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Unfortunately none of the original reviewers were able to review the revised manuscript. However I evaluated the revisions and rebuttal myself, and determined that you satisfactorily addressed all the reviewers' concerns and implemented significant changes in your manuscript. Your manuscript is thus much improved.
The reviewers had several major concerns about your manuscript. With regard to your introduction, the knowledge gap that your manuscript addresses, needs to be clearly described and your hypothesis needs to be better developed. A more clear distinction between alpha vs beta thalassemia needs to be made throughout your manuscript, and your references need to be checked and updated. There were also major concerns regarding your methods, results and conclusions. First, the inclusion and exclusion criteria for subject selection, details on how your volunteers were recruited need to be described, and IRB information as well as the raw data need to be provided. You need to more clearly explain how and which genotypes were used for subject stratification, whether the control group was matched for sex and age, as well as whether and how you accounted for sources of variability and confounders. Additionally, more information on genetic testing needs to be provided. In your data analysis, a correction for multiple comparisons needs to be made. The fact that some of your statistically significant findings are within the normal reference range, thus may not be clinically significant, needs to be addressed. Some of your conclusions seem to not be supported by your evidence, and the terms clinical evidence and clinical phenotype are used, even though your manuscript only provides of biochemical evidence. Lastly, your discussion is a reiteration of your results, and does not put your findings in context or address limitations of your study, thus needs to be revised.
Please, submit a detailed rebuttal that shows where and how you have taken all comments and suggestions into consideration. If you do not agree with some of the reviewers’ comments or suggestions, please explain why. Your rebuttal will be critical in making a final decision on your manuscript. Please note also that your revised version may enter a new round of review by the same or by different reviewers. Therefore, I cannot guarantee that your manuscript will eventually be accepted.
The manuscript is written in generally understandable English, but there are frequent issues with clarity and phrasing that affect readability. The introduction lacks depth in contextualizing α-thalassemia, particularly in distinguishing it from β-thalassemia, which is frequently and inappropriately cited throughout the discussion. This reflects a limited understanding of disease-specific pathophysiology and weakens the scientific foundation of the manuscript.
The article structure is acceptable, and figures and tables are presented clearly. However, reference pediatric clinical ranges are missing from tables, making the interpretation of clinical relevance difficult. The discussion does not sufficiently relate findings to the stated aim, namely, the clinical heterogeneity of HbH disease in Hainan, or how the study contributes to improved management strategies. Overall, the manuscript lacks the clarity, context, and self-contained analysis expected for publication.
Although the study presents original data from a pediatric α-thalassemia cohort in Hainan, the research question is not clearly developed, and the study does not address a meaningful knowledge gap, as clinical features of HbH disease are already well-documented. The investigation lacks methodological rigor—data are pooled across a wide age range (1–18 years) and both sexes without adjustment, and statistically significant findings often remain within normal pediatric reference ranges, limiting clinical relevance.
Key methodological details are missing, including how participants were selected, grouped by genotype, and whether confounders like iron deficiency or infection were excluded. As written, the study does not meet the standards for clarity, rigor, or reproducibility expected for publication.
The study lacks clear novelty, as the clinical characteristics of pediatric HbH disease are already well-documented. The rationale for the study, to explore clinical heterogeneity in Hainan, is not convincingly developed, and the findings are not interpreted in a way that advances current knowledge or clinical practice.
Although statistical comparisons are provided, many reported differences remain within normal pediatric reference ranges and are not clinically significant. This distinction is not acknowledged, leading to potentially misleading conclusions. Furthermore, the authors do not stratify by age, sex, or genotype, and they group participants across a broad 1–18-year age range, reducing the validity of pooled comparisons.
Underlying data are presented in summary form but lack reference ranges and raw values, limiting transparency. Conclusions are overstated, not well-aligned with the research question, and not sufficiently supported by the data.
This manuscript describes the clinical data of pediatric α-thalassemia, including hematological parameters, liver function, renal function, and coagulation functions. The authors reported that among 160 children with various α-thalassemia genotypes, those with HbH disease have more severe anemia, altered platelet profile, liver and kidney function, higher serum ferritin levels, and poorer growth outcomes compared to silent and mild carriers. Although the clinical parameters for pediatric HbH disease are well-documented, the authors aim to provide insights into the clinical heterogeneity in Hainan to guide more tailored management strategies. The findings are consistent with what is already described in the literature. There are several concerns.
Major concerns
1. Materials and methods:
It would be helpful to provide more details in the Materials and Methods section.
1.1. The inclusion and exclusion criteria require further clarification. Specifically, was the normal group matched for age and sex? Additionally, how were potential confounders controlled?
1.2. Were iron depletion or iron deficiency considered in the exclusion criteria? If not, how might these factors have influenced the findings?
1.3. How were the volunteers recruited? Did they visit the hospital due to illness, or were they healthy controls recruited separately? Were current infections assessed and accounted for?
1.4. What specific α-globin genotypes were used to classify participants into the four
groups: silent carrier, mild α-thalassemia, HbH disease, and normal?
2. Results:
2.1. Concern regarding age and sex Grouping:
The manuscript presents data from a wide pediatric age range (1–18 years) and combines male and female participants without apparent stratification. Given that many clinical parameters, such as hemoglobin levels, iron status, growth indices, and organ function, vary significantly with age and sex, pooling all data together may obscure meaningful physiological differences. Addressing these concerns would strengthen the manuscript's conclusions and improve its clinical relevance. I recommend that the authors:
- Provide age-stratified analysis (e.g., grouping by early childhood, middle childhood, and adolescence) to account for developmental changes in clinical parameters.
- Consider analyzing data separately for males and females, particularly for parameters known to differ by sex in pediatric populations.
- Discuss how age-related and sex-related variations might impact the interpretation of their findings, especially in comparison to reference values from previous studies.
2.2. Concern regarding genotypic information:
The manuscript presents valuable clinical data on pediatric α-thalassemia patients. However, the severity of HbH disease can vary significantly depending on the specific α-thalassemia genotypes, particularly between deletional and nondeletional forms. For instance, patients with nondeletional HbH disease, such as those with the HbH Constant Spring (HbH-CS) mutation, often experience more severe anemia and
Related complications compared to those with deletional HbH disease. To better understand the clinical implications and variability among the study groups, I recommend that the authors provide detailed genotypic information for each participant group. Specifically:
- Genotypic Classification: Clearly delineate which α-thalassemia genotypes were
classified as silent carriers, mild α-thalassemia, and HbH disease within the study.
- Genotype Distribution: Present the distribution of specific genotypes within the HbH group, highlighting the prevalence of deletional versus nondeletional mutations.
- Correlation Analysis: If data permits, analyze and discuss the correlation between specific genotypes and observed clinical parameters, such as hemoglobin levels, organ function, and growth outcomes.
- Providing this genotypic information will significantly enhance the manuscript's contribution by elucidating the relationship between α-thalassemia genotypes and clinical severity, thereby informing more tailored management strategies for affected children.
2.3. Concern regarding growth analysis:
Utilizing growth charts to evaluate the growth of the study population allows for determining their positions relative to established percentiles, providing a clear assessment of their growth status. It is important to identify the number of individuals whose measurements fall below the 2nd or 3rd percentile for age and sex, as this analysis is crucial for detecting potential growth concerns.
2.4. The results section emphasizes numerous statistically significant differences in clinical parameters between the HbH group and the normal group. However, a closer examination of Tables 1–7 reveals that many of these parameters, despite being statistically different, remain within established pediatric reference ranges. This suggests that while the differences are statistically significant, they may not be clinically meaningful. It is important to distinguish between statistical significance, which indicates the likelihood that an observed difference is not due to chance, and clinical significance, which assesses the practical importance of that difference in patient care. A statistically significant result does not necessarily imply a clinically relevant finding, especially when the observed differences are within normal physiological limits. To enhance the clarity and clinical applicability of the manuscript, I recommend that the authors include normal reference ranges in the results tables. This addition would help readers discern which abnormalities are both statistically significant and clinically relevant, thereby providing a more nuanced interpretation of the data.
3. Discussion:
3.1. In the discussion section, there is a notable reliance on β-thalassemia literature to interpret findings related to HbH disease. Given the distinct pathophysiological mechanisms between β-thalassemia and α-thalassemia (HbH disease), this approach may not provide accurate insights. Specifically, β-thalassemia is characterized by ineffective erythropoiesis due to the precipitation of excess unpaired α-globin chains, leading to increased apoptosis of erythroid precursors in the bone marrow. In contrast, HbH disease involves the formation of β-globin tetramers (HbH) from excess β-globin chains, resulting predominantly in hemolysis rather than ineffective erythropoiesis. I recommend the authors:
- Focus on α-thalassemia-specific Literature: Reframe the discussion to ensure interpretations are grounded in α-thalassemia-specific literature, ensuring that interpretations are grounded in the appropriate context.
3.2. Furthermore, several parameters are reported as significantly different from the normal group; however, these values remain within established clinical reference ranges. While statistically significant, such differences may lack clinical relevance. It is important to distinguish between statistical significance and clinical significance to avoid overinterpreting findings. I recommend the authors:
- Clarify Clinical vs. Statistical Significance: Clearly differentiate between statistical significance and clinical relevance, emphasizing only those findings that have meaningful clinical implications for patient care.
- Contextualize Findings Within Clinical Reference Ranges: Provide context for the reported differences by discussing whether the observed values fall within normal clinical ranges, thereby aiding in the assessment of their true clinical impact.
3.3. The clinical parameters for pediatric HbH disease are well-documented. While the authors state that the aim of the study is to explore clinical heterogeneity in pediatric α-thalassemia in Hainan to inform tailored management strategies, the discussion does not adequately address this objective. I recommend revising the discussion to highlight the observed variability within the HbH group and how these findings may inform individualized care. Drawing clearer connections between the data and regional considerations in Hainan would also strengthen the discussion and better align it with the study’s stated aim.
The Discussion is the weakest part of the article. The beginning is repetitive and contains lots of descriptive words that are not needed. It has to be more targeted and directed to the exact work with an analysis of the results.
The study group needs to be described in a better way with more clinical data and differentiation between the different participants.
The control group should be described to confirm that they are a completely healthy group.
Lots of descriptive data should be changed into figures to show the results in a better way. The cardiac enzymes were not mentioned in your analysis, and the reason for including them in the study was not mentioned.
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The authors described the hematological and biochemical changes among normal controls, α-thalassemia trait, and Hb H disease. I have comments as follows.
1. Most of the data is laboratory-based. The term “clinical phenotype” in the title and “clinical data” in the whole manuscript should not be used.
2. Hematological and biochemical changes in thalassemia diseases have been described in many studies. A “gap of knowledge” should be provided in the Introduction.
3. How many healthy children’s subjects? 105 or 119? Tables 3-7 showed 119 subjects.
4. The HbH disease group should be divided into deletional and non-deletional HbH diseases.
The publication has a standard scientific organization. It has an introduction, methods, results, discussion, and conclusions.
This topic is important for public health, especially in places where α-thalassemia is common.
The language and grammar need a lot of work. The sentences are not written clearly. This is because of mistakes in the way the sentences are put together, the wording, and the syntax. For example, using characters like "³-thalassemia" instead of "α-thalassemia" shows that there are problems with encoding that need to be fixed everywhere in text.
The phrase "Hb H disease also showed worse physical indicators..." is unclear and not understandable. The text uses medical terminology and acronyms inconsistently. For example, it uses "GLU" instead of "glucose" and "PLT" instead of "platelets." It also doesn't define these terms the first time they are used. Please make it clear.
The introduction doesn't clearly explain what information is missing from the literature and what you are going to add. The setting is broad, and there is no clear reason given for why the study is needed.
References need a second look and update.
Suggestion: It's very important to have a professional English language and copyediting review.
The introduction should clearly state the difference between the clinical and scientific aspects.
Make sure that all references are up to date and match the in-text citations after corrections.
The study uses an appropriate design that looks at retrospective data.
The researchers did not provide a power analysis to justify the sample size.
The genotype classification is unclear. The definitions of "silent," "mild," and "Hb H" need to be explained more clearly, especially how the specific changes in genes are related to these categories.
The term "Hb H disease" is used a lot without an explanation of what it means, which makes it hard for people who are not experts to understand.
The description of what genetic testing is done is not clear. It is unclear what platform is used or what mutation panels are used.
Recommendation:
Include a table that summarizes all the genotypes for each group of patients.
Add detailed information about the process of genetic diagnostics (lab protocols, mutation classification criteria, etc ).
Explain why the sample size is as it is, or why you don't have enough data to do a power analysis- It is a local study, small population, etc, but please clarify.
A lot of clinical and biochemical data were collected and grouped by genotype.
Statistical analysis includes appropriate comparative tests (t-test, ANOVA, Mann-Whitney U).
But even though there are lots of p-values, no correction is made for multiple comparisons. This increases the chance of errors.
Some conclusions are based on data that is not reliable. For example, there is a link between lower glucose levels and diabetes, but this link is based only on lower glucose levels, not on a complete endocrine assessment.
The discussion section repeats what the results say. It doesn't explain the results of previous studies of the same age.
There is no long-term data, but there is speculation about possible long-term problems like liver damage or heart problems.
Please:
Use corrections for multiple testing (e.g., Bonferroni or FDR).
Explain the difference between statements that propose a hypothesis and statements that are based on evidence. Make clear what evidence is and what a hypothesis is.
Refer to the limitations of the study design, the short-term results, and the lack of confirmation of clinical features.
1. Tables and figures: Table 2 has too much information. Consider simplifying the structure or adding graphs to compare data.
2. The main submission mentions raw data availability but doesn't provide access. Please provide datasets per PeerJ's policy.
3. Ethical and legal compliance. Despite a generic ethical statement, the IRB protocol number is not disclosed.
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