ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene

ES2 is a gene deleted in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) which has homologs in species as distant as Caenorhabditis elegans and Drosophila . The function of ES2 is unknown, and the predicted protein sequence does not contain motifs which suggest a particular role in the developmental defects present in DGS and VCFS. Here we show that the mouse homolog, Es2 , is transcribed in two forms resulting from the use of alternative polyadenylation signals. Structural analysis programs predict that the Es2 -encoded peptide has a coiled-coil domain, and transfection experiments with an Es2 -green fluorescent protein (GFP) fusion construct show that the peptide is recruited into the nucleus. Es2 is highly expressed during mouse embryogenesis from E7 onwards. In situ hybridization with an RNA probe revealed that the gene is widely expressed; however, relatively higher expression was detected in the nervous system, with a particularly high area of expression in a sub-region of the pons. The Es2 expression domain in the pons is shared with a Goosecoid-like gene ( Gscl) which is located upstream of Es2 , and raises the possibility that the two genes share regulatory elements and/or interact in this region of the developing brain. This finding suggests that different genes in the deleted region may be functionally related and might explain the occurrence of the characteristic phenotype in patients with non-overlapping genetic lesions.      

Goosecoid-like (Gscl), a candidate gene for velocardiofacial syndrome, is not essential for normal mouse development

Velocardiofacial syndrome (VCFS) and DiGeorge syndrome (DGS) are characterized by a wide spectrum of abnormalities, including conotruncal heart defects, velopharyngeal insufficiency, craniofacial anomalies and learning disabilities. In addition, numerous other clinical features have been described, including frequent psychiatric illness. Hemizygosity for a 1.5-3 Mb region of chromosome 22q11 has been detected in >80% of VCFS/DGS patients. It is thought that a developmental field defect is responsible for many of the abnormalities seen in these patients and that the defect occurs due to reduced levels of a gene product active in early embryonic development. Goosecoid-like ( GSCL ) is a homeobox gene which is present in the VCFS/DGS commonly deleted region. The mouse homolog, Gscl, is expressed in mouse embryos as early as E8.5. Gscl is related to Goosecoid ( Gsc ), a gene required for proper craniofacial development in mice. GSCL has been considered an excellent candidate for contributing to the developmental defects in VCFS/DGS patients. To investigate the role of Goosecoid-like in VCFS/DGS etiology, we disrupted the Gscl gene in mouse embryonic stem cells and produced mice that transmit the disrupted allele. Mice that are homozygous for the disrupted allele appear to be normal and they do not exhibit any of the anatomical abnormalities seen in VCFS/DGS patients. RNA in situ hybridization to mouse embryo sections revealed that Gscl is expressed at E8.5 in the rostral region of the foregut and at E11.5 and E12.5 in the developing brain, in the pons region and in the choroid plexus of the fourth ventricle. Although the gene inactivation experiments indicate that haploinsufficiency for GSCL is unlikely to be the sole cause of the developmental field defect thought to be responsible for many of the abnormalities in VCFS/DGS patients, its localized expression during development could suggest that hemizygosity for GSCL, in combination with hemizygosity for other genes in 22q11, contributes to some of the developmental defects as well as the behavioral anomalies seen in these patients. The mice generated in this study should help in evaluating these possibilities.      

Functional analysis of Gscl in the pathogenesis of the DiGeorge and velocardiofacial syndromes

Gscl encodes a Goosecoid-related homeodomain protein that is expressed during mouse embryogenesis. In situ hybridization and immunohistochemistry studies show that Gscl is expressed in the pons region of the developing central nervous system and primordial germ cells. Gscl expression is also detected in a subset of adult tissues, including brain, eye, thymus, thyroid region, stomach, bladder and testis. Gscl is located within a region of the mouse genome that is syntenic with the region commonly deleted in DiGeorge and velocardiofacial syndrome (DGS/VCFS) patients. DGS/VCFS patients have craniofacial abnormalities, cardiac outflow defects and hypoplasia of the parathyroid gland and thymus due to haploinsufficiency of a gene or genes located within the deleted region. Thus, the genomic location of Gscl and its expression in a subset of the tissues affected in DGS/VCFS patients suggest that Gscl may contribute to the pathogenesis of DGS/VCFS. To determine the role of Gscl during mouse embryogenesis and in DGS/VCFS, we have deleted Gscl by gene targeting in mouse embryonic stem cells. Both Gscl heterozygous and Gscl null mice were normal and fertile, suggesting that Gscl is not a major factor in DGS/VCFS. Interestingly, expression of the adjacent Es2 gene in the pons region of Gscl null fetuses was absent, suggesting that mutations within the DGS/VCFS region can influence expression of adjacent genes. In addition, embryos that lacked both Gscl and the related Gsc gene appeared normal. These studies represent the first functional analysis of a DGS/VCFS candidate gene in vivo. These Gscl null mice will be an important genetic resource for crosses with other mouse models of the DGS/VCFS.      

Isolation of a gene expressed during early embryogenesis from the region of 22q11 commonly deleted in DiGeorge syndrome

DiGeorge syndrome (DGS) is one of several syndromes associatedwith deletions within the proximal long-arm of chromosome 22.The region of chromosome 22q11 responsible for the haplolnsufficiencysyndromes (the DiGeorge Critical Region or DGCR) has been mappedusing RFLPs, quantitative Southern blotting and FISH. Similardeletions are seen in the velo-cardio-facial syndrome (VCFS)and familial congenital heart defects. It Is not known whetherthe phenotypic spectrum is the result of the hemizygosity ofone gene or whether it is a consequence of contiguous genesbeing deleted. However, the majority of patients have a large(< =2Mb deletion). In this paper we report the isolationof a gene, lab name T10, encoding a serine/threonine rich proteinof unknown function which maps to the commonly deleted regionof chromosome 22q11. Studies in the mouse Indicate that it mapsto MMU16 and is expressed during early embryogenesis. Althoughnot mapping within the shortest region of overlap for DGS/VCFS,and therefore not the major gene Involved In DGS, the expressionpattern suggests that this gene may be involved in modifyingthe haplolnsufficient phenotype of hemizygous patients.     

UFD1L, a developmentally expressed ubiquitination gene, is deleted in CATCH 22 syndrome

The CATCH 22 acronym outlines the main clinical features of 22q11.2 deletions (cardiac defects, abnormal facies, thymic hypoplasia, cleft palate and hypocalcemia), usually found in DiGeorge (DGS) and velo- cardio-facial (VCFS) syndromes. Hemizygosity of this region may also be the cause of over 100 different clinical signs. The CATCH 22 locus maps within a 1.5 Mb region, which encompasses several genes. However, no single defect in 22q11.2 hemizygous patients can be ascribed to any gene so far isolated from the critical region of deletion. We have identified a gene in the CATCH 22 critical region, whose functional features and tissue-specific expression suggest a distinct role in embryogenesis. This gene, UFD1L, encodes the human homolog of the yeast ubiquitin fusion degradation 1 protein (UFD1p), involved in the degradation of ubiquitin fusion proteins. Cloning and characterization of the murine homolog (Ufd1l) showed it to be expressed during embryogenesis in the eyes and in the linear ear primordia. These data suggest that the proteolytic pathway that recognizes ubiquitin fusion proteins for degradation is conserved in vertebrates and that the UFD1L gene hemizygosity is the cause of some of the CATCH 22-associated developmental defects.      

Dlx5, the mouse homologue of the human-imprinted DLX5 gene,is biallelically expressed in the mouse brain

The mouse Dlx5 gene encodes a distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of mouse embryo and is located on chromosome 6, which is the syntenic region to the human chromosome 7q21–q31 imprinting cluster. Recently, its human homologue, DLX5, was identified to be imprinted and maternally expressed, at least in normal human lymphoblasts and in brain tissues. In our study, we analyzed the imprinting status of mouse Dlx5 by RT-PCR, first in the F1 of a reciprocal cross between two different mouse strains, and second in heterozygous Dlx5 mutant mice. Both approaches revealed that mouse Dlx5 followed a biallelic pattern of expression in brain tissue and in testis. Our findings suggest that the Dlx5 gene escapes genomic imprinting, at least in mice of certain genetic backgrounds.     

Isolation and characterization of a novel transcript embedded within HIRA, a gene deleted in DiGeorge syndrome.

We have isolated a few cDNAs from different human tissues, transcribed from the first intron of HIRA, a gene deleted in the DiGeorge syndrome. These cDNAs are produced by an intronic gene (22k48) which is transcribed by the HIRA opposite strand and is itself arranged in exons and subjected to alternative splicing. The longest continuum cDNA sequence we obtained is 3.6 kb long and contains 3 different exons and 2 introns. 22k48 cDNA is composed of several tandemly arranged repeated elements (Alu, LINEs, CAn) surrounding a unique sequence. In situ hybridization showed the presence of 22k48 RNA in the cytoplasm of CNS and PNS neurons. 22k48 RNA is able to bind cytoplasmic proteins in the range of 45 to 60 kDa. 22k48 is a new member of the small group of genes that are transcribed but not translated, and its haploinsufficiency could contribute to the pathogenesis of the DiGeorge syndrome.     

Unbalanced 15;22 translocation in a patient with manifestations of DiGeorge and velocardiofacial syndrome

We report on an 8-year-old girl with an unbalanced 15;22 translocation and manifestations of DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), and other abnormalities. The main manifestations of our patient were feeding difficulties, respiratory infections, short stature, peculiar face with hypertelorism, prominent nose, abnormal ears, microstomia and crowded teeth, short broad neck and shield chest with pectus deformity and widely spaced nipples with abnormal fat distribution, heart defect, scoliosis, asymmetric limb development, abnormal hands and feet, and hyperchromic skin patches. Cytogenetic studies demonstrated a 45,XX,der(15)t(15;22)(p11.2;q11.2), -22 karyotype. Fluorescence in situ hybridization (FISH) studies confirmed loss of the proximal DiGeorge chromosomal region (DGCR). This case adds to the diversity of clinical abnormalities caused by deletions within 22q11.2. Am. J. Med. Genet. 70:6–10, 1997. © 1997 Wiley-Liss, Inc.     

The necdin gene is deleted in Prader-Willi syndrome and is imprinted in human and mouse

Human chromosome 15q11-q13 contains genes that are imprinted and expressed from only one parental allele. Prader-Willi syndrome (PWS) is due to the loss of expression of one or more paternally expressed genes on proximal human chromosome 15q, most often by deletion or maternal uniparental disomy. Several candidate genes and a putative imprinting centre have been identified in the deletion region. We report that the human necdin-encoding gene (NDN) is within the centromeric portion of the PWS deletion region, between the two imprinted genes ZNF127 and SNRPN. Murine necdin is a nuclear protein expressed exclusively in differentiated neurons in the brain. Necdin is postulated to govern the permanent arrest of cell growth of post-mitotic neurons during murine nervous system development. We have localized the mouse locus Ndn encoding necdin to chromosome 7 in a region of conserved synteny with human chromosome 15q11-q13, by genetic mapping in an interspecific backcross panel. Furthermore, we demonstrate that expression of Ndn is limited to the paternal allele in RNA from newborn mouse brain. Expression of NDN is detected in many human tissues, with highest levels of expression in brain and placenta. NDN is expressed exclusively from the paternally inherited allele in human fibroblasts. Loss of necdin gene expression may contribute to the disorder of brain development in individuals with PWS.      

T cell receptor repertoire and function in patients with DiGeorge syndrome and velocardiofacial syndrome

DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) are associated with chromosome 22q11.2 deletion. Limited information is available on the T cell receptor (TCR) Vbeta repertoire. We therefore investigated TCR Vbeta families in lymphocytes isolated from blood and thymic samples of seven patients with DGS and seven patients with VCFS, all with 22q11.2 deletion. We also studied activities related to TCR signalling including in vitro proliferation, anti-CD3-induced protein tyrosine phosphorylation, and susceptibility to apoptosis. Reduced CD3+ T cells were observed in most patients. Spontaneous improvement of T cell numbers was detected in patients, 3 years after the first study. Analysis of CD4+ and CD8+ TCR Vbeta repertoire in peripheral and thymic cells showed a normal distribution of populations even if occasional deletions were observed. Lymphoproliferative responses to mitogens were comparable to controls as well as anti-CD3-induced protein tyrosine phosphorylation. Increased anti-CD3-mediated apoptosis was observed in thymic cells. Our data support the idea that in patients surviving the correction of cardiac anomalies, the immune defect appears milder than originally thought, suggesting development of a normal repertoire of mature T cells.     

Expression of Disrupted-In-Schizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development

DISC1 (Disrupted-In-Schizophrenia 1) has been associated with schizophrenia in multiple genetic studies. Studies from our laboratory have shown that Disc1, the mouse ortholog of DISC1, is highly expressed in the dentate gyrus of the hippocampus in the adult mouse brain. Because developmental dysfunction of the hippocampus is thought to play a major role in schizophrenia pathogenesis, and the dentate gyrus is a major locus for adult neurogenesis in the mouse, we investigated Disc1 expression during mouse brain development. Strikingly, Disc1 is strongly expressed in the hippocampus during all stages of hippocampal development, from embryonic day 14 through adulthood. Disc1 mRNA was detected in the dentate gyrus at all stages in which this structure was identifiable, as well as in the cornu ammonis (CA) fields of the hippocampus, the subiculum and adjacent entorhinal cortex, and the developing cerebral neocortex, hypothalamus, and olfactory bulbs, all of which also express Disc1 in the adult mouse brain. In addition, Disc1 mRNA was seen in regions of the developing mouse brain which do not express Disc1 during adulthood, regions including the bed nucleus of the stria terminalis, reticular thalamic nucleus and reuniens thalamic nucleus. These results demonstrate that Disc1 marks the hippocampus from its earliest stages, and suggest that developmental Disc1 dysfunction may lead to defects in hippocampal function that are associated with schizophrenia.     

C16orf5, a novel proline-rich gene at 16p13.3, is highly expressed in the brain

A novel gene has been characterized, designated C16orf5, with an unusually high content of proline residues (40% over 104 residues) at the N-terminus of the protein. The C-terminus of the protein is also cysteine rich with 14 cysteine residues present. Analysis using Northern and dot blots showed that the highest expression of this gene is in the brain. The gene was located on chromosome 16 at band p13.3 by FISH to metaphase chromosomes. Southern blot analysis with a human–rodent somatic cell hybrid panel showed a location between the somatic hybrid breakpoints 23HA and CY196. This gene comprises at least four exons and an open reading frame of 786 bp encoding a predicted protein of 261 amino acids. Analysis of this protein using PSORTII predicted a nuclear localization. Received: April 12, 1999 / Accepted: June 4, 1999     

Viral DNA detected by in situ hybridization in the developing mouse brain infected with murine cytomegalovirus.

To investigate the pathogenesis of brain abnormalities caused by congenital cytomegalovirus (CMV) infection, we previously reported experimental murine models of brain damage induced by intraventricular injection of murine CMV (MCMV) at the late stage of gestation. In the present study, viral DNA-positive cells in the damaged brain at different postnatal stages detected by in situ hybridization were compared with viral antigen-positive cells detected by an immunoperoxidase method using a monoclonal antibody against the immediate early antigen. At birth, the number of viral DNA-positive cells almost equalled that of viral antigen-positive cells. Seven to ten days after birth, the number of viral DNA-positive cells in the brain of MCMV-injected mice was one-fifth that of viral antigen-positive cells. Viral DNA-positive cells were more numerous in the hippocampus than in the cortex, and their density was dependent on the presence of viral antigen-positive cells. Dotted reaction products were observed in the nuclei of viral DNA-positive cells. These cells were rarely detected in lesions of later stages such as atrophy of the cortex and hippocampus, or the wall of the cystic lesions. These results suggest that viral DNA-positive cells detected by in situ hybridization are infected cells in which viral DNA replication is occurring actively.