Title: Identifying potential biomarkers of nonalcoholic fatty liver disease via genome-wide
analysis of copy number variation
Version: 2 Date: 12 Dec 2020
Reviewer's report:
In this original manuscript, the authors assessed the contribution of CNV to the evaluation of
NAFLD in by a case-control study. They used high-density comparative genomic hybridisation
microarrays (ACGH) to analyse CNV on 12 NAFLD patients and 12 healthy controls. They
found CNVs involving nine genes may be associated with the pathogenesis of NAFLD
progression. The manuscript does contain some interesting data, however, there are several
concerns should be addressed before it can be considered for publication.
1. In this study, ACGH was performed in peripheral blood samples. However, some CNV
variation only in liver or some (lipid metabolism) genes only expression in liver regulating
NAFLD development which cannot be detected in blood samples. It is important to validate or
perform the study in liver samples.
2. The number of cases enrolled is small.
3. Liver biopsy is the gold standard test for diagnosis, grading, and histological assessment of
NAFLD. The findings in this manuscript should be confirmed in the samples diagnosed by liver
biopsy.
4. Since genomic CNV data was presented by pair, which means selected matched control has
a great influence on the result. The authors should give detailed eligibility criteria for control.
5. Does the expression of NLRP4 correlate to the NAFLD grade? Does the expression of nine
(CNV regions) genes correlate to the NAFLD grade?
6. The authors presented a lot of data about the length of CNVs. What does those data contribute
to assess CNV of NAFLD? Does different size affect gene expression of NAFLD grade?
7. The authors mentioned “based on liver fatty accumulation diagnosed by abdominal
ultrasound, NAFLD was divided into three grades, and four cases were selected from each
grade.” What is the detailed division of each grade?
8. In the clinical characteristics of NAFLD patients, did the authors measure free fatty acids
levels?
9. The authors mentioned NLRP4 gene participate in inflammatory responses and is a
variation gene.  The 12 ACGH samples and 557 validation samples are simple steatosis or
including NSAH? The author should give detailed patient information. The grade and clinical
characteristics of samples should be showed in Table V.
Title: Identifying potential biomarkers of nonalcoholic fatty liver disease via genome-wide
analysis of copy number variation
Version: 2 Date: 12 Dec 2020
Reviewer's report:
In this original manuscript, the authors assessed the contribution of CNV to the evaluation of
NAFLD in by a case-control study. They used high-density comparative genomic hybridisation
microarrays (ACGH) to analyse CNV on 12 NAFLD patients and 12 healthy controls. They
found CNVs involving nine genes may be associated with the pathogenesis of NAFLD
progression. The manuscript does contain some interesting data, however, there are several
concerns should be addressed before it can be considered for publication.
1. In this study, ACGH was performed in peripheral blood samples. However, some CNV
variation only in liver or some (lipid metabolism) genes only expression in liver regulating
NAFLD development which cannot be detected in blood samples. It is important to validate or
perform the study in liver samples.
2. The number of cases enrolled is small.
3. Liver biopsy is the gold standard test for diagnosis, grading, and histological assessment of
NAFLD. The findings in this manuscript should be confirmed in the samples diagnosed by liver
biopsy.
4. Since genomic CNV data was presented by pair, which means selected matched control has
a great influence on the result. The authors should give detailed eligibility criteria for control.
5. Does the expression of NLRP4 correlate to the NAFLD grade? Does the expression of nine
(CNV regions) genes correlate to the NAFLD grade?
6. The authors presented a lot of data about the length of CNVs. What does those data contribute
to assess CNV of NAFLD? Does different size affect gene expression of NAFLD grade?
7. The authors mentioned “based on liver fatty accumulation diagnosed by abdominal
ultrasound, NAFLD was divided into three grades, and four cases were selected from each
grade.” What is the detailed division of each grade?
8. In the clinical characteristics of NAFLD patients, did the authors measure free fatty acids
levels?
9. The authors mentioned NLRP4 gene participate in inflammatory responses and is a
variation gene.  The 12 ACGH samples and 557 validation samples are simple steatosis or
including NSAH? The author should give detailed patient information. The grade and clinical
characteristics of samples should be showed in Table V.
Please review this paper.
user
Title: Identifying potential biomarkers of nonalcoholic fatty liver disease via genome-wide
analysis of copy number variation
Version: 2 Date: 12 Dec 2020
Reviewer's report:
In this original manuscript, the authors assessed the contribution of CNV to the evaluation of
NAFLD in by a case-control study. They used high-density comparative genomic hybridisation
microarrays (ACGH) to analyse CNV on 12 NAFLD patients and 12 healthy controls. They
found CNVs involving nine genes may be associated with the pathogenesis of NAFLD
progression. The manuscript does contain some interesting data, however, there are several
concerns should be addressed before it can be considered for publication.
1. In this study, ACGH was performed in peripheral blood samples. However, some CNV
variation only in liver or some (lipid metabolism) genes only expression in liver regulating
NAFLD development which cannot be detected in blood samples. It is important to validate or
perform the study in liver samples.
2. The number of cases enrolled is small.
3. Liver biopsy is the gold standard test for diagnosis, grading, and histological assessment of
NAFLD. The findings in this manuscript should be confirmed in the samples diagnosed by liver
biopsy.
4. Since genomic CNV data was presented by pair, which means selected matched control has
a great influence on the result. The authors should give detailed eligibility criteria for control.
5. Does the expression of NLRP4 correlate to the NAFLD grade? Does the expression of nine
(CNV regions) genes correlate to the NAFLD grade?
6. The authors presented a lot of data about the length of CNVs. What does those data contribute
to assess CNV of NAFLD? Does different size affect gene expression of NAFLD grade?
7. The authors mentioned “based on liver fatty accumulation diagnosed by abdominal
ultrasound, NAFLD was divided into three grades, and four cases were selected from each
grade.” What is the detailed division of each grade?
8. In the clinical characteristics of NAFLD patients, did the authors measure free fatty acids
levels?
9. The authors mentioned NLRP4 gene participate in inflammatory responses and is a
variation gene.  The 12 ACGH samples and 557 validation samples are simple steatosis or
including NSAH? The author should give detailed patient information. The grade and clinical
characteristics of samples should be showed in Table V.
assistant