A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families

Abstract

Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful, an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient-induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) transcriptome analysis. With this approach, we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.

Implementation of a registry and open access genetic testing program for inherited retinal diseases within a non‐profit foundation

“…As the increasing genetic diversity and overlap between RP and other IRD grew, the Foundation was renamed to the Foundation Fighting Blindness. The Foundation continues to invest ~25% of its funds in gene discovery and characterization, supporting increasingly sophisticated genetic tools to discover the genetic cause of the remaining 40–45% of unsolved IRD cases (Bronstein et al, 2020), and supports a nationwide Israeli IRD consortium performing clinical and genetic mapping of the entire Israeli IRD population (Sharon et al, 2020). In total the Foundation has raised over $760 M for IRD research with an annual research budget of over $20 M that supports over 73 investigators across 14 countries.…”

Noncanonical Splice Site and Deep Intronic FRMD7 Variants Activate Cryptic Exons in X-linked Infantile Nystagmus

“… 33 , 44 Obtaining a clinically relevant tissue in inherited eye diseases is impossible because the retina and lens cannot be biopsied without functional compromise. 45 Therefore, a patient-derived retinal organoid transcriptome can be used to detect cryptic splice sites in genetically unsolved patients with inherited retinal diseases. 45 Dynamic retinal expression pattern of FRMD7 during embryonic and fetal development has been reported using in situ hybridization techniques.…”

“… 45 Therefore, a patient-derived retinal organoid transcriptome can be used to detect cryptic splice sites in genetically unsolved patients with inherited retinal diseases. 45 Dynamic retinal expression pattern of FRMD7 during embryonic and fetal development has been reported using in situ hybridization techniques. 3 , 11 However, FRMD7 expression is scant or absent in both human normal retina ( https://oculargenomics.meei.harvard.edu/retinal-transcriptome/ ) and our in-house bulk RNA sequencing data of retinal organoids ( Supplementary Fig.…”

The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development

“…Treatment with BMP4 starting from day 0, however, did not at all promote retinal or neural differentiation [127]. The timely addition of BMP4 between day 6 and 18 has since been adopted into many methods of generating retinal organoids [48,[129][130][131].…”

“…(C) Alongside stand-alone protocols used solely by a single group (in blue), there were also a small number of manuscripts that did not relate to other methods. All papers included either produced retinal organoids, or were referenced as a method of producing retinal organoids[48,49,74,101,[107][108][109]113,127,[129][130][131]135,[143][144][145][146][148][149][150]152,.…”