A long noncoding RNA influences the choice of the X chromosome to be inactivated.

X chromosome inactivation (XCI) is the process of silencing one of the X chromosomes in cells of the female mammal which ensures dosage compensation between the sexes. Although theoretically random in somatic tissues, the choice of which X chromosome is chosen to be inactivated can be biased in mice by genetic element(s) associated with the so-called X-controlling element (Xce). Although the Xce was first described and genetically localized nearly 40 y ago, its mode of action remains elusive. In the approach presented here, we identify a single long noncoding RNA (lncRNA) within the Xce locus, Lppnx, which may be the driving factor in the choice of which X chromosome will be inactivated in the developing female mouse embryo. Comparing weak and strong Xce alleles we show that Lppnx modulates the expression of Xist lncRNA, one of the key factors in XCI, by controlling the occupancy of pluripotency factors at Intron1 of Xist. This effect is counteracted by enhanced binding of Rex1 in DxPas34, another key element in XCI regulating the activity of Tsix lncRNA, the main antagonist of Xist, in the strong but not in the weak Xce allele. These results suggest that the different susceptibility for XCI observed in weak and strong Xce alleles results from differential transcription factor binding of Xist Intron 1 and DxPas34, and that Lppnx represents a decisive factor in explaining the action of the Xce.

An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis.

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results: We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion: Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target.

Gene Dosage Effects at the Imprinted Gnas Cluster.

Genomic imprinting results in parent-of-origin-dependent monoallelic gene expression. Early work showed that distal mouse chromosome 2 is imprinted, as maternal and paternal duplications of the region (with corresponding paternal and maternal deficiencies) give rise to different anomalous phenotypes with early postnatal lethalities. Newborns with maternal duplication (MatDp(dist2)) are long, thin and hypoactive whereas those with paternal duplication (PatDp(dist2)) are chunky, oedematous, and hyperactive. Here we focus on PatDp(dist2). Loss of expression of the maternally expressed Gnas transcript at the Gnas cluster has been thought to account for the PatDp(dist2) phenotype. But PatDp(dist2) also have two expressed doses of the paternally expressed Gnasxl transcript. Through the use of targeted mutations, we have generated PatDp(dist2) mice predicted to have 1 or 2 expressed doses of Gnasxl, and 0, 1 or 2 expressed doses of Gnas. We confirm that oedema is due to lack of expression of imprinted Gnas alone. We show that it is the combination of a double dose of Gnasxl, with no dose of imprinted Gnas, that gives rise to the characteristic hyperactive, chunky, oedematous, lethal PatDp(dist2) phenotype, which is also hypoglycaemic. However PatDp(dist2) mice in which the dosage of the Gnasxl and Gnas is balanced (either 2∶2 or 1∶1) are neither dysmorphic nor hyperactive, have normal glucose levels, and are fully viable. But PatDp(dist2) with biallelic expression of both Gnasxl and Gnas show a marked postnatal growth retardation. Our results show that most of the PatDp(dist2) phenotype is due to overexpression of Gnasxl combined with loss of expression of Gnas, and suggest that Gnasxl and Gnas may act antagonistically in a number of tissues and to cause a wide range of phenotypic effects. It can be concluded that monoallelic expression of both Gnasxl and Gnas is a requirement for normal postnatal growth and development.

A -defensin mutation causes black coat color in domestic dogs.

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the K locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the K locus as beta-defensin 103 (CBD103) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of beta-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.

Interactions between imprinting effects in the mouse.

Mice with uniparental partial or complete disomies for any one of 11 identified chromosomes show abnormal phenotypes. The abnormalities, or imprinting effects, can be attributable to an incorrect dosage of maternal or paternal copies of imprinted gene(s) located within the regions involved. Here we show that combinations of partial disomies may result in interactions between imprinting effects that seemingly independently affect fetal and/or placental growth in different ways or modify neonatal and postnatal imprinting effects. Candidate genes within the regions have been identified. The findings are generally in accord with the "conflict hypothesis" for the evolution of genomic imprinting but do not clearly demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that represses or limits embryonic/fetal growth to the advantage of the mother--by any developmental means--will have been subject to evolutionary selection for paternal allele repression. Likewise, any gene that favors embryonic/fetal development at consequent cost to the mother--by any developmental means--will have faced selection for maternal allele repression. The classical Igf2-Igf2r axis may therefore be unique. The findings involve reinterpretation of older imprinting data and consequently revision of the mouse imprinting map.

Spontaneous and radiation-induced leukemogenesis of the mouse small eye mutant, Pax6(Sey3H).

Allelic loss on the chromosome 2 is associated with radiation-induced murine acute myeloid leukemia. However, the gene, which contributes mainly to the leukemogenesis has not yet been identified. Expecting any predisposition to acute myeloid leukemia, we performed a radiation leukemogenensis experiment with Pax6(Sey3H), one of the small eye mutants carrying a congenital hemizygosity of the chromosome 2 middle region. A deletion mapping of Pax6(Sey3H) with 50 STS markers indicated that the deleted segment extended between the 106.00 and 111.47 Mb site from the centromere with a length of 5.47 Mb. In the deleted segment, 6 known and 17 novel genes were located. Pax6(Sey3H) mutants that crossed back into C3H/He did not develop myeloid leukemia spontaneously, but they did when exposed to gamma-rays. The final incidence of myeloid leukemia in mutants (25.8%) was as high as that in normal sibs (21.4%). Survival curves of leukemia-bearing mutants shifted toward the left (p = 0.043 by the Log rank test). F1 hybrids of Pax6(Sey3H) with JF1 were less susceptible to radiation than Pax6(Sey3H) onto C3H/He in regard to survival (p = 0.003 and p < 0.00001 for mutants and normal sibs, respectively, by a test of the difference between two proportions). Congenital deletion of the 5.47 Mb segment at the middle region on chromosome 2 alone did not trigger myeloid stem cells to expand clonally in vivo; however, the deletion shortcut the latency of radiation-induced myeloid leukemia.

Disruption of ferroportin 1 regulation causes dynamic alterations in iron homeostasis and erythropoiesis in polycythaemia mice.

Coding region mutations in the principal basolateral iron transporter of the duodenal enterocyte, ferroportin 1 (FPN1), lead to autosomal dominant reticuloendothelial iron overload in humans. We report the positional cloning of a hypermorphic, regulatory mutation in Fpn1 from radiation-induced polycythaemia (Pcm) mice. A 58 bp microdeletion in the Fpn1 promoter region alters transcription start sites and eliminates the iron responsive element (IRE) in the 5' untranslated region, resulting in increased duodenal and hepatic Fpn1 protein levels during early postnatal development. Pcm mutants, which are iron deficient at birth, exhibited increased Fpn1-mediated iron uptake and reticuloendothelial iron overload as young adult mice. Additionally, Pcm mutants displayed an erythropoietin (Epo)-dependent polycythemia in heterozygotes and a hypochromic, microcytic anemia in homozygotes. Interestingly, both defects in erythropoiesis were transient, correcting by young adulthood. Delayed upregulation of the negative hormonal regulator of iron homeostasis, hepcidin (Hamp), during postnatal development correlates strongly with profound increases in Fpn1 protein levels and polycythemia in Pcm heterozygotes. Thus, our data suggest that a Hamp-mediated regulatory interference alleviates the defects in iron homeostasis and transient alterations in erythropoiesis caused by a regulatory mutation in Fpn1.

Identification of two novel mutations in the murine Nsdhl sterol dehydrogenase gene and development of a functional complementation assay in yeast.

Nsdhl is a 3beta-hydroxysterol dehydrogenase that is involved in the removal of C-4 methyl groups in the cholesterol biosynthetic pathway. Mutations in this gene are associated with the X-linked male lethal mouse mutations bare patches (Bpa) and striated (Str) and human CHILD syndrome. We have now detected the missense mutations V53D and A94T in conserved amino acids in two additional Bpa alleles. The latter alters the same amino acid as a missense mutation found in two unrelated CHILD patients, strongly suggesting that differences in the phenotype between Bpa mice and females with CHILD syndrome are unlikely to be explained by different types or sites of mutations. We have also demonstrated that the mouse NSDHL protein can rescue the lethality of erg26 deficient cells of Saccharomyces cerevisiae that lack the yeast ortholog, substantiating the role of NSDHL as a C-3 sterol dehydrogenase. Using this in vivo assay, we have demonstrated that two Str alleles function as hypomorphs, while three Bpa and one Str allele provide no complementation or rescue.

Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

The imprinted oedematous-small mutation on mouse chromosome 2 identifies new roles for Gnas and Gnasxl in development.

The Gnas locus is highly complex and encodes several oppositely imprinted and alternatively spliced transcripts. Gnas itself encodes Gsalpha, which is involved in endocrine function and bone development, but the roles for the other transcripts have not been established. Here we describe a mouse mutation that provides further biological functions for the Gnas locus. The mutation Oed-Sml, induced by ethylnitrosourea (ENU), has been mapped to the distal chromosome 2 imprinting region that includes Gnas. The mutation displays two distinct phenotypes dependent on parental origin. When the mutation is maternally transmitted, a microcardia with gross edema (Oed) results. By contrast, when the mutation is paternally transmitted, a growth retardation (Sml) is seen that becomes evident within 5 days of birth. Here we show Oed-Sml to be a point mutation in Gnas exon 6, resulting in a valine to glutamate substitution at residue 159 (V159E). Both maternal- and paternal-specific transcripts derive from this missense mutation. The maternally expressed mutant Gnas transcript is the candidate for Oed and the paternally expressed mutant Gnasxl transcript is the candidate for Sml. We propose a new role for Gnas in heart growth and a role for Gnasxl in postnatal growth. These findings potentially have implications for human Albright hereditary osteodystrophy, a condition caused by mutations in GNAS.

Disruption of mesodermal enhancers for Igf2 in the minute mutant.

The radiation-induced mutation minute (Mnt) in the mouse leads to intrauterine growth retardation with paternal transmission and has been linked to the distal chromosome 7 cluster of imprinted genes. We show that the mutation is an inversion, whose breakpoint distal to H19 disrupts and thus identifies an enhancer for Igf2 expression in skeletal muscle and tongue, and separates the gene from other mesodermal and extra-embryonic enhancers. Paternal transmission of Mnt leads to drastic downregulation of Igf2 transcripts in all mesodermal tissues and the placenta. Maternal transmission leads to methylation of the H19 differentially methylated region (DMR) and silencing of H19, showing that elements 3' of H19 can modify the maternal imprint. Methylation of the maternal DMR leads to biallelic expression of Igf2 in endodermal tissues and foetal overgrowth, demonstrating that methylation in vivo can open the chromatin boundary upstream of H19. Our work shows that most known enhancers for Igf2 are located 3' of H19 and establishes an important genetic paradigm for the inheritance of complex regulatory mutations in imprinted gene clusters.

Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19.

Dlk1 and Gtl2 are reciprocally imprinted genes located 80 kb apart on mouse chromosome 12. Similarities between this domain and that of the well characterized Igf2-H19 locus have been previously noted. Comparative genomic and epigenetic analysis of these two domains might help identify allele-specific epigenetic regulatory elements and common features involved in aspects of imprinting control. Here we describe a detailed methylation analysis of the Dlk1-Gtl2 domain on both parental alleles in the mouse. Like the Igf2-H19 domain, areas of differential methylation are hypermethylated on the paternal allele and hypomethylated on the maternal allele. Three differentially methylated regions (DMRs), each with different epigenetic characteristics, have been identified. One DMR is intergenic, contains tandem repeats and is the only region that inherits a paternal methylation mark from the germline. An intronic DMR contains a conserved putative CTCF-binding domain. All three DMRs have both unique and common features compared to those identified in the Igf2-H19 domain.