Molecular Heterogeneity of Hb H Disease in India

Round 1

Reviewer 1 Report

I find this review fascinating and very relevant. To a reader specially in a region where Hb H disease is not very common this gives tremendous insight about the genotypic variability.

Author Response

Thank you

Reviewer 2 Report

Thank you 

Author Response

Thank you

Reviewer 3 Report

The authors report a review on Hb H disease in India through bibliographic research and selection of manuscripts showing the molecular characterization of patients. In the text there are interesting data on the described genotypes, the cases are presented very well and clearly, but in my opinion the manuscript is a little bit simplistic in the definition of mutations as they distinguished only between deletional and non-deletional mutations. The manuscript is written in a clear way but at the end left me a sense of vague and the text should be more focused on the particularity of Indian genotypes. I think that a clearer message should be left to the readers performing some change in the text as reported below. For these reasons major revisions are required.

In the introduction the non-deletional mutations should be distinguish between thalassemic mutations and missense mutations causing the synthesis of unstable globin variants. The unstable variants (especially the Hb Sallanches) should be briefly described reporting the impact of the mutation on the interaction with the chaperone AHSP or with the beta chain, if known, and the epidemiology.  (I recommend consulting the review of Wajcman et al. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin. 2008;32(4):327-49).

Moreover, in the case of thalassemic mutations, the impact of the mutation on the expression of the gene should be indicated, at least for the most common as the Poly A, reporting that it is one of the a+thal mutations with the most severe phenotype infect it has been defined as a+/a⁰. Also, in this case please report the epidemiology of this mutation in India.

The characteristics of the Poly A and Hb variants Sallanches explains why Hb H disease has been reported in cases of homozygosity or compound heterozygosity for these mutations.

In my opinion in the conclusion should be focused on the particularity of Indian genotypes highlighting that also if in India the frequencies of alpha⁰ thal is low, there are some common non deletional variants, like Poly A and Hb Sallanches that, if co-inherited with another deletional or non-deletional mutations, can give rise to severe Hb H disease.

Please report the HGVS nomenclature for each deletional or non-deletional mutation at least once in the text.

Minor point

-       In the paper the mutations are written in a wrong way missing the alpha gene at the begin of the deletions like in the abstract, where they reported (-3.7, -4.2, ….) instead of -a^3.7, -a^4.2. Please revise and correct them in all the manuscript.

Abstract

-       the most common (-3.7, -4.2, --SA, Poly A, HbSallenches), rare [--SEA, --MED, IVS 1-1 (G→A), Hb Koya Dora, Hb Sun Prairie], very rare (Hb Iberia, Hb Seal Rock, Hb Zürich-Albisrieden) and novel [Codon 76 (+T) and --Kol]…Question: please correct the name of the deletions (-a^3.7, -a^4.2 instead of -3.7 and -4.2 ) and use the same brackets round (  ) or square [   ] to contain the mutations.  The same anomalies need to be corrected at the end of the discussion.

Introduction

-       In India, the common deletional α-globin gene mutations are -α3.7, - α4.2 and --SA while the non deletional α-globin gene mutations are Hb Constant Spring (α 142 T→C), Hb Sallanches (α 142 G→A) and the Polyadenylation (Poly A) signal mutations [8, 9].

Question: please report the poly mutation typical of India

Results

-       In the other two cases having unexplained anemia, the mutation was reported to be αα/--KOL , however, the other mutation remained uncharacterized [11]. Question: Please indicated if the authors performed the sequencing analysis of the alpha globin gene and the analysis of the enhancer regions.

-       In 16 cases (αSeal Rock α/--→1; -α3.7/--) only one mutation was characterized whereas the other large deletion involving α1 and α2 genes remained un-characterized [13]. Question: the sentence is not clear because the authors introduce 16 cases and then show only two cases. Please explain than out of two new deletions only one was characterized, if that's the case.

-       and the patient required regular blood transfusions every month and had then undergone splenectomy. Question: The sentence is not clear, please correct the form.

-       Another clarification should be made on the molecular characterizations. The authors report many cases of homozygosity for non-deletional mutations. Please report the procedures used to rule out that they were hemizygous, for example if MLPA or qRT-PCR analyzes have been performed to exclude the presence of unknown deletions, or if family analysis has been carried out to confirm that both parents were carriers of the same mutation.

Table I

-       The Table 1 could be arranged leaving the separation by region of origin, but by ordering the Hb H diseases starting from the genotypes in which at least one of the alleles is of non-deletion type and by type of mutation (for example homozygous Poly A, compound heterozygous for Poly A… etc), and then the Hb H disease with 2 deletional defects.

Discussion

-       Two cases had co-inherited three α-globin gene mutations --SA, -3.7 and Hb Sallanches and were clinically severe [12]. Question: the sentence is not clear: do you mean that 2 mutations were co-inherited in cis? Please specify.

The reference n. 19 must be aligned.

Author Response

Reply to the Reviewer 3 comments:

The authors report a review on Hb H disease in India through bibliographic research and selection of manuscripts showing the molecular characterization of patients. In the text there are interesting data on the described genotypes, the cases are presented very well and clearly, but in my opinion the manuscript is a little bit simplistic in the definition of mutations as they distinguished only between deletional and non-deletional mutations. The manuscript is written in a clear way but at the end left me a sense of vague and the text should be more focused on the particularity of Indian genotypes. I think that a clearer message should be left to the readers performing some change in the text as reported below. For these reasons major revisions are required.

 In the introduction the non-deletional mutations should be distinguish between thalassemic mutations and missense mutations causing the synthesis of unstable globin variants. The unstable variants (especially the Hb Sallanches) should be briefly described reporting the impact of the mutation on the interaction with the chaperone AHSP or with the beta chain, if known, and the epidemiology.  (I recommend consulting the review of Wajcman et al. Unstable and thalassemic alpha chain hemoglobin variants: a cause of Hb H disease and thalassemia intermedia. Hemoglobin. 2008;32(4):327-49).

Reply: As per the suggestion now we have described briefly about impact of the Hb Sallanches mutation on the interaction with AHSP or β-chains and the epidemiology in the introduction (Para No.2; line no. 4-7) along with the reference of Wajcman et al.

Moreover, in the case of thalassemic mutations, the impact of the mutation on the expression of the gene should be indicated, at least for the most common as the Poly A, reporting that it is one of the a+thal mutations with the most severe phenotype infect it has been defined as a+/a⁰. Also, in this case please report the epidemiology of this mutation in India.

The characteristics of the Poly A and Hb variants Sallanches explains why Hb H disease has been reported in cases of homozygosity or compound heterozygosity for these mutations.

Reply: As per the suggestion we have now included the Poly A mutation with the severe phenotype and the epidemiology in India (Para no. 2; Line no.: 7-12). The impact of the Poly A mutation on the expression of the gene has been incorporated in the discussion (Para no.:3; Line no.:1-8)

In my opinion in the conclusion should be focused on the particularity of Indian genotypes highlighting that also if in India the frequencies of alpha⁰ thal is low, there are some common non deletional variants, like Poly A and Hb Sallanches that, if co-inherited with another deletional or non-deletional mutations, can give rise to severe Hb H disease.

Reply: As per the suggestion we have now modified the conclusion by highlighting the Indian genotype which can give rise to severe Hb H disease (Line No.: 5-7)

 Please report the HGVS nomenclature for each deletional or non-deletional mutation at least once in the text.

Reply: We have now included the HGVS nomenclature in the Table 1

 Minor point

-     In the paper the mutations are written in a wrong way missing the alpha gene at the begin of the deletions like in the abstract, where they reported (-3.7, -4.2, ….) instead of -a^3.7, -a^4.2. Please revise and correct them in all the manuscript.

      Reply: As per the suggestion we have now made necessary corrections in the entire manuscript.

 Abstract

-     the most common (-3.7, -4.2, --SA, Poly A, HbSallenches), rare [--SEA, --MED, IVS 1-1 (G→A), Hb Koya Dora, Hb Sun Prairie], very rare (Hb Iberia, Hb Seal Rock, Hb Zürich-Albisrieden) and novel [Codon 76 (+T) and --Kol]…Question: please correct the name of the deletions (-a^3.7, -a^4.2 instead of -3.7 and -4.2 ) and use the same brackets round (  ) or square [   ] to contain the mutations.  The same anomalies need to be corrected at the end of the discussion.

      Reply: We have now corrected the name of deletions and used the same brackets to contain the mutation in the abstract (Line no.: 10-13) as well as at the  end of the  discussion (Last Para; Line no.: 24-26).

Introduction

-     In India, the common deletional α-globin gene mutations are -α3.7, - α4.2 and --SA while the non deletional α-globin gene mutations are Hb Constant Spring (α 142 T→C), Hb Sallanches (α 142 G→A) and the Polyadenylation (Poly A) signal mutations [8, 9].

Question: please report the poly mutation typical of India

Reply: We have now reported the Poly A Mutation typical in India in the Introduction (Para no.:  2; Line no.: 11)

 Results

-     In the other two cases having unexplained anemia, the mutation was reported to be αα/--KOL , however, the other mutation remained uncharacterized [11]. Question: Please indicated if the authors performed the sequencing analysis of the alpha globin gene and the analysis of the enhancer regions.

       Reply: The authors did not performed the sequencing analysis of a-globin gene and the analysis of the enhancer region.

-      In 16 cases (αSeal Rock α/--→1; -α3.7/--) only one mutation was characterized whereas the other large deletion involving α1 and α2 genes remained un-characterized [13]. Question: the sentence is not clear because the authors introduce 16 cases and then show only two cases. Please explain than out of two new deletions only one was characterized, if that's the case.

      Reply: The sentence has now been clarified (Para no.: 3; Line no.: 7). We are extremely sorry for the mistake.

-     and the patient required regular blood transfusions every month and had then undergone splenectomy. Question: The sentence is not clear, please correct the form.

      Reply: We have now made the necessary corrections in the sentence (Para no.: 4; Line no.: 15-16)

 -       Another clarification should be made on the molecular characterizations. The authors report many cases of homozygosity for non-deletional mutations. Please report the procedures used to rule out that they were hemizygous, for example if MLPA or qRT-PCR analyzes have been performed to exclude the presence of unknown deletions, or if family analysis has been carried out to confirm that both parents were carriers of the same mutation.

      Reply: As suggested, the Molecular techniques used to rule out zygosity have now been included in the text under material and methods (Para no.:2 ; Line no.:3-6)

Table I

-     The Table 1 could be arranged leaving the separation by region of origin, but by ordering the Hb H diseases starting from the genotypes in which at least one of the alleles is of non-deletion type and by type of mutation (for example homozygous Poly A, compound heterozygous for Poly A… etc), and then the Hb H disease with 2 deletional defects.

      Reply: As per the suggestion Table 1 has now been rearranged according to the  Hb H disease genotypes.

Discussion

-     Two cases had co-inherited three α-globin gene mutations --SA, -3.7 and Hb Sallanches and were clinically severe [12]. Question: the sentence is not clear: do you mean that 2 mutations were co-inherited in cis? Please specify.

     Reply: Yes, the mutations were co inherited in Cis and as suggested the same has now been incorporated in the discussion (Para no. 2; Line no.: 10-11)

      The reference n. 19 must be aligned.

Reply: We have now aligned the reference.

Author Response File: Author Response.docx

Reviewer 4 Report

None

Author Response

Thank you

Round 2

Reviewer 3 Report

The author have made the required clarifications, and answered the questions in an exhaustive manner, therefore the manuscript can be accepted in the current version.