Evidence for non-HFE linked hemochromatosis in Asian Indians.

Abstract

Hereditary hemochromatosis is commonly due to two HFE1 (Histone Family E1) gene mutations - H63D and C282Y. Mutations in the Asian Indians are less well studied.
The aim of this preliminary study was to find out the prevalence of HFE gene mutations in nonviral liver cirrhosis patients.
Unexplained liver cirrhosis cases with transferrin saturation> 45%, attending the gastroenterology clinic in the years 2004 and 2005 were subjects of the prospective study. Asymptomatic individuals with negative family history of hemolytic anemia or liver disease served as controls.
The clinical presentation was recorded in the patients. Transferrin saturation was estimated by standard colorimetric technique. The two common mutations in HFE1 gene and Y250X mutation of TFR (transferrin receptor) gene were studied by polymerase chain reaction based methods.
A majority of the cases were sporadic, but family history was positive in four patients. In one family with multiple affected members, there was clear evidence of autosomal dominant inheritance. Seven out of 31 (22.6%) of unexplained cirrhosis cases were positive for mutations. One was homozygous for H63D. In healthy controls, prevalence was 8.1% (6/74). None of the patients or controls was positive for C282Y mutation of HFE1 or Y250X of TFR gene.
Thus, in a number of cases of hemochromatosis in Indians, a gene with dominant inheritance may be involved in causation of the phenotype. The prevalence of HFE mutations in Indians is comparable to that reported from neighboring countries. It is worth studying other mutations in HFE gene and other iron overload genes in cryptogenic cirrhosis cases.

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H63D. In healthy controls, prevalence was 8.1% (6/74). None of the patients or
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RsaI digestion, the product was run on 7%
polyacrylamide gel electrophoresis (PAGE).
The Y250X mutation of the TFR gene was
studied by PCR-RFLP technique,[7]
Indian Journal of Medical SciencesIndian Journal of Medical Sciences
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Indian Journal of Medical Sciences
(INCORPORATING THE MEDICAL BULLETIN)
VOLUME 60 DECEMBER 2006 NUMBER 12
ORIGINAL CONTRIBUTIONS
491 492?EVIDENCE FOR NON-HFE LINKED HEMOCHROMATOSIS
Histone Family E (HFE) gene mutations are
important in causation
hemochromatosis in Caucasians[1] (HFE,
OMIM 235200). Mutation H63D (C→G
transversion) increases relative risk of
developing hemochromatosis in individuals
aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) levels were
measured using commercially available kits.
Controls were healthy staff of the hospital and
included the permanent staff, residents and
Ph.D. students with negative family history
of primary
heterozygous for C282Y mutation. Mutations
in the HFE protein alter affinity of the
transferrin receptor (TFR) for its ligand
transferrin and thus attenuate uptake of
transferrin-bound iron from plasma. There are
limited studies[2-4] on the role of HFE gene in
iron overload in Asian Indians and they have
identified mainly the H63D mutation. The aim
of this study was to determine the role of two
common HFE gene mutations and transferrin
receptor mutation Y250X in cryptogenic
cirrhosis cases.
of liver disease and hemolytic anemia.
Consent was taken while filling the pro forma.
Ethical clearance was obtained from the
Review Board.
EVIDENCE FOR NON-HFE LINKED HEMOCHROMATOSIS IN ASIAN?
INDIANS
I. PANIGRAHI, F. AHMAD, R. KAPOOR, P. K. SHARMA*, G. MAKHARIA*, R. SAXENA
The DNA (deoxyribonucleic acid) extraction
was done from citrated blood by standard
phenol chloroform method. The molecular
analyses for HFE mutations H63D and
C282Y were done by polymerase chain
reaction (PCR) using allele specific primers
described by Mullighan et al.[5]
cases were confirmed by haplotype analysis
and PCR-RFLP method,[6] with enzymes MboI
and RsaI respectively. The mutation 63D
abolishes the MboI site and the mutation
282Y creates an additional RsaI site in the
PCR product. Electrophoresis was done on
2% agarose gel for allele-specific PCR
products, following MboI digestion. Following
The positive
controls was positive for C282Y mutation of HFE1 or Y250X of TFR gene.
CONCLUSIONS: Thus, in a number of cases of hemochromatosis in Indians, a gene
with dominant inheritance may be involved in causation of the phenotype. The
prevalence of HFE mutations in Indians is comparable to that reported from
neighboring countries. It is worth studying other mutations in HFE gene and other
using
α1 anti-trypsin were normal. The diagnosis of
cirrhosis was based on clinical assessment,?
ultrasonographic and MRI findings. Patients
with history of chronic alcoholism and those?
positive for the viral hepatitis markers-
HbSAg, anti-HCV and autoimmune markers-
anticardiolipin antibody
anticoagulant were excluded from the study.
Transferrin saturation was quantitated by
standard colorimetric technique. The
restriction enzyme MaeI.?
RESULTS?
ABSTRACT
BACKGROUND: Hereditary hemochromatosis is commonly due to two HFE1 (Histone
Family E1) gene mutations – H63D and C282Y. Mutations in the Asian Indians are
less well studied. AIMS: The aim of this preliminary study was to find out the prevalence
of HFE gene mutations in nonviral liver cirrhosis patients. SETTINGS AND DESIGN:
Unexplained liver cirrhosis cases with transferrin saturation >45%, attending the
MATERIALS AND METHODS?
gastroenterology clinic in the years 2004 and 2005 were subjects of the prospective
study. Asymptomatic individuals with negative family history of hemolytic anemia or
liver disease served as controls. MATERIALS AND METHODS: The clinical
The cryptogenic cirrhosis cases were
consecutive nonviral hepatitis cases
attending the gastroenterology clinic in the
years 2004 and 2005 at a tertiary care center.
In the prospective study, the cases included
were those in which MRI was suggestive of
pigment deposition, transferrin saturation was
>45% and tests for autoimmune markers
presentation was recorded in the patients. Transferrin saturation was estimated by
standard colorimetric technique. The two common mutations in HFE1 gene and Y250X
mutation of TFR (transferrin receptor) gene were studied by polymerase chain reaction
based methods. RESULTS: A majority of the cases were sporadic, but family history
was positive in four patients. In one family with multiple affected members, there
was clear evidence of autosomal dominant inheritance. Seven out of 31 (22.6%) of
unexplained cirrhosis cases were positive for mutations. One was homozygous for
iron overload genes in cryptogenic cirrhosis cases.?
The age of the liver cirrhosis patients in the
present study ranged from 28 to 59 years
(median 38 years). There were 19 males and
12 females. There were 74 normal controls.
Most patients were symptomatic from the
second decade of life, with an average age
Key words: Chronic hepatitis, cirrhosis, ferroportin, mutations, transferrin saturation
and lupus
Correspondence
Inusha Panigrahi, 46/ Y1, C-block, Dilshad Garden,?
Shahdara, Delhi - 110 095, India. E-mail: inupan@yahoo.com
Departments of Hematology and *Gastroenterology,
All India Institute of Medical Sciences, Delhi, India
Indian J Med Sci, Vol. 60, No. 12, December 2006 Indian J Med Sci, Vol. 60, No. 12, December 2006

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1200 bp
1000 bp
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In haplotype analysis in the present study,
the product size was more than that
mentioned in reference.[5] The exact size was
found to be 1,997 bp by comparison with
sequence obtained from the NCBI blast site
(www.ncbi.nlm.nih.gov/BLAST). Since the
sizes of products obtained after digestion
with RsaI were not found from any available
reference, the sequence analysis of the gene
from above NCBI site revealed PCR product
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other genes or digenic inheritance of
mutations.
INDIAN JOURNAL OF MEDICAL SCIENCES?
493
of 26.5 years at onset. The transferrin
saturation (TS) was greater than 45% in all
cases (range 47-73%). The TS between 20
and 45% was taken as normal.
C282Y mutation are shown in Figure 2. There
was no difference in mean transferrin
saturation in the patients with and without the
H63D mutation (46.3 vs. 45.6%).
All the patients had hyperpigmentation of skin
and liver dysfunction with increased?
transaminase levels. AST and ALT levels
were 102.3 and 114.1 units respectively. In
four families, there was a strong family
history of liver disease; and in one, with
pedigree with multiple generations affected,
suggestive of autosomal
inheritance. Seven of 31 cases (22.5%) were
positive for H63D mutation. One was
homozygous for H63D. In contrast, in
controls, 6 of 74 (8.1%) were heterozygous
for H63D mutation. None of the patients or
controls showed the C282Y mutation of HFE
gene or Y250X mutation of TFR2 gene.
Figure 1 shows the size of HFE haplotype
product to be around 2 kb. The sizes of
products obtained after digestion with RsaI for
DISCUSSION
494?
EVIDENCE FOR NON-HFE LINKED HEMOCHROMATOSIS
of Thailand.[10]?
size to be 307 bp and sizes after enzymatic
digestion to be 185 and 122 bp for the normal
allele and 156, 122 and 29 bp for the mutant
allele.
In a previous report by Rees et al,[11] 1 of 47
thalassemia intermedia cases from this
region, a resident of Punjab, was positive for
C282Y mutation; however, more recent
studies have found absence of this
mutation,[3,4] which is comparable to the
present study.
Transferrin receptor mutations (OMIM,
604250) are also
hemochromatosis. None of the patients in the
present study showed Y250X mutation.
Camaschella et al first reported this mutation
from Sicily.[17] TFR2 mutations have also
been reported in Sardinians and Italians.[7]
The mutations are likely to be different in
different populations.
associated with
1 2 3 4 5
2600 bp?
2000 bp?
1500 bp?
dominant
The HFE1 gene at 6p21.3 is commonly
implicated in hereditary hemochromatosis,
with two mutations H63D and C282Y present
in majority of cases in the Western
population. In the present study, most
patients were heterozygous for H63D
mutation. Comparable frequency has been
reported from Italy, Finland, Africa, Australian
population[1,8] and Turkish population.[9]
previous study in liver disease from India
also did not find C282Y mutation.[3]
The H63D allele frequency was 4.1% (6/148
chromosomes) in control population in the
present study. This is lower than that reported
in North Europeans[6] but comparable to that
Figure 1: Lanes 1 and 2: HFE normal haplotype
products; lane 3: DNA molecular marker XIV (Roche
Diagnostics, GmBH, Germany); lanes 4 and 5: 2 kb
and 1.2 kb amplification products from factor VIII
gene. Sequence comparison from NCBI Blast site
revealed HFE haplotype product size to be 1,997 bp
In genetic hemochromatosis patients from
Western Australia, the C282Y mutation was
homozygous in 64 of 72 cases.[12] In an Italian
study on patients with increased transferrin
saturation (>50% in males and >45% in
females), overall C282Y allele frequency was
9% and that of H63D and S65C was 22.2 and
1.4% respectively.[13] In noncirrhotic HBV
patients, HFE mutations were not found to
have significant role in the causation of iron
overload,[14] though 27% patients had
elevated transferrin saturation. In another
study,[15] patients who were heterozygous for
C282Y and H63D mutations exhibited higher
iron serum parameters than subjects without
mutations. H63D mutation in
heterozygote state has also been reported in
NASH patients in an Indian study.[16]
The other genes causing severe iron-loading
disorder are hepcidin (HAMP) on 9q13 and
hemojuvelin (HJV) on 1q21.[18] Many cirrhotic
patients in the present study had severe
presentation and earlier onset of disease
manifestations. Mutations in ferroportin gene
[HFE4 (OMIM 606069)]
associated with autosomal
hemochromatosis. This gene needs to be
further studied on a larger patient population.
There is also a possibility of mutations in the
ACKNOWLEDGMENTS
A
these
have been
dominant
12
3
4
201 bp?
185 bp?
180 bp
Figure 2: Lane 1: pBR Msp I digest (Bangalore
Genei); lanes 2-4: digested products 185 and 122 bp.
None of the patients in the gel showed the 156 bp
product, suggestive of presence of mutant allele
160 bp
147 bp
122 bp
500 bp
Indian J Med Sci, Vol. 60, No. 12, December 2006 Indian J Med Sci, Vol. 60, No. 12, December 2006
We thank all the patients for participating in the study
and the residents in gastroenterology for providing
the samples.
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Source of Support: Nil, Conflict of Interest: None declared.
Indian J Med Sci, Vol. 60, No. 12, December 2006