WO2010042102A1 - Identification of subjects likely to benefit from copper treatment - Google Patents

Abstract

Disclosed herein are methods of identifying a subject as a candidate for treatment with copper. The described methods include determining the presence of at least one mutation in an ATP7 A gene and the presence of at least one biochemical marker of abnormal copper metabolism, wherein the presence of at least one ATP7A mutation and at least one biochemical marker of abnormal copper metabolism identifies an individual likely to benefit from copper treatment. The mutation in ATP7A is one or more mutation selected from the group consisting of Gln197Ter; Arg201Ter; Ala629Pro; Ser637Leu; Gly666Arg; Gly727Arg; Ser833Gly; Gly1019Asp; Asn1304Ser; Ala1362Asp; IVS8,AS,dup5; IVS9,DS,+6T>G; IVS21,DS,+3A>T; Del4246-4260; and Del 4284-4315. The biochemical marker of abnormal copper metabolism includes copper level, ceruloplasmin level, placental copper level, catecholamine level, or cellular copper egress.

Description

IDENTIFICATION OF SUBJECTS LIKELY TO BENEFIT FROM

COPPER TREATMENT

FIELD This application relates to methods of identifying individuals who may benefit from treatment with copper, particularly those having Menkes disease or Occipital Horn Syndrome.

BACKGROUND Menkes disease is an infantile onset X-linked recessive neurodegenerative disorder caused by deficiency or dysfunction of a copper-transporting ATPase, ATP7A. The clinical and pathologic features of this condition reflect decreased activities of enzymes that require copper as a cofactor, including dopamine-β- hydroxylase, cytochrome c oxidase, and lysyl oxidase. Recent studies indicate that ATP7 A normally responds to N-methyl-D-aspartate receptor activation in the brain, and an impaired response probably contributes to the neuropathology of Menkes disease. Affected infants appear healthy at birth and develop normally for 6 to 8 weeks. Subsequently, hypotonia, seizures, and failure to thrive occur, and death by 3 years of age is typical. Occipital horn syndrome (OHS) is also caused by mutations in the copper transporting ATPase ATP7A, although its symptoms are milder than Menkes syndrome, including occipital horns and lax skin and joints.

Treatment with daily copper injections may improve the outcome in Menkes disease if commenced within days after birth; however, newborn screening for this disorder is not available and early detection is difficult because clinical abnormalities in affected newborns are absent or subtle. Moreover, the usual biochemical markers (low serum copper and ceruloplasmin,) are unreliable predictors in the neonatal period, since levels in healthy newborns are low and overlap with those in infants with Menkes disease. Although molecular diagnosis is available, its use is complicated by the diversity of mutation types and the large size of ATP7A (about 140 kb). Thus, there is a need for improved methods for early detection of infants with Menkes disease or OHS in order to start immediate treatment and improve outcomes. SUMMARY

Disclosed herein are methods for identifying subjects likely to benefit from treatment with copper. The methods include determining the presence of at least one mutation in an ATP7A molecule from the subject and determining the presence of at least one biochemical marker of abnormal copper metabolism in a sample from the subject. Early detection and treatment of Menkes disease and OHS is critical to positive clinical outcome. The disclosed methods allow pre- symptomatic identification of individuals most likely to benefit from copper treatment and reduce morbidity and mortality due to these diseases.

The disclosed method includes determining the presence of at least one mutation in an ATP7A molecule (such as DNA, RNA, or protein), wherein the mutation includes one or more of Glnl97Ter; Arg201Ter; Ala629Pro; Ser637Leu; Gly666Arg; Gly727Arg; Ser833Gly; GlylO19Asp; Asnl304Ser; Alal362Asp; IVS8,AS,dup5; IVS9,DS,+6T>G; IVS21,DS,+3A>T; Del4246-4260; and Del 4284-4315. The method also includes determining the presence of at least one biochemical marker of abnormal copper metabolism, wherein the biochemical marker includes copper level, ceruloplasmin level, catecholamine levels, or cellular copper egress. In particular examples, the subject has Menkes disease or OHS. The sample from the subject is a biological sample which contains ATP7A

DNA, RNA or protein for use in determining the presence of at least one mutation in an ATP7A molecule. Such samples include, but are not limited to blood, serum, plasma, isolated cells or tissue, urine, and saliva. The sample from the subject also includes biological samples which contain biochemical markers of copper metabolism, for example copper, ceruloplasmin, or catecholamines. Such samples include, but are not limited to, blood, serum, plasma, cerebrospinal fluid (CSF), urine, placenta or other tissue biopsy, and isolated cells (such as fibroblast or lymphoblast cells). The presence of at least one mutation in an ATP7A molecule and the presence of at least one biochemical marker of abnormal copper metabolism may be determined in the same sample from the subject (such as one or more portions of a sample) or in more than one sample from the subject.

In some examples, the mutation in an ATP7A molecule is determined in a DNA molecule, for example by one or more of probe hybridization, nucleic acid amplification, heteroduplex analysis, size analysis, or nucleotide sequencing. In additional examples, the mutation in an ATP7A molecule is determined in an RNA sample, for example by reverse-transcriptase polymerase chain reaction or size analysis. The presence of a mutation in an ATP7A molecule may also be determined in a protein molecule, for example by Western blot or ELISA assay (for example using at least one ATP7A-specific antibody).

In some examples, the biochemical marker of abnormal copper metabolism includes reduced copper levels (such as serum or CSF copper level) as compared to a normal control or reference value or reduced ceruloplasmin levels (such as serum ceruloplasmin) in a sample from a subject as compared to a normal control or reference value, wherein reduced copper level or reduced ceruloplasmin level is an indicator that the subject is a candidate for copper treatment. Biochemical markers of abnormal copper metabolism also include increased placental copper level in a sample from a subject as compared to a normal control or reference value wherein increased placental copper is an indicator that the subject is a candidate for copper treatment and reduced cellular copper egress (such as from fibroblast or lymphoblast cells) in a sample from a subject as compared to a normal control or reference value, wherein reduced copper egress is an indicator that the subject is a candidate for copper treatment. In further examples, the biochemical marker of abnormal copper metabolism correlates with decreased dopamine β hydroxylase (DBH) activity, such as serum, plasma, or CSF catecholamine levels. In particular examples, catecholamine levels correlated with decreased DBH activity include an increased ratio of dopamine to norepinephrine or an increased ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol as compared to a normal control or reference, wherein an increased ratio of dopamine to norepinephrine or an increased ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol is an indicator that the subject is a candidate for copper treatment.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is a scatter plot of the ratio of plasma dopamine to norepinephrine versus the ratio of plasma dihydroxyphenylacetic acid to dihydroxyphenylglycol in 36 newborns at risk for Menkes disease. Figure IB is a receiver-operating-characteristic (ROC) curve for plasma dihydroxyphenylacetic acid in 36 newborns at risk for Menkes disease. The upper curve, plotted by the locally weighted scatter-plot smoothing technique, represents the sensitivity and specificity for the diagnosis of Menkes disease when different cutoff values for dihydroxyphenylacetic acid are applied. The area under the curve (C statistic) for the ROC shown is 0.96. The diagonal line indicates where the curve would rest if a test were completely unreliable (area under the curve, 0.5).

Figure 2A is a bar graph showing clinical neurodevelopmental outcomes at 36 months of age for patients 1 to 9. Patient 1 died at 19 months of age. The horizontal line shows normal level of development for 36 months. Figure 2B is a bar graph showing neurodevelopmental levels for patients 10, 11, and 12 at less than 36 months of age. The horizontal lines show normal developmental level for the patient's age.

SEQUENCE LISTING Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C. F. R. 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the genomic nucleic acid sequence of an exemplary human ATP7A gene.

SEQ ID NOs: 2 and 3 show the nucleic acid (cDNA) and amino acid sequences, respectively, of an exemplary human ATP7A. SEQ ID NOs: 4 and 5 show forward and reverse primers, respectively, used to amplify ATP7 A exon 1.

SEQ ID NOs: 6 and 7 show forward and reverse primers, respectively, used to amplify ATP7A exon 2. SEQ ID NOs: 8 and 9 show forward and reverse primers, respectively, used to amplify ATP7 A exon 3.

SEQ ID NOs: 10 and 11 show forward and reverse primers, respectively, used to amplify ATP7A exon 3 a. SEQ ID NOs: 12 and 13 show forward and reverse primers, respectively, used to amplify ATP7A exon 3b.

SEQ ID NOs: 14 and 15 show forward and reverse primers, respectively, used to amplify ATP7A exon 4.

SEQ ID NOs: 16 and 17 show forward and reverse primers, respectively, used to amplify ATP7A exon 4a.

SEQ ID NOs: 18 and 19 show forward and reverse primers, respectively, used to amplify ATP7A exon 4b.

SEQ ID NOs: 20 and 21 show forward and reverse primers, respectively, used to amplify ATP7A exon 4c. SEQ ID NOs: 22 and 23 show forward and reverse primers, respectively, used to amplify ATP7A exon 5.

SEQ ID NOs: 24 and 25 show forward and reverse primers, respectively, used to amplify ATP7A exon 6.

SEQ ID NOs: 26 and 27 show forward and reverse primers, respectively, used to amplify ATP7A exon 7.

SEQ ID NOs: 28 and 29 show forward and reverse primers, respectively, used to amplify ATP7A exon 8.

SEQ ID NOs: 30 and 31 show forward and reverse primers, respectively, used to amplify ATP7A exon 9. SEQ ID NOs: 32 and 33 show forward and reverse primers, respectively, used to amplify ATP7A exon 10.

SEQ ID NOs: 34 and 45 show forward and reverse primers, respectively, used to amplify ATP7A exon 11.

SEQ ID NOs: 36 and 37 show forward and reverse primers, respectively, used to amplify ATP7A exon 12.

SEQ ID NOs: 38 and 39 show forward and reverse primers, respectively, used to amplify ATP7A exon 13. SEQ ID NOs: 40 and 41 show forward and reverse primers, respectively, used to amplify ATP7A exon 14.

SEQ ID NOs: 42 and 43 show forward and reverse primers, respectively, used to amplify ATP7A exon 15. SEQ ID NOs: 44 and 45 show forward and reverse primers, respectively, used to amplify ATP7A exon 16.

SEQ ID NOs: 46 and 47 show forward and reverse primers, respectively, used to amplify ATP7A exon 17.

SEQ ID NOs: 48 and 49 show forward and reverse primers, respectively, used to amplify ATP7 A exon 18.

SEQ ID NOs: 50 and 51 show forward and reverse primers, respectively, used to amplify ATP7A exon 19.

SEQ ID NOs: 52 and 53 show forward and reverse primers, respectively, used to amplify ATP7A exon 20. SEQ ID NOs: 54 and 55 show forward and reverse primers, respectively, used to amplify ATP7A exon 21.

SEQ ID NOs: 56 and 57 show forward and reverse primers, respectively, used to amplify ATP7A exon 22.

SEQ ID NOs: 58 and 59 show forward and reverse primers, respectively, used to amplify ATP7 A exon 23.

DETAILED DESCRIPTION I. Abbreviations and Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0- 19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

ATP7A: copper transporting, alpha polypeptide ATPase (gene or protein)

CSF: cerebrospinal fluid DBH: dopamine β hydroxylase

DHPA: dihydroxyphenylacetic acid

DHPG: dihydroxyphenylglycol

DOPA: dihydroxyphenylalanine

OHS: occipital horn syndrome

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (V_L) and "variable heavy chain" (V_H) refer, respectively, to these light and heavy chains. As used herein, the term "antibodies" includes intact immunoglobulins as well as a number of well-characterized functional fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohl er and Milstein (Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.

ATP7A: The copper transporting ATPase alpha gene or protein. There are two copper-transporting ATPases in humans (ATP7A and ATP7B) which are members of the metal-transporting P-type ATPase family (see, e.g., Lutsenko et al, Physiol. Rev. 87: 1011-1046, 2007). The ATP7A protein has eight membrane spanning segments. The amino-terminal segment contains six copper binding sites. ATP7A also contains an A-domain, which is required for the phosphatase step of the catalytic cycle and an ATP binding domain consisting of a P domain and an N domain.

Copper plays a critical role in metabolism as a cofactor of key metabolic enzymes (such as dopamine β hydroxylase, lysyl oxidase, and cytochrome c oxidase). ATP7A transports copper from enterocytes (where it is taken up from dietary copper) into the blood. In Menkes disease and OHS, ATP7A activity is reduced or absent and copper export from the enterocytes is impaired. As a result, copper accumulates in intestinal cells and less copper is delivered to the blood, resulting in restricted copper supply to other tissues. ATP7A sequences are publicly available. For example, GenBank

Accession numbers NC_000023.9 (region 77052876...77192208; provided herein as SEQ ID NO: 1) and NC_000086 disclose human and mouse ATP 7 A gene sequences, respectively. GenBank Accession numbers NM_000052.4 (provided herein as SEQ ID NO: 2) and NP 000043 (provided herein as SEQ ID NO: 3) disclose exemplary human ATP7A cDNA and protein sequences, respectively and GenBank Accession numbers NM_009726 and NP_033856 disclose exemplary mouse Atp7a cDNA and protein sequences, respectively. All GenBank sequences described herein are incorporated by reference in their entirety on October 6, 2008. One skilled in the art will appreciate that ATP7A nucleic acid and protein molecules can vary from those publicly available, such as ATP7A sequences having one or more substitutions, deletions, insertions, or combinations thereof, while still retaining ATP7A biological activity. In addition, ATP7A molecules include alternatively spliced isoforms and fragments that retain the desired ATP7A biological activity. Catecholamine: A compound derived from the amino acid tyrosine containing a catechol group (dihydroxyphenol) and an amine group, or a derivative thereof. Dopamine, norepinephrine, and epinephrine are exemplary catecholamines. The synthesis of the catecholamines includes production of dihydroxyphenylalanine (DOPA) from tyrosine by the action of tyrosine hydroxylase. DOPA is then converted to dopamine by DOPA decarboxylase and aromatic amino acid decarboxylase. Dopamine β hydroxylase (DBH) converts dopamine to norepinephrine, which can be converted to epinephrine by phenylethanolamine N-methyltransferase. DBH is a copper-dependent enzyme, therefore disruption in copper metabolism (such as in Menkes disease and OHS) result in alterations of catecholamine biosynthesis and degradation.

Catecholamines also include derivatives of (such as the metabolites of) dopamine, norepinephrine, and epinephrine. Metabolites of dopamine include dihydroxyphenylacetic acid (DHPA or DOPAC), 3-methoxytyramine, and homovanillic acid. Metabolites of norepinephrine include dihydroxyphenylglycol (DHPG), normetanephrine, 3,4-dihydroxymandelic acid, 3-methoxy-4- hydroxymandelic acid, and 3-methoxy-4-hydroxyphenylethylene glycol. Ceruloplasmin: Also known as ferroxidase of iron(II): oxygen oxidoreductase, ceruloplasmin is the major copper-carrying protein in the blood. The protein is synthesized in hepatocytes and secreted into the serum with copper incorporated during biosynthesis. Failure to incorporate copper during synthesis results in the secretion of an apoprotein devoid of copper, termed apoceruloplasmin. In one example, decreased ceruloplasmin (such as plasma or serum ceruloplasmin) as compared to a normal control individual or reference value is a marker of abnormal copper metabolism (such as Menkes disease or OHS).

Copper histidine: A form of copper replacement that can be administered parenterally (such as subcutaneously) in a subject, bypassing the gastrointestinal system. Copper histidine is most often used clinically as a 1 :2 complex of copper (II) with L-histidine.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprises the genetic material of most living organisms (some viruses have genes comprising ribonucleic acid (RNA)). The repeating units in DNA polymers are four different nucleotides, each of which comprises one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides (referred to as codons) code for each amino acid in a polypeptide, or for a stop signal. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intended to include the reverse complement of that DNA molecule. Except where single- strandedness is required by the text herein, DNA molecules, though written to depict only a single strand, encompass both strands of a double-stranded DNA molecule. For example, a reference to the DNA molecule that encodes ATP7A, or a fragment thereof, encompasses both the sense strand and its reverse complement. Thus, for instance, it is appropriate to generate probes or primers from the reverse complement sequence of the disclosed nucleic acid molecules.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as "base pairing." More specifically, A will hydrogen bond to T or U, and G will bond to C. "Complementary" refers to the base pairing that occurs between to distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence.

"Specifically hybridizable" and "specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the oligonucleotide (or its analog) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na⁺ concentration) of the hybridization buffer will determine the stringency of hybridization, though waste times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, chapters 9 and 11, herein incorporated by reference. For present purposes, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize.

In vitro amplification: Techniques that increase the number of copies of a nucleic acid molecule in a sample or specimen. An example of in vitro amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions using a polymerase, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.

The product of in vitro amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.

Other examples of in vitro amplification techniques include strand displacement amplification (see U.S. Patent No. 5,744,311); transcription-free isothermal amplification (see U.S. Patent No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A- 320 308); gap filling ligase chain reaction amplification (see U.S. Patent No. 5,427,930); coupled ligase detection and PCR (see U.S. Patent No. 6,027,889); and NASBA™ RNA transcription-free amplification (see U.S. Patent No. 6,025,134).

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids or proteins.

Menkes disease (also known as kinky hair disease or steely hair disease): An infantile onset X-linked recessive neurodegenerative disorder caused by deficiency or dysfunction of the copper-transporting ATPase ATP7A (OMIM 309400). As an X-linked disease, Menkes disease typically occurs in males who present when aged 2-3 months with loss of previously obtained developmental milestones and the onset of hypotonia, seizures, and failure to thrive. Characteristic physical changes of the hair and facies, in conjunction with typical neurologic findings, often suggest the diagnosis. The scalp hair of infants with classic Menkes disease is short, sparse, coarse, and twisted. Light microscopy of patient hair illustrates pathognomonic pili torti (for example, 180° twisting of the hair shaft) and the hair tends to be lightly pigmented and may demonstrate unusual colors, such as white, silver, or gray. The face of the individual with Menkes disease has pronounced jowls, with sagging cheeks and ears that often appear large. The palate tends to be high-arched, and tooth eruption is delayed. The skin often appears loose and redundant, particularly at the nape of the neck and on the trunk. Neurologically, profound truncal hypotonia with poor head control is invariably present. Developmental skills are confined to occasional smiling and babbling in most patients. Growth failure commences shortly after the onset of neurodegeneration and is asymmetric, with linear growth relatively preserved in comparison to weight and head circumference.

The biochemical phenotype in Menkes disease involves (1) low levels of copper in plasma, liver, and brain because of impaired intestinal absorption, (2) reduced activities of numerous copper-dependent enzymes, and (3) paradoxical accumulation of copper in certain tissues (such as the duodenum, kidney, spleen, pancreas, skeletal muscle, and/or placenta). The copper-retention phenotype is also evident in cultured fibroblasts and lymphoblasts, in which reduced egress of radiolabeled copper is demonstrable in pulse-chase experiments.

Mouse models of Menkes disease are available and include the mottled mouse (Mercer, Am. J. Clin. Nutr. 76: 1022S-1028S, 1998; for example, brindled, tortoise, dappled, viable-brindled, and/or blotchy mice) and the macular mouse {e.g. Kodama et al, J. Histochem. Cytochem. 41 : 1529-1535, 1993). Mutation: Any change of the DNA sequence within a gene or chromosome. In some instances, a mutation will alter a characteristic or trait (phenotype), but this is not always the case. In some instances, mutations are referred to as variants; the terms are used interchangeably herein. Types of mutations include base substitution point mutations {e.g., transitions or transversions), deletions, and insertions. Missense mutations (or variants) are those that introduce a different amino acid into the sequence of the encoded protein; nonsense mutations are those that introduce a new stop codon. In the case of insertions or deletions, mutations can be in- frame (not changing the frame of the overall sequence) or frame shift mutations, which may result in the misreading of a large number of codons (and often leads to abnormal termination of the encoded product due to the presence of a stop codon in the alternative frame).

This term specifically encompasses mutations that arise through somatic mutation, for instance those that are found only in disease cells, but not constitutionally, in a given individual. This term also encompasses DNA alterations that are present constitutionally, that alter the function of the encoded protein in a readily demonstrable manner, and that can be inherited by the children of an affected individual. In this respect, the term overlaps with "polymorphism," but generally refers to the subset of constitutional alterations that have arisen within the past few generations in a kindred and that are not widely disseminated in a population group. In particular embodiments, the term is directed to those alterations that have major impact on the health of affected individuals.

Mutations or variants can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation (e.g., an alteration of a secondary structure such as a stem-loop, or an alteration of the binding affinity of the nucleic acid for associated molecules, such as polymerases, RNases, and so forth).

Occipital horn syndrome (OHS; also known as type IX Ehlers-Danlos syndrome or X-linked cutis laxa): An infantile onset disease caused by mutations in the copper transporting ATPase ATP7A (OMIM 304150). OHS is characterized by occipital horns, distinctive wedge-shaped calcifications at the sites of attachment of the trapezius muscle and the sternocleidomastoid muscle to the occipital bone. Occipital horns may be clinically palpable or observed on skull radiographs. Individuals with OHS also have lax skin and joints, bladder diverticula, inguinal hernias, and vascular tortuosity. Intellect is normal or slightly reduced and affected individuals typically live to at least mid-adulthood. Oligonucleotide: An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 70 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.

Probes and primers: A probe comprises an isolated nucleic acid capable of hybridizing to a target nucleic acid (such as an ATP7A nucleic acid molecule). A detectable label or reporter molecule can be attached to a probe or primer. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, for example in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

In a particular example, a probe includes at least one fluorophore, such as an acceptor fluorophore or donor fluorophore. For example, a fluorophore can be attached at the 5'- or 3'-end of the probe. In specific examples, the fluorophore is attached to the base at the 5'-end of the probe, the base at its 3'-end, the phosphate group at its 5'-end or a modified base, such as a T internal to the probe.

Probes are generally at least 15 nucleotides in length, such as at least 15, at least 16, at least 17, at least 18, at least 19, least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50 at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule, such as 20-70 nucleotides, 20-60 nucleotides, 20-50 nucleotides, 20-40 nucleotides, or 20-30 nucleotides.

Primers are short nucleic acid molecules, for instance DNA oligonucleotides 10 nucleotides or more in length, which can be annealed to a complementary target nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. A primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify a target nucleic acid molecule (such as a portion of an ATP7 A nucleic acid molecule).

The specificity of a primer increases with its length. Thus, for example, a primer that includes 30 consecutive nucleotides will anneal to a target sequence with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, to obtain greater specificity, probes and primers can be selected that include at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides. In particular examples, a primer is at least 15 nucleotides in length, such as at least 15 contiguous nucleotides complementary to a target nucleic acid molecule. Particular lengths of primers that can be used to practice the methods of the present disclosure (for example, to amplify a region of an ATP7 A nucleic acid molecule) include primers having at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or more contiguous nucleotides complementary to the target nucleic acid molecule to be amplified, such as a primer of 15-70 nucleotides, 15-60 nucleotides, 15-50 nucleotides, or 15-30 nucleotides. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by PCR, real-time PCR, or other nucleic-acid amplification methods known in the art. An "upstream" or "forward" primer is a primer 5' to a reference point on a nucleic acid sequence. A "downstream" or "reverse" primer is a primer 3' to a reference point on a nucleic acid sequence. In general, at least one forward and one reverse primer are included in an amplification reaction.

Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided herein. It is also appropriate to generate probes and primers based on fragments or portions of these disclosed nucleic acid molecules, for instance regions that encompass the identified mutations an ATP7A sequence, or the site of mutation in the genomic nucleic acid sequence of ATP7 A or a subsequence thereof.

PCR primer pairs can be derived from a known sequence (such as a gene encoding an ATP7A protein; such as an ATP7A protein as set forth in SEQ ID NO: 3), by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, MA) or PRIMER EXPRESS® Software (Applied Biosystems, AB, Foster City, CA).

Preventing, treating or ameliorating: "Preventing" a disease or condition refers to inhibiting the full development of a disease or condition.

"Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease or condition. In some embodiments, the disease or condition is Menkes disease or occipital horn syndrome.

Ratio: A quantitative relation between two amounts showing the number of times one value contains or is contained within the other; a proportional relationship between two different numbers or quantities. A ratio can be calculated by dividing one number or value by another. In some examples, the ratio of catecholamine levels is a biochemical marker of abnormal copper metabolism. Particular examples include the ratio of dopamine level to norepinephrine level and the ratio of DHPA level to DHPG level. Reference value: A value used as a reference for values obtained by examinations of patients or samples (such as blood, serum, plasma, CSF, or isolated cells) collected from individuals. A reference value generally consists of pooled data from a large number of samples. Reference data is commonly in the form of reference intervals for healthy individuals. A 95% reference interval usually is bounded by two limiting values and contains 95% of the values found in healthy individuals. Since test results often are dependent on sex and age, it is often necessary to have separate reference intervals for the two sexes and/or for different age groups.

In some examples a reference value is the value for serum or CSF copper in a normal population in a particular age group (such as less than 12 months or 12 months or older), the value for serum ceruloplasmin in a normal population in a particular age group (such as less than 12 months or 12 months or older), or plasma or CSF catecholamines in a normal population in a particular age group (such as less than 12 months or 12 months or older). Sample: A sample, such as a biological sample, is obtained from a plant or animal subject. As used herein, biological samples include all clinical samples useful for detection of ATP7A in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue (such as skin or placenta), including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates. In a particular example, a sample includes blood obtained from a human subject, such as whole blood or serum. In another particular example, a sample includes buccal cells, for example collected using a swab or by an oral rinse. In a further example, a sample includes cells from a subject, such as fibroblast cells obtained by a skin biopsy. Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of ATP7A protein, and the corresponding cDNA or gene sequence(s), will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or genes or cDNAs are derived from species that are more closely related (e.g., human and chimpanzee sequences), compared to species more distantly related (e.g., human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. MoI. Biol. 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp, Gene, 73: 237-244, 1988; Higgins & Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nuc. Acids Res. 16, 10881-90, 1988; Huang et al, Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al, Meth. MoI. Bio. 24:307-31, 1994. Altschul etal. (J. MoI. Biol. 215:403-410, 1990) presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J.

MoI. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. By way of example, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and are different under different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand. Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Tijssen {Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York, 1993). Nucleic acid molecules that hybridize under stringent conditions to a human ATP7 A protein-encoding sequence will typically hybridize to a probe based on either an entire human ATP7A protein-encoding sequence or selected portions of the encoding sequence under wash conditions of 2x SSC at 50° C.

Nucleic acid sequences that do not show a high degree of sequence identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as laboratory or veterinary subjects).

Therapeutically effective amount: A dose sufficient to prevent advancement, delay progression, or to cause regression of the disease, or which is capable of reducing symptoms caused by the disease, such as Menkes disease or occipital horn syndrome. IL Overview of Several Embodiments

Disclosed herein are methods of identifying a subject as a candidate for treatment with copper. The described methods include determining the presence of at least one mutation in an ATP7 A gene and determining the presence of at least one biochemical marker of abnormal copper metabolism, wherein the presence of at least one ATP7 A mutation and at least one biochemical marker of abnormal copper metabolism identifies an individual likely to benefit from copper treatment.

The mutation in ATP7A is one or more mutation selected from the group consisting of Glnl97Ter; Arg201Ter; Ala629Pro; Ser637Leu; Gly666Arg; Gly727Arg; Ser833Gly; GlylO19Asp; Asnl304Ser; Alal362Asp; IVS8,AS,dup5; IVS9,DS,+6T>G; IVS21,DS,+3A>T; Del4246-4260; and Del 4284-4315. The biochemical marker of abnormal copper metabolism includes copper level (such as serum copper level or CSF copper level), ceruloplasmin level (such as serum ceruloplasmin level), placental copper level, catecholamine level (such as plasma or CSF catecholamine level or one or more ratios of catecholamine levels), or cellular copper egress (such as copper egress from isolated fibroblast cells).

In some examples, the biochemical marker of abnormal copper metabolism includes reduced copper levels (such as serum or CSF copper level) as compared to a normal control or reference value or reduced ceruloplasmin levels (such as serum ceruloplasmin) in a sample from a subject as compared to a normal control or reference value, wherein reduced copper level or reduced ceruloplasmin level is an indicator that the subject is a candidate for copper treatment. Biochemical markers of abnormal copper metabolism also include increased placental copper level in a sample from a subject as compared to a normal control or reference value wherein increased placental copper is an indicator that the subject is a candidate for copper treatment and reduced cellular copper egress (such as from fibroblast or lymphoblast cells) in a sample from a subject as compared to a normal control or reference value, wherein reduced copper egress is an indicator that the subject is a candidate for copper treatment. In further examples, the biochemical marker of abnormal copper metabolism correlates with decreased dopamine β hydroxylase (DBH) activity, such as serum, plasma, or CSF catecholamine levels. In particular examples, catecholamine levels correlated with decreased DBH activity include an increased ratio of dopamine to norepinephrine or an increased ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol as compared to a normal control or reference, wherein an increased ratio of dopamine to norepinephrine or an increased ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol is an indicator that the subject is a candidate for copper treatment. Menkes disease and OHS are caused by a deficiency or dysfunction of the copper transporting ATPase ATP7 A. Although affected infants appear healthy at birth and develop normally for about six to eight weeks, subsequent hypotonia, seizures, and failure to thrive occur and death by three years of age is typical. Treatment with daily copper injections may improve outcome if treatment is started within days after birth. However, early detection is difficult because affected individuals do not typically exhibit obvious clinical abnormalities. Even when Menkes disease or OHS is diagnosed very early in life and treatment with copper injections is started, neurodevelopmental outcome is highly variable.

The present invention is a method for identifying subjects most likely to benefit from treatment with copper and to have the greatest neurodevelopmental benefit from such treatment. The present invention is based on the observation that individuals with at least one of a particular set of ATP7A mutations who also exhibit at least one biochemical marker of abnormal copper metabolism are most likely to have successful neurodevelopmental outcomes upon treatment with copper injections. Successful neurodevelopmental outcome include age appropriate neurodevelopment (for example, gross motor skills, language skills, fine motor-adaptive skills, and/or personal-social skills) or improved neurodevelopment as compared to an untreated individual with an ATP7A mutation. The particular ATP7A mutations, which can be used in conjunction with biochemical markers of abnormal copper metabolism to identify copper-responsive individuals, may result in an ATP7A protein having at least some copper transport activity. Examples of ATP7A mutants which retain at least some copper transport activity include Glnl97Ter, Arg201Ter, Gly666Arg, Gly727Arg, and Asnl304Ser. By identifying individuals most likely to respond to copper treatment, the methods provided herein allow treatment with copper injections to begin as early as possible, leading to the best neurodevelopmental outcomes. Although copper treatment can successfully prevent the morbidity and mortality associated with Menkes disease and OHS in some cases, daily copper injections may cause proximal renal tubule kidney damage. Therefore, the described methods may allow treatment decisions to administer copper injections despite the associated risks. ATP 7 A Activity

In some examples, the mutant ATP7A protein retains at least some copper transport activity (such as reduced copper transport activity compared to the wild type ATP7A protein). Methods of determining ATP7A copper transport activity are well known in the art (see, e.g., Kaler et ah, N. Eng. J. Med. 358:605-614, 2008). In a particular example, ATP7A copper transport activity is measured by the ability of an ATP7 A protein to complement a yeast copper transport knockout strain (such as the S. cerevisiae Δccc2 strain). The Δccc2 strain is unable to grow on nutritionally restricted (low copper and low iron) medium; however, transformation with wild type ATP7A cDNA restores normal growth. In some examples, complementation of the Δccc2 mutation is determined qualitatively by assessing the ability of an ATP7A protein (wild type or mutant) to restore growth on nutritionally restricted medium.

In other examples, ATP7A copper transport activity is determined quantitatively as a percentage of the function of wild type ATP7A in the complementation assay in a timed growth assay. In the timed growth assay, normal Δccc2 strains transfected with mutant or wild type ATP7A constructs, and mock transfected Δccc2 strains are grown in copper/iron limited medium. The OD₆₀₀ is determined at time points (such as after 0, 2, 4, and/or 8 hours of growth). Copper transport activity is expressed as a percentage of growth of wild type ATP7A cells (for example, OD₆₀₀ mutant/OD₆₀₀ wild type x 100). In particular examples, a mutant ATP7A protein retains at least some copper transport activity when the mutant ATP7A protein has at least about 5% to about 40% (such as about 5%, about 10%, about 20%, about 30%, or about 40%) activity in the Δcc2 complementation assay as compared to wild type ATP7A. Examples of ATP7A mutants which retain at least some copper transport activity include Glnl97Ter, Arg201Ter, Gly666Arg, Gly727Arg, and Asnl304Ser.

In some examples, mutant ATP7A proteins containing a premature termination codon may retain at least some copper transport activity as a result of translational read through or translation reinitiation downstream of the termination codon. In the case of a premature termination codon, residual ATP7 A copper transport activity may be assessed by detecting the presence of a full length protein (such as by Western blotting) or by detecting the presence of a full length RNA (such as by RT-PCR). Examples of ATP7 A proteins containing a premature termination codon which have reduced copper transport activity include Glnl97Ter and Arg201Ter.

In another example, the activity of ATP7A proteins (such as mutant ATP7A proteins) can be determined by measuring cellular copper egress in a cell (such as a fibroblast cell) expressing the ATP7A mutant protein. Methods of determining cellular copper egress are well known to one of skill in the art (see, e.g. La Fontaine et al, J. Biol. Chem. 273:31375-31380, 1998; Goka et α/, Proc. Natl. Acad. Sci. USA 73:604-606, 1976). Briefly, cells (such as fibroblast cells or lymphoblast cells) expressing an ATP7A protein (either natively or by transformation with an ATP7 A construct) are loaded with radioisotopic copper and copper efflux is measured by determining the decrease in intracellular copper over time. Alternatively, total cellular copper can be determined by atomic absorption spectroscopy. Reduced cellular copper egress can be measured indirectly and includes increased retention of copper as compared to a control cell or cell population (such as an increase of about 30% to about 20-fold, for example, about 30%, about 50%, about 70%, about 90%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold) or increased copper accumulation as compared to a control cell or cell population (such as an increase of about 50% to about 3-fold, for example, about 50%, about 60%, about 70%, about 80%, about 90%, about 2-fold, about 2.5-fold, or about 3-fold).

In additional examples, activity of ATP7 A proteins can be determined by a functional assay which measures the ability of the ATP7A protein to form a transient acylphosphate intermediate. ATP7A activity can be measured in membrane vesicles enriched for ATP7A protein from yeast or mammalian cells expressing ATP7A (see, e.g., Voskoboinik et al, J. Biol. Chem. 276:28620-28627, 2001) or using purified and membrane-reconstituted ATP7A protein (see, e.g., Hung et al, Biochem. J. 401 :569-579, 2007). ATP7A activity can be expressed as the percentage of phosphorylation of ATP7A protein. Mutant ATP7A proteins may have reduced ATP7A phosphorylation as compared to a control or wild type ATP7A protein (such as a decrease of about 10% to about 20-fold, for example, about 20%, about 30%, about 40%, about 50%, about 70%, about 90%, about 2- fold, about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold). Copper Treatment

The methods disclosed herein are useful for identifying a subject who will benefit from copper treatment, such as an individual having Menkes disease or OHS. The copper used for treatment may be in any form that can be conveniently administered and having an acceptable level of adverse effects (such as proximal renal tubular damage). In some examples, copper is in the form of copper histidine, copper chloride, and/or copper sulfate. In preferred embodiments, the copper is in the form of copper histidine, such as freeze-dried copper histidine.

The copper can be administered for therapeutic treatment of a subject selected by the method described herein, such as an individual with Menkes disease or OHS. Thus, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to improve a sign or a symptom of the disorder. Generally a suitable dose is about 250 μg to about 500 μg of copper (such as copper histidine, copper chloride, or copper sulfate) per day. However, other higher or lower dosages also could be used, such as from about lOOμg to about 500 μg per day.

Single or multiple administrations of the composition can be carried out with dose levels and pattern being selected by the treating physician. Generally, multiple doses are administered. In a particular example, the composition is administered parenterally once per day. However, the composition can be administered twice per day, three times per day, four times per day, six times per day, every other day, twice a week, weekly, or monthly. Treatment will typically continue for at least a month, more often for two or three months, sometimes for six months or a year, and may even continue indefinitely, i.e., chronically. In a particular example, 250 μg of copper histidine is administered by subcutaneous injection twice per day to a subject less than 12 months of age. In another example, 250 μg of copper histidine is administered by subcutaneous injection once per day to a subject 12 months of age or older. In some examples, the copper composition is administered until the subject reaches 3 years of age, at which time treatment is discontinued.

The copper is administered parenterally, such as subcutaneous, intravenous, intraperitoneal, intrathecal, or intramuscular injection. The copper may be combined with one or more pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers useful in copper treatment of individuals identified using the methods disclosed herein are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the compositions herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween®, sodium acetate or sorbitan monolaurate. The copper can be administered in combination with a therapeutically effective amount of at least one other agent for the treatment of Menkes disease or OHS. For example, the copper can be administered with a therapeutically effective amount of a catecholamine (such as L-threo-dihydroxyphenylserine (L-DOPS)). The combined administration of the copper and L-DOPS includes administering L- DOPS either sequentially with the copper, for example, treatment with one agent first and then the second agent, or administering both agents at substantially the same time, such as an overlap in performing the administration. With sequential administration a subject is exposed to the agents at different times so long as some amount of the first agent remains in the subject (or has a therapeutic effect) when the other agent is administered. The treatment with both agents at the same time can be in the same dose, i.e., physically mixed, or in separate doses administered at the same time. III. ATP7A Mutations

Mutations useful for the methods described herein include missense mutations, nonsense mutations, deletions, insertions, duplications, and frame shift mutations in an ATP7A molecule. Mutations can be referred to, for instance, by the nucleotide position at which the variation exists, by the change in amino acid sequence caused by the nucleotide variation, or by a change in some other characteristic of the nucleic acid molecule or protein that is linked to the variation.

In some examples, the ATP7A molecule is an ATP7A DNA molecule (such as a genomic ATP7A sequence or an ATP7A cDNA sequence). The mutation can be described by the nucleotide position at which the change exists and the change in the nucleotide. For example, a 77,987C>T mutation in an ATP7A gene refers to a substitution of the C at position 77,987 of the ATP7A gene sequence for a T. In some examples, the mutations in an ATP7A DNA include a mutation in an ATP7A gene, such as a variant nucleic acid at position(s) 77,987 (such as 77,987OT), 77,999 (such as 77,999OT), 100,469 (such as

100,469G>C), 100,494 (such as 100,494OT), 100,776 (such as 100,776G>C or 100,776G>A), 100,958 (such as 100,958T>G), 102,163 (such as 102,163G>C or 102,163G>A), 104,030 (such as 104,030A>G), 118,667 (such as 118,667G>A), 131,954 (such as 131,954A>G), 132,675 (such as 132,675OA), and/or 132,716 (such as 132,716A>T) of SEQ ID NO: 1. In additional examples, the mutation in an ATP7A DNA includes a deletion or one or more nucleotides; for example, deletion of nucleotides 4246-4260 of SEQ ID NO: 2 (positions 132,676-132,690 of SEQ ID NO: 1) and/or Del 4284-4315 of SEQ ID NO: 2 (nucleotides 134,748- 134,779 of SEQ ID NO: 1). In another example, the mutation in an ATP7A DNA includes a duplication of one or more nucleotides (such as IVS8,AS,dup5; duplication of position 100,722-100,726 of SEQ ID NO: 1).

In some examples, the ATP7A mutation includes a mutation in an ATP7A cDNA molecule, such as a variant nucleic acid at position(s) 749 (such as 749OT), 761 (such as 761 OT), 2045 (such as 2045G>C), 2070 (such as 2070OT), 2156 (such as 2156G>C or 2156G>A), 2339 (such as 2339G>C or 2339G>A), 2657 (such as 2657A>G), 3216 (such as 3216G>A), 4052 (such as 4052A>G), and/or 4245 (such as 4245OA) of SEQ ID NO: 2. In additional examples, the ATP7A mutation includes a deletion of nucleotides, such as Del4246-4260 of SEQ ID NO: 2 (nucleotides 132,676-132,690 of SEQ ID NO: 1) or Del4284-4315 of SEQ ID NO: 2 (nucleotides 134,748-134,779 of SEQ ID NO: 1), each of which results in a frame shift mutation and a premature termination codon. In other examples, the ATP7A mutation is located in an intron (for example within an intron, such as at or near a splice site). The mutation can be described by the nucleotide position relative to the intron splice site and the change in the nucleotide. For example, an IVS9,DS,+6T>G mutation in an ATP7A gene refers to a substitution of the T at the position six nucleotides from the intron 9 donor splice site for G. In some examples, an ATP7A mutation in an intron includes IVS8,AS,dup5 (duplication of the five nucleotides at the splice acceptor site of intron 8 (ATAAG); position 100,722-100,726 of SEQ ID NO: 1), IVS9,DS,+6T>G (mutation of the nucleotide six bases downstream of the intron 9 splice donor site from T to G; position 100,958 of SEQ ID NO: 1), and/or IVS21,DS,+3A>T (mutation of the nucleotide three bases downstream of the intron 21 splice donor site from A to T; position 132,716 of SEQ ID NO: 1).

In some examples, the ATP7A mutation results in the introduction of a premature stop codon. Examples of ATP7A mutations which result in introduction of a premature stop codon include point mutations that introduce a stop codon (such as Glnl97Ter and Arg201Ter) and frameshift mutations (such as an insertion, deletion, or an alteration in exon/intron splicing) which result in a premature stop codon downstream of the mutation (such as Del4284-4315 and IVS21,DS,+3A>T). In further examples, the ATP7A results in exon skipping (such as IVS21,DS,+3A>T, which results in skipping of exon 21). In additional examples, the ATP7A molecule is an ATP7A polypeptide or fragment thereof. The mutation can be described by the amino acid position at which the change exists and the change in the amino acid. For example, a Glnl97Ter mutation in an ATP7A protein refers to a substitution of the glutamine residue at amino acid 197 of an ATP7A protein for a termination (stop) codon. Similarly, an Ala629Pro mutation in an ATP7A protein refers to a substitution of the alanine residue at amino acid 629 of an ATP7A protein for a proline residue. In some examples, the ATP7A mutation includes a variant amino acid sequence at position(s) 197, 201, 629, 637, 666, 727, 833, 1019, 1304 and/or 1362 of SEQ ID NO: 3. In particular examples, the mutations in an ATP7A polypeptide include the mutations Glnl97Ter, Arg201Ter, Ala629Pro, Ser637Leu, Gly666Arg, Gly727Arg, Ser833Gly, GlylO19Asp, Asnl304Ser, and/or Ala 1362 Asp. Biological Samples The sample from a subject may be any, which is conveniently taken from the patient and contains sufficient information to yield reliable results. Typically, the sample will be a biological fluid or a tissue sample that contains, for example about 1 to about 10,000,000 cells. In one embodiment, the sample contains about 1000 to about 10,000,000 cells, or from about 1,000,000 to 10,000,000 somatic cells. It is possible to obtain samples which contain smaller numbers of cells (for example about 1 to about 1,000 cells) and then enrich the cells. In addition, with certain highly sensitive assays (such as reverse transcriptase polymerase chain reaction (RT-PCR)) or by use of whole genome amplification, it is possible for the sample size to be reduced down to single cell level. The sample need not contain any intact cells, so long as it contains sufficient biological material (for example a nucleic acid, such as DNA or RNA) to assess the presence or absence of a mutation or variant in nucleic acid molecules obtained from the subject.

The biological or tissue sample can be drawn from the tissue which is susceptible to the type of disease to which the detection test is directed. For example, the tissue may be obtained by surgery, biopsy, swab, or other collection method from the tissue of interest. In some examples, the tissue is skin cells, such as fibroblasts, obtained from a skin biopsy. In another example, the tissue is placenta. In addition, a blood sample, serum, skin scrape, buccal cell, urine, or a sputum sample can be used. In one embodiment, the biological sample is a blood or serum sample. The blood sample may be obtained in any conventional way, such as finger prick or phlebotomy. Suitably, the blood sample is approximately 0.1 to 20 ml, or from about 1 to 15 ml, or about 10 ml of blood. In another example the sample is a buccal cell sample obtained in any conventional way, such as by a cheek swab or an oral rinse. In another example, the sample can be previously isolated DNA. In one example, a sample from a subject is a placenta or a biopsy from a placenta which is associated with the affected or potentially affected individual (such as the fetal portion of the placenta, for example, the chorion, or the maternal portion of the placenta, for example, the decidua). The presence of at least one mutation in an ATP7 A molecule and the presence of at least one biochemical marker of abnormal copper metabolism may be determined in the same sample from the subject (such as one or more portions of a sample) or in more than one sample from the subject. In some examples, a first sample from the subject (such as, blood, serum, isolated cells, and/or saliva) is utilized to determine the presence of an ATP7 A mutation and a second sample from the subject (such as serum, plasma, CSF, isolated cells, and/or placenta) is utilized to determine the presence of one or more biochemical marker of abnormal copper metabolism. In a particular example, a blood sample from a subject is used for DNA analysis to determine the presence of at least one ATP7A mutation. A CSF or plasma sample from the same subject is used for catecholamine level determination in order to detect the presence of a biochemical marker of abnormal copper metabolism.

In other examples, the same sample from the subject is used to determine the presence of at least one ATP7 A mutation and the presence of at least one biochemical marker of abnormal copper metabolism. The sample may be divided into multiple portions prior to identifying an ATP7 A mutation and determining a biochemical marker of abnormal copper metabolism. In a particular example, the sample is a blood sample from a subject. The blood sample can be divided into two or more portions; at least one portion may be used to determine the presence of at least one ATP7A mutation (for example, by DNA analysis, RNA analysis, and/or protein analysis) and at least one other portion may be used to determine the presence of at least one biochemical marker of abnormal copper metabolism (such as by determining catecholamine levels). Mutation Detection in DNA

Methods of detecting mutations in a gene are well known in the art. In some examples, the presence of a mutation is detected in DNA from a subject. Southern hybridization is one method of identifying differences in sequences. In particular, Southern hybridization may be useful to identify the presence of large deletions in a gene of interest, such as ATP7A. Hybridization conditions, such as salt concentration and temperature can be adjusted for the sequence to be screened. Southern blotting and hybridization protocols are described, for example in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). Very high specific activity probe can be obtained using commercially available kits such as the Ready-To-Go DNA Labeling Beads (Pharmacia Biotech), following the manufacturer's protocol. Restriction enzyme polymorphism is an additional method of identifying differences in sequences. Restriction enzyme polymorphism allows differences to be established by comparing the characteristic polymorphic patterns that are obtained when certain regions of DNA are cut with various restriction enzymes. In one embodiment, the DNA is amplified prior to being cut with the restriction enzymes.

In one embodiment, an ATP7 A nucleic acid, or a portion thereof is amplified. Amplification of a selected, or target, nucleic acid sequence from an ATP7A gene or cDNA can be carried out by any suitable means (see for example Kwoh and Kwoh, Am Biotechnol Lab, 8: 14-25, 1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction (see for example Barany, Proc Natl Acad Sci USA 88: 189- 193, 1991), strand displacement amplification (see for example Walker et al., Nucleic Acids Res. 20: 1691-1696, 1992; Walker et al., Proc Natl Acad Sci USA 89:392-396, 1992), transcription-based amplification (see for example Kwoh et al., Proc Natl Acad Sci USA 86: 1173-1177, 1989), self-sustained sequence replication (or "3SR") (see for example Guatelli et al, Proc Natl Acad Sci USA 87: 1874-1878, 1990), the Q β-replicase system (see for example Lizardi et al., Biotechnology, 6: 1197-1202, 1988), nucleic acid sequence-based amplification (NASBA) (see for example Lewis, Genetic Engineering News, 12(9): 1-9, 1992), the repair chain reaction (RCR) (see for example Lewis, Genetic Engineering News, 12(9): 1-9,

1992), and boomerang DNA amplification (BDA) (see for example Lewis, Genetic Engineering News, 12(9): 1-9, 1992). In one specific non-limiting example, polymerase chain reaction is utilized. In another example, multiplex PCR is used. In some examples, the occurrence of an amplification product of a size different than that expected (such as a smaller product or a larger product than expected) indicates the presence of a mutation (such as a deletion, insertion, or duplication). Single strand polymorphism assay (SSPA) analysis and the closely related heteroduplex analysis methods can be used as effective methods for screening for single-base polymorphisms (Orita et al, Proc. Natl. Acad. ScL USA, 86:2766- 2770, 1989). In these methods, the mobility of PCR-amplified test DNA from clinical specimens is compared with the mobility of DNA amplified from normal sources by direct electrophoresis of samples in adjacent lanes of native polyacrylamide or other types of matrix gels. Single-base changes often alter the secondary structure of the molecule sufficiently to cause slight mobility differences between the normal and mutant PCR products after prolonged electrophoresis. In one example, the heteroduplex analysis method is denaturing high-pressure liquid chromatography (DHPLC). In another example, the heteroduplex analysis method is single strand polymorphism analysis.

In some examples, mutations in an ATP7A gene are detected by a sequential analysis. Multiplex PCR may be performed to detect deletions of one or more exons (such as the absence of one or more expected PCR products) and heteroduplex analysis (such as SSPA) can be performed to detect small mutations or alterations (such as one or more missense mutation, nonsense mutation, or small deletion or insertion). The presence of a mutation can be confirmed by direct DNA sequencing. These techniques may be performed in any order; in a particular example, a sample is analyzed by multiplex PCR, followed by heteroduplex analysis, followed by direct DNA sequencing. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (PCR Cloning Protocols, Methods in Molecular Biology, Vol. 67, 1997) and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or VENT® polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites (see also U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188). Specific embodiments involve in vitro amplification of an ATP7 A nucleic acid prior to detecting the ATP7 A mutation. By way of example, the ATP7 A nucleic acid is in some cases in vitro amplified using at least one oligonucleotide primer derived from an ATP7A-protein encoding sequence, such as the specific oligonucleotide primers listed herein. Other specific oligonucleotide primers comprise at least 15 contiguous nucleotides (such as about 15 to about 50 nucleotides, for example, about 20 to about 30 nucleotides) from SEQ ID NO: 1 or 2. In particular examples, such primers include a sequence that amplifies an exon, a portion of an exon, or the exon/intron boundaries of an ATP7A-encoding gene, such as represented by SEQ ID NOs: 4-59. Following in vitro amplification, the amplified nucleic acid fragments can be analyzed by size separation (such as agarose gel or polyacrylamide gel electrophoresis), by heteroduplex analysis (such as SSPA or DHPLC) and/or by direct nucleotide sequencing to determine the presence of at least one ATP7 A mutation. Mutation Detection in RNA

In some examples, the presence of a mutation is detected in RNA or mRNA from a subject. Northern hybridization is one method of identifying differences in sequences. In particular, Northern hybridization may be useful to identify the presence of large deletions in a gene of interest, such as ATP7 A. Hybridization conditions, such as salt concentration and temperature can be adjusted for the sequence to be screened. Northern blotting and hybridization protocols are described, for example in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). For amplification of mRNAs, it is within the scope of the present disclosure to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al. PCR Methods Appl. 4:80-84, 1994. AGLCR is a modification of GLCR that allows the amplification of RNA.

In some examples, deletions or splicing abnormalities in the ATP7A gene can be detected by RT-PCR. For example, mRNA from an individual can be amplified by RT-PCR using primers that amplify the full-length mRNA or at least a portion of the mRNA (such as a 5' portion or a 3' portion). The resulting PCR product is analyzed, for example by gel electrophoresis (such as agarose gel or polyacrylamide gel electrophoresis). The products can be compared to products from a normal control individual and the presence of products of different sizes from the control or the absence of a product indicates a deletion, insertion, or a splicing abnormality in the sample. Alternatively, the RT-PCR product can be analyzed by direct sequencing to determine the presence or absence of a mutation in a sample. Mutation Detection in Proteins In other methods of detection, the ATP7A molecule is an ATP7A protein, for instance an ATP7A protein comprising a sequence as shown in SEQ ID NO: 3, or variants thereof, such as those described herein. In examples of such methods, the ATP7A protein is detected by Western blot assay, ELISA, or immunohistochemistry. In the case of Western blotting, in some examples, the detected ATP7 A protein can be compared to an ATP7 A protein from a normal control individual and the presence of a protein of different size from the control or the absence of a protein indicates a deletion or insertion in the sample.

In particular examples, the Western blot, ELISA, or immunohistochemistry utilizes at least one ATP7A-specific antibody or a functional fragment thereof, for instance monoclonal antibodies or fragments of monoclonal antibodies.

Optionally, such monoclonal antibodies recognize an epitope of a mutant ATP7 A protein (such as an epitope of a mutant ATP7 A having an amino acid sequence) and not (or to a lesser extent) an epitope of wild type ATP7A, such as that shown in SEQ ID NO: 3. In particular methods, the antibody is reactive to an epitope including a mutant amino acid at position(s) 197, 201, 629, 637, 666, 727, 833, 1019, 1304 and/or 1362 of SEQ ID NO: 3.

IV. Biochemical Markers of Copper Metabolism

The methods disclosed herein are useful for identifying a subject likely to benefit from treatment with copper by determining the presence of at least one biochemical marker of abnormal copper metabolism in a sample from a subject (such as reduced serum or CSF copper level, reduced ceruloplasmin level, increased placental copper, altered plasma or CSF catecholamine levels, or reduced copper egress from isolated cells). Methods of detecting these biochemical markers of abnormal copper metabolism are well known in the art.

In some examples, a value for a biochemical marker of copper metabolism (such as copper level, ceruloplasmin level, placental copper level, catecholamine level, or cellular copper egress) in a sample from a subject is compared to a value for the same marker from a control sample (such as a reference value, a control population, or a control individual). Control samples are samples (such as blood, serum, plasma, CSF, placenta, tissue, or isolated cells) from an individual or individuals that are not affected by a disease that alters copper metabolism, such as Menkes disease or OHS (or any other disease that alters copper metabolism, for example Wilson disease).

In some examples, the biochemical marker of copper metabolism in a sample from a subject is compared to a value obtained from a control sample from a single individual. In other examples, the biochemical marker of copper metabolism in a sample from a subject is compared to a value obtained from a control population, such as control samples from more than one individual (such as two or more individuals, five or more individuals, ten or more individuals, or even 100 or more individuals). In the case of a control population, the value of the sample from the subject can be compared to the mean of the control population values or to the range of the values from the control population. In further examples, the biochemical marker of copper metabolism in a sample from a subject is compared to a reference value, such as a standard value obtained from a population of normal individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval). Biological Samples

The sample from a subject may be any, which is conveniently taken from the patient and contains sufficient information to yield reliable results, such as a biological or tissue sample. The biological or tissue sample can be drawn from the tissue which is susceptible to the type of disease to which the detection test is directed. For example, the tissue may be obtained by surgery, biopsy, swab, or other collection method from the tissue of interest. In some examples, the tissue is skin cells, such as fibroblasts, obtained from a skin biopsy. In another example, the tissue is placenta. In addition, a blood sample, plasma, serum, skin scrape, cerebrospinal fluid (CSF), urine, or a sputum sample can be used. In one embodiment, the biological sample is a blood or serum sample. The blood sample may be obtained in any conventional way, such as finger prick or phlebotomy.

Suitably, the blood sample is approximately 0.1 to 20 ml, or from about 1 to 15 ml, or about 10 ml of blood. In another example, the sample is CSF, such as fluid collected from a spinal tap. In one example, a sample from a subject is a placenta or a biopsy from a placenta which is associated with the affected or potentially affected individual (such as the fetal portion of the placenta, for example, the chorion).

The presence of at least one mutation in an ATP7 A molecule and the presence of at least one biochemical marker of abnormal copper metabolism may be determined in the same sample from the subject (such as one or more portions of a sample) or in more than one sample from the subject. In some examples, a first sample from the subject (such as, blood, serum, isolated cells, and/or saliva) is utilized to determine the presence of an ATP7 A mutation and a second sample from the subject (such as serum, plasma, CSF, isolated cells, and/or placenta) is utilized to determine the presence of one or more biochemical marker of abnormal copper metabolism. In a particular example, a blood sample from a subject is used for DNA analysis to determine the presence of at least one ATP7A mutation. A CSF or plasma sample from the same subject is used for catecholamine level determination in order to detect the presence of a biochemical marker of abnormal copper metabolism. In other examples, the same sample from the subject is used to determine the presence of at least one ATP7 A mutation and the presence of at least one biochemical marker of abnormal copper metabolism. The sample may be divided into multiple portions prior to identifying an ATP7 A mutation and determining a biochemical marker of abnormal copper metabolism. In a particular example, the sample is a blood sample from a subject. The blood sample can be divided into two or more portions; at least one portion may be used to determine the presence of at least one ATP7A mutation (for example, by DNA analysis, RNA analysis, and/or protein analysis) and at least one other portion may be used to determine the presence of at least one biochemical marker of abnormal copper metabolism (such as by determining catecholamine levels). Copper Levels

One biochemical marker of copper metabolism is the level of copper in a sample from a subject (such as serum, plasma, or CSF). In some examples, reduced copper level as compared to a normal control individual or normal control population is a marker of abnormal copper metabolism.

Methods of determining copper levels in a sample (such as serum, plasma, or CSF) are well known to one of skill in the art. In some examples, methods for determining copper levels in a sample include flame atomic absorption spectrometry, anodic stripping voltammetry, graphite furnace atomic absorption, electrothermal atomic absorption spectrophotometry, inductively coupled plasma- atomic emission spectroscopy, and inductively coupled plasma-mass spectrometry. See, e.g. Evenson and Warren, Clin. Chem. 21 :619-625, 1975; Weinstock and Uhlemann, Clin. Chem. 27: 1438-1440, 1981; WO 93/017321. In a particular example, copper levels are determined by electrothermal atomic absorption spectrophotometry.

In particular examples, reduced copper levels in a sample from a subject as compared to a normal control individual or population or reference value are a biochemical marker of abnormal copper metabolism. In some examples, copper levels are reduced by about 10% to about 95%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to a normal control sample or population or a reference value. In some examples, values of serum copper level for a normal control population are about 5 μg/dl to about 45 μg/dl in newborns and about 70 μg/dl to about 150 μg/dl in adults. Ceruloplasmin Levels

Ceruloplasmin is the major copper-carrying protein in the blood. This protein has ferroxidase and amine oxidase activity and catalyzes the enzymatic oxidation of p-phenylenediamine (PPD) and Fe(II). Levels of ceruloplasmin in a sample from a subject (such as serum, plasma, or CSF) are a biochemical marker of copper metabolism. In some examples, reduced ceruloplasmin level as compared to normal control sample or population or a reference value is a marker of abnormal copper metabolism.

Methods of determining ceruloplasmin levels in a sample (such as serum, plasma, or CSF) are well known to one of skill in the art. In one example, ceruloplasmin levels in a sample are determined by measuring ceruloplasmin oxidase activity (such as PPD-oxidase activity or ferroxidase activity). See, e.g., Sunderman and Nomoto, Clin. Chem. 16:903-910, 1970). The rate of formation of oxidation product is proportional to the concentration of serum ceruloplasmin (with a correction for non-enzymatic oxidation of substrate). In another example, ceruloplasmin levels in a sample are determined by immunoassay, such as ELISA, dissociation-enhanced time-resolved fluoroimmunoassay, or turbidimetric immunoassay (see, e.g., U.S. Pat. Nos. 6,806,044; 6,010,903; 5,491,066). In a further example, ceruloplasmin levels are determined by purifying ceruloplasmin and analyzing the copper content using inductively coupled plasma mass spectroscopy to provide a copper ion specific signal; and the sample is evaluated for ceruloplasmin based on the copper ion specific signal (see, e.g., U.S. Pat. Publication No. 2007/0161120).

In particular examples, reduced ceruloplasmin levels in a sample from a subject as compared to a normal control sample or population or a reference value are a biochemical marker of abnormal copper metabolism. In some examples, ceruloplasmin levels are reduced by about 10% to about 95%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% as compared to a normal control sample or population or a reference value. In some examples, values of serum ceruloplasmin level for a normal control population are about 5 mg/dl to about 25 mg/dl in newborns and about 15 mg/dl to about 50 mg/dl in adults. Placental Copper Levels

In pregnancies affected by a mutation in ATP7A in the fetus, reduced copper export by placental cells results in placental copper accumulation and low hepatic copper levels in the fetus. In some examples, increased placental copper level as compared to a normal control sample or population or a reference value is a marker of abnormal copper metabolism. Methods of determining copper levels are well known in the art and are described above. Placental copper levels are determined utilizing a small tissue biopsy sample from a placenta. In some examples, placental copper is increased by about 50% to about 10-fold, such as about 50%, about 75%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9- fold, about 10-fold as compared to a normal control individual or population or a reference value. Catecholamine Levels

Copper is required for activity of metabolic enzymes (such as dopamine β hydroxylase, lysyl oxidase, and cytochrome c oxidase). For example, copper is required for the activity of the enzyme dopamine β hydroxylase (DBH), which converts dopamine to norepinephrine. In some examples, catecholamine levels (such as plasma or CSF catecholamine levels) are correlated with DBH activity. A portion of the metabolic pathway of dopamine and norepinephrine is shown below.

If there is decreased copper available (such as in Menkes disease or OHS), activity of DBH is decreased, resulting in decreased norepinephrine levels and increased dopamine levels. Substrates and metabolites of dopamine and norepinephrine are also affected.

Catecholamine levels can be informative to changes in copper metabolism. As the activity of one or more enzymes (such as DBH) change, the ratio of substrate metabolites used to product metabolites can indicate the direction of the change. In some examples, increases in enzyme activity will be reflected by decreases in the ratio of one or more substrates to its products. Likewise, decreases in enzyme activity will be reflected by increases in the ratio of one or more substrates to its product (such as the ratio of dopamine to norepinephrine). These ratios can be ratios of two metabolites or ratios of complex relationships among metabolites. Further, the metabolites do not need to be direct product- substrate metabolites of specific enzymes (or a specific enzyme), but can be ratios of any two or more metabolites (such as the ratio of DHPA to DHPG). In some examples the assessment of catecholamine levels in a sample from a subject is compared to an assessment of catecholamine levels from a normal control sample or population or a reference value.

In some examples, the quantity of one or more catecholamines (reported in either an absolute or relative concentration, or a ratio thereof) is correlated to DBH activity. Thus, some of the provided methods involve quantifying one or more catecholamine in a biological sample from a subject. Catecholamines include compounds that include a catechol group (such as the classical catecholamines, for example, dopamine, norepinephrine, and epinephrine). In additional examples, catecholamines include metabolites or substrates of the classical catecholamines. In some examples, catecholamines include metabolites of dopamine (for example, DHPA, 3-methoxytyramine, and homovanillic acid) and metabolites of norepinephrine (for example, DHPG, normetanephrine, 3,4-dihydroxymandelic acid, 3-methoxy-4-hydroxymandelic acid, and 3-methoxy-4- hydroxyphenylethylene glycol). Methods of determining catecholamine levels are well known to one of skill in the art. These methods include fluorometric, radioenzymatic, immunologic (such as radioimmunoassay), electrophoretic, electrochemical (such as fast scan cyclic voltammetry) and chromatographic (such as high pressure liquid chromatography (HPLC)) methods. See, e.g., Raum, Am. J. Physiol. Endocrinol. Metab. 247:E4-E12, 1984; Robinson et al., Clin. Chem., 49: 1763-1773, 2003;

Holmes et al, J. Chromatogr. B Biomed. Appl. 653: 131-138, 1994. In a particular example, catecholamine levels are determined by reverse-phase liquid chromatography with electrochemical detection (see, e.g., Eisenhofer et al, Clin. Chem. 32:2030-2033, 1986). In one example, the catecholamine level is correlated with decreased DBH activity. In particular examples, catecholamines include dopamine, norepinephrine, dihydroxyphenylacetic acid (DHPA), dihydroxyphenylglycol (DHPG), or a combination of two or more thereof. For example, decreased DBH activity can be reflected by an increase in the substrate of the enzyme (such as dopamine) as compared to a normal control sample or population or a reference value (for example, an increase of about 50% to about 25-fold, such as about 50%, about 75%, about 2-fold, about 3 -fold, about 4-fold, about 5-fold, about 10-fold or about 20-fold compared to a control). Decreased DBH activity can also be reflected by a decrease in the product of the enzyme (such as norepinephrine) as compared to normal control sample or population or a reference value (for example, a decrease of about 10% to about 95%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to a control). In other examples, decreased DBH activity can be reflected by an increase in a metabolite of the substrate of the enzyme (such as DHPA, 3-methoxytyramine, and homovanillic acid) as compared to a normal control sample or population or a reference value (for example, an increase of about 50% to about 10-fold, such as about 50%, about 75%, about 2-fold, about 3- fold, about 4-fold, about 5-fold, or about 10-fold compared to a control). In some examples, decreased DBH activity can be reflected by a decrease in a metabolite of the product of the enzyme (such as DHPG, normetanephrine, 3,4- dihydroxymandelic acid, 3-methoxy-4-hydroxymandelic acid, and 3-methoxy-4- hydroxyphenylethylene glycol) as compared to a normal control sample or population or a reference value (for example, a decrease of about 10% to about 95%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to a control).

In additional examples, a ratio of catecholamine levels correlates with decreased DBH activity. Decreased DBH activity can be reflected by an increase in the ratio of a substrate of the enzyme (such as dopamine) to a product of the enzyme (such as norepinephrine) as compared to a normal control sample or population or a reference value. In some examples, decreased DBH activity is correlated to an increase in the ratio of dopamine to norepinephrine compared to a normal control sample or population or a reference value (such as an increase of about 2-fold to about 50-fold, such as about 2-fold, about 5 -fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold or about 50-fold). Decreased DBH activity can also be reflected by a change in the ratio of catecholamine metabolites. In one example, decreased DBH activity is correlated to an increase in the ratio of DHPA to DHPG compared to a normal control sample or population or a reference value (such as an increase of about 2-fold to about 20- fold, such as about 2-fold, about 3 -fold, about 4-fold, about 5 -fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, or about 20- fold). In another example, decreased DBH activity is correlated to an increase in the ratio of DOPA to DHPG compared to a normal control sample or population or a reference value (such as an increase of about 2-fold to about 20-fold, such as about 2-fold, about 3 -fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, or about 20-fold). Cellular Copper Egress

ATP7A is responsible for the transport of copper across the plasma membrane from the cell cytoplasm to the external environment. As a result, cells expressing an ATP7A with reduced or absent copper transport activity accumulate copper inside the cell and exhibit reduced cellular copper egress. In some examples, reduced cellular copper egress can be used as a biochemical marker of abnormal copper metabolism.

Methods for determining cellular copper egress are well known in the art. Classically, egress of radiolabeled copper is measured in pulse-chase experiments utilizing isolated cells (such as isolated fibroblast or lymphoblast cells), such as cells from an individual having Menkes disease or OHS. See, e.g., La Fontaine et al, J. Biol. Chem. 273:31375-31380, 1998; Goka et al, Proc. Natl. Acad. ScL USA 73:604-606, 1976. In some examples, cells (such as fibroblast or lymphoblast cells) are incubated with radioisotopic copper (such as ⁶⁴Cu or ⁶⁷Cu) and the cells are allowed to take up the copper. Uptake of copper by the cells can be determined by measuring the radioisotope in the cells (such as by scintillation counting). Once the cellular copper is in equilibrium with the extracellular copper, copper egress can be measured (such as loss of copper from the cells in a period of time). In other examples, cells are cultured in standard medium and total intracellular copper is measured in isolated cells, for example by atomic absorption spectroscopy. Reduced cellular copper egress can be reflected by increased copper accumulation of cells (such as cells expressing a mutant ATP7A protein, for example cells from an individual with Menkes disease or OHS) as compared to a normal control sample or population or a reference value (for example cells expressing a wild type ATP7A protein, such as cells from a healthy individual). Accumulation of copper is the amount of copper accumulated in cells following a period of incubation (such as in standard medium containing unlabeled copper or in medium containing added radioisotopic copper). In some examples, cells expressing a mutant ATP7A protein, such as the mutants described herein, have increased copper accumulation as compared to normal control sample or population or a reference value, such as an increase of about 50% to about 3-fold, for example, about 50%, about 60%, about 70%, about 80%, about 90%, about 2- fold, about 2.5-fold, or about 3-fold. Reduced cellular copper egress can also be reflected by increased copper retention of cells (such as cells expressing a mutant ATP7A protein, for example cells from an individual with Menkes disease or OHS) as compared to a normal control sample or population or a reference value (for example cells expressing a wild type ATP7A protein, such as cells from a healthy individual). Copper retention is the amount of copper remaining in cells (for example cells labeled with radioisotopic copper) following a period of time in medium lacking the radioisotopic copper. In some examples, cells expressing a mutant ATP7A protein, such as the mutants described herein, have increased copper accumulation as compared to a normal control sample or population or a reference value, such as an increase of about 30% to about 40-fold, for example, about 30%, about 50%, about 70%, about 90%, about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, or about 40-fold.

The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1

Detection of ATP7A Mutations in Subjects with Menkes Disease and Occipital

Horn Syndrome This example describes methods for detection of ATP7A mutations in individuals with Menkes disease or OHS. Methods

81 infants who were at risk for Menkes disease because of a positive family history or suggestive clinical or biochemical findings were evaluated. A multiplex PCR assay was used to screen for large deletions in the ATP7A gene. Oligonucleotide primers for PCR amplification (Table 1) were designed from the published sequence of the human Menkes/OHS gene (Dierick et al., Genomics 28:462-469, 1995; Turner et al., Genomics 26:437-442, 1995). All 23 exons of the ATP7A gene were amplified in eight multiplex PCR reactions A-H (Table 2). Each multiplex PCR reaction contained a final concentration of IX optimized PCR buffer D (Invitrogen), 500 nM each PCR primer, 200 μM dNTP, and 1.5 U Taq DNA polymerase (Life Technologies Inc.). A total of 200 ng of genomic DNA was added to the total volume of 50 μl multiplex PCR reaction. These PCR reactions were then denatured for 5 min at 95°C in a Perkin-Elmer 9700 thermocycler; followed by 30 cycles of 95°C for 30 sec, 55°C for 45 sec, and 72°C for 2 min; with a final extension of 72°C for 7 min. The products were electrophoresed on a 2% agarose gel. The gel was stained in ethidium bromide and photographed under ultraviolet light, and deletions were identified by the absence of expected bands.

Each of the 23 ATP7A exons and flanking sequences were amplified from genomic DNA and subjected to heteroduplex analysis as a screen to identify small alterations in patients whose multiplex PCR pattern was normal. PCR amplification was performed under the following conditions using a Perkin-Elmer 9700 thermocycler: 95°C for 5 min; 30 cycles at 95°C for 30 sec, 62°C for 45 sec, and 72°C for 90 sec, and a final extension of 72°C for 7 min. Aliquots of PCR products were first electrophoresed on agarose gels to confirm that each reaction contained a single band of the expected size. Five μl aliquots of PCR products from patients were then mixed with an equal volume of PCR product from a normal male control, heated at 95°C for 4 min, and slowly cooled to room temperature. The samples were then electrophoresed on a IX MDN™ gel (FMC) in 1% TBE buffer at 500 volts for 6 h on a vertical single adjustable slab gel system (CBC Scientific). The gel was subsequently stained with ethidium bromide, and conformers were identified and photographed under ultraviolet light. (OS ON (JI 03S) 9X39X9XXXXVXXXX3XX9VV33XXVX pjBΛUOj 6 T uoxg 6 T uoxa ON αi όas) xvvxxvvvxvvvxxvvvvvDXDXDxggvgv 9SJ9Λ9-g g x uoxg

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aDue to their large sizes, exons 3 and 4 were amplified in several fragments for heteroduplex analysis.

Results

This serial approach to mutation detection identified molecular alterations in twelve subjects (Table 3). This assay was designed by grouping together exons from the ATP7A gene based on PCR product size, to enable simultaneous visualization after electrophoresis through agarose gels. Alterations detected by heteroduplex analysis were confirmed by DNA sequencing.

Mutation analysis was also performed in an infant with a family history of Menkes disease (a prior affected sibling). The infant underwent a biochemical evaluation in the immediate postnatal period and was diagnosed as having Menkes disease based on elevated placental copper and abnormal plasma and cerebrospinal catecholamine levels. Mutation analysis identified a mutation that introduced a premature stop codon (Arg201Ter). Mutation analysis of an additional infant with no family history of Menkes disease, but low serum copper at 6 months of age identified a mutation of Gly727Arg.

Example 2

Preparation and Administration of Copper Histidine This example describes preparation of copper histidine for use in treating individuals diagnosed with Menkes disease or OHS and administration to subjects. Copper histidine was prepared by dissolving 1.345 g CuCl₂ dihydrate in about 500 ml water which had been bubbled with nitrogen for about 20 minutes and dissolving 2.45 g L-histidine in about 500 ml water which had been bubbled with nitrogen for about 20 minutes. The CuCl₂ and L-histidine solutions were mixed, pH was adjusted to 7.30-7.40 with 0.1 N NaOH, and final volume was adjusted to 1000 ml. The solution was filtered through a 0.22 micron filter

(Sterivex-GV; Millipore, Billerica, MA, aliquoted gravimetrically 2 g (2 ml) into 5 ml vials, and freeze-dried (Virtis; Gardiner, NY). Vials were stoppered under vacuum and stored at -2O°C until use.

For injection, the contents of the vial were reconstituted with 2.0 ml of preservative-free, 0.9% Sodium Chloride Injection, USP. When reconstituted, each ml contained copper histidine equivalent to 500 μg copper (II) as a clear blue solution. Starting dose was 500 μg/day in two divided subcutaneous doses for infants less than 12 months of age. Patients older than one year of age received a starting dose of 250 μg per day as a single subcutaneous injection. Example 3 Analysis of Plasma Neurochemical Levels in At-Risk Infants

This example describes analysis of several plasma neurochemical markers in a group of infants at risk of Menkes disease. Methods

81 infants who were at risk for Menkes disease because of a positive family history or suggestive clinical or biochemical findings were evaluated. Follow-up included physical examination and neurodevelopmental assessment, or communication with referring physicians, other health care providers, or the infants' parents.

Plasma catecholamine levels were determined by high performance liquid chromatography with electrochemical detection, as described in Eisenhofer et al, Clin. Chem. 32:2030-2033, 1986 (incorporated herein by reference).

Three paired comparisons were made of mean neurochemical levels and ratios: all affected versus all unaffected infants (46 and 35 infants, respectively), older (>1 month) affected versus older unaffected infants (32 and 13 infants, respectively), and newborn (<1 month) affected versus newborn unaffected infants (14 and 22 infants, respectively). Group means were compared by nonparametric Wilcoxon rank-sum tests. A two tailed P value less than 0.05 was considered to indicate statistical significance. Receiver-operating-characteristic (ROC) curves were determined for plasma dopamine, plasma dihydroxyphenylacetic acid, the ratio of dopamine to norepinephrine, and the ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol as independent diagnostic tests for Menkes disease in the 36 at-risk newborns (<1 month of age at screening). A ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol of 4.0 or less was predefined as a negative test result and a value of more than 4.0 was predefined as a positive test result.

Results

The mean plasma concentrations of dihydroxyphenylacetic acid (DHPA), dopamine, norepinephrine, and dihydroxyphenylglycol (DHPG) differed between infants at risk in whom the Menkes disease phenotype did not develop (35 infants) and those subsequently determined to have Menkes disease according to clinical, biochemical (low serum copper and ceruloplasmin at >3 months of age), or molecular criteria (46 infants) (P<0.001 for all comparisons) (Table 4). In affected as compared with unaffected patients, dopamine and dihydroxyphenylacetic acid levels were higher and norepinephrine and dihydroxyphenylglycol levels were lower. The ratios of dopamine to norepinephrine and dihydroxyphenylacetic acid to dihydroxyphenylglycol differed between affected and unaffected infants (P<0.001 for both comparisons).

Among at-risk newborns 1 month old or less, a scatter plot of dopamine-to- norepinephrine versus dihydroxyphenylacetic acid to dihydroxyphenylglycol ratios (Fig. IA) clearly distinguished affected from unaffected infants. ROC curves show the relationship between true positive and false positive rates for a test across various threshold values used to diagnose a condition. ROC curve analyses showed a C statistic of 1.0 for plasma dopamine and 0.96 for plasma dihydroxyphenylacetic acid (Fig. IB), indicating high sensitivity and specificity. Example 4 Copper Histidine Treatment of Infants with Menkes Disease

This example describes treatment of infants diagnosed with Menkes disease, neurological outcomes, and correlation with ATP7A genotype. Methods

Twelve of the infants described in Example 3 who met eligibility criteria (1 month of age or less with no neurologic symptoms) were enrolled in a clinical trial of early copper treatment. Written informed consent was obtained from parents. The study drug was copper histidine, prepared and administered as described in Example 2. Clinical and biochemical follow-up occurred every six months. Eight patients received treatment for 3 years; one patient, who died during the trial, received treatment for 1.6 years; and three patients under 3 years of age are still being treated. In a historical control group of 15 late-diagnosis patients (mean age±SD at diagnosis, 163±113 days; range, 42 to 390) also treated with the same copper regimen, identification was based on classic clinical and biochemical findings (low serum copper and ceruloplasmin after the age of 3 months). The interval from diagnosis to initiation of copper treatment in these patients varied. Five patients received treatment for 3 years, and 10 died before 3 years of treatment could be completed. The mean length of treatment for the latter patients was 12.2 months (range, 4.0 to 18.0).

ATP7 A mutation analysis was performed as described in Example 1. Baseline and follow-up electroencephalograms were obtained for the 12 infants enrolled in the early treatment trial. Most patients underwent four or more electroencephalograms. Tracings were considered abnormal if any of the following findings were present: background slowing or disorganization, spike waves or polyspikes, or diffuse spike and slow-wave complexes. Magnetic resonance scans of the brain were performed using Tl- and T2-weighted techniques as well as flow-attenuated inversion recovery. Patients underwent up to three scans, which were evaluated for the presence of structural brain abnormalities and progression of white-matter myelination. Denver Developmental Screening Test II was used to track neurodevelopment in four areas. These were gross motor, language, fine motor-adaptive, and personal-social. Results

Of fourteen newborn infants with high ratios of both dopamine to norepinephrine and DHPA to DHPG, twelve were enrolled in a clinical trial of daily copper injections. Table 5 summarizes the mutations and post-treatment biochemical data for the 12 patients in the clinical trial. Ten different ATP7A mutations were identified in the 12 enrolled patients. The serum copper levels achieved in the treated infants were higher than those in infants with untreated Menkes disease in all but two patients (Patients 2 and 11). All patients tested showed increased levels of urinary β2-microglobulin, a sensitive marker of renal tubular damage.

Survival among the cohort receiving early diagnosis and treatment (mean age at first treatment, 10±4 days) was compared with that among a historical control group of 15 patients with Menkes disease who received a diagnosis and were treated later in infancy (mean age at diagnosis, 163±113 days). Survival was 92% after a median follow-up of 4.6 years among patients treated with copper early and 13% after a median follow-up of 1.8 years among the historical control group of patients who were treated late.

Electroencephalographic abnormalities were detected in 7 of 12 patients (58%), although only two had evidence of clinical seizures. Abnormal findings included background slowing or disorganization, focal slowing (mainly in the posterior head region), focal spike waves or polyspikes (predominantly in the occipital region), and diffuse spike and slow-wave complexes. Distinct abnormalities on brain magnetic resonance imaging (MRI), including cortical cerebral and cerebellar atrophy and delayed myelination, were evident in six of nine patients studied. Two patients (Patients 5 and 8) had normal serial brain MRI scans. There was a range of neurodevelopmental outcomes in the early-treatment cohort (Fig. 2). Two patients had completely normal neurodevelopment during the 3 -year treatment period and beyond. At ages 7 and 5 years, they had normal neurologic function, were bright and active children, and did not require special education. The mutations associated with these outcomes are IVS9,DS,+6T→G (Patient 5) and G666R (Patient 8). An unrelated patient with the G666R mutation (Patient 4) had a less successful clinical response (Fig. 2A), although his neurodevelopmental achievements exceeded those attained by his deceased maternal relative. At his age 8 years, he could ride a tricycle, walk with support, and was very socially interactive. He showed no distinctive electroencephalographic or brain MRI abnormalities. In contrast, patients with large or small deletions that disrupted the ATP7A translational reading frame (Patients 1, 2, 3, 6, 7, 9, 11, and 12) and one with a mutation that introduced a premature termination (or nonsense) codon (Patient 10) had less dramatic responses to early treatment, and one died (Patient 1).

Example 5

Measurement of Biochemical Markers of Copper Metabolism This example describes representative methods for measuring copper levels, ceruloplasmin levels, and cellular copper egress, which are biochemical markers of copper metabolism. Copper Levels

Copper levels in placenta and brain tissues are determined employing electrothermal atomic absorption spectrophotometry (ETAAS) equipped with a Zeeman-background corrector (Model AAnalyst 800, Perkin-Elmer, New Jersey). Specimens are prepared in acid-washed plastic labware and digested in a mixture of 1 mL of 70% HNO₃ (Ultrex, JT Baker), 1 mL of 30% H₂O₂ (JT Baker), and 1 mL deionized water. Experimental measurements are compared using certified standard reference material controls from the National Institute of Standards and Technology (Gaithersburg, MD). Tissue copper levels are calculated as micrograms of the element per gram of dry tissue.

Copper levels in serum or cerebrospinal fluid are determined via atomic absorption spectrometry in a graphite furnace (Model AAnalyst 800). Prior to analysis, samples are thawed from storage at -70°C to room temperature. Samples are diluted twofold with analytical grade 0.1% HNO₃ (JT Baker). Deionized water from a Milli-Q system (Millipore) is used in the analyses. The method for determining the calibration curve (linear, through zero) uses certified standard reference materials at 2, 5, and 10 ppb from High Purity Standards (Charleston, SC). Copper levels are calculated in μg per liter, equivalent to parts-per-billion. Two dilutions are made for each specimen, and each dilution is measured in triplicate.

Elevated placental copper and/or reduced serum or CSF copper level as compared to a control sample or population confirm the diagnosis of Menkes disease.

Ceruloplasmin Levels

Ceruloplasmin levels in serum are determined by immunoassay, such as ELISA or nephelometry as described in Lockitch et ah, Clin. Chem. 34: 1618-1621, 1988. Reduced ceruloplasmin level as compared to a control sample or population confirms the diagnosis of Menkes disease. Cellular Copper Egress

Fibroblast skin cells at low passage number (fewer than ten) established from patients with clinical and/or biochemical features of Menkes disease and normal fibroblast cell lines (such as GM5400, GM8398, and GM9503, Coriell Institute) are grown to confluence in 75 cm² flasks. Cells are rinsed once with 10 ml DMEM without additives and then pulsed for 21 hours with 10 μCi ⁶⁴Cu(NO₃)₂ (peak specific activity 0.82 mCi/mg, McMaster University Nuclear Reactor, Hamilton, ON) in 10 ml DMEM with 2 mM glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin and without fetal bovine serum. Following removal of pulse medium, cultures are rinsed twice with DMEM prior to addition of chase medium (DMEM with 10% fetal bovine serum, and antibiotics and glutamine as above). After a 25 hour chase period, cells are harvested by trypsinization. The centrifuged cell pellets are resuspended in 500 μl phosphate buffered saline and sonicated on ice for 30 seconds. Radioactivity is determined using triplicate 50 μl aliquots from each sample with a model 1219 Rack Beta liquid scintillation counter (Pharmacia LKB Technology). Protein is determined by bicinchoninic acid method with bovine serum albumin as standard (Pierce). Fibroblasts from patients with clinical and biochemical features of Menkes disease have increased copper retention as compared with the normal fibroblast cell lines.

Example 6 Measurement of ATP7A Activity

This example describes representative methods for measuring ATP7A activity by a yeast complementation assay.

Wild type Saccharomyces cerevisiae strains (such as strain BY4743) and a copper transport mutant (Δccc2, such as strain YDR270W) are used. The genotypes, transformation procedures, and growth conditions are those reported previously (Tang et al., Genet. Med. 8:711-718, 2006). All cultures are grown to saturation in conventional synthetic medium (YPD) and washed three times with sterile, deionized ice-cold water. For complementation assays, washed cells are resuspended in induction medium containing 2% galactose (Sigma) and 1% raffinose and 75 μg/ml Blasticidin (SC), and incubated at 30°C by shaking at 220 rpm for 16 h. The cells are harvested, washed, resuspended in copper/iron-limited medium and then cultured for 16 hours. Cells are washed three times in sterile, deionized water, diluted to an OD₆₀₀ of 0.1 and streaked onto experimental plates. All plates are incubated at 3O°C for 48 hrs and photographed. To assess the residual activity of copper transport as a percentage of normal, Δccc2 strains transfected with mutant and wild-type human ATP 7 A, mock transfected Δccc2 and non-transfected Δccc2 are grown overnight in copper/iron limited media and diluted to an OD₆₀₀ of 0.1 in triplicate 10 ml cultures. Aliquots (800 μl) are withdrawn after 0, 2 and 4 hours of growth at 30°C. To adjust for background, the mean of triplicate OD₆₀₀ absorbance for Δccc2 is subtracted from those of the mutant and WT-transfected strains at each time point. Growth in copper/iron-limited media is assessed at each time point by comparison to growth by the ATP7A wild type-transformed strain (OD₆₀₀ mutant/ OD₆₀₀wild type x 100). The mean value at 0, 2, and 4 hours is calculated as an estimate of functional copper transport activity.

Example 7 Identification of Subjects Likely to Benefit from Copper Treatment

This example describes methods of identifying a subject likely to benefit from copper treatment by determining the presence of at least one ATP7 A mutation and at least one biochemical marker of abnormal copper metabolism in one or more samples from the subject. Individuals to be tested are identified based on a positive family history of

Menkes disease or OHS and/or suggestive clinical findings (for example, characteristic hair phenotype, lax skin, pectus excavatum, and/or umbilical or inguinal hernia).

One or more biological samples are collected from the subject for ATP7A mutation analysis and analysis of marker(s) of copper metabolism. If available, a sample of the placenta is collected at the time of birth for analysis of placental copper levels.

Peripheral blood is collected from the subject for ATP7A mutation analysis and genomic DNA is isolated according to standard procedures (such as Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, WI)). The presence of one or more ATP7A mutations is identified as described in Example 1.

Plasma samples are obtained by collecting peripheral blood and separating the plasma by refrigerated centrifugation. The peripheral blood used for plasma preparation may be a portion of the sample collected for DNA isolation and mutation analysis, or may be a separately collected sample. Plasma samples are stored at -70°C for up to one month prior to assaying for catecholamine levels as described in Example 3. Alternatively, CSF is obtained by lumbar puncture, immediately frozen on dry ice and stored at -70°C for up to one month prior to assaying for catecholamine levels as described in Example 3. The ratio of dopamine levels to norepinephrine levels and/or the ratio of DHPA levels to DHPG levels are determined.

Individuals with an ATP7A mutation of Glnl97Ter; Arg201Ter; Ala629Pro; Ser637Leu; Gly666Arg; Gly727Arg; Ser833Gly; GlylO19Asp;

Asnl304Ser; Alal362Asp; IVS8,AS,dup5; IVS9,DS,+6T>G; IVS21,DS,+3A>T; Del4246-4260 and/or Del 4284-4315 and with an increased ratio of dopamine to norepinephrine compared to a normal control sample or population or a reference value and/or an increased ratio of DHPA to DHPG compared to a normal control sample or population or a reference value are immediately treated with daily injections of copper.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for identifying a subject as a candidate for copper treatment, comprising determining the presence of at least one mutation in a copper transporting, alpha polypeptide ATPase (ATP7A) molecule in a sample from the subject, wherein the mutation is selected from the group consisting of Gly727Arg; Glnl97Ter; Arg201Ter; Ala629Pro; Ser637Leu; Gly666Arg; Ser833Gly; GlylO19Asp; Asnl304Ser; Alal362Asp; IVS8,AS,dup5; IVS9,DS,+6T>G; IVS21,DS,+3A>T; Del4246-4260; and Del 4284-4315; and determining the presence of at least one biochemical marker of abnormal copper metabolism in a sample from the subject, wherein the presence of at least one mutation in the ATP7 A molecule and at least one biochemical marker of abnormal copper metabolism indicates the subject is a candidate for copper treatment.

2. The method of claim 1, wherein the copper treatment comprises administering a therapeutically effective amount of copper to the subject.

3. The method of claim 2, wherein the copper comprises copper histidine, copper chloride, or copper sulfate. 4. The method of claim 1, wherein the subject has Menkes disease or occiptal horn syndrome.

5. The method of claim 1, wherein the sample from the subject comprises one or more of blood, serum, plasma, isolated cells, saliva, cerebrospinal fluid, or placenta. 6. The method of claim 1, wherein the biochemical marker of abnormal copper metabolism comprises one or more of copper level, ceruloplasmin level, placenta copper level, catecholamine level, or cellular copper egress.

7. The method of claim 6, wherein the biochemical marker of abnormal copper metabolism is copper level, and wherein reduced copper level as compared to a reference value of copper is an indicator that the subject is a candidate for copper treatment.

8. The method of claim 7, wherein the copper level comprises serum copper level or cerebrospinal fluid copper level.

10. The method of claim 6, wherein the biochemical marker of abnormal copper metabolism is ceruloplasmin level, and wherein reduced ceruloplasmin level as compared to a reference value of ceruloplasmin is an indicator that the subject is a candidate for copper treatment. 11. The method of claim 10, wherein the ceruloplasmin level comprises serum ceruloplasmin level.

12. The method of claim 6, wherein the biochemical marker of abnormal copper metabolism is placenta copper level, and wherein increased placenta copper level as compared to a reference value of placenta copper is an indicator that the subject is a candidate for copper treatment.

13. The method of claim 12, wherein the biochemical marker of abnormal copper metabolism is catecholamine level, wherein the catecholamine level is correlated with decreased dopamine-β-hydroxylase activity.

14. The method of claim 13, wherein the catecholamine level correlated with decreased dopamine-β-hydroxylase activity is selected from the group consisting of an increased ratio of dopamine to norepinephrine and an increased ratio of dihydroxyphenylacetic acid to dihydroxyphenylglycol as compared to a reference value ratio and wherein an increased ratio of dopamine to norepinephrine and/or dihydroxyphenylacetic acid to dihydroxyphenylglycol is an indicator that the subject is a candidate for copper treatment.

15. The method of claim 6, wherein the biochemical marker of abnormal copper metabolism is cellular copper egress from isolated cells from the subject, and wherein increased cellular copper egress as compared to copper egress from isolated normal cells is an indicator that the subject is a candidate for copper treatment.

16. The method of claim 15, wherein the isolated cells comprise fibroblast cells or lymphoblast cells.

17. The method of claim 1, wherein determining the presence of at least one mutation in an ATP7A molecule comprises: obtaining a test sample of DNA containing an ATP7A sequence of the subject; and determining the presence of the at least one mutation of the ATP7A molecule in the DNA.

18. The method of claim 17, wherein determining the presence of the at least one mutation in the ATP7A molecule comprises using one or more of restriction digestion, probe hybridization, nucleic acid amplification, heteroduplex analysis, or nucleotide sequencing. 19. The method of claim 18, wherein nucleic acid amplification comprises using at least one oligonucleotide primer comprising at least 15 contiguous nucleotides of SEQ ID NO: 1.

20. The method of claim 18, wherein probe hybridization comprises a probe comprising at least 15 contiguous nucleotides of SEQ ID NO: 1. 21. The method of claim 1, wherein determining the presence of at least one mutation in an ATP7A molecule comprises: obtaining a test sample of RNA containing an ATP7A sequence of the subject; and determining the presence of the at least one mutation of the ATP7A molecule in the RNA.

22. The method of claim 21, wherein determining the presence of the at least one mutation in the ATP7A molecule comprises a reverse-transcriptase polymerase chain reaction.

23. The method of claim 1, wherein determining the presence of at least one mutation in an ATP7A molecule comprises: obtaining a test sample containing an ATP7A protein of the subject; and determining the presence of the at least one mutation of the ATP7A protein in the test sample.

24. The method of claim 23, wherein determining the presence of the at least one mutation comprises using one or more of Western blot assay or ELISA.

25. The method of claim 24, wherein the Western blot assay or ELISA comprises using at least one ATP7A-specific antibody or a functional fragment thereof.

26. The method of claim 24, wherein the antibody is a monoclonal antibody.

27. The method of claim 26, wherein the monoclonal antibody recognizes an epitope of the mutant ATP7A and no epitope of wild type ATP7A.