A quantitative trait locus on 13q14.2 for trunk strength.

Previous findings show strong evidence for the role of retinoblastoma (Rb) in myoblast proliferation and differentiation. However, it is not known whether variation in the retinoblastoma gene (RB1 ) is responsible for normal variation in human muscle strength. Therefore, a linkage analysis for quantitative traits was performed on 329 young male siblings from 146 families with muscle strength, using a polymorphic marker in RB1 (D13S153 on 13q14.2). Trunk strength, a general strength indicator that requires activation of large muscle groups, was measured on a Cybex TEF isokinetic dynamometer. We found evidence for linkage between locus D13S153 at 13q14.2 and several measurements of trunk flexion with LOD scores between 1.62 and 2.78 (.002< p <.0002). No evidence for linkage was found with trunk extension. This first exploration of the relationship between RB1 and human muscle strength through linkage analysis warrants efforts for further fine mapping of this region.     

Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single-sperm typing

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.     

Studies on the human retinoblastoma susceptibility gene

The retinoblastoma susceptibility (RB) gene is unique among other cloned cancer genes because its causal role in a human cancer, retinoblastoma, was established by classical genetic methods before its isolation. Earlier hypotheses and experimental data suggested that inactivation of a gene in chromosome band 13q14 resulted in retinoblastoma formation. A gene in this region was identified as the RB gene on the basis of mutations found specifically in retinoblastoma tumors; however, its proposed biological activity in suppressing neoplasia has yet to be demonstrated. The RB gene product was identified as a nuclear phosphoprotein of 110 kD associated with DNA binding activity, suggesting that the RB protein may regulate other genes. Probes for the RB gene and gene product will be useful for genetic diagnosis of retinoblastoma susceptibility in affected families; for direct detection of mutant RB alleles; and, potentially, for genetic diagnosis of susceptibility to osteosarcoma and other tumors tentatively linked to RB-gene dysfunction. Continued study of the RB gene should yield further insight into mechanisms of oncogenesis, development, and gene regulation.     

Detection of small RB1 gene deletions in retinoblastoma by multiplex PCR and high-resolution gel electrophoresis

Summary 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.     

Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity

Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.     

Expression of the RB gene under the control of MuLV-LTR suppresses tumorigenicity of WERI-Rb-27 retinoblastoma cells in immunodefective mice

Retinoblastomas arise by the loss of the retinoblastoma (RB) gene. The isolation of the RB gene and its expression in RB protein defective tumor cells permits direct tests of the ability of the protein to act as a tumor suppressor. We demonstrate that a functional RB gene introduced into WERI-Rb-27 retinoblastoma cells by retrovirally mediated gene transfer can suppress their tumorigenicity in immunodefective mice.     

Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.

Most sporadic cases of retinoblastoma, malignant eye tumor of children, may require the identification of a mutation of the retinoblastoma gene (RB1 gene) for precise genetic counseling. We established a mutation detection system of and screened for the RB1 gene mutation in 24 patients with retinoblastoma--12 bilateral patients and 12 unilateral patients. Mutation analysis was performed by PCR-mediated SSCP analysis in the entire coding region and promoter region, as an initial screening method, followed by direct genomic sequencing. Possible oncogenic mutations were identified in 14 (58%) of 24 tumors, of which 6 were single base substitutions, 4 were small deletions, 3 were small insertions, and 1 was a complex alteration due to deletion-insertion. A constitutional somatic mosaicism was suggested in one bilateral patient. A majority (57%) of mutations were found in E1A binding domains, and all were presumed to truncate the normal gene products. The mutation analysis presented here may provide a basis for the screening system of RB1 gene mutations in retinoblastoma patients.     

RB基因与肿瘤抑制

RB基因位于13q14,全长150kb,编码一个由928个氨基酸组成的分子量为110 000蛋白(pp110RB).它能特异性与SV40大T,E1A和E7结合.在视网膜细胞中,RB呈衡定组成性表达,其缺陷除引起RB外,在骨肉瘤、乳腺癌、小细胞肺癌、软组织肉瘤及造血系统增生性疾病也有RB基因的突变.把RB基因导入到基因缺陷的恶性肿瘤细胞能全部或部分抑制其恶性表现.     

Molecular detection of chromosomal translocations that disrupt the putative retinoblastoma susceptibility locus.

A candidate DNA sequence with many of the properties predicted for the retinoblastoma susceptibility (RB1) locus has been cloned (S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature [London] 323:643-645, 1986). The large size of this gene (ca. 200 kilobases [kb]) and its multiple dispersed exons (Wiggs et al., N. Engl. J. Med. 318:151-157, 1988) complicate molecular screening strategies important in prenatal and presymptomatic diagnosis and in carrier detection. Here we used field inversion gel electrophoresis (FIGE) to construct a restriction map of approximately 1,000 kb of DNA surrounding the RB1 locus and to detect the translocation breakpoints in three retinoblastoma patients. DNA probes from either the 5' or 3' end of the gene were used to detect a 250-kb EagI restriction fragment in DNA from unaffected individuals. Both probes identified an additional hybridizing fragment in the DNA from each patient, permitting the breakpoints in all three to be mapped within the cloned RB1 gene. Analysis of the breakpoint in one translocation cell line allowed the RB1 gene to be oriented with its 5' end toward the centromere. The 5' end of the gene also appeared to be associated with a clustering of sites for several infrequently cleaving restriction enzymes, indicating the presence of an HpaII tiny fragment island. The detection and mapping of the translocation breakpoints of all three retinoblastoma patients to within the putative RB1 gene substantiated the authenticity of this candidate sequence and demonstrated the utility of FIGE in detecting chromosomal rearrangements affecting this locus.     

Molecular-genetic analysis of two cases with retinoblastoma: Benefits for disease management

Effective counselling and management of retinoblastoma families using genetic information is presently practised in many parts of the world. We studied histopathological, chromosomal and molecular-genetic data of two retinoblastoma patients from India. The two patients, one with bilateral and the other with unilateral retinoblastoma, underwent complete ophthalmic examination, cytogenetic study, retinoblastoma gene (RB1) mutational analysis andRB1 promoter region methylation screening. In the bilateral retinoblastoma patient deletion of chromosome region 13q14 in peripheral blood lymphocytes and a hemizygous novel 8-bp deletion in exon 4 ofRB1 in tumour sample were observed. In the unilaterally affected patient CGA to TGA transition protein truncation mutations were observed in exons 8 and 14 ofRB1.     

How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein

The retinoblastoma tumor suppressor gene (RB1) is currently the only known gene whose mutation is necessary and sufficient for the development of a human cancer. Mutation or deregulation of RB1 is observed so frequently in other tumor types that compromising RB1 function may be a prerequisite for malignant transformation. Identifying the molecular mechanisms that provide the basis for RB1-mediated tumor suppression has become an important goal in the quest to understand and treat cancer. The lion's share of research on these mechanisms has focused on the carboxy-terminal half of the RB1 encoded protein (pRB). This focus is with good reason since this part of the protein, now called the "large pocket," is required for most of its known activities identified in vitro and in vivo. Large pocket mediated mechanisms alone, however, cannot account for all observed properties of pRB. The thesis presented here is that the relatively uncharacterized amino-terminal half of the protein makes important contributions to pRB-mediated tumor suppression. The goals of this review are to summarize evidence indicating that an amino-terminal structural domain is important for pRB function and to suggest a general hypothesis as to how this domain can be integrated with current models of pRB function.