Title: PNPLA3 and TM6SF2 genetic variants and hepatic fibrosis and cirrhosis in Pakistani
chronic hepatitis C patients: a genetic association study
Version: 0 Date: 09 Jun 2022
Reviewer's report:
Rauff et al evaluated PNPLA3 and TM6SF2 polymorphisms association with liver fibrosis
in 502 patients with chronic hepatitis C from Pakistan. The authors reported that the
aforementioned polymorphisms were not associated with liver fibrosis grades #F2 or cirrhosis in
any of the genetic models tested. Despite interesting, some concerns were raised while reviewing
the manuscript:
Major concerns:
1- Since the study evaluated two single nucleotide polymorphisms related to liver fat
accumulation, data regarding liver steatosis (Controlled Attenuation Parameter or Liver
imaging) and metabolic syndrome components are missing and would have been important,
as the authors have also recognized. In addition, TM6SF2 polymorphisms may also have a
dual net effect, since the common allele (C allele) seems to be associated with a lower risk of
liver steatosis, but with higher serum levels of total cholesterol, low-density-lipoprotein and
triglycerides. Therefore, the evaluation of serum lipid profile would add relevant insights to the
study. If these data are not available, this issue should be mentioned as a limitation of the study.
2- Since BMI was independently associated with #F2 and cirrhosis (Table 5), it would have
been valuable to stratify patients according to BMI and look at SNPs associations. For example,
normal weight versus overweight / obese.
3- In the abstract it is stated that “no association with hepatic fibrosis or cirrhosis persisted in the
multivariate logistic regression models adjusting for age, gender, body mass index, HCV viral
load and liver enzymes”. However these data were not presented in the manuscript.
4- The GTEx evaluation rationale was not clear in the study objectives, nor its relation to other
aspects of the manuscript. The rationale for this evaluation and the association with the described
study findings is missing.  If it is not related to the study procedures, I suggest removing it from
the current manuscript.
Minor considerations:
Abstract section:
1- CHC abbreviation meaning should be presented.
2- The genotypic distributions frequencies should be reported in the results.
Introduction section:
3- Page 3 Line 61: HCV is written twice.
Methods section:
4- The patients were prospectively recruited, but it seems that the study was cross-section by
design. The type of study should be clarified.
5- Please specify the Fibroscan model and the utilized probe.
6- The adopted Fibroscan liver stiffness measurement cut-offs according to Metavir
classification should be described.
7- Page 6 Line 112-113: Please explain “based on clinical indications”, to improve clarity.
8- In addition to the aforementioned lack of comorbidities data, do you have HOMA-IR results
available?
9- The variable selection criterion for the multivariate logistic regression analysis should be
reported.
Results section:
10- In Table 1: Please include the meaning of the abbreviations BMI, ALT, AST (please check
all tables and additional files regarding the abbreviations).
11- Supplementary Table 1: Would be valuable to understand why p values are significant for
each variable (specify which Metavir group comparison led to this positive statistical test (age,
BMI, etc), i. e. Metavir F1 vs Metavir F4). Only p values for multiple group comparisons were
provided.
12- Page 8 Line 170: Since the current study design seems to be cross-sectional and the moment
of acquisition of HCV infection are not presented, I suggest remove the term “developing”.
13- The % described in Tables 3 and 4 are incompatible with the absolute numbers presented.
Please verify.
14- Please describe the definition of advanced and severe hepatic fibrosis in Methods section.
Usually advanced hepatic fibrosis is defined as #F3, and significant hepatic fibrosis as #F2. I
suggest using these most applied terms and definitions throughout your manuscript / Figures.
15- Page 8 Line 179: Is this information regarding cirrhosis? If so, please rewrite to improve
clarity.
16- Page 9 Line 183: As the study design seems to be cross-sectional, the term "risk" throughout
the manuscript should be avoided. The term "association" would be more appropriate.
Discussion section:
17- Page 11 Line 228-231: In the transplant setting, the cited references (37 and 38) are related
to liver steatosis, and not HCC. Please rewrite the whole paragraph to improve clarity.
18- Page 11 Line 245 and 247: Please consider changing the colloquial term “a couple of”.
19- Page 11 Line 250: Please correct the word “observant”.
20- Page 12 Line 262: Is there difference between steatosis and hepatosteatosis? Please clarify.
21- Page 12 Line 269 to 273: Please include references for these data.
22- Please standardize all the references (for example, references 31, 34, 38, 40, 42 are
unformatted).
Title: PNPLA3 and TM6SF2 genetic variants and hepatic fibrosis and cirrhosis in Pakistani
chronic hepatitis C patients: a genetic association study
Version: 0 Date: 09 Jun 2022
Reviewer's report:
Rauff et al evaluated PNPLA3 and TM6SF2 polymorphisms association with liver fibrosis
in 502 patients with chronic hepatitis C from Pakistan. The authors reported that the
aforementioned polymorphisms were not associated with liver fibrosis grades #F2 or cirrhosis in
any of the genetic models tested. Despite interesting, some concerns were raised while reviewing
the manuscript:
Major concerns:
1- Since the study evaluated two single nucleotide polymorphisms related to liver fat
accumulation, data regarding liver steatosis (Controlled Attenuation Parameter or Liver
imaging) and metabolic syndrome components are missing and would have been important,
as the authors have also recognized. In addition, TM6SF2 polymorphisms may also have a
dual net effect, since the common allele (C allele) seems to be associated with a lower risk of
liver steatosis, but with higher serum levels of total cholesterol, low-density-lipoprotein and
triglycerides. Therefore, the evaluation of serum lipid profile would add relevant insights to the
study. If these data are not available, this issue should be mentioned as a limitation of the study.
2- Since BMI was independently associated with #F2 and cirrhosis (Table 5), it would have
been valuable to stratify patients according to BMI and look at SNPs associations. For example,
normal weight versus overweight / obese.
3- In the abstract it is stated that “no association with hepatic fibrosis or cirrhosis persisted in the
multivariate logistic regression models adjusting for age, gender, body mass index, HCV viral
load and liver enzymes”. However these data were not presented in the manuscript.
4- The GTEx evaluation rationale was not clear in the study objectives, nor its relation to other
aspects of the manuscript. The rationale for this evaluation and the association with the described
study findings is missing.  If it is not related to the study procedures, I suggest removing it from
the current manuscript.
Minor considerations:
Abstract section:
1- CHC abbreviation meaning should be presented.
2- The genotypic distributions frequencies should be reported in the results.
Introduction section:
3- Page 3 Line 61: HCV is written twice.
Methods section:
4- The patients were prospectively recruited, but it seems that the study was cross-section by
design. The type of study should be clarified.
5- Please specify the Fibroscan model and the utilized probe.
6- The adopted Fibroscan liver stiffness measurement cut-offs according to Metavir
classification should be described.
7- Page 6 Line 112-113: Please explain “based on clinical indications”, to improve clarity.
8- In addition to the aforementioned lack of comorbidities data, do you have HOMA-IR results
available?
9- The variable selection criterion for the multivariate logistic regression analysis should be
reported.
Results section:
10- In Table 1: Please include the meaning of the abbreviations BMI, ALT, AST (please check
all tables and additional files regarding the abbreviations).
11- Supplementary Table 1: Would be valuable to understand why p values are significant for
each variable (specify which Metavir group comparison led to this positive statistical test (age,
BMI, etc), i. e. Metavir F1 vs Metavir F4). Only p values for multiple group comparisons were
provided.
12- Page 8 Line 170: Since the current study design seems to be cross-sectional and the moment
of acquisition of HCV infection are not presented, I suggest remove the term “developing”.
13- The % described in Tables 3 and 4 are incompatible with the absolute numbers presented.
Please verify.
14- Please describe the definition of advanced and severe hepatic fibrosis in Methods section.
Usually advanced hepatic fibrosis is defined as #F3, and significant hepatic fibrosis as #F2. I
suggest using these most applied terms and definitions throughout your manuscript / Figures.
15- Page 8 Line 179: Is this information regarding cirrhosis? If so, please rewrite to improve
clarity.
16- Page 9 Line 183: As the study design seems to be cross-sectional, the term "risk" throughout
the manuscript should be avoided. The term "association" would be more appropriate.
Discussion section:
17- Page 11 Line 228-231: In the transplant setting, the cited references (37 and 38) are related
to liver steatosis, and not HCC. Please rewrite the whole paragraph to improve clarity.
18- Page 11 Line 245 and 247: Please consider changing the colloquial term “a couple of”.
19- Page 11 Line 250: Please correct the word “observant”.
20- Page 12 Line 262: Is there difference between steatosis and hepatosteatosis? Please clarify.
21- Page 12 Line 269 to 273: Please include references for these data.
22- Please standardize all the references (for example, references 31, 34, 38, 40, 42 are
unformatted).
Please review this paper.
user
Title: PNPLA3 and TM6SF2 genetic variants and hepatic fibrosis and cirrhosis in Pakistani
chronic hepatitis C patients: a genetic association study
Version: 0 Date: 09 Jun 2022
Reviewer's report:
Rauff et al evaluated PNPLA3 and TM6SF2 polymorphisms association with liver fibrosis
in 502 patients with chronic hepatitis C from Pakistan. The authors reported that the
aforementioned polymorphisms were not associated with liver fibrosis grades #F2 or cirrhosis in
any of the genetic models tested. Despite interesting, some concerns were raised while reviewing
the manuscript:
Major concerns:
1- Since the study evaluated two single nucleotide polymorphisms related to liver fat
accumulation, data regarding liver steatosis (Controlled Attenuation Parameter or Liver
imaging) and metabolic syndrome components are missing and would have been important,
as the authors have also recognized. In addition, TM6SF2 polymorphisms may also have a
dual net effect, since the common allele (C allele) seems to be associated with a lower risk of
liver steatosis, but with higher serum levels of total cholesterol, low-density-lipoprotein and
triglycerides. Therefore, the evaluation of serum lipid profile would add relevant insights to the
study. If these data are not available, this issue should be mentioned as a limitation of the study.
2- Since BMI was independently associated with #F2 and cirrhosis (Table 5), it would have
been valuable to stratify patients according to BMI and look at SNPs associations. For example,
normal weight versus overweight / obese.
3- In the abstract it is stated that “no association with hepatic fibrosis or cirrhosis persisted in the
multivariate logistic regression models adjusting for age, gender, body mass index, HCV viral
load and liver enzymes”. However these data were not presented in the manuscript.
4- The GTEx evaluation rationale was not clear in the study objectives, nor its relation to other
aspects of the manuscript. The rationale for this evaluation and the association with the described
study findings is missing.  If it is not related to the study procedures, I suggest removing it from
the current manuscript.
Minor considerations:
Abstract section:
1- CHC abbreviation meaning should be presented.
2- The genotypic distributions frequencies should be reported in the results.
Introduction section:
3- Page 3 Line 61: HCV is written twice.
Methods section:
4- The patients were prospectively recruited, but it seems that the study was cross-section by
design. The type of study should be clarified.
5- Please specify the Fibroscan model and the utilized probe.
6- The adopted Fibroscan liver stiffness measurement cut-offs according to Metavir
classification should be described.
7- Page 6 Line 112-113: Please explain “based on clinical indications”, to improve clarity.
8- In addition to the aforementioned lack of comorbidities data, do you have HOMA-IR results
available?
9- The variable selection criterion for the multivariate logistic regression analysis should be
reported.
Results section:
10- In Table 1: Please include the meaning of the abbreviations BMI, ALT, AST (please check
all tables and additional files regarding the abbreviations).
11- Supplementary Table 1: Would be valuable to understand why p values are significant for
each variable (specify which Metavir group comparison led to this positive statistical test (age,
BMI, etc), i. e. Metavir F1 vs Metavir F4). Only p values for multiple group comparisons were
provided.
12- Page 8 Line 170: Since the current study design seems to be cross-sectional and the moment
of acquisition of HCV infection are not presented, I suggest remove the term “developing”.
13- The % described in Tables 3 and 4 are incompatible with the absolute numbers presented.
Please verify.
14- Please describe the definition of advanced and severe hepatic fibrosis in Methods section.
Usually advanced hepatic fibrosis is defined as #F3, and significant hepatic fibrosis as #F2. I
suggest using these most applied terms and definitions throughout your manuscript / Figures.
15- Page 8 Line 179: Is this information regarding cirrhosis? If so, please rewrite to improve
clarity.
16- Page 9 Line 183: As the study design seems to be cross-sectional, the term "risk" throughout
the manuscript should be avoided. The term "association" would be more appropriate.
Discussion section:
17- Page 11 Line 228-231: In the transplant setting, the cited references (37 and 38) are related
to liver steatosis, and not HCC. Please rewrite the whole paragraph to improve clarity.
18- Page 11 Line 245 and 247: Please consider changing the colloquial term “a couple of”.
19- Page 11 Line 250: Please correct the word “observant”.
20- Page 12 Line 262: Is there difference between steatosis and hepatosteatosis? Please clarify.
21- Page 12 Line 269 to 273: Please include references for these data.
22- Please standardize all the references (for example, references 31, 34, 38, 40, 42 are
unformatted).
assistant