Nonalcoholic steatohepatitis in Asian Indians is neither associated with iron overload nor with HFE gene mutations.

Abstract

The pathogenesis of occurrence of liver inflammation and fibrosis in patients with nonalcoholic steatohepatitis (NASH) is not completely understood. Other than insulin resistance, iron abnormalities have been thought to be one of the triggering factors. Therefore, our aim was to study the role of iron abnormalities and HFE gene mutations in patients with NASH.
Thirty-one patients of NASH diagnosed on the basis of clinical examination biochemistry, ultrasonography and liver biopsy (n = 14) were included in the study. Serum iron parameters (n = 23) (iron, ferritin, total iron-binding capacity and transferrin saturation), Perls' iron staining on liver biopsies (n = 14) and HFE gene mutations (C282Y and H63D) (n = 16) were studied in these patients. The association between iron staining, necroinflammatory activity and fibrosis stage on liver biopsies was also determined.
Elevated serum iron, ferritin and transferrin saturation above 55% were observed in 4.3% of patients. On histology, 71% of the patients had negative iron staining, 21.4% had 1+ staining, 7.2% had 2+ staining and none had 3+ or 4+ staining. There was no association between the degree of iron staining and necroinflammatory activity (P = 0.55) and fibrosis stage (P = 0.09) on histology. None of the patients had C282Y HFE gene mutation and four patients (25%) were found to be heterozygotes for H63D gene mutation.
Our study does not favor iron overload and HFE gene mutations as major factors in the pathogenesis of NASH in Asian Indians.

Full text

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• BRIEF REPORTS •
Nonalcoholic steatohepatitis in Asian Indians is neither associated
with iron overload nor with HFE gene mutations
Ajay Duseja, Reena Das, Mohit Nanda, Ashim Das, Gurjeewan Garewal, Yogesh Chawla
PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(3):393-395
Fax: +86-10-85381893 World Journal of Gastroenterology ISSN 1007-9327
E-mail: wjg@wjgnet.com www.wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
ELSEVIER
Ajay Duseja, Mohit Nanda, Yogesh Chawla, Department of
Hepatology, Postgraduate Institute of Medical Education and Research,
Chandigarh, India
Reena Das, Gurjeewan Garewal, Department of Hematology,
Postgraduate Institute of Medical Education and Research, Chandigarh,
India
Ashim Das, Department of Histopathology, Postgraduate Institute
of Medical Education & Research, Chandigarh, India
Correspondence to: Dr. Yogesh Chawla, Professor and Head, Department
of Hepatology, PGIMER, Chandigarh, India. pgihepat@glide.net.in
Telephone: +172-2747585-6334 Fax: +172-2744401
Received: 2003-11-26 Accepted: 2004-03-10
Abstract
AIM: The pathogenesis of occurrence of liver inflammation
and fibrosis in patients with nonalcoholic steatohepatitis
(NASH) is not completely understood. Other than insulin
resistance, iron abnormalities have been thought to be one
of the triggering factors. Therefore, our aim was to study
the role of iron abnormalities and HFE gene mutations in
patients with NASH.
METHODS: Thirty-one patients of NASH diagnosed on the
basis of clinical examination biochemistry, ultrasonography
and liver biopsy (n = 14) were included in the study. Serum
iron parameters (n = 23) (iron, ferritin, total iron-binding
capacity and transferrin saturation), Perls’ iron staining on
liver biopsies (n = 14) and HFE gene mutations (C282Y and
H63D) (n = 16) were studied in these patients. The
association between iron staining, necroinflammatory activity
and fibrosis stage on liver biopsies was also determined.
RESULTS: Elevated serum iron, ferritin and transferrin
saturation above 55% were observed in 4.3% of patients.
On histology, 71% of the patients had negative iron staining,
21.4% had 1+ staining, 7.2% had 2+ staining and none
had 3+ or 4+ staining. There was no association between
the degree of iron staining and necroinflammatory activity
(P = 0.55) and fibrosis stage (P = 0.09) on histology. None
of the patients had C282Y HFE gene mutation and four
patients (25%) were found to be heterozygotes for H63D
gene mutation.
CONCLUSION: Our study does not favor iron overload and
HFE gene mutations as major factors in the pathogenesis of
NASH in Asian Indians.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Nonalcoholic steatohepatitis; Iron overload; HFE
gene; Mutation
Duseja A, Das R, Nanda M, Das A, Garewal G, Chawla Y.
Nonalcoholic steatohepatitis in Asian Indians is neither
associated with iron overload nor with HFE gene mutations.
World J Gastroenterol 2005; 11(3): 393-395
http://www.wjgnet.com/1007-9327/11/393.asp
INTRODUCTION
Non-alcoholic steatohepatitis (NASH) is a clinicopathological
entity that occurs in non alcoholics but histologically simulates
alcoholic hepatitis[1]. Mechanisms leading to inflammation,
fibrosis, and cirrhosis from a relatively benign stage of only
steatosis are poorly understood[2]. For this purpose, NASH is
considered a two-hit process[3]. Initial hit leads to excessive fat
accumulation in the liver parenchyma and the second hit
initiated by an oxidative stress may lead to lipid peroxidation,
thus damaging the cells and producing a variety of proinflammatory
and fibrogenic byproducts[2,3]. This second hit may be initiated
by various factors including insulin resistance and hepatic iron
with or without HFE gene mutation of hemochromatosis[2,4].
The prevalence of HFE gene mutations and iron abnormalities
in NASH patients have been linked to the ethnicity[5]. There is
no study from India[6,7] on the role of iron abnormalities and
HFE gene mutations in patients with NASH. We retrospectively
studied 31 patients of NASH and analyzed the serum and liver
iron abnormalities in them and also determined the mutations
in the HFE gene.
MATERIALS AND METHODS
Thirty-one patients with NASH attending liver clinic of our
Institute over a period of two years (April 2001- March 2003)
were diagnosed on the basis of history, clinical examination,
serum biochemistry, ultrasonography, upper gastrointestinal
(GI) endoscopy, and were included in the study. Since liver
biopsy in patients with NASH is controversial, it was carried
out only in 14 patients who failed to respond to a treatment
protocol. All patients gave an informed consent and the
Institute’s Ethics Committee approved the study. None of the
patients consumed more than 20 g/d of alcohol, which was
confirmed by at least two family members or relatives. All these
patients had over two times elevated transaminases for more
than six mo and had a hyperechoic liver on ultrasound
examination. They were all negative for hepatitis B virus surface
antigen (HBsAg-Monolisa Ag HBs plus, Biorad), antibodies
to hepatitis C virus (anti HCV -3rd generation, LG HCD 3.0) and
autoimmune markers including anti-nuclear antibody (ANA),
anti-smooth muscle antibody (ASMA), anti-liver kidney
microsomal antibody (LKM1), antimitochondrial antibody
(AMA). They all had normal serum ceruloplasmin levels and
no evidence of Kayser-Fleischer rings on slit lamp examination.
Overnight fasting samples were collected in iron free tubes
from 23 patients to perform serum iron, total iron-binding
capacity (TIBC) and transferrin saturation (TS)[8]. Serum ferritin
was performed using ELISA kits (Orgentec, USA). Normal
values of serum iron parameters are mentioned in Table 1. For
studying HFE gene mutations (C282Y and H63D), genomic DNA
was extracted from peripheral blood leucocytes of 16 patients
and polymerase chain reaction-restriction fragment length
polymorphism (PCR-RFLP) was performed by using the primers
as described by Feder et al.[9] Grading, staging and Perls
Prussian blue staining for iron were done on liver biopsy
specimens from 14 patients as described by Brunt et al.[10].
All patients underwent anthropometric measurements to
calculate the body mass index (BMI) (body weight in Kg/height

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in meter2) while waist hip ratio (W/H) for the central obesity
was determined in 13 patients. Overweight, obesity, and
abnormal waist hip ratio were defined as per the international
criteria[11,12] (Table 1). A fasting plasma glucose
on more than one occasion or a random plasma glucose of more
than 200 mg/dL in a symptomatic patient or a two-hour plasma
glucose more than 200 mg/dL on glucose tolerance test (GTT)
was defined as diabetes[13]. Impaired glucose tolerance (2 h plasma
glucose after 75 g of oral glucose between 140-200 mg/dL)
and impaired fasting glucose (fasting plasma glucose between
110-126 mg/dL) were also defined[13]. Dose and duration of
drugs were recorded in patients who were known diabetics for
a long duration. Lipid profiles including serum cholesterol, high-
density lipoprotein (HDL), low-density-lipoprotein (LDL) and
serum triglycerides were done in all patients (Table 1).
126 mg/dL
Table 1 Anthropometric, biochemical, serum iron parameters,
liver iron staining and HFE gene mutations in 31 patients
with NASH
Parameter (n=31) Reference value Abnormal-n (%)
1 BMI
I
II Obesity
W-H ratio (n = 13)
I. Males (n = 10)
II. Females (n = 3)
S cholesterol
HDL
L DL
S Triglycerides
Diabetes
Impaired glucose tolerance
Serum iron parameters (n = 23)
9Serum iron
10 Transferrin saturation
11Serum Ferritin
Perls Prussian blue staining on liver biopsy (n = 14)
12 Iron staining positivity
<25 kg/ m2
25-29
30
23 (74.2)
20 (64.5)
3 (9.7)
12 (92)
10 (100)
2 (67)
13 (41.9)
8 (25.8)
13 (41.9)
10 (32.3)
3 (9.7)
7 (22.6)
Overweight
2
<0.9
<0.85
130-200 mg/ dL
35-60 mg/ dL
<130 mg/ dL
30-200 mg/ dL
3
4
5
6
7
8
65-170 µg/ dL
<55%
20-250 ng/ mL
1 (4.3)
1 (4.3)
1 (4.3)
0
1+
2+
3+
4+
10 (71.4)
3 (21.4)
1 (7.2)
0
0
13 HFE gene mutations (n = 16)
I. Normal/ normal
II. C282Y/ C282Y
III. C282Y / H63D
IV. H63D/ H63D
V. H63D/ Normal
12 (75)
0
0
0
4 (25)
Statistical analysis
Chi square test was used to determine the association between
the grade of iron staining and necroinflammatory grade and the
stage of fibrosis on liver biopsies. P<0.05 was considered
statistically significant.
RESULTS
There were 31 patients (M-25, F-6) with a mean age of 38.4 years
(Range 19-70 years). Their clinical presentations included
asymptomatic transaminases detected incidentally either during
work up for other non-hepatic diseases (17 patients), generalized
weakness and body aches (8 patients), and non-specific upper
abdominal discomfort (6 patients). The rest of liver function
tests were normal and upper GI endoscopy did not show varices
in any patient. Results of the lipid profile, presence of diabetes
and impaired glucose tolerance (IGT) are shown in Table 1.
Abnormalities found in serum iron studies and on Perls Prussian
blue staining of liver biopsies are also shown in Table 1. None of
the 16 patients studied for HFE gene mutations revealed C282Y
mutations, while four (25%) patients were found to be
heterozygotes for H63D mutations. The relation between the
grading, staging, and iron positivity on Perls staining of liver
biopsies is shown in Table 2. There was no association between
the iron staining and degree of inflammation (P = 0.55) and
fibrosis stage (P = 0.09).
Table 2 Grade of iron staining, necroinflammatory grade and
stage of fibrosis in liver biopsies of 14 patients with NASH
Iron grade Necroinflammatory grade Fibrosis stage
0
1
0
0
0
0
0
0
1
0
2
0
0
1
Mild
Mild
Mild
Mild
Moderate
Mild
Mild
Mild
Mild
Mild
Mild
Mild
Mild
Moderate
2
3
3
2
2
2
2
2
3
0
1
1
2
2
No association between iron grade and necroinflammatory
grade (P = 0.55) and stage of fibrosis (P = 0.09).
DISCUSSION
We did not find significant abnormalities of serum and liver
iron in our patients with NASH. Only one patient had a high
serum iron level, one having a high ferritin level and one having
a high TS level above 55%, but the values were not high enough
to either suggest hereditary hemochromatosis (HHC) or an iron
overload state. Though we did not measure the hepatic iron
concentration (HIC) and did not calculate the hepatic iron index
(HII = HIC÷age), no significant positivity for Perls stain was
found on liver histology (Table 1). Moreover, Perls staining
on liver biopsies had no association with the degree of
necroinflammatory activity (grade) and fibrosis (stage) (Table 2).
The two-hit hypothesis[3] in the pathogenesis of NASH
includes deposition of excessive fat in hepatocytes (first hit)
and development of oxidative stress and lipid peroxidation
(second hit). In addition to other factors like insulin resistance,[14]
oxidative stress may be initiated by excessive hepatic iron.
With the saturation of mitochondrial β-oxidation by excess free
fatty acids in the liver, peroxisomal β-oxidation generates
hydrogen peroxide. In the presence of excess iron, hydrogen
peroxide is converted to hydroxyl radicals, thus leading to
oxidative stress and release of various proinflammatory and
fibrogenic products[15]. There are studies both favoring and
refuting the role of iron in the pathogenesis of NASH[4,16]. Fifty-
eight percent patients in a series by Bacon et al.[17] had elevated
iron indices, while some patients had stainable iron in their liver
biopsy specimens and an elevated hepatic iron concentration.
Another study[18] showed the presence of hyperferritinemia
with normal transferrin saturation in patients with NASH and
suggested that a simultaneous disorder of iron and glucose
and/or lipid metabolism in most cases associated with insulin
resistance was responsible for persistent hyperferritinemia. On
the other hand, Younossi et al.[19] studied 15 patients with NAFLD
including NASH and found that all patients had a normal HII and
there was no correlation between the degree of iron accumulation
and different degrees of clinically aggressive forms of NAFLD
and NASH. The relationship between the iron overload and NASH
was further complicated by not finding changes of NASH in
patients who were homozygotes for hereditary hemochromatosis
394 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 21, 2005 Volume 11 Number 3

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Duseja A et al. Iron in NASH395
and had massive iron accumulation[20]. Iron abnormalities in
patients with NASH may occur as a secondary phenomenon to
insulin resistance syndrome and non-hereditary hemochromatosis
iron overload may be one of the components of insulin resistance
syndrome or as suggested by Mendler et al.[21] the “insulin
resistance-associated hepatic iron overload” syndrome.
Earlier studies have shown the prevalence of homozygosity
and heterozygosity for either of the C282Y or H63D mutations
to be significantly higher (69%) in patients with NASH than
controls (40%)[22]. In one study, the presence of C282Y mutation
in patients with NASH was significantly associated with the
degree of iron staining, HIC and the degree of fibrosis on liver
biopsy specimens[16]. Since the C282Y HFE gene mutation was
observed almost exclusively among Caucasian subjects of
Northern European (Anglo-Celtic) ancestry, its influence on
the progression of disease was dependent on the population
studied. H63D gene mutation on the other hand is more widely
distributed but has a minor role in the pathogenesis of iron
overload. As expected, none of our patients was heterozygotes
for the C282Y gene mutation and the finding that four patients
were heterozygotes for H63D mutation might not be significant.
Chitturi et al.[5] addressed this issue of ethnicity in studying
the iron abnormalities and HFE mutation in patients with NASH.
They found that the frequency of C282Y heterozygosity was
increased only in Anglo-Celtic patients with NASH compared
to ethnic blood donors. There were no C282Y homozygotes in
NASH group and the frequency of compound C282Y/H63D
and H63D heterozygotes were not increased in NASH. In
addition, they found that hepatic iron was not a factor linked to
the hepatic fibrogenesis in patients with NASH[5]. We studied
58 normal controls and 154 individuals with β thalassemia trait
and found that no subject was either heterozygous or
homozygous for the C282Y mutation in the past few years[23].
However, the prevalence of H63D mutation was 16.5% in these
212 subjects, with an allele frequency of 8.73% with two
individuals being homozygous for the mutation. Even in
patients with hereditary hemochromatosis, both of these
mutations were negative in eight patients and one patient was
heterozygous for H63D mutation[23] suggesting that ethnic
background has influences on the frequency of C282Y HFE
gene mutation and H63D mutation can be found in normal
individuals and non-liver disease patients.
It seems that iron contributes to the pathogenesis of NASH
only in individuals with excess serum and hepatic iron. In
patients with NASH, who had no serum and hepatic iron
abnormalities, other factors might play a part in the pathogenesis
of NASH. Since C282Y HFE gene mutation has been found
almost exclusively in Caucasians of North-European descent,
it is not an important contributor in patients with NASH in
other populations including Asian Indians. On the other hand,
H63D HFE gene mutation is less specific and its presence in
patients with NASH may not be significant.
In conclusion, our study does not favor iron overload and
HFE gene mutations as major factors in the pathogenesis of
NASH in Asian Indians, but would require further studies to
confirm the findings.
REFERENCES
1 Ludwig J, V iggiano TR, McGill DB, Oh BJ. Nonalcoholic
steatohepatitis. Mayo Clinic experience with a hitherto unnamed
disease. Mayo Clin Proc 1980; 55: 434-438
2 Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic
steatohepatitis. Semin Liver Dis 2001; 21: 27-41
3 D ay CP, James OF. Steatohepatitis: a tale of two “hits”?
(editorial). Gastroenterology 1998; 114: 842-845
4 Blendis L, Oren R, Halpern Z. NA SH: Can we iron out the
pathogenesis? Gastroenterology 2000; 118: 981-983
Chitturi S, Weltman M, Farrell GC, McDonald D, Kench J,
Liddle C, Samarasinghe D, Lin R, Abeygunasekera S, George J.
HFE mutations, hepatic iron, and fibrosis: Ethnic specific asso-
ciation of NA SH with C282Y but not with fibrotic severity.
Hepatology 2002; 36: 142-149
Amrapurkar DN, Amrapurkar AD. Nonalcoholic steatohepaitis:
clinicopathological profile. J Assoc Physicians India 2000; 48:
311-313
Agarwal SR, Malhotra V, Sakhuja P, Sarin SK. Clinical, bio-
chemical and histological profile of nonalcoholic steatohepatitis.
Indian J Gastroenterol 2001; 20: 183-186
Dacie JV, Lewis SM, Hoffbrand AV. Investigation of megaloblas-
tic and iron deficiency anemias. In Dacie JV, Lewis SM (eds): Prac-
tical Hematology Churchil Livingstone: Singapore 1984: 313-336
Feder JN, Gnirke A , Thomas W, Tsuchihashi Z, Ruddy DA ,
Basava A, Dormishian F, Domingo R Jr, Ellis MC, Fullan A,
Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee
VK, Loeb DB, Mapa FA, McClelland E, Meyer NC, Mintier GA,
Moeller N, Moore T, Morikang E, Wolff RK. A novel MHC class I-
like gene is mutated in patients with hereditary haemochromatosis.
Clinical and biochemical abnormalities in people heterozygous
for hemochromatosis. Nat Genet 1996; 13: 399-408
Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri
BA , Bacon BR. Nonalcoholic steatohepatitis: a proposal for
grading and staging the histological lesions. Am J Gastroenterol
1999; 94: 2467-2474
Willett WC, Dietz WH, Colditz GA . Guidelines for healthy
weight. N Eng J Med 1999; 341: 427-434
World Health Organisation. Definition, diagnosis and classifi-
cation of diabetes mellitus and its complications–Part 1: Diag-
nosis and classification of diabetes mellitus. Geneva: World
Health Organisation 1999: 31-33
Diabetes care: report of the expert committee on the diagnosis and
classification of diabetes mellitus. Diabetes Care 2002; 25: S5-S20
Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J,
Hui JM, Fung C, Karim R, Lin R, Samarasinghe D, Liddle C,
Weltman M, George J. NASH and insulin resistance: insulin
hypersecretion and specific association with the insulin resis-
tance syndrome. Hepatology 2002; 35: 373-379
James OF, Day CP. Non-alcoholic steatohepatitis (NASH): a
disease of emerging identity and importance. J Hepatol 1998;
29: 495-501
George DK , Goldwurm S, MacDonald GA, Cowley LL, Walker
NI, Ward PJ, Jazwinska EC, Powell LW. Increased hepatic iron
concentration in nonalcoholic steatohepatitis is associated with
increased fibrosis. Gastroenterology 1998; 114: 311-318
Bacon BR, Farahvash MJ, Janney CG, Neuschwander–Tetri
BA. Nonalcoholic steatohepatitis: an expanded clinical entity.
Gastroenterology 1994; 107: 1103-1109
Fargion S, Mattioli M, Fracanzani AL, Sampietro M, Tavazzi
D, Fociani P, Taioli E, Valenti L, Fiorelli G. Hyperferritinemia,
iron overload, and multiple metabolic alterations identify pa-
tients at risk for nonalcoholic steatohepatitis. Am J Gastroenterol
2001; 96: 2448-2455
Younossi ZM, Gramlich T, Bacon BR, Matteoni CA, Boparai
N, O’Neill R, McCullough AJ. Hepatic iron and nonalcoholic
fatty liver disease. Hepatology 1999; 30: 847-850
Bacon BR. Hemochromatosis: diagnosis and management.
Gastroenterology 2001; 120: 718-725
Mendler MH, Turlin B, Moirand R, Jouanolle AM, Sapey T,
Guyader D, Le Gall JY, Brissot P, David V, Deugnier Y. Insulin
resistance-associated hepatic iron overload. Gastroenterology
1999; 117: 1155-1163
Bonkovsky HL, Jawaid Q, Tortorelli K, LeClair P, Cobb J,
Lambrecht RW, Banner BF. Non-alcoholic steatohepatitis and
iron: increased prevalence of mutations of the HFE gene in non-
alcoholic steatohepatitis. J Hepatol 1999; 31: 421-429
Garewal G, Das R, Kaur J, Chawla Y. H63D mutation of the
HFE gene in beta thalassamia traits does not cause iron over-
load and the hereditary haemochromatosis in North India is of
the non-HFE gene type. Blood 2002; 100: 46
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Edited by Wang XL