Absence of hemochromatosis associated Cys282Tyr HFE gene mutation and low frequency of hemochromatosis phenotype in nonalcoholic chronic liver disease patients in India

Absence of hemochromatosis associated Cys282Tyr HFE gene mutation and low frequency of hemochromatosis phenotype in nonalcoholic chronic liver disease patients in India
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   Journal of Gastroenterology and Hepatology (2004) 19, 86–90 Blackwell Science, LtdOxford, UKJGHJournal of Gastroenterology and Hepatology0815-93192004 Blackwell Publishing Asia Pty LtdJanuary 20041918690Original Article Absence of hemochromatosis in chronic liver diseaseV Thakur et al. Correspondence: Dr SK Sarin, Professor and Head, Department of Gastroenterology, G.B. Pant Hospital, New Delhi110 002, India.Accepted for publication 1 June 2003. HEPATOLOGY Absence of hemochromatosis associated Cys282Tyr HFE gene mutation and low frequency of hemochromatosis phenotype in nonalcoholic chronic liver disease patients in India V THAKUR,* RC GUPTAN,* AZ HASHMI,* P SAKHUJA, †  V MALHOTRA †  AND SK SARIN* Departments of * Gastroenterology and † Pathology, G.B. Pant Hospital, New Delhi, India Abstract  Background and Aim : Hereditary hemochromatosis (HHC) is an autosomal recessive disorder caus-ing primary iron overload syndrome and chronic liver disease (CLD). This genetic disease is commonlyassociated with C282Y mutation of the HFE gene, commonly seen in the Northern European popula-tion. Minor reports on HHC are available from Asia, however, so far no genetic study is available fromIndia. We prospectively studied the prevalence of C282Y mutation in CLD patients and healthy subjectsin a tertiary care referral center in India.  Methods : A total of 249 consecutive biopsy proven CLD (HBV =  112, HCV =  72, cryptogenic =  65)patients and 134 age matched healthy controls were included. Cases of secondary iron overload, preg-nancy, chronic alcoholism, age <  30 years and hepatocellular carcinoma (HCC) were excluded. A trans-ferrin saturation index (TSI) of > 60% was suggestive of a phenotypic presentation of HHC. C282Ymutation was studied by restriction fragment length polymorphism (RFLP) using genomic DNA. The387 bp fragment obtained after polymerase chain reaction was digested with 10 units of endonucleaseRsa1. The mutation was detected by creation of an additional restriction site, giving rise to fragments of 247 111 and 29 bp.  Results : While the mean TSI was comparable, serum ferritin was significantly higher in CLD patientscompared to controls (38 ±  16% vs  28 ±  13%; p =  not significant (NS), and 125 ±  18 vs  42 ±  25 ng/mL;p <  0.001). A TSI of > 60% was detected in 24 (9.64%) patients. Only one restriction site was found forendonuclease Rsa1, giving rise to two fragments of 247 and 140 bp, suggesting absence of C282Y muta-tion in the HFE gene in all patients. Conclusions : Almost 10% of nonalcoholic CLD patients in India have iron overload, but this is inde-pendent of C282Y mutation of the HFE gene. Large population based studies are recommended toinvestigate the prevalence of this rare disorder in India.© 2004 Blackwell Publishing Asia Pty Ltd INTRODUCTION Iron is an essential nutrient for the human body andhepatic parenchyma is the major site of iron storage.The amount absorbed by the body is well regulated anddepends on body iron stores and dietary iron. 1  Hered-itary hemochromatosis (HHC) is an autosomal reces-sive single gene disorder resulting in iron overloadsyndrome and accounts for clinical complications suchas cirrhosis, diabetes mellitus, cardiomyopathy andendocrine dysfunction in the third or fourth decade of life. 2,3  This disease is not uncommon in the Caucasianpopulation. 2 The etiological basis of HHC has been poorlyunderstood. In 1996, a candidate gene was identi-fied on chromosome 6 4  and was named the HFEgene. 5  It is located 4.5 mega bases telometric to theHLA-A locus and encodes for a protein of 343amino acids. Two mutations, C282Y and H63D,have been found to be associated with the HHCphenotype. The C282Y mutation is a missensemutation which changes the amino acid cystine totyrosine (C282Y). Many studies have confirmed thatmost, but not all, patients with a phenotypic diagno-sis of hemochromatosis are homozygous for C282Yin 60–100% of cases in white patients of European   Absence of hemochromatosis in chronic liver disease 87and Australian descent. 4,6–8  Population studies havesuggested that the disease prevalence is approxi-mately 1 in 300 by phenotyping and as high as 1 in150 by genotyping. 8  The frequency of heterozygousstate for the hemachromatosis gene is as high as 1in 10 in patients of Northern European ancestry. 9 Limited data is available on Asian populationsregarding HFE gene mutation. An earlier reportfrom a random sample of 106 cases revealed 0.2%frequency of C282Y mutation in the general popu-lation in India, 10  although this study could notpredict the proportionate population with HH phe-notype. We investigated the prevalence of HHCphenotype and C282Y mutation in chronic liverdisease patients, without otherwise obvious reasonsfor iron overload. METHODS Patients Consecutive patients with liver biopsy proven chronicliver disease seen in the Department of Gastroenter-ology in a tertiary care liver diseases referral hospi-tal were studied prospectively. The patients wereexcluded if they were less than 30 years of age, hada history of multiple blood transfusion or blood lossin the past 60 days, hemolytic anemia, hemoglobin-opathy, pregnancy, oral or i.v. iron supplementation,chronic alcoholism ( >  80 g/day ¥  5 years) or if hepa-tocellular carcinoma (HCC) was present. The diag-nosis of chronic liver disease was based on clinical,serological and biochemical criteria and confirmedon histology. A total of 249 patients with CLD seenbetween January 2000 and February 2003 wereenrolled in the present study. The etiology of liverdisease was due to chronic hepatitis B viral (HBV)infection ( n   =  112), chronic hepatitis C viral (HCV)infection ( n   =  72), or of cryptogenic srcin ( n   =  65).Cryptogenic chronic liver disease was diagnosed if there was no evidence of HBV (HBsAg, HBeAg,HBeAb, anti-HBc and HBV DNA negative), HCV(anti-HCV and HCV RNA negative), autoimmunehepatitis (antinuclear antibody, antimicrosomal anti-body negative), Wilson’s disease or alfa-1 antitrypsindeficiency. Wilson’s disease was diagnosed if serumceruloplasmin was more than 20 m g/dL and K.F. ringsseen and liver biopsy suggestive of significant copperdeposition. Alfa-1 antitrypsin deficiency was excludedby measuring the serum levels of alfa-1 antitrypsinusing commercially available kits. Controls Controls comprised 134 healthy adults with compara-ble age and sex. All patients and control subjectswere of Asian Indian srcin. Approval for the study wasobtained from the Institutional Ethical Committee andinformed consent for participation was obtained fromeach subject. Dietary assessment The 72 h dietary recall method was used to assess thedaily iron intake. Subjects were asked to record whatthey had consumed for two working days and one non-working day. A food frequency checklist consisting of iron-rich food commonly consumed by the local popu-lation was used to supplement information about ironintake. The iron content of the food was determined bythe standard nutritive value tables from the NationalInstitute of Nutrition, Hyderabad, India and was com-pared with the recommended dietary allowances(28 mg/day for men and 30 mg/day for women). 11 Serum iron and transferrin estimation A fasting morning blood sample was collected in PVCtubes for iron studies using stainless steel needles.Serum was separated in iron-free PVC tubes. Investiga-tions were performed within one week of collection of ablood sample. The serum iron and total iron bindingcapacity (TIBC) were measured by the colorimetricmethod with ferrozine chromogen and the transferrinconcentration by immunoturbidometry (Randox Labo-ratory, San Diego, CA, USA). Deionized water wasused to avoid a false increase in the iron levels. Total iron binding capacity Total iron binding capacity was determined using thecolorimetric method (Randox Laboratory) and theknown concentration of iron solution was added to sat-urate transferrin in patient’s serum. The unbound ironwas precipitated with basic magnesium carbonate. Aftercentrifugation, the iron in the supernatant was deter-mined. The TSI was calculated as serum iron ¥  100divided by TIBC. Serum ferritin Serum ferritin was measured using an enzyme immunoassay kit (Melotec Biotechnology, Madrid, Spain). Foranalytical accuracy and precision, both normal and ele-vated serum levels were assayed for internal quality con-trol (IQC). QC multisera (Randox Laboratory) wererun simultaneously along with the test samples. Thus allthe iron parameters tested were standardized. Themean ±  1 SD was taken to define the normal range forall of the iron parameters. A patient with values beyond ± 1 SD was considered abnormal, as describedpreviously. 12 DNA amplifications Genomic DNA was isolated from peripheral blood leu-kocytes by the phenol-chloroform method using thewhole blood. Positive and a negative control DNA werekindly provided by Professor HE Blum, Frieburg,  88 V Thakur et al.Germany. Patient’s DNA was amplified by polymerasechain reaction (PCR) in a 25 m L reaction mixture. Theoligonucleotide primer sequences used were thosedescribed previously. 4  In brief, these were: forward 5 ¢ -TGG CAA GGG TAA ACA GAT CC3 ¢  and reverse5 ¢ CTC AGG CAC TCC TCT CAA CC3 ¢ . The reactionconditions used for the PCR were 96 ∞ C for 15 s, 60 ∞ Cfor 45 s and 72 ∞ C for 45 s for 35 cycles. The amplifiedfragment of 387 bp was resolved on 3% agarose gel tocheck the amplification. Restriction fragment length polymorphism The mis-sense C282Y mutation creates a new RsaIrestriction site. 13  In normal subjects, the 387 base pairPCR product digested with enzyme RsaI produces twoDNA fragments of 247 and 140 bp (Fig. 1). In the pres-ence of C282Y mutation, the mutant DNA generatesthree fragments of 247 111 and 29 bp. To detect theC282Y mutation, the amplified product was digestedwith 10 U endonuclease RsaI at 37 ∞ C for 4 h andresolved on 3% agarose gel to check the amplification. Hepatic iron staining Iron was stained in the liver tissue using Pearl’s Prussianblue reaction for ferric iron. 14  Ferric iron is released bytreatment with dilute hydrochloric acid and reacts witha dilute solution of potassium ferrocyanide to producean insoluble blue compound, ferric ferro cyanide(Prussian blue). The iron staining was graded 1–4 incoded biopsy samples by two independent pathologists.A grade of 4 represented clinical suspicion for HHCphenotype. Statistics Results were expressed as the mean ±  SD. The modified t  -test and chi-squared test were used to determine thestatistical significance of different variables. A P  -value of  < 0.05 was considered significant. RESULTS Demographic profile The mean age of chronic liver disease patients andhealthy controls was comparable (42 ±  16 vs  40 ±  14years; p =  not significant [NS]). There was male pre-ponderance in both groups. Mean height and bodyweight of subjects in the two groups were comparable(Table 1). Dietary iron profile Majority of the subjects in the chronic liver disease(74%) and the control group (69%) were vegetarian.The 24 h iron intake was also comparable in bothgroups, although the mean intake was only approxi-mately 50% of the recommended dietary allowance setfor this population (Table 1). Serum iron profile and the transferrin saturation index Mean serum iron levels were found to be within the nor-mal range (75–175 m g/dL) in subjects belonging to bothgroups. The mean levels in the chronic liver diseasegroup were higher than the control group, however, thedifference was not significant (123 ±  48 vs  95 ±  51 m g/dL). Twenty-four (9.64%) CLD patients had serum Figure 1 Restriction fragment length polymorphism, Rsa1digestion for Cys282Tyr mutation in patients with transferrinsaturation index (TSI) > 60% .  Lane 1, negative control; lane 2,positive control; lanes 3–12, patient samples with TSI > 60%;lane 13, molecular weight marker. Table 1 Demographic profile of the study groupsParameterChronic liver disease ( n   = 249)Healthy controls ( n   = 134) P  -valueMean age (years)42 ± 1640 ± 14NSMale: female ratio195 : 5488 : 50NSDietary iron intake (mg/day)13.2 ± 5.212.1 ± 6.4NSBody weight (kg)58.3 ± 1060.2 ± 14NSHeight (m)1.67 ± 1.81.69 ± 2.6NSVegetarian diet 185 (74%)92 (69%)NSNS, not significant.   Absence of hemochromatosis in chronic liver disease 89iron levels higher than normal. Overall mean TSI in thechronic liver disease patients was comparable to thecontrol group (38 ±  16% vs  28 ±  13%; p =  NS). The 24cases with high serum iron also had a TSI of more than60%. Serum ferritin Mean serum ferritin levels were found to be within thenormal range (8–270 ng/mL). In chronic liver diseasepatients, the mean serum ferritin level was higher com-pared to the controls (125 ±  18 vs  42 ±  25 ng/mL;p <  0.001). Hepatic iron staining Hepatic iron staining was negative or negligible (traces)in almost all patients. However, detailed analysis of the24 patients with a TSI of > 60% revealed grade 1 and 2staining in 15 and 3 patients, respectively. The remain-ing 6 patients had only traces of stainable iron in theliver tissue. None demonstrated grade 4 iron stainingsuggestive of the HHC phenotype. Comparative analysis of patients with a transferrin saturation index < 60% and > 60% Of the 249 patients studied, 24 (9.64%) had a meanTSI > 60%. Demographically these patients were com-parable. Serum iron was found to be significantly higherin patients with TSI >  60% (197 ±  28 m g/dL vs 73 ±  25 m g/dL). Although serum ferritin levels werehigher in these patients and were statistically compara-ble to patients with TSI <  60% (Table 2). The etiologyof chronic liver disease in patients with high TSI wasHBV related in 12, HCV related in 6 and cryptogenic in6. Genotypes for hemochromatosis Only one restriction site for the enzyme RsaI was foundin amplicons of liver disease as well as the control groupsubjects, giving rise to two fragments of 247 and140 bp. This suggested absence of C282Y mutation inany group. Importantly, even in the 24 patients with TSI > 60% and positive hepatic iron staining, no mutationwas observed at amino acid 282 (Fig. 1). DISCUSSION The results of the present study demonstrate theabsence of C282Y HFE gene mutation in a subset of CLD patients and control subjects in India. Our resultsare similar to the report from an earlier small seriesfrom this region. 15  In the present study, patients withchronic liver disease had significantly higher mean fer-ritin levels ( P    <  0.001) compared to age matched con-trols, suggestive of altered iron metabolism. Secondaryiron overload is a well recognized feature of end-stageliver disease of various etiologies, notably, alcoholic cir-rhosis, hepatitis C and alpha-1 antitrypsin deficiency. 8,16 We found a high TSI ( >  60%) and grade 1–2 hepaticiron staining in approximately 10% of CLD patients.Elevated TSI is the earliest phenotypic abnormality inHHC. A recent report from Australia found that TSI > 45% could identify 98% of homozygotes without mis-identifying a healthy person or heterozygous carrier. 17 Thus TSI is a useful marker suggestive of C282Y HFEgene mutation in iron overload syndrome. However,serum ferritin levels and TSI are often elevated inpatients with acute or chronic liver disease and nonal-coholic steatohepatitis, 18  creating an unrealistic pheno-typic presentation of HHC in a clinical setting. Severalinvestigators have examined the role of HFE gene muta-tions in borderline liver disease cases associated withmild iron overload. An increased prevalence of C282Yin porphyria cutanea tarda is a recent example. 19 Similarly, C282Y mutation was demonstrated to beassociated with mild iron overload in nonalcoholicsteatohepatitis (NASH). 20 A severe case of HHC has never been reported fromthe Indian subcontinent except for one isolated casereport. 21  Ironically, in this patient only iron stainingstudies were performed. There is also a major belief thatHHC phenotype possibly has low expression in theIndian population due to a low dietary iron intake, espe-cially the heme form. 21  This could have been a theoret-ical possibility explaining low prevalence of HHCphenotypes in India, however, the study by Feder et al  . 4 demonstrated that C282Y genetic mutation of chromo-some 6 results in a two-fold increase in intestinal ironabsorption,   mostly   independent   of    dietary   intake. 4  Hom-ozygosity for C282Y was known to be associated withrisk and the odds ratio of 23 : 00 to develop HHC. 15 Thus HFE gene testing is a very sensitive and specificapproach for diagnosing cases of HHC.In the present study, almost 10% of CLD patientshad raised TSI, ferritin and mild stainable hepatic iron.However, none had C282Y mutation. This data sug-gests that HHC is not an important cause of cirrhosis oriron overload in India. Another candidate gene forHHC is H63D, which is frequent in Europe and rangesbetween 6 and 30% cases. 15  We have not studied thismutation in our patients and cannot rule out the possi- Table 2 Comparison of chronic liver disease patients with atransferrin saturation index (TSI) < 60% and > 60%ParametersTSI <  60%( n   = 225)TSI >  60%( n   = 24)Mean age (years)41 ± 1442 ± 11Height (m)1.65 ± 1.01.68 ± 1.5Weight (kg)61 ± 1560 ± 12Serum iron ( m g/dL)73 ± 25197 ± 28*Serum ferritin (ng/mL)72 ± 54115 ± 60TIBC (g/dL)282 ± 72251 ± 68* P    <  0.05. TIBC, total iron binding capacity.  90 V Thakur et al.bility of this genetic defect in them. However, with theknowledge that patients with this mutation are also het-erozygous for C282Y (compound heterozygote), 22  thepossibility of H63D existing alone seems unlikely inIndia. An earlier global survey from the Asian regionhad suggested the absence of C282Y mutation in Chi-nese, Japanese, Micronesian and Nepalese subjects. 22 Moreover, a recent report of low prevalence of C282Ymutation in the Chinese population has generated newinterest. Our results suggest that the cause of iron over-load syndrome in Indian patients with CLD is possiblynot C282Y mutation related. Furthermore, the fre-quency of C282Y mutation in the general populationmay also be negligible. The results of the present studyfurther substantiate the fact that C282Y is absent inIndia and a rare occurrence in heterozygous state is onlydue to admixture with European alleles. 23  This is imper-ative information on the HHC phenotype in a subcon-tinent but does not dampen the spirits for a largepopulation based study to detect this rare genetic dis-order in India. ACKNOWLEDGMENTS The authors acknowledge the support of Professor HEBlum, Chairman, Department of Internal Medicine,University of Frieburg, Germany, for providing posi-tive/negative control DNA and primers and the IndianCouncil of Medical Research for providing financialassistance for the completion of the present study. Weare also thankful to the Venous Research Foundation(IL, USA) for allowing Dr RC Guptan, Director, tocomplete this research. REFERENCES 1Guptan RC, Thakur V, Sarin SK. Iron and liver: Currentconcepts in the pathogenesis and management of chronicliver disease.  J. Assoc. Phys. India  1996; 44 : 198–202.2Edwards CQ, Griffen LM, Goldgar D, Drummond C,Skolnick MH, Kusher JP. Prevalence of hemochromatosisamong 11065 presumably healthy blood donors.  N. Engl. J. Med.  1988; 318 : 1355–62.3Bacon BR, Tavill AS. Hemochromatosis and the ironoverload syndromes. In: Zakim D, Boyer TD, eds. 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