Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing

Abstract

BACKGROUND. Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS. One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network (UDN) clinical site from 2014-2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS. The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCKassociated encephalopathy, NSD2and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSION. For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA. 4 TRIAL REGISTRATION. Not applicable.

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy

“…Moreover, all patients with congenital myopathy with unsolved clinical exomes should undergo reanalyses of extant ES data and consider WGS studies and perhaps RNA-seq on muscle biopsy. 20 - 22 …”

MRSD: a novel quantitative approach for assessing suitability of RNA-seq in the clinical investigation of mis-splicing in Mendelian disease

“…Other approaches to select genes amenable to functional analysis through RNA-seq include leveraging relative gene expression metrics (14, 20), or tools which assess the similarity of transcript isoforms between tissues, e.g. MAGIQ-CAT (7).…”

The Role of RNA-Sequencing as a New Genetic Diagnosis Tool

“…This provided a MEPAN disorder diagnosis for both patients [30] and demonstrated the advantage of combining RNA-Seq with WGS to answer the challenges of variant prioritisation. Murdock et al [28] support the idea of using a multi-omics approach rather than solely WGS or WES for intronic variants after a deeply intronic variant was observed in a 3-yr-old male patient with multiple congenital abnormalities. Here, a 50% reduction in the expression of PQBP1 from whole blood was associated to a hemizygous variant in PQBP1, resulting in an activated cryptic splice donor, abnormal splicing pattern and intron retention.…”

“…In contrast, Rentas et al [26] explore the option of using B-lymphoblastoid cell lines (LCL) from patients to analyse expression and splicing. Additionally, Lee et al [27] find an 18% diagnostic yield in a heterogenous cohort, while Murdock et al [28] suggest a novel method of integrating RNA-Seq data that contrasts the commonly used candidate gene approach used in most studies. Instead, Murdock et al suggest starting with RNA-Seq and, thus excitingly, advocate its role in research and gene discovery.…”