JP7558511B2 - Method for predicting risk of severe cerebral microbleeds in a subject, method for screening subjects at high risk of severe cerebral microbleeds, and kit for use therein - Google Patents

Description

The present invention relates to a method for predicting a subject's risk of developing severe cerebral microbleeds, a method for screening subjects at high risk of developing severe cerebral microbleeds, and a kit for use therein.

Cerebral microbleeds (CMBs, MBs) are a symptom in which a small amount of red blood cells leak out of blood vessels from ruptured capillaries. They occur at a significantly higher frequency than healthy individuals (5%), in approximately 60% of cerebral hemorrhage patients, approximately 35% of cerebral infarction patients, approximately 30% of Alzheimer's disease patients, and approximately 15% of mild cognitive impairment patients. It is also known that patients with multiple cerebral microbleeds have a higher incidence and recurrence rate of stroke (cerebral hemorrhage) due to bleeding. Therefore, the severity of cerebral microbleeds, such as the number and frequency, is considered to be one of the necessary factors for predicting the risk of developing these diseases and understanding their pathology.

The main causes of cerebral microbleeds are hypertensive arteriopathy (HA) and cerebral amyloid angiopathy (CAA), but in recent years, it has been reported that certain strains of Streptococcus mutans (S. mutans) are associated with cerebral microbleeds and cerebral hemorrhage.

For example, Japanese Patent Publication No. 2010-233552 (Patent Document 1) describes that CNM-positive S. mutans carrying the cnm gene encoding the CNM (collagen-binding adheren) protein is associated with hemorrhagic stroke, and that the cause is the CNM protein. In addition, S. Tonomura et al., Scientific Reports, 6:20074, DOI:10.1038/srep20074 (2016) (Non-Patent Document 1) describes that CNM-positive S. mutans carrying the cnm gene bind to collagen in the blood vessel wall, gather at the wound of the blood vessel, inhibit the hemostatic action of platelets, and cause inflammation in the brain, which contributes to the onset of cerebral hemorrhage.

JP 2010-233552 A

S. Tonomura et al., Scientific Reports, 6:20074, DOI:10.1038/srep20074 (2016)

As mentioned above, it has been reported that CNM-positive S. mutans is involved in the development of cerebral microbleeds. However, even among CNM-positive S. mutans carriers, there are differences in the severity of cerebral microbleeds, and the cause of this remains unknown. Therefore, until now, there has been no method for predicting the likelihood that cerebral microbleeds will become severe, i.e., the risk of cerebral microbleeds becoming severe, or for screening subjects at high risk of developing such conditions.

The present invention has been made in consideration of the above problems, and aims to provide a method for predicting a subject's risk of developing severe cerebral microbleeds, a method for screening subjects at high risk of developing severe cerebral microbleeds, and a kit for use in these methods.

In order to solve the above problem, the inventors conducted intensive research and first collected samples from patients whose number of cerebral microbleeds had been confirmed by diagnosis, and obtained the full length of the amino acid sequence of the CNM protein (sometimes referred to as the "CNM amino acid sequence" in this specification) from the whole genome sequence of Streptococcus mutans contained in the samples. Next, multiple alignment was performed on the CNM amino acid sequences obtained from each sample. It is known that there are many polymorphisms in the B-repeat region of the CNM protein, but the inventors found that, as a result of the multiple alignment, there is a clear correlation between the amino acid sequence length of a specific repeat region that overlaps partially or completely with the B-repeat region and the severity of the cerebral microbleeds of the sample (here, the number of cerebral microbleeds), and the longer the amino acid sequence length, the more severe the severity of the cerebral microbleeds. Therefore, the inventors discovered that it is possible to predict a subject's risk of developing severe cerebral microbleeds and to screen subjects at high risk of developing severe cerebral microbleeds by using the amino acid sequence length of the specific repeat region of the CNM protein as an index, thereby completing the present invention.

The present invention, based on these findings, has the following features.
[1]
A method for predicting a subject's risk of developing severe cerebral microbleeds,
A method for predicting the risk of severity of cerebral microbleeds in a subject using as an indicator the amino acid sequence length of a repeat region in the CNM protein of Streptococcus mutans contained in a sample from the subject.
[2]
The method according to [1], characterized in that a subject in which the amino acid sequence length of the repeat region is 111 residues or more is predicted to be at high risk of developing severe cerebral microbleeds.
[3]
A method for screening subjects at high risk of developing severe cerebral microbleeds,
A method for screening subjects, comprising using as an index the amino acid sequence length of a repeat region in a CNM protein of Streptococcus mutans contained in a sample derived from the subject.
[4]
The method according to [3], characterized in that subjects in which the amino acid sequence length of the repeat region is 111 residues or more are selected as being at high risk of developing severe cerebral microbleeds.
[5]
The method according to any one of [1] to [4], wherein the repeat region is a region consisting of a plurality of repeat units, each of which has 4 to 10 amino acid residues.
[6]
The method according to [5], wherein the repeat units are each independently a unit containing at least two threonine residues.
[7]
The method according to [5] or [6], wherein the repeat units are each independently a unit in which the first amino acid residue is threonine or alanine.
[8]
The method according to any one of [5] to [7], wherein the repeat units are each independently a unit whose last amino acid residue is proline or serine.
[9]
A kit for use in the method according to any one of [1] to [8], the kit comprising an oligonucleotide primer capable of amplifying a repeat region in the CNM protein of Streptococcus mutans.

Although the reason why the above object is achieved by the configuration of the present invention is not necessarily clear, the inventors speculate as follows. That is, it is believed that when Streptococcus mutans migrates into the brain, it binds to cells in the brain via the CNM protein, remains in the brain, and induces cerebral microbleeds. The CNM protein binds to the bacterial cell wall via the LPXTG motif located on the C-terminus side, and binds to cells in the brain via the collagen binding domain located on the N-terminus side (Nomura et al., Journal of Pediatric Dentistry, 52(1):1-11, 2014), and the specific repeat domain according to the present invention is located between these LPXTG motifs and the collagen binding domain. Therefore, the inventors speculate that the longer the length of the repeat domain, the easier it is for Streptococcus mutans to bind flexibly to cells in the brain and remain in the brain, which results in the more severe cerebral microbleeds.

The present invention makes it possible to provide a method for predicting a subject's risk of developing severe cerebral microbleeds, a method for screening subjects at high risk of developing severe cerebral microbleeds, and a kit for use in these methods.

FIG. 1 shows the results of multiple alignment of the CNM amino acid sequences obtained from each sample (N-terminal side of the repeat region). FIG. 1 shows the results of multiple alignment of the CNM amino acid sequences obtained from each sample (middle of the repeat region). FIG. 1 shows the results of multiple alignment of the CNM amino acid sequences obtained from each sample (C-terminal side of the repeat region). FIG. 13 is a heat map diagram showing clustering based on the gene ID and the number of gene regions obtained from each sample. FIG. 13 is a heat map diagram showing clustering based on the gene ID of gene regions obtained from each sample and their presence or absence. FIG. 1 shows electrophoretic images of 11 samples in a reproduction test for predicting the risk of severe illness.

The present invention will be described in detail below based on preferred embodiments thereof.
A method for predicting a subject's risk of developing severe cerebral microbleeds,
A method for predicting the risk of cerebral microbleeds becoming severe in a subject using the amino acid sequence length of the repeat region in the CNM protein of Streptococcus mutans contained in a sample from the subject as an index (hereinafter, sometimes referred to as a "method for predicting the risk of cerebral microbleeds becoming severe" or simply "prediction method"); and
A method for screening subjects at high risk of developing severe cerebral microbleeds,
A method for screening subjects using the amino acid sequence length of a repeat region in a CNM protein of Streptococcus mutans contained in a sample from the subject as an index (hereinafter sometimes referred to as a "screening method").
to provide.

(Cerebral Microbleeds)
"Cerebral microbleeds" refers to a symptom in which a small amount of red blood cells leak out of blood vessels from ruptured capillaries. The cerebral microbleeds according to the present invention may be deep cerebral microbleeds or cortical-subcortical cerebral microbleeds, but are preferably deep cerebral microbleeds. In the present invention, the "risk of aggravation of cerebral microbleeds" refers to the possibility that the severity of cerebral microbleeds that have already developed will become severe, or the possibility that the severity of cerebral microbleeds will become severe if they develop.

In the present invention, the "severity of cerebral microbleeds" is preferably truly determined by the number of cerebral microbleeds and/or their traces, and the number of cerebral microbleeds and their traces is typically confirmed by head imaging diagnosis. Examples of such head imaging diagnosis include head MRI and head CT. The criteria for the severity of cerebral microbleeds can be appropriately set depending on the disease associated with cerebral microbleeds and the purpose of the method, and for example, the severity of cerebral microbleeds can be mild when the total number of cerebral microbleeds and their traces is 0 to 1, and the severity of cerebral microbleeds can be severe when the total number of cerebral microbleeds and their traces is 2 or more. Examples of diseases associated with cerebral microbleeds include cerebral hemorrhage, cerebral infarction, Alzheimer's disease, and cognitive impairment.

(subject)
In the present invention, the term "subject" refers to a human being on whom the prediction method or screening method of the present invention is carried out, and may be a healthy individual or an individual without subjective symptoms, or may be a patient who is already suffering from or is known to have suffered from cerebral microhemorrhage in the past, for the purpose of determining the prognosis or confirming the effectiveness of treatment, or a patient who is known to be suffering from a disease associated with cerebral microhemorrhage.

(Specimen)
In the present invention, the "specimen" derived from the subject is not particularly limited as long as it is collected from the subject and can contain the following Streptococcus mutans, but generally includes tissues, body fluids, secretions, excretions, etc. collected from the subject (human). Among these, tissues, body fluids, secretions, or excretions collected from the oral cavity or pharynx are preferred, and dental plaque, saliva, oral mucosa, or pharyngeal mucosa are more preferred. These specimens may be appropriately diluted or suspended in a diluent or may have been pretreated as necessary.

(Streptococcus mutans)
Streptococcus mutans (sometimes referred to as "S. mutans" in this specification) is a type of Gram-positive streptococcus, and is known as an oral bacterium that is a major cause of dental caries. Four serotypes (c, e, f, and k) are known to exist as S. mutans. The prediction method and screening method of the present invention are directed to S. mutans that possess the cnm gene encoding the CNM protein described below (herein referred to as "CNM-positive S. mutans"). CNM-positive S. mutans have been reported to account for approximately 10-20% of all S. mutans, and are known to exist in large numbers as serotype k, but are not limited to this.

(CNM protein)
"CNM (Collagen-binding adhesive) protein" is also called "CBP (Collagen Binding Protein)". CNM protein is a collagen-binding protein with a molecular weight of about 120 kDa, and is composed of a collagen-binding region located on the N-terminus side, an LPXTG motif located on the C-terminus side for covalently binding the protein to the peptidoglycan of the cell wall of the fungus, and a B-repeat region, which is a repeat region located between them. The amino acid sequence of CNM protein is typically represented by Entry_Name: C4B6T3_STRMG in the known database UniprotKB (Universal Protein Resource Knowledgebase) that lists the amino acid sequences of proteins.

(Repeat Region)
In the prediction and screening methods of the present invention, a region of the amino acid sequence of the CNM protein that overlaps partially or completely with the B-repeat region is defined as a "repeat region," and the length of this amino acid sequence is used as an index for prediction or selection.

The "repeat region" according to the present invention specifically refers to the region between the first residue adjacent to the C-terminus of the amino acid sequence shown in SEQ ID NO:1 (AAGGVDGR) and the first residue adjacent to the N-terminus of the amino acid sequence shown in SEQ ID NO:2 (TTTEVSSE), and the "amino acid sequence length of the repeat region" refers to the number of amino acid residues, with the first residue adjacent to the C-terminus of the amino acid sequence shown in SEQ ID NO:1 being the first residue and the first residue adjacent to the N-terminus of the amino acid sequence shown in SEQ ID NO:2 being the last residue. More preferably, the "repeat region" according to the present invention refers to the region starting from the amino acid sequence shown in SEQ ID NO:3 (TTTTTEKP).

The amino acid sequence shown in SEQ ID NO:1 may have one amino acid residue deleted or substituted (preferably one of the amino acid residues 1 to 7 from the N-terminus) or one amino acid residue inserted (preferably inserted before the 8th residue from the N-terminus). The amino acid sequence shown in SEQ ID NO:2 may have one amino acid residue deleted or substituted (preferably one of the amino acid residues 3 or later from the N-terminus) or one amino acid residue inserted (preferably inserted after the 3rd residue from the N-terminus). Furthermore, the amino acid sequence shown in SEQ ID NO:3 may have one amino acid residue deleted or substituted (preferably one of the amino acid residues 3 or later from the N-terminus) or one amino acid residue inserted (preferably inserted after the 3rd residue from the N-terminus).

The repeat region according to the present invention is a region consisting of a plurality of repeat units which are common or similar amino acid sequences. The repeat units which are the constituent units of such a repeat region preferably each independently have 4 to 10 amino acid residues, more preferably 5 to 8 amino acid residues. Furthermore, the repeat units according to the present invention preferably each independently have at least two amino acid residues which are threonine (T), more preferably two consecutive threonine (T) residues, and even more preferably three consecutive threonine (T) residues. Furthermore, the repeat units according to the present invention preferably each independently have the first amino acid residue which is threonine (T) or alanine (A), more preferably threonine (T). Furthermore, the repeat units according to the present invention preferably each independently have the last amino acid residue which is proline (P) or serine (S), more preferably proline (P).

More specifically, examples of the amino acid sequences of such repeat units include, but are not limited to, the amino acid sequence shown in SEQ ID NO:3, the amino acid sequence shown in SEQ ID NO:4 (TTTTE(K/A)P), the amino acid sequence shown in SEQ ID NO:5 (TTTE(A/S/T)P), the amino acid sequence shown in SEQ ID NO:6 (TTEAP), the amino acid sequence shown in SEQ ID NO:7 (TTTGAP), the amino acid sequence shown in SEQ ID NO:8 (TTTEAS), the amino acid sequence shown in SEQ ID NO:9 (TITEAP), and the amino acid sequence shown in SEQ ID NO:10 (ATTEAP).

The combination of repeat units contained in the repeat region of the present invention is not particularly limited and may be one type or a combination of two or more types, but typically the repeat region contains two or more types of repeat units.

The amino acid sequence length of the repeat region of the present invention, which is composed of such repeat units, is preferably 35 residues or more.

(Method for predicting the risk of developing severe cerebral microbleeds and screening method)
In the prediction method of the present invention, the risk of cerebral microbleeds becoming severe in a subject is predicted using the amino acid sequence length of the repeat region as an index. In the screening method of the present invention, the risk of cerebral microbleeds becoming severe in a subject is determined using the amino acid sequence length of the repeat region as an index, and the subject is screened.

In the present invention, "predicting the risk of cerebral microbleeds becoming severe" includes not only determining whether or not there is a possibility that the severity of cerebral microbleeds will become severe, but also evaluating the degree of severity if there is a possibility (evaluating as high/medium/low, etc.).

In the present invention, "screening" specifically refers to determining that a subject is likely to have severe cerebral microbleeds and selecting the subject from a group with no or low possibility of the subject having severe cerebral microbleeds. In the screening method of the present invention, subjects may be selected at a level where a moderate possibility is expected, for example, depending on the disease associated with cerebral microbleeds and the purpose of the method.

[Length of amino acid sequence of repeat region]
The inventors have found that the longer the amino acid sequence length of the repeat region, i.e., the longer the amino acid sequence length of the repeat region in the CNM protein of S. mutans contained in a sample collected from a subject, the more severe the severity of the cerebral microbleeds of the subject. Therefore, the longer the amino acid sequence length of the repeat region, the higher the risk of the cerebral microbleeds becoming severe for that subject can be predicted or determined. On the other hand, the absence of the repeat region (i.e., the absence of CNM-positive S. mutans in the sample) or the shorter the amino acid sequence length of the repeat region in the sample, the lower the risk of the cerebral microbleeds becoming severe can be predicted or determined.

The standard for the amino acid sequence length of the repeat region can be set appropriately depending on the disease associated with cerebral microbleeds, the purpose of the method, etc., but the positive match rate (the proportion of subjects predicted or judged to be in the severe group by the standard who are truly severe or will become truly severe when subjects are divided into two groups, a severe group (positive) and a mild group (negative)) is preferably 90% or more, and more preferably 100%. In addition, for example, if the severity of cerebral microbleeds is determined to be mild when the total number of cerebral microbleeds and their traces is 0 to 1, and the severity of cerebral microbleeds is determined to be severe when the total number of cerebral microbleeds and their traces is 2 or more, subjects whose amino acid sequence length is 111 residues or more, preferably 113 residues or more, more preferably 116 residues or more, and even more preferably 120 residues or more can be predicted or judged to be at high risk of developing severe cerebral microbleeds.

In addition, since the longer the amino acid sequence length of the repeat region, the greater the number of repeat units constituting the repeat region (hereinafter sometimes referred to as "repeat number"), the repeat number may be used as an index for prediction or judgment in the prediction method and screening method of the present invention. As with the amino acid sequence length criterion, the repeat number criterion can be appropriately set according to the disease associated with cerebral microbleeds and the purpose of the method. For example, if the total number of cerebral microbleeds and their traces is 0 to 1, and the total number of cerebral microbleeds and their traces is 2 or more, the severity of cerebral microbleeds is considered to be mild, and if the total number of cerebral microbleeds and their traces is considered to be 2 or more, the severity of cerebral microbleeds is considered to be severe, subjects with the repeat number of 19 or more, preferably 20 or more, more preferably 21 or more can be predicted or judged to be at high risk of developing severe cerebral microbleeds.

[Method of obtaining amino acid sequence length information of repeat regions]
In the prediction method and screening method of the present invention, the information on the amino acid sequence length of the repeat region serving as an index may be direct information obtained by obtaining the amino acid sequence length itself, or, since S. mutans does not have introns, the base sequence length of the DNA encoding the repeat region may be indirectly used as information on the amino acid sequence length. Furthermore, since the amino acid sequence of the CNM protein other than the repeat region tends to be conserved among S. mutans strains, and the mass of the CNM protein increases correspondingly as the amino acid sequence length of the repeat region increases, the mass of the CNM protein may be indirectly used as information on the amino acid sequence length.

The method for obtaining the amino acid sequence length itself is not particularly limited, but for example, a method is available in which DNA containing the repeat region is isolated from S. mutans contained in a sample based on the base sequence information of DNA or cDNA encoding a CNM protein, and the amino acid sequence length of the encoded repeat region is obtained from the base sequence of the DNA. In this case, the number of repeats contained in the repeat region can also be obtained. The base sequence of the isolated DNA can be determined by a method known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method, and a next-generation sequencer capable of analyzing gene base sequences quickly and comprehensively can also be used. The base sequence information of the DNA or cDNA encoding the CNM protein for isolating the DNA can be obtained from a known database, in addition to the base sequence encoding the typical amino acid sequence of the CNM protein listed above.

Other methods for obtaining the amino acid sequence length itself include, for example, fragmenting the total genomic DNA recovered from S. mutans contained in a sample, obtaining the total genome sequence information using a single molecule real-time sequencer or the like, obtaining the amino acid sequence therefrom, and then performing a homology search based on the amino acid sequence information of the CNM protein to select an amino acid sequence with a homology (preferably identity) of 85% or more (preferably 90% or more) as the amino acid sequence of the CNM protein of the sample, and obtaining the amino acid sequence length of the repeat region. In this case, the number of repeats contained in the repeat region can also be obtained. The amino acid sequence information of the CNM protein for the homology search can be obtained from a known database, in addition to the typical amino acid sequences of the CNM proteins listed above. In addition, the homology search can be appropriately performed by a person skilled in the art using a known method (e.g., BLAST (NCBI)).

In addition, when the base sequence length of the DNA encoding the repeat region is used indirectly as information on the amino acid sequence length, for example, DNA containing the repeat region can be isolated from S. mutans contained in a sample based on the base sequence information of the DNA or cDNA encoding the CNM protein, and its length can be confirmed to indirectly confirm the length of the amino acid sequence encoded by the DNA. The length of the isolated DNA may be confirmed by determining the base sequence as described above, but it can also be confirmed more simply by cutting the isolated DNA with an appropriate enzyme as necessary, performing electrophoresis, or the like, and comparing the amino acid sequence length of the repeat region with known DNA, etc.

In addition, when the mass of the CNM protein is indirectly used as information on the amino acid sequence length, a protein sample is prepared from S. mutans contained in a specimen, the protein is labeled and fractionated, and the fractionated protein is subjected to mass spectrometry (MS, LC/MS, etc.), and the CNM protein and its mass are identified from the mass spectrometry value, thereby indirectly confirming the amino acid sequence length of the repeat region in the CNM protein. As the label, an isotope labeling reagent known in the art can be used, and suitable labeling reagents are commercially available. Fractionation can also be performed by a method known in the art, for example, using a commercially available ion exchange column, etc.

As a method for isolating DNA containing the repeat region from S. mutans contained in the above sample, a conventionally known method or a method similar thereto can be appropriately adopted. For example, the method can be performed by PCR using genomic DNA or RNA as a template, with a pair of oligonucleotide primers designed to sandwich all or part (preferably all) of the region encoding the repeat region.

The oligonucleotide primers can be designed based on the base sequence information of the DNA or cDNA encoding the CNM protein and the base sequence information of the start and end positions of the repeat region, so that they are primers that are appropriate for the above-mentioned method and the repeat region to be amplified, so that they have a sequence that is conserved among S. mutans strains, and so that amplification products of genes other than the region encoding the repeat region are minimized. Such oligonucleotide primers can be designed by a person skilled in the art using conventionally known methods or methods similar thereto.

The length of the oligonucleotide primer is usually 15 to 50 bases, preferably 15 to 30 bases, but may be longer or shorter depending on the method and purpose. An example of such an oligonucleotide primer is a combination of a forward primer set forth in SEQ ID NO: 11 and a reverse primer set forth in SEQ ID NO: 12, but is not limited thereto.

The oligonucleotide primer can be prepared, for example, by a commercially available oligonucleotide synthesizer. The oligonucleotide primer does not have to be composed of only natural nucleotides, and may be composed in part or entirely of non-natural nucleotides. Examples of non-natural nucleotides include PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), and complexes thereof.

The amino acid sequence length of the repeat region itself or the base sequence length of the DNA encoding the repeat region can be obtained directly from the sample. The target samples may include samples that do not contain S. mutans, and samples that contain S. mutans but not CNM-positive S. mutans. In such cases, the DNA encoding the repeat region is not isolated, or the amino acid sequence of the CNM protein cannot be obtained, so it can be determined that the repeat region is absent. Therefore, in this case, the prediction method and screening method of the present invention predict or determine that the risk of the cerebral microbleeds becoming severe is low.

In addition, the amino acid sequence length information of the repeat region may be directly or indirectly obtained by first isolating S. mutans from the sample, and then performing the direct or indirect acquisition on the isolated S. mutans, in order to prevent the accuracy of prediction or selection from being reduced due to contamination with amplification products of genes other than the region encoding the repeat region (such as genes of other organisms). The method for isolating S. mutans from the sample is not particularly limited, and a conventionally known method or a method similar thereto may be appropriately adopted, for example, a method in which the sample is cultured in a medium capable of detecting streptococci, such as MS medium, and a colony recognized as S. mutans is collected. Note that, in the prediction method and screening method of the present invention, if S. mutans cannot be isolated from the sample, the risk of the cerebral microbleed becoming severe is predicted or determined to be low.

Using the thus obtained information on the amino acid sequence length of the repeat region as an index, the prediction method of the present invention can predict that a subject has a high risk of developing severe cerebral microbleeds. Furthermore, the screening method of the present invention can select subjects who are likely to develop severe cerebral microbleeds from a normal group.

Therefore, according to the prediction method and screening method of the present invention, it is possible to predict or judge the risk of cerebral microbleeds becoming severe using easily available samples such as saliva and dental plaque. As a result, subjects at high risk of developing severe cerebral microbleeds can be identified, and by taking measures such as eradicating the causative bacteria and providing dental hygiene guidance, it is possible to effectively prevent the cerebral microbleeds from becoming severe. In addition, subjects selected by screening can be subjects for further testing, treatment, observation, etc. for cerebral microbleeds or diseases associated with cerebral microbleeds. Therefore, the prediction method and screening method of the present invention are useful for assisting in the determination of a method for preventing cerebral microbleeds or diseases associated with cerebral microbleeds, the determination of a treatment policy, confirmation of the effectiveness of treatment, etc. in subjects. The "prediction method" of the present invention can also be expressed as a method for assisting diagnosis by a physician or the like, or a method for providing information for diagnosis by a physician or the like. In other words, it can also be expressed as a "method for assisting diagnosis of the risk of developing severe cerebral microbleeds" or a "method for providing information for diagnosis of the risk of developing severe cerebral microbleeds", which is characterized by making the prediction.

(kit)
The present invention also provides a kit for use in the above-mentioned prediction method or screening method of the present invention. Such a kit preferably contains an oligonucleotide primer capable of amplifying the repeat region in the CNM protein of Streptococcus mutans.

Examples of oligonucleotide primers capable of amplifying the repeat region include the oligonucleotide primers listed in the above method for obtaining amino acid sequence length information of the repeat region, including preferred embodiments thereof.

The kit of the present invention may also include one or a combination of two or more of the following: specimen collection tools such as saliva collection spitzes, droppers, and swabs; equipment for selecting S. mutans (e.g., sterile substrates such as plates, MSB agar medium, gram-positive bacteria detection reagents, antibiotics, etc.); washing and dilution buffers; and equipment for PCR (e.g., tubes, enzymes, buffers, etc.).

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

1. Obtaining genome sequence information and gene annotation The samples used were dental plaque samples taken from the tooth surfaces of 14 patients who had been treated for stroke at the National Cerebral and Cardiovascular Center Hospital. These patients had been confirmed to have deep cerebral microbleeds by head MRI.

First, each specimen was spread on Mitis-Salivarius (MS) agar medium supplemented with bacitracin and sucrose, and cultured anaerobically at 37°C for 48 hours. Rock-like colonies morphologically recognized as S. mutans were selected and cultured anaerobically in BHI (brain heart infusion) medium at 37°C for an additional 48 hours. Next, the specimen was centrifuged at 7,000 rpm for 10 minutes using a centrifuge (Sorvall ST 8FR, Thermo Fisher Scientific), the supernatant was removed, and the bacterial cells (precipitate) were collected.

Genomic DNA was extracted from the collected cells using a DNA extraction kit ("smart DNA prep (m)", Analytik Yena). The extracted genomic DNA was fragmented using a DNA Shearing Tube ("g-TUBE", Covaris), and a DNA library for sequence analysis was prepared using the SMR Tbell Template Prep Kit (PacBio).

The prepared DNA library was used to obtain whole genome sequence information using a single molecule real-time DNA sequencer (Sequel, PacBio). The obtained sequence information was subjected to assembly analysis using an assembler (in this example, "HGAP (Hierarchical Genome Assembly Process) 4", PacBio) to reconstruct the genome sequence. Gene prediction software (in this example, "Prokka") was used to predict gene regions on the constructed whole genome sequence, and their amino acid sequences were obtained.

2. Acquisition of CNM amino acid sequence and multiple alignment From the UniProt database, the amino acid sequence of collagen-binding adheren protein (CNM protein), UniprotKB (Universal Protein Resource Knowledgebase) Entry_Name: C4B6T3_STRMG) of S. mutans was obtained, and a homology search was performed using Blast+ for the amino acid sequence obtained in 1 above, and the amino acid sequence output at the top was used as the amino acid sequence of the CNM protein in that sample (CNM amino acid sequence). In all samples, a sequence (CNM amino acid sequence) with a homology (identity) of 93.6% or more to the amino acid sequence of the CNM protein of S. mutans was confirmed. From the CNM amino acid sequence, the full length (amino acid sequence length) was obtained.

Next, multiple alignment was performed on the CNM amino acid sequences obtained from each sample using base sequence analysis software (in this example, "CLC Sequence Viewer", Qiagen). The results are shown in Figures 1A to 1C. Figures 1A to 1C show only the area surrounding the repeat region of each CNM amino acid sequence, with the repeat region surrounded by a dotted line (however, in the sequence of sample No. 12, TTTE from residue 413 onwards within the dotted line frame is not included in the repeat region (the repeat region ends with proline (P) at residue 413)). In this example, TTTTE(K/A)P (SEQ ID NO: 4), TTTE(A/S/T)P (SEQ ID NO: 5), TTEAP (SEQ ID NO: 6), and TTTTTEKP (SEQ ID NO: 3) were used as the building blocks (repeat units) that make up the repeat region.

The length of the repeat region in the CNM amino acid sequence obtained from each sample, the number of repeats contained in the repeat region, and the severity of deep cerebral microbleeds of the patient from whom the sample was taken (0-1 deep cerebral microbleeds: mild (L), 2 or more deep cerebral microbleeds: severe (H)) are shown in Table 1 below. In Table 1, the length of the repeat region was evaluated as "long" or "medium" when it was 111 residues or more, and "short" when it was less than 111 residues. The relationship between the length of the amino acid sequence and the number of samples of each severity of deep cerebral microbleeds (contingency table) is shown in Table 2 below. When Fisher's exact test was performed on Table 2 using statistical analysis software (in this example, "R"), it was determined that there was a significant difference when the significance level was set at 0.05 (P value = 0.015).

3. Heat map analysis For the genome sequences obtained from 12 of the 14 samples, a homology search software (in this example, "DIAMOND") was used to search for homology of the sequences of the gene regions predicted in 1 above against a gene sequence database (in this example, "KEGG"), and IDs were assigned for each functional classification. Table 3 below shows some of the gene IDs and the number of genes possessed in each sample.

Using the tabulated results in Table 3, a heat map analysis was performed using a statistical analysis program (in this example, the "heatmap.2 function" of the statistical analysis software "R"). Figure 2 shows a heat map diagram clustered by gene ID and its number, and Figure 3 shows a heat map diagram clustered by gene ID and its presence or absence. A clear correlation was confirmed between the amino acid sequence length of the repeat region in the above CNM amino acid sequence and severity (Tables 1-2), whereas no clear correlation was confirmed between the presence or absence of genes and severity, as shown in the heat map analysis in Figures 2-3.

4. Reproduction test of risk prediction of severe disease It was confirmed that the amino acid sequence length can be obtained by amplifying the DNA encoding the repeat region, and the risk of severe cerebral microbleeds can be predicted using the amino acid sequence length as an index. As samples, dental plaque collected using a swab from the tooth surface of patients (60 people) who were treated for stroke at the National Cerebral and Cardiovascular Center Hospital was used. These patients were patients whose number of deep cerebral microbleeds had been confirmed by head MRI and who were already known to have CNM-positive S. mutans in their oral cavity.

First, each specimen was spread on Mitis-Salivarius (MS) agar medium supplemented with bacitracin and sucrose, and cultured anaerobically at 37°C for 48 hours. Blue rock-like colonies morphologically recognized as S. mutans were selected and further cultured anaerobically at 37°C for 24 to 48 hours in BHI (brain heart infusion) medium. Next, the specimens were centrifuged at 800 x g for 10 minutes, the supernatant was removed, and the bacterial cells (precipitate) were collected. The collected cells were washed once with Phosphate Buffered Saline, suspended in a DNA extraction reagent (InstaGene DNA Purification Matrix, BioRad), heated at 56°C for 20 minutes, vortexed, and further heated at 100°C for 8 minutes to extract genomic DNA, and the supernatant was used in the PCR described below.

The DNA encoding the repeat region was amplified by PCR using "GeneAtlas 482" (ASTEC). "Emerald Amp Max PCR Master Mix" (Takara Bio) and two types of primers (forward primer: an example of a forward primer shown in SEQ ID NO: 11; reverse primer: an example of a reverse primer shown in SEQ ID NO: 12) were used under the following conditions:
Initial denaturation: 94°C 2 minutes,
Cycle:
Denaturation: 94℃ 30 seconds,
Annealing: 66℃ 30 seconds,
Number of cycles: 27 times,
Final extension: The target DNA fragment was amplified at 72° C. for 5 minutes.

The obtained DNA fragments (PCR products) were electrophoresed for 90 minutes on a 3% agarose gel using "Mupid-2x" (ADVANCE) while cooling. The 3% agarose gel was made using agarose (low electro-osmosis, high gel strength, Nacalai Tesque), "Midori Green Advance DNA Stain" (Nihon Genetics), and Tris-Borate-EDTA Buffer (TBE), and a 100 bp DNA ladder ("dye plus", Takara Bio) was used as a size marker. After electrophoresis, the gel was photographed using a UV sample photographing device "FAS-III" (TOYOBO), and the S. The length of the DNA sequence encoding the repeat region in S. mutans was measured.

Part of the electrophoretic images (for 11 samples) is shown in Figure 4. Among the samples shown in Figure 4, sample "H205-4 (the leftmost sample, the eighth sample from the left excluding the blank lane, and the rightmost sample)" is a sample known by sequencing to have an amino acid sequence length of 110 residues in the repeat region of the CNM amino acid sequence. In this case, in the above PCR example, a band is detected at the position of 486 bp. Therefore, bands detected in the electrophoretic image that are longer than 486 bp (i.e., the amino acid sequence length of the repeat region is 111 residues or more) were evaluated as having an amino acid sequence length of "long/medium," and bands that are 486 bp or less (i.e., the amino acid sequence length of the repeat region is 110 residues or less) were evaluated as having an amino acid sequence length of "short." The other samples were also evaluated in the same way.

The relationship (contingency table) between the amino acid sequence length and the number of samples with each severity of deep cerebral microbleeds (0-1 deep cerebral microbleeds: mild (L) and 2 or more deep cerebral microbleeds: severe (H)) is shown in Table 4 below. When Fisher's exact test was performed on Table 4, the P value was 0.015, confirming a significant difference. From the above, it was confirmed that the amino acid sequence length can be obtained by amplifying the DNA encoding the repeat region, and that the risk of severe cerebral microbleeds can be predicted using the obtained amino acid sequence length as an index.

The present invention makes it possible to provide a method for predicting a subject's risk of developing severe cerebral microbleeds, a method for screening subjects at high risk of developing severe cerebral microbleeds, and a kit for use in these methods. Therefore, the present invention is useful for developing methods for preventing and treating the development of severe cerebral microbleeds, as well as for preventing and treating diseases such as dementia and stroke that are thought to be associated with cerebral microbleeds.

SEQ ID NO:4
<223> SEQ ID NO: 5, where Xaa represents K or A
<223> SEQ ID NO: 11, where Xaa represents A, S, or T
<223> Example of forward primer SEQ ID NO: 12
<223> Reverse primer example

Claims (9)

A method for providing information that serves as an index for predicting a subject's risk of developing severe cerebral microbleeds,
A method comprising obtaining the amino acid sequence length of a repeat region in a CNM protein of Streptococcus mutans contained in a sample derived from a subject, and providing the length as the indicator. The method according to claim 1, characterized in that it provides information that serves as an indicator for predicting that subjects whose amino acid sequence length of the repeat region is 111 residues or more are at high risk of developing severe cerebral microbleeds. A method for providing information that serves as an index for screening subjects at high risk of developing severe cerebral microbleeds,
A method comprising obtaining the amino acid sequence length of a repeat region in a CNM protein of Streptococcus mutans contained in a sample derived from a subject, and providing the length as the indicator. The method according to claim 3, characterized in that it provides information that serves as an index for selecting subjects whose amino acid sequence length of the repeat region is 111 residues or more as being at high risk of developing severe cerebral microbleeds. The method according to any one of claims 1 to 4, characterized in that the repeat region is a region consisting of multiple repeat units, each of which has 4 to 10 amino acid residues. The method according to claim 5, characterized in that each of the repeat units independently contains at least two threonine residues. The method according to claim 5 or 6, characterized in that the repeat units are each independently a unit in which the first amino acid residue is threonine or alanine. The method according to any one of claims 5 to 7, characterized in that the repeat units are each independently a unit whose last amino acid residue is proline or serine. A kit for use in the method according to any one of claims 1 to 8, the kit comprising an oligonucleotide primer capable of amplifying a repeat region in the CNM protein of Streptococcus mutans.