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Biomedical subjects

V van Heyningen

Publications and source records attributed to V van Heyningen.

At least 55 records · Page 3Linked to original sources
PubMed1995

DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations.

Constitutional point mutations in the zinc finger (ZF) region of the Wilms' tumour suppressor gene 1 (WT1) lead to Denys-Drash syndrome (DDS). Patients with this syndrome display renal failure, Wilms' tumour (WT) and pseudohermaphroditism. DDS WT1 mutations fall into three major categories: (a) missense mutations altering amino acids which directly interact with the DNA target; (b) substitution of amino acids involved in zinc complexing; and (c) nonsense mutations leading to the removal of at least two zinc fingers. We have expressed the WT1 zinc fingers as glutathione-S-transferase fusion proteins, with the lysine-threonine-serine (KTS) alternate splice between ZF3 and ZF4 either present or absent. WT1 fusion constructs with all three classes of DDS mutation were also created. Wild-type and mutant fusion proteins were assayed for their DNA-binding affinity using four previously identified WT1 DNA targets: an EGR1 consensus site; murine insulin-like growth factor 2 promoter 2 (IGF2P2); a (TCC)n motif from the PDGFA-chain promoter; and +P5, a genomic fragment isolated by its affinity for WT1 + KTS. WT1-KTS bound all four targets, but WT1 + KTS only bound +P5. All three classes of DDS mutation investigated, with or without KTS, abolished binding to all four targets. This provides evidence that DDS mutations act either as dominant-negative antimorphs, or elicit their effect through disturbed isoform dosage balance.

Abnormalities, Multiple↗
PubMed1995

Micronucleus frequencies in lymphoblastoid cell lines measured with the cytokinesis-block technique and flow cytometry.

In these experiments, the cytokinesis-block technique was used to measure the spontaneous and radiation-induced micronucleus frequencies in lymphoblastoid cell lines from normal individuals and individuals with radiation-sensitive syndromes. The radiation-induced micronucleus frequencies were measured at different harvest times to investigate the optimum time to measure radiation-induced cytogenetic damage with the cytokinesis-block technique. The frequency of radiation-induced micronuclei was found to be independent of the frequency of binucleate cells in these cell lines. Longer incubation times were complicated by increasing numbers of mononucleate and multinucleate cells, and scoring slides at an early incubation time after the maximum frequency of binucleate cells is reached was recommended. Two of the cell lines (one established from an individual with ataxia telangiectasia, and one from an infant with mitotic instability) had significantly higher radiation-induced micronucleus frequencies than the control cell lines. Finally, the radiation-induced micronucleus frequencies in three of the cell lines were measured by flow cytometry, and the results ranked the cell lines in the same order of radiosensitivity as the cytokinesis block technique.

Cell Division↗
PubMed1995

A new PAX6 mutation in familial aniridia.

Aniridia (lack of iris) is caused by loss of function mutations in one copy of the PAX6 gene. Here we present a new PAX6 splice mutation in a family with autosomal dominant aniridia. The mutation is a single nucleotide change which, although occurring within an exon, affects the splice junction consensus and results in skipping of that exon.

Aniridia↗
PubMed1995

Placement and refined mapping of established and new markers on human chromosome 11q using a small panel of somatic cell hybrids.

Constructing detailed chromosome maps relies on the ability to place new markers rapidly and accurately. Here we map two new anonymous DNA markers precisely on chromosome 11q and refine the location of established markers using a panel of just six fragmentation hybrids, with reference to four translocation chromosomes. As several of the hybrids retain multiple segments of chromosome 11, it is possible to quickly assign new markers to subintervals using this modest set of hybrid DNAs.

Base Sequence↗
PubMed1995

Does the Wilms' tumour suppressor gene, WT1, play roles in both splicing and transcription?

The Wilms' tumour suppressor gene (WT1) encodes a protein(s) with 4 zinc fingers that is essential for the development of the genitourinary system. A considerable body of evidence exists to support the idea that WT1 binds DNA and functions as a transcription factor. However, we have shown recently by confocal microscopy and immunoprecipitation studies that a significant proportion of WT1 is associated with splice factors in kidney cell lines, fetal tissues and transfected Cos cells. Different isoforms of WT1 are produced by an alternative splice that leads to the presence or absence of a 3 amino acid insertion (KTS) between zinc fingers 3 and 4. We have shown that these different forms localise differently in the nucleus. The +KTS form mainly localises with splice factors, the -KTS form mainly with transcription factors. Here we propose a model to account for these different localisations. Also, we discuss the possible significance of these findings.

Amino Acid Sequence↗
PubMed1994

Assignment of 112 microsatellite markers to 23 chromosome 11 subregions delineated by somatic hybrids: comparison with the genetic map.

Using a panel of 25 somatic cell hybrids, we have regionally localized 112 microsatellite markers generated by Généthon and assigned to chromosome 11. A genetic map of 74 of them was produced using linkage analysis of the eight largest CEPH (Centre d'Etude du Polymorphisme Humain) families. They could be ordered on chromosome 11 with an average distance of 2.1 cM. The tight correlation observed between the genetic order and the physical assignment of these microsatellites reinforces the genetic map data. These newly localized markers identified by the PCR method using a standardized protocol represent useful tools for mapping YAC clones and establishing YAC contigs and for studying genetic diseases or cancers associated with specific genes and/or germinal/somatic rearrangements of chromosome 11.

Animals↗
PubMed1994

The human serum amyloid A protein (SAA) superfamily gene cluster: mapping to chromosome 11p15.1 by physical and genetic linkage analysis.

The human serum amyloid A protein (SAA) family comprises a number of small, hepatically produced, differentially expressed apolipoproteins encoded by genes localized on the short arm of chromosome 11.SAA1 and SAA2 are highly related genes that together encode the acute-phase SAAs; SAA3 is a pseudogene; and SAA4 is a low-level constitutively expressed gene encoding constitutive SAA. We have used a combination of physical and genetic mapping techniques to provide evidence that the SAA gene superfamily comprises a cluster of closely linked genes localized to 11p15.1. Pulsed-field gel electrophoresis placed SAA1 to within 350 kb of the previously linked SAA2 and SAA4 genes. SAA locus-specific polymerase chain reaction amplification from a panel of somatic cell hybrids carrying defined regions of chromosome 11p mapped all four loci to 11p15.1-pter. Fluorescence in situ hybridization analysis using a cosmid probe carrying the SAA2 and SAA4 genes refined the localization of these genes (and SAA1) to 11p15.1. To order SAA3 on the genetic map, a highly polymorphic (CA)n dinucleotide repeat within SAA3 was typed through the CEPH reference families. In accordance with the physical localization of SAAs 1, 2, and 4, SAA3 maps to the 11p15.1 region proximal to the parathyroid hormone (PTH) locus (theta = 0.02; lod = 12.020) and distal to D11S455 (theta = 0.058, lod = 8.274). To provide further evidence of an SAA superfamily gene cluster, an NcoI restriction fragment length polymorphism in the SAA2 gene was also typed through the CEPH reference panel.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1994

Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.

Mutation or deletion of the PAX6 gene underlies many cases of aniridia. Three lines of evidence now converge to implicate PAX6 more widely in anterior segment malformations including Peters' anomaly. First, a child with Peters' anomaly is deleted for one copy of PAX6. Second, affected members of a family with dominantly inherited anterior segment malformations, including Peters' anomaly are heterozygous for an R26G mutation in the PAX6 paired box. Third, a proportion of Sey/+ Smalleye mice, heterozygous for a nonsense mutation in murine Pax-6, have an ocular phenotype resembling Peters' anomaly. We therefore propose that a variety of anterior segment anomalies may be associated with PAX6 mutations.

Amino Acid Sequence↗
PubMed1994

Wilms' tumour--a case of disrupted development.

Wilms' tumour is a paediatric kidney malignancy that arises through aberrant differentiation of nephric stem cells. We are studying the role of one Wilms' tumour predisposition gene, WT1. This is a tumour suppressor gene whose function is required for normal development of the genitourinary system. WT1 encodes a putative transcriptional repressor of the zinc finger family. Here we discuss how one of the normal functions of WT1 may be to suppress myogenesis during kidney development. Furthermore, we describe how we are proposing to use YAC (yeast artificial chromosome) transgenesis to analyse WT1 regulation and function in mice. We also discuss the evolution of the WT1 gene amongst different vertebrate classes and how this may provide insights into genitourinary evolution.

Animals↗
PubMed1993

Transmeiotic differentiation of male germ cells in culture.

A cell culture system that supports the differentiation of male germ cells through meiosis is described. It takes advantage of the properties of a cell line, 15P-1, established from testicular cells of transgenic mice that express the large T protein of polyoma virus in the seminiferous epithelium. This line exhibits features characteristics of Sertoli cells, including transcription of the Wilms' tumor (WT1) and Steel genes. Cells of the 15P-1 type support the meiotic and postmeiotic differentiation in cocultures of diploid premeiotic germ cells into haploid spermatids expressing the protamine (Prm-1) gene. When cocultured with 15P-1 cells, testicular cells explanted from immature 9-day-old animals, before the onset of the first meiosis, generated tetrads of haploid cells with the morphology of round spermatids and initiated protamine transcription.

Animals↗
PubMed1993

Colocalization of the human CD59 gene to 11p13 with the MIC11 cell surface antigen.

The human CD59 gene encodes a cell surface antigen detected by MEM43 and other antibodies. It has homology to the mouse Ly-6 genes that map on mouse chromosome 15 and are involved in lymphocyte signal transduction. CD59 may play a role in protecting against complement-mediated lysis. The human CD59 gene had been previously localized to 11p13-p14. We expected, on the basis of synteny arguments, that CD59 would map in 11p14. However, we have precisely localized the human CD59 gene to band p13 of chromosome 11 by somatic cell genetics and by pulsed-field gel electrophoresis; indeed the gene is often deleted in WAGR individuals. This region of chromosome 11 is syntenic with mouse chromosome 2. This suggests that CD59 is not a homolog of the mouse Ly-6 genes on mouse chromosome 15, but rather is a related gene. The human CD59 gene is shown to map within 500 kb of another cell surface marker, MIC11, the gene for which has not been cloned. This localization and the results of immunoprecipitation experiments suggest that the CD59 gene could encode the MIC11 antigen; alternatively this region of 11p13 may contain a cluster of genes encoding cell surface molecules.

Animals↗
PubMed1993

Assignment of the human RT6 gene to 11q13 by PCR screening of somatic cell hybrids and in situ hybridization.

RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, we have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin beta chain locus (HBB) and its neighboring conserved linkage group at 11p15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents.

Animals↗
PubMed1993

Human olfactory marker protein maps close to tyrosinase and is a candidate gene for Usher syndrome type I.

Olfactory marker protein (OMP) shows olfactory neuron-specific expression in rodents. We recently reported tight linkage on mouse chromosome 7 of OMP to the shaker-1 deafness mutant, between the tyrosinase and globin loci. Here we isolate and map the human homologue. Our results show that OMP maps immediately centromeric to tyrosinase on the long arm of human chromosome 11. Genetic linkage to this region has recently been established for Usher Syndrome Type I, an autosomal recessive blindness and deafness disorder and a putative homologue of the shaker-1 mutant. OMP is thus a candidate gene for both congenital deafness defects.

Animals↗
PubMed1993

Evidence that WT1 mutations in Denys-Drash syndrome patients may act in a dominant-negative fashion.

The triad of nephropathy, partial gonadal dysgenesis and Wilms' tumour (WT) is known as Denys-Drash syndrome (DDS). The WT predisposition gene WT1, which plays a vital role in both genital and renal development, is known to be mutated in DDS patients. The WT1 mutations in these patients are constitutional point mutations clustered in the zinc finger (ZF) encoding exons, particularly the exons encoding ZF2 and ZF3. The predicted functional alteration in WT1 is thought to underlie DDS aetiology either by abolishing binding of the WT1 ZF domain to its normal target DNA binding site(s), perhaps blocking the binding of the wild type WT1 present (dominant negative mutation), and/or by conferring the ability to recognise novel but inappropriate DNA binding sites (dominant mutation). We report here on the analysis of WT1 in a further five cases of DDS. In each case a constitutional point mutation was detected in either ZF2 or ZF3. Three of these mutations are novel, with two affecting the conserved histidine and cysteine residues crucial for ZF tertiary structure. The protein product of the third is predicted to lack ZF2, 3 and 4 as a result of a chain termination mutation, and is presumably incapable of binding DNA. However since the DDS phenotype is only elicited by mutations which lead to loss or alteration of ZF function (presumably DNA binding) while the N-terminal upstream portion of the gene remains intact, we suggest that a dominant negative mechanism is at work here.

Amino Acid Sequence↗