Publications by authors named "Robert D Nicholls"

27 Publications

  • Page 1 of 1

Novel GUCY2C variant causing familial diarrhea in a Mennonite kindred and a potential therapeutic approach.

Am J Med Genet A 2021 May 5. Epub 2021 May 5.

Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Guanylate cyclase 2C (GC-C), encoded by the GUCY2C gene, is implicated in hereditary early onset chronic diarrhea. Several families with chronic diarrhea symptoms have been identified with autosomal dominant, gain-of-function mutations in GUCY2C. We have identified a Mennonite patient with a novel GUCY2C variant (c.2381A > T; p.Asp794Val) with chronic diarrhea and an extensive maternal family history of chronic diarrhea and bowel dilatation. Functional studies including co-segregation analysis showed that all family members who were heterozygous for this variant had GI-related symptoms. HEK-293 T cells expressing the Asp794Val GC-C variant showed increased cGMP production when stimulated with Escherichia coli heat-stable enterotoxin STp (HST), which was reversed when 5-(3-Bromophenyl)-5,11-dihydro-1,3-dimethyl-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (BPIPP; a GC-C inhibitor) was used. In addition, cystic fibrosis transmembrane conductance regulator (CFTR) activity measured with SPQ fluorescence assay was increased in these cells after treatment with HST, indicating a crucial role for CFTR activity in the pathogenesis of this disorder. These results support pathogenicity of the GC-C Asp794Val variant as a cause of chronic diarrhea in this family. Furthermore, this work identifies potential candidate drug, GC-C inhibitor BPIPP, to treat diarrhea caused by this syndrome.
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http://dx.doi.org/10.1002/ajmg.a.62207DOI Listing
May 2021

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

JCI Insight 2020 10 15;5(20). Epub 2020 Oct 15.

Division of Medical Genetics, Department of Pediatrics, University of Pittsburgh School of Medicine, and Universityof Pittsburgh Medical Center (UPMC) Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA.

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.
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http://dx.doi.org/10.1172/jci.insight.141523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605535PMC
October 2020

A newborn screening pilot study using methylation-sensitive high resolution melting on dried blood spots to detect Prader-Willi and Angelman syndromes.

Sci Rep 2020 08 3;10(1):13026. Epub 2020 Aug 3.

Laboratório de Alta Complexidade, Instituto Nacional da Saúde da Mulher, da Criança E Do Adolescente Fernandes Figueira, Fiocruz, Avenida Rui Barbosa 716, Flamengo, Rio de Janeiro, RJ, 22250-020, Brazil.

Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct imprinted disorders characterized by genetic abnormalities at 15q11-q13. Early diagnosis of both syndromes provides improved treatment and accurate genetic counseling. Whole blood (WB) is the most common DNA source of many methodologies to detect PWS and AS, however, the need of WB makes a massive screening difficult in newborns due to economic and technical limitations. The aim of this study was to adapt a Methylation-sensitive High-Resolution Melting (MS-HRM) approach from dried blood spot (DBS) samples, assessing the different DNA isolation techniques and diagnostic performance. Over a 1-year period, we collected 125 DBS cards, of which 45 had already been diagnosed by MS-HRM (20 PWS, 1 AS, and 24 healthy individuals). We tested three different DBS-DNA extraction techniques assessing the DNA concentration and quality, followed by MS-HRM and statistical comparison. Each DBS-DNA extraction method was capable of accuracy in detecting all PWS and AS individuals. However, the efficiency to detect healthy individuals varied according to methodology. In our experience, DNA extracted from DBS analyzed by the MS-HRM methodology provides an accurate approach for genetic screening of imprinting related disorders in newborns, offering several benefits compared to traditional whole blood methods.
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http://dx.doi.org/10.1038/s41598-020-69750-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400512PMC
August 2020

Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons.

Am J Hum Genet 2019 05 25;104(5):815-834. Epub 2019 Apr 25.

Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, WC1N 3BG London, UK.

We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.
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http://dx.doi.org/10.1016/j.ajhg.2019.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6507050PMC
May 2019

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency.

Hum Mol Genet 2015 Jun 26;24(11):3238-47. Epub 2015 Feb 26.

Department of Pediatrics, University of Pittsburgh School of Medicine, University of Pittsburgh, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224, USA Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15224, USA

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.
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http://dx.doi.org/10.1093/hmg/ddv074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424958PMC
June 2015

Recommendations for the investigation of animal models of Prader-Willi syndrome.

Mamm Genome 2013 Jun 23;24(5-6):165-78. Epub 2013 Apr 23.

Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.

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.
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http://dx.doi.org/10.1007/s00335-013-9454-2DOI Listing
June 2013

In vivo evolution of tumor-derived endothelial cells.

PLoS One 2012 18;7(5):e37138. Epub 2012 May 18.

Division of Hematology/Oncology, Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania, United States of America.

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037138PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356387PMC
September 2012

Transcriptional and post-transcriptional regulation of SPAST, the gene most frequently mutated in hereditary spastic paraplegia.

PLoS One 2012 4;7(5):e36505. Epub 2012 May 4.

Birth Defects Laboratories, Division of Medical Genetics, Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036505PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3344893PMC
September 2012

Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome.

Am J Physiol Endocrinol Metab 2011 May 22;300(5):E909-22. Epub 2011 Feb 22.

Dept. of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Rangos Research Bldg., 4401 Penn Ave., Pittsburgh, PA 15224, USA.

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.
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http://dx.doi.org/10.1152/ajpendo.00185.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093973PMC
May 2011

The snoRNA MBII-52 (SNORD 115) is processed into smaller RNAs and regulates alternative splicing.

Hum Mol Genet 2010 Apr 6;19(7):1153-64. Epub 2010 Jan 6.

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.

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.
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http://dx.doi.org/10.1093/hmg/ddp585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838533PMC
April 2010

Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1.

J Clin Invest 2008 Jun;118(6):2316-24

Department of Pediatrics, Children's Hospital of Philadelphia, Department of Medicine, and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

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.
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http://dx.doi.org/10.1172/JCI33655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373422PMC
June 2008

Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences.

Nature 2007 May;447(7141):167-77

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.

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.
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http://dx.doi.org/10.1038/nature05805DOI Listing
May 2007

Recent assembly of an imprinted domain from non-imprinted components.

PLoS Genet 2006 Oct;2(10):e182

Australian Research Council Center for Kangaroo Genomics and Research School of Biological Sciences, Australian National University, Canberra, Australia.

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how--and especially why--epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105-180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B'. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.
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http://dx.doi.org/10.1371/journal.pgen.0020182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1626109PMC
October 2006

The putatively functional Mkrn1-p1 pseudogene is neither expressed nor imprinted, nor does it regulate its source gene in trans.

Proc Natl Acad Sci U S A 2006 Aug 1;103(32):12039-44. Epub 2006 Aug 1.

Wadsworth Center, David Axelrod Institute, Albany, NY 12208, USA.

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 approximately 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.
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http://dx.doi.org/10.1073/pnas.0602216103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567693PMC
August 2006

The gene encoding the fragile X RNA-binding protein is controlled by nuclear respiratory factor 2 and the CREB family of transcription factors.

Nucleic Acids Res 2006 25;34(4):1205-15. Epub 2006 Feb 25.

Program in Genetics & Molecular Biology and Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

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.
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http://dx.doi.org/10.1093/nar/gkj521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1383620PMC
March 2006

Genetic mapping of putative Chrna7 and Luzp2 neuronal transcriptional enhancers due to impact of a transgene-insertion and 6.8 Mb deletion in a mouse model of Prader-Willi and Angelman syndromes.

BMC Genomics 2005 Nov 9;6:157. Epub 2005 Nov 9.

Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, PA 19104, USA.

Background: Prader-Willi and Angelman syndrome (PWS and AS) patients typically have an approximately 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 > or =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 approximately 33% of allelic transcriptional activity. The Chrna7 results are explained by hypothesizing loss of an essential neuronal transcriptional enhancer required for approximately 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.
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http://dx.doi.org/10.1186/1471-2164-6-157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1322230PMC
November 2005

Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus.

Nucleic Acids Res 2005 22;33(15):4740-53. Epub 2005 Aug 22.

Department of Biochemistry and Molecular Biology, University of Florida College of Medicine Gainesville, FL 32610, USA.

The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5' region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5' region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function.
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http://dx.doi.org/10.1093/nar/gki786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1188517PMC
September 2005

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader-Willi syndrome mouse models.

Mamm Genome 2005 Jun;16(6):424-31

Department of Genetics, Stanford University, Stanford, California 94305, USA.

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 p30PUb 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 p30PUb 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 p30PUb 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.
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http://dx.doi.org/10.1007/s00335-005-2460-2DOI Listing
June 2005

A nonimprinted Prader-Willi Syndrome (PWS)-region gene regulates a different chromosomal domain in trans but the imprinted pws loci do not alter genome-wide mRNA levels.

Genomics 2005 May;85(5):630-40

Department of Psychiatry, Center for Neurobiology and Behavior, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104-6140, USA.

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.
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http://dx.doi.org/10.1016/j.ygeno.2005.02.004DOI Listing
May 2005

Possible genomic imprinting of three human obesity-related genetic loci.

Am J Hum Genet 2005 Mar 12;76(3):427-37. Epub 2005 Jan 12.

Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, PA 19104-6140, USA.

To detect potentially imprinted, obesity-related genetic loci, we performed genomewide parent-of-origin linkage analyses under an allele-sharing model for discrete traits and under a family regression model for obesity-related quantitative traits, using a European American sample of 1,297 individuals from 260 families, with 391 microsatellite markers. We also used two smaller, independent samples for replication (a sample of 370 German individuals from 89 families and a sample of 277 African American individuals from 52 families). For discrete-trait analysis, we found evidence for a maternal effect in chromosome region 10p12 across the three samples, with LOD scores of 5.69 (single-point) and 4.52 (multipoint) for the pooled sample. For quantitative-trait analysis, we found the strongest evidence for a maternal effect (single-point LOD of 2.85; multipoint LOD of 4.01 for body mass index [BMI] and 3.69 for waist circumference) in region 12q24 and for a paternal effect (single-point LOD of 4.79; multipoint LOD of 3.72 for BMI) in region 13q32, in the European American sample. The results suggest that parent-of-origin effects, perhaps including genomic imprinting, may play a role in human obesity.
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http://dx.doi.org/10.1086/428438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196395PMC
March 2005

Physical mapping of the pink-eyed dilution complex in mouse chromosome 7 shows that Atp10c is the only transcript between Gabrb3 and Ube3a.

DNA Seq 2004 Aug;15(4):306-9

318 Jessie Harris Building, Department of Nutrition, University of Tennessee, 1215 Cumberland Avenue, Knoxville, TN 37996, USA.

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.
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http://dx.doi.org/10.1080/10425170412331279855DOI Listing
August 2004

Cellular source of the poxviral N1R/p28 gene family.

Virus Genes 2004 Dec;29(3):359-64

Center for Neurobiology and Behavior, Department of Psychiatry, CRB528, University of Pennsylvania, 415 Curie Blvd, PA 19104-6140, USA.

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.
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http://dx.doi.org/10.1007/s11262-004-7440-1DOI Listing
December 2004

What have rare genetic syndromes taught us about the pathophysiology of the common forms of obesity?

Curr Diab Rep 2004 Apr;4(2):143-50

Department of Psychiatry, University of Pennsylvania, Clinical Research Building, Room 528, 415 Curie Boulevard, Philadelphia, PA 19104-6140, USA.

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.
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http://dx.doi.org/10.1007/s11892-004-0070-0DOI Listing
April 2004

NIPA1 gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6).

Am J Hum Genet 2003 Oct 23;73(4):967-71. Epub 2003 Sep 23.

Department of Neurology, University of Michigan, Ann Arbor, MI 48109, USA.

The hereditary spastic paraplegias (HSPs) are genetically heterogeneous disorders characterized by progressive lower-extremity weakness and spasticity. The molecular pathogenesis is poorly understood. We report discovery of a dominant negative mutation in the NIPA1 gene in a kindred with autosomal dominant HSP (ADHSP), linked to chromosome 15q11-q13 (SPG6 locus); and precisely the same mutation in an unrelated kindred with ADHSP that was too small for meaningful linkage analysis. NIPA1 is highly expressed in neuronal tissues and encodes a putative membrane transporter or receptor. Identification of the NIPA1 function and ligand will aid an understanding of axonal neurodegeneration in HSP and may have important therapeutic implications.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180617PMC
http://dx.doi.org/10.1086/378817DOI Listing
October 2003

2002 Curt Stern Award address. Introductory speech for James R. Lupski.

Am J Hum Genet 2003 Feb;72(2):244-5

Center for Neurobiology and Behavior, Departments of Psychiatry and Genetics, University of Pennsylvania, Philadelphia, PA 19104-6140, USA .

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC379219PMC
http://dx.doi.org/10.1086/346216DOI Listing
February 2003

Anorexigenic melanocortin signaling in the hypothalamus is augmented in association with failure-to-thrive in a transgenic mouse model for Prader-Willi syndrome.

Brain Res 2002 Dec;957(1):42-5

University of Florida McKnight Brain Institute, College of Medicine, Department of Neuroscience, PO Box 100244, Gainesville, FL 32610, USA.

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 alpha-melanocyte stimulating hormone (alpha-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, alpha-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.
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http://dx.doi.org/10.1016/s0006-8993(02)03583-7DOI Listing
December 2002

A 46 kDa integral membrane protein from Mycobacterium leprae resembles a number of bacterial and mammalian membrane transport proteins.

Microbiology (Reading) 1995 Aug;141 ( Pt 8):1963-1968

Royal Tropical Institute, Department of Biomedical Research,Meibergdreef 39, NL-1105AZ Amsterdam,The Netherlands.

In this paper we describe the nucleotide sequence of a 3.4 kbp region of the Mycobacterium leprae genome. This region contains an open reading frame of 1290 bp with a coding capacity for a protein of 46,179 Da, designated the 38L protein. Using antibodies against part of the 38L protein, we were able to demonstrate that the 38L protein is present in the membrane protein fraction of M. leprae. The 38L protein showed significant matches with a number of integral membrane proteins involved in the transport of small molecules through the cellular membrane. Among these are a human and a murine protein involved in melanin biosynthesis. The 38L protein might play a role in the hypopigmentation observed in leprosy patients.
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http://dx.doi.org/10.1099/13500872-141-8-1963DOI Listing
August 1995