JP2006121961A - Causative gene gdd1 of gnathodiaphyseal dysplasia gdd and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a causative gene of gnathodiaphyseal dysplasia (GDD), capable of being used in cloning and clarifying mechanism of action thereof and therefore capable of solving important problems in clinical medicine related to diagnosis, treatment, and prevention of the GDD, regeneration of a bone, reconstruction of a hard tissue, development of a substitutive material for the hard tissue, etc., and further in basic research for differentiation of the bone, formation of the bone, etc., while the GDD is a systemic bone disease which causes osteopsathyrosis, easy fracture of the bone, sclerosis of a long bone, a cementification lesion of a mandible bone, etc. <P>SOLUTION: A full-length DNA base sequence of the causative gene GDD1 of the GDD, a DNA including the base sequence and a fragment thereof, an amino acid sequence encoded by the gene, a protein including the amino acid sequence and a fragment thereof, an RNA complementary to the DNA, a testing agent for a bone disease gene, which uses the DNA, the RNA, and the protein, a diagnosing agent for the GDD, a bone deficiency symptom, and deossification disease, two of missense mutations of C356R and C356G used as a gene diagnosing marker, a treating agent for the bone disease, a treating agent for the bone disease gene, and the like are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to GDD1 (hereinafter referred to as “GDD1 gene”), which is a causative gene or responsible gene of jaw bone dysplasia (hereinafter abbreviated as “GDD”), and bone disease including GDD as its use. The present invention relates to reagents for basic research relating to bone formation, bone tissue regeneration, hard tissue reconstruction, etc., diagnostics, clinical tests, diagnostic agents, clinical test agents, gene diagnostic agents, therapeutic agents, gene therapeutic agents, and the like.

GDD was first reported by Akasaka et al. In 1969 as a new family with systemic bone disease and hereditary jawbone sclerosis (Non-patent Document 1). According to this report, clinical symptoms of GDD include repeated fractures, normal healing of fractures, replacement of the mandible and maxilla with fibrous connective tissue, various ossifications of varying degrees, propensity to infect the jaw, sporadic Suppurative osteomyelitis, rare face deformation, etc. This disease is further classified as osteodysplasia with abnormal bone lesions by Levin et al. In 1985 (Non-patent Document 2), and as chin bone dysplasia (GDD) by Riminucci et al. In 2001 (Non-patent Documents). 3) Each was reported. The invention relating to GDD and its causal gene or responsible gene (patent document) is not yet known.
Japanese Biochemical Society, 43, 381-394, 1969. Am. J. et al. Med. Genet. 21, 257-269, 1985. J. et al. Bone Miner. Res. 16: 1710-1718, 2001.

As described above, GDD is a bone system disease that causes bone fragility, easy fracture, hardening of long bones, cement bone lesions of the mandible, etc. -It is an important issue not only in clinical medicine related to treatment / prevention, bone regeneration, hard tissue reconstruction, and development of hard tissue substitute materials, but also in basic research such as bone differentiation and bone formation.

According to this invention, as means for solving the above problems, a GDD causative gene clone, the full-length nucleotide sequence of the gene DNA and the DNA of the sequence, the full-length amino acid sequence encoded by the gene, the protein of the sequence, GDD, Missense mutations specific to patients, their use, etc. are provided.
First, in order to enhance the understanding of the present invention, the following is an outline of the process leading to the completion of the present invention by outstanding ideas based on the deep knowledge and experience of the present inventors and concentrated constant diligence:

The present inventors conducted a linkage analysis on the Japanese GDD family reported by Akasaka et al., And found that the GDD disease-causing gene falls within the range of the short arm 14.3 to 15.1 of chromosome 11 and D11S1308. It was clarified that a disease-causing gene exists in a region of 8.7 centmorgan (recombination distance) sandwiched between two microsatellite markers of D11s980 (FIG. 1). Linkage analysis uses a phenomenon in which a disease-causing gene and the following markers are linked and transmitted to offspring, and about 400 types existing in the whole human genome for affected individuals and healthy controls in the family. This is a method of determining the locus of a disease-causing gene by analyzing which of the microsatellite markers exists in the vicinity of the disease-causing gene.
According to the gene analysis, there were 8 known genes (NAV2, HJATLP2, PRMT2, SLC6A5, NELL1, SLC17A6, FANCF, GAS2) in the above-mentioned 8.7 centmorgan region. However, no mutation was observed in any exon or exon-intron boundary region (FIG. 1).
Therefore, the inventors expressed 6.7 messenger RNA of about 6.7 kb as one of ESTs (Expressed Sequence Tags) that are expressed as messenger RNA in this region, but the function of the gene has not been identified yet. Paying attention to cDNA base sequence, GenBank Accession No. AL833271, various studies were repeated. As a result, since the cDNA has a 3 ′ polyadenylation signal, it is suggested that it may be expressed as a gene, and a sequence of 2,742 bases including a reading frame of a polypeptide consisting of 913 amino acids. Therefore, it was considered to be a gene encoding a 107.20 kD protein. Furthermore, by confirming that no other sequences existed upstream of 5 ′ by the method of RACE (Rapid Amplification of cDNA Ends), this cDNA base sequence was considered to be a full-length sequence.
Based on the above findings, the cDNA base sequence was determined to be a novel gene consisting of 22 exons, corresponding to a human genomic DNA base sequence of about 90 kb. The inventors named this novel gene GDD1 (FIG. 1).

Furthermore, the present inventors have confirmed the following (1) to (3): (1) The presence of a novel gene, GDD1, composed of 913 amino acids in the above-mentioned region; (2) Recognize a missense mutation at the 356th amino acid of GDD1 not only in Japanese GDD families but also in African American GDD families; and (3) the 356th amino acid of GDD1 is human, mouse, Conserved across species in zebrafish, Drosophila, and mosquitoes, and considered to play a biologically important role. (4) It was predicted that GDD1 protein was localized in the endoplasmic reticulum and involved in maintaining homeostasis of intracellular calcium concentration. Furthermore, (5) in two families with different onset of GDD, the cysteine (C) highly conserved in GDD1 is mutated to arginine (R) or glycine (G) specifically in the affected person (C → R and C → G) It was proved that the novel GDD1 gene is a causative gene of GDD disease.

Based on the above circumstances, according to the present invention, the following technologies (1) to (13) are provided:
(1) A base consisting of the GDD1 gene DNA full-length base sequence described in SEQ ID NO: 1, a partial base sequence that is a fragment thereof, and a base sequence in which at least one of these base sequences is substituted based on the degeneracy of codons DNA or polynucleotide having at least one base sequence selected from the sequence group.
In addition, the polynucleotide as used in this specification is used as a general term for nucleotide compounds composed of two or more nucleotides.
In addition, since the non-coding region of the full-length base sequence includes regulatory genes such as promoters and operators, expression of GDD1 and other genes by genetic manipulation, elucidation of gene functions and regulation, etc. Useful for.
The bases of the coding region can then be synonymously substituted based on codon degeneracy, optionally matching, for example, codon usage.
(2) RNA or polynucleotide having an RNA base sequence complementary to the DNA base sequence of (1) above.
Such RNA and its base sequence can be used, for example, for the design and synthesis of antisense RNA and RNAi (RNA interference) as a reagent for bone differentiation research.
(3) A bone disease gene test drug containing an amount of reaction using the DNA of (1) or the RNA of (2) as a reaction reagent.
Here, the genetic test drug means a gene diagnostic agent for clinical tests in the medical and dental field, a reagent for analysis of gene functions for drug discovery and basic research, and the like.
As a marker for genetic diagnosis of GDD, two missense mutations, C356R and C365G described later in the column of the best mode, can be used.
In addition, the polynucleotide as used in this specification is used as a general term for nucleotide compounds composed of two or more nucleotides.

(4) The bone disease genetic test drug according to (3), wherein the bone disease is GDD and / or bone deficiency and / or bone resorbable disease.
As specific examples of the bone disease referred to in this specification, alveolar bone deficiency, osteoporosis and the like can be raised in addition to GDD.
(5) A primer designed based on the full-length base sequence of (1) above.
As primers for PCR, for example, as described later in Examples 1 and 2,
A pair consisting of 5′-TTACCTAAACACCCTACTCACTG-3 ′ and 5′-TATACGAGGCAAAAACAACAC-3 ′;
One set consisting of 5′-TTACCTAAACACCCTACTACTCG-3 ′ and 5′-CAAGTCTTTTCAACATGTTAC-3 ′ can be raised.
(6) A probe designed based on the full-length base sequence of (1).
The probe according to the present invention can be used for Southern blotting, Northern blotting, markers for gene expression, and the like.

(7) A bone disease gene therapy agent designed and constructed so that the DNA of (1) is expressed in vivo.
For example, the GDD1 gene coding region DNA, its fragment DNA, and the like can be used as an expression vector prepared by inserting these into a viral vector, a eukaryotic cell vector, or the like.
(8) The bone disease gene therapy drug according to (7), wherein the bone disease is GDD and / or bone deficiency and / or bone resorbable disease. The bone disease is as described in (4) above.
(9) Full-length amino acid sequence encoded by the GDD1 gene described in SEQ ID NO: 2, a partial amino acid sequence that is a fragment thereof, and an amino acid sequence in which at least one amino acid residue of these amino acid sequences is substituted, deleted or inserted A protein having at least one amino acid sequence selected from the group consisting of amino acid sequences.
(10) The protein of the above (9) having the amino acid sequence of the following SEQ ID NO: This protein is a fragment of a protein having a full-length amino acid sequence encoded by the GDD1 gene, and is an active domain of GDD1 function:
SEQ ID NO: 3 (amino acids 312 to 332), SEQ ID NO: 4 (381 to 401),
SEQ ID NO: 5 (Nos. 463 to 483), SEQ ID NO: 6 (Nos. 512 to 532),
SEQ ID NO: 7 (Nos. 558 to 578), SEQ ID NO: 8 (Nos. 680 to 700),
Sequence number 9 (No. 733-753) and sequence number 10 (No. 833-853).
In the above (amino acid number), the N-terminal methionine (M) residue of the amino acid sequence shown in SEQ ID NO: 2 is the first amino acid, and the amino acid residues are counted from the left to the right from the C-terminal direction. Ordinal numbers.

(11) A bone disease therapeutic agent comprising the protein of (9) or (10) above as an active ingredient in an amount having a medicinal effect.
In addition, the protein as used in this specification is used as a general term for peptide compounds composed of two or more amino acids.
(12) The bone disease therapeutic agent according to (11), wherein the bone disease is GDD and / or bone deficiency and / or bone resorbable disease. The bone disease is as described in (4) above.
(13) An antibody against the protein of (9) or (10).
Examples of the antibody include a monoclonal antibody and a polyclonal antibody that can be prepared by a conventional method. Such antibodies can be used in diagnostic agents, antigen-antibody reaction reagents, therapeutic agents and the like.

The present invention relates to fields of application such as clinical medicine, dentistry, regenerative medicine, pharmaceuticals, etc. related to diagnosis, treatment, prevention, hard tissue reconstruction, development of hard tissue substitute materials, etc., and basics related to bone differentiation, bone formation, etc. Contributes greatly to research.

The embodiment of the present invention will be described in detail as follows:
(1) Physical positional relationship between the existence region of GDD disease-causing gene and GDD1:
See FIG. Exons are indicated by filled boxes, and untranslated regions are indicated by gray boxes.
(2) Missense mutation of cysteine (C) at position 356 of GDD1:
See FIG. In Japanese GDD families, the codon base is replaced with “T → C”, and C is a arginine (R) “C → R” missense mutation (C356R) [FIG. 1 (a)]. In African-American GDD families, the codon base is replaced by “T → G” and C is a missense mutation (C365G) in glycine (G) “C → G” [FIG. 1 (b)].
It shows that the above-mentioned missense mutation was recognized in the affected person by the method of SSCP (Single Strand Conformation Polymorphism; single chain conformation polymorphism). Analyzed cases are indicated by arrows.
(3) Hydrophobic plot of GDD1 protein: see FIG. Eight hydrophobic transmembrane sites are shown.

(4) Positional relationship of GDD1 protein with respect to membrane:
Refer to FIG. N-glycosylation sites, 8 cysteines fully conserved across species, endoplasmic reticulum localization signal (grey box), and mutated cysteines are shown, respectively.
(5) Amino acid sequences of GDD1 of various organisms:
See FIG. The amino acid sequence identity with humans, mice, zebrafish, Drosophila, and mosquitoes is 79%, 56%, 40%, and 41%. The eight cysteines present in the extracellular loop are conserved in all these species. Expected transmembrane sites are indicated by boxes. As a comparison of amino acid sequences between species, the same (*), highly conserved (:), and conserved (•) are shown.
(6) Expression analysis of GDD1 messenger RNA in human and mouse tissues:
See FIG. Northern blot analysis of messenger RNA in human multiple tissues [FIG. 5 (a)]. A 581 bp (base pair) GDD1 fragment labeled with ³² P of hGDD1 was analyzed as a probe, and further analyzed with beta actin labeled with ³² P.
FIG. 5 shows the results of RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for detecting messenger RNA of human tissue, healthy human osteoblasts, and healthy human periodontal ligament cells [FIG. 5 (b)]. . HOB1-4 is an osteoblast obtained from the mandible, HOB5 is an osteoblast obtained from the femur, and hPDL is a periodontal ligament cell obtained from the extracted tooth. These cells were cultured in α-minimum essential medium containing 10% fetal bovine serum, 50 μg / ml L-ascorbic acid, 50 U / ml penicillin, and 50 μg / ml streptomycin. Total RNA was extracted from cells subcultured 3-4 times. The 348 bp fragment of hGDD1 was PCR amplified with primers set in exons 9 and 12. The PCR amplification was performed by heating at 95 ° C. for 2 minutes, followed by 30 to 40 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds. hGAPDH messenger RNA analyzed with a 452 bp fragment of hGAPDH (human Glycero-Aldehyde Phosphate Dehydrogenase) cDNA was used as an internal standard.
The relative expression level of mGDD1 messenger RNA normalized with mGAPDH [FIG. 5 (c)]. A real time RT-PCR using a TaqMan system called 7900HT manufactured by ABI and Cyber Green Master Mix was used. Total RNA was isolated from 8-week-old mice and primary strand DNA was prepared using random primers. Spliced mGDD1 and mGAPDH were detected. The amplification product amplified from genomic DNA was excluded and examined. Standardized as relative amounts of mGDD1 messenger RNA to mGAPDH messenger RNA.

(7) Expression of GDD1 protein in COS-7 cells:
See FIG. The expression of wild type and mutant GDD1 was confirmed by Western blot [FIG. 6 (a)]. Plasmid hGDD-1-V5 expressing wild type GDD1, and a mutant GDD1 (meaning that cysteine at position 356 of C356R or C356G was mutated to arginine or glycine) were transfected with a plasmid with a −V5 epitope. Whole cell lysates from the introduced COS-7 cells were subjected to SDS-polyacrylamide gel electrophoresis. Western blotting was performed using an anti-V5 antibody [manufactured by Invitrogen (USA)]. As a control protein unrelated to GDD1, pcDNA6 / lacZ-V5 plasmid expressing lacZ-V5 was used.
Intracellular localization of wild-type GDD1 [FIG. 6 (b)]. The hGDD1 protein labeled with V5 clearly showed a mesh-like distribution pattern around the nucleus (green). The distribution pattern stained with an antibody against calreticulin present in the endoplasmic reticulum completely overlapped with the distribution pattern indicating the intracellular localization of wild-type GDD1. It was also shown that hGDD1 is localized in the endoplasmic reticulum.
Morphological change of COS-7 cells expressing mutant GDD1 [FIG. 6 (c)]. COS-7 cells that expressed mutant GDD1 showed a spherical morphology and cell adhesion was attenuated.
Hereinafter, the present invention will be specifically described with reference to experimental examples, reference examples, and examples. However, the present invention is not limited only to these experimental examples and examples.
(Experimental example 1)

Identification of genetic mutations that cause disease:
As a result of determining the base sequence of all the exons and the exon-intron boundary region of GDD1, the codon encoding the 356th cysteine present in the 11th exon was mutated from TGT to CGT, so that cysteine was replaced with arginine. Was confirmed in all 14 affected individuals in the Japanese GDD family, and none of the 8 healthy controls in the family had this mutation. In addition, in the African-American GDD family, the codon encoding cysteine of the 356th gene was mutated from TGT to GGT, and as a result, it was confirmed that cysteine was replaced with glycine in an affected person-specific manner [FIG. a) and (b)]. On the other hand, these mutations were not observed in a population of 488 healthy Japanese or 80 healthy African Americans. Since two different GDD families found GDD1 gene mutations specifically in affected individuals, it was confirmed that GDD1 is a disease-causing gene for GDD.
(Experimental example 2)

GDD1 protein structure, motif, and homology with mouse genes:
There is no known protein showing homology to the GDD1 protein. There are 8 transmembrane regions showing hydrophobicity, and the N-terminus and C-terminus of the protein are both present in the cell, and there are sites that may cause N-glycosylation of 5 proteins. It has an endoplasmic reticulum localization signal (alanine-lysine-serine-threonine) at the C-terminus [FIG. 3 (b)].
The mouse GDD1 cDNA sequence and the human GDD1 cDNA sequence show 79% identity at the amino acid level and are encoded as amino acids 342, 353, 356, 360, 369, 601, 606, and 804. Two cysteines are conserved in humans, mice, zebrafish, Drosophila, and mosquitoes, and are thought to be involved in the extracellular protein loop structure (FIG. 4).
(Experimental example 3)

Gene expression analysis:
As a result of Northern blotting of human multi-tissue, the fragment of 3 ′ end 581 bp (base pair) of human GDD1 cDNA was used as a labeled probe. As a result, two signals of 7.5 kb and 4.5 kb were detected as skeletal muscle, cardiac muscle, pancreas. To admit. RT-PCR (Reverse Transcription Polymerase Chain Reaction) that reverse-transcribes messenger RNA to cDNA and quantifies the amount of messenger RNA. Compares human GDD1 to brain, myocardium, kidney, lung, and skeletal muscle in 30 cycles of PCR. Detected strongly. Osteoblasts collected and cultured from humans and periodontal ligament cells were detected relatively weakly in 40 cycles of PCR. In RT-PCR using mouse tissue, strong expression of mouse GDD1 gene was observed in the calvaria, femur, and mandible [FIGS. 5 (a) to (c)].
(Experimental example 4)

Artificial expression and subcellular localization of GDD1:
As a result of analyzing the human GDD1 expressed in COS-7 cells by Western blot, the GDD1 protein was about 110 kDa. Since the intracellular expression of calreticulin, which is specifically expressed in the endoplasmic reticulum, overlapped with the intracellular expression of GDD1, it was revealed that GDD1 is expressed in the endoplasmic reticulum. Moreover, unlike the expression of wild-type GDD1, the expression of mutant GDD1 spheroidized COS-7 cells and reduced cell adhesion to the culture dish [FIGS. 6 (a) to (c). ].
(Reference Example 1)

Function prediction: Functions of the GDD1 protein present in the endoplasmic reticulum membrane may be related to protein processing, protein folding, calcium homeostasis, and the like. Although there is no ATP binding site in GDD1 protein, it may be involved in transport of low molecular weight substances such as calcium ions through the membrane.

Detection of two missense mutations (1):
Detection was performed by the direct sequencing method. The following pairs were used as primers:
Forward: 5'-TTACCTAAAACCACTCATCACTG-3 'and Reverse: 5'-TATATGGAGGCAAAAACAACAC-3'.
The PCR product of the above primer pair was enzymatically treated with ExoSAP-IT [Amersham Biosciences (USA)], and then ABI PRISM 3100 Genetic Analyzer and BigDye Terminator v1.1 Cycle Sequencing Kit (American Sequencing Kit, USA) were used. Sequenced directly.

Detection of two missense mutations (2):
Detection was performed by PCR-SSCP method. The following pair of primers labeled with 6-FAM was used:
5'-TTACCTAAAACCACTCATCACTG-3 'and 5'-CAAGTCTTTTCAACATGTTAC-3'
The PCR product using the above primers was electrophoresed on ABI377 (Applied Biosystems (USA))].

The present invention includes bone diseases including GDD, bone formation, bone tissue regeneration, hard tissue reconstruction, regulation and mechanism elucidation of bone differentiation, cell membrane transport of small molecules, protein processing and folding, drug discovery, etc. Available to: Applications include basic research reagents, clinical tests, diagnostic agents, clinical test agents, genetic diagnostic agents, therapeutic agents, gene therapeutic agents, bone filling materials, bone tissue regeneration promoting materials, etc., pharmaceuticals, dental materials, regenerative medicine It is useful in the manufacture of fields such as materials and experimental reagents.

Indicates the physical positional relationship between the GDD disease-causing gene existing region and GDD1. Exons are shown as filled boxes and untranslated regions are shown as gray boxes. A missense mutation of cysteine (C) at position 356 of GDD1 is shown. Analyzed cases are indicated by arrows. The hydrophobicity plot of the GDD1 protein [FIG. 3 (a)], the positional relationship of the GDD1 protein to the membrane [FIG. 3 (b)], and the endoplasmic reticulum localization signal are shown in gray boxes. The amino acid sequences of GDD1 of various organisms are shown. Expected transmembrane sites are indicated by boxes. As comparison of amino acid sequences between species, the same (*), highly conserved (:), and conserved (•) are shown respectively. Figure 3 shows expression analysis of GDD1 messenger RNA in human and mouse tissues. Northern blot analysis of messenger RNA in human multiple tissues [FIG. 5 (a)], results of RT-PCR method for detecting messenger RNA in human tissues, healthy human osteoblasts, and healthy human periodontal ligament cells [FIG. (B)], and the relative expression level of mGDD1 messenger RNA normalized with mGAPDH [FIG. 5 (c)]. The expression of GDD1 protein in COS-7 cells is shown. Western blot image of wild type and mutant GDD1 expression [FIG. 6 (a)], microscopic image of intracellular localization of wild type GDD1 [FIG. 6 (b)], and COS-7 cells expressing mutant GDD1. Microscopic image of morphological change [FIG. 6 (c)].

Claims (13)

  Selected from the sequence group consisting of the full-length nucleotide sequence of the gene DNA described in SEQ ID NO: 1, a partial nucleotide sequence that is a fragment thereof, and a nucleotide sequence in which at least one of these nucleotide sequences has been substituted based on degeneracy of codons DNA having at least one base sequence. An RNA having an RNA base sequence complementary to the DNA base sequence according to claim 1.   A bone disease gene test agent containing an amount of reaction using the DNA according to claim 1 or the RNA according to claim 2 as a reaction reagent.   The bone disease genetic test drug according to claim 3, wherein the bone disease is GDD and / or bone defect and / or bone resorbable disease. A primer designed based on the full-length base sequence according to claim 1. A probe designed on the basis of the full-length base sequence according to claim 1. A bone disease gene therapeutic agent designed and constructed so that the DNA of claim 1 is expressed in vivo.   The bone disease gene therapeutic agent according to claim 7, wherein the bone disease is GDD and / or bone deficiency and / or bone resorbable disease. At least selected from the group consisting of the full-length amino acid sequence set forth in SEQ ID NO: 2, a partial amino acid sequence that is a fragment thereof, and an amino acid sequence in which at least one amino acid residue of these amino acid sequences is substituted, deleted, or inserted A protein having one amino acid sequence.   The protein according to claim 9 having the amino acid sequence of the following SEQ ID NO: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 . A bone disease therapeutic agent comprising the protein according to claim 9 or 10 as an active ingredient in an amount having a medicinal effect. The bone disease therapeutic agent according to claim 11, wherein the bone disease is GDD and / or bone defect and / or bone resorbable disease. An antibody against the protein according to claim 9 or 10.

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