A novel de-novo RB1 mutation identified in a patient with bilateral retinoblastoma

Abstract

Retinoblastoma manifests as ocular malignancy due to mutations in the RB1 gene. A 17-month-old girl with bilateral retinoblastoma having no family history was admitted to our hospital. The right eye was enucleated but the other was preserved with systemic chemotherapy and topical treatment. The patient has been tumor-free for over 7 years since diagnosis. All exons of RB1 were sequenced and a novel 1-base pair deletion (NM_000321.2:c.2409del, p.Asn803Lysfs*7) was detected.

retinoblastoma, novel mutation, heritable tumor

Introduction

Retinoblastoma, especially familial retinoblastoma, (MIM: #180200) is marked by germline mutations in the RB1 gene, which is inherited in an autosomal-dominant fashion. It is the most frequent intraocular malignancy among children, manifesting mostly as bilateral retinoblastoma. Retinoblastoma occurs at a frequency of 1 in 15 000 births with 60% of the patients developing unilaterally and 40% bilaterally.

Most of the children with bilateral retinoblastoma carry germline mutation in RB1 and ⁓10% of these mutations are inherited from one of their parents (1). A total of 10–15% of the children with unilateral retinoblastoma carry germline RB1 mutation. Patients with chromosome 13q14 deletion variant have congenital anomalies and mental retardation in addition to retinoblastoma. Furthermore, age at diagnosis varies by the type of retinoblastoma: bilateral tumors are usually found before 15 months of age while unilateral tumors are usually found ⁓24 months of age. Genotype–phenotype correlation has not been clearly identified, but the patients with bilateral retinoblastoma and the splice variant tended to result in mean diagnosis age of 19.3 month that is older than the patients with bilateral and other mutation (2). Other reported that patients with bilateral retinoblastoma have truncating mutation at 93% and the patients with the unilateral retinoblastoma were at 57% (3).

While the eyes of affected individuals can be preserved by early tumor detection and treatment (4), the RB1 mutation predisposes sufferers to secondary cancers such as sarcoma or other malignancies (5). Therefore, vigilance against these secondary tumors must be clinically maintained even after successful retinoblastoma therapy.

Figure 1

RB1 1-base pair deletion by Sanger sequencing. One-base pair deletion was confirmed by Sanger sequencing. A base of ‘C’ (c.2409) pointed by black arrow was deleted and frameshift was detected.

Figure 2

Schematic representation of the Retinoblastoma protein and the mutation of our patient. Retinoblastoma protein consists of pocket (A and B) and Rb C-terminal domain (Rb C). The mutation of our patient was located in Rb C that is a highly conserved domain.

Case description

A 17-month-old girl presented to the University of Tsukuba Hospital complaining of strabismus. She was diagnosed with bilateral retinoblastoma after ophthalmologic testing and magnetic resonance imaging. She had no family history of any type of cancer. A metastatic survey by computed tomography, spinal tap and bone marrow aspiration revealed no evidence of disease outside of the eyes. The right eye was enucleated because it was classified as group Va by the Reese-Ellsworth classification (R-E group) and group E of the international classification for intraocular retinoblastoma (IC group). The left eye was classified as group IVa/C and managed with systemic chemotherapy and topical treatment. A total of six cycles of VEC (Vincristine 0.05 mg/kg, Etoposide 10 mg/kg, Carboplatin 18.6 mg/kg) were conducted as a systemic chemotherapy while three cycles of selective ophthalmic arterial injection of melphalan (7.5 mg/m²) and seven cycles of topical ablation were conducted at the National Cancer Center Hospital as previously described (4). The combined therapy was successfully conducted without severe adverse events and led to complete remission. There have been no tumor relapses for over 7 years from diagnosis and 6 years from completion of the treatment. Furthermore, uncorrected visual acuity is currently 0.1.

Deoxyribonucleic acid was extracted from peripheral blood. Cancer-related genes, including RB1, were amplified with primers designed by Ion AmpliSeq™ Designer (ThermoFisher Scientific, MA, USA, https://www.ampliseq.com/browse.action) which covered 99.7% of RB1 exons. The library was generated with an Ion AmpliSeq™ library preparation kit and sequencing was run on an Ion PGM™ (ThermoFisher Scientific) system according to the manufacturer’s instructions. Obtained reads were mapped against human reference sequence hg19 and annotated using Ion Torrent Suite v.5.2.2. The mean mapped read depth for the total region was 187.2 and mean aligned read per amplicon for RB1 was 123.7. A mutation of NM_000321.2:c.2409del,p.Asn803Lysfs*7 was detected in 23 of 27 exons of RB1 and confirmed by Sanger sequencing (Fig. 1). The aligned total read depth of this position and alternative variant count were 197 and 96 (variant allele frequency is 0.51), respectively. The same mutation was not detected in her parents.

Discussion

There have been many papers describing the genetics of retinoblastoma (2,6), with mutational hotspots existing at exons 8, 10, 11, 14, 15, 17, 18 and 23. Many of these are reported as frameshift, nonsense, missense or splicing mutations. The mutation of our patient was located in Rb C-terminal domain that is highly conserved domain and is required for high-affinity binding to E2F-DP complexes resulting in a growth suppress by blocking the G1-S transition of the cell cycle (Fig. 2) (7). Hence, at least, this deletion may affect the efficacy of growth-suppression of Retinoblastoma 1. Furthermore, this mutation resulted in truncated mutation, thus the protein of retinoblastoma 1 may not be formed. Most of the patients with 1-base pair deletion in exon 23 of RB1 had bilateral retinoblastoma (8–10) and a patient with mosaic mutation of 1-base pair deletion of exon 23 developed unilateral retinoblastoma at 7 months old (11). Our patient who had truncated mutation in exon 23 developed bilateral retinoblastoma at young age (<24 months old) was similar to the previous reports. Additionally, the variant found in our patient is not likely to be mosaic mutation due to the variant allele frequency of 0.51.

The cited number of distinct, causative RB1 mutations in ClinVar and HGMD were 604 and 1167, respectively (12,13). Among them, small deletions were 255. Although another missense variation in the same position (c.2409C > G, p. Asn803Lys, HGMD #CM952104) was reported previously (14), the variation of our patient (NM_000321.2:c.2409del, p.Asn803Lysfs*7), to the best of our knowledge, had not been previously reported. We hope this report of the novel Rb1 mutation may have some contribution for the diagnosis of retinoblastoma.

Acknowledgments

We would like to thank Dr. Bryan J. Mathis of the University of Tsukuba Medical English Communication Center for providing critical scientific writing assistance.

Funding

This work was partially supported by Grant-in-Aid for Young Scientist (B) from Japan Society for the Promotion of Science (17K16239).

Conflict of interest

The authors have no conflict of interest to declare with regards to this work.