Association of a Promoter DNA Methyltransferase 3 Gene Variant with DNA Methylation and Anthropometrics in Children from 4 to 12 Years Old

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The message of the paper is clear I recommend to accept after changes addressed below. Especially to explain in better why DNA methylation is relevant at 4 years and not anymore at later ages.

 

The paper is well written and the main message is clear.

however I have problems to interept the methylation data.

It would be great if authors can change the style of presenting the DNA methylation data (independently from genetic data).

Can you discuss why methylation is signficant at 4 years and not in later ages?

Can the authors verify the mechanism by which this genetic variance in the promoter region of DNMT3B expression?

Can this snp alter the binding of specific transcription factors?

Can the authors discuss how DNMT3B an enzyme for methylation reaction could trigger  obesity?

 

Author Response

Comments 1: The paper is well written and the main message is clear. however I have problems to interept the methylation data. It would be great if authors can change the style of presenting the DNA methylation data (independently from genetic data).

Response 1: In brief, sample DNA is bound to strip wells with high-affinity to DNA, and methylated DNA is detected using antibodies specific to 5-methyl cytosine (5-mC). The methylation level is quantified colorimetrically by measuring absorbance at 450 nm with a spectrophotometer. The amount of methylated DNA is directly proportional to the measured OD intensity. The absolute quantity of methylated DNA was determined using a standard curve generated by plotting OD values against five serial dilutions of control methylated DNA (0.5–10 ng), following the protocol. DNA methylation results were expressed in terms of percent methylation (percentage of deoxy-methyl cytosine; % dmC). This information was included in the Materials and Methods section, page 3.

Deeper details on the DNA methylation data were already published in our paper “Impact of maternal dietary counseling in the first year of life on DNA methylation in a cohort of children” (Genetics and Molecular Biology, 44, 4, e20200330, 2021,  https://doi.org/10.1590/1678-4685-GMB-2020-0330), so we believe it is better not to repeat them here, due to the risk of committing self-plagiarism.

Comments 2: Can you discuss why methylation is signficant at 4 years and not in later ages?

Response 2: Unfortunately, we were only able to measure DNA methylation at 4 years old, because we did not have blood samples in later ages of our children. Therefore, we could not estimate the genotype associations with DNA methylation in later ages. We were able to verify that the BMI Z score at age 4 exhibited a small but significant correlation with methylation (rSpearman = 0.152, p = 0.018). However, this correlation disappeared at 8 (rSpearman = 0.057, p = 0.401) and 12 years old (rSpearman = -0.055, p = 0.497). As the correlation is not very strong at the age of 4, we cannot rule out that this loss of significance is due to the smaller number of samples at ages 8 and 12, due to the expected losses in follow-up of some children during the study. These information were included in the Discussion section, page 7

 

Comments 3: Can the authors verify the mechanism by which this genetic variance in the promoter region of DNMT3B expression? Can this snp alter the binding of specific transcription factors?

Response 3 – We agree with the referee that this is in important issue. We were not able to find studies reporting whether the investigated SNP can alter the binding of specific transcription factors. However, according to Liu et al. (2008), in vitro assays have shown that this SNP results in a 30% increase in the promoter activity of DNMT3B. The authors hypothesized that the T allele enhances DNMT3B expression, leading to an increased tendency for abnormal de novo methylation of CpG sites in target genes. Xiao et al. (2011) assessed DNMT3B promoter activity in two pancreatic cancer cell lines. Transient transfection experiments using luciferase assays revealed that the promoter activity of the DNMT3B construct with the 149T allele was greater than that of the construct containing the 149C allele. Several other studies associated the T-allele with deleterious phenotypes, as childhood chronic immune thrombocytopenia (Gouda et al., 2016), lung cancer (Shen et al., 2002), and prostate cancer (R. Singal, P.M. Das, M. Manoharan, I.M. Reis, J.J. Schlesselman, Polymorphisms in the DNA methyltransferase 3B gene and prostate cancer risk, Oncol. Rep. 14 (2005) 569–573) .We believe that these evidence strongly argue for a functional effect of the SNP in the expression of the DNMT3B gene, although the transcription factors involved remain to be elucidated.

These information were included in the Discussion section, page 7

Comments 4: Can the authors discuss how DNMT3B an enzyme for methylation reaction could trigger  obesity?

Response 4 - Hervouet et al. (2009) demonstrated experimentally that DNMT3b interacts with 52 transcription factors fixed on membranes and hypothesized that this interaction is crucial for this enzyme to exert its function catalyzing site-specific hypermethylation in target genes. Among the 27 transcription factors with stronger interactions with DNMT3b is PPARγ. By using a cellular assay, they also confirmed the presence in glioma cells of direct interaction between Dnmt3b and PPARγ. This transcription factor has been strongly associated with obesity-related phenotypes, including a study performed by our group (Mattevi VS, Zembrzuski VM, Hutz MH. Effects of a PPARG gene variant on obesity characteristics in Brazil. Braz J Med Biol Res. 2007 40(7):927-32). Therefore, it is possible that DNMT3b, acting together with different transcription factors, regulates the expression of target genes involved in the regulation of body fat accumulation. As more than 500 gene variants that influence BMI have been associated with this phenotype through genome-wide association studies, most of them non-coding and likely to act through regulating genes nearby (Hemerich, D.; Svenstrup,V.; Obrero, V.D.; Preuss, M.; Moscati, A.; Hirschhorn, J.N.; Loos, R.J.F. An integrative framework to prioritize genes in more than 500 loci associated with body mass index. Am J Hum Genet 111, 1035–1046, 2024), functional studies regarding these interactions would bring clarity to these mechanisms.

These information were included in the discussion section.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript "Association of a promoter DNA methyltransferase 3 gene variant with DNA methylation and anthropometrics in children from 4 to 12 years old" is well design and well written. It is bringing new and interesting results for the scientific community regarding the association between rs2424913 (TT) genotype with obesity and global DNA methylation changes. 

In my opition the manuscript is ready to be published. I coudn't find any flaws. 

Congratulations for the high quality study presentation!

 

Author Response

Comments: Dear Authors,

 

The manuscript "Association of a promoter DNA methyltransferase 3 gene variant with DNA methylation and anthropometrics in children from 4 to 12 years old" is well design and well written. It is bringing new and interesting results for the scientific community regarding the association between rs2424913 (TT) genotype with obesity and global DNA methylation changes. In my opition the manuscript is ready to be published. I coudn't find any flaws.

Congratulations for the high quality study presentation!

Response: Thank you!