Review Reports - Association of Bone Disorder and Gene Polymorphism of PPAR-γ Pro12 Ala in Egyptian Children with β-Thalassemia

Round 1

Reviewer 1 Report

In their manuscript „Association of Bone disorder and gene polymorphism of PPAR-ɤPro12Ala in Egyptian children with ß-thalassemia“ Hamied et al discuss the importance of PPAR ɤ polymorphisms as underlying risk factor for developing osteopenia in ß-thalassemia patients.

Major:

The small case number precludes a meaningful interpretation of the genotype data. The representation of the literature, in Discussion, is incomplete and not entirely accurate. For example, the statement on lines 330-331 is not supported by the cited reference (#37). Sahmani M et al decribed a significant that the risk of osteopenia was significantly (p <0.05) higher in the homozygous wild-type genotype than carriers of the rare alleles. Hamied et al did not correlate Z-score to wild type or c/g or g/g allele in thalassemia patients vs healty individuals. The data are all archival and correlative but lack power due to small number, and thus could be "true-true and not related" in terms of the actually underlying causes for bone disorders.

The review by Youssry I et al (doi.org/10.1002/pbc.29377) as well as Wong P et al (doi.org/10.1210/er.2015-1105) should be mentioned to provide a more comprehensive representation of the literature.

Minor:

Table 1 needs reconfiguration as layout is not displayed properly. This also includes falsely displayed content (BMD -> male/female in contrast to 11+46 males, 57 in total vs 7+36 females (43 in total )).

Table 3 + 4 These two tables do not really tell anything. They need to be either deleted or specified what the authors want to tell.

Comments for author File: Comments.docx

Author Response

Major:

The small case number precludes a meaningful interpretation of the genotype data.

Dear Reviewer , thanks for your review and your valuable points , according to major points , small case number in our study , we concentrate on the patients of β-thalassemia at specific period ( from 8 to 18 years ) age of mineralization to age of complete process of puberty that is critical period in the patient for affordable bone growth and puberty ,however, we look for another studies of β-thalassemia patient with more number and extended age time .

The representation of the literature, in Discussion, is incomplete and not entirely accurate. For example, the statement on lines 330-331 is not supported by the cited reference (#37). Sahmani M et al described a significant that the risk of osteopenia was significantly (p <0.05) higher in the homozygous wild-type genotype than carriers of the rare alleles. Hamied et al did not correlate Z-score to wild type or c/g or g/g allele in thalassemia patients vs healthy individuals.

I changed the representation of the discussion to complete and accurate form and thanks for your good note . Z-score Numbers are index for Bone mineral density of the pateinets and control groups .In table 3 , there is a comparison between the two sex groups as regarding PPAR-γ gene polymorphism . In table 4 , There are a comparison between PPAR-γ homozygous and PPAR-γ heterozygous gene groups regarding the BMD values .

The data are all archival and correlative but lack power due to small number, and thus could be "true-true and not related" in terms of the actually underlying causes for bone disorders.

The review by Youssry I et al (doi.org/10.1002/pbc.29377) as well as Wong P et al (doi.org/10.1210/er.2015-1105) should be mentioned to provide a more comprehensive representation of the literature.

As I mentioned above we concentrate on the patients of β-thalassemia at specific period ( from 8 to 18 years ) age of mineralization to age of complete process of puberty that is critical period in the patient , and we focus on important laboratory data related to bone disorder including measurements of serum calcium , phosphorus , ALP enzyme ,Hb and ferritin .

Minor:

According to minor points , I reconfigured the table 1 and removed all false contents ,

Table 3 + 4 These two tables do not really tell anything. They need to be either deleted or specified what the authors want to tell.

besides I removed table 3 . Tables 4 showed the different isotypes of PPAR-γ gene polymorphism in males and females of patients and control group , thanks for your attention .

I thought our study with 50 Egyptian children of β-thalassemia at critical age of life with measurements of laboratory data , BMD levels and searching for the possibility of using PPAR-γ polymorphisms as marker for osteopenia in these patients was helpful work for the other researchers in the world for the increasing the proper management and cure of β-thalassemia patients .

Reviewer 2 Report

In this paper, the authors present the association of bone disorder and gene polymorphism of 2 PPAR-γ Pro12 Ala in Egyptian children with β-thalassemia.
The introduction focuses on the general knowledge on the subject. The information provided is useful in understanding the topic and the importance of the subject in nowadays medicine.
The methods used are complex and reliable.
The inclusion and exclusion criteria were wisely chosen.
The authors conducted a proper collection of the data. The information presented is up to date, suitable, and substantial.
The conclusions are coherent and sustain the findings.
The figures and tables presented are easy to interpret and understand.
Good English level.
I recommend it for publication.

Author Response

Thanks for your review and valuable sentences , I appreciate hardly your wisdom and idea about my study , I hope it will be helpful study for all researcher all over the world , thanks my dear reviewer .

Reviewer 3 Report

β-Thalassemic patients exhibit an unbalance in bone mineral turnover resulting in diminished bone mineral density (BMD) more evident in the lumbar spine. According to the authors, the purpose of their study was to investigate the association between genetic polymorphism (which?) of the PPAR-γ gene and the presence of osteopenia or osteoporosis in children with Thalassemia.

The authors found that eighteen (11 males and 7 females) of fifty patients (representing 36 % of the patient group) had osteopenia with low bone mineral density (Z-score is -1 or less than 1). The authors did not find statistically significant BMD differences between males and females. By comparing the frequency of ??12 Ala gene polymorphism between the patient group and control group, the authors found that no statistically significant differences were detected. Furthermore, the authors stated that the BMD values were not significantly different between the groups of PPAR-γ Pro12Ala gene polymorphism (which groups??). In conclusion, according to the authors, decreased bone mineral density is frequent in β-Thalassemia patients but PPAR-γ Pro12Ala gene polymorphism is not common in patients with β-thalassemia. And no significant relationship between the groups of PPAR-γ Pro12Ala gene polymorphism and BMD level or osteopenia in Egyptian β -thalassemia patient

The paper is potentially interesting, however, in its present form is hard to follow.

In particular:

The methods section is quite poor and more information should be provided by the authors. I do suggest that the authors should be supported by a professional in statistics and methods writing.

Recruited patients were alpha or beta?

Was delta thalassemia excluded?

The Genotyping identification section is very confusing and must be improved.

There is no sense in comparing the BMD of an 8-years old thalassemic patient with an 18-years healthy subject. The authors should take into account the age effect.

The authors should correlate also the PPAR-γ Pro12Ala gene polymorphisms with the number of missing genes for alpha and beta globins.

The authors should correlate also the PPAR-γ Pro12Ala gene polymorphisms with the iron overload.

There is no description of the treatments given to the patients and their correlation with the PPAR-γ Pro12Ala gene polymorphisms.

The editing of the tables is very poor

Moderate editing of the English language required

Author Response

The paper is potentially interesting, however, in its present form is hard to follow.

In particular:

The methods section is quite poor and more information should be provided by the authors. I do suggest that the authors should be supported by a professional in statistics and methods writing.

Dear reviewer , thanks for your valuable points on my study and sure I appreciate your words and really it helps me to configure and modify my paper to acceptable form .

Recruited patients were alpha or beta?

Our study was related to patients with β-thalassemia as they suffer from chronicity of this disease with continuous needing to regular blood transfusion with a lot of complications including osteopenia and osteoporosis that have more chance to be occurred in these patients in comparing with α-thalassemia that most viable patients of them have mild or no complications .

Was delta thalassemia excluded?

Besides , patients with delta thalassemia are clinically silent with no complications and no needing for blood transfusion .

The Genotyping identification section is very confusing and must be improved.

There is no sense in comparing the BMD of an 8-years old thalassemic patient with an 18-years healthy subject. The authors should take into account the age effect.

Thanks for your attention , I improved the genotyping identification . we concentrate on the patients of β-thalassemia at specific period ( from 8 to 18 years ) age of mineralization to age of complete process of puberty that is critical period in the patient for affordable bone growth and puberty ,however, we look for another studies of β-thalassemia patient with more number and extended age time .

The authors should correlate also the PPAR-γ Pro12Ala gene polymorphisms with the number of missing genes for alpha and beta globins.

PPAR-γ gene is located on chromosome 3 and β-gene of hemoglobin is located on chromosome 11 , so we concentrate on the relation between the relation of PPAR-γ gene polymorphism mainly to osteopenia and labs markers and not to complicated molecular relations between the two genes to care of clinical details and relation in β-thalassemia patient , however , your fantastic idea could be a point for recent research on thalassemia patients .

The authors should correlate also the PPAR-γ Pro12Ala gene polymorphisms with the iron overload.

Moreover ,really I correlate the PPAR-γ Pro12Ala gene polymorphisms with ferritin level , that show not significant difference between them , and that may be explained by the regular use of chelation therapy to the patients with β-thalassemia and the younger age of our patients .

,No doubt , I thought recent research of extended age of patients of β-thalassemia with collaboration between pediatrics and internal medicine departments will add interesting details and notes

There is no description of the treatments given to the patients and their correlation with the PPAR-γ Pro12Ala gene polymorphisms.

.All patients received regular blood transfusion and chelation therapy that was mentioned in the inclusion and exclusion criteria .

The editing of the tables is very poor

I have edited the tables and all the paper to better form . hanks a lot for your wonderful notes , and I hope our study was helpful work for the other researchers in the world for the increasing the proper management and cure of β-thalassemia patients .

Round 2

Reviewer 1 Report

The revision conspicuously does not correct its most critical weakness. The authors state “ This study suggests that the Pro12 Ala gene polymorphism of the PPARγ gene was unrelated to low BMD levels and osteopenia in Egyptian children of β-Thalassemia. However larger scale number of patients trials for better assessment.” (Lines 408-411) Nevertheless, the genotype results are buried in lengthy descriptions of methodology, and the results have been presented before e.g.

El-Dafrawy, M., A. M. Hamed, N., Ghallab, O., Elneely, D., & Khalifa, M. (2016). Preliminary Report on the Frequency of Pro12Ala Polymorphism of the Peroxisome Proliferator-Activated Receptor-gamma Gene in Egyptian β-Thalassemia Major Patients. International Blood Research & Reviews, 6(3), 1–7. https://doi.org/10.9734/IBRR/2016/28458

Mehdi Sahmani 1, Abdollah Gholami, Azita Azarkeivan, Masoud Darabi, Mohammad Hossein Ahmadi, Majid Sirati Sabet, Reza Najafipour (2013) Peroxisome proliferator-activated receptor-γ Pro12Ala polymorphism and risk of osteopenia in β-thalassemia major patients. Hemoglobin, 2013;37(6):564-73. doi: 10.3109/03630269.2013.814035

Moderate improvements have already been applied.

Author Response

Author: Thanks for your effort and great comment , I add more details to the discussion and conclusion of the study to show more explanations and interpretations that help the researchers and open a gate for recent researches about osteopenia in β-thalassemia patients and the possibility of using laboratory markers and PPAR gene polymorphism in screening and monitoring of osteopenic β-thalassemia patients

Sahmani, M., et al., Peroxisome proliferator-activated receptor-γ Pro12Ala polymorphism and risk of osteopenia in β-thalassemia major patients. Hemoglobin, 2013. 37(6): p. 564-573.

Author: This previous study was different , it focused on patients of Iranian β-thalassemia patients with an age range of 20-45 years and searching for the relationship between the Pro12Ala polymorphism and low BMD in β-Thalassemia patients. But our study focus on the Egyptian children with different genotypes than Iranian patients . Moreover, we concentrated our study

on the earlier age group from 8 years to 18 years to try to find any earlier changes in these children helping us in earlier screening and diagnosis of osteopenia in these children so prompt earlier prevention or management may be available . Besides our study look for the relationships between the laboratory markers including serum calcium , phosphorus and ALP to the osteopenia and low BMD in the children with β-thalassemia , also searching for the relation between these markers and the PPAR gene polymorphism ( that wasn't present in the previous study that search only for the relation between the osteopenia and the PPAR gene polymorphisms.

Author: On the other side , however this study was nearly done in the same period of our study and on Egyptian β-Thalassemia patients with the same genotypes , but that study also was different , it focused on only 30 patients and 10 subjects as control group ( our study was done on 50 patients and 50 subjects as the control group ) . The patients of that study were with an age range of 16-39 years in the internal medicine department but ( our study focused on younger age in the pediatric department ) to try to find any earlier changes in these children helping us in earlier screening and diagnosis of osteopenia in these children as I said above , so we need study with young age to detect any earlier changes and exclusion of factors related to the chronicity of the diseases and other interrelated complications .

Besides our study look for the relationships between the laboratory markers including serum calcium , phosphorus and ALP to the osteopenia and low BMD in the children with β-thalassemia , also searching for the relation between these markers and the PPAR gene polymorphism ( that wasn't present in that previous study that search for the relation between the osteopenia and the PPAR gene polymorphisms and other markers not important in pediatrics or osteopenia as serum ALT , AST, Fasting blood sugar , urea and creatinine , So I think our study add a new interpretation or note helping other searchers in study of osteopenia in children β-thalassemia and the relation between PPAR gene polymorphism and the laboratory markers of these patients opening a gate for more researches about that .

Reviewer 3 Report

The age effect should (must) be considered.

The paper needs careful work of editing....

Minor editing of the English language required

Author Response

The age effect should (must) be considered.

Author: These were two previous studies about osteopenic β-thalassemia patients with older ages with other interrelated factors as chronicity , liver cirrhosis , complications of blood transfusion or other treatments , so in our study we focused on younger age of β-thalassemia patients to dismiss these factors and to try to find any earlier changes in these children helping us in earlier screening and diagnosis of osteopenia in these children so prompt earlier prevention or management may be available

Sahmani, M., et al., Peroxisome proliferator-activated receptor-γ Pro12Ala polymorphism and risk of osteopenia in β-thalassemia major patients. Hemoglobin, 2013. 37(6): p. 564-573.

The paper needs careful work of editing....

Round 3

Reviewer 1 Report

Major improvements have been applied. This includes a much more concise recapitulation of current literature. I think that this article is suitable for publication in its current version.

Minor spelling improvements can be applied.

Reviewer 3 Report

It is clear that I and the authors have different opinions about the data on the age effect.

Minor editing of the English language required