intensity of linkage disequilibrium, the location of the FCMD locus has been identified to a region of less than 100 kb including D9S2107, and most of the current FCMD chromosomes are derived from one ancestor. (Toda, T., et al., Am. J. Hum. Genet., 59,
1313-1320 (1996)), and more than 80% of the FCMD chromosomes contain a founder haplotype (D
9S2105-D9S2170-D9S2171-D9
138-192-147-183 for S2107)
And found that this was not found on the chromosomes of normal individuals. D9S2105, D9S217
Cosmid-aligned clones (contigs) containing the 0, D9S2171 and D9S2107 loci have also been constructed and reported (Miyake, M., et al., Genomics, 40, 284-2).
93 (1997)).

However, the identification of the FCMD-causing gene has not yet been achieved, and its analysis and identification have been strongly desired in the art.

[0012]

SUMMARY OF THE INVENTION The present invention has been developed in view of the above circumstances, and has as its object to provide a Fukuyama-type congenital muscular dystrophy causative gene (FCMD causative gene). In the present invention, the FCMD causative gene and its cDNA are collectively referred to as FCMD DNA.

[0013] Another object of the present invention is to provide a protein encoded by the FCMD-causing gene (FCMD-causing protein) and its specific antibody.

It is a further object of the present invention to provide a vector used for expression and production of an FCMD-causing protein, a technique for expressing and producing the protein by using the vector, and a medical use thereof.

Still another object of the present invention is to provide information on an abnormality (mutation) of the above-mentioned FCMD-causing gene associated with the development of Fukuyama-type congenital muscular dystrophy.

Furthermore, the present invention provides a technique for detecting the gene expression of the FCMD-causing gene and a technique for detecting the gene abnormality of the FCMD-causing gene, and diagnoses, predicts, and prevents Fukuyama-type congenital muscular dystrophy by using the techniques. The purpose is to provide technology related to.

[0017]

According to the present invention, (a)
An FCMD-causing protein having the amino acid sequence shown in SEQ ID NO: 1; and (b) an amino acid sequence in which one or more (or several) amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Nari, FC
According to the present invention, there is provided a protein having a biological function equivalent to that of the MD-causing protein. Further, the present invention provides a DNA encoding the protein, particularly a DNA having the nucleotide sequence shown in SEQ ID NO: 2 or 3. You.

Further, according to the present invention, there is provided a mutant FCMD-causing DNA characterized by having a mutation in the base sequence shown in SEQ ID NO: 3 that causes a dysfunction of the encoded FCMD-causing protein. You.

In addition, according to the present invention, a method for detecting a gene abnormality for FCMD diagnosis, which comprises detecting the presence of the above-mentioned mutant FCMD-causing DNA in the gene of a subject, In the base sequence shown, (i) substitution of cytosine at base number 250 to thymine, (ii) deletion of base numbers 298-299, (iii) 1 or 5 between base numbers 5889 and 5890 A method for detecting an abnormality of the same gene comprising detecting at least one mutation of at least one of insertion of a plurality of (or several) nucleotides, a step of preparing a DNA fragment containing the mutation position, and a base of the DNA fragment There is provided a method for detecting an abnormality in the gene, comprising a step of analyzing the sequence.

In the present specification, the abbreviations for amino acids, peptides, base sequences, nucleic acids, restriction enzymes, etc. are used to refer to the nomenclature according to IUPAC and IUPAC-IUB or its definition, and to include “the base sequence or amino acid sequence. Guidelines for Preparation of Specifications, etc. ”(Heisei 9
March 2010, JPO Coordination Division Examination Standards Office).

In the present invention, (C → T25)
0) means base number 250 in the FCMD causative gene.
Means that the position cytosine (C) is point-mutated to thymine (T). Also, (del: 298-299) means that nucleotide positions 298 and 299 in the FCMD-causing gene are deleted. The nucleotide numbers and nucleotide positions used in the present invention are described with reference to the nucleotide sequence of the FCMD-causing gene shown in SEQ ID NO: 3 derived from a healthy human for both the mutant gene and the DNA fragment. The amino acid numbers are expressed based on the amino acid sequence of the FCMD-causing protein shown in SEQ ID NO: 1.

The DNA is intended to include not only double-stranded DNA but also single-stranded DNAs such as a sense strand and an antisense strand which constitute the double-stranded DNA, and is not limited to any length. Absent. Therefore, the DN of the present invention
A contains double-stranded DNA containing human genomic DNA, and c
Includes single-stranded DNA (sense strand) containing DNA, single-stranded DNA (antisense strand) having a sequence complementary to the sense strand, and fragments thereof.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Specific examples of the FCMD-causing protein of the present invention include, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, which is the long arm of chromosome 9 of a healthy human. Is identified as a protein encoded by a gene (about 1383 bp) located very close to the centromere side of the microsatellite marker D9S2170.

As will be described in detail in an embodiment described later, this F
The CMD causative protein is a pathogenic protein involved in the development of FCMD, and is considered to cause the development of FCMD due to its dysfunction, and plays an important role in the development and pathophysiology of FCMD, and further, the formation and growth of the brain. it is conceivable that.

Thus, the provision of the FCMD-causing protein and the gene encoding the same (FCMD-causing gene) according to the present invention are intended to elucidate FCMD and its onset or pathological condition,
It provides extremely useful information and means in the pharmaceutical field such as grasping and diagnosis, prevention and treatment, and in basic research and application of brain formation and growth.

For example, gene therapy for utilizing or expressing the FCMD-causing gene of the present invention or administration of the FCMD-causing protein of the present invention to a living body will be useful for treating FCMD. In addition, the present invention F in an individual or tissue
CMD causative gene or its product (FCMD causative protein)
The detection of the expression of, and the detection of mutation or abnormal expression of the gene may be suitably used in the elucidation and diagnosis of FCMD.

Hereinafter, the present invention will be described in more detail according to its embodiments.

The FCMD-causing protein of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 1 has the following structural features or characteristics: (a) consisting of a sequence of 461 amino acids; (b) estimation of 53.6 kDa (C) has an estimated isoelectric point of 8.29; (d) hydrophobic amino acid residues (Lys, Val, Phe,
(Ile and Met) account for 30.8% of total amino acids; (e) has one putative N-glycosylation region; (f) has four putative protein kinase C phosphorylation sites; (g) 1 (H) has five N-myristorylation putative sites; (i) has a signal sequence putative region.

(J) Has zero or one transmembrane estimation region.

Specifically, (e) the predicted N-glycosylation region is located at amino acid positions 92 to 95 in the amino acid sequence shown in SEQ ID NO: 1 (the same applies hereinafter) (in FIG. 4, Underlined). (F)
Putative protein kinase C phosphorylation sites that can be phosphorylated by protein kinase C are located at serine residues at amino acid positions 33 and 276, and at threonine residues at amino acid positions 386 and 426 (in FIG. 4, the symbol %). (G) The putative tyrosine kinase phosphorylation site that can be phosphorylated by tyrosine kinase is located at the tyrosine residue at amino acid number 252 (indicated by the symbol ＾ in FIG. 4). (H) N-myristolation predicted sites include amino acid positions 37, 39, 45,
It is located at glycine residues at positions 300 and 380 (FIG. 4).
, Is indicated by the symbol *). (I) The predicted signal sequence region is located at amino acid positions 1 to 21 (indicated by a solid underline in FIG. 4). It is also suggested that (J) the transmembrane region may not be present or may be present at amino acid positions 288 to 306 (indicated by a solid underline in FIG. 4).

Database search (GeneBank, dbEST, Swi
According to ssProt, PIR), the amino acid sequence of this FCMD-causing protein is not similar to the amino acid sequence of a known protein, and the gene encoding the same (the FCMD-causing gene having the nucleotide sequence shown in SEQ ID NO: 3) ) Also hardly resembles a gene having a known function, which confirms that the FCMD-causing protein and its DNA are completely novel.

The DNA of the present invention is, for example, a DNA encoding the above-mentioned FCMD-causing protein, and more specifically, a DNA having the nucleotide sequence shown in SEQ ID NO: 2 or 3.
Examples of A include, but are not limited to, DNAs encoding functional equivalents of FCMD-causing proteins.

That is, the DNA of the present invention has a nucleotide sequence encoding a protein consisting of an amino acid sequence in which one or more (or several) amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. In this case, the degree of “deletion, substitution or addition of amino acids” and the positions thereof are the same as those of the FCMD-causing protein comprising the amino acid sequence represented by SEQ ID NO: 1. There is no particular limitation as long as it is a functional equivalent having a biological function.

The modification (mutation) of the amino acid sequence may occur naturally, for example, due to mutation or post-translational modification. However, the modification (mutation) of the amino acid sequence is based on a naturally-occurring gene (for example, a specific example gene of the present invention). It can also be modified artificially. The present invention encompasses all modified DNAs having the above characteristics regardless of the cause and means of such modification / mutation.

As the above-mentioned artificial means, for example, site-specific mutagenesis (Methods in Enzym
ology, 154: 350, 367-382 (1987); id. 100: 468 (198
3); Nucleic Acids Res., 12: 9441 (1984); Lecture on Sequent Chemistry Experiment 1 "Genetic Research Method II", edited by The Japanese Biochemical Society, p105
(1986)] and chemical synthesis methods such as the phosphate triester method and the phosphate amidite method (J. Am. Che.
m. Soc., 89: 4801 (1967); 91: 3350 (1969); Scien
ce, 150: 178 (1968); Tetrahedron Lett., 22: 1859 (1
981); ibid. 24: 245 (1983)) and methods of combining them.

Further, the DNA of the present invention has SEQ ID NO: 2 or 3
This is a specific example of a codon that encodes each amino acid residue in the above amino acid sequence (SEQ ID NO: 1), and the codon degeneracy is taken into consideration. For example, the DNA of the present invention is not limited to these, and it is of course possible to have a base sequence in which arbitrary codons are combined and selected for each amino acid residue. The selection of codons can be performed according to a conventional method, for example, by taking into account the frequency of codon usage of the host to be used (Ncle
ic Acids Res., 9: 43 (1981)).

It is to be noted that the present invention relates to the deletion of one or more (or several) amino acids in the functional equivalent of the FCMD-causing protein encoded by the DNA of the present invention, ie, the amino acid sequence shown in SEQ ID NO: 1. , A modified protein comprising a substituted or added amino acid sequence,
It also provides a protein that is a functional equivalent having the same biological function as the CMD-causing protein. These proteins have the same utility as the FCMD-causing protein.

In the present invention, the FCMD causative gene is
As described below, a human cDNA library was screened using a clone capable of detecting the region near D9S2170 as a probe, and the resulting clone was identified by gene mapping. The DNA of the present invention is based on the sequence information thereof. Can be easily manufactured and obtained by general genetic engineering techniques (Molecular Clonin
g 2nd.Ed., Cold Spring Harbor Laboratory Press (1
989); Seismic Chemistry Laboratory Course “Gene Research Methods I, II, II
I ", edited by The Biochemical Society of Japan (1986)).

For obtaining the FCMD causative gene, specifically, for example, a cDNA library is prepared according to a conventional method from a suitable source in which the gene is expressed, and a suitable probe or antibody specific to the FCMD causative gene is prepared from the library. (Proc. Natl. Acad. Sci. USA, 78, 6613 (1981); Sc).
ience, 222, 778 (1983)). In the above method, the cD
Examples of the origin of NA include human tissues such as human heart, brain, skeletal muscle, and pancreas, lymphoblasts, and the like. Separation of total RNA, separation and purification of mRNA, cDN
Acquisition of A and cloning thereof can be performed according to a conventional method. Also, cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, Clontech Lab.
Various cDNA libraries commercially available from c.) can also be used.

The method of screening the FCMD-causing gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used. More specifically, for example, a method for selecting a corresponding cDNA clone by immunoscreening using a protein-specific antibody for a protein produced by cDNA,
Examples include plaque hybridization, colony hybridization, and the like using a probe that selectively binds to a sequence, and combinations thereof.

The probe used herein generally includes a DNA sequence chemically synthesized based on the information on the base sequence of the FCMD-causing gene of the present invention. Fragments and the like can also be suitably used.

When the FCMD causative gene of the present invention is obtained, the PCR method (Science, 230, 1350-1354 (198
The DNA / RNA amplification method according to 5)) can be suitably used.
In particular, when it is difficult to obtain a full-length cDNA from a library, the lace method (RACE: Rapid amplification)
of cDNA ends; Experimental Medicine, 12 (6), 35-38 (1994)), especially the 5'-race (RACE) method (Frohman, MA, et a).
l., Proc. Natl. Acad. Sci., USA, 8, 8998-9002 (198
8)) is preferred. Primers used for adopting such a PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention.

The isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method, and specific examples include gel electrophoresis.

The means for chemically synthesizing DNA and modifying it using the FCMD-causing gene are as described above.
Can be obtained.

The nucleotide sequence of the FCMD-causing gene or DNA of the present invention obtained as described above can be determined according to a conventional method. Specifically, the dideoxy method (Proc. Nat.
l. Acad. Sci. USA, 74, 5463-5467 (1977)) and the Maxam-Gilbert method (Method in Enzymology, 65, 499 (19)
80)) and the like, and for convenience, a commercially available sequence kit or the like may be used.

The use of the DNA of the present invention makes it possible to easily and stably produce a large amount of a code product containing a FCMD-causing protein by using a general genetic engineering technique. Accordingly, the present invention also provides a vector (expression vector) containing the DNA of the present invention, a host cell transformed with the vector, and a method for producing the protein of the present invention containing an FCMD-causing protein by culturing the host cell. To provide.

The production method can be carried out by a conventional gene recombination technique (Science, 224: 1431 (1984); Biochem. Biophys. Re.
s. Comm., 130: 692 (1985); Proc. Natl. Acad. Sci.,
USA., 80: 5990 (1983) and the above-cited references).

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts widely include commonly used ones such as Escherichia coli and Bacillus subtilis. Can be exemplified by Escherichia coli, especially those contained in the Escherichia coli K12 strain. In addition, eukaryotic host cells include vertebrates, yeast and other cells, and as the former,
For example, COS cells (Cell, 23: 175 (1
981)) and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77: 4216 (1980)). As the latter, yeast cells of the genus Saccharomyces are preferably used, but the invention is not limited thereto.

In the case of using a vertebrate cell as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like located upstream of the DNA of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr (Mol. Cell. Biol., 1: 854 (1981)) and the like. Further, as a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 (Proc.
Acad. Sci., USA., 80: 1 (1983)).

When a prokaryotic cell is used as a host, a promoter and an SD (Shine & Sand) are used upstream of the DNA so that the DNA of the present invention can be expressed in the vector using a vector that can be replicated in the host cell.・ Dargano)
An expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis are added can be suitably used. As the above-mentioned vector, pBR322 and its improved vector are often used in general, but are not limited thereto, and various vectors can be used. There is no particular limitation on the promoter, for example, tryptophan (trp)
Promoter, lpp promoter, lac promoter,
Any of the PL / PR promoter and the like can be suitably used.

As an expression vector for the DNA of the present invention, an ordinary fusion protein expression vector can also be preferably used.
Specific examples of the vector include pGEX (Promega) for expression as a fusion protein with glutathione-S-transferase (GST).

The desired recombinant DNA (expression vector)
There is no particular limitation on the method of introducing and transforming the host into a host cell, and various general methods can be employed. The obtained transformant can also be cultured according to a conventional method, whereby the target protein encoded by the DNA of the present invention is expressed and produced, and accumulated in the transformant, extracellularly, or on the cell membrane of the transformant. Or secreted.

As the medium used in the above-mentioned culture, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cells.

The thus obtained recombinant protein (FCMD)
The protein of the present invention including the causative protein) can be separated and purified according to various separation operations utilizing its physical properties, chemical properties, and the like (“Biochemical Data Book II”, pp. 1175-1259), if desired. , 1st edition, 1st printing, June 23, 1980
Published by Tokyo Chemical Industry Co., Ltd .; Biochemistry, 25 (25):
8274 (1986); Eur. J. Biochem., 163: 313 (1987), etc.). Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Various liquid chromatography such as C), dialysis, combinations thereof, and the like.

The protein of the present invention obtained as described above is
As described above, it is useful, for example, in the pharmaceutical field.

The FCMD-causing protein can also be used as an immunizing antigen for preparing a specific antibody of the protein. The component used as an antigen here can be, for example, a protein or a fragment thereof produced in large amounts according to the above-described genetic engineering technique. By using these antigens, a desired antiserum (polyclonal antibody) can be obtained.
And a monoclonal antibody. The method for producing the antibody itself is well understood by those skilled in the art, and these methods can be followed in the present invention (sequence chemistry experiment course “Immunobiochemical research method”, edited by The Japanese Biochemical Society (1986)). ) Etc.).

For example, a normal animal such as a rabbit, a guinea pig, a rat, a mouse or a chicken can be arbitrarily selected as an immunized animal used for obtaining an antiserum. It can be implemented according to the law.

In addition, a monoclonal antibody can be obtained according to a conventional method by preparing a fusion cell of a plasma cell (immunocyte) and a plasmacytoma cell of an animal immunized with the above-mentioned immunizing antigen, and cloning a clone producing the desired antibody therefrom. And culturing the clone. Immunized animals are generally selected in consideration of compatibility with plasmacytoma cells used for cell fusion, and mice and rats are usually advantageously used. Immunization is the same as in the case of the above antiserum, and can be carried out in combination with a usual adjuvant if desired. In addition, as a plasmacytoma cell used for fusion,
There is no particular limitation, for example, p3 (p3 / x63-Ag8) (Nature, 25
6: 495-497 (1975)), p3-U1 (Current Topics in
Microbiology and Immunology, 81: 1-7 (1978)), N
S-1 (Eur. J. Immunol., 6: 511-519 (1976)), M
PC-11 (Cell, 8: 405-415 (1976)), SP2 / 0
(Nature, 276: 269-271 (1978)) and various myeloma cells such as R210 (Nature, 277: 131-133 (1979)) in rats and cells derived therefrom can be used.

Fusion of the above immune cells and plasmacytoma cells can be carried out in the presence of a conventional fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ) according to a known method. Separation of the desired hybridoma can also be performed (Meth. In Enzym
ol., 73: 3 (1981);

Further, the search for the antibody-producing strain of interest and the monocloning are also carried out by a conventional method. For example, the search for the antibody-producing strain is carried out by the ELISA method using the antigen of the present invention (Meth. In Enzymol. 70: 419-439 (1980)), plaque method, spot method, agglutination method, ochterony (Ouchte
rlony) method, radioimmunoassay and the like, and can be carried out according to various methods generally used for detection of antibodies.

The target antibody can be collected from the hybridoma thus obtained by culturing the hybridoma by a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal compatible with the hybridoma and growing it. It is carried out by a method for obtaining ascites or the like. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies. The antibody thus obtained can be further purified by ordinary means such as salting out, gel filtration, and affinity chromatography.

The antibody thus obtained is the FCM of the present invention.
It is characterized by having a binding property to the D causative protein, which can be advantageously used for purification of the FCMD causative protein and its measurement or identification by immunological techniques.

Thus, the present invention also provides such a novel antibody.

Further, the FC disclosed by the present invention
Based on the sequence information of the MD-causing gene, it is possible to detect the expression of the gene of the present invention in an individual or various tissues, for example, by utilizing the DNA of the present invention having a partial or entire nucleotide sequence of the gene. it can.

Such detection can be performed according to a conventional method, for example, RT-PCR (Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego,
21-27 (1991)) and Northern blot analysis (Molecular Cloning, Cold Spring Harbor Lab.
(1989)), in situ RT-PCR (Nucl. Acids Res.,
21: 3159-3166 (1993)) and their measurement at the cellular level, such as in situ hybridization, and the NASBA method (Nucleic acid sequence-based amplification, Natur
e, 350: 91-92 (1991)) and various other methods.

In the case of employing the PCR method, the primers used are not particularly limited as long as they are specific to the FCMD-causing gene of the present invention and can specifically amplify only the FCMD-causing gene of the present invention. It can be set appropriately based on the sequence. This is usually 15-
The DNA of the present invention can be a DNA of the present invention consisting of a partial sequence of about 30, preferably about 20 to 25 nucleotides, and the present invention F comprising a desired region set by the primers
It can be used for amplifying a CMD causative gene or a part thereof.

The probe for detecting the FCMD-causing gene of the present invention is not particularly limited as long as it is unique to the gene. The probe of the present invention comprises the FCMD-causing gene of the present invention or a partial sequence thereof. Can be.
Examples of the partial sequence include the DNA of the present invention, which comprises a sequence of usually about 10 to 500, preferably about 100 to 500 nucleotides.

Incidentally, such primers and probes are
It can be easily prepared by utilizing the FCMD causative gene of the present invention or by chemically synthesizing the nucleotide sequence disclosed in the present invention using an automatic DNA synthesizer or the like.

Thus, the present invention also provides a DNA fragment used as a specific primer and / or a specific probe for detecting an FCMD-causing gene.

According to the measurement or detection of the expression of the FCMD-causing gene by using the DNA of the present invention, or the measurement or detection of the FCMD-causing protein by using the above-mentioned specific antibody, the expression of the FCMD-causing gene of the present invention in a subject or a test tissue can be determined. The expression level and its abnormality can be grasped.
It can be used and useful in the diagnosis of MD.

Furthermore, according to the present invention, it has been clarified that the onset of FCMD is associated with abnormal expression of the FCMD-causing protein based on mutation of the FCMD-causing gene. According to the detection of the presence of a mutant FCMD causative gene having a mutation that causes deficiency (detection of a genetic abnormality of the FCMD causative gene),
It enables a desired diagnosis of FCMD.

Thus, the present invention provides a method for detecting a gene abnormality of an FCMD-causing gene useful for such diagnosis,
The present invention also provides the mutant FCMD causative gene.

Here, “mutation” refers to a dysfunction of the FCMD-causing protein due to the mutation of the FCMD-causing gene, ie,
The position and degree of the protein are not limited as long as it causes loss or reduction of the function of the protein and / or loss or reduction of the expression of the protein. It can be located in any of the untranslated regions, and can include deletions, substitutions and / or additions (insertions) of one or more (or several) nucleotides.

As a more specific example of such a mutant FCMD-causing gene, a DNA which has been confirmed by the present inventors to cause dysfunction of the FCMD-causing protein, that is, the nucleotide sequence shown in SEQ ID NO: 3 , (I) substitution of cytosine at base number 250 with thymine, (ii) deletion of base numbers 298-299, (iii) base number 5889
DNA having at least one kind of mutation of one or more (or several) nucleotides between the positions 5890.

In the insertion mutation (iii), the nucleotide sequence inserted between nucleotide positions 5889 and 5890 and its length are not particularly limited.
C) ₄₁ ; 49 bp sequence (5′-GGGAGGGAGGGT
GGGGGGGTCAGCCCCCCGCCTGGCC
27 copies of AGCCGCCCCATCC-3 ') (tandem repeat); SINE (short interspersed sequ
ence) type human retroposon sequence; poly-A addition signal (AATAAA); and poly (A), about 3 kb
Can be mentioned.

Detection of such a mutation in a subject's gene, preferably detection of at least one of the above-mentioned mutations (i) to (iii), means a genetic abnormality of the FCMD-causing gene of the subject, It is extremely useful for diagnosis of Such a diagnosis indicates that the subject has both the normal FCMD causative gene and the mutant FCMD causative gene (heterozygotes) or has only the mutant gene (mutant FCMD causative gene).
(A homozygote of the causative gene) or only a normal FCMD causative gene.

The detection of the gene abnormality in the FCMD causative gene is not limited to any particular method or means, and various methods known or available in the future can be widely used. For example, such detection methods include Southern hybridization and dot hybridization (both in Southern, EM, J. Mol. Biol., 98:
503-517 (1975), etc.), dideoxy base sequencing, or various detection methods combining DNA amplification techniques, such as PCR-RFLP (Restriction fragment l).
ength polymorphism: PCR-restriction enzyme fragment length polymorphism analysis), PCR-single-chain higher order structural polymorphism analysis (Orita, M., et al.)
al., Proc. Natl. Acad. Sci. USA, 86: 2766-2770 (19
89)), PCR-SSO method (Specific sequence o
ligonucleotide: PCR-specific sequence oligonucleotide method), allele specific oligomer method using PCR-SSO and dot hybridization (Saiki, RK, et al., Na)
, 324: 163-166 (1986)) and the like.

For example, a more specific example of a method for detecting the above-mentioned mutations (i) to (iii) is the step of preparing a DNA fragment containing the mutated region and the method of preparing the DNA.
This is exemplified as a method comprising a step of analyzing the base sequence of the fragment.

Preparation of a DNA fragment containing a mutated region from a test DNA can be carried out by amplification according to the above-described PCR method or a modification thereof, or by a conventional method such as digestion of genomic DNA with restriction enzymes.

The primers used for the amplification of the DNA fragment are as described above, and are set so that a DNA fragment of a fixed base length having at least a region containing the above mutation position can be synthesized. Preferably, the sequence in the upstream region of the mutation position to be detected is used as a sense primer, and the sequence in the downstream region of the mutation position is designed as an antisense primer.

The region (base length) of the DNA fragment synthesized here is not particularly limited.
It is desirable to set in consideration of the convenience of the analysis step of the nucleotide sequence of the A fragment. For example, when the RFLP method is used for the analysis, a difference in base length is provided so that cleavage by a restriction enzyme can be confirmed. It is good to set. The term “synthesis of DNA” as used herein is a concept that broadly includes extending and amplifying a DNA having a sequence complementary to a specific DNA (sense strand, antisense strand) as a template.

The analysis of the base sequence of the DNA fragment thus prepared can also be performed according to the above-mentioned various methods including sequencing by sequencing. Preferably,
The mutations (i) and (ii) are performed by methods usually used for detection of point mutations, such as the RFLP method and the allele-specific oligonucleotide method. A method using a probe specific to is preferably exemplified.

In the examples described below, the detection of the mutation relating to the point mutation (C → T250) of the above (i) is performed by PCR-RFL
It is performed according to the P method. The mutation position is located in exon 3 of the FCMD causative gene. To amplify a region including this position, a primer “e” for amplifying exon 3 was used.
x3F "and" ex3R ". According to such primer setting, 142 bp amplified DNA
The desired area is provided as a fragment. The point mutation (C → T250) in (i) generates a specific cleavage site (AGCT) for the restriction enzyme AluI at base positions 247 to 250. Therefore, the DNA fragment containing the mutation (14
2 bp), by subjecting it to restriction enzyme AluI digestion to give three fragments of 113 bp, 25 bp and 4 bp, whereas the DNA fragment without the mutation does not show this mode of cleavage and its detection is easier. It is possible. The generated fragment or DNA fragment can be confirmed as a specific band according to a conventional method.

The RFLP method can be carried out by utilizing restriction enzyme sites generated or eliminated by mutation. However, when the mutation of the present invention alone cannot be used as a restriction enzyme recognition site, For example, a desired restriction enzyme site can be artificially introduced by introducing a mismatch into the primer.

In the detection of the mutation (del: 298-299) according to the above (ii), similarly, exon 4 containing the mutation position
Primers ("ex4F" and "ex4R1") are used to amplify a part of the DNA fragment, and the mutation is detected by directly sequencing (sequencing) the amplified DNA fragment.

In the detection of the insertion mutation according to the above (iii), a genomic DNA digested with an appropriate restriction enzyme giving a fragment containing the mutation position, for example, PvuII or the like, is used, and a probe capable of detecting the region is used. The mutation is detected by Southern hybridization. As the probe, any probe can be used as long as it can detect the region, such as the entire insert of cosmid clone cE6, but preferably a probe that detects an inserted sequence of about 3 kb related to the mutation, for example, the cE6
1.4 kb EcoRI fragment and the like. In the case of the above PvuII digestion, the corresponding DNA
The fragment can be confirmed by giving a band of about 8 kb when the mutation is present (about 5 kb when there is no insertion mutation of about 3 kb).

Note that these detection methods merely exemplify typical modes for detecting a genetic abnormality according to the present invention, and the present invention is not limited to these. Based on the sequence information of the FCMD causative gene provided by the present invention, these detection methods and the various methods described above are appropriately adopted, or the methods are appropriately modified according to the gene mutation to be detected. It can be easily adopted by those skilled in the art.

In addition, various operations which can be employed in these mutation detection methods, such as synthesis of DNA or DNA fragment, enzymatic treatment for cutting, deleting, adding or binding DNA, isolation and purification of DNA, Replication, selection, DNA
Amplification of the fragment and the like can be performed according to a conventional method (Molecular Genetics Experiment, published by Kyoritsu Shuppan Co., Ltd., 1983;
Technology, Takara Shuzo Co., Ltd. published in 1990),
It can be appropriately modified as needed.

Further, in the method for detecting a genetic abnormality according to the present invention, the gene or DNA to be measured is not particularly limited. For example, blood, muscle biopsy, various autopsy including human-derived FCMD-causing gene, etc. Widely collected from biological samples such as tissues, villi, amniotic fluid, biomaterial tissues, and various cell lines.

When detecting an abnormality in the FCMD-causing gene as described above and performing a genetic diagnosis of FCMD based on such a method, a reagent kit containing a reagent for detecting a mutation in the FCMD gene as an active ingredient It is preferable to use.

Such a reagent kit is characterized by containing, as an active ingredient, a reagent for specifically detecting the mutation according to the present invention, and the active ingredient is appropriately set according to the detection means to be employed. be able to. Preferably, a reagent for preparing a DNA fragment containing a mutated region to be detected, for example, D
A primer of the present invention designed to be able to synthesize the DNA fragment using NA as a template, a restriction enzyme for digesting the DNA of a subject to prepare a desired DNA fragment, and / or a base of the DNA fragment It contains a reagent for analyzing the sequence to confirm the presence of the mutation, such as a restriction enzyme or the probe of the present invention, as an essential component.

Further, one or several reagents corresponding to the detection can be combined in the reagent kit of the present invention. Examples of such combination reagents include dATP and dCT.
Reagents can be appropriately selected and adopted according to the detection method employed, such as P, dTTP, dGTP, DNA synthase, and RNA synthase. In addition, the reagent kit may contain an appropriate buffer, washing solution, or the like for the benefit of performing the measurement.

[0093]

The present invention provides the nucleotide sequence of the gene responsible for Fukuyama-type congenital muscular dystrophy and the amino acid sequence of the gene product. Also, the present invention
Mechanism of onset of Fukuyama-type congenital muscular dystrophy, that is, Fukuyama-type congenital muscular dystrophy, for the first time elucidated that onset is caused by deficiency of the gene product (FCMD-causing protein) based on abnormality of the FCMD-causing gene of the present invention. It is.

The detection method or diagnostic method of the present invention for discriminating the abnormality of the FCMD causative gene enables a genetic or biochemical diagnosis of Fukuyama-type congenital muscular dystrophy, particularly a prenatal or carrier diagnosis. It is useful in predicting the predisposition, course, and prognosis.

Further, according to the genetic diagnosis using the FCMD causative gene of the present invention, the Fukuyama-type congenital muscular dystrophy and Walker-Warburg syndrome (Dobyns , W.
B., et al, Am. J. Med.Genet., 32, 195-210 (198
9)), cerebrum-eye-dysplasia-muscular dystrophy syndrome (Towf
ighi, J., et al, Acta.Neuropath., 65, 110-123 (19
84)) and Fukuyama-type congenital muscular dystrophy-related diseases, including diseases sharing the characteristics of Fukuyama-type congenital muscular dystrophy.

The FCMD-causing protein of the present invention is also useful for preparing a specific antibody thereof. Further, such a specific antibody can be used for immunological measurement of an FCMD-causing protein expressed and produced in a living body. It is useful for diagnosis of Fukuyama-type congenital muscular dystrophy, elucidation of pathological conditions, and the like.

Further, the information on the FCMD-causing gene and the gene product thereof in the present invention is important for knowing the onset mechanism of Fukuyama-type congenital muscular dystrophy, which causes complicated damage to nerves and brain. It is considered to be extremely important in understanding development and development of the whole brain.

[0098]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the contents of the present invention will be specifically described using embodiments. However, the present invention is not limited to these.

Materials and experimental methods in the examples are shown below.

1. DNA sample of FCMD patient Previous report (Toda, T., et al., Nat. Genet., 5, 283-286
(1993); Toda, T., etal., Am. J. Hum. Genet., 55, 9
46-950 (1994); Toda, T., et al., Am. J. Hum. Gene
t., 59, 1313-1320 (1996)) to 63 FCMD families.
A total of 86 FCMD ancestry DNs with an additional 23 ancestry
The A sample was analyzed, and the DNA of 304 persons including 115 persons having FCMD was examined. FCMD was diagnosed based on standard diagnostic criteria (Fukuyama, Y., et.
al., Brain Dev., 3, 1-30 (1981)).

2. Southern analysis Membranes were prehybridized in 10% SDS and 7% PEG containing 200 μg / ml sonicated human placental DNA at 65 ° C. overnight. The cosmid was digested with the restriction enzyme NotI, and the inserted DNA was separated. The probe DNA was radiolabeled with α ³² P-dATP using a commercially available kit (Megaprime labeling kit, Amersham), prehybridized in the same solution at 65 ° C. for 1 hour, and then hybridized with the membrane (Tokino, T). ., et al., Am. J. Hum. Genet., 4
8, 258-268 (1991)). The washing conditions were 0.1 × SSC,
Performed at 63-65 ° C. using 0.1% SDS.

3. Sequencing analysis Sequencing is performed using a commercial kit (PRISM ReadyReactio
n DyeDeoxy Terminator Cycle-sequencing Kit and Ampl
iTaq-FS, Perkin-Elmer) and analyzed with an ABI model 377 sequencer (Perkin-Elmer).

Further, the measurement was manually performed by a deoxy chain termination method using α ³⁵ S-dATP. In this case, the sequencing reaction was electrophoresed on a 6% polyacrylamide denaturing gel and exposed to X-ray film.

4. Northern analysis RNA blots from various human tissues (Multiple-tis
sue Northern blot (Clontech) was purchased from Clontech and analyzed according to the manual.

Total RN from patient and normal lymphoblasts
A was separated by acid phenol extraction (Trizolm, Gibco-BRL). Poly (A) + RNA (Poly (A) + RNA)
It was separated from total RNA by oligo dT chromatography (Oligo-dT Latex, Takara Shuzo) according to standard methods. Poly (A) + RNA (2 μg) was electrophoresed on a formamide-agarose gel, and the membrane (Hybond N + membran) was analyzed.
e, Amersham). The membrane is 0.1 × SSC,
Washed at 50 ° C. using 0.1% SDS.

5. PCR PCR is a commercially available polymerase (AmpliTaq polymerase, P
erkin-Elmer).

A long PCR assay for the founder insertion mutation was performed using 500 ng of chromosomal DNA, 10 pmo of each primer.
l, 1 × LA PCR buffer II (Takara Shuzo), each dNT
P0.4 mM (for dGTP, dGTP: deaza
GTP = 1: 1), 2.5 mM and 2.5 mM of MgCl ₂
The reaction was performed in a 50 μl reaction solution containing the unit polymerase (EX Taq polymerase, Takara Shuzo).

The samples were incubated in a DNA thermocycler (GeneAmp 9600, Perkin-Elmer) under the following conditions: 94 ° C. for 1 minute 98 ° C. for 10 seconds and 68 ° C. for 20 minutes 10 cycles at 98 ° C. 5. 20 cycles at 68 ° C. for 20 minutes and an extension of 20 seconds per cycle. SSCP analysis The amplification of the template DNA was performed by intron primers ex3F and ex3R for exon 3 and intron primer ex4F and exon primer ex4 for exon 4.
Performed using R1.

Ex3F: 5'-GTTGCATGCTGGACTTTGAA-3 'ex3R: 5'-CATAAAGCACTTGGTAAAGGGC-3' ex4F: 5'-GACTGTTGTGTTGGCTTACTGG-3 'ex4R1: 5'-GATACTGCAGTGCAAATGCAG-3' SSCP analysis contains 5% by volume of glycerol. Using a 10% polyacrylamide gel, with or without, the method of Sakauchi et al. (Bannai, M., et al., Eur. J. Immunogenet.,
21, 1-9 (1994)). Each sample (~ 1
/ 8 μl) is ~ 60 m in the instrument (ResolMax, Atto)
Ah, subjected to electrophoresis at 10 ° C. or 20 ° C.
The NA fragment was separated. Detection was performed by the silver staining method, and PCR products showing a band shift were sequenced by the above method 3.

Example 1 Identification and Solution of FCMD Causal Gene
Analysis (1) In order to examine genomic rearrangements such as large deletions and insertions in the DNA of FCMD patients, marker loci D9
Using each cosmid clone (FIG. 1) of a cosmid contig having S2105 and D9S2107 (Miyake, M., Genomics, 40, 284-293 (1997)) as a probe,
Genomic DNA from MD patients was screened by Southern analysis.

When the insert of cosmid clone cE6 containing D9S2170 as a whole was used as a probe, FCMD DNA digested with PvuII was used.
Was found to have a new 8 kb band without a normal 5 kb band (FIG. 2, left column). In addition, the same DNA is replaced with PstI or Bst instead of PvuII.
Upon digestion with glII, one normal band disappeared and a new band larger than the disappeared band appeared (FIG. 2). These facts suggested that most genomic DNA of FCMD patients had a 3 kb fragment insertion sequence.

The small fragment E in the insert of cE6
When 6f3 was used as a probe, most patients homozygously displayed the abnormal 8 kb band, and the remaining patients displayed both the normal 5 kb band and the abnormal 8 kb band (FIG. 3). In the figure, N represents normal control, F represents FCM.
D shows the results obtained from the patient. . A more detailed analysis shows that in most FCMD patients, 1.4 kb of cE6
Insertion of a 3 kb sequence was confirmed in the EcoRI fragment (see FIG. 1).

Of the 88 unrelated normal individuals, only one had the insert heterozygously. The ratio of 1/88 is the Japanese FCM
This is in good agreement with the reported values for the proportion of D carriers (Fukuyama, Y., et al., Brain Dev., 6, 373-390).
(1984)).

Furthermore, this inserted allele is compatible with the Japanese FC
D9S2105-D9 possessed by 80% or more of MD chromosome
FCMD founder haplotype 138-192-147 for S2170-D9S2171-D9S2107
It was found to segregate with -183.

These results suggest that insertion of the 3 kb fragment into genomic DNA specific to FCMD patients is directly related to the FCMD phenotype, and even if it is polymorphic, This suggests that it is located very close to the FCMD causative gene.

(2) Using a 1.4 kb EcoRI fragment of cE6 capable of detecting the above 3 kb inserted sequence as a probe, screening a human adult brain (caudate nucleus) cDNA library (1.4 × 10 6 clones) , 2.1 kb and 3.2 kb cDNA with poly (A)
I got The human adult brain cDNA library used was obtained from Dr. Triangle at Fukuoka University.

Using the inserted sequence at the 5 ′ end of this cDNA, another cDNA library was screened, and in parallel with this, RACE (rapid amplification of
By conducting cDNAends experiments, 13 additional clones (of which 7 were fetal brain cDNA libraries (Clonte
ch), and the remainder is prepared heart cDNA for RACE
).

The RACE experiment was performed using a cDNA amplification kit (Marathon cDNA Amplification: Clontech) and a prepared cDNA (Marathon Ready cDN) derived from an adult heart.
A: Clontech).

By sequencing a total of 15 cDNA clones obtained, the full-length nucleotide sequence of the continuous cDNA was determined (FIGS. 4 and 5). The cDNA is 7349
bp and a 40 base poly (A) tail. Open reading frame (1383bp)
Starts with a start ATG codon located at base positions 112-114 and ends with a stop codon TGA located at base positions 1495-1497. Also, a stop codon is present in-frame at 42 nucleotides upstream of the start site, and the common poly (A) addition site is at base number 410
It is located at 1, 4337, 6266 and 7327. The two original cDNA clones obtained from the human adult brain cDNA library were obtained by adding poly (A) at the latter two sites (base Nos. 6266 and 7327), and Appears to correspond to two bands on the blot (FIGS. 6 and 7).

The cDNA clone containing the translation region (II-
By Northern blotting using 5) as a probe,
Poly (A) mRNA prepared from relatively various human tissues
Revealed the presence of 6.5 kb and 7.5 kb transcripts. Significant signals are found in the heart, brain, placenta,
Obtained from skeletal muscle and pancreas, but not from lung, liver and kidney (FIG. 6). Furthermore, the transcript was also detected in cultured lymphoblasts (FIG. 7).

The predicted protein product of the cDNA (FCMD-causing gene) thus obtained is composed of 461 amino acids, has a molecular weight of about 53.6 kDa and an isoelectric point of 8.29.
Is estimated. The main hydrophobic amino acid residues (Lys, Val, Ile, Phe and Met) account for 30.8% of the total amino acids.

(3) Gen using BLASTN and FASTA sequence alignment algorithms
By searching the Bank and dbEST databases, F
The CMD-causing gene was hardly similar to a gene having a known function. However, seven different c
DNA library (fetal heart, brain, neuroepithelial cell line, embryo, parathyroid tumor, melanocyte and fibroblast)
CDNA end registration sequence (EST: expressed
sequence tag) 11 pieces (R57783, N41001,
45012, AA082552, AA328556, W
44740, W60550, W37098, N3539
8, AA235981 and N94327), R57783 corresponds to the base number 95-318 region, and the other 10 ESTs are 3 ′ of the FCMD cDNA.
Corresponded to the terminal sequence.

The deduced protein sequence was determined using BLASTP and FAS.
SwissProt and PIR databases using TA and GenBank using TBLASTN.
As a result of comparison with the predicted translation products of the k, EMBL, DDBJ and PDB databases, no significant similarity to the known protein was observed in any case. In addition, nematodes (C.el
egans) cosmids W02B3 and T07A5 (GenB
An accession numbers: U22833 and Z48055) showed weak similarity to the predicted translation products.

The protein motif of the FCMD protein was changed to ProSi
te database (Bairoch, A., etal., Nucl. Acids R
es., 25, 217-221 (1997)).
It was found to have one N-glycosylation site, four protein kinase C phosphorylation sites, one tyrosine kinase phosphorylation site, and five N-myristorylation sites (FIGS. 4-5). . Hydropathy calculation: Kyte, J., et a
l., J. Mol. Biol., 157, 105-132 (1982))
It was predicted that there would be 16 hydrophobic regions. PSORT program (Nakai, K., et al., Genomics, 14, 897-911 (1
992)), the location of the protein was estimated.
The CMD protein was found to have an N-terminal signal sequence with a cleavage site at codon 21, but no transmembrane region.

However, other programs, SAPS
(Brendel, V., et al., Proc. Natl. Acad. Sci. USA,
89, 2002-2006 (1992)), SOSUI (Hirokawa, T., e
t al., http://www.tuat.ac.jp/-mitaku/adv_sosui/.)
And TMpred (Hofmann, K., et al., Biol. Chem.
According to Hoppe. Seyler, 374, 166 (1993)),
One (amino acids 8-23), one (amino acids 6-27)
And the presence of two (amino acids 8-28 and 288-306) transmembrane segments was predicted. If a "transmembrane" segment starting at amino acids 6 or 8 is interpreted as a signal sequence, the FCMD protein will have a signal sequence and zero or one transmembrane domain.

Example 2 Mutation of FCMD Causal Gene
-1 (insertion of 3 kb fragment) (1) Comparison of the FCMD cDNA and the genomic sequence revealed that the 3 ′ untranslated region of the 3 ′
b was confirmed to be inserted (FIGS. 1, 4 to
(Fig. 5).

[0127] Chromosomal DNA (144 lines) of FCMD patients who are not related to each other is digested with restriction enzymes and subjected to Southern blotting analysis, or long PCR method, so that the FCMD chromosome has a high probability of insertion (125/144). It turns out that there is an array. On the other hand, in a normal person, this inserted sequence was observed only in one of 176 chromosomes.

FIG. 8 shows 138-192-147-183.
Two generation FCM showing separation with founder haplotype
FIG. 4 shows Mendelian inheritance of this inserted allele in D family.

(2) To examine the effect of this inserted sequence on the transcript of the FCMD-causing gene, Northern (B) analysis of poly (A) mRNA isolated from cultured lymphoblasts of patients was performed.

As can be seen from the results shown in FIG. 7, FCMD patients homozygous for the inserted sequence
Almost no transcript of the causative gene could be detected. Also, in some FCMD patients heterozygous for the inserted sequence and other haplotypes, the amount of the transcript was significantly lower. This result suggests that the insertion or other type of mutation in the FCMD-causing gene causes a decrease in the transcription level of the FCMD-causing gene and / or a decrease in mRNA stability, leading to a loss of function of the FCMD-causing protein. .

(3) In order to identify the inserted DNA (3 kb), a clone containing the inserted sequence was recovered from a genomic library constructed from an FCMD patient having the inserted sequence homozygously.

By sequencing analysis, the inserted DN
The A fragment is 3062 bp long and (TCTCCC) ₄₁ ;
49 bp sequence (5'-GGGAGGGAGGTGGGGG
GGGTCAGCCCCCCGCCTGGCCAGCC
GCCCCATCC-3 '), 27 copies (tandem repeat); SINE (short interspersed sequence) type human retroposon sequence; Poly A addition signal (AATA
AA); and poly (A). In addition, AAGAAAAAAAAAAATTG is added to both ends.
The presence of "target site duplication" consisting of tandem repeats of T suggested that insertion of this 3 kb fragment was caused by a retrotransposon mechanism.

According to the homology search, the tandem repeat sequence in the inserted sequence was found to be the human gene encoding complement component C2 (Z11739), Huntington's disease (Huntington's disease).
disease) near the genomic region (Z69654) and the repetitive sequences found in other sites.

Embodiment 3 FIG . Mutation of FCMD causative gene
-2 (C → T250) In Example 2, the patient having the inserted chromosome heterozygously also showed a remarkably low amount of the transcript (FIG. 7), but did not have such a 3 kb inserted sequence. FCM
To investigate the mechanism of inactivation on the D chromosome, single-stranded structural polymorphism (SSCP: single-strande
The sequence of exon and exon-intron boundary regions was screened over the entire coding region by d conformation polymorphism analysis.

As a result, the founder-inserted sequence derived from the paternal and D9S2105-D9S2170-
130-20 for D9S2171-D9S2107
It was found that the mobility of the PCR product corresponding to exon 3 of the gene of the patient HM having the 1-157-183 haplotype (lane 3 in FIG. 7 and the left panel in FIG. 9) was shifted. Sequence analysis of this PCR product revealed that cytosine (C) at nucleotide position 250, a maternal allele, had been replaced with thymine (T), which resulted in translation termination (from CGA). T
GA, R47X: FIG. 10, left panel).

Since a new recognition site for the restriction enzyme AluI is generated by this base substitution (from AGCC to AGCT),
This can be used to generate PCR-R for other families of patient HM.
FLP analysis was performed to confirm that this point mutation segregated with the disease and with the microsatellite haplotype. Furthermore, it was confirmed that the other five kindreds (YS, RM, AG, AT and AY) having the same haplotype had the same nonsense mutation (FIG. 1).
1, left panel).

Embodiment 4 FIG . Mutation of FCMD causative gene
-3 (del: 298-299) In the same manner as in Example 3, 2 bp at nucleotide positions 298-299 (codon 63) of the FCMD-causing gene were added to the patient TI.
A deletion was found, confirming that this resulted in a frameshift and incomplete termination at codon 75 (FIGS. 9 and 10, right panels).

The Japanese mother of this patient had the inserted chromosome, and the American father (mixed English and German) did not. The 2 bp deletion was present in the chromosome inherited from the paternal (FIG. 11, right panel).

Example 5 Discussion According to the present invention, a novel gene involved in the development of FCMD,
An FCMD causative gene was provided.

According to haplotype analysis of the FCMD81 kindred, the major mutation in the FCMD-causing gene was D9S21
05-D9S2170-D9S2171-D9S210
It is thought to have occurred on a chromosome of one ancestor with a 138-192-147-183 haplotype for chromosome 7.
The present invention has provided an important finding that the mutation is an insertion of a 3 kb retrotransposon in the 3 ′ untranslated region, which causes loss of function of the FCMD-causing gene.

The mutation was found in 87% of the chromosomes of the FCMD patients tested.

By Northern blot analysis, it was confirmed that the normal transcript of the FCMD-causing gene was widely expressed in various human tissues. In particular, the remarkable expression in heart, brain and skeletal muscle indicates that FCMD patients present together with congenital muscular dystrophy associated with cerebral malformation, and often myocardial fibrosis (Miura, K., et al., Acta. Path.
Jpn. 37, 1823-1835 (1987)).

The inserted DNA contains a tandem repeat of the 49 bp sequence commonly found in the human genome, and this repeat is linked to a SINE-type retroposon.
Since this inserted sequence has a poly (A) addition signal and contains poly (A) which has been elongated, the functional sequence is different from that transcribed and integrated into the FCMD gene by the mechanism of retrotransposon. Conceivable. The existence of a 17 bp "target site duplication" supports this idea (Lewin, B.,
In Genes, VI, B. Lewin, ed. (Oxford: OxfordUniversi
ty Press), pp. 597-619 (1997)). Some examples of disease-related somatic or de novo germline retrotransposal integration are known in humans. They include hemophilia A (Kazazian, HH, et al., Nature, 332, 164-16).
6 (1988)) and colorectal cancer (Miki, Y., et al., Cancer Re
s., 52, 643-645 (1992)) and hemophilia B (Vidaud, D., et al., Eur. J. Hum. Genet., 1, 30-).
36 (1993)), neurofibromatosis type 1 (Wallace, MR, et a
l., Nature, 353, 864-866 (1991)) and breast cancer (Miki,
Y., et al., Nat. Genet., 13, 245-247 (1996)). FCMD is thought to be the first human disease in which tandem repeats inserted by retrotransposon mechanisms are transmitted from distant ancestors.

This insertion in the FCMD causative gene (3
kb) is thought to result in a significant decrease in the corresponding mRNA, leading to an FCMD phenotype due to its protein dysfunction. There are at least three possible mechanisms by which the insertion may cause such a phenomenon: (1) the transcriptional efficiency is suppressed by the inserted sequence; (2) the mutant FCMD RNA does not become a mature transcript (3) the stability of mRNA transcribed from the inserted allele varies.

For the development of myotonic dystrophy, myotonin-protein kinase (myotonin-protein kinase)
se) It has been found that an increase in (CTG) repeat sequences in the 3 ′ untranslated region of the gene is involved (Broo
k, JD, et al., Cell, 68, 799-808 (1992)), and it has been described that this increase in repeats prevents poly (A) + RNA accumulation (Wang, J., et al., Hum. Mol. Genet.,
4, 599-606 (1995)). Also, the development of Friedreich's ataxia involves an abnormal increase in (GAA) repeats in the first intron of the gene encoding frataxin (Campuzano, V., et al., Science, 271, 1423). -1
427 (1996)), and patients homozygous for this increased sequence are thought to have a corresponding decrease in their mRNA due to inhibition of transcription elongation (Bidichandani, SI, et al., Am. J. Hum. Genet., 59
(suppl.), A246 (1996)). On the other hand, it has been shown that the 3 'untranslated region affects mRNA stability (Sachs, AB, Cell, 78, 625-633 (1993)). For example I
It has been reported that binding of a RE (iron-responsive element) -binding protein to transferrin mRNA stabilizes mRNA containing a stem-loop structure capable of binding this protein (Klausner, RD, Cell, 7).
2, 19-28 (1993)). Therefore, the FCMD in the present invention
Insertion of a 3 kb fragment into the 3 'untranslated region of the causative gene may change the secondary structure of mRNA and render it unstable.

In addition, in some FCMD patients, a point mutation (deletion or substitution) that causes premature termination of the FCMD causative gene product has been found separately from the above-mentioned insertion mutation. Expression levels were reduced to 10-20% of normals.

This strongly supports the conclusion that the FCMD causative gene of the present invention corresponds to the FCMD pathogenic gene.

The function of the FCMD-causing protein according to the present invention can be considered from comparison with known information on the defective gene product of muscular dystrophy and onset mechanism.

Duchenne muscular dystrophy (DM
D), a defective gene product, dystrophin (Dystro
phin) is a large rod-shaped protein that exists directly below the muscle cell membrane and is involved in the formation of a large oligomeric complex (dystrophin-glycoprotein complex) composed of muscle cell membrane glycoproteins (Ervasti, JM, et al. ., Cell, 66, 1121-1
131 (1991)). Recent studies have shown that the deficiency of each component of this complex impairs a series of linkages from laminin α2 (merosin), present in the extracellular matrix, to actin in the cytoskeleton just below the muscle cell membrane. On the basis of various muscular dystrophy [DMD and Becker muscular dystrophy (Ohlendieck, K., Neurology, 43, 795-80)
0 (1993)); α, β, γ and δ-sarcoglycanopathy (Roberds, sl, et al., Cell, 78, 625-633 (199
4), Lim, LE, et al., Nat.Genet., 11, 257-265 (199
5), Bonnemann, CG, et al., Nat.Genet., 11, 266-2.
73 (1995), Noguchi, S., et al., Science, 270, 819-8
21 (1995), Nigro, V., et al., Nat.Genet., 14, 195-
198 (1996)); Melosin-deficient congenital muscular dystrophy (Helbling-Leclerc, A., et al., Nat. Genet., 11,
216-218 (1995)].

On the other hand, in the muscles of FCMD patients, β-dystroglycan (Matsumura, K., et al., Lancet, 34), one of the dystrophin-binding glycoproteins.
1, 521-522 (1993)) and laminin α, an extracellular matrix that binds to α-dystroglycan.
A decrease in immunostaining of the two chains has been observed. The genes encoding these proteins are located on chromosome 3p21 (lbraghimov
-Beskrovnaya, O., Etal., Hum. Mol. Genet., 2, 1651-
1657 (1993)) and chromosome 6q22-23 (Tryggvason,
K., Cell Biol., 5, 877-882 (1993)), and their abnormal expression in FCMD muscle is considered to be a secondary change. However, abnormalities of FCMD muscle and brain basement membranes have also been observed by electron microscopy (Nakano, I., et al., Acta. Neuropathol.,
91, 3131-321 (1996); Ishii, H., et al., Neuromuscu
l. Disord., 7, 191-197 (1997); Yamamoto, T., et a
l., Ultrastruct. Pathol., 21, 355-350 (1997); Yama
moto, T., et al., Brain Dev., 19, 35-42 (1997)).

From these findings, the presence of a signal sequence and zero or one transmembrane region in the FCMD-causing protein indicates that the FCMD-causing protein is present in the extracellular matrix or in the sarcoplasmic membrane. Interacts with a large complex surrounding the exterior and interior of the plant. Some putative phosphorylation sites are also in the F
This suggests that the CMD-causing protein binds to one or more other proteins and undergoes phosphorylation.

Further, FCMD is known to be accompanied by brain malformation (brain formation disorder), so-called cerebellar gyrus, which is attributed to a defect in migration of nerve cells (Ta).
kada, K., et al., J. Neuropathol. Exp. Neurol., 43,
395-407 (1984)). Therefore, it is considered that the FCMD-causing protein of the present invention plays an important role in the development of nerves, particularly in the migration of nerve cells. Mouse mutant "reel
"er" indicates abnormalities in the cerebral layer structure as a result of defects in neuronal migration during neurogenesis.
n ″ is a secreted glycoprotein with some structural properties that are characteristic of extracellular matrix proteins, similar to the FCMD causative protein (D'Arcangelo, G., et al., Nature, 374, 7).
19-723 (1995); D'Arcangelo, G., et al., J. Neurosc
i., 17, 23-31 (1997)).

Therefore, the information on the FCMD-causing gene and its gene product in the present invention is important for understanding the development of the nervous system and the development of the whole brain.
Elucidating their role in D is considered to be extremely important in understanding the mechanism of the onset of FCMD that causes complex damage to nerves and brain.

[0154]

[Sequence list] SEQ ID NO: 1 Sequence length: 461 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Ser Arg Ile Asn Lys Asn Val Val Leu Ala Leu Leu Thr Leu Thr 1 5 10 15 Ser Ser Ala Phe Leu Leu Phe Gln Leu Tyr Tyr Tyr Lys His Tyr Leu 20 25 30 Ser Thr Lys Asn Gly Ala Gly Leu Ser Lys Ser Lys Gly Ser Arg Ile 35 40 45 Gly Phe Asp Ser Thr Gln Trp Arg Ala Val Lys Lys Phe Ile Met Leu 50 55 60 Thr Ser Asn Gln Asn Val Pro Val Phe Leu Ile Asp Pro Leu Ile Leu 65 70 75 80 Glu Leu Ile Asn Lys Asn Phe Glu Gln Val Lys Asn Thr Ser His Gly 85 90 95 Ser Thr Ser Gln Cys Lys Phe Phe Cys Val Pro Arg Asp Phe Thr Ala 100 105 110 Phe Ala Leu Gln Tyr His Leu Trp Lys Asn Glu Glu Gly Trp Phe Arg 115 120 125 Ile Ala Glu Asn Met Gly Phe Gln Cys Leu Lys Ile Glu Ser Lys Asp 130 135 140 Pro Arg Leu Asp Gly Ile Asp Ser Leu Ser Gly Thr Glu Ile Pro Leu 145 150 155 160 His Tyr Ile Cys Lys Leu Ala Thr His Ala Ile His Leu Val Val Phe 165 170 175 His Glu Arg Ser Gly Asn Tyr Leu Trp His Gly His Leu Arg Leu Lys 180 185 190 Glu His Ile Asp Arg Lys Phe Val Pro Phe Gln Lys Leu Gln Phe Gly 195 200 205 Arg Tyr Pro Gly Ala Phe Asp Arg Pro Glu Leu Gln Gln Val Thr Val 210 215 220 Asp Gly Leu Glu Val Leu Ile Pro Lys Asp Pro Met His Phe Val Glu 225 230 235 240 Glu Val Pro His Ser Arg Phe Ile Glu Cys Arg Tyr Lys Glu Ala Arg 245 250 255 Ala Phe Phe Gln Gln Tyr Leu Asp Asp Asn Thr Val Glu Ala Val Ala 260 265 270 Phe Arg Lys Ser Ala Lys Glu Leu Leu Gln Leu Ala Ala Lys Thr Leu 275 280 285 Asn Lys Leu Gly Val Pro Phe Trp Leu Ser Ser Gly Thr Cys Leu Gly 290 295 300 Trp Tyr Arg Gln Cys Asn Ile Ile Pro Tyr Ser Lys Asp Val Asp Leu 305 310 315 320 Gly Ile Phe Ile Gln Asp Tyr Lys Ser Asp Ile Ile Leu Ala Phe Gln 325 330 335 Asp Ala Gly Leu Pro Leu Lys His Lys Phe Gly Lys Val Glu Asp Ser 340 345 350 Leu Glu Leu Ser Phe Gln Gly Lys Asp Asp Val Lys Leu Asp Val Phe 355 360 365 Phe Phe Tyr Glu Glu Thr Asp His Met Trp Asn Gly Gly Thr Gln Ala 370 375 380 Lys Thr Gly Lys Lys Phe Lys Ty r Leu Phe Pro Lys Phe Thr Leu Cys 385 390 395 400 Trp Thr Glu Phe Val Asp Met Lys Val His Val Pro Cys Glu Thr Leu 405 410 415 Glu Tyr Ile Glu Ala Asn Tyr Gly Lys Thr Trp Lys Ile Pro Val Lys 420 425 430 Thr Trp Asp Trp Lys Arg Ser Pro Pro Asn Val Gln Pro Asn Gly Ile 435 440 445 Trp Pro Ile Ser Glu Trp Asp Glu Val Ile Gln Leu Tyr 450 455 460 SEQ ID NO: 2 Sequence length: 1383 Sequence type: Number of nucleic acid strands: single strand Topology: linear Sequence type: cDNA to mRNA origin Organism name: human Sequence characteristics Characteristic symbol: CDS Location: 1..1383 Sequence ATGAGTAGAA TCAATAAGAA CGTGGTTTTG GCCCTTTTAA CGCTGACAAG TTCTGCATTT 60 CTGCTGTTTC AGTTGTACTA CTACAAGCAC TATTTATCAA CAAAGAATGG AGCTGGTTTG 120 TCAAAATCCA AAGGAAGCCG AATTGGATTT GATAGCACAC AGTGGCGTGC AGTTAAAAAA 180 TTTATTATGT TAACATCCAA CCAAAATGTA CCAGTGTTTC TTATTGATCC TTTGATACTG 240 GAATTGATTA ATAAGAACTT TGAACAAGTC AAAAATACTT CTCATGGCTC TACTTCACAA 300 TGCAAGTTTT TCTGTGTTCC AAGAGACTTT ACTGCATTTG CACTGCAGTA TCACCTATGG 360 AAGAATGAGG AAGGCTGGTT TCGGATAGCT GAGAATATGG GATTTCAGTG CCTAAAGATT 420 GAGAGTAAAG ATCCCCGGCT AGACGGGATA GACTCACTCT CTGGAACTGA AATCCCCCTG 480 CACTATATCT GCAAACTGGC CACTCATGCG ATCCACTTGG TAGTCTTTCA TGAGAGGAGT 540 GGCAACTACC TCTGGCACGG CCACTTGAGA CTTAAAGAAC ACATTGACAG GAAATTTGTT 600 CCCTTCCAAA AGTTACAGTT TGGTCGTTAT CCAGGAGCTT TTGACAGGCC AGAGTTACAG 660 CAAGTTACTG TTGATGGACT GGAAGTTCTC ATTCCAAAGG ATCCAATGCA CTTTGTAGAA 720 GAAGTACCAC ACTCTAGGTT TATTGAGTGT AGGTATAAAG AAGCTCGAGC ATTCTTTCAG 780 CAGTACCTTG ATGATAACAC TGTGGAAGCT GTGGCCTTTC GGAAGAGTGC AAAGGAATTA 840 CTGCAACTAG CAGCGAAAAC ATTAAACAAA TTGGGAGTAC CATTCTGGCT GAGCAGTGGA 900 ACTTGTCTAG GATGGTATCG ACAATGCAAC ATTATTCCTT ATAGCAAAGA TGTTGACCTA 960 GGAATTTTTA TACAAGATTA CAAATCTGAT ATTATTTTAG CATTTCAGGA TGCAGGACTT 1020 CCGCTCAAAC ACAAATTTGG GAAGGTAGAA GACAGCTTGG AACTATCCTT CCAGGGAAAA 1080 GATGATGTAA AACTTGATGT TTTTTTCTTC TATGAAGAAA CTGATCACAT GTGGAATGGA 1140 GGCACTCAGG CCAAAACAGG AAAAAAATTC AAATACCTGT TTCCGAAGTT TACACTGTGC 1200 TGGACTGAGT T TGTAGACAT GAAGGTCCAT GTACCCTGTG AAACCCTCGA ATACATTGAA 1260 GCCAACTATG GTAAGACCTG GAAGATTCCT GTAAAGACGT GGGACTGGAA GCGCTCTCCT 1320 CCCAATGTGC AACCCAATGG AATCTGGCCT ATTTCTGAGT GGGATGAGGT TATCCAGTTA 1 Nucleotide Sequence: No. Type: cDNA to mRNA Origin Organism name: Human Sequence characteristics Characteristic code: CDS Location: 112..1494 Sequence GCTGCAGCCT GCTGTTGAGT GAGAAAACAA AATTATCTTC CTTTCCAAAT CCAAAAAGAT 60 GAAAACGACT GAGATACTTT CAAAAGTGCATGATCATGATCATGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGATCGATGCTT ACTACAAGCA CTATTTATCA ACAAAGAATG GAGCTGGTTT GTCAAAATCC 240 AAAGGAAGCC GAATTGGATT TGATAGCACA CAGTGGCGTG CAGTTAAAAA ATTTATTATG 300 TTAACATCCA ACCAAAATGT ACCAGTGTTT CTTATCT GCTATCTAGTC TAGTATCTAGTC TGATCTAGTC TGATCTAGTC TAGTATCTAGTC AAGAATGAG 480 GAAGGCTGGT TTCGGATAGC TGAGAATATG GGATTTCAGT GCCTAAAGAT TGAGAGTAAA 540 GATCCCCGGC TAGACGGGAT AGACTCACTC TCTGGAACTG AAATCCCCCT GCACTATATC 600 TGCAAACTGG CCACTCATGC GATCCACTTG GTAGTCTTTC ATGAGAGGAG TGGCAACTAC 660 CTCTGGCACG GCCACTTGAG ACTTAAAGAA CACATTGACA GGAAATTTGT TCCCTTCCAA 720 AAGTTACAGT TTGGTCGTTA TCCAGGAGCT TTTGACAGGC CAGAGTTACA GCAAGTTACT 780 GTTGATGGAC TGGAAGTTCT CATTCCAAAG GATCCAATGC ACTTTGTAGA AGAAGTACCA 840 CACTCTAGGT TTATTGAGTG TAGGTATAAA GAAGCTCGAG CATTCTTTCA GCAGTACCTT 900 GATGATAACA CTGTGGAAGC TGTGGCCTTT CGGAAGAGTG CAAAGGAATT ACTGCAACTA 960 GCAGCGAAAA CATTAAACAA ATTGGGAGTA CCATTCTGGC TGAGCAGTGG AACTTGTCTA 1020 GGATGGTATC GACAATGCAA CATTATTCCT TATAGCAAAG ATGTTGACCT AGGAATTTTT 1080 ATACAAGATT ACAAATCTGA TATTATTTTA GCATTTCAGG ATGCAGGACT TCCGCTCAAA 1140 CACAAATTTG GGAAGGTAGA AGACAGCTTG GAACTATCCT TCCAGGGAAA AGATGATGTA 1200 AAACTTGATG TTTTTTTCTT CTATGAAGAA ACTGATCACA TGTGGAATGG AGGCACTCAG 1260 GCCAAAACAG GAAAAAAATT CAAATACCTG TTTCCGAAGT TTACACTGTG CTGGACTGAG 1320TTTGTAGACA TGAAGGTCCA TGTACCCTGT GAAACCCTCG AATACATTGA AGCCAACTAT 1380 GGTAAGACCT GGAAGATTCC TGTAAAGACG TGGGACTGGA AGCGCTCTCC TCCCAATGTG 1440 CAACCCAATG GAATCTGGCC TATTTCTGAG TGGGATGAGG TTATCCAGTT ATATTGAGAT 1500 AGTAGGTTGA AATGGGAGAA TTTCTCTTTT GGAAAAAAAG GTAGATAACT GTTTAAAAAA 1560 TACATGTCTA TTTGTCAAAC ATAAGTGGGA ACCAAAGAAA AAATGTGACA AGTTTGAAGA 1620 CACAGAAAGA GTCATCTGAT GTAATTCTCT CACTTAGTAC TGAGGAATTT TCATGTGCCA 1680 CATACAATGC TAGGTTACAG TGGAGAAGCC AAGATGAATG AGACAAATAC CTACTTCTTT 1740 TATTCCTCCT TTTGGTAAAC AACTCAATTT TCCTTTGAGG GAACCCTCCC CCACCCTTTG 1800 AAGAGTTCAA GTTCTGTACA GGTTTTTAAA ACGTGAAGTA ATGTTTGAAC TGGAAGATGA 1860 GCTCACCAGG CAAAGCTAAG GAAGGATATA CTAGTTGAAA AGAATAACCC ACTCCTCTTC 1920 TGGGCATTTA AGCTGGTATG TTAGTGCTAC TTTTAAGATT GTGGAGTCTG AGTTAATATT 1980 CAAGTGATCA GACTTTGAGT GACATCAAGA AAAGATGATA TCAGGTTCAT TTTTCAACTA 2040 ATCTTATGTG GAATTGAATT AGAGAACAAG GCATTATTCT TTTAGGGAAG GTGAGAGCTT 2100 ATTTGTATCA GAGCTTATTA CTTGTCAGGA TAAGTAAATT TCTGTACATG TACTGTTTTC 2160 ATATGT GAAG TGAGAAGAAA CTTTATGCTT GTTTAATGCT TAAATTTCCA TCCATTGTGA 2220 GAATATTTTC ACTGACCTCT GATGGCACTT GTTGACAAAT CATTCAAGTG AGACCATGTT 2280 ACTAGACATG ATCTTGAAAG AGGCCATGAT TTCACAAAAC TCATTTTTAT TTTATTCTCA 2340 GACAGTCTGT TAGGTAAAAA TATGAGAAAT TCATGTACAT TTTATATTTT CTGAATTTAT 2400 AATCTGTGCA CTCCCAATTT TAATGACACT AAAATATTAA TAGAATTTTT TAAATATACT 2460 CTAATTTTTA AAACATACTC TTTATTATCT TCATTTATCA ATTACAGCTT CGTATCTCTA 2520 ATTTATGGTC TATATACCAA TTTAAATGGC ATGTAAACCT GCCTGTTTCT TCTCCTCTTC 2580 TAATATATCA GATCTCCAAA ATGGAAGCTA AATGGTGGAC TTGACAACTA TTCACCCTAC 2640 CTCAGATCAT AGAGTTTGTA AAGTTTGTGT CCCTCCCCAG TTTTTCAGTC TGTTGAATGT 2700 CGATGGGGCA AGAACTCAGA CTTCTACTTT ACCAAGTACC ACACACTCTG GAATGCTTTA 2760 GTTTCCTTTT CCCCTCAAGA TGTCTGAGTC AGCTAGGATG CTGTTTACCC CATCTCTCTC 2820 TTATATCACT TGAATGATAT ATTGTAAGTG AGAGGTAAAG GAAAATGTAG GCACAATAAG 2880 CACTGCTATT TTTCTCTTTG TCTAGGAAAG GAAGCTGAAT CTTATATCTT ATCTATGCTA 2940 TTTAGGACTA CTTTCTGGAG CTTGGCAGAT TTTCCTCTGA CACCAGAGAA CAATAGTAGT 3000 CTCAAGAATG G AAACCTGAA TGTCTGAGGG AATGGGCTGG TAGACTTTTT CGAAAACAAA 3060 TTAGAGAAAG TAACTTACAA CCACCCATTC CGGATTTGTA AAGCAACATG AAAACCTTTG 3120 ATAAATGATA ACCAACAGTC TTCTGTCCTA ATTTGCATTC TCAATGCAGA ATTATTGGGT 3180 CTTTCATAAA CAACATGAGT GGTTTCTGGA GACATTTAAG ATTGTCAGCA AACTTGTCCC 3240 AAAATGGCAC ATCATCATAA TCCATTTTCT CTTTGCTAGA AAATCAGCCC ATAGAGTGCA 3300 TTCCCAAATT CTAAATAGCT GACCCTAAAT TCATTTTTCA TGCTTAAAAA TAATAGAACA 3360 AATAATAATA CTATTTTTGG GCAAAATCCA CCCTACTTGA TGAGGACCAT TTGCTTGTGC 3420 TCAGTATTTA GAAATGCATA CACTCCACAT TTCTCCCCAT TTCCAATTGG GACTCCCATT 3480 CTTTGCCAGG AGATCTTACC CATCCCTTTT CAGATTAATC TTTTATCCTT TCCTGAAGTA 3540 CAAAGTCTTA AGAGTGTCTG AAATCCAAGA CATCTTGGCC CAATTGACAC AGGTTCTATA 3600 TTCTTCCCTA CATAGACCAC TGGCTGCTGA AATACTTTTC TTGTCTTCTG GTTTCACAGG 3660 CTTCAGCTCA TGGGTTCCAC CCTAACTTGG GGAAACAAAA GCCTTCTCTA GAATCTGAAG 3720 GCCAGGCTTA CCTCTGATTC TGATTCATCC ATACTTCATC TTATGTATTT TAACAGTTGG 3780 GTTCTGTGGA GTGTCCAGAG ACCTTGGGTT AGGTATATCC ATCTTCAGTA CCTCATTGGA 3840 TCACTTTTCT TTCATCA CTT GAGTATTCTA GCAGTCATTC TCCTAATCTG ACCACTTTCA 3900 GGTTTAGTTT ACCTGGCTTA CCAGGGGAAG TTGACAACTT GTTGGTAGTT AGGCACCCAT 3960 GAATGTCTCC AGAGACATCC TGAGAGGCAA GATTCCTCTT AATTGATAAC CAGGACAACC 4020 AGGTAGTCAC CCAGTCCTCT CTAAGCAGGG AGCACTTGTC CTTCTCTCCT CTGCTGCAGC 4080 TACTGATATC TGGCCCCTGG AATAAAACCA TAGTTCCTAA AATTGAGCAT CCCTAAGAGT 4140 AGCTGCTTGG TGGGACAGCT GATTTCTTTG ATTCCCTAGC TGCAAAACCT GAAATTGACC 4200 ACTAGGTGAC AGAATGTTTG CCAGTATCCC CACAAAACAA GTTAAAACTT AAGTGAAAAT 4260 CATTCTGCTT TTGAATCTTA AAAGCTAGAA AAACCATAAT GTAATATTCT TTTTAAACCC 4320 TCTTATTTTA TAACTAAATA AATAATCACA GAAAAGAATG ATCTGAACAA GAACAGCTGA 4380 GGCTAAAACC CAAGACTGCC GTGACTCCTA GTCCAATGTC TGTTTCGCAT CACAACACAG 4440 TATCTTTAAC AGTGCTTGAG ATGCTTAACA GAGAAGGCAT CCAGTGCTTC ATTGAGGCTA 4500 AGTCTCAGGG TGTTTCTGCC GCTTAGTATC TTTTTGTTCA GATTGAGAAC CTAAGGCGAC 4560 AGAGAGGTCA AGGGGAAGTA TTTTCTAGTT CAGAATTCAG AATTCTTAGG CAACCTTCAT 4620 ACCTTTATCT GGAAAATGCC TTCTCTCCAG ACAATAAGAC AGTTCACATC TGGAGACATC 4680 AGCCTTTGGT ACCTAATTCT CT AAGCTCAA GATGCATCCC ATTGCCACTT TACCATTCTA 4740 TTTCCCAAGG GAACCATCAG CACCAACCTG CTAAATGCCT GTATTTGAAG TCTCTCCCCT 4800 TCCTGAAGTG ACCTTTTATG AGGCTCTTTC CTTCCTAAGG GACCTTTCCC TGGTAGTAGT 4860 GGTCTCATTC ACAAAAAAGA ATAATAGATG TAACTGGAGT CACTTTGCAG TTTTCAAGAT 4920 TTTTTTTTTA TCTGCTTGGC TGGAAAGGAG ACTAGAGTAT CTAGTTTTTA GTATTTAAAC 4980 TACTTTTAAT TATTTATATT GATACTCTCT GATAAATGCA GGTAGTTAAG AATAGATTGT 5040 ACTCATTCCT TTTTGAGTTT GTGACCTCAA ACCATTGTTT TTATTCTTAC ACGACTACAG 5100 TACTTCAAAT GGCACATAGA TAGGCAGGAT ATTACATAGG TAAGCAGGAA TTTATACATT 5160 GGGTACCAGA GCTAAATCTG GAAAACACAT TTGGAATGAG CTTCCACACT TCAGTCTAAA 5220 ATCTCACCCA AACTTATGGC TACATATTTT TCTAAGTTTC CCCCCTAGTT TCACTTTCTG 5280 GAAAGTGAAC AGTTTTTTAA ATCCTAAATG TTATAAATCC TTAGGAGAAA TATTCCCTCA 5340 AAACCTAGTC AAGAAAGTGC CACATTCCCA CCTTCTAACA GGTAATTATT AGTTGTAATA 5400 GCTTTAATGC ATGGAAAAAC TTCAGTTACT GACCAGTCAA GAGAATTTCT CAAGAAAAAT 5460 CCCAGCAACG TTCCCTCTTT CCTCCTTGTC TTCCACTATC ATTAAGACCT GGGCTAGATC 5520 ACCTCTAACA TCTCACTCAG GGAAGGCA AT GTGTTGCCAT AAAAAGACCA CAAGCTTTTG 5580 AGTTAGGCCT GAATTTGAAT TTCAGCTTAA TCATGTGGGA TTTATGGTGG GGGCCCTTTC 5640 AGTCTCAATT TCCACATTAG TGAACTGGGG AAATGATACA TACTTCTAAG GGTTTTTAGG 5700 GGGATTAAAT GAAGTATACA GTTCTTAGCC TCAGGCCTAG GATTAAGTAA GTACTCAAGA 5760 AATGTGCAAT TTTCTAGTTC CATGTTTCTT TTCTAATCTT CAAATGGAAT TATAGAAACC 5820 ATGGGTCAGA ACATATTCCT TTACTCAAAA GATTGCATGA CTGAATTTGC TTAAGAAAAA 5880 AAAAATTGTA TCAAGTCATT AATACAATTA TACATTAATT ATATTACATT AATACAATAT 5940 ATGGTTTGTG AATTCAGAGA CATTACCAGT TTGCCTCCTT CTCTCAATAG AACTTGTATT 6000 TTCATTTTCT TGGTTAAGCA GTTGTCTCCT AATATTATCC CATATGCTAC CTAGTTTGCT 6060 GGTCCCAAGC AGTTTACTGT ACTTCACTAG ATTTGGTACC TGCTCTCCCC TGGACTTCTT 6120 TTTCAATATT CTAGCCTTTC CTAGATGTAA ATCTTTACCT CCTTGTTAGT GAAATTAGAT 6180 ATAAGCCATG ATTTGGAGAG GGAAGAAATC TGGAATACTT AATTTCATTT AATTATCTAT 6240 GCTGATGAAT GCCTGTATCA TTGTTAATAA AGGAGAATTG AAAATACTCA TTTCTACTTT 6300 CTGCCCTCAA ATTTCTGTTT CTATCTCAAC TAGGCAAGAA TCAGCAGGGT GCATGATGCC 6360 ATTTTAAGCT GCTTCACATC AGACTGAAAT CCT AATTACA GTTCATAAGT GAAACAGACT 6420 AATTCAATGG CAATACCTTT TGTATAGGTC CTGTGCTTAA AGGAGGCAAG TATAAATTTT 6480 CTAATAAGAA ATCCCTGCTT CTTTTGGGTG CAGTGGCTCA CACCTGTAAT ACCAGCAGTT 6540 TGAGAAGCTA AGGCAGGTGG ATCACTTGAG GCCAGGAGTT CGAGACCAGC CTGGCCAACA 6600 TAGTGAAACC CCGTCTCTTC TAAAAATACA AAAAAATTAG CTGGGCATGG TGGTGCACGC 6660 CTATAGTCCC AGCTACACAG GAGGCTGAGG CAGGAGAATC ACTTGAACCC GGGAAGCAGA 6720 GTCTGCAGTG AGCCAAGATC GCGGCATTGC ACTTCAGCCT GGGCGATAGA GCAAGACTCT 6780 GTCTCAGAAA AAAAAAAAAG GAAAGAAAGA AATCTCTGCT TCCGTACTAT CAAAACTTCT 6840 ACCCTAGAAT ACCCCCTGGC ACCTTCTAAC CCAACCTAAT ACAGAGATTT TGTAGGGACC 6900 CATTAACAAG CTCCTATATA ATTATAAGCA GCTCTCACAG TAGGGTTGAA AGAGAATATA 6960 AGGAAAATTA GAAGTACTGT ATTTTTGCTA TTGGAAATGA AATATTGTTT CATGCACCTT 7020 TGAAAAAAAT AACAGGATTT TACAGCCTTC TGATGATTCA TTCAAAGCAT GGGGAATAGT 7080 TCATATGTTT GTTAAATGAA AATCTTATGT AGCAATTTGT GTGTCCCTCC CACTTCATAA 7140 CTACAAAAAC ATATATATGA ATTTCTAAAC AAAGTGATAT TTTAAAGATG AATTGATTCA 7200 ATGTGTACTT ACCAGTTTAC TGTGTGGTTT TATCTTCAG G TACAGATAGT TTGTGGTACT 7260 ACTTGAAAAT ACCTTTAATA TTATTTTCAT CAGAATTTGT AATATATAAT CCAGTTTGGG 7320 ATGATGAATA AAATTTTTCT AATCTCTTGA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA 7380 AAAAAAAAA 7389 Sequence length: 7 types : CDNA to mRNA origin Organism name: Human Sequence characteristics Characteristic symbol: CDS Location: 112..1494 Characteristic determination method: E Characteristic symbol: mat peptide Location: 112..1494 Characteristic determined Method: E sequence GCTGCAGCCT GCTGTTGAGT GAGAAAACAA AATTATCTTC CTTTCCAAAT CCAAAAAGAT 60 GAAAACGACT GAGATACTTT CAAAAGACAA CCAAGTGAGC AGCACAGACT A ATG AGT 117 Met Ser 1 AGA ATC AAT AAG AAC GTG GTT TTG ACC TTT ACA Val TCG Leu Thr Leu Thr Ser Ser 5 10 15 GCA TTT CTG CTG TTT CAG TTG TAC TAC TAC AAG CAC TAT TTA TCA ACA 213 Ala Phe Leu Leu Phe Gln Leu Tyr Tyr Tyr Lys His Tyr Leu Ser Thr 20 25 30 AAG AAT GGA GCT GGT TTG TCA AAA TCC AAA GGA AGC CGA ATT GGA TTT 261 Lys Asn Gly Ala Gly Leu Ser Lys Ser Lys Gly Ser Arg Ile Gly Phe 35 40 45 50 GAT AGC ACA CAG TGG CGT GCA GTT AAA AAA TTT ATT ATG TTA ACA TCC 309 Asp Ser Thr Gln Trp Arg Ala Val Lys Lys Phe Ile Met Leu Thr Ser 55 60 65 AAC CAA AAT GTA CCA GTG TTT CTT ATT GAT CCT TTG ATA CTG GAA TTG 357 Asn Gln Asn Val Pro Val Phe Leu Ile Asp Pro Leu Ile Leu Glu Leu 70 75 80 ATT AAT AAG AAC TTT GAA CAA GTC AAA AAT ACT TCT CAT GGC TCT ACT 405 Ile Asn Lys Asn Phe Glu Gln Val Lys Asn Thr Ser His Gly Ser Thr 85 90 95 TCA CAA TGC AAG TTT TTC TGT GTT CCA AGA GAC TTT ACT GCA TTT GCA 453 Ser Gln Cys Lys Phe Phe Cys Val Pro Arg Asp Phe Thr Ala Phe Ala 100 105 110 CTG CAG TAT CAC CTA TGG AAG AAT GAG GAA GGC TGG TTT CGG ATA GCT 501 Leu Gln Tyr His Leu Trp Lys Asn Glu Glu Gly Trp Phe Arg Ile Ala 115 120 125 130 GAG AAT ATG GGA TTT CAG TGC CTA AAG ATT GAG AGT AAA GAT CCC CGG 549 Glu Asn Met Gly Phe Gln Cys Leu Lys Ile Glu Ser Lys Asp Pro Arg 135 140 145 CTA GAC GGG ATA GAC TCA CTC TCT GGA ACT GAA ATC CCC CTG CAC TAT 597 Leu Asp Gly Ile Asp Ser Leu Ser Gly Thr Glu Ile Pro Leu His Tyr 150 155 160 ATC TGC AAA CTG GCC ACT CAT GCG ATC CAC TTG GTA GTC TTT CAT GAG 645 Ile Cys Lys Leu Ala Thr His Ala Ile His Leu Val Val Phe His Glu 165 170 175 AGG AGT GGC AAC TAC CTC TGG CAC GGC CAC TTG AGA CTT AAA GAA CAC 693 Arg Ser Gly Asn Tyr Leu Trp His Gly His Leu Arg Leu Lys Glu His 180 185 190 ATT GAC AGG AAA TTT GTT CCC TTC CAA AAG TTA CAG TTT GGT CGT TAT 741 Ile Asp Arg Lys Phe Val Pro Phe Gln Lys Leu Gln Phe Gly Arg Tyr 195 200 205 210 CCA GGA GCT TTT GAC AGG CCA GAG TTA CAG CAA GTT ACT GTT GAT GGA 789 Pro Gly Ala Phe Asp Arg Pro Glu Leu Gln Gln Val Thr Val Asp Gly 215 220 225 CTG GAA GTT CTC ATT CCA AAG GAT CCA ATG CAC TTT GTA GAA GAA GTA 837 Leu Glu Val Leu Ile Pro Lys Asp Pro Met His Phe Val Glu Glu Val 230 235 240 CCA CAC TCT AGG TTT ATT GAG TGT AGG TAT AAA GAA GCT CGA GCA TTC 885 Pro His Ser Arg Phe Ile Glu Cys Arg Tyr Lys Glu Ala Arg Ala Phe 245 250 255TTT CAG CAG TAC CTT GAT GAT AAC ACT GTG GAA GCT GTG GCC TTT CGG 933 Phe Gln Gln Tyr Leu Asp Asp Asn Thr Val Glu Ala Val Ala Phe Arg 260 265 270 AAG AGT GCA AAG GAA TTA CTG CAA CTA GCA GCG AAA ACA TTA AAC AAA 981 Lys Ser Ala Lys Glu Leu Leu Gln Leu Ala Ala Lys Thr Leu Asn Lys 275 280 285 290 TTG GGA GTA CCA TTC TGG CTG AGC AGT GGA ACT TGT CTA GGA TGG TAT 1029 Leu Gly Val Pro Phe Trp Leu Ser Ser Gly Thr Cys Leu Gly Trp Tyr 295 300 305 CGA CAA TGC AAC ATT ATT CCT TAT AGC AAA GAT GTT GAC CTA GGA ATT 1077 Arg Gln Cys Asn Ile Ile Pro Tyr Ser Lys Asp Val Asp Leu Gly Ile 310 315 320 TTT ATA CAA GAT TAC AAA TCT GAT ATT ATT TTA GCA TTT CAG GAT GCA 1125 Phe Ile Gln Asp Tyr Lys Ser Asp Ile Ile Leu Ala Phe Gln Asp Ala 325 330 335 GGA CTT CCG CTC AAA CAC AAA TTT GGG AAG GTA GAA GAC AGC TTG GAA 1173 Gly Leu Pro Leu Lys His Lys Phe Gly Lys Val Glu Asp Ser Leu Glu 340 345 350 CTA TCC TTC CAG GGA AAA GAT GAT GTA AAA CTT GAT GTT TTT TTC TTC 1221 Leu Ser Phe Gln Gly Lys Asp Asp Val Lys Leu Asp Val Phe Phe Phe 355 360 365 370 TAT GAA GAA ACT GAT CAC ATG TGG AAT GGA GGC ACT CAG GCC AAA ACA 1269 Tyr Glu Glu Thr Asp His Met Trp Asn Gly Gly Thr Gln Ala Lys Thr 375 380 385 GGA AAA AAA TTC AAA TAC CTG TTT CCG AAG TTT ACA CTG TGC TGG ACT 1317 Gly Lys Lys Phe Lys Tyr Leu Phe Pro Lys Phe Thr Leu Cys Trp Thr 390 395 400 GAG TTT GTA GAC ATG AAG GTC CAT GTA CCC TGT GAA ACC CTC GAA TAC 1365 Glu Phe Val Asp Met Lys Val His Val Pro Cys Glu Thr Leu Glu Tyr 405 410 415 ATT GAA GCC AAC TAT GGT AAG ACC TGG AAG ATT CCT GTA AAG ACG TGG 1413 Ile Glu Ala Asn Tyr Gly Lys Thr Trp Lys Ile Pro Val Lys Thr Trp 420 425 430 GAC TGG AAG CGC TCT CCT CCC AAT GTG CAA CCC AAT GGA ATC TGG CCT 1461 Asp Trp Lys Arg Ser Pro Pro Asn Val Gln Pro Asn Gly Ile Trp Pro 435 440 445 445 450 ATT TCT GAG TGG GAT GAG GTT ATC CAG TTA TAT TGAGATAGTA GGTTGATG Ile Ser Glu Trp Asp Glu Val Ile Gln Leu Tyr 455 460 GGAGAATTTC TCTTTTGGAA AAAAAGGTAG ATAACTGTTT AAAAAATACA TGTCTATTTG 1574 TCAAACATAA GTGGGAACCA AAGAAAAAAT GTGACAAGTT TGAAGACACA GAAAGAGTCA 1634 TCTGATGTAA TTCTCTCACT TAGTACTGAG GAATTTTCAT GTGCCACATA CAATGCTAGG 1694 TTACAGTGGA GAAGCCAAGA TGAATGAGAC AAATACCTAC TTCTTTTATT CCTCCTTTTG 1754 GTAAACAACT CAATTTTCCT TTGAGGGAAC CCTCCCCCAC CCTTTGAAGA GTTCAAGTTC 1814 TGTACAGGTT TTTAAAACGT GAAGTAATGT TTGAACTGGA AGATGAGCTC ACCAGGCAAA 1874 GCTAAGGAAG GATATACTAG TTGAAAAGAA TAACCCACTC CTCTTCTGGG CATTTAAGCT 1934 GGTATGTTAG TGCTACTTTT AAGATTGTGG AGTCTGAGTT AATATTCAAG TGATCAGACT 1994 TTGAGTGACA TCAAGAAAAG ATGATATCAG GTTCATTTTT CAACTAATCT TATGTGGAAT 2054 TGAATTAGAG AACAAGGCAT TATTCTTTTA GGGAAGGTGA GAGCTTATTT GTATCAGAGC 2114 TTATTACTTG TCAGGATAAG TAAATTTCTG TACATGTACT GTTTTCATAT GTGAAGTGAG 2174 AAGAAACTTT ATGCTTGTTT AATGCTTAAA TTTCCATCCA TTGTGAGAAT ATTTTCACTG 2234 ACCTCTGATG GCACTTGTTG ACAAATCATT CAAGTGAGAC CATGTTACTA GACATGATCT 2294 TGAAAGAGGC CATGATTTCA CAAAACTCAT TTTTATTTTA TTCTCAGACA GTCTGTTAGG 2354 TAAAAATATG AGAAATTCAT GTACATTTTA TATTTTCTGA ATTTATAATC TGTGCACTCC 2414 CAATTTTAAT GACACTAAAA TATTAATAGA ATTTTTTAAA TATACTCTAA TTTTTA AAAC 2474 ATACTCTTTA TTATCTTCAT TTATCAATTA CAGCTTCGTA TCTCTAATTT ATGGTCTATA 2534 TACCAATTTA AATGGCATGT AAACCTGCCT GTTTCTTCTC CTCTTCTAAT ATATCAGATC 2594 TCCAAAATGG AAGCTAAATG GTGGACTTGA CAACTATTCA CCCTACCTCA GATCATAGAG 2654 TTTGTAAAGT TTGTGTCCCT CCCCAGTTTT TCAGTCTGTT GAATGTCGAT GGGGCAAGAA 2714 CTCAGACTTC TACTTTACCA AGTACCACAC ACTCTGGAAT GCTTTAGTTT CCTTTTCCCC 2774 TCAAGATGTC TGAGTCAGCT AGGATGCTGT TTACCCCATC TCTCTCTTAT ATCACTTGAA 2834 TGATATATTG TAAGTGAGAG GTAAAGGAAA ATGTAGGCAC AATAAGCACT GCTATTTTTC 2894 TCTTTGTCTA GGAAAGGAAG CTGAATCTTA TATCTTATCT ATGCTATTTA GGACTACTTT 2954 CTGGAGCTTG GCAGATTTTC CTCTGACACC AGAGAACAAT AGTAGTCTCA AGAATGGAAA 3014 CCTGAATGTC TGAGGGAATG GGCTGGTAGA CTTTTTCGAA AACAAATTAG AGAAAGTAAC 3074 TTACAACCAC CCATTCCGGA TTTGTAAAGC AACATGAAAA CCTTTGATAA ATGATAACCA 3134 ACAGTCTTCT GTCCTAATTT GCATTCTCAA TGCAGAATTA TTGGGTCTTT CATAAACAAC 3194 ATGAGTGGTT TCTGGAGACA TTTAAGATTG TCAGCAAACT TGTCCCAAAA TGGCACATCA 3254 TCATAATCCA TTTTCTCTTT GCTAGAAAAT CAGCCCATAG AGTGCATTCC CAAATTCTAA 3 314 ATAGCTGACC CTAAATTCAT TTTTCATGCT TAAAAATAAT AGAACAAATA ATAATACTAT 3374 TTTTGGGCAA AATCCACCCT ACTTGATGAG GACCATTTGC TTGTGCTCAG TATTTAGAAA 3434 TGCATACACT CCACATTTCT CCCCATTTCC AATTGGGACT CCCATTCTTT GCCAGGAGAT 3494 CTTACCCATC CCTTTTCAGA TTAATCTTTT ATCCTTTCCT GAAGTACAAA GTCTTAAGAG 3554 TGTCTGAAAT CCAAGACATC TTGGCCCAAT TGACACAGGT TCTATATTCT TCCCTACATA 3614 GACCACTGGC TGCTGAAATA CTTTTCTTGT CTTCTGGTTT CACAGGCTTC AGCTCATGGG 3674 TTCCACCCTA ACTTGGGGAA ACAAAAGCCT TCTCTAGAAT CTGAAGGCCA GGCTTACCTC 3734 TGATTCTGAT TCATCCATAC TTCATCTTAT GTATTTTAAC AGTTGGGTTC TGTGGAGTGT 3794 CCAGAGACCT TGGGTTAGGT ATATCCATCT TCAGTACCTC ATTGGATCAC TTTTCTTTCA 3854 TCACTTGAGT ATTCTAGCAG TCATTCTCCT AATCTGACCA CTTTCAGGTT TAGTTTACCT 3914 GGCTTACCAG GGGAAGTTGA CAACTTGTTG GTAGTTAGGC ACCCATGAAT GTCTCCAGAG 3974 ACATCCTGAG AGGCAAGATT CCTCTTAATT GATAACCAGG ACAACCAGGT AGTCACCCAG 4034 TCCTCTCTAA GCAGGGAGCA CTTGTCCTTC TCTCCTCTGC TGCAGCTACT GATATCTGGC 4094 CCCTGGAATA AAACCATAGT TCCTAAAATT GAGCATCCCT AAGAGTAGCT GCTTGGTGGG 4154 AC AGCTGATT TCTTTGATTC CCTAGCTGCA AAACCTGAAA TTGACCACTA GGTGACAGAA 4214 TGTTTGCCAG TATCCCCACA AAACAAGTTA AAACTTAAGT GAAAATCATT CTGCTTTTGA 4274 ATCTTAAAAG CTAGAAAAAC CATAATGTAA TATTCTTTTT AAACCCTCTT ATTTTATAAC 4334 TAAATAAATA ATCACAGAAA AGAATGATCT GAACAAGAAC AGCTGAGGCT AAAACCCAAG 4394 ACTGCCGTGA CTCCTAGTCC AATGTCTGTT TCGCATCACA ACACAGTATC TTTAACAGTG 4454 CTTGAGATGC TTAACAGAGA AGGCATCCAG TGCTTCATTG AGGCTAAGTC TCAGGGTGTT 4514 TCTGCCGCTT AGTATCTTTT TGTTCAGATT GAGAACCTAA GGCGACAGAG AGGTCAAGGG 4574 GAAGTATTTT CTAGTTCAGA ATTCAGAATT CTTAGGCAAC CTTCATACCT TTATCTGGAA 4634 AATGCCTTCT CTCCAGACAA TAAGACAGTT CACATCTGGA GACATCAGCC TTTGGTACCT 4694 AATTCTCTAA GCTCAAGATG CATCCCATTG CCACTTTACC ATTCTATTTC CCAAGGGAAC 4754 CATCAGCACC AACCTGCTAA ATGCCTGTAT TTGAAGTCTC TCCCCTTCCT GAAGTGACCT 4814 TTTATGAGGC TCTTTCCTTC CTAAGGGACC TTTCCCTGGT AGTAGTGGTC TCATTCACAA 4874 AAAAGAATAA TAGATGTAAC TGGAGTCACT TTGCAGTTTT CAAGATTTTT TTTTTATCTG 4934 CTTGGCTGGA AAGGAGACTA GAGTATCTAG TTTTTAGTAT TTAAACTACT TTTAATTATT 4994 TATATTGA TA CTCTCTGATA AATGCAGGTA GTTAAGAATA GATTGTACTC ATTCCTTTTT 5054 GAGTTTGTGA CCTCAAACCA TTGTTTTTAT TCTTACACGA CTACAGTACT TCAAATGGCA 5114 CATAGATAGG CAGGATATTA CATAGGTAAG CAGGAATTTA TACATTGGGT ACCAGAGCTA 5174 AATCTGGAAA ACACATTTGG AATGAGCTTC CACACTTCAG TCTAAAATCT CACCCAAACT 5234 TATGGCTACA TATTTTTCTA AGTTTCCCCC CTAGTTTCAC TTTCTGGAAA GTGAACAGTT 5294 TTTTAAATCC TAAATGTTAT AAATCCTTAG GAGAAATATT CCCTCAAAAC CTAGTCAAGA 5354 AAGTGCCACA TTCCCACCTT CTAACAGGTA ATTATTAGTT GTAATAGCTT TAATGCATGG 5414 AAAAACTTCA GTTACTGACC AGTCAAGAGA ATTTCTCAAG AAAAATCCCA GCAACGTTCC 5474 CTCTTTCCTC CTTGTCTTCC ACTATCATTA AGACCTGGGC TAGATCACCT CTAACATCTC 5534 ACTCAGGGAA GGCAATGTGT TGCCATAAAA AGACCACAAG CTTTTGAGTT AGGCCTGAAT 5594 TTGAATTTCA GCTTAATCAT GTGGGATTTA TGGTGGGGGC CCTTTCAGTC TCAATTTCCA 5654 CATTAGTGAA CTGGGGAAAT GATACATACT TCTAAGGGTT TTTAGGGGGA TTAAATGAAG 5714 TATACAGTTC TTAGCCTCAG GCCTAGGATT AAGTAAGTAC TCAAGAAATG TGCAATTTTC 5774 TAGTTCCATG TTTCTTTTCT AATCTTCAAA TGGAATTATA GAAACCATGG GTCAGAACAT 5834 ATTCCTTTAC TCA AAAGATT GCATGACTGA ATTTGCTTAA GAAAAAAAAA ATTGTATCAA 5894 GTCATTAATA CAATTATACA TTAATTATAT TACATTAATA CAATATATGG TTTGTGAATT 5954 CAGAGACATT ACCAGTTTGC CTCCTTCTCT CAATAGAACT TGTATTTTCA TTTTCTTGGT 6014 TAAGCAGTTG TCTCCTAATA TTATCCCATA TGCTACCTAG TTTGCTGGTC CCAAGCAGTT 6074 TACTGTACTT CACTAGATTT GGTACCTGCT CTCCCCTGGA CTTCTTTTTC AATATTCTAG 6134 CCTTTCCTAG ATGTAAATCT TTACCTCCTT GTTAGTGAAA TTAGATATAA GCCATGATTT 6194 GGAGAGGGAA GAAATCTGGA ATACTTAATT TCATTTAATT ATCTATGCTG ATGAATGCCT 6254 GTATCATTGT TAATAAAGGA GAATTGAAAA TACTCATTTC TACTTTCTGC CCTCAAATTT 6314 CTGTTTCTAT CTCAACTAGG CAAGAATCAG CAGGGTGCAT GATGCCATTT TAAGCTGCTT 6374 CACATCAGAC TGAAATCCTA ATTACAGTTC ATAAGTGAAA CAGACTAATT CAATGGCAAT 6434 ACCTTTTGTA TAGGTCCTGT GCTTAAAGGA GGCAAGTATA AATTTTCTAA TAAGAAATCC 6494 CTGCTTCTTT TGGGTGCAGT GGCTCACACC TGTAATACCA GCAGTTTGAG AAGCTAAGGC 6554 AGGTGGATCA CTTGAGGCCA GGAGTTCGAG ACCAGCCTGG CCAACATAGT GAAACCCCGT 6614 CTCTTCTAAA AATACAAAAA AATTAGCTGG GCATGGTGGT GCACGCCTAT AGTCCCAGCT 6674 ACACAGGAGG CTGAGGCAG G AGAATCACTT GAACCCGGGA AGCAGAGTCT GCAGTGAGCC 6734 AAGATCGCGG CATTGCACTT CAGCCTGGGC GATAGAGCAA GACTCTGTCT CAGAAAAAAA 6794 AAAAAGGAAA GAAAGAAATC TCTGCTTCCG TACTATCAAA ACTTCTACCC TAGAATACCC 6854 CCTGGCACCT TCTAACCCAA CCTAATACAG AGATTTTGTA GGGACCCATT AACAAGCTCC 6914 TATATAATTA TAAGCAGCTC TCACAGTAGG GTTGAAAGAG AATATAAGGA AAATTAGAAG 6974 TACTGTATTT TTGCTATTGG AAATGAAATA TTGTTTCATG CACCTTTGAA AAAAATAACA 7034 GGATTTTACA GCCTTCTGAT GATTCATTCA AAGCATGGGG AATAGTTCAT ATGTTTGTTA 7094 AATGAAAATC TTATGTAGCA ATTTGTGTGT CCCTCCCACT TCATAACTAC AAAAACATAT 7154 ATATGAATTT CTAAACAAAG TGATATTTTA AAGATGAATT GATTCAATGT GTACTTACCA 7214 GTTTACTGTG TGGTTTTATC TTCAGGTACA GATAGTTTGT GGTACTACTT GAAAATACCT 7274 TTAATATTAT TTTCATCAGA ATTTGTAATA TATAATCCAG TTTGGGATGA TGAATAAAAT 7334 TTTTCTAATC TCTTGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 7389

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an FCMD candidate region on chromosome 9q31. Figure 3 shows the location of DNA microsatellites used for linkage disequilibrium mapping and founder haplotype mapping. The FCMD causative gene is D9S21
It was estimated to be located very close to the 70 centromere side.
The individual cosmids that make up the cosmid contig are indicated by horizontal lines, and the EcoRI site is indicated by a short vertical line. In the figure, N means NotI, which is a common mutation ~
The position of the 3 kb insertion is also shown (3-kb insertion).
The spread of the FCMD cDNA on the genome is shown below the map with arrows indicating the direction of transcription.

FIG. 2 ~

FIG. 3 is a photograph instead of a drawing, showing Southern analysis (electrophoresis image) in each FCMD patient.

FIG. 2 is a photograph instead of a drawing, showing a result (electrophoresis image) of genomic DNA digested with PvuII, PstI or BglII. Lanes 1-3 are 138-192-1
47-183 founder haplotype homozygous patient; lane 4 is founder haplotype heterozygous; lane 5 is normal control.
The entire insert of cosmid clone cE6 was used as a probe. The band observed in the normal control was lost in the FCMD patient (arrow), and it was observed that the band shifted to ８8 kb (PvuII). Similarly,
As for DNA digested with PstI or BglII, one of the normal bands disappeared, and a new band larger than the disappeared band appeared.

FIG. 3 is a photograph instead of a drawing, showing the result (electrophoresis image) of Southern hybridization of a PvuII digest using a partial probe E6f3 in the cE6 insert. N is normal person control, F is FCM
D shows a patient.

FIG. 4

FIG. 5 shows the nucleotide sequence of FCMD cDNA and the amino acid sequence deduced from the sequence.

FIG. 4. Nucleotide sequence of FCMD cDNA (1-2
FIG. 10 shows the amino acid sequence deduced from the sequence (position 100) and its sequence. The putative hydrophobic signal sequence region and transmembrane main region are underlined (solid line) and N
-Binding glycosylation sites are dashed, possible N-myristolation sites are "*", and serine / threonine and tyrosine residues that can potentially be phosphorylated by protein kinase C and tyrosine kinase are represented by "% ”And“ ＾ ”. In addition, the nucleotide (250%) related to the mutation identified by the present invention.
298-299) are shown in oblique lines.

FIG. 5 is a diagram showing the nucleotide sequence of the FCMD cDNA (positions 2101 to 7381), following FIG. 4; The poly (A) addition site used is indicated by “Δ”, and the founder insertion mutation site is indicated by an arrow.

FIG. 6

FIG. 7 is a photograph instead of a drawing, showing Northern analysis (electrophoresis image) of the FCMD-causing gene.

FIG. 6 is a photograph instead of a drawing showing tissue-specific expression of FCMD mRNA in human tissues. 8 displayed
(A) + RNA (2 μg) derived from human tissues of different species
RNA blot (Clontech) containing FCMD cD
The results of hybridization with NA probe (clone II-5, upper row) and β-actin cDNA probe (lower row) are shown.

FIG. 7: R containing poly (A) + RNA (2 μg) derived from lymphoblasts of FCMD patients and healthy controls
It is a photograph replacing a drawing which shows the result of having hybridized with the same probe as above using NA blot. FCM having insertion sequence homozygously or heterozygously
In D patients, the signal was almost zero or abnormally low.

FIG. 8 is a drawing instead of a photograph, showing Mendelian inheritance of an inserted allele in a two-generation FCMD kindred showing the 138-192-147-183 founder haplotype (electrophoresis image).

FIG. 9

FIG. 11 shows in-family segregation of point mutations in FCMD patients.

FIG. 9 is a photograph instead of a drawing showing Southern analysis (electrophoresis image) showing in-family segregation of point mutations in FCMD patients (HM, TI). Southern analysis was performed for patients HM and TI
Showed heterozygosity for the founder-inserted allele inherited from the father and mother, respectively.
Patient TI is the daughter of an American father and a Japanese mother.

FIG. 10 is a diagram showing the results of fluorescence sequence analysis for in-family isolation of point mutations in FCMD patients. By such an analysis, the patient's sequence could be replaced (C → T250) and deleted (de
l: 298-299).
All of these mutations show incomplete translation termination (R
47X and frame shift). The sequence of the sense strand is shown.

FIG. 11 shows the HM pedigree and the other five pedigrees with the same haplotype (YS, RM, AG, A) using the endonuclease AluI, which cuts DNA at the AGCT sequence site.
T, and AY) are photographs in place of drawings showing the results (electrophoretic images) of confirming mutations. The C → T250 mutant allele can be distinguished by AluI digestion since it has the AGCT sequence, whereas the normal allele is AGCC. In this case, the size of the normal PCR product is 142 bp, and the A of the mutant allele
The luI digest splits into 113 bp, 25 bp and 4 bp fragments (small fragments could not be detected due to the presence of excess primer). In HM families, the mutated allele (without the insertion mutation) was from the mother and this mutation was inherited along with the microsatellite haplotype. On the other hand, SSCP analysis showed that 2
The bp deletion was shown to be from a Western father (right, arrow).

Claims (8)

[Claims] 1. A protein represented by the following (a) or (b): (a) a Fukuyama-type congenital muscular dystrophy (FCMD) causative protein having the amino acid sequence represented by SEQ ID NO: 1;
(B) In the amino acid sequence represented by SEQ ID NO: 1,
Alternatively, a protein comprising an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added, and having a biological function equivalent to that of the FCMD-causing protein. 2. A DNA encoding the protein according to claim 1.
A. 3. The DNA according to claim 2, which has the nucleotide sequence shown in SEQ ID NO: 2. 4. The DNA according to claim 2, which has the nucleotide sequence shown in SEQ ID NO: 3. 5. The nucleotide sequence of SEQ ID NO: 3,
A mutant FCMD-causing DNA characterized by having a mutation that causes dysfunction of the encoded FCMD-causing protein. 6. A method for detecting a gene abnormality for FCMD diagnosis, comprising detecting the presence of the mutant FCMD-causing DNA according to claim 5 in a gene of a subject. 7. The nucleotide sequence of SEQ ID NO: 3,
(i) substitution of cytosine at position 250 with thymine;
detecting at least one mutation of (ii) a deletion at base positions 298-299 and (iii) insertion of one or more nucleotides between base positions 5889 and 5890. 7. The detection method according to 6. 8. The method for detecting a genetic abnormality according to claim 7, comprising a step of preparing a DNA fragment containing the mutation position and a step of analyzing the base sequence of the DNA fragment.