JP4830101B2 - Costello syndrome-causing gene mutation and detection or diagnosis method thereof - Google Patents

Description

  The present invention relates to a mutation in the base sequence of the H-Ras gene associated with Costello syndrome and the amino acid sequence encoded thereby, a method for detecting the mutation, a method for diagnosing Costello syndrome using the detection method, and the like.

Costello syndrome is a disease first reported in 1971 by JM Costello, New Zealand (Non-Patent Document 1). Characteristic facial features, mental retardation, curled hair, neonatal feeding disorders, cardiac complications (hypertrophic cardiomyopathy / arrhythmia), loose skin on the limbs, abnormal position of the ankle joint, congenital syndrome showing tumor favor It is. About 115 cases have been reported by 2003, but there are few siblings, and autosomal dominant inheritance with mutations is predicted, but the cause is unknown (Non-patent Document 2).

The causative gene of Noonan syndrome, a related disease of Costello syndrome, was identified in 2001 (Non-patent Document 3). The PTPN11 gene, the causative gene of Noonan syndrome, encodes cytoplasmic tyrosine phosphatase SHP-2, and a mutation was identified in about 50% of Noonan syndrome patients. The identified mutation was a gain-of-function mutation that increased phosphatase activity (Non-Patent Documents 4 to 6). Since the PTPN11 mutation was not identified in Costello syndrome, the causes of these related diseases were predicted to be different (Non-patent Documents 7 and 8).
Costello, J. A new syndrome.NZ Med J 74, 397 (1971) Hennekam, RC Costello syndrome: an overview. Am J Med Genet C Semin Med Genet 117, 42-8 (2003) Tartaglia, M. et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 29, 465-8 (2001) Araki, T. et al. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 10, 849-57 (2004) Niihori, T. et al. Functional analysis of PTPN11 / SHP-2 mutants identified in Noonan syndrome and childhood leukemia. J Hum Genet 50, 192-202 (2005) Tartaglia, M. et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 34, 148-50 (2003) Troger, B. et al. No mutation in the gene for Noonan syndrome, PTPN11, in 18 patients with Costello syndrome. Am J Med Genet A 121, 82-4 (2003) Tartaglia, M., Cotter, PD, Zampino, G., Gelb, BD & Rauen, KA Exclusion of PTPN11 mutations in Costello syndrome: further evidence for distinct genetic etiologies for Noonan, cardio-facio-cutaneous and Costello syndromes.Clin Genet 63 , 423-6 (2003)

Therefore, an object of the present invention is to find a genetic mutation that causes Costello syndrome and to provide a detection method, a diagnostic method, and the like based thereon.

In order to solve the above problems, the present inventor paid attention to the fact that SHP-2 activates RAS / MAP kinase in signal transduction such as growth factors, and the cause of Costello syndrome is upstream of SHP-2 in the signal transduction system, Or we hypothesized that it is a downstream molecule. Based on this, the sequence of H-RAS, K-RAS, N-RAS and E-RAS, which are the superfamily of Ras, which is an oncogene, was compared against the genomic DNA of 28 Noonan syndrome and 13 Costello syndrome without PTPN11 mutation. went. As a result, the inventors found a genetic mutation that causes Costello syndrome and completed the present invention.

That is, the present invention relates to the following aspects.
[1] A polynucleotide or oligonucleotide comprising at least a part of the base sequence of the H-Ras gene shown in SEQ ID NO: 1 and having at least one of the following mutations:
(A) the 34th g is replaced with a;
(B) the 35th g is replaced with c;
(C) 35th g is replaced with t, 36th c is replaced with t; and (d) 38th g is replaced with a.
[2] A polynucleotide or a polynucleotide according to aspect 1 that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of the polynucleotide or oligonucleotide according to aspect 1 A polynucleotide or oligonucleotide consisting of DNA that can be used to detect the presence of an oligonucleotide.
[3] The polynucleotide or oligonucleotide according to the above aspect 1 or 2, consisting of 10 to 100 consecutive base sequences.
[4] A gene amplification primer comprising the polynucleotide or oligonucleotide according to any one of the above aspects 1 to 3.
[5] A hybridization probe comprising the polynucleotide or oligonucleotide according to any one of the above aspects 1 to 3.
[6] A polypeptide or oligopeptide comprising at least part of the expression product of the H-Ras gene and having at least one of the following mutations:
(A) the 12th G (glycine) is replaced with A (alanine);
(B) the 12th G (glycine) is replaced with S (serine);
(C) 12th G (glycine) is replaced with V (valine); and (d) 13th G (glycine) is replaced with D (aspartic acid).
[7] The polypeptide or oligopeptide according to aspect 6 above, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added except for the four substitution sites described in aspect 6 above. And the polypeptide or oligopeptide which functions as an antigen of the antibody which recognizes the polypeptide or oligopeptide of the said aspect 6.
[8] An antibody that recognizes the polypeptide or oligopeptide according to the above aspect 6 or 7.
[9] A method for detecting an H-Ras gene mutation by detecting the presence of the polynucleotide or oligonucleotide according to any one of Embodiments 1 to 3.
[10] A method for detecting an H-Ras gene mutation by detecting the presence of the polypeptide or oligopeptide according to the above aspect 6 or 7.
[11] A measurement system for carrying out the detection method according to the above aspect 9 or 10.
[12] The measurement system according to the above aspect 11, comprising the gene amplification primer according to the above aspect 4.
[13] The measurement system according to the above aspect 11, which includes the hybridization probe according to the above aspect 5.
[14] The measurement system according to the above aspect 11, comprising the antibody according to the above aspect 8.
[15] A method for diagnosing Costello syndrome, which uses the detection method according to the above aspect 9 or 10.

The present invention revealed that germline mutations in H-RAS cause Costello syndrome, a congenital malformation syndrome, and have been found to play an important role in human development of H-RAS. Furthermore, the present invention has established a genetic diagnosis method for Costello syndrome, which enables a definitive diagnosis of Costello syndrome.

  The inventor has identified H-RAS gene mutations found in patients with Costello syndrome, as shown in Table 2 herein. Specific mutations at the base level of these mutations are included in the polynucleotide or oligonucleotide according to the first aspect of the present invention, and specific mutations at the amino acid level are the polymorphs according to the sixth aspect of the present invention. It is contained in a peptide or oligopeptide. The positions of these mutations are such that the first base of the “atg” start codon in the coding region of the gene has a base number of 1, and the corresponding methionine has an amino acid number of 1.

In the present invention, “polynucleotide” and “oligonucleotide” refer to a phosphate ester of a nucleoside in which purine or pyrimidine is β-N-glycoside-linked to a sugar (nucleotide ATP, GTP, CTP, UTP; ) Means a molecule in which multiple bonds are bound. In general, a combination of several to several tens of nucleotides is called an oligonucleotide, and a combination of more nucleotides is called a polynucleotide. There is no strict distinction. The polynucleotide and oligonucleotide in the present invention are single-stranded or double-stranded, and in the case of single-stranded, it is either a sense strand or an antisense strand.

The polynucleotide or oligonucleotide of the present invention uses genomic DNA or mRNA prepared from an appropriate sample such as blood cells obtained from a Costello syndrome patient as a template. For example, for exon 1 of the H-RAS gene shown in Table 1 PCR (Polymerase Chain Reaction) method or RT-PCR method using appropriate primers, NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method and SDA (Strand Displacement Amplification) method, etc. The gene can be amplified by any gene amplification method known to those skilled in the art.

Furthermore, the polynucleotide or oligonucleotide of the present invention is screened by a method well known to those skilled in the art using a cDNA library prepared from the total mRNA of an appropriate sample obtained from a Costello syndrome patient using the oligonucleotide of the present invention as a probe. Can be isolated. Alternatively, it is also possible to prepare a cDNA of a wild type H-RAS gene by introducing a base mutation using a commercially available mutation system or the like based on site-directed mutagenesis known to those skilled in the art.

In addition, the oligonucleotide of the present invention can be obtained by a known method (for example, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47: 411-418; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; US Pat. No. 4,458,066). Can also be synthesized. It can also be produced by a method such as cleaving the polynucleotide of the present invention with an appropriate restriction enzyme.

The polynucleotide or oligonucleotide of the present invention is hybridized under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of the above-described polynucleotide or oligonucleotide, and described in the above aspect 1. Also included are polynucleotides or oligonucleotides consisting of DNA that can be used to detect the presence of a polynucleotide or oligonucleotide. In the present specification, “stringent conditions” in hybridization are conditions that enable selective and detectable specific binding between the polynucleotide or oligonucleotide and genomic DNA. Stringent conditions are defined by a suitable combination of salt concentration, organic solvent (eg, formamide), temperature, and other known conditions. That is, stringency increases depending on whether the salt concentration is reduced, the organic solvent concentration is increased, or the hybridization temperature is increased. In addition, washing conditions after hybridization also affect stringency. This wash condition is also defined by salt concentration and temperature, and the stringency of the wash increases with decreasing salt concentration and increasing temperature. Therefore, a preferred specific example of stringent conditions is that hybridization is performed under the conditions of 750 mM NaCl, 75 mM trisodium citrate, 1% SDS, and 30 ° C., and 30 mM NaCl, 3 mM tricitrate. Washing is performed under conditions of sodium, 0.1% SDS, and 25 ° C.

In addition, such “polynucleotide or oligonucleotide comprising DNA that can be used to detect the presence of a polynucleotide or oligonucleotide” refers to amplification primers and hybridization probes in the various detection methods described below. It means that it can be used as a probe to detect and identify H-RAS gene mutations found in patients with Costello syndrome.

The polynucleotide or oligonucleotide of the present invention is useful as a component of various measurement systems used in the method for detecting an H-Ras gene mutation. Typical examples include gene (DNA) amplification primers and hybridization probes. Therefore, the polynucleotide or oligonucleotide of the present invention comprises an appropriate length, for example, 10 to 100 consecutive base sequences, depending on the application.

In the present invention, “polypeptide” and “oligopeptide” mean molecules composed of a plurality of amino acid residues bonded to each other by amide bonds (peptide bonds). Usually, a combination of several to several tens of amino acid residues is called an oligopeptide, and a combination of more than one amino acid residue is called a polypeptide. There is no strict distinction according to the degree of polymerization.

These polypeptides or oligopeptides are then isolated according to any method known to those skilled in the art when included in an appropriate sample obtained from a Costello syndrome patient as the expression product of the polynucleotide or oligonucleotide of the invention. A method for preparing a peptide by any chemical synthesis known to those skilled in the art based on information on the amino acid sequence including each mutated amino acid residue, or a suitable recombinant vector containing the polynucleotide or oligonucleotide of the present invention It can be prepared by a method in which an appropriate host cell is transformed by a recombinant DNA technique using the above and the like and produced by the transformed cell. Furthermore, an oligopeptide can be produced by digesting the polypeptide of the present invention with an appropriate protease.

As long as the polypeptide or oligopeptide of the present invention functions as an antigen of an antibody that recognizes the above-mentioned polypeptide or oligopeptide, in addition to the four types of mutations derived from H-Ras gene mutations specific to Costello syndrome patients, In addition, one or several amino acids may be deleted, substituted, or added. That is, such a mutation may be contained in a site other than the epitope recognized by the antibody. Such a polypeptide or oligopeptide is prepared not only by a method of directly preparing a peptide by chemical synthesis, but also by a site-directed mutagenesis method or the like which has already been described for a polynucleotide or oligonucleotide encoding the same. It can also be prepared by expressing in an appropriate system known to those skilled in the art.

The antibody of the present invention specifically recognizes the polypeptide or oligopeptide of the present invention. The antibody of the present invention is a polyclonal antibody or a monoclonal antibody, and is not limited to an antibody having the original complete structure as long as it has such a binding activity, for example, Fab, F (ab ′) ₂ , Fv All the various derivatives derived from complete antibodies such as fragments are included. These various antibodies or derivatives thereof are useful as components of various measurement systems used in the detection method of H-Ras gene mutation.

The antibody of the present invention can be prepared by any method known to those skilled in the art using the above polypeptide or oligopeptide, or a complex of these and an appropriate adjuvant as an immunogen. For example, in the case of a polyclonal antibody, it can be obtained from serum after immunizing the animal with the above immunogen. Alternatively, it can be prepared by introducing an expression vector encoding the above-mentioned immunogen into the muscle or skin of an animal by injection or gene gun and then collecting the serum. As the animal, mouse, rat, rabbit, goat, chicken and the like are used. Monoclonal antibodies are known monoclonal antibody production methods ("monoclonal antibodies", Kamei Nagamune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996) Can be created according to.

  The H-Ras gene mutation detection method of the present invention is characterized by detecting the presence of the above-mentioned polynucleotide or oligonucleotide, or polypeptide or oligopeptide in various samples such as blood of a subject. This detection can be performed by any conditions and methods known to those skilled in the art depending on the measurement target.

  For example, the presence of the polynucleotide or oligonucleotide of the present invention is detected in amplification products obtained by using various PCR methods such as the PCR method and RT-PCR method already described, and various gene amplification methods such as the ICAN method. can do. Such detection in the amplified product can be performed by, for example, PCR-SSCP method, PCR-CFLP method, PCR-PHFA method, etc. in addition to the method for directly determining the base sequence (sequence method). Alternatively, using various methods used in snip typing such as Taqman PCR, Invader method, RCA method, and microarray (DNA chip) method using a hybridization reaction, for example, The presence of the polynucleotide or oligonucleotide of the present invention can also be detected.

The system (apparatus or kit) of the present invention used for such a detection method can take an appropriate configuration depending on the specific method and means for measuring the mutation in the base sequence. The system can include, for example, a gene amplification primer and a hybridization probe comprising the polynucleotide or oligonucleotide of the present invention. Or it contains the primer which consists of a base sequence of the appropriate length used in order to amplify the oligonucleotide or polynucleotide of this invention. These consist of a continuous base sequence of an appropriate length, for example, 10 to 100, depending on the application.

In addition, the presence of the polypeptide or oligopeptide of the present invention can be determined, for example, by a method using an immunological specific reaction such as EIA or ELISA using the antibody of the present invention, an amino acid of a peptide such as a gas phase sequencer using the Edman method, etc. It can be detected by sequence analysis, and further by mass spectrometry represented by MALDI-TOF / MS and ESI Q-TOF / MS methods.

Various primers, probes, or antibodies included as constituent elements in the above system may be labeled with an appropriate labeling substance such as any radioactive substance, fluorescent substance, and dye known to those skilled in the art. Further, the system includes other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), and the like, depending on the configuration and purpose of use.

  When the detection method of the present invention detects the presence of a polynucleotide or oligonucleotide containing a specific mutation, or a corresponding polypeptide or oligopeptide in a sample derived from a subject, the subject has Costello syndrome. You can diagnose it.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. Also, unless otherwise stated, the following examples are described in, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols. It can be performed according to standard genetic engineering and molecular biology techniques known to those skilled in the art, as described in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. Moreover, the description content of the literature referred in this specification comprises some disclosure content of this specification.

Method 1) Purification of genomic DNA Genomic DNA was purified from leukocytes, cultured cells, and tissues in patient blood according to a conventional method (Promega DNA extraction kit).
2) PCR of all exons of RAS gene
PCR of exons in the protein translation region of H-RAS, K-RAS, N-RAS and E-RAS was performed using 100 ng of patient genomic DNA. Table 1 shows the deposit number (GenBank: NCBI) exon of each gene, the base sequence of each primer used, and the aryling temperature. The composition of the reaction solution (30 μl) is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM dNTP, 10% (v / v) DMSO, 0.4 pmol of each primer, 100 ng genomic DNA and 2.5 units of Taq DNA polymerase. The PCR reaction was repeated 35 times with an amplification reaction cycle consisting of denaturation at 94 ° C. for 15 seconds; temperature shown in Table 1, annealing at 15 seconds; and extension at 72 ° C. for 30 minutes.
3) Sequence reaction
The PCR product was run on an agarose gel and cut out and purified. The purified product was sequenced by ABI PRISM 310 automated DNA sequencer (Applied Biosystems) and the nucleotide sequence was determined. Gene mutations were found by comparing the sequence results with the registered sequences in Genebank.

Results and discussion H-RAS gene mutations were identified in 12 out of 13 Costello syndromes (Table 2). Mutations were concentrated in codons 12 and 13, found in 7 patients with G12S, 2 with G12A, 1 with G12V, and 2 with G13D. These mutations exist in exon 1 of the H-RAS gene. It has been reported so far that Costello syndrome is mostly sporadic and less hereditary. In order to investigate whether these mutations were caused by mutations, when parents' genomic DNA was searched in four families, no mutations were identified in the parents (Table 2). Next, in order to verify whether the mutation was a germ cell mutation, the mutation was examined in buccal cells other than blood cells (buccal cells) or fibroblasts (COS38, 62, 37, 64). ). As a result, mutations were identified in two tissues, and it was confirmed that the mutation was a germ cell mutation caused by the mutation (Table 2). Incidentally, the RAS mutation was the first oncogene discovered in 1982, and somatic mutations have been identified in about 30% of all cancers (Takai, Y., Sasaki, T. & Matozaki, T Small GTP-binding proteins. Physiol Rev 81, 153-208 (2001))

No mutations were identified in H-RAS, K-RAS, N-RAS, or E-RAS in 28 Noonan syndrome patients without PTPN11 mutation and 1 Costello syndrome. Noonan syndrome and Costello syndrome were known as related diseases with overlapping phenotypes, but the present invention identified two mutations in molecular genetics by identifying H-RAS gene mutations in Costello syndrome alone. Proved to be a different disease. In addition, it was revealed that activation of RAS / MAP kinase causes multiple malformation syndrome because both causative genes are mutations that activate the RAS / MAP kinase system of the signal transduction system.

Until now, Costello syndrome has been diagnosed with a combination of clinical symptoms, so the exact frequency and natural history were unknown. Establishing a genetic diagnosis method for Costello syndrome according to the present invention enables a definitive diagnosis of Costello syndrome. Although it has been suggested that about 17% of Costello syndromes have tumors, it is difficult to study tumor screening methods due to lack of an accurate diagnosis (Gripp, KW et al. Five additional Costello syndrome patients with rhabdomyosarcoma : proposal for a tumor screening protocol. Am J Med Genet 108, 80-7 (2002)). In the future, an accurate diagnosis will enable the study of the rate of tumor complications of Costello syndrome, screening methods for its prevention, and approaches to treatment.

Claims (7)

H-Ras by detecting the presence of a polynucleotide or oligonucleotide comprising at least part of the base sequence of the H-Ras gene shown in SEQ ID NO: 1 and having any one mutation selected from the following group: A method for detecting a gene mutation and determining that a patient having the H-Ras gene mutation is likely to have Costello syndrome based on a criterion that the H-Ras gene mutation is identified in Costello syndrome but not in Noonan syndrome :
(A) the 34th g is replaced with a;
(B) the 35th g is replaced with c;
(C) 35th g is replaced with t, 36th c is replaced with t; and (d) 38th g is replaced with a. Hybridization with DNA comprising a base sequence complementary to the DNA comprising the base sequence of the polynucleotide or oligonucleotide according to claim 1 under stringent conditions, and detecting the H-Ras gene mutation according to claim 1 The method according to claim 1, wherein a polynucleotide or oligonucleotide comprising DNA that can be used is used. Used as polynucleotide or oligonucleotide primers for gene amplification comprising DNA of claim 2 which is capable of detecting the H-Ras gene mutations of claim 1, method of claim 2. Used as polynucleotide or probe for hybridization to an oligonucleotide comprising DNA of claim 2 which is capable of detecting the H-Ras gene mutations of claim 1, method of claim 2. Detecting the presence of a polypeptide or oligopeptide comprising at least part of the expression product of the H-Ras gene comprising the amino acid sequence shown in SEQ ID NO: 2 and having any one mutation selected from the following group: Detects a H-Ras gene mutation, and based on the criteria that the H-Ras gene mutation is identified in Costello syndrome but not in Noonan syndrome, patients with the H-Ras gene mutation are likely to have Costello syndrome How to determine:
(A) the 12th G (glycine) is replaced with A (alanine);
(B) the 12th G (glycine) is replaced with S (serine);
(C) 12th G (glycine) is replaced with V (valine); and (d) 13th G (glycine) is replaced with D (aspartic acid). Including gene amplification primer of claim 3, the measuring system for carrying out the method of claim 3. Including hybridization probe according to claim 4, measurement system for implementing the method according to claim 4.