Review article Menkes advances newrequiring enzymes, such as cytochrome c oxidase (electron transport), superoxide dis-mutase (free radical detoxication), dopamine,B hydroxylase (catecholamine

JMed Genet 1997;34:265-274

Review article

Menkes disease: recent advances and new aspects

Zeynep Tiimer, Nina Horn

Copper is the third most abundant traceelement in the body, after iron and zinc, and itis required for the normal function of severalimportant copper enzymes. However, the sameelement in excess is highly toxic and has detri-mental effects. Fine regulation of intracellularcopper homeostasis is therefore vitally impor-tant and disturbance of this balance is reflectedin two hereditary disorders, Menkes diseaseand Wilson disease. In recent years, remarkableprogress has been made in this field followingthe isolation of the defective gene in Menkesdisease (MD), which will be the main focus ofthis review.

Progressive neurodegeneration and connec-

tive tissue disturbances are the main manifesta-tions ofX linked recessive Menkes disease andmost of the clinical features can be explainedby malfunction of one or more copperenzymes. The disease locus has been mappedto Xql3.3 and the gene (MNK) defective inMenkes disease has been isolated by positionalcloning. The protein product is predicted to bea copper binding P type ATPase (ATP7A), thefirst intracellular copper transporter describedin eukaryotes. Identification ofMNK led to a

series of advances in a very short time. Themouse homologue of MNK has been isolatedand the allelic relationship between MD andthe occipital horn syndrome confirmed. Mostimportantly, the gene defective in Wilsondisease was isolated using sequences specific toMNK and the predicted protein productshowed high homology to ATP7A. In thisreview we will outline these recent advancesand their consequences with special referenceto Menkes disease. Wilson disease will bedescribed briefly, and the defective copper

metabolism in both diseases will be summa-

rised in the light of the new insights. Finally,copper translocating ATPases identified inother species and their significance in under-standing copper metabolism will be discussed.

The John F Kennedy (7Med Genet 1997;34:265-274)Institute, Gl Landevej7, 2600 Glostrup, Keywords: Menkes disease; Wilson disease; copper

Denmark metabolism.Z TurmerN Horn

Menkes diseaseCorrespondence to:Dr Tumer, Department of Menkes disease (MD) is a multisystemic lethalMedical Genetics, Panum disorder, dominated by neurodegenerativeInstitute, University of symptoms and connective tissue mani-Copenhagen, Blegdamsvej, festations. " A striking and pathognomonic2200 Copenhagen,Denmark. feature is the sparse, coarse, and depigmented

Table 1 Main symptoms ofpatients with the classicalform ofMenkes disease and OHS

MD OHS

Neurological symptomsMental retardation ++ +/-Convulsions ++Hypothermia + +Feeding difficulties + +Muscle tone changes + ND

Connective tissue symptomsTortuous vessels + +Skeletal changes + ++Bladder diverticulae + ++Loose skin + +Loose joints + +

Other symptomsFacial dysmorphism + +Abnormal hair, pili torti + +Hypopigmentation + +

Laboratory findingsSerum copper 14. J1Serum ceruloplasmin .14. .1Intracellular Cu accumulation + +

Life span Death before *3 years

*Except for four patients, all the known OHS patients (about22) are still alive, the oldest patient being 52 years old. Twopatients have died in car accidents (I Kaitila and P Byers,personal communication), one patient has died of an unknowncause at 40 years of age (I Kaitila, personal communication),and one patient has died aged 49 years because of a bladderrupture (D Weaver, personal communication).ND, not determined.

hair, the reason why the disease has also beencalled "kinky or steely hair disease" (fig 1).Though most patients (90-95%) have a severeclinical course (table 1), various forms of thedisease exhibiting different degrees of nervoussystem or connective tissue involvement can bedistinguished.4 The occipital horn syndrome(OHS), mainly characterised by connective tis-sue manifestations, has been suggested to be avery mild allelic form ofMD5 (table 1). BesidesOHS, a late onset mild form6 7 and a moderateform can also be distinguished from the severeclassical form.4 Variation also exists withinthese groups and there are, for example, a sig-nificant number of patients surviving morethan six years despite the severe clinicalsymptoms.4 It is thus conceivable that MDcovers a clinical continuum from the severeclassical form to the mild OHS.4

Involvement of copper metabolism in MDwas first described by Danks et al,' showing lowserum levels and defective intestinal absorptionof the metal. At first, copper malabsorption wasthought to be the primary cause, but laterinvestigations showing copper accumulation inextrahepatic tissues, apart from the brain, indi-cated a multisystemic involvement.9 10 Most of

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6Tumer, Horn

Figure 1 A 9 month old Menkes disease patient with thesevere lethalform. (Photograph reproduced withpermission.)

the clinical features of MD are attributable todeficiency of one or more important copperrequiring enzymes, such as cytochrome c

oxidase (electron transport), superoxide dis-mutase (free radical detoxication), dopamine ,Bhydroxylase (catecholamine production), lysyloxidase (cross linking of collagen and elastin),and peptidyl-glycine a-amidating mono-

oxygenase (PAM, bioactivation of peptide hor-mones).

Genetics ofMenkes diseaseLOCALISATION OF THE MENKES LOCUS

Menkes disease was recognised to segregate as

an X linked recessive trait when it was firstdescribed in 1962 by Menkes et al.' Cellculture studies in phenotypically normal fe-male Menkes carriers indicated that theMenkes disease gene (MNK) was subject torandom X inactivation, also supporting an Xlinked mode of inheritance." Later, MNK was

linked to the centromeric region of the X chro-mosome by C banding polymorphism studies'2and further linkage analyses, exclusion map-ping, and comparative gene mapping betweenman and mouse suggested proximal Xq as thecandidate region.13-17The first physical evidence for the location of

MNK was the finding of a female Menkespatient with a balanced X;2 translocation'8 andthe X chromosome breakpoint was localised toXql 3.2-qi 3.3.19 This finding suggested thatthe X chromosome breakpoint was at or verynear the Menkes locus, directly affecting thefunction of the gene. Description of anotherMenkes patient with an X chromosome abnor-mality involving Xql 3 not only supported this

region as the candidate locus for MNK, butalso finely localised it to Xql3.3.20 This patientwas a male carrying a unique intrachromo-somal rearrangement where the segmentXql3.3-q21.2 was inserted into the shortarm.20 21

ISOLATION OF THE GENE DEFECTIVE IN MENKESDISEASEThe finding of two Menkes patients with Xchromosome breakpoints within the candidateregion expedited the efforts in cloning MNK.Three groups, including ours, isolated MNKusing slightly different positional cloningstrategies.22-24 The X chromosome breakpointof the X;2 translocation patient was the startingpoint for all three groups, though our groupalso investigated the patient with the intrachro-mosomal rearrangement.20 The common stepwas to construct a physical map of the genomicregion encompassing the site ofthe breakpointsand to isolate YACs (yeast artificial chromo-somes) covering this region.2225 ScreeningcDNA libraries with different kinds of probesenabled all three groups to isolate candidatecDNA sequences which were mapping to thebreakpoint region and were identical to eachother.2224 Southern blot analysis of 16 unre-lated Menkes patients referred to our instituteshowed partial gene deletions of different sizesand locations, indicating that this candidategene was defective in MD, and hence was "the"Menkes disease gene (MNK).23 Furthermore,in line with the disturbed copper metabolism,the predicted protein sequence showed a P typeATPase with striking sequence homology to abacterial heavy metal binding protein.22 Thepredicted protein is now designated ATP7Aand the MNK gene is occasionally calledATP7A.The mRNA transcript of MNK is 8.5 kb

long and the 3' end contains a 3.8 kb untrans-lated region.22 An open reading frame of 4500bp is identified encoding a predicted protein of1500 amino acids. The mRNA transcript isexpressed in heart, brain, lung, liver, skeletalmuscle, kidney, and placenta, but the level islow in pancreas and hardly detectable in liver.MNK is organised in 23 exons spanning abouta 150 kb genomic region26 (fig 2). The firstexon is a leader exon containing only untrans-lated sequences and the ATG start codon is inthe second exon. The exons in generalcorrespond to the predicted functional/structural domains of the protein product witha few exceptions.26

About the protein (ATP7A)Comparison of the predicted protein sequence(ATP7A)22 with other sequences in proteindatabases indicated a close similarity to cationtransporting P type ATPases, especially a heavymetal ion ATPase, involved in copper homeo-stasis in Enterococcus hirae.27 It was also closelyrelated to the cadmium resistance ATPasesfound in Staphylococcus aureus.28P type ATPases are energy using membrane

proteins, functioning as cation pumps either foruptake, efflux, or cation exchange. Theseenzymes are called "P type" ATPases, because

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A

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Figure 2 (A) Exon structure ofMNK and corresponding protein domains.26 The vertical lines indicate the positions of theintrons and the exons are indicated by numbers. (B) Predicted protein structure ofATP7A.22 23 The structure ofATP7B isalso predicted to be similar to ATP7A.86 87 Transmembrane domain is a common structuralfeature ofP type ATPases andanchors the protein into the membrane. ATP7A is predicted to have eight transmembrane domains (indicated with whitebars in fig 2A). ATP binding domain (ATP) is an extramembranous segment, responsible forATP binding in P typeATPases and it is one of the most conserved sites. Phosphorylation domain (D) contains an invariant cytoplasmic DKTGTmotif The aspartate residue (D) is crucialfor the enzyme activity and it is phosphorylated with the terminal phosphate ofATP in the cation transport cycle. CPC is the predicted cation channel with a proline residue (P) highly conserved amongP type ATPases. This residue is proposed to participate in the transduction of energy from the phosphorylation site to cationtransport. In ATP7A proline is surrounded by cysteine residues, which may provide specificity for heavy metals.Phosphatase domain (PD) contains the TGE motif, which may have a role in removing the phosphate from thephosphorylated aspartic acid (D) as part of the cation transport. The amino-termini ofP type ATPases is one of the mostdivergent domains. The GMXCXXC motif with a pair of conserved cysteine residues is repeated six times in ATP7A andthis motif is suggested to be binding copper (Cu).

of a conserved aspartate residue (D) that istransiently phosphorylated with the terminalphosphate of ATP during the transport of thecations across a membrane. The overallsequence similarity among prokaryotic andeukaryotic cation transporting ATPases sug-gest that these proteins have been modifiedthroughout evolution in response to the needfor translocation ofvarious cations. ATP7A hasall the features common to P typeATPases22 29-33 (fig 2B). A remarkable feature ofthis protein is the presence of six successiverepeats at the amino terminal. These repeatscontain the consensus GMXCXXC motif andthe presence of paired cysteine residuessuggests that these domains are the copperbinding sites.

The mottled mouseSoon after the isolation ofMNK the status ofmottled mouse, which was long thought to bethe murine model for Menkes disease, wasestablished. In mouse, 23 known mutations (YBoyd, personal communication) at the same Xlinked mottled locus (Mo) lead to a mottledcoat pigmentation in the female heterozygotesand the affected males show neurological andconnective tissue abnormalities, differinggreatly in severity.34 Phenotypic and biochemi-cal similarities between MD patients and mot-tled mutants,3537 along with the conserved mappositions of MNK and Mo,'5 16 38 have longsuggested that Mo phenotypes were caused bymutations in Mnk, the mouse homologue ofMNK. Following the isolation of MNK, Mnk

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Figure 3 Chromosome painting of the metaphase and interphase chromosomes of thefemale Menkes patient with X;2 translocation, using hamster cell hybrid containing a singlehuman X chromosome (CL-20, kindly provided by T Kruse). The normalX chromosomeis indicated with an arrow and the derivative chromosomes with arrow heads. (Thephotograph was taken by Y Hindkjcer on a confocal laser microscope.)

was cloned using the human sequences." 40The predicted protein product was also a Ptype ATPase (atp7a), showing 89% sequence

homology to ATP7A, and the tissue expressionprofile was also similar to the human counter-part. In the two mottled mutants, mottledblotchy (Mob'o) and mottled dappled (ModP),altered levels of the Mnk transcript were

detected."9 4 Recently a mutation in Mnk was

reported in Mo6'1 confirming the status of theMo mouse as an animal model for MD4' (seebelow).

The occipital horn syndromeCloning ofMNK provided the opportunity toanalyse the occipital horn syndrome (OHS)patients for an allelic mutation. These two Xlinked recessive disorders of copper metabo-lism, were suggested to be allelic based on theirbiochemical and clinical resemblances (table1), and their homology with the possibly allelicforms of the mottled mouse. Two well charac-terised mottled mutants, the mottled brindled(Mob) and the mottled blotchy (Mo6'°), were

suggested as the murine models for the classi-cal form of MD and OHS, respectively. TheMobr phenotype and classical MD have simi-larly disturbed copper homeostasis leading tosevere neurological impairment and death atan early age.3 The Mobl° phenotype resemblesOHS showing predominantly connective tissuemanifestations.'6 Both in MD and OHS, serum

copper and ceruloplasmin are low, though usu-

ally lower in MD and occasionally normal inOHS.42 Cultured fibroblasts of OHS and MDpatients show increased copper accumulationand markedly low lysyl oxidase activity.5"' InOHS the major effect is on lysyl oxidase, thecopper dependent enzyme that initiates cross

linking of collagen and elastin in connectivetissues. However, the possibility of a primarydefect in the lysyl oxidase gene was excluded bythe assignment of the gene to an autosome.44

Following the isolation ofMNK, Levinson etaP5 detected markedly reduced levels of themRNA transcript in two unrelated OHS

patients, suggesting that MNK had a role inOHS. Recently, impairment of MNK in threepatients with OHS has been reported, givingdirect molecular evidence for the allelic rela-tionship between these two diseases4" 46 (un-published data). These mutations are base pairsubstitutions affecting the normal mRNAsplicing and the Moblo also has a similar splicingdefect.4' Further studies, elucidating the effectsof the gene mutations on the structure andfunction of the protein, are required to under-stand the cellular pathology resulting in differ-ent phenotypes in mice and humans.

Females with Menkes diseaseA total of eight females affected with Menkesdisease have been described (Tsukahara, per-sonal communication).'8 47-52 In twopatients47 48 diagnosis was suggested clinically,and cytogenetic and biochemical analyses werenot performed. In three cases,495' diagnosis wasconfirmed by significantly increased copperaccumulation in cultured fibroblasts.5' Two ofthese patients505' had normal karyotypes andthe most likely explanation for the expressionof the disease was odd X inactivation. The thirdpatient49 had a 46,XX/45,X(31.5%) mosaicismin cultured fibroblasts and expression of thedisease could be explained by monosomy of theX chromosome harbouring the defective geneor by odd X inactivation.5' Three femalepatients had balanced translocations, t(X;2)'8(fig 3), t(X; 1),52 and t(X;21) (Tsukahara,personal communication) and in each case theX chromosome breakpoint was shown to be atthe MNK locus by fluorescence in situ hybridi-sation analyses25 52 (unpublished data). Asexpected, the normal X chromosomes werepreferentially inactivated in these patients,resulting in the expression of the disease, whichwas also confirmed by copper uptakestudies'8 52 (unpublished data).

Mutation spectrum in Menkes diseaseMenkes disease is one of the so called "newmutation disorders". These are usually Xlinked diseases, which are prone to new muta-tions, and almost every affected family shows adifferent alteration.5" Results to date indicatethat the mutations leading to MD show a widevariety from cytogenetic abnormalities tosingle base pair changes.

Cytogenetically visible chromosome abnor-malities comprise about 1% of the underlyinggenetic defect in MD. Three of these patientsare females with balanced X;autosomal trans-locations as described above (fig 3). The onlymale patient with a cytogenetic aberration20was detected during a systematic screening of180 unrelated Menkes disease patients.2' Wehave recently localised the Xql3.3 breakpointof the male patient and one of the femalepatients52 within MNK using Southern blothybridisation (unpublished data).

In about 15-20% of patients the mutationsare gross deletions or rearrangements. WithSouthern blot analysis we have identified rear-rangements or partial gene deletions in about45 unrelated Menkes patients2' 54 (unpublisheddata) (fig 4). The boundaries of 30 partial gene

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2 3

C * *.''

j_

Figure 4 Prenatal diagnosis ofMD by Southern blothybridisation63 (reproducedfrom Tiumer et al, Med Genet,1994, with permission). Genomic DNA is digested with therestriction enzyme HindIII and hybridised with a genomicprobe including exons 3-5 ofMNK. The lowerfragment(10 kb) is the normal band representing the normalXchromosome and it can be observed in the control (C).Index patient 1. 1 has only an abnormal band (23 kb)representing the defective gene. The mother (I) is a carrierof the mutation as indicated by the presence of both thenormal and the abnormalfragments. We performedprenatal diagnosis by mutation analysis and the fetus(I.3) had only the abnormalfragment, indicating that hewas affected. The biochemical analysis was in line with theDNA results and the family decided to terminate thepregnancy. The male fetus of the mother's previouslyterminated pregnancy (I.2) shows the normal bandpattern, indicating that he was not affected, and exogenouscopper contamination had resulted in a false positivebiochemical diagnosis. This analysis also shows clearly thatchorionic villi (1. 2, 3) were not contaminated bynmaternal tissue.

deletions were further delimited by PCRamplification of the individual exons withintron specific primers (unpublished data).The deletions show great variability in size andlocation. The largest defect observed is thedeletion of the whole gene except for the firsttwo exons, the leader exon, and the secondexon coding for the ATG start codon and thefirst metal binding domain (fig 2A). The small-

r. ~

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Figure 5 Carrier diagnosis ofMD by SSCP and subsequent direct sequencing using

genomic DNA. The index patient has a single base pair substitution (CT) in exon 10introducing a premature termination codon (R795X) within the fourth putativetransmembrane domain. The mother is heterozygous for this mutation and the abnormal Xchromosome is preferentially inactivated in line with her normal phenotype (data not

shown). The father represents the normal control (N).

est mutation shown by this approach is thedeletion of the leader exon (unpublished data).The remaining genetic defects found in

MNK are thus base pair changes or very smallrearrangements which can be analysed by vari-ous PCR based methodologies. Das et ar5 havecharacterised the mutations in 10 patients byRT-PCR (reverse transcription-PCR) andchemical cleavage mismatch detection usingRNA. To screen the mutations in our vastpatient population, we first determined thegenomic organisation of MNK26 and con-structed primers to analyse each exon usinggenomic DNA. Using SSCP analysis (singlestrand conformation polymorphism) and sub-sequent direct sequencing of the exons (fig 5)we identified the genetic defect in 41 unrelatedpatients.56 All these point mutations show greatvariety, including missense and nonsensemutations, deletions and insertions of a singleor more base pairs resulting in frameshifts andsplice site mutations.

Diagnosis of Menkes diseaseInitial diagnosis of Menkes disease is suggestedby the clinical features (especially the typicalhair changes) and supported by the reducedlevels of serum copper and ceruloplasmin.However, interpretation of these markers maybe difficult in the first months of life, as serumcopper and ceruloplasmin levels may also below in normal infants in this period.57 A defini-tive biochemical diagnosis exists and is basedon the intracellular accumulation of copperowing to impaired efflux. Accumulation isevaluated in cultured cells by measuring radio-active copper (64Cu) retention after a 20 hourpulse, and impaired efflux is directly deter-mined after a 24 hour pulse chase.5' However,these analyses demand expertise and arecarried out only in a few specialised centres inthe world.' 3 5 Postnatal diagnosis is performedon cultured fibroblasts and though a clear dis-crimination between affected and unaffectedmales is possible, milder phenotypes cannot bedistinguished from the classical form.4

Isolation of MNK and characterisation ofthe genomic organisation now opens up a newdiagnostic possibility, mutation analysis. Dem-onstration of a defect in MNK will be the ulti-mate diagnostic proof and as our knowledgeabout the mutations in the different forms ofMD increases, it may also be possible to distin-guish the different clinical forms genotypically.However, mutation detection in Menkes dis-ease is quite challenging; the large MNK tran-script is organised in 23 exons, the geneticdefect shows great variety, and each family hasits own unique mutation.

PRENATAL DIAGNOSISAmniocentesisPrenatal diagnosis ofMD was initiated in 1974by measuring 64Cu accumulation in culturedamniotic fluid cells.60 61 However, copper accu-mulation in these cells is less pronounced thanin fibroblasts and may occasionally give rise todiagnostic difficulties. Standardisation of thecell culture conditions is therefore crucial andneeds expertise.6' Amniocentesis should be

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performed before the 18th week of gestation asthe cell growth and hence copper accumulationis negatively correlated to the gestational age.

Chorionic villus samplingFirst trimester prenatal diagnosis is carried outby determining the total copper content inchorionic villi by neutron activation analysisand was initiated in 1983.62 As the normal cop-per content in this tissue is very low (1 ng cop-per per mg tissue), the test is highly susceptibleto exogenous copper contamination, whichmay give false positive results. The whole pro-cedure of chorionic villus sampling must there-fore be absolutely free of copper contaminationand, as this condition is not always fulfilled,DNA analysis become a valuable supplement.63False positive or negative results in biochemicalanalysis may also occur when the separation ofthe maternal tissue from the chorionic villi isnot optimal.58 Verification of the prenatal diag-nosis can be carried out by direct measurementof copper accumulation in the placenta64 andthis method has also proven to be valuable forneonatal diagnosis of males at risk.

DNA analysisDNA based prenatal diagnosis of MD is nowpossible63 65 and in some cases may be superiorto biochemical diagnosis63 (fig 4). However,when the mutation in the family has not yetbeen identified, biochemical diagnosis will stillbe preferred as mutation detection in MD isquite challenging for the reasons mentionedabove. Even in the cases where the geneticdefect is known, the mutation detectionmethod used has to be optimised beforeperforming the prenatal diagnosis, whereasbiochemical diagnosis is already well estab-lished. Diagnostic possibilities and risk factorsshould therefore be evaluated critically for eachcase.

CARRIER DIAGNOSISCarrier determination by measuring radioac-tive copper accumulation in cultured fibro-blasts is possible.'1 59 66 However, owing to ran-dom inactivation of one of the X chromo-somes, negative results are not reliable andmutation analyses will therefore provide theultimate proof of carriership54 (figs 4 and 5).Identification of carriers will also have animpact on the number of the prenatal diag-noses referred to our institute, as about 80% ofthe male fetuses tested are not affected,indicating that a substantial number of thefemales are not carriers. Using mutation analy-ses we have performed carrier diagnosis inabout 15 families and in 1 1 of these families themother was heterozygous for the mutationfound in the index patient (unpublished data).

In families where the mutation is unknown,intragenic polymorphic markers may alsoenable carrier diagnosis. A BfaI polymorphismhas been identified within the coding region ofMNK"5 and it is localised to exon 10(unpublished data). Furthermore, two poly-morphic CA repeats within the gene (introns 2and 5) were identified by us and others67 andthese are currently being used for carrier diag-

nosis in the families where the mutation is asyet unknown (unpublished data).

Treatment ofMenkes diseaseIn MD copper uptake is normal, but a defect inMNK disturbs the intracellular copper home-ostasis and copper requiring enzymes cannotreceive the copper necessary for their normalfunction. The objective of a treatment is thus toprovide copper to the intracellular compart-ments where the copper enzymes are synthe-sised. However, parenteral administration ofvarious copper preparations (as copper sul-phate or copper-EDTA) did not produce sub-stantial clinical improvement in MD patients.66On the other hand, copper-histidine, the physi-ological copper complex found in humanserum," had a positive effect in four unrelatedMD patients, who are the oldest survivingpatients receiving this therapy.3 70-73 They arenow alive between the ages of 7 and 19 yearswith a milder clinical course resembling OHS.In two of these patients70 71 we have identifiedthe genetic defect and they both have severemutations (premature stop codons in exon 4and exon 12) which would have resulted in aprogressive clinical course if untreated.77 How-ever, patients receiving copper-histidine afterthe first few months of age do not benefit in thesame way (though survival may be prolonged),4suggesting that the therapy should be initiatedvery early, before the occurrence of irreparableneurodegeneration.7476 Studies with the mousemodel ofMD imply that there is a critical stagein brain development at which copper is essen-tial, suggesting that induced premature deliv-ery of affected infants for early treatment mightbe beneficial.3

In the four patients mentioned above,though the neurological symptoms improved,the connective tissue abnormalities persisted.As lysyl oxidase is the enzyme involved in crosslinking of collagen and elastin in connectivetissues, these results suggest that the functionof this enzyme does not appear to be correctedwith copper-histidine administration. How-ever, as indicated by the improvement of theneurological symptoms, copper seems to bedelivered to some of the copper requiringenzymes using copper-histidine.

Wilson diseaseWilson disease (WD) is an autosomal recessivedisorder of copper metabolism resulting fromthe toxic effects of copper, in contrast to MDmimicking copper deficiency (table 2). Thoughthese two diseases have different clinicalprogression, the serum copper and ceruloplas-min are low in both cases. WD is mainly char-acterised by different degrees of liver disease,neurological or psychiatric symptoms, andclinical variability.78 79 The clinical features ofWD are attributable to the toxic accumulationof copper in the liver and other tissues, such askidney, brain, and cornea (Kayser-Fleischerrings).The Wilson disease locus (WND) has been

assigned to chromosome 1380 and was localisedwithin an approximately 1.6 cM region at13ql4-21 by extensive linkage studies.81-83 In

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contrast to MD, visible cytogenetic rearrange-ments, which could lead to straightforward iso-lation of the gene, were not reported for WD.Identification of MNK encoding a putativecopper transporting ATPase, and its hardlydetectable expression in liver, led to thesuggestion that WD might be caused by adefective liver specific copper transporter.22Soon after, WND was cloned using sequencesspecific to MNK84 85 and to a heavy metalbinding site within the amyloid 1 proteinprecursor.86 The 7.5 kb mRNA transcript isexpressed predominantly in liver, kidney, andplacenta, while the message is low in heart,brain, lung, muscle, and pancreas.886 Theputative protein product is also a copper bind-ing P type ATPase (designated ATP7B), highlyhomologous to ATP7A (57%).87 The WND-gene (or ATP7B) has 21 exons in livertranscripts88 and 22 exons in kidneytranscripts89 and its genomic structure shows aremarkable similarity to MNK, organised in 23exons.26The Long-Evans Cinnamon (LEC) rat

showing liver disturbances is suggested to be ananimal model for Wilson disease.90 The rathomologue ofWND has recently been isolatedusing human specific sequences and it has beenshown to be defective in the LEC rat, confirm-ing its status as a murine model for WD.9' Thepredicted protein product of the rat gene is alsoa copper binding P type ATPase (atp7b) show-ing 82% sequence similarity to ATP7B.

Isolation ofWND now enables DNA baseddiagnosis of Wilson disease. Differing fromMenkes disease, only point mutations or verysmall rearrangements are detected in Wilsondisease.84 86 89 92-94 Mutation detection in Wilsondisease is also challenging because of the largenumber ofmutations in a 4.1 kb coding region.However, the presence ofhaplotypes specific toWND chromosomes may facilitate the muta-tion analyses as each haplotype is shown to begenerally associated with a specific mutation.92Mutation analysis in WD would be of greatimportance especially in diagnosing potentialpatients with no family history of Wilson

disease and in early diagnosis of the sibs ofaffected patients.

New insights into normal and defectivecopper metabolismCopper homeostasis depends on a balancebetween intestinal absorption and biliary ex-cretion. Copper absorbed from the intestine isattached mainly to albumin and transported tothe liver, the central organ of copper homeosta-sis, where storage and biliary excretion of cop-per and ceruloplasmin synthesis take place.Copper is secreted from the hepatocytes to theplasma bound to ceruloplasmin, but the mech-anism by which it is delivered to differenttissues is not fully understood. Regulation ofintracellular copper homeostasis, from uptaketo transport of the metal to its functional desti-nation and export from the cell, is not wellknown either.

In Menkes disease, as in Wilson disease,serum copper and ceruloplasmin levels are low.In MD intestinal absorption of copper isgrossly diminished.8 Copper accumulates inintestinal mucosa, kidney, spleen, lung, pan-creas, muscle, and skin, while in the liver andbrain the copper levels are below normal.9 10The finding of potential CpG islands has beenthe first molecular indication that MNK mightbe a housekeeping gene, required by all or mosttissues, in line with the multisystemic characterof the disorder.25 Consistent with the tissuedistribution of copper in Menkes patients, theMNK transcript is also widely expressed.However, two organs, brain and liver, wherethe copper levels are low, need special atten-tion. The MNK transcript is expressed inbrain, supporting the suggestion that in MDlow copper levels in this organ might be a sec-ondary effect, owing to diminished transportacross the blood-brain barrier.2 In liver theMNK transcript is hardly detectable and astraightforward explanation for the involve-ment of liver in MD awaits further studies.

In MD cellular copper uptake is normal anda defect in MNK causes intracellular accumu-lation of the metal. Copper enzymes are

Table 2 Comparison ofMenkes disease with Wilson disease

InheritanceChromosome locationIncidencePathognomonic featureMain clinical features

Basic pathology

Diagnostic markers

PrognosisAnimal model

Predicted proteinTissue expressionGenomic organisationGene defect

Menkes disease

X linked recessiveXql 3.31:300 000Kinky hairNeurological symptomsConnective tissue symptomsHypopigmentationFacial dysmorphismImpaired function of copper enzymes

Serum Cu 4Serum ceruloplasmin 4-Cu accumulation in cultured cells 1

Lethal in severe casesMottled mouse

1500 AA copper binding ATPaseAll tissues, hardly detectable in liver23 exons spanning 150 kb genomic region1% cytogenetic abnormalities20% gross rearrangements80% small base pair changes

Wilson disease

Autosomal recessive13q14.31:100 000Kayser-Fleischer ringHepatic diseaseNeurological dysfunction

Impaired biliary excretion of CuImpaired incorporation of Cu into ceruloplasminSerum Cu 4Serum ceruloplasmin 1Liver Cu TUrinary Cu excretion TEffective treatment with chelatorsLEC ratsToxic milk miceBedlington terriers141 1 AA copper binding ATPaseMainly in liver and kidney, low in other tissues22 exons spanning 80 kb genomic regionPoint mutations or small rearrangements

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deprived of copper necessary for their normalfunction,3 while the metal is bound to metal-lothionein, a cysteine rich heavy metal bindingprotein, which protects the cell from toxiceffects of the free ion. It is thus conceivable thatATP7A delivers copper to the enzymes and isalso involved in copper efflux. However, copperaccumulation is likely to result from a defect incopper translocation across an intracellularcompartment, rather than a defective copperexport across the plasma membrane.3 9'ATP7A is thus likely to be located in one ormore intracellular compartments or organelles,possibly endoplasmic reticulum or golgi appa-ratus or both, and is involved in the regulationof the intracellular copper pool by translocatingthe metal through membranes.'6

In WD it is likely that ATP7B plays a directrole in incorporation of copper into ceruloplas-min and in biliary copper excretion. A defect inWND results in deficient production ofceruloplasmin and copper starts accumulatingin the hepatocytes bound to metallothionein.However, in later stages copper accumulationreaches a toxic level and subsequent overflowfrom liver to other tissues, like brain, cornea,and kidney, leads to a variety of damage tothese organs also. Expression of the WNDtranscript predominantly in liver is in line withthe clinical course and the underlying bio-chemical defect.MD is characterised by an overall copper

deficiency, while WD is associated with coppertoxicity. However, the functions ofATP7A andATP7B appear to be quite similar. They areboth predicted to be copper translocatingmembrane proteins likely to be located in theintracellular compartments or organelles.96 7

ATP7A has a role in the delivery of copper todifferent enzymes, while ATP7B is involved insupplying copper to ceruloplasmin. Further-more, both proteins are likely to be involved inthe excretion of copper from the cell viaintracellular compartments or organelles.96Though an important step has been taken by

the cloning of the genes defective in Wilson andMenkes diseases, and their murine homo-logues, the enigma of intracellular coppermetabolism is not solved yet. Identification ofother potential proteins involved in the copperuptake and intracellular transport will un-doubtedly increase our understanding of cellu-lar copper homeostasis.

Copper translocating P type ATPases inother speciesATP7A shows a remarkable sequence similar-ity to P type ATPases involved in copper trans-location in Enterococcus hirae,57 leading to theprediction of the first intracellular copperbinding P type ATPase in eukaryotes."2 Sincethen, a number of related genes have beendescribed in eukaryotes and prokaryotes.These are all predicted to encode P typeATPases with one or more copper bindingdomains at the amino-terminus. The humanmembers of this copper associated subfamily ofthe P type ATPases, ATP7A and ATP7B, showhigh sequence homology. They both have sixpredicted copper binding domains, including

the consensus GMXCXXC motif, and copperbinding of one of these domains has recentlybeen reported for ATP7B.9" The two othereukaryotic members of this family are themouse protein atp7a with six copper bindingdomains39 40 and the rat protein atp7b withfive.9' Both proteins show remarkable sequencehomology to their human counterparts.

In Saccharomyces cerevisiae two genes map-ping to different chromosomes were identifiedand they were predicted to encode for proteins(Pcal and Ccc2) belonging to this family.99 100Ccc2 contains two copper binding motifs andmay provide copper from the cytosol into anextracytosolic compartment."' 101 This is in-deed analogous to the mechanism suggestedfor ATP7A and ATP7B.96 Pcal has a singlecopper binding motif and may have a role incopper extrusion from the cell.99 Among thebacterial members of this family, there arethree copper ATPases, Cop A and Cop Bdescribed in Enterococcus hirae27 and CtaAdescribed in Synechococcus 7942.102 All theseproteins are involved in copper homeostasis,where CopA and CtaA are serving in theuptake and CopB in the extrusion of copper, inthe respective bacteria. It is likely that not onlythe eukaryotic cells but most of the prokaryoticcells as well have copper enzymes involved inelectron transfer and therefore require coppertransport systems. These systems in bacteriaand yeast may thus provide an experimentallyaccessible model for understanding the overallintracellular copper homeostasis in highereukaryotes. Recently, a role of Ccc2 in ironmetabolism has been described in yeast and itwas shown to be providing copper to aceruloplasmin-like oxidase required for ironuptake.10' Identification of other potential cop-per proteins in bacteria or yeast, and under-standing their function, may thus provideinsight also into the connection of copper andiron homeostasis in man.

Despite recent advances, copper homeosta-sis is still not fully understood. However, tosolve this enigma we now have the most neces-sary tools, the animal models, and simple bio-logical systems such as bacteria and yeast.

1 Menkes JH, Alter M, Steigleder G, Weakley DR, Sung JH. Asex-linked recessive disorder with retardation of growth,peculiar hair and focal cerebral and cerebellar degenera-tion. Pediatrics 1962;29:764-79.

2 Horn N, Tonnesen T, Tulmer Z. Menkes disease: anX-linked neurological disorder of the copper metabolism.Brain Pat 1992,2:351-62.

3 Danks DM. Disorders of copper transport. In: Scriver JR,Beaudet AL, Sly WS, Valle D, eds. The metabolic basis ofinherited disease. New York: McGraw-Hill, 1995:2211-35.

4 Horn N, Tonnesen T, Tulmer Z. Clinical variability of theMenkes disease. In: Sarkar B, ed. Metals and genetics. NewYork: Marcel and Dekker, 1995:285-303.

5 Peltonen L, Kuivaniemi H, Palotie A, Horn N, Kaitila I,Kivirikko KI. Alterations in copper and collagen metabo-lism in the Menkes syndrome and a new subtype of theEhlers-Danlos syndrome. Biochemistry 1983;22:6156-63.

6 Procopis P, Camakaris J, Danks DM. A mild form ofMenkes' syndrome. 7 Pediatr 1981 ;98:97-9.

7 Danks DM. The mild form of Menkes disease: progressreport on the original case. Am J Med Genet 1988;30:859-64.

8 Danks DM, Stevens BJ, Campbell PE, et al. Menkes' kinky-hair syndrome. Lancet 1972;i: 1100-2.

9 Heydorn K, Damsgaard E, Horn N, et al. Extra-hepaticstorage of copper. A male foetus suspected of Menkes' dis-ease. Humangenetik 1975;29:171-5.

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21 Tommerup N, Tiumer Z, Tonnesen T, Horn N. Acytogenetic survey in Menkes disease: implications of chro-mosomal rearrangements in X-linked disorders. .7 MedGenet 1993;30:314-15.

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99 Rad MR, Kirchrath L, Hollenberg CP. A putative P-typeCu2'-transporting ATPase gene on chromosome II of Sac-charonmyces cerevisiae. Yeast 1994; 10: 1217-25.

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