Mutation analysis provides additional proof that mottled is the mouse homologue of Menkes' disease
Mutation analysis provides additional proof that mottled is the mouse homologue of Menkes' diseaseVivienne Reed and Yvonne Boyd*
MRC Mammalian Genetics Unit, Harwell, Oxon OX11 ORD, UK
Received October 28, 1996;Revised and Accepted November 28, 1996
Menkes' disease (MD) and occipital horn syndrome (OHS) are allelic X-linked disorders caused by mutations in the copper ion transporting ATPase, ATP7A. Genetic, phenotypic and biochemical data suggest that mottled mutants in the mouse, which range in severity and phenotype, are caused by mutations in Atp7a, the mouse homologue of ATP7A. As the only causal mutation in Atp7a has been reported in one very mild allele thought to be a model for OHS, Atp7aMo-blo (mottled blotchy), we sequenced the entire 4.5 kb coding region of three other mottled mutants, two of which are thought to be models for classical MD (AtpaMo-br, AtpaMo-13H) and one with a slightly milder phenotype (Atp7aMo-vbr). Although no causal mutation was found in Atp7aMo-13H, mutations which can be predicted to affect Atp7a function were identified in Atp7aMo-br and Atp7aMo-vbr. A 6 bp deletion of nucleotides 2478-2483, which can be predicted to affect the correct processing of the protein, was found in Atp7aMo-br and an A3189 -> C nucleotide change, which results in lysine -> threonine amino acid substitution in the phosphorylation domain, was found in Atp7aMo-vbr. Thus we provide further proof that mottled mutants will provide excellent models for MD as well as OHS.
Menkes disease (MD) is an X-linked disorder, in which affected males die during the first few years of life, and is characterised by growth retardation, peculiar hair, hypopigmentation and neurological and connective tissue abnormalities (1 ,2 ). The underlying cause is a defect in the process by which dietary copper is distributed to dependent enzymes and proteins and as a result the overall clinical picture is one of copper deficiency. In 1993, exploitation of X chromosome rearrangements which disrupted the MD locus led to the cloning of the gene responsible (3 -5 ). It was shown to encode a P-type metal ion ATPase which had considerable homology to bacterial transporting ATPases. Qualitative and quantitative changes in ATP7A RNA levels were reported in many patients (3 -5 ) and 20% of patients were found to possess large gene deletions (6 ). The ATP7A transcript is 8.5 kb and covers >100 kb of genomic DNA (3 -5 ). The 4.5 kb coding region is organised into 23 exons with the first exon, which is untranslated, separated from exon 2 by an intron of ~40 kb (6 ,7 ). Mutation analyses of 12 patients with severe MD identified 10 independent lesions, six splicing abnormalities which were distributed throughout the gene and four basepair substitutions or small deletions (8 ). Aberrant splicing of ATP7A was also shown to be responsible for the phenotype of three independent patients with the milder X-linked disorder cutis laxa, or occipital horn syndrome (OHS), thus providing formal proof that MD and OHS are allelic (9 ,10 ).
Because of phenotypic and biochemical similarities and of comparative map position, the X-linked mouse mutant mottled was proposed as the homologue of MD (11 ,12 ). Many mottled mutants have arisen both spontaneously and after chemical and radiation mutagenesis and all are assumed to be alleles on the basis of their X-linked inheritance and their similar phenotypes (13 ). The one exception is yellow mottled (Ym) which lies close to mottled but which has been separated from it by recombination (14 ). However, there are phenotypic and severity differences between the various mottled alleles which enables them to be assigned into one of three groups. The first group of alleles have a phenotype similar to OHS in that affected males suffer mainly from connective tissue problems and are viable and fertile, for example mottled blotchy (13 ). The second group of alleles, of which mottled brindled (13 ) is an example, have severe neurological problems and are thought to be the most similar to classical MD as affected males die ~7-10 days after birth, with the exception of mottled viable brindled, in which affected males survive beyond weaning. The third group of alleles can be represented by mottled dappled (15 ), have been reported to have skeletal abnormalities and are the most severely affected with mutant males dying in utero. Although phenotypic symptoms vary between alleles, copper tissue levels and the kinetics of cellular copper accumulation are similar in all alleles tested and are the same as seen in MD patients (12 ,16 , Masson et al., in preparation). As mouse mutants can be maintained on identical genetic backgrounds, mottled alleles provide an unprecedented opportunity to analyse the phenotypic effect of different mutationsin Atp7a.
Atp7a, the mouse homologue of ATP7A, was cloned in 1994 and has a high degree of homology to the human locus (17 -19 ). A genomic deletion was reported as the mutational event in the dappled allele (17 ,18 ); however, this was pointed out to be a strain-specific restriction fragment length variant (20 ). Furthermore, a search for deletions in a range of 12 mottled alleles using both pulsed field and conventional gel electrophoresis failed to detect any genomic rearrangements in Atp7a despite the fact that several of the mutations arose in radiation mutagenesis experiments (21 ,22 ). There is some molecular evidence that mutations in Atp7a are the cause of the mottled phenotype. A splice site mutation, which leads to the occasional skipping of exon 11 and a subsequent frameshift of the remainder of the transcript, has been demonstrated in mottled blotchy (9 ) and Atp7a transcripts cannot be detected in embryonic fibroblasts of mottled dappled origin (17 ,18 ). However, a significant, although reduced amount of the normal transcript is present in addition to the truncated form in mottled blotchy (7 -9 ), and no genomic mutation has yet been described in mottled dappled. To provide additional proof that mutations in Atp7a cause the mottled phenotype, we have sequenced the entire Atp7a coding region in three alleles of mottled and confirmed that the splice site mutation reported in mottled blotchy is not present in 15 other strains. The alleles chosen for sequencing were one which resembles the clinical picture seen in mild Menkes' disease and two in which affected males die ~10 days after birth and are thought to most closely resemble classical Menkes' disease.
The Atp7a coding region was amplified in four overlapping segments using RT-PCR and primers designed from the published mouse cDNA sequence (accession number U03434). For all four segments, products of equal size and intensity were amplified from RT products prepared from affected males and control male littermates from stocks that carried eight independent mottled alleles: Atp7aMo-blo (mottled blotchy), Atp7aMo-pew (mottled pewter), Atp7aMo-vbr (mottled viable brindled), Atp7aMo-br (mottled brindled), Atp7aMo-brJ (mottled brindledJ), Atp7aMo-8J (mottled 8J), Atp7aMo-13H (mottled 13H) (Fig. 1 ). In addition to the expected product, smaller allele-specific products were consistently seen in segment 1 reactions amplified from mottled blotchy, brindled and 13H (Fig. 1 ). Both larger and smaller products were amplified in segment 3 reactions when mottled blotchy cDNA was used as a template as described previously (9 ). All RT-PCR products were shown to hybridise to relevant portions of human ATP7A cDNA and this (and subsequent sequence analysis) confirmed that they had been amplified from the Atp7a locus (data not shown). When the smaller segment 1 products were cloned and sequenced they were found to contain deletions which ran from nucleotides 137-991 (blotchy), 267-335 (brindled) and 440-1162 (mottled 13H). In each case, a unique sequence identity of 4-9 bp was found at both ends of the deletion. Sequence analysis of these products, the normal RT-PCR product and the full genomic exonic and intronic DNA regions deleted revealed that there were no causal genomic changes in the deleted region and it was assumed that these RNA fragments resulted from a perturbation of the normal splicing of the primary transcript by a downstream mutation. A similar situation has been described at the DHPR locus (23 ). It is unclear whether any of the aberrant segment 1 products, which were never observed in controls, has a direct effect on Atp7a transcription.
As Atp7a was transcribed in all alleles and no products which could be attributed to aberrant splicing were observed other than in blotchy (see below), it was likely that any mutational lesions would be small changes in the coding sequence. We therefore undertook to sequence the entire Atp7a coding region in four mutants, two of which were thought to be appropriate models for classical MD (brindled and 13H), one of intermediate severity (viable brindled) and mottled blotchy which is thought to be an appropriate model for OHS.
Discrepancies from the published mouse Atp7a sequence were considered significant if they led to nonsense or frameshift mutations, or when they led to amino acid substitutions different from those published in the mouse or human protein sequences. Five such changes from the published sequence were found with two of these associated with strain-specific amino acid substitutions. A T1491 -> C nucleotide change which led to the substitution of a leucine by a proline at residue 71 was found in stocks of C3H/HeH, 101/H and Mus spretus origin and an A3840 -> G nucleotide change which led to the substitution of a glutamine by an arginine at residue 1254 was found in stocks of C57BL/6 origin. The remaining three significant changes were specific to the mottled stock being analysed.
Two significant nucleotide changes were found in RT-PCR and genomic DNA products amplified from mottled brindled. A G1619 -> A nucleotide change was found which results in the substitution of alanine by threonine at position 514. This change lies in the interval between the last two copper binding domains which is significantly different in mouse and man and therefore is unlikely to be a critical region for protein function. A better candidate for the causal mutation is the 6 bp deletion found when sequencing the cloned RT-PCR containing exon 11 from mottled brindled (Fig. 2 ). The deletion removes nucleotides 2478-2483, which encode a leucine and an alanine, but leaves the remainder of the transcript in frame. To verify that this change was a genuine mutation and unique to mottled brindled, primers (MO3eF and MO3int) were designed to amplify exon 11 from genomic DNA. Amplification products of the expected size of 195 bp were generated in PCR reactions from DNA prepared from affected males of the five other mottled alleles and three strains of mice, including the strain of origin C57BL/6 (21 ). Only when DNA from mottled brindled males was used as a template was a smaller size (189 bp) fragment produced (Fig. 2 ).
An A -> C substitution was found at nucleotide 3189 when the amplification product of segment 3 from mottled viable brindled was sequenced (Fig. 2 ). Nucleotide 3189 lies in exon 16 which can be amplified directly from genomic DNA using primers Moex16F and Moex16R. This nucleotide change was present in no other sequence data obtained from RT-PCR or genomic DNA products. It was also found in genomic DNA amplified from mottled viable brindled, and as no other unique change was found in the entire coding sequence, we suggest that this is the molecular lesion responsible for the mottled brindled phenotype. This transition results in a lysine (K) residue being replaced by a threonine (T) in the phosphorylation domain of the ATPase (see below).
The strain-specific change T1491 -> C, which caused the substitution of a leucine by a proline, was identified during the sequencing of clones from two independent RT-PCR reactions using DNA from mottled 13H as a template and was initially a candidate for a causal mutation until it was found to be present in the inbred mouse strains C3H/HeH and 101/H (data not shown). As this amino acid substitution introduces a proline into the region between the 4th and 5th metal binding sites of Atp7a, we can conclude that the tertiary structure of protein in this region is not critical for efficient copper binding and delivery. No other sequence alteration was found in the entire coding region of mottled 13H and we therefore suggest that the causal mutation lies outside the coding region.
During the course of sequencing cloned RT-PCR products from the mottled blotchy mutant, a +3 splice-donor mutation was reported in exon 11 of this mutant (9 ). As the strain of origin for mottled blotchy is unknown, we confirmed that the published A -> C substitution was unique to mottled blotchy by testing for the presence of the diagnostic AvaII site in 15 additional mottled alleles (data not shown) and 12 additional strains/species of mice (Fig. 2 ). We therefore did not sequence segments 2-4 from this mutant.
For some time, mottled has been thought to be the mouse homologue of Menkes disease and the identification of a molecular lesion in Atp7a in three independent mottled mutants now provides further proof that this is the case (Table 1 ). The two novel lesions described here can be predicted to have an effect on the function of this P-type ATPase. The characteristic feature of the superfamily of P-type ATPases is the use of the energy of ATP hydrolysis to pump substrate across a membrane (24 -26 ). Atp7a has the basic domain structure of this group of proteins, with multiple copper binding motifs at the N-terminal end of the protein, followed by a transmembranous region and two cytoplasmic domains, a small one thought to be responsible for energy transduction and a larger one which contains highly conserved phosphorylation and ATP binding motifs (Fig. 3 ). The new mutations reported here lie in these two cytoplasmic domains.
Mottled blotchy (Atp7aMo-blo), mottled brindled (Atp7aMo-br), mottled viable brindled (Atp7aMo-vbr), and mottled 13H (Atp7aMo-13H), were maintained by mating Mo/+ females to 3H1 males, where 3H1 is an F1 hybrid stock produced by mating C3H/HeH females to 101/H males. Frozen tissues dissected from mottled pewter (Atp7aMo-pew), mottled brindled J (Atp7aMo-brJ), and mottled 8J (Atp7aMo-8J), were obtained from Hope Sweet and Muriel Davisson of the Jackson laboratory. Affected males from Atp7aMo-blo and AtpMo-pew have normal viability and fertility, and those from Atp7aMo-vbr have a reduced viability and are generally infertile. Affected males from the other mutant stocks (Atp7aMo-br, Atp7aMo-brJ, Atp7aMo-8J) all die ~7-12 days after birth. Mutant mottled alleles are described in refs 13 and 21 , except for mottled 8J which is a recent remutation discovered at the Jackson Laboratory.
Individual mouse tissues were dissected, flash frozen and stored in liquid nitrogen until needed. RNA was prepared from pulverised tissues by treatment with RNAzol (Biogenesis Ltd.). Nested RT-PCR was performed using brain RNA as a template for four overlapping segments of Atp7a (Fig. 1 ) using the appropriate primers (Table 2 ) in a standard PCR buffer (1.5 mM MgCl2) containing AmpliTaq (Perkin Elmer).
An initial round of PCR (30 cycles) was carried out using primers equivalent to the first and last ~20 bp for each segment, i.e. Mnk1aF/Mnk1bR for segment 1, Mnk2aF/Mnk2bR for segment 2, Mnk3aF/Mnk3bR for segment 3 and Mnk 4aF/Mnk4bR for segment 4. Amplification products from these reactions were then re-amplified (20 cycles of PCR) using nested primers as follows: Mnk1cF/Mnk1dR for segment 1, Mnk2cF/Mnk2bR for segment 2, Mnk3cF/Mnk3dR for segment 3 and Mnk4cF/Mnk4bR for segment 4.
RT-PCR products from sections 1 and 4 were cloned directly into the EcoRV site of pBluescript but segments 2 and 3 could not be cloned directly therefore additional RT-PCR reactions were designed to yield smaller products which were cloned and sequenced in the same way as the larger segments. Sequencing was performed on purified double-stranded DNA using a Pharmacia T7 sequencing kit and both universal primers and internal primers designed from the mouse cDNA sequence.
All significant nucleotide changes (i.e. all those which would lead to nonsense, frameshift or missense mutations) were checked routinely by two or more of the following methods: an independent clone from the same PCR reaction was sequenced to identify PCR errors which occurred late in the amplification reaction, products from independent RT-PCR reactions were cloned and sequenced to identify PCR errors which occurred early in the amplification reaction, the same RT-PCR product was cloned and sequenced from control strains including the known or probable strain of origin and finally, primers were designed to amplify the equivalent genomic region, and the amplification product was cloned and sequenced. During the sequencing of a total of ~14 kb of DNA, 16 significant changes could be attributed to infidelities of the PCR reaction. Five nucleotide changes (C214 -> A, G389 -> A, G597 -> T, C1626 -> T and G3733 -> T) were found in all mottled stocks and control strains sequenced. The resultant amino acid changes substituted residues which were the same as that reported in the human protein sequence, and therefore these nucleotide changes were assumed to be errors in the published sequence or strain specific differences between Balb/c, the origin of the Atp7a published sequence, and the control strains used in this study (C57BL/6 and C3H/HeH). Four nucleotide changes were found which caused no change to the amino acid residues and these were assumed to be conservative strain/stock-specific changes.
Probes were labelled with [32P]dCTP by nick-translation or multipriming using commercial kits (Amersham International) and hybridised to DNA blotted onto Hybond-N+ membranes using a standard hybridisation solution.
We are grateful to Drs Steve Hyde and Deborah Gill for helpful discussions and to Hope Sweet and Dr Muriel Davisson (Jackson Laboratory) for supplying frozen tissues from Atp7aMo-pew, Atp7aMo-brJ and Atp7aMo-8J animals. We thank Hester Hughes for assistance with animal care and Kevin Glover for assistance with photography.
1 Menkes,J.H., Alter,M., Steigleder,G.K., Weakley,D.R. and Sung,J.H. (1962) A sex-linked recessive disorder with retardation of growth, peculiar hair and focal cerebral and cerebellar degeneration. Pediatrics29, 764-769.
Horn,N., Tønnesen,T. and Tümer,Z. (1992) Menkes disease: an X-linked neurological disorder of the copper metabolism. Brain Pathol.2, 351-362.
Chelly, J., Tümer,Z., Tønnesen,T., Petterson,A., Ishikawa-Brush,Y., Tommerup,N., Horn,N. and Monaco,A.P. (1993) Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. NatureGenet. 3, 14-19.
Mercer,J.F.B., Livingston,J., Hall,B., Paynter,J.A., Begy,C., Chandrasekharappa,S., Lockhart,P., Grimes,A., Bhave,M., Siemieniak,D. and Glover,T.W. (1993) Isolation of a partial candidate gene for Menkes' disease by positional cloning. NatureGenet. 3, 20-25.
Vulpe,C., Levinson,B., Whitney,S., Packman,S. and Gitschier,J. (1993) Isolation of a candidate gene for Menkes disease and evidence that encodes a copper-transporting ATPase. NatureGenet. 3, 7-13.
Tümer,Z., Vural,B., Tønnesen,T., Chelly,J., Monaco,A.P. and Horn,N. (1995) Characterization of the exon structure of the Menkes disease gene using vectorette PCR. Genomics26, 437-442.
Dierick,H.A., Ambrosini,L., Spencer,J., Glover,T.W. and Mercer,J.F. (1995) Molecular structure of the Menkes Disease gene (ATP7A). Genomics28, 462-469.
Das,S., Levinson,B., Whitney,S., Vulpe,C., Packman,S. and Gitschier,J. (1994) Diverse mutations in patients with Menkes disease often lead to exon skipping. Am. J. Hum. Genet. 55, 883-889.
Das,S., Levinson,B., Vulpe,C., Whitney,S., Gitschier,J. and Packman,S. (1995) Similar splicing mutations of the Menkes/mottled copper transporting ATPase gene in occipital horn syndrome and the blotchy mouse. Am. J. Hum. Genet. 56, 570-576.
10 Kaler,S.G., Gallo,L.K., Proud,V.K., Percy,A.K., Mark, Y., Segal,N.A., Goldstein,D.S., Holmes,C.S. and Gahl,W.A. (1994) Occipital horn syndrome and mild Menkes phenotype associated with splice site mutations at the MNK locus. Nature Genet. 8, 195-202.
11 Danks,D.M. (1977) Copper transport and utilisation in Menkes' syndrome and in mottled mice. Inorg. Perspect. Biol. Med. 1, 73-100.
12 Hunt,D.M. (1974) Primary defect in copper transport underlies mottled mutants in the mouse. Nature249, 852-854.
13 Green,M.C. (1995) Catalog of mutant genes and polymorphic loci. In Rastan,S., Lyon,M.F. and Brown,S.D.M. (Eds) Genetic Variants and Strains of the Laboratory Mouse, Oxford University Press, Oxford, UK.
15 Phillips,R.J.S. (1961) `Dappled', a new allele at the mottled locus in the house mouse. Genet. Res. 2, 290-295.
16 Horn, N. (1989) Menkes X-linked disease: prenatal diagnosis of hemizygous males and heterozygous females. Prenat. Diagn. 1, 107-120.
17 Levinson,B., Vulpe,C., Elder,B., Martin,C., Verley,F., Packman,S. and Gitschier,J. (1994) The mottled gene is the mouse homologue of the Menkes disease gene. Nature Genet. 6, 369-373.
18 Mercer,J.F.B., Grimes,A., Ambrosini,L., Lockhart,P., Paynter,J.A., Dierick,H. and Glover,T.W. (1994) Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. NatureGenet. 6, 374-378.
19 Cecchi,C. and Avner,P. (1996) Genomic organisation of the mottled gene, the mouse homologue of the human Menkes' disease gene. Genomics 37, 96-104.
20 Reed,V. and Boyd,Y. (1994) Mutations reported in mottled dappled are RFLVs. NatureGenet.8, 11-12.
21 George,A.M., Reed,V., Glenister,P., Tümer,Z., Horn,N., Chelly,J., Monaco,A.P. and Boyd,Y. (1994) Analysis of Mnk, the murine homologue of the locus for Menkes disease, in normal and mottled (Mo) mice. Genomics22, 27-35.
22 Masson,W., Holt,S., Reed,V. and Boyd,Y. (1996) The use of compound heterozygotes and Hprt selection to analyze X-linked mottled alleles associated with prenatal male lethality. Mamm. Genome7, 486-489.
23 Smooker,P.M., Christodoulou,J., McInnes,R.R. and Cotton,R.G.H. (1995) A mutation causing DHPR deficiency results in a frameshift and a secondary splicing defect. J. Med. Genet. 32, 220-223.
24 Higgins,C.F. (1992) ABC transporters: from microorgansims to man. Annu. Rev. Cell Biol.8, 67-113.
25 Solioz,M. and Vulpe,C. (1996) Cpx-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biol. Sci.521, 237-241.
26 Møller,J.V., Juul,B., le Maire,M. (1996) Structural organisation, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta1286, 1-51.
27 Kerem,B-S., Zielenski,J., Markiewicz,D., Bozon,D., Gazit,E., Yahav,J., Kennedy,D., Riordan,J.R., Collins,F.S., Rommens,J.M. and Tsui,L-C. (1990) Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the ccystic fibrosis gene. Proc. Natl. Acad. Sci. USA87, 8447-84451.
28 Welsh,M.E. and Smith,A.E. (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell73, 1251-1254.
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