From Suspected Congenital Cytomegalovirus Infection to Malan Syndrome: Delayed Genetic Diagnosis Due to Diagnostic Anchoring

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article by Kovacevic et al is a case report describing a child with sensorineural hearing loss, visual impairment and developmental delay.  The child was initially treated for cytomegalovirus infection and several years later through chromosomal microarray analysis confirmed to have Malan syndrome which explains the clinical features observed.  The main conclusion from the authors is that early genetic testing is warranted even when viral infections that may explain symptoms are present.

The article is well written and clearly describes the case presentation and results of the genetic analysis.  My only comment would be that maybe the authors could comment, with regard to this particular case, what the consequences for clinical care might have been had genetic testing been performed at an earlier age.  Presumably since sensorineural hearing loss is relative uncommon in cases of Malan syndrome and common in congenital CMV infection would antiviral therapy still be warranted?  Where any interventions for developmental delay missed due to the late diagnosis?  Are there implications for the parents and clinicians treating the child due to the delay in diagnosis of Malan syndrome?

Author Response

Dear Reviewer,

We sincerely thank you for your careful review and valuable comments, which helped us improve the clarity and clinical relevance of our manuscript. We have revised the manuscript accordingly, and our detailed responses are provided below.

Comment 1.

With regard to this particular case, what the consequences for clinical care might have been had genetic testing been performed at an earlier age?

Response:

We thank the reviewer for this important question. Earlier genetic testing would probably not have altered the overall neurodevelopmental outcome, as no disease-specific treatment currently exists for Malan syndrome. However, an earlier diagnosis could have provided a unifying explanation for the patient’s multisystem phenotype, reduced diagnostic uncertainty, and avoided prolonged attribution of clinical findings solely to presumed CMV infection. Earlier recognition may also have facilitated more targeted multidisciplinary follow-up, anticipatory guidance, genetic counseling for the family, and a clearer understanding of prognosis.

Comment 2.

Presumably since sensorineural hearing loss is relative uncommon in cases of Malan syndrome and common in congenital CMV infection would antiviral therapy still be warranted?  

Response:

We agree that sensorineural hearing loss is uncommon in Malan syndrome and is a well-recognized manifestation of congenital CMV infection. At the time of presentation, the combination of hearing loss together with positive CMV serology and detection of CMV DNA in serum and urine supported the clinical suspicion of CMV-associated disease. Therefore, antiviral treatment was considered justified based on the available evidence and existing clinical practice. Our intention is not to suggest that antiviral therapy was inappropriate, but rather to highlight that the persistence of atypical features and the evolution of the phenotype should prompt reconsideration of the initial diagnostic assumption and consideration of genetic testing.

Comment 3.

Where any interventions for developmental delay missed due to the late diagnosis?  

Response:

The patient received supportive multidisciplinary care, including developmental rehabilitation and audiological follow-up, independent of the final genetic diagnosis. Therefore, we do not believe that major therapeutic interventions were missed due to the delayed diagnosis. Nevertheless, an earlier diagnosis of Malan syndrome could have improved care coordination, enabled syndrome-specific surveillance, and reduced uncertainty for both clinicians and the family.

Comment 4.

Are there implications for the parents and clinicians treating the child due to the delay in diagnosis of Malan syndrome?

Response:

The delayed diagnosis had important implications for both the family and treating clinicians. For the parents, prolonged diagnostic uncertainty may have contributed to emotional burden and limited the opportunity for earlier genetic counseling regarding recurrence risk, which was ultimately considered low due to the de novo deletion. For clinicians, this case illustrates the risk of diagnostic anchoring when an initially plausible infectious diagnosis is identified. We believe the case emphasizes the importance of re-evaluating the diagnosis when the clinical course or phenotype is not fully explained by the presumed etiology, and of considering early chromosomal microarray testing in children with complex neurodevelopmental presentations.

We summarize these responses and added it in the manuscript.

“If a diagnosis of NFIX-related Malan syndrome had been established earlier by CMA analysis, several aspects of long-term management and genetic counseling might have been improved. Valganciclovir therapy for presumed congenital CMV infection would likely still have been appropriate given the significant sensorineural hearing loss, a common manifestation of cCMV infection. However, earlier recognition of the under-lying genetic syndrome could have enabled earlier multidisciplinary developmental follow-up, individualized rehabilitation planning, and reduced diagnostic uncertainty for both clinicians and the family. Confirmation of the deletion would have enabled accurate genetic counseling. Although the recurrence risk is considered low because of the apparently de novo origin of the CNV, it is not negligible due to rare reported cases of parental gonadal mosaicism involving NFIX alterations. Therefore, prenatal CMA testing may be offered in future pregnancies following appropriate genetic counseling. Overall, this case highlights how diagnostic anchoring to an infectious etiology may delay recognition of an underlying genetic syndrome and supports the role of early genomic testing in complex neurodevelopmental phenotypes”.

Reviewer 2 Report

Comments and Suggestions for Authors

This case highlights the risk of diagnostic anchoring in the context of a presumed infectious etiology and underscores the importance of early genetic testing, including chromosomal microarray analysis, in children with complex neurodevelopmental phenotypes, even when an alternative diagnosis appears likely. However, there are some concerns.

Major concerns

1. The abstract section is somewhat lengthy. Please simplify the background section, highlight the core concept of ‘diagnostic anchoring’, and clearly point out the suggestion that ‘CMA should be performed as early as possible despite evidence of infection’.
2. The evidence for the diagnosis of congenital CMV is insufficient. The patient was only tested for CMV DNA at 6 months, making it impossible to distinguish between congenital and acquired infections.
3. The article did not mention whether CMA validation (de novo confirmation) was conducted on parents, nor did it discuss the risk of recurrence.
4. Page 4, please supplement the ACMG classification of the 19p13.2p13.13 microdeletion (already mentioned in Discussion, move to Results for completeness).
5. The patient received 6 months of treatment with valganciclovir, but it is unclear whether there has been any improvement in hearing or development.
6. Discussion section, please strengthen the clinical recommendation part, propose a clear diagnostic workflow for children with hearing loss + developmental delay + suspected CMV infection.
7. English grammar and writing should be checked and revised throughout the manuscript.

Minor concerns

1. Abstract section, the sentence ‘Retrospective PCR testing for CMV using a dried blood spot was negative; however, this result was considered inconclusive due to the known limitations of the method’ can be slightly condensed to improve conciseness.
2. Abstract section, please explain ‘NFIX’.
3. Keywords, in general, abbreviation should not be used as keyword.
4. Pages 2 and 4, there are two ‘Congenital cytomegalovirus (cCMV)’. Please check and revise others throughout the manuscript. For example, three ‘Chromosomal microarray (CMA)’ in the text.
5. Page 3, please clarify the duration and dosage of valganciclovir more explicitly (currently stated as 16 mg/kg BW over six months; specify once or twice daily).
6. Page 5, revise ‘(7,8,9)’ to ‘[7-9]’. Please check and revise others throughout the manuscript.
7. Page 5, revise ‘(12,5)’ to ‘[5,12]’. Please check and revise others throughout the manuscript.
8. Page 5, revise ‘ZSWIM4, NANOS3, BRME1’ to ‘ZSWIM4, NANOS3, and BRME1’. Please check and revise others throughout the manuscript.
9. The format of references is inconsistent.

For example,

Ref. 1-5, have doi, but Ref. 7-10, without doi.

Ref. 9, revise ‘2026 Feb;118(2):e70025’.

Ref. 23, revise ‘2024 Jul 29;19(1):282. doi: 10.1186/s13023-024-03288-6. PMID: 39075508; PMCID: PMC11288048.’.

Please check others.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Dear Reviewer,

We sincerely thank you for your careful review and valuable comments, which helped us improve the clarity and clinical relevance of our manuscript. We have revised the manuscript accordingly, and our detailed responses are provided below.

Major concerns

Comment 1.

The abstract section is somewhat lengthy. Please simplify the background section, highlight the core concept of ‘diagnostic anchoring’, and clearly point out the suggestion that ‘CMA should be performed as early as possible despite evidence of infection’.

Response:

We thank the reviewer for this valuable comment. The abstract has been revised to improve conciseness and clarity. The background section was shortened, and the concept of diagnostic anchoring in the setting of presumed infection was emphasized more explicitly. In addition, we clarified our main message that chromosomal microarray analysis should be considered early in children with complex neurodevelopmental phenotypes, even when infectious findings suggest an alternative diagnosis.

Background: Diagnostic anchoring to a presumed infectious etiology may delay recognition of underlying genetic disorders in children with neurodevelopmental impairment.
Case presentation: We present a child with sensorineural hearing loss, visual impairment, and developmental delay, in whom CMV infection was suspected at 6 months of age based on positive serology and detection of viral DNA in serum and urine. Given the timing of testing, congenital CMV infection could not be definitively confirmed. Antiviral therapy with valganciclovir was administered. Despite antiviral severe neurodevelopmental impairment and hearing loss persisted, associated with facial dysmorphism, bilateral cryptorchidism, pectus excavatum, and optic nerve hypoplasia, findings not fully explained by CMV infection. Brain MRI showed nonspecific abnormalities. Chromosomal microarray analysis performed at 4.5 years of age identified a heterozygous 19p13.2p13.13 microdeletion consistent with NFIX-related Malan syndrome.
Conclusions: This case highlights the risk of diagnostic anchoring in the context of presumed infection and underscores the importance of early chromosomal microarray analysis in children with complex neurodevelopmental phenotypes, even when infectious findings are present

Comment 2

The evidence for the diagnosis of congenital CMV is insufficient. The patient was only tested for CMV DNA at 6 months, making it impossible to distinguish between congenital and acquired infections.

Response:

We thank the reviewer for this important comment and fully agree that congenital CMV infection could not be definitively confirmed in this patient. As correctly noted, CMV DNA testing was performed only at 6 months of age, which does not allow reliable differentiation between congenital and postnatal infection.

We would like to emphasize that throughout the manuscript we intentionally refer to the diagnosis as “presumed” or “unconfirmed” congenital CMV infection and explicitly discuss this limitation in both the Case Presentation and Discussion sections. We have further revised the text to make this point clearer and avoid any possible ambiguity regarding the interpretation of the CMV findings.

            “A positive CMV PCR in serum and urine at 6 months of age confirmed CMV infection;                  however, due to the delayed timing of testing, it was not possible to distinguish between congenital and postnatal acquisition. Therefore, cCMV infection could not be             definitively established in this patient. According to current diagnostic criteria      confirmation of cCMV requires virological testing within the first 2–3 weeks of life.

            In contrast, CMV infection in our patient most likely represented either a postnatal infection or an unconfirmed congenital infection with uncertain contribution to the               overall clinical phenotype. Rather than representing a straightforward dual pathology,             this report demonstrates how the presence of a plausible infectious finding can lead to diagnostic anchoring and delay consideration of an underlying genetic diagnosis”.

Comment 3.

The article did not mention whether CMA validation (de novo confirmation) was conducted on parents, nor did it discuss the risk of recurrence.

Thank Reviewer for this comment. Parental CMA testing was performed during the revision process and confirmed the de novo origin of the 19p13.2p13.13 microdeletion. This information has now been incorporated into the Abstract , Case presentation, and Discussion section.

Abstract:

“Chromosomal microarray analysis (CMA) performed at 4.5 years of age, identified a heterozygous 908 kb de novo microdeletion at 19p13.2p13.13 containing NFIX (MIM *164005) and other morbid genes. The de novo variant was confirmed by parental testing and the unifying genetic diagnosis of NFIX-related Malan syndrome (MIM#614753) was established”.

Case presentation

“Subsequent CMA testing of both parents confirmed that deletion occurred de novo (final criteria: 1A, 2A, 3A, 5A) [4]”.

Discussion:

“Although the recurrence risk is considered low because of the apparently de novo origin of the CNV, it is not negligible due to rare reported cases of parental gonadal mosaicism in-volving NFIX alterations [27,28]”.

Comment 4

Page 4, please supplement the ACMG classification of the 19p13.2p13.13 microdeletion (already mentioned in Discussion, move to Results for completeness).

Response

We are grateful for addressing this. The ACMG/ClinGen classification of the 19p13.2p13.13 variant has been moved from the Discussion to Case presentation (Results) section (page 4) for completeness. The variant is now explicitly stated as pathogenic according to the ACMG/ClinGen technical standards (initial criteria (before parental testing): 1A, 2A, 3A, 5G; final criteria (after parental testing): 1A, 2A, 3A, 5A).

“The variant was classified as pathogenic according to ACMG/ClinGen technical stand-ards for constitutional copy number loss (initial criteria: 1A, 2A, 3A, 5G) [4]. Subsequent CMA testing of both parents confirmed that deletion occurred de novo (final criteria: 1A, 2A, 3A, 5A) [4]”.

Comment 5

The patient received 6 months of treatment with valganciclovir, but it is unclear whether there has been any improvement in hearing or development.

Response

We thank the reviewer for this important comment. Although the patient received six months of valganciclovir therapy, no substantial improvement in hearing or neurodevelopmental outcome was observed. Severe global developmental delay persisted despite treatment, with independent sitting achieved at four years of age and assisted ambulation at five years. In addition, severe hearing impairment remained present. A hearing aid was initially prescribed; however, due to insufficient benefit, cochlear implantation was subsequently performed. These findings have now been clarified in both the Abstract and Discussion sections of the manuscript.

Abstract

“The treatment was administered over six months and completed without complications. However, severe global developmental delay and hearing impairment persisted without improvement following antiviral therapy. Independent sitting was achieved at four years of age, and assisted ambulation with a few steps at five years. The child remained nonverbal and showed limited interaction with the environment. Due to severe sensorineural hearing loss, hearing amplification was initially introduced, followed by cochlear implantation because of insufficient benefit”.

Discussion:

“The persistence of severe developmental delay and hearing impairment despite antiviral treatment along with atypical clinical features further increased suspicion of an additional underlying etiology and prompted further genetic evaluation”.

Comment 5.

Discussion section, please strengthen the clinical recommendation part, propose a clear diagnostic workflow for children with hearing loss + developmental delay + suspected CMV infection.

Response

We thank the reviewer for this valuable suggestion. We agree that the clinical implications of this case can be strengthened by proposing a practical diagnostic approach. We have revised the Discussion section to emphasize that, in children presenting with hearing loss, developmental delay, and suspected CMV infection, the presence of congenital anomalies, dysmorphic features, disproportionate growth, severe neurodevelopmental impairment, or findings not fully explained by CMV infection should prompt early parallel genetic evaluation, including chromosomal microarray analysis. We aimed to highlight the importance of avoiding diagnostic anchoring and maintaining a multidisciplinary, phenotype-driven diagnostic approach.

“Based on this case, we suggest that children presenting with hearing impairment, developmental delay, and suspected CMV infection should undergo careful phenotypic reassessment throughout follow-up. While evaluation for congenital CMV remains essential, the presence of congenital anomalies, dysmorphic features, disproportionate growth, severe developmental impairment, atypical neuroimaging findings, or clinical evolution not fully explained by CMV should prompt early parallel genetic investigation, particularly chromosomal microarray analysis. A multidisciplinary and phenotype-driven diagnostic approach may help avoid diagnostic anchoring and improve diagnostic accuracy in complex neurodevelopmental disorders”.

We added a proposed diagnostic workflow for children with hearing loss + developmental delay + suspected CMV infection.

Comment 7

English grammar and writing should be checked and revised throughout the manuscript.

Response

We carefully checked and revise the manuscript to improve English grammar and writing.

Minor concerns

Regarding the minor changes recommendations, the following changes were implemented:

Abstract section, the sentence ‘Retrospective PCR testing for CMV using a dried blood spot was negative; however, this result was considered inconclusive due to the known limitations of the method’ can be slightly condensed to improve conciseness.

Comment

Thank you for this comment. We condensed the sentence to improve clearness.

Retrospective CMV PCR testing from a dried blood spot was negative but could not exclude congenital infection because of the limited sensitivity of this method.

Comment 2.

Abstract section, please explain ‘NFIX’.

Response

We appreciate the reviewer’s comment. We have now explained in the revised Abstract that NFIX denotes the gene name: “…high-resolution CMA showed a heterozygous 908 kb de novo microdeletion at 19p13.2p13.13 containing NFIX (MIM *164005) and other morbid genes.”

Comment 3

Keywords, in general, abbreviation should not be used as keyword.

Response

Thank you for this comment. Keywords were revised to avoid abbreviations where appropriate, in accordance with the journal style.

Comment 4

Pages 2 and 4, there are two ‘Congenital cytomegalovirus (cCMV)’. Please check and revise others throughout the manuscript. For example, three ‘Chromosomal microarray (CMA)’ in the text.

Response

Thank you for this observation. Repeated definitions of abbreviations such as "congenital cytomegalovirus (cCMV)" and "chromosomal microarray analysis (CMA)" were corrected throughout the manuscript to ensure consistency.

Comment 5

Page 3, please clarify the duration and dosage of valganciclovir more explicitly (currently stated as 16 mg/kg BW over six months; specify once or twice daily).

Response

We revise the manuscript and clarified the duration and dosage of valganciclovir more explicitly.

“Despite being beyond the neonatal period, antiviral therapy with valganciclovir at the dose of 16 mg/kg BW twice daily was initiated due to significant hearing loss and developmental delay”

Comment 6

Page 5, revise ‘(7,8,9)’ to ‘[7-9]’. Please check and revise others throughout the manuscript.

Response

Thank you for this observation. We have revised the citation from (7,8,9) to (7-9) on page 5 and carefully checked and standardized all similar page ranges throughout the manuscript.

Comment 7

 Page 5, revise ‘(12,5)’ to ‘[5,12]’. Please check and revise others throughout the manuscript.

Response

Thank you for this comment. The citation (12,5) has been corrected to [5,12] on page 5, and all other numerical citations in the manuscript have been reviewed and formatted consistently.

Comment 8

Page 5, revise ‘ZSWIM4, NANOS3, BRME1’ to ‘ZSWIM4, NANOS3, and BRME1’. Please check and revise others throughout the manuscript.

Response

Thank you for this suggestion. We have revised the gene list on the page 5 to “ZSWIM4, NANOS3, and BRME1” and ensured consistent use of the serial comma in similar lists throughout the manuscript.

Comment 9

The format of references is inconsistent.

Response

We are grateful for this observation. The reference list has been reformatted for consistency and all missing DOI numbers have been added. We also revised a reference order and added 5 new references in order to illustrate the significance and utility of DBS PCR CMV testing as well as the need for prenatal testing in further pregnancies in a family with Malan syndrome (the added references are highlighted in the references list.

• Typographical inconsistencies and word-splitting artifacts introduced during formatting were corrected throughout the manuscript and tables.

• In addition, the clinical photograph presented in Figure 1 was revised to further protect patient identity. The patient's eyes have been anonymized in the updated figure. We would like to emphasize that this modification does not compromise the phenotypic interpretability or illustrative value of the image, which was included solely to demonstrate relevant clinical features associated with the reported syndrome.

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer Comments to Author

The manuscript entitled “From Suspected Congenital Cytomegalovirus Infection to Malan syndrome: Delayed Genetic Diagnosis Due to Diagnostic Anchoring” is well written, and the quality of the English is proficient. The study presents an interesting case report of a child who was initially diagnosed with cytomegalovirus (CMV) infection, which partially explained the clinical findings. However, the subsequent identification of an underlying chromosomal abnormality provided a more comprehensive explanation for the overall phenotype. This case report underlines the risk of misdiagnosis when the appropriate genetic testing is not ordered. Overall, the manuscript after some minor revisions is suitable for publication; however, several points must be addressed and be answered for further improvement:

1. Page 4, 2^nd paragraph: the reevaluation of congenital CMV infection through a blood spot on a Guthrie card is not entirely valid as CMV is mainly detected in blood and urine (and amniotic fluid in a prenatal level). So, either way the results were expected to be negative. This must be stated clearly in the text.
2. Page 6, 3^rd paragraph: There should be a notice in the manuscript that though the clinical features of the patient were not entirely precluding the diagnosis of Malan syndrome, the whole spectrum of the clinical characteristics could lead in the investigation through a more high-throughput method such as array-CGH.
3. Page 5, paragraphs 7 and 8: the authors must point out that the whole symptomatology could lead directly to further genetic evaluation besides the investigation of CMV infection.
4. Page 6: Since hearing impairment is not common in Malan syndrome and since this symptom remained after the administration of antiviral therapy, the authors could suggest hearing impairment to be included in the classical symptomatology of Malan syndrome as an additional characteristic.
5. Page 7, 2^nd paragraph: The authors should suggest that a more detailed evaluation and genetic counseling must be applied in all cases in order to improve the diagnosis.
6. Page 7, Conclusion: there is a space missing between words: “glance” and “apparent”.

Author Response

Dear Reviewer,

We sincerely thank you for your careful review and valuable comments, which helped us improve the clarity and clinical relevance of our manuscript. We have revised the manuscript accordingly, and our detailed responses are provided below.

Comment 1

Page 4, 2^nd paragraph: the reevaluation of congenital CMV infection through a blood spot on a Guthrie card is not entirely valid as CMV is mainly detected in blood and urine (and amniotic fluid in a prenatal level). So, either way the results were expected to be negative. This must be stated clearly in the text.

Response

We thank the reviewer for this important comment. We agree that a negative DBS PCR result cannot definitively exclude congenital CMV infection. We have therefore revised the manuscript to clarify that the negative Guthrie card result should be interpreted cautiously and was not considered definitive evidence against congenital CMV infection, while preserving the discussion regarding the recognized clinical utility of retrospective DBS testing.

“Furthermore, by retrospective testing using PCR on the archived patient’s Guthrie card, congenital CMV infection could not be confirmed, since the result was negative. However, this finding should be interpreted cautiously, as a negative DBS PCR result does not definitively exclude congenital CMV infection. In spite of some possible limitations of this method, considering viral DNA stability under storage conditions and potential risk of cross-contamination, testing neonatal DBS samples continues to be a valid and useful approach for both screening and clinical purposes [13-17].”

Comment 2.

Page 6, 3^rd paragraph: There should be a notice in the manuscript that though the clinical features of the patient were not entirely precluding the diagnosis of Malan syndrome, the whole spectrum of the clinical characteristics could lead in the investigation through a more high-throughput method such as array-CGH.

Response

We thank the reviewer for this important comment. We agree that although the patient did not exhibit the full classical phenotype of Malan syndrome, the combination of developmental delay, congenital anomalies, dysmorphic features, and multisystem involvement should have prompted earlier investigation using a high-throughput genomic method such as chromosomal microarray analysis (array-CGH/CMA). We have revised the manuscript to emphasize that atypical or incomplete phenotypic presentation should not delay comprehensive genetic evaluation.

“In our patient, the absence of marked overgrowth did not exclude the diagnosis of Malan syndrome, but contributed to diagnostic uncertainty and delay. Nevertheless, the coexistence of developmental delay, facial features, multiple congenital anomalies, and hearing impairment suggested a complex neurodevelopmental phenotype that should prompted earlier investigation using a high-throughput genomic approach such as CMA”.

Comment 3

Page 5, paragraphs 7 and 8: the authors must point out that the whole symptomatology could lead directly to further genetic evaluation besides the investigation of CMV infection.

Response

We thank the reviewer for this insightful comment. We agree that the overall clinical presentation, including multiple congenital anomalies, dysmorphic features, skeletal abnormalities, and severe developmental impairment, could itself have justified earlier genetic evaluation regardless of the CMV findings. We have revised the Discussion section to emphasize that the phenotype was not fully consistent with CMV infection alone and that these atypical features should have prompted earlier consideration of a genetic etiology and chromosomal microarray analysis.

“Although CMV infection was confirmed, several clinical features in our patient were not fully explained by either congenital or acquired CMV infection. These included multiple congenital anomalies (bilateral cryptorchidism, rectal stenosis), dysmorphic facial features, skeletal abnormalities (pectus excavatum, kyphosis), disproportionate growth, and profound developmental impairment. Collectively, this phenotype could itself have justified earlier genetic evaluation, irrespective of the CMV findings.

In addition, neuroimaging findings, including a subarachnoid cyst and mild ventricular dilatation, were nonspecific and insufficient to explain the severity of the neurodevelopmental phenotype. The persistence of severe developmental delay and hearing impairment following antiviral therapy along with atypical clinical features increased suspicion of an underlying genetic disorder, ultimately prompting further genetic analysis”.

Comment 4

Page 6: Since hearing impairment is not common in Malan syndrome and since this symptom remained after the administration of antiviral therapy, the authors could suggest hearing impairment to be included in the classical symptomatology of Malan syndrome as an additional characteristic.

We thank the reviewer for this thoughtful suggestion. We agree that the persistence of sensorineural hearing loss despite antiviral therapy, together with previous reports of hearing impairment in Malan syndrome, raises the possibility that hearing loss may represent an underrecognized feature of the syndrome in at least a subset of patients. However, given the concomitant CMV infection and the inability to definitively distinguish congenital from acquired CMV infection in our patient, we believe caution is warranted in attributing the hearing impairment solely to Malan syndrome. We have therefore revised the Discussion to acknowledge that this case may further expand the phenotypic spectrum of NFIX-related Malan syndrome.

“Hearing impairment in Malan syndrome is considered uncommon but has been previously reported, including bilateral moderate-to-severe sensorineural hearing loss (17). In adults with Malan syndrome, hearing abnormalities, including sensorineural, conductive, and progressive forms, have been described in approximately 20% of patients, while altered auditory processing and hypersensitivity to noise appear even more frequent (23).

In our patient, persistent sensorineural hearing loss despite antiviral therapy raised the possibility that hearing impairment may represent an underrecognized feature within the phenotypic spectrum of Malan syndrome. However, as the CMV infection could not be definitively classified as congenital or postnatal, it remains unclear to what extent the hearing loss was related to the CMV infection, Malan syndrome, or both”.

Comment 5

Page 7, 2^nd paragraph: The authors should suggest that a more detailed evaluation and genetic counseling must be applied in all cases in order to improve the diagnosis.

We thank the reviewer for this valuable suggestion. We agree that comprehensive clinical evaluation and early genetic counseling are important in children with complex neurodevelopmental phenotypes, particularly when infectious findings may initially appear explanatory. We have revised the Discussion to emphasize the importance of detailed phenotypic reassessment, multidisciplinary evaluation, and timely genetic counseling in order to improve diagnostic accuracy and support appropriate family counseling. We proposed a diagnostic workflow for children with sensorineural hearing loss, developmental delay, and suspected CMV infection,

“Based on this case, we suggest that children presenting with hearing impairment, de-velopmental delay, and suspected CMV infection should undergo careful phenotypic reassessment throughout follow-up. While evaluation for congenital CMV remains es-sential, the presence of congenital anomalies, dysmorphic features, disproportionate growth, severe developmental impairment, atypical neuroimaging findings, or clinical evolution not fully explained by CMV should prompt early parallel genetic investiga-tion, particularly chromosomal microarray analysis. A multidisciplinary and phenotype-driven diagnostic approach may help avoid diagnostic anchoring and improve diagnostic accuracy in complex neurodevelopmental disorders”

Comment 6

Page 7, Conclusion: there is a space missing between words: “glance” and “apparent”.

Response

Thank you for this observation. We added a space between words: “glance” and “apparent”.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for author’s responses. However, there are STILL some concerns.

1. There are some ‘we’ and ‘our’. As a scientific paper, the writing style should adopt the third person style.
2. Page 1, please explain ‘CMV’ first.
3. Page 5, delete ” in one paragraphs.
4. The format of references should be based on MDPI style, and checked one by one.

Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Dear Reviewer,

We sincerely thank you for your careful review and important suggestions, which helped us improve the clarity and clinical relevance of our manuscript. We have revised the manuscript accordingly, and our detailed responses are provided below.

Comment 1

There are some ‘we’ and ‘our’. As a scientific paper, the writing style should adopt the third person style.

Response

Thank the Reviewer for this important comment. We carefully checked the manuscript and changed “we” and “our” to the third person style. All changes are highlighted in green.

Comment 2

Page 1, please explain ‘CMV’ first

Response

Thank the Reviewer for this suggestion. We explain “CMV” in the Page 1 and add abbreviations for congenital cytomegalovirus infection (cCMV) and chromosomal microarray analysis (CMA) in the Abstract session.

Comment 3

Page 5, delete ” in one paragraph.

Response

Thank the Reviewer for this observation. We deleted “in paragraph 8.

Comment 4

The format of references should be based on MDPI style, and checked one by one.
Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range 

Response

Thank you for this valuable comment.

The references have been reformatted based on MDPI style, and checked one by one.