KR20220126512A - Single Nucleotide Polymorphisms Implicated in External Apical Root Resorption and Use Thereof - Google Patents

Abstract

The present invention relates to a composition for diagnosing or predicting root disease, specifically, root external resorption, and a diagnostic method using the same. The present invention was first to identify a significant correlation between root external resorption and a single nucleotide polymorphism (rs3810765) in an SFRP2 gene such that the possibility of root external resorption can be predicted with high reliability only by a simple process of detecting the SNP by a known method. By providing important clinical information on the genetic risk of root tissue damage during orthodontic tooth movement in Asians, especially Koreans, the present invention can be usefully used to early establish a customized prevention/treatment strategy for patients who are undergoing orthodontic treatment or who are scheduled to undergo orthodontic treatment in the future.

Description

Single Nucleotide Polymorphisms Implicated in External Apical Root Resorption and Use Thereof

The present invention relates to a method for predicting the genetic risk of extra-root resorption in a patient due to orthodontic treatment by detecting a single nucleotide polymorphism (SNP) on the SFRP2 gene.

External apical root resorption (EARR) is one of the most frequent side effects of orthodontic treatment and causes loss of tooth structure. About one-third of patients who received orthodontic treatment showed moderate extra-root resorption (3-5 mm), and 2-5% showed severe extra-root resorption (more than 5 mm). known (Killiany, 1999). Clinical characteristics related to EARR are diverse, and the degree of orthodontic force, direction of force, root shape, allergy, trauma history, orthodontic treatment start age, excessive horizontal overjet, tooth extraction, gender, treatment period, etc. occurred during orthodontic treatment. are known to be risk factors for EARR (Acar et al., 1999; Martins et al., 2012; Nanekrungsan et al., 2012; Parker and Harris, 1998; Picanco et al., 2013; Segal et al., 2004; Weltman et al., 2010). Significant individual differences in orthodontic treatment-related EARR suggest that individual tendencies and multiple etiologies are involved (Harris et al., 1997; Hartsfield et al., 2004). The response to orthodontic treatment depends on the patient's genetic characteristics, as the patient's genetic information determines the proteins and signal processing processes involved in the absorption and restoration of cementum and dentin during orthodontic treatment (Abass and Hartsfield, 2007; Harris, 2008).

Research results on genetic factors that influence EARR continue to be reported, but early studies were mainly related to heritability. It has been reported that EARR and genetic mutations accompanying orthodontic treatment show a familial clustering phenomenon (Newman, 1975). Studies have shown that mutations are observed in genes related to the inflammatory response required for orthodontic tooth movement. In addition, as a result of investigating the genetic relevance of EARR using the Sib-pair model, it was found to have high heritability (Harris et al., 1997), showing the importance of genetic predisposition in EARR.

Recently, a paper has been reported to reveal specific genes related to EARR using a genetic analysis method through saliva extraction. Iglesias-Linares et al. (2012) reported that there was a significant relationship between mutations in IL-1β and IL-1 receptor antagonist (IL-1RN) genes and extra-root resorption in 54 Caucasian orthodontic patients in Spain ( Iglesias-Linares et al., 2012a), the association between mutations in the purine receptor P2X (P2RX7) gene associated with the IL-1β pathway and extra-root resorption was also reported in 134 patients in the United States (Sharab et al., 2015). In addition, the association between mutations in the IL-1 receptor-related kinase 1 (IRAK1) gene and extraroot resorption related to inflammation was reported in 195 orthodontic patients in Portugal (Pereira et al., 2016; Sharab et al., 2015). . There is also a study that the osteoprotezerin (OPG)/receptor activator nuclear factor kappa B ligand (RANKL) pathway affects cementitious formation and thus is involved in root resorption in 124 American Caucasian orthodontists (Al- Qawasmi et al., 2003).

Until now, studies on the genetic factors of extraroot resorption caused by orthodontic treatment have been limited to Westerners, and few studies have been conducted on Asians including Koreans. Accordingly, the present inventors tried to investigate the relationship between factors (age, sex, angle classification, correction period, traction amount) related to extra-root resorption caused by orthodontic treatment, and to explore genetic variations related to extra-root resorption.

Numerous papers and patent documents are referenced throughout this specification and citations thereof are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Non-Patent Literature 1. Journal of Bone and Mineral Research 35(11): 2252-2264 (2013)

The present inventors predict in advance the possibility of occurrence of extra-root resorption (EARR), which is a high frequency side effect accompanying orthodontic treatment, in order to develop a method for pre-selecting a patient with a potential for damage to the root structure during orthodontic treatment. Careful research efforts were made. As a result, the presence of rs3810765, a single nucleotide polymorphism in the SFRP2 gene, has a significant relationship with root resorption, and it is possible to establish a patient-specific treatment strategy early by predicting with high reliability the genetic risk of root resorption through detection. It has been found that the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for diagnosing or predicting a periodontal disease, specifically, extra-root resorption.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for diagnosing or predicting periodontal disease comprising an agent detecting the rs3810765 single nucleotide polymorphism (SNP) site on the SFRP2 gene.

The present inventors predict in advance the possibility of occurrence of extra-root resorption (EARR), which is a high frequency side effect accompanying orthodontic treatment, in order to develop a method for pre-selecting a patient with a potential for damage to the root structure during orthodontic treatment. Careful research efforts were made. As a result, the presence of rs3810765, a single nucleotide polymorphism in the SFRP2 gene, has a significant relationship with root resorption, and it is possible to establish a patient-specific treatment strategy early by predicting with high reliability the genetic risk of root resorption through detection. found that there is

As used herein, the term “dental root disorder” refers to a series of diseases that directly or indirectly cause quantitative loss or dysfunction of the tissue of the tooth root. Periodontal disease can be caused by a developmental defect in the root tissue, physical and chemical stress stimulation, or tissue damage caused by inflammatory stimulation. Early diagnosis or prevention through advance prediction is of the utmost importance because there is no fundamental treatment method that depends only on transplantation treatment.

According to a specific embodiment of the present invention, the root disease that can be diagnosed or predicted by the composition of the present invention is external apical root resorption (EARR).

As used herein, the term “diagnosis” refers to determination of an individual's susceptibility to a specific disease, determination of whether an individual currently has a specific disease, determination of the prognosis of an individual with a specific disease, and determination of the individual's Including the prediction of the risk of developing a particular disease in the future. For the purpose of the present invention, the diagnosis of the present invention means predicting the risk of developing a root disease in the future, although the subject has a current periodontal disease, for example, extra-root resorption, or a current periodontal disease has not been clinically confirmed. can

As used herein, the term “diagnostic composition” refers to an integrated mixture or device including a means for detecting SNP (rs3810765) in order to determine whether or not a subject develops a periodontal disease or predict the onset possibility, and It may also be expressed as a “diagnostic kit”.

As used herein, the term “nucleotide” has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules that exist in single-stranded or double-stranded form, and nucleotides, which are the basic structural units in nucleic acid molecules, include natural nucleotides as well as natural nucleotides. but also analogs in which sugar or base sites have been modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

As used herein, the term “single nucleotide polymorphism (SNP)” refers to a DNA sequence that occurs when single nucleotides (A, T, C, G) in the genome differ between members of a species or between paired chromosomes of an individual. means the diversity of For example, three DNA fragments from different individuals (e.g., When it contains a difference in a single base, such as AAGT[A/A]AG, AAGT[A/G]AG, AAGT[G/G]AG), it is called two alleles, and in general, almost all SNPs have two alleles. have genes Within a population, SNPs can be assigned a minor allele frequency (MAF; the lowest allele frequency at a locus found in a particular population). Variations exist within human populations, and single SNP alleles common in geological or ethnic groups are very rare.

The presence of a SNP may cause a change in the protein amino acid sequence through a change in the translation frame, but the SNP in the coding sequence of a gene does not necessarily cause a change in the amino acid sequence of the target protein due to codon degeneracy.

According to a specific embodiment of the present invention, the agent for detecting the SNP site of the present invention is specific for the rs3810765 single nucleotide polymorphism on the SFRP2 gene, that is, a sequence of 10-100 consecutive nucleotides containing the position of the 153,788,328 base of human chromosome 4 It is a primer or probe that binds positively.

According to one embodiment of the present invention, the SNP of the present invention can be detected according to a gene amplification method, for example, polymerase chain reaction (PCR) using primers.

As used herein, the term “primer” refers to conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (template), that is, the presence of nucleotides and a polymerization agent such as DNA polymerase, and a suitable temperature and pH. It refers to an oligonucleotide that acts as a starting point of Specifically, the primer is a single-stranded deoxyribonucleotide. The primer used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides, and may also include ribonucleotides.

The primer of the present invention may be an extension primer that is annealed to a target nucleic acid to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which is extended to the position where the immobilized probe is annealed so that the probe is It occupies the annealed area.

The extension primer used in the present invention includes a hybridization nucleotide sequence complementary to a target nucleic acid, for example, 10-100 consecutive nucleotide sequences including the rs3810765 mononucleotide polymorphism site of the SFRP2 gene. The term “complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under certain annealing or hybridization conditions, and when substantially complementary and perfectly complementary ), and specifically means a completely complementary case. As used herein, the term "substantially complementary sequence" is meant to include not only a completely identical sequence, but also a sequence that is partially mismatched with a sequence to be compared within a range that can function as a primer by annealing to a specific sequence.

The primer should be long enough to prime the synthesis of the extension product in the presence of a polymerizer. A suitable length of a primer depends on a number of factors, such as temperature, pH and source of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The design of such a primer can be easily performed by those skilled in the art with reference to the target nucleotide sequence, for example, using a primer design program (eg, PRIMER 3 program).

According to another embodiment of the present invention, the SNP of the present invention can be detected according to a microarray method using a probe immobilized on a solid surface.

As used herein, the term “probe” refers to a natural or modified monomer or a linear oligomer including deoxyribonucleotides and ribonucleotides capable of hybridizing to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, more specifically deoxyribonucleotides. As the probe used in the present invention, a sequence perfectly complementary to the target region of the SFRP2 gene may be used, but a substantially complementary sequence may be used within a range that does not interfere with specific hybridization. may be In general, since the stability of the duplex formed by hybridization tends to be determined by the match of the sequences at the ends, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence. do.

Suitable conditions for hybridization are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington , DC (1985) can be determined with reference to the disclosure.

According to a specific embodiment of the present invention, the composition of the present invention is a single nucleotide polymorphism (SNP) site (rs4754) having a T at the 321st nucleotide position of SEQ ID NO: 2, at the 789th nucleotide position of SEQ ID NO:2 One or more SPP1 genes selected from the group consisting of a single polymorphic site having C (rs1126616) and a single polymorphic site having A at the 294 nucleotide position of SEQ ID NO: 2 (rs9138) To detect a single polymorphism site Additional agents are included.

More specifically, the agent specifically binds to a sequence of 10-100 contiguous nucleotides comprising at least one nucleotide position selected from the group consisting of nucleotide 321, nucleotide 789 and nucleotide 294 of SEQ ID NO: 2 primers or probes.

According to a specific embodiment of the present invention, the composition of the present invention is applied to an Asian.

In the present invention, the term “Asia” refers to the Far East region where Mongolian races, including Korea, China, and Japan, reside. “Asian” means a population of Asian ancestry, preferably a population of Asian ancestry for at least 10 generations. More specifically, the Asian is Korean.

According to another aspect of the present invention, the present invention provides information necessary for the diagnosis or prediction of periodontal disease, comprising the step of detecting the rs3810765 single nucleotide polymorphism (SNP) site on the SFRP2 gene in a biological sample isolated from an individual. provides

Since the single nucleotide polymorphism as a diagnostic marker used in the present invention and a root disease that can be diagnosed or predicted using the same have already been described above, description thereof will be omitted to avoid excessive duplication.

As used herein, the term “individual” refers to an individual that provides a sample for detecting the above-described single nucleotide polymorphism of the present invention, and is ultimately analyzed for the diagnosis of dental root disease or the prediction of the onset possibility. Subjects include, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons or rhesus monkeys, specifically humans. The composition of the present invention contains information for predicting the genetic risk of not only the onset of current periodontal disease, specifically extra-root resorption, but also the occurrence of extra-root resorption due to orthodontic treatment in the future, and quantitative loss and loss of function of the root tissue. Because it provides, the subject of the present invention may be a patient with periodontal disease or a healthy subject that has not yet developed.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for diagnosing or predicting a periodontal disease, specifically, extra-root resorption, and a diagnostic method using the same.

(b) The present invention is the first to identify a significant correlation between extra-root resorption and the single nucleotide polymorphism (rs3810765) of the SFRP2 gene. can

(c) The present invention provides important clinical information on the genetic risk of root tissue damage during orthodontic movement in Asians, especially Koreans, tailored to patients undergoing orthodontic treatment or scheduled for future orthodontic treatment It can be usefully used to establish prevention and treatment strategies at an early stage.

1 is a diagram schematically illustrating each baseline and reference point according to Linge's research. 1, apical; 2, terminal CEJ; 3, Central CEJ; 4, cut edge (cut edge). CEJ was determined through a straight line between the two points of the central and distal CEJ. Crown length (C) and root length (R) were measured as the longest distance from the apex to the incisal edge while perpendicular to the CEJ.
2 is a diagram showing landmarks and baselines for cranial measurement analysis. Horizontal (HRP) and vertical (VRP) reference planes were used to measure the movement of each landmark: sella (S), non-proximal (N), A point (A), A point 3 mm below (A-3) , B point (B), mandibular point (Pog), mandrel (Me), apical end of maxillary central incisor (U1r), cervical point of maxillary central incisor (U1c), distal end of maxillary central incisor (U1t), distal end of mandibular central incisor (L1t) , cervical point of mandibular central incisor (L1c), apical end of mandibular central incisor (L1r), anteriormost point of nose (Pn), subnasal point (Sn), soft tissue A point (A'), upper labial apex (Hendrix et al.), Upper labial fusion point (Stms), lower labial fusion point (Stmi), lower labial apex (Li), soft tissue B point (B'), soft tissue mandibular point (Pog'), soft tissue mandibular (Me').
3 is a diagram showing the EARR level according to the genotype of each SNP of rs4754 and rs1126616 (FIG. 3A), rs9138 and rs3810765 (FIG. 3B). The y-axis represents the EARR value, and the horizontal bar in the scatter plot represents the mean value. Statistical analysis was performed Statistical analysis was performed through Kruskal-Wallis test or Mann-Whitney U-test. *P<0.05.
4 is a diagram illustrating NGS data statistics for target sequencing, and shows the average sequencing depth of the target region ( FIG. 4a ) and the coverage (cover age) of the target region in each sample ( FIG. 4b ), respectively.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

Research subject

This study was conducted retrospectively and was approved by the Clinical Trial Review Committee (IRB: 2-2016-0023) of Yonsei University Dental Hospital. All clinical examinations were performed in accordance with the Declaration of Helsinki, and written informed consent was obtained with a description of the study prior to enrollment of all study subjects. Between February 2008 and April 2020, 122 patients who completed orthodontic treatment at the Department of Orthodontics at Yonsei University Dental Hospital were selected. 118 patients (25 males, 93 females) with an average age of 19.8±6.2 years were selected, excluding patients with difficult genetic analysis. The mean duration of orthodontic treatment was 33.6±8.3 months, and the selection criteria were as follows:

(1) In patients 12 years of age or older, the root formation of the maxillary 4th incisors must be complete before orthodontic treatment;

(2) Panoramic and lateral radiographs were taken before and after treatment;

(3) The case should be a case in which both maxillary premolars were extracted and the maxillary anterior teeth were pulled posteriorly;

(4) The tooth to be measured EARR must have radiographic and clinically normal apical anatomy before and after orthodontic treatment; and

(5) The tooth to be measured EARR must have no change in the incisal edge on the cast before and after orthodontic treatment, and there must be no history of caries treatment during orthodontic treatment.

Patients with systemic diseases affecting root resorption, patients with maxillofacial deformity or orthognathic surgery, and patients with impaction teeth were excluded.

All 118 patients were treated with conventional archwire sequences from 0.016-in nickel-titanium to 0.019 x 0.025-in stainless steel (G&H Orthodontics, Franklin, Ind) using conventional brackets with .018 or .022 slots. The chart was reviewed to confirm the clinical test results of each patient.

Subjects were divided into two groups according to whether the EARR obtained from radiographic measurements was greater than or equal to 2 mm. Group 1 was classified as an EARR-occurring group (EARR greater than or equal to 2 mm), and group 2 was classified as a non-occurring group (EARR less than 2 mm). (Bastos Lages et al., 2009; Iglesias-Linares et al., 2017; Iglesias-Linares et al., 2014; Iglesias-Linares et al., 2012b). The classified sample groups are as follows.

(1) Group 1 (EARR occurrence group; EARR ≥ 2 mm): 59 patients. The average age at the start of orthodontic treatment was 19.8 years, and the average orthodontic treatment period was 33.7 months.

(2) Group 2 (EARR non-occurring group; EARR < 2 mm): 59 patients. The average age at the start of orthodontic treatment was 19.7 years, and the average orthodontic treatment period was 33.5 months.

Panoramic radiographic measurement and metrology

EARR was measured through panoramic radiographs taken before (T1) and after (T2) orthodontic treatment of the study subjects. Panoramic radiographs were taken by a trained radiologist using Cranex3+ (Soredex, Helsinki, Finland) at the Department of Imaging Dentistry, Yonsei University College of Dentistry. The patient was placed in an upright position with the midsagittal plane perpendicular to the floor and the Frankfurt horizontal plane parallel to the floor. In the panoramic radiograph, the root resorption amount was measured for the tooth showing the highest EARR among the maxillary 4 incisors and adopted as an independent variable. The crown and root lengths were measured using the ZeTTA PACS Viewer program. Base points and baselines before and after treatment are indicated in FIG. 1 . For measurement of crown and root length, the Linge and Linge (FIG. 1) method was referred to (Linge and Linge, 1991; Lee and Lee, 2016). First, a straight line connecting two points of mesiodistal CEJ was set as a cementoenamel junction (CEJ). The crown length was measured from the incisal edge to the CEJ, and the root length was measured as the straight line distance from the CEJ to the root apex. EARR was measured in units of 0.01 mm as follows:

EARR = Root length before orthodontic treatment - Root length after orthodontic treatment x (Crown length before orthodontic treatment / Crown length after orthodontic treatment)

Measurement and measurement of lateral head standard radiograph

Lateral head radiographs were taken by a trained radiographer using Cranex3+ (Soredex, Helsinki, Finland) at the Department of Imaging Dentistry, Yonsei University College of Dentistry Hospital, at the time of the initial examination and after treatment. ) and induced to naturally close the lips by relaxing the lips during filming. It was measured by one person using V-Ceph ^TM 5.5 (Osstem, Seoul, South Korea) and the length was measured in units of 0.1 mm.

The horizontal reference line (HRP) was taken as the reference line from the Sella-non-proximal line to the Sella by 7 degrees (Burstone et al., 1978). ) was made. The horizontal traction amount was determined as the difference between the maxillary anterior tip parallel to the HRP before and after treatment on the pre-treatment (T1) lateral head radiograph and the post-treatment (T2) radiograph, and the vertical traction amount was the difference between the maxillary anterior tip parallel to the VRP. .

Within-Investigator Error and Method Error

In order to prevent inter-investigator errors, all measurements and analyzes were performed by the same investigator, and for 10 randomly selected subjects to check intra-investigator errors, the EARR was 0.01 unit and the traction amount was 0.1 mm in the ZeTTA PACS Viewer program. and V-ceph 5.5 (Osstem Inc., Korea) program were repeated twice for measurement.

Sample collection and DNA extraction

DNA 수집 킷(DNA Genotek) 또는 Oragene DNA 자가-수집 킷(Genotek, Ottawa, Ontario, Canada)을 이용하여 제조사의 지시에 따라서 킷에 침(2ml)을 뱉도록 하였다. 킷의 뚜껑을 닫고 대상자의 침이 고정액(DNA-보존 용액, 2 ml)과 섞이도록 하였다. Oragene 타액 시료는 DNA 추출 전까지 냉장 보관하였다. Genomic DNA는 prepIT-L2P DNA 추출 킷(Genotek, Ottawa, Ontario, Canada)을 사용하여 분리시켰다. DNA는 Qubit을 사용하여 정량화하였다.Subjects were asked to rinse their mouths once with water, and after 30 seconds, OG-500 Oragene

A DNA collection kit (DNA Genotek) or an Oragene DNA self-collection kit (Genotek, Ottawa, Ontario, Canada) was used to spit (2 ml) into the kit according to the manufacturer's instructions. The kit was capped and the subject's saliva was allowed to mix with the fixative (DNA-preserving solution, 2 ml). Oragene saliva samples were refrigerated until DNA extraction. Genomic DNA was isolated using a prepIT-L2P DNA extraction kit (Genotek, Ottawa, Ontario, Canada). DNA was quantified using Qubit.

Target gene selection and targeted next-generation sequencing

The target gene included only the gene of the coding sequence (CDS) region, and additional target probes for single nucleotide polymorphisms (SNPs) were designed (Table 1). Targeted sequencing was carried out based on hybridized capture-based next-generation sequencing. For next-generation sequencing data, genotype data were obtained using an enome analysis tool kit (GATK) for positions with an average depth of 20 or more. A total of 299 mutations were found, among which allele frequencies of 0 or 100 were excluded because they were monotypes. Variants with a minor allele frequency of less than 3% were also excluded from analysis. According to the stated criteria, 6 and 151 variants were excluded, respectively, and the final 142 variants were analyzed in the present invention.

gene additional area ALPL CASP1 rs530537 (intron) CASP5 rs554344 (2KB upstream) DKK1 DKK2 DKK3 FRZB FZD7 IL-17A rs2275913 (2KB upstream) IL1A rs1800587 (5'-UTR) IL1B IL1RN IL-6 rs1800796 (intron) IL-8 IRAK1 LRP1 LRP5 LRP6 P2RX7 SFRP1 rs16890444 (intron) SFRP2 rs3242 (3'UTR) SFRP4 SFRP5 SOST rs1230399 (upstream), rs851054 (2KB upstream), rs851056 (2KB upstream) SPP1 rs11730582 (2KB upstream), rs9138 (3'UTR region) TNF rs1800629 (2KB upstream) TNFRSF11A rs12455775, rs12956925, rs12959396, rs12970081, rs17069845, rs17069898, rs17069902, rs17069904, rs17720953, rs3826620, rs4426449, rs4485469, rs4500848, rs4524034, rs4941125, rs4941129, rs6567272, rs7233197, rs7236060, rs7237982, rs7239667, rs8083511, rs8086340, rs8089829, rs8099222, rs9951012 (above, intron) TNFRSF11B rs3102735 (2KB upstream), rs1032128 (hereafter intron), rs11573856, rs11573884, rs11573901, rs11573938, rs1485289, rs2875845, rs3102724, rs3102728, rs3134057, rs3134060, rs7010267 TNFSF11 rs1038434, rs12585229, rs3742257, rs931273 (above, intron) VDR WIF1 WISP3 WNT10B WNT3A rs4653533 (intron), rs752107 (intron) WNT7B

statistical analysis

Statistical analysis was performed using the R 3.5.2 (R Foundation) program. Comparison of EARR occurrence and non-occurrence groups of clinical variables was analyzed by x2 test or t-test. The association between EARR and clinical variables was analyzed using linear regression analysis. The comparison of genotype and allele frequency between EARR occurrence and non-occurrence groups was analyzed through multiple linear regression analysis with age and sex as covariates. The genotype frequency for each SNP was verified by Hardy-Weinberg equilibrium. The degree of association between SPP1 (rs4754, rs1126616, rs9138) and SFRP2 (rs3810765) with clinical variables was evaluated by OR (odds ratio) obtained through logistic regression analysis. EARR values according to SPP1 (rs4754, rs1126616, rs9138) and SFRP2 (rs3810765) mutations were analyzed by Kruskal-Wallis test or Mann-Whitney U-test. All P values were based on two-sided comparisons and were considered statistically significant if P<0.05.

Experiment result

Within-Investigator Error Test

In order to evaluate the reliability of the measurement of the present invention, the EARR and traction amount of 10 people randomly selected from each group were re-measured at 2-week intervals. As a result of testing reliability with the intraclass correlation coefficient (ICC), all of them were 0.9 or higher, indicating a high degree of agreement between repeated measurements.

Clinical parameters of study subjects

Of the total 118 patients, 93 were female and 25 were male. The average age at the start of orthodontic treatment was 19.8 years (12-47 years) (Table 2).

The average orthodontic treatment period was 33.6±8.3 months, the average traction amount was 4.4±2.8mm, and the average EARR was 2.9±2.4mm. As for the angle classification, Class I was 45 (38.1%), Class II was 57 (48.3%), and Class III was 16 (13.6%). The study subjects were divided into two groups (EARR occurrence and non-EARR groups) and analyzed, and the analysis results of each group are as follows:

(1) Group 1 (patients with EARR, EARR ≥ 2 mm): 59 patients (43 female, 16 male). The average age at the start of orthodontic treatment was 19.8 years (12-37 years), the average orthodontic treatment period was 33.7±8.3 months, the average traction amount was 4.2±3mm, and the average EARR was 4.8±1.8mm. As for the angle classification, Class I was 19 (32.2%), Class II was 31 (52.5%), and Class III was 9 (15.3%).

(2) Group 2 (patients without EARR, EARR < 2 mm): 59 patients (50 female, 9 male). The average age at the start of orthodontic treatment was 19.7 years (12-47 years), the average length of orthodontic treatment was 33.5±8.4 months, the average traction amount was 4.6±2.6 mm, and the average EARR was 0.9±0.6 mm. As for the angle classification, there were 26 (44.1%) Class I, 26 (44.1%) Class II, and 7 (11.9%) Class III. There were no significant differences between the EARR groups with respect to clinical variables (age, sex, angle classification, correction period, and traction amount).

clinical parameters all patients
(n=118) EARR occurrence
group (n=59) EARR non-occurring group (n=59) *P value (EARR occurrence vs non-occurrence) starting age (years),
Median (range) 19.8 (12-47) 19.8 (12-37) 19.7 (12-47) 0.91 Gender, n (%) female 93 (78.8) 43 (72.9) 50 (84.7) 0.12 male 25 (21.2) 16 (27.1) 9 (15.3) Angle classification, n (%) Class I 45 (38.1) 19 (32.2) 26 (44.1) 0.41 Class II 57 (48.3) 31 (52.5) 26 (44.1) Class III 16 (13.6) 9 (15.3) 7 (11.9) Treatment period (months),
mean ± standard deviation 33.6±8.3 33.7±8.3 33.5±8.4 0.90 Horizontal anterior retraction (mm), mean ± standard deviation 4.4±2.8 4.2±3.0 4.6±2.6 0.39 EARR(mm),
mean ± standard deviation 2.9±2.4 4.8±1.8 0.9±0.6 <0.01

Analysis of association between EARR and clinical variables

There was no significant association with EARR for clinical variables (age, gender, angle classification, correction period, and traction amount) (Table 3).

clinical parameters r relation P value Age at start(years) 0.0075 0.0865 0.36 gender 0.0048 0.0695 0.46 Angle classification 0.0198 0.1407 0.14 Treatment period (months) 0.0069 0.0832 0.37 Horizontal anterior retraction (mm) 0.0000 -0.0008 0.99

Targeted sequencing results

As a result of genetic analysis, a total of 4 SNPs on 2 genes showed a significant difference in frequency between the group with and without EARR (Table 4). Accordingly, it was found that SPP1 (rs4754, rs1126616, rs9138) and SFRP2 (rs3810765) can be markers between the EARR groups (P<0.05).

Additionally, as a result of analyzing the EARR level for each genotype, rs9138 on SPP1 showed higher EARR than CC genotype in AA and AC genotypes (P=0.0275), and rs3810765 on SFRP2 TT genotype in CC and CT genotypes. It was found that the degree of EARR was higher than that (P=0.0284) (FIG. 3b). In the two SNPs of SPP1 (rs4754, rs1126616), there was no significant difference in EARR levels according to each genotype ( FIG. 3A ).

gene
(rs no.) * base change
(AA change) genotype group. No. (%) **P value [OR (95% CI)] EARR
Occur EARR
non-occurring dominant zeal Gong Woo-sung allele release isomorphic SPP1
(rs4754) c.321 T >C
(p.D107D) T/T 7
(11.9) 4
(6.8) 0.02 [2.45
(1.14-5.26)] 0.38 [1.79]
(0.49-6.58)] 0.03 [2.43
(1.07-5.54)] 0.18 [2.51]
(0.66-9.59)] 0.03 [1.97
(1.08-3.58)] T/C 25
(42.4) 16
(27.1) C/C 27
(45.8) 39
(66.1) SPP1
(rs1126616) c.789 C > T
(p.A263A) C/C 7
(11.9) 4
(6.8) 0.02 [2.45
(1.14-5.26)] 0.38 [1.79]
(0.49-6.58)] 0.03 [2.43
(1.07-5.54)] 0.18 [2.51]
(0.66-9.59)] 0.03 [1.97
(1.08-3.58)] C/T 25
(42.4) 16
(27.1) T/T 27
(45.8) 39
(66.1) SPP1
(rs9138) c.*294 A >C A/A 11
(18.6) 4
(6.8) 0.003 [3.16]
(1.44-6.90)] 0.06 [3.25]
(0.96-10.99)] 0.02 [2.71
(1.16-6.32)] 0.01 [4.9
(1.36-17.56)] 0.001 [2.64]
(1.46-4.78)] A/C 24
(40.7) 16
(27.1) C/C 24
(40.7) 39
(66.1) SFRP2
(rs3810765) c.502+6C> T C/C 23
(39) 14
(23.7) 0.07 [2.49
(0.92-6.7)] 0.10 [2.00
(0.88-4.57)] 0.17 [2.07
(0.73-5.84)] 0.03 [3.43
(1.1-10.74)] 0.04 [1.77
(1.04-3.02)] C/T 28
(47.5) 30
(50.8) T/T 8
(13.6) 15
(25.4)

*Nucleotide position numbers were assigned based on SPP1 (Transcript ID: NM_001251830.1) and SFRP2 (Transcript ID: NM_003013.2) mRNA sequences. Minor allele sequences are underlined.

Multivariate Logistic Regression

The results of multivariate logistic regression analysis for each SNP of EARR and clinical variables (age, sex, angle classification, treatment period, and traction amount) also showed significant differences between the groups with and without EARR at rs4754, rs1126616, rs9138 on SPP1 and rs3810765 on SFRP2. Differences were observed (Table 5, P<0.05).

gene (rs number) OR *P value Presence of the T allele 1.903 (1.035-3.502) 0.038 gender 1.758 (0.886-3.486) 0.106 age 1.009 (0.965-1.054) 0.699 SPP1 (rs4754) Angle classification 1.215 (0.799-1.846) 0.362 duration of treatment 1.005 (0.974-1.038) 0.749 horizontal anterior retraction 0.961 (0.872-1.059) 0.423 Presence of the C allele 1.903 (1.035-3.502) 0.038 gender 1.758 (0.886-3.486) 0.106 age 1.009 (0.965-1.054) 0.699 SPP1 (rs1126616) Angle classification 1.215 (0.799-1.846) 0.362 duration of treatment 1.005 (0.974-1.038) 0.749 horizontal anterior retraction 0.961 (0.872-1.059) 0.423 The presence of the A allele 2.595 (1.42-4.743) 0.002 gender 1.859 (0.929-3.717) 0.08 age 1.004 (0.96-1.05) 0.865 SPP1 (rs9138) Angle classification 1.173 (0.768-1.791) 0.461 duration of treatment 1.006 (0.974-1.04) 0.699 horizontal anterior retraction 0.959 (0.869-1.059) 0.407 Presence of the T allele 0.575 (0.334-0.988) 0.045 gender 1.704 (0.859-3.381) 0.127 age 1.022 (0.978-1.069) 0.334 SFRP2 (rs3810765) Angle classification 1.255 (0.828-1.901) 0.285 duration of treatment 0.999 (0.968-1.032) 0.965 horizontal anterior retraction 0.955 (0.866-1.053) 0.356

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry-academic cooperation foundation yonsei university <120> Single Nucleotide Polymorphisms Implicated in External Apical Root Resorption and Use Thereof <130> HPC9798 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 2005 <212> DNA <213> Homo sapiens <400> 1 caacggctca ttctgctccc ccgggtcgga gccccccgga gctgcgcgcg ggcttgcagc 60 gcctcgcccg cgctgtcctc ccggtgtccc gcttctccgc gccccagccg ccggctgcca 120 gcttttcggg gccccgagtc gcacccagcg aagagagcgg gcccgggaca agctcgaact 180 ccggccgcct cgcccttccc cggctccgct ccctctgccc cctcggggtc gcgcgcccac 240 gatgctgcag ggccctggct cgctgctgct gctcttcctc gcctcgcact gctgcctggg 300 ctcggcgcgc gggctcttcc tctttggcca gcccgacttc tcctacaagc gcagcaattg 360 caagcccatc cctgccaacc tgcagctgtg ccacggcatc gaataccaga acatgcggct 420 gcccaacctg ctgggccacg agaccatgaa ggaggtgctg gagcaggccg gcgcttggat 480 cccgctggtc atgaagcagt gccacccgga caccaagaag ttcctgtgct cgctcttcgc 540 ccccgtctgc ctcgatgacc tagacgagac catccagcca tgccactcgc tctgcgtgca 600 ggtgaaggac cgctgcgccc cggtcatgtc cgccttcggc ttcccctggc ccgacatgct 660 tgagtgcgac cgtttccccc aggacaacga cctttgcatc cccctcgcta gcagcgacca 720 cctcctgcca gccaccgagg aagctccaaa ggtatgtgaa gcctgcaaaa ataaaaatga 780 tgatgacaac gacataatgg aaacgctttg taaaaatgat tttgcactga aaataaaagt 840 gaaggagata acctacatca accgagatac caaaatcatc ctggagacca agagcaagac 900 catttacaag ctgaacggtg tgtccgaaag ggacctgaag aaatcggtgc tgtggctcaa 960 agacagcttg cagtgcacct gtgaggagat gaacgacatc aacgcgccct atctggtcat 1020 gggacagaaa cagggtgggg agctggtgat cacctcggtg aagcggtggc agaaggggca 1080 gagagagttc aagcgcatct cccgcagcat ccgcaagctg cagtgctagt cccggcatcc 1140 tgatggctcc gacaggcctg ctccagagca cggctgacca tttctgctcc gggatctcag 1200 ctcccgttcc ccaagcacac tcctagctgc tccagtctca gcctgggcag cttccccctg 1260 ccttttgcac gtttgcatcc ccagcatttc ctgagttata aggccacagg agtggatagc 1320 tgttttcacc taaaggaaaa gcccacccga atcttgtaga aatattcaaa ctaataaaat 1380 catgaatatt tttatgaagt ttaaaaatag ctcactttaa agctagtttt gaataggtgc 1440 aactgtgact tgggtctggt tggttgttgt ttgttgtttt gagtcagctg attttcactt 1500 cccactgagg ttgtcataac atgcaaattg cttcaatttt ctctgtggcc caaacttgtg 1560 ggtcacaaac cctgttgaga taaagctggc tgttatctca acatcttcat cagctccaga 1620 ctgagactca gtgtctaagt cttacaacaa ttcatcattt tataccttca atgggaactt 1680 aaactgttac atgtatcaca ttccagctac aatacttcca tttattagaa gcacattaac 1740 catttctata gcatgatttc ttcaagtaaa aggcaaaaga tataaatttt ataattgact 1800 tgagtacttt aagccttgtt taaaacattt cttacttaac ttttgcaaat taaacccatt 1860 gtagcttacc tgtaatatac atagtagttt acctttaaaa gttgtaaaaa tattgcttta 1920 accaacactg taaatatttc agataaacat tatattcttg tatataaact ttacatcctg 1980 ttttacctat aaaaaaaaaa aaaaa 2005 <210> 2 <211> 1823 <212> DNA <213> Homo sapiens <400> 2 ctccctgtgt tggtggagga tgtctgcagc agcatttaaa ttctgggagg gcttggttgt 60 cagcagcagc aggaggaggc agagcacagc atcgtcggga ccagactcgt ctcaggccag 120 ttgcagcctt ctcagccaaa cgccgaccaa ggaaaactca ctaccatgag aattgcagtg 180 atttgctttt gcctcctagg catcacctgt gccataccag ttaaacaggc tgattctgga 240 agttctgagg aaaagcagca ctaaagatgt acctacccct ccacaacaga tgaaactgtg 300 ccagccaaac aacaaatggg cattgtcccc agaagcttgg acaaaaaggc acacagagtt 360 caattccagt tgaacagaat aaaggccaaa atagagctgc cttgggggtc actgcaatta 420 gactgcttaa tgaagacatt aaaagaactt tacaacaaat acccagatgc tgtggccaca 480 tggctaaacc ctgacccatc tcagaagcag aatctcctag ccccacagaa tgctgtgtcc 540 tctgaagaaa ccaatgactt taaacaagag acccttccaa gtaagtccaa cgaaagccat 600 gaccacatgg atgatatgga tgatgaagat gatgatgacc atgtggacag ccaggactcc 660 attgactcga acgactctga tgatgtagat gacactgatg attctcacca gtctgatgag 720 tctcaccatt ctgatgaatc tgatgaactg gtcactgatt ttcccacgga cctgccagca 780 accgaagttt tcactccagt tgtccccaca gtagacacat atgatggccg aggtgatagt 840 gtggtttatg gactgaggtc aaaatctaag aagtttcgca gacctgacat ccagtaccct 900 gatgctacag acgaggacat cacctcacac atggaaagcg aggagttgaa tggtgcatac 960 aaggccatcc ccgttgccca ggacctgaac gcgccttctg attgggacag ccgtgggaag 1020 gacagttatg aaacgagtca gctggatgac cagagtgctg aaacccacag ccacaagcag 1080 tccagattat ataagcggaa agccaatgat gagagcaatg agcattccga tgtgattgat 1140 agtcaggaac tttccaaagt cagccgtgaa ttccacagcc atgaatttca cagccatgaa 1200 gatatgctgg ttgtagaccc caaaagtaag gaagaagata aacacctgaa atttcgtatt 1260 tctcatgaat tagatagtgc atcttctgag gtcaattaaa aggagaaaaa atacaatttc 1320 tcactttgca tttagtcaaa agaaaaaatg ctttatagca aaatgaaaga gaacatgaaa 1380 tgcttctttc tcagtttatt ggttgaatgt gtatctattt gagtctggaa ataactaatg 1440 tgtttgataa ttagtttagt ttgtggcttc atggaaactc cctgtaaact aaaagcttca 1500 gggttatgtc tatgttcatt ctatagaaga aatgcaaact atcactgtat tttaatattt 1560 gttattctct catgaataga aatttatgta gaagcaaaca aaatactttt acccacttaa 1620 aaagagaata taacatttta tgtcactata atcttttgtt ttttaagtta gtgtatattt 1680 tgttgtgatt atctttttgt ggtgtgaata aatcttttat cttgaatgta ataagaattt 1740 ggtggtgtca attgcttatt tgttttccca cggttgtcca gcaattaata aaacataacc 1800 ttttttactg cctaaaaaaa aaa 1823

Claims (10)

A composition for diagnosing or predicting periodontal disease, comprising an agent for detecting the rs3810765 single nucleotide polymorphism (SNP) site on the SFRP2 gene.
The composition according to claim 1, wherein the agent is a primer or probe that specifically binds to a sequence of 10-100 consecutive nucleotides including the rs3810765 single nucleotide polymorphism (SNP) site on the SFRP2 gene.
According to claim 1, wherein the composition is a single nucleotide polymorphism (SNP) site (rs4754) having a T at the 321th nucleotide position of SEQ ID NO: 2, a single base having a C at the 789th nucleotide position of SEQ ID NO:2 A polymorphic site (rs1126616) and a single polymorphic site (rs9138) having an A at the 294 nucleotide position of SEQ ID NO: 2 An agent for detecting a single polymorphic site on one or more SPP1 genes additionally comprising Composition, characterized in that.
The method of claim 3, wherein the agent is specifically for a sequence of 10-100 consecutive nucleotides comprising one or more nucleotide positions selected from the group consisting of nucleotide 321, nucleotide 789, and nucleotide 294 of SEQ ID NO: 2 A composition characterized in that it is a primer or probe that binds.
The composition of claim 1, wherein the root disease is external apical root resorption (EARR).
The composition according to claim 1, wherein the composition is applied to Asians.
A method for providing information necessary for the diagnosis or prediction of periodontal disease, comprising the step of detecting the rs3810765 single nucleotide polymorphism (SNP) site on the SFRP2 gene in a biological sample isolated from an individual.
The method according to claim 7, wherein the method comprises a single nucleotide polymorphism (SNP) site (rs4754) having a T at the 321 nucleotide position of SEQ ID NO: 2, and a single base having a C at the 789 nucleotide position of SEQ ID NO: 2 A polymorphic site (rs1126616) and a single polymorphic site (rs9138) having an A at the 294 nucleotide position of SEQ ID NO: 2 In one or more SPP1 genes selected from the group consisting of additionally detecting a single polymorphism site A method characterized in that.
The composition of claim 7, wherein the root disease is external apical root resorption (EARR).
8. The composition of claim 7, wherein said subject is Asian.

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