Abstract
Purpose :
Retinoblastoma (RB) is the most common pediatric intraocular neoplasm. Over 98% of RB is due to biallelic inactivation of the retinoblastoma (RB1) gene. Individuals with pathogenic germline RB1 variants are at an increased risk for bilateral disease, as well as extra-ocular malignancies, making a prompt diagnosis crucial to reduce morbidity and mortality. Germline variant analysis can take weeks with clinical testing platforms, which use short-read sequencing (SRS). Moreover, complex variant analysis may require multiple sequencing runs, further delaying diagnosis. In comparison, long-read sequencing (LRS) can drastically reduce turnaround time (TAT) and provide comprehensive profiling of complex genomic and epigenomic variation with a single sequencing run.
Methods :
Eleven patients with RB from 10 families were recruited for retrospective case reviews. Peripheral blood samples were obtained from all 11 patients during routine exams under anesthesia, as well as from 7 parents (from 4 families), for DNA isolation and targeted LRS. A neural network based analysis pipeline of LRS data provided haplotagged variant calling and tiered ranking of deleterious variants.
Results :
The 10 families recruited for participation included 7 families with familial or sporadic RB and prior positive genetic testing, 1 family with sporadic RB and prior negative genetic testing, 1 family with familial RB but no prior genetic testing, and 1 family with sporadic RB and a previously identified mosaic variant. LRS of each proband’s sample (n = 11) validated the prior clinical genetic test results (n=9), solved a previously unsolved case (n=1), and identified a mosaic variant (n=1) within a single sequencing run with a TAT of ~72 hours. Parental samples were analyzed to confirm the de novo or inherited nature of the identified variants.
Conclusions :
LRS was able to validate prior genetic testing results in a cohort of 9 probands with RB as well as identify a pathogenic RB1 variant in an individual without prior genetic testing, all with faster TATs compared to SRS methods. More importantly, LRS was able to identify a mosaic variant in a complex case within days compared to months with clinical SRS. Targeted LRS represents a new paradigm in an evolving era of genetic testing that can provide rapid and comprehensive germline variant information for RB patients and their families.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.