Mutation spectrum of RB1 gene in Korean bilateral retinoblastoma patients using direct sequencing and gene dosage analysis

To the Editor : Retinoblastoma is the most common form of childhood intraocular tumor. Tumor initiation is known to be associated with a double mutation in the RB1 gene, requiring mutations in both alleles. Approximately 40% of retinoblastoma patients carry predisposing germline mutations that are usually associated with bilateral tumors. In contrast, nearly 15% of patients affected by unilateral tumors are known to have germline RB1 mutations (1). The detection rate of RB1 mutations is often variable, even when using the same screening method. Various reports using single-strand conformation polymorphism as the primary screening method have shown relatively low detection rates in sporadic bilateral retinoblastoma patients. Here we carried out mutational studies in 21 unrelated patients with retinoblastoma. DNA was extracted from peripheral blood leukocytes, and RB1 gene mutations were screened via sequence analysis for all exons using an ABI 3730 analyzer and multiple ligation probe-dependent amplification (MLPA) method (SALSA P047-B1 RB1 kit; MRCHolland, Amsterdam, the Netherlands). RNA analysis was performed in order to identify aberrant splicing products associated with a novel splicing mutation. Among 21 patients, 16 had bilateral tumors and 5 had unilateral tumors. None of the patients had a family history of retinoblastoma. The most common clinical presentation was leukocoria (66.7% in all patients and 62.5% in bilateral tumor patients). Germline RB1 mutations were detected in 15 of 16 bilateral retinoblastoma patients (Table 1). No mutations were detected in unilateral patients. We identified 10 known mutations in 11 patients, and 4 novel mutations in 4 patients (Table 1). Four novel mutations included two frame-shifts (c.2191delC and c.1448delA), one splicing (c.1814+3A>C), and one indel (c.1969_1981delinsT). Subsequent reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis revealed that the splicing mutation led to an abnormal transcript by skipping of exon 18 (Fig. 1). No large deletion or duplication mutation was found in this series, but we identified one false positive MLPA deletion case, resulting from a point mutation (c.1448delA) positioned in the eighth base pair from the 3′-end of the left one of two sister halfprobes. In this study, a detection rate of 93.8% (15 out of 16) was achieved in bilateral retinoblastoma patients. A study of RB1 mutations in New Zealand had a detection rate of 100% in eight bilateral patients using both sequencing and MLPA methods (2). A large-scale study reported a mutation detection limit of 89.3% in sporadic bilateral patients using quantitative multiplex-PCR and sequencing without considering low-level mosaicisms; however, when low-level mosaicisms were detected in an additional 22 patients, the detection rate increased to 94.5% (3). The patient with bilateral tumors lacking evidence of a germline mutation, as well as the five unilateral tumor patients in this study might be candidates for further investigation with respect to low-level mosaicisms. A recent study showed that mosaicism was evident in 5.5% of bilateral tumor probands and in 3.8% of unilateral tumor probands; half of the mosaic mutations were only detectable by allele-specific PCR and not by standard sequencing (3). Exome sequencing using next-generation sequencing techniques was also found to be an effective method for the detection of low-level mosaicism (4). Systemic treatment for retinoblastoma improves management of patients and reduces the frequency of enoculation; therefore, the development of sensitive assays for germline mutation detection in blood samples becomes increasingly more important. Germline mutations in the RB1 gene are also implicated in increased mortality associated with other second non-ocular primary tumors. In this study, two cases developed osteosarcoma several years after diagnosis. A patient with the c.1333C>T mutation, who did not receive external beam irradiation during treatment, developed osteosarcoma in the right distal femur at the age of six, which was 5 years after the diagnosis of retinoblastoma. Another patient with the c.265-2A>G mutation also developed osteosarcoma 7 years after receiving external beam radiation therapy in her right eye. Compared to previous studies, this study shows high detection rate of germline mutations in patients with