Integrative Analysis of ENAM rs3796704 Polymorphism and Eugenol–Cinnamic Acid Docking/ADMET Against Biofilm-Forming Streptococcus Mutans: Genetic–Phytochemical Links to Oral Dysbiosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Hello, dear colleagues!

This article is a comprehensive study combining clinical genetic analysis (association of the rs3796704 ENAM gene polymorphism with caries) with a bioinformatics approach (molecular docking and ADMET analysis of phytochemicals targeting Sortase A in Streptococcus mutans).
The strengths of the study include its integrative approach, relevant population, and high-quality bioinformatics analysis.

Nevertheless, there are a few aspects I would like to highlight.
1. The Abstract and Results sections indicate that the study included 120 patients and 120 controls. However, in the Materials and Methods section (3. Materials and Methods), the very first sentence reads: *"The researchers chose to conduct a case-control study involving 60 patients and 60 individuals in the control group.
2. The text does not mention a power analysis to determine the required sample size.
3. There are numerous sources older than 10 years (e.g., [1] 2018, [2] 2001, [3] 2006, [4] 2011, [16] 2019, [22] 2015, [23] 2021, [43] 1997), which need to be replaced with ones more relevant to the current understanding of the problem.
4. The Results section begins with general phrases about the function of the ENAM gene, which is more suitable for an introduction, but this section should be presented directly with the data.
5. The work is purely in silico in its phytochemical part. The absence of even in vitro confirmation of the inhibitory activity of eugenol and The use of cinnamic acid against Sortase A or S. mutans biofilm formation weakens the practical significance of the docking results. This should be clearly identified in the "Limitations" section as a priority for future research.

Author Response

Dear Reviewr 1 :

Thank you for your valuable comments, respected reviewer. All points have been carefully addressed one by one, and all suggested modifications have been implemented. The revised sections have been highlighted in turquoise for your convenience and easy identification.

• The manuscript structure has been revised to strictly follow the journal style, including clearly separated sections: Introduction, Materials and Methods, Results, and Discussion.
• A detailed study design schematic was prepared using a licensed BioRender subscription and is presented in Figure 1.
• Molecular dynamics simulations were performed and the results are presented in Figures 7 and 8.
• A dedicated subsection on dental caries diagnosis and scoring criteria has been added within the Materials and Methods section.
• A consolidated Limitations section has been added immediately after the Discussion.
• The English language of the manuscript has been carefully reviewed and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

Nevertheless, there are a few aspects I would like to highlight.
1. The Abstract and Results sections indicate that the study included 120 patients and 120 controls. However, in the Materials and Methods section (3. Materials and Methods), the very first sentence reads: *"The researchers chose to conduct a case-control study involving 60 patients and 60 individuals in the control group.

Response : Two hundred and forty (240) women aged between 25 and 30 years were recruited and separated into two groups 120 patients with dental caries and 120 caries-free healthy controls
2. The text does not mention a power analysis to determine the required sample size

Response :

1. Sample Size and Power Considerations : The sample population was pragmatically calculated with the limit of the number of eligible participants that could be recruited in the study period with strict inclusion/exclusion criteria and to ensure homogeneity in the demography of the sample (age range, ethnicity, and residency) to minimize the confounding. We further considered statistical power of the case-control genetic association with the typical assumption of a biallelic SNP with a two sided significance level of 0.05 to enhance the transparency of reporting. Post hoc analysis of the effect size was carried out based on the distribution of allele frequencies observed and estimated effect size (odds ratio) of the allele, rs3796704, which showed that the given sample (120 cases and 120 controls) will be sufficient to support the association on the observed scale. Further multicenter research, which will involve bigger samples, will be beneficial in the future in order to become more precise and perform stratified analyses.
2. Limitation : One of the limitations of this study is that the sample size was not pre-determined through an a priori calculation of power, instead of it being more of a feasibility recruitment within one center and within a restricted cohort. Even with our post hoc power consideration, which is dependent on the observed allele frequencies and effect estimates, it will be impossible to prove the association unless larger multicenter cohorts are conducted to enhance the confidence interval, and also to perform subgroup/interaction analyses (e.g., environmental exposures and microbial variables).
3. 
   There are numerous sources older than 10 years (e.g., [1] 2018, [2] 2001, [3] 2006, [4] 2011, [16] 2019, [22] 2015, [23] 2021, [43] 1997), which need to be replaced with ones more relevant to the current understanding of the problem.

Response : It is done . Most of the references have been updated and recent, topic-relevant sources have been incorporated. However, a limited number of older references were retained because they represent foundational studies that are central to the conceptual framework of our work, particularly in the genetic field. These studies remain scientifically valid and are still widely cited. Moreover, our manuscript integrates both genetic and computational approaches to address this multifactorial problem, and the selected foundational references are essential to support this integrative perspective.

4. 
   The Results section begins with general phrases about the function of the ENAM gene, which is more suitable for an introduction, but this section should be presented directly with the data.
   5. The work is purely in silico in its phytochemical part. The absence of even in vitro confirmation of the inhibitory activity of eugenol and The use of cinnamic acid against Sortase A or S. mutans biofilm formation weakens the practical significance of the docking results. This should be clearly identified in the "Limitations" section as a priority for future research.

Response : The Results section has been carefully revised and reorganized to begin directly with the presentation of experimental and statistical data. Introductory statements regarding the biological function of the ENAM gene have been relocated to the Introduction section to ensure a clearer and more methodologically appropriate structure.

Regarding the phytochemical component, we acknowledge that this aspect of the study was based on in silico approaches (molecular docking and molecular dynamics), which provided mechanistic insight into ligand–Sortase A interactions. However, no direct experimental validation, such as biofilm inhibition assays or enzymatic activity assays, was performed to confirm the proposed inhibitory effects.

Accordingly, this limitation has been explicitly addressed in the “Limitations” section. We have emphasized that conducting in vitro validation studies represents a priority for future research to enhance translational relevance and to bridge computational findings with biological confirmation.

We added in limitation section :

Though molecular docking and molecular dynamics have been able to give mechanistic data on the ligand-Sortase A interactions, there is no experimental biofilm or enzymatic assays to directly validate the proposed inhibitory effect. The need to include in vitro validation in future studies is to enhance translational relevance.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thanks for the opportunity to review the current manuscript.

Here are a few considerations for your reference to improve it further:

• In the introduction section please consider adding some information regarding the gaps in the body of knowledge on the basis of gender and population groups.
• Please consider adding a comment on the novelty of the study.
• Please consider updating the text in tables from “Patients No. Controls No.” to either “controls” or “patients” and use “n” for sample size.
• In the methods section, please consider adding information about the indices used for dental caries scoring.
• Please move some information from the discussion section to the introduction section to highlight the gaps in the body of knowledge.

Author Response

Dear Reviewr 2 :

Thank you for your valuable comments, respected reviewer. All points have been carefully addressed one by one, and all suggested modifications have been implemented. The revised sections have been highlighted in yellow for your convenience and easy identification.

• The manuscript structure has been revised to strictly follow the journal style, including clearly separated sections: Introduction, Materials and Methods, Results, and Discussion.
• A detailed study design schematic was prepared using a licensed BioRender subscription and is presented in Figure 1.
• Molecular dynamics simulations were performed and the results are presented in Figures 7 and 8.
• A dedicated subsection on dental caries diagnosis and scoring criteria has been added within the Materials and Methods section.
• A consolidated Limitations section has been added immediately after the Discussion.
• The English language of the manuscript has been carefully reviewed and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

• In the introduction section please consider adding some information regarding the gaps in the body of knowledge on the basis of gender and population groups.

Response : it is done .. first section revised in highlighted in yellow and turquoise

• Please consider adding a comment on the novelty of the study.

Response : The present study is novel in several key aspects. First, it represents the first investigation of the ENAM rs3796704 polymorphism in relation to dental caries susceptibility in the Iraqi population, thereby addressing a clear geographical and ethnic knowledge gap. Second, by focusing exclusively on adult Iraqi Arab women, this study provides insight into potential gender-specific genetic contributions to caries risk—an aspect that has been largely overlooked in previous genetic association studies[3].

Importantly, this gender-focused approach is biologically justified, as previous anthropological and biomedical evidence has consistently demonstrated higher caries prevalence among females, attributed not only to behavioral factors but also to hormone-mediated alterations in saliva composition and flow across life-history stages, including puberty, menstruation, and pregnancy[4]. These physiological differences create a more cariogenic oral environment in women, underscoring the relevance of investigating female-specific genetic susceptibility.

Third, this work uniquely integrates host genetic data (ENAM polymorphism) with in silico anti-virulence targeting of Streptococcus mutans Sortase A, thereby bridging human enamel genetics with bacterial biofilm-forming mechanisms. Finally, the combined use of molecular docking and ADMET profiling to evaluate the therapeutic potential of eugenol and cinnamic acid against Sortase A provides a mechanistically informed and translational framework for personalized preventive strategies in dental caries management, particularly in genetically susceptible female populations.

• Please consider updating the text in tables from “Patients No. Controls No.” to either “controls” or “patients” and use “n” for sample size.

Response : I t is done . Two hundred and forty (n=240) women aged between 25 and 30 years were recruited and separated into two groups (n=120) patients with dental caries and (n=120) caries-free healthy controls.

• In the methods section, please consider adding information about the indices used for dental caries scoring.

Response : Dental caries scoring.

In a bid to assure standardized and reproducible measurement of the severity of dental caries, the severity of dental caries was measured with the help of validated clinical indices that have been used in both epidemiological and genetic studies. Precisely, data on caries experience was captured as the Decayed, Missing and Filled Teeth (DMFT) index of permanent dentition as per the world health organization (WHO) recommendations. The DMFT index measures the cumulative burden of caries by adding together the number of decayed (D), caries-related lost (M) and restored (F) teeth and has been acknowledged to be both reliable and clinically significant in adults. Incidental clinical examination was conducted under standardised conditions with the help of sterile dental mirrors and explorers. Teeth were considered to be in a decayed condition when these cavities or enamel/dentin softness were visible. The scoring was not included in the teeth that had been lost to reasons other than caries (e.g., orthodontic extraction or trauma). The index was chosen because it has been significantly used in genetic association studies of dental caries, and therefore can be compared with the already published data. The study was performed by the Assistant Professor Dr. Elham Hazeim Abdulkareem, PhD (UK) majoring in Oral Health, one of the contributors of the study, and being the first author.

• Please move some information from the discussion section to the introduction section to highlight the gaps in the body of knowledge.
• Response : it is done .. first section revised in highlighted in yellow and turquoise

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Integrative Analysis of ENAM rs3796704 Polymorphism and 2 Eugenol-Cinnamic Acid Docking/ADMET Against Biofilm 3 Forming-Streptococcus Mutans: Genetic–Phytochemical Links 4 to Oral Dysbiosis

This paper looks into the presence of SNPs in a women’s population in Iraq and following that, the authors build a digital connection between Eugenol and Cinnamic acid to SrtA protein from Streptococcus mutans . In its current state the paper requires major revisions. It seem like it is from completely two separate projects that were combined to be one. The paper is in need of professional language review as well.

 Language is not unified within Materials and Methods, introduction and discussion. Please have a language review done and provide certification of language editing.

Line 311: Why women only?  There was no mention for any reasons in the text of the manuscript. This should be cleared.

Line 327: IRB approval number and blood withdraw, and storage method should be added. DNA quality cut-offs should be added. DNA concentration and purity. Also, no gel images were provided. Please provide a sample image from the gel electrophoresis

Line 337: using the word “crafting” primer design in highly unusual, please change it to a more commonly used word.

Line: 496: Talks about some limitations. Line 545 talks about limitations again. Limitation should be combined into one paragraph.

Several mention of Streptococcus mutans have not been italicized, for example line 503. Please go over the manuscript and correct all bacteria names. Also make sure to be consistent in terms of naming. Some places it is S. mutans and other the full name is written.

I do not get the connection between the docking study and SNP identification study. Is it a SNP identification study or is a molecular docking? Why did you choose the Eugenol and Cinnamic Acid? What is its connection between the SNPs and Eugenol and Cinnamic acid? The connection between them should have been made in the introduction, but nothing was there. Starting at line 538, the connection is briefly mentioned but without any mention of significant findings. The discussion is unnecessarily long and has many components of an introduction. Please rewrite and remove introduction elements and focus on analysing the results while making connection to previous work and the application importance of the finding

I believe the value of the paper would be found in rewriting it and describe and clinical condition of patients in relation to SNP presence and type of SNP. Molecular docking, although valuable information, has no role in this study in terms of adding any value.

Comments on the Quality of English Language

Needs professional review 

Author Response

Dear Reveiwer 3,

Thank you for your valuable comments, respected reviewer. All points have been carefully addressed one by one, and all suggested modifications have been implemented. The revised sections have been highlighted in pink for your convenience and easy identification.

• The manuscript structure has been revised to strictly follow the journal style, including clearly separated sections: Introduction, Materials and Methods, Results, and Discussion.
• A detailed study design schematic was prepared using a licensed BioRender subscription and is presented in Figure 1.
• Molecular dynamics simulations were performed and the results are presented in Figures 7 and 8.
• A dedicated subsection on dental caries diagnosis and scoring criteria has been added within the Materials and Methods section.
• A consolidated Limitations section has been added immediately after the Discussion.
• The English language of the manuscript has been carefully reviewed and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

Open Review

Comments and Suggestions for Authors

Integrative Analysis of ENAM rs3796704 Polymorphism and 2 Eugenol-Cinnamic Acid Docking/ADMET Against Biofilm 3 Forming-Streptococcus Mutans: Genetic–Phytochemical Links 4 to Oral Dysbiosis

This paper looks into the presence of SNPs in a women’s population in Iraq and following that, the authors build a digital connection between Eugenol and Cinnamic acid to SrtA protein from Streptococcus mutans . In its current state the paper requires major revisions. It seem like it is from completely two separate projects that were combined to be one. The paper is in need of professional language review as well.

Response : We sincerely thank the reviewer for this important observation. We agree that the manuscript requires clearer conceptual integration. We have therefore revised the Introduction and Discussion to explicitly articulate the unifying hypothesis: host enamel genetic variation (ENAM rs3796704) may increase susceptibility to caries by altering enamel resistance and facilitating S. mutans colonization/biofilm establishment; in parallel, inhibiting a key S. mutans virulence determinant (Sortase A) with safe phytochemicals may represent an adjunct preventive strategy—particularly relevant for genetically susceptible individuals.

 Language is not unified within Materials and Methods, introduction and discussion. Please have a language review done and provide certification of language editing.

Response : The language of the manuscript has been carefully reviewed and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

Line 311: Why women only?  There was no mention for any reasons in the text of the manuscript. This should be cleared.

Response : Thank you for this important observation. The study was intentionally restricted to women in order to reduce biological heterogeneity and avoid sex-related confounding factors in genetic association analysis. Previous epidemiological and biological evidence indicates that hormonal fluctuations, salivary composition, and life-history–related physiological changes may influence dental caries susceptibility in females. By limiting recruitment to a single sex and narrow age range, we aimed to improve cohort homogeneity and enhance the interpretability of the ENAM polymorphism association. This rationale has now been clearly stated in the manuscript.

Line 327: IRB approval number and blood withdraw, and storage method should be added. DNA quality cut-offs should be added. DNA concentration and purity. Also, no gel images were provided. Please provide a sample image from the gel electrophoresis

                                        Ethical approval :

The Scientific Research Ethics Committee at the University of Anbar gave the approval to the study on 12 April 2023 (Approval No. 124). Every process was done on the basis of the Declaration of Helsinki (1975, revised in 2013).

Estimation DNA concentration and Purity :

The concentration and purity of the DNA were determined as a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The purity of the A260/A280 varied within a range of 1.7 to 2.1, which is deemed to have a good quality of DNA to undergo downstream molecular tests.

Integrity of DNA :

In order to detect the quality of the bands, the extracted DNA integrity was first verified using electrophoresis in a 1% agarose gel as shown in figure.

Line 337: using the word “crafting” primer design in highly unusual, please change it to a more commonly used word.

Response : Two primers (forward and reverse) were designed for High-Resolution Melting (HRM) analysis after scrutinizing the ENAM gene sequence from the SNP database on NCBI. Primer characteristics are outlined in Table 9. DNA extracted from venous blood was subsequently subjected to HRM, a real-time PCR-based technique.

Line: 496: Talks about some limitations. Line 545 talks about limitations again. Limitation should be combined into one paragraph.

Response : we merged in limitation section

Several mention of Streptococcus mutans have not been italicized, for example line 503. Please go over the manuscript and correct all bacteria names. Also make sure to be consistent in terms of naming. Some places it is S. mutans and other the full name is written.

Response : I t is done with highlighted in pink  and grey .

I do not get the connection between the docking study and SNP identification study. Is it a SNP identification study or is a molecular docking? Why did you choose the Eugenol and Cinnamic Acid? What is its connection between the SNPs and Eugenol and Cinnamic acid? The connection between them should have been made in the introduction, but nothing was there. Starting at line 538, the connection is briefly mentioned but without any mention of significant findings. The discussion is unnecessarily long and has many components of an introduction. Please rewrite and remove introduction elements and focus on analysing the results while making connection to previous work and the application importance of the finding

I believe the value of the paper would be found in rewriting it and describe and clinical condition of patients in relation to SNP presence and type of SNP. Molecular docking, although valuable information, has no role in this study in terms of adding any value.

Response : We sincerely thank you for your critical and insightful comments regarding the conceptual relationship between the SNP identification and the molecular docking components of our study. We agree that this connection required clearer articulation in the original manuscript.

Our investigation was designed within an integrative host–microbe framework rather than as an isolated SNP study or a purely computational docking analysis. The ENAM rs3796704 polymorphism was selected because ENAM encodes enamelin, a key structural protein essential for enamel biomineralization. A previous case–control study demonstrated that while individual SNPs were not independently associated with caries susceptibility, significant gene–microbe interactions, particularly between tuftelin polymorphism and S. mutans, accounted for 26.8% of dmfs variation, underscoring the multifactorial and interactive nature of caries pathogenesis [1]. A significant association between ENAM rs12640848 polymorphism and primary tooth caries has been reported in Polish children, supporting the role of enamel-related genetic variation as a predictive factor for caries susceptibility [2]. Structural compromise of enamel may facilitate bacterial adhesion and colonization, thereby predisposing individuals to cariogenic biofilm formation.

Dental caries is a multifactorial disease involving both host susceptibility and microbial virulence determinants[3].Dental caries is recognized as a biofilm-mediated disease governed by the classical four-factor model involving microorganisms, host susceptibility, dietary carbohydrates, and time. Accumulation of acidogenic and aciduric bacteria following excessive sugar intake results in ecological dysbiosis within the dental biofilm, ultimately leading to enamel demineralization. Recent evidence supports the therapeutic value of natural bioactive compounds as anti-biofilm and anti-cariogenic agents with potentially fewer adverse effects than synthetic antimicrobials [4].

Furthermore, eugenol significantly suppressed biofilm formation at the early developmental stage and reduced preformed biofilms. Mechanistically, this antibiofilm activity was associated with the down-regulation of key virulence genes involved in adhesion and biofilm maturation, including fimA, hagA, hagB, rgpA, rgpB, and kgp. These findings further support the therapeutic potential of eugenol as a natural antibiofilm agent and reinforce its applicability in oral infection control strategies [5], Recent evidence suggests that cinnamic acid may represent a promising antibiofilm agent against complex oral biofilms. Al-Darwish et al. reported that cinnamic acid significantly reduced biofilm formation, extracellular polymeric substance (EPS) production, and total biomass in both single-species and multispecies oral biofilms at higher concentrations (≥600 mg/L). Additionally, chemoinformatics analysis indicated that cinnamic acid complies with Lipinski’s rule of five, supporting its drug-likeness profile. These findings highlight cinnamic acid as a potential plant-derived compound for targeting polymicrobial biofilm-associated oral infections, although further validation through peer-reviewed and in vivo studies is required [6].

Thus, the docking analysis complements the genetic findings by providing a mechanistic therapeutic perspective within a precision dentistry framework.

References :

[1]        R. L. Slayton, M. E. Cooper, and M. L. Marazita, “Tuftelin, mutans streptococci, and dental caries susceptibility,” J. Dent. Res., vol. 84, no. 8, pp. 711–714, 2005.

[2]        K. Gerreth, K. Zaorska, M. Zabel, M. Borysewicz-Lewicka, and M. Nowicki, “Association of ENAM gene single nucleotide polymorphisms with dental caries in Polish children,” Clin. Oral Investig., vol. 20, pp. 631–636, 2016.

[3]        G. Spatafora, Y. Li, X. He, A. Cowan, and A. C. R. Tanner, “The evolving microbiome of dental caries,” Microorganisms, vol. 12, no. 1, p. 121, 2024.

[4]        X. Chen, E. B.-M. Daliri, N. Kim, J.-R. Kim, D. Yoo, and D.-H. Oh, “Microbial etiology and prevention of dental caries: exploiting natural products to inhibit cariogenic biofilms,” Pathogens, vol. 9, no. 7, p. 569, 2020.

[5]        Y. Zhang, Y. Wang, X. Zhu, P. Cao, S. Wei, and Y. Lu, “Antibacterial and antibiofilm activities of eugenol from essential oil of Syzygium aromaticum (L.) Merr. & LM Perry (clove) leaf against periodontal pathogen Porphyromonas gingivalis,” Microb. Pathog., vol. 113, pp. 396–402, 2017.

[6]        L. Al_Darwish, M. Alajlani, and S. Alashkar, “Beyond Antibiotics: Cinnamic Acid’s role in combatting complex biofilms in the oral cavity,” bioRxiv, pp. 2001–2024, 2024.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

I reviewed the manuscript "Integrative Analysis of ENAM rs3796704 Polymorphism and Eugenol-cinnamic acid Docking/ADMET against biofilm forming-Streptococcus mutans: Genetic–Phytochemical Links to Oral Dysbiosis". 

The manuscript addresses a potentially relevant topic; however, in its current form it presents several substantive and technical issues that limit its clarity, methodological rigor, and compliance with the journal’s guidelines. The following comments are intended to help the authors improve the scientific quality, coherence, and presentation of the manuscript.

Abstract: The abstract in its current form does not comply with the limits defined by the journal's guidelines. Furthermore, acronyms such as “ADDIE” are used without being explained in full. A reassessment is necessary following its revision.

Materials and Methods: The Materials and Methods section presents several methodological inconsistencies, clarity issues that need to be addressed to ensure scientific rigor and reproducibility.

First, there is a major inconsistency in the study design and sample size. The authors initially state that the study is a case–control design involving 60 patients and 60 controls; however, later in Section 3.1, the study population is described as including 120 women with dental caries and 120 healthy women. This discrepancy must be clarified, and the final sample size for each group should be consistently reported throughout the manuscript. Additionally, no justification or power calculation is provided to support the chosen sample size.

Second, the definition of cases and controls is insufficiently detailed. Although dental examinations and radiographs are mentioned, the diagnostic criteria for dental caries are not specified. Similarly, the criteria used to define “healthy controls” are not clearly stated, raising concerns about potential misclassification bias.

Third, regarding the docking methodology, validation is insufficient. Using chlorhexidine as a positive control without redocking a co-crystallized ligand or reporting RMSD values does not fully validate the docking protocol. Additionally, the reported binding affinity for chlorhexidine is relatively weak, and no discussion is provided to justify its suitability as a benchmark.

Results: The following sentences are not adequate for the results section "The enamelin (ENAM) gene encodes a critical protein involved in the formation and
mineralization of dental enamel, which is the hardest and most highly mineralized substance in the human body. Proper functioning of the ENAM gene is essential for the development of strong and healthy enamel, which serves as a protective barrier for teeth. Variations in this gene, particularly single nucleotide polymorphisms (SNPs), can potentially affect enamel quality and, consequently, an individual's susceptibility to tooth decay (dental caries). The rs3796704 SNP in the ENAM gene (chr4:70643714) has been studied to understand its association with tooth decay. This paper addresses observed genotype and allele frequencies of the SNP at rs3796704 in patients with tooth decay and control subjects, Hardy-Weinberg Equilibrium (HWE) and the implications of the recessive and dominant genetic models. " Please move these sentences to the Introduction or the Discussion section.

Discussion: These sentences in the Discussion were repeated twice: "Several limitations should be recognized. The case–control design limits causal inference, and although the sample was ethnically homogeneous, it may not reflect broader population diversity. Docking predictions must be validated experimentally through enzyme inhibition assays, biofilm quantification, and in vivo models."

Write the limitations only once. Furthermore, I ask authors to justify the decision to include only women in the study and to explicitly consider this a limitation of the study.

Technical aspects: references in both the text and bibliography are not formatted according to the journal guidelines.

Furthermore, the names of microorganisms should be written in italics.

Section 2 is the Materials and Methods section, not the Results section. Please reverse the sections.

Table 10 is indicated as Table 8 in the main text.

Author Response

Dear Revewer 4  ,

Thank you for your valuable comments, respected reviewer. All points have been carefully addressed one by one, and all suggested modifications have been implemented. The revised sections have been highlighted in  grey for your convenience and easy identification.

• The manuscript structure has been revised to strictly follow the journal style, including clearly separated sections: Introduction, Materials and Methods, Results, and Discussion.
• A detailed study design schematic was prepared using a licensed BioRender subscription and is presented in Figure 1.
• Molecular dynamics simulations were performed and the results are presented in Figures 7 and 8.
• A dedicated subsection on dental caries diagnosis and scoring criteria has been added within the Materials and Methods section.
• A consolidated Limitations section has been added immediately after the Discussion.
• The English language of the manuscript has been carefully reviewed and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

I reviewed the manuscript "Integrative Analysis of ENAM rs3796704 Polymorphism and Eugenol-cinnamic acid Docking/ADMET against biofilm forming-Streptococcus mutans: Genetic–Phytochemical Links to Oral Dysbiosis". 

The manuscript addresses a potentially relevant topic; however, in its current form it presents several substantive and technical issues that limit its clarity, methodological rigor, and compliance with the journal’s guidelines. The following comments are intended to help the authors improve the scientific quality, coherence, and presentation of the manuscript.

Abstract: The abstract in its current form does not comply with the limits defined by the journal's guidelines. Furthermore, acronyms such as “ADDIE” are used without being explained in full. A reassessment is necessary following its revision.

Response : it is done with highlighted in grey .

Abstract :

Background : Dental caries is a chronic illness mediated by biofilm, in which Streptococcus mutans plays a role in acidogenic plaque formation, and host enamel heredity may mediate the predisposition. The variants of enamelin (ENAM) can modify the microstructure of enamel and so, affect bacterial attachment and cariogenic biofilm formation. Sortase A (SrtA), a cysteine transpeptidase involved in anchoring the LPXTG-containing adhesins is a rational anti-virulence antigen to reduce S. mutans colonization without necessarily affecting viability. Aim: To test the relationship between ENAM rs3796704 and dental caries susceptible in adult Iraqi Arab women, and to test the antibiofilm potential of eugenol and cinnamic acid with S. mutans SrtA by molecular docking, ADMET profiling, and molecular dynamics. Methods: A case-control cohort of 240 women (25-30 years) was recruited (120 caries; 120 healthy control). Genomic DNA extraction and quality assessment of the samples were done on peripheral blood through NanoDrop (A260/A280: 1.7-2.1) and agarose gel electrophoresis. Dental caries scoring and healthy control was performed by the Assistant Professor Dr. Elham Hazeim Abdulkareem, PhD (UK) majoring in Oral Health. ENAM rs3796704 was typed using the high-resolution melting (HRM) real time PCR. Chi-square test and odds ratios (p < 0.05) were used to analyze allele/genotype distributions, HardyWeinberg equilibrium, and genetic models. In silico treatment was directed against S. mutans Sortase A (PDB: 4TQX). AutoDock Vina was used to dock eugenol, cinnamic acid, and chlorhexidine (positive reference) with grid center: 18, 18, -2; 20x20x20A; exhaustiveness: 16) and interaction mapping and ADMET prediction (ADMET-AI). The analysis was performed using complex stability 50 ns of molecular dynamics simulation (OPLS4/TIP3P, NPT 300 K/1 atm) with RMSD evaluation. Results: The G allele occurred less often in the cases than in the controls (60 % vs. 70 %; OR = 0.6429; p = 0.02) whereas the A allele was overrepresented in the cases (40 % vs. 30 %; OR = 1.5556; p = 0.02). A fixed GG and AA in cases were less in genotype frequencies (30% vs. 43.3% and 10% vs. 3.3% respectively). According to Docking, the affinities of eugenol (-4.961 kcal/mol) and cinnamic acid (-4.939 kcal/mol) were a little higher than that of chlorhexidine (-4.692 kcal/mol). Eugenol selectively bound the hydrophobic channel with an H-bond stabilizer to Ala92, and wide hydrophobic / 9 -alkyl interactions (e.g. Val166/Val168), whereas cinnamic acid oriented its carboxyl group to the catalytic microenvironment, and polar interactions with His120 and Arg197 and 9 -alkyl stacks with Trp194 close to Cys184. Phytochemicals were predicted to be superior to chlorhexidine and the predictive properties of the phytochemicals include lower molecular weight, complete Lipinski compliance, high predicted intestinal absorption, and reduced risk of cardiotoxicity (hERG). Both complexes were found to have a persistent binding and a stable protein backbone over a duration of 50 ns under molecular dynamics sup-port. Conclusion: ENAM rs3796704 be-tween enamel and caries in this Iraqi female cohort, suggesting a contributory role in risk formation in enamel genetics. Eugenol and cinnamic acid demonstrated mechanistically different, but convergent inhibitory interactions with S. mutans Sortase A and displayed positive in silico pharmacokinetic and safety profiles relative to chlorhexidine. These results would suggest a genotype-informed hypothesis that relates the variation in host enamel to bacterial adhesion biology and indicate that phytochemical SrtA inhibition is a possible adjunct treatment approach to caries prevention and should be validated on an enzymatic and biofilm-based basis and in larger multicenter cohort studies.

Materials and Methods: The Materials and Methods section presents several methodological inconsistencies, clarity issues that need to be addressed to ensure scientific rigor and reproducibility.

First, there is a major inconsistency in the study design and sample size. The authors initially state that the study is a case–control design involving 60 patients and 60 controls; however, later in Section 3.1, the study population is described as including 120 women with dental caries and 120 healthy women. This discrepancy must be clarified, and the final sample size for each group should be consistently reported throughout the manuscript. Additionally, no justification or power calculation is provided to support the chosen sample size.

Response : it is done .

• Two hundred and forty (n=240) women aged between 25 and 30 years were recruited and separated into two groups (n=120) patients with dental caries and (n=120) caries-free healthy controls.
• Sample Size and Power Considerations : The sample population was pragmatically calculated with the limit of the number of eligible participants that could be recruited in the study period with strict inclusion/exclusion criteria and to ensure homogeneity in the demography of the sample (age range, ethnicity, and residency) to minimize the confounding. We further considered statistical power of the case-control genetic association with the typical assumption of a biallelic SNP with a two sided significance level of 0.05 to enhance the transparency of reporting. Post hoc analysis of the effect size was carried out based on the distribution of allele frequencies observed and estimated effect size (odds ratio) of the allele, rs3796704, which showed that the given sample (120 cases and 120 controls) will be sufficient to support the association on the observed scale. Further multicenter research, which will involve bigger samples, will be beneficial in the future in order to become more precise and perform stratified analyses.Also, add point about these in limitation section : One of the limitations of this study is that the sample size was not pre-determined through an a priori calculation of power, instead of it being more of a feasibility recruitment within one center and within a restricted cohort. Even with our post hoc power consideration, which is dependent on the observed allele frequencies and effect estimates, it will be impossible to prove the association unless larger multicenter cohorts are conducted to enhance the confidence interval, and also to perform subgroup/interaction analyses (e.g., environmental exposures and microbial variables).
•  

Second, the definition of cases and controls is insufficiently detailed. Although dental examinations and radiographs are mentioned, the diagnostic criteria for dental caries are not specified. Similarly, the criteria used to define “healthy controls” are not clearly stated, raising concerns about potential misclassification bias. :

Response : we added “Dental caries scoring and healthy control ”in methodology section :

Dental caries scoring and healthy control .

In a bid to assure standardized and reproducible measurement of the severity of dental caries, the severity of dental caries was measured with the help of validated clinical indices that have been used in both epidemiological and genetic studies. Precisely, data on caries experience was captured as the Decayed, Missing and Filled Teeth (DMFT) index of permanent dentition as per the world health organization (WHO) recommendations. The DMFT index measures the cumulative burden of caries by adding together the number of decayed (D), caries-related lost (M) and restored (F) teeth and has been acknowledged to be both reliable and clinically significant in adults. Incidental clinical examination was conducted under standardised conditions with the help of sterile dental mirrors and explorers. Teeth were considered to be in a decayed condition when these cavities or enamel/dentin softness were visible. The scoring was not included in the teeth that had been lost to reasons other than caries (e.g., orthodontic extraction or trauma). The index was chosen because it has been significantly used in genetic association studies of dental caries, and therefore can be compared with the already published data. The study was performed by the Assistant Professor Dr. Elham Hazeim Abdulkareem, PhD (UK) majoring in Oral Health, one of the contributors of the study, and being the first author.

Third, regarding the docking methodology, validation is insufficient. Using chlorhexidine as a positive control without redocking a co-crystallized ligand or reporting RMSD values does not fully validate the docking protocol.

Response : we treat those in two section : methods and results

2.11. Molecular Dynamics Simulation

The simulation engine of molecular dynamics was conducted with the Desmond simulation engine in the Maestro interface (Maestro interface, Schrodinger Release 2023-4, Schrodinger LLC, New York, USA). The simulated pro-tein-ligand complexes with the docked complexes inserted into the simulation workspace and the explicit water en-vironment were solvated using the TIP3P model. An orthorhombic simulation cell was created, which was 10 A bigger than the outer atoms of the complex in order to fully solvate the complex.

System neutrality was obtained by adding suitable counter-ions and physiological ionic strength was obtained by the addition of NaCl up to the final concentration of 0.15 M. The OPLS4 force field was used to de-describe all molecular interactions. Before production run, the system was subject to energy minimization and then to staged equilibration procedure as per default relaxation protocol given in Des-mond.

The simulations of production were conducted under periodic boundary conditions in the NPT ensemble at 300 K and 1 atm temperature and pressure respectively. The temperature control by the NoseHoover chain thermostat and pressure control by MartynaTobiasKlein barostat were used. The overall simulation time was fixed at 50ns and an integration time step was fixed at 2fs. Trajectory analy-sis was done at intervals of 100 ps.

Conformational stability and dynamic properties were studied with post-simulation analyses. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were used to measure structural deviation and structural flexibility. The measure of compactness compared to radius of gyration (Rg) was done and the pattern of hydrogen bond formation was also measured over the lifespan of the simulation.

2.12. Preparation of Protein Structures Using Swiss-PdbViewer

The protein structures were retrieved in PDB format in a Protein Data Bank and analyzed by Swiss-PdbViewer (version 4.1). The structures were all analyzed to check the absence of atoms, side chains or inconsistencies in structure. Missing atoms were rebuilt around rotamer conformations that were found in the software library, and incomplete residues were fixed with the side-chain repair tools. Hydrogen atomation was done to provide the correct valency of the molecules and stabilize the molecular structure before docking. Local energy minimization was done where required to eliminate steric interaction and poor geometries. The completed structures were exported and then used in molecular docking and molecular dynamics simulations.

2.13. Re-Docking Validation

Reliability of the docking protocol was evaluated with re-docking procedure. The native ligand was removed out of the crystallographic protein-ligand complex and placed back into the respective active site with the same docking parameters used in the initial experiments with the docking. The root mean square deviation (RMSD) was used to determine the similarity between the predicted and experimentally determined conformation. A value of RMSD less than 2.0 A was deemed to be representative of good reproducibility and methodological reliability. The calculations of re-docking were done with AutoDock Vina version 1.2.0 with the same grid box size and scor-ing parameter as used in the virtual screening step.

Molecular Dynamics Simulation and Conformational Stability of the Cinnamic Acid–Sortase A Complex

To validate the structural stability and dynamic behavior of the cinnamic acid–Streptococcus mutans Sortase A complex (PDB ID: 4TQX), a 50 ns molecular dynamics (MD) simulation was performed, and the root mean square deviation (RMSD) of both the protein backbone (Cα atoms) and the bound ligand was systematically analyzed. RMSD analysis is a critical indicator of system equilibration, conformational integrity, and binding persistence under simulated physiological conditions.

The protein backbone RMSD exhibited a rapid increase during the initial equilibration phase (0–5 ns), reflecting necessary structural relaxation of the Sortase A enzyme within the solvated environment. Following this phase, the RMSD trajectory reached a stable plateau, fluctuating within a narrow range of approximately 1.6–2.0 Å for the remainder of the 50 ns simulation. This stable RMSD profile indicates the absence of major conformational rearrangements and confirms that Sortase A maintained a structurally conserved fold throughout the simulation, thereby demonstrating high intrinsic stability of the protein in complex with the ligand.

In parallel, ligand RMSD analysis, calculated by fitting the cinnamic acid molecule to the protein backbone, revealed an initial adaptive phase characterized by moderate fluctuations during the early simulation interval. These fluctuations correspond to ligand reorientation and optimization of intermolecular contacts within the binding pocket. Importantly, after approximately 25–30 ns, the ligand RMSD converged and remained consistently stable until the end of the simulation, with no evidence of ligand displacement or unbinding events. This stabilization indicates that cinnamic acid achieved a favorable binding conformation and remained securely anchored within the Sortase A active site.

Notably, the synchronized stabilization patterns observed for both protein and ligand RMSD profiles suggest a well-equilibrated and dynamically stable protein–ligand complex. Such coupled stability reflects strong intermolecular compatibility and supports the persistence of non-covalent interactions governing ligand retention. From a mechanistic perspective, the sustained RMSD stability over an extended 50 ns simulation strongly reinforces the reliability of the docking predictions and suggests that cinnamic acid effectively stabilizes the Sortase A binding pocket.

Overall, the MD simulation results demonstrate that cinnamic acid forms a conformationally stable and dynamically persistent complex with S. mutans Sortase A. These findings provide compelling computational evidence supporting cinnamic acid as a promising Sortase A inhibitor, thereby highlighting its potential utility as a natural anti-virulence agent targeting bacterial adhesion and biofilm formation. Further energetic analyses, such as MM-PBSA/MM-GBSA calculations, may provide additional quantitative insights into binding free energy contributions.

Figure :Molecular Dynamics Simulation and Conformational Stability of the Cinnamic Acid–Sortase A Complex

Molecular Dynamics Simulation and Stability Assessment of the Eugenol–Sortase A Complex

The dynamic stability and conformational behavior of the eugenol–Streptococcus mutans Sortase A complex (PDB ID: 4TQX) were rigorously evaluated through a 50 ns molecular dynamics (MD) simulation. Protein backbone (Cα atoms) and ligand RMSD trajectories were analyzed to assess system equilibration, structural integrity, and binding persistence under solvated, near-physiological conditions.

The protein backbone RMSD exhibited a sharp rise during the initial equilibration phase (0–2 ns), followed by stabilization within a narrow range of approximately 1.2–1.6 Å for the majority of the simulation. This early adjustment reflects relaxation of the Sortase A structure upon solvation and temperature equilibration. Importantly, the absence of long-term RMSD drift or abrupt fluctuations throughout the 50 ns trajectory indicates that Sortase A maintained a highly stable global fold in the presence of eugenol, confirming that ligand binding did not induce destabilizing conformational changes.

Ligand RMSD analysis, calculated by fitting eugenol to the protein backbone, revealed a distinct multi-phase behavior. During the early simulation period (0–15 ns), the ligand RMSD increased gradually, corresponding to initial reorientation and optimization of intermolecular contacts within the binding pocket. A transient increase in ligand RMSD observed around 15–25 ns suggests localized rearrangements within the binding site, likely driven by adaptive side-chain movements and solvent-mediated interactions. Following this phase, the ligand RMSD converged and remained relatively stable for the remainder of the simulation (approximately 25–45 ns), indicating sustained ligand retention within the active site. A modest rise toward the end of the simulation did not coincide with protein destabilization, suggesting conformational flexibility rather than ligand dissociation.

The parallel stabilization trends observed between protein and ligand RMSD profiles strongly support the formation of a dynamically equilibrated protein–ligand complex. The ability of eugenol to maintain stable association with Sortase A despite minor adaptive fluctuations highlights favorable binding compatibility and resilience of the interaction under dynamic conditions. Such behavior is characteristic of biologically relevant ligand binding, where moderate flexibility facilitates interaction persistence without compromising complex stability.

Overall, the 50 ns MD simulation provides compelling evidence that eugenol forms a structurally stable and dynamically persistent complex with S. mutans Sortase A. These findings corroborate the docking results and suggest that eugenol effectively stabilizes the Sortase A binding environment, reinforcing its potential as a natural anti-virulence agent targeting bacterial adhesion and biofilm-associated pathogenicity. Further free-energy calculations and residue-level fluctuation analyses would provide additional mechanistic insights into the energetic contributions governing this interaction.

Figure : Molecular Dynamics Simulation and Stability Assessment of the Eugenol–Sortase A Complex

Based on 50 ns molecular dynamics simulations, eugenol demonstrates superior dynamic stability and binding adaptability toward Streptococcus mutans Sortase A compared to cinnamic acid. The lower and more stable protein RMSD, coupled with sustained ligand retention and adaptive flexibility, indicates that eugenol forms a more dynamically favorable and resilient complex.

Additionally, the reported binding affinity for chlorhexidine is relatively weak, and no discussion is provided to justify its suitability as a benchmark.

Response : We thank the reviewer for this important observation. We agree that the predicted docking affinity of chlorhexidine (CHX) toward S. mutans Sortase A (SrtA) is modest in our Vina simulations. However, we selected CHX as a benchmark comparator for many reasons:The docking score of chlorhexidine (CHX) with regard to S. mutans Sortase A was only moderate in the current Vina simulations, but with an objective to use this as a benchmark in a purely clinical context. CHX is commonly accepted as the gold standard chemical antiplaque agent in the dentistry field and it is generally used as a comparator reference point when novel antibiofilm interventions are being tested[42]. Mechanistically, CHX works by cationic membrane- active antimicrobial action and high substantivity i.e. adsorption on pellicle-covered enamel and oral tissues which allows it to be active long after rinsing[43]. Since these prevailing in vivo actions need not involve high affinity binding to the comparatively small SrtA catalytic groove, a mediocre predicted docking score is feasible. In this way, CHX was applied in the present study as a clinically familiar positive-control comparator and not as a SrtA-specific inhibitor, thus permitting the binding of eugenol and cinnamic acid to be explained in comparison to a standard oral antiseptic. Other similar modeling studies have also used CHX as a reference control in SrtA docking comparisons[44].

Results: The following sentences are not adequate for the results section "The enamelin (ENAM) gene encodes a critical protein involved in the formation and
mineralization of dental enamel, which is the hardest and most highly mineralized substance in the human body. Proper functioning of the ENAM gene is essential for the development of strong and healthy enamel, which serves as a protective barrier for teeth. Variations in this gene, particularly single nucleotide polymorphisms (SNPs), can potentially affect enamel quality and, consequently, an individual's susceptibility to tooth decay (dental caries). The rs3796704 SNP in the ENAM gene (chr4:70643714) has been studied to understand its association with tooth decay. This paper addresses observed genotype and allele frequencies of the SNP at rs3796704 in patients with tooth decay and control subjects, Hardy-Weinberg Equilibrium (HWE) and the implications of the recessive and dominant genetic models. " Please move these sentences to the Introduction or the Discussion section.

Response : These sentences were deleted .

Discussion: These sentences in the Discussion were repeated twice: "Several limitations should be recognized. The case–control design limits causal inference, and although the sample was ethnically homogeneous, it may not reflect broader population diversity. Docking predictions must be validated experimentally through enzyme inhibition assays, biofilm quantification, and in vivo models."

Write the limitations only once. Furthermore, I ask authors to justify the decision to include only women in the study and to explicitly consider this a limitation of the study.

Response : it is done and we added limitation section .

Limitation :

1. One of the limitations of this study is that the sample size was not pre-determined through an a priori calculation of power, instead of it being more of a feasibility recruitment within one center and within a restricted cohort. Even with our post hoc power consideration, which is dependent on the observed allele frequencies and effect estimates, it will be impossible to prove the association unless larger multicenter cohorts are conducted to enhance the confidence interval, and also to perform subgroup/interaction analyses (e.g., environmental exposures and microbial variables).
2. Though molecular docking and molecular dynamics have been able to give mechanistic data on the ligand-Sortase A interactions, there is no experimental biofilm or enzymatic assays to directly validate the proposed inhibitory effect. The need to include in vitro validation in future studies is to enhance translational relevance.
3. The case–control design does not allow causal inference, and although the cohort was demographically homogeneous, it may not represent broader population diversity. Docking results require experimental confirmation through SrtA inhibition assays, biofilm studies, and in vivo validation. Despite these limitations, the findings highlight a promising interaction between ENAM genetic variation, mutans virulence, and phytochemical inhibitors, warranting further investigation into genotype-informed caries prevention strategies.
4. Females respondents were used to minimize biological heterogeneity and confounding effects of sex in genetic study. This sex-restricted design, however, discourages a wide-range generalization of the findings to the rest of the population especially males.

Technical aspects: references in both the text and bibliography are not formatted according to the journal guidelines.Furthermore, the names of microorganisms should be written in italics.

Response : it is done

Section 2 is the Materials and Methods section, not the Results section. Please reverse the sections.

Response : It is done

Table 10 is indicated as Table 8 in the main text.

Response : It is done.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Im satisfied

Author Response

Dear Reviewer4,

We sincerely thank you for your careful evaluation and constructive comments. All points raised by you have been addressed point-by-point in the revised manuscript. All modifications are clearly highlighted in light orange, and language revisions are highlighted in teal color, as indicated.

The language of the manuscript has been carefully reviewed, highlighted with teal-colored and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

Reviewer 3 Report

Comments and Suggestions for Authors

I guess its suitable in its current format 

Comments on the Quality of English Language

Needs professional review 

Author Response

Dear Reviewer4,

We sincerely thank you for your careful evaluation and constructive comments. All points raised by you have been addressed point-by-point in the revised manuscript. All modifications are clearly highlighted in light orange, and language revisions are highlighted in teal color, as indicated.

The language of the manuscript has been carefully reviewed, highlighted with teal-colored and edited by the first author, Dr. Elham Hazem Abdul Kareem (Ph.D., United Kingdom), as well as by Assistant Professor Dr. Sameer Ahmed Awad (Ph.D., Australia), Professor Dr. Mohammed Oday (Ph.D., Malaysia), and Professor Dr. Safaa Abed Latef Al-Meani (Postdoctoral Researcher, Sweden).

Reviewer 4 Report

Comments and Suggestions for Authors

I thank the Authors for the answers.

Some aspects have not been fulfilled, and some considerations must be made following the responses received.

The abstract is still not acceptable. Its length is more than double the character limit accepted by the journal.
In order to review its conformity with the manuscript, the character limit must be respected. It makes no sense for reviewers to review an abstract that is more than twice as long as the acceptable length.

While the Authors clarified the sample size discrepancy and added a limitation regarding the pragmatic recruitment, it should be noted that a post hoc power analysis does not compensate for the lack of an a priori calculation in genetic association studies. The sample size remains relatively small for SNP associations."

• Diagnostic Criteria: The inclusion of the DMFT index is an improvement. However, the Authors should explicitly state the cut-off scores used to categorize 'cases' vs 'controls' (e.g., DMFT=0 for healthy controls). Furthermore, providing the intra-examiner reliability (Kappa score) for the clinical examinations is essential to ensure data reproducibility.

• Radiographic clarification: The Authors mentioned radiographs in the previous version, but the response focuses mainly on clinical examination. It should be clarified if radiographs were used for all participants to detect interproximal caries that might be missed by visual inspection alone (the DMFT index can underreport caries without imaging).

Please do not use the full names of authors/reviewers in the methodology, nor their university positions. This was not requested by the reviewer and is not in line with scientific articles. Use only initials if necessary.

Author Response

Dear Reviewer4,

We sincerely thank you for your careful evaluation and constructive comments. All points raised by you have been addressed point-by-point in the revised manuscript. All modifications are clearly highlighted in light orange, and language revisions are highlighted in teal color, as indicated.

1. Abstract Length:
   The abstract has been substantially revised and shortened to fully comply with the journal’s character limit requirements. The revised version maintains scientific clarity and consistency with the manuscript.
2. Sample Size and Power Consideration:
   We have clearly stated that the sample size was feasibility-based rather than determined by an a priori power calculation. We explicitly acknowledged that post hoc power analysis does not substitute prospective calculation and clarified that the genetic findings should be interpreted as exploratory and hypothesis-generating. The limitations section has been strengthened accordingly.
3. Diagnostic Criteria (DMFT):
   The DMFT cut-off criteria have now been explicitly stated (controls: DMFT = 0; cases: DMFT ≥ 1). The calculation method has been clearly described in the Methods section.
4. Intra-Examiner Reliability:
   We clarified that formal kappa statistics were not calculated; however, all examinations were conducted by a single experienced examiner under standardized clinical conditions to ensure consistency. This has been added to the manuscript.
5. Radiographic Assessment:
   We clarified that radiographic examination was not performed and that diagnosis was based solely on clinical evaluation. We acknowledged that this may have led to underestimation of interproximal caries and added this explicitly to the limitations section.
6. Limitations Section:
   We have expanded the limitations to include:

• Feasibility-based sample size without a priori power calculation.
• Absence of formal kappa reliability assessment.
• Lack of radiographic evaluation and its potential impact on DMFT scoring.

7. Methodology Presentation:
   As recommended, full author names and academic positions have been removed from the methodology section. The text has been revised to comply with standard scientific reporting practices.

We sincerely appreciate your valuable comments, which have significantly improved the scientific clarity and methodological transparency of our manuscript.

Respectfully,
The Authors

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

In the M&M Authors declared that "Bitewing radiographs and panoramic radiographs were employed to verify clinically detected lesions, as well as enhance the diagnosis of the interproximal caries that might not be detected in the course of visual-tactile examination. The radiographic findings were added to the DMFT scoring."

However, in the limitations section Authors declared that " Furthermore, radiographic examination was not done, and the diagnosis of caries was based on the clinical determined assessment only, which could have resulted in underestimation of interproximal caries, and therefore, low DMFT scores."

This inconsistency needs to be resolved. It is recommended that the statement in the restrictions not be removed. 

Author Response

Dear reviewer :

Thank you for your valuable comments and suggestions. All the reviewer’s comments have been carefully addressed and revised point-by-point in the manuscript. The corresponding corrections and clarifications have been incorporated in the revised version of the paper. We believe that these revisions have improved the clarity and scientific quality of the manuscript.

1. English editing .

Response : The English language of the manuscript has been carefully revised and edited by the co-authors of the study, who hold PhD degrees and have academic experience in international institutions in the United Kingdom, Australia, Malaysia, and Sweden. The manuscript has been thoroughly checked to improve clarity, grammar, and scientific expression throughout the text.

2. Introduction and research design :

Response :Thank you for your valuable comments and suggestions. All the reviewer’s comments have been carefully addressed and revised point-by-point in the manuscript. The corresponding corrections and clarifications have been incorporated in the revised version of the paper. We believe that these revisions have improved the clarity and scientific quality of the manuscript.

3. In the M&M Authors declared that "Bitewing radiographs and panoramic radiographs were employed to verify clinically detected lesions, as well as enhance the diagnosis of the interproximal caries that might not be detected in the course of visual-tactile examination. The radiographic findings were added to the DMFT scoring."

However, in the limitations section Authors declared that " Furthermore, radiographic examination was not done, and the diagnosis of caries was based on the clinical determined assessment only, which could have resulted in underestimation of interproximal caries, and therefore, low DMFT scores."

This inconsistency needs to be resolved. It is recommended that the statement in the restrictions not be removed. 

Response :

Thank you for carefully identifying this inconsistency. The radiographic examination was not performed in the present study, and the diagnosis of dental caries was based solely on clinical visual–tactile examination using the WHO-recommended DMFT index. The sentence referring to the use of bitewing and panoramic radiographs in the Materials and Methods section was included inadvertently during manuscript preparation. This statement has now been removed to eliminate any confusion. The limitation section correctly reflects the study design and has been retained to clarify that the absence of radiographic examination may have resulted in underestimation of interproximal caries and consequently lower DMFT scores.