An overview of RB1 transcript alterations detected during retinoblastoma genetic screening

ABSTRACT

Identification of pathogenic RB1 variants aids in the clinical management of families with retinoblastoma. We routinely screen DNA for RB1 variants, but transcript analysis can also be used for variant screening, and to help decide variant pathogenicity. DNA was screened by conformation analysis followed by Sanger sequencing. Large deletion/insertions were detected by polymorphism analysis, MLPA and quantitative-PCR. Methylation-specific PCR was used to detect hypermethylation. RNA screening was performed when a DNA pathogenic variant was missing, or to determine effects on splicing.

Two hundred and thirteen small coding variants were predicted to affect splicing in 207 patients. Splice donor (sd) variants were nearly twice as frequent as splice acceptor (sa) with the most affected positions being sd + 1 and sa−1. Some missense and nonsense codons altered splicing, while some splice consensus variants did not. Large deletion/insertions can disrupt splicing, but RNA analysis showed that some of these are more complex than indicated by DNA testing. RNA screening found pathogenic variants in 53.8% of samples where DNA analysis did not. RB1 splicing is altered by changes at consensus splice sites, some missense and nonsense codons, deep intronic changes and large deletion/insertions. Common alternatively spliced transcripts may complicate analysis. An effective molecular screening strategy would include RNA analysis to help determine pathogenicity.

KEYWORDS:

• Retinoblastoma
• RB1
• splicing
• pathogenic
• genetic
• transcript

Acknowledgments

The authors gratefully acknowledge the contributions of the late Mr. John Hungerford (Consultant Ophthalmologist) and Dr. Judith Kingston (Consultant Pediatric Oncologist) for the referral, provision of clinical information and material for analysis. We also thank the Barts Health Rb clinical nurse specialists and pathologists for their help in collecting samples, and RGSU genetic technologists for sample processing and testing.

Author contributors

EAP and ZO performed RB1 variant analysis, interpreted the data and drafted the article. MSS and MAR referred cases, provided clinical information and material for analysis, and revised/approved the article. All authors critically read, revised, and approved the final document.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Ethics approval

Barts Health Clinical Effectiveness Unit (audit no. 12614).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13816810.2023.2270570.

Additional information

Funding

The authors reported there is no funding associated with the work featured in this article.