JP2019216658A - Method for determining presence or risk of invasive periodontitis - Google Patents

Abstract

To identify the causative gene of invasive periodontitis and provide a method for diagnosing invasive periodontitis and a model animal of invasive periodontitis.SOLUTION: The present disclosure provides a method for determining the presence or risk of invasive periodontitis in a subject, the method comprising detecting a mutation in the MMD2 gene in a sample obtained from a subject, and/or measuring the number of neutrophils in the sample obtained from the subject. The present disclosure also provides a model animal of invasive periodontitis, which is a non-human animal having a mutation deficient in the function of MMD2 or a non-human animal having a c.347C<T mutation of the MMD2 gene.SELECTED DRAWING: None

Description

  The present disclosure relates to the diagnosis of invasive periodontitis. The present disclosure also relates to an animal model of invasive periodontitis.

  Invasive periodontitis is a periodontitis that affects young people in their 10s and 30s, and is characterized by rapid and severe periodontal tissue destruction. It is known that there are cases where invasive periodontitis accumulates in families, but the pathogenesis mechanism at the molecular or gene level has not been sufficiently elucidated. The current treatment for invasive periodontitis is symptomatic treatment, and there may be cases of poor progression.Therefore, there is a strong demand for diagnosis as early as possible and development of treatments based on the pathogenesis. . For example, among patients with invasive periodontitis, there are patients with neutrophil dysfunction, and it is proposed to diagnose invasive periodontitis using genes highly expressed in neutrophils in such patients (Patent Document 1).

JP 2008-206483 A

  An object of the present disclosure is to identify a causative gene of invasive periodontitis, and to provide a method of diagnosing invasive periodontitis, a model animal of invasive periodontitis, and the like.

The present disclosure is a method of determining the presence or risk of invasive periodontitis in a subject, comprising:
Detecting a mutation in the MMD2 gene in a sample obtained from the subject, and / or determining a neutrophil count in the sample obtained from the subject;
A method comprising:

  The present disclosure also provides a primer for amplifying a region containing the mutation position of the MMD2 gene, a probe that specifically binds to the region containing the mutation position of the MMD2 gene, and a kit containing the primer and / or the probe.

  The present disclosure also provides a diagnostic marker for invasive periodontitis comprising a mutant MMD2 gene or mutant MMD2, and the use of a mutant MMD2 gene or mutant MMD2 as a diagnostic marker for invasive periodontitis.

  The present disclosure also relates to a non-human animal having a mutation deficient in MMD2 function or a non-human animal having a c.347C <T mutation of the MMD2 gene, a model animal of invasive periodontitis, and the non-human animal. Provided is a use as a model animal for invasive periodontitis.

  INDUSTRIAL APPLICABILITY The present disclosure is useful for early diagnosis of invasive periodontitis, elucidation of the pathogenesis, development of prophylactic or therapeutic agents, and the like.

FIG. 1 shows family information of an invasive periodontitis patient analyzed in the example. Black indicates a patient with invasive periodontitis, and white indicates a non-patient. C / C and C / T indicate the genotype at position 347 of the coding DNA of the MMD2 gene. Squares indicate men, circles indicate women, and diagonal lines indicate deaths. * Indicates the subject of the exome sequence. Arrows indicate probands. FIG. 2 shows the TALEN target site on mouse chromosome 5. Bases corresponding to the c.347C <T mutation of the human MMD2 gene are boxed and the TALEN target sequence is underlined. FIG. 3 shows the sequence of the ssODN and the sequence of the knock-in (KI) allele. T: Target base substitution, TAG: Base substitution for RFLP (Restriction Fragment Length Polymorphism). The TALEN target sequence is underlined. FIG. 4 shows genomic sequences of a wild-type (WT) mouse and a genetically modified mouse obtained by genome editing. Underlining indicates base substitution or insertion. Squares indicate TALEN target sequences. FIG. 5 shows the evaluation site of experimental periodontitis. From the left, the measurement points of the third molar mesial, the second molar distal and mesial, and the first molar distal are shown. FIG. 6 shows alveolar bone resorption during periodontitis induction in WT mice (WT / WT), KI mice (KI / WT, KI / KI), and knockout (KO) mice (KO / KO). Non-treated refers to untreated mice, Treated refers to mice ligated with silk. FIG. 7 shows the distance (CEJ-ABC, mm) from the cement enamel junction to the alveolar bone dehiscence (sum of four measurement points). FIG. 8 shows the number of colonies induced by granulocyte colony stimulating factor (G-CSF) stimulation from bone marrow nucleated cells of WT mice, KI mice (KI / KI), and KO mice (KO / KO).

  Unless otherwise specified, terms used herein have the meanings as commonly understood by one of ordinary skill in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, and the like. The following provides definitions for some of the terms used herein, but these definitions supersede general understanding herein.

  Periodontitis (Periodontitis) is an inflammatory destructive disease that occurs in periodontal tissue due to bacteria and the like. Aggressive periodontitis is a periodontitis that is systemically healthy, but is characterized by rapid periodontal tissue destruction (alveolar bone resorption, loss of adhesion) and family accumulation. It mainly occurs in the teens and thirties. The rate of tissue destruction is relatively slow, distinguishing it from chronic periodontitis, which mainly develops after age 35 years.

  MMD2 (monocyte to macrophage differentiation-associated 2) is also called PAQR (progestin and adipoQ receptor) 10. The genomic DNA sequence, mRNA sequence, and amino acid sequence of MMD2 are known, and are published in databases (http://www.ncbi.nlm.nih.gov/) provided by NCIB (National Center for Biotechnology Information). . For example, the GenBank accession numbers for the genomic DNA sequence, mRNA sequence, and amino acid sequence of human MMD2 are CH471144.1, NM_001100600.1, and AAH37881.2, respectively.

  In the present specification, a mutation in the MMD2 gene means a mutation that results in an amino acid sequence different from the amino acid sequence encoded by the wild-type MMD2 gene. Mutations in the MMD2 gene also include mutations in non-coding regions that inhibit expression of the MMD2 gene. In the present specification, the MMD2 gene having such a mutation is referred to as a mutant MMD2 gene, and the MMD2 encoded by the mutant MMD2 gene is referred to as a mutant MMD2. Mutations include base deletions, substitutions, and insertions. The mutation in the MMD2 gene can be a loss-of-function mutation of MMD2. The loss of function may be a complete or partial loss of function. The mutation in the MMD2 gene can be a mutation that results in a decrease in neutrophil count in the subject.

The cDNA and amino acid sequences of a typical human and mouse wild type MMD2 are shown in SEQ ID NOs: 1-4. The 347th position of SEQ ID NO: 1 and the corresponding position in SEQ ID NO: 3 (350th position), and the 116th position of SEQ ID NO: 2 and the corresponding position in SEQ ID NO: 4 (117th position) are indicated by squares.

  As used herein, the term “c.347C <T mutation” in the MMD2 gene means a C to T mutation in the MMD2 gene at a position corresponding to position 347 of the coding DNA of the human MMD2 gene. The position corresponding to position 347 of the coding DNA of the human MMD2 gene means the position corresponding to position 347 of SEQ ID NO: 1. The sequence of SEQ ID NO: 1 and the coding DNA of a certain MMD2 gene are optimally matched (sequence matching). Means the position in the coding DNA of the MMD2 gene corresponding to position 347 of SEQ ID NO: 1 when the alignment is performed (to a state where the maximum value is obtained). For example, in the present specification, the sequence of SEQ ID NO: 3 containing the c.347C <T mutation means a sequence in which C at position 350 in SEQ ID NO: 3 is mutated to T.

  In the present specification, the p.Ala116Val mutation of MMD2 means a mutation from Ala to Val at a position corresponding to the 116th amino acid sequence of human MMD2 in the MMD2. The position corresponding to position 116 in the amino acid sequence of human MMD2 refers to the position corresponding to position 116 in SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of a certain MMD2 are optimally matched. Means the position in the amino acid sequence of MMD2 corresponding to position 116 of SEQ ID NO: 2 when the alignment is performed (to a state where is the maximum). For example, in the present specification, the sequence of SEQ ID NO: 4 containing p.Ala116Val mutation means a sequence in which Ala at position 117 in SEQ ID NO: 4 is mutated to Val.

  As used herein, a subject includes a subject who has developed periodontitis and a subject who has not. Subjects who have not developed periodontitis include those who are predisposed to invasive periodontitis but have not yet developed periodontitis. Subjects with a predisposition to invasive periodontitis include those in a family with patients with invasive periodontitis. Subjects with periodontitis include those with periodontitis but who have not been diagnosed with invasive periodontitis. In certain embodiments, the subject is a subject without a systemic disease.

  In certain embodiments, if the subject has a mutation in the MMD2 gene, i.e., if a mutation in the MMD2 gene is detected in a sample obtained from the subject, the subject is suffering from invasive periodontitis or is invasive. It is determined that there is a risk of developing periodontitis. For example, if a mutation is detected in a subject that is predisposed to invasive periodontitis but has not yet developed periodontitis, the subject is determined to be at risk for developing invasive periodontitis. When a mutation is detected in a subject who has developed periodontitis but has not been diagnosed with invasive periodontitis, the subject is determined to be suffering from invasive periodontitis.

  That is, the mutant MMD2 gene and the mutant MMD2 are diagnostic markers for invasive periodontitis. In certain embodiments, the mutant MMD2 gene comprises a c.347C <T mutation, wherein the sequence of SEQ ID NO: 1 or 3 is combined with 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. % Or a sequence having a sequence identity of 99% or more. In another embodiment, the mutant MMD2 gene comprises a c.347C <T mutation, wherein in the sequence of SEQ ID NO: 1 or 3, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0-60, 0-50, 0-40, 0-30, 0-20, 0-10, 0-5, 0-3, 0-2, or 0-1 Comprises or consists of a sequence in which the bases of SEQ ID NO: have been deleted, substituted, inserted or added. In a further embodiment, the mutant MMD2 gene comprises or consists of the sequence of SEQ ID NO: 1 or 3 comprising a c.347C <T mutation.

  In certain embodiments, the mutated MMD2 gene comprises a p.Ala116Val mutation and 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or a sequence of SEQ ID NO: 2 or 4. It comprises or consists of a sequence encoding a mutant MMD2 containing a sequence having 99% or more sequence identity. In another embodiment, the mutant MMD2 gene comprises a p.Ala116Val mutation, and in the sequence of SEQ ID NO: 2 or 4, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 5, It comprises or consists of a sequence encoding a mutant MMD2 containing a sequence in which 3, 0 to 2 or 0 to 1 amino acids have been deleted, substituted, inserted or added. In a further embodiment, the mutant MMD2 gene comprises or consists of a sequence encoding a mutant MMD2 comprising the sequence of SEQ ID NO: 2 or 4 comprising the p.Ala116Val mutation.

  In certain embodiments, the mutant MMD2 comprises the p.Ala116Val mutation and has 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 2 or 4. % Or has a sequence identity of at least%. In another embodiment, the mutant MMD2 comprises a p.Ala116Val mutation, wherein in the sequence of SEQ ID NO: 2 or 4, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, , 0-2, or 0-1 amino acids, including or consisting of a deleted, substituted, inserted or added sequence. In a further embodiment, the mutant MMD2 comprises or consists of the sequence of SEQ ID NO: 2 or 4 comprising the p.Ala116Val mutation.

  Sequence identity refers to the degree of similarity between two sequences and is determined by comparing two sequences that are optimally aligned (maximum sequence identity) over the region of the sequence being compared. Is done. The sequence identity number (%) is obtained by determining the number of matching bases or amino acid residues between the two sequences and dividing this number by the total number of bases or amino acid residues in the sequence region to be compared. It is calculated by multiplying the calculated value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those of skill in the art (eg, BLAST algorithm, FASTA algorithm, etc.). Sequence identity is determined using sequence analysis software such as, for example, BLAST, FASTA.

  The sample obtained from the subject for detecting the mutation is not particularly limited as long as the nucleic acid or polypeptide derived from the subject can be obtained. Samples include blood, plasma, serum, excised tissue during surgery, saliva, and the like. Usually, from these samples, samples containing nucleic acids (eg, genomic DNA, mRNA, or cDNA prepared from mRNA) or polypeptides from the subject are obtained and used to detect mutations.

  Mutations in the MMD2 gene can be detected by various methods. For example, the sequence of a nucleic acid such as genomic DNA or cDNA or an amplification product thereof is determined; an allele-specific probe is bound to the nucleic acid; or the nucleic acid is amplified using an allele-specific primer. , Can be detected. Examples of mutation detection methods include Sanger sequencing, PCR-RFLP (restriction fragment length polymorphism), PCR-SSCP (single strand conformation polymorphism), PCR-SSO (sequence oligonucleotide), and ASP-PCR (Allele Specific Primer- PCR) method, TaqMan (registered trademark) -PCR method, Invader (registered trademark) method, cyclin probe method, a method using a DNA chip or a microarray, and the like. Mutations in the MMD2 gene can also be detected by detecting amino acid mutations caused by the mutation (ie, by detecting mutant MMD2), for example, an antibody that specifically binds to mutant MMD2 ELISA, Western blotting, and protein sequencing can be used.

  In one embodiment, the c.347C <T mutation in the MMD2 gene is detected. The c.347C <T mutation of the MMD2 gene may be obtained, for example, by amplifying a region containing the position corresponding to position 347 of SEQ ID NO: 1 from the nucleic acid of the subject, or in the nucleic acid of the subject or its amplified product according to SEQ ID NO: 1. Can be detected by determining the sequence of a region including the position corresponding to the 347th position, or by binding a probe that specifically binds to the region to a nucleic acid derived from a subject or an amplification product thereof. The c.347C <T mutation of the MMD2 gene may be detected by detecting a mutant MMD2 containing a p.Ala116Val mutation. The mutant MMD2 containing the p.Ala116Val mutation can be detected, for example, by using an antibody prepared using a region containing the p.Ala116Val mutation as an epitope.

  Primers used for mutation detection amplify a region containing the mutation position of the MMD2 gene. The primer usually has a length of 10 to 70 bases, preferably 15 to 60 bases, and more preferably 20 to 50 bases. The region to be amplified is appropriately determined depending on the detection method, and is, for example, 10 to 1000 bases, 20 to 500 bases, 30 to 300 bases, or 50 to 100 bases. Primers are usually complementary to a portion of the sequence (including coding and non-coding sequences) of the wild-type or mutant MMD2 gene or its complement, but contain mismatches as long as the region of interest can be amplified. May be. The number of mismatches is, for example, 1-3, 1-2 or 1. Primers should be designed to amplify the region containing the mutation position only when the mutation to be detected exists or only when the mutation does not exist (that is, only when the mutation position to be detected is a specific base). You can also. When a fluorescent reagent such as SYBR (registered trademark) Green is intercalated into the amplification product, the presence or absence of amplification can be confirmed by fluorescence.

  In certain embodiments, the primer comprises a c.347C <T mutation, and 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 1 or 3. %, Or a part of a sequence having a sequence identity of not less than%, and optionally 1 to 3, 1, 2 or 1 mismatches. In another embodiment, the primer comprises a c.347C <T mutation, wherein in the sequence of SEQ ID NO: 1 or 3, 0-100, 0-90, 0-80, 0-70, 0-60 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or 0 to 1 bases Complementary to a part of the deleted, substituted, inserted or added sequence or its complementary sequence, optionally containing 1-3, 1-2 or 1 mismatches. In a further embodiment, the primer is complementary to a portion of the sequence of SEQ ID NO: 1 or 3 comprising the c.347C <T mutation, or a portion thereof, and optionally 1-3, 1-2 or 1 Including mismatches.

  The probe used for detecting the mutation specifically binds to a region containing the mutation position of the MMD2 gene. In the present specification, the term “specifically binds” means that a region containing a mutation can be distinguished from the same region not containing the mutation under hybridization conditions usually used for detection by a probe. The length of the probe is appropriately determined depending on the detection method, but is, for example, 10 to 500 bases or 16 to 300 bases, and may be 20 to 60 bases. Probes are usually designed to bind to a region containing the mutation position when the mutation position to be detected is a specific base, and are designed to bind when a mutation is present, or when they are not present. It may be designed to be combined. The probe is usually complementary to a sequence of the wild-type or mutant MMD2 gene (including the coding sequence and the non-coding sequence) or a part of its complementary sequence, but as long as it can specifically bind to the target region. It may include a mismatch. The number of mismatches is, for example, 1-3, 1-2 or 1.

  In certain embodiments, the probe comprises a c.347C <T mutation and comprises 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 1 or 3. % Or more of a sequence having sequence identity or a complementary sequence thereof, and optionally contains 1-3, 1-2 or 1 mismatches, and specifically binds to a region containing the mutation. Join. In another embodiment, the probe comprises a c.347C <T mutation and 0-100, 0-90, 0-80, 0-70, 0-60 in the sequence of SEQ ID NO: 1 or 3. 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or 0 to 1 bases It is complementary to a part of the deleted, substituted, inserted or added sequence or its complementary sequence, and optionally contains 1-3, 1-2 or 1 mismatches, and is specific for the region containing the mutation. To combine. In a further embodiment, the probe is complementary to a portion of the sequence of SEQ ID NO: 1 or 3 comprising the c.347C <T mutation, or a portion thereof, and optionally 1-3, 1-2 or 1 And specifically binds to the region containing the mutation.

  Primers and probes are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or pepeptide nucleic acid (PNA), cross-linked artificial nucleic acid (LNA), 2'-O, 4'-C-ethylene cross-linked nucleic acid (ENA), morpholino It may consist of artificial nucleic acids such as nucleic acids. Primers and probes can be synthesized by a known method such as the phosphodiester method. The primer and the probe may be modified according to the detection method, and for example, a fluorescent substance, a quenching substance, a luminescent substance and the like may be bound.

  Primers or probes may be included in a diagnostic kit for invasive periodontitis. The diagnostic kit may include, in addition to the primers and / or probes, other reagents such as enzymes for reverse transcription and PCR, dNTPs, buffers, and control nucleic acids.

  Neutrophil counts, alone or with mutations in the MMD2 gene, can be used to determine the presence or risk of invasive periodontitis. In certain embodiments, if the subject's neutrophil count is lower than the reference value, the subject is determined to have or be at risk for developing invasive periodontitis. The reference value can be the lower limit of the normal range at or near the age (or age and gender) of the subject (eg, the lower limit ± 10%). For example, in the case of a human adult, there may be 2000, 1800, or 1500 cells per μL of peripheral blood. In a preferred embodiment, the subject's neutrophil count is below a reference value (eg, 2000 / μL, 1800 / μL, or 1500 / μL, preferably 1800 / μL) and greater than 1000 / μL. In some cases, the subject is determined to be suffering from or at risk for developing invasive periodontitis. In another embodiment, a subject determined to be at risk for developing invasive periodontitis by detecting a mutation in the MMD2 gene and / or measuring neutrophil counts, the lower the neutrophil count, the more invasive It is determined that the risk of developing periodontitis is high or that the symptoms of invasive periodontitis are expected to be severe.

A sample obtained from a subject for measurement of neutrophil count may be neutrophils or progenitor cells thereof (eg,
Myeloblasts, promyelocytes, myelocytes, metamyelocytes, rod-shaped nuclei), and blood or bone marrow fluid. The number of neutrophils in a sample can be measured by a hemocytometer equipped with a leukocyte classification mechanism usually used for blood tests, such as electrical resistance detection or flow cytometry, or by a hemocytometer. Further, as described in Examples, nucleated cells may be obtained from these samples, cultured in the presence of granulocyte colony stimulating factor (G-CSF), and the number of obtained colonies may be measured. Nucleated cells can be obtained by commonly used methods such as centrifugation.

  If it is determined that the subject has or is at risk for developing invasive periodontitis, the subject can be treated for invasive periodontitis. Treatments include plaque control, removal of plaque retention factor, scaling, root planing, periodontal surgery (including periodontal tissue regeneration therapy), and the like. As used herein, the treatment of invasive periodontitis includes a treatment for treating onset periodontitis and a treatment for preventing the onset periodontitis. For example, prophylactic treatments include plaque control, removal of plaque retention factor, and scaling, while therapeutic treatments include plaque control, removal of plaque retention factor, scaling, root planing, and periodontal surgery (Including periodontal tissue regeneration therapy).

  An animal having a mutant MMD2 gene has a high degree of periodontal tissue destruction when periodontitis is induced, as compared with the wild-type animal, and can be used as a model animal for invasive periodontitis. In one embodiment, the model animal of invasive periodontitis is a non-human animal having a mutation that deletes the function of MMD2. In another embodiment, the model animal of invasive periodontitis is a non-human animal having a c.347C <T mutation in the MMD2 gene. The model animal may be a naturally occurring non-human animal or a non-human animal having genetic information artificially modified by genetic engineering techniques (hereinafter referred to as a genetically modified non-human animal). You may. In one embodiment, the model animal of invasive periodontitis is a non-human animal into which a mutation that deletes the function of MMD2 has been introduced. In another embodiment, the model animal of invasive periodontitis is a non-human animal into which the c.347C <T mutation of the MMD2 gene has been introduced.

  In certain embodiments, the non-human animal is a non-human mammal. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, sheep, pigs, cows, monkeys, and the like. In certain embodiments, the non-human mammal is a rodent, such as a mouse, rat, hamster, guinea pig, and preferably a mouse.

  Genetically modified non-human animals into which a mutation that deletes the function of MMD2 has been introduced include knock-in animals and knock-out animals. In the present specification, a knock-in animal in which a mutation deficient in the function of MMD2 has been introduced is a specific sequence introduced into the genome, whereby the function of MMD2 is deleted or the expression of MMD2 is inhibited. Means an animal. A knockout animal in which a mutation that deletes the function of MMD2 has been introduced refers to the deletion, substitution, or insertion of bases in the process of repairing a double-strand break in the genome, and the function of MMD2 is reduced by a frame shift or amino acid substitution. It means an animal that is deleted or whose expression of MMD2 is inhibited. The lack of MMD2 function can be confirmed by, for example, a decrease in the number of neutrophils. The neutrophil count of the genetically modified non-human animal is, for example, 70%, 60%, 50%, 40%, 30%, or 20% or less of the neutrophil count of the corresponding wild-type animal.

  In certain embodiments, the non-human animal having a mutation that deletes the function of MMD2 has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, It has a mutant MMD2 gene encoding an amino acid sequence lacking 90% or 100%. In certain embodiments, the non-human animal having a mutation that deletes the function of MMD2 has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the coding sequence. Or a mutant MMD2 gene lacking 100%.

  In certain embodiments, the non-human animal having the c.347C <T mutation in the MMD2 gene comprises the c.347C <T mutation and has at least 70%, 80%, 85%, 90% , A mutant MMD2 gene containing a sequence having a sequence identity of 95%, 96%, 97%, 98% or 99% or more; c. Containing a 347C <T mutation; 100, 0-90, 0-80, 0-70, 0-60, 0-50, 0-40, 0-30, 0-20, 0-10, 0- A mutant MMD2 gene containing a sequence in which 5, 0 to 3, 0 to 2, or 0 to 1 bases are deleted, substituted, inserted or added; or c. A sequence number containing a 347C <T mutation It has a mutant MMD2 gene containing one or three sequences. In a further embodiment, the non-human animal having the c.347C <T mutation in the MMD2 gene comprises the p.Ala116Val mutation and has at least 70%, 80%, 85%, 90%, 95% %, 96%, 97%, 98% or a mutant MMD2 gene encoding a mutant MMD2 comprising a sequence having at least 99% sequence identity; including the p.Ala116Val mutation, the sequence of SEQ ID NO: 2 or 4, A sequence in which 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or 0 to 1 amino acids are deleted, substituted, inserted or added. Or a mutant MMD2 gene encoding a mutant MMD2 comprising the sequence of SEQ ID NO: 2 or 4 containing the p.Ala116Val mutation.

  Genetically modified non-human animals can be prepared by known methods. For example, an ES cell or a fertilized egg in which a desired mutation has been introduced into the genome is obtained by homologous recombination using a targeting vector or by a genome editing technique such as CRISPR / Cas9, TALEN, or ZFN. In genome editing, a chimeric protein obtained by fusing a DNA binding domain designed to recognize a specific genomic sequence with a functional domain having nuclease activity is introduced into cells or fertilized eggs, and the genome is cut at a desired site. For example, in TALEN, a target cleavage site is present in a pair of a DNA binding domain that binds to a region of about 15 to 30 bases on the 5 'side of a spacer region of about 12 to 20 bases and a functional domain having nuclease activity. The mRNA encoding TALEN or a vector expressing the same is introduced into cells or fertilized eggs. Base replacement, deletion, or insertion occurs during the repair of a double-stranded break in the genome. When the donor DNA is introduced together with the mRNA or vector encoding TALEN, cells or fertilized eggs in which the sequence of the donor has been inserted into the genome can be obtained.

  By transplanting the ES cell into which the mutation has been introduced into an ES cell-transplanted embryo at the 8-cell stage or in the blastocyst embryo, or by transplanting the fertilized egg into which the mutation has been introduced into the uterus of a pseudopregnant foster parent, and giving birth, A chimeric animal is obtained. When a chimeric animal in which the desired sequence has been introduced into the germ line is bred to a wild-type animal, a heterozygote is obtained, and when the heterozygotes are bred to each other, a homozygote is obtained. The fact that heterozygotes or homozygotes have been obtained can be confirmed by Southern blot or PCR. Genetically modified non-human animals herein include heterozygotes, homozygotes and their progeny.

  The model animal for invasive periodontitis can be used for screening for a preventive or therapeutic agent for invasive periodontitis, screening for causative bacteria of invasive periodontitis, and the like. When screening for a preventive or therapeutic agent for invasive periodontitis, a candidate substance may be administered to an animal before or after the onset of periodontitis, and the periodontitis symptom presented by the animal may be evaluated. When screening for the causative bacteria of invasive periodontitis, an animal may be inoculated with candidate bacteria and the periodontitis symptoms exhibited by the animal may be evaluated.

  In the model animal of the present disclosure, experimentally inducing periodontitis by ligating a silk thread or a metal wire to the cervical region or by inoculating a periodontal disease-causing bacterium such as PG (Porphyromonas gingivalis). Can be.

  Candidate substances include, but are not limited to, for example, small molecule compounds, nucleic acids, proteins, peptides, or antibodies, or microbial, plant or animal cell extracts or culture supernatants. The candidate substance may be provided in the form of a library, such as a synthetic low-molecular compound library, a synthetic peptide library, or an antibody library.

  The candidate substance may be administered orally or parenterally (eg, intravenous, pulmonary, subcutaneous, intramuscular, intraperitoneal). The number of administrations and the administration period are not particularly limited, and it is conceivable that the administration is performed once to several times per day every day to several days or several weeks, every day or every several days.

  Evaluation of the response of a model animal to a candidate substance is usually done by comparing the symptoms of invasive periodontitis in the animal to which the candidate substance was administered with a control (usually except for the absence of the candidate substance and the presence or absence of the candidate substance). Symptoms of invasive periodontitis in animals under the same conditions). Candidate substances that improve the symptoms of invasive periodontitis as compared to controls are selected as prophylactic or therapeutic agents for invasive periodontitis. Improvement of symptoms includes improvement in degree and delay of onset. For example, if a candidate substance is administered before the onset of invasive periodontitis and the onset of invasive periodontitis is delayed in an animal to which the candidate substance is administered compared to a control, or the symptoms of invasive periodontitis are reduced. If mild, the candidate substance may be selected as a prophylactic for invasive periodontitis. In addition, if the candidate substance is administered after the onset of invasive periodontitis, and the symptoms of invasive periodontitis improve in the animal to which the candidate substance is administered compared to the control, the candidate substance is treated for invasive periodontitis. Can be selected as medicine. Symptoms of invasive periodontitis include gum inflammation and alveolar bone resorption.

Exemplary embodiments of the present invention are described below.
[1]
A method of determining the presence or risk of invasive periodontitis in a subject, comprising:
Detecting a mutation in the MMD2 gene in a sample obtained from the subject, and / or determining a neutrophil count in the sample obtained from the subject;
A method that includes
[2]
Detecting a mutation in the MMD2 gene in a sample obtained from the subject, and, if the mutation is detected, the subject is suffering from invasive periodontitis, or at risk of developing invasive periodontitis; The method according to the above [1], comprising determining that there is.
[3]
The method according to the above [1] or [2], further comprising measuring a neutrophil count in a sample obtained from the subject.
[4]
Measuring a neutrophil count in a sample obtained from the subject, and if the neutrophil count is lower than a reference value, the subject is suffering from invasive periodontitis, or The method according to any one of [1] to [3], including determining that there is a risk of developing the disease.
[5]
Detecting a mutation in the MMD2 gene in a sample obtained from the subject,
Measuring the neutrophil count in a sample obtained from the subject, and wherein the mutation is detected and the neutrophil count is lower than a reference value, wherein the subject is suffering from invasive periodontitis, Alternatively, the method according to any one of [1] to [4], including determining that there is a risk of developing invasive periodontitis.
[6]
The method according to any one of [1] to [5] above, wherein the mutation is a c.347C <T mutation.
[7]
The method according to any one of [1] to [6] above, wherein the detection of the mutation includes determining a sequence of a region including a position corresponding to position 347 of SEQ ID NO: 1 in a sample containing the nucleic acid derived from the subject. .
[8]
The method according to any one of [1] to [6], wherein detecting the mutation comprises amplifying a region containing a position corresponding to position 347 of SEQ ID NO: 1 in a sample containing a nucleic acid derived from the subject.
[9]
The method according to the above [8], wherein the detection of the mutation further comprises determining a sequence of a region including a position corresponding to the 347th position of SEQ ID NO: 1 in the amplification product.
[10]
The method according to any one of [1] to [6], wherein the detection of the mutation includes detecting a mutant MMD2 containing a p.Ala116Val mutation in a sample containing the polypeptide derived from the subject.
[11]
The method according to any one of the above [1] to [10], wherein the subject is a human.
[12]
The method according to any one of [1] to [11], wherein the sample is blood.
[13]
[1] The method of [1], further comprising recommending a subject suffering from or determined to be at risk for developing invasive periodontitis to be treated for invasive periodontitis. ] The method according to any one of [12].
[14]
A method of treating invasive periodontitis in a subject, comprising:
Determining the presence or risk of invasive periodontitis in a subject by the method according to any one of [1] to [13], and suffering from or invasive periodontitis Subjecting the subject determined to be at risk of developing inflammation to be treated for invasive periodontitis,
A method that includes
[15]
A method for treating invasive periodontitis in a subject, wherein the subject is suffering from or develops invasive periodontitis by the method according to any one of [1] to [13]. Treating a subject determined to be at risk for invasive periodontitis.
[16]
Primer for amplifying the region containing the mutation position of MMD2 gene.
[17]
The primer according to the above [16], wherein the mutation position is a position corresponding to position 347 of SEQ ID NO: 1.
[18]
The primer according to the above [16] or [17], wherein the region has a length of 10 to 1000 bases.
[19]
The primer according to any of [16] to [18], wherein the primer has a length of 10 to 70 bases.
[20]
c. The primer according to any of [16] to [19] above, which is complementary to a part of the sequence of SEQ ID NO: 1 containing the 347C <T mutation or a part of the complementary sequence thereof.
[21]
Probe that specifically binds to the region containing the mutation position of the MMD2 gene.
[22]
The probe according to [21], wherein the mutation position is a position corresponding to position 347 of SEQ ID NO: 1.
[23]
The probe according to the above [21] or [22], wherein the probe has a length of 10 to 500 bases.
[24]
c. any of the above-mentioned [21] to [23], which is complementary to the sequence of SEQ ID NO: 1 containing the 347C <T mutation or a part of the complementary sequence thereof and specifically binds to the region containing the mutation; Probe as described.
[25]
A diagnostic kit for invasive periodontitis, comprising the primer according to any of [16] to [20] or the probe according to any of [21] to [24].
[26]
A diagnostic marker for invasive periodontitis, comprising the mutant MMD2 gene or mutant MMD2.
[27]
The diagnostic marker according to [26], wherein the mutant MMD2 gene contains a c.347C <T mutation.
[28]
The mutant MMD2 gene contains the p.Ala116Val mutation and has 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more than 99% sequence identity with the sequence of SEQ ID NO: 2 The diagnostic marker according to the above [26] or [27], which encodes a mutant MMD2 comprising a sequence having the following sequence:
[29]
Mutant MMD2 gene contains a p.Ala116Val mutation, and in the sequence of SEQ ID NO: 2, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, Alternatively, the diagnostic marker according to any one of the above [26] to [28], which encodes a mutant MMD2 containing a sequence in which 0 to 1 amino acid has been deleted, substituted, inserted or added.
[30]
The diagnostic marker according to any one of [26] to [29], wherein the mutant MMD2 gene encodes a mutant MMD2 comprising a sequence of SEQ ID NO: 2 containing a p.Ala116Val mutation.
[31]
The mutant MMD2 gene contains the c.347C <T mutation, and has a sequence of SEQ ID NO: 1 and a sequence of 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more The diagnostic marker according to any one of [26] to [30], comprising a sequence having identity.
[32]
Mutant MMD2 gene contains c.347C <T mutation, and in the sequence of SEQ ID NO: 1, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50 0 to 40, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or 0 to 1 bases deleted, substituted, The diagnostic marker according to any one of the above [26] to [31], which comprises an inserted or added sequence.
[33]
The diagnostic marker according to any one of [26] to [32], wherein the mutant MMD2 gene includes the sequence of SEQ ID NO: 1 including a c.347C <T mutation.
[34]
The diagnostic marker according to any one of the above [26] to [33], wherein the mutant MMD2 contains a p.Ala116Val mutation.
[35]
Mutant MMD2 contains the p.Ala116Val mutation and has 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more than 99% sequence identity with the sequence of SEQ ID NO: 2. The diagnostic marker according to any one of the above [26] to [34], comprising a sequence having the diagnostic marker.
[36]
Mutant MMD2 contains a p.Ala116Val mutation, and in the sequence of SEQ ID NO: 2, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or The diagnostic marker according to any one of the above [26] to [35], which comprises a sequence in which 0 to 1 amino acid has been deleted, substituted, inserted or added.
[37]
The diagnostic marker according to any one of [26] to [36], wherein the mutant MMD2 comprises the sequence of SEQ ID NO: 2 containing the p.Ala116Val mutation.
[38]
Use of the mutant MMD2 gene or the mutant MMD2 defined in any one of the above [26] to [37] as a diagnostic marker for invasive periodontitis.
[39]
A model animal of invasive periodontitis, which is a non-human animal having a mutation deficient in the function of MMD2.
[40]
The model animal according to [39], which is a genetically modified animal into which the mutation has been introduced.
[41]
Having a mutant MMD2 gene encoding an amino acid sequence lacking at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the amino acid sequence of MMD2; The model animal according to the above [39] or [40].
[42]
A model animal of invasive periodontitis, which is a non-human animal having a c.347C <T mutation in the MMD2 gene.
[43]
The model animal according to [42], which is a genetically modified animal into which the mutation has been introduced.
[44]
A mutation containing the p.Ala116Val mutation and a sequence having a sequence identity of 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more with the sequence of SEQ ID NO: 4 The model animal according to [42] or [43], which has a mutant MMD2 gene encoding type MMD2.
[45]
Including the p.Ala116Val mutation, in the sequence of SEQ ID NO: 4, 0 to 30, 0 to 20, 0 to 10, 0 to 5, 0 to 3, 0 to 2, or 0 to 1 The model animal according to any of [42] to [44] above, which has a mutant MMD2 gene encoding a mutant MMD2 containing a sequence in which amino acids have been deleted, substituted, inserted or added.
[46]
The model animal according to any of [42] to [45], which has a mutant MMD2 gene encoding a mutant MMD2 comprising the sequence of SEQ ID NO: 4 containing the p.Ala116Val mutation.
[47]
c. a sequence containing the 347C <T mutation and having a sequence identity of 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more with the sequence of SEQ ID NO: 3 The model animal according to any one of the above [42] to [46], comprising the mutant MMD2 gene.
[48]
c. Contains the 347C <T mutation, and in the sequence of SEQ ID NO: 3, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40 0-30, 0-20, 0-10, 0-5, 0-3, 0-2, or 0-1 bases deleted, substituted, inserted or added sequence The model animal according to any one of the above [42] to [47], having a mutant MMD2 gene comprising:
[49]
c. The model animal according to any of [42] to [48] above, which has a mutant MMD2 gene comprising the sequence of SEQ ID NO: 3 containing the 347C <T mutation.
[50]
Use of the non-human animal defined in any of [39] to [49] above as a model animal for invasive periodontitis.
[51]
A method for screening a prophylactic or therapeutic agent for invasive periodontitis,
Administering a candidate substance to the model animal of invasive periodontitis according to any of [39] to [49], and suppressing the onset of invasive periodontitis, or a symptom of invasive periodontitis Selecting a candidate substance that reduces the inflammation as a prophylactic or therapeutic agent for invasive periodontitis,
A method that includes

Patient information A family with four invasive periodontitis patients was extracted from a family of patients visiting Hiroshima University Hospital. Blood was collected and DNA was extracted from four affected and four non-affected individuals within the family. The family information of the patient is shown in FIG.

Linkage analysis
DNAs of II-1, II-6, II-9, III-2, III-3, III-4, and III-5 (see FIG. 1) were used for linkage analysis. Genome-typing of single nucleotide polymorphisms (SNPs) was performed using Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA), and analysis was performed using Allegro software. Regions with high rod scores were present on chromosomes 3 and 7. The respective rod scores were 1.8047 and 1.8058.

Identification of candidate genes using exome sequence The exome sequence was variant called with GATK and Samtools, and annotated with Annovar. PolyPhen-2, Mutation Taster and SIFT were used to predict the damage of amino acid mutations. A genomic DNA library was prepared using SeqCap EZ Human Exome Library v2.0 (Roche, Basel, Switzerland). The sequence was performed using a HiSeq2000 sequencer (Illumina, San Diego, CA, USA) with a 100-bp pair end. The resulting gene mutation was determined by Sanger sequencing using Applied Biosystems 3130 DNA sequencer (Thermo Fisher Scientific, Waltham, Mass., USA).

More than 90,000 gene mutations were detected in the DNA of three affected individuals. Using an open database (dbSNP, 1000 Genomes, and EPS 6400), candidate gene mutations were narrowed down, and 282 mutations common to three individuals were extracted. Further narrowing was performed with reference to the original database of central degenerative diseases, and finally seven heterogene mutations were extracted (Tables 2 and 3).

  Gene mutations predicted to have high amino acid damage were present in NID1, MUC4, MUC21, MMD2, and LOC100288778. The only mutation in the family that was shared by affected individuals and not shared by non-affected individuals was the mutation present in MMD2. This mutation was present in a candidate region of human chromosome 7, which showed a high rod score even in linkage analysis. In this mutation, C at position 347 of the coding DNA of the human MMD2 gene was replaced with T (c.347C <T mutation), and Ala at position 116 in the amino acid sequence was replaced with Val (c.347C <T, p.Ala116Val mutation).

Generation of MMD2 knock-in mouse by genome editing using TALEN c.347C <T, p.Ala116Val mutation of human MMD2 was generated in mice using the TALEN genome editing tool. TALEN was designed so that double-strand breaks occur near the position of the c.347C <T mutation in the MMD2 gene present on mouse chromosome 5 (FIG. 2). Using the MEGAscript T7 transcription kit (Thermo Fisher Scientific, Yokohama, JAPAN), TALEN-encoding mRNA was synthesized, and four nucleotide mutations (targeting C> T substitution and restriction enzyme PstI to confirm introduction of oligonucleotide) Was injected into a fertilized egg of a C57BL / 6 mouse together with 87 ssODN containing a substitution to introduce a recognition site (Fig. 3). In addition to the mutation of interest, deletions and insertions of 5 bp, 7 bp, and 11 bp with frame shift were obtained by genome editing (FIG. 4). In the following experiments, mice having a C> T substitution were used as knock-in (KI) mice, and mice having a 7 bp deletion were used as knock-out (KO) mice.

Silk-ligated periodontitis induction model Experimental periodontitis was induced in wild-type (WT) mice, KI mice (KI / WT, KI / KI), and KO mice (KO / KO). Periodontitis and concomitant alveolar bone resorption were induced by applying 5-0 silk to the second molars of 8-week-old male mice. Seven days after the ligation, the distance from the cement enamel junction to the alveolar bone dehiscence was measured and evaluated at four points: the first molar distal, the second molar mesial and distal, and the third molar mesial (FIG. 5). Higher alveolar bone resorption was observed in KI and KO mice compared to WT mice (FIGS. 6 and 7).

Colony analysis Remove femurs and tibias from wild-type (WT) mice, KI mice (KI / WT, KI / KI), and KO mice (KO / KO), flush and extract bone marrow cells, then nucleated cells Collected. The cells were diluted to 5 × 10 ³ cells / dish, mixed with MethoCult (M3231), and seeded in 3.5 cm dishes at a final G-CSF concentration of 10 ng / ml. After 7 days, colonies were counted. As a result, under G-CSF stimulation, a decrease in the number of colonies was observed in both KI and KO mice as compared to WT mice. These results indicate that MMD2 mutant mice show abnormalities in the number of neutrophils corresponding to the pathogen at the forefront of the immune system.

Analysis of neutrophil count The neutrophil count of patients of this kindred was measured by an automatic hematology analyzer equipped with a leukocyte classification mechanism. The white blood cell count in the normal range of human adults is about 4000 to 8000 / μL, and the neutrophil count is about 1800 to 7500 / μL, while the white blood cell count of patients of this kindred is about 2500 to 4000 / μL. The number of neutrophils was about 1300-1900 / μL. Similarly, when the neutrophil counts of WT mice, KI mice (KI / KI), and KO mice (KO / KO) were measured, they were 490 / μL, 200 / μL, and 72 / μL, respectively. A decrease in the sphere was observed.

Claims (16)

A method of determining the presence or risk of invasive periodontitis in a subject, comprising:
Detecting a mutation in the MMD2 gene in a sample obtained from the subject, and, if the mutation is detected, the subject is suffering from invasive periodontitis, or at risk of developing invasive periodontitis; Determining that there is,
A method that includes   The method according to claim 1, wherein the mutation is a c.347C <T mutation.   3. The method of claim 1 or 2, further comprising measuring neutrophil count in a sample obtained from the subject.   Primer for amplifying the region containing the mutation position of MMD2 gene.   The primer according to claim 4, wherein the mutation position is a position corresponding to position 347 of SEQ ID NO: 1.   Probe that specifically binds to the region containing the mutation position of the MMD2 gene.   The probe according to claim 6, wherein the mutation position is a position corresponding to position 347 of SEQ ID NO: 1.   A diagnostic kit for invasive periodontitis, comprising the primer according to claim 4 or 5 or the probe according to claim 6 or 7.   A diagnostic marker for invasive periodontitis, comprising the mutant MMD2 gene or mutant MMD2.   The diagnostic marker according to claim 9, wherein the mutant MMD2 gene contains a c.347C <T mutation.   The diagnostic marker according to claim 9 or 10, wherein the mutant MMD2 contains a p.Ala116Val mutation.   Use of the mutant MMD2 gene or mutant MMD2 defined in any one of claims 9 to 11 as a diagnostic marker for invasive periodontitis.   A model animal of invasive periodontitis, which is a non-human animal having a mutation deficient in the function of MMD2.   A model animal of invasive periodontitis, which is a non-human animal having a c.347C <T mutation in the MMD2 gene.   The model animal according to claim 14, which has a mutant MMD2 gene encoding a mutant MMD2 comprising the sequence of SEQ ID NO: 4 containing the p.Ala116Val mutation.   Use of a non-human animal as defined in any of claims 13 to 15 as a model animal for invasive periodontitis.