ATP7B mutations in families in a predominantly southern Indian cohort of Wilson s disease patients

Transcription

1 Original Article ATP7B mutations in families in a predominantly southern Indian cohort of Wilson s disease patients S Santhosh, R V Shaji,* C E Eapen, V Jayanthi, S Malathi, # M Chandy,* M Stanley, S Selvi, G Kurian, G M Chandy Departments of GI Sciences and *Hematology, Christian Medical College, Vellore; Department of Medical Gastroenterology, Stanley Medical College, Chennai; and #Department of Pediatrics, Institute of Child Health, Chennai Objective: To analyze ATP7B mutations in Wilson s disease (WD) patients from the Indian subcontinent and to correlate these with WD phenotype. Methods: We studied 27 WD patients from 25 unrelated families. Twenty-two families were from three southern Indian states Tamil Nadu, Andhra Pradesh and Kerala. We applied conformation- sensitive gel electrophoresis (CSGE) to screen for the mutations in patients and their families. PCR products exhibiting aberrant patterns in CSGE were subjected to direct DNA sequencing. As siblings affected by WD within a family share identical ATP7B genotype, we compared WD phenotype among affected siblings within families. Results: ATP7B mutations were detected in 22 of the 25 probands 13 were homozygotes and 9 were compound heterozygotes. Eleven novel mutations were detected. Only two common mutations were found: G3182A in 4 (16%) and C813A in 3 (12%) probands. Hot spots for ATP7B mutations were exons 18 and 13. Lack of common dominant mutations prevented correlation of individual ATP7B mutations with WD phenotype. Symptomatic WD in a live sibling was not found in any family. In 8 families, a sibling died of presumed WD in 6 of these, WD phenotype was identical to that in the proband. Conclusions: We describe the spectrum of ATP7B mutations including 11 novel mutations in Indian WD patients and document lack of a single dominant mutation. Identical WD phenotype among siblings in only 6 of 8 families with >1 child affected by WD suggests that factors other than ATP7B mutations influence WD phenotype. [Indian J Gastroenterol 2006;25: ] Wilson s disease (WD) is an autosomal recessive disorder of copper metabolism caused by defective biliary excretion of copper, leading to hepatic and/or neuropsychiatric disease in children and young adults. It is caused by mutations in the ATP7B gene, which is located on chromosome 13 and consists of 21 exons. The ATP7B gene product is postulated to transport copper out of the hepatocyte into the biliary Copyright 2006 by Indian Society of Gastroenterology canaliculus and plasma. 1 We studied the spectrum of ATP7B mutations in patients with WD, mostly from three southern Indian states, and in their family members. Methods The diagnosis of WD was based on the presence of low serum ceruloplasmin (normal: U/L) and of Kayser-Fleischer (KF) ring on slit-lamp examination of the cornea. In patients who had only one of these, elevation of 24-hour urine copper (normal value: <150 µg) was needed to diagnose WD. Liver involvement was ascertained based on clinical evaluation, liver function tests and ultrasonography; liver biopsy was done when necessary. Neuropsychiatric manifestations were assessed at clinical examination. WD phenotype was assigned as per an international consensus. 2 ATP7B mutation analysis DNA was isolated from peripheral blood leukocytes of patients, their family members, and 45 healthy controls, using the phenol-chloroform method. 3 All the 21 exons of the ATP7B gene and their flanking intronic regions were amplified by polymerase chain reaction (PCR) using previously described primers. 4,5 Each 25-µL reaction contained 1x concentration of a ready reaction mix (ABgene; Epsom, UK): 75 mm Tris-HCl (ph 8.8), 20 mm ammonium sulfate, 0.2 mm each of deoxynucleotide triphosphate, 0.01% (v/ v) Tween-20, 1.25 U of Thermoprime Plus DNA polymerase (ABgene), pmol of each primer and 50 ng of genomic DNA. We used conformation-sensitive gel electrophoresis (CSGE) as a mutation screening tool. 6 In brief, 3 µl of each PCR product was mixed with an equal volume of PCR product from a healthy person with ATP7B gene sequences similar to those included in the GenBank database. The mixture was denatured at 95 o C for 5 min, followed by incubation at 55 o C for 45 min to allow heteroduplex formation, and then loaded (3 µl; mixed with 2 µl of loading buffer) on

2 a 10% CSGE gel (50 cm long, 20 cm wide) that had been pre-run for 1 hour at 750 V/cm. Following electrophoresis at 450 V/cm for 17 hours, the gel was examined after ethidium bromide staining; PCR products with band pattern different from that of the healthy control were sequenced. Family studies For each family, medical histories of all the members (patients, and available parents, siblings and children) and the presence or absence of consanguinity was recorded. The family members were screened for WD using clinical examination, copper chemistry and mutation screening by CSGE. For deceased siblings of patients with WD, detailed history of any illnesses was obtained by interviewing the parents and reviewing any available medical records. The deceased sibling was considered to have had presumed hepatic WD in case of death in childhood of an illness associated with jaun- dice, ascites, with or without hematemesis, or presumed neurological WD in case of disabling writing disabilities, gait abnormalities or spasticity. Phenotype-to-mutation correlation Failure to find a dominant ATP7B mutation in our patients and lack of symptomatic disease among living family members in their families limited such analysis to comparison of disease phenotype of patients with that of deceased siblings. The study was approved by the institutional Ethics Committee. All study subjects provided informed consent. Results Between June 2000 and May 2001, we enrolled 27 patients (18 male) with WD from 25 unrelated families. Most patients belonged to three southern Indian states (Tamil Nadu 15, Kerala 4, Andhra Pradesh 3); one patient each belonged to Punjab, West Bengal, Table 1: ATP7B mutations and Wilson s disease phenotype in Indian WD patients Patient Age (y) at onset ATP7B mutation Disease phenotype number of symptoms / gender 1 12/F EX2D C813A(+/-) Hepatic (H2) 2 6/F EX2D C813A(+/+) Hepatic (H2), hemolytic anemia 3 # 8/M EX2D C813A(+/+) Hepatic (H2) 4 13/M EX 2E G997A(+/-) EX 8 C2302T(+/+) Hepatic (H2), renal tubular acidosis 5 13/M EX 5 G1847A(+/+) Hepatic (H2) 6 # 9/F EX delc(+/+) Neurological (N2) 7 9/M EX 8 C2145A(+/-) EX 14 G3182A(+/-) Neuropsychiatric (N2) 8 # 35/F EX8 C2302T(+/+) Hepatic (H2) 9 9/M EX13 G2906A(+/+) Hepatic (H2) /M EX 13A3029G(+/+) Hepatic (H2), arthropathy 10.2* 16/M EX 13A3029G(+/+) Asymptomatic (H2) 11 # 21/M EX 13 C3008T(+/+) Hepatic (H2) 12 # 20/F EX 13 C3008T(+/-) Hepatic (H2) 13 # 4/M EX14 G3182A(+/+) Hepatic (H2) 14 9/M EX14 G3182A (+/-) EX18 A3809G(+/-) Neuropsychiatric (N2) 15.1 # 10/M EX 15 C3282G(+/-) Hepatic (H2) 15.2 # * 11/M EX 15 C3282G(+/-) Asymptomatic (H2) 16 # 6/M EX 18 A3809G(+/+) Hepatic (N1), neurological 17 # 13/M EX (6T>C) (+/-) / T3890A(+/+) Hepatic, neurological (N1) 18 4/F EX18 C3722T(+/-) Neurological (N2) 19 # 9/F EX delc(+/-) Hepatic, neurological (N1) 20 35/M EX18 C3895T(+/-) Hepatic, neurological (N1) 21 # 6/M EX 19 G4021A (+/+) Hepatic, neurological (N1) 22 11/F EX14 G3182A(+/-) EX (3A>G)(+/-) EX9 C2383T(+/-) Hepatic (H2) 23 7/F Not identified Hepatic (H2) 24 9/M Not identified Hepatic (H2) 25 9/M Not identified Neurological (N2), amino aciduria EX: exon; H2, N1 and N2 refer to international consensus classification of WD phenotype 2 # Patient born of consanguineous marriage * Pre-symptomatic WD detected on family screening in patient 10.2 (son of patient 10.1) and 15.2 (brother of patient 15.1); all other patients were probands with symptomatic WD (+/+) and (+/-) denote homozygous and heterozygous mutation, respectively 278 Indian Journal of Gastroenterology 2006 Vol 25 November - December

3 Table 2: Mutations in ATP7B gene in Indian Wilson s disease (WD) patients Mutation Codon Exon Amino acid Domain No. of WD change change chromosomes detected in patients Mis-sense WD phenotype and ATP7B mutations in symptomatic patients Age at onset of symp G997A GGG-AGG 2 Gly333Arg Cu3 1 tomatic WD (n=25) was C2302T* CCC-CTC 8 Pro768Leu Tm4 4 median (range) 9 (4-48) C2383T CTC-TTC 9 Leu795Phe Tm4/Td 2 years, being <5 years G2906A CGG-CAG 13 Arg969Gln Ch/Tm6 4 in 2 patients, 5-10 years A3029G* AAG-AGG 13 Lys1010Arg Ch/Tm6 3 in 13 patients, C3008T GCG-GTG 13 Ala1003Val Ch/Tm6 5 years in 6 patients, and G3182A GGG-GAG 14 Gly1061Glu ATP Loop 2 C3282G TTC-TTG 15 Phe1094Leu ATP Loop 2 >20 years in 4 patients. A3809G AAT-AGT 18 Asp1270Ser ATP Hinge 3 Fifteen patients had T3890A* GTC-GAC 18 Val1296Asp ATP Hinge 1 chronic hepatic presen C3722T* GCA-GTA 18 Ala1241Val ATP Hinge 1 tation (H2), 5 had C3895T* CTT-TTT 18 Leu1299Phe ATP Hinge 1 symptomatic liver and Non-sense neurological involve C813A TGC-TGA 2 Cys271ter Cu3 5 ment (N1), and 5 had C2145A* TAC-TAA 8 Tyr715ter Tm4 1 symptomatic neurologi- Deletion cal involvement alone 1962delC* 7 Tm1 2 (N2) (Table 1). 3895delC* 18 ATP Hinge 1 Splice site Mutations in the G4021A* 19 Splice site acceptor 2 ATP7B gene were iden 4021(3A>G)* 19 Splice site acceptor 1 tified in 22 (88%) of 3903 (6T>C)* 18 Splice site donor 1 the 25 symptomatic pa- Putative # tients (Table 1); of G1847A CGG-CAG 5 Arg616Glu After Cu6 2 these, 13 had homozy * Novel mutations identified with respect to particular nucleotide change were absent in 90 gous mutations and 9 normal chromsosomes tested # had compound het This putative mutation was absent in 90 normal chromosomes tested Tm: transmembrane domain, Ch: channel membrane, Cu: copper-binding domain, Td: transduc- erozygous mutations tion domain (both alleles identified in 2 patients, only one Bihar, Chattisgarh and Bangladesh. Twelve patients allele identified in 6, and triple heterozygous mutawere born of consanguineous unions; of these, 9 tion in 1). The patient with triple heterozygous mutation were marriages between first cousins (including one (patient no. 22 in Table 1) had 3 different heterozythat led to two sons with WD), one between second gous mutations in exons 9, 14 and 19. cousins, and one uncle-niece marriage. Of the 27 Table 2 shows details of mutations detected and patients, 25 were symptomatic, whereas 2 patients their effects. Most of the mutations were mis-sense were asymptomatic and were diagnosed on family in nature, 2 were non-sense in nature leading to screening. formation of premature termination, 2 deletion mu- Of these 27 patients, 17 had low serum ceru- tations led to truncation of the protein, and 3 afloplasmin (0-32 U/L) and KF rings. Five patients fected splice sites. G1847A in exon 5 has been prehad low serum ceruloplasmin (6-56 U/L), no KF viously described as a putative mutation, as it crerings, and elevated 24-hour urine copper ( ates a conservative amino acid change that is not µg). Five other patients had normal serum cerulo- predicted to severely disrupt tertiary protein strucplasmin ( U/L), detectable KF rings, and high ture and was absent in the normal population. 7 Eleven 24-hour urine copper ( µg). In 10 patients, mutations were novel. repeat measurement of serum ceruloplasmin level Exons 18 and 13 were mutation hot spots, was done median (range) 25 (8-86) months after with 6 (27%) and 3 (14%) mutations, respectively starting penicillamine therapy; these levels were low (Table 1). The commonest mutations were G3182A in all of them, including in two patients who had and C813A; these were detected in 4 (16%; honormal serum ceruloplasmin prior to the start of mozygous 1, heterozygous 3) and 3 (12%; homozytreatment. gous 2, heterozygous 1) probands, respectively. Indian Journal of Gastroenterology 2006 Vol 25 November - December 279

4 Table 3: Polymorphisms and nucleotide changes of unknown significance (NCUS) of ATP7B gene in Indian Wilson s disease patients Polymorphism Sequence / Location Amino acid Reason for not considering / NCUS codon change change as mutation -80A>C CCACG 5 UTR Found in normal controls b -128C>A CCGCG 5 UTR Found in normal controls b -30del5bp GAGCCccgag 5 UTR Found in normal controls b T1216G TCT-GCT Exon 2 Ser 406 Ala Conservative substitution a G1366C GTG-CTG Exon 3 Leu456Val Conservative substitution a A2495G AAG-AGG Exon 10 Arg832Lys Conservative substitution a A2855G AAA-AGA Exon 12 Lys952Arg Conservative substitution a G3009A GCG-GCA Exon 13 Ala1003Ala No amino acid change 5 UTR: 5 untranslated region, a Not found in normal population 7, b normal controls tested Of 24 WD patients with ATP7B mutations (22 probands, 2 affected family members), 12 were born of consanguineous marriages (Table 1); of these, 8 had homozygous ATP7B mutations and 4 had heterozygous mutations. Of the remaining 12 patients born of non-consanguineous marriages, 6 had homozygous mutations and 6 had heterozygous mutations. ATP7B polymorphisms and nucleotide changes of unknown significance (Table 3) Nucleotide sequence changes leading to no amino acid change or conservative substitution have been designated as nucleotide changes of unknown significance. 7 Applying this definition, we found 5 nucleotide sequence changes of unknown significance all 5 of these have been reported previously. 7 Further study is needed to determine if the nucleotide changes causing conservative substitution are putative mutations. The 3 commonest nucleotide changes, G3009A, A2495G and A2855G, were present in 20, 9 and 7 patients, respectively. Three novel polymorphisms in the 5 -untranslated region, including two substitution polymorphisms ( 80A>C and -128C>A) and a deletion polymorphism (-30del5bp), were found in one pa- tient each. Heterozygous nature of this deletion polymorphism was confirmed by sequencing parents samples. WD phenotype and ATP7B mutations within families Conventional tests (copper chemistry and slit-lamp ex- amination for KF ring) led to detection of asymptomatic WD in two families patient numbers 10.1, 10.2 (father and son) and 15.1, 15.2 (brothers) in Table 1. Symptomatic WD was not found in any live family member. In 8 families, other siblings died of hepatic and/or neurological or neuropsychiatric disease ( presumed WD ) at age 11 (7-22 years) (Table 4). We were able to review medical records of 5 deceased siblings; investigations for WD were not done in any of them. Excluding one family with father as proband (patient no in Table 1), of 41 live siblings of probands from 24 families, 17 (from 11 families) could be accessed for study. Heterozygote mutations were identified in all these 17 (carrier state in 16, WD diagnosed on conventional tests for WD in one patient no. 15.2). In the family of the fatherson pair with WD (patient numbers 10.1, 10.2 in Table 1), both father and son were homozygous for A3029G mutation on exon 13, while mother and daughter were heterozygote carriers. Genotype-phenotype correlation WD phenotypes for different ATP7B mutations are given in Table 1. Lack of a dominant mutation prevented phenotypic correlation with individual mutations. Symptomatic WD was not found in live siblings of probands. In 6 of 8 families wherein siblings died Table 4: Phenotype of presumed Wilson s disease (WD) in deceased siblings of probands with WD Proband Deceased sibling(s) Family Age (y) WD Relationship Age (y) at onset Phenotype of Age (y) number at onset phenotype of symptoms of presumed WD at death of symptoms presumed WD / gender 1 6 M Hepatic and Brother 9 Hepatic and 18 neurological neurological 2 9 M Neuro- psychiatric Brother 7 Neuro - psychiatric F Hepatic Sister 11 Neuro -psychiatric F Hepatic Sister 9 Hepatic M Hepatic Sister 6 Hepatic F Hepatic 2 sisters 17, Not known Hepatic, 12, 22 in other neuro-psychiatric 7 8 M Hepatic Brother Not known Hepatic M Hepatic Brother Not known Hepatic Indian Journal of Gastroenterology 2006 Vol 25 November - December

5 of presumed WD, phenotype of presumed WD in deceased sibling within each family was identical to WD phenotype in the proband (Table 4). Discussion We studied ATP7B mutations and WD phenotypes in families of WD patients, belonging mostly to three southern Indian states. The previous two reports of WD genotype from India are from Chandigarh 8 (patients from Punjab, Haryana, Jammu and Kashmir, western Uttar Pradesh, and Chandigarh) and from Kolkata. 9 The spectra of ATP7B mutations in these three studies from different parts of India were quite different. Fifty-one ATP7B mutations were identified in 130 WD patients altogether, but only one mutation (C813A) was found in common between the three centers (accounting for 12%, 19% and 0% of mutations in our center, Kolkata and Chandigarh, respectively). A number of novel ATP7B mutations were found 11 (current study), 18 (Chandigarh study) and 5 (Kolkata study). In all three studies, a single predominant ATP7B mutation was not identified. The two commonest ATP7B mutations in our study, G3182A and C813A, occurred in 16% and 12% of probands, respectively. The commonest ATP7B mutations in the Chandigarh study were T3305C, C2975A, 2977insA and 3031insC (6% each), while C813A, the commonest mutation, was noted in 19% of mutations in the Kolkata study. In contrast, H1069Q was the single predominant ATP7B mutation (38%) in Caucasian WD patients, 10 while R778L occurred in 46% of Chinese WD patients. 11 Exons 18 and 13 of ATP7B gene were hot spots in our study, while exons 8, 12, 13, 15, 16 and 18 were hot spots in the Chandigarh study and exon 2 in the Kolkata study. We have not done studies to determine the effect of ATP7B mutations on WD protein function. Functional studies would be particularly useful to confirm novel mis-sense mutations and to determine if splice mutation 3903 (6T>C) where the mutation is located 6 nucleotides away from the splice site and the putative mutation, G1847A, are indeed disease-causing. It is intriguing that despite 44% of parents being in consanguineous marriage, a single dominant mutation was not found in our study population. A possible explanation is that marriages in India are largely within ethnic subgroups. Studies of Indian WD patients within different ethnic subgroups are needed to look for dominant mutations and exonic hot spots. In autosomal recessive disorders, consanguin- ity is a risk factor for homozygosity. 12 However, we did not find any difference in the frequency of homozygous mutations in WD patients born of consanguineous and non-consanguineous marriages; this may be related to the small sample size studied. Serum levels of ceruloplasmin, an acute-phase reactant, may fall as liver disease improves with copper-chelation therapy. 13 This may explain the decrease in serum ceruloplasmin levels after treatment with penicillamine in two of our patients who had normal baseline ceruloplasmin levels. The oldest age of onset of symptomatic WD in our study was 48 years. This is a bit unusual; however, symptomatic WD has been reported up to 72 years of age. 14 Lack of single dominant mutation prevented us from correlating WD phenotype with individual ATP7B mutations. As affected siblings in a family share common ATP7B genotype, we studied how closely the disease repeated itself within affected siblings in a family. We did not detect symptomatic WD in any live family member other than in the proband. However, in 8 of the 25 families (32%), a sibling had died of presumed (undiagnosed) WD. In 6 of 8 families, siblings who died of presumed WD shared identical disease phenotype with the proband. It is of interest that this was noted even in the unusual presentation of neurological WD in children (Table 4: family no. 2). In the remaining two families, WD phenotype was different from the proband (Table 4: family numbers 3, 6). This suggests that genetic factors outside ATP7B gene and/or acquired/environmental factors also influence WD phenotype. Different WD phenotypes within siblings, though uncommon, have been reported before. 9,15 As >250 ATP7B mutations have been reported in WD and a majority of patients are compound heterozygotes, genotype-phenotype correlation is difficult in WD. 16 Analyzing WD phenotype in unrelated patients sharing a common mutation is one approach to study this, but has yielded disparate results. 10,11 Study of siblings with symptomatic WD within each family (who will share the same ATP7B genotype) is another approach that may be useful in this situation. However, family screening of probands usually detects pre-symptomatic WD in siblings (as in our series), which prevents genotype-phenotype correlation. Symptomatic WD in live siblings is uncommon. Thus, study of deceased siblings with presumed WD (with its inherent limitations) and probands provides an opportunity to compare phenotype of symptomatic WD in families. In our study, in 32% of families, one other Indian Journal of Gastroenterology 2006 Vol 25 November - December 281

6 sibling died of presumed (undiagnosed) WD. This highlights lack of awareness of this treatable disease in India. Any child or young adult with unexplained liver or neuropsychiatric disease should be tested for WD. The whole family should be screened for WD by tests of copper chemistry (serum ceruloplasmin, urinary copper) and slit-lamp corneal examination. Genetic tests are mainly used to rule out disease among family members of an index WD patient, especially when diagnostic uncertainty occurs in a heterozygote carrier. Heterozygote carriers can have low serum ceruloplasmin and genetic tests can establish this diagnosis and thus rule out WD. What then is the optimal genetic screening strategy for WD in India? DNA linkage analysis is cheap and easy to perform; however, as flanking dinucleotide repeats are prone to recombination, direct analysis of ATP7B gene may be preferable. 8,17 Disorders of copper overload and liver disease other than WD reported from the Indian subcontinent, like Indian childhood cirrhosis 18 and atypical copper-associated cirrhosis, 19 may not be associated with ATP7B mutations and could be caused by disorders of a copper-transport gene or other epigenetic factors. 20 This provides another reason to prefer direct analysis of ATP7B gene over DNA linkage analysis to screen for WD in India. References 1. Gitlin JD. Wilson disease. Gastroenterology 2003;125: Ferenci P, Caca K, Loudianos G, Mieli-Vergani G, Tanner S, Sternlieb I, et al. Diagnosis and phenotypic classification of Wilson disease. Liver Int 2003;23: Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, Vol. 1, 3rd ed. New York: Cold Spring Harbor Laboratory Press. 2001: p Thomas GR, Forbes JR, Roberts EA, Walshe JM, Cox DW. The Wilson disease gene: the spectrum of mutations and their consequences. Nat Genet 1995;9: Loudianos G, Dessi V, Lovicu M, Angius A, Altuntas B, Giacchino R, et al. Mutation analysis in patients of Mediterranean descent with Wilson s disease: identification of 19 novel mutation. J Med Genet 1999;36: Ganguly A, Rock, MJ, Prockop DJ. Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent induced bends in DNA heteroduplexes. Proc Natl Acad Sci USA 1993;90:10,325-10, Curtis D, Durkie M, Balac (Morris) P, Sheard D, Goodeve A, Peake I, et al. A study of Wilson disease mutations in Britain. Hum Mutat 1999;14: Kumar S, Thapa BR, Kaur G, Prasad R. Identification and molecular characterization of 18 novel mutations in the ATP7B gene from Indian Wilson disease patients: genotype. Clin Genet 2005; 67: Gupta A, Aikath D, Neogi R, Datta S, Basu K, Maity B, et al. Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotypephenotype correlation in Indian patients. Hum Genet 2005; 118: Shah AB, Chernov I, Zhang HT, Ross BM, Das K, Lutsenko S, et al. Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses. Am J Hum Genet 1997;61: Liu XQ, Zhang YF, Liu TT, Hsiao KJ, Zhang JM, Gu XF, et al. Correlation of ATP7B genotype with phenotype in Chinese patients with Wilson disease. World J Gastroenterol 2004;10: Woods CG, Cox J, Springell K, Hampshire DJ, Mohamed MD, McKibbin M, et al.. Quantification of homozygosity in consanguineous individuals with autosomal recessive disease. Am J Hum Genet 2006;78: Ferenci P. Wilson s disease. In: Bacon BR, Di Bisceglie AM, Eds. Liver Disease: Diagnosis and Management. Pennsylvania: Churchill Livingstone. 2000: p Ala A, Borjigin J, Rochwarger A, Schilsky M. Wilson disease in septuagenarian siblings: raising the bar for diagnosis. Hepatology 2005;41: Takeshita Y, Shimizu N, Yamaguchi Y, Nakazono H, Saitou M, Fujikawa Y, et al. Two families with Wilson disease in which siblings showed different phenotypes. J Hum Genet 2002;47: Santhosh S, Shaji RV, Eapen CE, Chandy GM. ATP7B gene and Wilson s disease. J Gastroenterol Hepatol 2004;19: Gupta A, Neogi R, Mukherjea M, Mukhopadhyay A, Roychoudhury S, Senapati A, et al. DNA linkage based diagnosis of Wilson disease in asymptomatic siblings. Indian J Med Res 2003;118: Pediatric Liver Study Group of India. Metabolic liver diseases in childhood: Indian scenario. Indian J Pediatr 1999;66(1 Suppl):S97-S Ramakrishna B, Date A, Kirubakaran C, Raghupathy P. Atypical copper cirrhosis in Indian children. Ann Trop Paediatr 1995;15: Tanner MS. Indian childhood cirrhosis and Tyrolean childhood cirrhosis. Disorders of copper transport gene? Adv Exp Med Biol 1999;448: Correspondence to: Dr. Eapen. Fax: (416) , eapen@cmcvellore.ac.in Acknowledgements: We acknowledge funding support from the Department of Science and Technology, Government of India, and Fluid Research Fund, Christian Medical College, Vellore Received April 11, Received in final revised form September 25, Accepted October 7, Indian Journal of Gastroenterology 2006 Vol 25 November - December