Next-Generation Sequencing Analysis in Greek Patients with Predominantly Antibody Deficiencies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, Galanopoulos et al. aim to identify genetic defects associated with predominantly antibody deficiencies (PAD) through next-generation sequencing (NGS) analysis. The authors examined twenty Greek patients using a custom-designed NGS panel targeting 30 genes relevant to PAD. A total of 103 genetic variants were identified, including 3 pathogenic, 6 likely pathogenic, 12 variants of uncertain significance, 5 likely benign, and 77 benign variants. All pathogenic and likely pathogenic variants, as well as novel mutations, were further validated by PCR and Sanger sequencing. To assess the potential structural and functional impact of the identified mutations, the authors employed AlphaFold3-based protein structure prediction.

The manuscript is well written and presents a thorough analysis, highlighting several pathogenic variants across 30 candidate genes associated with PAD. Notably, the study reports novel mutations in NFKB1, NFKB2, and IKZF1 (IKAROS), contributing valuable insights to the ongoing effort to delineate the genetic basis of PAD.

While functional validation of the mutant proteins would further strengthen the findings, such experiments might be beyond the scope of the present study and may be pursued in future studies.

Author Response

“In this manuscript, Galanopoulos et al. aim to identify genetic defects associated with predominantly antibody deficiencies (PAD) through next-generation sequencing (NGS) analysis. The authors examined twenty Greek patients using a custom-designed NGS panel targeting 30 genes relevant to PAD. A total of 103 genetic variants were identified, including 3 pathogenic, 6 likely pathogenic, 12 variants of uncertain significance, 5 likely benign, and 77 benign variants. All pathogenic and likely pathogenic variants, as well as novel mutations, were further validated by PCR and Sanger sequencing. To assess the potential structural and functional impact of the identified mutations, the authors employed AlphaFold3-based protein structure prediction.

The manuscript is well written and presents a thorough analysis, highlighting several pathogenic variants across 30 candidate genes associated with PAD. Notably, the study reports novel mutations in NFKB1, NFKB2, and IKZF1 (IKAROS), contributing valuable insights to the ongoing effort to delineate the genetic basis of PAD.

While functional validation of the mutant proteins would further strengthen the findings, such experiments might be beyond the scope of the present study and may be pursued in future studies”.

Response: We appreciate the reviewer’s insightful comment. Indeed, we fully agree that functional validation of the mutant proteins would significantly enhance our findings and these experiments are part of our future research plans.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents molecular findings from 20 Greek patients with primary antibody deficiencies, primarily CVID, using a custom-designed targeted gene panel. The stated aim is to develop and assess a screening procedure for identifying genetic etiologies in PAD. Among the 20 cases, 9 were included as positive controls for their known molecular etiologies. Through the use of this custom panel, an additional 3 cases were solved, each involving variants in AD genes, with corresponding family histories provided and variable clinical manifestations observed. Another 3 cases carried either VUS in AD genes or a single pathogenic variant in an AR gene, warranting further investigation.

 

While the study provides relevant data and reflects clinical genetic heterogeneity, further clarification is needed regarding the scope and implications of the panel as a screening versus diagnostic tool. A more focused narrative aligned with this primary goal would benefit the discussion.

 

Overall

Study Aim and Interpretation

The manuscript states the goal is to develop a screening procedure, but much of the discussion centers on genetic diagnosis in individual cases.

If the main purpose is screening, please emphasize the diagnostic yield, technical performance, and efficiency of the panel.

If the purpose is diagnostic, then the discussion should explore the clinical implications of findings, such as immune profiles in family members, inheritance patterns, and incomplete penetrance.

Consider reorganizing the Results and Discussion to align clearly with the primary aim of the study.

 

Results:

Table 1 – Molecular Diagnosis Details

• Please consider to list the individual molecular etiologies identified for each patient (e.g., gene name, variant type, inheritance mode). This will help readers interpret the diagnostic value of the panel more effectively.

 

TACI Variant

• Several patients are reported with a common TACI variant. Please clarify whether these individuals belong to the same family or are unrelated. This will affect the interpretation of allele frequency, penetrance, and inheritance patterns.
• The same TACI variant appears in both autosomal dominant and autosomal recessive contexts. Please compare the clinical presentations and immune profiles between these two groups.

Author Response

“The manuscript presents molecular findings from 20 Greek patients with primary antibody deficiencies, primarily CVID, using a custom-designed targeted gene panel. The stated aim is to develop and assess a screening procedure for identifying genetic etiologies in PAD. Among the 20 cases, 9 were included as positive controls for their known molecular etiologies. Through the use of this custom panel, an additional 3 cases were solved, each involving variants in AD genes, with corresponding family histories provided and variable clinical manifestations observed. Another 3 cases carried either VUS in AD genes or a single pathogenic variant in an AR gene, warranting further investigation. 

While the study provides relevant data and reflects clinical genetic heterogeneity, further clarification is needed regarding the scope and implications of the panel as a screening versus diagnostic tool. A more focused narrative aligned with this primary goal would benefit the discussion

Overall

Study Aim and Interpretation

The manuscript states the goal is to develop a screening procedure, but much of the discussion centers on genetic diagnosis in individual cases.

If the main purpose is screening, please emphasize the diagnostic yield, technical performance, and efficiency of the panel.

If the purpose is diagnostic, then the discussion should explore the clinical implications of findings, such as immune profiles in family members, inheritance patterns, and incomplete penetrance.

Consider reorganizing the Results and Discussion to align clearly with the primary aim of the study”.

 Response: We thank the reviewer for this insightful comment. We acknowledge that the term "screening" was inaccurately used in the manuscript. Our actual aim was to develop and apply a rapid, targeted diagnostic tool for the genetic evaluation of patients with predominantly antibody deficiencies (PAD). Accordingly, the focus of our study is on genetic diagnosis, and we have now revised the manuscript to ensure consistent use of terminology reflecting this diagnostic purpose.

“Results:

Table 1 – Molecular Diagnosis Details

• Please consider to list the individual molecular etiologies identified for each patient (e.g., gene name, variant type, inheritance mode). This will help readers interpret the diagnostic value of the panel more effectively”

Response: According to the reviewer’s recommendation, we have included a heatmap (Figure 2) that illustrates the detection of pathogenic, likely pathogenic, and variants of unknown significance (VUS) across the patient cohort. In this figure, the x-axis represents individual patients (identified by their study IDs), and the y-axis lists the genetic variants, grouped by their corresponding genes. Additionally, in the revised version we provide a supplementary table (Supplementary Table 3, line 260) detailing the clinical and demographic characteristics of each patient alongside the detected variants to facilitate a comprehensive interpretation of the diagnostic findings.

“TACI Variant

• Several patients are reported with a common TACI variant. Please clarify whether these individuals belong to the same family or are unrelated. This will affect the interpretation of allele frequency, penetrance, and inheritance patterns.
• The same TACI variant appears in both autosomal dominant and autosomal recessive contexts. Please compare the clinical presentations and immune profiles between these two groups”.

Response: (A) The patients carrying TACI (TNFRSF13B) variants are not related, as confirmed by their ancestor profiles.

(B) There are several reports in the literature suggesting that TACI (TNFRSF13B) defects mainly affect the phenotype of CVID (Salzer & Grimbacher B, Curr Opin Immunol 2021; Kakkas et al., Medicina 2021; Sathkumara et al., Int J Immunogenet 2015) although the biallelic defects seem to have a more profound effect in disease pathogenesis/phenotype (Salzer et al., Blood 2009). In our manuscript (lines 118-126), we describe a patient (#18) with biallelic TACI defects (p.C104R/p.I87N), and several patients with monoallelic variants (p.C104R in patients #4, #15, #16, #19; p.I87N in patient #8; and p.E236X in patient #5). A comparison of their clinical presentations and immunologic profiles revealed a high degree of variability, regardless of zygosity, which were consistent with previous findings in the literature (Salzer & Grimbacher B, Curr Opin Immunol 2021; Kakkas et al., Medicina 2021; Sathkumara et al., Int J Immunogenet 2015), suggesting an incomplete penetrance of TACI defects in CVID.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents conclusive data about mutant variants in a panel of 30 genes associated with PAD, detected by NGS and their possible disease phenotype.

The study has a well-defined objective, the methodology used in this investigation is suitable for reaching the results and it is well described; the findings are clearly presented and well illustrated; the data analysis and interpretation are suitable; the discussion is pertinent and clearly written, the conclusion is proper; the references cited are relevant and up-to-date.

I have some minor concerns:

The authors should clearly state which of the mutations they have identified are already known and which are newly identified.

For nonsense mutations, information should be provided on which exon they are located, how many amino acids the protein is shortened by, and what the potential effect is, classifying the mutation in terms of change in function.

As a limitation, the small number of patients should also be mentioned.

Author Response

Reviewer #3

“The manuscript presents conclusive data about mutant variants in a panel of 30 genes associated with PAD, detected by NGS and their possible disease phenotype.

The study has a well-defined objective, the methodology used in this investigation is suitable for reaching the results and it is well described; the findings are clearly presented and well illustrated; the data analysis and interpretation are suitable; the discussion is pertinent and clearly written, the conclusion is proper; the references cited are relevant and up-to-date”

Response: We are grateful for these supporting comments.

“I have some minor concerns:

The authors should clearly state which of the mutations they have identified are already known and which are newly identified”.

Response: We have clarified this point according to reviewer’s recommendation (lines 30, 303).  

“For nonsense mutations, information should be provided on which exon they are located, how many amino acids the protein is shortened by, and what the potential effect is, classifying the mutation in terms of change in function”.

Response: We thank the reviewer for this important observation. The nonsense mutations identified in NFKB2, NFKB1, and LRBA are described in Sections 3.2.1, 3.2.2, and 3.2.4 of the manuscript, respectively. Specifically:

– The p.R313X mutation in NFKB2 is located in exon 11 (line 280), and its potential impact on protein functionality, including the resulting protein truncation, is discussed in lines 372–381.

– The p.Y350X mutation in NFKB1 is located in exon 11 (line 303), with its functional implications and truncation details addressed in lines 404–409.

– The p.Q717X mutation in LRBA is located in exon 17 (line 345), and its potential effect on protein function is discussed in lines 445–453.

In all cases, we have included information on the affected exon, the extent of protein shortening, and the predicted functional consequences of the mutation, according to the reviewer’s recommendation.

“As a limitation, the small number of patients should also be mentioned”.

Response: We acknowledge the limited sample size as a potential limitation of our study and have now included this point in the revised manuscript (lines 464).