RB1 gene mutation up-date, a meta-analysis based on 932 reported mutations available in a searchable database
© Valverde et al; licensee BioMed Central Ltd. 2005
Received: 25 April 2005
Accepted: 04 November 2005
Published: 04 November 2005
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.
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.
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.
Retinoblastoma (MIM# 180200), a rare embryonic neoplasm of retinal origin, is the most common intraocular tumor in children, with a relative incidence of 3% of all pediatric tumors. Although current therapeutic strategies have led to dramatic improvement of individual prognosis, retinoblastoma is still life-threatening when leaved untreated or in cases of late diagnosis, a condition of concern in developing countries . The frequency estimates of retinoblastoma in different populations range between 1:34.000 and 1:10.000 live-born, with the most reliable figures between 1:28.000 and 1:15.000. An increasing incidence observed in recent studies can result from more complete ascertainment and also from population-genetic reasons, due to the increased survival of retinoblastoma patients . Most of the clinical phenotypes can be explained by the double mutational inactivation of the retinoblastoma susceptibility gene , the prototype tumor suppressor gene that controls cell cycle progression . However, additional mutations in apoptosis signaling may well be involved in tumor development , a hypothesis that has been in the cell-of-origin studies in mice . In addition, a detailed analysis of the relations between genotype and phenotypic expression suggest that the hereditary retinoblastoma has features of a complex trait . In the hereditary form of the disease, a germ line mutation is transmitted as a high penetrance (90%) autonomic dominant trait, resulting in a 45% risk in offspring of patients with hereditary retinoblastoma; the second inactivating mutation occurs in retinal cell precursors . Most of these patients have bilateral retinoblastoma and a mean age at diagnosis of 12 months. In the non-hereditary form of the disease, both inactivating events occur during somatic development of retinal cells and result in the relatively late onset of a single tumor in one eye . However, nearly 15% of the unilaterally affected patients have germ line RB1 mutations, representing a 45% risk for their offspring, and these patients cannot be clinically distinguished from patients with true somatic unilateral retinoblastoma, who present a negligible risk for siblings and offspring. Taking these situations together, the hereditary form represents nearly 50% of all the retinoblastoma patients, according to recent epidemiological figures . The presence of RB1 germ line mutations confers an increased risk for development of second primary tumors in the survivors of hereditary retinoblastoma, with a cumulative incidence of 22% at the age of 25 years. Most of these second tumors were osteosarcomas (37.0%), other sarcomas (16.8%) and melanomas (7.4%), while brain tumors (4.5%), leukemia (2.4%) and non-Hodkin lymphomas (1.6%) were less frequent . In addition, hereditary retinoblastoma survivors have a lifetime risk of developing common epithelial cancers .
The development of sensitive and reliable genetic tests to detect RB1 mutations has greatly improved the identification of carriers, facilitating accurate genetic counseling. In addition, the detection of children at risk among siblings would obviate the need for many routine examinations and potentially decrease the economic impact of the disease . Attempts by several groups to define the mutations resulting in the inactivation of the RB1 gene in retinoblastoma have led to the identification of a broad spectrum of mutations. To date, the most comprehensive report of RB1 mutations corresponds to Lohman's database  which contains 228 different mutations and 130 recurrences. In this article we describe an up-dated, searchable database containing 500 distinct somatic or germ line RB1 mutations and more than 400 recurrences that we have retrieved from publications. In addition, we analyze the spectrum of RB1 mutations, with emphasis in molecular epidemiology and phenotype-genotype relationships. This information is important for the development of rapid procedures to detect mutations in patients and also to understanding the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity.
Results and discussion
Distribution of germ line and somatic RB1 mutations by phenotype of the patients.
Type of mutation
Number of entries by mutation type as compared with HGMD data. Statistical analysis of distinct RBGMdbDB vs. HGMD using χ2 test.
P < 0.0001
P < 0.0001
P < 0.0001
P = 0.6267
P < 0.0001
Hot spots of RB1 gene mutations
In this sense it is worth to mention the different fate of three CGG-codons in RB1 gene: two of unknown methylated status in exon 20, comprising the highly recurrent R661W (20 mutations as shown in Additional File 2) and R656W (1 mutation); the third, in exon 8 (R262) is frequently methylated although no mutations have been so far observed. In these cases, the differences in mutability cannot be explained by the methylation status not by differences in tumorogenity (oncogenity), since both R656W and R661W lie in the folded and hydrogen-bond rich structural domain of the A-B interface, together with other missense mutations . Alternatively, the hyper mutability found in R661 can be explained by neighboring-nucleotide effects (T CGG in R661 versus A CGG in R656 and R 262) and the known differences in mutability of these tetranucleotides . In the case of other non-CpG hot spots in RB1 gene, such as the nonsense E137X and the three splicing mutations affecting the first invariant nucleotide in introns 6, 12 and 19, the presence of short quasi-repeat sequences could be documented (see Additional file 2). These sequence motifs would favor replication errors such as misinsertions or misalignments leading to base substitution or single-base frameshift with the mismatch repair machinery . Although this DNA environment offers an attractive explanation for the presence of hot spots in non CpG sites, no preponderance of direct or inverted repeats has been observed in the spectrum of single-base-pair substitutions logged in the HGMD . However, the HGDM only includes distinct mutations and therefore an association of repeated sequence motifs with recurrent mutations cannot be excluded.
Mutational spectrum of RB1 by exon
Spectrum of RB1 gene mutations by country of origin
Age and type of mutations
Age at diagnosis in retinoblastoma patients according to phenotype and type of mutation. Statistical analysis using the Welch's unpaired t test.
SP vs. NS-MS-FS
P = 0.7237
P = 0.0025
P = 0.0044
Unilateral vs. Bilateral
P = 0.0073
P = 0.0147
P = 0.3321
Low penetrance retinoblastoma
In 27 out of the 133 (20%) familial retinoblastoma entries in RBGMdb, the presence of unaffected carriers (reduced penetrance) or unilateral retinoblastoma or benign retinoma (reduced expressivity) were documented (see Additional file 4 for a complete description of the mutations and references). This figure probably represents an overestimation of the true incidence of the low penetrance (LP) phenotype, biased by its notorious scientific interest. As shown in Table 3, most of the reported mutations associated to LP families (23/28 = 82%) correspond to MS and SP mutations, with hot spots in g.156713 C>T (R661W), g.160757T>C (C712R) and g.45867G>T (IVS6+1G>T). Different mechanisms have been proposed to explain this rare phenotype, including epigenetic events, delayed mutation, involvement of a second retinoblastoma locus ("three-hit hypothesis") or host resistant factors. However, most low-penetrance retinoblastoma can be explained by mutations at the RB1 locus . Mutations affecting regulatory sequences in the RB1 promoter are known to reduce the expression of normal Rb protein below a threshold level, necessary for tumor suppression functions [29, 30]. Missense single-nucleotide substitutions can, under certain circumstances, partially inactivate the retinoblastoma function. In the case of the highly recurrent R661W mutant allele, Otterson et al. have shown that the mutant retinoblastoma protein has a temperature-sensitive pocket activity whose reversible fluctuations may result in the low penetrance phenotype. Temperature-sensitive Rb pocket activity may also explain the low penetrance of C712R and delN480 . In the case of the large in frame deletions outside the pocket domain, such as Del: E04  and Del: E24–25 , unessential functions of Rb protein seems to be affected.
After the report of a splicing mutation (c.2211G>A) affecting the last nucleotide of exon 21 , a new category of low-penetrance genotype has been proposed to occur through alternative splicing mechanisms. In the case reported by these authors, the RNA analysis showed skipping of exon 21 and a low amount (10%) of normally spliced RNA (E732E) which can explain the variable expressivity observed in that family. A similar mechanism can explain the low-penetrance of other splicing mutations affecting the last exonic nucleotide, such as c.1331A>G and c.1960G>C. In these cases, the splicing machinery could alternate between defective missense splicing (Q443P and V654L, respectively) and inactivating skipping of exons 13 or 21, both in the pocket box domain of Rb. In the low-penetrance family studied by Lefevre et al., a T>C substitution in the polypyrimidine tract of intron 8 (IVS9-10T>C) was shown to be at the origin of the in frame deletion of exon 9 giving a defective or inactive Rb protein lacking 26 amino acids from the N-terminal region. It is also possible that this mutation only partially affected spliceosome assembly and that the mutated allele could be in part correctly spliced. While this alternative splicing mechanism would better explain the low-penetrance phenotype, no supporting evidence is available . Similar alternative mechanism could also explain the low-penetrance of c.2325+5G>A, causing in frame skipping of exon 22 . In this case, the G>C transversion makes a slight reduction of the consensus value of the 5' splice site  from 88 to 75 compatible with the presence of a fraction of correctly spliced mutant allele.
Alternative splicing mechanisms might also be involved in the recurrent c.607+1G>T transversion, firstly described in a low-expressivity and delayed onset phenotype in one Spanish family , and thoroughly analyzed by Klutz et al  in two low-penetrance German pedigrees. These authors made the interesting observation of a posttranscriptional mechanism that reduces the level of the mutant transcripts (skipping of exon 6 giving a stop codon in exon 7) when the mutant allele was received from the father. However this parent-of-origin effect does not explain the low-penetrance phenotype, since all but one unaffected carriers have maternally inherited mutant alleles and high level of expression, while most of the tumor bearing carriers have paternally inherited mutant alleles and low level of expression of the mutant allele. In a search for alternative splicing mechanisms, using the electronic exon-search facilities at HGMP, we have observed the presence of a cryptic exon whose usage in mutant alleles (see Additional File 5) could result in a defective Rb protein lacking 12 amino acids in the amino terminal region. Since no evidence of in-frame restoring mechanisms in lymphoblastoid cell line derived from unaffected carriers was observed , the hypothetical alternative splicing mechanism should be explored in retinoblastoma derived cell lines. In the only nonsense substitution (Q675X) observed in a low-penetrance phenotype, the G>T transversion in c.2023 could also activate a cryptic splicing site involving the stop codon, with the result of a defective Rb lacking 22 amino acids .
Molecular basis of low penetrance retinoblastoma
Type of mutation
Number of LP families
Description of mutations and frequency a
Low expression of normal Rb protein
MS point mutations
Partial inactivation of Rb protein
Partial inactivation of Rb protein
539+1delG, del E05 (1/1)
2325+5G>A, del E:22 (1/1)
Alternative splicing and/or unessential exon skipping resulting in low expression or partial inactivation of Rb protein
NS point mutation
Alternative splicing involving the stop codon
Materials and methods
Primary bibliographic resources were retrieved from Entrez-PubMed, searching for human retinoblastoma (RB1) mutations. Reprints of all these articles were obtained and additional articles describing RB1 mutations were picked up from its reference lists. In addition, all the articles from the same authors or research group were thoroughly scrutinized in order to avoid repetitions of mutations present in one patient. In all, data from 68 research articles have been compiled in the data base. These articles, together with the number of mutation they contributed and the country of origin of the main research group are supplied in the Additional file 1.
Description of the flat-file format
All the mutations were thoroughly revised according the recommended nomenclature for sequence variations  using the genomic sequence GenBank: L11910.1, the cDNA sequence NCBI: NM_000321.1, and the protein sequence NCBI: NP_000312.1. The mutations were annotated in a Microsoft Excel data sheet containing 15 columns (Shown in Additional File 6).
Structure and management of the database
The public interface of RBGMdb is located at EMBnet Spanish node . As we have already mentioned, it provides resources for issuing simple queries for records containing a search term, as well as for complex queries where up to four search terms may be looked up on user-specified fields each combined with its logical operator. Searches produce their results as an HTML table with online help that is displayed whenever the cursor moves over any field. Results may be further sorted by any field by just clicking on the field name in the table header. The user is offered the possibility of generating PDF reports for download and/or printing at every step of the process. In addition to the search forms, we have created a submission form to facilitate the public addition of new data. This form sends an e-mail to the database coordinator who effectively acts as supervisor for all new additions. Restricted access database update forms are also available for the database coordinator(s) to actually modify the data in the database.
Computer analysis and statistics
When indicated, normal and mutated RB1 sequences were analyzed with the programs for exon identification available at the Computing Services of UK HUMAN GENOME MAPPING PROJECT . Statistical significance tests used in this study included Chi square and Fisher exact test for 2 × 2 tables, and the Welch's unpaired t test and nonparametric Mann-Whitney test for the analysis of means. Statistical summaries from the Human Gene Mutation Database (HGMD) were retrieved from the web.
List of abbreviations used
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