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D W Cox

Publications and source records attributed to D W Cox.

At least 37 records · Page 2Linked to original sources
PubMed1999

Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease.

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.

Adenosine Triphosphatases↗
PubMed1999

Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16.

Abnormal hepatic copper accumulation is recognized as an inherited disorder in man, mouse, rat and dog. The major cause of hepatic copper accumulation in man is a dysfunctional ATP7B gene, causing Wilson disease (WD). Mutations in the ATP7B genes have also been demonstrated in mouse and rat. The ATP7B gene has been excluded in the much rarer human copper overload disease non-Indian childhood cirrhosis, indicating genetic heterogeneity. By investigating the common autosomal recessive copper toxicosis (CT) in Bedlington terriers, we have identified a new locus involved in progressive liver disease. We examined whether the WD gene ATP7B was also causative for CT by investigating the chromosomal co-localization of ATP7B and C04107, using fluorescence in situ hybridization (FISH). C04107 is an anonymous microsatellite marker closely linked to CT. However, BAC clones containing ATP7B and C04107 mapped to the canine chromosome regions CFA22q11 and CFA10q26, respectively, demonstrating that WD cannot be homologous to CT. The copper transport genes CTR1 and CTR2 were also excluded as candidate genes for CT since they both mapped to canine chromosome region CFA11q22. 2-22.5. A transcribed sequence identified from the C04107-containing BAC was found to be homologous to a gene expressed from human chromosome 2p13-p16, a region devoid of any positional candidate genes.

Adenosine Triphosphatases↗
PubMed1999

Functional and antigenic concentrations of alpha-1-proteinase inhibitor after administration for the prevention of chronic lung disease of prematurity.

OBJECTIVE AND METHODS: Alpha-1-proteinase inhibitor (A1PI) supplementation has been used in adults with inherited alpha-1-antitrypsin (A1AT) deficiency to impede the development of emphysema. A1PI supplementation may also be useful for protecting premature neonates who receive mechanical ventilation from the development of chronic lung disease (CLD). However, the pharmacokinetics of exogenous A1PI in this population are unknown. We attempted to determine the disposition of A1PI in premature infants with birth weight 600-1,250 g who received 60 mg/kg on days 0, 4, 7 and 14 in a randomized, placebo-controlled, double-blind trial. Functional and antigenic plasma concentrations of A1PI were measured at specified time points. RESULTS: On both functional and antigenic assays, concentrations began in the normal adult range and rose from day 0 to 10 then fell slightly, but remained above initial values. The concentrations were not significantly different between the treatment and placebo groups. CONCLUSIONS: The results of this study indicate that neonatal pharmacokinetics of A1PI differ markedly from those of the adult. Total plasma clearance of exogenous A1PI seems high in the ventilated premature neonate. Higher or more frequent doses may be necessary to maintain A1PI plasma concentrations above baseline.

Adrenal Cortex Hormones↗
PubMed1999

Disorders of copper transport.

Copper is an essential component of a number of important enzymes. Efficient systems have developed for providing sufficient copper for essential functions, while eliminating excess to avoid tissue toxicity. Copper transport is disrupted in two human diseases: Wilson disease and Menkes disease. Both have defects in copper transporting membrane proteins. Many other proteins are involved in copper transport. Some of these proteins have been identified through a study of the similar copper pathway in yeast. This suggests other copper transport diseases are yet to be discovered. Molecular diagnosis holds promise for reliable diagnosis of patients. Testing of flanking markers is a reliable way to detect presymptomatic sibs of a definite patient.

Cation Transport Proteins↗
PubMed1998

Wilson disease.

Wilson disease is a recessively inherited disorder of copper transport. Clinical features are highly variable, with any combination of neurological, hepatic or psychiatric illness. The age of onset varies from 3 to 50 years of age. Diagnosis is challenging because no specific combination of clinical or biochemical features is necessarily definitive. The genetic defect is due to a variety of abnormalities in a copper-transporting membrane ATPase. Most of the more than 80 mutations are present at a low frequency, and mutations differ between ethnic groups. At least two mutations are sufficiently common to aid in rapid diagnosis, in European and Asian populations respectively. Molecular analysis can provide a definitive diagnosis for asymptomatic sibs. Treatment, using chelating agents or zinc, is most effective when started before permanent tissue damage occurs.

Adolescent↗
PubMed1998

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.

Adenosine Triphosphatases↗
PubMed1998

Elevated LDL triglyceride concentrations in subjects heterozygous for the hepatic lipase S267F variant.

Although naturally occurring loss-of-function mutations in human hepatic lipase (HL) have been described, the biochemical phenotype of heterozygous HL deficiency remains ill defined. This may be due to the relatively small numbers of heterozygous adult carriers of HL mutations in index kindreds. We have identified several new heterozygotes for the catalytically inactive, nonsecreted HL variant S267F in the kindred that was originally ascertained because of hypertriglyceridemia due to the mutant, secreted, circulating apolipoprotein (apo) CII variant apo CII-T. Pairwise comparisons with family controls showed that only the plasma low density lipoprotein triglycerides (LDL TGs) were higher in 11 simple heterozygotes for HL S267F (P=0.002). In contrast, both plasma total TGs and LDL TGs were significantly higher in 12 simple heterozygotes for apo CII-T than in family-matched control subjects (P=0.005 and 0.009, respectively). These findings suggest that the TG content of LDL is increased by heterozygosity for 2 different mutations that affect different proteins involved in lipolysis. However, the mechanisms underlying this compositional change in LDL appear to be different for the 2 mutations, because the total TGs are also elevated in subjects heterozygous for apo CII-T but not in subjects heterozygous for HL S267F.

Adult↗
PubMed1998

alpha1-Proteinase inhibitor therapy for the prevention of chronic lung disease of prematurity: a randomized, controlled trial.

BACKGROUND: An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD). We hypothesized that administration of alpha1-proteinase inhibitor (A1PI), also known as alpha1-antitrypsin, to premature neonates would prevent CLD. DESIGN: A randomized, placebo-controlled, prospective study of A1PI supplementation was performed. Neonates <24 hours of age with birth weights 600-1000 g on respiratory support, and 1001-1250 g with respiratory distress syndrome (RDS) were eligible. Intravenous A1PI (60 mg/kg) or placebo was infused on days 0, 4, 7, and 14. Primary outcome was CLD in survivors, defined as the need for supplemental oxygen on day 28. RESULTS: A total of 106 patients were recruited. There were no significant differences between groups in birth weight or incidence of RDS. The incidence of CLD in survivors was lower in the treated group, but the difference did not reach statistical significance (relative risk [RR], 0.79; confidence interval [CI], 0.60-1.02). This beneficial trend persisted at 36 weeks corrected gestational age (RR, 0.48; CI, 0.23-1.00). The incidence of pulmonary hemorrhage was lower in the treated group (RR, 0.22; CI, 0.05-0.98). Other complications were not significantly different between groups. CONCLUSIONS: In this, the first trial of a protease inhibitor for the prevention of CLD in premature infants, the infusions were well-tolerated. A1PI therapy may impede the development of CLD and appears to reduce the incidence of pulmonary hemorrhage in some neonates born prematurely.

Birth Weight↗
PubMed1997

Expression, purification, and metal binding properties of the N-terminal domain from the wilson disease putative copper-transporting ATPase (ATP7B).

The putative copper binding domain from the copper-transporting ATPase implicated in Wilson disease (ATP7B) has been expressed and purified as a fusion to glutathione S-transferase. Immobilized metal ion affinity chromatography revealed that the fusion protein is able to bind to columns charged with different transition metals with varying affinities as follows: Cu(II)>>Zn(II)>Ni(II)>Co(II). The fusion protein did not bind to columns charged with Fe(II) or Fe(III). 65Zinc(II) blotting analysis showed that the domain is able to bind Zn(II) over a range of pH values from 6.5 to 9.0. Competition 65Zn(II) blotting showed that Cd(II), Hg(II), Au(III), and Fe(III) can successfully compete with Zn(II), at comparable concentrations, for binding to the domain. In contrast, the domain had little or no affinity for Ca(II), Mg(II), Mn(II), and Ni(II) relative to copper. Neutron activation analysis of the copper bound to the domain showed a copper:protein ratio of 6.5-7.3:1. Both Cu(II) and Cu(I) were found to have a higher affinity for the domain relative to Zn(II). In addition, a sharp, reproducible transition was only observed in competition experiments with copper, which may suggest that copper binding has some degree of cooperativity.

Adenosine Triphosphatases↗
PubMed1997

Physical and transcription map in the region 14q24.3: identification of six novel transcripts.

The region of chromosome 14q24 has been of particular interest as it is known to contain one of the early-onset Alzheimer disease genes (AD3). Other genes of medical interest, such as arrhythmogenic right ventricular cardiomyopathy, have been mapped to this region by linkage analysis or chromosome rearrangements. We have focused on the region of a balanced translocation (2;14)(p25;q24). Members of a family with this translocation all have anterior polar cataracts, suggesting the presence of a gene involved in lens integrity at the vicinity of the breakpoint. The chromosome 14 breakpoint has been defined between the short tandem repeats D14S289 and D14S277, a region of overlap for yeast artificial chromosomes (YACs) 888b2 and 934d4. We have extended the study of the region to 2 Mb on chromosome 14 and present a physical map of this region, including several sequence-tagged sites. New probes were generated using several end clones and inter-Alu PCR fragments from YACs. cDNA selection was used to identify transcribed sequences. Mapping and alignment of 17 nonoverlapping cDNAs completed by sequence and expression pattern analysis suggested that a minimum of eight putative transcription units is present in this region: six of these units correspond to five new genes and one member of a new gene family.

Blotting, Northern↗
PubMed1997

Genetic polymorphism and recombination in the subtelomeric region of chromosome 14q.

Subtelomeric regions of human chromosomes are the sites of increased meiotic recombination and have a male-to-female recombination ratio that is higher than elsewhere in the genome. We isolated two novel, polymorphic CA repeat markers from the distal part of the immunoglobulin heavy chain gene cluster, approximately 90 and 200 kb from the telomere of chromosome 14q. The 14q telomere was unambiguously located by physical mapping of telomeric YACs and Bal31 exonuclease digestion of genomic DNA. We then constructed haplotypes using genotype data from these markers and data from sCAW1 (D14S826) for use as a highly polymorphic genetic marker. Linkage analysis using the 40 pedigree CEPH reference panel and genotype data from these and other loci physically mapped to the terminal 1.5 Mb of chromosome 14q revealed an apparent increase in meiotic recombination within this region, relative to the average rate for the genome. Further, we found that recombination was higher in females than in males, indicating that the subtelomeric region of 14q differs from other human subtelomeric regions.

Chromosomes, Artificial, Yeast↗
PubMed1997

Haplotype and mutation analysis in Japanese patients with Wilson disease.

Wilson disease (WD), an autosomal recessive disorder of copper transport, is characterized by impaired biliary excretion and by impaired incorporation of copper into ceruloplasmin. Toxic accumulation of copper causes tissue damage, primarily in the liver, brain, and kidneys. The gene for WD (ATP7B) has been cloned, and the protein product is predicted to be a copper-transporting P-type ATPase with high amino acid identity with that for Menkes disease, an X-linked disorder of copper transport. Mutation screening in WD patients has led to the identification of at least 40 mutations. In addition, haplotype analysis using three dinucleotide-repeat markers, D13S314, D13S301, and D13S316, has been a useful indicator of specific mutations. We have determined haplotypes for the patients and their parents and sibs, in 21 unrelated WD families from Japan. Twenty-eight different haplotypes were observed on 42 WD chromosomes. In all the patients, the ATP7B coding sequence, including the intron-exon boundaries, was screened for mutations, by SSCP, followed by direct-sequence analysis of the shifted fragments. We identified 13 mutations, of which 11 mutations are novel, including 7 mutations-1 insertion, 4 deletions, and 2 missense mutations-in the coding region. The mutations reported in previous studies are 2299insC and Arg778Leu. Two patients were shown to have the 2299insC mutation, which has occurred in many different haplotypes in several populations, indicating a mutation hot spot. Primer-extension analysis of ATP7B mRNA has revealed multiple transcription start sites. Four of the novel mutations (three 1-bp changes and one 5-bp deletion) occur in the 5' UTR and may result in altered expression of the WD gene.

Adenosine Triphosphatases↗
PubMed1997

Review: molecular approaches to inherited liver disease. Focus on Wilson disease.

Although infectious agents account for a large proportion of liver disease, inherited forms of liver disease, often treatable, must not be overlooked. New approaches to the cloning of disease genes are allowing an increased understanding of the basic defect of human diseases. Identification of disease genes can have practical applications for diagnosis, can provide information on prognosis and can lead to new therapies. This review focuses on selected inherited liver diseases and the knowledge to be gained from molecular studies. Three genetic diseases affecting the liver, Wilson disease, haemochromatosis and alpha 1-antitrypsin deficiency, are selected as examples of current approaches to the study of genetic liver disease.

Hemochromatosis↗
PubMed1996

Tandem repeats 3' of the IGHA genes in the human immunoglobulin heavy chain gene cluster.

The human IGH constant region spans 350 kb and includes nine genes and two pseudogenes. All of the constant region gene cluster has been cloned except for sequences between the IGHD and IGHG3 genes, between the IGHA1 and IGHG2 genes, and the 3' region downstream of the IGHA2 gene. The regions 3' of the IGHA genes, which are not cloned, are of interest since transcriptional control elements were found downstream of the IGHA genes in the rat and the mouse IGH loci. In addition, by pulsed-field gel electrophoresis mapping, CpG islands were identified approximately 30 kb downstream of each IGHA gene, within the uncloned portion of the human IGH. These findings indicate that the regions 3' of the IGHA genes are candidate regions for additional transcriptional elements of the human IGH genes. In an effort to characterize these regions, we screened five different libraries and determined the regions 3' of the IGHA genes to be unclonable by standard cloning methods. Therefore, we applied the Inverse-PCR technique to amplify the sequences flanking the IGHA genes. We obtained 1418 bp of new sequence 3' of the IGHA1 gene. The new sequence included tandem repeats of 20 bp, which we propose is the cause of the unclonability of this region.

Animals↗
PubMed1996

The toxic milk mouse is a murine model of Wilson disease.

Wilson disease (WD) is an autosomal recessive defect of copper transport characterized by massive accumulation of copper in the liver, which can lead to liver failure. Mutations in a copper transporting ATPase (WND or ATP7B) have been shown to cause the disease. The toxic milk mouse mutant (tx) accumulates copper in the liver in a manner similar to that observed in patients with WD. However, some physiological differences between tx mice and human WD patients have cast doubts on whether this mutant mouse is a valid model for WD. In this paper we report the isolation of cDNA clones encoding the murine homologue of WND. The predicted amino acid sequence is 1462 amino acids and contains the same functional domains identified in human and rat WND. As in the rat, the fourth metal binding domain is apparently non-functional. Similar levels of a 7.5 kb WND mRNA were detected in liver and kidney from normal and tx mice, indicating that transcription of this gene was unaffected in the mutant mice. The coding sequence of WND cDNA from the tx mouse liver identified a single nucleotide difference between the normal DL mouse and the tx which is predicted to change methionine 1356 in the eighth transmembrane domain to valine. This methionine is conserved in all copper ATPases including those from bacteria and yeast. The conclusion that this is the causative mutation is supported by the recent mapping of tx and WND to the same region of mouse chromosome 8. Thus the tx mouse is presented as a valid model for studies of the role of WND in copper transport and for investigation of different treatment strategies for WD.

Adenosine Triphosphatases↗
PubMed1996

High frequency of two mutations in codon 778 in exon 8 of the ATP7B gene in Taiwanese families with Wilson disease.

The gene for Wilson disease (WD) has been cloned as a P type copper transporter gene (ATP7B). To elucidate the possible genetic mechanism for the diversity of clinical manifestations, we characterised 22 Taiwanese families with WD by microsatellite haplotyping of close DNA markers D13S314-D13S301-D13S316. We also screened for mutations of codon 778 in the transmembrane region. There were at least 15 haplotypes within eight broad subgroups observed among 44 WD chromosomes. Among the 22 unrelated patients, we found that six patients (27%) carried a codon 778 mutation. Nucleotide sequence analysis showed there were two different mutations: the previously reported Arg778Leu mutation in four patients and Arg778Gln, a new mutation, in two patients. The two different mutations of the same codon occurred in two distinct microsatellite haplotypes.

Codon↗