Genetic analysis of genomic imprinting: an Imprintor-1 gene controls inactivation of the paternal copy of the mouse Tme locus.

The Thp deletion on mouse chromosome 17 is lethal when inherited from the mother, because it deletes the T-associated maternal effect (Tme) locus, the paternal copy of which is inactivated by genomic imprinting. We have found a paternally nonimprinted Tme variant in crosses of Thp females with Mus m. musculus males. The data are consistent with the existence of a single Tme-unlinked gene, Imprintor-1 (Imp-1), with two alleles, one of which only causes imprinting at the Tme locus. Imp-1 is unlinked to the gene for cation-dependent Man-6-P receptor and acts prezygotically. Although Tme and Igf2r were thought to be identical, they show different patterns of imprinting in interspecies hybrids. The apparent nonequivalence of the Igf2r gene and Tme results in occurrence of viable mice lacking an active Igf2r gene. These mice are bigger at birth than their normal littermates, in accord with the proposed function of the IGF-II/Man-6-P receptor.     

Paternal transmission of the mouse Thp mutation is lethal in some genetic backgrounds

T^hp is a large deletion on chromosome 17 which includes the maternal lethal gene Tme. Documentation of inheritance patterns suggests that Tme is an imprinted gene which is required for viability; maternal deletion is lethal while paternal deletion is viable. However, paternal transmission of T^hp is rarely the expected 50%. We show here that paternally inherited T^hp is lethal in some strains, providing evidence of an incompletely penetrant, dosage sensitive lethal allele of a locus that probably maps to the hairpin tail region of chr. 17. Interpretation of the various phenotypes associated with loss of the putative Tme gene, lgf2r, may need to be revised in view of these observations. Dev Genet 20:23–28, 1997. © 1997 Wiley-Liss, Inc.     

Escape from Genomic Imprinting at the Mouse T-Associated Maternal Effect (Tme) Locus

Genomic imprinting occurs at the paternally inherited allele of the mouse T-associated maternal effect (Tme) locus. As a consequence, maternal transmission of a functional Tme gene is normally required for viability and individuals that receive a Tme-deleted chromosome (Thp or tlub2) from their mother die late in gestation or shortly thereafter. Here we report that a rearranged paternally derived chromosome duplicated for the Tme locus can act to rescue animals that have not received a maternal copy of the Tme locus. Unexpectedly, all rescued animals display an abnormal short/kinky tail phenotype. Somatic transfer of genomic imprinting between homologs by means of a transvection-like process between paired Tme and T loci is proposed as a model to explain the results obtained.     

Functional Analysis of Mutations of Murine Chromosome 17 with the Use of Tertiary Trisomy

Analysis of the functional nature of mutations can be based on comparisons of their manifestation in organisms with a deletion or duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H, it is feasible to produce mice with tertiary trisomy of the proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t complex tail interaction), and tcl (t complex lethal) that are specific to t haplotypes. The set of dominant and recessive mutations was assigned to two groups: one obligatory, manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes. It seeks to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.     

Functional analysis of mutations on murine chromosome 17 using tertiary trisomy

Analysis of the functional nature of mutations can be based on their manifestation in organisms with a deletion or a duplication of a particular chromosome segment. With the use of reciprocal translocation T(16;17)43H it is feasible to produce mice with tertiary trisomy for proximal region of chromosome 17. The mutations on chromosome 17 we tested included brachyury (T), hairpin tail (Thp), kinky (Fuki), quaking (qk), tufted (tf), as well as tct (t-complex tail interaction) and tcl (t complex lethal), that are specific for t haplotypes. The set of dominant and recessive mutations was assigned to two groups, one obligatory manifesting itself in the phenotype independently of the number of normal alleles in di- and trisomics, and the other facultative, phenotypically manifesting itself, depending upon the dosage of mutant alleles. A model was derived from analysis of the interaction of the T and Thp mutations with t haplotypes which is to explain the morphogenetic effects of the mutations observed in mice of different genotypes. The tir gene is postulated to reside on chromosome 17 within its framework. It is suggested that the gene dosage ratio at the tir and tct loci determines tail length.     

Unequal meiotic crossover: a frequent cause of NF1 microdeletions

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.     

Rare etiology of autosomal recessive disease in a child with noncarrier parents

A child with maple syrup urine disease type 2 (MSUD2) was found to be homozygous for a 10-bp MSUD2-gene deletion on chromosome 1. Both purported parents were tested, and neither carries the gene deletion. Polymorphic simple-sequence repeat analyses at 15 loci on chromosome 1 and at 16 loci on other chromosomes confirmed parentage and revealed that a de novo mutation prior to maternal meiosis I, followed by nondisjunction in maternal meiosis II, resulted in an oocyte with two copies of the de novo mutant allele. Fertilization by a sperm that did not carry a paternal chromosome 1 or subsequent mitotic loss of the paternal chromosome 1 resulted in the propositus inheriting two mutant MSUD2 alleles on two maternal number 1 chromosomes.     

A genetic model for the Prader-Willi syndrome and its implication for angelman syndrome

Summary Sporadic cases of Prader-Willi syndrome (PWS) are associated with the physical absence of the paternal Prader-Willi chromosome region (PWCR) by deletion 15q11–13, by segmental maternal heterodisomy or by chromosome rearrangements resulting in homozygosity for maternal PWCR. In isolated/familial cases, it is proposed that the expression of PWS depends on the functional absence caused by mutated gene(s) within the paternal PWCR. The same mutation on a maternally derived chromosome 15 is not able to express PWS. An epigenetic mechanism associated with the paternal meiosis is essential. In the Angelman syndrome (AS), inverse mechanisms are postulated. There is convincing evidence for specific PWS and AS genes or alleles within PWCR. This is compatible with the observations of interstitial chromosome deletions of the critical region in normal individuals or in probands with phenotypes other than PWS or AS. The new ideas of the model stated here are: (1) the proposed epigenetic mechanism in PWCR is obviously common in humans, but is usually of no phenotypic relevance; (2) interactions with specific chromosomal or gene mutations are required for the clinical expression of PWS or AS; (3) each factor alone is not able to produce an abnormal phenotype.     

Expression of size-polymorphic androgen receptor (AR) gene in ovarian endometriosis according to the number of cytosine, adenine, and guanine (CAG) repeats in AR alleles.

An increase in androgen receptor (AR) caused by estrogen is recognized as one of the biological phenomena related to estrogen-induced growth in uterine endometrium. The A/B region of AR gene in X chromosome involves the cytosine, adenine, and guanine (CAG) repeats. Random X chromosome inactivation with AR alleles in individual cells occurs in females. Therefore, approximately either paternal or maternal single dominant polymorphic AR mRNA must be expressed in neoplastic tissue originated from monoclone. This prompted us to determine deviated number of CAG repeats in AR mRNA to understand clonality in ovarian endometriosis. In all cases of heterozygous AR alleles, although paternal and maternal AR mRNAs from normal eutopic uterine endometrium were consistently expressed as AR alleles, either paternal or maternal single dominant AR mRNA expression was found in an individual ovarian endometrioma. Therefore, an individual ovarian endometrioma might be formed from an independent monoclonal ovarian endometriotic endometrial cell after inactivation of either AR allele in X chromosome.     

Sporadic imprinting defects in Prader-Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counseling, and prenatal diagnosis.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are caused by the loss of function of imprinted genes in proximal 15q. In approximately 2%-4% of patients, this loss of function is due to an imprinting defect. In some cases, the imprinting defect is the result of a parental imprint-switch failure caused by a microdeletion of the imprinting center (IC). Here we describe the molecular analysis of 13 PWS patients and 17 AS patients who have an imprinting defect but no IC deletion. Heteroduplex and partial sequence analysis did not reveal any point mutations of the known IC elements, either. Interestingly, all of these patients represent sporadic cases, and some share the paternal (PWS) or the maternal (AS) 15q11-q13 haplotype with an unaffected sib. In each of five PWS patients informative for the grandparental origin of the incorrectly imprinted chromosome region and four cases described elsewhere, the maternally imprinted paternal chromosome region was inherited from the paternal grandmother. This suggests that the grandmaternal imprint was not erased in the father's germ line. In seven informative AS patients reported here and in three previously reported patients, the paternally imprinted maternal chromosome region was inherited from either the maternal grandfather or the maternal grandmother. The latter finding is not compatible with an imprint-switch failure, but it suggests that a paternal imprint developed either in the maternal germ line or postzygotically. We conclude (1) that the incorrect imprint in non-IC-deletion cases is the result of a spontaneous prezygotic or postzygotic error, (2) that these cases have a low recurrence risk, and (3) that the paternal imprint may be the default imprint.     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

Parental effect of DNA (Cytosine-5) methyltransferase 1 on grandparental-origin-dependent transmission ratio distortion in mouse crosses and human families

Transmission ratio distortion (TRD) is a deviation from the expected Mendelian 1:1 ratio of alleles transmitted from parents to offspring and may arise by different mechanisms. Earlier we described a grandparental-origin-dependent sex-of-offspring-specific TRD of maternal chromosome 12 alleles closely linked to an imprinted region and hypothesized that it resulted from imprint resetting errors in the maternal germline. Here, we report that the genotype of the parents for loss-of-function mutations in the Dnmt1 gene influences the transmission of grandparental chromosome 12 alleles. More specifically, maternal Dnmt1 mutations restore Mendelian transmission ratios of chromosome 12 alleles. Transmission of maternal alleles depends upon the presence of the Dnmt1 mutation in the mother rather than upon the Dnmt1 genotype of the offspring. Paternal transmission mirrors the maternal one: live-born offspring of wild-type fathers display 1:1 transmission ratios, whereas offspring of heterozygous Dnmt1 mutant fathers tend to inherit grandpaternal alleles. Analysis of allelic transmission in the homologous region of human chromosome 14q32 detected preferential transmission of alleles from the paternal grandfather to grandsons. Thus, parental Dnmt1 is a modifier of transmission of alleles at an unlinked chromosomal region and perhaps has a role in the genesis of TRD.     

The paternal gene of the DDK syndrome maps to the Schlafen gene cluster on mouse chromosome 11

The DDK syndrome is an early embryonic lethal phenotype observed in crosses between females of the DDK inbred mouse strain and many non-DDK males. Lethality results from an incompatibility between a maternal DDK factor and a non-DDK paternal gene, both of which have been mapped to the Ovum mutant (Om) locus on mouse chromosome 11. Here we define a 465-kb candidate interval for the paternal gene by recombinant progeny testing. To further refine the candidate interval we determined whether males from 17 classical and wild-derived inbred strains are interfertile with DDK females. We conclude that the incompatible paternal allele arose in the Mus musculus domesticus lineage and that incompatible strains should share a common haplotype spanning the paternal gene. We tested for association between paternal allele compatibility/incompatibility and 167 genetic variants located in the candidate interval. Two diallelic SNPs, located in the Schlafen gene cluster, are completely predictive of the polar-lethal phenotype. These SNPs also predict the compatible or incompatible status of males of five additional strains.     

An unexpected function of the Prader-Willi syndrome imprinting center in maternal imprinting in mice

Genomic imprinting is a phenomenon that some genes are expressed differentially according to the parent of origin. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurobehavioral disorders caused by deficiency of imprinted gene expression from paternal and maternal chromosome 15q11-q13, respectively. Imprinted genes at the PWS/AS domain are regulated through a bipartite imprinting center, the PWS-IC and AS-IC. The PWS-IC activates paternal-specific gene expression and is responsible for the paternal imprint, whereas the AS-IC functions in the maternal imprint by allele-specific repression of the PWS-IC to prevent the paternal imprinting program. Although mouse chromosome 7C has a conserved PWS/AS imprinted domain, the mouse equivalent of the human AS-IC element has not yet been identified. Here, we suggest another dimension that the PWS-IC also functions in maternal imprinting by negatively regulating the paternally expressed imprinted genes in mice, in contrast to its known function as a positive regulator for paternal-specific gene expression. Using a mouse model carrying a 4.8-kb deletion at the PWS-IC, we demonstrated that maternal transmission of the PWS-IC deletion resulted in a maternal imprinting defect with activation of the paternally expressed imprinted genes and decreased expression of the maternally expressed imprinted gene on the maternal chromosome, accompanied by alteration of the maternal epigenotype toward a paternal state spread over the PWS/AS domain. The functional significance of this acquired paternal pattern of gene expression was demonstrated by the ability to complement PWS phenotypes by maternal inheritance of the PWS-IC deletion, which is in stark contrast to paternal inheritance of the PWS-IC deletion that resulted in the PWS phenotypes. Importantly, low levels of expression of the paternally expressed imprinted genes are sufficient to rescue postnatal lethality and growth retardation in two PWS mouse models. These findings open the opportunity for a novel approach to the treatment of PWS.     

The evolution of X-linked genomic imprinting

We develop a quantitative genetic model to investigate the evolution of X-imprinting. The model compares two forces that select for X-imprinting: genomic conflict caused by polygamy and sex-specific selection. Genomic conflict can only explain small reductions in maternal X gene expression and cannot explain silencing of the maternal X. In contrast, sex-specific selection can cause extreme differences in gene expression, in either direction (lowered maternal or paternal gene expression), even to the point of gene silencing of either the maternal or paternal copy. These conclusions assume that the Y chromosome lacks genetic activity. The presence of an active Y homologue makes imprinting resemble the autosomal pattern, with active paternal alleles (X- and Y-linked) and silenced maternal alleles. This outcome is likely to be restricted as Y-linked alleles are subject to the accumulation of deleterious mutations. Experimental evidence concerning X-imprinting in mouse and human is interpreted in the light of these predictions and is shown to be far more easily explained by sex-specific selection.     

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.     

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11-q13 deletions. Both also can result from uniparental disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11-q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11-q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, postnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11-q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.