About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

暂无摘要。

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3\' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3\' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3\' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

Retinoblastoma is the most common paediatric neoplasm of the retina, and one of the earliest model of cancer genetics since the identification of the master tumour suppressor gene RB1. Tumorigenesis has been shown to be driven by pathogenic variants of the RB1 locus, but also genomic and epigenomic alterations outside the locus. The increasing knowledge on this \"mutational landscape\" is used in current practice for precise genetic testing and counselling. Novel methods provide access to pre-therapeutic tumour DNA, by isolating cell-free DNA from aqueous humour or plasma. This is expected to facilitate assessment of the constitutional status of RB1, to provide an early risk stratification using molecular prognostic markers, to follow the response to the treatment in longitudinal studies, and to predict the response to targeted therapies. The aim of this review is to show how molecular genetics of retinoblastoma drives diagnosis, treatment, monitoring of the disease and surveillance of the patients and relatives. We first recap the current knowledge on retinoblastoma genetics and its use in every-day practice. We then focus on retinoblastoma subgrouping at the era of molecular biology, and the expected input of cell-free DNA in the field.

In patients with retinoblastoma, gains of chromosome 6p have been associated with less differentiated tumors. In cell-free DNA from the aqueous humor (AH), 6p gain has been associated with an increased risk of enucleation. While the identification of somatic copy number alterations (SCNAs) via the AH has been well established, these alterations are not routinely identified in the blood due to low tumor fraction.
SCNAs were considered positive at 20% deflection from the baseline. Somatic RB1 pathogenic variants were identified with targeted sequencing using a panel including all RB1 exons.
A 24-month-old patient presented with unilateral retinoblastoma (Group D/AJCC Stage cT2B) and was treated with primary enucleation. In the peripheral blood, a heterozygous mutation (c.3920T>A) in the APC gene was reported. Genomic analysis of the tumor and AH revealed two novel somatic RB1 mutations (c.1589_1590del and c.2330dupC). Both also demonstrated highly recurrent RB-related SCNAs. Chromosome 6p gain was detected in the blood with an amplitude suggesting approximately 12% tumor fraction. At a follow-up of 24 months, there has been no evidence of metastatic disease.
To our knowledge, this is the first time an SCNA has been detected in the blood of an RB patient, suggesting in some advanced eyes there may be a high enough tumor fraction to detect these alterations (>5% needed). It remains unclear whether 6p gain or increased tumor fraction in the blood is indicative of increased risk of metastatic disease or new primary cancer; studies to address this are ongoing.

Identification of disease-causing variants of the retinoblastoma gene (RB1), the predominant cause of retinoblastoma, is challenging. Targeted long-read genome sequencing offers a novel approach to resolve the diverse range of pathogenic variants in RB1 and provides haplotype information rapidly.
Genomic DNA was isolated from a venipuncture blood draw of a retinoblastoma patient. Whole genome sequencing (WGS) was carried out using the short-read Ilumina platform. WGS and targeted sequencing of RB1 was accomplished using the long-read Oxford Nanopore Technologies (ONT) platform. Deep-learning frameworks allowed haplotagging, variant calling, and variant annotation of both short- and long-read data.
Targeted long-read sequencing of the RB1 gene allowed for enhanced depth of read coverage for discovery of rare variants and haplotype analysis. A duplication leading to a frameshift and early termination in RB1 was identified as the most deleterious variant by all sequencing methods, with long-read technology providing additional information of methylation signal and haplotype information. More importantly, there was greater than 98% concordance of RB1 variants identified between short-read and targeted long-read sequencing modalities.
Targeted long-read technology allows for focused sequencing effort for variant discovery. Application of this for the first time in a retinoblastoma patient allowed haplotagged variant identification and demonstrated excellent concordance with benchmark short-read sequencing. The added benefit of targeted long-read sequencing to resolve disease-causing genomic variation in RB1 rapidly from a blood draw will provide a more definitive diagnosis of heritable RB and guide management decisions for patients and their families.

Since the discovery of the retinoblastoma susceptibility gene (RB1) decades ago, RB1 has been regarded as a prototype tumor suppressor gene providing a paradigm for tumor genetic research. Constant research has updated the understanding of RB1-related pathways and their impact on tumor and nontumor diseases. Mutation of RB1 gene has been observed in multiple types of malignant tumors including prostate cancer, lung cancer, breast cancer, and almost every familial and sporadic case of retinoblastoma. Even if well-known and long-investigated, the application potential of RB1 mutation has not been fully tapped. In this review, we focus on the mechanism underlying RB1 mutation during oncogenesis. Therapeutically, we have further discussed potential clinical strategies by targeting RB1-mutated cancers. The unsolved problems and prospects of RB1 mutation are also discussed.

Retinoblastoma (RB) is initiated by mutation in both alleles of RB1 gene. However, few cases may occur even in the absence of RB1 mutation suggesting the role of genes other than RB1.
The current study was planned to utilize targeted exome sequencing in Indian RB patients affected with unilateral non-familial RB. 75 unilateral RB patients below 5 years of age were enrolled. Genomic DNA was extracted from blood and tumor tissue. From peripheral blood DNA, all coding and exon/intron regions were amplified using PCR and direct sequencing. Cases which did not harbor pathogenic variants in peripheral blood DNA were further screened for mutations in their tumor tissue DNA using targeted exome sequencing. Three pathogenicity prediction tools (Mutation Taster, SIFT, and PolyPhen-2) were used to determine the pathogenicity of non-synonymous variations. An in-house bioinformatics pipeline was devised for the mutation screening by targeted exome sequencing. Protein modeling studies were also done to predict the effect of the mutations on the protein structure and function.
Using the mentioned approach, we found two novel variants (g.69673_69674insT and g.48373314C>A) in RB1 gene in peripheral blood DNA. We also found novel variants in eight genes (RB1, ACAD11, GPR151, KCNA1, OTOR, SOX30, ARL11, and MYCT1) that may be associated with RB pathogenesis.
The present study expands our current knowledge regarding the genomic landscape of RB and also highlights the importance of NGS technologies to detect genes and novel variants that may play an important role in cancer initiation, progression, and prognosis.

UNASSIGNED: Retinoblastoma (RB) is the most common malignant intraocular tumor in children; it affects their eyes often even prenatally. RB may be sporadic or familial, due to germinal mutation in RB1 gene or by abnormal chromosomal abnormalities involving RB1 gene, located in 13q14. Monosomy of subband 13q14 as a partial deletion can also be responsible for RB with additional symptoms. The latter may be RB associated with psychomotor retardation, macrocephaly, broad forehead, thick earlobes, and bulbous nose.
UNASSIGNED: We present here the case of a boy from a consanguineous marriage with bilateral retinoblastoma, intellectual disability and facial dysmorphic features. Classical and molecular cytogenetics were used to recognize genotype-phenotype association.
UNASSIGNED: The karyotype showed a three way translocation involving chromosomes 5, 12 and 13. Further molecular cytogenetics analysis revealed a deletion of 13q14 involving the tumor suppressor gene RB1.
UNASSIGNED: This case highlights the impact of classical and molecular cytogenetics in diagnosis of rare genetic syndromes and for the genetic counselling of patients and their families. Pure molecular karyotyping analyses would miss the underlying chromosomal mechanism leading to the rearrangement.

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.