Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE p.C282Y/p.C282Y
Version: 3 Date: 15 Feb 2023
Reviewer's report:
This manuscript by Drs. Barton and Acton compares HFE C282Y homozygotes with fatty
liver with homozygotes without fatty liver.  They report that the homozygotes with fatty
liver had higher median serum ferritin those without fatty liver despite similar amount of
mobilizable iron, were more likely to have elevated aminotransferases and were marginally more
likely to have Type 2 DM.  These are interesting results, but the overall significance of the
observations is limited by the relatively small sample size and the heterogeneous nature of the
data/characterization of the patients.
Major comments
1. It would be of interest to know whether elevated aminotransferases resolved with de-ironing
in either group of C282Y homozygotes.
2.  Table 1 shows the frequency of common conditions associated with fatty liver (obesity, DM,
etc.) in the homozygotes with or without fatty liver.  Assuming that none of 16 subjects with
fatty liver had more than 1 of the associated conditions, there were 3 patients with fatty liver
(nearly 20%) who had no NAFLD risk factors.   Is this correct?  (This number may be higher if
any of the subjects had more than one associated condition.)  This should be clarified.
3.  The prevalence of NAFLD risk factors was appreciable in the “No NAFLD” group (60%!).
This raises concern about the accuracy of the characterization of these subjects as not having
fatty liver.  A smaller proportion of the “No NAFLD” patients underwent biopsy than in the
NAFLD group, and while ultrasound is a reasonably good way to identify steatotic-appearing
livers, its sensitivity is not perfect (can be as low as the mid-80%-range).
4.  In any case, it is worth considering that the lack of significant differences in the prevalence
of the NAFLD risk factors other than Type 2 DM in the 2 groups may reflect type 2 error.
5.  In the Discussion regarding the higher ferritin levels in the fatty liver group, the authors
cite a study that showed lower hepcidin levels in C282Y homozygotes than in patients with
NAFLD alone, and suggest that low hepcidin levels associated with C282Y homozygosity “have
a greater influence on SF, a surrogate marker of iron overload severity, than NAFLD.”  I found
this statement a bit confusing, perhaps because it does not track with the authors’ observations.
First, the presence of NAFLD seems to contribute quite a bit to the hyperferritinemia in the
NAFLD group, although the extent to which this reflects inflammation vs low hepcidin levels is
not known.  Secondly, in the homozygotes with fatty liver, it seems fair to say that serum ferritin
has limited value as a “surrogate marker of iron overload severity,” since median SF was twice
as high in this group as in the homozygotes without fatty liver, despite similar mobilizable iron
burdens.  This is, in fact, the most interesting observation of this study.
Minor comments
1.  The Methods indicate that the subjects included in the study were evaluated in the interval
1989-2005.  In at least 2 places in the manuscript (pg 5, pg 12), the authors make reference
to fibroelastography.  It would be surprising indeed if many subjects underwent elastography
during this time interval.  Although ultrasound elastography may have been available in the
latter part of the study interval, Fibroscan was not approved by the FDA until 2013.   Did any of
the subjects have elastography performed during their initial evaluations?  If not, the manuscript
should be modified to make clear that this test was not available at that time.
2. In the second sentence of the second paragraph on pg. 12, which discusses liver biopsy
findings, the text states, “Of these, 11 had NAFLD (68.6% (11/15)) . . .”  This proportion should
be 11/16, should it not?
Title: Non-alcoholic fatty liver disease in hemochromatosis probands with iron overload and
HFE p.C282Y/p.C282Y
Version: 3 Date: 15 Feb 2023
Reviewer's report:
This manuscript by Drs. Barton and Acton compares HFE C282Y homozygotes with fatty
liver with homozygotes without fatty liver.  They report that the homozygotes with fatty
liver had higher median serum ferritin those without fatty liver despite similar amount of
mobilizable iron, were more likely to have elevated aminotransferases and were marginally more
likely to have Type 2 DM.  These are interesting results, but the overall significance of the
observations is limited by the relatively small sample size and the heterogeneous nature of the
data/characterization of the patients.
Major comments
1. It would be of interest to know whether elevated aminotransferases resolved with de-ironing
in either group of C282Y homozygotes.
2.  Table 1 shows the frequency of common conditions associated with fatty liver (obesity, DM,
etc.) in the homozygotes with or without fatty liver.  Assuming that none of 16 subjects with
fatty liver had more than 1 of the associated conditions, there were 3 patients with fatty liver
(nearly 20%) who had no NAFLD risk factors.   Is this correct?  (This number may be higher if
any of the subjects had more than one associated condition.)  This should be clarified.
3.  The prevalence of NAFLD risk factors was appreciable in the “No NAFLD” group (60%!).
This raises concern about the accuracy of the characterization of these subjects as not having
fatty liver.  A smaller proportion of the “No NAFLD” patients underwent biopsy than in the
NAFLD group, and while ultrasound is a reasonably good way to identify steatotic-appearing
livers, its sensitivity is not perfect (can be as low as the mid-80%-range).
4.  In any case, it is worth considering that the lack of significant differences in the prevalence
of the NAFLD risk factors other than Type 2 DM in the 2 groups may reflect type 2 error.
5.  In the Discussion regarding the higher ferritin levels in the fatty liver group, the authors
cite a study that showed lower hepcidin levels in C282Y homozygotes than in patients with
NAFLD alone, and suggest that low hepcidin levels associated with C282Y homozygosity “have
a greater influence on SF, a surrogate marker of iron overload severity, than NAFLD.”  I found
this statement a bit confusing, perhaps because it does not track with the authors’ observations.
First, the presence of NAFLD seems to contribute quite a bit to the hyperferritinemia in the
NAFLD group, although the extent to which this reflects inflammation vs low hepcidin levels is
not known.  Secondly, in the homozygotes with fatty liver, it seems fair to say that serum ferritin
has limited value as a “surrogate marker of iron overload severity,” since median SF was twice
as high in this group as in the homozygotes without fatty liver, despite similar mobilizable iron
burdens.  This is, in fact, the most interesting observation of this study.
Minor comments
1.  The Methods indicate that the subjects included in the study were evaluated in the interval
1989-2005.  In at least 2 places in the manuscript (pg 5, pg 12), the authors make reference
to fibroelastography.  It would be surprising indeed if many subjects underwent elastography
during this time interval.  Although ultrasound elastography may have been available in the
latter part of the study interval, Fibroscan was not approved by the FDA until 2013.   Did any of
the subjects have elastography performed during their initial evaluations?  If not, the manuscript
should be modified to make clear that this test was not available at that time.
2. In the second sentence of the second paragraph on pg. 12, which discusses liver biopsy
findings, the text states, “Of these, 11 had NAFLD (68.6% (11/15)) . . .”  This proportion should
be 11/16, should it not?
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: 15 Feb 2023
Reviewer's report:
This manuscript by Drs. Barton and Acton compares HFE C282Y homozygotes with fatty
liver with homozygotes without fatty liver.  They report that the homozygotes with fatty
liver had higher median serum ferritin those without fatty liver despite similar amount of
mobilizable iron, were more likely to have elevated aminotransferases and were marginally more
likely to have Type 2 DM.  These are interesting results, but the overall significance of the
observations is limited by the relatively small sample size and the heterogeneous nature of the
data/characterization of the patients.
Major comments
1. It would be of interest to know whether elevated aminotransferases resolved with de-ironing
in either group of C282Y homozygotes.
2.  Table 1 shows the frequency of common conditions associated with fatty liver (obesity, DM,
etc.) in the homozygotes with or without fatty liver.  Assuming that none of 16 subjects with
fatty liver had more than 1 of the associated conditions, there were 3 patients with fatty liver
(nearly 20%) who had no NAFLD risk factors.   Is this correct?  (This number may be higher if
any of the subjects had more than one associated condition.)  This should be clarified.
3.  The prevalence of NAFLD risk factors was appreciable in the “No NAFLD” group (60%!).
This raises concern about the accuracy of the characterization of these subjects as not having
fatty liver.  A smaller proportion of the “No NAFLD” patients underwent biopsy than in the
NAFLD group, and while ultrasound is a reasonably good way to identify steatotic-appearing
livers, its sensitivity is not perfect (can be as low as the mid-80%-range).
4.  In any case, it is worth considering that the lack of significant differences in the prevalence
of the NAFLD risk factors other than Type 2 DM in the 2 groups may reflect type 2 error.
5.  In the Discussion regarding the higher ferritin levels in the fatty liver group, the authors
cite a study that showed lower hepcidin levels in C282Y homozygotes than in patients with
NAFLD alone, and suggest that low hepcidin levels associated with C282Y homozygosity “have
a greater influence on SF, a surrogate marker of iron overload severity, than NAFLD.”  I found
this statement a bit confusing, perhaps because it does not track with the authors’ observations.
First, the presence of NAFLD seems to contribute quite a bit to the hyperferritinemia in the
NAFLD group, although the extent to which this reflects inflammation vs low hepcidin levels is
not known.  Secondly, in the homozygotes with fatty liver, it seems fair to say that serum ferritin
has limited value as a “surrogate marker of iron overload severity,” since median SF was twice
as high in this group as in the homozygotes without fatty liver, despite similar mobilizable iron
burdens.  This is, in fact, the most interesting observation of this study.
Minor comments
1.  The Methods indicate that the subjects included in the study were evaluated in the interval
1989-2005.  In at least 2 places in the manuscript (pg 5, pg 12), the authors make reference
to fibroelastography.  It would be surprising indeed if many subjects underwent elastography
during this time interval.  Although ultrasound elastography may have been available in the
latter part of the study interval, Fibroscan was not approved by the FDA until 2013.   Did any of
the subjects have elastography performed during their initial evaluations?  If not, the manuscript
should be modified to make clear that this test was not available at that time.
2. In the second sentence of the second paragraph on pg. 12, which discusses liver biopsy
findings, the text states, “Of these, 11 had NAFLD (68.6% (11/15)) . . .”  This proportion should
be 11/16, should it not?
assistant