Genetic Determinants of Trace Element Metabolism

DOUGLAS M. TEMPLETON, Ph.D., M.D.

Department of Laboratory Medicine and Pathobiology, Faculty o f Medicine, University o f Toronto, Toronto M5G 1L5, Canada.

The final years of the millennium have wit similar copper transporters, yet whereas Men nessed a breakthrough in our understanding of kes is a disease of copper deficiency presenting a number of metal-related disease processes as in infancy, Wilson disease is one of copper the responsible genes and their corresponding overload generally developing later in adult proteins have been identified. These events hood. Both are characterized by decreased lev have paralleled, and no doubt contributed to, a els of ceruloplasmin. change of perspective in metallobiochemistiy.

Whereas the metal ion was formerly the focus M e n k e s D is e a s e of attention, today we tend to think first about the proteins that permissively or actively gov Menkes disease is a syndrome of abnormal ern the trafficking and utilization of the ion. neurodevelopment which runs a natural The chemistry of the metal, once the focus of course of developmental regression, seizures, the bioinorganic chemist, has taken second and death in infancy or early childhood. It place to the molecular biology of the metal occurs as an X-linked disorder with an inci ion’s transporters, channels and chaperones. dence of between 1/100,000 and 1/250,000 live The result has been insight into metal-related births.8,9 It was recognized by Danks10 to diseases, as distinct from metal toxicology. Per result from copper deficiency and a genetic haps topping the list of successes in under defect in copper transport was suspected. The standing such diseases are the cloning of the responsible gene was identified by positional Menkes disease gene in 1993,1,2>3 the Wilson cloning by three groups in 1993.1>2'3 The gene, disease gene in 1993,4,5,6 and the gene for named ATP7A, includes a coding region of hereditary haemochromatosis in 1996.7 This 8.5 kb with nearly 4 kb of 3' untranslated overview will thus address mainly genetic dis sequence. It encodes a 1500-amino acid mem orders of iron and copper metabolism, but will ber of the P-type ATPase transporter family. It also have a few words to say about our pros is arranged in 23 exons spanning 150 kb of pects of recognizing genetic determinants of genomic DNA.11 In about 20% of cases dele other metal-related pathologies in the future. tions or rearrangements are found that are large enough to detect by Southern blot. Symptomatic point mutations occur through Copper out the sequence, but are clustered in exon 8, a region coding the stalk that connects the Wilson and Menkes diseases illustrate the metal-binding N-terminal to the remainder of extent of phenotypic divergence that can arise the protein.12 from closely related genetic determinants. The ATP7A protein has cystosolic N- and Both result from mutations in structurally C-terminal domains and eight intervening trans 24 0091-7370/99/0100-0024 $00.00 © Institute for Clinical Science, Inc. GENETIC DETERMINANTS OF TRACE ELEMENT METABOLISM 25 membrane regions. It has four signature ATP7B encodes a 1411-amino acid protein domains of the P-type ATPases13,14 that with an identical domain structure to the prod include a phosphatase domain (Thr-Gly-Glu- uct of ATP7A, ie, six copper-binding sites in Ala), a Cys-Pro-Cys domain in the putative cat the N-terminus, eight transmembrane regions, ion channel, and a conserved Asp residue and a phosphatase domain, a Cys-Pro-Cys domain ATP-binding domain in the cytosolic loop in the ion channel, and an Asp residue and between transmembrane regions 6 and 7. The ATP-binding domain in the cytosolic loop Asp residue is phosphorylated and subse between transmembrane regions 6 and 7. The quently dephosphorylated by the phosphatase gene spans over 80 kb of genomic DNA. In domain to effect a conformational change dur contrast to the large deletions found in ing the transport cycle. In the large cytosolic ATP7A, Wilson disease is associated only with N-terminal domain are found six copies of point mutations or small deletions.13 The Cys-X-X-Cys sequence that appear to be cop- mutation Hisl069Glu accounts for 38 per per-binding sites. They may serve to deliver cent of cases, but more than half the mutations copper to the transport machinery, or to main are rare.18 tain copper in the cuprous state to facilitate Recent evidence suggests that ATP- transport. Alternatively, a role in sensing cop independent transport of copper as Cu(I) per levels has been proposed (c/15). If three or occurs on the plasma membrane whereas the more of these metal-binding sites are lacking, ATP7B gene product transports Cu(II) in an the protein is non-functional.14 Various cul ATP-dependent manner and is located on the tured cells not expressing ATP7A take up cop Golgi membrane.15 This is consistent with a per at rates comparable to other cells but show role in incorporation of copper into ceruloplas delayed rates of copper efflux after copper min and is analogous to the iron transport sys loading.16 ATP7A is expressed in most cells tem of Saccharomyces cerevisiae, in which the except liver. The copper-deficient phenotype ATP-dependent copper transporter pumps in Menkes is explained by failure of the intes copper into an intracellular organelle for incor tinal epithelium to release absorbed dietary poration into the multicopper ferrooxidase, copper into the blood. FET3, required for iron acquisition by the cell.19 This is consistent with a role for ceru loplasmin in iron transport, as demonstrated W il s o n D is e a se by the aceruloplasminemic phenotype (see below) and explains the low levels of cerulo Dr. S. A. K. Wilson identified a syndrome plasmin generally observed in Wilson disease. of hepatolenticular degeneration in 1912.17 In cultured liver cells, increasing copper in the Typically, adults present with hepatic and medium caused the ATP7B protein to redis (or) neurological dysfunction secondary to tribute to a cytosolic vesicular compartment.20 copper accumulation, and may have asymp This is in contrast to the Menkes ATP7A pro tomatic Kayser-Fleischer rings reflecting tein, where increasing copper causes translo copper deposition in the cornea. World cation from the Golgi to the plasma mem wide, the disease affects about 30 people per brane.21 The cytosolic location is necessary for million population. The absence of ATP7A ceruloplasmin synthesis and for copper export. expression in liver suggested that a homolo There is also evidence that cleavage of about gous but unique protein might be expressed 20 kDa of the ATP7B N-terminal copper- there, perhaps reflecting the role of liver in binding region results in localization to the exercising homeostatic control over copper mitochondrion, suggesting a role in mitochon through biliary excretion. This suggested in drial copper homeostasis.22 An alternatively turn homology cloning strategies to identify spliced form of the protein lacking the regions the gene causing Wilson disease, and in 1993 coded by exons 6, 7, 8 and 12 is expressed in ATP7B was identified.4’5,6 the brain, where it localizes to cytosol.23 26 TEMPLETON

This complex functional regulation suggests some initial concern that it represented an arti that mutations affecting protein trafficking, fact of the linkage disequilibrium strategy by post-translational modification or alternative which it had been located. splicing could all occur in addition to loss-of- It was known that presentation of various function mutations. Their future identification MHC class I molecules on the cell surface may help to explain the clinical variability of required expression of (32-microglobulin. This the disease. suggested that investigating iron metabolism in P2-microglobulin-deficient mice might shed Iron light on the role of HFE in HH and, indeed, |32-microglobulin-nuIl mice recapitulated the H aemochromatosis pathology of iron overload in H H .25 Intestinal mucosal iron uptake involves exposure of the Hereditary or idiopathic haemochromatosis epithelium to both redox states of iron in a (HH) is one of the most common genetic dis number of inorganic, organic and macro- eases, although a marked variability in the molecular species. Indiscriminate uptake degree of penetrance means the true inci of iron could still allow intracellular chemistry dence is unknown. Perhaps a good estimate in to control release to the plasma, or elimina the Caucasian population is 1/400, with a car tion through desquamation of the epithelia, so rier frequency proposed as high as one in release might be a more logical point of ten.24 The disease has long been known to control than uptake. As this reasoning would result from an inability to limit intestinal predict, the (32-microglobulin-null mice fail absorption of iron in response to replete iron to regulate release of iron from the intesti stores, and in its severest manifestations HH is nal epithelium.25 characterized by cirrhosis and hepatocellular The role of fi2-microglobulin is becoming carcinoma, cardiomyopathy and insulin- clearer. Mutant Cys282Tyr HFE protein fails dependent diabetes mellitus, each the conse to bind |32-microglobulin, does not translocate quence of iron overload in the respective to the cell surface and becomes prematurely organ. The paradoxically high prevalence of degraded in the Golgi.26,27 As in cystic fibrosis HH may indicate that enhanced absorption of and the cystic fibrosis transmembrane con iron has afforded an evolutionary advantage, ductance regulator, the most common muta particularly in women. Because the disease is tion in HH probably results in a disorder of common, potentially fatal, not typically protein trafficking. screened for and fully manageable by periodic The genetic basis of the variability of HH phlebotomy, the search for the genetic basis phenotypes is also becoming clearer. The was intense. The gene was found by Feder and Cys282Tyr mutation is non-functional; hetero colleagues in 1996.7 It encodes a 34-amino zygotes will have half the amount of functional acid member of the major histocompatibility protein and homozygotes none. A second com complex (MHC) class I family denoted mon mutation, His63Asp, appears to worsen HLA-H. Over 80 percent of patients in the the phenotype in the compound heterozygote initial studies had a Cys282Tyr mutation in this without itself causing disease.28 The high fre protein. It has since come to be referred to as quency of the major mutation among affected HFE. In contrast to the obvious identification individuals is allowing genotyping to replace of ATP7A and ATP7B as copper-transporting hepatic iron index as a diagnostic test.29 molecules, it was not understood how an MHC Interestingly, high frequencies of both class I molecule could be involved in ion trans mutations have also been reported in cohorts port. Identification of HFE did not immedi with porphyria cutanea tarda,30 although in an ately contribute to our understanding of the Italian population only the His63Asp mutation pathogenesis of HH; a number of interesting was present.31 Most cases of porphyria cutanea hypotheses were advanced, but there was also tarda result from reversible inhibition of uro GENETIC DETERMINANTS OF TRACE ELEMENT METABOLISM 27 porphyrinogen decarboxylase by an iron- be in time. A mutation in mouse Nramp2 con dependent process, and although affected fers hereditary microcytic anemia with individuals do not develop frank haemo- decreased iron uptake by the erythron and chromatosis, subtle changes in iron metabo decreased intestinal iron absorption.42 A simi lism in concert with other precipitating fac lar pathology results from an Nramp2 muta tors such as alcohol and hepatitis C appear to tion in the Belgrade rat.43 Because other diva be involved.32 lent cations are transported by this system, other trace element abnormalities may occur

M a m m a l ia n I r o n T ransporters in association with Nramp2 mutations. A skin disorder in the microcytic anemic mouse is The delivery of iron to tissues from transfer reminiscent of zinc deficiency-acrodermatitis rin through transferrin receptor-mediated enteropathica44 and the Belgrade rat has been endocytosis is a well-established and widely- reported to have abnormalities in manganese studied process.33,34 However, non-trans- metabolism.45 In a current model, Nramp2 ferrin-bound iron (NTBI) can reach a concen transports Fe2+ into the intestinal epithelium tration of several micromolar in patients with and then the HFE gene product, the protein iron overload,35,36 and until recently very little responsible for HH, regulates egress into the has been known about the mechanisms by plasma in response to iron stores.46 Nramp2 which it enters cells. Increased intracellular also appears to play a role in release of iron iron increases the rate of NTBI uptake in cul from acidified endosomes after internalization tured hepatocytes,37 whereas it decreases the of transferrin. This release is impaired in the expression of transferrin receptor.38 There red cell precursor and diferric transferrin is fore, NTBI is likely to become an important cycled back outside the cell in the Belgrade source of hepatic iron in iron overload, and we rat43 Transport of Fe2+ is consistent with our hypothesized up-regulation of NTBI transport conclusion of obligatory reduction of Fe3+ for may serve to remove harmful circulating NTBI uptake by HepG2 cells. However, our obser and sequester it in tissues for short-term vation of increased iron uptake in response to detoxification.37 Uptake of NTBI dependent iron-loading in HepG2 cells remains to be rec upon intracellular iron concentration involves onciled with increased expression of Nramp2 reduction of Fe3+ and obligatory translocation in iron deficiency in vivo. of the nascent Fe2+.37-39-'10 Ceruloplasmin stimulates NTBI uptake in Several proteins involved in mammalian cultured HepG2 cells, a line of human hepa NTBI transport were recently described. The tocytes.47 A homologous protein plays a similar divalent cation transporter (DCT-1) is a 561- role in iron acquisition by yeast.48 Endogenous amino acid protein with 12 putative transmem- ceruloplasmin secreted by HepG2 cells medi brane-spanning regions that was cloned from a ates iron uptake from conditioned medium. duodenal cDNA library derived from iron- The transport is independent of transferrin deficient rats41 and confers upon Xenopus and, like Nramp2 expression, increased by iron oocytes the ability to take up Fe2+. Although deprivation or chelation. Synthesis of a new apparently playing an important role in intes protein is required for this response, leading to tinal iron absorption, DCT-1 mRNA is the idea that induction of a transporter that expressed in a broad range of tissues, including interacts with ceruloplasmin is involved.47 The liver. DCT-1 is a member of the “natural- possibility that this transporter is Nramp2 itself resistance-associated macrophage protein” should be considered. Thus, there seem to be (Nramp) family; rat DCT-1 has 92 percent opposing mechanisms of NTBI uptake in sequence identity to human Nramp2. HepG2 cells, one involving oxidation of Fe2+ Although no mutations associated with and increased by iron depletion,47 and one Nramp2 have been identified in man as yet, involving reduction of Fe3+ and increased by experience with rodents suggests that they will iron loading.37 28 TEMPLETON

A 41 kDa protein has been cloned from iron in soft tissues is reminiscent of haemo- K562 erythrocyte precursors that also confers chromatosis, with diabetes and liver damage iron uptake upon Xenopus oocytes.49 Named prominent. Interestingly, neurological symp ‘stimulator of Fe transport’ (SFT), the protein toms are most prominent with death from neu has six transmembrane-spanning regions and rodegeneration. The basal ganglia are a pri provides phorbol ester-stimulated, energy- mary target for iron accumulation. Iron is also dependent specific iron transport, characteris deposited in the retina leading to retinal tics of uptake in K562 cells. It is structurally degeneration, a phenomenon unknown in hae- and functionally distinct from Nramp2, and mochromatosis. The explanation for the may be a member of either the cation diffusion prominence of central nervous system involve facilitator (CDF)50 or ATP-binding cassette ment appears to be that ceruloplasmin does (ABC) metal transporters,51 though clear evi not cross the blood-brain barrier; rather, the dence of membership in either family is lack central nervous system synthesizes its own ing. The SFT transcript is present in a variety ceruloplasmin in astrocytes, particularly in the of human tissues.49 basal ganglia and substantia nigra.56 Frataxin, the product of the gene mutated in The current model of the pathogenesis of Friedreich’s ataxia,52,53 is a regulator of intra aceruloplasminemia is that stored iron is cellular iron transport. It maintains mitochon released from ferritin by reduction to Fe2+, drial iron homeostasis by promoting egress of following which oxidation by ceruloplasmin in iron, and the pathogenesis of Friedrich’s ataxia the plasma provides Fe3+ for formation of fer can now be explained by mitochondrial iron ric transferrin and delivery of Fe3+ to the mar toxicity. As noted, Nramp2 may also be row.54 In the absence of efficient transferrin involved intracellularly in transport of transfer- binding, excess iron is cleared from the circu rin-derived iron out of the endosome.43,46 lation by other tissues that then develop iron Likewise, SFT cycles with endosomes and overload. Iron crossing the blood-brain barrier facilitates iron uptake from transferrin.49 CDF by receptor-mediated endocytosis of transfer and ABC transporters are found in organelles rin is normally resequestered in the central as well as on the cell surface, and this dual nervous system by transferrin synthesized by functionality seems to be a general property of oligodendrocytes. It appears that in the metal transport proteins. This suggests again absence of ceruloplasmin, it is cleared by that many metal-related diseases are not sim parenchymal cells as non-transferrin-bound ply caused by decreased or increased cellular iron just as occurs in the periphery. concentrations of the metal but also by its Patients with aceruloplasminemia are not inappropriate intracellular trafficking. anemic because enough non-enzymatic oxida tion occurs to produce sufficient ferric trans

A caeruloplasminemia ferrin to supply the erythron. Nor do Wilson disease patients develop iron overload. In that Aceruloplasminemia is a rare autosomal disease, enough holoceruloplasmin presum recessive disorder resulting in a lack of func ably forms in extrahepatic tissue to allow the tional ceruloplasmin.54’55 Five mutations have successful scavenging of mobilized iron stores been described in the ceruloplasmin gene so by transferrin. far and all result in failure of its three-copper cluster to form.54 These individuals have nor I r o n -d e p e n d e n t G e n e E x p r e s s io n a n d mal copper metabolism, arguing against a role H e r e d it a r y C a ta racts for ceruloplasmin in copper transport or storage. Rather, the absence of the copper Iron metabolism is under pleiotropic regu cluster of the holoenzyme results in absent lation at the level of translation. The iron- plasma ferro-oxidase activity and a disorder responsive element (IRE) is a stem-loop struc of iron metabolism. The accumulation of ture in some mRNAs that interacts with iron GENETIC DETERMINANTS OF TRACE ELEMENT METABOLISM 29

regulatory proteins (IRPs) to influence tran develop cataracts and it has been named the script translation or stability.38'57 When cellu hereditary hyperferritinemia-cataract syn lar iron levels are low, IRPs occupy IREs, and drome. All families had point mutations in the when iron is high, the IRE is free. Occupancy L-ferritin IRE, affecting IRP binding. Further of an IRE in the 5' untranslated region of fer analyses have demonstrated point mutations in ritin mRNA blocks translation when occupied stem, bulge and loop regions of the IRE.65-66 by interfering with binding of the translational Affected individuals cannot down-regulate machinery. In contrast, occupancy of multiple L-ferritin production and express inappropri copies of IRE in the 3' untranslated region of ately high levels of the protein. Lens epithelia transferrin receptor mRNA does not influence actively synthesize ferritin in response to oxi translation but affects steady state message dative stress-a protective response. Accumula levels by increasing message stability. As a tion of L-rich apoferritin may alter the equi consequence, in iron deficiency ferritin syn libria among the crystalline proteins of the thesis is decreased and TfR expression lens, leading to loss of transparency. It is increases, whereas in iron overload transferrin not clear why only mutations in the L-ferritin receptor levels decrease and ferritin increases IRE have been identified to date; per to minimize further uptake and facilitate stor haps mutations in IREs of other transcripts age. Our earlier observation that ferritin syn await identification. thesis does not keep up with iron load in iron-

overloaded patients who are chelated58 may H aemoglobinopathies be accounted for by this mechanism; chela tion of the labile iron pool sensed by the IRE/ Thalassemias and other haemoglobinopa IRP system results in apparent iron deficiency thies remain a major global genetic problem even in the face of increased iron stores. Other at the close of the millenium. The World transcripts regulated by IREs include 8-ami- Health Organization predicts that in the year nolevulinate synthase (the rate limiting 2000, 7 percent of the world’s popula enzyme in heme synthesis) and mitochondrial tion, some 350 million individuals, will carry aconitase (a control point in the Krebs cycle).38 a gene for some form of haemoglobinop- Nramp2 mRNA contains a putative IRE in athy. Presently, 500,000 individuals have trans- the 3' UTR41 that could account for increased fusion-dependent thalassemia and another Nramp2 transporter in iron deficiency by a 300,000, sickle cell disease. Although gene mechanism analogous to transferrin recep therapy and control of globin gene switching to tor modulation. preserve expression of fetal haemoglobin are Two IRPs have been identified in mamma potential treatments, the complexity of regula lian tissues that sense iron concentrations in tion of the balanced expression of globin different ways.59,60,61 IRP-1 contains iron in a chains67 suggests that such therapies are still 4Fe-4S cluster. Removal of a labile iron atom some time away. Ironically, although the glo from this cluster converts the constitutively bin genes were among the first investigated by expressed protein from its nonbinding to its molecular techniques, they may be among the IRE-binding state, affording a rapid response last to yield to gene therapy. In the meantime, to changing iron levels. IRP-2 does not contain multiple transfusions and the resulting need iron. Rather, increasing cellular iron favors for iron chelation will continue to exacerbate degradation of the protein and thus decreased the iron overload associated with these genetic IRE binding activity. IRP-1 is the predomi disorders.68,69 nant IRP in most tissues.62 In 1995 there were independent reports Future Directions of two Italian families and a French family affected by hyperferritinemia.63,64 The disor Genetic disorders involving other metal ions der is benign except that affected individuals are generally not as well understood as the 30 TEMPLETON above examples. For instance, the first mam overexpression could occur. Arsenic-induced malian Zn2+ transporters have only recently malignant transformation of a cultured cell been identified. They are denoted ZnT-1 and line has been associated with DNA hypometh ZnT-2.70’71 In contrast to the Wilson and Men ylation and c-myc oncogene overexpression.76 kes transporters that shuttle between cellular It will be interesting to study the methyltrans- compartments, ZnT-1 resides on the plasma ferase genotype of arsenic-resistant and sensi membrane and supports Zn efflux, whereas tive human populations. ZnT-2 pumps zinc into an intracellular vesicu lar compartment, probably lysosomal. Both are R eferences thought to serve the common function of low ering cytoplasmic zinc. No pathology has yet 1. Chelly J, Tümer Z, Tonnesen T, Petterson A, Ishi- been associated with defects in ZnT-1 or kawa-Brush Y, Tommerup N, Horn N, Monaco AP. Isolation of a candidate gene for Menkes disease that ZnT-2, but as transporters for zinc and other encodes a potential heavy metal binding protein. elements are identified, it seems inevitable Nature Genet 1993;3:14-19. that additional genetic disorders will emerge. 2. Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Isolation of a candidate gene for Menkes An emerging and exciting concept in trace disease by positional cloning. Nature Genet 1993;3: element utilization is the recognition that, 7-13. instead of simple diffusion, chaperones may be 3. Mercer JFB, Livingston J, Hall B, Paynter JA, Begy C, widely involved in directing metal ions to their Chandrasekharappa S, Lockhart P, Grimes A, Bhave M, Siemieniak D. Isolation of a partial candidate gene specific molecular targets.72,73 For example, for Menkes disease by positional cloning. Nature three yeast proteins, Coxl7, Lys7 and Atxl Genet 1993;3:20-25. deliver copper to cytochrome c oxidase, super 4. Bull PC, Thomas GR, Rommens JM, Forbes JR, Cox DW. The Wilson disease gene is a putative copper oxide dismutase and the yeast homologue of transporting P-type ATPase similar to the Menkes the Wilson/Menkes proteins, respectively. gene. Nature Genet 1993;5:327-337. Each has a human counterpart. Such chaper 5. Petrukhin K, Fischer SG, Pirastu M, Tanzi RE, Cher nov I, Devoto M, Brzustowicz LM, Cayanis E, Vitale ones may prove to be involved in many unex E, Russo JJ, Matseoane D, Boukhgalter B, Wasco W, plained disease processes. For example, CCS, Figus AL, Loudianos J, Cao A, Stemlieb I, Evgrafov the human homologue of Lys7, is necessary for O, Parano E, Pavone L, Warburton D, Ott J, Pen- chaszadeh GK, Scheinberg IH. Mapping, cloning and correct insertion of copper into superoxide dis genetic characterization of the region containing the mutase. Aberrant binding of copper to super Wilson disease gene. Nature Genet 1993;5:338-343. oxide dismutase is implicated in some cases of 6 . Yamaguchi Y, Heiny ME, Gitlin JD. Isolation and characterization of a human liver cDNA as a candidate amyotrophic lateral sclerosis. gene for Wilson disease. Biochem Biophys Res Com- Finally, genetic determinants of toxic metal mun 1993;197:271-277. metabolism represent a rich area for study. 7. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo Jr. R, For example, arsenic toxicity varies among dif Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel ferent populations. In India and Taiwan, con BE, Kronmal GS, Lauer P, Lee VK, Loeb DB, Mapa sumption of water contaminated with arsenic FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E. A novel MHC class I-like is linked to development of cancers represent gene is mutated in patients with hereditary haemo- ing a major public health concern. Similar con chromatosis. Nature Genet 1996;13:399-108. sumption by indigent people in Chile and 8 . Kaler SG. Menkes disease. Adv Pediatr 1994;41:263- 304. Mexico is without effect.74 Arsenic is detoxi 9. Kaler SG. Diagnosis and therapy of Menkes syn fied in man by stepwise methylation to mono- drome, a genetic form of copper deficiency. Am J Clin methylarsonic and dimethylarsinic acids with Nutr 1998;67 Suppl:1029S-1034S. S-adenosyl methionine serving as the methyl 10. Danks DM, Cartwright E, Stephens BJ, Townley RR. Menkes’ kinky hair disease: further definition of the donor.75 However, S-adenosyl methionine is defect in copper transport. Science 1973;179:1140- also the methyl donor regulating gene expres 1142. sion through DNA methylation. If S-adenosyl 11. Tümer Z, Vural B, Tonnesen T, Chelly J, Monaco AP, Horn N. Characterization of the exon structure of the methionine is depleted by enzymatic metabo Menkes disease gene using vectorette PCR. Genomics lism of arsenic, DNA hypomethylation and 1995;26:437-442. GENETIC DETERMINANTS OF TRACE ELEMENT METABOLISM 31

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