Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Assessment

This manuscript investigates the heritability and transcriptional impact of rare germline JAK3, STAT5A, and STAT6 variants within a single extended Tyrolean family. By integrating WES/WGS, RNA-seq, in silico pathogenicity prediction, and AlphaFold3 structural modeling, the study addresses an important and underexplored question: how rare germline JAK/STAT variants act as genetic modifiers rather than deterministic drivers of immune phenotypes.

The work is conceptually sound, technically solid, and well aligned with recent literature on variant penetrance, epistasis, and immune transcriptome variability. The family-based design is a major strength, as is the integration of baseline and post–SARS-CoV-2 immune responses. However, the study remains largely descriptive, and several interpretations would benefit from clearer framing, stronger statistical justification, and more cautious causal language. The manuscript is publishable after moderate revision.

 

Major Comments

• Novelty and Conceptual Contribution (Clarify More Explicitly)

While the manuscript is interesting, the novel conceptual advance should be more explicitly articulated in the Introduction and Discussion. The study reiterates that rare variants may act as modifiers, but this concept is already discussed in prior work by the same group. The unique contribution of this family (beyond replication) is not sufficiently emphasized.

Recommendations

Clearly state what is new:

• Multigenerational inheritance of multiple co-occurring JAK/STAT variants
• Evidence for context-dependent immune transcriptome activation
• Integration of AF3 structural neutrality vs. transcriptomic impact

Add a short paragraph in the Introduction outlining why family-based combinatorial variant analysis is uniquely informative compared to population cohorts.

• Interpretation of Transcriptomic Findings (Avoid Overinterpretation)

The association between variants and enhanced basal or virus-induced transcriptomes is intriguing but should be interpreted more cautiously.

Recommendations

Consider adding a pathway-level summary (e.g., interferon, cytokine signaling) instead of relying mainly on gene-level volcano plots.

• TYK2

TYK2 variants appear repeatedly but remain underdeveloped analytically. TYK2 variants are acknowledged as confounders but not analyzed systematically.This weakens causal interpretation of JAK/STAT effects.

Recommendations

Either:

• Include a brief TYK2-focused supplementary analysis, or
• More explicitly frame the study as an epistatic landscape analysis, not a JAK/STAT-only study

Consider adding a schematic showing gene-variant interaction complexity.

• Conclusions:

The conclusions are strong but slightly overstated given the dataset size.

Recommendations

Replace deterministic language (“demonstrates”, “indicates”) with probabilistic language (“suggests”, “supports”).

Minor Comments

• Abstract

Sentence “The presence of activated basal transcriptomes was limited to some, but not all, individuals carrying the above variants above” → remove redundancy (“above” twice).

• Methods

Correct “Whole genome sequencing” section title if WES and WGS are both used.

Editorial and Language Suggestions

Overall English quality is high. Minor points:

• “Identifcation” → “Identification” (Figure 1)
• “be explained be age differences” → “be explained by age differences”
• “his study demonstrates” → “This study demonstrates”

 

Final Recommendation

Decision: Minor to Moderate Revision

This is a well-designed, timely, and methodologically sound manuscript that fits the scope of International Journal of Molecular Sciences. With clearer framing of novelty, more cautious interpretation of transcriptomic associations, and minor clarifications, it will make a valuable contribution to the literature on rare germline immune variants and epistasis.

Author Response

This manuscript investigates the heritability and transcriptional impact of rare germline JAK3, STAT5A, and STAT6 variants within a single extended Tyrolean family. By integrating WES/WGS, RNA-seq, in silico pathogenicity prediction, and AlphaFold3 structural modeling, the study addresses an important and underexplored question: how rare germline JAK/STAT variants act as genetic modifiers rather than deterministic drivers of immune phenotypes.

 

The work is conceptually sound, technically solid, and well aligned with recent literature on variant penetrance, epistasis, and immune transcriptome variability. The family-based design is a major strength, as is the integration of baseline and post–SARS-CoV-2 immune responses. However, the study remains largely descriptive, and several interpretations would benefit from clearer framing, stronger statistical justification, and more cautious causal language. The manuscript is publishable after moderate revision.

 

Response

We thank the reviewer for the positive assessment.

 

Major Comments

1. Novelty and Conceptual Contribution (Clarify More Explicitly)

While the manuscript is interesting, the novel conceptual advance should be more explicitly articulated in the Introduction and Discussion. The study reiterates that rare variants may act as modifiers, but this concept is already discussed in prior work by the same group. The unique contribution of this family (beyond replication) is not sufficiently emphasized.

 

Response

We have now expanded the Introduction and Discussion and emphasized the unique contributions of our study (lines 51-59, 62-75, 279-281). Specifically, we further emphasized the combinatorial arrangements of JAK/STAT variants in the extended family and current knowledge of these variants.

 

2. Clearly state what is new:

• Multigenerational inheritance of multiple co-occurring JAK/STAT variants
• Evidence for context-dependent immune transcriptome activation
• Integration of AF3 structural neutrality vs. transcriptomic impact

Add a short paragraph in the Introduction outlining why family-based combinatorial variant analysis is uniquely informative compared to population cohorts.

• Interpretation of Transcriptomic Findings (Avoid Overinterpretation)

The association between variants and enhanced basal or virus-induced transcriptomes is intriguing but should be interpreted more cautiously.

 

Response

We have added detailed information to the introduction, addressing the points raised by the reviewer (lines 62-75).

 

3. Consider adding a pathway-level summary (e.g., interferon, cytokine signaling) instead of relying mainly on gene-level volcano plots.

 

Response

We have conducted additional GSEA pathway analyses that are now included in Figures 3B and 4B as well as Table S3 and S4 (lines 197-200, 212-215).

 

4. TYK2

TYK2 variants appear repeatedly but remain underdeveloped analytically. TYK2 variants are acknowledged as confounders but not analyzed systematically. This weakens causal interpretation of JAK/STAT effects.

 

Either:

• Include a brief TYK2-focused supplementary analysis, or
• More explicitly frame the study as an epistatic landscape analysis, not a JAK/STAT-only study

Consider adding a schematic showing gene-variant interaction complexity.

 

Response

We provided additional details about the four TYK2 variants identified in our cohort (lines 108-112 and 121-130). No epistatic interactions were detected between the JAK/STAT variants identified in this study and other immune-related genes; epistasis analyses of the JAK/STAT variants are summarized in Table S2.

 

• Conclusions:

The conclusions are strong but slightly overstated given the dataset size.

 

Response

We agree with the reviewer that the dataset size is small, and we toned down the conclusions. We would like to emphasize that due the rarity of these variants and the large number of possible combinations it is unlikely that additional individuals carrying specific combinations observed in our study cohort can be found.

 

5. Replace deterministic language (“demonstrates”, “indicates”) with probabilistic language (“suggests”, “supports”).

 

Response

When appropriate and justified we modified the deterministic language.

 

Minor Comments

• Abstract

Sentence “The presence of activated basal transcriptomes was limited to some, but not all, individuals carrying the above variants above” → remove redundancy (“above” twice).

 

• Methods

Correct “Whole genome sequencing” section title if WES and WGS are both used.

 

• Editorial and Language Suggestions

 

• Overall English quality is high. Minor points:

“Identifcation” → “Identification” (Figure 1)

“be explained be age differences” → “be explained by age differences”

“his study demonstrates” → “This study demonstrates”

 

Response

We addressed these points.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

I read the work entitled “Heritability and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family” with interest. The manuscript submitted for review presents a family-based genetic and transcriptomic study of rare germline variants in the JAK3, STAT5A, and STAT6 genes, combining inheritance analysis, in silico pathogenicity prediction, AlphaFold3 structural modelling, and RNA sequencing of PBMCs collected at baseline and after SARS-CoV-2 Omicron infection. The study is technically and methodologically sound and addresses an important issue in population genetics: the context-dependent effects and incomplete penetrance of rare genetic variants in immune pathways. Overall, the received data are of good quality, and the analyses are appropriate to this study. However, several aspects of interpretation and presentation should be revised to ensure that the conclusions remain fully supported by the available evidence.

1. In the manuscript, there was no clear answer as to why this particular family was chosen for analysis. From the provided information, the family was initially recruited into a broader SARS-CoV-2 cohort, and an enriched-for-rare-JAK/STAT variant was identified post hoc through NGS analysis. There is no indication that the family was selected due to severe COVID-19 or any adverse events after vaccination. Explicitly stating this in the manuscript would improve transparency and help readers avoid assumptions of phenotype-driven selection.
2. The RNA-seq analysis is based on a small number of individuals per genotype variant compilation, often just a single individual. Although this is an inherent family-based feature of rare variants, it limits statistical power. In molecular genetics, single-individual RNA-seq comparisons, even when visualized in volcano plots, do not constitute statistically robust differential expression analyses. While the authors acknowledge this limitation, the Results and Discussion sometimes treat these patterns as biologically decisive.
3. Most family members carry additional variants in TYK2, whose product belongs to the same functional signal pathway as JAK and STAT. Although acknowledged, the discussion occasionally suggests combinatorial or synergistic effects that cannot be conclusively demonstrated. From a molecular and population genetics perspective, these observations should be consistently framed as context-dependent associations compatible with incomplete penetrance and modifier-gene effects.
4. The study depends on in silico predictions, structural modelling, and transcriptomic associations. These methods cannot determine gain- or loss-of-function effects. Therefore, any statements suggesting functional consequences should be tempered, and the lack of functional assays should be more clearly highlighted as a limitation.
5. Figures 3 and 4 contain multiple small volcano plots corresponding to different individuals and conditions. The current layout is visually dense and somewhat challenging to interpret, particularly for readers unfamiliar with the study design. Simplifying the layout and enlarging labels would substantially improve readability.

Minor points:

1. The term “activated basal immune transcriptome” should be more clearly defined.
2. Potential age- and sex-related effects on immune transcriptomes should be acknowledged, even if underpowered.

Recommendation: Major revision.

This recommendation reflects the need to constrain interpretation further, clarify family selection, and improve figure readability, rather than any fundamental methodological shortcomings. With these revisions, the manuscript would represent a solid and credible contribution to the literature on rare germline immune variants and genetic context effects.

Author Response

I read the work entitled “Heritability and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family” with interest. The manuscript submitted for review presents a family-based genetic and transcriptomic study of rare germline variants in the JAK3, STAT5A, and STAT6 genes, combining inheritance analysis, in silico pathogenicity prediction, AlphaFold3 structural modelling, and RNA sequencing of PBMCs collected at baseline and after SARS-CoV-2 Omicron infection. The study is technically and methodologically sound and addresses an important issue in population genetics: the context-dependent effects and incomplete penetrance of rare genetic variants in immune pathways. Overall, the received data are of good quality, and the analyses are appropriate to this study. However, several aspects of interpretation and presentation should be revised to ensure that the conclusions remain fully supported by the available evidence.

 

1. In the manuscript, there was no clear answer as to why this particular family was chosen for analysis. From the provided information, the family was initially recruited into a broader SARS-CoV-2 cohort, and an enriched-for-rare-JAK/STATvariant was identified post hoc through NGS analysis. There is no indication that the family was selected due to severe COVID-19 or any adverse events after vaccination. Explicitly stating this in the manuscript would improve transparency and help readers avoid assumptions of phenotype-driven selection.

 

Response

We now provided a clear rationale why this family was chosen for our study (lines 81-91).

 

2. The RNA-seq analysis is based on a small number of individuals per genotype variant compilation, often just a single individual. Although this is an inherent family-based feature of rare variants, it limits statistical power. In molecular genetics, single-individual RNA-seq comparisons, even when visualized in volcano plots, do not constitute statistically robust differential expression analyses. While the authors acknowledge this limitation, the Results and Discussion sometimes treat these patterns as biologically decisive.

 

Response

The reviewer addresses a very important issue, the small sample number of individuals carrying various combinations of JAK/STAT variants. The low frequency of these variants makes it difficult, if not impossible, to identify additional individuals displaying these variant combinations. We highlighted this challenge in the Discussion (lines 272-277). Specifically, this complexity is further expanded by including variants in TYK2 and additional proteins acting upstream of JAK/STAT, such as receptors, and downstream, such as SOCS proteins. We discussed this throughout the manuscript.

 

3. Most family members carry additional variants in TYK2, whose product belongs to the same functional signal pathway as JAK and STAT. Although acknowledged, the discussion occasionally suggests combinatorial or synergistic effects that cannot be conclusively demonstrated. From a molecular and population genetics perspective, these observations should be consistently framed as context-dependent associations compatible with incomplete penetrance and modifier-gene effects.

 

Response

We completely agree with the reviewer, and we included this concept in the Discussion (lines 246-249).

 

4. The study depends on in silicopredictions, structural modelling, and transcriptomic associations. These methods cannot determine gain- or loss-of-function effects. Therefore, any statements suggesting functional consequences should be tempered, and the lack of functional assays should be more clearly highlighted as a limitation.

 

Response

We concur with the reviewer and added the following sentence to the limitation section: The study depends on in silico predictions, structural modelling, and transcriptomic associations. These methods by themselves cannot determine gain- or loss-of-function effects. Functional in vitro assays could provide some biological information on individual JAK/STAT variants but would not yield significant information on the combinatorial impact of several co-inherited variants. (lines 382-389).

 

5. Figures 3 and 4 contain multiple small volcano plots corresponding to different individuals and conditions. The current layout is visually dense and somewhat challenging to interpret, particularly for readers unfamiliar with the study design. Simplifying the layout and enlarging labels would substantially improve readability.

 

Response

As requested by Reviewer #1, outlines were added to clearly delineate individual variants, mutation carriers, and associated pathway-level information.

 

Minor points:

1. The term “activated basal immune transcriptome” should be more clearly defined.
2. Potential age- and sex-related effects on immune transcriptomes should be acknowledged, even if underpowered.

 

Response

#1: we defined this in line 181.

#2: we added this to the limitation section.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript addresses an important topic: the germline inheritance and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family. The integration of family-based genomics, RNA-seq, and AI-based structural modeling is a major strength. The manuscript is clear, and the conclusions are generally supported by the results. I have a few suggestions that could further improve clarity and strengthen the manuscript before publication.

 

1. Please correct the sentence in the Abstract: “but not all, individuals carrying the above variants above”, as the word “above” is used twice.

 

2. Introduction is short. Nothing is said about what we know about JAK3, STAT5A and STAT6 at the cellular and molecular level.

 

 

3. In the Results section, the manuscript states that whole exome sequencing (WES) was performed (lines 57–58). In the Methods section (lines 239–249), the authors clearly describe whole genome sequencing (WGS). This is a factual inconsistency and must be corrected (either clarify that both were done, or correct one).

 

4. The authors state that AF3 provided insight into pathogenicity, but later admit it provided limited biological insight. This is internally contradictory. Structural predictions are descriptive, not functional; please have a look and do correction.

 

5. Some abbreviations (e.g., GOF, AF3, PBMC) are used before being clearly defined in the text.

 

6. Provide a brief justification for using hg19 for RNA-seq alignment while WES uses hg38.

 

7. Ensure consistent gene and protein formatting (e.g., JAK3 vs JAK3) throughout the manuscript.

 

8. Please carefully check the reference list, as references 14 and 43 are duplicated. Ensure that all references are consistently cited throughout the manuscript.

Author Response

This manuscript addresses an important topic: the germline inheritance and transcriptional impact of JAK3, STAT5A and STAT6 variants in a Tyrolean family. The integration of family-based genomics, RNA-seq, and AI-based structural modeling is a major strength. The manuscript is clear, and the conclusions are generally supported by the results. I have a few suggestions that could further improve clarity and strengthen the manuscript before publication.

 

1. Please correct the sentence in the Abstract: “but not all, individuals carrying the above variants above”, as the word “above” is used twice.

 

Response

We corrected this.

 

2. Introduction is short. Nothing is said about what we know about JAK3, STAT5A andSTAT6at the cellular and molecular level.

 

Response

We significantly expanded the introduction to address these issues (lines 51-59). We also introduced current knowledge about these variants in the Result section (lines 121-130).

 

3. In the Results section, the manuscript states that whole exome sequencing (WES) was performed (lines 57–58). In the Methods section (lines 239–249), the authors clearly describe whole genome sequencing (WGS). This is a factual inconsistency and must be corrected (either clarify that both were done, or correct one).

 

Response

We conducted WES in addition to employing data from our previous RNA-seq experiments. This has now been clearly stated.

 

4. The authors state that AF3 provided insight into pathogenicity, but later admit it provided limited biological insight. This is internally contradictory. Structural predictions are descriptive, not functional; please have a look and do correction.

 

Response

We have reviewed the manuscript and believe that we have clarified that “…structural predictions are descriptive, not functional…”, while still providing “…insight into pathogenicity…”. Please see lines 71-75, 242-249.

 

5. Some abbreviations (e.g., GOF, AF3, PBMC) are used before being clearly defined in the text.

 

Response

We addressed this issue.

 

6. Provide a brief justification for using hg19 for RNA-seq alignment while WES uses hg38.

 

Response

In our previously published Omicron infection study, RNA-seq data were aligned to hg19 and deposited in GEO; variants identified from these data were subsequently converted to hg38 to enable direct comparison with the newly generated whole-exome sequencing data, which were aligned to hg38 in the current study.

 

7. Ensure consistent gene and protein formatting (e.g., JAK3vs JAK3) throughout the manuscript.

 

Response

Random JAK/STAT variants were denoted using gene-level nomenclature, whereas variants with defined amino acid substitutions were described using protein-level notation.

 

8. Please carefully check the reference list, as references 14 and 43 are duplicated. Ensure that all references are consistently cited throughout the manuscript.

 

Response

We reviewed the reference list and removed the duplicate entry.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

The authors investigate the heritability and transcriptional impact of four rare germline JAK/STAT variants (JAK3⁹¹⁵¹ᴿ, JAK3ᴿ⁹²⁵⁵, STAT5Aⱽ⁴⁹⁴ᴸ, STAT6Q⁶³³ᴴ) in a large Tyrolean family across three generations. Using whole-exome sequencing, RNA-seq, and structural predictions (AlphaFold 3), the study finds that these variants are inherited without overt clinical phenotypes but associate with enhanced basal and SARS-CoV-2-induced immune transcriptomes in a subset of carriers. The work highlights the combinatorial and context-dependent nature of germline variant effects.

1. The RNA-seq analysis lacks a clear control group. Are “non-carrier” family members used as controls? If not, how was differential expression determined?

2. The volcano plots (Figures 3–4) are descriptive but not statistically rigorous. A multi-group comparison (e.g., ANOVA-like approach) or linear modeling adjusted for age and sex would strengthen the findings.

3. Gene set enrichment analysis (GSEA) is mentioned but not shown. The authors should include a table or figure summarizing enriched pathways in carriers with activated transcriptomes.

4. The structural analysis using AlphaFold 3 is a strength, but the functional interpretation remains speculative. The authors should suggest or cite experimental studies (e.g., kinase assays, STAT dimerization assays) that could validate their predictions.

5. The contradictory in silico pathogenicity scores (Table 1) are noted but not sufficiently discussed. A brief commentary on the limitations of these tools in assessing germline variants would be valuable.

6. The role of TYK2 variants is mentioned but underexplored. Given that most carriers also harbor TYK2 mutations, how might these interact with JAK/STAT variants? A network or correlation analysis could be informative.

7. The discussion on “epistasis” is appropriately highlighted but remains theoretical. The authors could propose a testable model for how JAK/STAT variants interact with other immune modifiers.

Author Response

The authors investigate the heritability and transcriptional impact of four rare germline JAK/STAT variants (JAK3⁹¹⁵¹ᴿ, JAK3ᴿ⁹²⁵⁵, STAT5Aⱽ⁴⁹⁴ᴸ, STAT6Q⁶³³ᴴ) in a large Tyrolean family across three generations. Using whole-exome sequencing, RNA-seq, and structural predictions (AlphaFold 3), the study finds that these variants are inherited without overt clinical phenotypes but associate with enhanced basal and SARS-CoV-2-induced immune transcriptomes in a subset of carriers. The work highlights the combinatorial and context-dependent nature of germline variant effects.

 

1. The RNA-seq analysis lacks a clear control group. Are “non-carrier” family members used as controls? If not, how was differential expression determined?

 

Response

Yes, non-carrier family members were used as controls. Differential expression was determined by comparing RNA-seq profiles of family members carrying JAK and/or STAT variants with those of family members without JAK/STAT variants at the same time point (lines 188-189, 204-205).

 

2. The volcano plots (Figures 3 and 4) are descriptive but not statistically rigorous. A multi-group comparison (e.g., ANOVA-like approach) or linear modeling adjusted for age and sex would strengthen the findings.

 

Response

We agree that multigroup or linear modeling approaches would strengthen the analysis; however, the cohort size is very small and lacks sufficient variability in age, sex, and genotype combinations to support such analyses with adequate statistical power. Therefore, we do not consider these approaches appropriate for the present study and have interpreted the volcano plots as descriptive rather than inferential.

 

3. Gene set enrichment analysis (GSEA) is mentioned but not shown. The authors should include a table or figure summarizing enriched pathways in carriers with activated transcriptomes.

 

Response

We now included GSEA analyses in Figures 3B and 4B (lines 197-200, 212-215).

 

4. The structural analysis using AlphaFold 3 is a strength, but the functional interpretation remains speculative. The authors should suggest or cite experimental studies (e.g., kinase assays, STAT dimerization assays) that could validate their predictions.

 

Response

We included this in the ‘limitation’ section (lines 382-389).

 

5. The contradictory in silico pathogenicity scores (Table 1) are noted but not sufficiently discussed. A brief commentary on the limitations of these tools in assessing germline variants would be valuable.

 

Response

We addressed this issue in the discussion (lines 246-249).

 

6. The role of TYK2 variants is mentioned but underexplored. Given that most carriers also harbor TYK2 mutations, how might these interact with JAK/STAT variants? A network or correlation analysis could be informative.

 

Response

We agree that TYK2 variants may contribute to the immune transcriptome context in this family. However, epistasis analyses using the Epistasis Disease Atlas did not identify significant interactions between the JAK/STAT variants and TYK2 or other immune-related genes (Table S2). Given the limited sample size and the rarity of specific variant combinations, more complex network or correlation analyses would be underpowered. We therefore interpret the TYK2 variants as potential modifier background factors and have framed the findings accordingly in the Result and Discussion (lines 108-112, 121-130, 272-277).

 

7. The discussion on “epistasis” is appropriately highlighted but remains theoretical. The authors could propose a testable model for how JAK/STAT variants interact with other immune modifiers.

 

Response

We agree with the reviewer and now added that our epistasis findings in in the Result and Discussion (Table S2 and lines 108-112, 121-130, 272-277).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have thoroughly and effectively addressed all previous comments. The revised manuscript clearly explains the rationale behind family selection and appropriately limits the interpretation of the transcriptomic findings. It also transparently discusses the limitations related to sample size, variant combinations, and the lack of functional validation. The discussion consistently frames the results as context-dependent and hypothesis-generating, aligning with current principles of molecular and population genetics. Improvements to the figures and terminology enhance clarity. Although the figures remain information-dense. Overall, the manuscript is scientifically sound, well-presented, and suitable for publication in its current form.

Reviewer 4 Report

Comments and Suggestions for Authors

The revised manuscript (v2) represents a substantial improvement over the initial submission (v1). The structure has been significantly strengthened with a complete, standard scientific manuscript format. Key methodological sections, particularly for WES, RNA-seq, and variant analysis, are now comprehensively detailed, enhancing reproducibility. The inclusion of gene enrichment analyses (GSEA) and more rigorous statistical reporting adds important functional and quantitative depth to the results. The discussion is more nuanced, effectively addressing the complexities of variant interpretation, combinatorial effects, and study limitations. Overall, the v2 version is well-structured, methodologically sound, and presents a compelling narrative. It is recommended for acceptance, pending any minor editorial corrections.