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The authors report the occurrence of pulsus alternans, a condition characterised by the alternance of pulses of higher and lower amplitude, in two dogs and one cat under general anaesthesia. The presence of an underlying cardiac disease was confirmed in the cat but not in either dog, which - based on history and clinical findings - had presumably normal cardiovascular function before the anaesthetic. Possible mechanisms, including negative inotropy and haemodynamic and Frank-Starling effects, as well as the role of general anaesthesia as the potential triggering factor, are discussed in this report. Ephedrine resulted in the successful treatment of pulsus alternans, as demonstrated by the return of normal pulse and synchronisation of heart and pulse rates in the cat and in one dog. In the other dog, pulse pattern and frequency returned to normal once the guidewire for central line placement was withdrawn.

Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions., A phenylalanine hydroxylase–deficient pig model of phenylketonuria is established and characterized.

Prader-Willi syndrome (PWS) is a multisystem disorder caused by genetic loss of function of a cluster of imprinted, paternally expressed genes. Neonatal failure to thrive in PWS is followed by childhood-onset hyperphagia and obesity among other endocrine and behavioral abnormalities. PWS is typically assumed to be caused by an unknown hypothalamic-pituitary dysfunction, but the underlying pathogenesis remains unknown. A transgenic deletion mouse model (TgPWS) has severe failure to thrive, with very low levels of plasma insulin and glucagon in fetal and neonatal life prior to and following onset of progressive hypoglycemia. In this study, we tested the hypothesis that primary deficits in pancreatic islet development or function may play a fundamental role in the TgPWS neonatal phenotype. Major pancreatic islet hormones (insulin, glucagon) were decreased in TgPWS mice, consistent with plasma levels. Immunohistochemical analysis of the pancreas demonstrated disrupted morphology of TgPWS islets, with reduced α- and β-cell mass arising from an increase in apoptosis. Furthermore, in vivo and in vitro studies show that the rate of insulin secretion is significantly impaired in TgPWS β-cells. In TgPWS pancreas, mRNA levels for genes encoding all pancreatic hormones, other secretory factors, and the ISL1 transcription factor are upregulated by either a compensatory response to plasma hormone deficiencies or a primary effect of a deleted gene. Our findings identify a cluster of imprinted genes required for the development, survival, coordinate regulation of genes encoding hormones, and secretory function of pancreatic endocrine cells, which may underlie the neonatal phenotype of the TgPWS mouse model.

To characterize a novel splice variant of the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMRalpha), which we discovered in human neutrophils.We used reverse transcriptase polymerase chain reaction to identify, characterize, and examine the expression of a novel splice variant of the GMRalpha transcript. At the protein level, surface plasmon resonance was used to measure the affinity of a recombinant soluble form of the novel GMRalpha protein for GM-CSF ligand. The full-length novel GMRalpha protein was expressed in a recombinant cell culture system, and its expression and localization were examined using Western blotting, I(125) GM-CSF binding assays, flow cytometry, and a soluble GMRalpha enzyme-linked immunosorbent assay.The novel GMRalpha transcript identified herein contains a previously undescribed exon of the GMRalpha gene; this exon derives from an Alu DNA repeat element, and is alternatively spliced in the novel GMRalpha transcript. Inclusion of this 102 nucleotide exon results in translation of a protein product, which we have named Alu-GMRalpha. Alu-GMRalpha is identical to cell surface GMRalpha, but additionally contains a 34 amino-acid insert in the juxtamembrane region of the extracellular domain of GMRalpha. Functionally, the Alu-GMRalpha-specific epitope does not modify the ability of the protein to bind GM-CSF, but rather appears to be preferentially targeted by ectodomain proteases to mediate the release of a third soluble GM-CSF receptor into the extracellular space.This study provides the first example of a cytokine receptor system in which soluble receptors are produced by three distinct mechanisms. Our results highlight the importance of soluble GMRalpha proteins in regulation of GM-CSF signaling.

We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d 2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS.

Prader–Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11–13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%–100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p 30PUb deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p 30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p 30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p 30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.

Prader-Willi syndrome (PWS) is a complex neurobehavioral disorder that results from loss of function of 10 clustered, paternally expressed genes in a 1.5-Mb region of chromosome 15q11-q13. Many of the primary PWS region genes appear to have nuclear RNA regulatory functions, suggesting that multiple genetic pathways could be secondarily affected in PWS. Using a transgenic mouse model of PWS (TgPWS) with an approximately 4-Mb chromosome 7C deletion of paternal origin that models the neonatal phenotype of the human syndrome we compared by oligonucleotide microarrays expression levels of approximately 12,000 genes and ESTs in TgPWS and wild-type brain. Hybridization data were processed with two distinct statistical algorithms and revealed a dramatically reduced expression of 4 imprinted genes within the deletion region in TgPWS mice, with 2 nonimprinted, codeleted genes reduced twofold. However, only 3 genes outside the deletion were significantly altered in TgPWS mouse brain, with approximately 1.5-fold up-regulation of mRNA levels. Remarkably, these genes map to a single chromosome domain (18B3), and by quantitative RT-PCR we show that 8 genes in this domain are up-regulated in TgPWS brain. These 18B3 genes were up-regulated in an equivalent manner in Angelman syndrome mouse (TgAS) brain, which has the same deletion but of maternal origin. Therefore, the trans-regulation of the chromosome 18B3 domain is due to decreased expression of a nonimprinted gene within the TgPWS/AS mouse deletion in mouse chromosome 7C. Most surprisingly, since 48-60% of the genome was screened, it appears that the imprinted mouse PWS loci do not widely regulate mRNA levels of other genes and may regulate RNA structure.

Obesity is a central feature for several congenital syndromes, including Prader-Willi, Angelman, Bardet-Biedl, Cohen, Alström, and Börjeson-Forssman-Lehmann syndromes, and Albright's hereditary osteodystrophy. Although a role for the central nervous system, including the hypothalamus-pituitary axis, has been suggested for the etiology of obesity in these syndromes, the pathophysiologic pathways are as yet not well defined, and in many cases may identify currently unknown mechanisms. Nevertheless, many of the causative genes and unusual mechanisms, including parental imprinting of genes and complex patterns of inheritance, have been identified. We review the latest advances in understanding congenital syndromes in which obesity is purely genetic, drawing on comparisons to genetic studies of obesity in the human population as well as to those in experimental and agricultural animal models. An understanding of the genetic basis for these syndromes will provide a more comprehensive picture of the mechanisms that control food intake and energy balance in humans.

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions.

As in Prader–Willi syndrome (PWS) infants, mouse models of PWS display failure-to-thrive during the neonatal period. In rodents, the hypothalamic neuropeptide, Neuropeptide Y (NPY) and Agouti-related peptide (AgrP) stimulate while α-melanocyte stimulating hormone (α-MSH) inhibits appetite. We hypothesized that altered expression of these neuropeptides in the hypothalamus may underlie the failure-to-thrive in PWS neonatal mice. To test this hypothesis we evaluated mRNA expression of Npy , Agrp , and Pomc by in situ hybridization in the hypothalamic arcuate nucleus (ARC) of 3-day-old female and male PWS neonates. The results showed that Agrp mRNA expression was decreased relative to wild-type (WT) controls in neonates of both sexes, while mRNA expression of Pomc was upregulated in PWS neonates. Since AgrP and the Pomc -derived peptide, α-MSH, are functional antagonists at melanocortin 4 receptors in the hypothalamic regulation of appetitive behavior, these results show that robust anorexigenic melanocortin signaling, may contribute to the failure-to-thrive in PWS neonatal mice.

Prader-Willi syndrome (PWS) results from loss of function of a 1.0- to 1.5-Mb domain of imprinted, paternally expressed genes in human Chromosome (Chr) 15q11-q13. The loss of imprinted gene expression in the homologous region in mouse Chr 7C leads to a similar neonatal PWS phenotype. Several protein-coding genes in the human PWS region are intronless, possibly arising by retrotransposition. Here we present evidence for continued acquisition of genes by the mouse PWS region during evolution. Bioinformatic analyses identified a BAC containing four genes, Mkrn3, Magel2, Ndn, Frat3, and the Atp5l-ps1 pseudogene, the latter two genes derived from recent L1-mediated retrotransposition. Analyses of eight overlapping BACs indicate that these genes are clustered within 120 kb in two inbred strains, in the order tel-Atp5l-ps1-Frat3-Mkrn3-Magel2-Ndn-cen. Imprinting analyses show that Frat3 is differentially methylated and expressed solely from the paternal allele in a transgenic mouse model of Angelman syndrome, with no expression from the maternal allele in a mouse model of PWS. Loss of Frat3 expression may, therefore, contribute to the phenotype of mouse models of PWS. The identification of five intronless genes in a small genomic interval suggests that this region is prone to retroposition in germ cells or their zygotic and embryonic cell precursors, and that it allows the subsequent functional expression of these foreign sequences. The recent evolutionary acquisition of genes that adopt the same imprint as older, flanking genes indicates that the newly acquired genes become 'innocent bystanders' of a primary epigenetic signal causing imprinting in the PWS domain.

Comparative mapping between the human and chicken genomes has revealed a striking conservation of synteny between the genomes of these two species, but the results have been based on low-resolution comparative maps. To address this conserved synteny in much more detail, a high-resolution human-chicken comparative map was constructed from human chromosome 15. Mapping, sequencing, and ordering of specific chicken bacterial artificial chromosomes has improved the comparative map of chromosome 15 (Hsa15) and the homologous regions in chicken with almost 100 new genes and/or expressed sequence tags. A comparison of Hsa15 with chicken identified seven conserved chromosomal segments between the two species. In chicken, these were on chromosome 1 (Gga1; two segments), Gga5 (two segments), and Gga10 (three segments). Although four conserved segments were also observed between Hsa15 and mouse, only one of the underlying rearrangement breakpoints was located at the same position as in chicken, indicating that the rearrangements generating the other three breakpoints occurred after the divergence of the rodent and the primate lineages. A high-resolution comparison of Gga10 with Hsa15 identified 19 conserved blocks, indicating the presence of at least 16 intrachromosomal rearrangement breakpoints in the bird lineage after the separation of birds and mammals. These results improve our knowledge of the evolution and dynamics of the vertebrate genomes and will aid in the clarification of the mechanisms that underlie the differentiation between the vertebrate species.

Molybdenum is an essential cofactor in many enzymes, but must first be complexed by molybdopterin, whose synthesis requires four enzymatic activities. The first two enzymes of this pathway are encoded by the MOCS1 locus in humans. We describe here a remarkably well-conserved novel mRNA splicing phenomenon that produces both an apparently bicistronic MOCS1AM-OCS1B transcript, as well as a distinct class of monocistronic transcript. The latter are created by a variety of splicing mechanisms (alternative splice donors, alternative splice acceptors, and exon-skipping) to bypass the normal termination nonsense codon of MOCS1A resulting in fusion of the MOCS1A and MOCS1B open reading frames. Therefore, these "no-nonsense" transcripts encode a single bifunctional protein embodying both MOCS1A and MOCS1B activities. This coexpression profile was observed in vertebrates (human, mouse, cow, rabbit, opossum, and chicken) and invertebrates (fruit fly and nematode) spanning at least 700 million years of evolution. Our phylogenetic data also provide evidence that the bicistronic form of MOCS1 mRNA is likely to only produce MOCS1A protein and, combined with Northern analyses, suggests that MOCS1B is translated only as a fusion with MOCS1A. Taken together, the data presented here demonstrate a very highly conserved and physiologically relevant dynamic splicing scheme that profoundly influences the protein-coding potential of the MOCS1 locus.

Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C(3)H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor beta cluster (TCRB) in chromosome 7q34-q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.

Recombination between chromosome-specific low-copy repeats (duplicons) is an underlying mechanism for several genetic disorders. Recently, a chromosome 15 duplicon was discovered in the common breakpoint regions of Prader–Willi and Angelman syndrome deletions. We identified previously the large HERC2 transcript as an ancestral gene in this duplicon, with ∼11 HERC2-containing duplicons, and demonstrated that recessive mutations in mouseHerc2 lead to a developmental syndrome, juvenile development and fertility 2 (jdf2). We have now constructed and sequenced a genomic contig of HERC2, revealing a total of 93 exons spanning ∼250 kb and a CpG island promoter. A processed ribosomal protein L41 pseudogene occurs in intron 2 of HERC2, and putative VNTRs occur in intron 70 (28 copies, ∼76-bp repeat) and 3′ exon 40 through intron 40 (6 copies, ∼62-bp repeat). Sequence comparisons show that HERC2-containing duplicons have undergone several deletion, inversion, and dispersion events to form complex duplicons in 15q11, 15q13, and 16p11. To further understand the developmental role of HERC2, a highly conservedDrosophila ortholog was characterized, with 70% amino acid sequence identity to human HERC2 over the carboxy-terminal 743 residues. Combined, these studies provide significant insights into the structure of complex duplicons and into the evolutionary pathways of formation, dispersal, and genomic instability of duplicons. Our results establish that some genes not only have a protein coding function but can also play a structural role in the genome.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF189221 (Drosophila HERC2 partial cDNA),AC004583 (human HERC2 exons 1–52, genomic);AF224242–AF224257 (human HERC2 exons 54–70, partial genomic sequences); AF225400–AF225409 (human HERC2 exons 71–93, partial genomic sequences). The exon-intron boundaries for exons 53–93 are derived from BACs R-142A11 and 263O22. Additional information is available as a supplementary table at www.genome.org.]

The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mutations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mutations were probably caused by disruption of a single gene. Recent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemically induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11–q13. The central part of mouse chromosome 7 is homologous to human 15q11–q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein–Barr virus Latent Membrane Protein 2A, LMP2A ) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Summary Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurobehavioral disorders that most often arise from a 4-Mb deletion of chromosome 15q11-q13 during paternal or maternal gametogenesis, respectively. At a de novo frequency of ∼.67–1/10,000 births, these deletions represent a common structural chromosome change in the human genome. To elucidate the mechanism underlying these events, we characterized the regions that contain two proximal breakpoint clusters and a distal cluster. Novel DNA sequences potentially associated with the breakpoints were positionally cloned from YACs within or near these regions. Analyses of rodent-human somatic-cell hybrids, YAC contigs, and FISH of normal or rearranged chromosomes 15 identified duplicated sequences (the END repeats) at or near the breakpoints. The END -repeat units are derived from large genomic duplications of a novel gene ( HERC2 ), many copies of which are transcriptionally active in germline tissues. One of five PWS/AS patients analyzed to date has an identifiable, rearranged HERC2 transcript derived from the deletion event. We postulate that the END repeats flanking 15q11-q13 mediate homologous recombination resulting in deletion. Furthermore, we propose that active transcription of these repeats in male and female germ cells may facilitate the homologous recombination process.

Loss of imprinting at IGF2 , generally through an H19 -independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith–Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island ( KvDMR1 ) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2 . Reverse transcription–PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1 -associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA ( KvLQT1-AS ) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

SummaryMicrodeletions of a region termed the “imprinting center” (IC) in chromosome 15q11-q13 have been identified in several families with Prader-Willi syndrome (PWS) or Angelman syndrome who show epigenetic inheritance for this region that is consistent with a mutation in the imprinting process. The IC controls resetting of parental imprints in 15q11-q13 during gametogenesis. We have identified a larger series of cases of familial PWS, including one case with a deletion of only 7.5 kb, that narrows the PWS critical region to

Imprinted genes are marked in the germline and retain molecular memory of their parental origin, resulting in allelic expression differences during development. Abnormalities in imprinted inheritance occur in several genetic diseases and cancer, and are exemplified by the diverse genetic defects involving chromosome 15q11-q13 in Prader-Willi (PWS) and Angelman (AS) syndromes. PWS involves loss of function of multiple paternally expressed genes, while mutations in a single gene, UBE3A, which is subject to spatially restricted imprinting, occur in some AS patients. Identification of mutations in the imprinting process in PWS and AS has led to a definition of an imprinting center (IC), involving the promoter (in PWS) or an alternative transcript of the SNRPN gene (in AS). The IC regulates initiation of imprint switching for all genes in a 2 Mb imprinted domain during gametogenesis. Imprinting mutations define a novel mechanism of genetic disease because they have no direct effect in the affected patient but, rather, it is the parental germline effect of an IC mutation that leads to disease in the offspring.

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.

We have determined the structure, nucleotide sequence, and polymorphisms of the human P gene. Mutations of the P gene result in type II oculocutaneous albinism (OCA2) in humans and pink-eyed dilution (p) in mice. We find that the human P gene is quite large, consisting of 25 exons spanning 250 to 600 kb in chromosome segment 15q11-q13. The P polypeptide appears to define a novel family of small molecule transporters and may be involved in transport of tyrosine, the precursor to melanin synthesis, within the melanocyte. These results provide the basis for analyses of patients with OCA2 and may point toward eventual pharmacologic treatment of this and related disorders of pigmentation.

Prader–Willi syndrome (PWS) occurs in about 1 in 15,000 individuals and is a contiguous gene disorder causing developmental disability, hyperphagia usually with obesity, and behavioral problems, including an increased incidence of psychiatric illness. The genomic imprinting that regulates allele-specific expression of PWS candidate genes, the fact that multiple genes are typically inactivated, and the presence of many genes that produce functional RNAs rather than proteins has complicated the identification of the underlying genetic pathophysiology of PWS. Over 30 genetically modified mouse strains that have been developed and characterized have been instrumental in elucidating the genetic and epigenetic mechanisms for the regulation of PWS genes and in discovering their physiological functions. In 2011, a PWS Animal Models Working Group (AMWG) was established to generate discussions and facilitate exchange of ideas regarding the best use of PWS animal models. Here, we summarize the goals of the AMWG, describe current animal models of PWS, and make recommendations for strategies to maximize the utility of animal models and for the development and use of new animal models of PWS.

Hereditary spastic paraplegias (HSPs) comprise a group of neurodegenerative disorders that are characterized by progressive spasticity of the lower extremities, due to axonal degeneration in the corticospinal motor tracts. HSPs are genetically heterogeneous and show autosomal dominant inheritance in ∼70–80% of cases, with additional cases being recessive or X-linked. The most common type of HSP is SPG4 with mutations in the SPAST gene, encoding spastin, which occurs in 40% of dominantly inherited cases and in ∼10% of sporadic cases. Both loss-of-function and dominant-negative mutation mechanisms have been described for SPG4, suggesting that precise or stoichiometric levels of spastin are necessary for biological function. Therefore, we hypothesized that regulatory mechanisms controlling expression of SPAST are important determinants of spastin biology, and if altered, could contribute to the development and progression of the disease. To examine the transcriptional and post-transcriptional regulation of SPAST, we used molecular phylogenetic methods to identify conserved sequences for putative transcription factor binding sites and miRNA targeting motifs in the SPAST promoter and 3′-UTR, respectively. By a variety of molecular methods, we demonstrate that SPAST transcription is positively regulated by NRF1 and SOX11. Furthermore, we show that miR-96 and miR-182 negatively regulate SPAST by effects on mRNA stability and protein level. These transcriptional and miRNA regulatory mechanisms provide new functional targets for mutation screening and therapeutic targeting in HSP.

Genetic and molecular analyses of a number of radiation-induced deletion mutations of the pink-eyed dilution (p) locus in mouse chromosome 7 have identified a specific interval on the genetic map associated with a neonatally lethal mutation that results in cleft palate. This interval, closely linked and distal to p, and bracketed by the genes encoding the alpha 5 and beta 3 subunits of the type A gamma-aminobutyric acid receptor (Gabra5 and Gabrb3, respectively), contains a gene(s) (cp1; cleft palate 1) necessary for normal palate development. The cp1 interval extends from the distal breakpoint of the prenatally lethal p83FBFo deletion to the Gabrb3 locus. Among 20 p deletions tested, there was complete concordance between alterations at the Gabrb3 transcription unit and inability to complement the cleft-palate defect. These mapping data, along with previously described in vivo and in vitro teratological effects of gamma-aminobutyric acid or its agonists on palate development, suggest the possibility that a particular type A gamma-aminobutyric acid receptor that includes the beta 3 subunit may be necessary for normal palate development. The placement of the cp1 gene within a defined segment of the larger D15S12h (p)-D15S9h-1 interval in the mouse suggests that the highly homologous region of the human genome, 15q11-q13, be evaluated for a role(s) in human fetal facial development.

The Prader-Willi and Angelman syndromes are now well established as the paradigm of genomic imprinting in human disease. Over the past year, much has been learnt about the mechanisms by which these syndromes arise and molecular diagnostics for the majority of patients are now available. Mouse models for aspects of the syndromes have been established, and the first association between a gene, located in chromosome 15, at 15q11-q13, and a phenotype (albinism) has been proven. Large parts of the critical regions have been cloned and at least six genes identified. Three genes or DNA sequences may be imprinted: two of these demonstrate DNA-methylation imprints and one is functionally imprinted in mouse. While the molecular mechanism of imprinting is not yet understood, it is beginning to yield its secrets to DNA methylation, replication, and chromatin structure studies of the phenomenon.

Although Angelman (AS) and Prader-Willi (PWS) syndromes are human genetic disorders with distinctly different developmental and neurobehavioural phenotypes, they both have abnormalities in inheritance of chromosome 15q11-q13. Whether AS or PWS arises depends on the parental origin of a deletion or uniparental disomy (the inheritance of 2 copies of a genetic locus from only one parent) for 15q11-q13. Normal development requires a genetic contribution for this genetic region from both a male and a female parent. The dependence on parental origin implies that genes in human 15q11-q13 have distinct functions depending upon epigenetic, parent-of-origin differences, known as genomic imprinting. Here, I review the role of uniparental disomy and genomic imprinting in the pathogenesis of AS and PWS, and briefly discuss phenotype-genotype correlations using candidate genes and mouse models, in particular for hypopigmentation.

THE mouse pink-eyed dilution (p) locus on chromosome 7 is associated with defects of skin, eye and coat pigmentation1. Mutations at p cause a reduction of eumelanin (black-brown) pigment and altered morphology of black pigment granules (eumelano-somes), but have little effect on pheomelanin (yellow-red) pigment2. We show here that the human complementary DNA DN10, linked to thep locus in mice3–5, identifies the human homologue (P) of the mouse pgene, and appears to encode an integral membrane transporter protein. The expression pattern of this gene in various p mutant mice correlates with the pigmentation phenotype; moreover, an abnormally sized messenger RNA is detected in one mutant,pun, which reverts to the normal size in pun revertants. The human P gene corresponds to the D15S12locus within the chromosome segment 15qll–ql3, which is typically deleted in patients with Prader–Willi and Angelman syndrome (see ref. 5 for review). These disorders are phenotypically distinct, depending on the parent of origin of the deleted chromosome5–7, but both syndromes are often associated with hypopigmentation of the skin, hair and eyes (see ref. 8 for review), and deletion of theP gene may be responsible for this hypopigmentation. In addition, we report a mutation in both copies of the human P gene in one case of tyrosinase-positive (type II) oculocutaneous albinism, recently linked to 15qll–ql3 (ref. 9).

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader–Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

Intrauterine growth retardation (IUGR) has been linked to the onset of diseases in adulthood, including type 2 diabetes, and has been proposed to result from altered gene regulation patterns due to epigenetic modifications of developmental genes. To determine whether epigenetic modifications may play a role in the development of adult diabetes following IUGR, we used a rodent model of IUGR that expresses lower levels of Pdx1, a pancreatic and duodenal homeobox 1 transcription factor critical for beta cell function and development, which develops diabetes in adulthood. We found that expression of Pdx1 was permanently reduced in IUGR beta cells and underwent epigenetic modifications throughout development. The fetal IUGR state was characterized by loss of USF-1 binding at the proximal promoter of Pdx1, recruitment of the histone deacetylase 1 (HDAC1) and the corepressor Sin3A, and deacetylation of histones H3 and H4. Following birth, histone 3 lysine 4 (H3K4) was demethylated and histone 3 lysine 9 (H3K9) was methylated. During the neonatal period, these epigenetic changes and the reduction in Pdx1 expression could be reversed by HDAC inhibition. After the onset of diabetes in adulthood, the CpG island in the proximal promoter was methylated, resulting in permanent silencing of the Pdx1 locus. These results provide insight into the development of type 2 diabetes following IUGR and we believe they are the first to describe the ontogeny of chromatin remodeling in vivo from the fetus to the onset of disease in adulthood.

A recently promoted genome evolution model posits that mammalian pseudogenes can regulate their founding source genes, and it thereby ascribes an important function to “junk DNA.” This model arose from analysis of a serendipitous mouse mutant in which a transgene insertion/deletion caused severe polycystic kidney disease and osteogenesis imperfecta with ≈80% perinatal lethality, when inherited paternally [Hirotsune, S., et al . (2003) Nature 423, 91–96]. The authors concluded that the transgene reduced the expression of a nearby transcribed and imprinted pseudogene, Mkrn1-p1 . This reduction in chromosome 5-imprinted Mkrn1-p1 transcripts was proposed to destabilize the cognate chromosome 6 Mkrn1 source gene mRNA, with a partial reduction in one Mkrn1 isoform leading to the imprinted phenotype. Here, we show that 5’ Mkrn1-p1 is fully methylated on both alleles, a pattern indicative of silenced chromatin, and that Mkrn1-p1 is not transcribed and therefore cannot stabilize Mkrn1 transcripts in trans. A small, truncated, rodent-specific Mkrn1 transcript explains the product erroneously attributed to Mkrn1-p1 . Additionally, Mkrn1 expression is not imprinted, and 5’ Mkrn1 is fully unmethylated. Finally, mice in which Mkrn1 has been directly disrupted show none of the phenotypes attributed to a partial reduction of Mkrn1 . These data contradict the previous suggestions that Mkrn1-p1 is imprinted, and that either it or its source Mkrn1 gene relates to the original imprinted transgene phenotype. This study invalidates the data upon which the pseudogene trans-regulation model is based and therefore strongly supports the view that mammalian pseudogenes are evolutionary relics.

FMR1 encodes an RNA-binding protein whose absence results in fragile X mental retardation. In most patients, the FMR1 gene is cytosine-methylated and transcriptionally inactive. NRF-1 and Sp1 are known to bind and stimulate the active, but not the methylated/silenced, FMR1 promoter. Prior analysis has implicated a CRE site in regulation of FMR1 in neural cells but the role of this site is controversial. We now show that a phospho-CREB/ATF family member is bound to this site in vivo. We also find that the histone acetyltransferases CBP and p300 are associated with active FMR1 but are lost at the hypoacetylated fragile X allele. Surprisingly, FMR1 is not cAMP-inducible and resides in a newly recognized subclass of CREB-regulated genes. We have also elucidated a role for NRF-2 as a regulator of FMR1 in vivo through a previously unrecognized and highly conserved recognition site in FMR1. NRF-1 and NRF-2 act additively while NRF-2 synergizes with CREB/ATF at FMR1's promoter. These data add FMR1 to the collection of genes controlled by both NRF-1 and NRF-2 and disfavor its membership in the immediate early response group of genes.

Background Prader-Willi and Angelman syndrome (PWS and AS) patients typically have an ~5 Mb deletion of human chromosome 15q11-q13, of opposite parental origin. A mouse model of PWS and AS has a transgenic insertion-deletion (TgPWS/TgAS) of chromosome 7B/C subsequent to paternal or maternal inheritance, respectively. In this study, we define the deletion endpoints and examine the impact on expression of flanking genes. Results Using molecular and cytological methods we demonstrate that 13 imprinted and 11 non-imprinted genes are included in the TgPWS/TgAS deletion. Normal expression levels were found in TgPWS brain for genes extending 9.1- or 5.6-Mb centromeric or telomeric of the deletion, respectively. Our molecular cytological studies map the proximal deletion breakpoint between the Luzp2 and Siglec-H loci, and we show that overall mRNA levels of Luzp2 in TgPWS and TgAS brain are significantly reduced by 17%. Intriguingly, 5' Chrna7 shows 1.7-fold decreased levels in TgPWS and TgAS brain whereas there is a ≥15-fold increase in expression in neonatal liver and spleen of these mouse models. By isolating a Chrna7-Tg fusion transcript from TgAS mice, we mapped the telomeric deletion breakpoint in Chrna7 intron 4. Conclusion Based on the extent of the deletion, TgPWS/TgAS mice are models for PWS/AS class I deletions. Other than for the first gene promoters immediately outside the deletion, since genes extending 5.6–9.1 Mb away from each end of the deletion show normal expression levels in TgPWS brain, this indicates that the transgene array does not induce silencing and there are no additional linked rearrangements. Using gene expression, non-coding conserved sequence (NCCS) and synteny data, we have genetically mapped a putative Luzp2 neuronal enhancer responsible for ~33% of allelic transcriptional activity. The Chrna7 results are explained by hypothesizing loss of an essential neuronal transcriptional enhancer required for ~80% of allelic Chrna7 promoter activity, while the Chrna7 promoter is upregulated in B lymphocytes by the transgene immunoglobulin enhancer. The mapping of a putative Chrna7 neuronal enhancer inside the deletion has significant implications for understanding the transcriptional regulation of this schizophrenia-susceptibility candidate gene.

Our studies aim to identify the mechanisms and genes involved in genomic imprinting in mammalian development and human disease. Imprinting refers to an epigenetic modification of DNA that results in parent-of-origin specific expression during embryogenesis and in the adult. This imprint is reset at each generation, depending on the sex of the parental gametogenesis. Prader-Willi (PWS) and Angelman (AS) syndromes are excellent models for the study of genomic imprinting in humans, since these distinct neurobehavioural disorders are both associated with genetic abnormalities (large deletions, uniparental disomy, and imprinting mutations) of inheritance in chromosome 15q11-q13, dependent on the parental origin (reviewed in ref. 1). Some AS patients have biparental inheritance, consistent with a single imprinted gene (active on the maternal chromosome), whereas similar PWS patients are not found suggesting that at least two imprinted genes (active on the paternal allele) may be necessary for classical PWS. We have previously shown that the small ribonucleoprotein associated protein SmN gene (SNRPN), located in the PWS critical region [2], is only expressed from the paternal allele and is differentially methylated on parental alleles [3]. Therefore, SNRPN may have a role in PWS. Methylation imprints have also been found at two other loci in 15q11-q13, PW71 [4] and D15S9 [5], which map 120 kb and 1.5 Mb proximal to SNRPN, respectively. We have now characterized in detail the gene structure and expression from two imprinted loci within 15q11-q13, SNRPN and D15S9, which suggests that both loci are surprisingly complex, with important implications for the pathogenesis of PWS.

Phenotypic analyses of a set of homozygous-lethal deletion mutants at the pink-eyed dilution (p) locus has resulted in the identification of p-linked obesity locus 1 (plo 1), distal to the p locus, as a locus involved in the modulation of body fat and/or affecting lipid metabolism in these mice. The plo 1 region maps to mouse chromosome 7 (MMU 7) between two genes, Gabrb3 and Ube3a, which have been used as anchor points to generate an integrated deletion and physical map of plo 1 that encompasses about 1.2-1.3 Mb. A deletion/physical map was constructed and the genomic DNA between the two loci was sequenced to identify genes mapping to this region. Data show that Atp10c, a novel type IV ATPase a putative phospholipid transporter, is the only coding unit in this region of the chromosome.

Full-length poxvirus N1R/p28 orthologous proteins feature a prominent C-terminal RING zinc-finger motif. The RING moiety is conspicuously mutated in a number of vaccinia virus strains relative to variola virus. This, together with empirical data, suggests that N1R/p28 proteins promote virulence by suppressing apoptosis. Poxvirus N1R/p28 orthologues are strikingly similar to the RING motif of the cellular Makorin family of zinc-finger proteins, suggesting a homologous relationship connecting the viral and cellular genes. Recently identified avipox N1R/p28 orthologues further encode additional Makorin-like zinc-finger motifs, consistent with this suggestion. Phylogenetic analysis supports a model of poxviral capture of a MKRN cDNA and fusion with an existing viral gene. Establishing an evolutionary link between the viral and cellular genes will facilitate the elucidation of their respective cellular functions, and of how they interact in modulating virulence.

Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.

The chromosomal region, 15q11-q13, involved in Prader-Willi and Angelman syndromes (PWS and AS) represents a paradigm for understanding the relationships between genome structure, epigenetics, evolution, and function. The PWS/AS region is conserved in organization and function with the homologous mouse chromosome 7C region. However, the primate 4 Mb PWS/AS region is bounded by duplicons derived from an ancestral HERC2 gene and other sequences that may predispose to chromosome rearrangements. Within a 2 Mb imprinted domain, gene function depends on parental origin. Genetic evidence suggests that PWS arises from functional loss of several paternally expressed genes, including those that function as RNAs, and that AS results from loss of maternal UBE3A brain-specific expression. Imprinted expression is coordinately controlled in cis by an imprinting center (IC), a genetic element functional in germline and/or early postzygotic development that regulates the establishment of parental specific allelic differences in replication timing, DNA methylation, and chromatin structure.

Natural endogenous antisense RNAs have been reported in multiple loci, with evidence in some cases supporting a regulatory role for the antisense transcript. Here, we describe a novel gene, makorin RING zinc finger-2 (MKRN2), that overlaps and is antisense to the gene RAF1 in mammals. Phylogenetic analysis of the 3′ untranslated region of RAF1 orthologues suggests that this relationship may have existed for up to 450 million years. We have also identified MKRN2 orthologues in two species of fish. This places the gene duplication event that created this locus from an ancestral MKRN1 gene early in vertebrate evolution, over 450 million years ago. Northern blot analyses show that human MKRN2 and RAF1 are co-expressed in tissues and cell lines, raising the possibility of mRNA duplex formation. The encoded makorin-2 protein is likely a ribonucleoprotein of unknown function, but its conservation suggests an important cellular role. The data presented here describe a conserved vertebrate MKRN2 gene that is closely associated with the RAF1 locus.

Prader-Willi syndrome is a multigenic disorder with developmental and neurobehavioural abnormalities. There are multiple genetic causes, although all ultimately involve the loss of paternally derived gene expression of chromosome region 15q11-q13. Multiple imprinted genes expressed only from the paternal allele have been identified in the specific region of human chromosome 15q associated with Prader-Willi syndrome and in the syntenic mouse chromosome 7C region, including a novel polycistronic gene (SNURF-SNRPN) that encodes two independent proteins. The latter genetic locus may play a key role in Prader-Willi syndrome and the evolution of imprinting in this domain, because it is uniquely involved with mutations in the imprinting process and balanced translocations in this syndrome. Indeed, based on the co-localization of SNURF and SNRPN within the imprinting control region critical to Prader-Willi syndrome, evolutionary arguments would suggest that this genetic locus is a prime candidate for mutations producing the failure-to-thrive phenotype of neonates with this syndrome and of corresponding mouse models. Hence, the SNURF-SNRPN gene may encode a paternally derived postnatal growth factor.

Nuclear matrix binding assays (NMBAs) define certain DNA sequences as matrix attachment regions (MARs), which often have cis-acting epigenetic regulatory functions. We used NMBAs to analyze the functionally important 15q11-q13 imprinting center (IC). We find that the IC is composed of an unusually high density of MARs, located in close proximity to the germ line elements that are proposed to direct imprint switching in this region. Moreover, we find that the organization of MARs is the same at the homologous mouse locus, despite extensive divergence of DNA sequence. MARs of this size are not usually associated with genes but rather with heterochromatin-forming areas of the genome. In contrast, the 15q11-q13 region contains multiple transcribed genes and is unusual for being subject to genomic imprinting, causing the maternal chromosome to be more transcriptionally silent, methylated, and late replicating than the paternal chromosome. We suggest that the extensive MAR sequences at the IC are organized as heterochromatin during oogenesis, an organization disrupted during spermatogenesis. Consistent with this model, multicolor fluorescence in situ hybridization to halo nuclei demonstrates a strong matrix association of the maternal IC, whereas the paternal IC is more decondensed, extending into the nuclear halo. This model also provides a mechanism for spreading of the imprinting signal, because heterochromatin at the IC on the maternal chromosome may exert a suppressive position effect in cis. We propose that the germ line elements at the 15q11-q13 IC mediate their effects through the candidate heterochromatin-forming DNA identified in this study.

Polycistronic transcripts are common in prokaryotes but rare in eukaryotes. Phylogenetic analysis of the SNRPN (SmN) mRNA in five eutherian mammals reveals a second highly conserved coding sequence, termed SNURF ( SNRPN u pstream r eading f rame). The vast majority of nucleotide substitutions in SNURF occur in the wobble codon position, providing strong evolutionary evidence for selection for protein-coding function. Because SNURF–SNRPN maps to human chromosome 15q11–q13 and is paternally expressed, each cistron is a candidate for a role in the imprinted Prader-Willi syndrome (PWS) and PWS mouse models. SNURF encodes a highly basic 71-aa protein that is nuclear-localized (as is SmN). Because SNURF is the only protein-coding sequence within the imprinting regulatory region in 15q11–q13, it may have provided the original selection for imprinting in this domain. Whereas some human tissues express a minor SNURF -only transcript, mouse tissues express only the bicistronic Snurf–Snrpn transcript. We show that both SNURF and SNRPN are translated in normal, but not PWS, human, and mouse tissues and cell lines. These findings identify SNURF as a protein that is produced along with SmN from a bicistronic transcript; polycistronic mRNAs therefore are encoded in mammalian genomes where they may form functional operons.

A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C 3 HC 4 ) and three C 3 H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculusxM.spretus F 1 interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS.

Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

The human SNRPN gene maps within Chromosome (Chr) 15q11-q13, the region responsible for Prader-Willi syndrome (PWS) and Angelman syndrome (AS). As one of several 15q11-q13 transcripts expressed from the paternal allele-only, SNRPN is a candidate gene to explain at least some of the PWS phenotype in human and in genetic mouse models. The promoter and first exon of the SNRPN gene also correspond to an imprinting center element responsible for resetting of the maternal to paternal imprints within 15q11-q13 during spermatogenesis. Through characterization of the imprinted murine Snrpn locus in mouse Chr 7C, we have found that the gene structure is very similar to the human, with ten conserved exons spanning 22 kb, the last seven of which are tightly clustered. The promoter of Snrpn is differentially methylated in ES cells and adult tissues, supporting a role for DNA methylation at this site in somatic establishment and/or maintenance of Snrpn imprinting. The first intron of the mouse and human genes contains structurally conserved G-rich clustered repeats which may play a role in establishing DNA methylation patterns associated with imprinting of this gene. On the basis of the conserved structural and imprinted features of the human SNRPN and mouse Snrpn genes, we suggest that imprinting mechanisms are conserved between human and mouse.