A 37-year-old Menkes disease patient-Residual ATP7A activity and early copper administration as key factors in beneficial treatment.

Menkes disease (MD) is a lethal disorder characterized by severe neurological symptoms and connective tissue abnormalities; and results from malfunctioning of cuproenzymes, which cannot receive copper due to a defective intracellular copper transporting protein, ATP7A. Early parenteral copper-histidine supplementation may modify disease progression substantially but beneficial effects of long-term treatment have been recorded in only a few patients. Here we report on the eldest surviving MD patient (37 years) receiving early-onset and long-term copper treatment. He has few neurological symptoms without connective tissue disturbances; and a missense ATP7A variant, p.(Pro852Leu), which results in impaired protein trafficking while the copper transport function is spared. These findings suggest that some cuproenzymes maintain their function when sufficient copper is provided to the cells; and underline the importance of early initiated copper treatment, efficiency of which is likely to be dependent on the mutant ATP7A function.

Mutational and phenotypical spectrum of phenylalanine hydroxylase deficiency in Denmark.

We describe the genotypes of the complete cohort, from 1967 to 2014, of phenylketonuria (PKU) patients in Denmark, in total 376 patients. A total of 752 independent alleles were investigated. Mutations were identified on 744 PKU alleles (98.9%). In total, 82 different mutations were present in the cohort. The most frequent mutation c.1315+1G>A (IVS12+1G>A) was found on 25.80% of the 744 alleles. Other very frequent mutations were c.1222C>T (p.R408W) (16.93%) and c.1241A>G (p.Y414C) (11.15%). Among the identified mutations, five mutations; c.532G>A (p.E178K), c.730C>T (p.P244S), c.925G>A (p.A309T), c.1228T>A (p.F410I), and c.1199+4A>G (IVS11+4A>G) have not been reported previously. The metabolic phenotypes of PKU are classified into four categories; 'classical PKU', 'moderate PKU', 'mild PKU' and 'mild hyperphenylalaninemia'. In this study, we assigned the phenotypic outcome of three of the five novel mutations and furthermore six not previously classified mutations to one of the four PKU categories.

Identification of a novel locus for a USH3 like syndrome combined with congenital cataract.

Usher syndrome (USH) is the most common genetic disease that causes both deafness and blindness. USH is divided into three types, USH1, USH2 and USH3, depending on the age of onset, the course of the disease, and on the degree of vestibular dysfunction. By homozygosity mapping of a consanguineous Danish family of Dutch descent, we have identified a novel locus for a rare USH3-like syndrome. The affected family members have a unique association of retinitis pigmentosa, progressive hearing impairment, vestibular dysfunction, and congenital cataract. The phenotype is similar, but not identical to that of USH3 patients, as congenital cataract has not been reported for USH3. By homozygosity mapping, we identified a 7.3 Mb locus on chromosome 15q22.2-23 with a maximum multipoint LOD score of 2.0. The locus partially overlaps with the USH1 locus, USH1H, a novel unnamed USH2 locus, and the non-syndromic deafness locus DFNB48.

Low frequency of Parkin, Tyrosine Hydroxylase, and GTP Cyclohydrolase I gene mutations in a Danish population of early-onset Parkinson's Disease.

Autosomal recessive Parkinson's disease (PD) with early-onset may be caused by mutations in the parkin gene (PARK2). We have ascertained 87 Danish patients with an early-onset form of PD (age at onset < or =40 years, or < or =50 years if family history is positive) in a multicenter study in order to determine the frequency of PARK2 mutations. Analysis of the GTP cyclohydrolase I gene (GCH1) and the tyrosine hydroxylase gene (TH), mutated in dopa-responsive dystonia and juvenile PD, have also been included. Ten different PARK2 mutations were identified in 10 patients. Two of the patients (2.3%) were found to have homozygous or compound heterozygous mutations, and eight of the patients (9.2%) were found to be heterozygous. A mutation has been identified in 10.4% of the sporadic cases and in 15.0% of cases with a positive family history of PD. One patient was found to be heterozygous for both a PARK2 mutation and a missense mutation (A6T) in TH of unknown significance. It cannot be excluded that both mutations contribute to the phenotype. No other putative disease causing TH or GCH1 mutations were found. In conclusion, homozygous, or compound heterozygous PARK2 mutations, and mutations in GCH1 and TH, are rare even in a population of PD patients with early-onset of the disease.

Expanded motor and psychiatric phenotype in autosomal dominant Segawa syndrome due to GTP cyclohydrolase deficiency.

BACKGROUND: Segawa syndrome due to GTP cyclohydrolase deficiency is an autosomal dominant disorder with variable expression, that is clinically characterised by l-dopa responsive, diurnally fluctuating dystonia and parkinsonian symptoms. OBJECTIVE: To delineate the neurological and psychiatric phenotype in all affected individuals of three extended families. METHODS: GTP cyclohydrolase deficiency was documented by biochemical analyses, enzymatic measurements in fibroblasts, and molecular investigations. All affected individuals were examined neurologically, and psychiatric data were systematically reviewed. RESULTS: Eighteen affected patients from three families with proven GTP cyclohydrolase deficiency were identified. Eight patients presenting at less than 20 years of age had typical motor symptoms of dystonia with diurnal variation. Five family members had late-presenting mild dopa-responsive symptoms of rigidity, frequent falls, and tendonitis. Among mutation carriers older than 20 years of age, major depressive disorder, often recurrent, and obsessive-compulsive disorder were strikingly more frequent than observed in the general population. Patients responded well to medication increasing serotonergic neurotransmission and to l-dopa substitution. Sleep disorders including difficulty in sleep onset and maintenance, excessive sleepiness, and frequent disturbing nightmares were present in 55% of patients. CONCLUSION: Physicians should be aware of this expanded phenotype in affected members of families with GTP cyclohydrolase deficiency.

Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.

Deficiencies of different proteins involved in copper metabolism have been reported to cause human diseases. Well-known syndromes, for example, are Menkes and Wilson diseases. Here we report a patient presenting with congenital cataract, severe muscular hypotonia, developmental delay, sensorineural hearing loss and cytochrome-c oxidase deficiency with repeatedly low copper and ceruloplasmin levels. These findings were suggestive of a copper metabolism disorder. In support of this, the patient's fibroblasts showed an increased copper uptake with normal retention. Detailed follow-up examinations were performed. Immunoblotting for several proteins including ATP7A (MNK or Menkes protein), ATP7B (Wilson protein) and SOD1 showed normal results, implying a copper metabolism defect other than Wilson or Menkes disease. Sequence analysis of ATOX1 and genes coding for proteins that are known to play a role in the mitochondrial copper metabolism (COI-III, SCO1, SCO2, COX11, COX17, COX19) revealed no mutations. Additional disease genes that have been associated with cytochrome-c oxidase deficiency were negative for mutations as well. As beneficial effects of copper histidinate supplementation have been reported in selected disorders of copper metabolism presenting with low serum copper and ceruloplasmin levels, we initiated a copper histidinate supplementation. Remarkable improvement of clinical symptoms was observed, with complete restoration of cytochrome-c oxidase activity in skeletal muscle.

X-linked recessive Menkes disease: carrier detection in the case of a partial gene deletion.

X-linked recessive Menkes disease is a lethal disorder of copper metabolism, caused by defects in the ATP7A gene. About 15% of the mutations causing Menkes disease are partial gene deletions. We have previously demonstrated carrier diagnosis of deletions in heterozygotes by Southern blot analysis. As this technique is very time-consuming alternative methods are obviously of high value. Multiplex polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR) and spanning the deletion on genomic DNA can all be used for detection of partial gene deletions in male patients, but only spanning of the deletion can be applied for carrier detection. Simple multiplex PCR is not applicable for carrier detection because the normal allele of ATP7A will be PCR amplified thus masking the deletion. Here, we demonstrate, in addition to spanning of the deletion on genomic DNA, carrier detection based on the use of a previously unrecognized polymorphism in intron 13 of ATP7A in combination with previously identified intragenic polymorphic markers. We show that these intragenic markers can be used for carrier detection, not only indirectly by determining segregation of the disease related allele but also directly if located within the deleted region. We demonstrate determination of the carrier status of 21 at-risk carriers.

X-linked recessive Menkes disease: identification of partial gene deletions in affected males.

Menkes disease is an X-linked recessive lethal disorder of copper metabolism, caused by defects in the ATP7A gene. Partial gene deletions comprise about 15% of the mutations causing Menkes disease. We have previously demonstrated identification of partial ATP7A deletions in patients by Southern blot analysis. In the present study, we report the use of three fast and reliable polymerase chain reaction (PCR)-based methods for the identification of partial ATP7A deletions in Menkes disease patients. First we demonstrate the use of multiplex PCR, a fast method for identification and rough localization of partial gene deletions, in which two exons of ATP7A are coamplified. Second, we present PCR amplification of genomic DNA across the deletion junctions, a method enabling identification of the deletion breakpoints and hence the exact size of the deletion. Finally, application of reverse transcription PCR (RT-PCR) for identification and localization of gene deletions at the cDNA level is demonstrated. By studying the mutation at the cDNA level the predicted effect of the mutation on the amino acid sequence and consequently the protein structure and function can be inferred. We demonstrate characterization of partial gene deletions in five patients, and in three of these we were able to determine the breakpoint sequences.

The RihA, RihB, and RihC ribonucleoside hydrolases of Escherichia coli. Substrate specificity, gene expression, and regulation.

Pyrimidine-requiring cdd mutants of Escherichia coli deficient in cytidine deaminase utilize cytidine as a pyrimidine source by an alternative pathway. This has been presumed to involve phosphorylation of cytidine to CMP by cytidine/uridine kinase and subsequent hydrolysis of CMP to cytosine and ribose 5-phosphate by a putative CMP hydrolase. Here we show that cytidine, in cdd strains, is converted directly to cytosine and ribose by a ribonucleoside hydrolase encoded by the previously uncharacterized gene ybeK, which we have renamed rihA. The RihA enzyme is homologous to the products of two unlinked genes, yeiK and yaaF, which have been renamed rihB and rihC, respectively. The RihB enzyme was shown to be a pyrimidine-specific ribonucleoside hydrolase like RihA, whereas RihC hydrolyzed both pyrimidine and purine ribonucleosides. The physiological function of the ribonucleoside hydrolases in wild-type E. coli strains is enigmatic, as their activities are paralleled by the phosphorolytic activities of the nucleoside phosphorylases, and a triple mutant lacking all three hydrolytic activities grew normally. Furthermore, enzyme assays and lacZ gene fusion analysis indicated that rihB was essentially silent unless activated by mutation, whereas rihA and rihC were poorly expressed in glucose medium due to catabolite repression.

Disturbed copper transport in humans. Part 1: mutations of the ATP7A gene lead to Menkes disease and occipital horn syndrome.

Mutations of the ATP7A gene (OMIM 300011) lead to the Menkes disease (MD, OMIM 309400) involving impaired brain development, neurological degeneration, connective tissue abnormalities, and high lethality in early infancy. Occipital horn syndrome (OHS, OMIM 304150), a milder phenotype, is also caused by ATP7A gene mutations. In MD patients, an early copper-histidine treatment may prevent the neurological impairment and prolong survival leading to an OHS phenotype. To demonstrate the genotype/phenotype correlation, two male patients are reported with different ATP7A gene mutations and several phenotypes. In the first patient with the MD phenotype, a mutation within the exon 20 (Gln1288Ter) was found producing a stop codon just prior to the highly conserved ATP binding domain. The OHS phenotype of the second patient was caused by a splice site mutation involving the position +6 of intron 6 within a copper binding domain. Small amounts of correctly spliced ATP7A transcript were sufficient to develop the milder OHS phenotype in this patient (OMIM 30001.0006). In conclusion, mutations of the copper transporting P-type ATPase ATP7A gene cause distinct human diseases showing some genotype/phenotype correlation and implications for treatment.

Characterization of the hCTR1 gene: genomic organization, functional expression, and identification of a highly homologous processed gene.

The human hCTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997. A human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. USA 94, 7481-7486). Here, we have determined the DNA sequence of the exon-intron borders of the hCTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule. Furthermore, human fibroblasts, transfected with hCTR1 cDNA, were shown to have a dramatically increased capacity for (64)Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells. In contrast, no evidence was found for involvement of the hCTR2 gene product in copper uptake. Finally, we have identified a highly homologous processed pseudogene, hCTR1psi, which was localized to chromosome 3q25/26. The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with hCTR1psi DNA showed no increase of (64)Cu uptake.

Similar splice-site mutations of the ATP7A gene lead to different phenotypes: classical Menkes disease or occipital horn syndrome.

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

Invariance of the nucleoside triphosphate pools of Escherichia coli with growth rate.

The ATP and GTP pools of Escherichia coli have recently been reported to increase approximately 10-fold with increasing growth rates in the range from 0.4 to 1.4 generations/hour (Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L., and Gourse, R. L. (1997) Science 278, 2092-2097). Moreover, it was proposed that this variation of the nucleotide pools, particularly the ATP pool, might be responsible for the well known growth rate-dependent regulation of rRNA synthesis in E. coli. To test this hypothesis we have measured the nucleoside triphosphate pools as a function of growth rate for several E. coli strains. We found that the size of all four RNA precursor pools are essentially invariant with growth rate, in the range from 0.5 to 2.3 generations/hour. Nevertheless we observed the expected growth rate-dependent increase of RNA accumulation in these strains. In light of these results, it seems unlikely that nucleotide pool variations should be responsible for the growth rate-dependent regulation of rRNA synthesis.

Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR.

We have isolated and characterized a copper sensitive Escherichia coli mutant that is deficient in the copper transporting P-type ATPase encoded by the copA gene (previously ybaR). Measurements of uptake and efflux of 64Cu by wild-type and mutant cells implicated the CopA protein in copper efflux from the cytoplasm, and further demonstrated that cell-associated copper in intact E. coli cells is distributed between two kinetically distinguishable pools, the ratio of which was dramatically disturbed by the copA mutation. Using a copA-lacZ gene fusion the copA promoter was found to be specifically induced by copper, and this induction was shown to be dependent on a MerR-like transcriptional activator encoded by a previously uncharacterized gene, copR (previously ybbI). In the copA deficient background the copA-lacZ fusion was super induced to very high levels even in the absence of copper addition to the medium, and this induction was dependent on CopR. These results indicated that the cytoplasmic copper concentration was dramatically increased in the copA mutant, in agreement with the 64Cu uptake experiments. Moreover, they implied, that the copper concentration in wild type cells is determined primarily by the CopA efflux pump, while copper is taken up by an essentially constitutive mechanism.

Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein.

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.

Mutation spectrum of ATP7A, the gene defective in Menkes disease.

Our knowledge about Menkes disease (MD) has expanded greatly since its description in 1962 as a new X-linked recessive neurodegenerative disorder of early infancy. Ten years later a defect in copper metabolism was established as the underlying biochemical deficiency. In the beginning of 1990s efforts were concentrated on the molecular genetic aspects. The disease locus was mapped to Xq13.3 and the gene has been isolated by means of positional cloning. This was the beginning of a series of new findings which have greatly enhanced our understanding of copper metabolism not only in human, but also in other species. This review will focus on the molecular genetic aspects of Menkes disease and its allelic form occipital horn syndrome. The mutations will be compared briefly with those described in the animal model mottled mouse, and in Wilson disease, the autosomal recessive disorder of copper metabolism.

Mannose 6-phosphate/insulin-like growth factor-II receptor targets the urokinase receptor to lysosomes via a novel binding interaction.

The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-beta, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor-II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution.