Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1).

Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: "cen-WEE1-D11S2043-ZNF143-RANBP7-CEGF1- ST5-D11S932-LMO1-D11S572-TUB-tel", with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.

LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.

Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.

Functional characterization of ORCTL2--an organic cation transporter expressed in the renal proximal tubules.

Chromosome 11p15.5 harbors a gene or genes involved in Beckwith-Wiedemann syndrome that confer(s) susceptibility to Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma. We have previously identified a transcript at 11p15.5 which encodes a putative membrane transport protein, designated organic cation transporter-like 2 (ORCTL2), that shares homology with tetracycline resistance proteins and bacterial multidrug resistance proteins. In this report, we have investigated the transport properties of ORCTL2 and show that this protein can confer resistance to chloroquine and quinidine when overexpressed in bacteria. Immunohistochemistry analyses performed with anti-ORCTL2 polyclonal antibodies on human renal sections indicate that ORCTL2 is localized on the apical membrane surface of the proximal tubules. These results suggest that ORCTL2 may play a role in the transport of chloroquine and quinidine related compounds in the kidney.

Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain.

Human chromosomal band 11p15.5 has been shown to contain genes involved in the development of several pediatric and adult tumors and in Beckwith-Wiedemann syndrome (BWS). Overlapping P1 artificial chromosome clones from this region have been used as templates for genomic sequencing in an effort to identify candidate genes for these disorders. PowerBLAST identified several matches with expressed sequence tags (ESTs) from fetal brain and liver cDNA libraries. Northern blot analysis indicated that two of the genes identified by these ESTs encode transcripts of 1-1.5 kb with predominant expression in fetal and adult liver and kidney. With RT-PCR and RACE, full-length transcripts were isolated for these two genes, with the largest open reading frames encoding putative proteins of 253 and 424 amino acids. Database comparison of the predicted amino acid sequence of the larger transcript indicated homology to integral membrane organic cation transporters; hence, we designate this gene ORCTL2 (organic cation transporter-like 2). An expressed sequence polymorphism provided evidence that the ORCTL2 gene exhibits "leaky" imprinting in both human fetal kidney and human fetal liver. The mouse orthologue (Orctl2) was identified, and a similar polymorphism was used to demonstrate maternal-specific expression of this gene in fetal liver from interspecific F1 mice. The predicted protein of the smaller gene showed no significant similarity in the database. Northern and RACE analyses suggest that this gene may have multiple transcription start sites. Determination of the genomic structure in humans indicated that the 5'-end of this transcript overlaps in divergent orientation with the first two exons of ORCTL2, suggesting a possible role for antisense regulation of one gene by the other. We, therefore, provisionally name this second transcript ORCTL2S (ORCTL2-antisense). The expression patterns of these genes and the imprinted expression of ORCTL2 are suggestive of a possible role in the development of Wilms tumor (WT) and hepatoblastoma. Although SSCP analysis of 62 WT samples and 10 BWS patients did not result in the identification of any mutations in ORCTL2 or ORCTL2S, it will be important to examine their expression pattern in tumors and BWS patients, since epigenetic alteration at these loci may play a role in the etiology of these diseases.

Cloning and characterization of a novel gene (TM7SF1) encoding a putative seven-pass transmembrane protein that is upregulated during kidney development.

We have used the cDNA differential display of mRNA technique to isolate genes differentially regulated during kidney development. Here we report the identification of a novel gene, TM7SF1, which is upregulated in the course of kidney development. The full-length cDNA of TM7SF1 is about 2.4 kb and contains an open reading frame of 1197 nucleotides. The predicted secondary structure of the corresponding protein displays seven putative helical transmembrane domains, a structural feature shared by all members of the G-protein-coupled receptor class of transmembrane proteins. Two minor alternatively spliced versions of approximately 2.3 and approximately 2.2 kb could be detected, one of which contains a nearly identical open reading frame with a truncated carboxy-terminus of the deduced protein. The second alternatively spliced version harbors a completely shifted open reading frame with a potential new ATG start codon. By the use of single-chromosome hybrid cells and fluorescence in situ hybridization experiments, TM7SF1 could be localized to chromosome 1q42-q43. Human multiple tissue Northern blot analysis revealed TM7SF1 transcripts in human kidney, heart, brain, and placenta tissue. Studies on Wilms tumor samples showed variable TM7SF1 expression, ranging from nearly undetectable levels to an abundant level of expression comparable to that of adult kidney tissue.

Novel WT1 mutation, 11p LOH, and t(7;12) (p22;q22) chromosomal translocation identified in a Wilms' tumor case.

About 5-10% of sporadic Wilms' tumors (WT) are associated with mutations in the Wilms' tumor 1 gene (WT1). More than 90% of patients with Denys-Drash syndrome (DDS; characterized by renal nephropathy, gonadal anomaly, and predisposition to WT) show constitutional intragenic WT1 mutations. We describe a novel WT1 stop-mutation in exon 2. This heterozygous germline mutation was detected in a one-year-old girl who was bilaterally affected with Wilms' tumor but without any other clinical manifestations of DDS. The C-to-A transversion is predicted to result in a polypeptide comprising only the first 165 amino acids of the WT1 protein. Loss of heterozygosity (LOH) studies comparing tumor DNA with lymphocyte DNA revealed LOH for the entire short arm of chromosome 11 in tumor tissue. In addition to the chromosome 11 lesions, the tumor showed a seemingly balanced chromosomal translocation t(7;12) (p22;q22) as the only visible cytogenetic aberration.

Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal-dominant condition characterized by hypoplasia/aplasia of clavicles, patent fontanelles, supernumerary teeth, short stature, and other changes in skeletal patterning and growth. In some families, the phenotype segregates with deletions resulting in heterozygous loss of CBFA1, a member of the runt family of transcription factors. In other families, insertion, deletion, and missense mutations lead to translational stop codons in the DNA binding domain or in the C-terminal transactivating region. In-frame expansion of a polyalanine stretch segregates in an affected family with brachydactyly and minor clinical findings of CCD. We conclude that CBFA1 mutations cause CCD and that heterozygous loss of function is sufficient to produce the disorder.

Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome.

Rieger syndrome (RIEG) is an autosomal-dominant human disorder that includes anomalies of the anterior chamber of the eye, dental hypoplasia and a protuberant umbilicus. We report the human cDNA and genomic characterization of a new homeobox gene, RIEG, causing this disorder. Six mutations in RIEG were found in individuals with the disorder. The cDNA sequence of Rieg, the murine homologue of RIEG, has also been isolated and shows strong homology with the human sequence. In mouse embryos Rieg mRNA localized in the periocular mesenchyme, maxillary and mandibular epithelia, and umbilicus, all consistent with RIEG abnormalities. The gene is also expressed in Rathke's pouch, vitelline vessels and the limb mesenchyme. RIEG characterization provides opportunities for understanding ocular, dental and umbilical development and the pleiotropic interactions of pituitary and limb morphogenesis.

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.

Molecular cloning and chromosomal assignment of the human homologue of the rat cGMP-inhibited phosphodiesterase 1 (PDE3A)--a gene involved in fat metabolism located at 11p 15.1.

We have cloned the coding region of a human gene, whose predicted amino acid sequence shows 88% homology and higher correspondence in functional domains to the rat cGMP inhibited phosphodiesterase gene (PDE3A). In concordance with the expression data of the rat PDE3A gene, a 5.3-kb transcript of the human cGMP-inhibited phosphodiesterase gene is shown in Northern blot analysis to be highly expressed in adipose tissue. In addition, weaker expression is seen in pancreas, skeletal muscle, liver, placenta, and heart. cDNA clones from the homologue mouse gene were isolated and sequenced spanning a highly conserved region coding for a C-terminal located catalytic core region of this enzyme family. Using a genomic cosmid clone of human PDE3A for fluorescence in situ hybridization, the gene was mapped to chromosomal region 11p15 and regionally sublocalized by PCR on a human-hamster somatic hybrid-cell mapping panel to 11p15.1-p2. Based on comparative linkage data in mouse and rat this chromosomal location is suggested to contain genes involved in complex diseases like obesity and diabetes mellitus type II. Therefore, a possible involvement of the human PDE3A gene in these polygenic traits is discussed, taking into account the prominent role of the rat PDE3A gene product in the antilipolytic action of insulin in adipocytes.

Kniest and Stickler dysplasia phenotypes caused by collagen type II gene (COL2A1) defect.

Kniest and Stickler dysplasia are two chondrodysplasias characterized by specific phenotypes. No basic defect has been found in patients with Kniest dysplasia, whereas Stickler dysplasia is one of four chondrodysplasias for which mutations of type II procollagen gene (COL2A1) have been identified. We studied a 2-year-old girl presenting with manifestations of Kniest dysplasia and her mother showing a Stickler phenotype. Analysing COL2A1 in both patients, we detected the same 28 basepair deletion spanning the 3'-exon/intron boundary of exon 12 in mother and daughter. We were able to prove a somatic mosaic status for this mutation in the mother which accounts for her milder Stickler-like phenotype.

The human ribonuclease/angiogenin inhibitor is encoded by a gene mapped to chromosome 11p15.5, within 90 kb of the HRAS protooncogene.

Ribonuclease/angiogenin inhibitor (RAI) is a tight-binding inhibitor of ribonucleolytic and angiogenic activities involved in tumor progression. It is translated from various mRNAs differing in their 5 regions and originating from a single gene locus. Recently, this gene (RNH) has been assigned to 11p15.5, the terminal part of the short arm of chromosome 11. The regional chromosomal localization was confirmed by somatic cell and in situ hybridization and further refined by long-range restriction mapping. The data place RNH within 90 kb of the Harvey-ras protooncogene (HRAS), so far the most telomeric gene on 11p, in a region involved in growth regulation and tumor development.

Human nuclear NAD+ ADP-ribosyltransferase: localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli.

The gene for human nuclear NAD+ ADP-ribosyltransferase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosetransferase, EC 2.4.2.30; pADPRT] was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pADPRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at internal translational start signals located within the pADPRT cDNA.

Mapping of the DNA locus D4S10 and the linked Huntington's disease gene to 4p16----p15.

An anonymous DNA fragment (G8) detects two restriction fragment length polymorphic alleles (RFLPs) called D4S10 in HindIII-digested human genomic DNA. This segment had been assigned to chromosome 4 and shows close linkage to the Huntington's disease gene. With in situ hybridization, we mapped D4S10 to the terminal region of the short arm of chromosome 4, localizing the Huntington's disease gene to bands 4p16----p15. This information may prove useful for the development of strategies to clone the Huntington's disease gene.

Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.

Chromosome mapping of genes on the short arm of human chromosome 11: parathyroid hormone gene is at 11p15 together with the genes for insulin, c-Harvey-ras 1, and beta-hemoglobin.

The human parathyroid hormone gene (PTH) was mapped to the 11p15 chromosomal band by in situ hybridization. Using the same procedures and cells, the closely linked beta-hemoglobin gene (HBB), the Harvey-ras 1 proto-oncogene (HRAS1), and the insulin gene (INS) were also mapped to this same region. Some reports have demonstrated differences in regional localization of the latter three genes, and linkage and molecular studies have not resolved how far this linkage group extends from p15 toward the centromere on the physical gene map. Our results show that all of these genes are localized at 11p15, a region of one chromosomal band that appears to comprise a genetic distance of more than 20 cM.

Cellular homologs of the avian erythroblastosis virus erb-A and erb-B genes are syntenic in mouse but asyntenic in man.

Avian erythroblastosis virus, a retrovirus that causes erythroblastosis and sarcomas in infected birds, possesses two host cell-derived genes [viral (v) erb-A and erb-B]. Although v-erb-B seems to be responsible for oncogenic transformation, v-erb-A might have an enhancing effect on transformation. In chickens, the natural host for avian erythroblastosis virus, cellular (c) erb-A and erb-B genes appear to be unlinked, but their chromosomal locations in other species are unknown. To ascertain the chromosomal location of c-erb genes in man and mouse, we analyzed interspecies somatic cell and microcell hybrids by Southern filter hybridization techniques using specific v-erb-A and v-erb-B probes. We found c-erb-A sequences on human chromosome 17 (17p11----qter) and located c-erb-B on human chromosome 7 (7pter----q22). In contrast, both c-erb-A and c-erb-B reside on mouse chromosome 11.

Regional localization of two human cellular Kirsten ras genes on chromosomes 6 and 12.

Human cellular Kirsten ras1 and ras2 genes were localized to chromosomes 6p23 ----q12 and 12p12 .05----pter, respectively, using human-rodent cell hybrids. Thus, the short arms of human chromosomes 11 (encoding lactate dehydrogenase-A and the proto-oncogene c-Ha- ras1 ) and 12 (encoding lactate dehydrogenase B and c-Ki- ras2 ) share at least two pairs of genes that probably evolved from common ancestral genes.

Localization of human U1 small nuclear RNA genes to band p36.3 of chromosome 1 by in situ hybridization.

U1 small nuclear RNA (U1 snRNA) is encoded by a large family of genes (30-125 copies/haploid genome) which are transcribed by RNA polymerase II. U1 snRNA is thought to function in gene splicing. Since the U1 genes were found to be greater than 20 kb apart by analyzing genomic phage clones, the chromosomal location of U1 genes in the human genome was determined using Southern filter analysis of DNA isolated from human-rodent somatic cell hybrids and by in situ hybridization. Human DNA digested with PvuII and probed with a U1-specific probe, pD2, show several major hybridizing fragments. Of these, two human PvuII fragments of 1.4 kb and 2.4 kb had unique mobilities compared to mouse fragments. In a study of 19 cell hybrids, the human-specific U1 fragments segregated with the chromosome 1 markers peptidase C and adenylate kinase 2. All other chromosomes showed greater than or equal to 19% discordancy . An additional 13 karyotyped cell hybrids, analyzed by Southern filter analysis, confirmed the assignment of this class of U1 genes to chromosome 1. Additional digests with MspI and PstI indicated that most U1 genes are located on chromosome 1. To determine if the U1 RNAs are located predominantly at one site or dispersed over chromosome 1, a tritium-labeled U1 probe was hybridized in situ to metaphase chromosomes. The majority of the grains were at band 1p36 .3, suggesting that most of the U1 genes are located in this region.