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All comments of the reviewers were adequately addressed, and the manuscript was amended accordingly. Therefore, the revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Paula Soares, a PeerJ Section Editor covering this Section #]
The authors have adequately addressed the issues raised and revised the manuscript accordingly. I have no further major concerns.
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Please address the concerns of the reviewer and revise the manuscript accordingly.
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1. Case 6 (Table 1) demonstrates an abnormally low HbA2 level (0.6%), a phenomenon rarely documented in published literature or our laboratory data. Further investigations are required to exclude additional contributing factors, such as potential co-inheritance of other δ-globin chain variants through Sanger sequencing. This clinically significant case warrants explicit discussion in the manuscript's dedicated discussion section.
The authors characterize the hematological and genotypic profiles of two novel mutations (δ-276 [A>G] and δ-77). The primary evidentiary support derives from the δ-77 mutation spanning Thailand, Myanmar, and Laos, which holds significance for regional researchers. In contrast, the δ-276 mutation was identified as a de novo variant whose pathogenic classification remains uncertain due to insufficient familial segregation evidence.
1.The proband carrying the δ-276 (A>G) mutation exhibited reduced HbA2, though unaffected family members showed normal values. Considering the advanced age (72 years) and anemia status (RDW 24%) of the proband, potential confounding from undiagnosed iron deficiency cannot be excluded. Supplemental iron status assessments (serum ferritin, transferrin saturation) would strengthen these conclusions.
2. The current evidence does not substantiate that the δ-276 mutation reduces HbA2 values, despite predictive software indicating a polymorphism. Even when combined with HbE in the first son, reduced A2 values cannot be assumed, as HbA2 levels in HbE heterozygotes can occasionally fall within the normal reference range in clinical observations. Therefore, the δ-276 mutation detected in this proband might represent incidental identification secondary to iron deficiency anemia.
The manuscript by Panyasai et al. explores the allele frequency and pathogenicity of two δ-globin gene variants, δ-276(A>G) and δ-77(T/C), in the Southeast Asian population. The writing is generally clear, and the manuscript is well-structured. Figures and tables are appropriately used, although a few issues need attention to improve clarity and consistency.
• In Figure 3, digestion with XmnI should yield two fragments (839 bp and 137 bp), but only the 839 bp band is shown. To avoid concerns about potential image manipulation or data omission, the authors should include the full gel image that shows the 137 bp band, or explain why it is not visible.
• In Figure 5 and the Materials and Methods section, the description of primer G218 is unclear. It should be revised to indicate whether G218 is specific to the wild-type or mutant allele.
• Avoid referencing figures and tables within the Materials and Methods section (lines 90, 104, 110, 117, and 132), unless necessary for methodological clarity. These should generally be cited in the Results section.
• The supplier name and location should be provided consistently (lines 103, 112, and 122).
• In the Results section, phrases such as “as shown in Figure X” are redundant and may be omitted in favor of simply citing the figure.
• The sentence in lines 159–160 is awkward and repetitive; a revision for clarity is recommended.
The study is based on a reasonable sample size and employs appropriate genetic and hematological analyses to address the research questions. The authors used PCR-RFLP for δ-276(A>G) and allele-specific PCR for δ-77(T/C). However, clarification is needed:
• What is the rationale for using different detection methods for the two variants? A brief explanation would enhance methodological transparency.
•The study includes in silico analyses for transcription factor binding and pathogenicity prediction, but the findings from these analyses are insufficiently described. The authors should explain the significance of the results, clarify what the prediction scores represent, and highlight any notable transcription factor binding sites. Additionally, it is unclear what the sequences shown in bold indicate—please explain their significance or selection criteria.
• Regarding the allele frequencies of rs3813726 in Table 1, the number of alleles reported for the Asian population (3,174) appears inconsistent with the NCBI dbSNP database cited in the manuscript. This number seems to correspond to the African American population instead. Please double-check the source data and correct the table accordingly.
The study provides compelling evidence that δ-276(A>G) is a common, non-pathogenic variant in the Thai population. However, a key issue remains unresolved: the reduced Hb A2 levels observed in the proband. This was the initial motivation for the study, and its lack of resolution is a limitation.
• Even if the etiology remains unknown, the authors should discuss potential explanations or alternative hypotheses. This would be helpful for clinicians encountering similar hematological profiles.
• The identification of δ-276(A>G) as a benign variant raises a clinically relevant point: should clinicians disregard this variant and search for other pathological mutations when it is detected? The authors are encouraged to elaborate on the practical implications of their findings.
• The study makes a valuable contribution to our understanding of δ-globin gene variants in Southeast Asia.
• To improve clarity and transparency, the authors should address the issues noted in the figures, provide better interpretation of the in silico data, and refine some aspects of the manuscript presentation.
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