Identification and evaluation of a non-coding variant located in the 5'-UTR of TMEM216 as an underlying cause of non-syndromic recessive retinal degeneration (RD) in Pakistani families.

Abstract

Purpose : To identify and characterize the underlying cause of recessive retinal degeneration (arRD) in families that remained unresolved after whole genome sequencing (WGS) and coding sequence analysis.

Methods : Available individuals from seven genetically unresolved Pakistani pedigrees underwent ophthalmic evaluation including fundoscopy and ERG. Homozygosity mapping and segregation of rare non-coding variants with disease was performed. CRISPR-Cas9 was utilized to generate hTERT-RPE1 cells with the candidate non-coding variant for functional assessment. Luciferase assay, cell morphology evaluation, gene expression via qRT-PCR, immunostaining, bulk RNA-seq, and CUT&RUN using cells with A/A and G/A genotypes were performed.

Results : Analysis of WGS identified a rare variant (GRChg38 chr11-61392563G>A; MAF: 0.00095 in South Asians) in a homozygous intergenic region of TMEM216 and TMEM138 segregating with arRD in 7 families of Pakistani Origin with 3-8 affected members. This variant is located upstream of theTMEM216 (c.-69G>A) gene transcription start site and in an ATAC-seq peak in RPE and photoreceptor cells suggesting a regulatory role. Based on FIMO transcription factor (TF) motif analysis, the variant is predicted to impact binding of CHIP-seq validated TFs. Genome editing of hTERT-RPE1 cells resulted in cells homozygous (A/A) for the variant (with an additional C>T off target edit two bases distal) and heterozygous (G/A) without off target changes. qRT-PCR analysis of TMEM216 and TMEM138 transcripts in these cells revealed significantly reduced expression of both transcripts. Further, the morphology of cilia in these cells was abnormal compared to wild type control cells. Luciferase assay, bulk RNA-sequencing data, and CUT&RUN sequencing results of candidate TF are being analyzed to further examine the impact of G>A change on TF binding and gene expression.

Conclusions : Genome analysis identified a rare non-coding variant GRChg38 chr11:61392563G>A segregating with early-onset non-syndromic arRD in 7 Pakistani families. hTERT-RPE1 cells with this variant showed significantly lower levels of TMEM216 and TMEM138 transcripts and abnormal ciliary morphology. These results suggest that reduced expression of TMEM216 and TMEM138 leads to abnormal photoreceptor ciliogenesis and in non-syndromic RD in Pakistani patients.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.