Molecular Vision 2007; 13:1740-1745 <>
Received 14 December 2006 | Accepted 17 August 2007 | Published 19 September 2007

Ten novel RB1 gene mutations in patients with retinoblastoma

Hana Abouzeid,1 Francis L. Munier,1,2 Francine Thonney,3 Daniel F. Schorderet2,4,5

1Jules-Gonin Eye Hospital, Lausanne, Switzerland, 2Department of Ophthalmology, University of Lausanne, Lausanne, 3Service of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, 4IRO-Institut de Recherche en Ophtalmologie, Sion, 5EPFL-Ecole polytechnique fédérale de Lausanne, Lausanne, Switzerland

Correspondence to: Francis Munier, Hôpital Ophtalmique Jules-Gonin, 15, Av de France, 1004 Lausanne, Switzerland; Phone: +41 21 626 81 11; FAX: +41 21 626 88 88; email:


Purpose: To study phenotype-genotype correlations in 65 retinoblastoma patients, who were seen between March 2004 and January 2006 and to report undescribed retinoblastoma 1 (RB1) mutations identified in ten additional patients in whom mutations were detected before 2004.

Methods: Complete ophthalmic examinations were performed in all patients and their parents. DNA was extracted from peripheral blood leukocytes, and the RB1 gene was screened by denaturing high-performance liquid chromatography and direct sequencing of the promoter and all the exons.

Results: Seven cases were familial, 30 were sporadic bilateral, and 28 were sporadic unilateral. Screening of the RB1 gene resulted in the identification of four mutations in the familial cases (57%), 22 in the sporadic bilateral cases (73%), and three in the sporadic unilateral cases (10.7%). Twenty-two mutations, were single-base substitutions (76%). Of these mutations, 68% were of the nonsense type (15 cases). Ten patients with bilateral retinoblastoma in whom ten mutations were detected in a non-systematic approach between 1995 and 1998 were added to our recent series. In total, ten novel mutations were identified, including four single base substitutions, four small deletions and two small duplications. These are g.39445G>A, g.41924A>G, g.56851A>G, g.156795T>G, g.41983delT, g.44699_44706delAGCAGTTC, g.73788_73789delAA, g.78253delA, g.2157dupC, and g.2179_2183dupGGACC. Two patients had dysmorphic features associated with 13q14 large deletions.

Conclusions: The detection rates of 73% in the sporadic bilateral cases and of 10.7% in the sporadic unilateral cases in our series are in accordance with recently published literature. Our pattern of mutations confirms the predominantly gene-inactivating mutations, i.e. single-base non-sense mutations and splice site mutations.


RB (OMIM 180200) is the most frequent intraocular tumor of childhood and is caused by mutations in the RB1 gene. The predisposition to develop RB is inherited as an autosomal dominant trait but mutations in both alleles are necessary to cause the disease [1]. Mutations of RB1 gene are highly heterogeneous and spread in promoter and exons 1-25 [2-6]. Previous reports described a wide range of detection rate, from 20 to 80%, according to the case selection and the screening technique [2,3,6]. While more than 900 RB1 mutations (see Retinoblastoma Genetics and [7]) the rate of mutation detection remains relatively low. This is attributable to the large size of the RB1 gene, to the significant mutational heterogeneity of the disease, and to limitations of currently available screening techniques. Furthermore, unusual mutation location can also impede on mutation detection. Single base substitutions represent the most frequent mutations and among them, nonsense mutations predominate [3,4,6]. Studies on genotype-phenotype correlation concluded there was an association between nonsense or frameshift mutations and severity of the disease defined as bilateral multifocal RB [3-5]. Meanwhile, variable expression of RB is well-known with description of unaffected carriers, unilateral RB or benign retinoma [8,9]. Thus, low-penetrant phenotype was associated with the p.R661W mutation of the RB1 gene [10], with 4-kb deletion spanning exons 24 and 25 [11], alternative splicing mutation in exon 21 [12], p. L662P [13], promoter mutations [14], in-frame deletions affecting the N-terminal region of pRB [15], or alternative translation initiation associated with nonsense mutations in exon 1 [16] among other molecular changes. Genetic modifying factors or residual protein function due to either missense mutation or alternative translation initiation may influence phenotype expression, particularly in low-penetrant RB.

Studies of patients from different parts of the world can help explain the spectrum of RB1 mutations and thus improve detection rate. Risk prediction is mandatory for current RB management [5,17] and justifies the continuous search for RB1 novel mutations and for phenotypic correlations. To characterize the spectrum of RB1 mutations and to analyze genotype-phenotype correlation, we performed a phenotype and mutation analysis of 65 patients with isolated or familial RB who underwent treatment in our institution. Ten patients with sporadic and familial bilateral RB in whom novel mutations were detected during a non-systematic mutation screening program occurring between 1995 and 1998 are reported separately.


Between March 2004 and January 2006, we performed a mutational screening of the RB1 gene in 65 consecutive probands with RB. Ten additional patients in whom mutations were detected before 2004 were included in the study.

Patients were referred to us either from university eye clinics and private Swiss practitioners or from neighboring European countries. All patients were examined and treated at the Retinoblastoma Clinic of the Jules-Gonin Eye Hospital, Lausanne, Switzerland. All patients underwent physical examination at our institution with special attention for dysmorphic features. Age ranged from 2 months to 12 years. Informed consent was obtained from all parents to draw blood and perform genetic analysis. Control DNA were obtained from 96 ethnically matched anonymous blood donors after informed consent. They were all above 18 years of age, had a history of good health but were not investigated by us. The study was conducted in accordance with the tenets of the Declaration of Helsinki. Specific clinical features such as tumor laterality, type (endophytic or exophytic), number of tumor foci, and systemic dysmorphic features were obtained from charts and photographs of the patients. Genetic analysis was performed at the Institute for Research in Ophthalmology (IRO) at Sion, Switzerland, between March 2004 and January 2006 for the 65 probands, and at the Service of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, between 1995 and 1998 for the ten added patients. DNA was extracted from blood leukocytes and used for PCR amplification. Denaturing high-performance liquid chromatography (DHPLC) and sequencing were used. Primers, and PCR reactions were described by Houdayer et al. (2004). In short, amplification was performed in a thermal cycler (GeneAmp 9700, Applied Biosystems, Foster City, CA), in a total volume of 30 μl. Each polymerase chain reaction (PCR) contained 100 ng genomic DNA, 0.9 nanomoles of each primer, and 15 μl master mix 2X (Qiagen, Hombrechtikon, Switzerland), with or without betaine. Reactions were subjected to 35 cycles of 94 °C for 1 m, annealing at the specific temperature for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 10 min (Table 1).

After PCR amplification, products were screened for mutations using DHPLC on a WAVE system (TEAA, Transgenomics, Crewe, Cheshire, UK). Buffer A contained 0.1 M triethylammonium acetate (TEAA, Transgenomics). Buffer B contained 0.1 M TEAA (Transgenomic) and 25% acetonitrile HPLC grade (Sigma-Aldrich, Suffolk, UK). The flow rate was set at 1.5 ml/min and the Buffer B gradient increased by 5% per minute for 2 min. The optimum temperature was determined by the Wavemaker software (Transgenomic) for each DNA fragment, and a time shift was applied as needed (Table 2). When multiple melting domains were established, each domain was analyzed at the appropriate temperature. Initial Buffer B concentrations and temperatures for each fragment are listed in Table 2.

PCR fragments displaying DHPLC abnormal retention times were further sequenced on both strands using ABI Dye Terminator, version 1 or 3, in a final reaction volume of 10 μl and electrophoresed on a 3130XL ABI genetic analyzer (Applied Biosystems). Sequences were aligned using the Chromas version 2.23 (Technelysium, Tewantin, Australia).

Denaturing gradient gel electrophoresis was used in the ten added patients studied at the Service of Medical Genetics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland, between 1995 and 1998.

Screening for large deletions was performed by haplotype analysis using RB1 flanking microsatellites D13S161, D13S164, D13S153, D13S1307, and D13S273. One primer was fluorescently labeled, and the product was separated on an automated sequencer (ABI XL3100; Applied Biosystems).


Out of the of 65 RB patients participating in this study, we found seven familial cases, 30 sporadic bilateral cases, and 28 sporadic unilateral cases (Table 3). Screening of the RB1 gene resulted in the identification of four mutations in the familial cases (57%), 22 in the sporadic bilateral cases (73%) and three in the sporadic unilateral cases (10.7%), as detailed in Table 3. We discovered 22 mutations which were the single-base substitution type and four (14%) showed single-base deletion (3) or duplication (1; Table 4).

Figure 1 shows the distribution of the mutations. In addition, one small deletion (8 bp) was detected as well as two large deletions associated with dysmorphic features of 13q-deletion syndrome [18-20].

Of the 22 single-base substitutions, 15 (68%) were nonsense mutations, six (27%) were missense mutations affecting splice sites and one (5%) was a missense mutation in the coding region of the gene. In the 65 index patients, four recurrent mutations were observed once; they were all of the nonsense type (g.59683C>T, g.78238C>T, g.150037C>T, and g.162237C>T). This recurrence is attributed to the fact that the C>T transition that changes arginine codons (CGA) to stop codons is the most common point mutation in the RB1 gene due to spontaneous deamination of methylcytosine to thymine in CpG dinucleotides [15,21].

In the ten patients screened during a non-systematic approach and added to our series, four of the ten detected mutations were nonsense mutations (one redundant), two missense, two small deletions, one small duplication, and one paracentric inversion (Table 4). Four out of these 10 mutations were not previously reported. We suggest that the two missense mutations (g.156795 and g.41924A>G) are pathogenic. Indeed, each new mutation has only been observed in single families and has not been found in any other patients.

The g.156795 mutation was located at amino acid L688, in a region that is conserved in many species from Canis familiaris to Mus musculus, Cavia porcellus, Rattus norvegicus, Gallus gallus, and even Takifugu rubripes and Oryzias latipes. The g.41924A>G mutation is part of the two conserved nucleotides of the 3' cononical splice site.

In total, ten novel mutations were identified that were not detected in 96 ethnically matched healthy individuals. They included a majority of single base mutations with four missense mutations, two single base deletion, two small deletions, one single base insertion, and one small duplication (Table 4). The previously reported g.156713C>T (R661W) mutation known to induce low-penetrant RB was also identified in our cohort (Patient 22, Table 4), and again showed reduced penetrance as mutations carriers of the family harbored only unilateral RB or were unaffected carriers (Figure 2) [10]. The nonsense mutation g.2157dupC in exon 1 detected in Patient 1 (Table 4) was also carried by his father, who presented unilateral RB but not by his mother (Figure 2). Patient 30 (Table 4) harboured a duplication mutation in exon 1, g.2179_2183dupGGACC, which was also carried by his father who had retinoblastoma in one eye and retinocytoma in the other and by his only sister, who had a unilateral RB (Figure 2). It has been reported that nonsense mutations in the first exon could be associated with low-penetrance by alternative in-frame translation involving methionines at position 113 or 233 [16,22]. Modulation of disease penetrance in these two families could be due to the described phenomenon, although we did not perform expression analysis to confirm this hypothesis.

No correlation between the type or localization of mutation and the number of tumor foci per eye, the laterality or the type of RB (endophytic or exophytic) could be established neither in the 65 patients nor in the 10 added patients.


The detection rates of 73% in the sporadic bilateral cases of 10.7% in the sporadic unilateral cases in our series are similar to previous reports [2,3,6]. Our pattern of mutations confirms the predominantly gene-inactivating mutations, i.e. single-base non-sense mutations and splice site mutations [3,6].

The reasons why not all mutations were detected remains controversial. Aside from technical limitations of mutation detection, epigenetic changes could contribute to this phenomenon to an underestimated degree [23]. Meanwhile, other described causes such as mosaicism or non-coding sequence variants may play a role in lowering mutation detection rate as well [24].

Clinical care of RB families includes disease prediction, which carries a significant socio-economic impact. Mutation detection in the family members helps differentiate those who will need specific care and follow-up from those who can avoid invasive and costly procedures. Carriers of germline mutation can benefit from early management, especially with prenatal diagnosis. This is of particular importance when considering tumor growth. For sporadic cases, it is mandatory to perform mutational analysis to know if the patient has a constitutive mutation that can potentially be transmitted to offspring. In sporadic cases, disease expression may be modified by the secondary somatic mutation. Therefore it is important to perform molecular studies on tumor material whenever available.


We thank Isabelle Favre, Tatiana Favez and Natacha Nanchen for technical help.


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