Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors describe the identification in Indonesia, of a less common mutation in the HBB gene. The proband was found through routine school-based anemia screening and presented with mild to moderate anemia but normal MCV.

Pedigree construction noted the mother was previously diagnosed with thalassemia-related anemia but results were “lost in the system”- an aspect that is a challenge in Indonesia. Standard testing of the index case revealed the Hb Malay mutation. This resulted in the extended family seeking confirmation of their individual status and provision of genetic counselling. Family members underwent general genetic screening with a commercial kit and And then into the mutation specific confirmation via Sanger sequencing

The authors suggest that the case highlighted a current lack of precise genetic information in Indonesia where even hemoglobinopathy screening is not systematic, thus leading to lack of awareness of the clinical implications of inherited thalassemia.

Comments

There are some aspects of the family pedigree that while not problematic, possibly require some comment, especially given the suggested rarity (line 15) of the mutation in Indonesia. Every tested person (13/13 with an inferred carrier (F2 HNF) making it 14 of 14) in the direct family line and an outside individual (F2 SA) is a carrier of the mutation. This is indirectly commented on as “familial clustering when both parental lineages carry the mutation” (line185-6) but conflicts with the concept that it is a rare mutation (it is 25% of carriers from Sumatra (ref 21). Sumatra is part of Indonesia and this is a very common mutation from that region and suggesting novel findings from family migration are a little weak. Maybe the authors might consider describing the Indonesian population as more diverse due to admixture from migration? This is suggested in cautionary advice (lines 187-9) but potentially deserves a bit more emphasis since it is a key point in the value of the presentation?  Without a key finding, what are the authors presenting? Mendelian inheritance is a well established phenomenon for this particular gene and family structure has little relevance without all persons being typed so that risk can be discussed. Typing of 45 to 77 year old females is of little relevance as far as genetic counselling for reduced risks in pregnancy. The true value of the study is held in the inadequacy of the common approach rather than the appearance of Hb Malay.

Absence of comment of any phenotype in the homozygous female (F3 KA 14) also detracts a little from the presentation since the authors keep citing other published sources.

Author Response

RESPONSE TO REVIEWER 1

Manuscript ID: thalassrep-4249004

Title: Familial Segregation and Molecular Identification of Hb Malay (HBB:c.59A>G) in a Three-Generation Indonesian Family

Dear R1,

Thank you very much for your careful and thoughtful review of our manuscript. We have greatly appreciated your comments, which have improved the quality and scientific rigor of this report. Here we provide a point by point response to each of your concerns. All changes in the revised manuscript are indicated by track changes.

Thank you for your fair read of the case. You are correct that the proband had mild anemia and near-normal MCV, that the mother’s previous diagnosis was “lost in the system,” and that the case illustrates the broader problem of inadequate genetic information infrastructure in Indonesia. Such contextual elements are now more clearly presented in the revised manuscript.

Comment 1: All tested individuals (13/13 with an inferred carrier F2 HNF making it 14 of 14) in the direct family line and one outside individual (F2 SA) are carriers of the mutation. This is in indirect conflict with the idea that it is a rare mutation (it is 25% of carriers from Sumatra, ref 21). This is a very common mutation from that region Sumatra is part of Indonesia Maybe the authors could consider describing the Indonesian population as more heterogeneous due to migration admixture? The value of the study is not the appearance of Hb Malay, but the inadequacy of the common approach.

Response: We thank the reviewer for this very important observation, with which we fully agree. You have correctly observed the apparent inconsistency between the high ascertainment rate in this family and the statement of rarity. This has now been covered extensively in the revised Discussion section with two additions:

(a) Ascertainment bias paragraph (new, Discussion Section 5):  We added an explicit paragraph explaining that the 81.25% variant-positive rate (13/16) is a direct consequence of the cascade family screening design, which by definition enriches for the shared variant. We explicitly state that this finding should not be extrapolated to population-level carrier frequency in Indonesia. It is also noted that Hb Malay carrier frequency in South Sumatran Malay populations has been reported to be around 5.3% (Sari et al., 2022) which is significantly lower than the family ascertainment rate in our cohort.

(b) Paragraph on the diversity of the Indonesian population : We have highlighted the ethnic and genomic diversity of the Indonesian population, reflecting the historical migration and inter-ethnic admixture across the archipelago. Older relatives of F1 individuals (ages 62-77 years) were included to establish the pattern of inheritance for genetic counseling, not to provide epidemiologic information.

(c) Refocusing of the key message: The key message paragraph has been re-written in its entirety to read: "The main contribution of this report is not the novelty of the Hb Malay mutation per se, but the demonstration that conventional hematological triage is insufficient to detect this variant. This is now the main message of the Discussion, as you rightly suggested it should be.

Comment 2: The typing of 45 to 77 year old females has little relevance as far as genetic counselling for reduced risks in pregnancy is concerned.

Response: We concur with this observation. Pregnancy risk counseling was not offered to members of the F1 generation (ages 62–77 years). The reasons for their inclusion were threefold: (1) to determine the parental lineages in which the mutation was present, so that the three-generation pedigree could be reconstructed; (2) to verify the familial origin of the variant for genetic counseling of F2 and F3 individuals of reproductive age; and (3) to follow the path of inheritance, as needed for pedigree analysis.

We have clarified this in the revised manuscript to avoid implying that these individuals were screened for reproductive risk reduction: “The inclusion of older relatives in the F1 generation (ages 62–77 years) was intended to map the inheritance trajectory across three generations for genetic counseling purposes, not to provide epidemiological data.”

Comment 3: The absence of any comment on the phenotype of the homozygous female (F3 KA 14) also detracts somewhat from the presentation as the authors keep referencing other published sources.

Response: We agree that this is the most important clinical gap in the manuscript. We have added a dedicated paragraph in the revised Discussion explicitly stating this as a major limitation:
Subject KA, the only homozygous individual for the Hb Malay variant (G/G, 15 years old), was not clinically examined in this study as the participants only agreed to a molecular genetic screening. This is a major shortcoming of this present report. KA did not undergo complete hematological phenotyping including hemoglobin level, MCV, MCH, HbA2, HbF, reticulocyte count, RDW, iron status and clinical assessment.

We recognize that an indirect characterization of the homozygous phenotype, via published references, is an imperfect substitute for the collection of clinical data. Both in the Discussion and in the Conclusions, prospective clinical and hematological evaluation of KA is now explicitly recommended as a follow-up priority.

We hope these changes sufficiently address your concerns. We believe the manuscript is substantially improved now with your guidance especially in refocusing the key message and addressing the ascertainment bias issue.

Reviewer 2 Report

Comments and Suggestions for Authors

This study is of particular relevance because, unlike in Malaysia and Thailand, the Hb Malay mutation is rarely documented in Indonesia. Based on the results obtained (screening and family history), the authors conclude that it would be advisable to incorporate molecular diagnostics and family screening into national thalassaemia prevention programmes in Indonesia. Whilst generally agreeing with the need for a national screening programme, the conclusion drawn in this article nevertheless seems rather categorical, as the authors have not provided the results of haematological phenotyping (this is the main point of criticism!). The diagnosis of thalassaemia, particularly β⁺ variants, is based on a combination of haematological parameters (MCV, MCH, Hb level, HbA2 content) and molecular confirmation.

There are no haematological analysis data for all other family members, so it cannot be confirmed that the identified genotype actually corresponds to the carrier phenotype. The authors state that consent was signed only for molecular screening, but if the issue is merely one of formal consent, might there be a chance to overcome the formalities and provide the results of a general blood test (Hb, MCV, HbA2, ferritin)?

The authors state that “this is the first report of the Hb Malay mutation in a large Indonesian family”. This may well be the case, but without a systematic review of previous publications (there have already been isolated cases in Indonesia, including those mentioned in the discussion), the claim of a “first case” should be made with caution. It would be more accurate to describe it as one of the few documented cases of familial carrier status/accumulation.

The article may be published as submitted

Author Response

RESPONSE TO REVIEWER 2

 

Manuscript ID: thalassrep-4249004

Title: Molecular Identification and Familial Segregation of the Hb Malay (HBB:c.59A>G) Variant in a Three-Generation Indonesian Family.

Dear Reviewer 2,

We thank you very much for your careful and constructive review and for the positive overall assessment of the manuscript. We appreciate your conclusion that our article could be published when submitted. However, we have taken your specific points of criticism seriously and have revised the manuscript accordingly. We also discuss below the additional revisions undertaken in response to comments from the Academic Editor and Reviewer 1 which we believe further strengthen the manuscript.

Comment 1: “The authors did not report the results of haematological phenotyping (this is the main point of criticism!) and therefore the conclusion drawn in this article seems too categorical, although we agree in general with the need of a national screening programme. The diagnosis of thalassaemia, especially β⁺ variants, is made on a combination of haematological indices (MCV, MCH, Hb level, HbA2 level) and molecular confirmation."

Response: We fully take this criticism on board, which we consider to be the most substantial scientific point raised in both reviews. You are right that the diagnosis of β⁺-thalassemia, especially carrier status, must be confirmed by both haematological parameters (MCV, MCH, Hb, HbA2) and molecular analysis. However, it is impossible to ensure that each identified genotype corresponds to the expected hematological carrier phenotype without HbA2 quantification and complete blood counts of all family members.

To address this we have modified the manuscript in three ways:

(a) The MCV of the proband is near-normal (80.5 fL), which is discussed as an atypical finding in classic β-thalassemia heterozygotes and the possibility of co-existing iron deficiency anemia has been explicitly acknowledged in both the Case Description and Discussion sections.

(b) The hematological findings of the proband are now discussed in a separate paragraph in the Discussion, acknowledging that the molecular finding of heterozygous Hb Malay confirms genetic carrier status but does not determine the hematological phenotype of this individual.

(c) The Conclusions have been thoroughly rewritten to eliminate any categorical policy recommendations. The conclusion has been altered to read that these findings “demonstrate the potential value of cascade molecular screening” and to include the following: “Population-based molecular epidemiological studies are needed to determine the true prevalence of Hb Malay in the diverse ethnic landscape of Indonesia and to evaluate the cost-effectiveness of its inclusion in national thalassemia screening panels prior to broader prevention policy recommendations being made. 

Comment 2: No data on haematological analysis are available for all other family members, therefore it cannot be confirmed that the identified genotype actually corresponds to the carrier phenotype. The authors state that signed consent was only for molecular screening, but if the issue is only formal consent, is there any chance to overcome the formalities and give the results of a general blood test (Hb, MCV, HbA2, ferritin)?

Response: We thank this constructive suggestion. As for the possibility of offering retrospective blood test data:

Unfortunately the consent obtained was specifically and explicitly for molecular genetic screening. Only the blood samples required for DNA extraction were taken and stored, and no hematological analyses were performed at the time of sample collection. The limitation of consent was not a procedural formality but the actual scope of the study protocol approved by our Institutional Ethics Committee (No. B-019/F12/KEPK/TL.00/08/2025). Hence, it was not possible to provide the retrospective hematological data of all the family members.

We have been open about this limitation in the revised manuscript, where we specifically state: “No further clinical examination or complete haematological profiling was conducted, as the participants consented solely to molecular genetic screening.”

As a priority KA (the homozygous individual) is now specifically recommended for prospective clinical and haematological assessment. We have added to the Conclusions: "Complete hematological phenotyping of family members, especially the homozygote, is an important goal for future study.” 

We acknowledge that this is a real limitation of the present report, but emphasize that the value of the present report is to demonstrate the feasibility and findings of cascade molecular screening in a resource-limited setting where hemoglobin fractionation was not available.

Comment 3: The authors report that "this is the first report of the Hb Malay mutation in a large family of Indonesian origin. While this may be the case, the statement of a ‘first case’ should be made with caution without a systematic review of previous publications (there have been already isolated cases in Indonesia, including those cited in the discussion). It would be more accurate to label it as one of the few documented cases of familial carrier state/accumulation.

Response: We agree completely with this important correction. The word “first report” has been deleted throughout the manuscript. In the Abstract, Introduction, Discussion, and Conclusions this is replaced by the following wording: “To the best of our knowledge, this is one of the few documented reports of cascade molecular screening for the Hb Malay (HBB:c.59A>G) variant in a large extended Indonesian family, confirmed by dual molecular methods.”

In the Discussion, we also note that isolated cases in Indonesia have been reported before (as cited in our Discussion section) and that the novelty of the present report is not in the variant itself, but rather in the cascade screening approach and the pedigree work-up.

We thank you for your positive evaluation of the manuscript and hope that the above revisions, which respond directly to your three specific criticisms, will make the article an even stronger contribution to the literature. We have maintained the scientific accuracy and proper epistemic humility that you have asked for.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the authors have presented a study that highlights the importance of understanding the admixture of genetics in a population considered high risk for thalassemia carrier status. While it was a limited study group, it does demonstrate what happens in the modern era where mutation carriers from one region migrate into another new genetic population and can impact on any screening program. It is a timely demonstration informing agencies involved in screening programs, that limited screening approaches may neglect minority groups in targeted areas and expose these families to unnecessary higher risks in offspring.

Reviewer 2 Report

Comments and Suggestions for Authors

I would like to thank the authors for their constructive responses. Thank you for highlighting the amended sections in a different colour.

1. 58-64 I fully agree with the authors - the absence of a diagnosis does not equate to the absence of the disease. The authors have phrased this addition absolutely correctly.
2. 75-82 The authors have made an important addition. Despite the lack of clear data on the presence of thalassemia, the authors carried out molecular screening and were correct in their assumptions. This is the very essence of research work.
3. 110-116 The authors have provided a relevant addition regarding the study’s approval by the ethics committee.
4. 126-138 The authors have provided the necessary information on diagnostic kits
5. 144-149 The authors have provided explanations for the terms used, which will aid understanding of the material
6. 207-228 A completely correct addition
7. 229-240 A completely correct addition
8. 249-260 an important addition aimed at optimising the logistics of genetic screening
9. 273-287 an excellent conclusion, fully based on the research findings and offering prospects for further work

The authors have responded to all the comments raised and made comprehensive additions to the text. The manuscript may be published as submitted.

I wish you every success!