Human Molecular Genetics, 2000, Vol. 9, No. 15 2263-2273
© 2000 Oxford University Press
Two imprinted gene mutations: three phenotypes
Medical Research Council, Mammalian Genetics Unit, Harwell, Didcot, Oxfordshire OX11 0RD, UK
Received 11 May 2000; Revised and Accepted 4 August 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting in mammals specifies a germ line marking of parental alleles that results in their differential expression in the zygote. This has been most widely demonstrated in the mouse through the creation of uniparental disomies and partial disomies, which have two copies of genes within the chromosomes/regions from one parent and none from the other (1). Such studies have shown imprinting to affect genes in at least 11 regions of the mouse genome, with the imprinting anomalies or phenotypes ranging from early embryonic lethalities, through neonatal abnormalities, to postnatal growth retardations. In a number of cases, the genes responsible for the abnormalities have been identified and following genetic modification, imprinted gene inheritances, as first hypothesized by Hall (2), have been demonstrated. These include those for Igf2 (3,4), Rasgrf1 (5), Gnas (6) and Ube3a (7) knockouts and the targeted central chromosome 7 deletions which involve several imprinted genes (8,9). The T maternal effect (Tme) lethality, associated with the long-recognized Thp mutation (10,11) which is attributable to the deletion of the maternally imprinted Igf2r locus (12), also shows an imprinted gene inheritance.
In humans, two inherited and imprinted forms of pseudo hypo-parathyroidism have been recognized. One, PHP-Ia, is associated with Albright’s hereditary osteodystrophy (AHO) and involves the GNAS locus (13) on chromosome 20. The other, PHP-Ib, is not associated with AHO but maps to the same region (14). Isolated examples of familial Prader–Willi and Angelman syndromes resulting from mutations at putative imprinting control loci have also been described (15). However, up until now imprinted gene inheritances, other than those of genetic modifications of imprinted genes (3–7) and that attributable to the Igf2r deletion (12), have not been described in the mouse. Here we report two mouse mutations which show imprinted gene inheritances. The first mutant, called minute (Mnt), lies within the distal chromosome 7 imprinting region and may involve the imprinted Igf2 locus. The phenotype resembles the Igf2 knockout (3,4) but there are differences. The second, provisionally called oedematous-small (Oed-Sml), lies within the distal chromosome 2 imprinting region and uniquely shows two distinct phenotypes according to the parental route of transmission. One of the latter resembles a distal chromosome 2 imprinting effect (16).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Minute (Mnt)
Origin.
The original mutant derived from one of a series of specific locus mutation experiments (17) in which spermatogonially irradiated (6 Gy X-rays) F1 hybrid C3H/HeH x 101/H males were mated to females homozygous for seven different recessive mutations. All offspring of the cross were screened not only for mutations at the tester loci but also for new dominant mutations, many of which, with radiation, have been found to represent chromosomal changes, notably deletions and duplications (18). The Mnt mutation was detected in a male through its severe growth retardation (60% of normal) observed initially at birth, but this was accompanied by an abnormal domed head effect (Fig. 1). No evidence of a chromosomal change has been seen in detailed G-band analysis (E.P. Evans, personal communication).
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Inheritance.
On crossing the original animal with normal (+) females, a proportion of the progeny (of both sexes) expressed the mutant phenotype (Table 1). A simple dominant inheritance was therefore suggested and this appeared to be confirmed on crossing affected sons with + females. Elevated levels of neonatal loss were encountered, notably in the second generation, but survival was enhanced in Mus mus castaneus crosses, perhaps because of the low litter size of this subspecies (Table 1). Remarkably, none of the progeny of mutant females were affected (Table 1), yet clearly the mutation was transmitted as approximately half their sons proved capable of producing Mnt young (Table 2). Mutant expression was therefore obtained only with transmission through males. This represents an imprinted gene inheritance as illustrated in Figure 2 and implies that the locus affected by Mnt is normally expressed only when transmitted through the male germ line; the maternal allele is silent.
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Mutant animals recovered from the phenotypically + males that had inherited the mutation from their mothers (MntM) were not detectably different from those (MntP) that had inherited the mutation directly from their affected fathers (Table 2).
Further characterization of Mnt.
Prenatal growth rates, as assessed by ratios of weights of mutant to + sibs within litters declined from the earliest prenatal age at which the size differences could be distinguished (13 days gestation) up until the time of birth when they approximated <50% of normal (Fig. 3). The size was significantly different from that of the sibs throughout (P < 0.1 x 10–9). Extrapolating the data backwards, the straight line of best fit crossed the age axis at embryonic day (E) 10.7 (data not shown), possibly defining the age at which the size difference in the fetus might first be initiated. Placental weight ratios were more affected than fetal weight ratios (see legend to Fig. 3) and the straight line of best fit crossed the age axis 
3 days earlier. This might suggest that the reduced fetal growth is at least in part mediated through the placenta.
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Postnatal Mnt growth rates suggested that the size difference declined further to reach a minimum at
4 days of age (Fig. 3) when most inviability occurred. It seems likely that the lethality is only a consequence of difficulties that these small animals have in competing with their larger, normal sibs, as growth and survival improve when litter sizes are artificially or naturally reduced, as in M.m.castaneus crosses which have low litter sizes (Table 1). At later ages there appeared to be a gradual recovery or ‘catch up’ (Fig. 3) but this could be due to the surviving animals being the least affected. Thus, differences between the weight ratios at birth of non-survivors (beyond 4 days) and survivors to later ages were highly significant [F(5,11) = 6.14; P = 0.000046]. Mnt adults were still 60% of the weight of their + sibs. The domed head phenotype was most clearly evident shortly after birth (Fig. 1) but could be observed at all ages.
Mapping of Mnt.
Almost all imprinted genes identified so far have been mapped within the imprinting regions defined with the use of chromosomal translocations (1). It was therefore anticipated that the Mnt locus would lie within one of these regions, several of which give growth retardation imprinting effects (1). On the basis that the maternal allele is normally silent and only the paternal allele is expressed, proximal chromosome 11 was initially considered the most likely site of the Mnt mutation but linkage tests (data not shown) with appropriate genetic markers negated this possibility. Distal chromosome 7 provided a further candidate region as a paternally inherited knockout of one of the known imprinted genes in this region, Igf2, also bringing about a growth retardation (3,4) that closely resembles that of Mnt. This gene therefore became the most likely candidate gene.
Close linkage with Mit markers located within the distal chromosome 7 imprinting region (19) established that this was the location of the mutation. Thus, with D7Mit108 and the more distal D7Mit47, which span the imprinted Igf2 locus and lie 12 units apart, 13 and 2 recombinants, respectively, were detected among 100 backcross progeny, suggesting that Mnt maps between these loci and therefore close to Igf2. This possibility was confirmed in linkage tests with D7Mit291, which lies between D7Mit108 and Igf2. Thus, using a further 96 backcross animals, two recombinants with the phenotype were detected and a further four were found with D7Mit47. Finally, when all 22 recombinants were tested with other markers in the region (D7Mit167, D7Mit259, D7Nds4 and specifically D7Mit46, which primes up part of the Igf2 gene itself), co-segregation with D7Mit46 and D7Mit 167 was demonstrated (Fig. 4). Mnt therefore involves, or lies very close to, Igf2 within the distal chromosome 7 imprinting region. All Mit markers selected could be studied. No evidence of a deletion on the Mnt chromosome was therefore indicated.
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Fate of Mnt homozygotes.
Investigations into the fate of the Mnt homozygotes were initiated before the map positions were known and did not provide a clear conclusion. The litter size of Mnt x Mnt intercrosses were notably low (mean = 3.9, n = 78), suggesting that the homozygote dies prenatally. On the other hand, openings of pregnant females from intercross matings failed to demonstrate a novel or lethal class at 17.5 days gestation, pre- and post-implantation losses approximating normal levels (2.4 and 9.2%, respectively). Notably, litter size was within the normal range at this time (mean = 6.5, n = 35) and Mnt and + embryos were detected in near-equal frequencies (124 and 102, respectively). It therefore seemed that at this stage of development Mnt homozygotes are viable and indistinguishable from heterozygotes. On this basis, the Mnt classes would comprise MntP/MntM and MntP/+ and the + classes MntM/+ and +/+.
Confirmation that the homozygote indeed survived until at least shortly before birth was finally obtained by opening females from Mnt F1 M.m castaneus intercrosses and screening fetuses for the closely linked Mit46(Igf2) and/or Mit167 markers. At 16–17 days gestation, 21 MntM/MntP, 28 MntP, 21 MntM and 18 + fetuses were identified. Seven of the homozygotes had recently died, and from the failure to find any of this class at birth it may be concluded that Mnt is homozygous lethal and that the loss of this class occurs late in gestation to around the time of birth. The homozygotes did not show any features that could enable them to be distinguished from heterozygotes. While the abnormal Mnt head phenotype provided the first indication that the mutant and the Igf2 knockout (3,4) were not identical, the homozygous inviability illustrated a second key difference.
Effect of Mnt on brachyury (T) deletion lethality.
The combination of a paternally inherited Igf2 knockout, in which the gene is not expressed, with maternally inherited brachyury deletions, which encompass the Igf2r receptor locus, allows partial rescue of the fetuses with the T maternal effect (Tme) lethality (20). The lethality, which typically occurs late in gestation (10,11), has been attributed to an excess of the Igf2 ligand in the absence of the receptor and the rescue being achieved by the deficit of Igf2 (20). To determine whether Mnt could similarly effect such a rescue, females heterozygous for the brachyury mutation, T37H, which causes the typical, variable Tme lethality when maternally inherited (B.M. Cattanach, unpublished data), were crossed with Mnt males. Some T37H survival to birth was found in control crosses of such females with + males (five dead T37H among 92 + young born), as occasionally found with the Thp deletion (10,11) on the same genetic background (B.M. Cattanach, unpublished data). However, when mated with Mnt males, no T37H young were recovered among 61 offspring born to T37H mothers, although Mnt young were obtained in the expected frequency. No suggestion of a rescue was therefore identified.
Further investigation of T37H x Mnt crosses at 17.5 days gestation indicated a high incidence of late deaths and early (small mole) losses and, perhaps as a result, the segregation of both T and Mnt were skewed. However, there was a clear shortage of both T and T Mnt offspring relative to their + and Mnt sibs (19:29 and 27:43, respectively), again showing no indication of rescue. Similar results were obtained with a small sample of equivalent crosses using Thp females (3:4 and 5:8, respectively). Weight ratios, however, indicated that the T37H fetuses were significantly heavier (due to oedema or overgrowth) than their + sibs (1.274 ± 0.041; t33 = 7.53, P = 8.1 x 10–9), but this was not found when T Mnt and Mnt fetuses were compared (1.039 ± 0.03; t35 = 0.437, P = 0.20). The latter finding might suggest that some element of the T maternal effect might be modified by the presence of Mnt, but it may just be more difficult to validly assess weight differences in these already severely compromised fetuses. The overall inability of Mnt to rescue the Tme lethality suggests a third difference from the Igf2 knockout (3,4).
Oedematous-small (Oed-Sml)
Origin.
As with Mnt, the Oed-Sml mutation derived from a specific locus mutation experiment and was detected because of a growth retardation evident at birth and later ages. In this case the mutagen used was ethylnitrosourea (ENU) (250 mg/kg body wt; B.M. Cattanach, unpublished data) and the original variant animal was female. As ENU is known to cause mutations by AT
GC base pair transitions or ATTA transversions (21), Oed-Sml was not expected to be a chromosomal mutation. No evidence of a chromosomal change has been seen in detailed G-band analysis (E.P. Evans, personal communication).
Inheritance.
On being subjected to breeding tests with a + male, the variant female did not produce growth-retarded young like herself. Instead, a new variant phenotype appeared and clearly had a genetic basis as near-identical examples (n = 17) were found in 11 litters (Table 3). The novel phenotype principally comprised a gross oedema, hereafter denoted Oed. None of the affected young lived for more than a few days.
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Ovary transplantation from neonates was used to try and achieve survival of the mutant stock. This yielded further affected animals, but the risk of losing the mutation remained high. Only the survival of a single Oed male assured the future of the mutation and this had to be accomplished using in vitro fertilization and embryo transfer, the male seemingly being unable to breed normally. Litters were produced by 16 normal recipient females and, on classification, these yielded a further unexpected finding. Of the 116 progeny born, none showed the Oed phenotype but, within 5–7 days of birth, approximately half became obviously growth retarded, like the original variant female. Subsequent studies established that when females showing this growth-retarded or small phenotype (denoted Sml) were mated with + males, the Oed phenotype reappeared among their offspring. On the other hand, when their Sml male sibs, or surviving Oed males, were crossed with + females, the Sml phenotype was produced, appearing in about half the offspring. Data establishing the inheritance of the double, parental source-dependent phenotypes are presented in Table 3 and illustrated diagrammatically in Figure 5. The Oed-Sml mutation therefore presents a second type of imprinting inheritance.
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Further characterization of the Oed phenotype.
The Oed phenotype was readily detectable at birth but the recovered frequency of affected young at this age was only half that expected (Table 3). Prenatal studies indicated an elevated level of resorption sites (9.9%) and dying Oed fetuses (15.3%) at 17.5–19.5 days gestation, accounting for the shortage.
At birth, Oed animals resembled mice with the oedematous imprinting phenotype caused by paternal duplication/maternal deficiency (partial disomy) for distal chromosome 2 (PatDp.dist2) (13). However, in addition, brown fat accumulations could be seen through the skin, localized at the scapular region on the back (Fig. 6A) and around the throat. The oedema, as seen at birth [36% difference between wet and dry weights (Fig. 6B)], was similar to that of the PatDp genotype at its peak at 16 days gestation (40% difference) (22), but was notably more evident before birth (56% difference). With both genetic conditions the oedema appears to decline just before and also subsequent to birth (Fig. 7A) and the few adult Oed survivors (obtained only following crosses with M.m.castaneus) were not overtly oedematous. However, in sharp contrast to the PatDp class, Oed neonates showed no indication of the hyperkinetic behaviour that characterizes the PatDp class (16).
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The ongoing loss of Oed young after birth was accompanied by a decline in the weight ratios (clearly observable beyond the loss of oedema). This reached minimum values over the second and third weeks and then, among the few that survived longer, appeared to rise (Fig. 7A). In view of the small numbers of survivors, the significance of the rise is not clear.
Pathological examination of those Oed young that survived for several days after birth showed inflammatory reactions in the larynx and pharynx and, in the lungs, there was a haemoraghagic reaction with an infiltration of blood cells. Perhaps consistent with the latter finding was the observation of breathing difficulties in the few animals that survived to such ages. The aetiology of these effects was not clear; they may be only secondary consequences of a primary defect. Of more significance was the finding that heart size was severely reduced, typically being less than half that of normal. This was evident from 16 days gestation to 10 days after birth during which time heart weight ratios declined further (Fig. 7B). However, two surviving adults had normal heart sizes. The early effect appeared that of a heart-specific growth retardation as no other abnormality within the heart was detected. Other organs appeared normal.
Further anomalies noted in Oed mice that survived beyond 2 weeks of age included sparse, wiry coats that became dense and rough in adults. Adult Oed animals also looked shorter bodied than their sibs, but body weights were not notably affected. Similar effects have been noted with those PatDp.dist2 mice that survive to adulthood (B.M. Cattanach, unpublished data). Oed mice of both sexes can be fertile.
Further characterization of the Sml phenotype.
Weight ratios based on whole litters indicated a statistical difference [P < 0.1 x 10–8] from one over the ages tested [birth to 8 weeks (Fig. 7C)]. Some Sml individuals were visibly growth retarded shortly after birth but classification became reliable only at 5–7 days. Weight ratios were minimum at 10–14 days after birth. Subsequently there was some indication of recovery, but comparisons of surviving and non-surviving Sml mice at earlier ages showed that the survivors were marginally bigger [F(14,121) = 1.64; P = 0.07]. The apparent recovery may therefore be attributable to survivors being the least affected. Losses of the Sml class were substantial with most occurring around the ages when weight ratios were at their lowest. The size difference appeared to be a whole animal effect as weight ratios of their organs (heart, kidney, liver), as a proportion of body weight, did not differ from 1 (data not shown).
Mapping of the Oed-Sml mutation.
The similarity between the Oed and PatDp.dist2 phenotypes suggested that the Oed-Sml locus might lie within the distal chromosome 2 imprinting region. Linkage back-crosses using Sml males and Mit markers which lie close to the region (D2Mit226 and D2Mit52 proximal and D2Mit174 distal) or within it (D2Mit25 and D2Mit504) (19, and J. Peters, unpublished data) confirmed that the mutation lies distally on chromosome 2. Thus, in the first screen of 30 animals there was complete linkage with D2Mit174, although there was a remarkably high recombination frequency (3/30, 10%) with D2Mit25 (Fig. 8) considering the distal location of these markers on the chromosome. In a second screen with 35 further animals, complete linkage was found with D2Mit504, but a high recombination frequency (8/35, 22.8%) was again found with D2Mit25 (Fig. 8). The high recombination frequencies are puzzling but the data nevertheless establish that the Oed-Sml mutation lies distally in chromosome 2 and probably at the distal end of the imprinting region. As such it becomes the candidate locus for at least the oedema component of the PatDp.dist2 imprinting phenotype.
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Fate of the Oed-Sml homozygote.
Intercrosses of phenotypically Sml animals generated 310 young among which, at birth, there were 54 Oed and 256 +. Of 224 + progeny that were reclassified at 5–7 days of age, 92 proved to be Sml and 132 were +. Twenty of the + class were test-mated and all proved to be genotypically +. A novel phenotype that might identify the homozygote was not therefore evident, and the proportions of Sml and Oed classes in relation to + were in accord with expectation (Table 3). Prenatal lethality of the homozygote was thus suggested. Consistent with this, the mean litter size of intercrosses was notably low (4.13/female, n = 75), especially in comparison with that of outcrosses of Sml females (5.13/female) (Table 3) in which the low viability Oed class is twice as numerous.
Openings of females derived from Sml M.m.castaneus F1 intercrosses at 17–19 days gestation again failed to identify a novel class that could represent the homozygote. On typing 90 fetuses using the two closely linked Mit markers, D2Mit25 and D2Mit174, 13 were recognized as Oed, 46 were identified as heterozygotes and 31 were deduced to be +. None were homozygous for the M.m.musculus alleles, indicating that the homozygote must die early in development. Post-implantation losses were marginally higher than that in Sml female x + male crosses (28 cf. 22%). Given that the frequency of Oed fetuses in the intercrosses should be only half that in the outcrosses, it would seem likely that much of the post-implantation loss in intercross matings is attributable to lethality of the homozygotes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mnt provides a classic example of an imprinted gene inheritance as postulated by Hall (2). Thus, in some crosses a clear dominant inheritance is evident and yet in others the phenotype disappears as though inherited as a recessive. The parental origin of the mutation provides the explanation. Expression is only seen with transmission through males; when maternally transmitted the phenotype is wild-type. The only other example in the mouse is seen with brachyury (T) mutations that are associated with deletions that encompass the paternally imprinted/repressed Igf2r locus (12). Described long before imprinting in mammals was recognized (10,11) these give the Tme lethality when transmitted though females; a functional maternal Igf2r copy is required (12). Knockouts of imprinted genes have in more recent times also shown imprinted gene inheritances (3–7), as have targeted deletions (8,9). The Oed-Sml mutation provides a variation on the theme. However, rather than giving a mutant phenotype when transmitted through one sex and wild-type or normal when transmitted through the other, two different mutant phenotypes are produced according to parental route of transmission. The double phenotype therefore resembles the seemingly ‘opposite’ phenotypes of distal chromosome 2 and proximal chromosome 11 reciprocal uniparental duplications/partial disomies (16).
Consistent with their imprinting inheritances, both Mnt and Oed-Sml map within known imprinting regions of mouse chromosomes (1). Mnt maps close to Igf2 within the distal chromosome 7 imprinting domain (1,19), and Oed-Sml maps within the distal chromosome 2 imprinting domain in the vicinity of the Gnas imprinted gene cluster (22–24).
In the case of Mnt, it is possible that the mutation involves the imprinted Igf2 locus itself. Thus, recombinants with D7Mit46, which is intrinsic to Igf2, have not yet been found, and the growth retardation phenotype closely resembles that of the Igf2 knockout (3,4). Furthermore, the time in development when the growth retardation is first seen accords with that observed with the Igf2 knockout (25). Placental weight growth retardation was seen with Mnt fetuses and this has also been found with the knockout (25). The knockout data show the growth effects to appear within the 24 h immediately prior to E13.5 and remain almost constant thereafter (25), whereas with Mnt both the fetal and placental growth ratios showed a significant decrease with increasing age (slopes = –0.0832 ± 0.0077 and –0.0570 ± 0.0149, respectively). Whether this represents a real difference between the mutant and the knockout is not clear. Extrapolation of the Mnt data backwards showed that the lines of best fit to cross the age axis at E10.7 and E14.0, respectively, providing estimates of when the fetal and placental growth differences might first be initiated. The approach assumes linearity of growth, which in view of the knockout data (25) may not be valid.
In contrast, at least two and possibly three observations clearly distinguish the mutant from the knockout. The first is the head phenotype. This has not been described with Igf2 knockout mice (3,4). The Mnt mutation, however, occurred following parental radiation exposure. It is therefore likely to represent a chromosomal change. A deletion could therefore account for the compound growth retardation and head anomaly phenotype, just as the Thp and many other brachyury deletions bring about the Tme lethality attributable to the absence of Igf2r (12), as well as the tail effect (10,11).
The second difference between Mnt and the Igf2 knockout is that the mutant is lethal in the homozygous condition. Here again, chromosomal deletion could be responsible as most deletions are homozygous lethal, suggesting that genes within them are haplo-insufficient (18).
The third and less firmly established difference between the mutant and the knockout is that Mnt appears to be unable to rescue a Tme lethality as does the knockout (20). Genetic background is unlikely to be responsible for the difference as the brachyury deletion mutant used (T37H) is not distinguishable from that employed with the knockout (Thp) on the same genetic background; with both, affected mice also occasionally survive to birth without rescue. A failure to effect Tme rescue suggests that at least some level of Igf2 expression remains in Mnt mice. If verified, this would rule out deletion of the Igf2 locus itself as the basis for the mutation and would suggest that either some other paternally expressed locus in the region is responsible for the growth effects, or that it is the regulation of Igf2 expression that is affected. Ongoing Igf2 expression studies and detailed analysis of the region are providing evidence that the mutation is complex. The findings will be the subject of a further paper.
In seeking interpretation of the double Oed-Sml phenotype, it may be noted that similar parent of origin-dependent phenotypes are brought about by uniparental duplications/partial disomy of the distal chromosome 2 imprinting region (16) and also with a knockout of exon 2 of Gnas (6). Comparison of the three phenomena suggests that the effects of all three conditions can be interpreted in terms of an absence of functional maternal/paternal alleles of distal chromosome 2 loci.
With absence of a functional maternal allele, oedema is found with all three genotypes. Microcardia is characteristically seen with the Oed-Sml mutation and we have since detected it in lower degree in paternal duplication (PatDp) mice derived in studies using the T30H translocation (weight ratio = 0.796 ± 0.050; P = 0.0024 compared with 0.542 ± 0.03 for Oed mice). Microcardia has not, however, been reported with the maternally inherited knockout (6). Postnatal viability is poor to zero with all three classes. On the other hand, no trace of the abnormal hyperkinetic behaviour seen with the PatDp genotype (16) is found in Oed mice, whereas some possibly different form of ‘ataxia’ has been described with the maternally inherited knockout (6).
In the reciprocal situation with functional paternal allele absence, clear similarities are seen for two of the three genotypes. With the MatDp (16) and the paternally derived knockout (6), neonates show thin flat-sided bodies and typically fail to suckle well if at all. They are hypokinetic. The only recognized difference between the two phenotypes is that the maternal duplication (MatDp) class invariably dies within a few hours, whereas a significant number of the knockouts survive despite the feeding difficulties.
The Sml phenotype obtained with paternally Oed-Sml transmission differs considerably. They do not have detectably abnormal body shapes, nor the hypokinetic behaviour with near-absent suckling ability. Generally they are normal at birth although there appears to be some early postnatal inviability that is detectable by a shortage in their numbers at 5–7 days of age. They cannot be reliably identified earlier and the only recognized effect is the growth retardation. The paternally inherited knockout is also reported to show growth retardation, but it is possible that this is due to the suckling difficulties. Overall, the Sml phenotype differs considerably from the hypokinetic MatDp and paternally derived knockout classes but, like the homozygous knockout (6), the Oed-Sml homozygote is an embryonic lethal.
It has recently been shown (6) that the loss of exon 2 of Gnas disrupts expression of the neighbouring imprinted genes, Nesp and Gnasxl. Both genes could therefore have roles in producing the phenotypes brought about in the uniparental duplication (16) and in the knockout (6) mice. For the Oed-Sml mutation, the expectation is that it represents a point mutation, but it also gives two parent of origin-dependent phenotypes. However, taking everything together, a hypothesis can be constructed that can explain the clearly established findings (Fig. 9).
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If Nesp and the newly identified imprinted gene Nespas, which runs antisense to Nesp (23), are taken as candidate loci for the Oed-Sml mutation, a single point mutation could disrupt expression of both to give two phenotypes that are parent of origin dependent. The absence of a maternal Nesp transcript in Oed mice, in PatDp and in maternally inherited knockout mice, could then account for the oedema and the absence of the paternal Nespas transcript could cause the Sml growth retardation phenotype (Fig. 9). The Sml phenotype would also be expected in MatDp mice as these animals can have no Nespas transcript, but these hypokinetic non-suckling mice die before this effect can be detected. In contrast, no such growth retardation would be expected with the paternally inherited knockout, as the Nespas transcript would be expressed normally. The hypothesis could be equally well applied to any gene within the region which has sense and anti-sense transcripts and which is disrupted by Gnas exon 2 knockouts.
If Gnasxl is taken as the candidate locus for the behavioural effects the transcript, which splices onto exon 2 of Gnas, would be disrupted in paternally inherited knockout mice. The absence of this transcript could then account for the hypokinetic second phenotype of the knockout and MatDp animals. On this basis, the hyperkinetic behaviour of the PatDp class, which is absent in Oed mice, could be explained by the presence of two expressed copies of Gnasxl (Fig. 9). The hypothesis ignores the ambiguous growth retardation suggested for the maternally inherited knockout and also the possibility that the late onset ‘ataxia’ described for the paternally inherited knockout (6) might correspond to the neonatal hyperkinetic behaviour of PatDp animals (16). Further studies with knockouts of the Nesp, Gnasxl and other genes in the region should elucidate the genetic complexities of this imprinting domain in the mouse. Further such studies in the mouse may provide a better understanding of imprinting in the homologous human chromosome domain.
Appraisal of the imprinting effects attributable to genes within the imprinting regions has suggested from the first that imprinted genes have important roles in development and commonly affect growth (26). Whether or not Mnt affects Igf2, the prenatal growth effects of the mutation accords with this observation. In the case of Oed-Sml, two such growth effects are evident. The first, seen in Oed mice, is a growth retardation of the heart. It might be expected that this organ will be one site of the responsible mutant gene expression. The second, seen in Sml mice, is the postnatal growth retardation, which no doubt is growth hormone-dependent.
It seems remarkable that all three phenotypes described, Mnt, Oed and Sml, show similarities with imprinting effects associated with other chromosomal regions. Thus, the prenatal growth reduction of the paternally inherited Mnt (and the Igf2 knockout) closely resembles the prenatal growth effect caused by an absence of a paternal copy of proximal chromosome 11 (27). An oedema resembling that caused by the maternally inherited Oed-Sml mutation, with congenital heart abnormality (although with enlargement rather than reduction), also occurs with absence of a maternal copy of Igf2r (20,27). Postnatal growth retardation effects that are similar to that illustrated with the paternally inherited Oed-Sml mutation occur with both a paternally transmitted knockout of the imprinted Rasgrf1 locus on chromosome 9 (5) and with an absence of a maternal copy of central chromosome 7 (with mouse AS) (28). This duplication of phenotypes is also seen with some other chromosome regions. Thus, suckling problems are seen with absences of a paternal copies of both distal chromosome 2 (16) and central chromosome 7 (mouse PWS) (28) and neurological abnormalities are seen with absence of maternal copies of both distal chromosome 2 (16) and central chromosome 7 (mouse AS) (28). More detailed characterization of these imprinting effects are needed to establish how well these similarities really correspond. Most of them fit with the ‘conflict hypothesis’ of Moore and Haig (29), but elucidation of this seeming phenotype duplication may help to resolve further the intriguing puzzle of the role of imprinting in mammalian development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Breeding and experimental procedures
The specific locus tests from which the mutants derived utilized F1 hybrid males deriving from the cross of the C3H/HeH and 101/H inbred strains. The tester females used were PT random breeds which are homozygous for seven different recessive mutations: non-agouti (a), brown (b), pink-eyed dilution (p), chinchilla (cch), short-ear (se), dilute (d) and piebald spotting (s). All genetic analyses of the two mutants, other than mapping, was carried out in crosses using the F1 hybrid, or with animals of an equivalent but mixed genetic background.
Growth of the mutants relative to their + sibs was made on the basis of weight ratios to allow for variation between litters and was assessed by Student’s t-tests. Wet and dry weights of neonates were similarly investigated. Dry weights were measured after freeze-drying chopped-up carcasses for 30 h. The brachyury mutation, T37H, used in crosses with Mnt, derived from the same series of specific locus mutation radiation experiments as Mnt (17). Like the well-studied Thp allele, T37H represents a chromsome 17 deletion (E.P. Evans, personal communication) and shows a Tme lethality when transmitted through females. Affected young display an oedema and overgrowth with occasional omphalocoele or hare-lip (B.M. Cattanach, unpublished data) and most die prior to birth. Mice with PatDp.dist2 used for comparison with Oed-Sml mutants were generated using the T(2;11)30H translocation.
All animal studies were carried out under the guidance issued by the Medical Research Council in The Use of Animals for Medical Research (July 1993) and Home Office Project Licence nos. 30/00875 and 30/01518.
Mapping procedures
Mnt and Oed-Sml mapping was carried by crossing mutant males with M.m.castaneus females, backcrossing to C3H/HeH and typing the backcross progeny for M.musculus–M.m.castaneus polymorphisms at Mit marker loci in the candidate regions. DNA was extracted from tail tissue using standard procedures. For PCR analysis, 50 ng of DNA was added to a final volume of 25 µl of PCR master mix (Advanced Biotechnologies, Epsom, UK), including 2.5 mM MgCl2 and 0.4 µM of relevant primers. PCR was performed at a 55°C annealing temperature and with 35 cycles. Products were run out on 3% agarose gels with TBE buffer.
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ACKNOWLEDGEMENTS |
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We thank Peter Glenister for achieving the rescue of the Oed-Sml mutation by in vitro fertilization and embryo transfer. We are also grateful to Dr Leon Cobb for helping with the pathology, Colin Beechey for preparing the diagrams, David Papworth for his detailed statistical analysis of the data and also Lynnette Hobbs, Anne-Marie Woodward and Vicky Savage for their animal husbandry.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1235 834393: Fax: +44 1235 834776; Email: b.cattanach@har.mrc.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Cattanach, B.M. and Beechey, C.V. (1997) Genomic imprinting in the mouse: possible final analysis. In Reik, W. and Surani, A. (eds), Genomic Imprinting: Frontiers in Molecular Biology, Vol. 18. IRL Press, Oxford University Press, Oxford, NY, Tokyo, pp. 118–145.
2 Hall, J.G. (1990) Genomic imprinting: review and relevance to human diseases. Am. J. Hum. Genet., 46, 857–873.[Web of Science][Medline]
3 DeChiara, T.M., Efstratiadis, A. and Robertson, E.J. (1990) A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature, 345, 78–80.[Medline]
4 DeChiara, T.M., Robertson, E.J. and Efstratiadis, A. (1991) Parental imprinting of the mouse insulin-like growth factor II gene. Cell, 64, 849–859.[Web of Science][Medline]
5 Plass, C., Shibata, H., Kalcheva, I., Mullins, L., Kotelevtseva, N., Mullins, J., Kato, R., Sasaki, H., Hirotsune, S., Okazaki, Y. et al (1966) Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M. Nature Genet., 14, 106–109.[Web of Science][Medline]
6 Yu, S., Yu, D., Lee, E., Eckhaus, M., Lee, R., Corria, Z., Accli, D., Westphal, H. and Weinstein, L.S. (1998) Variable and tissue-specific hormone resistance in heterotrimeric Gs protein 
-subunit (Gs) knockout mice is due to tissue-specific imprinting of the Gs gene. Proc. Natl Acad. Sci. USA, 95, 8715–8720.
7 Yang, T., Adamson, T.E., Resnick, J.L., Leff.S, Wevrick, R., Francke, U., Jenkins, N.A., Copeland, N.G. and Brannan, C.I. (1998) A mouse model for Prader-Willi syndrome imprinting-centre mutations. Nature Genet., 19, 25–31.[Web of Science][Medline]
8 Jiang, Y-H., Armstrong, D., Albrecht, U., Atkins, CM., Noebels, J.L., Eichele, G., Sweatt, J.D. and Beaudet, A.L. (1998) Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron, 21, 799–811.[Web of Science][Medline]
9 Gabriel, J.M., Merchant, M., Ohta, T., Ji, Y., Caldwell, R.G., Ramsey, M.J., Tucker, J.D., Longnecker, T. and Nicholls, R.D (1999) A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and Angelman syndromes. Proc. Natl Acad. Sci. USA, 96, 9258–9263.
10 Johnson, D.R. (1974) Hairpin-tail: a case of post reductional gene action in the mouse egg? Genetics, 76, 795–805.
11 Johnson, D.R. (1975) Further observations on the hairpin-tail (Thp) mutation in the mouse. Genet. Res., 24, 207–213.
12 Barlow, D.P., Stoger, R., Herrmann, B.G., Saito, K. and Schweifer, N. (1991) The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature, 349, 84–87.[Medline]
13 Wilson, L.C. and Trembath, R.C. (1994) Albright’s Hereditary Osteodystrophy. J. Med. Genet., 31, 779–794.
14 Juppner, H., Schipani, E., Bastepe, M., Cole, D.E.C., Lawson, M.L., Mannstadt, M., Hendy, G.N., Plotkin, H., Koshyami, H., Koh, T. et al (1998) The gene responsible for pseudohypoparathyroidism type Ib is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc. Natl Acad. Sci. USA, 95, 11798–11803.
15 Nicholls, R.D., Saitoh, S. and Horsthemke, B. (1998) Imprinting in Prader-Willi and Angelman syndromes. Trends Genet., 14, 194–200.[Web of Science][Medline]
16 Cattanach, B.M. and Kirk, M. (1985) Differential activity of maternally and paternally derived chromosome regions in mice. Nature, 315, 496–498.[Medline]
17 Cattanach, B.M., Peters, J. and Rasberry, C. (1989) Induction of specific locus mutations in mouse spermatogonial stem cells by combined chemical X-ray treatments. Mutat. Res., 212, 91–101.[Web of Science][Medline]
18 Cattanach, B.M., Burtenshaw, M.D., Rasbery, C. and Evans, E.P. (1993) Large deletions and other gross forms of chromosome imbalance compatible with viability and fertility in the mouse. Nature Genet., 3, 56–61.[Web of Science][Medline]
19 Mouse Genome Database (MGD), Mouse Genome Informatics Web Site, The Jackson Laboratory, Bar Harbor, ME (http://www.informatics.jax.org/May2000 ).
20 Filson, A.J., Louvi, A., Efsratiadis, A. and Robertson, E.J. (1993) Rescue of the T-associated maternal effect in mice carrying null mutations in Igf-2 and Igf2r, two reciprocally imprinted genes. Development, 118, 731–736.[Abstract]
21 Justice, M.J., Noveroska, J.K., Weber, J.S., Zheng, B. and Bradley, A. (1999) Mouse ENU mutagenesis. Hum. Mol. Genet., 8, 1855–1963.
22 Williamson, C.M., Beechey, C.V., Papworth, D., Wroe, S.F., Wells, C.A., Cobb, L. and Peters, J. (1998) Imprinting of distal mouse chromosome 2 is associated with phenotypic anomalies in utero. Genet. Res. Camb., 72, 255–265.[Web of Science][Medline]
23 Peters, J., Wroe, S.F., Wells, C.A., Miller, H.J., Bodle, D., Beechey, C.V., Williamson, C.M. and Kelsey, G. (1999) A cluster of oppositely imprinted transcripts at the Gnas locus in the distal imprinting region of mouse chromosome 2. Proc. Natl Acad. Sci. USA, 96, 3830–3835.
24 Wroe, S.F., Kelsey, G., Skinner, J.A., Bodle, D., Ball, S.T., Beechey, C.V., Peters, J. and Williamson, C.M. (2000) An imprinted transcript, antisense to Nesp, adds complexity to the cluster of imprinted genes at the mouse Gnas locus. Proc. Natl Acad. Sci. USA, 97, 5203–5208.
25 Baker, J., Liu, J.-P., Robertson, E.J. and Efstratiadis, A. (1993) Role of insulin-like growth factors in embryonic and postnatal growth. Cell, 75, 73–82.[Web of Science][Medline]
26 Cattanach, B.M. (1991) Gene expression and its control. In Davies, K. and Tighleman, S. (eds), ChromosomeIimprinting and its Significance for Mammalian Development. Genome Analysis, Vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 41–71.
27 Miyabara, S, Bogart, M.H., Winking, H. and Sugihara, H. (1993) Overgrowth and enlarged heart in mouse fetuses with maternally inherited Thp and twLub2. An example of genomic imprinting in animals. Cong. Aonom., 33, 63–75.
28 Cattanach, B.M., Barr, J.A., Beechey, C.V., Martin, J., Noebels, J. and Jones, J. (1997) A candidate model for Angelman syndrome in the mouse. Mamm. Genome, 8, 472–478.[Web of Science][Medline]
29 Moore, T. and Haig, D. (1991) Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet., 7, 45–49.[Web of Science][Medline]
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