Evaluation of Beta-Defensin 1 and Mannose-Binding Lectin 2 Polymorphisms in Children with Dental Caries Compared to Caries-Free Controls: A Systematic Review and Meta-Analysis

Background and objective: Some variants in defensin beta 1 (DEFB1) and mannose-binding lectin 2 (MBL2) genes can be associated with oral diseases. Herein, we designed a systematic review and meta-analysis to evaluate the association of DEFB1 (rs11362, rs1799946, and rs1800972) and MBL2 (rs7096206 and rs1800450) polymorphisms with the susceptibility to dental caries (DC) in children. Materials and methods: A systematic literature search was conducted in the PubMed/Medline, Web of Science, Scopus, and Cochrane Library databases until 3 December 2022, without any restrictions. The odds ratio (OR), along with a 95% confidence interval (CI) of the effect sizes, are reported. Analyses including a subgroup analysis, a sensitivity analysis, and funnel plot analyses were conducted. Results: A total of 416 records were identified among the databases, and nine articles were entered into the meta-analysis. A significant relationship was found between the T allele of DEFB1 rs11362 polymorphism and DC susceptibility, and the T allele was related to an elevated risk of DC in children (OR = 1.225; 95%CI: 1.022, 1.469; p = 0.028; I2 = 0%). No other polymorphisms were associated with DC. All articles were of moderate quality. Egger’s test in homozygous and dominant models demonstrated a significant publication bias for the association of DEFB1 rs1799946 polymorphism with DC risk. Conclusions: The results demonstrated that the T allele of DEFB1 rs11362 polymorphism had an elevated risk for DC in children. However, there were only few studies that evaluated this association.


Introduction
Oral health is important for a child's overall health and development [1]. Dental caries (DC) or tooth decay involves damage to tooth enamel [2] and is a chronic disease that is widely prevalent [2,3]. DC is the most prevalent chronic disease among children [4][5][6], with 0.5 billion prevalent cases of caries in deciduous teeth among 0-14-year-old children [7].

Materials and Methods
To design the study, the PRISMA guidelines provided in the Supplementary file were followed [33]. The PECO (Population, Exposure, Comparator, and Outcome) question [34,35] was as follows: Is there an association between DEFB1 and MBL2 polymorphisms and susceptibility to DC? (P: Children with DC (CDC), E: DEFB1 and MBL2 polymorphisms, C: Children free of DC (CFC); O: DC).

Search Strategy and Study Selection
The Scopus, PubMed/Medline, Cochrane Library, and Web of Science databases were searched by one author (M.S.) to retrieve records published until December 3, 2022, without any restrictions (e.g., language). The keywords or search terms were ("beta defensin*" or "β-defensin*" or "beta-defensin 1" or "β-defensin-1" or "beta-defensin-1" or "defensin beta 1" or "DEFB1" or "human beta-defensin-1" or "HBD-1" or "mannose-binding lectin" or "MBL" or "mannose binding lectin 2" or "MBL2" or "mannose binding lectin-2" or "MBL-2" or "mannan-binding lectin" or "mannan-binding protein" or "MBP") and ("tooth decay" or "dental caries" or "caries"). Moreover, the citations of the retrieved original articles/reviews/meta-analyses linked to the subject were searched to ensure that no study was missed. A second reviewer (G.H.) evaluated the titles/abstracts of the articles linked to the subject; afterwards, the full texts of the articles that met the inclusion criteria were downloaded and screened. Any study that was excluded was tagged with the reason for exclusion. In the event of a lack of agreement among the authors, a third reviewer (M.M.I) was involved.

Quality Assessment
The quality of studies was evaluated based on the modified Newcastle-Ottawa scale (NOS) [19] by two reviewers independently (G.H. and M.S.). The scores ranged from 0 Children 2023, 10, 232 3 of 16 to 10 points, with >7 points being considered as "high quality", 4 to 7 points denoting "moderate quality", and less than 4 points being "low quality". Disagreement between the authors was resolved by a third reviewer (M.M.I.).

Eligibility Criteria
The inclusion criteria were as follows: (I) any type of articles including two independent groups (CDC and CFCs); (II) studies with any defined DMFT/dmft score for CDC and CFCs; (III) studies including polymorphisms of DEFB1 or MBL2 genes including minimum two studies for the analysis with any amount of the Hardy-Weinberg equilibrium (HWE); and (IV) CDC and CFCs had no chronic illnesses, genetic diseases, or other disorders. Irrelevant studies, meta-analyses, studies without a control group, studies with insufficient data for analysis, case reports, and conference papers were excluded.

Data Extraction
Two authors (S.B. and R.S.) independently extracted the data of the studies. Disagreement between the authors was resolved by a third author (P.C.). The extracted data were the name of first author, publication year of the study, country of origin of the study, number of CDC and CFCs, ethnicity, age range of individuals, investigated dentition, DMFT/dmft score of the CDC and CFCs, type of reported polymorphism(s), the quality score of each study, and effect sizes (odds ratio (OR) and 95% confidence interval (CI)) for DC occurrence of each polymorphism according to five genetic models.

Statistical Analysis
To compute the effect sizes (ORs and 95% CIs) and the rest of the analyses, two authors (G.H. and M.S.) independently used comprehensive meta-analysis version 2.0 (CMA 2.0) software. Disagreement between the authors was resolved by a third author (M.M.I.). A p-value (two-sided) less than 0.05 was considered significant. The I 2 statistic was used to estimate heterogeneity, with I 2 > 50% (P heterogeneity < 0.1) recommending a significant heterogeneity, and we used the fixed-effects model [36]. The publication bias across or among the studies was evaluated using Egger's and Begg's tests [37,38]; if p-value (two-sided) was less than 0.10 (two-sided) for one and both tests, a significant publication bias was considered to be present. With regard to the stability of the results, two sensitivity analyses, including 'one-study-removed' and 'cumulative analyses' were carried out. A subgroup analysis based on ethnicity, type of dentition, and sample size was carried out for DEFB1 rs11362 polymorphism while such analysis wasn't possible for MBL2 polymorphisms due to an insufficient number of studies.

Study Selection
By searching the databases and electronic resources, 416 records were identified. After removing duplicates and irrelevant records, 16 full-text articles were obtained and, after that, assessed. Among the evaluated full-text articles, seven were excluded for different reasons ( Figure 1). Finally, nine articles [14,27,[39][40][41][42][43][44][45] involving analyses of 17 studies (several articles included more than one polymorphism, and each polymorphism was considered one independent study) were involved in the meta-analysis. studies (several articles included more than one polymorphism, and each polymorphism was considered one independent study) were involved in the meta-analysis.

Sensitivity Analysis
The sensitivity analyses demonstrated stability of the results for all explorations where there were a minimum of three studies (results are not presented).

Publication Bias
The results of Egger's and Begg's tests were checked to evaluate the publication bias across the studies. The funnel plots are illustrated in the Supplementary file. The findings reported that just Egger's test in homozygous and dominant models for association between DEFB1 rs1799946 polymorphism and DC risk showed a significant publication bias.

Discussion
A meta-analysis [28] reported that among DEFB1 (rs11362, rs1799946, and rs1800972) and MBL2 (rs7096206, rs11003125, and rs1800450) polymorphisms, just MBL2 rs11003125 had an association with the risk of DC, while another meta-analysis [19] found DEFB1 rs11362 polymorphism to be associated with the risk of DC in permanent dentition, not deciduous or mixed dentitions. This systematic review evaluated the association of DEFB1 (rs11362, rs1799946, and rs1800972) and MBL2 (rs7096206 and rs1800450) polymorphisms with DC risk; the findings suggest that DEFB1 rs11362 polymorphism in T allele is related to an increased likelihood of DC occurrence. In addition, ethnicity and type of dentition were significant factors in the subgroup analysis checking the relationship of DEFB1 rs11362 polymorphism with DC risk.
DC is a chronic disease that is usually affected by environmental and host agents and even genetic factors [2,[46][47][48][49]. Therefore, early detection, early diagnosis, and early treatment are the main considerations for the prevention and treatment of DC [50,51].
There are many genetic agents that probably contribute to DC susceptibility and resistance, such as salivary agents, taste preference, tooth morphology, immune system, enamel structure and composition, organic and inorganic substances, and behavior [16,17,46,[52][53][54]. In addition, the likelihood of DC occurrence is high in the first months after the tooth eruption but is much lower in adulthood and later stages of life, and at different ages, the DC intensity may be different [32].
Two studies [27,40] reported that the differences between the results of studies reporting the relationship between polymorphisms and susceptibility to DC can be a result of the variation in the sample sizes, experimental methods, and ethnicities. Another study [55] that included 53 genes reported as being involved in DC susceptibility showed that cytokine network relevant genes, the transforming growth factor-beta family, and the matrix metalloproteinases family had important roles in tooth development and carious lesions. It is believed that the flow of saliva, pH, and chemical composition of saliva are among other important factors in the occurrence and progress of DC [56][57][58]. The present meta-analysis reported that ethnicity and type of dentition were important factors for the relationship of DEFB1 rs11362 polymorphism with susceptibility to DC in children. Other reports showed that Africans had 32% and Mixed ethnicities had 69% more DC experience than Whites [59] and that DC prevalence was 30.4%, 39.0%, and 51.7% for White, Black, and Hispanic students, respectively [60]. For DEFB1 (rs1799946 and rs1800972) and MBL2 (rs7096206 and rs1800450) polymorphisms, we could not perform a subgroup analysis due to the limited number of reported studies. Therefore, a large number of studies are needed to prove the role of ethnicity in the prevalence of tooth decay. This can be due to the difference in ethnical factors (geographical conditions, bone structure, nutrition, etc.) that have affected dental genetics over time. To find these relationships, further studies and further emphasis on ethnicity and the risk of DC in the future can be discussed in further possible mechanisms. With regard to the type of dentition, the etiology of dental anomalies is partly environmental and partly genetic [61] and the DC phenotypes in the deciduous dentition were highly heritable [62]. Therefore, the role of type of dentition can be affected by genetics, but more studies are needed to find the possible mechanisms between type of dentition and risk of DC.
DEFB1 as an oral antimicrobial peptide gives the first line of defense against an extensive range of pathogens [63,64]. The high variability of defensin levels in oral tissues can be attributed to genetic changes in the host [65,66]. The present meta-analysis reported that DEFB1 rs1799946 polymorphism was related to the elevated risk of DC in children, and therefore, this can cause a reduction in DEFB1 and then oral infections. DC is caused by bacteria that destroy the enamel and dentin [67][68][69]. Therefore, the role of DEFB1 polymorphisms could be considered in future studies for reaching better and more accurate results.
The MBL2 plays an important role in the innate immune system and few polymorphisms in this gene can be responsible for increased susceptibility to some infectious diseases [70][71][72]; therefore, MBL2 insufficiency is related to bacterial infection [73]. The present meta-analysis could not find any association between MBL2 (rs7096206 and rs1800450) polymorphisms, perhaps due to a limited number of included studies. Therefore, more studies are needed to support or reject the present meta-analysis results.
The role of oral peptides as therapeutic agents and for clinical assessment of an individual's susceptibility to DC can be promising in the future [74][75][76]. Oral antimicrobial peptides give the first line of defense against an extensive range of pathogens [75,77]. Their expression in saliva and all over the oral cavity denotes their role in preserving the tooth structure from DC, as well as preserving the oral mucosa, in spite of the fact that the amount of antimicrobial peptides expressed in saliva varies among people [66,76,78,79]. Therefore, paying attention to the metabolism pathways of peptides, their genetic mutations, the values of these peptides in blood and saliva, and their expression can greatly help future research in finding factors associated with susceptibility to DC.
This meta-analysis has three limitations: (1) There were a limited number of published studies, therefore, an inability to conduct subgroup analyses for most polymorphisms; therefore, more studies with more cases are needed to confirm the association of these polymorphisms with DC risk. (2) There was possibly a publication bias for some analyses, which could also be due to fewer studies being included in the analyses. (3) None of the studies were of high quality. In contrast, the stability of the results and low/lack of heterogeneity across the studies were the strengths of the meta-analysis.

Conclusions
The findings suggest that the T allele of DEFB1 rs11362 polymorphism is associated with an increased likelihood of DC. Therefore, this polymorphism could have a significant role in the pathogenesis of DC. The limited number of studies and the moderate quality of the included studies demonstrate that well-designed studies with more cases are needed to confirm or reject the results.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.