A pedigree-based genetic appraisal of Boxer ARVC and the role of the Striatin mutation.

The objective of this paper was to investigate by pedigree-based genetic means the origins and inheritance of arrhythmogenic right ventricular cardiomyopathy (ARVC) in UK Boxers and assess the role of the proposed causal mutation in the gene, Striatin (STRN). All ARVC cases traced back to a small number of imported American dogs deriving from the group of Boxers studied by Harpster (1983) to define the disease, strongly suggesting that the disease is the same in the two countries. Dogs with and without the STRN mutation were found in both ARVC affected and normal Boxers showing that the mutation is not responsible for the disease. Evidence was found that the STRN mutation is, however, genetically linked with the gene responsible on the same chromosome. The linkage implies that the two genes can separate by meiotic recombination such that both ARVC-affected and ARVC-unaffected lines of dogs may carry either the STRN mutation or its wild-type allele. These have been found. Homozygotes for the STRN mutation tended to be severely affected at early ages, suggesting that there is an interaction between the known effects of the STRN mutation on the cardiomyocyte and ARVC.

Interactions between imprinting effects: summary and review.

Mice with uniparental disomies (uniparental duplications) for defined regions of certain chromosomes, or certain disomies, show a range of developmental abnormalities most of which affect growth. These defects can be attributed to incorrect dosages of maternal or paternal copies of imprinted genes lying within the regions involved. Combinations of certain partial disomies result in interactions between the imprinting effects that seemingly independently affect foetal and/or placental growth in different ways or modify neonatal and postnatal development. The findings are generally in accord with the 'conflict hypothesis' for the evolution of genomic imprinting but do not demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that favours embryonic/foetal development, at consequent cost to the mother, will have been subject to evolutionary selection for only paternal allele expression. Reciprocally, any gene that reduces embryonic/foetal growth to limit disadvantage to the mother will have been selected for only maternal allele expression. It is concluded that survival of the placenta is core to the evolution of imprinting.

Two imprinted gene mutations: three phenotypes.

Genetic modifications of imprinted genes have been generated in the mouse to investigate the regulation of their expression. They show classical imprinted gene inheritances. Here we describe two imprinted gene mutations deriving from mutagenesis experiments. One is expressed only when transmitted through males. It causes a prenatal growth retardation which resembles that of the Igf2 knockout and maps close to the locus on chromosome 7. Differences from the knockout, which include an abnormal head phenotype, homozygous lethality, and an inability to rescue a TME: (Igf2r-deficient) lethality, suggest that Igf2 itself may not be directly affected. The second mutation maps close to the GNAS: cluster of imprinted genes on distal chromosome 2. It gives two distinct phenotypes according to parental origin, a gross neonatal oedema with microcardia and a postnatal growth retardation. The oedema phenotype is effectively lethal and resembles that of mice with paternal partial disomy for distal chromosome 2, as well as that of mice having a maternally derived GNAS: exon 2 knockout. However, the second growth retardation phenotype differs from that of the maternal partial disomy and the paternal knockout. A hypothesis to explain the phenotypes associated with the three genotypes based on the NESP:/NESPAS: sense/antisense and GNASXL: transcripts in the GNAS: cluster is offered.

Organization and parent-of-origin-specific methylation of imprinted Peg3 gene on mouse proximal chromosome 7.

Peg3 is the first imprinted gene to be identified on mouse proximal chromosome 7; the human PEG3 homologue is on chromosome 19q13.4. Peg3 encodes a C(2)H(2)-type zinc finger protein that is expressed only from the paternal allele in embryos and adult brain. The gene has been shown to regulate maternal behavior and offspring growth and has been implicated in the TNF-NFkappaB signal pathway. Here we show that Peg3 consists of nine exons spanning 26 kb. The 5' region of the gene contains a region rich in repeated sequences and a CpG island. Analysis of expressed sequence tags revealed a transcript present upstream of the island and on the strand opposite to Peg3. These structural features and DNA sequences are conserved in mouse and human. The 5' region of Peg3 is preferentially methylated on the inactive maternal allele, as shown by comparing embryos with paternal (PatDp. prox7) and maternal (MatDp.prox7) duplication of proximal chromosome 7. Recently, a new maternally expressed Zim1 gene located downstream of Peg3 was identified, which suggested that another imprinted cluster is present on proximal chromosome 7.

Embryonic inheritance of the chromatin organisation of the imprinted H19 domain in mouse spermatozoa.

Insulin-like growth factor 2 (Igf 2) and H19 genes are oppositely imprinted and as such have been most extensively studied imprinted genes both genetically and at the molecular level. Imprints of the H19 gene, being established during spermatogenesis, are epigenetically transmitted to the somatic cells of the embryo. Current hypotheses attempting to explain the allele-specific silence of the H19 gene include DNA methylation and chromatin condensation. In order to understand the molecular basis of H19 epigenesis, it is crucial to identify the markings in the chromatin organising the imprinted domain in spermatozoa. Using Micrococcal nuclease (MNase), DNase I and Methidiumpropyl-EDTA. iron II (MPE.Fe(II)) as chromatin probes, we demonstrate that in mouse epididymal spermatozoa, at least 4kb DNA upstream of the H19 'cap' site, containing the imprinted and differentially methylated domain (DMD), is heterochromatic. The cleavage sites in this domain (-2 to -4kb) exhibit approximately 425bp periodicity. This structure is maintained in the paternal allele of normal embryos and is disrupted at -2.2, -2.65 and at -3.5kb in embryos maternally disomic for the distal end of chromosome 7 (MatDp 7). The hypersensitive sites in chromatin precisely register the MPE.Fe(II) cleavage sites in chromosomal DNA. Therefore, the DNA sequences in the imprinted domain constrain the chromatin structure in a way similar to that of 1.688g/cm(3) Drosophila satellite chromatin. In addition, we find that condensation of the paternal allele correlates with methylation-dependent alteration in the structure of DNA sequences in DMD. These results suggest that CpG-methylation induces localised changes in DNA conformation and these facilitate consequent remodelling of chromatin thereby allowing the paternal and maternal H19 alleles to be distinguished.

Investigation of lung tumour induction in C3H/HeH mice, with and without tumour promotion with urethane, following paternal X-irradiation.

In series of papers Nomura has reported that parental irradiation can lead to an enhanced incidence of lung and other tumours. However, in a recent study with BALB/cJ mice, using optimum conditions as defined by Nomura, we were unable to confirm this. We have now repeated the investigation using a different inbred strain, C3H/HeH, with and without tumour promotion in the F1 by urethane, again using protocols defined by Nomura. In a series of replicate studies spanning over 2 years, males were exposed to single, acute doses of 0, 250 and 500 cGy X-rays and thereafter placed with two females each in each of two consecutive weeks. Half the offspring from each treatment group and each week of mating were given 5 mmol/kg body weight of the urethane, while the remainder remained untreated. Most of the offspring produced were killed and scored for lung tumours at 6 months of age, while the rest were examined at 12 months of age. The proportion of fertile females and litter size provided evidence of a dose-dependent mutational response to the paternal irradiation, but no trace of a radiation-enhanced lung tumour incidence was detected among the progeny, whether in the urethane or non-urethane groups at 6 or 12 months of age, and whether assessed by numbers of mice with tumours, clusters of tumours, or cluster size. As seen in the BALB/cJ study, significant differences among different replicates were found, again suggesting a cyclical or seasonal variation in tumour incidence, but the variations seen with the two strains were not the same. The need for concurrent controls for tumour work was, nevertheless, again indicated. The overall findings do not therefore accord with those of Nomura. Furthermore, they do not support the causal association between the raised incidence of childhood leukaemia and non-Hodgkins lymphoma near Sellafield and the father's recorded radiation exposure during employment in the nuclear industry, as suggested by the Gardner report.

Localisation of the imprinted gene neuronatin, Nnat, confirms and refines the location of a second imprinting region on mouse chromosome 2.

Nine regions on six mouse autosomes are subject to imprinting and uniparental inheritance of any one of these regions results in mice with phenotypic anomalies. So far on distal Chromosome (Chr) 2 there is a unique imprinting region between 2H3 and 2H4 associated with two behavioural disorders and neonatal lethality. A maternally imprinted gene, Nnat, has been identified which is expressed in the nervous system and maps to distal Chr 2. Nnat has been excluded as a candidate for either or both the behavioural phenotypes as it lies proximal to the 2H3-2H4 imprinting region. Here we have mapped Nnat to band 2H1 which is at least 18 Mb proximal to the previously described imprinting region. It maps close to agouti, some alleles of which show differential expression according to parental origin. The localisation of Nnat to band H1 confirms and refines the map location of a second imprinting region on mouse Chr 2.

Association of a redefined proximal mouse chromosome 11 imprinting region and U2afbp-rs/U2af1-rs1 expression.

Mice with maternal and paternal disomy for chromosome 11 (Chr 11) show growth retarded and overgrowth phenotypes, respectively, which can be attributed to genomic imprinting. Previous studies have defined the region of Chr 11 responsible (the Chr 11 imprinting region) as lying proximal to the T30H translocation breakpoint at the borders of G-bands 11B1.2 and 11B1.3. Evidence is presented here with two new translocations, T57H and T41Ad, which sequentially reduce the size of the imprinting region and locate it proximal to the T41Ad breakpoint in G-band 11A3.2. It therefore lies close to the centromere. The imprinted gene, U2af1-rs1, is known to be located within the original region and has been regarded as a candidate for the imprinting effects. Meiotic and mitotic chromosome FISH analysis, together with U2af1-rs1 expression studies are now described which show that the gene lies within the newly defined imprinting region and that its expression levels relate to the presence/absence and number of functional paternal alleles. U2af1-rs1 therefore remains a candidate gene for the Chr 11 imprinting effects. However, another recently reported imprinted gene, Meg1/Grb10, that lies within the region is also a good candidate, as it encodes a growth factor receptor. Meg1/Grb10 maps about 15 cM from U2af1-rs1 and is separated by conserved regions showing homology with two different human chromosomes. For these reasons, and because the two human homologues of U2af1-rs1 and Meg1/Grb10 also lie on different chromosomes, it would seem likely that the two genes identify two distinct imprinting domains within the small proximal region of mouse Chr 11.

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities.

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.

Genetic and functional analysis of neuronatin in mice with maternal or paternal duplication of distal Chr 2.

Functional differences between parental genomes are due to differential expression of parental alleles of imprinted genes. Neuronatin (Nnat) is a recently identified paternally expressed imprinted gene that is initially expressed in the rhombomeres and pituitary gland and later more widely in the central and peripheral nervous system mainly in postmitotic and differentiating neuroepithelial cells. Nnat maps to distal chromosome (Chr) 2, which contains an imprinting region that causes morphological abnormalities and early neonatal lethality. More detailed mapping analysis of Nnat showed that it is located between the T26H and T2Wa translocation breakpoints which is, surprisingly, proximal to the reported imprinting region between the T2Wa and T28H translocation breakpoints, suggesting that there may be two distinct imprinting regions on distal chromosome 2. To investigate the potential role of Nnat, we compared normal embryos with those which were PatDp.dist2.T26H (paternal duplication/maternal deficiency of chromosome 2 distal to the translocation breakpoint T26H) and MatDp.dist2.T26H. Expression of Nnat was detected in the PatDp.dist2.T26H embryos, where both copies of Nnat are paternally inherited, and normal embryos but no expression was detected in the MatDp.dist2.T26H embryos with the two maternally inherited copies. The differential expression of Nnat was supported by DNA methylation analysis with the paternally inherited alleles being unmethylated and the maternal alleles fully methylated. Although experimental embryos appeared grossly similar phenotypically in the structures where expression of Nnat was detected, differences in folding of the cerebellum were observed in neonates, and other more subtle developmental or behavioral effects due to gain or loss of Nnat cannot be ruled out.

Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.

Angelman syndrome (AS) is a human genetic disorder characterized by mental retardation, seizures, inappropriate laughter, abnormal galt, tremor and ataxia. There is strong genetic evidence that the disorder is associated with a maternally expressed, imprinted gene mapping to chromosome 15q11-13. Affected patients demonstrate varied molecular abnormalities, including large maternal deletions, uniparental paternal disomy (UPD). Imprinting mutations and loss of function mutations of E6-associated-protein (E6-AP) ubiquitin-protein ligase (UBE3A). All of these abnormalities are associated with loss of maternal expression of UBE3A. Although mutations in UBE3A cause AS, indicating that maternal-specific expression of UBE3A is essential for a normal phenotype, evidence for maternal-specific expression of UBE3A has been lacking. Using mice with partial paternal UPD encompassing Ube3a to differentiate maternal and paternal expression, we found by in situ hybridization that expression of Ube3a in Purkinje cells, hippocampal neurons and mitral cells of the olfactory bulb in UPD mice was markedly reduced compared to non-UPD littermates. In contrast, expression of Ube3a in other regions of the brain was only moderately or not at all reduced in UPD mice. The major phenotypic features of AS correlate with the loss of maternal-specific expression of Ube3a in hippocampus and cerebellum as revealed in the mouse model.

Hindshaker, a novel myelin mutant showing hypomyelination preferentially affecting the spinal cord.

Animals with spontaneous mutations affecting myelin formation have provided useful information about the genetic and cellular mechanisms regulating normal and abnormal myelination. In this paper we describe a novel murine mutation termed hindshaker (hsh), which is inherited in an autosomal recessive manner. Affected mice are characterised by a variable tremor of the hind end which commences at about 2 weeks of age and largely disappears in animals older than 6 weeks. There is hypomyelination affecting predominantly the spinal cord, although the optic nerves and brain are involved to a much lesser degree. The defect of thinly myelinated and naked axons is maximal at 20 days of age and largely resolves with time so that in the adult most axons are myelinated. The myelin structure appears normal and immunostains for the major proteins. Although the distribution of oligodendrocytes in the spinal cord is similar to normal during the period of hypomyelination, there are fewer mature cells. The hsh mutation appears to delay the maturation of oligodendrocytes, particularly in the spinal cord. Additionally, there is a considerable variation in phenotypic expression and in penetrance when the mutation is expressed on different genetic backgrounds, suggesting the hsh locus is subject to the influence of modifying gene(s). Identification of the hsh gene should identify a factor important in the development of oligodendrocytes, particularly those in the spinal cord.

A candidate model for Angelman syndrome in the mouse.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15q11-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated.

Time of initiation and site of action of the mouse chromosome 11 imprinting effects.

Previous studies have shown that mice with paternal disomy for chromosome 11 are consistently larger at birth than their normal sibs, whereas mice with the maternal disomy are consistently smaller. An imprinting effect with monoallelic expression of some gene/s affecting growth was indicated. Here we show that the size differences become established prior to birth and are only maintained subsequently, indicating that the gene repression is limited to prenatal development. Fetal analysis was limited to 12.5-17.5 days post coitum. However by extrapolating the data backwards it could be calculated that both the maternal and paternal size effects might commence as early as 7 days post coitum, although possibly slightly later. It may be deduced that initiation of expression of the gene/s responsible may occur at about this time in development. The two disomy growth rates were mirror-images of each other, suggesting that expressed gene dosage is the underlying cause. Differential growth of the placentas of the two disomies was also found, and extrapolation of these data backwards suggested that the placental size differences were initiated later in development than those for the fetuses. The differential placental growth of the maternal and paternal disomies may therefore have developed independently or emerged as a consequence of the differential fetal growth. In either event it would seem that the expression of the responsible gene occurs in the fetus itself to cause the anomalies of growth. The data therefore provide information on the temporal and tissue specificity of the gene/s responsible for the chromosome 11 imprinting effects. Possible candidate genes are discussed.

Y chromosome short arm-Sxr recombination in XSxr/Y males causes deletion of Rbm and XY female sex reversal.

We earlier described three lines of sex-reversed XY female mice deleted for sequences believed close to the testes-determining gene (Sry) on the Y chromosome short arm (Yp). The original sex-reversed females appeared among the offspring of XY males that carried the Yp duplication Sxr on their X chromosome. Earlier cytogenetic observations had suggested that the deletions resulted from asymmetrical meiotic recombination between the Y and the homologous Sxr region, but no direct evidence for this hypothesis was available. We have now analyzed the offspring of XSxr/Y males carrying an evolutionarily divergent Mus musculus domesticus Y chromosome, which permits detection and characterization of such recombination events. This analysis has enabled the derivation of a recombination map of Yp and Sxr, also demonstrating the orientation of Yp with respect to the Y centromere. The mapping data have established that Rbm, the murine homologue of a gene family cloned from the human Y chromosome, lies between Sry and the centromere. Analysis of two additional XY female lines shows that asymmetrical Yp-Sxr recombination leading to XY female sex reversal results in deletion of Rbm sequences. The deletions bring Sry closer to Y centromere, consistent with the hypothesis that position-effect inactivation of Sry is the basis for the sex reversal.

Ubiquitous expression and imprinting of Snrpn in the mouse.

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.

Investigation of lung tumour induction in BALB/cJ mice following paternal X-irradiation.

Evidence of an enhanced incidence of lung tumours (benign adenomas and adenocarcinomas) was sought in the BALB/cJ mouse following paternal germ cell X-irradiation. In a series of replicate studies spanning approximately 1 year, males were exposed to single, acute X-ray doses of 0, 250 and 500 cGy. In each of the 2 consecutive weeks immediately thereafter they were placed with two females to generate progeny that were derived from irradiated post-meiotic cells (spermatozoa to late spermatids). These animals were then examined at 8 or 12 months for lung tumours. While the proportion of fertile females and mean litter size was affected by the radiation, showing a dose-dependent, dominant lethal response, and while cases of mutant offspring were detected, the paternal radiation did not affect lung tumour incidence in the offspring. The incidence did not vary significantly between germ cell stages irradiated (week of mating), sex of offspring, or radiation dose. However, significant differences between lung tumour incidence (mostly representing benign adenomas) were found between different replicates, these being high at the start of the study, declining and then rising to yet higher levels at its close. The finding that lung tumour incidence in BALB/cJ mice is not affected by paternal germ cell irradiation does not accord with Nomura's reports using other strains of mice. This, in turn, weakens biological support for a causal association between the raised incidence of childhood leukaemia and non-Hodgkin lymphoma near Sellafield and the father's recorded radiation exposure during employment by the nuclear industry.

Molecular genetic characterization of six recessive viable alleles of the mouse agouti locus.

The agouti locus on mouse chromosome 2 encodes a secreted cysteine-rich protein of 131 amino acids that acts as a molecular switch to instruct the melanocyte to make either yellow pigment (phaeomelanin) or black pigment (eumelanin). Mutations that up-regulate agouti expression are dominant to those causing decreased expression and result in yellow coat color. Other associated effects are obesity, diabetes, and increased susceptibility to tumors. To try to define important functional domains of the agouti protein, we have analyzed the molecular defects present in a series of recessive viable agouti mutations. In total, six alleles (amJ, au, ada, a16H, a18H, ae) were examined at both the RNA and DNA level. Two of the alleles, a16H and ae, result from mutations in the agouti coding region. Four alleles (amJ, au, a18H, and ada) appear to represent regulatory mutations that down-regulate agouti expression. Interestingly, one of these mutations, a18H, also appears to cause an immunological defect in the homozygous condition. This immunological defect is somewhat analogous to that observed in motheaten (me) mutant mice. Short and long-range restriction enzyme analyses of homozygous a18H DNA are consistent with the hypothesis that a18H results from a paracentric inversion where one end of the inversion maps in the 5' regulatory region of agouti and the other end in or near a gene that is required for normal immunological function. Cloning the breakpoints of this putative inversion should allow us to identify the gene that confers this interesting immunological disorder.

Enhanced specific-locus mutation response of 101/H male mice to single, acute X-irradiation.

The specific-locus mutation yield from 101/H mice following single, acute 6 Gy spermatogonial X-irradiation was significantly higher than both a concurrent C3H/HeH x 101/H F1 hybrid control and historical data obtained with mice of equivalent genotype. There is therefore discord with the reduced translocation response to single X-ray doses previously identified in this strain. By contrast, the mutation yield following a 24-h interval 3 + 3 Gy fractionated X-ray dose was not significantly different from that of its concurrent hybrid control, nor from results obtained by others with mice of the equivalent or different genotypes. Here there is no discord with the translocation response obtained with a 1 + 5 Gy 24-h interval fractionated regime. Appraisal of comparable specific-locus and translocation data indicates that the differing results obtained with the two genetic end-points are not without precedence. This, together with the observation that the responses for cytologically visible deletions and specific-locus mutations are similar, suggests that the latter two events predominantly derive from one-hit events, with translocations deriving from two-hit events and that the probabilities of induction of each type of lesion vary according to the organisation of the nucleus in different phases of the cell cycle.

Genetic imprinting in the mouse: implications for gene regulation.

Genetic imprinting specifies a germline marking that subsequently results in the repression of one or other parental allele at some point in development. Genetic manipulations to generate maternal and paternal duplications of specific chromosome regions have been used to screen almost the entire mouse genome for evidence of imprinting. As a result, 15 imprinting effects involving 10 regions on 6 different chromosomes have been detected that range from early embryonic lethalities to various growth and developmental defects seen only after birth. Genes with important roles in development therefore appear to be involved. Diverse studies have identified four imprinted genes, all of which show monoallelic expression in some, but not necessarily all, tissues. A correlation with methylation is indicated but the pattern of methylation is not consistent for each of the genes; methylation is therefore unlikely to be the imprinting signal. Methods being used to identify further imprinted genes are summarized and some of the difficulties posed are indicated.