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ORIGINAL ARTICLE

Wilson disease in offspring of affected patients: Report of four French families

Fabienne Dufernez a,h, Alain Lachaux b,h, Philippe Chappuis c,h, Lionel De Lumley d, Muriel Bost e,h, France Woimant f,h, Micheline Misrahi a,h, Dominique Debray g,h,
a

Laboratoire de gntique molculaire, pharmacogntique, hormonologie, universit Paris Sud, hpital Bictre, APHP, 94270 Le Kremlin-Bictre, France b Inserm U851, IFR-128, service de gastroentrologie, hpatologie et nutrition pdiatriques, HFME, facult de mdecine Lyon Est, universit de Lyon 1, CHU de Lyon, 69677 Bron, France c Ple biologie pathologie physiologie (B2P), service de biochimie et biologie molculaire, hpital Lariboisire, APHP, 75475 Paris, France d Service de pdiatrie, CHU de Dupuytren, 87042 Limoges, France e Centre de biologie et pathologie Est, laboratoires des maladies hrditaires du mtabolisme et de neurogntique molculaire, 69677 Bron, France f Ple neurosciences, service de neurologie, hpital Lariboisire, APHP, 75475 Paris, France g Service de hpatologie pdiatrique, hpital Bictre, APHP, 94270, Le Kremlin-Bictre, France h National Reference Center for Wilson Disease, hpital Lariboisire, APHP, 75475 Paris, France

Summary Background: Wilson disease (WD) is an autosomal recessive genetic disorder caused by mutations in the ATP7B gene resulting in toxic accumulation of copper mainly in the liver and brain. Early treatment may prevent irreversible tissue damage. Aim: We report on four families with an occurrence of WD in two consecutive generations in order to highlight the need for screening offspring of affected parents. Results: In all families, one parent was known to be affected with WD. Screening for the disease was not performed in children from two families until occurrence of liver disease in one and of neurological symptoms in the other. In two other families, screening of children as soon as diagnosis was performed in the affected parent allowed a timely rescue of advanced liver disease in one while two affected children were asymptomatic. In three children, diagnosis required direct sequencing of the ATP7B gene. Two novel disease-causing mutations are reported.

Corresponding author. Ple mdicochirurgical, service de hpatologie pdiatrique, hpital Necker, 149, rue de Svres, 75015 Paris, France. Tel.: +33 1 44 49 41 52; fax: +33 1 44 49 41 60. E-mail address: dominique.debray@nck.aphp.fr (D. Debray).

2210-7401/$ see front matter 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.clinre.2013.01.001

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001

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Conclusion: Patients with WD should be offered genetic counselling when considering pregnancy and offspring should always be screened for the disease. Diagnostic difculties based on copper disturbances in asymptomatic children that are obligate carriers of the Wilson gene and the usefulness of molecular diagnosis are discussed. 2013 Elsevier Masson SAS. All rights reserved.

Introduction
Wilson disease (WD) is an autosomal recessive genetic disorder of copper metabolism resulting in toxic accumulation of copper in several tissues, mainly the liver, brain and kidney. This disorder is caused by mutations in the ATP7B gene encoding a copper transporting P-type ATPase [1]. Because early treatment with chelating agents may prevent irreversible tissue damage, there is a strong rationale for screening siblings of WD subjects who have a 25% risk of being affected [2]. In this setting, DNA linkage studies using microsatellites within and around the ATP7B gene have greatly facilitated the diagnosis in asymptomatic siblings, although less often performed nowadays owing to the efciency of direct molecular testing [35]. According to the gene frequency, the probability of having offspring affected with WD is estimated to be less than 0.6% in the US and Europe. Although it is recommended, screening offspring of an affected parent with WD is not systematically performed [1,2]. In order to highlight the need for screening children of an affected parent for WD, we report here four families living in France with an occurrence of WD in two consecutive generations. This report also discusses the usefulness of molecular studies as a diagnostic tool in this setting.

Indirect linkage studies were performed using ve highly polymorphic microsatellite markers anking the gene D13S314, D13S316 [6] and D13S301, D13S133, D13S294 [7].

Results
Case reports
Family 1 Both parents originated from France. The affected mother with WD presented with neurological manifestations, hemolytic anemia, and Kayser-Fleischer rings at slit-lamp examination at the age of 15 years and was treated with dpenicillamine since then. The father was not affected with WD and unrelated to the mother. There was no known history of hepatic or neurological diseases in his family. Elevated serum aminotranferase activities were found incidentally in their daughter (patient 1) at the age of 10. Screening for hepatitis B and C viruses, cytomegalovirus and Epstein Barr virus infections, and for autoimmune hepatitis was negative. Abnormalities in copper metabolism were indistinguishable from those seen in a heterozygous carrier (Table 1). The child carried one mutation inherited from her mother while the other was undetermined. Measurement of hepatic parenchymal copper content did not provide clear evidence for the diagnosis of WD. At this point of the work-up, diagnosis of WD was not established according to the Ferencis score. Diagnosis of WD was unequivocally conrmed 1 year later by complete gene sequencing identifying the second mutation inherited from her father. Once the child was given d-penicillamine, serum aminotransferase activities returned to normal values within 4 months and remained normal at last follow-up 6 years later. Family 2 Both parents originated from France. The affected father with WD presented with liver cirrhosis, leucopenia, thrombocytopenia and Kayser-Fleischer rings at slit-lamp examination but no neurological symptoms at the age of 50, and was treated with d-penicillamine. The mother was not affected with WD and apparently not related to the father. There was no history of hepatic or neurological disease in the mothers family. Their two sons were subsequently screened for WD. The oldest boy (patient 2) was 16 years old and had a totally uneventful past medical history. Physical examination was normal. No Kayser-Fleischer ring was seen at slit-lamp examination. Abnormalities in copper metabolism suggested the diagnosis of WD (Table 1). According to the Ferencis score, diagnosis of WD was probable and nally established upon molecular studies. Once d-penicillamine

Patients and methods

Between 1997 and 2010, 80 children less than 18 years of age were newly diagnosed with WD in the two pediatric centers in France (Bictre and Lyon National reference centers). Among them, ve children from four distinct families had an affected parent with WD. The case history of these four families is reported. The Ferencis score, devised to aid diagnosis of WD, was calculated for each child prior to and after mutation analysis in order to evaluate its diagnostic accuracy [1]. For the molecular analysis of the ATP7B gene, DNA extraction from peripherical blood and polymerase chain reaction using an automated sequencer Applied Biosystems were carried out with standard methods using previously described primers [3]. The proband and family members of families 1, 2 and 3 were analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and denaturing high performance liquid chromatography (dHPLC) as screening tools for mutation detection followed by sequencing on both DNA strands on an ABI 3100 automated sequencer (PE Applied Biosystems, Foster City, CA). Family 4 was analysed by direct bi-directional sequencing on an ABI 3730 Genetic Analyser (PE Applied Biosystems, Foster City, CA).

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001

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Wilson disease in offspring of affected patients: Report of four French families

Table 1 Clinical, biochemical and genetic characteristics in ve patients with Wilson disease. Family 1 Patient 1 Hepatomegaly Total serum bilirubin (N < 0.1 mg/dl) ALT (N < 40 IU/l) PT (N > 70% of normal) (%) Kayser-Fleischer Rings Serum ceruloplasmin (N, 2040 mg/dl) Basal 24 h-urinary copper (N < 50 g) After d-penicillamine challenge (g/24 h) Hepatic copper content (N < 50 g/g dry weight) Ferencis score [1]a Without mutation analysis With mutation analysisb ATP7B mutation analysis No < 0.1 120 90 No 15 50 130 62.5 Family 2 Patient 2 No < 0.1 149 70 No 11 170 Not done Not done Family 2 Patient 3 No 0.25 310 33 Yes 3 1960 Not done Not done Family 3 Patient 4 Yes < 0.1 39 54 Yes 3 1275 Not done Not done Family 4 Patient 5 No < 0.1 27 100 No <3 14.4 72.2 Not done

2 6 p.His1069Gln / c.4125-1G>A

3 7 p.Arg778Trp / p.Arg778Trp

6 7 p.Arg778Trp / undetermined

6 10 p.Thr569del / p.Thr569del

2 6 p.Leu708Pro / p.Gly1099Ser

ALT: alanine aminotransferase; PT: prothrombin time. a Assessment of the WD score: 4 points or more, diagnosis of WD is highly likely; 23 points, diagnosis of WD is probable; 01 point, diagnosis of WD is unlikely. b Mutation analysis adds 1 point if one mutation is found on one chromosome or 4 points if two mutations are found on both chromosomes.

was started, serum aminotransferase activities returned to normal values within 3 months and remained normal until last follow-up 5 years later. The younger (patient 3) was 13 years old and presented with liver cirrhosis and liver failure. Kayser-Fleischer rings were seen at slit-lamp examination, associated with denite biochemical abnormalities in copper metabolism (Table 1). Treatment with d-penicillamine was immediately initiated. Liver function tests returned to normal values within 1 year of treatment. He remains well 5 years later. Family 3 Both parents originated from France. The affected mother with WD presented with fulminant liver failure in 1988 at the age of 17, and underwent an emergency liver transplantation. Fifteen years later, she underwent a second liver transplant for chronic graft rejection. She is doing well after 8 years follow-up. The father was not affected with WD and not known to be related to the mother. He died from myocardial infarction in 1999. There was no history of hepatic or neurological disease in the fathers family. Their older son (patient 4), born in 1992, presented at the age of 17 with dysphonia and learning difculties. Neurological examination revealed dystonia of the upper limbs, gait disorders and dysarthria. Kayser-Fleischer rings were seen at slit-lamp examination associated with biochemical abnormalities in copper metabolism (Table 1). Treatment

with d-penicillamine was immediately initiated. The patient improved dramatically within 6 months from onset of therapy. After 2 years of follow-up, mild residual dysarthria remains, and liver function tests are normal. The younger son, born in 1996, was screened at the time of diagnosis of WD in his brother, and was found not to be affected with WD. Family 4 Both parents originated from Portugal but lived in France. The affected father was screened for WD at the age of 34 years old when his 20-year-old sister underwent an emergency liver transplantation for fulminant WD in 2010. He was totally asymptomatic without any abnormalities of liver function tests. Molecular studies allowed the diagnosis of WD to be performed in the father. He remains well after 1 year of zinc therapy. The mother was not affected with WD and not known to be related to the father. There was no history of hepatic or neurological disease in the mothers family. Their daughter, born in 2005, was subsequently screened for WD at the age of 6 years. Physical examination was normal. No Kayser-Fleischer ring was seen at slit-lamp examination. Abnormalities in copper metabolism suggested the diagnosis of WD (Table 1). According to the Ferencis score, diagnosis of WD was probable and nally established upon molecular testing. Zinc therapy was initiated.

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001

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Figure 1 Mutation analysis of family 1. The arrow indicates the proband with secure diagnosis of WD. ? indicates that mutation analysis was not performed.

Familial genetic analysis

In family 1 (Fig. 1), the proband (i.e. the mother) carried the common p.His1069Gln mutation in exon 14 together with a heterozygous novel frame shift mutation (c.670dup, p.Ile224AsnfsX2) in exon 2. The father carried a known disease-causing mutation in exon 21 located within the consensus splice site (c.4125-1G>A). The child was a compound heterozygote and inherited the fathers splice site mutation together with the mothers p.His1069Gln mutation. In family 2 (Fig. 2), the proband (i.e. the father) and his younger son (patient 3) were both carriers of the heterozygous c.2332C>T, p.Arg778Trp mutation in exon 8, but the other mutation remained undetermined despite whole sequencing of the coding regions and adjacent intron/exon junctions of the gene. The older son (patient 2) was found to be homozygous for the p.Arg778Trp mutation. Microsatellite analysis conrmed that patients 2 and 3 had inherited the mothers allele carrying the p.Arg778Trp mutation. Patient 2 had inherited the fathers allele also carrying the p.Arg778Trp mutation and was therefore, homozygous for this mutation. Patient 3 had inherited the fathers second allele carrying an undetected mutation. Haplotype studies using microsatellite markers within and anking the ATP7B gene revealed that the mother and the father shared an identical haplotype of the allele carrying the p.Arg778Trp mutation suggesting that they shared a common ancestor. No

Figure 3 Mutation analysis of family 3. The arrow indicates the proband with secure diagnosis of WD. ? indicates that mutation analysis was not performed.

Figure 4 Mutation analysis of family 4. The black arrow indicates the proband with secure diagnosis of WD. ? indicates that mutation analysis was not performed.

Figure 2 Mutation analysis of family 2. The arrow indicates the proband with secure diagnosis of WD.

consanguinity in the family was recognized through clinical interrogation. In family 3 (Fig. 3), the proband (i.e. the mother) was homozygous for a novel frameshift mutation c.1705 1707+10del, p.Thr569del in exon 4. Her older son (patient 4) was homozygous for this same rare mutation, strongly suggesting consanguinity. Her younger son was heterozygous for this mutation. No genetic analysis could be undertaken in the father who died prior to the diagnosis of WD in his sons. A detailed pedigree analysis obtained through clinical interrogation revealed that the affected mother with WD and the father were rst cousins. In family 4 (Fig. 4), the proband (i.e. the father) was a compound heterozygote carrying the c.3061-12T>A mutation in intron 13 and the c.3295G>A, p.Gly1099Ser mutation in exon 15. The mother carried a heterozygous c.2123T<C, p.Leu708Pro mutation in exon 8. The child was a compound heterozygote and has inherited the fathers p.Gly1099Ser mutation together with the mothers p.Leu708Pro mutation.

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001

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5 decreased levels of serum ceruloplasmin below 20 mg/dl and an increase in basal urinary copper in the range of 40 to 100 g/24 hours (1.6 mol/24 hours) [2]. On the other hand, normal cerulopslasmin concentration and normal basal daily urinaty copper excretion do not exclude the diagnosis of WD. In clinically asymptomatic children except for elevated serum aminotransferases compared to children with liver disease other than WD, Nicastro et al. reported that serum ceruloplasmin below the cut-off value of 20 mg/dl showed a sensitivity of 95% and a specicity of only 84.5% for the diagnosis of WD, and that the optimal basal urinary copper diagnostic cutoff value was 40 g (0.65 mol)/24 hours with a sensitivity of 78.9% and a specicity of 87.9% [11]. A lower serum ceruloplasmin concentration threshold of 14 mg/dl was shown to render 100% specicity for the diagnosis of WD [12]. The diagnostic accuracy of daily urinary copper measurements after chelation with d-penicillamine is also poor in asymptomatic children (sensitivity of only 46%) [13]. In these equivocal cases, a liver biopsy for measurement of hepatic copper content has been recommended, but due to heterogeneity of copper content throughout the liver, it may not always provide evidence for the diagnosis of WD such as in one child (patient 1) reported here. Moreover, the concentration of hepatic copper in heterozygotes is also frequently elevated above normal, although it does not exceed 250 g/g dry weight [2]. Recently, relative exchangeable copper (namely exchangeable copper-to-total copper ratio) has been reported to provide 100% sensitivity and 100% specicity for the diagnosis of WD in adults [14]. Further studies are, however, needed to evaluate its diagnostic accuracy in adults and children. To improve diagnosis, a scoring system was proposed by an international consensus of experts in 2001, that includes biochemical parameters and molecular diagnostic [1]. This scoring system, recently reevaluated in WD children with mild liver disease restricted to elevated serum aminotransferases, proved to have positive and negative predictive values of 93% and 91.6% respectively [11]. The identication of only one disease-causing mutation appears adequate to conrm the diagnosis of WD only in the presence of denite clinical symptoms and abnormal copper metabolism. Therefore, in asymptomatic children of an affected parent who are obligate heterozygous carriers, identication of the two disease-causing mutations becomes necessary to allow diagnosis of WD as reported in patient 1 [1,15]. Taking into account the diagnostic difculties of WD, especially in young asymptomatic children, molecular testing for ATP7B mutations should be obtained and may be used as primary screening. More than 500 mutations within the ATP7B gene have been identied, and most affected individuals carry two different mutations, thus making genetic diagnosis difcult. Owing to the progress of sequencing techniques (high throughput sequencing methods), sequencing of the 21 exons and adjacent intron/exons junctions of the ATP7B gene should become available in most investigating laboratories in the near future, and increase the chance of identifying two mutant alleles in affected subjects to above 95% [16]. Indeed, diagnosis of WD in patients 1 and 5 was established using this approach. Two novel disease-causing mutations have been found, a frameshift mutation in exon 2 (c.670dup, p.Ile224AsnfsX2) and a 13 bp deletion in exon 4

Wilson disease in offspring of affected patients: Report of four French families

Discussion
Children of an affected parent with WD can only be affected if the other parent is at least a heterozygous carrier. Considering that the prevalence of WD is estimated to be on the order of 30 per 1 million with a carrier frequency of about 1 in 90, the likelihood of occurrence of disease in offspring is estimated to be 0.56% (0.5 times the carrier rate frequency) in the US and Europe [1,2]. However, the prevalence of WD and the carrier frequency are possibly under-estimated. Indeed, although age limits for consideration of WD have for long been stated within 5 to 40 years, WD is being nowadays increasingly diagnosed in children younger than 5 years old, and in older patients such as reported in the 50-year-old father of family 2 [2]. Moreover, the probability of occurrence of WD is increased in consanguineous families and in specic populations with a high carrier frequency [8]. In Europe, the occurrence of WD in two consecutive generations has been rarely reported in the Caucasian population [5,9,10]. However, the four families reported here provide evidence for screening WD in offspring of an affected parent, even in apparently non-consanguineous families. In two families, screening of children as soon as the diagnosis was performed in the affected parent allowed a timely rescue of advanced liver disease with liver failure in one while two affected children were asymptomatic. On the other hand, screening for the disease was not performed in children from two other families until occurrence of mild liver disease in one and of severe neurological symptoms, which could have been prevented with initiation of therapy in early childhood in the other. Moreover, consanguinity was conrmed through clinical interrogation in one family while pedigree analysis through haplotypes studies using microsatellites surrounding or within the WD gene suggested that the parents in another family, if not apparently consanguineous, shared a common ancestor. Importantly, all patients with WD should be informed about the risk, although low, of occurrence of WD in offspring. Screening for abnormalities in copper metabolism may be offered to the partner of an affected patient when the couple is considering pregnancy. Whether carrier testing should be offered to the partner may be discussed, acknowledging that it should be reasonably declined. Firstly, a negative result (i.e. no ATP7B disease-causing mutation is identied in the partner) would not exclude the possibility of being a heterozygous carrier of a molecular defect outside the coding regions and adjacent intron/exon junctions of the gene. Secondly, the nding of gene molecular changes that have not been established as disease-causing, especially missense mutations, could be rare normal variants. Finally and most importantly, the nding of an ATP7B disease-causing mutation in the partner would have limited clinical application because antenatal diagnosis of WD would not be offered since screening for WD in infancy will allow appropriate timing for treatment recommended from the age of 3 years old [2]. Diagnosis of WD based on abnormalities of copper metabolism (i.e. ceruloplasmin concentration and basal daily urinary copper excretion) may be however difcult in young asymptomatic offspring, who are obligate carriers of one mutation transmitted by the affected parent. Indeed, approximately 20% of heterozygotes subjects have

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001

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[6] Thomas GR, Bull PC, Roberts EA, Walshe JM, Cox DW. Haplotype studies in Wilson disease. Am J Hum Genet 1994;54:718. [7] Petrukhin K, Fischer SG, Pirastu M, Tanzi RE, Chernov I, Devoto M, et al. Mapping, cloning and genetic characterization of the region containing the Wilson disease gene. Nat Genet 1993;5:33843. [8] Garcia-Villarreal L, Daniels S, Shaw SH, Cotton D, Galvin M, Geskes J, et al. High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study. Hepatology 2000;32:132936. [9] Firneisz G, Szonyi L, Ferenci P, Wilheim C, Horvath A, Folhoffer A, et al. The other mutation is found: follow-up of an exceptional family with Wilson disease. Am J Gastroenterol 2004;99:25045. [10] Hofer H, Willheim-Polli C, Knoach P, Gabriel C, Vogel W, Trauner M, et al. Identication of a novel Wilson disease gene mutation frequent in Upper Austria: a genetic and clinical study. J Hum Genet 2012;57:5647. [11] Nicastro E, Ranucci G, Vajro P, Vegnente A, Iorio R. Reevaluation of the diagnostic criteria for Wilson disease in children with mild liver disease. Hepatology 2010;52:194856. [12] Mak CM, Lam CW, Tam S. Diagnostic accuracy of serum ceruloplasmin in Wilson disease: determination of sensitivity and specicity by ROC curve analysis among ATP7B-genotyped subjects. Clin Chem 2008;54:135662. [13] Mller T, Koppikar S, Taylor RM, Carragher F, Schlenck B, HeinzErian P, et al. Re-evaluation of the penicillamine challenge test in the diagnosis of Wilsons disease in children. J Hepatol 2007;47:2706. [14] El Balkhi S, Trocello JM, Poupon J, Chappuis P, Massicot F, Girardot-Tinant N, et al. Relative exchangeable copper: a new highly sensitive and highly specic biomarker for Wilsons disease diagnosis. Clin Chim Acta 2011;412(2324):225460. [15] Caprai S, Loudianos G, Massei F, Gori L, Lovicu M, Maggiore G. Direct diagnosis of Wilson disease by molecular genetics. J Pediatr 2006;148:13840. [16] Glenn TC. Field guide to next-generation DNA sequencers. Mol Ecol Resour 2011;11:75969.

6 (c.1705 1707+10del, p.Thr569del) that removes the natural splice site and is considered as a protein-negative mutation. In conclusion, all patients with WD should be informed about the need for screening their children for WD and offered genetic counselling when considering pregnancy. Diagnosis of WD based on abnormalities of copper metabolism only may be difcult in offspring because of the unreliability of distinguishing heterozygotes carriers from asymptomatic but affected children. In these cases, genetic studies should be performed. Molecular testing using latest generation DNA-sequencing platforms will become a powerful diagnostic tool in determining gene mutations associated with WD.

Disclosure of interest
The authors declare that they have no conicts of interest concerning this article.

References
[1] Ferenci P, Czlonkowska A, Stremmel W, Houwen R, Rosenberg W, Schilsky M, et al. EASL clinical practice guidelines: Wilsons disease. J Hepatol 2012;56:67185. [2] Roberts EA, Schilsky ML. Diagnosis and treatment of Wilson disease: an update. Hepatology 2008;47:2089111. [3] Thomas GR, Roberts EA, Walshe JM, Cox DW. Haplotypes and mutations in Wilson disease. Am J Hum Genet 1995;56: 13159. [4] Vidaud D, Assouline B, Lecoz P, Cadranel JF, Chappuis P. Misdiagnosis revealed by genetic linkage analysis in a family with Wilson disease. Neurology 1996;46:14856. [5] Maier-Dobersberger T, Mannhalter C, Rack S, Granditsch G, Kaserer K, Korninger L, et al. Diagnosis of Wilsons disease in an asymptomatic sibling by DNA linkage analysis. Gastroenterology 1995;109:20158.

Please cite this article in press as: Dufernez F, et al. Wilson disease in offspring of affected patients: Report of four French families. Clin Res Hepatol Gastroenterol (2013), http://dx.doi.org/10.1016/j.clinre.2013.01.001