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 HANDBOOK OF CLINICAL
NEUROLOGY

Series Editors

MICHAEL J. AMINOFF, FRANÇOIS BOLLER, AND DICK F. SWAAB

VOLUME 142
ELSEVIER
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 Foreword

It is a pleasure to present this volume of the Handbook of Clinical Neurology (HCN), which is dedicated to Wilson
disease. There are plenty of eponyms in medicine and particularly in neurology, but very few HCN volumes include a
person’s name in their title and this is only the third in the present series, after Parkinson and Huntington. Kinnier
Wilson (1878–1937) fully deserves this honor. In 1912, he published in Brain what had been his MD thesis:
“Progressive lenticular degeneration: a familial nervous system disease associated with cirrhosis of the liver.” As is
almost always the case, similar conditions associating liver and brain disease had been described previously, going
as far back as Morgagni and, more recently, by Westphal and Strumpell. However, the Wilson monograph included
the main clinical and pathologic features of the disease, and it opened the way to a new chapter in the history of
neurology, namely that of extrapyramidal system disorders, a term Wilson introduced.
The volume opens with a chapter describing in detail the history of the disease written by one of its protagonists,
John M. Walshe, who in 1956 proposed the use of penicillamine, one of the first disease-modifying drugs in neurology.
The volume includes chapters dealing with the epidemiology and genetics of the disease. It focuses on the two main
organs affected, the brain and the liver, with detailed descriptions of the pathology and pathogenesis of the disease,
including animal models, as well as its diagnosis and the various available forms of management and treatment. Novel
perspectives are discussed in a chapter that introduces a new chelating agent, tetrathiomolybdate, as well as nonchelat-
ing drugs currently under clinical investigation. As is appropriate for such a rare yet highly complex hereditary disease,
the volume concludes with a chapter on the groups that advocate for and support patients with Wilson disease.
We have been fortunate to have as volume editors two distinguished scholars and clinicians, Anna Członkowska,
Professor at the Second Department of Neurology, Institute of Psychiatry and Neurology in Warsaw, and Michael L.
Schilsky, Associate Professor of Medicine and Surgery and Medical Director of Adult Liver Transplantation at the
Yale-New Haven Transplantation Center. Both have been on the forefront of research on Wilson disease for many
years. They have assembled a truly international group of authors with acknowledged expertise and together they have
produced this authoritative, comprehensive, and up-to-date volume. Its availability electronically on Elsevier’s Science
Direct site as well as in print format should ensure its ready accessibility and facilitate searches for specific information.
We are grateful to them and to all the contributors for their efforts in creating such an invaluable resource. As series
editors we read and commented on each of the chapters with great interest. We are therefore confident that both
clinicians and researchers in many different medical disciplines will find much in this volume to appeal to them.
And last, but not least, we thank Elsevier, our publisher – and in particular Michael Parkinson in Scotland, and Mara
Conner and Kristi Anderson in San Diego – for their unfailing and expert assistance in the development and production
of this volume.
Michael J. Aminoff
François Boller
Dick F. Swaab
 Preface

There is almost no other treatable disorder with as wide a clinical spectrum for presentation as Wilson disease. From
child to septuagenarian at diagnosis, patients may be asymptomatic or present with advanced cirrhosis or liver failure,
with a movement disorder ranging from mild tremor or bradykinesia to severe dystonia, or with an affective disorder or
psychosis. Although this is a volume in a series focused on neurologic and neuropsychiatric disorders, it was inescap-
able that we cross between disciplines and consider the full spectrum of the disease, the underlying hepatic disease that
accompanies the disorder, the diverse neurologic presentations, and the fascinating history of the discovery of its basis
and treatment.
Recent advances in molecular genetics testing allow easier and faster diagnosis of Wilson disease, for which treat-
ment may prevent, stop, or even reverse tissue injury. Also introduced more recently are imaging techniques that help
estimate the stage of the disease at the time of diagnosis and which are helpful for monitoring the progress of treatment.
With a better understanding of the disease pathogenesis, there are new opportunities for safer and even more effective
therapy.
The aim of this text was to provide a thorough examination of Wilson disease in an easy-to-digest format. To accom-
plish this, we engaged a group of international experts from Europe and North America and asked for their help in
covering a wide range of relevant topics important for all caregivers of patients with Wilson disease, whether pediatric
or adult physicians, neurologists, psychiatrists, or hepatologists.
The text begins with a historic perspective, and then provides overviews of disease pathogenesis and its underlying
genetic basis, the effects of the disease on both the central nervous system and liver, its diagnosis, and the range of
clinical disease and treatments. Unusually, we have included the viewpoint of a lay patient organization on what is
important from a patient and advocacy perspective.
We thank all our contributors for their time and help in putting together this volume. A number of revisions were
necessary to make the handbook more cohesive, and we appreciate their patience and forbearance, which made this
possible.
We would like particularly to acknowledge John M. Walshe for his historic perspective and his contributions to the
field, and Mary L. Graper for her advocacy for all patients with Wilson disease. We especially thank all of our
coworkers, with whom we have worked over the years to assist hundreds of patients, broaden our personal experience,
and share it with others. We are also grateful for the cooperation of our patients and their families who put their trust in
us and from whom we continue to learn.
Anna Członkowska and Michael L. Schilsky
 Contributors
A. Ahmad
Section of Transplantation and Immunology,
Department of Surgery, Yale University School of
Medicine, New Haven, CT, USA
A. Ala
Department of Clinical and Experimental Medicine,
Faculty of Health and Medical Sciences, University of
Surrey and Department of Gastroenterology and
Hepatology, Royal Surrey County Hospital NHS
Foundation Trust, Guildford, Surrey, UK
O. Bandmann
Sheffield Institute for Translational Neuroscience
(SITraN), University of Sheffield, Sheffield, UK
S. Boga
Department of Gastroenterology, Sisli Etfal Education
and Research Hospital, Istanbul, Turkey
R. Brůha
Fourth Department of Internal Medicine, Charles
University in Prague, First Faculty of Medicine and General
University Hospital in Prague, Prague, Czech Republic
G. Chabik
Second Department of Neurology, Institute of Psychiatry
and Neurology, Warsaw, Poland
I.J. Chang
Division of Medical Genetics, Department of Medicine,
University of Washington School of Medicine, Seattle,
WA, USA
A. Członkowska
Second Department of Neurology, Institute of Psychiatry and
Neurology and Department of Experimental and Clinical
Pharmacology, Medical University of Warsaw, Poland
P. Dušek
Department of Neurology and Center of Clinical
Neuroscience, Charles University in Prague,
First Faculty of Medicine and General University
Hospital in Prague, Prague, Czech Republic and Institute
of Neuroradiology, University Medicine Goettingen,
Goettingen, Germany

K. Dziezyc
Second Department of Neurology, Institute of Psychiatry
and Neurology, Warsaw, Poland
P. Ferenci
Department of Internal Medicine 3, Gastroenterology
and Hepatology, Medical University of Vienna, Vienna,
Austria
M.L. Graper
Wilson Disease Association, Milwaukee, WI, USA
S.H. Hahn
Division of Genetic Medicine, Department of Pediatrics,
University of Washington School of Medicine, Seattle
Children’s Hospital, Seattle, WA, USA
D. Huster
Department of Gastroenterology and Oncology,
Deaconess Hospital Leipzig, Leipzig, Germany
T. Litwin
Second Department of Neurology, Institute of Psychiatry
and Neurology, Warsaw, Poland
C. Lo
Sheffield Institute for Translational Neuroscience
(SITraN), University of Sheffield, Sheffield, UK
V. Medici
Division of Gastroenterology and Hepatology,
Department of Internal Medicine, University of
California Davis, Sacramento, CA, USA
J. Mikol
Pathology Department, Paris Diderot University, Paris,
France
xii CONTRIBUTORS
J. Pfeiffenberger
Department of Gastroenterology and Hepatology,
University Hospital of Heidelberg, Heidelberg,
Germany
A. Poujois
French National Reference Centre for Wilson Disease,
Neurology Department, Lariboisière Hospital, Paris,
France
M. Pronicki
Department of Pathology, The Children’s Memorial
Health Institute, Warsaw, Poland
E.A. Roberts
Departments of Paediatrics, Medicine and Pharmacology
and Toxicology, University of Toronto, Toronto, Canada
C. Rupp
Department of Internal Medicine, University Hospital
Heidelberg, Heidelberg, Germany
I.F. Scheiber
Department of Parasitology, Faculty of Science, Charles
University, Prague, Czech Republic
M.L. Schilsky
Section of Digestive Diseases and Transplantation and
Immunology, Department of Medicine and Surgery, Yale
University School of Medicine, New Haven, CT, USA
J. Seniów
Second Department of Neurology, Institute of Psychiatry
and Neurology, Warsaw, Poland
P. Socha
Departments of Gastroenterology, Hepatology,
Nutritional Disorders and Pediatrics, The Children’s
Memorial Health Institute, Warsaw, Poland
W. Stremmel
Department of Gastroenterology and Hepatology,
University Hospital of Heidelberg, Heidelberg,
Germany
E. Torrazza-Perez
Division of Gastroenterology and Hepatology,
Department of Internal Medicine, University of New
Mexico, Albuquerque, USA
J.M. Walshe
Formerly of Addenbrookes Hospital, Cambridge and the
Middlesex Hospital, London, UK
K.-H. Weiss
Department of Gastroenterology and Hepatology,
University Hospital of Heidelberg, Heidelberg, Germany
F. Woimant
French National Reference Centre for Wilson Disease,
Neurology Department, Lariboisière Hospital, Paris,
France
P. Zimbrean
Departments of Psychiatry and Surgery (Transplant),
Yale University, New Haven, CT, USA
Handbook of Clinical Neurology, Vol. 142 (3rd series)
Wilson Disease
A. Członkowska and M.L. Schilsky, Editors
http://dx.doi.org/10.1016/B978-0-444-63625-6.00001-X
© 2017 Elsevier B.V. All rights reserved

Chapter 1

History of Wilson disease: a personal account

JOHN M. WALSHE*
Formerly of Addenbrookes Hospital, Cambridge and the Middlesex Hospital, London, UK

Abstract
This chapter focuses on the historic aspects of the development of much of our current knowledge of the
diagnosis and treatment of Wilson disease. Included are descriptions of the clinical signs of neurologic and
hepatic disease, the natural history of disease progression, studies of disease pathogenesis and a unique
perspective on the development of diagnostic testing and pharmacological therapy.

“Begin at the beginning,” the King said very gravely,
“then go on until the end, then stop.”
Alice’s Adventures Through the Looking Glass,
Lewis Carroll
This admirable advice for any story teller is honored
more often in the breach than the observance. The diffi-
culty remains, where is the beginning and does the story
have any definitive end? With the history of disease the
probability is that there is, in all likelihood, no end.
Knowledge and theories will continue to develop almost
indefinitely. Well then, is there a beginning? Probably,
but exactly where that is, is not always obvious. In this
case, it might seem self-evident that the story begins with
Wilson’s original article in Brain in 1912, when he
described four cases of what he called “progressive len-
ticular degeneration: a familial nervous disease associ-
ated with cirrhosis of the liver.” (Fig. 1.1) In addition
he found four cases in the literature which fitted his
description, one of which was dated back to 1890. Fur-
thermore, there is a case report in Frerichs’ book of
1860 that was almost certainly a case of Wilson disease.
Indeed, going back even further, Morgagni, in 1761,
described several cases of liver disease associated with
nervous symptoms which may possibly have been cases
of Wilson disease.
It is interesting that one of the defining physical signs
of Wilson disease, the Kayser–Fleischer corneal rings,
had been described 10 years before the disease to which
they later became associated (Kayser, 1902). It is surpris-
ing, in view of the very detailed nature of his descriptions
of his patients, that Wilson did not observe the corneal
rings and indeed he refused to admit of their relationship
to his disease for 10 years after his original publication
(Wilson, 1922). These rings were for many years thought
to pathognomonic of Wilson disease but it is now known
that they can be found in other forms of liver disease,
such as primary biliary cirrhosis and chronic cholestasis
(Fleming et al., 1975).
Only 1 year after Wilson’s original publication in
Brain, the Austrian pathologist, Rumpel, reported find-
ing an excess of copper in the liver of a patient dying
of Wilson disease, an observation whose importance
was completely missed (Rumpel, 1913). He also sug-
gested that there was excess of silver in the eyes.
Another important observation which passed unno-
ticed was by Bramwell, whose house physician Wilson
had once been (Bramwell, 1916). Bramwell described
a family in which four siblings died, between the ages
of 9 and 14 years, of rapid-onset liver failure and sug-
gested that this might be related to Wilson disease. It
would be interesting to see if any descendants of this fam-
ily still exist so that they could be tested to see if they
carry, in single dose, the mutation for Wilson disease.
The next important addition to knowledge came when
Hall (1921) showed that this disease was inherited in a
recessive mode, later confirmed in more detail by Bearn
(1960), who studied 30 families he saw in New York

*Correspondence to: J.M. Walshe, MD, ScD, FRCP, 58 High Street Hemingford Grey, Huntingdon PE 28 9BN, UK.
E-mail: penicillamine@waitrose.com
2 J.M. WALSHE
Fig. 1.1. Dr. John Walshe (right) with Dr. James Kinner
Wilson, son of neurologist Samuel Alexander Kinner Wilson,
whose 1912 publication on hepatolenticular degeneration was
being celebrated at the Centennial Symposium on Wilson’s
disease in London, October 5–6, 2012.
whose ancestry could be traced back to Eastern Europe or
Southern Italy.
It must be noted that in Wilson’s original publication
of only 4 patients, he described almost all of the signs and
symptoms which we now know are characteristic of this
disease (Wilson, 1912). He carried out his own patho-
logic studies and even traveled to Switzerland to collect
the brain of a patient dying of his disease. Wilson did not
believe that the liver pathology was a significant factor in
the natural history of the disease, although one of his own
patients actually died of bleeding esophageal varices.
Over the next 30 years there were only minor
advances in the understanding of the disease, whose
course remained remorseless and invariably fatal. In
1922 Siemerling and Oloff described the association of
sunflower cataracts with Kayser–Fleischer rings and
noted the similarity to lesions caused by intraocular dam-
age with intraocular copper fragments. Vogt (1929) and
Haurowitz (1930) reported finding excess copper in the
brain and liver of Wilson disease patients – an observa-
tion which was not followed up.
In 1945 Sir Rudolph Peters and his team in
Oxford were able to report their earlier work in the devel-
opment of the antiarsenical drug, dimercaprol (British
anti-Lewisite, BAL). This was developed during the Sec-
ond World War to combat anticipated attack by Hitler
with the arsenical gas Lewisite. The importance of this
became apparent in 1948 when Cumings showed that
Wilson disease was indeed due to accumulation of excess
copper in the brain and liver of all patients dying of this
disease and postulated that treatment with BAL might
halt the progress of the illness (Cumings, 1951). By coin-
cidence, at the same time, Mandelbrote et al. (1948)
reported their findings on the effect of BAL on copper
excretion on a wide spectrum of patients with neurologic
diseases; one of these patients had Wilson disease and in
this particular patient there was a great increase in the
copper excreted in the urine. This important observation
was followed up in 1951 by Cumings himself in London
and by Denny Brown and Porter (1951) in Boston. Both
teams showed that courses of BAL resulted in significant
improvement in their patients’ neurologic symptoms.
However it soon became apparent that patients needed
repeated courses of the drug and subsequent courses
were less effective than the initial one. In addition the
injections were painful and associated with a wide vari-
ety of toxic reactions. This was clearly not an ideal
treatment.
The picture changed when, in 1956, I reported that
penicillamine promoted a much larger excretion of cop-
per in the urine than either BAL or the other medical che-
lating agent ethylenediamine tetraacetic acid (EDTA)
(Walshe, 1956, 1960). Penicillamine is a breakdown
product of penicillin and is excreted in the urine of all
patients treated with penicillin, an observation I had
made some years earlier when studying changes in amino
acid metabolism in patients with liver damage (Walshe,
1956). This idea came to me when working in the Liver
Unit under Dr. Charles Davidson, at the Boston City
Hospital. Having seen one of Professor Denny Brown’s
Wilson disease patients who was not prospering on treat-
ment with BAL, it occurred to me that penicillamine,
with its –SH and –NH3 groups, might have the right
chemical structure to chelate copper and promote its
excretion in the urine. Dr. Davidson was able to obtain
for me 2 grams of penicillamine from Professor Sheehan
at the Massachusetts Institute of Technology. Having
taken 1 gram myself to prove its safety, I administered
the second gram to Professor Denny Brown’s patient
and recorded the very satisfactory cupresis so induced.
With this simple procedure the start of penicillamine
therapy was launched.
In those far-off days there were no ethical committees
to be approached for permission; life was a lot easier
when trying out new ideas. By 1960 I was able to publish
the first account of a patient with Wilson disease improv-
ing significantly as a result of this new therapy (Walshe,
1960). This was confirmed by similar studies by
Scheinberg and Sternlieb, also in 1960.
Whilst these important advances in the treatment of
this disease were progressing, so also was our under-
standing of its pathogenesis. In 1948 Holmberg and
Laurell described the finding of a copper-carrying pro-
tein in the plasma which they named ceruloplasmin. In
1952, working independently in New York, both Bearn
and Kunkel and Scheinberg and Gitlin showed that
patients with Wilson disease all had low or absent con-
centrations of this protein (Bearn and Kunkel,1952;
Scheinberg and Gitlin, 1952). Shortly after this
Cartwright and his team (1954) in Salt Lake City pub-
lished an important article on copper metabolism in
 HISTORY OF WILSON DISEASE: A PERSONAL ACCOUNT
Wilson disease, an article which inspired my own interest
in this malady, in which they discussed the various ther-
apies then available and their shortcomings. Despite
these real advances Uzman, working in Denny Brown’s
department in Boston, believed that Wilson disease was
due to an abnormality of peptide metabolism and that
copper deposition was a side-effect and not the directly
causative etiology of the disease (Uzman et al., 1956).
This hypothesis was later firmly disproved by Asator
et al. (1976) using a technique more sophisticated than
that available to Uzman. Although Uzman was wrong
on this point, he was able to confirm Bramwell’s hypoth-
esis that Wilson disease could present as liver disease in
children before the onset of neurologic signs (Bramwell,
1916; Uzman et al., 1956; Chalmers et al., 1957).
The clinical picture of the disease was widened by
Bearn, who showed that there were abnormalities of
renal function and skeletal lesions in many patients
(Bearn, 1957). He and Kunkel also introduced the idea
of using radiolabeled copper, the short half-isotope, to
study copper metabolism in Wilson disease patients
(Bearn and Kunkel, 1954). About the same time the role
of ceruloplasmin in the pathogenesis was hotly debated
and Uzman and Hood (1952) rightly pointed out that
the concentrations of ceruloplasmin found in Wilson dis-
ease patients was quite variable and frequently over-
lapped that found in symptomless carriers of the
Wilson disease gene in single dose.
In 1959 Cumings published a book, Heavy Metals
and the Brain, in which he tried to cover the entire liter-
ature of Wilson disease to date and this remains an
invaluable historical source of information for those
interested in the history of the disease, as also does
Scheinberg and Sternlieb’s (1984) monograph.
Beginning in the 1960s I realized the potential of
using radiocopper in the investigation of Wilson disease,
working first with Osborn, and later with Potter (Osborn
and Walshe, 1965, 1967; Walshe and Potter, 1977).
Together we devised a method of determining the uptake
of copper by the liver and its further distribution through-
out the body (Osborn and Walshe, 1967). It was thus pos-
sible to show that in the presymptomatic stages of the
disease the liver had a high affinity for the metal and
90% of the injected dose was present in the liver within
24 hours. As the disease progressed the liver uptake of
radiocopper became less and less effective and the
isotope could be found distributed through many other
tissues. However treatment with chelating agents effec-
tively decoppered the liver and restored its ability to
sequester the metal. But in no patient was it possible to
show, as in normal individuals, a peak in the lower abdo-
men which represented copper excreted via the bile in to
the intestine (Walshe and Potter, 1977). Later it was
possible to show, in patients and controls, who had
3
undergone cholecystectomy for gallstones, that the dif-
ference in copper excretion in the bile was of an order
of magnitude between normal individuals and patients
(Gibbs and Walshe, 1980). Furthermore, by studying
the difference in urine copper concentration in pigment
stones the mean figure for copper in patients was
50 mg/g compared with the figure of 1072.5 mg/g in con-
trols (Walshe, 1998).
The first description of hemolysis and a serious com-
plication of Wilson disease was first described by Profes-
sor Sherlock and her team at the Royal Free Hospital in
London (Sherlock et al., 1968), whilst Roche-Sicot and
Benhamou (1977) described the first case of massive
hepatic necrosis and hemolysis, thus proposing the idea
of acute fatal Wilson disease. Recently I showed that
hemolysis was to be found in 6.9% of 321 patients and
that the hemolysis was extravascular, the age of onset
was 12.6 years, and there was a female-to-male ratio of
2:1 (Walshe, 2013).
Again, in the 1960s, in addition to our increased
understanding of the pathogenesis of Wilson disease
was an increased range of therapies which favorably
influenced the prognosis. In 1961 Schouwink reported
that zinc salts could block the uptake of copper from
the intestine and thereby induce, although very slowly,
a negative copper balance and this was later taken up
enthusiastically by Hoogenraad et al. (1979) in the
Netherlands and by Brewer et al. (1981) in the United
States.
In an attempt to overcome the toxic side-effects of
penicillamine, which were becoming apparent by the
late 1960s (Walshe, 1968), with the invaluable help of
Dr. Hal Dixon, I proposed the use of triethylene tetramine
2HCl as an alternative chelating agent and this proved to
be an effective therapy and remarkably free of side-
effects (Walshe, 1982). The next advance was the
introduction of liver transplantation by Starzl and his
team in 1971 (DuBois et al., 1971). This approach actu-
ally cured the Wilson disease so patients no longer
needed chelation treatment, but they did require lifelong
immunosuppression.
The final advance came with the use of ammonium
tetrathiomolybdate (Walshe, 1986). I first took this
myself for a week to test its safety before giving it to a
patient who had proved intolerant to penicillamine and
trientine and had found the side-effects of zinc salts intol-
erable. Within a year it had effectively decoppered her
liver and changed the histology from a fatty liver with
cellular infiltrates to normal (Walshe, 1986). It is of inter-
est that Bickel et al. (1957), had tried the use of molyb-
date in the 1950s, but they had not used the tetrathio salt
and their preparation had no anticopper properties so the
possibilities of this form of therapy were set back some
30 years. They had relied on the observation that sheep
4 J.M. WALSHE
fed on pasture contaminated with molybdenum devel-
oped copper deficiency but failed to realize that in the
rumen of the sheep the MoO4 was converted, by reducing
conditions, to MoS4, an action which does not take place
in the human gut.
The most recent, and perhaps the most important,
breakthrough came in 1993 when three separate groups
(Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al.,
1993) reported in Nature Genetics that they had
identified the Wilson disease gene as a P-type APTase,
140-kDa copper-transporting enzyme located on chro-
mosome 13q14. There have been no further important
advances since then, except for the identification of more
than 500 mutations of this gene. As most patients are
compound heterozygotes the number of permutations
and combinations is enormous and will make
phenotype–genotype correlations almost impossible.
Having brought the story up to date the question
remains, what of the future? It would seem that, as far
as natural history and biochemistry and genetics are con-
cerned, the disease is worked out. However therapy is far
from ideal. The initial therapy with BAL still has a place
for severely dystonic patients but it has too many
side-effects and its administration is something of an
ordeal for the patient. Penicillamine is effective in many
cases but also has too many side-effects, mostly immu-
nogenic in nature and, in a small percentage of patients,
can induce a sudden deterioration which is, unfortu-
nately, unpredictable (Walshe, 2011). Trientine has far
fewer side-effects and is a very useful alternative
(Walshe, 1982) but, like all forms of treatment, is some-
times associated with initial worsening of symptoms,
which is always a worrying state of affairs. Tetrathiomo-
lybdate is yet another alternative but is not yet in the
pharmacopeia so is not readily available. Zinc salts are
effective in blocking copper absorption from the gut
but can only produce a very slow negative copper bal-
ance and are probably more useful for maintenance than
initial therapy. Many zinc salts are gastric irritants and are
probably contraindicated in patients with prominent
esophageal varices. Finally there is liver transplantation,
in effect a cure, but which condemns the patient to
long-term immunosuppression with all its possible com-
plications. Its use suggests a failure of either diagnosis or
initial treatment. One day it may be possible to offer gene
replacement but this is not yet even on the horizon.
It remains to be seen what the future holds for patients
with Wilson disease.
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Handbook of Clinical Neurology, Vol. 142 (3rd series)
Wilson Disease
A. Członkowska and M.L. Schilsky, Editors
http://dx.doi.org/10.1016/B978-0-444-63625-6.00002-1
© 2017 Elsevier B.V. All rights reserved

Chapter 2

Epidemiology and introduction to the clinical presentation

of Wilson disease

CHRISTINE LO AND OLIVER BANDMANN*

Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK

Abstract
Our understanding of the epidemiology of Wilson disease has steadily grown since Sternlieb and
Scheinberg’s first prevalence estimate of 5 per million individuals in 1968. Increasingly sophisticated genetic
techniques have led to revised genetic prevalence estimates of 142 per million. Various population isolates
exist where the prevalence of Wilson disease is higher still, the highest being 885 per million from within the
mountainous region of Rucar in Romania. In Sardinia, where the prevalence of Wilson disease has been cal-
culated at 370 per million births, six mutations account for around 85% of Wilson disease chromosomes
identified. Significant variation in the patterns of presentation may however exist, even between individuals
carrying the same mutations. At either extremes of presentation are an 8-month-old infant with abnormal liver
function tests and individuals diagnosed in their eighth decade of life. Three main patterns of presentation
have been recognized – hepatic, neurologic, and psychiatric – prompting their presentation to a diverse range
of specialists. Deviations in the family history from the anticipated autosomal-recessive mode of inheritance,
with apparent “pseudodominance” and mechanisms of inheritance that include uniparental isodisomy (the
inheritance of both chromosomal copies from a single parent), may all further cloud the diagnosis. It can
therefore take the efforts of an astute clinician with a high clinical index of suspicion to clinch the diagnosis
of this eminently treatable condition.

INTRODUCTION
Sternlieb and Scheinberg (1968) first estimated the prev-
alence of Wilson disease in 1968 to be 5 per million indi-
viduals. Subsequent work by Bachmann et al. (1979a, b)
at the Leipzig Centre for Wilson’s Disease in East
Germany between 1949 and 1977 calculated a birth prev-
alence of Wilson disease of 29 per million. Saito (1981)
reported a similar birth prevalence of 33 per million in
1981. With this expanded data set and taking into
account mortality figures from the USA pertaining to
the number of deaths from Wilson disease, which they
assumed to be half the true number, Scheinberg and
Sternlieb (1984) revised their worldwide prevalence esti-
mate to 30 per million with a heterozygote carrier fre-
quency of 1 in 90, whilst acknowledging the existence
of population isolates in which a higher prevalence
was likely.
Park and coworkers (1991), concerned by the potential
for a significant number of undiagnosed individuals with
Wilson disease, sought to determine the prevalence of
Wilson disease in Scotland. Examining computerized hos-
pital statistical records, death certificates, and the results of
a postal questionnaire sent to relevant clinicians, they iden-
tified 21 patients with Wilson disease alive at the point of
analysis in a population of 5 090 700 individuals, equating
to a prevalence of 4 per million. The authors re-examined
the data presented by Bachmann et al. (1979a, b) from East
Germany and calculated a revised prevalence rate of 4.6
per million, which was comparable with their own figure
(Park et al., 1991). This rate was still higher than that of

*Correspondence to: Oliver Bandmann, SITraN, 385a Glossop Road, Sheffield S10 2HQ, UK. Tel: +44-114-2222262,
E-mail: o.bandmann@sheffield.ac.uk
8 C. LO AND O. BANDMANN
2.7 per million reported by Przuntek and Hoffmann (1987)
from West Germany, notwithstanding their less than ideal
response rate of 45%. Reilly et al. (1993) adopted a meth-
odologic approach similar to that taken by Park et al. in
order to determine the prevalence of Wilson disease in
the Republic of Ireland. Twenty-six cases of Wilson
disease over a 19-year period were identified, 5 of
which had died of an illness consistent with the disease
before its formal diagnosis. An adjusted birth incidence
rate of 17 per million was calculated for the period
of 1950–1969, corresponding to a gene frequency of
0.41% and heterozygote incidence of 0.82%. To allow
for the maximal degree of anticipated consanguinity,
the gene frequency and heterozygote incidence esti-
mates were revised to 0.36% and 0.72% respectively,
providing minimum disease estimates.
In this chapter we will provide an overview of clinical
genetic and biochemical studies to date and examine
their roles in further delineating the epidemiology of
Wilson disease. We will explore factors which may help
to explain the disparity between the number of individ-
uals predicted to be affected on the basis of screening
studies and those diagnosed clinically. Finally, we will
review the clinical course of Wilson disease and the
diverse ways in which it may present.
BIOCHEMICAL SCREENING FOR THE
STUDY OF PREVALENCE
Many of the early epidemiologic studies are thought to
have underestimated the prevalence of Wilson disease,
not least due to their reliance on symptomatic patients
having received the correct diagnosis. Considerable
interest lies in the early diagnosis of presymptomatic
individuals with Wilson disease so that the early instiga-
tion of treatment may prevent future complications. The
UK NHS newborn blood spot screening program is
already utilized to screen for six inherited metabolic dis-
eases. A number of pilot studies have investigated the
possibility of mass screening for Wilson disease. Serum
copper is typically reduced in Wilson disease. However,
its utility as a target agent for dried blood spot screening
is limited by its environmental abundance and presence
in unpredictable amounts in dried filter paper itself
(Hahn, 2014). Instead an enzyme-linked immunosorbent
assay using a monoclonal antibody specify to holoceru-
loplasmin (ceruloplasmin bound with copper) has been
developed to measure ceruloplasmin in dried blood spots
(Ohura et al., 1999; Yamaguchi et al., 1999; Hahn et al.,
2002; Kroll et al., 2006).
Screening of 1045 anonymous newborn screening
dried blood spots failed to identify any cases of Wilson
disease (Kroll et al., 2006). The results of screening a
total of 126 810 newborns by Yamaguchi et al. (1999)
were similarly disappointing. They achieved greater suc-
cess when blood samples from a slightly older population
of 24 165 children aged 6 months to 9 years old were used
to measure ceruloplasmin with a combination of oxidase
activity, particle-coated fluorescence immunoassay, and
specific monoclonal antibody. Three patients with Wil-
son disease were identified, the youngest patient being
8 months old, equating to a prevalence of 124 per million.
The authors suggested that, considered in combination
with existing screening programs, serum ceruloplasmin
level stability and ease of blood collection meant that
the age of 3 years old was the most opportune time to
undertake any population-wide ceruloplasmin-based
mass screening in children (Yamaguchi et al., 1999).
In apparent support of their supposition, a presympto-
matic 32-month-old boy was the sole positive case iden-
tified through a pilot study in which ceruloplasmin levels
in dried blood spots were assessed in 3667 asymptomatic
Korean children aged between 3 months and 15 years old
(Hahn et al., 2002). A similar study, with a 2.6-fold
higher prevalence of 717 per million, screened the dried
blood spots of 2789 children, identifying 2 aged
30 and 39 months old, with markedly low ceruloplasmin
levels (0.24 and 0.40 mg/dL compared to an average
of 12.4 mg/dL). The diagnoses were later confirmed
genetically by the identification of compound heterozy-
gous A803T/2871delC and R778L/G1035V mutations
(Ohura et al., 1999).
Noninvasive techniques to assess the amount of uri-
nary holoceruloplasmin protein have also been developed.
Screening of urine from 48 819 children led to the identi-
fication of 36 children with very low urinary and serum
holoceruloplasmin levels, 2 of whom additionally demon-
strated low serum copper and increased urinary copper
excretion. Both children were otherwise asymptomatic
with no signs on physical examination. Yet on further
genetic testing they were found to be compound heterozy-
gotes for mutations recognized in Japanese patients with
Wilson disease (Owada et al., 2002).
The calculated prevalence of 41 per million was less
than half that derived from another Japanese study, where
a further 11 362 children underwent measurement of uri-
nary ceruloplasmin concentration. An immunologic latex
agglutination assay kit was applied to an automated ana-
lyzer and results were shown to correlate closely with uri-
nary holoceruloplasmin level measurements. Three
successive screening stages led to the identification of
9 children as positive for low urinary ceruloplasmin levels
from an initial 668 positive subjects. A diagnosis of
Wilson disease was excluded in 8 children in the context
of normal biochemical data. Ultimately, in the absence of
symptoms or clinical signs, genetic confirmation was
necessary to diagnose 1 child, aged 3 years old, as a com-
pound heterozygote (Nakayama et al., 2008).
 EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE
MODERN GENETICS STUDIES
As techniques in molecular genetics have developed,
attempts have been made to better characterize the under-
lying ATP7B mutations in patients with Wilson disease
from different populations. In 1999 Curtis et al. studied
Wilson disease mutations in Britain in 52 patients by
screening parts of the ATP7B gene using single-strand
conformational polymorphism (SSCP) and subsequent
sequencing. At the time SSCP screening of three exons
was predicted to identify around 60% of mutations.
Indeed, complete or partial genetic characterization
was achieved in 37 individuals, with 4 patients having
a homozygous ATP7B mutation, 18 being compound
heterozygotes, and 15 in whom a mutation in only one
allele was detected (Curtis et al., 1999). An earlier study
had similarly managed to characterize around 60% of
disease alleles (Nanji et al., 1997). A significant
improvement in the overall mutation detection frequency
to 98% was reported in a subsequent study from the UK
by Coffey et al. in 2013. Genetic confirmation was
achieved in 177 out of 181 patients with Wilson disease
diagnosed on the basis of clinical and biochemical find-
ings. Of the remaining 4 patients, 2 were found to have a
single ATP7B mutation whilst, in the others, no muta-
tions were identified (Coffey et al., 2013). The improved
detection rate was largely due to improved mutation tech-
niques as well as the analysis of the entire ATP7B coding
region.
Though population estimates vary, in Europe the most
common mutation observed is the H1069Q missense
mutation (Shah et al., 1997; Stapelbroek et al., 2004;
Gomes and Dedoussis, 2015). Ivanova-Somlenskya
et al. (1999) described a cohort of 40 unrelated patients
with Wilson disease from Slavonic families from the
European part of Russia to have H1069Q mutations,
representing 48.7% of Wilson disease chromosomes.
Tarnacka et al. in 2000 described 148 Polish patients with
Wilson disease from 95 families. The H1069Q mutation
was found in 57% of chromosomes studied appearing in
the homozygous and heterozygous states in 39.9% and
30.4% of cases respectively. Though they attempted
genotype–phenotype correlation, no relationship was
found either with symptomatology or age of onset. In
contrast, a study by Shah et al. (1997) reported a younger
age of onset in individuals heterozygous for the H1069Q
mutation at 15.4 years of age compared to 20 in homozy-
gotes. Through their use of the Hardy–Weinberg equilib-
rium, they estimated that the ratio of H1069Q
heterozygotes to homozygotes was in the order of 3.26.
In a study of 70 symptomatic Dutch patients from
59 unrelated families with predominant hepatic presenta-
tion, the H1069Q mutation was reported to account for
33% of alleles, with 23% of patients homozygous for
9
the mutation compared to 20% being heterozygous.
Homozygotes were reported to more often present with
neurologic symptoms, though this did not reach statisti-
cal significance. However, compared to patients without
the H1069Q mutation, homozygotes and heterozygotes
were 3.5 and 2.13 times more likely to present neurolog-
ically (Stapelbroek et al., 2004). Mirroring studies by
Shah et al. (1997) and Gromadzka et al. (2006), patients
heterozygote for the H1069Q mutation were found to
present at a slightly earlier age than homozygotes but pre-
sented at a later age than patients harboring non-H1069Q
mutations (Stapelbroek et al., 2004).
A greater disparity in the existence of heterozygous
and homozygous H1069Q mutations was found in
Romanian patients, where an allelic frequency of
38.1% was reported, accounting for 34.2% of mutations
in the heterozygous and 21.1% in the homozygous state
(Iacob et al., 2012). In a German cohort described
by Merle et al. in 2010, patients with homozygous
H1069Q mutations occurred at an even greater fre-
quency, being identified in 50.9% of patients with a fur-
ther 25.4% of patients being compound heterozygotes
with one H1069Q mutant allele.
Though the H1069Q mutation is considered to be
the most common mutation in Eastern and Northern
European populations, this is not necessarily the case
worldwide. Indeed, in a Japanese study by Nanji et al.
in 1997, out of 21 unrelated families the H1069Q muta-
tion was not detected in a single case. Instead, the R778L
mutation occurred at the greatest frequency, accounting
for 12% of the identified Wilson disease mutations.
The R778L mutation has also been reported in Taiwanese
families with Wilson disease (Chuang et al., 1996), as
well as in Korean patients (Kim et al., 1998) and in the
Chinese Han population, in whom the allele frequency
is considerably higher, at 45.6% (Liu et al., 2004).
A Spanish study identified the M645R missense muta-
tion as the most frequent mutation; its detection in the
heterozygous state was reported in 22 out of 40 unrelated
patients with Wilson disease (Margarit et al., 2005). In
Costa Rica another missense mutation, the N1270S
mutation, also observed in Sicilian and continental Ital-
ian and Turkish populations, was found to represent
61% of all mutations (Tanzi et al., 1993; Figus et al.,
1995; Shah et al., 1997).
Whilst interpatient variability may in part be
explained by differences in the underlying genotype,
studies have examined rates of intrafamilial concordance
between individuals sharing similar genetic characteris-
tics. In a study involving 73 index cases from 73 unre-
lated families and 95 of their siblings, with an overall
H1069Q allelic frequency of 77%, an 86% rate of con-
cordance of presenting symptoms was noted among indi-
viduals first presenting with hepatic symptoms. A lower
10 C. LO AND O. BANDMANN
concordance rate of 66% was noted between index cases
presenting primarily with neurologic symptoms and their
symptomatic siblings (Chabik et al., 2014).
In a survey of Wilson disease in Iceland over a 40-year
period, 8 patients, from two kindreds, were diagnosed
with Wilson disease. All patients were found to be homo-
zygous for the same 2010del7 mutation, presumably
inherited from a shared common progenitor. Despite
their common genotype and primarily neurologic symp-
tomatology, differences in their presentation were noted,
with 3 out of the 8 manifesting with psychiatric symp-
toms (Thomas et al., 1995). Differing phenotypes were
also reported by Wang et al. (2011) in two siblings, both
compound heterozygous for the I1148T missense muta-
tion and the S105Stop mutation. Whilst neuropsychiatric
symptoms were manifested by the older proband, her
younger brother’s presentation was of the hepatic type.
Członkowska et al. (2009) furthermore described phe-
notypic discordance in two pairs of monozygotic twins.
In the first pair of twins, both sisters were compound het-
erozygous for the H1069Q and N404Kfs mutations and
were found to have evidence of previous hepatitis
B infection, with active infection having been excluded.
The proposita had presented relatively late at the age of
38, with a 4-year history of fatigue followed by progres-
sive hepatic decompensation and then the onset on neu-
ropsychiatric symptoms. In contrast, her twin was
asymptomatic, with normal brain and liver imaging,
the only biochemical abnormality detected being
decreased serum levels of copper and ceruloplasmin.
The second pair of twins described was homozygous
for the common H1069Q mutation. The results of their
diagnostic evaluation at the age of 28 were similar. Imag-
ing revealed T2-weighted changes on brain magnetic res-
onance imaging (MRI) and hepatosplenomegaly on
ultrasound scanning. Laboratory investigations identi-
fied evidence of abnormal copper metabolism, liver
function test derangement, leukopenia, and thrombocy-
topenia. Whilst one twin had presented with neurologic
symptoms, the other described problems with menstrual
irregularity and recurrent nose bleeding but barring the
finding of Kayser–Fleischer rings on examination was
otherwise devoid of neurologic signs (Członkowska
et al., 2009).
POPULATION ISOLATES
Various population isolates exist in which Wilson disease
occurs at a greater incidence and prevalence than would
otherwise be expected based on worldwide figures. In a
small mountain village next to Heraklion city in the
island of Crete, the reported incidence of clinically
and/or biochemically diagnosed Wilson disease was 6
out of 90 births over a 25-year period. Screening of
200 unrelated habitants revealed a carrier frequency of
p.Q289X and 398delT mutations of 1 in 11 (Dedoussis
et al., 2005).
The relative genetic homogeneity within the Sardin-
ian population is thought to arise from its marked isola-
tion and history of inbreeding propagating a possible
founder effect (Loudianos et al., 1999b). Figus et al.
(1995) postulated a higher prevalence of Wilson disease
within the Sardinian population in 1995 based on their
identification of 10–12 new cases per year. Sixteen
different haplotypes associated with Wilson disease
chromosomes were identified in 39 individuals of Sar-
dinian descent, of which haplotype IX (5 10 3 3) was
the most common, observed at a frequency of 55% of
Wilson disease chromosomes. Further analysis of the
promoter and the 5’ UTR of the Wilson disease gene
sequence from patients with the most common haplotype
revealed a single mutation consisting of a 15-nt deletion
from position –441 to position –427 relative to the trans-
lation start site, which accounted for 60.5% of the studied
Wilson disease chromosomes (Loudianos et al., 1999b).
These mutations are uncommon in patients of European
ancestry outside of Sardinia (Cullen et al., 2003). How-
ever, screening of 5290 newborns in Sardinia revealed
the presence of 122 individuals heterozygous for the –
441/–427 deletion, equating to an allelic frequency of
1.15%. With the inclusion of an allelic frequency of
0.77%, accounting for non- –441/–427 deletions, an
overall Wilson disease mutation frequency of 1.92%
was inferred. Assuming Hardy–Weinberg equilibrium,
the prevalence of Wilson disease was calculated at 370
per million births, one of the highest in the world and
considerably higher than that first quoted by
Bachmann et al. (1979b) (Zappu et al., 2008). Yet it is
comparable with the estimated prevalence of 366 per mil-
lion calculated from a homozygosity index of 0.476 and
inbreeding coefficient of 7.8  10–4 and applied to a set
of 178 Sardinian individuals with clinically and molecu-
larly diagnosed Wilson disease (Gialluisi et al., 2013). As
six mutations account for around 85% of Wilson disease
chromosomes within the Sardinian population, further
efforts have included the targeted screening for these
mutations using automated TaqMan screening (Zappu
et al., 2010). The identification of heterozygotes through
this initial screening process is followed by screening for
the remaining 16 mutations detected in the Sardinian
population, yielding a sensitivity of 94.6% with a spec-
ificity of 100% and potentially resulting in the diagnosis
of an additional 5 cases per year (Lovicu et al., 2003;
Zappu et al., 2008, 2010).
A similarly high prevalence of Wilson disease of 385
per million was reported for the island of Gran Canaria,
where the rare Leu708Pro mutation was observed in
18 out of 24 individuals with Wilson disease; 12 in the
 EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE
homozygous state, 4 as compound heterozygotes in
conjunction with another established Wilson disease
mutation, and 2 further individuals who only had one
identifiable ATP7B mutation. A further 3 individuals
who were compound heterozygous for previously
established Wilson disease mutations were detected
and no mutations were identified in 3 affected
individuals from the same pedigree. Despite their com-
mon genotype, affected individuals with homozygous
Leu708Pro mutations had marked phenotypic variability
(Garcia-Villarreal et al., 2000).
The same group who initiated newborn screening of
the Sardinian population also screened 396 newborns
on the similarly isolated Greek island of Kalymnos.
The screening of 60% of newborns between 1999 and
2003 identified 18 newborns heterozygous for the
H1069Q mutation, 9 heterozygous for the R969Q muta-
tion, and 1 compound heterozygote. The allelic frequen-
cies of the H1069Q and R969Q mutations were 2.4% and
1.3% respectively, yielding a carrier rate for the two
mutations of 7% and, assuming Hardy–Weinberg equi-
librium, an even higher prevalence than in Sardinia of
135 per million births (Zappu et al., 2008).
The highest prevalence of genetically confirmed
Wilson disease ever reported is 885 per million from
within the mountainous region of Rucar in Romania.
Its isolation and tradition of intraclan marriage are
thought to have contributed to substantial levels of con-
sanguinity. Screening for ATP7B mutations in 50 individ-
uals from two large families, spanning six generations,
identified compound heterozygous H1069Q/M769H–
mutations in 5 symptomatic adults and 2 clinically
asymptomatic children. Symptomatic individuals dem-
onstrated strong phenotypic concordance with an age
of symptom onset of 18  1 years. Initial symptoms
included dysarthria and dysphagia and, in all symptom-
atic individuals, the presence of Kayser–Fleischer rings
was observed (Cocos et al., 2014).
GENETIC VERSUS CLINICAL
PREVALENCE
Sequencing of all 21 ATP7B exons in over 1000 DNA
samples and exons 8, 14, and 18 in over 5000 samples
by Coffey et al. led to a revised figure of 0.040 or 1:25
as the frequency of heterozygote mutation carriers in
the UK. Even with the exclusion of four class 2
single-nucleotide variants in the absence of in silico
(computer-simulated) evidence of pathogenicity, the fre-
quency of individuals predicted to carry two pathogenic
ATP7B mutations was 142 per million, over four times
the often-quoted prevalence figure of 30 per million
for Wilson disease (Scheinberg and Sternlieb, 1984;
Coffey et al., 2013).
11
There is a significant discrepancy between the number
of individuals predicted on the basis of genetic studies to
be affected with Wilson disease and those clinically diag-
nosed. Reduced penetrance of ATP7B mutations may
offer an alternative explanation for the lower than
expected number of individuals diagnosed clinically
with Wilson disease. Indeed, Członkowska et al.
(2008) described the indolent course of Wilson disease
first diagnosed in a woman at the age of 54 when she
was assessed during familial screening. At the time of
her initial assessment she was asymptomatic, the only
sign being Kayser–Fleischer rings, and she elected to
refuse treatment. Genetic confirmation was later
achieved when she was found to be homozygous for
the H1069Q mutation. It was not until the age of 74 that
she started to demonstrate evidence of liver dysfunction
with a slight elevation in her liver enzymes and reduction
in serum albumin. Thirty years after her original diagno-
sis, at the age of 84, she remained symptom free
(Członkowska et al., 2008).
Identification of such individuals may still be war-
ranted with respect to the risk of transmission to potential
children that they may have. Of greatest concern is the
potential for there to be a large number of patients
affected by Wilson disease who remain undiagnosed
and without effective treatment, having either never pre-
sented to their medical practitioner or having presented
with seemingly atypical features (Coffey et al., 2013;
Bandmann et al., 2015). The absence of a “typical” fam-
ily history suggestive of autosomal-recessive inheritance
may also dissuade a clinician from considering the diag-
nosis of Wilson disease. It is important to be aware of less
common mechanisms of inheritance, including the inher-
itance of three different ATP7B mutations, the potential
for Wilson disease in successive generations, and the role
of segmental uniparental isodisomy, as will now be
outlined.
The majority of patients with Wilson disease are com-
pound heterozygotes with one mutation on each ATP7B
allele (Schilsky, 2014a). There are nevertheless a number
of patients who carry three different mutations.
Dedoussis et al. (2005) described a 15-year-old boy with
Wilson disease from a consanguineous pedigree who
was compound heterozygous for the Q289X, I1148T,
and G1176R mutations; the latter two mutations cosegre-
gating in cis (i.e., in the same copy of the gene). Although
his clinically asymptomatic younger brother carried a
wild-type allele alongside the I1148T/G1176R muta-
tions, biochemical indices demonstrated significantly
lower copper and ceruloplasmin levels.
In an ethnic Han Chinese population, with one out of
65 families reporting consanguinity, Mak et al. (2008)
identified 4 patients who carried the Q1142H nonsense
and I1148T missense mutations in cis, with another
12 C. LO AND O. BANDMANN
mutation on the trans allele (i.e., in the other copy of the
gene). All mutations had previously been identified inde-
pendently as disease-causing mutations (Loudianos et al.,
1998, 1999a). Due to the frequency of their cosegregation
in cis, the authors recommended further parental genotyp-
ing or exon sequencing upon the simultaneous discovery
of Q1142H and I1148T mutations, in order to distinguish
individuals bearing two mutations from those bearing
three (Mak et al., 2008).
Wang et al. (2011) subsequently described 2 patients
who presented with neuropsychiatric symptoms and
were found to carry three mutations. Fascinatingly, hav-
ing previously been reported to cosegregate with another
mutation in cis, the I1148T and Q1142H mutations were
identified on the same allele, a finding also reported in
another patient by Coffey et al. (2013). The I381S/
I1184T mutation and the N41S/I1021V mutations have
also been reported to cosegregate in cis (Coffey
et al., 2013).
As an autosomal-recessively inherited condition,
Wilson disease would normally not be expected in sub-
sequent generations within a family. The phenomenon of
“pseudodominance” is one that has been observed in
other conditions, including Friedreich’s ataxia (Lamont
et al., 1997) and ataxia with ocular apraxia type 2
(Schols et al., 2008). Cases of Wilson disease in two suc-
cessive generations have also been reported. Loudianos
et al. (2013) described two families from Sardinia. In the
first family, Wilson disease was diagnosed after the pro-
band presented following a suicide attempt. She was
found to be compound heterozygous for the R919W
and H1069Q mutations. Screening of her triplet siblings
led to the diagnosis of her dizygotic sibling with Wilson
disease, carrying both the R919W and T993W muta-
tions. Their mother carried the R919W mutation in addi-
tion to a wild-type allele. A posthumous diagnosis was
made in their father, who was postulated to have trans-
mitted the H1069Q and T993W mutations, having died
at the age of 39 with liver failure. The affected mother of
another family was homozygous for the –441/–427 dele-
tion, the most common regional mutation. Both of her
monozygotic twins had inherited an allele with
the –441/–427 deletion as well as the G869R mutation,
which they inherited from their father. Despite being
compound heterozygotes, the twins were clinically
asymptomatic when they were diagnosed at the age of
33 (Loudianos et al., 2013).
A similar pattern of inheritance, whereby an affected
parent unknowingly had children with a heterozygous
carrier resulting in a child with Wilson disease, was
reported in three out of four French families described
by Dufernez et al. (2013). In the fourth family, the iden-
tification of both the mother and her affected child as
being homozygous for the same T569del mutation raised
the strong suspicion of consanguinity, later confirmed
when the mother and father were found to be first
cousins.
Based on the much-quoted heterozygote carrier fre-
quency of 1 in 90 (Scheinberg and Sternlieb, 1984),
Bennett et al. (2013) calculated the probability of an indi-
vidual with Wilson disease having an affected grandchild
to be 0.003%. Yet such a family did they describe, with
the proband’s mother, maternal aunt, and son having Wil-
son disease; this was due to all their reproductive partners
having been heterozygote carriers. Dziezyc et al. (2014)
identified nine nonconsanguineous families comprising
12 affected offspring from 9 probands from their screen
of 294 individuals. A risk of 4.08% of Wilson disease in
the offspring of probands was calculated, higher than the
figure of 0.5% that had previously been estimated (Ala
et al., 2007; Dziezyc et al., 2014).
In the study by Coffey et al. (2013), three families
with “pseudodominant” Wilson disease were identified.
In one family, the proband and his wife remained con-
cerned about the genetic risk to their asymptomatic chil-
dren in spite of a very low risk having been quoted to
them. Rather surprisingly, a novel variant, predicted by
in silico analysis to be pathogenic, was identified in
the proband’s wife, leading to the subsequent diagnosis
of one of their two children as a compound heterozygote
for the V1234F and R778W mutations (Coffey et al.,
2013). Taken together, the comparatively frequent obser-
vation of families with Wilson disease in subsequent gen-
erations in different populations lends support to the
assumption that heterozygote carrier status for ATP7B
mutations may be more common than previously
thought.
Segmental uniparental isodisomy has also been pro-
posed as a mechanism by which the direct transmission
of a normally monogenically inherited disorder may
occur between a single parent and his or her offspring
(Loudianos et al., 1999a). Uniparental isodisomy for
any chromosome is estimated to affect up to 1 in 3500
births and occurs when both copies of a chromosome
or part thereof are inherited from a single parent with
no contribution from the other. From their cohort of
181 index cases with Wilson disease, Coffey et al.
(2013) reported the finding of 2 patients homozygous
for different mutations, in the absence of biparental
homologue contribution. Multiple microsatellite markers
subsequently confirmed segmental isodisomy giving rise
to autozygosity for mutations inherited from one parent,
with potential significance with respect to genetic
counseling of individuals (Coffey et al., 2013).
Heterozygote carriers are often clinically asymptom-
atic. However, detailed examination may reveal subtle
abnormalities. Ultrastructural examination of liver tissue
using electron microscopy has revealed alterations in the
 EPIDEMIOLOGY AND INTRODUCTION TO THE CLINICAL PRESENTATION OF WILSON DISEASE
mitochondria and endoplasmic reticulum of heterozy-
gous individuals, in keeping with copper toxicity
(Lough and Wiglesworth, 1976). Biochemical studies
have reported various abnormalities of copper metabo-
lism, including decreased levels of serum ceruloplasmin
and copper and an increase in the biologic halftime for
the clearance of the isotope 65Cu from the plasma pool
(Marecek and Nevsimalova, 1984; Lyon et al., 1995;
Merli et al., 1998; Tarnacka et al., 2009).
Relative exchangeable copper determination may be
better at distinguishing patients with Wilson disease from
heterozygotes and controls (Trocello et al., 2014). In a
study involving 12 heterozygous carriers, without path-
ologic changes on plain MRI, significantly higher ratios
of Glx/Cr and Lip/Cr in 1H magnetic resonance spectros-
copy in the pallidum and thalami were reported com-
pared to controls, suggesting possible asymptomatic
copper deposition (Tarnacka et al., 2009). Electroen-
cephalographic abnormalities have also been described
in heterozygous children. However, such studies, per-
formed prior to the discovery of the ATP7B gene, relied
upon the inferred heterozygous carrier state in immediate
relatives of affected individuals. Out of 16 presumed
heterozygotes, neurologic abnormalities were detected
in 5 children. Abnormalities on electroencephalography
were detected in 12 children (75%), 5 of which demon-
strated epileptic features which did not correlate with
clinical symptomatology (Marecek and Nevsimalova,
1984). The findings were replicated in a subsequent
study but interestingly appeared to resolve by adulthood,
at which time no significant pathologic electroencepha-
lographic abnormalities were detected above that of the
control group (Nevsimalova et al., 1986).
The potential for heterozygous mutations in the
Wilson disease gene to influence other, apparently unre-
lated disease processes has been suggested. Cocco et al.
(2009) described a 37-year-old man with liver cirrhosis
secondary to chronic hepatitis C presenting with enceph-
alopathy, in whom treatment with penicillamine and tri-
hexyphenidyl resulted in a marked improvement of his
neuropsychiatric symptoms. The patient was later found
to be a heterozygous carrier of the G1111A missense
mutation, his response to treatment suggesting a subtle
defect in brain copper metabolism (Cocco et al., 2009).
In another case, screening of an otherwise asymptom-
atic 18-year-old man, the sibling of a patient recently
diagnosed with Wilson disease, revealed deranged liver
function tests. Indices of copper metabolism were normal
and further investigation led to a diagnosis of Alagille
syndrome, a rare autosomal dominantly inherited disor-
der of embryogenesis, affecting multiple organ systems,
in which abnormalities in bile duct formation led to liver
dysfunction. The associated JAG1 gene mutation was
found to have arisen de novo but, interestingly, one
13
H1069Q mutation was also identified. The authors spec-
ulated on the unusual occurrence of two rare conditions
within a single family and postulated a potential interplay
between the mutations that might account for an unusu-
ally late presentation of Alagille syndrome in the absence
of bile duct paucity typically observed on liver biopsy
(Amson et al., 2012).
CLINICAL COURSE
Three main patterns of presentation have been recog-
nized: hepatic, neurologic, and psychiatric, the symp-
toms of the latter two groups sometimes being
combined. The presentations observed by clinicians
may be skewed by the specialties to which patients pre-
sent. Unsurprisingly, neurologists report a greater pro-
portion of patients presenting with neurologic
symptoms (69.1%) compared to hepatic symptoms
(14.9%), whereas the opposite is true with patients pre-
senting to gastroenterologists, where hepatic features
predominate (68.1%) (Taly et al., 2007; Weiss et al.,
2011). In a registry of 627 patients with Wilson disease
seen at a neurology department in Poland, 510 of whom
were symptomatic at the point of diagnosis, a male pre-
ponderance was observed (55%). Neurologic and hepatic
symptoms and signs were seen more frequently in men
(60%) and women (57%) respectively. Overall, symp-
toms and signs were found to precede a diagnosis of
Wilson disease by around 2 years. The mean age of
symptom onset in the hepatic group was about 4 years
earlier than in the group with neuropsychiatric symptoms
(23.7  8 years versus 28.0  8 years) (Litwin et al.,
2012). Similar findings were reported in an independent
German cohort of 163 patients with Wilson disease, 137
of whom were symptomatic. Though the mean age of
symptom onset was younger than that described in the
Polish cohort, hepatic symptoms were again found to
precede neurologic symptoms (15.5 compared to
20.2 years of age). The interval between symptom onset
and diagnosis was reported to be almost three times lon-
ger in patients presenting with neurologic symptoms
compared to hepatic symptoms (44.4 months vs.
14.4 months) (Merle et al., 2007).
Individuals may present with a spectrum of hepatic
symptoms, with 25.4% of patients described by
Gheorghe et al. (2004) having clinically asymptomatic
disease, 21.8% fulminant hepatic failure, and 52.8%
chronic liver disease at presentation . Untreated, hepatic
symptoms may remain self-limiting, yet may also pro-
gress and precede the development of neurologic
sequelae. Reassuringly, in treated cases, the prognosis
of hepatic Wilson disease is good (Schilsky, 2014b).
Of patients presenting with neurologic symptoms,
improvement in 53.5–58.2%, stability in 22.4–27.3%,
14 C. LO AND O. BANDMANN
and worsening in 3.6–24.1% may be expected after
pharmacologic treatment (Merle et al., 2007; Bruha
et al., 2011). Psychiatric symptoms have been reported
in up to 51% of patients upon their initial presentation
and 72% of patients established on treatment, although
a pure psychiatric presentation is rare (Dening and
Berrios, 1989; Svetel et al., 2009). The most commonly
identified symptoms on the neuropsychiatric inventory
include anxiety, depression, irritability, and apathy
(Svetel et al., 2009). Cognitive changes, when present,
may be subtle and prone to influence by changes in
affect (Lang et al., 1990). Normalization may occur
in response to treatment (Rosselli et al., 1987). Whilst
symptomatic individuals typically present with hepatic,
neurologic, or psychiatric manifestations of Wilson dis-
ease, less common presentations have been recognized,
including presentation with an osseomuscular pheno-
type as well as renal, cardiovascular, endocrine, hema-
tologic, and dermatologic involvement. The varied
presentations of Wilson disease and the role of genetic
and environmental modifiers will be detailed in subse-
quent chapters.
With the ability to screen for mutations in the ATP7B
gene, a diagnosis of Wilson disease is possible even in
the absence of symptoms or definitive biochemical
abnormalities. Diagnosis is therefore possible in the pre-
symptomatic stage, even in the newborn. Shimizu et al.
(1997) described an asymptomatic 8-month-old boy who
was found on opportunistic screening to have low ceru-
loplasmin levels in the context of otherwise normal bio-
chemical parameters. No signs were demonstrated on
clinical examination. A diagnosis of Wilson disease
was made on the basis of DNA sequencing analysis,
which revealed him to be homozygous for the frameshift
mutation 2302insC (Shimizu et al., 1997). A child of the
same age was also reported to be the youngest individual
to present with evidence of liver dysfunction, detected
during a hospital admission with diarrhea. Clinical exam-
ination was normal, yet low ceruloplasmin levels raised
the suspicion of Wilson disease. Other causes of elevated
alanine aminotransferase and aspartate aminotransferase
were excluded. Genetic testing identified him to be com-
pound heterozygous for the missense mutations A874V
and N1270S (Abuduxikuer et al., 2015). A similar pre-
sentation in a 9-month-old boy was reported by Kim
et al. (2013). Biochemical evidence of liver dysfunction
was investigated further with percutaneous liver biopsy.
Elevated hepatic copper content (748 mg/g dry weight of
liver tissue) was accompanied by histologic findings of
mild lobular activity, mild portoperiportal activity, and
periportal fibrosis. Molecular analysis confirmed the
presence of the missense mutation G1186S and the
frameshift mutation c.4006delA on separate alleles
(Kim et al., 2013).
Although classically considered to be associated with
an onset in the second or third decade of life, an increas-
ing number of cases of late-onset Wilson disease are
being reported (Członkowska and Rodo, 1981; Ala
et al., 2005; Ferenci et al., 2007; Sohtaoglu et al.,
2007; Członkowska et al., 2008). Two siblings, diag-
nosed in their eighth decade of life, were described by
Ala et al. (2005). The elder sibling had presented at the
age of 72 with a 5-year history of progressive neurologic
disability. Her younger brother had had a mild hand
tremor at the age of 45 that had been treated with a
beta-blocker. He was diagnosed at the age of 70 during
screening for Wilson disease; his only symptom was mild
gait abnormalities. Both siblings were found to be com-
pound heterozygous for the E1064A and H1069Q muta-
tions with stabilization of their clinical course following
treatment (Ala et al., 2005).
Another similar case, described by Sohtaoglu et al.
(2007), was of a woman who developed slurred speech,
tremor, and postural instability at the age of 75, though
her first symptoms of mild tremor and forgetfulness
had begun 8 years earlier. The salient message from
her case was of a marked deterioration following the ini-
tiation of penicillamine, prompting the authors to caution
with respect to its use in older patients with neurologic
Wilson disease.
CONCLUSION
Considerable advances have been made since the first esti-
mate of the prevalence of Wilson disease in 1968
(Scheinberg and Sternlieb, 1984). As diagnostic tech-
niques have become increasingly sophisticated, so too
has our ability to identify the causative mutations in indi-
viduals with Wilsondisease. Nonetheless, work from our
own recent genetic prevalence study and others has raised
concerns that Wilson disease may be considerably more
common than previously thought (Ohura et al., 1999;
Coffey et al., 2013; Gialluisi et al., 2013). Considerable
phenotypic variation, the potential for reduced penetrance,
and the diversity in the number of mutations and mecha-
nisms of inheritance may all complicate the diagnosis.
Thus, it is necessary to maintain an index of suspicion in
individuals presenting with symptoms consistent with a
diagnosis of Wilson disease, even though they may not
conform to the stereotypic presentation of this disorder.
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Handbook of Clinical Neurology, Vol. 142 (3rd series)
Wilson Disease
A. Członkowska and M.L. Schilsky, Editors
http://dx.doi.org/10.1016/B978-0-444-63625-6.00003-3
© 2017 Elsevier B.V. All rights reserved

Chapter 3

The genetics of Wilson disease

IRENE J. CHANG1 AND SI HOUN HAHN2*
1
Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
2
Division of Genetic Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle Children’s Hospital,
Seattle, WA, USA

Abstract
Wilson disease (WD) is an autosomal-recessive disorder of hepatocellular copper deposition caused by
pathogenic variants in the copper-transporting gene, ATP7B. Early detection and treatment are critical
to prevent lifelong neuropsychiatric, hepatic, and systemic disabilities. Due to the marked heterogeneity
in age of onset and clinical presentation, the diagnosis of Wilson disease remains challenging to physicians
today. Direct sequencing of the ATP7B gene is the most sensitive and widely used confirmatory testing
method, and concurrent biochemical testing improves diagnostic accuracy. More than 600 pathogenic var-
iants in ATP7B have been identified, with single-nucleotide missense and nonsense mutations being the
most common, followed by insertions/deletions, and, rarely, splice site mutations. The prevalence of Wil-
son disease varies by geographic region, with higher frequency of certain mutations occurring in specific
ethnic groups. Wilson disease has poor genotype–phenotype correlation, although a few possible modi-
fiers have been proposed. Improving molecular genetic studies continue to advance our understanding of
the pathogenesis, diagnosis, and screening for Wilson disease.

INTRODUCTION excluded simply due to a misleading family history con-

In this chapter, we will discuss the inheritance, gene fre-
quency, variants, genotype–phenotype correlation, and
modifiers of the ATP7B gene, and the clinical molecular
diagnosis and population screening for Wilson disease.
INHERITANCE
Wilson disease is a monogenic autosomal-recessive con-
dition and carriers do not manifest any symptoms.
Autosomal-recessive conditions are not usually present
in consecutive generations, but may occur in populations
with particularly high carrier frequency of Wilson dis-
ease (Wu et al., 2015). Our group and others have
reported the presence of Wilson disease in two or more
successive generations within the same family, reflecting
a “pseudo-dominant” inheritance (Dziezyc et al., 2011,
2014; Bennett et al., 2013; H. Park et al., 2015). There-
fore, the diagnosis of Wilson disease should not be
sistent with an autosomal-dominant inheritance pattern.
Furthermore, recent studies have also identified Wilson
disease due to atypical forms of inheritance, such as
the presence of three concurrent mutations in a single
patient or segmental uniparental disomy (Coffey et al.,
2013). Uniparental disomy occurs when both homologs
of a chromosome originate from a single parent. These
findings have implications for clinical practice and
genetic counseling, as clinicians may need to consider
genotyping asymptomatic parents or obtaining full
sequencing of ATP7B to confirm that pathogenic variants
occur in trans.
ATP7B GENE AND ATPASE
Wilson disease is caused by homozygous or compound
heterozygous mutations in the ATP7B gene (OMIM#
606882), which encodes a transmembrane copper-
transporting P-type ATPase of the same name. Currently,

*Correspondence to: Si Houn Hahn, MD, PhD, Department of Pediatrics, University of Washington School of Medicine, Seattle
Children’s Hospital, Seattle WA 98105, USA. Tel: +1-206-987-7610, Fax: +1-206-987-5329, E-mail: sihahn@uw.edu
20 I.J. CHANG AND S.H. HAHN

Fig. 3.1. Schematic representation of copper-induced relocalization of ATP7A and ATP7B. The left side of the diagram represents
an enterocyte and the right side represents a hepatocyte. On both sides, copper enters the cell through copper transporter 1 (CTR1)
and is escorted by copper chaperone antioxidant protein 1 (ATOX1) to ATP7A or ATP7B in the trans-Golgi network (TGN). When
copper levels rise above a certain threshold, ATP7A and ATP7B excrete copper into the plasma on the basolateral side of the
enterocyte and into the bile on the apical side of the hepatocyte. Defects in localization of ATP7B may lead to copper accumulation
at the (1) TGN due to unresponsiveness, (2) cell periphery, and (3) endoplasmic reticulum (ER) due to misfolding. (Reproduced
from de Bie et al., 2007.)

ATP7B is the only identified gene known to cause Wil
son disease (Bull et al., 1993; Petrukhin et al., 1993;
Tanzi et al., 1993). Mutations in the ATP7B gene have
been reported in almost all exons. Previous studies have
reported individuals with both biochemical and clinical
diagnosis of Wilson disease in the absence of two
ATP7B mutations, raising the possibility of a second
causative gene (Lovicu et al., 2006; Kenney and Cox,
2007; S. Park et al., 2007; Mak and Lam, 2008;
Nicastro et al., 2010; Coffey et al., 2013). Nonetheless,
ATP7B remains the only known gene responsible for
Wilson disease.
Human dietary intake of copper is about
1.5–2.5 mg/day, which is absorbed in the stomach and
duodenum, bound to circulating albumin, and trans-
ported to the liver for regulation and excretion (Culotta
and Scott, 2016). The uptake of copper occurs on the
basolateral side of hepatocytes via copper transporter 1
(CTR1), as illustrated in Figure 3.1. A specific copper
chaperone, antioxidant protein 1 (ATOX1), delivers cop-
per to the Wilson disease protein, ATP7B, by copper-
dependent protein–protein interactions (Walker et al.,
2004). Within hepatocytes, ATP7B performs two impor-
tant functions in either the trans-Golgi network (TGN) or
in cytoplasmic vesicles. In the TGN, ATP7B activates
ceruloplasmin by packaging six copper molecules into
apoceruloplasmin, which is then secreted into the
plasma. In the cytoplasm, ATP7B sequesters excess
copper into vesicles and excretes it via exocytosis across
the apical canalicular membrane into bile (Bull et al.,
1993; Tanzi et al., 1993; Yamaguchi et al., 1999; Cater
et al., 2007). Due to the binary role of the ATP7B trans-
porter in both the synthesis and excretion of copper,
defects in its function lead to copper accumulation and
the progressive features of Wilson disease (Fig. 3.1).
MOLECULAR STRUCTURE OF ATP7B
ATP7B is located on 13q14.3 and contains 20 introns and
21 exons, for a total genomic length of 80 kb (Bull et al.,
1993; Petrukhin et al., 1993; Tanzi et al., 1993). The gene
is synthesized in the endoplasmic reticulum, then relo-
cated to the TGN within hepatocytes. ATP7B is most
highly expressed in the liver, but is also found in the kid-
ney, placenta, mammary glands, brain, and lung.
ATP7B (P-TYPE ATPASE) PROTEIN
STRUCTURE AND FUNCTION
ATP7B belongs to class 1B (PIB) of the highly conserved
P-type ATPase superfamily, which is responsible for
the transport of copper and other heavy metals across
cellular membranes (Gourdon et al., 2011). The protein
contains 1465 amino acids, a phosphatase domain
(A-domain), phosphorylation domain (P-domain, amino
acid residues 971–1035), nucleotide-binding domain
(N-domain, amino acid residues 1240–1291), and
 THE GENETICS OF WILSON DISEASE 21

5’UTR 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

M Tm Tm Tm Tm Tm Tm Tm Tm

GD
Cu PD
D COOH

GV
K

ND
Cu T
Cu G
NH2 Hinge region
Cu Phosphorylation
Cu
domain

Cu N
SEHPL D
TG
ATP binding site

Fig. 3.2. Schematic representation of ATP7B gene and corresponding human ATP7B protein. Top diagram shows 5’UTR pro-
moter region and exons separated by introns. Bottom diagram shows the domain organization of human copper ATPase. Conserved
amino acid motifs are present at the core structure of each functional domain, i.e., TGDN and GDGVND at the A-domain, DKTG at
the P-domain, and SEHPL in the N-domain. M, phospholipidic bilayer of the membrane; Cu, the metal-binding domains of the
trasmembrane cation channel; Tm, transmembrane domains; PD, phosphatase domain. (Reproduced from Fanni et al., 2005.)

M-domain, which comprises eight transmembrane ion
channels (Fig. 3.2) (Cater et al., 2004, 2007;
Lenartowicz and Krzeptowski, 2010).
Unique amino acid motifs are present at the core struc-
ture of each domain, such as TGEA at the A-domain,
DKTGT at the P-domain, and SEHPL in the
N-domain. Specifically, the N-terminal metal-binding
domain (MBD) is composed of six copper-binding sites,
each with the conserved sequence motif GMXCXXC
(Fatemi and Sarkar, 2002; Sazinsky et al., 2006). These
MBDs play a central role in accepting copper from cop-
per chaperone ATOX1 through protein–protein interac-
tions. Previous studies have demonstrated unequal
impact of MBDs on ATP7B activity, with MBD 5 and
6 having stronger effects on the catalytic activation of
ATP7B than MBDs 1–4 (Lutsenko et al.,1997).
The active transport of copper across membranes is a
complex process that begins with ATP7B binding copper
at the N-terminal domain and transporting it across cel-
lular membranes, using ATP as an energy source
(Fig. 3.2). Next, free copper binds intracellularly to
GG motifs in the MBDs, followed by transport on to
the Cys-Pro-Cys (CPC) sequence motifs in MBD 6.
Finally, dephosphorylation of acyl-phosphate at the
A-domain discharges copper across the cellular mem-
brane. Mutations causing copper accumulation may
occur at any of these steps (Huster et al., 2006;
Schushan et al., 2012).
Although the mechanism by which the histadine-
containing SEHPL motif affects copper transport
remains to be elucidated, it is clear that histidine-to-
glutamate substitution at amino acid 1069 (p.H1069Q)
in this motif is the most common cause of Wilson disease
in northern Europeans. In the hepatocytes of patients
homozygous for p.H1069Q, ATP7B was found in the
endoplasmic reticulum instead of its usual TGN location,
suggesting abnormal protein trafficking (Huster et al.,
2003). Insect models with the p.H1069Q mutation in
SF9 cells showed decreased ATP-mediated catalytic
phosphorylation but no major protein misfolding, sug-
gesting a role for p.H1069Q in the orientation of the
ATP7B catalytic site for ATP binding prior to hydrolysis
(Tsivkovskii et al., 2003).
VARIANTS IN THE ATP7B GENE
More than 600 pathogenic variants in ATP7B have been
identified, with single-nucleotide missense and nonsense
mutations being the most common, followed by inser-
tions/deletions and splice site mutations (Human Gene
Mutation Database, accessed 29 April 2016; Stenson
et al., 2014). Other rare genetic mechanisms that have
been reported in the literature include whole-exon dele-
tions, promoter region mutations, three concurrent path-
ogenic variants, and monogenic disomy (Coffey et al.,
2013; Bandmann et al., 2015). Mutation “hotspots” in
22 I.J. CHANG AND S.H. HAHN
Transmembrane
Cu binding Transmembrane (1-6) ATP binding (7-8)

2 3 4 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

p.C271* p.M645R p.R778L p.L795F p.G943D c.3400delC p.N1270S c.4193delC

p.E122fs p.Q457* p.G710S p.A874V p.L1083F p.L1273S p.Q1399R
p.c.1708-1G>C p.N1270S
c.2299insC p.P992H p.A1135Q p.Q1399R
c.-441_-427del
p.G691R p.I1002T p.G1266R p.S774R
p.S774R p.A1003V
p.Y670*
p.W779X p.H1069Q c.3903+6C>T
p.L708P p.V845fs p.E1064K
p.K832R p.G1351*
p.M769fs p.G1061E p.V1146M
p.L1371P
p.W779* p.A11140V
p.R969Q
p.A1003T p.A1003T
p.P992L
p.P992H
p.T977M
p.N958fs
Fig. 3.3. Schematic of the ATP7B gene with common mutation sites, including p.H1069Q (rs76151636), p.R778L (rs28942074),
p.E1064K (rs376910645), c.3400delC, and p.Ala1135fs (rs137853281). Please refer to Table 3.1 for more details.

ATP7B have also been reported to vary by geographic
region (see regional gene frequency section, below).
The majority of pathogenic mutations are located in
the M- and N-domains in presymptomatic patients or
in those with hepatic symptoms (S. Park et al., 2007).
The common mutations in ATP7B seen in various popu-
lations are listed in Figure 3.3.
The p.H1069Q mutation is one of the most common
mutations, with a population allelic frequency of
10–40% (30–70% among Caucasians). Most patients
are compound heterozygotes, carrying different muta-
tions on each copy of the chromosome (Usta et al.,
2014). The p.H1069Q mutation occurs when histidine
of the conserved SEHPL motif in the N-domain of
ATP7B is replaced by glutamic acid, resulting in
N-domain protein misfolding, abnormal phosphoryla-
tion in the P-domain, and decreased ATP binding affinity
(Rodriguez-Granillo et al., 2008). This mutation also
leads to decreased heat stability and abnormal localiza-
tion of the protein to the TGN (Ralle et al., 2010).
Other common mutations in ATP7B include p.
E1064A, p.R778L, p.G943S, and p.M769V. Mutations
in p.E1064A, also found in the SEHPL motif, completely
disable ATP binding affinity but do not result in protein
misfolding, transport abnormalities, or thermal instabil-
ity. The p.R778L mutation affects transmembrane trans-
port of copper (Dmitriev et al., 2011). The p.G943S and
p.M769V mutations result in defective copper metabo-
lism but preserved ceruloplasmin levels (Okada
et al., 2010).
A substantial proportion of Wilson disease-associated
missense mutations, including p.H1069Q and p.R778L,
result in markedly decreased level of the protein caused
by enhanced degradation (Payne et al., 1998; de Bie
et al., 2007; van den Berghe et al., 2009). Other preva-
lent mutations, such as protein-truncating nonsense
mutations (13% of known point mutations) (Merle
et al., 2010) and frameshift mutations (Vrabelova
et al., 2005), are predicted to cause decay of mRNA
(Mendell et al., 2004; Chang et al., 2007) or a severely
truncated protein, resulting in absent or diminished levels
of protein. It is therefore expected that most patients with
Wilson disease have absent or significantly reduced
levels of ATP7B.
REGIONAL GENE FREQUENCY
The prevalence of Wilson disease varies by geographic
region, with higher prevalence of specific mutations
reported in certain populations (Ferenci, 2006) (see
Chapter 2 for more details). A list of the common
regional variants of ATP7B mutations and geographic
clustering of mutations are shown in Table 3.1 and
Figure 3.4, respectively.
GENOTYPE–PHENOTYPE CORRELATION
Direct genotype–phenotype relationships in Wilson dis-
ease have been difficult to establish, despite several stud-
ies examining correlation (Panagiotakaki et al., 2004;
Vrabelova et al., 2005; Nicastro et al., 2010; Cocoş
et al., 2014; Usta et al., 2014). The numerous
low-frequency and compound heterozygous nature of
Wilson disease obfuscate the process of characterizing
its numerous genetic variants and their clinical conse-
quences. Descriptions of phenotypes are limited to age
of onset and presenting symptoms, both of which may
be affected by inaccurate diagnostic criteria, delayed
diagnosis, and practitioner selection bias. Therefore,
Table 3.1
Regional distribution of common Wilson disease mutations by geographic location

Prevalent mutations

Region AF (%) Protein Nucleotide RS Exon Type Domain

Europe
Austria (Ferenci, 2006) 34.1 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
6.4 p.Gly710Ser c.2128G > A 8 Missense TM2
3.6 p.Met769fs c.2298_2299insC rs137853287 8 Premature stop TM4
Benelux (Ferenci, 2006) 53 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
Bulgaria (Todorov et al., 2005) 58.8 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
Canary Islands 64 p.Leu708Pro c.2123 T > C 8 Missense TM2
(García Villarreal et al., 2000)
Czech Republic 57 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Vrabelova et al., 2005)
Denmark 18 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Møller et al., 2011)
16 p.Trp779* c.2336G > A rs137853283 8 Nonsense TM4
France 15 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Bost et al., 2012)
Germany 47.9 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Ferenci, 2006)
Germany (East, former) 63 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Caca et al., 2000)
Greece 35 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Panagiotakaki et al., 2004;
Dedoussis et al., 2005; Gomes
and Dedoussis, 2016)
12 p.Arg969Gln c.2906G > A rs774028495 13 Missense TM6
Hungary 42.9 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Firneisz et al., 2002; Folhoffer et al., 2007)
Iceland 100 p.Tyr670* c.2007_2013del 7 Nonsense TM1
(Thomas et al., 1995a; Hofer et al., 2012)
Italy 17.5 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Loudianos et al., 1999)
9 p.Val845fs c.2530delA rs755709270 10 Premature stop Td
6 p.Met769fs c.2298_2299insC rs137853287 8 Premature stop TM4
Netherlands 33 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Stapelbroek et al., 2004)

Continued
Table 3.1
Continued

Prevalent mutations

Region AF (%) Protein Nucleotide RS Exon Type Domain

Poland 72 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop

(Gromadzka et al., 2005)
7.3 p.Ala1135fs c.3400delC rs137853281 15 Premature stop ATP loop
3.7 p.Gln1351* c.4051C > T 20 Nonsense
Romania (Iacob et al., 2012) 38.1 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
Russia 49 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Ivanova-Smolenskaya et al., 1997)
Sardinia 60.5 c.-441_-427del 5prime Unknown Promoter
(Figus et al., 1995)
8.5 p.Met822fs c.2463delC 10 Deletion TM4/Td
7.9 p.Val1146Met c.3436G > A 16 Missense ATP loop
Serbia 38.4 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Tomi
c et al., 2013)
11.6 p.Met769fs c.2304dupC 8 Missense TM4
9.3 p.Ala1003Thr c.3007G > A rs1801247 13 Missense TM6/Ph
Spain 27 p.Met645Arg c.1934 T > G rs121907998 6 Missense Cu6/TM1
(Margarit et al., 2005)
Sweden 38 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Shah et al., 1997)
Turkey 17.4 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Ferenci, 2006; Simsek Papur et al., 2013)
5.3 p.Gly710Ser c.2128G > A rs772595172 8 Missense TM2
4.53 p.Gln457* c.1369C > T 3 Nonsense Cu4/Cu5
UK 19 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Coffey et al., 2013)
8 p.Met769Val c.2305A > G 8 Missense TM4
Yugoslavia (former) 48.9 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Loudianos et al., 1999)
11.4 p.Met769fs c.2298_2299insC rs137853287 8 Premature stop TM4
Asia
China 31 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4
(Gu et al., 2003; Z.-Y. Wu et al., 2003;
Wang et al., 2011; Wei et al., 2014)
10 p.Pro992Leu c.2975C > T rs201038679 13 Missense TM6/Ph
9.6 p.Ile1148Thr c.3443 T > C rs60431989 16 Missense ATP loop
3.3 p.Thr935Met c.2804C > T 12 Missense TM5
19 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4
North India
(S. Kumar et al., 2006; Gupta et al., 2007)
12 p.Ile1102Thr c.3305 T > C rs560952220 15 Missense ATP loop
9 p.Pro992His c.2975C > A 13 Missense TM6/Ph
South India 11 p.Ala1003Val c.3008C > T 13 Missense TM6/Ph
(Santhosh et al., 2006;
S. S. Kumar et al., 2012)
11 p.Cys271* c.813C > A rs572147914 2 Nonsense Cu3
9 p.Pro768Leu c.2303C > T 8 Missense TM4
9 p.Arg969Gln c.2906G > A rs121907996 13 Missense TM6
East India 16 p.Cys271* c.813C > A rs572147914 2 Nonsense Cu3
(Gupta et al., 2005)
11 p.Gly1061Glu c.3182G > A 14 Missense ATP loop
8.5 c.1708-1G > C rs137853280 5 Splice Cu6
West India 20 p.Cys271* c.813C > A rs572147914 2 Nonsense Cu3
(Aggarwal and Bhatt, 2013;
Aggarwal et al., 2013)
11 p.Glu122fs c.365_366delins 2 Ins/Del Cu1
TTCGAAGC
6 p.Thr977Met c.2930C > T rs72552255 13 Missense TM6
6 p.Leu795Phe c.2383C > T 9 Missense TM4/Td
Japan 17.95 p.Asn958fs c.2871delC 13 Premature stop TM5/TM6
(Okada et al., 2000; Tatsumi et al., 2010)
16.7 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4
10.5 c.1708-5 T > G 5 Splice Cu6
Korea 37.9 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4
(E. K. Kim et al., 1998; Yoo, 2002;
G.-H. Kim et al., 2008; Song et al., 2012)
12.1 p.Asn1270Ser c.3809A > G rs121907990 18 Missense ATP hinge
9.4 p.Ala874Val c.2621C > T rs376355660 11 Missense TM5
8 p.Leu1083Phe c.3247C > T 15 Missense ATP loop
Lebanon 44.7 p. Ala1003Thr c.2299insC rs137853287 8 Missense TM4
(Usta et al., 2014)
Saudi Arabia 32 p.Gln1399Arg c.4196A > G 21 Missense After TM8
(Al Jumah et al., 2004; Majumdar et al., 2004)
16 p.Ser774Arg c.2230 T > C rs535217574 21 Missense TM3
Taiwan 29.6 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4
(Lee et al., 2000; Wan et al., 2006)
8.9 p.Pro992Leu c.2975C > T rs201038679 13 Missense TM6
4.8 p.Gly943Asp c.2828G > A 12 Missense TM5

Continued
Table 3.1
Continued

Prevalent mutations

Region AF (%) Protein Nucleotide RS Exon Type Domain

Thailand 10.52 p.Arg778Leu c.2332C > T rs28942074 8 Missense TM4

(Panichareon et al., 2011)
7.89 p.Leu1371Pro c.4112 T > C 20 Missense TM8
Iran 19 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Zali et al., 2011)
Africa
Egypt 42.2 IVS18 + 6 T > C c.3903 + 6C > T rs2282057 18 Splice
(Abdelghaffar et al., 2008;
Abdel Ghaffar et al., 2011)
40.6 p.Ala11140Val c.3419C > T 16 Missense ATP loop
26.5 p.Lys832Arg c.2495A > G rs1061472 10 Missense TM4/Td
Americas
USA 40.3 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Kuppala et al., 2009)
1.9 p.Asn1270Ser c.3809A > G rs121907990 18 Missense ATP hinge
1.9 p.Gly1266Arg c.3796G > A rs121907992 18 Missense ATP hinge
Brazil 37.1 p.His1069Gln c.3207C > A rs76151636 14 Missense ATP loop
(Deguti et al., 2004; Machado et al., 2008;
Bem et al., 2013)
31.25 p.Ala1135fs c.3400delC rs137853281 15 Premature stop ATP loop
11.4 p.Ala1135GlnfsX13 c.3402delC rs137853281 15 Premature stop ATP loop
p.Leu708Pro c.2123 T > C 8 Missense TM2
Costa Rica 61 p.Asn1270Ser c.3809A > G rs121907990 18 Missense ATP hinge
(Shah et al., 1997)
Venezuela 26.9 p.Ala1135GlnfsX13 c.3402delC rs137853281 15 Premature stop ATP loop
(Paradisi et al., 2015)
9.6 p.Gly691Arg c.2071G > A 7 Missense TM2

AF, allelic frequency; RS, Reference single nucleotide polymorphism (SNP) cluster identification number.
 THE GENETICS OF WILSON DISEASE 27

Fig. 3.4. Prevalence of ATP7B mutation by geographic region; the darker the gradient, the higher the allelic frequency. (Repro-
duced from Gomes and Dedoussis, 2016, with permission from Taylor and Francis.)

the marked variability in phenotype of Wilson disease is
likely attributable to an amalgamation of genetic, meta-
bolic, and environmental factors (Leggio et al., 2006).
The most consistent genotype–phenotype correlation
in Wilson disease is that the most severe, early-onset dis-
ease with predominantly hepatic presentation is associ-
ated with mutations causing absent ATPase activity.
Convincing studies have demonstrated fulminant hepatic
disease in mouse models such as the toxic milk (tx)
mouse and the Jackson tx mouse (txj), which harbor point
mutations causing loss of ATP7B function, but not affect-
ing ATP7B synthesis (Theophilos et al., 1996; Coronado
et al., 2001; La Fontaine et al., 2001; Huster et al., 2006).
Genetic polymorphisms in ATP7B, other genes, and
epigenetic factors have been shown to impact disease
phenotype by affecting ATP7B protein structure and
function. Of the over 600 mutations associated with Wil-
son disease, the majority are missense mutations that
completely inactivate the copper-transporting function
of ATP7B (Lutsenko, 2014). In general, individuals with
protein-truncating mutations have earlier onset of disease
due to decreased protein stability and quantity (Merle
et al., 2010). However, other studies have demonstrated
partial preservation of copper-transporting function, per-
haps explaining the milder phenotypes associated with
certain mutations (Rodriguez-Granillo et al., 2008;
Dmitriev et al., 2011; Huster et al., 2012). Individuals
with the R778L mutation have been shown to have an
earlier onset of disease and predominantly hepatic pre-
sentation (Z. Y. Wu et al., 2003). In contrast, individuals
with the H1069Q mutation have a mean onset of symp-
toms between 20–22 years old and predominantly neuro-
logic phenotype (Stapelbroek et al., 2004; Kalita et al.,
2010). There is also some evidence that Kayser–
Fleischer rings are more common in H1069Q homozy-
gous patients in Hungary at time of diagnosis than in
compound heterozygous individuals (Folhoffer
et al., 2007).
Moreover, pathogenic variants may affect ATP7B tar-
geting from the TGN to cytosolic vesicles. For instance,
the p.Met875Val mutation results in a less stable protein
and causes reversible ATP7B localization defects. Under
a low-copper environment, the p.Gly875Arg variant is
sequestered in the endoplasmic reticulum. However,
addition of exogenous copper to the cellular growth
medium stabilizes the protein, allowing it to complete
28 I.J. CHANG AND S.H. HAHN
its intended journey to the TGN and overcoming its
disease-causing phenotype. Theoretically, patients with
this specific variant may be more sensitive to dietary cop-
per deficiency (Gupta et al., 2011).
The timing and location of copper buildup can also
preferentially alter the hepatic transcriptome, based
on homozygous ATP7B–/– mouse models. Proteomic
analyses of mRNA profiles at each of these disease
stages reflect unique patterns (Huster et al., 2006;
Ralle et al., 2010). In the initial stage, mRNA for pro-
teins responsible for cell cycle regulation, splicing,
and cholesterol synthesis is present (Burkhead et al.,
2011). This leads to early accumulation of copper
bound to metallothioneins in the cytosol and free copper
in the nuclei. In the progressive stage, mRNA changes
throughout the cell are present, including the endoplas-
mic reticulum, mitochondria, and endocytic pathways,
causing copper to pathologically accumulate within
hepatocytes. In the later stages, mRNA for lysosomal
and endosomal proteins is upregulated. In these final
stages, copper concentrations decrease in the cytosol
and nuclei, and accumulate in the membranous cellular
compartment, causing bile duct proliferation and
hepatic neoplastic changes. Therefore, the location of
copper accumulation may convey more specific prog-
nostic information about disease progression rather
than total copper levels.
Other studies have compared homozygotes to com-
pound heterozygotes of the same mutation to establish
genotype–phenotype correlations. A study of 76 mem-
bers of a large, consanguineous Lebanese family showed
an association between c.2299insC and hepatic disease
and between the p.Ala1003Thr mutation and neurologic
disease (Usta et al., 2014).
Other candidate polymorphisms that are thought to
modify the clinical phenotype of Wilson disease include
MTHFR (Gromadzka et al., 2005), COMMD1 (Weiss,
2006), ATOX1 (Simon, 2008), XIAP (Weiss et al.,
2010), PNPLA3 and hepatic steatosis (St€attermayer
et al., 2012), and DMT1 (Przybyłkowski et al., 2014),
although none of these genes has been demonstrated to
have significant diagnostic or predictive value.
Significant phenotypic variation of Wilson disease
exists between individuals with the same mutation, indi-
viduals within the same family, and even between mono-
zygotic twins (Członkowska et al., 2009; Kegley et al.,
2010). While some studies have documented high intra-
familial concordance of clinical symptoms and biochem-
ical results (Hofer et al., 2012; Chabik et al., 2014;
Ferenci et al., 2015), others have reported a wide range
in age of onset and presenting symptoms amongst sib-
lings (Ala et al., 2007; Taly et al., 2007) and families car-
rying the same mutation (Takeshita et al., 2002). Indeed,
disparate clinical presentations in monozygotic twins
raise the suspicion for epigenetic modifiers in Wilson
disease. See Chapter 4 for more details about the genetic
and environmental modifiers of Wilson disease.
CLINICAL MOLECULAR DIAGNOSIS
The current gold standard of diagnosis for Wilson disease
is direct Sanger sequencing of the ATP7B gene or molec-
ular testing for previously identified familial mutations.
Historically, most pathogenic variants in ATP7B were
identified using a combination of polymerase chain reac-
tion (PCR)/restriction fragment length polymorphism
(RFLP), single-strand conformation polymorphism
(SSCP), denaturing gradient gel electrophoresis
(DGGE), temporal temperature gradient electrophoresis
(TTGE), denaturing high-performance liquid chroma-
tography (DHPLC), and Sanger sequencing
(Loudianos et al., 1999; Shimizu et al., 1999; Margarit
et al., 2005; Vrabelova et al., 2005; G. H. Kim et al.,
2008). The critical demerits of this complex tiered
approach are that the detection rate is not high enough
to find most mutations and the turnaround time is often
extended. Although regional clusters of specific muta-
tions have been well described, a customized screening
approach taking into account these regional variants
may be complicated by ethnically diverse populations
and inaccurate clinical information provided with
samples. Biochemical results are often imprecise, as ele-
vations in urinary copper excretion tend to occur late in
the disease process and fewer than 40% of presympto-
matic patients excrete copper less than 100 mg/day
(Sternlieb and Scheinberg, 1968; Nakayama et al.,
2008). For these reasons, direct sequencing of the ATP7B
gene has become the preferred standard and provides the
greatest yield in clinical molecular diagnosis. Please refer
to Chapter 14 for details about the diagnosis of Wilson
disease.
Starting the diagnostic process with molecular testing
may significantly reduce the need for invasive liver
biopsy. Liver copper content alone was found to be insuf-
ficient to exclude Wilson disease, as levels may not be
elevated in some affected patients. Based on several
previous studies, biallelic pathogenic variants were
identified in about 80% of patients with biochemical
and clinical tests suggestive of Wilson disease. Currently
available screening tests may not definitively rule out the
disease, and no single test could permit de novo diagno-
sis. Of note, many patients may not possess the charac-
teristic findings and may present when their clinical
disease is relatively mild. Inappropriate treatment for
false-positive cases has the potential of inducing copper
deficiency, which can result in hematologic and neuro-
logic sequelae (N. Kumar et al., 2003). These findings
reinforce the need for reliable clinical diagnostic criteria
 THE GENETICS OF WILSON DISEASE
and underscore the benefits of DNA testing prior to inva-
sive procedures (Ferenci, 2005).
Multiplex PCR is used to amplify all 21 exons and
splice sites of ATP7B, including promoter regions.
Although the large deletions or duplications cannot be
detected with this conventional Sanger sequencing
method, the chance of these being present in Wilson
disease appears low (Stenson et al., 2012). If clinical
suspicion is still high with only one pathogenic variant
identified, then multiplex ligation-dependent probe
amplification (MLPA) test should be considered. Micro-
array-based comparative genomic hybridization is
another option to evaluate partial or full gene deletions
or duplications with higher sensitivity. Cases with only
one pathogenic variant present should be carefully
reviewed in the context of other biochemical and clinical
findings. Molecular genetic testing using direct mutation
analysis is very effective in identifying affected patients
and presymptomatic siblings of probands (Manolaki
et al., 2009).
Wilson disease is an autosomal-recessive disorder,
which means that there is a 25% chance that a full sibling
of the index case is also affected. Once homozygous or
compound heterozygous mutations in ATP7B have been
established in the index patient, mutation detection
becomes valuable in family screening. The same geno-
type in asymptomatic family members confirms diagno-
sis of the disease, thus allowing for early treatment before
the onset of complications. In family members in whom
clinical and biochemical features are uncertain, the dem-
onstration of either heterozygous (carrier) or wild-type
gene sequence prevents unnecessary treatment (Chang
et al., 2007).
If the proband has secured a diagnosis of Wilson dis-
ease on the basis of clinical and biochemical evidence,
but testing for ATP7B mutations is not available, family
screening can be done by haplotype analysis of polymor-
phic markers flanking the disease gene (Thomas et al.,
1995b; Gupta et al., 2005; Przybyłkowski et al., 2014).
In this instance, the rare possibility of recombination
events (typically 0.5–5% of cases) needs to be consid-
ered. The rate of recombination is dependent on which
flanking markers are studied. Microsatellite or single-
nucleotide polymorphisms in the ATP7B lateral wing
are used for haplotyping, which is useful for screening
relatives of patients with previously identified familial
mutations. False-positive results may occur if hap-
lotyping is used on patients with low-probability gene
recombinations.
Genetic testing for ATP7B mutations can be valuable
to confirm a diagnosis of Wilson disease, especially
when presentation is unusual (Caprai et al., 2006). Atten-
tion has been drawn to this situation by the molecular
confirmation of early-onset hepatic disease in a
29
3-year-old child (Wilson et al., 2000). Mutation analysis
has also confirmed late-onset disease, including the case
of two siblings in their 70s – the oldest reported patients
so far at time of diagnosis (Nanji et al., 1997; Gupta et al.,
2005; Perri et al., 2005; Weitzman et al., 2014).
ATP7B mutation analysis makes an important con-
tribution to clinical practice. Unfortunately, systematic
genetic testing for Wilson disease is still difficult and
fairly expensive due to the plethora of different muta-
tions, the occurrence of regulatory mutations in non-
coding sequence, the large size of the gene, and the
limitations of currently available methods. However,
technical advances allowing high-throughput screen-
ing could be applied to the disease (Bost et al.,
2012; Lepori et al., 2012). This new apparatus can
sequence six million basepairs of DNA per hour with
accuracy greater than 99%. Such advances might per-
mit specialized laboratories to detect all variants by
sequencing the entire genomic Wilson disease gene
from patients, including not only the translated exons,
but also the important noncoding sequences that are
not normally investigated.
Interpretation of variants of uncertain significance has
become a major challenge for accurate interpretation,
genetic counseling, and prevention. Screening family
members may help with the interpretation of variants
of uncertain significance, but not all variants can be
resolved with this approach. Functional analysis is often
necessary; however, no clinical functional analysis is
currently available. A computational approach to predict
significance of mutations is often helpful, but a further
concrete model is required to demonstrate the efficacy
in guiding clinical decisions.
POPULATION SCREENING
The purpose of newborn screening is to identify treatable
congenital conditions that can affect a child’s long-term
health and development. Recent tandem mass spectrom-
etry (MS/MS) applications have markedly expanded the
ability to screen for >50 metabolic diseases from a single
dried blood spot. In addition to the original Wilson–
Jungner classic screening criteria (Wilson and Jungner,
1968), the American College of Medical Genetics con-
vened the Newborn Screening Expert Group to develop
a uniform screening panel in 2006 (American College of
Medical Genetics Newborn Screening Expert Group,
2006). Of the primary tenants, Wilson disease is an ideal
target for screening, given its relatively high prevalence
and availability of effective treatment (Hahn et al., 2002;
Roberts et al., 2008). Unfortunately, despite extensive
discussion on the need for population screening, no
cost-effective biomarkers or methods for early detection
have been developed for Wilson disease yet. Several
30 I.J. CHANG AND S.H. HAHN
small pilot studies have been conducted using cerulo-
plasmin as a biomarker for screening, with limited find-
ings (Yamaguchi et al., 1999; Hahn et al., 2002; Owada
et al., 2002; Schilsky and Shneider, 2002; Kroll et al.,
2006). Ceruloplasmin alone is not sufficient to screen
for Wilson disease in newborns, as a substantial number
of newborns present with physiologically low cerulo-
plasmin. Ceruloplasmin assay around 3 years of age
may be the most appropriate population-screening
method, but mandatory health checkups at this age are
not universally available in the USA and worldwide.
Many treatable congenital disorders are caused by
mutations that result in absent or diminished levels of
proteins; thus, protein biomarkers have enormous poten-
tial in the diagnosis/screening of congenital disorders.
Liquid chromatography mass spectrometry with multiple
reaction monitoring (LC-MRM-MS) has emerged as a
robust technology that enables highly precise, specific,
multiplex quantification of signature proteotypic pep-
tides as stoichiometric surrogates of biomarker proteins.
Our lab is currently exploring the use of peptide
immunoaffinity enrichment (Whiteaker et al., 2010,
2011) to quantify ATP7B in dried blood spot (DBS).
These promising proof-of-concept data open up the pos-
sibility of screening for Wilson disease in newborns. Fur-
ther clinical validation on a large-scale study will be
required to determine the efficacy of the assay.
CONCLUSION
Wilson disease is an autosomal-recessive disease due to
pathogenic mutations in ATP7B. ATP7B is the only iden-
tified gene known to cause Wilson disease, and encodes a
transmembrane copper-transporting ATPase of the same
name. While biochemical testing and clinical criteria
may assist in the early diagnosis and treatment, the cur-
rent gold standard for Wilson disease diagnosis is direct
Sanger sequencing of ATP7B or molecular testing for
known familial mutations. Genotype–phenotype correla-
tions have been studied extensively but direct causations
remain nebulous. Modifier genes may affect the pene-
trance and phenotypes but a large-scale study for clinical
validation is warranted. The overall worldwide preva-
lence of Wilson disease is 1 in 30 000 individuals, with
significant geographic variation. The most common
mutation in Northern America and Europe is the mis-
sense mutation p.H1069Q and the most common muta-
tion in East Asian populations is the missense p.R778L.
Ceruloplasmin alone is insufficient to screen for Wilson
disease in newborns. While peptide immunoaffinity
assays show promise for newborn screening, further
large-scale clinical studies are required to determine effi-
cacy of these population-based screening methods for
Wilson’s disease.
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Handbook of Clinical Neurology, Vol. 142 (3rd series)
Wilson Disease
A. Członkowska and M.L. Schilsky, Editors
http://dx.doi.org/10.1016/B978-0-444-63625-6.00004-5
© 2017 Elsevier B.V. All rights reserved

Chapter 4

Genetic and environmental modifiers of Wilson disease

VALENTINA MEDICI1* AND KARL-HEINZ WEISS2
1
Division of Gastroenterology and Hepatology, Department of Internal Medicine, University of California Davis,
Sacramento, CA, USA
2
Department of Gastroenterology and Hepatology, University Hospital of Heidelberg, Heidelberg, Germany

Abstract
Wilson disease (WD) is characterized by remarkable variety in its phenotypic presentation. Patients with
WD can present with hepatic, neurologic, and psychiatric symptoms combined in different and unpredict-
able ways. Importantly, no convincing phenotype–genotype correlation has ever been identified, opening
the possibility that other genes, aside from ATPase copper-transporting beta (ATP7B), are involved in the
pathogenesis of this condition. In addition, modifier genes, or genes that can affect the expression of other
genes, may be involved. Clinical and basic science data indicate that environmental and dietary factors can
potentially modify gene expression in WD and, consequently, its clinical presentation and course. In par-
ticular, previously studied genes include copper metabolism domain-containing 1 (COMMD1), antioxi-
dant 1 copper chaperone (ATOX1), X-linked inhibitor of apoptosis (XIAP), apolipoprotein E (APOE),
hemochromatosis (HFE), and 5,10-methylenetetrahydrofolate reductase (MTHFR). Dietary factors
include iron and methyl group donors which could affect methionine metabolism and epigenetic mecha-
nisms of gene expression regulation. Most of the work conducted in this field is in its initial stages but it has
the potential to change the diagnosis and treatment of WD.

INTRODUCTION1 COMMD1 GENE

The major knowledge gap in Wilson disease (WD) is the
lack of mechanistic understanding of the phenotype diver-
sity and the responses to treatment despite the genetic
inheritance of ATPase copper-transporting beta (ATP7B)
mutations. Proteins other than ATP7B contribute to the
molecular mechanism of copper excretion and mutations
or polymorphisms of these proteins might contribute to
WD, perhaps explaining the highly variable clinical pre-
sentation (Steindl et al., 1997; Riordan and Williams,
2001; Ala and Schilsky, 2011) and course of this disease.
This chapter will review the published data on concomi-
tant genes and modifier genes as well as environmental
factors that can have a role in WD phenotypic expression.
An interesting candidate for a modifier gene is copper
metabolism domain-containing 1 (COMMD1). Defi-
ciency of COMMD1 causes canine copper toxicosis of
Bedlington terriers (Twedt et al., 1979; van de Sluis
et al., 2002; Klomp et al., 2003) that resembles WD,
although ceruloplasmin levels are not decreased
(Su et al., 1982) and there are no evident neurologic
symptoms. The human orthologous gene was identified
on chromosome 2p13-16 and is distinct from the ATP7B
gene locus (van de Sluis et al., 1999).
As several studies confirmed a physical interaction of
COMMD1 with ATP7B (Tao et al., 2003; de Bie et al.,
2006; Donadio et al., 2007), cohorts of WD patients were

1
Abbreviations used in the chapter are listed at the end of the chapter before References section.

*Correspondence to: Valentina Medici, MD, Department of Internal Medicine, Division of Gastroenterology and Hepatology,
University of California Davis, 4150 V St., Suite 3500, Sacramento CA 95817, USA. Tel: +1-916-734-3751, E-mail:
vmedici@ucdavis.edu
36 V. MEDICI AND K.-H. WEISS
screened for alterations in COMMD1. So far no exonic
mutations of COMMD1 have been found in patients
with WD (Stuehler et al., 2004) or other rare human cop-
per overload diseases like Indian childhood cirrhosis,
endemic Tyrolean infantile cirrhosis, or idiopathic cop-
per toxicosis (Muller et al., 2003). Studies on known
polymorphisms of COMMD1 in patients with WD
revealed conflicting results. In WD patients homozygous
for the most common ATP7B mutation H1069Q, an
association between a COMMD1 polymorphism and
onset of neurologic and hepatic symptoms was reported.
Onset of disease was significantly earlier for heterozy-
gous patients at codon Asn 164 (GAT/GAC) than for
wild-type (GAT/GAT) (Stuehler et al., 2004). This could
only partially be confirmed by others (Weiss et al., 2006;
Bost et al., 2012).
ATOX1 GENE
The human homologue antioxidant 1 copper chaperone
(ATOX1) is an 8-kDa cytosolic protein that contains a
single copy of the highly conserved MxCxxC motif in
the amino-terminus which is repeated sixfold in ATP7B.
This metallochaperone interacts directly with ATP7B
(Hamza et al., 1999) and can regulate its copper occu-
pancy (Walker et al., 2002). By modulating the amount
of copper bound to the protein, ATOX1 has an impact
on intracellular localization (DiDonato et al., 2000), as
well as posttranslational modification and enzymatic
activity of ATP7B as a copper-dependent transcription
factor involved in cell proliferation (Vanderwerf et al.,
2001; Itoh et al., 2008). However, in human cohort stud-
ies, no significant nonsynonymous coding variations in
the ATOX1 gene were evident (Simon et al., 2008;
Bost et al., 2012). A common polymorphism within
ATOX1 (5’UTR -99 T > C) was detected at expected fre-
quencies of 49–57%. Based on these data, no major role
can be attributed to ATOX1 in the pathophysiology or
clinical variation of WD.
XIAP GENE
XIAP (X-linked inhibitor of apoptosis protein), a
well-characterized antiapoptotic protein, has been pro-
posed as a regulator of copper-induced cell injury. XIAP
binds copper directly, followed by enhanced degradation
and blunted inhibition of caspases. Therefore, in addition
to copper overload, copper binding to XIAP sensitizes
hepatocytes for apoptosis (Mufti et al., 2006), which sug-
gests an unexpected new pathogenic mechanism for
copper-induced cell damage (Mufti et al., 2007).
It has also been hypothesized that XIAP plays a role
in maintaining cellular copper homeostasis by interacting
with COMMD1 and promoting its degradation (Burstein
et al., 2004; Prohaska, 2008; Maine et al., 2009).
Correspondingly, reduced copper levels are found in
cells and tissues of XIAP-deficient knockout mice
(Burstein et al., 2004). Four nonsynonymous coding
single-nucleotide polymorphisms (SNPs) have previously
been described in the coding region of the XIAP gene
(Salzer et al., 2008; Serre et al., 2008); their physiologic
role however remains unclear. In 98 WD patients
(Weiss et al., 2010), overall allele frequency did not differ
significantly from previously reported panels. For all
SNPs, statistical analysis did not reveal any correlation
to age of onset, clinical presentation (neurologic vs.
hepatic vs. mixed vs. asymptomatic), or presentation as
fulminant WD (Weiss et al., 2010).
APOE GENE
Similarly to WD, several studies suggest that copper dys-
function and ATP7B variants influence the phenotypes of
neurodegenerative disorders such as Alzheimer’s disease
(Squitti et al., 2013). The presence of apolipoprotein E
(APOE) allele ε4 is associated with an increased vulner-
ability of the brain to disease effects, whereas the pres-
ence of APOE genotype ε3/3 appears to provide
moderate neuroprotection. In line with this concept,
Schiefermeier et al. (2000) reported that the homozygous
APOE ε3 genotype is associated with delayed onset of
WD signs and symptoms. In an independent cohort,
Litwin et al. (2012b) reported that women with APOE
ε4-positive genotypes presented earlier onset of WD
symptoms, particularly among ATP7B p.H1069Q homo-
zygous patients. However, data on an association
between APOE genotype and WD clinical expression
conflict with other authors who do not confirm these
findings (Gu et al., 2005; Kocabay et al., 2009).
HFE GENE AND METAL TRANSPORTER
GENES DMT1 AND ATP7A
Data in patients and in animal models of WD indicate
iron accumulation may contribute to the phenotype of
this condition. HFE (hemochromatosis) gene mutations
are associated with hereditary hemochromatosis and are
related to increased intestinal iron uptake. Two initial
case reports described concomitant presence of HFE
and ATP7B gene polymorphisms with corresponding
hepatic iron and copper accumulation (Hafkemeyer
et al., 1994; Walshe and Cox, 1998). A study of
32 WD patients from Sardinia described iron and copper
metabolism indices, HFE mutations, and liver biopsies
(Sorbello et al., 2010). Six of the studied patients were
heterozygous for the H63D mutation; none presented
C282Y and S65C mutations. Patients with HFE poly-
morphisms presented with approximately double the
hepatic iron concentration compared to patients without
HFE polymorphisms. Long-term anticopper treatment,
 GENETIC AND ENVIRONMENTAL MODIFIERS OF WILSON DISEASE
both zinc salts and chelation, was associated with
improved alanine aminotransferase (ALT) levels and
decreased hepatic iron concentration in HFE wild-type
patients, whereas ALT levels and hepatic iron remained
stable in patients with HFE polymorphism. HFE muta-
tions were also studied in 40 patients with WD compared
to 295 healthy controls and there was no difference in
allele frequencies for the C282Y and H63D mutations
between the two populations (Erhardt et al., 2002). Sim-
ilarly, a larger study on 143 WD patients explored HFE
allele frequencies and did not find any significant differ-
ence between WD patients and the general population
(Pfeiffenberger et al., 2012).
The prevalence of divalent metal transporter 1
(DMT1) and ATPase copper-transporting alpha (ATP7A)
mutations was also studied in patients with WD
(Przybyłkowski et al., 2014), as DMT1 is involved in
iron transport and ATP7A is involved in copper transport
in the intestinal epithelium. The study, which included
more than 100 patients with WD, found an increased fre-
quency of the DMT1 IVS4 C(+) allele in patients with
WD compared to healthy controls, but no differences
according to hepatic or neurologic phenotype. ATP7A
alleles did not present different prevalence between the
two studied populations. The rationale for studying these
genes resides in two previous findings: increased tran-
script levels of DMT1 in the brains of patients with
Parkinson’s disease and in animal models of this condi-
tion (Salazar et al., 2008), and increased mRNA tran-
script and protein levels of duodenal ATP7A as a
possible compensatory mechanism after copper accumu-
lation (Przybyłkowski et al., 2013).
MTHFR GENE
A key enzyme in folate and methionine me-
tabolism is 5,10-methylenetetrahydrofolate reductase
(MTHFR), which catalyzes the conversion of 5,10-
methylenetetrahydrofolate to 5-methyltetrahydrofolate,
a co-substrate for homocysteine remethylation to
methionine. Mutations of MTHFR are associated with
increased homocysteine levels, which may contribute
to greater severity of WD or to its phenotypic variability.
Two-hundred and forty-five patients with WD were gen-
otyped for the two MTHFR polymorphisms, C677T and
A1298C, and genotype–phenotype correlations were
studied. The C667T genotype was more frequent than
expected according to Hardy–Weinberg equilibrium.
The C677T allele was associated more frequently with
hepatic onset. The A1298C allele was associated with
younger age at presentation. MTHFR polymorphisms
were not associated with any difference in copper metab-
olism (Gromadzka et al., 2011). Even though these initial
observations were not explored in other populations and
37
hyperhomocysteinemia has not been described in WD,
the possibility that homocysteine or aberrant methionine
metabolism can affect the phenotype is interesting, as
homocysteine can pass the blood–brain barrier and can
also have neurotoxic effects by interacting with copper
(White et al., 2001; Linnebank et al., 2006). In addition,
methionine metabolism is closely related to mechanisms
of DNA and histone methylation with potential conse-
quences for gene expression regulation.
HUMAN PRION GENE
Prion protein (PRNP) binds copper ion with low affinity
(Brown et al., 1997) and may affect copper metabolism,
especially in the central nervous system, where this pro-
tein is highly expressed (Ford et al., 2002). The interac-
tion between copper and PRNP may have a protective
effect on neurons (Gasperini et al., 2015). Given this
background, Merle et al. (2006) studied the prevalence
of the PRNP polymorphism at codon 129 (M129V) asso-
ciated with age at disease onset and subsequent disease
course in 134 patients with WD. The prevalence of the
M129V genotype was similar in WD patients compared
to a healthy control population. With respect to their clin-
ical presentation, patients with PRNP codon 129 result-
ing in homozygous methionine (129 M/M) were about
5 years older at disease onset and had neurologic presen-
tation an average of 7 years later compared to carriers of
PRNP 129 V(+).
GENDER
As pointed out by a study from Litwin et al. (2012a), neu-
ropsychiatric presentation in WD is more common in
men than women and women develop these symptoms
at a later age than men. In a complex study on more than
1000 WD patients, Ferenci (2014) reported that gender
and age modify disease presentation specifically. Young
females present more often with fulminant liver disease
(Ferenci et al., 2011) and males present more frequently
with neurologic disease, whereas neurologic disease is
rare among children with WD (Ferenci, 2014). These
findings may be related to hormonal changes but also
to differences in iron accumulation and metabolism
(by menstrual iron loss), which are discussed below.
IRON
Studies in animal models and in humans show evidence
that copper accumulation and low ceruloplasmin levels
as well as long-term treatment of WD are associated with
iron accumulation. There is a close relation between iron
and copper accumulation primarily derived from the fact
that ceruloplasmin is a potent ferroxidase, catalyzing the
conversion of Fe2+ to Fe3+, and this activity is required
38 V. MEDICI AND K.-H. WEISS
for iron cellular uptake (Attieh et al., 1999). Low cerulo-
plasmin levels as presented in WD may thus result in iron
accumulation. The other mechanism possibly underlying
iron accumulation in WD is related to long-term anticop-
per treatment or overtreatment, probably associated with
reduced copper bioavailability, worsening hypocerulo-
plasminemia, and, ultimately, iron overload (Schilsky,
2001). In a genetic animal model of WD, the Long–
Evans Cinnamon (LEC) rats, iron deprivation or phlebot-
omies prevented the development of acute liver failure
and hepatocellular carcinoma, despite absence of hepatic
copper treatment (Kato et al., 1996). In WD patients trea-
ted with penicillamine for 3–8.5 years, there was evi-
dence of hepatic iron accumulation and reduced
hepatic copper content over time. Phlebotomies in 2
patients achieved lower serum ferritin and ALT levels
(Shiono et al., 2001). Iron parameters were studied for
a group of 40 patients with WD compared to healthy con-
trol subjects. WD patients presented higher serum ferritin
levels than controls and a subgroup of WD patients with
low ceruloplasmin presented higher ferritin levels than
those with normal ceruloplasmin (Erhardt et al., 2002).
A larger study of hepatic iron concentrations on
follow-up liver biopsies in patients with WD confirmed
penicillamine treatment was associated with 3–10 times
increased hepatic iron content compared to baseline,
whereas zinc treatment was not associated with signifi-
cant increase in hepatic iron concentration (Medici
et al., 2007). However, iron studies were conducted on
a subsequent larger study of 143 patients with WD
(Pfeiffenberger et al., 2012). Interestingly, serum ferritin
was lower in males with WD who also presented lower
ceruloplasmin levels. Only 3 out of 27 patients who
underwent liver biopsy presented hepatic iron concentra-
tion higher than the upper limit, all of them on chelating
agents at the time of the biopsy. Collectively, the direct
and indirect evidence points to the fact that iron may con-
tribute to the phenotype of WD and monitoring of iron
metabolism parameters should be considered in the
long-term management of these patients.
METHYL GROUPS
Hepatic methionine metabolism regulates DNA and
histone methylation reactions with potential conse-
quences on gene expression regulation and disease
presentation and course. The central players in methio-
nine metabolism are S-adenosylmethionine (SAM),
S-adenosylhomocysteine (SAH), and the bidirectional
SAH hydrolase (SAHH). SAM is irreversibly converted
to SAH by donating its methyl moiety to DNA methyl-
transferases. The SAM-to-SAH ratio is considered a
useful index of methylation capacity, as SAM is the uni-
versal methyl donor for methyltransferase reactions and
SAH is an inhibitor of almost all methylation reactions.
SAHH is a bidirectional enzyme that regulates the pro-
duction of homocysteine in the forward direction, while
excess homocysteine promotes SAH in the reverse direc-
tion. Thus, SAHH may play a critical role in the regula-
tion of methylation pathways; its inhibition could result
in an increase of its substrate SAH, with resultant inhibi-
tion of gene methylation reactions, thereby potentially
regulating the expression of genes involved in liver
injury. Dietary methyl groups are provided by either
betaine or choline which are abundant in spinach, beet,
cereals or eggs, liver, almonds, and broccoli.
Copper is a major regulator of methionine metabolism
through its effect on SAHH (Bethin et al., 1995). Copper
inhibits SAHH in a noncompetitive manner since its
binding to this enzyme results in the release of NAD+
cofactors (Li et al., 2007). Sahh hepatic transcript levels
are downregulated in a murine model of WD character-
ized by spontaneous hepatic copper accumulation
(Medici et al., 2013; Le et al., 2014). In addition, mater-
nal provision of methyl groups in the form of choline
was able to increase Sahh transcripts to control levels,
indicating maternal diet can have an effect on gene
expression and epigenetic mechanisms of phenotype reg-
ulation in WD (Medici et al. 2014).
OTHER DIETARY FACTORS
Another study on the LEC rat examined the effects of die-
tary fatty acids on peroxidative stress and histologic dam-
age. Polyunsaturated fatty acids, both n-6 and n-3 type,
were associated with fewer histologic features of hepati-
tis and lower liver enzymes. Cyclooxygenase-2 hepatic
transcript levels, a marker of inflammation, were down-
regulated in the group of rats receiving polyunsaturated
fatty acids (Du et al., 2004).
Another study explored the effects of mild zinc defi-
ciency on the same animal model and the major finding
was that LEC rats exposed to a zinc-deficient diet for
4 weeks developed acute liver failure at a younger age
compared to control rats (94 vs. 136 days of age)
(Saito et al., 2007). A diet rich in histidine was associated
with reduced hepatic copper levels by 47% and increased
urinary copper excretion in LEC rats, with correspond-
ing reduced activity of the antioxidant enzyme Cu,
Zn-superoxide dismutase (Xu et al., 2003). LEC rats
fed a soy protein-rich diet starting at 6 weeks of age pre-
sented 80% higher hepatic copper concentration and
reduced survival compared to control mice (Yonezawa
et al., 2003).
SUMMARYAND CONCLUSION
It is becoming evident from clinical and basic science
research that the phenotype of WD is affected by
 GENETIC AND ENVIRONMENTAL MODIFIERS OF WILSON DISEASE
genetic, epigenetic, and environmental factors. This can
have implications for the neonatal screening, early diag-
nosis, and treatment of this condition. The fact that
maternal diet can affect the onset and progression of
disease supports the indication of neonatal screening
of WD. In addition, current anticopper treatments could
be optimized by modifying dietary factors and could be
tailored according to the presence of certain mutations
and gene modifiers, ultimately improving disease
course and prognosis.
ABBREVIATIONS
APOE, apolipoprotein E; ATOX1, antioxidant 1
copper chaperone; ATP7A, ATPase copper-transporting
alpha; ATP7B, ATPase copper-transporting beta;
COMMD1, copper metabolism domain-containing 1;
DMT1, divalent metal transporter 1; HFE, hemochromato-
sis; LEC, Long–Evans Cinnamon; MTHFR, 5,10-
methylenetetrahydrofolate reductase; PRNP, prion protein;
SAH, S-adenosylhomocysteine; SAHH, S-adenosyl-
homocysteine hydrolase; SAM, S-adenosylmethionine;
WD, Wilson disease.
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