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  • Yasuyo Kashiwagi 1,*,
  • Hironobu Okuno 1 and
  • Gaku Yamanaka 1
  • et al.

Reviewer 1: Anonymous Reviewer 2: Tamara Nikuseva Martic

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article is interesting and highlights the differential diagnosis of Alport syndrome, also emphasizing possible differences in the evolution between the different forms of this disease. In the family pedigree of Figure 1, I would represent the maternal grandfather of the proband (individual II-3) differently (not as an empty square) to draw attention to the finding of end-stage renal failure of unknown etiology with dialysis, since this is important in the evaluation and understanding of the disease within the family. I believe that a table with the differential diagnoses addressed in the case, highlighting the main findings of each condition, would further enrich the message of the article. It would be important to detail the description of the genetic analysis of the patient and her mother, including information such as the technique and kit used in the evaluations. In the Discussion, it would also be important, perhaps in the fourth paragraph, when discussing the identified COL4A4 variant, to state whether it has been previously described in the literature or even in databases such as ClinVar, even to highlight its possible rarity. In the Discussion section, I think it would also be interesting to comment at some point on the ocular changes associated with Alport syndrome, especially since they are rare among patients with the autosomal dominant form of the disease, which is also consistent with the observations described in the family in this report.

Author Response

  1. Pedigree revision: In Figure 1, we modified the representation of the maternal grandfather (II-3) to clearly indicate his history of end-stage renal disease requiring dialysis. This change highlights his clinical relevance in understanding the familial pattern of disease.
  2. Addition of a differential diagnosis table: As suggested, we added a new table summarizing the differential diagnoses considered in this case (benign familial hematuria/TBMN, IgA nephropathy, and autosomal dominant Alport syndrome), including key supporting and arguing features for each condition.
  3. Detailed description of genetic analysis: We expanded the Methods/Discussion to include detailed information on the genetic testing methodology, including the targeted NGS panel, sequencing platform, genomic regions analyzed, variant annotation pipeline, and databases used for frequency assessment.
  4. Rarity and database status of the COL4A4 variant: We added information indicating that the COL4A4 frameshift variant (c.2317_2318del; p.Arg773GlyfsTer14) is not listed in ClinVar, is absent from gnomAD v3.1, and has an extremely low allele frequency in the Japanese population (ToMMo 0.000013). We also noted that it has been reported as a pathogenic loss-of-function variant in a recent publication.
  5. Ocular manifestations in ADAS: We expanded the Discussion to comment on ocular findings in Alport syndrome, emphasizing that such manifestations are rare in autosomal dominant disease. We also noted that no ocular abnormalities were observed in either the proband or her mother, consistent with the expected phenotype of COL4A4-related ADAS.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a clinically relevant case of delayed diagnosis of autosomal dominant Alport syndrome (ADAS) in a patient with long-standing microscopic hematuria initially interpreted as benign familial hematuria. The longitudinal follow-up is valuable, and the case highlights the importance of revisiting diagnoses when new clinical or family information emerges. However, the manuscript reflects an outdated and overly simplified view of persistent microscopic hematuria and its clinical significance, and does not adequately incorporate current knowledge or guideline-based recommendations regarding genetic testing in such patients. This significantly weakens the scientific and educational value of the report. The statement:“Microhematuria is common in children and is generally associated with a favorable prognosis” is overly simplistic and potentially misleading. While microscopic hematuria is indeed common, current evidence clearly shows that a substantial proportion of children with persistent hematuria have underlying glomerular disease, particularly collagen IV-related nephropathies (COL4A3/COL4A4/COL4A5 spectrum). The statement reflects an outdated, overly reassuring perspective and underestimates the clinical relevance of persistent hematuria. The statement: “The clinical, genetic, and pathologic backgrounds of patients with ADAS remain unclear” is not accurate and should be revised. The clinical and genetic spectrum of autosomal dominant Alport syndrome is now well described, with extensive literature on: genotype–phenotype correlations, variability in disease progression and association with COL4A3/COL4A4 variants.  This sentence suggests insufficient familiarity with current literature and should be corrected accordingly. A major limitation of the manuscript is the lack of alignment with current guideline-based practice. Recent literature and international recommendations clearly support a much lower threshold for genetic testing in patients with persistent microscopic hematuria: genetic testing of COL4A3/COL4A4/COL4A5 is increasingly considered a key diagnostic step, this applies even in the absence of a clear family history, persistent hematuria is now widely recognized as part of the collagen IV disease spectrum. The manuscript currently presents genetic testing as a late or optional step, which does not reflect contemporary practice. The discussion should be revised to emphasize that diagnostic delay is not only due to missing history, but also due to outdated diagnostic strategies. The case itself is clinically sound but not highly novel, as delayed diagnosis of Alport syndrome in patients with presumed benign hematuria is well described. To justify publication, the authors should: better position the case within the modern diagnostic paradigm and explicitly highlight how this case supports earlier genetic testing strategies. The identified COL4A4 variant should be more thoroughly described: ACMG classification, database presence (ClinVar, gnomAD) and supporting evidence for pathogenicity.

In conclusion this case report addresses a clinically relevant scenario but is currently weakened by an outdated conceptual framework regarding persistent hematuria and genetic testing. Substantial revision is required to align the manuscript with contemporary nephrology practice and literature.

Comments on the Quality of English Language

Minor language editing is recommended to improve clarity and reduce repetition.

Author Response

  1. Updated conceptual framework for persistent microscopic hematuria We revised the Introduction and Discussion to remove outdated statements suggesting that persistent microscopic hematuria is generally benign. We incorporated current evidence demonstrating that a significant proportion of children with persistent hematuria have underlying glomerular disease, particularly COL4A3–COL4A5–related nephropathies. We also added recent epidemiologic data and guideline-based recommendations supporting early genetic evaluation.
  2. Revised description of ADAS to reflect current knowledge The statement that “the clinical, genetic, and pathologic backgrounds of ADAS remain unclear” has been removed. We added a detailed discussion summarizing recent advances in understanding the clinical spectrum, genotype–phenotype correlations, and variability associated with COL4A3/COL4A4 variants. This revision aligns the manuscript with contemporary literature.
  3. Alignment with modern diagnostic paradigms and genetic testing recommendations We revised the Discussion to emphasize that diagnostic delay in this case was not solely due to incomplete family history, but also reflected older diagnostic approaches. We now highlight that current practice supports a low threshold for genetic testing in patients with persistent glomerular hematuria, even in the absence of a clear family history. The manuscript now explicitly states that earlier genetic testing would likely have led to a timelier diagnosis.
  4. Clarification of the clinical relevance and educational value of the case We strengthened the framing of the case by discussing how it illustrates the importance of early genetic evaluation, reassessment of presumed benign hematuria, and the evolving understanding of collagen IV–related disease. This positions the case within the modern diagnostic landscape and enhances its educational contribution.
  5. Expanded description of the COL4A4 variant We added detailed information regarding the identified COL4A4 frameshift variant, including:
    1. ACMG/AMP classification (PVS1, PM2, PP4 → Pathogenic)
    2. Absence from ClinVar and gnomAD v3.1
    3. Extremely low allele frequency in the Japanese population (ToMMo 0.000013)
    4. Prior publication reporting this variant as pathogenic These additions address the need for a more comprehensive genetic interpretation.
  6. Additional structural improvements
    1. Added a differential diagnosis table comparing TBMN, IgA nephropathy, and ADAS.
    2. Revised the pedigree to highlight the maternal grandfather’s end-stage renal disease.
    3. Expanded the discussion of extrarenal manifestations, including ocular findings and their rarity in ADAS.
    4. Added a clinical timeline figure to clarify key decision points.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you to the authors for the corrections made, including the addition of the table and figure, as well as for the answers to the questions. I suggest only using capital letters and italics for the gene names, including in the abstract, table, and figures, following standard nomenclature. The last paragraph on page 2 is separate from the previous paragraph, but I believe it is a continuation of the same. Regarding Table 1, I suggest clarifying the meaning of the acronym ESKD. Furthermore, I suggest including other forms of Alport syndrome, specifically autosomal recessive and X-linked, among the differential diagnoses. I believe that in this review the authors can find information and references that can help with this: https://www.ncbi.nlm.nih.gov/books/NBK1207/. I also suggest reviewing the citation format for references at the end of the article. For example, some journal names are complete and written out in full, while others are abbreviated. Additionally, some have a DOI number and others do not.

Author Response

1. Use of capital letters and italics for gene names

Thank you for pointing this out. Following standard gene nomenclature, we have revised all gene names to capital letters and italics throughout the manuscript, including the abstract, main text, Table 1, and figures.

2. Paragraph structure on page 2

We appreciate the reviewer’s observation. The last paragraph on page 2 has now been merged with the preceding paragraph, as it is indeed a continuation of the same content. The revised structure improves the logical flow of the section.

3. Clarification of the acronym “ESKD” in Table 1

We have revised Table 1 to spell out the term at first use:

ESKD: end-stage kidney disease

This clarification has been added to the table footnote.

4. Inclusion of other forms of Alport syndrome in the differential diagnosis

Thank you for this important suggestion. We have now added autosomal recessive Alport syndrome (ARAS) and X-linked Alport syndrome (XLAS) to the differential diagnosis section. We also incorporated relevant information and references from the GeneReviews chapter suggested by the reviewer (https://www.ncbi.nlm.nih.gov/books/NBK1207/).

This addition strengthens the clinical context and improves the completeness of the discussion.

5. Review of reference formatting

We appreciate the reviewer’s careful attention to citation style. All references have now been revised to conform to the Pediatric Reports / MDPI reference format.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has improved substantially and addresses the most important conceptual concerns raised in the previous review. I have only a few minor comments. In the Introduction, the statement that benign familial hematuria is “untreatable” may be misleading because the term itself is increasingly being abandoned in favor of more precise genetic diagnoses.&nbsp.

Comments on the Quality of English Language

A careful language editing would improve readability, as a few sentences remain awkwardly phrased.

Author Response

Response to Reviewer2

We sincerely thank the reviewer for the positive evaluation of our revised manuscript and for the helpful additional comment.

Comment: “In the Introduction, the statement that benign familial hematuria is ‘untreatable’ may be misleading because the term itself is increasingly being abandoned in favor of more precise genetic diagnoses.”

Response: Thank you for this important clarification. We agree that the historical term benign familial hematuria is increasingly being replaced by genetically defined diagnoses, particularly COL4A3- and COL4A4-associated disease. To avoid implying that the condition is inherently “untreatable” or benign, we have revised the relevant section of the Introduction.

“The updated text now states that benign familial hematuria was historically regarded as a harmless condition for which invasive diagnostic procedures such as renal biopsy were considered unnecessary, but that this descriptive term is now being replaced by more precise molecular diagnoses due to the recognized potential for renal progression.”

This revision reflects current understanding and aligns with the reviewer’s suggestion.

We appreciate the reviewer’s insightful comment, which has improved the accuracy and clarity of our manuscript.