Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this publication authors described new variants in P2RY1, GP6, TBXAS1 genes and publish comprehensive review of the IPFD focused on disorders caused by pathological variants in these genes.

IPFD comprise a heterogenous group of very rare diseases, so characterising new alterations is beneficial since diagnosis is very limited due to their high clinical and laboratory heterogeneity.

Question to authors:

1. You are evaluation severity of bleeding in patients with novel variants. Did you exclude VWD in these patients. Did you screen VWF gene in your HTS panel?
2. In fig. 2 you presented all previously reported variants however databases LOVD3 and HGMD present father pathological variants: ( P2RY12- Met1Arg (NM_022788.3,NP_073625.1), which you wrong interpreted in reference (1) in supplement.     p.Val207Ala (NM_022788.3,NP_073625.1), GP6- c.471del,  p.Glu158ArgfsTer13 (NM_001083899.1), c.1288_1291dup, p.Thr431AsnfsTer34 (NM_001083899.1), TBXAS1- p.Arg249Ter (NM_001061.4, NP_001052.2).
3. I did not find the reference sequences that you use for numbering of your variants. It should be announced.

Author Response

In this publication authors described new variants in P2RY1, GP6, TBXAS1 genes and publish comprehensive review of the IPFD focused on disorders caused by pathological variants in these genes.

IPFD comprise a heterogenous group of very rare diseases, so characterising new alterations is beneficial since diagnosis is very limited due to their high clinical and laboratory heterogeneity.

 

Question to authors:

1. You are evaluation severity of bleeding in patients with novel variants. Did you exclude VWD in these patients. Did you screen VWF gene in your HTS panel?

RESPONSE: We would like to express our gratitude to the reviewer for his/her feedback.  In all cases where suspicion of a congenital platelet disorder was raised, we were able to exclude any alterations to coagulation parameters, vWF levels and function. Furthermore, our gene panel encompasses all exonic regions of VWF. In the patients documented in this manuscript, no pathogenic or likely pathogenic variants were identified in the gene.

To address the reviewer suggestion, we have amended the first paragraph of the result section (page 11) to state:  

“None of them presented alterations in coagulation parameters or in von Willebrand factor, nor other appreciable clinical complications”

In addition, the last paragraph in this section (page 12) states:

“Our genetic studies in these patients identified no likely pathogenic or pathogenic variants in other genes that are relevant to platelet function or the VWF gene.”

 

2. In fig. 2 you presented all previously reported variants however databases LOVD3 and HGMD present father pathological variants: (P2RY12-Met1Arg (NM_022788.3,NP_073625.1), which you wrong interpreted in reference (1) in supplement. p.Val207Ala (NM_022788.3,NP_073625.1), GP6-c.471del, p.Glu158ArgfsTer13 (NM_001083899.1), c.1288_1291dup, p.Thr431AsnfsTer34 (NM_001083899.1), TBXAS1-p.Arg249Ter (NM_001061.4, NP_001052.2).

RESPONSE: Thank you to the reviewer for this observation. Only patients with a clear diagnosis of IPFD and candidate variants in these genes have been included, as population databases such as GnomAD, LOVD3 and HGMD report variants for which the causality and phenotype are unknown. As noticed by the reviewer, we have change the first P2RY12 variant in Supplemental Table 1 to the corrected one p.Met1Arg. In addition, we have added the variant p.Val207Ala as correctly indicated by the reviewer. 

As regards the GP6 we will like to mention that the transcript indicated by the reviewer (NM_001083899.1) is not the one we used NM_016363.5 and it proposed in the current list of TIER1 gene for inherited platelet disorders (https://www.isth.org/page/GinTh_GeneLists). Yet, these two transcripts may be similar is not identical. The indicated variant GP6-c.471del [p.Glu158ArgfsTer13] is indeed present also in NM_016363.5, but c.1288_1291dup [p.Thr431AsnfsTer34] is not. Moreover, none of these variants are in gnomAD, and in LOVD the effect on function has not been curated.  As far we know, no patients have been described or reported with these variants.  We have therefore not to include in our Supplemental Table 2.

Lastly, for TBXAS1, we used the transcript indicated by the reviewer (NM_001061.4). However, we have found discrepancy between different database (LOVD, ClinVar, Varsome ) regarding the presence of and Arginine at this transcript position.  Clinvar does report p. R248*, with conflicting classifications (pathogenic or VUS).   We have neither found this variant reported in any patient. Therefore, we have decided not adding this variant in Supplemental Table 3.

 

3. I did not find the reference sequences that you use for numbering of your variants. It should be announced.

RESPONSE:  Thank you very much for this additional observation. To address this, we have added in the text the identification of the gene transcript that we have used in our study:   P2RY12: NM_022788.5 (page 11) GP6:  NM_016363.5 (page 12) / TBXAS1: NM_001061.7 (page 12).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Summary:

This is a combined case series and molecular/functional characterization of four novel heterozygous variants in the P2RY12, GP6, and TBXAS1 genes, identified in six patients with bleeding and platelet disorders. High-Throughput Sequencing (HTS), comprehensive platelet phenotyping (aggregation, flow cytometry), molecular assessment (mRNA, protein expression, and novel downstream signaling analysis of GPVI pathway), and functional validation in transfected cell line models were employed. Key findings revealed that heterozygous variants impaired clotting signaling.

The reporting quality overall is nuanced, with an appreciated level of careful reservation. Given the rarity of the disorder studied and the limited number of patients recruited and samples, the substantial efforts to conduct an impressive number of molecular assessments on patients holding on to ethical guidelines indicate expertise. The study provides novel and valuable data on the investigated genetic variants.

Core publishing ethics must be upheld by methodology and data transparency, fair authorship attribution, and full disclosure of any conflicts of interest. Publishable data should suffice the stated research objectives and be free from any signs of manipulation. Methods should be written in a reproducible manner. On the reviewer’s part, respectful conduct throughout the review process will be maintained for trust and integrity.

Comments:

61-62. Saying "techniques have been shown to provide a molecular diagnosis in 25% to 70% of cases" is as unclear as "sometimes it works a little, sometimes it works a lot." Raise scientific rigor by a definite accuracy rate.

Add 459-461 information to the caption of Figure 4. Add 466-467 information to the caption of Figure 5. Add any "unfortunately" information to the corresponding Figure's caption to enhance comprehensibility.

Figure 4. P4 and P5 carrying GP6 variants are assessed comprehensively across all platelet characterization parameters (4F–I) except for PAC1 binding. However, data for P1–P3 with P2RY12 variants appear more variable (if not selective); P1 and P2, but not P3, were assessed for Fg binding (4B), and only P3 was assessed for PAC1 binding (4C). While sample availability is acknowledged as a limitation, consider addressing the inconsistency to reduce misleadingness.

Figure 5. Statistical analyses are shown for 5D and 5G, indicated by error bars. The remaining histograms appear without statistical representation, and p-values are not provided. While the variant expression data are quite informative, and the limited patient sample is understandable, consider addressing the inconsistency to merit reproducibility.

Figure 7. Since this is cell work to verify the defect of the variants, replicate numbers and statistical details should be included in the Figure caption, and statistical analyses should be conducted for all subfigures.

Author Response

Reviewer 2

This is a combined case series and molecular/functional characterization of four novel heterozygous variants in the P2RY12, GP6, and TBXAS1 genes, identified in six patients with bleeding and platelet disorders. High-Throughput Sequencing (HTS), comprehensive platelet phenotyping (aggregation, flow cytometry), molecular assessment (mRNA, protein expression, and novel downstream signaling analysis of GPVI pathway), and functional validation in transfected cell line models were employed. Key findings revealed that heterozygous variants impaired clotting signaling.

The reporting quality overall is nuanced, with an appreciated level of careful reservation. Given the rarity of the disorder studied and the limited number of patients recruited and samples, the substantial efforts to conduct an impressive number of molecular assessments on patients holding on to ethical guidelines indicate expertise. The study provides novel and valuable data on the investigated genetic variants.

Core publishing ethics must be upheld by methodology and data transparency, fair authorship attribution, and full disclosure of any conflicts of interest. Publishable data should suffice the stated research objectives and be free from any signs of manipulation. Methods should be written in a reproducible manner. On the reviewer’s part, respectful conduct throughout the review process will be maintained for trust and integrity.

 

Comments:

61-62. Saying "techniques have been shown to provide a molecular diagnosis in 25% to 70% of cases" is as unclear as "sometimes it works a little, sometimes it works a lot." Raise scientific rigor by a definite accuracy rate.

RESPONSE: We thank the reviewer for highlighting the imprecise information in this paragraph. 

To clarify, it now states (page 2):

“Sanger sequencing has long been the standard assay used to discern the candidate variants [9]. However, we were only able to achieve genetic diagnosis in 40% of cases suspected to have an inherited platelet disorder using this candidate gene approach [10]. The recent incorporation of high-throughput sequencing (HTS) has enhanced the genetic diagnosis of IPDs, with many specialised laboratories now implementing this approach [11,12]. Using this novel approach, we achieved a genetic diagnosis rate of 70% [12]. In the largest series of cases described so far, the diagnostic rate was 47.8% for inherited thrombocytopenia, 26.1% for platelet dysfunction and 63.6% for coagulation disorders [11]. Moreover, the advent of third generation sequencing techniques, such as Nanopore sequencing, allows long-read sequencing of DNA and characterization of structural var-iants (SVs) [13]”.

 

Add 459-461 information to the caption of Figure 4. Add 466-467 information to the caption of Figure 5. Add any "unfortunately" information to the corresponding Figure's caption to enhance comprehensibility.

RESPONSE: As requested by the reviewer, we have added more information to the captions. In addition, as indicated, we have added 'unfortunately' in cases where we were unable to obtain samples. Furthermore, we have adjusted the size of the axes on all graphs to improve visibility. Figure 4F, 5E, 6B, 6C and 6D have also been updated, as these experiments were performed with P5 and not P4. We apologise for this error.  In our opinion, these changes have improved all the figures.

 

Figure 4. P4 and P5 carrying GP6 variants are assessed comprehensively across all platelet characterization parameters (4F–I) except for PAC1 binding. However, data for P1–P3 with P2RY12 variants appear more variable (if not selective); P1 and P2, but not P3, were assessed for Fg binding (4B), and only P3 was assessed for PAC1 binding (4C). While sample availability is acknowledged as a limitation, consider addressing the inconsistency to reduce misleadingness.

RESPONSE: We appreciate the reviewer's comment. The activation of αIIbβ3 integrin by an agonist can be assessed by measuring fibrinogen binding or by using the PAC1 antibody, which recognises the integrin conformational change. Both methods are valid and are referenced in the literature. In our laboratory, fibrinogen binding is the preferred method (as was used for P1, P2, P4 and P5). However, in P3, fibrinogen binding could not be used due to technical issues on the day of blood sampling. Consequently, we used PAC1 binding to assess αIIbβ3 integrin and used the same agonist concentrations as in the fibrinogen binding assays to obtain comparable results.

 

Figure 5. Statistical analyses are shown for 5D and 5G, indicated by error bars. The remaining histograms appear without statistical representation, and p-values are not provided. While the variant expression data are quite informative, and the limited patient sample is understandable, consider addressing the inconsistency to merit reproducibility.

RESPONSE: Thank you for your comment. Two controls were included in the 5D-G experiments. However, as the reviewer points out, this can lead to confusion since the other experiments were carried out with only one control. For this reason, we have removed the information on the second control (previously labelled C4) and retained only C3. 

 

Figure 7. Since this is cell work to verify the defect of the variants, replicate numbers and statistical details should be included in the Figure caption, and statistical analyses should be conducted for all subfigures.

Response: We thank the reviewer for their comment. As requested, we have performed a one-way ANOVA on the data presented in Figures 7A, 7B and 7C. This information has been added to the 'Materials and Methods' section on page 10 (379), as well as to the figure caption. We have also included the replicate numbers in the figure caption.

Author Response File: Author Response.pdf