Title: Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency: Clinical Features, Genetic
Characteristics, and Treatment Outcomes
Version: 4 Date: 12 Mar 2025
Reviewer's report:
Overall, this study provides valuable insights into NICCD; however, several aspects
require clarification and refinement. Key areas needing improvement include consistency in
methodology, clearer reporting of clinical findings, and a more precise discussion of genetic
and metabolic data. Certain claims in the discussion should be better supported with appropriate
references. Addressing these points will enhance the scientific rigor and clinical relevance of
the manuscript. Below are specific suggestions for revision.
• The gene SLC25A13 should be written in italics.
• To ensure consistency, the references should be formatted according to the journal's submission
guidelines.
• Clarification is needed regarding the selection of the control group in the Methods and Figure
1. It is important to specify which tests were conducted to exclude other causes and confirm
idiopathic neonatal cholestasis. There is no information on how citrin deficiency was ruled out
in the idiopathic group. Figure 1 seems to represent a general approach to patients with elevated
transaminases rather than illustrating the exclusion criteria.
• In Table 1, plasma citrulline levels are presented, whereas in the Methods section, the analysis
of urinary citrulline levels by ESI-MS/MS is mentioned. This appears to be inconsistent. If
plasma citrulline levels were measured, the Methods section should be revised accordingly to
avoid confusion.
• The statement regarding whole gene and/or exon sequencing is somewhat ambiguous. It would
be beneficial to specify whether these analyses were conducted for all patients or only for a
selected subgroup.
• The description of the tests performed could be more specific. For example, it would be
beneficial to clarify whether 'hepatitis virus' testing was limited to those listed in Figure 1 or if a
broader screening was performed. Similarly, specifying the parameters included in 'hepatic and
renal function' tests and the details of 'urinalysis' would enhance clarity.
• Given the significantly higher levels of bile acids reported, it would be helpful to include
information on whether patients experienced cholestatic symptoms such as pruritus. Adding this
detail would strengthen the clinical relevance of the findings.
• Diffuse fatty liver, parenchymal cellular infiltration associated with hepatic fibrosis, and
hemolytic anemia are among the other findings of NICCD. However, the authors have not
mentioned whether these manifestations were present in their patients. imaging findings have
not been discussed in the manuscript. Including this information would provide a more
comprehensive evaluation of NICCD-related hepatic involvement.
• The authors provide detailed amino acid profile differences between the NICCD and INC
groups. However, the threonine/serine ratio is an important metabolic marker for NICCD. It
would be beneficial to include this ratio in the analysis to enhance the diagnostic insights of
the study.
• Including the ACMG classification of the identified genetic variants in Table 3 would
improve the clarity and clinical significance of the reported findings. This would help in better
understanding the pathogenic impact of the mutations.
• In the results section, adding the percentage values when mentioning the most frequent
mutation would enhance the clarity and quantitative representation of the data.
• The paragraph of outcomes of treatment describes the follow-up and resolution of jaundice
but does not specify the treatment approach used for these patients. It would be important to
clarify whether dietary therapy for citrin deficiency or symptomatic treatment for cholestasis
was implemented. in addition, presenting the clinical features of all 15 patients in a table format
would be helpful for better understanding individual patient characteristics. This would also
allow for a clearer correlation between genotype and clinical course, providing valuable insights
into disease variability.
• T he reason why four patients did not achieve complete normalization of liver enzymes is not
clearly explained. Clarifying whether this was due to genetic differences, concomitant conditions
such as sepsis, or variations in treatment would provide valuable insights and address potential
uncertainties in the interpretation of the results.
• In the discussion section, it is mentioned that ALT levels did not normalize in four patients,
potentially due to concurrent cytomegalovirus infection or low birth weight. However, this
finding is not clearly presented in the results section. it remains unclear whether this persistent
ALT elevation was associated with a specific genotype. Including this information in the Results
section and clarifying potential genotype correlations would enhance the comprehensiveness of
the study.
• Since this study was conducted in China, it would be beneficial to include more region-
specific epidemiological and screening data in the discussion section. Providing information on
the prevalence of NICCD in China, whether it is included in the national newborn screening
program, and any region-specific genetic patterns would enhance the context and relevance of
the findings. Discussing how the study’s results align with or differ from other NICCD studies
conducted in China would provide a more comprehensive perspective.
• The statement suggesting that NICCD patients may progress to hyperammonemia and Type
II citrullinemia is unclear in the context of the present study’s findings. It is not evident how
the current data support this conclusion. Additionally, reference [22] primarily discusses the
clinical course and liver transplantation outcomes of adult-onset CTLN2 patients rather than
the progression from NICCD. If the authors aim to highlight a connection between NICCD
and CTLN2, they should support this statement with references that explicitly establish this
relationship. Otherwise, it would be more appropriate to remove this speculation, as it lacks a
strong scientific basis.
• Table 1 includes the patients' ages; however, the age of onset for symptoms such as jaundice
is not provided. Including this information would be valuable for understanding the disease
progression and potential differences between NICCD and INC patients.
• Since AFP reference values change according to the patient's age in days, it would be beneficial
to clarify whether all patients had AFP levels above the upper limit based on age-specific
reference ranges.
Title: Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency: Clinical Features, Genetic
Characteristics, and Treatment Outcomes
Version: 4 Date: 12 Mar 2025
Reviewer's report:
Overall, this study provides valuable insights into NICCD; however, several aspects
require clarification and refinement. Key areas needing improvement include consistency in
methodology, clearer reporting of clinical findings, and a more precise discussion of genetic
and metabolic data. Certain claims in the discussion should be better supported with appropriate
references. Addressing these points will enhance the scientific rigor and clinical relevance of
the manuscript. Below are specific suggestions for revision.
• The gene SLC25A13 should be written in italics.
• To ensure consistency, the references should be formatted according to the journal's submission
guidelines.
• Clarification is needed regarding the selection of the control group in the Methods and Figure
1. It is important to specify which tests were conducted to exclude other causes and confirm
idiopathic neonatal cholestasis. There is no information on how citrin deficiency was ruled out
in the idiopathic group. Figure 1 seems to represent a general approach to patients with elevated
transaminases rather than illustrating the exclusion criteria.
• In Table 1, plasma citrulline levels are presented, whereas in the Methods section, the analysis
of urinary citrulline levels by ESI-MS/MS is mentioned. This appears to be inconsistent. If
plasma citrulline levels were measured, the Methods section should be revised accordingly to
avoid confusion.
• The statement regarding whole gene and/or exon sequencing is somewhat ambiguous. It would
be beneficial to specify whether these analyses were conducted for all patients or only for a
selected subgroup.
• The description of the tests performed could be more specific. For example, it would be
beneficial to clarify whether 'hepatitis virus' testing was limited to those listed in Figure 1 or if a
broader screening was performed. Similarly, specifying the parameters included in 'hepatic and
renal function' tests and the details of 'urinalysis' would enhance clarity.
• Given the significantly higher levels of bile acids reported, it would be helpful to include
information on whether patients experienced cholestatic symptoms such as pruritus. Adding this
detail would strengthen the clinical relevance of the findings.
• Diffuse fatty liver, parenchymal cellular infiltration associated with hepatic fibrosis, and
hemolytic anemia are among the other findings of NICCD. However, the authors have not
mentioned whether these manifestations were present in their patients. imaging findings have
not been discussed in the manuscript. Including this information would provide a more
comprehensive evaluation of NICCD-related hepatic involvement.
• The authors provide detailed amino acid profile differences between the NICCD and INC
groups. However, the threonine/serine ratio is an important metabolic marker for NICCD. It
would be beneficial to include this ratio in the analysis to enhance the diagnostic insights of
the study.
• Including the ACMG classification of the identified genetic variants in Table 3 would
improve the clarity and clinical significance of the reported findings. This would help in better
understanding the pathogenic impact of the mutations.
• In the results section, adding the percentage values when mentioning the most frequent
mutation would enhance the clarity and quantitative representation of the data.
• The paragraph of outcomes of treatment describes the follow-up and resolution of jaundice
but does not specify the treatment approach used for these patients. It would be important to
clarify whether dietary therapy for citrin deficiency or symptomatic treatment for cholestasis
was implemented. in addition, presenting the clinical features of all 15 patients in a table format
would be helpful for better understanding individual patient characteristics. This would also
allow for a clearer correlation between genotype and clinical course, providing valuable insights
into disease variability.
• T he reason why four patients did not achieve complete normalization of liver enzymes is not
clearly explained. Clarifying whether this was due to genetic differences, concomitant conditions
such as sepsis, or variations in treatment would provide valuable insights and address potential
uncertainties in the interpretation of the results.
• In the discussion section, it is mentioned that ALT levels did not normalize in four patients,
potentially due to concurrent cytomegalovirus infection or low birth weight. However, this
finding is not clearly presented in the results section. it remains unclear whether this persistent
ALT elevation was associated with a specific genotype. Including this information in the Results
section and clarifying potential genotype correlations would enhance the comprehensiveness of
the study.
• Since this study was conducted in China, it would be beneficial to include more region-
specific epidemiological and screening data in the discussion section. Providing information on
the prevalence of NICCD in China, whether it is included in the national newborn screening
program, and any region-specific genetic patterns would enhance the context and relevance of
the findings. Discussing how the study’s results align with or differ from other NICCD studies
conducted in China would provide a more comprehensive perspective.
• The statement suggesting that NICCD patients may progress to hyperammonemia and Type
II citrullinemia is unclear in the context of the present study’s findings. It is not evident how
the current data support this conclusion. Additionally, reference [22] primarily discusses the
clinical course and liver transplantation outcomes of adult-onset CTLN2 patients rather than
the progression from NICCD. If the authors aim to highlight a connection between NICCD
and CTLN2, they should support this statement with references that explicitly establish this
relationship. Otherwise, it would be more appropriate to remove this speculation, as it lacks a
strong scientific basis.
• Table 1 includes the patients' ages; however, the age of onset for symptoms such as jaundice
is not provided. Including this information would be valuable for understanding the disease
progression and potential differences between NICCD and INC patients.
• Since AFP reference values change according to the patient's age in days, it would be beneficial
to clarify whether all patients had AFP levels above the upper limit based on age-specific
reference ranges.
Please review this paper.
user
Title: Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency: Clinical Features, Genetic
Characteristics, and Treatment Outcomes
Version: 4 Date: 12 Mar 2025
Reviewer's report:
Overall, this study provides valuable insights into NICCD; however, several aspects
require clarification and refinement. Key areas needing improvement include consistency in
methodology, clearer reporting of clinical findings, and a more precise discussion of genetic
and metabolic data. Certain claims in the discussion should be better supported with appropriate
references. Addressing these points will enhance the scientific rigor and clinical relevance of
the manuscript. Below are specific suggestions for revision.
• The gene SLC25A13 should be written in italics.
• To ensure consistency, the references should be formatted according to the journal's submission
guidelines.
• Clarification is needed regarding the selection of the control group in the Methods and Figure
1. It is important to specify which tests were conducted to exclude other causes and confirm
idiopathic neonatal cholestasis. There is no information on how citrin deficiency was ruled out
in the idiopathic group. Figure 1 seems to represent a general approach to patients with elevated
transaminases rather than illustrating the exclusion criteria.
• In Table 1, plasma citrulline levels are presented, whereas in the Methods section, the analysis
of urinary citrulline levels by ESI-MS/MS is mentioned. This appears to be inconsistent. If
plasma citrulline levels were measured, the Methods section should be revised accordingly to
avoid confusion.
• The statement regarding whole gene and/or exon sequencing is somewhat ambiguous. It would
be beneficial to specify whether these analyses were conducted for all patients or only for a
selected subgroup.
• The description of the tests performed could be more specific. For example, it would be
beneficial to clarify whether 'hepatitis virus' testing was limited to those listed in Figure 1 or if a
broader screening was performed. Similarly, specifying the parameters included in 'hepatic and
renal function' tests and the details of 'urinalysis' would enhance clarity.
• Given the significantly higher levels of bile acids reported, it would be helpful to include
information on whether patients experienced cholestatic symptoms such as pruritus. Adding this
detail would strengthen the clinical relevance of the findings.
• Diffuse fatty liver, parenchymal cellular infiltration associated with hepatic fibrosis, and
hemolytic anemia are among the other findings of NICCD. However, the authors have not
mentioned whether these manifestations were present in their patients. imaging findings have
not been discussed in the manuscript. Including this information would provide a more
comprehensive evaluation of NICCD-related hepatic involvement.
• The authors provide detailed amino acid profile differences between the NICCD and INC
groups. However, the threonine/serine ratio is an important metabolic marker for NICCD. It
would be beneficial to include this ratio in the analysis to enhance the diagnostic insights of
the study.
• Including the ACMG classification of the identified genetic variants in Table 3 would
improve the clarity and clinical significance of the reported findings. This would help in better
understanding the pathogenic impact of the mutations.
• In the results section, adding the percentage values when mentioning the most frequent
mutation would enhance the clarity and quantitative representation of the data.
• The paragraph of outcomes of treatment describes the follow-up and resolution of jaundice
but does not specify the treatment approach used for these patients. It would be important to
clarify whether dietary therapy for citrin deficiency or symptomatic treatment for cholestasis
was implemented. in addition, presenting the clinical features of all 15 patients in a table format
would be helpful for better understanding individual patient characteristics. This would also
allow for a clearer correlation between genotype and clinical course, providing valuable insights
into disease variability.
• T he reason why four patients did not achieve complete normalization of liver enzymes is not
clearly explained. Clarifying whether this was due to genetic differences, concomitant conditions
such as sepsis, or variations in treatment would provide valuable insights and address potential
uncertainties in the interpretation of the results.
• In the discussion section, it is mentioned that ALT levels did not normalize in four patients,
potentially due to concurrent cytomegalovirus infection or low birth weight. However, this
finding is not clearly presented in the results section. it remains unclear whether this persistent
ALT elevation was associated with a specific genotype. Including this information in the Results
section and clarifying potential genotype correlations would enhance the comprehensiveness of
the study.
• Since this study was conducted in China, it would be beneficial to include more region-
specific epidemiological and screening data in the discussion section. Providing information on
the prevalence of NICCD in China, whether it is included in the national newborn screening
program, and any region-specific genetic patterns would enhance the context and relevance of
the findings. Discussing how the study’s results align with or differ from other NICCD studies
conducted in China would provide a more comprehensive perspective.
• The statement suggesting that NICCD patients may progress to hyperammonemia and Type
II citrullinemia is unclear in the context of the present study’s findings. It is not evident how
the current data support this conclusion. Additionally, reference [22] primarily discusses the
clinical course and liver transplantation outcomes of adult-onset CTLN2 patients rather than
the progression from NICCD. If the authors aim to highlight a connection between NICCD
and CTLN2, they should support this statement with references that explicitly establish this
relationship. Otherwise, it would be more appropriate to remove this speculation, as it lacks a
strong scientific basis.
• Table 1 includes the patients' ages; however, the age of onset for symptoms such as jaundice
is not provided. Including this information would be valuable for understanding the disease
progression and potential differences between NICCD and INC patients.
• Since AFP reference values change according to the patient's age in days, it would be beneficial
to clarify whether all patients had AFP levels above the upper limit based on age-specific
reference ranges.
assistant