Upstream variants causing reduced transcription and unusual presentation of recessive inherited retinal dystrophies

Abstract

Purpose : To determine the effect of non-coding putative promoter/regulatory region variants in BBS10 and GUCY2D identified by whole genome sequencing (WGS) in trans with a protein altering variant in patients with atypical presentation of BBS10 and GUCY2D disease.

Methods : Patients underwent WGS as part of the UK’s 100,000 genomes project followed by clinical variant interrogation identifying a single candidate coding region variant in BBS10 (patient #1) and GUCY2D (patients #2 & #3). Non-coding variant analysis of the suspected gene was performed including promoter region interrogation (Eukaryotic Promoter Database; https://epd.epfl.ch). Promoters were amplified, cloned into pGL3 vector, and suspected variants were introduced by site-directed mutagenesis. Luciferase reporter assays in HEK293 cells were used to determine the effect of promoter variants.

Results : Patient #1 harboured variants in BBS10: c.2119_2120delGT (p.Val707Ter), and c.-80dupC within a predicted promoter region. Patients #2 and #3 harboured GUCY2D c.-148T>C in the TAAT core (5’-TAAT-3’ > 5’-TAGT-3’) of a predicted CRX binding element in trans to c.2837C>A [p.(Ala946Glu)], and c.3043+5G>A (proven pathogenic impact via minigene assay), respectively. Luciferase assays showed that BBS10 c.-80dupC caused a ~70% decrease in activity compared to the wild-type promoter. For GUCY2D, the c.-148T>C variant only altered the luciferase activity when co-transfected with a CRX expression plasmid. In this instance, the mutant GUCY2D promoter exhibited ~75% (p<0.02) activity of the wild-type promoter.

Conclusions : Heterologous expression of luciferase under the control of BBS10 and GUCY2D promoters indicated that the candidate variants are likely to affect intrinsic expression of these genes in the retina. Patient #1 has no extra-ocular features of Bardet-Biedl syndrome at the age of 71 years, and patient 2 presented atypically for GUCY2D-Leber congenital amaurosis with a stationary retinal dystrophy affecting both rods and cones but retaining some visual function (BCVA 1/60 OD, 2/60 OS) at age 46 years. Patient #3 presented with LCA (no further details available at the time of writing). These findings suggest that in patients #1 and #2, the genotypes may be hypomorphic, evidenced by the clinical presentation at the mild end of the disease spectrum and retained promotor function (predicting about 30% for BBS10 and 75% for GUCY2D).

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.