Search PubMed⌕ Search

Biomedical subjects

D Housman

Publications and source records attributed to D Housman.

At least 19 recordsLinked to original sources
PubMed1995

Antagonism of WT1 activity by protein self-association.

Germline loss-of-function mutations at the Wilms tumor (WT) suppressor locus WT1 are associated with a predisposition to WTs and mild genital system anomalies. In contrast, germ-line missense mutations within the WT1 gene encoding the DNA-binding domain often yield a more severe phenotype consisting of WT, sexual ambiguity, and renal nephropathy. In this report, we demonstrate that the products of mutant alleles that impair DNA recognition can antagonize WT1-mediated transcriptional repression. We demonstrate that WT1 can self-associate in vitro and in vivo and that the responsible domain maps to the amino-terminal region of the protein. Oligomers of full-length protein form less efficiently or produce less stable complexes than oligomers between truncated polypeptides and full-length protein. Our data suggest a molecular mechanism to explain how WT1 mutations may act in deregulating cellular proliferation and differentiation.

Alleles↗
PubMed1995

Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach.

We report here an efficient approach to the establishment of extended YAC contigs on human chromosome 2 by using an interspersed repetitive sequences (IRS)-PCR-based screening strategy for YAC DNA pools. Genomic DNA was extracted from 1152 YAC pools comprised of 55,296 YACs mostly derived from the CEPH Mark I library. Alu-element-mediated PCR was performed for each pool, and amplification products were spotted on hybridization membranes (IRS filters). IRS probes for the screening of the IRS filters were obtained by Alu-element-mediated PCR. Of 708 distinct probes obtained from chromosome 2-specific somatic cell hybrids, 85% were successfully used for library screening. Similarly, 80% of 80 YAC walking probes were successfully used for library screening. Each probe detected an average of 6.6 YACs, which is in good agreement with the 7- to 7.5-fold genome coverage provided by the library. In a preliminary analysis, we have identified 188 YAC groups that are the basis for building contigs for chromosome 2. The coverage of the telomeric half of chromosome 2q was considered to be good since 31 of 34 microsatellites and 22 of 23 expressed sequence tags that were chosen from chromosome region 2q13-q37 were contained in a chromosome 2 YAC sublibrary generated by our experiments. We have identified a minimum of 1610 distinct chromosome 2-specific YACs, which will be a valuable asset for the physical mapping of the second largest human chromosome.

Animals↗
PubMed1995

Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues.

We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.

Adult↗
PubMed1994

Sequential strategy to identify a susceptibility gene for schizophrenia: report of potential linkage on chromosome 22q12-q13.1: Part 1.

To identify genes responsible for the susceptibility for schizophrenia, and to test the hypothesis that schizophrenia is etiologically heterogeneous, we have studied 39 multiplex families from a systematic sample of schizophrenic patients. Using a complex autosomal dominant model, which considers only those with a diagnosis of schizophrenia or schizoaffective disorder as affected, a random search of the genome for detection of linkage was undertaken. Pairwise linkage analyses suggest a potential linkage (LRH = 34.7 or maximum lod score = 1.54) for one region (22q12-q13.1). Reanalyses, varying parameters in the dominant model, maximized the LRH at 660.7 (maximum lod score 2.82). This finding is of sufficient interest to warrant further investigation through collaborative studies.

Adult↗
PubMed1994

Follow-up of a report of a potential linkage for schizophrenia on chromosome 22q12-q13.1: Part 2.

A collaboration involving four groups of investigators (Johns Hopkins University/Massachusetts Institute of Technology; Medical College of Virginia/The Health Research Board, Dublin; Institute of Psychiatry, London/University of Wales, Cardiff; Centre National de la Recherche Scientifique, Paris) was organized to confirm results suggestive of a schizophrenia susceptibility locus on chromosome 22 identified by the JHU/MIT group after a random search of the genome. Diagnostic, laboratory, and analytical reliability exercises were conducted among the groups to ensure uniformity of procedures. Data from genotyping of 3 dinucleotide repeat polymorphisms (at the loci D22S268, IL2RB, D22S307) for a combined replication sample of 256 families, each having 2 or more affected individuals with DNA, were analysed using a complex autosomal dominant model. This study provided no evidence for linkage or heterogeneity for the region 22q12-q13 under this model. We conclude that if this region confers susceptibility to schizophrenia, it must be in only a small proportion of families. Collaborative efforts to obtain large samples must continue to play an important role in the genetic search for clues to complex psychiatric disorders such as schizophrenia.

Adult↗
PubMed1993

Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products.

An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.

Base Sequence↗
PubMed1993

Analysis of the 11p13 Wilms' tumor suppressor gene (WT1) in ovarian tumors.

We have examined the status of the Wilms' tumor suppressor gene (WT1), residing at chromosome 11 band p13, in a total of 40 cancers of the female reproductive tract. Northern blot analysis revealed that the WT1 gene is expressed in a large percentage of ovarian tumors (75%) analyzed. Single-strand conformation polymorphism analysis was performed on all the tumors in this study in an attempt to detect mutations within the WT1 gene. Only silent mutations were detected within intron 7 of WT1 using this method. Loss of heterozygosity studies were performed at the WT1 locus in several ovarian tumors and revealed that in the informative cases, heterozygosity was retained.

Base Sequence↗
PubMed1992

The recombination activating genes, RAG 1 and RAG 2, are on chromosome 11p in humans and chromosome 2p in mice.

The recombination activating genes RAG-1 and RAG-2 are adjacent genes that act synergistically to activate variable-diversity-joining (V(D)J) recombination. Southern analysis of hybrid cell lines derived from patients with the Wilms tumor-aniridia-genitourinary defects-mental retardation (WAGR) syndrome and from mutagenized cell hybrids selected for deletions in chromosome 11 has allowed us to map the chromosomal location of the human RAG locus. The RAG locus defines a new interval of human chromosome 11p, but is not associated with any genetically mapped human disease. Guided by the chromosomal localization of the human recombination activating genes, we have also mapped the location of the mouse Rag locus.

Animals↗
PubMed1992

High-frequency DNA sequence polymorphisms in the insulin receptor gene detected by denaturing gradient gel blots.

A limiting factor in the study of genetic determinants of human disorders is the availability of informative DNA markers. In this report, we describe an application of the denaturing gradient gel blot method for detecting high-frequency DNA sequence polymorphisms in the human insulin receptor locus. Using two restriction enzymes and cDNA probes for the insulin receptor, we found five DNA polymorphisms. The probe that contained exons 4-10 of the insulin receptor gene detected two two-allelic polymorphisms in HinfI digests, one at denaturant concentrations of 38%/39% and the other at 46%/48%. The probe that contained exons 14-22 detected three two-allelic polymorphisms in Sau96I digests, the first at denaturant concentrations of 34%/35%, the second at 38%/39%, and the third at 46%/47%. All these DNA polymorphisms segregated in families in a Mendelian fashion, and the allelic distribution for each of them did not deviate from Hardy-Weinberg equilibrium. The identified polymorphisms were in linkage equilibrium and provided sufficient genetic information to determine parental haplotypes at the insulin receptor locus in small two-generation families. The denaturing gradient gel blot method is a very sensitive technique for identifying sequence polymorphisms in genomic DNA; its application will facilitate the search for genes involved in the development of many inherited disorders.

DNA↗
PubMed1992

Depressive affect in "normal" adolescents: relationship to life stress, family, and friends.

Self-reported depressive affect was examined in high school students in relation to stress and the quality of relationships with family and friends. Higher levels of depressive affect were connected with stress around sexuality and achievement, lower levels of family cohesion, and more problematic peer relationships. The effects of high stress were buffered for boys by positive peer relationships, and for girls by cohesive family relationships.

Achievement↗
PubMed1992

Germline intronic and exonic mutations in the Wilms' tumour gene (WT1) affecting urogenital development.

Denys-Drash syndrome is a rare human developmental disorder affecting the urogenital system and leading to renal failure, intersex disorders and Wilms' tumour. In this report, four individuals with this syndrome are described carrying germline point mutations in the Wilms' tumour suppressor gene, WT1. Three of these mutations were in the zinc finger domains of WT1. The fourth occurred within intron 9, preventing splicing at one of the alternatively chosen splice donor sites of exon 9 when assayed in vitro. These results provide genetic evidence for distinct functional roles of the WT1 isoforms in urogenital development.

Alternative Splicing↗
PubMed1992

Molecular genetic approaches to the identification of genes involved in the development of nephropathy in insulin-dependent diabetes mellitus.

Genetic factors contribute significantly to the development of diabetic nephropathy in patients with insulin-dependent diabetes mellitus. This report discusses some models of diabetic nephropathy that incorporate genetic susceptibility and presents strategies for identifying the responsible genes. To identify variation at a locus, newly developed methods are discussed that employ denaturing gradient gel electrophoresis to study sequence differences in both polymerase chain reaction-amplified DNA fragments and genomic DNA. These techniques are illustrated with studies of the angiotensinogen gene and the insulin receptor gene. In preliminary data from a comparison between individuals with and without diabetic nephropathy, no DNA sequence difference in that part of the angiotensinogen gene that codes for angiotensin I was found. However, with a probe corresponding to exons 7 and 8 of the insulin receptor gene and denaturing gradient gel electrophoresis of Rsal digestions of genomic DNA, different distributions of a DNA polymorphism were found in patients with fast as compared with slowly progressing nephropathy. The interpretation of this finding and the need for further studies are discussed. In conclusion, the advent of methods of molecular genetics makes possible studies on genetic determinants of diabetic nephropathy. However, more clinical and epidemiologic data are needed to find out how many genes are involved and how they interact with exposure to diabetes. Foremost, DNA from families with two or more siblings with diabetic nephropathy must be collected to permit the necessary genetic studies.

Chromosome Mapping↗
PubMed1991

Direct pulsed field gel electrophoresis of Wilms' tumors shows that DNA deletions in 11p13 are rare.

In order to search for small tumor-specific deletions in 11p13 we analysed DNA isolated from 30 fresh Wilms' tumor (WT) samples with pulsed field gel electrophoresis. For these studies we have isolated new probes from the ends of several Notl fragments. Using these and previously described probes from 11p13 we first completed and extended the existing map of the 11p13 region. The analysis of the tumor material showed that (I) tumor-specific deletions were very rare: one homozygous deletion out of 30 tumors analysed, (2) hemizygous deletions were not observed in any of the tumors. The homozygous deletion in one patient spans 220 kb and is composed of a tumor-specific translocation associated with a deletion on one chromosome and a deletion of about 220 kb on the other chromosome at the same site. The WT-33 Wilms' tumor candidate gene maps to this deleted segment. A small constitutional deletion of 1,300 kb was identified in a patient with WT and genital tract malformations. These results suggest that in the majority of sporadic WT loss of gene function is due to subtle alterations in the gene, e.g., point mutations or very small deletions.

Alleles↗