Human Molecular Genetics, 2000, Vol. 9, No. 12 1865-1871
© 2000 Oxford University Press
Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy
Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK, 1Department of Neuroscience, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex CM19 5AW, UK, 2MRC Mammalian Genetics Unit and UK Mouse Genome Centre, Harwell, Oxon OX11 ORD, UK, 3Department of Biotechnology and Genetics, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Harlow, Essex CM19 5AW, UK, 4Centre for Cutaneous Research, Queen Mary and Westfield College, London E1 4NS, UK, 5Department of Neurogenetics, Imperial College, London SW7 2AZ, UK and 6Department of Histopathology, Queen Mary and Westfield College, London E1 4NS, UK
Received 7 April 2000; Revised and Accepted 29 May 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1–2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypomyelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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As the sequencing phase of the Human Genome Project nears completion, the need for new approaches to tackle gene function analysis in a mammalian system is abundantly clear. Large-scale mutagenesis in the mouse is set to offer a solution to this requirement by providing a comprehensive resource of mouse mutants linking gene with phenotype. We have recently initiated a large-scale N-ethyl-N-nitrosurea (ENU) mutagenesis project in the mouse for this purpose (1).
ENU has been shown to be a potent mutagen in the mouse (2) that creates mainly point mutations; a male mouse injected with ENU can produce many mutant offspring. A screen for dominant phenotypes allows mutants to be identified in the first generation of progeny of an injected male. To identify and characterize mutant phenotypes generated from our mutagenesis programme, the SHIRPA [SmithKline Beecham Pharmaceuticals, Harwell MRC Mouse Genome Centre and Mammalian Genetics Unit, Imperial College School of Medicine (St Mary’s) Royal London Hospital, St Bartholomew’s and the Royal London School of Medicine Phenotype Assessment] protocol, a wide-ranging and sensitive screen for mouse mutants (3), is used. If a phenotype is shown to be stably inherited, the gene involved can then be positionally cloned. The ability to rapidly identify the underlying genetic mutation will be crucial to the success of large-scale mutagenesis programmes as functional genomics tools. We have identified mutations in two mouse mutants generated from our programme using a mapping strategy involving in vitro fertilization (IVF) to quickly produce sufficient numbers of back-cross mice followed by pooling of the mouse DNA to facilitate a rapid genome scan.
Large-scale mutagenesis will be an important tool for generating better mouse models of human diseases. This phenotype-driven approach will produce models that closely resemble human disorders without any assumptions concerning the genes involved. This should allow new biological pathways involved in disease aetiology and pathogenesis to be uncovered. The mutants described here, trembler-m1H (Tr-m1H) and trembler-m2H (Tr-m2H), two mice with a clinically relevant resting tremor phenotype, are shown to have mutations in the peripheral myelin protein 22 (Pmp22) gene. Pmp22 is a constituent of peripheral nerve myelin, and mutations in human PMP22 can cause the peripheral neuropathies Charcot–Marie–Tooth disease (CMT), Dejerine Sottas syndrome (DSS) and hereditary neuropathy with liability to pressure palsies (HNPP) (4). The characterization of new mutations in Pmp22 indicates the potential of large-scale mutagenesis screens for developing animal models of human disease.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tr-m1H and Tr-m2H phenotype
Tr-m1H and Tr-m2H were identified from the progeny of two separate male BALB/c mice injected with ENU and mated with C3H/HeH females. Both mice exhibited a marked resting tremor and were smaller than littermates at weaning, remaining so into adulthood. Tr-m1H was more severely affected, having fits and a more marked tremor. Mutant lines were established by back-crossing both mutants to C3H/HeH, and the SHIRPA protocol (3) used to more fully assess the behavioural and functional phenotype of each mutant (Table 1). Both lines were inactive, showed low locomotor activity and had abnormal gaits with low body position and splayed legs. Both lines failed the wire manoeuvre, an indicator of truncal and limb strength and also performed poorly on an accelerating rotarod, an indicator of motor coordination (5), on which Tr-m1H showed a greater deficit. Both had defects in grip strength, with Tr-m1H again more severely affected. On tail suspension, both exhibited limb grasping, with Tr-m1H exhibiting a particularly vigorous extension and flexion of the hind limbs. No sensory deficit was evident, all digits were normal and the toe pinch response was unimpaired. These results indicate some impairment of muscle or motor function and provide a semi-quantitative measure of the difference in severity of the phenotypes of Tr-m1H and Tr-m2H.
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Hypomyelination of Tr-m1H and Tr-m2H peripheral nerve
Histopathological analysis of Tr-m1H and Tr-m2H showed the morphology of the brain and spinal cord to be normal and Luxol fast blue staining revealed no gross loss of central nervous system myelin (data not shown). Peripheral nerves were examined by light and electron microscopy (Fig. 1). Hypomyelination was apparent in both lines, the few myelin sheaths present were thin and surrounded axons of small diameter. There was a decrease in the number of axonal profiles and an increase in the amount of endoneurial connective tissue. The number and size of Schwann cell nuclei was also increased. The pathology of Tr-m1H was more severe than that of Tr-m2H, having fewer, generally thinner myelin sheaths, mirroring the severity of the phenotype.
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Tr-m1H and Tr-m2H map between D11Mit131 and D11Mit177
IVF (6) of C3H/HeH eggs with sperm from Tr-m1H and Tr-m2H mice was used to speed the generation of a significant number of back-cross progeny. The resulting offspring were scored for the tremor phenotype and an autosomal dominant inheritance pattern with complete penetrance confirmed in each case. Equimolar aliquots of DNA from Tr-m1H (n = 45) or Tr-m2H (n = 44) mutant back-cross mice were combined, and duplicate DNA pools scanned with 229 CA repeat markers multiplexed into 23 panels, spanning the mouse genome with an average spacing of 10 cM. By measuring the peak heights of fluorescently labelled alleles using a modified version of TrueAllele genotyping software (7), we compared BALB/c:C3H/HeH allele ratios in each DNA pool. The expected signal ratio for the two alleles of an unlinked marker is 1 BALB/c:3 C3H/HeH in back-cross mice. As a recombination fraction of zero between the mutant gene and marker is approached, the signal ratio approaches 1:1 (Fig. 2). Differential amplification of BALB/c and C3H/HeH alleles was accounted for by comparison with individual F1 heterozygote genotypes and appropriate adjustment of signal strength. Linkage for both Tr-m1H and Tr-m2H was detected on chromosome 11, with the minimal region bounded by markers D11Mit131 and D11Mit177. Genotyping of individual mice confirmed the pooled data and identified recombinants between the mutant phenotype and D11Mit131 [25.1 cM from the centromere (8)] and D11Mit177 [35.0 cM from the centromere (8)], thus defining a 9.9 cM minimal interval (Fig. 3). No recombinants were identified with intervening marker D11Mit242 (31.7 cM). Pmp22 lies 30.6 cM from the centromere (9).
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Tr-m1H and Tr-m2H have mutations in Pmp22
The genomic structure of Pmp22 was determined by direct bacterial artificial clone (BAC) clone sequencing across exon–intron junctions using the human PMP22 gene structure (10) to predict splice sites. Pmp22 exons 2–5 (exon 1 is non-coding) were PCR amplified and sequenced from three mutant back-cross mice from each tremor strain together with the parental BALB/c and C3H/HeH strains. An A
G transition in codon 12 resulting in a HisArg amino acid substitution was identified in all Tr-m1H mutant mice (Fig. 4). A HisGln mutation has been identified at this site in an individual suffering DSS (11), a demyelinating neuropathy similar to (but more severe than) CMT type 1 (CMT1), which also results from PMP22 point mutations in some cases (4). Tr-m1H may therefore prove a more accurate mouse model for DSS than those currently available. An AT transversion in codon 153 resulting in a premature stop codon truncating the Pmp22 protein by seven amino acids was identified in all Tr-m2H mutant mice (Fig. 4). The truncation removes the last three amino acids of the fourth transmembrane domain and final four amino acids of the intracellular C-terminal domain. Parental strains had wild-type alleles at both positions implying that both mutations were a result of ENU administration. Both mutations are different from the three previously identified spontaneous Pmp22 mouse mutants (12–14) (Table 2) and are the only Pmp22 mutants on a common genetic background.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PMP22 alterations are responsible for a significant proportion of cases of dominantly inherited hereditary peripheral neuropathy. CMT1 is the most common form of hereditary peripheral neuropathy and its largest subtype, CMT type 1A (CMT1A), is caused by either a 1.5 Mb duplication of a region of chromosome 17p11.2 that contains PMP22, or PMP22 point mutations (4). DSS, which is characterized by an early age of onset and a more severe phenotype than CMT1A, can also be caused by point mutations in PMP22 (4). This varying degree of severity in human peripheral neuropathies associated with PMP22 mutations is mirrored by both the Pmp22 mouse mutants described here and previously described mutants.
Pmp22Tr-m1H causes a severe tremor phenotype with seizures, as does Pmp22Tr (12). Pmp22Tr-m2H leads to a milder phenotype with less severe tremors and no seizures. Pmp22Tr-J results in a milder phenotype and pathology than Pmp22Tr and lacks seizures (15) and Pmp22Tr-Ncnp is reported to produce a gait abnormality (14). The severe Pmp22Tr and Pmp22Tr-m1H mutations affect the same amino acids altered in DSS (16), while the less severe Pmp22Tr-J mutation has been found in patients with CMT1A (17), indicating that the correlation between genotype and phenotype of PMP22 mutations is well conserved between mice and humans. How this genotype–phenotype correlation relates to the function of PMP22 remains unclear.
The existence of Schwann cells in early myelin forms, as reported here and in the other trembler mutants (14,15) together with the delay of onset of myelination followed by severe demyelination in Pmp22 deficient mice (18) suggests a role for Pmp22 in the initiation of myelination and myelin maintenance. Several studies have shown that Pmp22Tr, Pmp22Tr-J and several CMT1A and DSS mutations impair trafficking of mutant PMP22 leading to its accumulation in intracellular compartments, resulting in failure to reach its normal location at the plasma membrane (19–22). HNPP, the least severe of the human neuropathies discussed here, can result from complete loss of one copy of PMP22 (23) suggesting that PMP22 haploinsufficiency can cause disease. As CMT1A frequently results from a duplication resulting in an additional copy of PMP22 (24), PMP22 stoichiometric imbalance appears to be sufficient for pathogenesis, with excess PMP22 giving the more severe phenotype. It has been reported that PMP22 plays a structural role in the myelin membrane, where it binds peripheral myelin protein zero, a major constituent of peripheral nerve myelin (25); therefore an inappropriate level of PMP22 may result in loss of myelin membrane integrity and consequent neuropathy.
However, simple loss of function of one copy of PMP22 cannot explain why a more severe phenotype can result from a point mutation of one PMP22 allele than from complete loss of one copy of the PMP22 gene. It has been suggested that mutant PMP22 can dimerize with wild-type PMP22 in intracellular compartments, sequestering both the mutant and wild-type forms and resulting in further loss of PMP22 at the plasma membrane (22); however, the evidence for this is contentious (19). Abnormal PMP22 expression has also been shown to directly alter Schwann cell proliferation (26), adding to the complexity of the phenotype. To increase understanding of the relevance of Pmp22 sequestration in intracellular compartments to disease phenotype, we are currently determining the cellular localization of the Pmp22Tr-m2H mutant protein, which is truncated and gives a less severe phenotype than the missense mutations Pmp22Tr and Pmp22Tr-m1H.
Tr-m1H and Tr-m2H, which are the only Pmp22 mutants exhibiting differing disease severities on a common genetic background, should help to further define the genotype–phenotype correlation of Pmp22 mutants. By providing insights into the functional domains of Pmp22, these mutants may allow protein trafficking mechanisms and the binding of partners in the myelin membrane to be further dissected, thereby improving our understanding of the pathophysiological mechanism of peripheral neuropathy.
Tr-m1H and Tr-m2H are examples of disease-causing mutations identified from a large-scale mammalian mutagenesis programme. The identification and characterization of clinically relevant mutant phenotypes together with the identification of the underlying genetic lesion underlines the potential of phenotype-driven screens for the development of models of human disease. Moreover, the use of ENU mutagenesis for the generation of novel mouse mutants and eventually multiple mutations within a single gene, thereby increasing both the breadth and the depth of the mouse mutant resource, will be a major tool for systematic studies of mammalian gene function. However, the identification of the underlying genetic defect has undoubtedly been the largest bottleneck in phenotype-driven screens (27). Our rapid mapping strategy, which is optimized to scan several mutant mouse genomes at once, will go some way to reducing this bottleneck. In addition, we recently reported the use of IVF for the rapid generation of back-cross progeny for genetic mapping (6). These so-called ‘speed back-crosses’ enable the generation of large numbers of back-cross progeny for genotyping within 2 months. The use of speed back-crosses allied to rapid genotyping protocols of the type reported here will have a significant impact on throughput for mutant localization and move the bottleneck to candidate gene identification.
The rapid identification of mutations in Pmp22 in Tr-m1H and Tr-m2H was facilitated by the presence of a promising candidate gene. Although the positional candidate approach is still not feasible in every case, the continuing characterization of the mouse genome through publicly supported mapping and sequencing projects will render it increasingly viable. In conclusion, the implementation of rapid gene identification protocols in conjunction with large-scale mutagenesis programmes is a powerful strategy. Utilizing this strategy in tandem with sensitive phenotype assessment, which allows the identification of subtle phenotypes, should allow comprehensive functional analysis of a mammalian genome to become a reality.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
The animal studies described in this paper were carried out under the guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and Home Office Project Licence no. 30/1517. For mutagenesis, BALB/c males (Charles River, Margate, UK) were injected intraperitoneally with two doses of 100 mg/kg ENU (Sigma-Aldrich, Poole, UK) at ~10 weeks of age. Doses were separated by a 1 week period. Procedures for ENU administration, including detailed safety procedures, are described elsewhere (28). Phenotypic screens were carried out on F1 progeny of mutagenized males mice crossed to C3H/He females (Charles River). Mating cages contained one male and up to two female occupants. Following weaning at 3 weeks of age, mice were housed in cages of about five mice. For inheritance testing, F1 mice were back-crossed to the C3H/He strain and progeny classified for the phenotype identified in the founder. Mutant lines were subsequently maintained by back-crossing to C3H/He.
Phenotype assessment
The SHIRPA protocol was used to evaluate at least three Tr-m1H and Tr-m2H mice and three littermate controls. SHIRPA has been described previously (4) and at the website http://www.mgc.har.mrc.ac.uk/mutabase/shirpa_summary.html .
Histology
For brain and spinal cord histology, mice were given a lethal overdose of pentobarbitone, and intracardially perfused with 4% paraformaldehyde. Brain and spinal cord were removed and post-fixed in 4% paraformaldehyde overnight. Specimens were dehydrated through a series of alcohols, and embedded in paraffin wax. Sections (10 µm) were cut throughout the brain, and cervical and lumbar enlargements of the spinal cord. Sections were stained with cresyl violet, haematoxylin and eosin, and Luxol fast blue. For nerve histology, mice were intracardially perfused with 2% gluteraldehyde, 2% paraformaldehyde, and a section of the sciatic nerve removed and post-fixed in the same fixative overnight; or, mice were killed by cervical dislocation, and a portion of the sciatic nerve removed and immersion fixed in 2% gluteraldehyde, 2% paraformaldehyde for 2 h. All nerves were then post-fixed in 2% osmium tetroxide for 2 h, dehydrated through a series of alcohols, and embedded in araldite CY212. For light microscopy 1 µm semi-thin sections were cut, and stained with toludine blue. For electron microscopy 60–90 nm sections were cut, and stained with lead citrate and uranyl acetate.
Linkage analysis
Tail snips were taken from 45 Tr-m1H and 44 Tr-m2H mutant back-cross mice and DNA extracted using standard procedures. DNA concentrations were measured by UV absorbance and equimolar aliquots of DNA combined for each strain. The DNA pools, together with DNA from BALB/c, C3H/HeH and F1 mice, were screened with 229 simple tandem repeat markers polymorphic between BALB/c and C3H/HeH selected from the 410 marker set available from Research Genetics (Huntsville, AL), and arranged into 22 panels. Genotyping was performed using a PE Biosystems 377 PRISM system (Foster City, CA) in accordance with the manufacturer’s instructions. Genotype data were analysed using a modified version of TrueAllele software (Cybergenetics, Pittsburgh, PA).
Determination of Pmp22 genomic structure
PCR primers 5'-cactatgggtgacccagtc-3' and 5'-cgtgtggccccagtgctggc-3', derived from the 3' untranslated region of Pmp22, were used to identify clone 201-C-16 from a mouse BAC genomic library (Research Genetics). PCR conditions were as follows: 20 µl reactions containing 200 µM dNTPs, 1.5 mM MgCl2, 1 U of Taq platinum (PE Biosystems) together with the supplied buffer and the recommended amount of template were subjected to 35 cycles of 94°C for 30s, 65°C for 30s and 72°C for 30s. The product (169 bp) was identified by electrophoresis in a 2% agarose gel. A 250 ml culture of 201-C-16 was grown overnight and DNA prepared using a Qiagen plasmid maxi kit. Direct sequencing of the BAC clone DNA to determine intron–exon boundaries was performed using the protocol described at http://www.tree.caltech.edu/protocols/BAC_end_sequencing.html . Primers used for sequencing were: 5'-ctgcacatcgcggtgctagtgttgc-3' and 5'-gtacagttctgccagagatcagtcg-3' (intron B), 5'-cgccttgggagccgtccaacactgc-3' and 5'-gaagatgacagacaggatcatggtggc-3' (intron C) and 5'-ccaaaggcggccggttttacatcactgg-3' and 5'-ctgtgcctcactgtgtagatggccgc-3' (intron D).
Pmp22 mutation analysis
Flanking intronic and untranslated sequences were used to design primers for amplification of exons 2–5 from Tr-m1H, Tr-m2H, BALB/c and C3H/HeH and subsequent sequence analysis. Primer sequences were: 5'-gcaccgctcctctgatcccgagc-3' and gcactggctgggaacccagagc-3' (exon 2), 5'-ggaataccatcctttgccctgc-3' and 5'-cccagttaggaccaatctgc-3' (exon 3), 5'-ggcaggtcctcctggccattcc-3' and 5'-gcaggaatgaattgttttcccc-3' (exon 4) and 5'-ggaggctcagacacatcttcc-3' and 5'-cctgtggaccctatgcgcgc-3' (exon 5). PCR reaction conditions were as described above, using 30 ng of genomic DNA as template. PCR products were purified using Qiagen QIAquick columns and sequenced directly using an Applied Biosystems 377 PRISM system in accordance with the manufacturer’s instructions.
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ACKNOWLEDGEMENTS |
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We thank Lesley Rooke and Colin Clapham for technical assistance and See-Kiong Ng for assistance with software. This work was supported by SmithKline Beecham Pharmaceuticals and the Medical Research Council.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1279 627225; Fax: +44 1279 622500; Email: ian_gray-1@sbphrd.com
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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