Molecular mechanisms of copper metabolism and the role of the Menkes disease protein

Menkes disease is an X‐linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births. The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood. The symptoms of Menkes disease result from a deficiency of serum copper and copper‐dependent enzymes. A candidate gene for the disease has been isolated and designated MNK. The MNK gene codes for a P‐type cation transporting ATPase, based on homology to known P‐type ATPases and in vitro experimentation. cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments. The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal‐binding repeats in the amino‐terminal domain. The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease. Each of the Menkes protein amino‐terminal repeats contains a conserved ‐X‐Met‐X‐Cys‐X‐X‐Cys‐ motif (where X is any amino acid). These metal‐binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes. An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented. This forms the basis for a discussion of Menkes disease and the protein deficit in this disease. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 93–106, 1999     

Long and short repeats of sea urchin DNA and their evolution

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar numbers of long and short class members. Two were members of predominantly short class families. — The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.     

Multipoint linkage analysis in Menkes disease.

Linkage analyses were performed in 11 families with X-linked Menkes disease. In each family more than one affected patient had been diagnosed. Forty informative meioses were tested using 11 polymorphic DNA markers. From two-point linkage analyses high lod scores are seen for DXS146 (pTAK-8; maximal lod score 3.16 at recombination fraction [theta] = .0), for DXS1 (p-8; maximal lod score 3.44 at theta = .0), for PGK1 (maximal lod score 2.48 at theta = .0), and for DXS3 (p19-2; maximal lod score 2.90 at theta = .0). This indicates linkage to the pericentromeric region. Multilocus linkage analyses of the same data revealed a peak for the location score between DXS146(pTAK-8) and DXYS1X(pDP34). The most likely location is between DXS159 (cpX289) and DXYS1X(pDP34). Odds for this location relative to the second-best-supported region, between DXS146(pTAK-8) and DXS159 (cpX289), are better than 74:1. Visualization of individual recombinant X chromosomes in two of the Menkes families showed the Menkes locus to be situated between DXS159(cpX289) and DXS94(pXG-12). Combination of the present results with the reported absence of Menkes symptoms in male patients with deletions in Xq21 leads to the conclusion that the Menkes locus is proximal to DXSY1X(pDP34) and located in the region Xq12 to Xq13.3.     

Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd²⁺ sequence than to P-type ATPases from animal sources. Menkes syndrome is an X-chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P-type ATPase, whose properties satisfactorily explain the phenotype of the disease. P-type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K⁺ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca²⁺ ATPase), or for cation exchange (e.g. the animal cell Na⁺/K⁺ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P-type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P-type ATPases and also an N-terminal dithiol region that was proposed to be a ‘metal-binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.     

Structure and genomic organization of centromeric repeats in<Emphasis Type="Italic"> Arabidopsis</Emphasis> species

Centromeric repetitive sequences were isolated from Arabidopsis halleri ssp. gemmifera and A. lyrata ssp. kawasakiana. Two novel repeat families isolated from A. gemmifera were designated pAge1 and pAge2. These repeats are 180 bp in length and are organized in a head-to-tail manner. They are similar to the pAL1 repeats of A. thaliana and the pAa units of A. arenosa. Both A. gemmifera and A. kawasakiana possess the pAa, pAge1 and pAge2 repeat families. Sequence comparisons of different centromeric repeats revealed that these families share a highly conserved region of approximately 50 bp. Within each of the four repeat families, two or three regions showed low levels of sequence variation. The average difference in nucleotide sequence was approximately 10% within families and 30% between families, which resulted in clear distinctions between families upon phylogenetic analysis. FISH analysis revealed that the localization patterns for the pAa, pAge1 and pAge2 families were chromosome specific in A. gemmifera and A. kawasakiana. In one pair of chromosomes in A. gemmifera, and three pairs of chromosomes in A. kawasakiana, two repeat families were present. The presence of three families of centromeric repeats in A. gemmifera and A. kawasakiana indicates that the first step toward homogenization of centromeric repeats occurred at the chromosome level.Communicated by W. R. McCombie     

High-resolution mapping of repetitive DNA by in situ hybridization: molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens

Members of three prominent DNA families of Beta procumbens have been isolated as Sau3A repeats. Two families consisting of repeats of about 158 bp and 312 bp are organized as satellite DNAs (Sau3A satellites I and II), whereas the third family with a repeat length of 202 bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differentially labelled repeats. The physical order of satellite subfamily arrays along a chromosome was visualized and provided evidence that large arrays of plant satellite repeats are not contiguous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau3A satellite family I. It is known that arrays of Sau3A satellite I repeats are tightly linked to a nematode (Heterodera schachtii) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau3A satellite II were found in centromeric regions of 16 chromosomes and, in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202 bp repeats is uniformly distributed over all chromosomes and largely excluded from the rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta.     

Rapid and robust screening of the Menkes disease/occipital horn syndrome gene

Menkes disease and occipital horn syndrome (OHS) are allelic neurogenetic disorders of copper transport associated with mutations in an X-linked gene, ATP7A. This gene encodes a copper-transporting P-type ATPase. The spectrum of mutations at the Menkes/OHS locus is estimated to include 1% chromosomal rearrangements and 15-20% large deletions, with the remaining defects involving small alterations. There is a compelling need for a rapid and reliable molecular diagnostic approach for patients and families impacted by these conditions. In addition to testing suspected affected males, carrier screening of females in Menkes/OHS families and prenatal evaluation of at-risk pregnancies will be enhanced by the wider availability of robust mutation analysis for this large (23-exon) locus. Here we describe a stepwise approach to mutation screening for these disorders that successfully identified molecular alterations in over 95% of our patient population (n = 49). This genomic DNA-based technique employs multiplex PCR, heteroduplex analysis, and direct sequencing, in a serial fashion. This approach should find application in molecular diagnostic laboratories in the United States and other countries. Currently, only a single European center provides commercial testing for unknown mutations in Menkes/OHS patients, even though these disorders occur worldwide.     

Wide distribution of related satellite DNA families within the genus <Emphasis Type="Italic">Pimelia</Emphasis> (Tenebrionidae)

Major satellites of species in the genus Pimelia comprise large portions of their genomes and belong to seven major satellite families which all originate from a common ancestral sequence. Here we present the results of comprehensive screening of 26 Pimelia species belonging to three distinct geographic groups (Ibero-Balearic, African and Canary Islands) for the presence of different Pimelia satellite families in their genomes. Dot-blot hybridization experiments suggest that together with one dominant, highly abundant satellite family, other families are also present in genomes of the majority of examined Pimelia species, but as low-copy number repeats. The estimated abundance of these underrepresented repeats is about 4,000 copies per haploid genome. Signals of highly abundant satellite family from P. scabrosa (PSCA) in examined congeneric species, obtained after PCR amplification and Southern hybridization under high stringency conditions, corroborate sequence preservation of low-copy representatives of satellite families. PRINS localized low-copy repeats within the pericentromeric regions of all chromosomes. These results point to the existence of an extensive library of repetitive DNAs that was already present in the genome of the common ancestor of extant Pimelia taxa, and shifts the period of diversification of Pimelia satellites far in the history of this genus.     

Two new families of tandem repeats isolated from genus Vicia using genomic self-priming PCR

A modified genomic self-priming technique was used for rapid isolation of tandem repeats from several Vicia species. Based on homologies of their nucleotide sequences the newly isolated clones were assigned to two repeat families named VicTR-A and VicTR-B. Both families are rich in AT (74%) and are organized as long blocks of tandemly repeated units. The VicTR-A repeats are characterized by a monomer size of 69 bp, whereas the VicTR-B repeat monomer is about 38 bp long, and the two families do not share significant sequence homology. VicTR sequences show different degrees of amplification (up to 10⁶–10⁷ copies/haploid genome) in individual Vicia species and are not amplified in other legumes. The abundances of these repeats do not correlate with genome sizes but are similar in species that belong to the same taxonomic section within the genus Vicia. Primed in situ (PRINS) labeling of metaphase chromosomes of V. pannonica revealed that VicTR-A sequences are located predominantly in the telomeric regions of the short arms of all chromosomes. In contrast, labeling of VicTR-B repeats in V. sativa resulted in mainly intercalary bands of various intensities and only weak telomeric signals. Received: 15 December 1999 / Accepted: 8 March 2000     

Relative Efficiencies of Plasma Catechol Levels and Ratios for Neonatal Diagnosis of Menkes Disease

Background Menkes disease is an X-linked recessive neurodevelopmental disorder resulting from mutation in a copper-transporting ATPase gene. Menkes disease can be detected by relatively high concentrations of dopamine (DA) and its metabolites compared to norepinephrine (NE) and its metabolites, presumably because dopamine-beta-hydroxylase (DBH) requires copper as a co-factor. The relative diagnostic efficiencies of levels of catechol analytes, alone or in combination, in neonates at genetic risk of Menkes disease have been unknown. Methods Plasma from 44 at-risk neonates less than 30 days old were assayed for DA, NE, and other catechols. Of the 44, 19 were diagnosed subsequently with Menkes disease, and 25 were unaffected. Results Compared to unaffected at-risk infants, those with Menkes disease had high plasma DA (P < 10⁻⁶) and low NE (P < 10⁻⁶) levels. Considered alone, neither DA nor NE levels had perfect sensitivity, whereas the ratio of DA:NE was higher in all affected than in all unaffected subjects (P = 2 × 10⁻⁸). Analogously, levels of the DA metabolite, dihydroxyphenylacetic acid (DOPAC), and the NE metabolite, dihydroxyphenylglycol (DHPG), were imperfectly sensitive, whereas the DOPAC:DHPG ratio was higher in all affected than in all unaffected subjects (P = 2 × 10⁻⁴). Plasma dihydroxyphenylalanine (DOPA) and the ratio of epinephrine (EPI):NE levels were higher in affected than in unaffected neonates (P = 0.0015; P = 0.013). Conclusions Plasma DA:NE and DOPAC:DHPG ratios are remarkably sensitive and specific for diagnosing Menkes disease in at-risk newborns. Affected newborns also have elevated DOPA and EPI:NE ratios, which decreased DBH activity alone cannot explain.     

X-linked Menkes disease: first documented report of germ-line mosaicism

This work investigated a three-generation Menkes disease family, where germ-line mosaicism was suspected in the maternal grandmother of the index patient. She had given birth to 2 boys who died of suspected Menkes disease on the basis of clinical and photographic evidence. Biochemical analysis of the index patient confirmed the diagnosis of Menkes disease, and DNA analysis established a partial gene deletion (EX11_EX23del), involving exons 11-23 and the 3'-untranslated region (UTR) of ATP7A. A junction fragment was detectable by Southern blot analysis, which enabled carrier analysis. The mother was demonstrated to be a carrier, whereas analysis of lymphoblasts and skin fibroblasts from the maternal grandmother gave no indication of a partial gene deletion. No materials were available from the possibly affected maternal uncles. Further genetic analyses, including biochemical testing of the grandmother and haplotype analysis using four intragenic markers on DNA from selected members of the family, corroborated this finding. The combined results from DNA analyses showed that the grandmother had transmitted three different ATP7A haplotypes to her offspring: (1) the at-risk allele (CA(B))-1 and the deletion; (2) the at-risk allele (CA(B))-1 without deletion; and (3) the second allele (CAB)-2 without deletion. In conclusion, our study demonstrated segregation of Menkes disease within the family investigated that can best be explained by extensive germ-line mosaicism in the maternal grandmother. The finding of germ-line mosaicism has obvious implications for genetic counseling of Menkes disease families.     

Unexpectedly slow homogenisation within a repetitive DNA family shared between two subspecies of tsetse fly

Summary Repetitive DNA families in sexual species are subject to a variety of turnover mechanisms capable of homogenising newly arising mutations. Very high levels of homogeneity in DNA families in some species ofDrosophila indicate that the rate of turnover is fast relative to that of mutation. To gauge the generality of such phenomena, we cloned and sequenced individual members of homologous repetitive DNA families from two subspecies of tsetse fly,Glossina morsitans centralis andG. morsitans morsitans. Unexpectedly high levels of variation were found within each subspecies, averaging 24% and 31%, respectively. Contiguous repeats and repeats cloned at random were comparably divergent. Nevertheless, it was possible to identify three instances of apparent homogenisation, each being, remarkably, of an insertion/deletion nature. We conclude that the rate of turnover in the tsetse families is comparable to that of most mutations, and discuss the possible parameters affecting flux in these families.     

Population and Family Studies of the CAG Repeats in the IT-15Gene

A simple and effective method for typing of CAG repeats in the IT-15gene has been suggested. This method was applied for examination of the CAG allele distribution in Huntington's disease (HD) patients in five different populations from the Commonwealth of Independent States. A total of 21 normal alleles with the sizes ranging from 9 to 32 triplet repeats units were revealed. Moreover, alleles with the sizes ranging from 16 to 20 repeats predominated constituting from 54.4 to 74.6% of all alleles in different populations. The number of repeats in one allele in HD patients exceeded 38 units (43 triplets on average). In two families an increase in the CAG repeat units number in the mutant allele upon its paternal transmission was recorded.     

Junctions between repetitive DNAs on the PSR chromosome of Nasonia vitripennis: Association of palindromes with recombination

The Paternal-Sex-Ratio (PSR) chromosome of Nasonia vitripennis contains several families of repetitive DNAs that show significant sequence divergence but share two palindromic regions. This study reports on the analysis of junctions between two of these repetitive DNA families (psr2 and psr18). Three lambda clones that hybridized to both repeat families were isolated from PSR-genomic DNA libraries through multiple screenings and analyzed by Southern blots. Analysis of clones showed a region in which the two repeat types are interspersed, flanked by uniform blocks of each repeat type. PCR amplification of genomic DNA confirmed the contiguous arrangement of psr2 and psr18 on PSR and identified an additional junction region between these repeats that was not present in the lambda inserts. We isolated and sequenced 41 clones from the lambda inserts and genomic PCR products containing junction sequences. Sequence analysis showed that all transitions between psr2 and psr18 repeats occurred near one of the two palindromes. Based on the inheritance pattern of PSR, recombination between repeats on this chromosome must be mitotic (rather than meiotic) in origin. The occurrence of exchanges near the palindromes suggests that these sequences enhance recombination between repeat units. Rapid amplification of repetitive DNA may have been an important factor in the evolution of the PSR chromosome. Correspondence to: John H. Werren     

Insights into the mutational history and prevalence of SCA1 in the Indian population through anchored polymorphisms

There is a wide variation in prevalence of spinocerebellar ataxia type 1 (SCA1) in different populations. In the present study, we observed SCA1 in ∼22% (37/167 families) of the autosomal dominant cerebellar ataxias (ADCAs) in the Indian population. We investigated the role of various genetic factors like repeat length, interruption pattern and chromosomal background in predisposing the repeats to instability in these families. We analyzed 12 markers (9 SNPs and 3 microsatellite markers) and found 3 of them, spanning a region of ∼65 kbp to be linked with the disease locus in the Indian population. The haplotype C-4-C defined by rs1476464 (SNP9)-D6S288-rs2075974 (SNP1), which was extremely rare in nonaffected chromosomes (∼3%), was observed to be significantly (P<0.0000) associated with the expanded chromosomes in ∼44% of SCA1 families. This haplotype was found in all nonhuman primates. SNP1 (C/T), which showed a skewed allelic distribution between large (LN > 30 repeats) and small normal (SN ≤ 30 repeats) alleles (P<0.0000) had similar allelic distribution (P=0.3477) in LN and expanded alleles. Our study suggested that LN and expanded chromosomes linked with the ancestral C allele of SNP1 might have originated simultaneously during evolution by the lengthening of repeats. The LN alleles might have accumulated repeat stabilizing non-CAG interruptions during this process. Similar proportions of T allele in SN with single interruptions, LN and expanded chromosomes lend credence to the origin of expanded alleles from singly-interrupted chromosomes. Our analyses using markers linked (anchoring) to SCA1 suggest that prevalence of SCA1 is correlated to both repeat length and number of interruptions in the Indian population. The spectrum of these alleles also points toward the antiquity of SCA1 mutation in the Indian population.Electronic Supplementary Material Supplementary material is available for this article at     

Incidence of Menkes disease

Summary We have calculated the incidence of Menkes disease for Denmark, France, The Netherlands, the United Kingdom and West Germany, based on known Menkes patients born during the time period 1976–87. Considering live-born Menkes patients, the combined incidence for these five countries is 1 Menkes patient per 298000 live-born babies. If the number of affected aborted fetuses are taken into account, the incidence is 1 Menkes per 254000 live-born babies. This incidence, which is 2–4 times lower than earlier published incidence figures, places Menkes disease as an extremely rare disease. The mutation rate for Menkes disease is estimated to be 1.96 × 10^–6, based on the number of isolated Menkes cases born during the time period 1976–87 and the total number of newborn males during this time.     

Alu Repeats in the Human Genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long and short interspersed elements. An Alu element is a dimer of similar, but not identical, fragments of total size about 300 bp, and originates from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3"-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats constitute more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently have been inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Association of CTG repeats and the 1-kbAlu insertion/deletion polymorphism at the myotonin protein kinase gene in the Japanese population suggests a common Eurasian origin of the myotonic dystrophy mutation

We have studied linkage disequilibrium between CTG repeats and anAlu insertion/deletion polymorphism at the myotonin protein kinase gene (DMPK) in 102 Japanese families, of which 93 were affected with myotonic dystrophy (DM). All of the affected chromosomes are in complete linkage disequilibrium with theAlu insertion allele. Among the normal chromosomes, alleles of CTG repeats 5 and 17 are exclusively associated with the insertion allele. On the other hand, intermediate alleles of 11-6 repeats show a significantly greater association with the deletion allele. A strikingly similar pattern of linkage disequilibrium observed in European populations suggests a common origin of the DM mutation in the Japanese and European populations.     

A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome

We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient’s genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat’s number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.