Camilynn I. Brannan

CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF

CD30 is a surface marker for neoplastic cells of Hodgkins lymphoma and shows sequence homology to members of the tumor necrosis factor (TNF) receptor superfamily. Using a chimeric probe consisting of the extracellular domain of CD30 fused to truncated immunoglobulin heavy chains, we expression cloned the cDNA cognate from the murine T cell clone 7B9. The encoded protein is a 239 amino acid type II membrane protein whose C-terminal domain shows significant homology to TNF alpha, TNF beta, and the CD40L. Cross-hybridization to an induced peripheral blood T cell cDNA library yielded the human homolog, which is 72% identical at the amino acid level. The recombinant human ligand enhances the proliferation of CD3-activated T cells yet induces differential responses, including cell death, in several CD30+ lymphoma-derived clones. The human and murine genes map to 9q33 and the proximal region of chromosome 4, respectively.

Nf1 deficiency causes Ras-mediated granulocyte/macrophage colony stimulating factor hypersensitivity and chronic myeloid leukaemia.

The Ras signal transduction pathway is often deregulated in human myeloid leukaemia. For example, activating point mutations in RAS genes are found in some patients with juvenile chronic myelogenous leukaemia (JCML), while other patients with JCML show loss of the neurofibromatosis type 1 (NF1) gene, a Ras GTPase activating protein. By generating mice whose haematopoietic system is reconsituted with NF1 deficient haematopoietic stem cells we show that NF1 gene loss, by itself, is sufficient to produce the myeloproliferative symptoms associated with human JCML. We also provide evidence to indicate that NF1 gene loss induces myeloproliferative disease through a Ras-mediated hypersensitivity to granulocyte/macrophage-colony stimulating factor (GM-CSF). Finally, we describe a genetic screen for identifying genes that cooperate with NF1 gene loss during progression to acute myeloid leukaemia.

A mouse model for Prader-Willi syndrome imprinting-centre mutations.

Imprinting in the 15q11–q13 region involves an ‘imprinting centre’ (IC), mapping in part to the promoter and first exon of SNRPN. Deletion of this IC abolishes local paternally derived gene expression and results in Prader-Willi syndrome (PWS). We have created two deletion mutations in mice to understand PWS and the mechanism of this IC. Mice harbouring an intragenic deletion in Snrpn are phenotypically normal, suggesting that mutations of SNRPN are not sufficient to induce PWS. Mice with a larger deletion involving both Snrpn and the putative PWS-IC lack expression of the imprinted genes Zfp127 (mouse homologue of ZNF127), Ndn and Ipw, and manifest several phenotypes common to PWS infants. These data demonstrate that both the position of the IC and its role in the coordinate expression of genes is conserved between mouse and human, and indicate that the mouse is a suitable model system in which to investigate the molecular mechanisms of imprinting in this region of the genome.

Maternal methylation imprints on human chromosome 15 are established during or after fertilization.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the lack of transcripts expressed from the paternal copy of the imprinted chromosomal region 15q11–q13 (refs. 1,2). In some patients, this is associated with a deletion of the SNURF-SNRPN exon 1 region inherited from the paternal grandmother and the presence of a maternal imprint on the paternal chromosome. Assuming that imprints are reset in the germ line, we and others have suggested that this region constitutes part of the 15q imprinting center (IC) and is important for the maternal to paternal imprint switch in the male germ line. Here we report that sperm DNA from two males with an IC deletion had a normal paternal methylation pattern along 15q11–q13. Similar findings were made in a mouse model. Our results indicate that the incorrect maternal methylation imprint in IC deletion patients is established de novo after fertilization. Moreover, we found that CpG-rich regions in SNURF-SNRPN and NDN, which in somatic tissues are methylated on the maternal allele, are hypomethylated in unfertilized human oocytes. Our results indicate that the normal maternal methylation imprints in 15q11–q13 also are established during or after fertilization.

Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor.

Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors. Each of the murine receptors shows strong sequence homology to the corresponding human receptor (approximately 65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves. The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-alpha and -beta with multiple affinity classes, and the ligands cross-compete. Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined. It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells. The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes. They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).

MECHANISMS OF GENOMIC IMPRINTING

A small number of mammalian genes undergo the process of genomic imprinting whereby the expression level of the alleles of a gene depends upon their parental origin. In the past year, attention has focused on the mechanisms that determine parental-specific expression patterns. Many imprinted genes are located in conserved clusters and, although it is apparent that imprinting of adjacent genes is jointly regulated, multiple mechanisms among and within clusters may operate. Recent developments have also refined the timing of the gametic imprints and further defined the mechanism by which DNA methyltransferases confer allelic methylation patterns.

Learning deficits, but normal development and tumor predisposition, in mice lacking exon 23a of Nf1

Neurofibromatosis type 1 (NF1) is a commonly inherited autosomal dominant disorder. Previous studies indicated that mice homozygous for a null mutation in Nf1 exhibit mid-gestation lethality, whereas heterozygous mice have an increased predisposition to tumors and learning impairments. Here we show that mice lacking the alternatively spliced exon 23a, which modifies the GTPase-activating protein (GAP) domain of Nf1, are viable and physically normal, and do not have an increased tumor predisposition, but show specific learning impairments. Our findings have implications for the development of a treatment for the learning disabilities associated with NF1 and indicate that the GAP domain of NF1 modulates learning and memory.

Loss of neurofibromin results in neurotrophin-independent survival of embryonic sensory and sympathetic neurons

Mutations at the neurofibromatosis 1 (NF1) locus in humans and mice result in abnormal growth of neural crest-derived cells, including melanocytes and Schwann cells. We have exploited a targeted disruption of the NF1 gene in mice to examine the role of neurofibromin in the acquisition of neurotrophin dependence in embryonic neurons. We show that both neural crest- and placode-derived sensory neurons isolated from NF1(-/-) embryos develop, extend neurites, and survive in the absence of neurotrophins, whereas their wild-type counterparts die rapidly unless nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) is added to the culture medium. Moreover, NF1 (-/-) sympathetic neurons survive for extended periods and acquire mature morphology in the presence of NGF-blocking antibodies. Our results are consistent with a model wherein neurofibromin acts as a negative regulator of neurotrophin-mediated signaling for survival of embryonic peripheral neurons.

De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch.

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11–q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6–8) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite ‘imprinting centre’ (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.

Neurofibromin Expression and Astrogliosis in Neurofibromatosis (Type 1) Brains

Abstract . Patients with type 1 neurofibromatosis (NF1) have mutations in the gene encoding the protein neurofibromin. Immunocytochemistry on sections of cortex and cerebellum of unaffected and NF1 individuals and wild-type and NF1-deficient mice showed that the distribution of neurofibromin was similar to that reported for rat. However, dystrophic neurofibromin-expressing neurons were found in human but not rodent brain. Intensity of anti-neurofibromin reactivity was reduced in NF1-deficient mice but not in human brains. GFAP was upregulated in three NF1 brains studied by immunocytochemistry; a 4–18-fold increase in GFAP levels was documented by Western blot analysis in three brains. GFAP content/ cell and the number of GFAP-immunoreactive astrocytes was increased in NF1 brains as compared to the controls. These results suggest that mutations in the NF1 gene do not grossly alter the pattern of neurofibromin expression, but activation of astrocytes may be common in NF1. Presence of degenerative debris in one of two brains using the cupric silver method suggests that degeneration is not always detectable in NF1 brains.