Introduction
Retinoblastoma (Rb, OMIM#180200)
is a malignant tumor of the developing retina that affects children
before the age of five years with an estimated incidence between 1 in
16,000 and 1 in 18,000 live births [1]. Rb occurs in both heritable
(25-30%) and non-heritable (70-75%) forms. A heritable form is defined
by the presence of a germline heterozygotic mutation in the RB1gene (Genbank accession number L11910.1; NCBI RefSeq NM_000321.2), wich
is followed by a second somatic hit in the developing retina. As a
result, tumors affecting either one (unilateral) or both (bilateral)
eyes may develop. In the non-heritable form both mutations occur in
somatic cells, usually leading to unilateral malignancy [2]. In
addition to the highly malignant early onset Rb, the risk of developing
second cancers, e.g. osteosarcomas and other soft-tissue sarcomas,
rarely melanomas is increased. Molecular diagnostics is required to
clear heredity status and to deliver the best options for the management
of the disease [3, 4]. Due to the genetic predisposition, second
primary malignancies may arise spontaneously or following radiotherapy,
which have become the leading cause of death in Rb survivors.
Osteosarcomas in retinoblastoma patients occurred 1.2 years earlier and
the latency period between radiotherapy and osteosarcoma onset was 1.3
years shorter inside than outside of the radiation field [5].
The RB1 gene shows a wide spectrum of mutations, including single
nucleotide variants (SNVs), small insertions/deletions (indels), and
large deletions/duplications. These mutations are distributed throughout
the entire length of the gene, spanning over 27 exons, and no hotspots
have been reported [6]. New advances in molecular genetic testing,
and especially next-generation sequencing (NGS), allow the comprehensive
demonstration of all SNVs and large aberrations throughout the full
length of the gene. Pathogenic mutations in both alleles of theRB1 gene are related to the development of this neoplasm in the
large majority of the cases. Alternatively, complex mutation patterns
missing the RB1 gene aberration were identified in rare cases of
manifest Rb, indicating to oncogenic interactions between different
signal transduction pathways [7].
The aim of our study was (i) to identify germline genetic aberrations in
a patient with bilateral Rb of non-parental origin, (ii) to exclude the
parental carrier status, (iii) to characterize histological and genetic
features of samples originating from the two Rb and the four
anatomically distinct osteosarcoma tumors (iv) to genetically
differentiate between the irradiation-related orbitofacial and the
non-related de novo osteosarcoma of the lower extremity, (v)
structural and functional prediction of the germline mutant proteins
reconstructed after DNA sequencing, and, finally (vi) identify potential
interaction between defected proteins using prediction analysis. For
this purpose, histology, including immunohistochemistry (IHC) and NGS
solid tumor gene panel (Illumina MiSeq platform) analysis were performed
using samples from both enucleated eyes and from the four available
osteosarcoma tissues. Autopsy sample of the skin was taken as normal
non-neoplastic control tissue. In addition, in silico prediction
methods were applied to analyse the secondary structure and
functionality of detected germline variants and to predict
protein-protein interaction. To exclude potential parental origin
conventional Sanger sequencing was used.