JP2005525079A - ALS2 gene and amyotrophic lateral sclerosis type 2 - Google Patents

Abstract

A human gene associated with amyotrophic lateral sclerosis type 2 (ALS2) is provided. This gene is located in the chromosome 2 q33 region. Also provided are variants of this gene, as well as isolated nucleic acids derived from the gene and peptides encoded thereby. Furthermore, a method for diagnosing ALS2 is provided.

Description

  This application is from U.S. Patent No. 60 / 267,723 filed on February 12, 2001, Japanese Patent No. 2001-116973 filed on Apr. 16, 2001, and U.S. Patent No. 60 / 318,352 filed on Sep. 12, 2001. Priority is claimed and these applications are incorporated herein by reference.

  This invention relates to the genetic cause of amyotrophic lateral sclerosis type 2 ("ALS2").

Amyotrophic lateral sclerosis ("ALS") is a progressive neurodegenerative disease that selectively degenerates distal and proximal motor neurons ¹ . The cause of the disease is unclear and most of the disease develops in the middle and later years. The incidence of the disease is approximately 2-6 cases per 100,000 people, and the onset is accompanied by muscle weakness and wrist muscle atrophy as a secondary neuron disorder, resulting in limb muscle atrophy, tongue atrophy, grammatical disorder Medullary palsy syndromes such as difficulty swallowing and breathing. Treatment has not been established and most patients die within 5 years of onset.

Juvenile amyotrophic lateral sclerosis type 2 ("ALS2"; OMIM215100 ² ) is a somatic recessive genetic disease. Incidence is rare, but limb, face and throat muscle spasms develop gradually in patients in their teens or 20s and become chronic due to medullary paralysis as described above.

Amyotrophic lateral sclerosis type 2 is mapped to the interval of 1.7 cM sandwiched on human chromosome 2q33 D2S116 and D2S2237 ^3,4. Within this interval, 391 exons from 43 non-overlapping transcripts and changes in their flanking regions are recorded ^5,6 .

  ALS is a very serious disease and it is necessary to develop means for early detection / diagnosis and treatment.

Summary of invention

  We have now identified a gene associated with juvenile amyotrophic lateral sclerosis type 2 and named it the ALS2 or ALS2CR6 gene. This gene is expressed in various human tissues including brain nerves and spinal cord, and encodes a protein having homology to RanGED and RhoGEF.

  The invention now provides mammalian ALS2 genes and variants thereof, and peptides (including proteins) encoded by such genes. Also included are fragments and nucleic acids derived from these genes, corresponding peptides and oligonucleotides suitable for use during amplification of primers and / or probes. Antibodies against the peptides of this invention are also provided.

  The invention also provides a method for diagnosing ALS2, which may include identification of a degenerated ALS2 gene or protein in a patient at risk. A patient can be examined to characterize one or more mutations in the gene or protein produced. Such mutations are composed of the A261del mutation or the AG1548del mutation described in this paper.

  The invention also provides a nucleic acid corresponding to a region of the ALS2 gene, but this nucleic acid is generally at least about 6, at least about 10, at least about 15, at least about 20, or at least about the ALS2 sequence described herein. Hybridization is performed on 25 contiguous nucleotides, or on the complement of such a sequence, or on naturally occurring variants or allelic variants thereof. Probes or primers can be selected (such as by amplification or hybridization) to make allelic variants including the A261del or AG1548del variants described herein distinguishable. Such probes or primers can further include a detectable label. The invention also provides a kit for identifying an ALS2 gene that includes a gene that constitutes an allele associated with an ALS2 disease state, which can be comprised of a probe or primer as described herein. The kit may further include guidance for using the probe or primer to identify alleles as described herein.

  The invention also provides vectors comprising the nucleic acids of the invention, including vectors adapted for expressing such nucleic acids in target cells or microorganisms. Such vectors may be composed of transcriptional regulatory elements suitable for the purpose of directing transcription of the nucleic acid in the target cell or microorganism. The nucleic acids and peptides of this invention can be expressed in nucleated cells including microorganisms and mammalian cells. The nucleic acid of the invention can be expressed in the reverse direction for the purpose of altering such vectors and generating antisense transcripts.

  The present invention also provides a non-human mammal comprising a gene in which the ALS2 gene is mutated due to deletion or the like. Such mammals are mice, for example, and methods for altering the mouse genome, such as producing ALS2 knockout mice, are described in this paper and are known in the art.

  In addition, the present invention provides a method of using a nucleic acid and a peptide as disclosed in this paper for preparing a drug intended for ALS2 treatment or used for ALS2 therapy.

  The present invention also presents a method for treating ALS2 patients, which may consist of patient testing to diagnose or characterize the pathology of ALS2. The patient can receive ALS2 treatment, for example by administering the drug to the patient, or the natural form or functional fragment or derivative of the ALS2 peptide described herein, or the protein function within the patient. It can be carried out by providing other therapeutic agents that restore the disease. Also included in this invention are vectors suitable for gene therapy applications and gene therapy methodologies, the latter of which is treated by the patient by producing or creating a functional gene for expression in the patient's body. This is a methodology for recovering ALS2 function. The gene therapy vector is, for example, an adeno-related vector known in the art. General methods of gene therapy are also known in the art.

  This invention includes the human ALS2 gene, which is present in the human chromosome 2 q33 region and may encode a GTPase regulatory factor. This gene may encode the amino acid sequence of SEQ ID NO: 2. A cDNA synthesized from mRNA that can be transcribed by this gene has the base sequence of SEQ ID NO: 1.

  This invention includes a human ALS2 mutated gene, which is related to amyotrophic lateral sclerosis type 2, and by deleting one or two bases of the above-mentioned human ALS2 gene. It encodes a mutated protein having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 84.

  This invention includes nucleic acids purified from genomic DNA, mRNA or cDNA, as well as synthesized nucleic acids.

  The invention includes oligonucleotides that hybridize with the ALS2 gene and variants that are desirable under severe conditions.

  The present invention includes a kit comprising an oligonucleotide or an oligonucleotide primer set, which can be used to amplify the nucleic acid encoded by ALS2 using, for example, polymerase chain reaction (PCR).

  The invention includes oligonucleotide probes that hybridize under severe conditions with regions containing base deletion sites (A261del and AG1548del) in ALS2.

  This invention includes an oligonucleotide primer set, which performs PCR amplification of a region that forms a base deletion site in ALS2, as described herein. A specific example of this primer is a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 6 and NO: 7, or a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 8 and NO: 9. is there.

  This invention includes a recombinant vector constituting the nucleic acid and a cell transcribed by the recombinant vector.

  This invention includes a GTPase regulator or GEF, which is characterized by being an expression product of the ALS2 gene as described herein.

  An example of such a GEF GTPase regulator is a recombinant protein produced by a transformed cell transformed according to this invention.

  The present invention includes a peptide comprising an amino acid sequence having 5 or more consecutive amino acid residues in the amino acid sequence from SEQ ID NO: 2 to 1st to 46th, and SEQ ID NO: 2, 47th to 1657 Peptides constituting an amino acid sequence having 5 or more consecutive amino acid residues among the amino acid sequences up to the th are included. These peptides can be used to produce antibodies.

  The present invention also provides a modified protein comprising the amino acid sequence of SEQ ID NO: 3 that can be an expression product of a mutant human ALS2 gene. An example of this modified protein is a recombinant protein produced by a transformed cell.

  This invention includes antibodies that recognize the peptides (including proteins) disclosed herein. An example of this antibody is an antibody prepared using a peptide as an antigen according to the present invention, and has 5 or more consecutive amino acid residues in the amino acid sequence from SEQ ID NO: 2 to 1st to 46th Peptides that constitute amino acid sequences are included. Further, an antibody prepared by using as an antigen a peptide constituting an amino acid sequence having 5 or more consecutive amino acid residues from the 47th to 1657th amino acid sequences of SEQ ID NO: 2 is also included.

  The invention further provides a method for diagnosing amyotrophic lateral sclerosis type 2, which is characterized by the detection of an ALS2 mutant gene. An example of this diagnostic method is a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 6 and NO: 7, or a pair of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 8 and NO: 9 The genomic DNA of the cells of the subject to be diagnosed is subjected to PCR amplification using the primer set that constitutes, and the resulting DNA fragment is treated with the restriction enzyme NarI, so that each DNA fragment is converted into two fragments. The divided subject is determined to be a patient with amyotrophic lateral sclerosis type 2.

  The present invention also provides a method for diagnosing amyotrophic lateral sclerosis type 2, which is characterized in that a transcript of the ALS2 gene or mutant gene is detected. In the embodiment of this diagnostic method, the transcript is cDNA of the ALS2 mutant gene or mRNA of the gene, or a modified protein expressed by the mutant gene. This modified protein detection example is a method aimed at protein detection, in which case antibodies that recognize amino acid sequences 1 to 46 of SEQ ID NO: 2 react, but SEQ ID NO: 2 An antibody that recognizes the amino acid sequence from 47th to 1657th of NO: 2 does not react.

  Furthermore, the present invention provides a mouse ALS2 gene that can have the amino acid sequence of SEQ ID NO: 5, as well as a nucleic acid resulting therefrom, wherein the nucleic acid comprises the genomic DNA, mRNA or cDNA of the mouse gene or a complementary sequence thereof. Nucleic acids synthesized or purified based on the above are included.

  The present invention also provides a non-human mammal deficient in a gene, such as a rodent, and preferably a mouse, which substantially lacks the function of the ALS2 gene. It is. Similarly, mouse tissue is also provided.

  The human ALS2 gene according to the present invention has 33 introns and 34 exons as genomic genes, and is present in the 80.3 kb genomic DNA adjacent to the polymorphic DNA marker D2S2309 in the human second chromosome q33 region. And encodes a human GTPase regulator having the amino acid sequence of SEQ ID NO: 2. In this ALS2 gene, the cDNA has the base sequence of SEQ ID NO: 1.

  The present invention provides an isolated nucleic acid from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 84; and amino acids 372-1657 of SEQ ID NO: 2 It encodes a peptide having at least about 75, 80, 85, 90, 95, 97 or 100% identity to all amino acid sequences selected from the composed group. Peptides encoded by these nucleic acids are provided as well.

  The present invention also provides an isolated nucleic acid, essentially consisting of SEQ ID NO: 1; SEQ ID NO: 4; SEQ ID NO: 1 nucleotides 124-5094; SEQ ID NO: 1 nucleotides And at least about 75, 80, 85, 90, 95, 97 or all nucleotide sequences selected from the group consisting of nucleotides 124-5076 of SEQ ID NO: 4 or their complementary sequences A nucleotide sequence with 100% identity. Peptides encoded by these nucleic acids are provided as well.

  The nucleic acid of this invention can be bound to a second nucleic acid that is not naturally related to the nucleic acid of this invention. Not related to nature means that the second nucleic acid is not part of the ALS2 gene and is not directly linked to the ALS2 gene in the mammalian genome.

  The invention also provides 6 to 75 oligonucleotides, which hybridize under stringent conditions to the nucleic acids of the invention or their complementary sequences. The oligonucleotides of this invention can be attached to a label, which can be any component suitable for the purpose of labeling a detectable nucleic acid or attaching a nucleic acid to a component other than a nucleic acid.

  Also according to the invention, amino acids 1-46 of SEQ ID NO: 2; amino acids 47-1657 of SEQ ID NO: 2, SEQ ID NO: 3; amino acids 43-49 of SEQ ID NO: 3; SEQ ID NO: 84; And a peptide consisting essentially of a sequence of at least 5 consecutive amino acids based on a sequence selected from the group consisting of amino acids 476-545 of SEQ ID NO: 84. These peptides are useful, for example, in preparing the antibody of the present invention and investigating the function of ALS2 protein.

  The invention also provides a non-human mammal carrying a mutated gene, wherein the gene associated only with the mutation is at least about 75 for all of SEQ ID NO: 2 or SEQ ID NO: 5, It encodes proteins with 80, 85, 90, 95, 97 or 100% sequence identity.

  The invention also provides a method for diagnosing amyotrophic lateral sclerosis type 2 in a patient, which is at least about 75, 80, as compared to SEQ ID NO: 2, in a patient or patient-derived biological sample. By detecting the presence of mutations in genes encoding proteins with 85, 90, 95, 97 or 100% sequence identity.

  The invention also provides a method for diagnosing amyotrophic lateral sclerosis type 2 in a patient, which is at least about 75, 80 relative to the entire SEQ ID NO: 2 in a patient or patient-derived biological sample. , 85, 90, 95, 97 or 100% by detecting the presence or absence of a protein having sequence identity.

  The present invention also provides a method for diagnosing amyotrophic lateral sclerosis type 2 in a patient, which can be applied to SEQ ID NO: 3 or SEQ ID NO: 84 as a whole in a patient or patient-derived biological sample. By detecting the presence or absence of a protein having at least about 75, 80, 85, 90, 95, 97 or 100% sequence identity.

  In the diagnostic method of the present invention, a sequence comparison is performed to determine the presence of a mutation, an oligonucleotide is used to detect hybridization to a patient's nucleic acid, a patient's nucleic acid is amplified, and a patient's protein is detected from the present invention. In some cases, the antibody is contacted with the antibody or the function of the ALS2 protein is evaluated for the protein produced in the patient.

  The present invention also provides a method for treating amyotrophic lateral sclerosis type 2, which comprises a method of administering a peptide, a nucleic acid, or a pharmacological component comprising a peptide or a nucleic acid to a patient with the disease. Wherein the peptide consists of an amino acid sequence having at least about 75, 80, 85, 90, 95, 97 or 100% sequence identity to SEQ ID NO: 2 or a fragment thereof, and the nucleic acid Is coding for the peptide.

  The present invention also provides a method for treating amyotrophic lateral sclerosis type 2, which comprises a method of administering a certain component to a patient of the disease, which component is SEQ ID NO: 2 or It mimics the biological activity of the fragment.

  The present invention also provides a method for preparing a drug for use in the treatment of amyotrophic lateral sclerosis type 2 using a peptide or nucleic acid, wherein the peptide is against EQ ID NO: 2 or a fragment thereof. It is composed of an amino acid sequence having at least about 75, 80, 85, 90, 95, 97 or 100% identity, and the nucleic acid encodes the peptide.

  The term “isolated” in reference to a nucleic acid or peptide in this specification means that the nucleic acid is separated from the genome of the cell, the peptide is separated from the cell, Does not mean that is obtained from the genome or cells. In some cases, the nucleic acids and peptides of this invention may be synthesized using conventional techniques.

  Two nucleic acid or protein sequences are considered substantially identical if they share at least about 70% sequence identity when they are most appropriately positioned. In other embodiments, the sequence identity may be, for example, at least 75%, at least 90%, or at least 95%. Optimal alignment of sequences for identity comparison can be performed using a variety of algorithms, such as the local identity of Smith and Waterman, 1981, Adv. Appl. Math 2: 482. Algorithm, Needleman and Wunsch, 1970, J. Mol. Biol, 48: 443 identity alignment algorithm, Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444 similarity search method, and these Computerized implementations of algorithms (such as GAP, BESTFIT, FASTA and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA). Sequence alignment can also be performed using the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 (using published default settings). ing).

In some embodiments, such as substantially identical gene targeting substrates and target sequences, the nucleic acid sequences of this invention can be substantially identical. The substantial identity of such sequences can be reflected in the ratio of identities when optimally aligned, for example 50%, 80% to 100%, at least 80%, at least 90%, Alternatively, it may be at least 95%, and this ratio may refer to the identity of the ratio of gene targeting substrate to the ratio of target sequence in the case of gene targeting substrate, in which case homology depends on the degree of identity Pairing and recombination and / or repair may be facilitated. Another indication that two nucleic acid sequences are substantially identical is substantially synonymous with two sequences hybridizing to each other under moderately harsh or desirable harsh conditions. is there. Hybridization to filter binding sequences under moderately stringent conditions, eg, 0.5M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C., and washing at 0.2 ° SSC / 0.1 at 42 ° C. % SDS (Ausubel, et al. (Eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York , See page 2.10.3). Alternatively, hybridization to filter binding sequences under stringent conditions can be achieved, for example, at 0.5 ° C NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, and at 0.1 ° C SSC / 0.1% SDS at 68 ° C. (See Ausubel, et al. (Eds), 1989, supra). Hybridization conditions can be modified according to known methods, depending on the sequence in question (Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays ", Elsevier, New York). Severe conditions are generally chosen to be about 5 ° C. below the melting temperature of the specific sequence at a given ionic strength and pH.

  It is a well-known fact in the art that a biologically equivalent polypeptide can be obtained by making some modification or change in the polypeptide structure without substantially altering the biological function of the peptide. Is possible. In one aspect of the invention, LPL S447X therapeutics may include peptides that differ from portions of the wild-type LPL sequence due to conservative amino acid substitutions. As used herein, the term “conservative amino acid substitution” refers to the substitution of one amino acid for another amino acid at a given position in the peptide, without the loss of function in that substitution. The case where it is possible to do. In order to make such changes, for example, substitution of similar amino acid residues may be performed based on relative similarities such as side chain substituents in terms of size, charge, hydrophobicity, hydrophilicity, similarity, etc. It is possible and the effect of such substitutions on peptide function can be analyzed by routine testing.

In some embodiments, conservative amino acid substitutions may be made by substituting one amino acid residue with another having a similar hydrophilicity value (eg, having a value in the range of plus or minus 2.0). In this case, the following hydrophilicity values have been assigned to amino acid residues (details are as in US Pat. No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (-0.5); Thr (- 0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3) Phe (-2.5); and Trp (-3.4).
In other examples, conservative amino acid substitutions may be made by substituting one amino acid residue with another having a similar hydropathic index (eg, having a value in the range of plus or minus 2.0). In these examples, based on hydrophobicity and charge properties, each amino acid residue is assigned the following hydropathic index: Ile (+4.5); Val (+4.2); Leu (+3.8) ; Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg ( -4.5).
In other examples, one amino acid residue may be replaced with another residue of the same class to make a conservative amino acid substitution, in which case the amino acid is nonpolar, acidic, basic or It is divided into neutral classes: nonpolar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidity: Asp, Glu; basicity: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.
^{3 and 4} were mapped to the gene locus of 1.7cM region defined by the human chromosome 2 q33 region microsatellite markers D2S116 and D2S2237 for. We have created a physical map based on YAC / BAC / PAC genomic regions of 3Mb covering the candidate region of FIG. 1 so far ^5,6. Sequences of cDNA clones and ESTs are currently analyzed and 42 non-replicating transcription units containing 10 new genes have been mapped. 411 pairs of primers were designed based on the genomic DNA of 14 humans from the ALS2 family and 6 normal individuals who were not related to this family, and were amplified by PCR. Sequencing of all PCR products revealed intron or exon polymorphisms of 77 base sequences. Among these, a gene having a base deletion related to the development of ALS2 was identified.

  The ALS2 gene includes a restriction region and a regulatory region (promoter / enhancer, suppressor, etc.), which function when the protein encoded thereby is expressed. Such restriction or regulatory regions are useful for elucidating the function of ALS2 gene products such as GEF or GTPase regulators.

  The ALS2 gene can be isolated, for example, by screening a human genomic library using as a probe a pure polynucleotide or oligonucleotide constituting the base sequence of SEQ ID NO: 1 or a partial sequence thereof. The resulting genomic genes can be amplified by commonly used gene amplification techniques. These techniques include, for example, the PCR (polymerase chain reaction) method and the NASBN (nucleic acid sequence amplification) method. , TMA (transcription-mediated amplification) method or SDA (strand displacement amplification) method.

  Similarly, pure polynucleotides (DNA fragments and RNA fragments) can be prepared based on this ALS2 genomic gene, mRNA transcribed from this gene, or cDNA synthesized from mRNA. For example, cDNA can be synthesized using poly (A) + RNA extracted from human cells as a template. Human cells are either excised from the human body by surgery or are cultured cells. cDNA can be synthesized by known methods (Mol. Cell Biol., 2, 161-170, 1982; J. Gene, 25, 263-269, 1983; Gene, 150, 243-250, 1994). CDNA can also be synthesized by RT-PCR using oligonucleotides as primers and mRNA isolated from human cells as a template. Specifically, the cDNA thus prepared has the base sequence of SEQ ID NO: 1. These polynucleotides can be used for recombinant expression of human GTPase regulatory factors.

  The oligonucleotide of the present invention is a DNA fragment or an RNA fragment, and hybridizes under severe conditions to the above-mentioned ALS2 or the above-mentioned nucleic acid. For example, it is a 10-100 bp continuous DNA fragment having the base sequence of SEQ ID NO: 1. Here, the strict conditions indicate that specific hybridization of the target to the probe is possible depending on the salt concentration, the concentration of organic solvent (formamide, etc.), or the temperature conditions during the hybridization and washing steps. The method is described in US Pat. No. 6,100,037.

One way to create stringent hybridization conditions is [B & K insertion]
The primer set of the present invention is a pair of oligonucleotides mainly used for amplification of ALS2 gene or related nucleic acid. Such a primer set is designed based on the base sequence of SEQ ID NO: 1 and will be synthesized and purified by known methods. The primer size (number of bases) is preferably 15 to 40 bases, and more preferably 15 to 30 bases that specifically anneal to the template DNA. However, when performing LA (long and accurate) PCR, it is effective to use primers with more than 30 bases. The pair of (two) primers constituting the sense strand (5 ′ end side) and the antisense strand (3 ′ end side) must not be complementary. In addition, self-complementary sequences must be avoided within the primer to prevent the formation of hairpin structures. Furthermore, in order to ensure stable binding to the template DNA, the GC content should be about 50%, so that there is no GC-rich region or AT-rich region in the primer. Since the annealing temperature depends on Tm (dissolution temperature), a primer with a Tm of 55-65 ° C. is selected so that a PCR product with high specificity is prepared. The final primer concentration used in PCR should be about 0.1 to about 1 (M. Oligo ^TM software [National Bioscience Inc. (USA) sales), Genetyx ^TM software [Software Development KK (Japan) sales], etc. Commercial software for primer design can also be used.

  Obtain a mutated ALS2 gene by screening a DNA library prepared from cells of a patient suspected of having ALS2 using a probe that hybridizes to a region containing a mutation (such as a base-deficient site) under severe conditions. be able to. A pure polynucleotide (DNA fragment or RNA fragment) is obtained from genomic DNA, mRNA or cDNA of the ALS mutant gene or its complementary sequence. For example, a certain ALS2 mutant gene constitutes a nucleic acid in which the 261st base a of SEQ ID NO: 1 is deleted. By using such a polynucleotide, it is possible to carry out gene recombinant production of ALS2 altered protein or to diagnose ALS2.

  Primers used for PCR amplification of ALS2, including various regions having a base-deficient site in mutant ALS2, are a pair of synthetic oligonucleotides constituting (for example) the base sequences of SEQ ID NO: 6 and NO: 7. With this primer set, PCR amplification of a region (339 bp) containing exon 3 in the ALS2 gene and introns before and after that can be performed. Other primer sets may be composed of synthetic oligonucleotides comprising the base sequences of SEQ ID NO: 8 and NO: 9, and using RNA as a template, exons 2-4 (302 bp) of the ALS2 gene PCR amplification can be performed. Although any PCR product that is not cleaved by the restriction enzyme NarI is derived from the normal ALS2 gene, the PCR product derived from the mutated ALS2 gene may be cleaved by NarI, resulting in two fragments (FIG. 2c).

  The recombinant vector of this invention may be a cloning vector or an expression vector. The construction of the vector will be carried out according to the type of polynucleotide as the insert or the intended use. For example, when cDNA or its ORF region is used as an insert to produce ALS2 protein or a modified protein thereof, prokaryotic cells such as Escherichia coli and Bacillus subtilus and true cells such as yeast, insect cells and mammalian cells are used. In vitro transcription or expression can be performed using expression vectors suitable for each nuclear cell. When genomic DNA of the ALS2 gene or a mutant gene thereof is used as an insert, a BAC (bacterial artificial chromosome) vector or a cosmid vector can also be used. Such a recombinant vector is also useful as a probe for diagnosing chromosomal abnormalities using hybridization such as fluorescence in situ hybridization (FISH). Furthermore, it is possible to bind a nucleic acid derived from a normal ALS2 gene in a viral vector such as an adenovirus and perform gene therapy using the product.

  The transformed cells in this invention when producing ALS2 peptides (including proteins) can be prokaryotic cells such as Escherichia coli and Bacillus subtilis, or eukaryotic cells such as yeast, insect cells and mammalian cells. In addition, cells derived from patients suffering from ALS2 (blood stem cells, etc.) are transformed with the viral vector of the present invention, and this vector is used when the nucleic acid derived from the normal ALS2 gene is incorporated in the vector. Thus, ALS2 gene therapy can be performed. Such a transformed cell can be prepared by introducing a recombinant vector into the cell using a known method such as electroporation, calcium phosphate method, liposome method or DEAE dextran method.

  The peptide of the present invention may be a normal ALS2 gene expression product or a mutated ALS2 gene expression product. The normal gene product is a GTPase transcription factor or GEF having the amino acid sequence of SEQ ID NO: 2. The peptide of the present invention is useful as an immunizing antigen for use in antibody preparation, or as a target molecule for the development of ALS2 therapeutics. These peptides can also be prepared by a method of isolating peptides from cells of healthy individuals or patients with ALS2. A chemical synthesis method based on the amino acid sequence of interest derived from SEQ ID NO: 2 or SEQ ID NO: 3, etc. (preferably) by production, isolation or purification in the above-described transformed cells. Incubating such transformed cells and isolating and purifying the culture by, for example, treatment with a denaturing agent such as urea, or using detergent, sonication, enzymatic digestion, precipitation by salting out, etc. Alternatively, it is performed by solution, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange column chromatography, hydrophobic chromatography, affinity chromatography and reverse phase chromatography. Such proteins may include fusion proteins with any other protein. Examples thereof include fusion proteins with glutathione-S-transferase (GST) and green fluorescent protein (GFP). In addition, proteins expressed in cells may undergo various types of modifications in cells after translation. Accordingly, the scope of the protein of the present invention includes modified proteins. Examples of such post-translational modifications include N-terminal methionine removal, N-terminal acetylation, sugar chain addition, limited degradation by intracellular proteases, myristoylation, isoprenylation and phosphorylation.

The antibody of this invention is a polyclonal antibody or a monoclonal antibody, and recognizes the peptide of this invention. Examples include antibodies prepared using peptides comprising a sequence of 5 or more consecutive amino acid residues of the first to 46th amino acid sequence of SEQ ID NO: 2, 47 of SEQ ID NO: 2, Examples thereof include an antibody prepared by using a peptide constituting an amino acid sequence of 5 or more consecutive amino acid residues from the 1st to 1657th amino acid sequences. When these two types of antibodies are used, it is possible to detect and distinguish between normal and A261del mutant proteins. The antibody of the present invention includes all molecules having the ability to bind to the epitope of ALS2 protein and other peptides of the present invention, and all Fab, F (ab ′) ₂ and Fv fragments of the molecule. Such an antibody can be obtained from serum after immunizing an animal using a protein or peptide derived from ALS2 as an antigen. Alternatively, the expression vector for eukaryotic cells described above can be introduced into the muscle or skin of an animal by injection or particle gun, and serum can then be collected therefrom. Examples of animals that can be used are mice, rats, rabbits, goats, chickens and the like. When B cells collected from the spleen of an immunized animal are fused with myeloma cells, monoclonal antibodies can be produced.

  According to the diagnostic method of the present invention, a gene having an ALS2 mutation or a transcription product of a gene having an ALS2 mutation is detected, whereby the risk of developing ALS2 can be evaluated. In particular, it can be analyzed for subjects of known ALS2 families, but diagnosis is not limited thereto.

  Any of the primer sets described above, or other oligonucleotides of the invention can be used to PCR amplify the genomic DNA of a subject to be diagnosed. The resulting DNA fragment can be treated with one or more restriction enzymes, such as NarI, but if the DNA fragment is cleaved and becomes a fragment different from the cleavage product generated from a non-ALS2 patient. In view of the presence of a mutation in the ALS2 gene, it is suggested that the patient is an ALS2 patient or a patient at some risk of ALS2.

  Similarly, detection of ALS2 mutant genes is (for example) allele-specific oligonucleotide probe method, oligonucleotide ligation assay method, PCR-SSCP method, PCR-CFLP method, PCR-PHFA method, invader method, RCA (rolling circle amplification) ) Method, primer oligo base extension method and the like.

  When detecting the transcript of the ALS2 mutant gene, the diagnosis can be performed by determining the sequence of the mRNA or cDNA of the subject. The diagnosis can also be performed by incorporating the ALS2 gene of the subject to be diagnosed or its cDNA into an expression vector, transfecting the cells, and measuring the expression product.

  Expression products of normal and mutant ALS2 genes can be evaluated by molecular weight measurement. For example, the absence of a single base in the normal ALS2 gene causes a frameshift, in which case the modified protein comprises amino acid residues 1 to 46 of SEQ ID NO: 2. (SEQ ID NO: 3), and the frameshift newly encoded a three amino acid residue (Pro-Ser-Glu), resulting in the naturally occurring gene product of the normal ALS2 gene. A product with a comparable molecular weight. Further, a diagnosis can be performed using the above-described antibody provided by the present invention, in which case the ALS2 correction protein is (for example) the amino acid sequence from the first to the 46th amino acid sequence of SEQ ID NO: 2, or It reacts with an antibody that recognizes the region consisting of amino acids 43-49 of SEQ ID NO: 3, but does not react with an antibody that recognizes the amino acid sequence from the 47th to 1657th amino acids of SEQ ID NO: 2. If an antibody that is specific for amino acids 476 to 545 of SEQ ID NO: 84 is used in the same manner as compared to any amino acid of SEQ ID NO: 2, it would also be possible to make a diagnosis of AG1548del. Diagnosis using an antibody can also be performed using, for example, the ELIZA method.

  The mouse ALS2 gene of the present invention is a mouse genomic gene isolated as a homologue of the human ALS2 gene, and encodes a mouse ALS2 protein constituting the amino acid sequence of SEQ ID NO: 5. The cDNA has the base sequence of SEQ ID NO: 4. This gene can be used to create “knockout” mice.

  Such “knockout” mice can be prepared according to known gene targeting methods (Science, 244: 1288-1292, 1989) or generally according to the following cases.

  First, a DNA fragment of the mouse ALS2 gene containing the start codon in the gene is corrected to obtain a defective DNA fragment lacking the expression of the ALS2 gene. Using this defective DNA fragment, a targeting vector is prepared, and its behavior is introduced into mouse totipotent cells (ES cells) using known methods (such as those described in Science 244, 1288-1292, 1989). . For example, a recombinant plasmid DNA in which a gene that has resistance to cytotoxins is substituted or inserted into the genomic gene constituting the ALS2 gene, and the deletion gene has been treated so that it has homologous sequences to the genomic DNA of the ALS2 gene at both ends. Is prepared (targeting vector). It is also possible to regulate expression by binding a resistance gene to a sequence such as a PGK1 promoter or a PGK1 polyadenylation signal. A method is preferred in which the genomic DNA region of the ALS2CR6 gene that is replaced or inserted by the resistance gene is a genomic DNA region that includes an exon region that includes the initiation codon.

  For such target vectors, it has a sequence homologous to the genomic DNA of the ALS2 gene and a resistance sequence or other sequence that can be used for cell sorting (for example, diphtheria toxin A gene and herpesvirus thymidine kinase gene). There is no particular limitation except that. Promoters and enhancers can be appropriately combined and used. Thereafter, the targeting vector is introduced into ES cells (embryonic stem cells) using a known method (Nature, 292: 154-156, etc.). Such methods include the electric pulse method, the liposome method, and the calcium phosphate method. The electric pulse method is preferred when transgene recombination efficiency becomes a problem. DNA in each ES cell into which the gene has been introduced is extracted, and homologous genetic recombination occurs between the wild-type ALS2 gene and the defective ALS2 gene fragment introduced by Southern blot analysis or PCR analysis. Select cells in which the defective gene fragment is located.

  The ES cells having the defective gene prepared above can be injected into the blastocyst of a wild type animal to obtain a chimeric embryo, which can be transplanted into the uterus of a foster parent. The resulting offspring are screened for ALS2-deficient genes and propagated. Selection can be performed by examining the difference in hair color, extracting DNA from a part of the body (such as the tail end), and performing Southern blot analysis or PCR assay after DNA extraction. DNA extracted from a part of the body (such as the tail end) of a progeny obtained by crossing a wild-type animal with a chimeric animal in which an ALS2-deficient gene is present in a sex cell. Can be used as a material for identifying a heterozygote into which an ALS2-deficient gene has been introduced. Heterozygotes carrying ALS2-deficient genes that are stable in all sex cells and somatic cells can be bred to produce offspring in which the ALS2 gene is completely “knocked out”.

  Using the animal thus prepared as an ALS2 model animal, it is possible to analyze the function of the ALS2 gene at the time of ALS2 onset, screen for therapeutic drugs, or develop a treatment method.

  The method and results of the procedure for cloning this ALS2 gene and analyzing its function are shown.

1. Method
1-1. ALS2 family We analyzed 16 cases (Reference 2) from a Tunisian family ALS2 family, including 8 individuals with this disease. ALS2 is characterized by progressive muscle spasms in the limbs and face, with peripheral limb disorders in the limbs. The age of onset is between 3 and 10 years (Reference 2). Nerve and muscle biopsy, as well as electromyography, confirmed peripheral motor neuron loss (Reference 2). ALS2 was clearly autosomal recessive when analyzed for genotypes of polymorphic DNA markers along with clinical trial data.

1-2. Transcription map Genome database (GDB) (http://www.gdb.org) and National Biotechnology Information Center (NCBI) UniGene (http://www.ncbi.nlm.nih.gov) are available. These were searched because they can be used to identify sequences of transcribed DNA mapped within the target region. Genomic DNA sequences overlapping with the target region of ALS2 were searched from Genbank's “nr” or “htgs” database, and this was used as a test target when performing a BLAST search on the dbEST database. RT-PCR, 5'-RACE and cDNA library screening were performed to isolate full length transcripts. In addition, EST clones were purchased from Research Genetics, DNAs were sequenced, and insertion of the entire clone was evaluated. Double-stranded DNA sequencing was performed by conducting dideoxy sequence analysis using the BigDye Terminator Cycle Sequencing Kit (ABI) and AB1377 DNA sequencer. The entire sequence of EST data, PCR products and DNA obtained from cDNA clones was determined, and the estimated independent transcription unit was established. Each unit was then mapped onto a physical map using PCR.

1-3. Exon Identification To determine the structure of introns and exons of transcribed DNA, use the Sequencer Version 3.0 (Gene Codes Corporation) program, as described in BLAST (Reference 28) and References (5 and 6), GenBank Genomic DNA sequence data published in the database was compared with cDNA sequences.

1-4. PCR
Exon and intron / exon boundaries were subjected to PCR amplification. Using ExTaq polymerase (Takara), a cycle of 95 ° C. for 15 seconds, 60 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 35 times, and about 50 mg of genomic DNA was amplified by PCR. RT-PCR was performed to detect the defective form of the transcribed DNA. Total RNA was isolated from lymphocytes from 4 patients and 2 carriers of ALS2 family. Total RNA extracted from healthy human brain was purchased from Clontech. RT-PCR was performed by using a SuperScript preamplification system (Gibco-BRL) according to the manufacturer's protocol. The oligonucleotide primer used for such PCR was designed using Primer 3.0 (http://www.genome.wi.mit.edu). Table 1 lists the primers used for ALS2 (ALS2CR6) amplification.

1-5. 変異の分析
DNA配列のエクソンまたはイントロン/エクソン境界における変異を検出するため、エクソンのPCR産物のDNAシーケンスを決定した。エクソンのPCR産物のDNA配列は、プライマと同じオリゴヌクレオチドを用いて分析した。公開されたデータベース中の配列を、患者、キャリアおよび健康人より得たDNA配列と比較し、ヌクレオチド中の変化を区別した。

1-5. Mutation analysis
In order to detect mutations in the exon or intron / exon boundary of the DNA sequence, the DNA sequence of the exon PCR product was determined. The DNA sequence of the exon PCR product was analyzed using the same oligonucleotide as the primer. Sequences in the published database were compared with DNA sequences obtained from patients, carriers and healthy people to distinguish changes in nucleotides.

  Similarly, RT-PCR amplification of exon 2-4 or PCR amplification of exon 3 confirmed that a new NarI site was formed after NarI treatment (A261del). Here, 5′-CCTAGTCATCCATGTGCTGG-3 ′ (SEQ ID NO: 6) and 5′-TCCCATACCTGACCTTCCAC-3 ′ (SEQ ID NO: 7) were used as primers for exon 3-PCR. Here, 5′-CTTGATAGACTTTCTGTAAAGAAG-3 ′ (SEQ ID NO: 8) and 5′-GGCTACTTGGACAAATCTCCACTG-3 ′ (SEQ ID NO: 9) were used as primers for RT-PCR of exon 2-4. NarI degradation products were separated by 1.5% agarose gel.

1-6. Northern blot analysis Northern (MTN) blots (Clontech) of a large number of human adult tissues were analyzed in Exon 4 labeled with ALS2CR6 with ³² P-dCTP in Perfect Hyb hybridization solution (Toyobo) or human (actin) Hybridization was performed using cDNA.The membrane was washed with 0.1 × SSC containing 1% SDS and subjected to X-ray film (Bio-MAX, Kodak).

1-7. MRNA in situ hybridization Antisense and sense cRNA probes were prepared from two mouse cDNA clones m2-as and m2-s. These mouse cDNA clones covered part of the mouse mALS2CR6 cDNA (bases 1732 to 2685 of SEQ ID NO: 4; 954 bp) and were inserted in the reverse direction into pCR2.1 (Invitrogen). The probe was prepared by an in vitro transcription reaction in which digoxigenin-labeled UTP and T7 polymerase were mixed according to the manufacturer's protocol (Roche Molecular Biochemicals). Sample preparation and in situ hybridization followed literature (29).

1-8. Searching the Database Each DNA and amino acid sequence was compared with a database of nucleotide and protein sequences that did not overlap each other using BLASTN and BLASTP. Protein domains and motifs include Genome Net Japan's MOTIF server (http://www.genome.ad.jp), BCM search launcher (http://www.hgse.hem.tmc.edu/Search.launcher) And NCBI CD search (http://www.ncbi.nlm.nih.gov).

2. Results Based on YAC / BAC / PAC, the inventor created a physical map of the 3Mb genomic region that covers the entire ALS2 candidate region (References 5 and 6). Sequence analysis of EST and cDNA clones was performed within a large region, and at the same time using this physical map, the 18 genes already analyzed (KIAA0005, CLK1, PP1L3, ORC2L, NDUFB3, CFLAR, CASP10, CASP8, FZD7 43 independent transcription units, including NOP5, UBL1, BMPR2, FLJ10881, LOC57404, AIP-1, CD28, CTLA4 and AILIM, and 10 new full-length transcripts (ALS2CR1, ALS2CR2, ALS2CR3, ALS2CR4, ALS2CR5 / MPP4, ALS2CR6, ALS2CR7, ALS2CR8, ALS2CR9 and ALS2CR12) were mapped. These genetic sequences are present at the ALS2 locus (Figure 1).

  Juvenile ALS2 is rare and has signs that muscle spasms of the limbs, face and pharynx develop gradually in teens or twenties. Since ALS2 is recessive, this ALS2 disease is expected to result from loss of functional mutation. Large deletions or translocations at the ALS2 locus were investigated by mapping STS / EST content on a PCR and Southern blot analysis basis, but this was not detected. Subsequent small deletions or base substitutions at the exon or intron-exon boundary were investigated. In order to detect these mutations, each gene was analyzed and its intron / exon boundaries were determined. At present, 395 exons have been identified from 42 genes. A total of 411 primers were designed to amplify the exon and its flanking sequences, including consensus sequences for splicing donors and acceptors, and using these primers, PCR was performed on 14 cases of the ALS2 family (Figure 2a). Amplified genomic DNA of 10 unrelated normal control subjects. Each of these PCR products was sequenced, revealing a total of 77 intron or exon sequence polymorphisms. Of these 77 polymorphisms, 8 mutations in 4 different genes were associated with ALS2 (Table 2).

これらの配列変異のうち、ALS2CR6のエクソン3中に記録された一つのヌクレオチド欠失（A261del）がリーディングフレームを壊しており、このような変異が蛋白質を変異させていることが示唆される。疑わしいヘテロ接合性キャリアは全て複製された配列パターンを示しており、これは不完全部位の後にある最初のヌクレオチドより始まっている（図2b）。この欠失は明らかにALS2発現型と共に移動しており（図2c）、様々な人種の正常対照個体533例においては記録されていない（データは示さず）。他の変異の中には、ALS2CR9遺伝子のエクソン4において、1個の塩基がCからTへ置換されている状態がある（C873T）。しかしこの変異は3番目のコドンに対応しており、そのためアミノ酸残基を変化させない。他の配列変異により潜在化または顕在化するスプライシングエラーを検出するため、患者と健常対照者のリンパ球より抽出した総RNAを用いてRT-PCRを実施したが、mRNAの配列変異は検出されなかった（データは示さず）。従って、イントロンまたはエクソン領域に関連する変異はスプライシングエラーを生じない。これらの結果から、ALS2CR6のエクソン3における1個の塩基の欠失（A261del；表1）がALS2に関連する変異であることが確認されている。

Of these sequence variations, one nucleotide deletion (A261del) recorded in exon 3 of ALS2CR6 disrupts the reading frame, suggesting that such a mutation mutates the protein. All suspicious heterozygous carriers show a replicated sequence pattern, starting with the first nucleotide after the incomplete site (Figure 2b). This deletion clearly migrated with the ALS2 phenotype (Figure 2c) and has not been recorded in 533 normal control individuals of various races (data not shown). Among other mutations, there is a state in which one base is replaced from C to T in exon 4 of the ALS2CR9 gene (C873T). However, this mutation corresponds to the third codon and therefore does not change the amino acid residue. RT-PCR was performed using total RNA extracted from lymphocytes of patients and healthy controls to detect splicing errors that were latent or manifested by other sequence variations, but no mRNA sequence variation was detected. (Data not shown). Thus, mutations associated with intron or exon regions do not cause splicing errors. From these results, it has been confirmed that the deletion of one base in exon 3 of ALS2CR6 (A261del; Table 1) is a mutation related to ALS2.

  The ALS2CR6 gene contains 33 introns and 34 exons and is present in the 80.3 kb genomic DNA adjacent to the polymorphic DNA marker D2S2309 (FIG. 1). The transcriptional polarity of the ALS2CR6 gene is from the telomere toward the central body. The ALS2CR6 transcript (mRNA) has a total length of 6394 bp (SEQ ID NO: 1), a single open reading frame (ORF) 4974 nucleotides long (124-5,097 nt), and 1,657 amino acid residues. It encodes a 184 kDa protein consisting of a base. The polyadenylation putative signal (AATAAA: 6,375-6,380 nt) and the poly (A) region are evident. In addition, a short ALS2CR6 transcript having an ORF of 2,651 bp and 1,191 bp in total length and encoding 396 amino acid sequences was identified. This short mutant splices the 5'-donor site after exon 4, resulting in a stop codon after 25 amino acid residues in intron 4. Corresponding to these results, Northern blot analysis identified two transcripts of approximately 6.5 kb and 2.6 kb in many adult tissues (FIG. 3a). Both transcripts showed similar expression patterns with the exception that the short transcript was expressed mainly in the liver. The 6.5 kb large transcript is expressed at a slightly higher level than the 2.6 kb transcript, confirming that it is expressed most abundantly in the cerebellum. It has been confirmed that this gene is also expressed in cells of ALS2 patients.

  In addition, a mouse ALS2CR6 homolog was isolated and designated mALS2CR6. The transcript of mALS2CR6 is 6,349 bp in length (SEQ ID NO: 4), has an ORF of 4,956 bp (124-5,076 nt), and encodes a 183 kDa protein consisting of 1651 amino acids (SEQ ID NO: 5). ing. Overall, this ORF is well conserved between human and mouse at the DNA level (87% identical) and protein level (91% identical; 94% similar; Figure 4), and the ALS2CR6 gene is well conserved in mammals It is suggested to be a gene.

  To examine the nature of mALS2CR6 transcript expression localization in the mouse brain and spinal cord, in situ hybridization was performed using a riboprobe corresponding to a portion of the mALS2CR6 cDNA. As a result, as shown in Fig. 5, mALS2CR6 transcripts are expressed at various levels in neurons from the brain to the spinal cord, especially neurons in the hippocampus and dentate gyrus, cerebellar Purkinje cells, and neurons in the cerebral cortex And it was prominent in the gray matter of the spinal cord containing anterior horn cells. In addition, significant expression was recorded in the olfactory bulb, basal ganglia and cranial nerve nucleus.

  Human ALS2CR6 protein showed many interesting properties (Fig. 6a). The first property appears in the A-terminal region of ALS2CR6, which is highly homologous to RCC1 (regulator of chromosome enrichment; ref. 7) and RPGR (GTPase associated with retinitis pigmentosa; ref. 8). (Figure 8). RCC1 and RPGR proteins act as guanine nucleotide exchange factors (GEF) for GTPases such as Ran. The second property is that ALS2CR6 has a Db1 homology (DH) domain and a pleckstrin homology (PH) domain, both of which are typical domains recorded as RhoGEF proteins ( References 9 and 10). In addition, it has been recorded that the VPS9 domain is present in the C-terminal region. The VPS9 domain is recorded in many GEFs such as Vps9 (Reference 11) and Rabex-5 (Reference 12), each of which is said to mediate vacuolar protein secretion and phagocytic transport. Two MORN motifs composed of 14 amino acids (Reference 14) are also recorded. Recent studies on Junctophilin containing the MORN motif have shown that this motif contributes to plasma membrane binding (Reference 13). GEF is related to the GDP-binding form of GTPase, and it is known that GTPase is activated by promoting the separation of GDP and the binding of GTP. GEF is known to play an important role in many signal transduction cascades (14), neuron formation (15), membrane transport (16), and actin cytoskeleton formation (17) (References 18 and 19), ALS2CR6 acts as a regulator / activator for Ran-related GTPase, regulates membrane formation, and acts in (membrane) transport of cells including neurons.

  According to RT-PCR analysis, the transcript of the mutated ALS2CR6 gene is transcribed from the patient's chromosome (Figure 2c) and consists of 49 amino acids, with 3 new residues at the C-terminus (Pro-Ser -Glu) is produced (FIG. 6a). Since this modified protein does not have a functional domain corresponding to the ALS2CR6 protein, it appears that its intrinsic function has been lost. Thus, this A261del mutation recorded in ALS2CR6 is related to the fact that ALS2 is recessive.

  Recent findings indicate that ALS is associated with blood vessels related to axonal transport and cytoskeletal formation (References 20, 21 and 22), from which the ALS2CR6 gene corresponds to ALS2 and the membrane structure Ran The hypothesis that ALS2 occurs due to a defect in membrane structure due to lack of regulatory function of the related GTPase is derived.

  The ALS2CR6 gene is the second ALS gene that follows the role that copper-zinc superoxide dismutase (SDS-1) plays in ALS. SOD-1 mutations are associated with late-onset autosomal dominant forms (Reference 23).

While the above invention has been described in some detail by way of illustration and example for clarity of understanding, changes and revisions will not depart from the spirit or scope of the appended claims in light of the teachings of the invention. The expert will easily understand what will be done to it. All patents, patent application documents and publications referenced herein are hereby incorporated by reference.
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Transcription map for 3Mb region of human chromosome 2q33 including ALS2 candidate region. Without rectangle in white is a section of D2S2237 from D2S116 ^2,3. The location of 7 STS markers, 12 polymorphic DNA markers and 42 independent transcription units are shown. The polarity of the 38 transcription units is indicated by arrows. The position of the ALS2 gene is called “ALS2CR6”, and this term may be used synonymously with ALS2 below. Figure 2 shows a process for detecting mutations associated with ALS2. “A” is an example of ALS2 family in Tunisia and Kuwait. Family members genotypes are shown on the basis of previous reports results ^3,4. “B” shows the sequencing result of the mutation (A261del) in genomic DNA in ALS2 family in Tunisia. Patient 10797 is homozygous A261del and carrier 10784 is heterozygous. Sequencing was performed on the PCR products. “C” shows the result of sequencing the mutation (AG1548del) in genomic DNA in ALS2 family in Kuwait. The sequence of the reverse strand of exon 5 in the region of interest is shown. Individual 18279 is a healthy sibling, unaffected by ALS2, and possesses two normal haplotypes. The box in this sequence indicates the site of the base that is deleted in the diseased patient. Individual 18281 is a non-symptomatic patient with one disease haplotype. Overlapping normal and mutated sequences are shown. Individual 18275 is sick, and the figure shows a homozygous CT deletion in the reverse strand of exon 5. The site of the deleted base is indicated by an arrow. Corresponding forward sequences and encoded normal amino acids and novel amino acids generated by frameshifting are shown. “D” indicates the segregation of the A261del mutation in the Tunisian ALS2 family. The presence of the deletion was determined by digestion with NarI, which only cleaves the mutant gene product. For the exon-PCR product, the 339 bp fragment representing the normal allele was cleaved into two fragments (225 bp and 113 bp) for the mutated allele. For the RT-PCR product, the 302 bp fragment representing the normal allele was cleaved into two fragments (195 bp and 106 bp) in the mutated allele. Figure 5 shows Northern blot analysis of ALS2 (ALS2CR6) mRNA. In “a”, a Northern blot containing poly (A + mRNA) 2 (g) from a number of adult human tissues was hybridized with exon 4 of the ALS2 cDNA. In the figure below, the same blot was hybridized with human (actin cDNA). In the left figure, the size of the ALS2 transcript is shown. “B” shows total RNA 10 (g, normal and normal (10788 cases) obtained from normal whole brain. ) Total DNA 20 (g) obtained from lymphocytes was hybridized to ALS2 cDNA exon 4. The right part shows agarose gel electrophoresis of the RNA sample. A comparison of amino acid sequences in human ALS2CR6 and mouse homology pair mALS2CR6. Identical residues are shown in boxes. This includes three additional amino acid residue sites in the Tunisian variant protein (starting from the 47th amino acid residue) and 25 amino acid residue sites in the short mutation part of the ALS2 gene (372nd amino acid residue) Residues), and an additional 70 amino acid residue sites (starting from the 476th residue) in the Kuwaiti mutant protein are shown. ALS2 mRNA expression in the brain and spinal cord of mature mice. “A” is an overall arrow-shaped image of RNA / RNA in situ hybridization using an antisense ALS2 riboprobe, and “b” is a control image using a sense strand probe. Prominent expression was recorded in spinal cords including neurons in the hippocampus and dentate gyrus (c and g), Purkinje cells in the cerebellum (d and h), neurons in the cerebral cortex (e and i), and anterior horn cells Gray matter (f and j). The scale bar indicates 10 (m length. It is the result of an amino acid sequence analysis. “A” is a schematic diagram of domains and motifs of normal and mutant ALS2 proteins. RCC1 is a regulator for chromosome enrichment, DH is a homology domain to Db1, PH is a pleckstrin homology domain, MORN is a membrane structure and recognition nexus, and VPS9 is a vacuolar protein for distinguishing 9 domains It is. “B” is a comparison of the amino acid sequences of the region containing the RCC1 repeat for human ALS2 (hALS2CR6), mouse ALS2 (mALS2CR6), human (h) RCC1, human (h) RPGR and mouse (m) RPGR. The amino acid residues shown in the open box are identical. Conserved amino acid residues are also abundant as well. The positions of the seven blades corresponding to RCC1 are indicated according to the literature ³⁰ . It is the figure which compared the coding product of the abbreviated human variant of a wild type human, mouse | mouth, and ALS2 protein, and A261del (Tunisia) and AG1548del (Kuwait) variant.

[Sequence Listing]

Claims (62)

  SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 84; and for all amino acid sequences selected from the group consisting of amino acids 372-1657 of SEQ ID NO: 2, An isolated nucleic acid encoding a peptide having at least 75% identity.   2. The nucleic acid of claim 1, which encodes a peptide having about 80% or more sequence identity to a selected sequence.   2. The nucleic acid of claim 1, which encodes a peptide having about 85% or more sequence identity to a selected sequence.   2. The nucleic acid of claim 1, which encodes a peptide having about 90% or more sequence identity to a selected sequence.   2. The nucleic acid of claim 1, which encodes a peptide having about 95% or more sequence identity to a selected sequence.   6. The nucleic acid of any of claims 1-5, wherein the selected sequence is SEQ ID NO: 2.   2. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO: 3.   2. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO: 5.   2. The nucleic acid of claim 1, wherein the selected sequence is SEQ ID NO: 84.   SEQ ID NO: 1; SEQ ID NO: 4; selected from the group consisting of nucleotides 124-5094 of SEQ ID NO: 1; nucleotides 1225-5094 of SEQ ID NO: 1; and nucleotides 124-5076 of SEQ ID NO: 4 An isolated nucleic acid comprising essentially a nucleotide sequence having at least 75% identity to the entire nucleotide sequence or its complementary sequence.   11. The nucleic acid of claim 10, having about 80% or more sequence identity to a selected sequence or its complementary sequence.   11. The nucleic acid of claim 10, having about 85% or more sequence identity to the selected sequence or its complementary sequence.   11. The nucleic acid of claim 10, having about 90% or more sequence identity to a selected sequence or its complementary sequence.   11. The nucleic acid of claim 10, having about 95% or more sequence identity to the selected sequence or its complementary sequence.   15. The nucleic acid of any of claims 10-14, wherein the selected sequence is SEQ ID NO: 1.   15. The nucleic acid of any of claims 10-14, wherein the selected sequence is SEQ ID NO: 4.   15. The nucleic acid of any of claims 10-14, wherein the selected sequence is nucleotides 124-5094 of SEQ ID NO: 1.   15. The nucleic acid of any of claims 10-14, wherein the selected sequence is nucleotides 124-5076 of SEQ ID NO: 4.   19. The isolated nucleic acid of any of claims 1-18, wherein a second nucleic acid that is not naturally associated with any of the isolated nucleic acids of claim 1-18 is linked.   A recombinant vector comprising the nucleic acid according to any one of claims 1-19.   A cell comprising the nucleic acid of claim 19 or the vector of claim 20.   An oligonucleotide consisting of 6 to 75 nucleotides that hybridizes under stringent conditions to the nucleic acid according to any one of claims 1-18 or a complementary sequence thereof.   23. The oligonucleotide of claim 22, consisting of about 10 to about 40 nucleotides.   23. The oligonucleotide of claim 22, consisting of about 15 to about 30 nucleotides.   23. The oligonucleotide of claim 22, consisting of about 15 to about 25 nucleotides.   Under stringent conditions, it hybridizes to a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO: 3 or a complementary nucleic acid sequence thereof, but a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO: 2 or The oligonucleotide of any one of claims 22-25, which does not hybridize to a complementary nucleic acid sequence.   Under stringent conditions, it hybridizes to a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO: 84 or a complementary nucleic acid sequence thereof, but a nucleic acid encoding a peptide consisting of the sequence of SEQ ID NO: 2 or its complement The oligonucleotide of any one of claims 22-25, which does not hybridize to a nucleic acid sequence.   28. The oligonucleotide of any of claims 22-27, to which a label is linked.   A kit for nucleic acid amplification comprising two or more different oligonucleotides of any of claims 22-27.   21. An isolated peptide comprising the amino acid sequence encoded by the nucleic acid of any of claims 1-19 or the recombinant vector of claim 20.   SEQ ID NO: 2 amino acids 1-46; SEQ ID NO: 2 amino acids 47-1657; SEQ ID NO: 3; SEQ ID NO: 3 amino acids 43-49; SEQ ID NO: 84; and SEQ ID NO: A peptide comprising essentially at least 5 consecutive amino acids of a sequence selected from the group consisting of 84 amino acids 476-545.   A peptide comprising essentially at least 5 consecutive amino acids of amino acids 43-49 of SEQ ID NO: 3, or amino acids 476-545 of SEQ ID NO: 84.   An antibody that binds to any of the peptides of claims 30-32.   34. The antibody of claim 33, which is prepared by using any of the peptides of claims 30-32 as an antigen.   A non-human mammal comprising a mutated gene of a gene encoding a protein having at least 75% sequence identity to all of SEQ ID NO: 2 or SEQ ID NO: 5.   36. The mammal of claim 35, wherein the protein has at least 85% sequence identity to all of SEQ ID NO: 1 or SEQ ID NO: 2.   37. The mammal of claim 35 or 36, wherein the mutated gene does not express a protein having biological activity.   38. The mammal of claim 35, 36 or 37, wherein the mutated gene does not have protein expression ability.   The mammal according to any one of claims 35 to 38, wherein the mammal is a rodent.   40. The mammal of claim 39, wherein the mammal is a mouse.   A method for diagnosing amyotrophic lateral sclerosis type 2 in a patient comprising detecting a mutation in a gene encoding a protein having at least 75% sequence identity to SEQ ID NO: 2.   42. The method of claim 41, wherein the protein has at least 90% sequence identity to SEQ ID NO: 2.   42. The method of claim 41, wherein the protein has at least 95% sequence identity to SEQ ID NO: 2.   42. The method of claim 41, wherein the protein has at least 97% sequence identity to SEQ ID NO: 2.   42. The method of claim 41, wherein the protein has the sequence of SEQ ID NO: 2 as essential, except for the presence of mutations.   46. The method of any of claims 41-45, comprising detecting the presence of the mutation in the biological sample from the patient.   Detection includes comparison of SEQ ID NO: 1 with the sequence of the gene, the RNA transcript of this gene, and the cDNA produced from this RNA, or the protein expressed from a gene obtained from a human patient, 47. The method according to any one of claims 41 to 46, wherein any sequence difference is used as an indicator of mutation.   49. The method of claim 46, comprising contacting the nucleic acid obtained from the biological sample or cDNA made from the nucleic acid with one or more oligonucleotides of any of claims 22-28.   47. The method of claim 46, comprising detecting whether one or more oligonucleotides hybridize under stringent conditions to the nucleic acid or cDNA.   48. The method of claim 46, comprising amplifying nucleic acid or cDNA to which two or more of said oligonucleotides hybridize and determining the presence of an amplification product.   A method of diagnosing amyotrophic lateral sclerosis type 2, comprising detecting in a patient the presence or absence of a protein having at least 85% sequence identity to all of SEQ ID NO: 2.   A method of diagnosing amyotrophic lateral sclerosis type 2, comprising detecting in a patient the presence or absence of a protein having at least 95% sequence identity to all of SEQ ID NO: 2.   52. The method of claim 51, wherein detecting comprises determining whether a protein having at least 85% sequence identity to all of SEQ ID NO: 2 is present in the biological sample from the patient.   53. The method of claim 52, wherein detecting comprises determining whether a protein having at least 95% sequence identity to all of SEQ ID NO: 2 is present in the biological sample from the patient.   A method for diagnosing amyotrophic lateral sclerosis type 2, comprising the presence or absence of a protein having at least 85% sequence identity to all of SEQ ID NO: 3 or SEQ ID NO: 84, from an organism from a patient Detecting in a biological sample.   A method for diagnosing amyotrophic lateral sclerosis type 2, wherein the presence or absence of a protein having at least 95% sequence identity to all of SEQ ID NO: 3 or SEQ ID NO: 84 Detecting in a biological sample.   57. The method of any of claims 51-56, comprising contacting the antibody of any of claims 33 or 34 with a biological sample from a patient and determining whether the antibody binds to a protein in the sample. .   A method for treating amyotrophic lateral sclerosis type 2, comprising a peptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2 or a fragment thereof, a nucleic acid encoding this peptide, or a method thereof Administering a pharmacological composition comprising a peptide or nucleic acid to a patient in need thereof.   A method of treating amyotrophic lateral sclerosis type 2, comprising a peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 2 or a fragment thereof, a nucleic acid encoding this peptide, or a method thereof Administering a pharmacological composition comprising a peptide or nucleic acid to a patient in need thereof.   A method for the treatment of amyotrophic lateral sclerosis type 2 comprising administering to a patient in need thereof a composition that mimics the biological activity of the peptide of SEQ ID NO.2.   A peptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2 or a fragment thereof for the preparation of a therapeutic drug for amyotrophic lateral sclerosis type 2, a nucleic acid encoding this peptide use.   A peptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 2 or a fragment thereof for the preparation of a therapeutic drug for amyotrophic lateral sclerosis type 2, a nucleic acid encoding this peptide use.

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