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.…”

Development and comparison of RNA-sequencing pipelines for more accurate SNP identification: practical example of functional SNP detection associated with feed efficiency in Nellore beef cattle

“…Use of whole genome data allows for the identification of variants in an individual or group of individuals, allowing for detection of potential causal variants in the whole genome that may be associated with a trait of interest. In addition, when using genome sequence data, more non-coding variants can be identified as they are more present in the genome compared to coding variants [26]. In contrast, evaluation of the transcriptome using RNA-Seq allows for detection of variants within coding regions which may provide functional information regarding a trait of interest [4].…”

Patient derived stem cells for discovery and validation of novel pathogenic variants in inherited retinal disease

“…In recent years, the potential for RNA-sequencing-based transcriptome analysis as a diagnostic tool has been raised in various contexts (Byron et al, 2016). In the setting of unsolved Mendelian disease, multiple groups have conducted studies that utilize transcriptomic analysis to identify candidate pathogenic variants in patients for whom exome sequencing has failed to identify a convincing disease-causing genotype (Table 1) (Bronstein et al, 2020;Chandrasekharappa et al, 2013;Cummings et al, 2017;Evrony et al, 2017;Fresard et al, 2019;Gonorazky et al, 2019;Hamanaka et al, 2019;Kernohan et al, 2017;Kremer et al, 2017;Lee et al, 2020). On average, the diagnostic yield of such studies is approximately 29% (Table 1), ranging from 7.5% (Fresard et al, 2019) to 60% (Rentas et al, 2020).…”

“…To date, the most notable venture away from testing easily biopsied tissues has been Bronstein and colleagues' 2020 study of a single family with inherited cone dysfunction (Bronstein et al, 2020). The authors performed RNA sequencing on both accessible samples of blood and skin, but also on retinal organoids differentiated from iPSCs that were ultimately derived from patient fibroblasts.…”

“…Detecting aberrant expression patterns is an important outcome in the transcriptome analysis of rare diseases (Brechtmann et al, 2018;Bronstein et al, 2020;Cummings et al, 2017;Gonorazky et al, 2016;Kremer et al, 2017Kremer et al, , 2018. We set out to determine if important genes in inherited retinal disease were expressed by the same cell types in human post mortem eyes (Voigt et al, 2019b) and retinal organoids (Sridhar et al, 2020).…”