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  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2018/04/05

    Dear Dr. Liu:

    I am very pleased to inform you that your manuscript entitled ""Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP"" (CGE-01082-2017.R2), has been received, accepted, and will be forwarded for publication in Clinical Genetics.

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    Author Response
    2018/04/03

    Dear Prof. Veitia:

    Thank you very much for giving us an opportunity to revise the manuscript entitled “Variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP” (CGE-01082-2017.R1). We would like to express our sincere gratitude to the editors and reviewers for their positive and constructive comments, which are valuable for improving the quality of our manuscript.

    We have revised the manuscript according to the reviewers’ comments point by point, and the corrections have been shown in red in the revised manuscript. Besides, we select Open Peer Review.

    With the modifications and improvements, we hope that the revised manuscript will be acceptable for publication in the Journal of Clinical Genetics.

    Looking forward to hearing from you at your earliest convenience.

    Yours sincerely, Hongxing Liu



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  • pre-publication peer review (ROUND 2)
    Decision Letter
    2018/03/30

    Dear Dr. Liu:

    Thank you for submitting your manuscript entitled “Variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP ”. It has now been carefully reviewed.

    Your manuscript is interesting. The reviewers have recommended Acceptance subject to Minor Revisions. For details, please see the commentary below.

    In keeping with the policy of rapid publication, revised manuscripts should be re-submitted within four weeks of the date on this letter. Manuscripts received after this date may be treated as new submissions. We cannot guarantee acceptance after re-submission, and your revised manuscript will be subject to further review.

    When you send your revised manuscript, do not forget to include: Ø a cover letter which responds to the reviewers’ comments, point by point Ø if your manuscript includes clinical photographs, include a copy of written informed consent for publication. Ø Please indicate in the text where you have made amendments to meet referees' concerns. This may be either in the form of 'Track Changes' or of differently coloured text.

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    If you feel that your paper could benefit from English language polishing, you may wish to consider having your paper professionally edited for English language by a service such as Wiley’s at http://wileyeditingservices.com. Please note that while this service will greatly improve the readability of your paper, it does not guarantee acceptance of your paper by the journal.

    Thank you for considering Clinical Genetics for the publication of your research. I look forward to hearing from you soon.

    Sincerely,

    Prof. Reiner A. Veitia, Ph.D. Acad. Europaea, French National Acad. Med. Editor-In-Chief, Clinical Genetics

    Reviewer: 1

    Comments to the Author The authors have convincingly addressed all the reviewers concerns and improved the quality of the manuscript. Manuscript provides a detailed genetic report of 265 FHL patients, which is one the largest and most complete one, and identifies racial/geographic genetic variation in FHL patients.

    Reviewer: 2

    Comments to the Author This manuscript describes the results from genetic analysis of 6 genes causative of hemophagocytic lymphohistiocytosis (HLH) in 265 patients of Chinese origin. Considering the large number of patients studied and the fact that 6 genes were tested in all patients, this manuscript provides a good overview of the genetic architecture of primary HLH in China, which might help future diagnostics of patients from this area.

    The authors have revised the manuscript and addressed most of this reviewer's comments. Additional comments: - gene names should be in italics - the word ""mutation"" was changed to ""variant"" as previously request, but since the authors do not use many modifiers (such as pathogenic, of unclear significance, rare, disease-causing, etc.), the term ""variant"" remains a bit vague and unclear in several occasions in the manuscript. The authors should review the text and add relevant modifiers to the Word ""variant"" to increase text readability. - ACMG classification: the fact that variants were classified according to ACMG should be added to the methods, inclusing a relevant reference. - Moreover, authors should clear state in the results section how many pathogenic/likely pathogenic or unclear variants were identify. Ideally, only patients with pathogenic/likely pathogenic biallelic variants should be considered as diagnosed with HLH. - in figure 3, variants of unclear significance should be distinguish from likely pathogenic/pathogenic variants according to the ACMG classification.

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    Reviewer report
    2018/03/29

    This manuscript describes the results from genetic analysis of 6 genes causative of hemophagocytic lymphohistiocytosis (HLH) in 265 patients of Chinese origin. Considering the large number of patients studied and the fact that 6 genes were tested in all patients, this manuscript provides a good overview of the genetic architecture of primary HLH in China, which might help future diagnostics of patients from this area.

    The authors have revised the manuscript and addressed most of this reviewer's comments. Additional comments: - gene names should be in italics - the word "mutation" was changed to "variant" as previously request, but since the authors do not use many modifiers (such as pathogenic, of unclear significance, rare, disease-causing, etc.), the term "variant" remains a bit vague and unclear in several occasions in the manuscript. The authors should review the text and add relevant modifiers to the Word "variant" to increase text readability. - ACMG classification: the fact that variants were classified according to ACMG should be added to the methods, inclusing a relevant reference. - Moreover, authors should clear state in the results section how many pathogenic/likely pathogenic or unclear variants were identify. Ideally, only patients with pathogenic/likely pathogenic biallelic variants should be considered as diagnosed with HLH. - in figure 3, variants of unclear significance should be distinguish from likely pathogenic/pathogenic variants according to the ACMG classification.

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    Reviewer report
    2018/03/25

    The authors have convincingly addressed all the reviewers concerns and improved the quality of the manuscript. Manuscript provides a detailed genetic report of 265 FHL patients, which is one the largest and most complete one, and identifies racial/geographic genetic variation in FHL patients.

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    Author Response
    2018/03/10

    Dear Prof. Veitia:

    Thank you very much for giving us an opportunity to revise the manuscript entitled “Mutation spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP” (CGE-01082-2017). We would like to express our sincere gratitude to the editors and reviewers for their positive and constructive comments, which are valuable for improving the quality of our manuscript.

    We have revised the manuscript according to the reviewers’ comments point by point, and the corrections have been shown in red in the revised manuscript. Besides, in the process of modifying the manuscript, we find the c.497C>T (p.T166M) variant in STXBP2 gene which was reported in our manuscript before has an allele frequency >1% in the updated 1000 Genomes Project and gnomAD databases. Therefore, this variant is now excluded in the revised manuscript and the corresponding data, figures and tables have all been modified.

    With the modifications and improvements, we hope that the revised manuscript will be acceptable for publication in the Journal of Clinical Genetics.

    Looking forward to hearing from you at your earliest convenience.

    Yours sincerely, Hongxing Liu

    Replies to the reviewers’ comments

    Reviewer #1: Comments 1:

    The authors in this manuscript describe the frequency of mutations in known genes associated with Familial HLH in a cohort of patients from China and it compares these frequencies with previous studies target to cohorts of patients from Europe, Japan and Turkey. In particular, they targeted the sequencing to the following genes: UN13-D, STXBP2, Perforin, STX11, XIAP and SH2D1A. They found geographic difference with previous studies, showing that UNC13-D is more frequently observed than Perforin. Surprisingly they do not find any mutation in the known genes in 66% of the patients. These population of patients would be very important to perform whole exome sequencing in order to find any new gene linked to these disorders. Response: Thanks for your good suggestion. Indeed, as the only cure for primary HLH is hematopoietic stem cell transplantation, it is very important to reveal the underlying genetic factors and obtain a molecular diagnosis in HLH patients. For this reason, we are planning to perform whole exome sequencing (WES) in some cases who are negative of variants in the present study. The rapid development of sequencing technology and the decline of sequencing cost have made WES much easier to achieve and we are looking forward to find some new pathogenesis of HLH.

    Comments 2 The novelty of these studies is incremental but the do found new mutations in the know genes that would of general interest for the audience of this journal. Authors would need to provide additional tables describing more the mutations found in Unc13-D and STXBP2, Perforin and STX11 as they did for XIAP: showing the sex/ age of diagnostic, and whether mutation is heterozygous or homozygous. Response: We have added a table (Table 2) in our revised manuscript which listed the results of gene sequencing in all patients with variants, including their sex, age at diagnosis, the zygosity and ACMG classification of their variants. The frequencies of variants in population databases (1000 Genomes Project, ExAC, gnomAD) and references in which the variants have been reported are also shown.

    Comments 3 Also the author could expand table 2 showing how were the clinical labs for each individual population of patient (UNC13-D; Perforin, STXBP2, XIAP/SH2D1A, STX11). These will provide more information about the severity, similarities and differences in clinical lab for the different populations. Response: The reviewer might refer to “table 1” here. In our revised manuscript, the age distribution and clinical manifestations and laboratory findings of patients are listed. It contains not only the whole cohort (n=265), but also patient groups including patients with no variants (n=178), patients with variants (n=87), patients with only UNC13D variants (n=36), patients with only PRF1 variants (n=18), patients with only SH2D1A/XIAP variants (n=16), patients with only STXBP2 variants (n=9), patients with only STX11 variants (n=1), and patients with digenic variants (n=7).

    Reviewer #2: Comments to the Author This manuscript describes the results from genetic analysis of 6 genes causative of hemophagocytic lymphohistiocytosis (HLH) in 265 patients of Chinese origin. Considering the large number of patients studied and the fact that 6 genes were tested in all patients, this manuscript provides a good overview of the genetic architecture of primary HLH in China, which might help future diagnostics of patients from this area. Response: We very much appreciate your positive and valuable comments on the scientific merit of our manuscript.

    Major comments: A. A table including all the variants identified should be provided. The table should include HGVS nomenclature, frequency in ExAC/gnomAD, ACMG classification (see comment below) and should indicate whether the variant has been previously reported in FHL. Response: We have added a table (Table 2) in our revised manuscript which listed the results of gene sequencing in all patients with variants (n=87), including their sex, age at diagnosis, the HGVS nomenclature, zygosity and ACMG classification of their variants. The frequencies of variants in population databases (1000 Genomes Project, ExAC, gnomAD) and references in which the variants have been reported in FHL are also shown.

    B. The authors state in the methods that a frequency of 1% was used as cut-off for separating SNPs from “mutations”. It is arguably whether variants with a frequency close to 1% can be considered rare enough to be pathogenic. Hence the importance to present frequency data for all variants reported. Response: Indeed, assessing the frequency of a variant in a control or general population is useful in assessing its potential pathogenicity. For example, absent from controls (or at extremely low frequency if recessive) in 1000 Genomes Project or ExAC is a moderate evidence of pathogenicity, while allele frequency >5% in 1000 Genomes Project or ExAC is a stand-alone evidence of benign impact1. Therefore, the frequency data in population databases (1000 Genomes Project, ExAC, gnomAD) for all variants detected in the present study are listed in Table 2 in the revised manuscript.

    C. The authors should avoid the term “mutation” and refer instead to “variants” which can be described as “pathogenic”, “likely pathogenic” and “of uncertain significance”. The reported variants should be classified according to ACMG recommendations in order to facilitate their clinical interpretation in case they would be found in additional patients once this paper will be. ACMG classification should be included in the table with all variants (see first comment). Response: Thanks for your suggestion. The terms “mutation” and “polymorphism” have been replaced by the term “variant” with some modifiers (pathogenic, likely pathogenic, uncertain significance, likely benign, or benign) in the revised manuscript. And the ACMG classification for all variants reported in the present study has been listed in Table 2 in our revised manuscript.

    D. Familial HLH caused by mutations in PRF1, UNC13D, STX11, and STXBP2 is traditionally autosomal recessive. The reviewer is aware of reports of monoallelic variants in patients with secondary HLH, but there is not yet enough evidence to define cases with monoallelic variants as primary HLH. The authors should therefore report the patients with biallelic pathogenic variants separately from patients with only monoallelic variants. The % of cases with primary HLH should only include cases with biallelic variants. Authors should also modify their figures accordingly. Response: We are sorry that we did not modify our manuscript according to this suggestion. Our reasons are as follows and relevant content are stated in the Discussion of the revised manuscript: 1. We did not emphasize primary HLH in this study because the distinction between primary and secondary forms of HLH is becoming blurred. Severity of disease and the identification of an infectious agent do not discriminate between genetic and acquired forms of HLH. Age is helpful to some extent, but a minority of children <1 year of age have acquired HLH, and also older age does not reliably exclude genetic HLH. Impaired NK cell cytotoxicity is a characteristic finding in FHL, however, normal activity does not exclude either. Moreover, decreased function of NK cells has been also observed in patients with acquired HLH2. 2. Although most FHL patients harbor biallelic germline mutations, some individuals harbor only a single mutant allele. In these latter individuals, it is unclear whether and how heterozygous mutations lead to disease. Consequently, these patients are often classified as having “secondary” HLH. However, such a classification is problematic because the therapeutic approaches required for primary and secondary HLH are widely divergent, with primary HLH requiring treatment with chemoimmunotherapeutic agents followed by allogeneic hematopoietic stem cell transplantation, and secondary HLH requiring a much less intensive approach or even no therapy3. 3. There is growing evidence that monoallelic variants can also contribute to HLH. For example, Spessott et al.3 reported that the R65W or R65Q variant in STXBP2 act as dominant negatives that interfere with the release of lytic granules and thus lead to FHL-5. 4. Some patients with FHL have been found to carry a monoallelic variant in one or more HLH-associated genes4,5. These observations suggest the possibility that other types of variants (eg, gross insertions/deletions, complex rearrangements, variants in noncoding regions) in the other “normal” allele are not able to be detected by normal sequencing of the exons and exon/intron boundaries or that these patients possess variants in additional genes that contribute to the development of HLH. The discovery of deep intronic variants (c.118-308C>T and c.118-307G>A) and 253-kb inversion straddling UNC13D in FHL patients who have monoallelic variants in UNC13D, and the digenic mode of inheritance of FHL suggested by Zhang et al. gradually confirm the above speculations, and provide new ideas for the study of genetic factors in the development of HLH5-7.

    E. The authors report in several occasions that they found variants with “synergistic” effects affecting two different genes. No functional data is presented to support this statement. The authors should rephrase their statements and modify their figures accordingly. Response: Indeed, no functional data is presented to support the “synergistic” effects of two distinct genes in the present study. So we have descripted patients who have variants in two different genes as patients with “digenic variants” in the revised manuscript. The corresponding figures and tables have also been modified.

    F. It would be of interest to see diagnostic rates according to age at diagnosis/onset of HLH. Response: We have divided our patients into 5 groups according to age at diagnosis/onset of HLH (0-1y, 1-5y, 5-12y, 12-18y, >18y). In the whole cohort (n=265), 37 patients are detected to have homozygous or hemizygous or compound heterozygous variants (13.96%). The percentage of patients who have homozygous or hemizygous or compound heterozygous variants in the above 5 age groups are 13.33% (12/90), 15.91% (14/88), 16.33% (8/49), 6.25% (1/16) and 9.09% (2/22), respectively. The diagnostic rate is higher in children 12 years of age or younger than that in patients older than 12 years of age, but the difference is not statistically significant (14.98% vs. 7.89%, P = 0.317). The corresponding content has been added in Materials and methods and Results in the revised manuscript.

    G. Although the authors state that patients were diagnosed according to HLH-2004 criteria, functional assays such as NK cell cytotoxicity and NK cell degranulation assays are not commented. “NK cell activity < 14%” is mentioned in table 1, but no information is provided concerning the method used. Were functional assays performed? How? Data from functional assays would be extremely useful to score variants of uncertain significance and understand the impact of monoallelic variants. Response: Yes, functional assays were performed in this study. We measured NK cell cytotoxicity using flow cytometric method as described by Wu et al.8. The plasmid pEGFP-N1 was transfected into K562 cells. After scanned with G4l8 and monoclone, the EGFP-K562 cell line stably expressing enhanced green fluorescent protein was obtained. PBMNC and EGFP-K562 were mixed at the effector to target ratio of 10:1. After incubation of 2 hours, propidium iodine (PI) was added to stain dead cells, and then cytotoxic activity was analyzed using flow cytometry. Values >14% were considered to be normal. As suggested by the reviewer, the methodology we used to detect NK cell cytotoxicity has been stated in Materials and methods in the revised manuscript.

    References 1. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405-424. 2. Janka GE, Lehmberg K. Hemophagocytic lymphohistiocytosis: pathogenesis and treatment. Hematology Am Soc Hematol Educ Program. 2013;2013:605-611. 3. Spessott WA, Sanmillan ML, McCormick ME, et al. Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion. Blood. 2015;125:1566-1577. 4. Zhang K, Jordan MB, Marsh RA, et al. Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH. Blood. 2011;118:5794-5798. 5. Zhang K, Chandrakasan S, Chapman H, et al. Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis. Blood. 2014;124:1331- 1334. 6. Entesarian M, Chiang SC, Schlums H, et al. Novel deep intronic and missense UNC13D mutations in familial haemophagocytic lymphohistiocytosis type 3. Br J Haematol. 2013;162:415-418. 7. Meeths M, Chiang SC, Wood SM, et al. Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D. Blood. 2011;118:5783-5793. 8. Wu L, Wang Z, Chen X, Wang YN, Wang JS. [Application of measuring human peripheral NK cell activity with flow cytometry in diagnosis for hemophagocytic lymphohistiocytosis]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2009;17:1497-1501.



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  • pre-publication peer review (ROUND 1)
    Decision Letter
    2018/02/12

    Dear Dr. Liu:

    Thank you for submitting your manuscript entitled “Mutation spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP ”. It has now been carefully reviewed.

    The reviewers have suggested Major revisions. For details, please see the commentary below.

    In keeping with the policy of rapid publication, revised manuscripts should be re-submitted within four weeks of the date on this letter. Manuscripts received after this date may be treated as new submissions. We cannot guarantee acceptance after re-submission, and your revised manuscript will be subject to further review.

    When you send your revised manuscript, do not forget to include: Ø a cover letter which responds to the reviewers’ comments, point by point Ø if your manuscript includes clinical photographs, include a copy of written informed consent for publication. Ø Please indicate in the text where you have made amendments to meet referees' concerns. This may be either in the form of 'Track Changes' or of differently coloured text.

    Clinical Genetics allows authors the option to select Open Peer Review. Where authors select Open Peer Review referee reports as well as author responses will be made available to readers of Clinical Genetics. Should you wish to select this option please state this in your cover letter when you submit your revised manuscript.

    Manuscripts must be within the specified word count and in the correct format before acceptance. Please ensure that the manuscript is formatted correctly according to Clinical Genetics' Author Guidelines at http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1399-0004/homepage/ForAuthors.html

    Word count limits are:

    Original Article: Manuscripts can be up to 6,000 words in length (including title page, abstract, references, figure legends and Acknowledgements) and can include up to seven figures and/or tables.

    Short Report: They should not exceed 2500 words in length (including title page, abstract, references, figure legends and Acknowledgements) and can include up to three figures and/or tables.

    Letter to the Editor: Letters can be up to 900 words in length (including title page, references, a figure legend and Acknowledgements) and can include one display item (figure or table) and five references. Letters are not subdivided into sections nor do they include an abstract or online supplements. They can include for-review-only supplements.

    Correspondence Word limit: 300 words maximum

    Mini-reviews and Reviews: Manuscripts can be up to 3,000 words in length for Mini-reviews and 7,500 words for Reviews, including title page, abstract, references, figure legends and Acknowledgements. A maximum of 4 or 7 display items (figures and/or tables) can be included for Mini-reviews and Reviews, respectively.

    If you feel that your paper could benefit from English language polishing, you may wish to consider having your paper professionally edited for English language by a service such as Wiley’s at http://wileyeditingservices.com. Please note that while this service will greatly improve the readability of your paper, it does not guarantee acceptance of your paper by the journal.

    Thank you for considering Clinical Genetics for the publication of your research. I look forward to hearing from you soon.

    Sincerely,

    Prof. Reiner A. Veitia, Ph.D. Acad. Europaea, French National Acad. Med. Editor-In-Chief, Clinical Genetics

    Reviewer: 1

    Comments to the Author The authors in this manuscript describe the frequency of mutations in known genes associated with Familial HLH in a cohort of patients from china and it compares these frequencies with previous studies target to cohorts of patients from Europe, Japan and Turkey. In particular they targeted the sequencing to the following genes: UN13-D, STXBP2, Perforin, STX11, XIAP and SH2D1A. They found geographic difference with previous studies, showing that UNC13-D is more frequently observed than Perforin. Surprisingly they do not find any mutation in the known genes in 66% of the patients. These population of patients would be very important to perform whole exome sequencing in order to find any new gene linked to these disorders. The novelty of these studies is incremental but the do found new mutations in the know genes that would of general interest for the audience of this journal. Authors would need to provide additional tables describing more the mutations found in Unc13-D and STXBP2, Perforin and STX11 as they did for XIAP : showing the sex/ age of diagnostic, and whether mutation is heterozygous or homozygous. Also the author could expand table 2 showing how were the clinical labs for each individual population of patient ( UNC-13-D; Perforin, STXBP2, XIAP/SH2D1A, STX11). These will provide more information about the severity, similarities and differences in clinical lab for the different populations.

    Reviewer: 2

    Comments to the Author Review of CGE-01082-2017 Mutation spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP

    This manuscript describes the results from genetic analysis of 6 genes causative of hemophagocytic lymphohistiocytosis (HLH) in 265 patients of Chinese origin. Considering the large number of patients studied and the fact that 6 genes were tested in all patients, this manuscript provides a good overview of the genetic architecture of primary HLH in China, which might help future diagnostics of patients from this area.

    Major comments:

    • A table including all the variants identified should be provided. The table should include HGVS nomenclature, frequency in ExAC/gnomAD, ACMG classification (see comment below) and should indicate whether the variant has been previously reported in FHL. • The authors state in the methods that a frequency of 1% was used as cut-off for separating SNPs from “mutations”. It is arguably whether variants with a frequency close to 1% can be considered rare enough to be pathogenic. Hence the importance to present frequency data for all variants reported. • The authors should avoid the term “mutation” and refer instead to “variants” which can be described as “pathogenic”, “likely pathogenic” and “of uncertain significance”. The reported variants should be classified according to ACMG recommendations in order to facilitate their clinical interpretation in case they would be found in additional patients once this paper will be. ACMG classification should be included in the table with all variants (see first comment). • Familial HLH caused by mutations in PRF1, UNC13D, STX11, and STXBP2 is traditionally autosomal recessive. The reviewer is aware of reports of monoallelic variants in patients with secondary HLH, but there is not yet enough evidence to define cases with monoallelic variants as primary HLH. The authors should therefore report the patients with biallelic pathogenic variants separately from patients with only monoallelic variants. The % of cases with primary HLH should only include cases with biallelic variants. Authors should also modify their figures accordingly. • The authors report in several occasions that they found variants with “synergistic” effects affecting two different genes. No functional data is presented to support this statement. The authors should rephrase their statements and modify their figures accordingly. • It would be of interest to see diagnostic rates according to age at diagnosis/onset of HLH. • Although the authors state that patients were diagnosed according to HLH-2004 criteria, functional assays such as NK cell cytotoxicity and NK cell degranulation assays are not commented. “NK cell activity < 14%” is mentioned in table 1, but no information is provided concerning the method used. Were functional assays performed? How? Data from functional assays would be extremely useful to score variants of uncertain significance and understand the impact of monoallelic variants.

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    Reviewer report
    2018/02/06

    Review of CGE-01082-2017 Mutation spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP

    This manuscript describes the results from genetic analysis of 6 genes causative of hemophagocytic lymphohistiocytosis (HLH) in 265 patients of Chinese origin. Considering the large number of patients studied and the fact that 6 genes were tested in all patients, this manuscript provides a good overview of the genetic architecture of primary HLH in China, which might help future diagnostics of patients from this area.

    Major comments:

    • A table including all the variants identified should be provided. The table should include HGVS nomenclature, frequency in ExAC/gnomAD, ACMG classification (see comment below) and should indicate whether the variant has been previously reported in FHL. • The authors state in the methods that a frequency of 1% was used as cut-off for separating SNPs from “mutations”. It is arguably whether variants with a frequency close to 1% can be considered rare enough to be pathogenic. Hence the importance to present frequency data for all variants reported. • The authors should avoid the term “mutation” and refer instead to “variants” which can be described as “pathogenic”, “likely pathogenic” and “of uncertain significance”. The reported variants should be classified according to ACMG recommendations in order to facilitate their clinical interpretation in case they would be found in additional patients once this paper will be. ACMG classification should be included in the table with all variants (see first comment). • Familial HLH caused by mutations in PRF1, UNC13D, STX11, and STXBP2 is traditionally autosomal recessive. The reviewer is aware of reports of monoallelic variants in patients with secondary HLH, but there is not yet enough evidence to define cases with monoallelic variants as primary HLH. The authors should therefore report the patients with biallelic pathogenic variants separately from patients with only monoallelic variants. The % of cases with primary HLH should only include cases with biallelic variants. Authors should also modify their figures accordingly. • The authors report in several occasions that they found variants with “synergistic” effects affecting two different genes. No functional data is presented to support this statement. The authors should rephrase their statements and modify their figures accordingly. • It would be of interest to see diagnostic rates according to age at diagnosis/onset of HLH. • Although the authors state that patients were diagnosed according to HLH-2004 criteria, functional assays such as NK cell cytotoxicity and NK cell degranulation assays are not commented. “NK cell activity < 14%” is mentioned in table 1, but no information is provided concerning the method used. Were functional assays performed? How? Data from functional assays would be extremely useful to score variants of uncertain significance and understand the impact of monoallelic variants.

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    Reviewer report
    2018/01/23

    The authors in this manuscript describe the frequency of mutations in known genes associated with Familial HLH in a cohort of patients from china and it compares these frequencies with previous studies target to cohorts of patients from Europe, Japan and Turkey. In particular they targeted the sequencing to the following genes: UN13-D, STXBP2, Perforin, STX11, XIAP and SH2D1A. They found geographic difference with previous studies, showing that UNC13-D is more frequently observed than Perforin. Surprisingly they do not find any mutation in the known genes in 66% of the patients. These population of patients would be very important to perform whole exome sequencing in order to find any new gene linked to these disorders. The novelty of these studies is incremental but the do found new mutations in the know genes that would of general interest for the audience of this journal. Authors would need to provide additional tables describing more the mutations found in Unc13-D and STXBP2, Perforin and STX11 as they did for XIAP : showing the sex/ age of diagnostic, and whether mutation is heterozygous or homozygous. Also the author could expand table 2 showing how were the clinical labs for each individual population of patient ( UNC-13-D; Perforin, STXBP2, XIAP/SH2D1A, STX11). These will provide more information about the severity, similarities and differences in clinical lab for the different populations.

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