Chromosomal aberrations defining uveal melanoma of poor prognosis.
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Chromosome Aberrations↗
Biomedical subjects
B Horsthemke
Publications and source records attributed to B Horsthemke.
Loss of chromosome 3 alleles and multiplication of chromosome 8 alleles in uveal melanoma.
Uveal melanoma is the most frequent primary intraocular tumor. The etiology is unknown. Using neutral DNA polymorphisms on chromosomes 2, 3, and 8, we have detected loss of chromosome 3 alleles in 8 of 13 tumors and multiplication of chromosome 8 alleles in 6 of 11 tumors. No anomalies at a locus on chromosome 2 were found in 10 of 10 tumors. These results confirm and extend previous cytogenetic findings and suggest that a tumor suppressor gene on chromosome 3 and an oncogene on chromosome 8 may be involved in the formation or progression of this tumor.
Adult↗
A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes.
We have used a standard protocol established for human chromosomes to create a chromosome-specific plasmid library from a Beta patellaris chromosome conferring nematode resistance. A monosomic addition line was chosen carrying 18 sugar-beet (Beta vulgaris L.) and one wild-beet (B. patellaris) chromosome. The wild-beet chromosome can readily be identified as a univalent during metaphase I of meiosis. Highly synchronized meiotic material was used to excise the univalents from four pollen mother cells. The chromatin was lysed in a 1 nl collection drop, the DNA purified and restricted with Rsa I, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. The amplified DNA was inserted into a standard plasmid vector and cloned. Approximately 23,000 recombinant plasmids were obtained of which 15,800 could be utilized. Their insert sizes ranged from 80 to 700 bp with an average of 130 bp. 61 clones were tested in more detail by genomic Southern hybridization with sugar-beet and wild-beet DNA. Of these 32 plasmids (52%) contained single-copy inserts, 11 (18%) were specific for wild-beet DNA indicating that the DNA cloned originates in the univalent chromosome. The application of this technique for establishing high-density RFLP maps for discrete regions of plant genomes is discussed.
Animals↗
Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis.
Loss of function of both copies of the RB1 gene is a causal event in the development of retinoblastoma. The predisposition to this tumor can be inherited as an autosomal dominant trait. Direct detection of the genetic defect is important for presymptomatic DNA diagnosis and genetic counseling in families with hereditary retinoblastoma. We have used multiplex polymerase chain reaction and high-resolution polyacrylamide gel electrophoresis to detect RB1 gene deletions as small as one base pair. By using three independent sets of amplification reactions, which cover 26% of the RB1 gene coding region, we identified RB1 gene deletions in the DNA of peripheral blood cells in 3 out of 24 (12.5%) unrelated patients with hereditary retinoblastoma. In one case, formalin-fixed paraffin-embedded tumor material was also used to detect the mutation. Sequencing of the mutated alleles revealed deletions of 1, 3 and 10 base pairs. Each deleted region was flanked by direct repeats.
Amino Acid Sequence↗
Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q11-13.
The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are distinct genetic disorders that are caused by a deletion of chromosome region 15q11-13 or by uniparental disomy for chromosome 15. Whereas PWS results from the absence of a paternal copy of 15q11-13, the absence of a maternal copy of 15q11-13 leads to AS. We have found that an MspI/HpaII restriction site at the D15S63 locus in 15q11-13 is methylated on the maternally derived chromosome, but unmethylated on the paternally derived chromosome. Based on this difference, we have devised a rapid diagnostic test for patients suspected of having PWS and AS.
Adult↗
Interstitial deletions in DiGeorge syndrome detected with microclones from 22q11.
DiGeorge syndrome in humans is characterized by immunodeficiency, heart defects, mental retardation and facial dysmorphism; cytogenetic analysis has shown that deletions at 22q11 occur in approximately 25% of cases. To generate DNA markers from this region, we have microdissected and microcloned band q11 of human Chromosome (Chr) 22. Nineteen thousand clones were obtained from material dissected from 20 chromosome fragments. Seventeen of 61 clones analyzed (28%) were repetitive, 27 (44%) gave no signal, and 17 (28%) detected single copy sequences of which ten mapped to Chr 22. Two of these were found to be deleted in patients with DiGeorge syndrome and either monosomy for 22q11-pter or visible interstitial deletions of 22q11. These two markers are also hemizygous in patients with no visible chromosomal abnormality, demonstrating that submicroscopic deletions are common in DiGeorge syndrome patients.
Blotting, Southern↗
Molecular detection of genetic defects in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a study of 27 families.
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (21-OHase) deficiency is inherited as an autosomal recessive trait. Patients can present with the salt wasting, simple virilizing or a non-classical form of the disease. The gene for P450C21, the enzyme carrying 21-OHase activity, has been mapped to the major histocompatibility complex on chromosome 6p. Using molecular hybridisation techniques we have studied the genetic defect in 27 families with one or more affected offspring diagnosed and treated at the University Hospital of Essen. DNA samples were digested with restriction endonuclease TaqI, PvuII, BglII, and EcoRI and analysed by Southern blot hybridisation with the cDNA probe pC21/3c. Eleven of 40 haplotypes associated with the salt wasting form were found to have a large deletion of 30 kb affecting the 5' end of the active 21-OHase gene and the 3' end of the closely linked pseudogene. Results in another 11 cases are compatible with gene conversion; 18 cases were not informative. The 30 kb deletion was associated with a combination of the HLA antigens Bw47 and DR7 in 7 of 11 cases. In the haplotypes with gene conversion, no linkage disequilibrium to HLA antigens was found. No apparent gene alterations were detected in simple virilizing and non-classical haplotypes. The direct detection of the genetic defect in 55% of the salt wasting haplotypes may help to improve predictive testing in families with CAH.
Adrenal Hyperplasia, Congenital↗
11p15.5-specific libraries for identification of potential gene sequences involved in Beckwith-Wiedemann syndrome and tumorigenesis.
Constitutional and somatic chromosomal abnormalities of the chromosome 11p15 region are involved in an overgrowth malformation syndrome, the Beckwith-Wiedemann syndrome (BWS), and in several types of associated tumors. The bias in parental origin for the different etiologic forms of this syndrome and for loss of heterozygosity in the tumors suggests that a gene (or genes) mapping to this region undergoes genomic imprinting. However, the precise localization of the locus (or loci) for the BWS and associated tumors is still unknown and more markers are required. We therefore isolated 11p15 markers from two libraries: the first one obtained by microdissection of the chromosome 11p15.5 region and the second one, a phage library, constructed from a hybrid cell line containing this region as its sole human DNA. Of 19 microclones isolated from the microdissection library, 11 were evolutionarily conserved. Four phage clones were isolated; one (D11S774) detected a highly informative variable number of tandem repeats (VNTR) and another (D11S773) a biallelic polymorphism. These clones were sublocalized using a panel of somatic cell hybrids that defines eight physical intervals in 11p15.5. Twenty-one clones map to the distal interval that harbors the BWS locus.
Animals↗
Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11-13) by YAC cloning and FISH analysis.
Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.
Angelman Syndrome↗
PCR-mediated cloning of HpaII tiny fragments from microdissected human chromosomes.
Vertebrate DNA contains a small fraction of unmethylated CpG-rich DNA sequences, many of which include the 5' end of a gene. This fraction can be detected by its cleavage to tiny fragments with the methylation-sensitive restriction enzyme HpaII. Thus, the isolation of HpaII tiny fragments (HTFs) from a specific chromosome region may be a useful approach for making an inventory of the genes contained in it. Using microdissection, we have isolated DNA from human chromosome band 8q24.1. The DNA was digested with HpaII, ligated to a ClaI-cut pUC plasmid, and amplified with Taq DNA polymerase and the standard M13/pUC forward and reverse sequencing primers. The amplification products were used to construct an HTF library, which is enriched for CpG-rich single-copy clones.
Animals↗
A new restriction fragment length polymorphism of the human TNF-B gene detected by AspHI digest.
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Base Sequence↗
A simple and nonradioactive method for detecting the Rb1.20 DNA polymorphism in the retinoblastoma gene.
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Base Sequence↗
Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma.
DNA samples from 92 unrelated patients with bilateral retinoblastoma were analyzed by Southern blot hybridization with cDNA and genomic clones of the retinoblastoma (RB-1) gene. Qualitative and quantitative evaluation of the Southern blot patterns showed a deletion of all or part of the RB-1 gene in 15 patients. Deletion hot spots were not detected. The study shows that 16% of germ cell mutations are detectable by Southern blot hybridization, but that densitometric analysis is required in most cases.
Blotting, Southern↗
Microdissection of chromosome band 11p15.5: characterization of probes mapping distal to the HBBC locus.
Both cytogenetic and molecular genetic analyses of the 11p15.5 subband suggest it may contain loci important in the genesis of a wide variety of tumors such as rhabdomyosarcomas and Wilms' tumors as well as the congenital tumors associated with the Beckwith-Wiedemann syndrome. As a first step in further defining the involvement of this chromosomal region in these various maladies, a library was constructed from the specific microdissection of chromosomal fragments representing 11p15.5-pter. Of 98 microclones analyzed, 31 identified single copy human DNA sequences, 21 of which mapped to 11p15.5 while 10 mapped proximal to the HBBC locus. Five of the 11p15.5-positioned microprobes detected restriction fragment length polymorphisms at their homologous genomic loci for various enzymes. These microprobes are now being utilized in several ways in order to address the underlying basis of the Beckwith-Wiedemann syndrome and its associated tumors.
Animals↗
Long-term treatment effects in patients with bilateral retinoblastoma: ocular and mid-facial findings.
A total of 99 patients with bilateral retinoblastoma who had been treated between 1965 and 1982 were reexamined in April 1988 to study the late effects of treatment. Their median age at the follow-up visit in 1988 was 16 years (range, 6-27 years), and the median follow-up was 15 years (range, 6-26 years). All patients underwent a full eye examination, morphometric measurements of the mid-face and genetic counselling. Each eye or orbit and the corresponding side of the patient's mid-face were evaluated separately, resulting in 198 data sets from 99 individuals. Subjects were divided into four treatment groups according to whether photo- and cryo-coagulation, enucleation, radiation therapy or various combinations thereof were used. In all, 81 eyes had a visual acuity of greater than 0.4 (in 23 of these, however, only with low-vision aids). Within a dose range of 36-51 Gy, the location of the tumor (36%) or cataract (15%) were the main factors responsible for poor visual acuity, whereas radiation retinopathy and/or optic neuropathy occurred in only three cases. Cataracts were more frequently observed following orthovoltage as compared with megavoltage therapy (P = 0.012). A total of 72 eyes had been enucleated and had not received radiation therapy at any time. Cosmetic results (as measured by several parameters) in these cases were significantly better then those in 28 subjects who underwent combined radiation therapy and enucleation. As defined by various subjective as well as objective findings, mid-facial hypoplasia occurred significantly more often following orthovoltage as compared with megavoltage therapy.
Adolescent↗
No evidence for sequences structurally related to the RB1 gene in the human genome.
The retinoblastoma (RB1) gene is a ubiquitously expressed gene encoding a cell-cycle control protein. Inactivation of this gene plays a crucial role in the development of retinoblastoma, osteosarcoma, and other tumors. In a search for structurally related gene sequences we identified a 5.5-kb BamHI fragment strongly cross-hybridizing with the 5' end of the RB1 cDNA. Molecular cloning, in situ hybridization, restriction mapping, and sequence analysis identified this DNA segment as the 28S rRNA gene. The absence of other cross-hybridizing sequences suggests that the RB1 gene is not part of a structurally related gene family.
Base Sequence↗
The syntenic relationship between the critical deletion region for the Prader-Willi/Angelman syndromes and proximal mouse chromosome 7.
The deleted region of the proximal long arm of human chromosome 15, common to a large group of patients with the Prader-Willi and Angelman syndromes, has recently been defined. We have mapped to the mouse genome segments homologous to human probes found within and flanking this deletional region. These probes define a region of conserved synteny on proximal chromosome 7 of the mouse. Because the Prader-Willi and Angelman syndromes are postulated to result from genomic imprinting within the common deletion, these probes may define a genomically imprinted region on mouse chromosome 7.
Animals↗
Characterization and mapping of microdissected genomic clones from the adenomatous polyposis coli (APC) region.
The gene associated with adenomatous polyposis coli (APC) has been mapped to the long arm of chromosome 5. To saturate the APC region with DNA markers, two independent microdissection libraries with an emphasis on 5q21.2-21.3 and 5q22 have been constructed from GTG-banded human metaphase chromosomes. PCR-amplified insert DNA of the primary amplificate used as a probe in chromosomal in situ suppression (CISS) hybridization of human metaphase spreads revealed region-specific signals at the chromosomal site that was excised for cloning. One hundred forty-two inserts, derived from both libraries, have been characterized in more detail. Deletion mapping analysis was performed with 17 single-copy clones on a hamster-human hybrid cell panel. Seven of these clones were located within two interstitial deletions of 6-8 Mb from APC-affected individuals around chromosome bands 5q21-22. The identification of new microclones mapping into these deletions and their use in isolating YAC clones should contribute to the construction of a contiguous physical map of the APC region.
Adenomatous Polyposis Coli↗