|List |< first << previous [record 41 of 511] next >> last >||
|Coordinate||106,088,491 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||ATPase, Cu++ transporting, alpha polypeptide|
|Synonym(s)||MNK, br, Menkes protein|
|Chromosomal Location||106,027,276-106,124,926 bp (+)|
|MGI Phenotype||Mutations in this gene affect copper metabolism and, depending on the allele, result in abnormal pigmentation, vibrissae, hair, and skeleton. Behavior may be abnormal and defects of collagen and elastin fibers are reported. Some alleles are hemizygous lethal.|
|Amino Acid Change||Isoleucine changed to Threonine|
|Institutional Source||Beutler Lab|
I483T in Ensembl: ENSMUSP00000109186 (fasta)
|Gene Model||predicted sequence gene model|
|Predicted Effect||possibly damaging
PolyPhen 2 Score 0.476 (Sensitivity: 0.89; Specificity: 0.90)
|Phenotypic Category||pigmentation, skin/coat/nails|
|Alleles Listed at MGI|
|Mode of Inheritance||X-linked Recessive|
|Local Stock||Sperm, gDNA|
|Last Updated||05/13/2016 3:09 PM by Stephen Lyon|
|Record Created||09/29/2009 12:00 AM|
The brown phenotype was originally discovered as a visible variant among G3 mice homozygous for mutations induced by N-ethyl-N-nitrosourea (ENU). Hemizygous males and homozygous females from this strain exhibited a brown coat and normal pigmentation of the skin and eyes (Figure 1) (1). Two additional phenotypes emerged while the pigmentation phenotype was being mapped; a wavy coat (see the record for woolly) and hyperactivity. These phenotypes are due to independent mutations. The heterozygous brown females displayed normal black coats.
Examination of the copper content in the brains of brown hemizygotes found that there was a 60% reduction when compared to the wild-type littermates (Figure 2A) (1). The brown mice displayed delayed onset of prion disease compared to wild type littermates following intracranial inoculation with the Rocky Mountain Laboratory (RML) strain of mouse scrapie (Figure 2B) (1). However, the brains of both brown mice and wild type mice contained the proteinase K-resistant, abnormally folded, disease-associated form of cellular prion protein (PrPres), as detected by Western blot analysis (Figure 2C) (1). In addition, the brains of both the wild-type and brown mice exhibited spongiosis (Figure 3, top), damage to pyramidal neurons (as observed from loss of MAP2-immunoreactive dendrites in the neocortex and hippocampus) (Figure 3, center), and astrogliosis (as observed from changes in GFAP staining) (Figure 3, bottom) (1). Further analysis by immunohistochemistry (Figure 4A) and Western blot (Figure 4B-D) demonstrated that the total levels of PrP, and the levels of PrPres were reduced in clinically ill brown animals when compared to wild-type animals (1).
|Nature of Mutation|
To map the brown mutation, the index brown male was outcrossed to C3H/HeN females, and then backcrossed to his F1 daughters. Among 17 offspring, 6 had black coats (1 female, 5 male), and 11 had brown coats (7 female, 4 male). Linkage mapping using 128 polymorphic sites across the genome demonstrated strongest linkage of the brown mutation with the marker DXMit172, flanked by the distal marker DXMit114 and proximal marker DXMit121 on chromosome X (LOD=3.86). This region contained three genes previously described to affect coat pigmentation when mutated (Atp7a, Eda, and Htr2c). Because brown mice did not display ectodermal dysplasia or postnatal grown retardation, which have been observed in Eda and Htr2c mutants, respectively, these two genes were not considered as candidates for causation of the brown phenotype. Capillary sequencing of coding exons and splice junctions of Atp7a revealed a T to C transition at position 1570 of the Atp7a transcript, in exon 5 of 23 total exons.
The mutated nucleotide is indicated in red lettering, and results in an isoleucine to threonine substitution at amino acid 483 of the ATP7A protein.
Please see the record for Tigrou-like for information about Atp7a.
In humans, ATP7A deficiency causes Menkes disease (MD; OMIM #309400) and the milder occipital horn syndrome (OHS; OMIM #304150). Mice with mutations in Atp7a are collectively known as mottled mice after the variegated pigmentation pattern present in heterozygous females. Mottled mice display a wide range of phenotypes, from prenatal death to more subtle defects in hemizygous males (2). Like brown, the classical Blotchy mouse mutant is viable (2). Blotchy mice are considered to be a model of OHS (2). The Blotchy mutation occurs at a splice site resulting in the production of both aberrant and wild type transcripts (3), and low levels of wild type functional protein likely explain the milder phenotype. Male mice carrying another allele of Atp7a on a CBA/J background, known as mottled pewter, display a light gray coat color and no other phenotypes. The Atp7a mutation in mottled pewter mice results in an alanine to threonine substitution at amino acid 998 of ATP7A (4). The mutated alanine resides in the highly conserved transduction sequence CPCSLGLA in the sixth transmembrane domain of ATP7A. The two cysteines in this sequence are responsible for copper binding and the proline mediates the conformational change associated with copper transport. The mild phenotype found in mottled pewter mice, suggests that the substitution of a threonine residue for an alanine residue interferes minimally with ATP7A copper transport.
The brown mutation substitutes a threonine for an isoleucine that lies close to the fifth metal-binding motif in the ATP7A N-terminal region. The mild phenotype found in brown males and absence of phenotype in heterozygous females suggests that the brown mutation results in an ATP7A protein that retains most of its function (1). Although each of the six N-terminal copper-binding motifs in ATP7A is capable of binding to a reduced Cu(I) ion, functional studies suggest that the entire N-terminal region of ATP7A binds a total of four copper ions and that the metal binding sites can be functionally redundant (5-7). However, the fifth and sixth of these motifs appear to be important for ATP7A function because at least one of these two sites needs to bind copper in order for copper transport to occur (8;9). The proximity of the residue affected by the brown mutation to the fifth metal-binding site may alter the efficiency of copper binding to this motif and consequently impair copper transport. In support of this hypothesis, the copper content of the brains of brown mice was reduced by 60% compared to wild type mice (1).
When inoculated with RML scrapie, the onset of clinical signs of scrapie were delayed in brown mice relative to wild type mice. Furthermore, levels of total PrP and PrPres were reduced in brown mice with clinical signs of scrapie compared to similarly affected wild type mice. These data support previous findings indicating that copper chelation delays the onset of scrapie (10), and that copper may induce proteinase resistance of PrP (11-13). However, brown mice displayed extensive neurodegeneration and scrapie symptoms similar in severity to those observed in infected wild type mice, indicating that the reduced levels of PrPres present in brown mice remain sufficient to cause disease and death.
|Primers||Primers cannot be located by automatic search.|
Brown genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change using the same primers used in Tigrou sequencing.
Primers for PCR amplification
Tig(F): 5’- TGATGCCAGGGTACAAATTGTCAGC -3’
Tig(R): 5’- GGTTAGGGCAGCCTAAGTACCAAAC -3’
1) 94°C 2:00
2) 94°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
Tig_seq(F): 5’- GCACAAGAACTGTCTAGTAACTG -3’
Tig_seq(R): 5’- CTAGGCAACTTGGATCTTACAAGG -3’
The following sequence of 1173 nucleotides (from Genbank genomic region NC_000086 for linear DNA sequence of Atp7a) is amplified:
60664 tgatgcc agggtacaaa ttgtcagcat gataaggcta gttaaataca agcacaagaa
60721 ctgtctagta actggttatt ttctactcgg ggttgggttt ggagataata gctagaagaa
60781 tctgacataa ctaaattttt tgtactcact tgagtcagat tttcttggga cccccctgag
60841 gtggacagta aagaaattca aagtcagtgt tgggaatggg taataacaat atatttgttg
60901 agagctttta ggaccttgct gattttatag aaatgcattg gcaggcctag aggtgtggct
60961 gtgacttttg acaaggtgta agctagagaa taaatgaaaa gaacctttct ctctccagca
61021 gacatgaaag agccactggt agtgatagct cagccctcac tggaaacacc tcttttgccc
61081 tcaagtaatg agctagaaaa tgtgatgacg tcagttcaga acaagtgtta catacaggtc
61141 tctgggatga cctgtgcttc ttgtgtagca aacattgaac gcaatttaag acgagaagaa
61201 ggtaagtgtt gttattttta tgtcccttat ttccagattc tgtccaatct gtgttttatg
61261 gccatgcttt gaagtctttc caaggcttcc ttcccaaaga tcatccttga tgaacaatac
61321 atccctgaat ttctggaagt ttttaattag tgttatttct tttacaatcc cattttagtg
61381 ccagtgaaat ctactaaaaa gtttatagca gaaggaaata catagcatta ctattatgag
61441 ccatggctat aatagccttt aagaaactaa attttttgtt aaagctgttt taaaaagtga
61501 taatgaataa gttaggtatt gtcttatcta gattaaacag cagagccaaa ccatatttgt
61561 gtagaattat attgtctcct ctagcagttt gacctctgat ctttccttgt aagatccaag
61621 ttgcctagta ctgtgtattt taacttcagg ttacaaaatc tttgaaaatc aacaccactg
61681 tttttctgta gttgctcaaa tgttttagtc tataaattta tatacatttt ataggtatat
61741 caataatata gcatagtacc ttagttaacc gtataaatat atgattatgt ttaatgagac
61801 caaagtctta agtttggtac ttaggctgcc ctaacc
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
1. Siggs, O. M., Cruite, J. T., Du, X., Rutschmann, S., Masliah, E., Beutler, B., and Oldstone, M. B. A. (2012) Disruption of Copper Homeostasis due to a Mutation of Atp7a Delays the Onset of Prion Disease. Proc Nat Assoc Sci U S A. Aug 6.
3. La Fontaine, S., Firth, S. D., Lockhart, P. J., Brooks, H., Camakaris, J., and Mercer, J. F. (1999) Intracellular Localization and Loss of Copper Responsiveness of Mnk, the Murine Homologue of the Menkes Protein, in Cells from Blotchy (Mo Blo) and Brindled (Mo Br) Mouse Mutants. Hum. Mol. Genet.. 8, 1069-1075.
4. Levinson, B., Packman, S., and Gitschier, J. (1997) Mutation Analysis of Mottled Pewter. Mouse Genome. 95, 163-165.
5. Jensen, P. Y., Bonander, N., Horn, N., Tumer, Z., and Farver, O. (1999) Expression, Purification and Copper-Binding Studies of the First Metal-Binding Domain of Menkes Protein. Eur. J. Biochem.. 264, 890-896.
6. Cobine, P. A., George, G. N., Winzor, D. J., Harrison, M. D., Mogahaddas, S., and Dameron, C. T. (2000) Stoichiometry of Complex Formation between Copper(I) and the N-Terminal Domain of the Menkes Protein. Biochemistry. 39, 6857-6863.
7. Voskoboinik, I., Strausak, D., Greenough, M., Brooks, H., Petris, M., Smith, S., Mercer, J. F., and Camakaris, J. (1999) Functional Analysis of the N-Terminal CXXC Metal-Binding Motifs in the Human Menkes Copper-Transporting P-Type ATPase Expressed in Cultured Mammalian Cells. J. Biol. Chem.. 274, 22008-22012.
8. Lutsenko, S., Barnes, N. L., Bartee, M. Y., and Dmitriev, O. Y. (2007) Function and Regulation of Human Copper-Transporting ATPases. Physiol. Rev.. 87, 1011-1046.
9. de Bie, P., Muller, P., Wijmenga, C., and Klomp, L. W. (2007) Molecular Pathogenesis of Wilson and Menkes Disease: Correlation of Mutations with Molecular Defects and Disease Phenotypes. J. Med. Genet.. 44, 673-688.
10. Sigurdsson, E. M., Brown, D. R., Alim, M. A., Scholtzova, H., Carp, R., Meeker, H. C., Prelli, F., Frangione, B., and Wisniewski, T. (2003) Copper Chelation Delays the Onset of Prion Disease. J. Biol. Chem.. 278, 46199-46202.
11. Hornshaw, M. P., McDermott, J. R., and Candy, J. M. (1995) Copper Binding to the N-Terminal Tandem Repeat Regions of Mammalian and Avian Prion Protein. Biochem. Biophys. Res. Commun.. 207, 621-629.
12. Hornshaw, M. P., McDermott, J. R., Candy, J. M., and Lakey, J. H. (1995) Copper Binding to the N-Terminal Tandem Repeat Region of Mammalian and Avian Prion Protein: Structural Studies using Synthetic Peptides. Biochem. Biophys. Res. Commun.. 214, 993-999.
|Science Writers||Nora G. Smart, Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||Owen M. Siggs, Bruce Beutler|
|List |< first << previous [record 41 of 511] next >> last >||