Deletion of RB exons 24 and 25 causes low-penetrance retinoblastoma.

Eye Research Institute of Canada, Department of Ophthalmology, University of Toronto, Ontario. rbremner@playfair.utoronto.ca

A deletion in the tumor-suppressor gene, RB, discovered by quantitative multiplex PCR, shows low penetrance (LP), since only 39% of eyes at risk in this family develop retinoblastoma. The 4-kb deletion spanning exons 24 and 25 (delta24-25) is the largest ever observed in an LP retinoblastoma family. Unlike the usual RB mutations, which cause retinoblastoma in 95% of at-risk eyes and yield no detectable protein, the delta24-25 allele transcribed a message splicing exon 23 to exon 26, resulting in a detectable protein (pRBdelta24-25) that lacks 58 amino acids from the C-terminal domain, proving that this domain is essential for suppression of retinoblastoma. Two functions were partially impaired by delta24-25-nuclear localization and repression of E2F-consistent with the idea that LP mutations generate "weak alleles" by reducing but not eliminating essential activities. However, delta24-25 ablated interaction of pRB with MDM2. Since a homozygous LP allele is considered nontumorigenic, the pRB/MDM2 interaction may be semi- or nonessential for suppressing retinoblastoma. Alternatively, some homozygous LP alleles may not cause tumorigenesis because an additional event is required (the "three-hit hypothesis"), or the resulting imbalance in pRB function may cause apoptosis (the "death allele hypothesis"). pRBdelta24-25 was also completely defective in suppressing growth of Saos-2 osteosarcoma cells. Targeting pRBdelta24-25 to the nucleus did not improve Saos-2 growth suppression, suggesting that C-terminal domain functions other than nuclear localization are essential for blocking proliferation in these cells. Since delta24-25 behaves like a null allele in these cells but like an LP allele in the retina, pRB may use different mechanisms to control growth in different cell types.

PMID: 9326321 [PubMed - indexed for MEDLINE]

PMCID: PMC1715941

[Low-penetrance retinoblastoma due to exons 24 and 25 deletions in the Rb1 gene]

Genetics Service Center, Guangzhou General Hospital of PLA, Guangzhou, Guangdong, 510010 P. R. China. wadewm@public.guangzhou.edu.cn

OBJECTIVE: Identification of Rb1 mutations permits accurate genetic counseling. Characterization of the causative mutation in a large low penetrance family is likely to provide important information for tumorigenesis of retinoblastoma(RB). METHODS: Quantitative fluorescent multiplex PCR QFM-PCR technique was used for mutation detection. Long fragment PCR, reverse transcriptase-PCR, subcloning, direct sequencing and Western blotting techniques were used for characterizing the mutation. RESULTS: A deletion covering exons 24 and 25 of Rb gene was found in a large family with 122 members in four generations. Of the 18 carries in the family, only 11 were delivered to either unilateral or bilateral RB. The family has much low-penetrance retinoblastoma, compared with the usual, high-penetrance RB (95%). An extent of 4 kb fragment deletion was detected in genomic deletion of the mutation. cDNA and sequence data showed a 174 bp shorter than the wild type message RNA resulting in an in-frame loss of 58 residues. Further analysis demonstrated the truncated protein expression of 6000 Da shorter than wild type RB1 protein. CONCLUSION: QFM-PCR technique has enabled the investigators to identify a large deletion covering entire exons 24 and 25 of the Rb1 gene. It is the largest deletion ever found in low penetrance RB families. The characterizations of the mutation in genomic DNA, RNA and protein have provided new evidences which enhance credence to the idea that low penetrance retinoblastoma is caused by only partially functional disable of Rb1. The data will be useful in genetic counseling, particularly significant for the unaffected carriers in RB low penetrance families.

PMID: 12362308 [PubMed - indexed for MEDLINE]

Attenuation of disease phenotype through alternative translation initiation in low-penetrance retinoblastoma.

Departamento de Genética, Facultad de Biología, Universidad de Valencia, Burjassot, Valencia, Spain. Francisco.SSanchez@uclm.es

Hereditary predisposition to retinoblastoma (RB) is caused by germline mutations in the retinoblastoma 1 (RB1) gene and transmits as an autosomal dominant trait. In the majority of cases disease develops in greater than 90% of carriers. However, reduced penetrance with a large portion of disease-free carrier is seen in some families. Unambiguous identification of the predisposing mutation in these families is important for accurate risk prediction in relatives and their genetic counseling but also provides conceptual information regarding the relationship between the RB1 genotype and the disease phenotype. In this study we report a novel mutation detected in 10 individuals of an extended family, only three of whom are affected by RB disease. The mutation comprises a 23-basepair (bp) duplication in the first exon of RB1 (c.43_65dup) producing a frameshift in exon 1 and premature chain termination in exon 2. Mutations resulting in premature chain termination classically are associated with high penetrance disease, as message translation may not generate functional product and nonsense mediated RNA decay (NMD) frequently eliminates the mutant transcript. However, appreciable NMD does not follow from the mutation described here and transcript expression in tissue culture cells and translation in vitro reveals that alternative in-frame translation start sites involving Met113 and possibly Met233 are used to generate truncated RB1 products (pRB94 and pRB80), known and suspected to exhibit tumor suppressor activity. These results strongly suggest that modulation of disease penetrance in this family is achieved by internal translation initiation. Our observations provide the first example for rescue of a chain-terminating mutation in RB1 through alternative translation initiation. (c) 2006 Wiley-Liss, Inc.

PMID: 16988938 [PubMed - indexed for MEDLINE]

RB1 germ-line deletions in Argentine retinoblastoma patients.

Genetics and Molecular Biology Department, Faculty of Pharmacy, Jose de San Martin Hospital, Buenos Aires University, Buenos Aires, Argentina.

BACKGROUND: Retinoblastoma (RB) is a malignant tumor originating in the retinal cell precursors and can be presented as a unilateral or bilateral form in childhood (one or both eyes affected). Development of this tumor is caused by mutations in the RB1 gene on chromosome 13q14; the first mutation may occur in the germ line (hereditary RB) or in somatic cells (non-hereditary RB). The hereditary form of RB is transmitted with a high penetrance to offspring (90%). Because early diagnosis is necessary for implementing effective treatment and preserving vision, it is important to identify the mutations in the affected family. AIM: The aim of this study was to identify large and small RB1 germ-line mutations and to correlate them with the RB phenotype. METHODS: Constitutional RB1 gene gross deletions were studied in 40 patients with bilateral or unilateral familial RB by a segregation assay of four intragenic polymorphisms located in introns 1, 4, 17, and 20 of the RB1 gene, along with fluorescence in situ hibridization (FISH) analysis. Small mutations were ascertained in a subgroup of ten patients by heteroduplex/sequence analysis of RB1-exons. RESULTS: In the course of our study, we have found three large deletions, which probably represent whole gene deletions, and two small deletions of 1bp in length. One large deletion was found in a family with several members affected. This represents a rare case of familial RB, which is usually caused by small mutations. Phenotype analysis of the family revealed a low penetrance inheritance, with an 'affected eyes : number of mutation-carriers' ratio of approximately 1.0, whereas this ratio in families with small loss-of-function mutations is 1.5-2.0. CONCLUSIONS: Our results emphasize the usefulness of a combined methodology that includes segregation of polymorphisms, FISH, and heteroduplex/sequence analyses for detection of gross and small DNA rearrangements in familial and sporadic RB. Identification of mutations in sporadic cases is important for risk-assessment in patients' relatives. The degree of penetrance in the inheritance of RB not only depends on the occurrence of the second mutation in the RB1 gene but also on the extent of inactivation of the first mutation.

PMID: 17286450 [PubMed - indexed for MEDLINE]

RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database.

Servicio de Informática, Centro Nacional de Biotecnología, CSIC, Campus de Cantoblanco, Madrid, Spain. jrvalverde@cnb.uam.es

BACKGROUND: Retinoblastoma, a prototype of hereditary cancer, is the most common intraocular tumour in children and potential cause of blindness from therapeutic eye ablation, second tumours in germ line carrier's survivors, and even death when left untreated. The molecular scanning of RB1 in search of germ line mutations lead to the publication of more than 900 mutations whose knowledge is important for genetic counselling and the characterization of phenotypic-genotypic relationships. RESULTS: A searchable database (RBGMdb) has been constructed with 932 published RB1 mutations. The spectrum of these mutations has been analyzed with the following results: 1) the retinoblastoma protein is frequently inactivated by deletions and nonsense mutations while missense mutations are the main inactivating event in most genetic diseases. 2) Near 40% of RB1 gene mutations are recurrent and gather in sixteen hot points, including twelve nonsense, two missense and three splicing mutations. The remainder mutations are scattered along RB1, being most frequent in exons 9, 10, 14, 17, 18, 20, and 23. 3) The analysis of RB1 mutations by country of origin of the patients identifies two groups in which the incidence of nonsense and splicing mutations show differences extremely significant, and suggest the involvement of predisposing ethnic backgrounds. 4) A significant association between late age at diagnosis and splicing mutations in bilateral retinoblastoma patients suggests the occurrence of a delayed-onset genotype. 5) Most of the reported mutations in low-penetrance families fall in three groups: a) Mutations in regulatory sequences at the promoter resulting in low expression of a normal Rb; b) Missense and in-frame deletions affecting non-essential sequence motifs which result in a partial inactivation of Rb functions; c) Splicing mutations leading to the reduction of normal mRNA splicing or to alternative splicing involving either true oncogenic or defective (weak) alleles. CONCLUSION: The analysis of RB1 gene mutations logged in the RBGMdb has shown relevant phenotype-genotype relationships and provided working hypothesis to ascertain mechanisms linking certain mutations to ethnicity, delayed onset of the disease and low-penetrance. Gene profiling of tumors will help to clarify the genetic background linked to ethnicity and variable expressivity or delayed onset phenotypes.

PMID: 16269091 [PubMed - indexed for MEDLINE]

PMCID: PMC1298292

Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function.

Genetics Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD 20889, USA.

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein "pocket" binding activity, and do not suppress colony growth of RB(-) cells. In contrast, two low-penetrant alleles (661W and "deletion of codon 480") retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, "deletion of RB exon 4," showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(-) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

PMID: 9342358 [PubMed - indexed for MEDLINE]

PMCID: PMC23695

Growth suppression by an E2F-binding-defective retinoblastoma protein (RB): contribution from the RB C pocket.

Department of Biology, Center for Molecular Genetics, and Cancer Center, University of California, San Diego, La Jolla, California 92093-0322, USA.

Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G1/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB's ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast, long-term growth arrest requires combined activities from both regions of RB. In addition, E2F and LXCXE binding are not the only mechanisms through which RB inhibits cell growth. The C pocket also contributes to RB-mediated growth suppression.

PMID: 9632788 [PubMed - indexed for MEDLINE]

PMCID: PMC108988

Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype.

Imperial Cancer Research Fund Oncology Group, Institute of Child Health, London.

The retinoblastoma-predisposition gene, RB1, segregates as an autosomal dominant trait with high (90%) penetrance. Certain families, however, show an unusual low-penetrance phenotype with many individuals being unaffected, unilaterally affected, or with evidence of spontaneously regressed tumors. We have used single-strand conformation polymorphism analysis and PCR sequencing to study two such families. Mutations were found in exon 20 of RB1 in both cases. In one family a C----T transition in codon 661 converts an arginine (CGG) to a tryptophan (TGG) codon. In this family, incomplete penetrance and mild phenotypic expression were observed in virtually all patients, possibly indicating that single amino acid changes may modify protein structure/function such that tumorigenesis is not inevitable. In the second family the mutation in codon 675 is a G----T transversion that converts a glutamine (GAA) to a stop (TAA) codon. However, this mutation also occurs near a potential cryptic splice acceptor site, raising the possibility of alternative splicing resulting in a less severely disrupted protein.

PMID: 1352883 [PubMed - indexed for MEDLINE]

PMCID: PMC402145

The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms.

Scripps Research Institute, Department of Molecular and Experimental Medicine, La Jolla, CA 92037, USA. plee@scripps.edu

Nramp2 is a gene encoding a transmembrane protein that is important in metal transport, in particular iron. Mutations in nramp2 have been shown to be associated with microcytic anemia in mk/mk mice and defective iron transport in Belgrade rats. Nramp2 contains a classical iron responsive element in the 3' untranslated region that confers iron dependent mRNA stabilization. In this report, we describe a splice variant form of human nramp2 that has the carboxyl terminal 18 amino acids substituted with 25 novel amino acids and has a new 3' untranslated region lacking a classical iron-responsive element. This splice form of nramp2, nramp2 non-IRE, was found to be derived from splicing of an additional exon into the terminal coding exon. The nramp2 gene is comprised of 17 exons and spans more than 36 kb. It contains an additional 5' exon and intron (exon and intron 1) and an additional 3' exon (exon 17) and intron (intron 16) as compared to nramp1, a homologous gene. The additional exons and introns account for much of the difference in length between nramp2 (> 36 kb) and nramp1 (12 kb). The exon-intron borders of nramp2 exons 3-15 are homologous to nramp1 exons 2-14. The nramp2 5' regulatory region contains two CCAAT boxes but lacks a TATA box. The 5' regulatory region of nramp2 also contains five potential metal response elements (MRE's) that are similar to the MRE's found in the metallothionein-IIA gene, three potential SP1 binding sites and a single gamma-interferon regulatory element. Five single nucleotide mutations or polymorphisms were identified within the nramp2 gene. One of these, 1303C-->A, occurs in the coding region of nramp2 and results in an amino acid change from leucine to isolecine. A polymorphism, 1254T/C, also occurs in the coding region of nramp2 but does not cause an amino acid change. The other 3 polymorphisms are within introns (IVS2 + 11A/G, IVS4 + 44C/A, and IVS6 + 538G/Gdel). In addition, a polymorphic microsatellite TATATCTATATATC (TA)6-7 (CA)10-11 CCCCCTATA (TATC)3 (TCTG)5 TCCG (TCTA)6 was identified in intron 3. Analysis of cDNA derived by direct amplification of reversed transcribed RNA or cDNA clones isolated from a library provide evidence of skipping of exons 10 and 12 of nramp2. Deletion of either of these exons would result in a sequence that remains in frame yet would generate a protein that would lack transmembrane spanning region 7 or 8 respectively. The deletion of a single transmembrane domain would have severe topological consequences. The coding region of the nramp2 gene of hemochromatosis patients with or without mutations in the hemochromatosis gene, HFE, were examined and found to be normal. One hemochromatosis patient, with a normal HFE genotype, was heterozygous for the 1303C-->A mutation. Furthermore, in an examination of hemochromatosis patients with mutant HFE and normal HFE genes, we did not observe a linkage disequilibrium of either group with a particular nramp2 haplotype. These data suggest that mutations in nramp2 are not commonly associated with hemochromatosis.

PMID: 9642100 [PubMed - indexed for MEDLINE]

Sensitive multistep clinical molecular screening of 180 unrelated individuals with retinoblastoma detects 36 novel mutations in the RB1 gene.

Division of Pediatric Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Retinoblastoma (RB) is a neoplasm of retinal origin caused by mutations in RB1, the retinoblastoma tumor suppressor gene. To facilitate genetics counseling and patient management, we adopted a multistep molecular screening assay for detecting RB1 mutations. This assay included DNA sequencing to identify mutations within coding exons and immediate flanking intronic regions, Southern blot analysis to characterize genomic rearrangements, and transcript analysis to characterize potential splicing mutations buried within introns. In a pilot investigation of 180 patients from North America, we identified germline RB1 mutations in 77 out of 85 bilateral RB patients (91%), 7 out of 10 familial unilateral (70%), and 6 out of 85 unilateral patients with no family history of RB (7%). Mutations included 36 novel alterations spanning the entire RB1 gene. Seven of these novel changes were missense or silent mutations. Sequence analysis predicted that, in five out of seven cases, the changes can cause aberrant splicing. This was confirmed by transcript analysis in four out of five cases. In addition, four intronic point mutations within nonconsensus sites activated cryptic splice sites. Without the transcript analysis, the significance of these 11 mutations would have remained undefined. In a separate investigation of a subset of unilateral RB tumors, we identified somatic biallelic RB1 gene inactivation in 34 out of 56 cases (61%) cases. In 14 tumors, only one of the two RB1 mutations could be detected, and in eight tumors, no mutations were detected. The absence of detectable RB1 mutations in eight bilateral cases and eight unilateral tumors suggests that alternative genetic mechanisms may underlie the development of RB in certain individuals.

PMID: 15884040 [PubMed - indexed for MEDLINE]

[Distinct Rb gene point mutations in families showing low penetrance of hereditary retinoblastoma]

Department of Ophthalmology, The First Affiliated Hospital, West China University of Medical Sciences, Chengdu, Sichuan Province, 610041 P.R. China.

OBJECTIVE: To investigate the possible cause and molecular mechanism of low penetrance in hereditary retinoblastoma kindred. METHODS: The DNAs from tumor or blood of affected and unaffected individuals in hereditary retinoblastoma families showing low penetrance were screened by SSCP analysis and further characterized by direct DNA sequencing. RESULTS: Eight from fifteen families showing low penetrance retinoblastoma were identified to have distinct Rb gene point mutations including Arg661-Trp661 in five families, aberrant splicing in two families and a G-T mutation at ATF binding site of Rb gene promoter in one family. CONCLUSION: The distribution of cases with low penetrance of retinoblastoma is not completely random. The low penetrance in the families described here was associated with several distinct Rb gene point mutations which did not result in complete disruption of the gene product,and the reduced penetrance of retinoblastoma is probably the result of a residual function of these alleles in retinoblastoma precursor cells.

PMID: 9621119 [PubMed - indexed for MEDLINE]

Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma.

Institut für Humangenetik, Universitätsklinikum Essen, Germany.

The interfamilial diversity in penetrance and expressivity of hereditary retinoblastoma was investigated in 29 families. By using a simple parameter for estimating the severity of the disease (diseased-eye-ratio), we were able to identify four families with a discrete low-penetrance phenotype. The underlying genetic defect was identified in three families. One family has a 3-bp deletion in exon 16 that results in the deletion of Asn480. In two further unrelated families, the identical missense mutation at codon 661 in exon 20 (CGG to TGG, Arg to Trp) was identified. These mutations are distinct from the majority of retinoblastoma gene alterations, as they do not result in the disruption of the gene product. We propose that reduced penetrance of retinoblastoma is the result of a residual function of these alleles in retinoblastoma precursor cells.

PMID: 7927327 [PubMed - indexed for MEDLINE]

The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated suppression of E2F and cell growth.

Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.

Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity.

PMID: 15485814 [PubMed - indexed for MEDLINE]

Spectrum of germline RB1 gene mutations in Spanish retinoblastoma patients: Phenotypic and molecular epidemiological implications.

OncoLab, Unidad de Biología Molecular y Celular del Cáncer, Instituto de Investigaciones Biomédicas "Alberto Sols," CSIC-UAM, Madrid, Spain.

Mutation analysis of retinoblastoma is considered important for genetic counseling purposes, as well as for understanding the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of an analysis of 43 hereditary retinoblastoma Spanish patients and kindred, using direct PCR sequencing, we have observed 29 mutations; most of them (62%) have not been reported previously. Of the mutations, 69% correspond to nonsense mutations (mainly CpG transitions) and frameshifts, with the expected outcome of a truncated Rb protein that lacks the functional pocket domains and tail. The remainder corresponds to splicing mutations, most of them (62%) targeted to invariant nucleotides, with the predicted consequence of out of frame exon skipping. Two of the splicing mutations in our study were found associated to families with a low-penetrance phenotype. Additionally, most of the mutations affecting splice junctions corresponded to retinoblastoma cases of either sporadic or hereditary nature with delayed onset (32 months on average). In contrast, most of the nonsense and frameshift mutations are associated with an early age at diagnosis (8.7 months on average). These differences are discussed in the context of the relationships between genotype and low expressivity phenotype. The differences in the spectrum of RB1 mutations found in this and other European surveys are also discussed in the context of alternate DNA methylation and mismatch repair phenotypes. Copyright 2001 Wiley-Liss, Inc.

PMID: 11317357 [PubMed - indexed for MEDLINE]

Structure of the human retinoblastoma gene.

Department of Pathology, University of California at San Diego, La Jolla 92093.

Complete inactivation of the human retinoblastoma gene (RB) is believed to be an essential step in tumorigenesis of several different cancers. To provide a framework for understanding inactivation mechanisms, the structure of RB was delineated. The RB transcript is encoded in 27 exons dispersed over about 200 kilobases (kb) of genomic DNA. The length of individual exons ranges from 31 to 1889 base pairs (bp). The largest intron spans greater than 60 kb and the smallest one has only 80 bp. Deletion of exons 13-17 is frequently observed in various types of tumors, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential "hot spot" for recombination in the region is predicted. A putative "leucine-zipper" motif is exclusively encoded by exon 20. The detailed RB structure presented here should prove useful in defining potential functional domains of its encoded protein. Transcription of RB is initiated at multiple positions and the sequences surrounding the initiation sites have a high G + C content. A typical upstream TATA box is not present. Localization of the RB promoter region was accomplished by utilizing a heterologous expression system containing a bacterial chloramphenicol acetyltransferase gene. Deletion analysis revealed that a region as small as 70 bp is sufficient for RB promoter activity, similar to other previously characterized G + C-rich gene promoters. Several direct repeats and possible stem-and-loop structures are found in the promoter region. No enhancer element was detected within the 7.3 kb of upstream sequence studied. Several features of the RB promoter are reminiscent of the characteristics associated with many "housekeeping" genes, consistent with its ubiquitous expression pattern.

PMID: 2748600 [PubMed - indexed for MEDLINE]

PMCID: PMC297651

A possible hot spot in exon 21 of the retinoblastoma gene predisposing to a low penetrant retinoblastoma phenotype?

Research Division, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

PURPOSE: To identify the mutation in the RB1 gene in a Syrian family showing incomplete penetrance of retinoblastoma (RB). METHODS: Genomic DNA was used as a template for the PCR reaction to amplify all exons as well as the promoter region of RB1 gene. These PCR products were screened by conformational sensitive gel electrophoresis and the 331-bp product containing exon 21 showing anomalous migration was sequenced directly to identify the mutation. RESULTS: We identified the missense point mutation in exon 21 of the RB1 gene converting a Cys-->Arg (codon 712) in one family with a low penetrant phenotype. The proband was unilaterally affected, whereas the paternal uncle was bilaterally affected and the mutation carrier father was unaffected. The T-->C substitution abolished a cleavage site for the Nde I restriction enzyme, enabling rapid detection of the mutant allele. CONCLUSION: Phenotypically, this family is different from the previously described low penetrant phenotype pedigree with the same mutation whose affected members all had unilateral tumors. These results suggest that codon 712 may represent a mutational 'hot spot' for the low penetrant phenotypes and that the mutation codes for retinoblastoma protein with an apparently residual tumor-suppressive function give rise to low penetrance. These results also raise the interesting question: what other factors influence the phenotype of mutation carriers in addition to the predisposing missense mutation.

PMID: 10617920 [PubMed - indexed for MEDLINE]

A bipartite nuclear localization signal in the retinoblastoma gene product and its importance for biological activity.

Division of Immunology and Cancer Research, Hospital for Sick Children, Toronto, Ontario, Canada.

The retinoblastoma gene product, p110RB1, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110RB1 into the nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110RB1, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS [to give mutant NLS(NQ)] or deletion of the entire NLS (delta NLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein [NLS(NQ)/delta 22] containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110RB1 with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli beta-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor delta NLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110RB1 and may be responsible for some retinoblastoma and other tumors.

PMID: 8336704 [PubMed - indexed for MEDLINE]

PMCID: PMC360081

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients.

Oncogenetic Laboratory (OncoLab), Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, 28029 Madrid, Spain.

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature. c) 2004 Wiley-Liss, Inc.

PMID: 15605413 [PubMed - indexed for MEDLINE]

Temperature-sensitive RB mutations linked to incomplete penetrance of familial retinoblastoma in 12 families.

Genetics Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD, USA.

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.

PMID: 10486322 [PubMed - indexed for MEDLINE]

PMCID: PMC1288236

Molecular basis of low-penetrance retinoblastoma.

Washington University, Campus Box 8069, 660 S Euclid Ave, St Louis, MO 63110, USA. harbour@vision.wustl.edu

Retinoblastoma is a malignant tumor of the retina that occurs primarily in young children as a result of mutations in the retinoblastoma gene (RB), the first tumor suppressor gene to be identified. In about 35% to 40% of patients with retinoblastoma, an RB gene mutation is present in the germline, resulting in hereditary transmission of the disease. Most families with hereditary retinoblastoma demonstrate autosomal dominant inheritance with almost complete penetrance and high expressivity. However, some families display an inheritance pattern characterized by reduced penetrance and expressivity. Recent advances in our understanding of the structure and function of the retinoblastoma protein (pRB) now provide new insights into the molecular basis of this low-penetrance form of retinoblastoma. Low-penetrance retinoblastoma mutations either cause a reduction in the amount of normal pRB that is produced (class 1 mutations) or result in a partially functional mutant pRB (class 2 mutations).

PMID: 11709023 [PubMed - indexed for MEDLINE]