Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is rich in data and might be interesting for geneticists/ physicians dealing with hemoglobinopathies.

However, the Reviewer has a strong  feeling that some data should be added to improve the validity of the study:

-In the ‘Introduction section the reason WHY the diagnosis of asymptomatic carriers of thalassemia traits is so crucial should  be stressed out. Is it because these individuals are endangered or there is a possibility to pass the condition onto the offspring?

-The authors stress ‘the limitations of traditional diagnostic methods—such as the osmotic fragility test’- however the test and its outcomes are not described. Apart from the description, it would be beneficial to include the results of  osmotic fragility test run on at least some of the patients to prove the limitation statement. If the Authors don’t have such results, they should restrain from comparing these two approaches.

- The last three paragraphs starting with “Clinically relevant forms of α-thalassaemia […]”, “A clinically significant condition is haemoglobin H disease […]” and “ The most severe form, which is incompatible with life […]” all require to be supplemented with references, where the given information were found.

The ‘Materials and methods’ section should be supplemented with patient information:

-inclusion/ exclusion criteria for the study,

-age (i.e. how many >20 y.o, 21-30 y.o, etc),

-sex

- ethnic background

 

Although the text is clear and easy to understand, there are some language/ editorial issues that should be corrected before publication:

1. The following expressions are hard to understand or linguistically incorrect and should be replaced :

• […] using a tiered diagnostic approach.” (found in the Abstract)
• “The most severe form, which is incompatible with life […]” (last-but-one paragraph of the Introduction ).

2. The ‘Reference list’ has some numeration errors at the end that should be corrected

The work is well-written and apart from the described shortcomings it does not have any major flaws.

Author Response

Comment 1
Questions	Answers
-In the ‘Introduction section the reason WHY the diagnosis of asymptomatic carriers of thalassemia traits is so crucial should  be stressed out. Is it because these individuals are endangered or there is a possibility to pass the condition onto the offspring?	As illustrated in the work, genetic diagnosis of asymptomatic carriers of the thalassemia trait is very important to have a differential diagnosis between thalassemia trait and iron deficiency anemia.
The authors stress ‘the limitations of traditional diagnostic methods—such as the osmotic fragility test’- however the test and its outcomes are not described. Apart from the description, it would be beneficial to include the results of  osmotic fragility test run on at least some of the patients to prove the limitation statement. If the Authors don’t have such results, they should restrain from comparing these two approaches.	Osmotic fragility testing has not been described because it is obsolete for identifying individuals with the thalassemia trait. This test has been replaced by HPLC analysis of the percentages of HbA, HbA2, HbF, and abnormal Hb, analysis of iron parameters, and blood count. These tests represent the first-level screening.
The last three paragraphs starting with “Clinically relevant forms of α-thalassaemia […]”, “A clinically significant condition is haemoglobin H disease […]” and “ The most severe form, which is incompatible with life […]” all require to be supplemented with references, where the given information were found.	The phrase “The most severe form, which is incompatible with life” has been removed from the manuscript.
Although the text is clear and easy to understand, there are some language/ editorial issues that should be corrected before publication: The following expressions are hard to understand or linguistically incorrect and should be replaced : ·         […] using a tiered diagnostic approach.” (found in the Abstract) ·         “The most severe form, which is incompatible with life […]” (last-but-one paragraph of the Introduction ). The ‘Reference list’ has some numeration errors at the end that should be corrected The work is well-written and apart from the described shortcomings it does not have any major flaws.	Although the text is clear and easy to understand, there are some language/ editorial issues that should be corrected before publication: The following expressions are hard to understand or linguistically incorrect and should be replaced […]using a tiered diagnostic approach.” (found in the Abstract) it has been replaced by “using both biochemical and genetic parameters”. “The most severe form, which is incompatible with life […]” (last-but-one paragraph of the Introduction ) it has been eliminated. The ‘Reference list’ has some numeration errors have been corrected

Reviewer 2 Report

Comments and Suggestions for Authors

The present paper examines genetic variants associated with haemoglobinopathy based on people in Southern Italy. Analysis on the correlation between biochemical parameters and specific genotype mutations made by molecular biology techniques is amazing. Results are very interesting and certainly worthy for publication. But this reviewer raises some concerns that need to be addressed before publication. If those concerns are adequately addressed in the revised manuscript, this interesting article would be significantly strengthened.

 

Concerns that need to be addressed are:

 

[1] Please make Abbreviation List on the first page or before References: Words in Abbreviations should be alphabetical order for general readers to easily pick up words.

 

Please put the following and other words into the List, if they appear more than twice in the text.

 

HPLC, NGS, HbG, HbA, HbH, MCV, EDTA, RBC, MCV, MCHC, gDNA, CNV, HGVS, HCT, MCH, HBA2, HBF, HB, NG, NC, NM, HBA2, HBF, HBB, HPFH, IVS1, IVS-I, RDW and others.

 

If those abbreviations are used only in tables, please insert abbreviation list into the legend for each table.

 

[2] Graphical figures would be beneficial for general readers to grasp points of this interesting paper. Please make two or more graphical figures and insert those into Introduction and Discussion.

 

[3] “3. Results” is composed of only five tables and just few comments/sentences. This pattern is not usual for the scientific paper. Five tables are great. But explanation/interpretation for each table is poor and too short. Only two lines are not enough to explain each table. Please describe the table contents more in detail. Maybe some parts in “4. Discussion” would be moved into 3. Result.

 

[4] References

Descriptions are not normalized. For example, some reference papers include PMID (Refs. 6, 9, 10, 11, 12 et al.) whereas others do not include PMID. Also, some reference papers include last page numbers (Refs. 3, 5, 6 ,7, 8 et al.), whereas other do not so. Please fix these issues.

 

Abbreviations of some journals are not appropriate. Please check Refs. 4, 9, 16.

 

Journal spellings are inappropriate: Refs. 7 and 9.

 

Please remedy Refs. 13-15.

 

Author Response

Commento 2
Domande	Risposte
[1] Si prega di creare un elenco delle abbreviazioni sulla prima pagina o prima dei riferimenti: le parole nelle abbreviazioni devono essere in ordine alfabetico affinché i lettori generici possano coglierle facilmente. Si prega di inserire le seguenti e altre parole nell'elenco, se compaiono più di due volte nel testo.	[1] L'elenco delle abbreviazioni è stato inserito prima dei riferimenti.
[3] “3. Risultati” è composto da sole cinque tabelle e pochi commenti/frasi. Questo schema non è usuale per un articolo scientifico. Cinque tabelle sono ottime. Ma la spiegazione/interpretazione per ogni tabella è scarsa e troppo breve. Solo due righe non sono sufficienti per spiegare ogni tabella. Si prega di descrivere il contenuto della tabella in modo più dettagliato. Forse alcune parti in “4. Discussione” potrebbero essere spostate in 3. Risultato.	[3] Abbiamo descritto più dettagliatamente il contenuto della tabella.
[4] Le descrizioni dei riferimenti non sono normalizzate. Ad esempio, alcuni documenti di riferimento includono PMID (rif. 6, 9, 10, 11, 12 et al.) mentre altri non includono PMID. Inoltre, alcuni documenti di riferimento includono i numeri delle ultime pagine (rif. 3, 5, 6, 7, 8 et al.), mentre altri no. Si prega di risolvere questi problemi.	[4] Le descrizioni di riferimento sono state normalizzate.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting your manuscript “Hemoglobinopathies: integrated biochemical and molecular diagnosis in 5,243 patients”. The manuscript reports an impressive dataset and underlines the importance of integrating biochemical and molecular method in diagnosing hemoglobinopathies. However, the study would benefit from clearer focus, statistical analysis of genotype-phenotype correlations, and deeper interpretation of rare variants. Presentation (table, terminology, references) also requires refinement. Below are the specific points you need to address to strengthening your work.

Major Comments

1. Novelty and contextualization

• The study provides valuable large-scale data (5243 patients), but the novelty is not fully highlighted. The authors should clarify how their findings significantly extend beyond existing prevalence studies and molecular diagnostic approaches already reported in Italy and the Mediterranean region.

 

2. Clarity of objectives

• The introduction states the aim is to correlate biochemical parameters with genotypes, but the discussion often shifts toward prevalence reporting. A more focused research question and hypothesis would improve clarity.

 

3. Data interpretation

• The manuscript sometimes presents results descriptively without deeper statistical analysis. For instance, differences in hematological parameters across genotypes are reported in tables, but no statistical comparisons (e.g., p-values, confidence intervals) are provided. This weakens conclusions about genotype-phenotype correlations.

 

4. Diagnostic Algorithm

• The proposed three-step diagnostic algorithm is interesting, but the justification lacks quantitative backing. How much did NGS improve detection beyond first and second level screening? A cost-effectiveness or sensitivity/specificity analysis would strengthen the argument.

 

5. Population Bias

• The cohort is described as including immigrants, but no stratified analysis is presented (e.g., prevalence among native Italians vs. non-Italians). This is important given the paper emphasizes the role of migration in genetic diversity.

 

6. Rare Variants

• The HbG Philadelphia and other rare findings are interesting, but discussion is limited. More details on clinical implications, family studies, or confirmatory analyses would enhance their relevance.

 

Minor Comments

1. Terminology Consistency

• Some terms are inconsistently used (e.g., “trait” vs. “carrier state”, “heterozygote” vs. “compound heterozygote”). Standardization would improve readability.

 

2. Formatting of Tables

• Tables are overcrowded with numbers, making interpretation difficult. Condensing or adding graphical summaries (box plots, bar graphs) would aid readers.

 

3. Language and Style

• The manuscript is understandable but occasionally wordy and repetitive. Sentences such as “This form is characterized by hemoglobin levels ranging from 7 to 10 g/dL and may present with jaundice and splenomegaly” could be streamlined.

 

4. Reference List

• Some references are duplicated (e.g., Mansoor et al. cited twice as [13] and [14]. A careful check is needed.

 

5. Ethical Considerations

• The authors mention informed consent and ethics approval, but there is no statement on whether genetic counselling was offered, which is standard in hemoglobinopathy carrier studies.

Author Response

Comment 3
Questions	Answers
Novelty and contextualization ·         The study provides valuable large-scale data (5243 patients), but the novelty is not fully highlighted. The authors should clarify how their findings significantly extend beyond existing prevalence studies and molecular diagnostic approaches already reported in Italy and the Mediterranean region.	The proposed study correlates biochemical data with genetic data. This study demonstrates how, in some cases, biochemical data are not exhaustive enough to delineate the thalassemia profile. For this reason, genetic testing is essential for a correct genotype-phenotype association.
Clarity of objectives ·         The introduction states the aim is to correlate biochemical parameters with genotypes, but the discussion often shifts toward prevalence reporting. A more focused research question and hypothesis would improve clarity.	In the discussion we reported the cases where the genetic data clarified the observed biochemical picture and the percentage of this deviation.
Data interpretation ·         The manuscript sometimes presents results descriptively without deeper statistical analysis. For instance, differences in hematological parameters across genotypes are reported in tables, but no statistical comparisons (e.g., p-values, confidence intervals) are provided. This weakens conclusions about genotype-phenotype correlations.	Our work emphasizes that whenever unclear biochemical data are present, molecular study of the globin genes is essential. For this reason, we compared the biochemical data with the genetic data, reporting the percentage of subjects in whom the genetic data served to establish a correct diagnosis.   . ·
Diagnostic Algorithm ·         The proposed three-step diagnostic algorithm is interesting, but the justification lacks quantitative backing. How much did NGS improve detection beyond first and second level screening? A cost-effectiveness or sensitivity/specificity analysis would strengthen the argument.	Given the presence of a multiethnic population, due to globalization, NGS is needed to obtain an exact genotype-phenotype correlation, which is often not possible with first-level screening, especially for some hemoglobin variants. Many hemoglobin variants are mistaken for iron deficiency anemia with first-level screening, and those affected undergo cycles of ineffective chemotherapy that burden healthcare costs. Therefore, the correct diagnosis achieved through NGS is useful in uncertain cases.
Population Bias ·         The cohort is described as including immigrants, but no stratified analysis is presented (e.g., prevalence among native Italians vs. non-Italians). This is important given the paper emphasizes the role of migration in genetic diversity.	Living in a multiethnic society (a result of globalization), ethnic differences could now lead to errors. For this reason, the authors of this paper analyzed the data in an aseptic manner, as mixed couples of different ethnic origins arrive at the medical genetics laboratory. Therefore, our primary concern is to make a correct diagnosis. For this reason, we no longer use the RDB technique, which detects only the most common mutations in the Italian population, but instead use the NGS technique, which sequences the alpha and beta globin genes. This increases the detection rate and eliminates the false negatives associated with the RDB method.
Rare Variants ·         The HbG Philadelphia and other rare findings are interesting, but discussion is limited. More details on clinical implications, family studies, or confirmatory analyses would enhance their relevance.	we have integrated this part
Minor Comments
Terminology Consistency ·         Some terms are inconsistently used (e.g., “trait” vs. “carrier state”, “heterozygote” vs. “compound heterozygote”). Standardization would improve readability. Formatting of Tables ·         Tables are overcrowded with numbers, making interpretation difficult. Condensing or adding graphical summaries (box plots, bar graphs) would aid readers. Language and Style ·         The manuscript is understandable but occasionally wordy and repetitive. Sentences such as “This form is characterized by hemoglobin levels ranging from 7 to 10 g/dL and may present with jaundice and splenomegaly” could be streamlined. Reference List ·         Some references are duplicated (e.g., Mansoor et al. cited twice as [13] and [14]. A careful check is needed. Ethical Considerations ·         The authors mention informed consent and ethics approval, but there is no statement on whether genetic counselling was offered, which is standard in hemoglobinopathy carrier studies.	1         we have eliminated these inconsistencies in the manuscript
	Notes have been added to the tables to better describe the results. Furthermore, before the references, we have added a section explaining the abbreviations in the manuscript we have improved the language style The ‘Reference list’ has some numeration errors have been corrected In the Informed Consent Statement session the sentence was inserted: All those who underwent this study received genetic counseling associated with the test.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Reviewer finds the answers to the comments satisfactory. However the authors have not added references required to back up statements found in the following parapgraphs of the Introduction:

"Clinically relevant forms of α-thalassaemia arise when at least three α-globin genes
are affected. When only two α-globin genes are involved—either by point mutations or
deletions—the resulting phenotype is mild and typically presents as microcytic anaemia
of low severity, usually requiring no treatment other than intermittent folic acid supplementation. Due to the associated biochemical profile, such cases are frequently misdiagnosed as iron deficiency anaemia. [REF]
A clinically significant condition is haemoglobin H disease (HbH), which occurs in
individuals with only one functional α-globin gene (genotype –/-α). This form is characterised by haemoglobin levels ranging from 7 to 10 g/dL and may present with jaundice
and splenomegaly. Microcytosis is typically more pronounced (MCV between 50 and 65
fL), HbA₂ levels are reduced (1–2%), and HbH may account for up to 8–10% of total haemoglobin. The presence of HbH inclusion bodies is characteristic and can be demonstrated using specific staining techniques.. [REF]
In the complete absence of α-chains, the fetus produces only Hb Bart’s (haemoglobin
composed of four γ-chains), a form of haemoglobin with extremely high oxygen affinity
but entirely ineffective at oxygen delivery to tissues. This results in severe intrauterine
hypoxia, rapidly progressing to hydrops fetalis, cardiac failure, and, in the vast majority
of cases, intrauterine or early neonatal death.. [REF]"

Author Response

"Clinically relevant forms of α-thalassaemia arise when at least three α-globin genes
are affected. When only two α-globin genes are involved—either by point mutations or
deletions—the resulting phenotype is mild and typically presents as microcytic anaemia
of low severity, usually requiring no treatment other than intermittent folic acid supplementation. Due to the associated biochemical profile, such cases are frequently misdiagnosed as iron deficiency anaemia. [Songdej, D.; Fucharoen, S. Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment. Thalass. Rep. 2022, 12, 157-172. https://doi.org/10.3390/thalassrep12040020; Vijian, D.; Wan Ab Rahman, W.S.; Ponnuraj, K.T.; Zulkafli, Z.; Bahar, R.; Yasin, N.; Hassan, S.; Esa, E. Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia. Diagnostics 2023, 13, 894. https://doi.org/10.3390/diagnostics13050894]
A clinically significant condition is haemoglobin H disease (HbH), which occurs in individuals with only one functional α-globin gene (genotype –/-α). This form is characterised by haemoglobin levels ranging from 7 to 10 g/dL and may present with jaundice and splenomegaly. Microcytosis is typically more pronounced (MCV between 50 and 65
fL), HbA₂ levels are reduced (1–2%), and HbH may account for up to 8–10% of total haemoglobin. The presence of HbH inclusion bodies is characteristic and can be demonstrated using specific staining techniques. [Songdej, D.; Fucharoen, S. Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment. Thalass. Rep. 2022, 12, 157-172. https://doi.org/10.3390/thalassrep12040020;Lal A, Vichinsky E. The Clinical Phenotypes of Alpha Thalassemia. Hematol Oncol Clin North Am. 2023 Apr;37(2):327-339. doi: 10.1016/j.hoc.2022.12.004. PMID: 36907606.]
In the complete absence of α-chains, the fetus produces only Hb Bart’s (haemoglobin composed of four γ-chains), a form of haemoglobin with extremely high oxygen affinity but entirely ineffective at oxygen delivery to tissues. This results in severe intrauterine hypoxia, rapidly progressing to hydrops fetalis, cardiac failure, and, in the vast majority of cases, intrauterine or early neonatal death.. [Chui DH. Alpha-thalassemia: Hb H disease and Hb Barts hydrops fetalis. Ann N Y Acad Sci. 2005;1054:25-32. doi: 10.1196/annals.1345.004. PMID: 16339648]

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have adequately addressed the concerns raised in the previous round of review. The revisions are clear, well-justified, and have improved the overall quality of the manuscript. I am satisfied with the changes made and have no further major comments.

Author Response

Risposta dell'autore al rapporto di revisione (revisore 3)
Domande	risposta
Le figure e le tabelle devono essere migliorate	Abbiamo descritto più dettagliatamente il contenuto della tabella.