Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE p.C282Y/p.C282Y
Version: 3 Date: 02 Feb 2023
Reviewer's report:
2nd February 2023
Review for BMC Gastroenterology
Ref: Submission ID 955cdc8e-2f62-44a8-8b99-1318dfbaea39
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE    p.C282Y/p.C282Y
Authors: James C Barton, J Clayborn Barton, and Ronald T. Acton
In this study the authors aimed to “identify characteristics of non-alcoholic fatty liver disease
(NAFLD) in adults with HFE p.C282Y/p.C282Y”. The design of the study was retrospective
(patients were recruited between 1989 and 2002), observational and small scale. The study
includes patients who were recruited before the hemochromatosis gene was identified and
presumably these were established as C282Y homozygotes retrospectively. The most common
approach to the question of whether there is an association between variants in HFE and risk of
NAFLD has been to determine the prevalence of C282Y (and H63D) in patients with NAFLD
and a number of meta-analyses have been published as documented in the study by Ye et
al. PLoS One. 2016 – the authors reference no. 33. The authors quote this paper in the first
paragraph of their Discussion (page 14) and state – “In a meta-analysis of 43 studies, NAFLD
risk in adults with p.C282Y/p.C282Y and adults with wt/wt (absence of p.C282Y and p.H63D
(rs1799945) did not differ significantly [33]”. Below are extracts from this quoted paper –
the Conclusion of the Abstract  - “Our meta-analysis provides evidence that the HFE C282Y
and H63D polymorphisms confer increased genetic susceptibility to NAFLD and HCC but not
liver cirrhosis. Additional well-powered studies are required to confirm our conclusion” and a
further statement from the results section “The pooled results in Fig 2A and Table 1 show that
compared with the control group, increased NAFLD risk was observed in the case group under
the allele (OR = 1.95, Passociation = 0.012), heterozygote (OR = 1.87, Passociation = 0.016)
and dominant models (OR = 1.95, Passociation = 0.014) but not in the other models. There is
clearly uncertainty here because the authors of the current paper under review select data from
Table 1 showing that the prevalence of the YY genotype is indeed not significantly different
from the CC genotype but there are other comparisons that are significant.
The approach taken in the present paper was to select 66 patient who were investigated for
hemochromatosis - not screen detected - and found to be  p.C282Y/p.C282Y –– and determine
whether NAFLD-associated characteristics were present. NAFLD was diagnosed in 16 patients
and as shown in Table 1, median serum ferritin, prevalences of elevated ALT/AST and type 2
diabetes were significantly greater in these patients as compared to the remaining 50 patients
who did not have NAFLD. When logistic regression was applied two positive associations
remained, serum ferritin and type 2 diabetes. The clinical utility of these findings are not clear
and indeed are not addressed by the authors. Should an elevated AST/ALT in a patient with
p.C282Y/p.C282Y haemochromatosis be investigated for the presence of NAFLD? As more
than half of such patients did not have an elevated AST/ALT the question arises of whether all
patients with hemochromatosis should be suspected of having NAFLD. As NAFLD is associated
with the development of HCC should this be considered a co-factor in the development of this
complication in hemochromatosis?
The authors do not consider the first paper to document steatosis as a cofactor in liver injury in
hemochromatosis – Powell et al. Gastroenterology 2005; 129:1937 – 1943. They found that in
subjects with steatosis the median serum ferritin was higher as were median AST/ALT levels
while hepatic iron concentration were significantly lower than in subjects without steatosis. The
findings in the present study replicate some of these findings and although the amount of iron
removed by phlebotomy in NAFLD patients was higher than in those without NAFLD this did
not reach statistical significance. 53% of the patients in the present study had a liver biopsy but
few details of the histological findings were presented other than the observation that no cases
of cirrhosis were reported.
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE p.C282Y/p.C282Y
Version: 3 Date: 02 Feb 2023
Reviewer's report:
2nd February 2023
Review for BMC Gastroenterology
Ref: Submission ID 955cdc8e-2f62-44a8-8b99-1318dfbaea39
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE    p.C282Y/p.C282Y
Authors: James C Barton, J Clayborn Barton, and Ronald T. Acton
In this study the authors aimed to “identify characteristics of non-alcoholic fatty liver disease
(NAFLD) in adults with HFE p.C282Y/p.C282Y”. The design of the study was retrospective
(patients were recruited between 1989 and 2002), observational and small scale. The study
includes patients who were recruited before the hemochromatosis gene was identified and
presumably these were established as C282Y homozygotes retrospectively. The most common
approach to the question of whether there is an association between variants in HFE and risk of
NAFLD has been to determine the prevalence of C282Y (and H63D) in patients with NAFLD
and a number of meta-analyses have been published as documented in the study by Ye et
al. PLoS One. 2016 – the authors reference no. 33. The authors quote this paper in the first
paragraph of their Discussion (page 14) and state – “In a meta-analysis of 43 studies, NAFLD
risk in adults with p.C282Y/p.C282Y and adults with wt/wt (absence of p.C282Y and p.H63D
(rs1799945) did not differ significantly [33]”. Below are extracts from this quoted paper –
the Conclusion of the Abstract  - “Our meta-analysis provides evidence that the HFE C282Y
and H63D polymorphisms confer increased genetic susceptibility to NAFLD and HCC but not
liver cirrhosis. Additional well-powered studies are required to confirm our conclusion” and a
further statement from the results section “The pooled results in Fig 2A and Table 1 show that
compared with the control group, increased NAFLD risk was observed in the case group under
the allele (OR = 1.95, Passociation = 0.012), heterozygote (OR = 1.87, Passociation = 0.016)
and dominant models (OR = 1.95, Passociation = 0.014) but not in the other models. There is
clearly uncertainty here because the authors of the current paper under review select data from
Table 1 showing that the prevalence of the YY genotype is indeed not significantly different
from the CC genotype but there are other comparisons that are significant.
The approach taken in the present paper was to select 66 patient who were investigated for
hemochromatosis - not screen detected - and found to be  p.C282Y/p.C282Y –– and determine
whether NAFLD-associated characteristics were present. NAFLD was diagnosed in 16 patients
and as shown in Table 1, median serum ferritin, prevalences of elevated ALT/AST and type 2
diabetes were significantly greater in these patients as compared to the remaining 50 patients
who did not have NAFLD. When logistic regression was applied two positive associations
remained, serum ferritin and type 2 diabetes. The clinical utility of these findings are not clear
and indeed are not addressed by the authors. Should an elevated AST/ALT in a patient with
p.C282Y/p.C282Y haemochromatosis be investigated for the presence of NAFLD? As more
than half of such patients did not have an elevated AST/ALT the question arises of whether all
patients with hemochromatosis should be suspected of having NAFLD. As NAFLD is associated
with the development of HCC should this be considered a co-factor in the development of this
complication in hemochromatosis?
The authors do not consider the first paper to document steatosis as a cofactor in liver injury in
hemochromatosis – Powell et al. Gastroenterology 2005; 129:1937 – 1943. They found that in
subjects with steatosis the median serum ferritin was higher as were median AST/ALT levels
while hepatic iron concentration were significantly lower than in subjects without steatosis. The
findings in the present study replicate some of these findings and although the amount of iron
removed by phlebotomy in NAFLD patients was higher than in those without NAFLD this did
not reach statistical significance. 53% of the patients in the present study had a liver biopsy but
few details of the histological findings were presented other than the observation that no cases
of cirrhosis were reported.
Please review this paper.
user
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE p.C282Y/p.C282Y
Version: 3 Date: 02 Feb 2023
Reviewer's report:
2nd February 2023
Review for BMC Gastroenterology
Ref: Submission ID 955cdc8e-2f62-44a8-8b99-1318dfbaea39
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE    p.C282Y/p.C282Y
Authors: James C Barton, J Clayborn Barton, and Ronald T. Acton
In this study the authors aimed to “identify characteristics of non-alcoholic fatty liver disease
(NAFLD) in adults with HFE p.C282Y/p.C282Y”. The design of the study was retrospective
(patients were recruited between 1989 and 2002), observational and small scale. The study
includes patients who were recruited before the hemochromatosis gene was identified and
presumably these were established as C282Y homozygotes retrospectively. The most common
approach to the question of whether there is an association between variants in HFE and risk of
NAFLD has been to determine the prevalence of C282Y (and H63D) in patients with NAFLD
and a number of meta-analyses have been published as documented in the study by Ye et
al. PLoS One. 2016 – the authors reference no. 33. The authors quote this paper in the first
paragraph of their Discussion (page 14) and state – “In a meta-analysis of 43 studies, NAFLD
risk in adults with p.C282Y/p.C282Y and adults with wt/wt (absence of p.C282Y and p.H63D
(rs1799945) did not differ significantly [33]”. Below are extracts from this quoted paper –
the Conclusion of the Abstract  - “Our meta-analysis provides evidence that the HFE C282Y
and H63D polymorphisms confer increased genetic susceptibility to NAFLD and HCC but not
liver cirrhosis. Additional well-powered studies are required to confirm our conclusion” and a
further statement from the results section “The pooled results in Fig 2A and Table 1 show that
compared with the control group, increased NAFLD risk was observed in the case group under
the allele (OR = 1.95, Passociation = 0.012), heterozygote (OR = 1.87, Passociation = 0.016)
and dominant models (OR = 1.95, Passociation = 0.014) but not in the other models. There is
clearly uncertainty here because the authors of the current paper under review select data from
Table 1 showing that the prevalence of the YY genotype is indeed not significantly different
from the CC genotype but there are other comparisons that are significant.
The approach taken in the present paper was to select 66 patient who were investigated for
hemochromatosis - not screen detected - and found to be  p.C282Y/p.C282Y –– and determine
whether NAFLD-associated characteristics were present. NAFLD was diagnosed in 16 patients
and as shown in Table 1, median serum ferritin, prevalences of elevated ALT/AST and type 2
diabetes were significantly greater in these patients as compared to the remaining 50 patients
who did not have NAFLD. When logistic regression was applied two positive associations
remained, serum ferritin and type 2 diabetes. The clinical utility of these findings are not clear
and indeed are not addressed by the authors. Should an elevated AST/ALT in a patient with
p.C282Y/p.C282Y haemochromatosis be investigated for the presence of NAFLD? As more
than half of such patients did not have an elevated AST/ALT the question arises of whether all
patients with hemochromatosis should be suspected of having NAFLD. As NAFLD is associated
with the development of HCC should this be considered a co-factor in the development of this
complication in hemochromatosis?
The authors do not consider the first paper to document steatosis as a cofactor in liver injury in
hemochromatosis – Powell et al. Gastroenterology 2005; 129:1937 – 1943. They found that in
subjects with steatosis the median serum ferritin was higher as were median AST/ALT levels
while hepatic iron concentration were significantly lower than in subjects without steatosis. The
findings in the present study replicate some of these findings and although the amount of iron
removed by phlebotomy in NAFLD patients was higher than in those without NAFLD this did
not reach statistical significance. 53% of the patients in the present study had a liver biopsy but
few details of the histological findings were presented other than the observation that no cases
of cirrhosis were reported.
assistant