June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Functional evaluation of rare deep intronic variants in patients with elusive genetic causality of inherited retinal degenerations
Author Affiliations & Notes
  • Marianna Weener
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Hilary Scott
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Emily Place
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Kit Sanderson
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Kevin Valestil
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Juerg Straubhaar
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Kinga Maria Bujakowska
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Eric A Pierce
    Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Marianna Weener None; Hilary Scott None; Emily Place None; Kit Sanderson None; Kevin Valestil None; Juerg Straubhaar None; Kinga Bujakowska None; Eric Pierce None
  • Footnotes
    Support  NEI Grant RO1 EY01920
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4527. doi:
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    • Get Citation

      Marianna Weener, Hilary Scott, Emily Place, Kit Sanderson, Kevin Valestil, Juerg Straubhaar, Kinga Maria Bujakowska, Eric A Pierce; Functional evaluation of rare deep intronic variants in patients with elusive genetic causality of inherited retinal degenerations. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4527.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : For ~30% of patients with inherited retinal degenerations (IRD) the genetic causality remains elusive. Part of the missing genetic causality are deep intronic (DI) variants which activate cryptic exons (CEs). Such variants have been reported as pathogenic in up to 22.5% of patients. It is challenging to determine which of the hundreds DI variants identified by whole genome sequencing (WGS) in IRD patients are causal pathogenic variants. The study goal was to use a novel high throughput splicing assay (HTSA) to empirically test the functional effects of rare DI variants.

Methods : We studied 38 IRD patients with 1 exonic pathogenic/likely pathogenic allele in a known recessive IRD gene. WGS identified 257 rare (AF<0.001) DI variants in 34 genes. Reference (REF) and variant (VAR) intronic sequences were cloned into a split GFP (green fluorescent protein) construct (Fig.1A) also containing an mCherry cassette. Activation of a CE disrupts GFP expression leading to GFP negative /mCherry positive cells, whereas lack of CE activation (1B) leads to the expression of both fluorophores (1C). The transfected modified HEK293 cells were FACS sorted, the GFP negative and GFP positive cells were separated, and the sequences they contained were analyzed by next generation sequencing. Exact matches to the REF and VAR in-silico library sequences were counted, and a CE inclusion index (CEII) calculated

Results : Molecular analyses showed 17 (6.6%) of the 257 interrogated deep intronic variants activated CEs, suggesting they are likely to be pathogenic. Variants with CEII of >0.6 showed a strong effect on CE inclusion, which are in ADGRV1 introns 70 c.14518-4989G>A, 85 c.18153-32774A>C, CDH23 intron 28 c.*4136A>G, CEP164 intron 25 c.3216+180G>A, EYS introns 12 c.2023+71410A>G, 29 c.6078+15034C>T, RPGRIP1L intron 22 c.3295-2113C>T, and USH2A intron 3 c.651+18339G>T genes. Variants with CEII of 0.3 to 0.6 showed a moderate effect which included genes: CEP83 intron 15, EYS introns 5,11,12,22,28,33, NR2E3 intron 7, USH2A intron 36. These results lead to genetic diagnoses for 11(29%) of the 38 IRD studied patients

Conclusions : The HTSA is an effective approach for functional analysis of rare DI variants and can be used to improve genetic testing for patients with no previous diagnosis. This will be especially valuable for patients who might be eligible to receive genetic therapies

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

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