ABSTRACT
PURPOSE: A 5-bp deletion in ELOVL4, a photoreceptor-specific gene, has been associated with autosomal dominant (ad) macular dystrophy phenotypes in five related families, in which phenotypes range from Stargardt-like macular dystrophy (STGD3; Mendelian Inheritance in Man 600110) to pattern dystrophy. This has been the only mutation identified in ELOVL4 to date, which is associated with macular dystrophy phenotypes. In the current study, the potential involvement was investigated of an ELOVL4 gene variation in adSTGD-like and other macular dystrophy phenotypes segregating in a large unrelated pedigree from Utah (K4175). METHODS: The entire open reading frame of the ELOVL4 gene was analyzed by direct sequencing in a proband from the K4175 family. The combination of denaturing high-performance liquid chromatography (DHPLC) analysis and direct sequencing of all available family members was used to further assess segregation of identified ELOVL4 variants in the pedigree. RESULTS: A complex mutation, two 1-bp deletions separated by four nucleotides, was detected in all affected members of the family. The mutation results in a frameshift and the truncation of the ELOVL4 protein, similar to the effect of the previously described 5-bp deletion. CONCLUSIONS: The discovery of a second mutation in the ELOVL4 gene segregating with macular dystrophy phenotypes confirms the role of this gene in a subset of dominant macular dystrophies with a wide range of clinical expressions and suggests a role for modifying genes and/or environmental factors in the disease process.
Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Membrane Proteins/genetics , Mutation/physiology , Adolescent , Adult , Aged , Child , Female , Genetic Linkage , Humans , Macular Degeneration/pathology , Male , Middle Aged , Pedigree , Penetrance , PhenotypeABSTRACT
PURPOSE: To study the presence of Best macular dystrophy (VMD2) gene mutations in patients diagnosed with maculopathies other than classic Best disease and to describe the clinical characteristics of these subjects. DESIGN: Case-comparison study of phenotype-genotype correlations. METHODS: Patients with either age-related maculopathy (ARM; n = 259) or maculopathies other than classic Best disease (n = 28) were screened for mutations in the Best gene (VMD2; OMIM 153700). These cases were compared with ethnically similar subjects in the same age range without maculopathy (n = 196). All patients underwent a complete dilated ocular examination, and all affected individuals underwent fundus photography. Phenotype-genotype comparisons were made. MAIN OUTCOME MEASURES: Presence of mutations in the Best gene (VMD2; OMIM 153700) and the clinical phenotype. RESULTS: Three of 259 patients (1%) with ARM and 2 of 28 patients (7%) with other maculopathies including 1 of 3 patients with adult-onset foveomacular vitelliform dystrophy and 1 of 5 patients with a bull's eye maculopathy, but none of the controls, were found to possess amino acid-changing variants in the VMD2 gene. These included a man with confluent drusen and retinal pigment epithelial detachments (variant in exon 6; T216I), a man with geographic atrophy and numerous soft drusen (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations (variant in exon 10; L567F), a woman with drusen and retinal pigment epithelial alterations resembling bull's-eye maculopathy (variant in exon 4; E119Q), and a woman diagnosed with adult-onset foveomacular vitelliform dystrophy (variant in exon 4; A146K). CONCLUSIONS: Novel mutations in the VMD2 gene were found in patients diagnosed with maculopathies other than classic Best disease. Some cases diagnosed as adult-onset vitelliform foveomacular dystrophy may represent a variant of Best disease with delayed onset. The VMD2 gene does not play a major role in the development of ARM.
Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Mutation , Aged , Bestrophins , Chloride Channels , DNA Mutational Analysis , Electrooculography , Female , Fluorescein Angiography , Fundus Oculi , Genotype , Humans , Male , Middle Aged , Pedigree , Phenotype , Visual AcuityABSTRACT
Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.
Subject(s)
Eye Proteins/genetics , Genes, Dominant/genetics , Macular Degeneration/genetics , Membrane Proteins/genetics , Sequence Deletion/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 6/genetics , Cloning, Molecular , DNA Mutational Analysis , Exons/genetics , Eye Proteins/chemistry , Eye Proteins/metabolism , Female , Humans , In Situ Hybridization , Introns/genetics , Lod Score , Macaca mulatta/genetics , Macular Degeneration/pathology , Male , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Pedigree , RNA, Messenger/analysis , RNA, Messenger/genetics , Retina/metabolism , Retina/pathology , Sequence AlignmentABSTRACT
Stargardt-like macular degeneration (STGD(3)) and autosomal dominant macular degeneration (adMD) share phenotypic characters with atrophic age-related macular degeneration (AMD). Mutations in a photoreceptor cell-specific factor involved in the elongation of very long chain fatty acids (ELOVL(4)) were shown to be associated with STGD(3), adMD, and pattern dystrophy. We screened 778 patients with AMD and 551 age-matched controls to define the role of sequence variants in the ELOVL(4) gene in age-related macular degeneration. We detected three sequence variants in the non-coding region and eight variants in the coding region. No statistically significant association was observed between sequence variants in the ELOVL(4) gene and susceptibility to AMD. However, for the detection of modest effects of multiple alleles in a complex disease, the analysis of larger cohorts of patients may be required.
Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Membrane Proteins/genetics , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Age Factors , Aged , DNA Primers/chemistry , Exons , Genetic Variation , Humans , Introns , Macular Degeneration/physiopathology , Middle Aged , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
Best vitelliform macular dystrophy is a dominantly inherited, early onset, macular degenerative disease that exhibits some histopathologic similarities to age-related macular degeneration. Although the vitelliform lesion is common in the fundus of individuals with Best disease, diagnosis is based on a reduced ratio of the light peak to dark trough in the electrooculogram. Recently, the VMD2 gene on chromosome 11q13, encoding the protein bestrophin, was identified. The function of bestrophin is unknown. To facilitate studies of bestrophin, we produced both rabbit polyclonal and mouse monoclonal antibodies that proved useful for Western blotting, immunoprecipitation, and immunocytochemistry. To characterize bestrophin, we initially probed the retinal pigment epithelium (RPE)-derived cell lines ARPE-19, D407, and RPE-J. All of the cell lines expressed bestrophin mRNA by reverse transcription-PCR, but not on Western blots. Bestrophin in human RPE partitioned in the detergent phase during Triton X-114 extraction and could be modified by biotin in intact cells, indicative of a plasma membrane localization. Immunocytochemical staining of macaque and porcine eyes indicated that bestrophin is localized at the basolateral plasma membrane of RPE cells. When expressed in RPE-J cells by adenovirus-mediated gene transfer, bestrophin again was determined by confocal microscopy and cell surface biotinylation to be a basolateral plasma membrane protein. The basolateral plasma membrane localization of bestrophin suggests the possibility that bestrophin plays a role in generating the altered electrooculogram of individuals with Best disease.
Subject(s)
Eye Proteins/analysis , Pigment Epithelium of Eye/chemistry , Retinal Degeneration/metabolism , Amino Acid Sequence , Animals , Antibody Formation , Bestrophins , Cell Line , Cell Membrane/chemistry , Chloride Channels , Eye Proteins/genetics , Gene Expression , Humans , Ion Channels , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pigment Epithelium of Eye/cytology , Rabbits , Retinal Degeneration/geneticsABSTRACT
In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814-4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.
Subject(s)
Carrier Proteins/biosynthesis , Neuroglia/metabolism , Pigment Epithelium of Eye/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Retina/metabolism , Transcription Factors , Alternative Splicing , Animals , Cloning, Molecular , Gene Expression Regulation , Humans , Macaca mulatta , Mice , Molecular Sequence Data , Orphan Nuclear Receptors , Promoter Regions, Genetic , Protein Binding , Receptors, Cytoplasmic and Nuclear/genetics , Sequence Analysis, DNA , Tissue Distribution , TransfectionABSTRACT
Vitelliform macular dystrophy (VMD2, Best disease, MIM153700) is an early onset, autosomal, dominant macular degeneration characterized by the deposition of lipofuscin-like material within and below the retinal pigment epithelium (RPE); it is associated with degeneration of the RPE and overlying photoreceptors. Recently, we cloned the gene bestrophin, which is responsible for the disease, and identified a number of causative mutations in families with VMD2. Here, we report that the analysis of bestrophin in a collection of 259 age-related macular degeneration (AMD) patients provides evidence that mutations in the Best disease gene do not play a significant role in the predisposition of individuals to AMD. However, our results suggest that, in addition to Best disease, mutations within the bestrophin gene could be responsible for other forms of maculopathy with phenotypic characteristics similar to Best disease and for other diseases not included in the VMD category.
Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Adult , Age Factors , Age of Onset , Aged , Alleles , Animals , Bestrophins , Chloride Channels , Genetic Variation , Humans , Ion Channels , Macular Degeneration/pathology , Mice , Phenotype , Point MutationABSTRACT
Best's macular dystrophy (BMD), also known as vitelliform macular degeneration type 2 (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration with mainly juvenile onset. BMD is characterized by the accumulation of lipofuscin within and beneath the retinal pigment epithelium. The gene causing the disease has been localized to 11q13 by recombination breakpoint mapping. Recently, we have identified the causative gene encoding a protein named bestrophin, and mutations have been found mainly to affect residues that are conserved from a family of genes in Caenorhabditis elegans. The function of bestrophin is so far unknown, and no reliable predictions can be made from sequence comparisons. We have investigated the bestrophin gene in 14 unrelated Swedish, Dutch, Danish, and Moroccan families affected with BMD and found eight new mutations. Including the previously published mutations, 15 different missense mutations have now been detected in 19 of the 22 families with BMD investigated by our laboratory. Interestingly, the mutations cluster in certain regions, and no nonsense mutations or mutations causing frame-shifts have been identified. Computer simulations of the structural elements in the bestrophin protein show that this protein is probably membrane bound, with four putative transmembrane regions.
Subject(s)
Chromosomes, Human, Pair 11 , Eye Proteins/genetics , Macular Degeneration/genetics , Mutation, Missense , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Bestrophins , Caenorhabditis elegans/genetics , Chloride Channels , Chromosome Mapping , Confidence Intervals , DNA Primers , Exons , Eye Proteins/chemistry , Humans , Molecular Sequence Data , Protein Conformation , Recombination, Genetic , Reference Values , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Zinc is an essential trace element required for structural integrity and functional activity of numerous proteins, yet mechanisms by which cells regulate zinc concentration are poorly understood. Here, we identified a gene from Proteus mirabilis that encodes a 135-amino acid residue protein, PMTR (P. mirabilis transcription regulator), a new member of the MerR family of transcription activators. Transformation of Escherichia coli with PMTR-carrying vectors specifically increases cell tolerance to zinc, suggesting the role of PMTR in zinc homeostasis. In response to zinc, PMTR-containing cells robustly accumulate a 12-kDa protein, the amount of which correlates with the cells' ability to grow at high zinc concentrations. The 12-kDa protein is not induced in the presence of Ni2+, Co2+, Cd2+, Mn2+, or Fe2+, indicating that the PMTR-dependent expression of the 12-kDa protein is specifically regulated by zinc. The 12-kDa protein was identified as the C-terminal fragment of E. coli protein YJAI, and was shown to contain two zinc-binding motifs. Metal-affinity chromatography and 65Zn blotting assay confirmed the ability of the 12-kDa protein to bind zinc specifically (zinc > cobalt >> cadmium). We propose that YJAI is an important component of the zinc-balancing mechanism in E. coli, the up-regulation of which with PMTR results in an increased tolerance to zinc.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Escherichia coli/metabolism , Proteus mirabilis/metabolism , Trans-Activators/metabolism , Zinc/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , DNA, Bacterial , Escherichia coli/growth & development , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Trans-Activators/chemistry , Trans-Activators/geneticsABSTRACT
Best macular dystrophy (BMD), also known as vitelliform macular dystrophy (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration characterized by an abnormal accumulation of lipofuscin within and beneath the retinal pigment epithelium cells. In pursuit of the disease gene, we limited the minimum genetic region by recombination breakpoint analysis and mapped to this region a novel retina-specific gene (VMD2). Genetic mapping data, identification of five independent disease-specific mutations and expression studies provide evidence that mutations within the candidate gene are a cause of BMD. The 3' UTR of the candidate gene contains a region of antisense complementarity to the 3' UTR of the ferritin heavy-chain gene (FTH1), indicating the possibility of antisense interaction between VMD2 and FTH1 transcripts.
Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Amino Acid Sequence , Animals , Base Sequence , Bestrophins , Caenorhabditis elegans/genetics , Chloride Channels , Chromosome Mapping , Cloning, Molecular , DNA Mutational Analysis , DNA, Complementary , Female , Ferritins/genetics , Genes, Overlapping , Humans , Ion Channels , Male , Mice , Molecular Sequence Data , PedigreeSubject(s)
ATP-Binding Cassette Transporters , Adenosine Triphosphatases/biosynthesis , Carrier Proteins/biosynthesis , Cation Transport Proteins , Escherichia coli Proteins , Monosaccharide Transport Proteins , Adenosine Triphosphatases/chemistry , Amino Acid Sequence , Binding Sites , Carrier Proteins/chemistry , Cloning, Molecular , Copper/metabolism , Copper-Transporting ATPases , Escherichia coli , Hepatolenticular Degeneration/enzymology , Humans , Maltose-Binding Proteins , Menkes Kinky Hair Syndrome/enzymology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistryABSTRACT
Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms. Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment. Recently a gene for Wilson disease (WD)(also named the ATP7B gene) was cloned. This gene encodes a copper transporter of the P-type ATPase. We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain. We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol. These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper.
Subject(s)
Adenosine Triphosphatases/genetics , Alternative Splicing , Carrier Proteins/genetics , Cation Transport Proteins , Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , Cell Compartmentation , Copper/metabolism , Copper-Transporting ATPases , Golgi Apparatus/metabolism , Humans , Tumor Cells, CulturedABSTRACT
Wilson disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver and subsequently in the brain and other organs. On the basis of sequence homology to known genes, the WD gene (ATP7B) appears to be a copper-transporting P-type ATPase. A search for ATP7B mutations in WD patients from five population samples, including 109 North American patients, revealed 27 distinct mutations, 18 of which are novel. A composite of published findings shows missense mutations in all exons-except in exons 1-5, which encode the six copper-binding motifs, and in exon 21, which spans the carboxy-terminus and the poly(A) tail. Over one-half of all WD mutations occur only rarely in any population sample. A splice-site mutation in exon 12 accounts for 3% of the WD mutations in our sample and produces an in-frame, 39-bp insertion in mRNA of patients homozygous, but not heterozygous, for the mutation. The most common WD mutation (His1069Glu) was represented in approximately 38% of all the WD chromosomes from the North American, Russian, and Swedish samples. In several population cohorts, this mutation deviated from Hardy-Weinberg equilibrium, with an overrepresentation of homozygotes. We did not find a significant correlation between His1069Glu homozygosity and several clinical indices, including age of onset, clinical manifestation, ceruloplasmin activity, hepatic copper levels, and the presence of Kayser-Fleischer rings. Finally, lymphoblast cell lines from individuals homozygous for His1069Glu and 4 other mutations all demonstrated significantly decreased copper-stimulated ATPase activity.
Subject(s)
Adenosine Triphosphatases/genetics , Carrier Proteins/genetics , Cation Transport Proteins , Hepatolenticular Degeneration/genetics , Mutation , Adult , Base Sequence , Child , Copper-Transporting ATPases , DNA Mutational Analysis , Frameshift Mutation , Gene Frequency , Genes , Genotype , Haplotypes , Hepatolenticular Degeneration/enzymology , Hepatolenticular Degeneration/ethnology , Humans , Molecular Epidemiology , Molecular Sequence Data , Mutagenesis, Insertional , Nucleic Acid Hybridization , Phenotype , Point Mutation , Polymorphism, Restriction Fragment Length , Polymorphism, Single-Stranded Conformational , RNA Splicing , Sequence DeletionABSTRACT
N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).
Subject(s)
Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , Cation Transport Proteins , Copper/metabolism , Recombinant Fusion Proteins , Binding Sites , Cells, Cultured , Copper-Transporting ATPases , Cysteine/metabolism , Hepatolenticular Degeneration/enzymology , Humans , Kinetics , Menkes Kinky Hair Syndrome/enzymology , Models, Molecular , Peptide Fragments/metabolism , Protein Structure, Secondary , SolubilityABSTRACT
The variant form of late infantile neuronal ceroid lipofuscinosis (vLINCL, locus definition CLN5) represents a progressive brain disease with autosomal recessive inheritance. We have previously assigned the CLN5 locus to chromosome 13q21.1-q32 between markers D13S160 and D13S162 by linkage analysis in Finnish families. The information on ancient recombination events obtained from linkage disequilibrium provided an efficient tool for further refining the assignment of the CLN5 locus. Isolation of two novel (CA)n markers, COLAC1 and AC224, resulted in a dramatic restriction of the critical DNA region. We utilized the Fiber-FISH technique to orient and order the large DNA clones isolated by STSs and were able to eliminate almost totally the restriction digestion and PFGE step in the construction of the long-range DNA contig. Both linkage disequilibrium data and Fiber-FISH analyses assigned the CLN5 locus to a well-defined 200-kb region. Here we report a complete physical map of about 350 kb covering the critical chromosomal region of CLN5, which will facilitate the final isolation of the CLN5 gene.
Subject(s)
Chromosome Mapping/methods , Chromosomes, Human, Pair 13/genetics , In Situ Hybridization, Fluorescence/methods , Neuronal Ceroid-Lipofuscinoses/genetics , Base Sequence , Child, Preschool , Chromosome Walking , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 13/ultrastructure , Female , Genetic Markers , Haplotypes , Humans , Image Processing, Computer-Assisted , Linkage Disequilibrium , Male , Molecular Sequence Data , Neuronal Ceroid-Lipofuscinoses/classification , Pedigree , Polymerase Chain Reaction , Recombination, GeneticABSTRACT
In this review we discuss four genetic disorders of copper metabolism. Wilson's disease and Indian childhood cirrhosis result from the toxic effects of copper accumulation in the liver. Menkes' disease and, most likely, occipital horn syndrome result from copper deficiency secondary to disturbances in copper transport. The recent cloning and sequencing of the genes defective in Wilson's disease and Menkes' disease provide the molecular basis for understanding the causes of the two major disorders of copper transport in humans. Mutations that result in Wilson's and Menkes' diseases were shown to disrupt the function of two related P-type copper transporting ATPases. Genetic analysis demonstrates that Wilson's disease and, probably, Menkes' disease are caused by a number of different mutations within a single gene (allelic heterogeneity), and that this occurrence likely explains the clinical heterogeneity of both diseases. The possibility that different mutations within the same gene account for the similar phenotypes of Wilson's disease and Indian childhood cirrhosis on the one hand and for Menkes' disease and occipital horn syndrome on the other are discussed.
Subject(s)
Copper/metabolism , Metabolism, Inborn Errors/genetics , Child , Hepatolenticular Degeneration/genetics , Humans , Menkes Kinky Hair Syndrome/geneticsABSTRACT
Wilson disease is an autosomal recessive disorder of copper transport. Disease symptoms develop from the toxic build-up of copper primarily in the liver, and subsequently in the brain, kidney, cornea and other tissues. A candidate gene for WD (ATP7B) has recently been identified based upon apparent disease-specific mutations and a striking amino acid homology to the gene (ATP7A) responsible for another human copper transport disorder, X-linked Menkes disease (MNK). The cloning of WD and MNK genes provides the first opportunity to study copper homeostasis in humans. A preliminary analysis of the WD gene is presented which includes: isolation and characterization of the 5'-end of the gene; construction of a genomic restriction map; identification of all 21 exon/intron boundaries; characterization of extensive alternative splicing in brain; prediction of structure/function features of the WD and MNK proteins which are unique to the subset of heavy metal-transporting P-type ATPases; and comparative analysis of the six metal-binding domains. The analysis indicates that WD and MNK proteins belong to a subset of transporting ATPases with several unique features presumably reflecting their specific regulation and function. It appears that the mechanism of alternative splicing serves to regulate the amount of functional WD protein produced in brain, kidney, placenta, and possibly in liver.
Subject(s)
Adenosine Triphosphatases/genetics , Cation Transport Proteins , Hepatolenticular Degeneration/genetics , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Alternative Splicing , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Biological Evolution , Brain/enzymology , Chromosome Mapping , Cloning, Molecular , Copper/metabolism , Copper-Transporting ATPases , DNA, Complementary/genetics , Female , Hepatolenticular Degeneration/enzymology , Humans , Ion Transport , Kidney/enzymology , Liver/enzymology , Molecular Sequence Data , Placenta/enzymology , Pregnancy , Structure-Activity Relationship , Tissue DistributionABSTRACT
Wilson disease (WD) is an autosomal recessive disorder of copper transport which map to chromosome 13q14.3. In pursuit of the WD gene, we developed yeast artificial chromosome and cosmid contigs, and microsatellite markers which span the WD gene region. Linkage disequilibrium and haplotype analysis of 115 WD families confined the disease locus to a single marker interval. A candidate cDNA clone was mapped to this interval which, as shown in the accompanying paper, is very likely the WD gene. Our haplotype and mutation analyses predict that approximately half of all WD mutations will be rare in the American and Russian populations.
Subject(s)
Chromosomes, Human, Pair 13 , Haplotypes/genetics , Hepatolenticular Degeneration/genetics , Base Sequence , Cosmids , Family , Female , Genetic Markers , Genomic Library , Genotype , Humans , Linkage Disequilibrium , Male , Molecular Sequence Data , MutationABSTRACT
Wilson disease (WD) is an autosomal recessive disorder characterized by the toxic accumulation of copper in a number of organs, particularly the liver and brain. As shown in the accompanying paper, linkage disequilibrium & haplotype analysis confirmed the disease locus to a single marker interval at 13q14.3. Here we describe a partial cDNA clone (pWD) which maps to this region and shows a particular 76% amino acid homology to the Menkes disease gene, Mc1. The predicted functional properties of the pWD gene together with its strong homology to Mc1, genetic mapping data and identification of four independent disease-specific mutations, provide convincing evidence that pWD is the Wilson disease gene.
Subject(s)
Adenosine Triphosphatases/genetics , Cation Transport Proteins , Copper/metabolism , Hepatolenticular Degeneration/genetics , Menkes Kinky Hair Syndrome/genetics , Sequence Homology, Amino Acid , Amino Acid Sequence , Base Sequence , Copper-Transporting ATPases , Gene Expression , Haplotypes , Humans , Linkage Disequilibrium , Molecular Sequence Data , MutationABSTRACT
We have characterized 21 polymorphic (CA)n microsatellites for the development of a genetic map of chromosome 13. Fifteen markers were isolated from a flow-sorted chromosome 13 library, four CA repeats were derived from NotI-containing cosmid clones, and two polymorphic markers were described previously (J. L. Weber, A. E. Kwitek, and P. E. May, 1990, Nucleic Acids Res. 18: 4638; L. Warnich, I. Groenwald, L. Laubscher, and A. E. Retief, 1991, Am. J. Hum. Genet. 49(Suppl.): 372 (Abstract)). Regional localization for all of the markers was performed by amplification of DNA from five somatic cell hybrids containing different deletions of chromosome 13. Genetic markers were shown to be distributed throughout 6 of the 11 resolvable chromosomal subregions. Using data from nine families provided by the Centre d'Etude du Polymorphisme Humain (CEPH), a framework map of 12 of these 21 markers was developed. Six of the 12 markers form three pairs, with each two members of a pair being tightly linked, such that nine systems of markers can be distinguished. The average heterozygosity of these 12 markers is 0.75. The total length of the sex-averaged map is 65.4 cM (Kosambi), with an average distance of 8.2 cM between systems of markers (eight intervals). Seven remaining markers were placed provisionally into the framework map.