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

Murdock, David R.; Dai, Hongzheng; Burrage, Lindsay C.; Rosenfeld, Jill A.; Ketkar, Shamika; Müller, Michaela; Yépez, Vicente A.; Gagneur, Julien; Liu, Pengfei; Chen, Shan; Jain, Mahim; Zapata, Gladys; Bacino, Carlos A.; Chao, Hsiao‐Tuan; Moretti, Paolo; Craigen, William J.; Hanchard, Neil A.; Lee, Brendan

doi:10.1172/jci141500

Cited by 111 publications

(143 citation statements)

References 54 publications

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“…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 …”

Section: Discussionmentioning

confidence: 99%

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy

et al. 2021

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ObjectivePathogenic variants in TNNT3, the gene encoding fast skeletal muscle troponin T, were first described in autosomal dominant distal arthrogryposis type 2B2. Recently, a homozygous splice site variant, c.681+1G>A, was identified in a patient with nemaline myopathy and distal arthrogryposis. Here, we describe the second individual with congenital myopathy associated with biallelic TNNT3 variants.MethodsClinical exome sequencing data from a patient with molecularly undiagnosed congenital myopathy underwent research reanalysis. Clinical and histopathologic data were collected and compared with the single reported patient with TNNT3-related congenital myopathy.ResultsA homozygous TNNT3 variant, c.481-1G>A, was identified. This variant alters a consensus splice acceptor and is predicted to affect splicing by multiple in silico prediction tools. Both the patient reported here and the previously published patient exhibited limb, bulbar, and respiratory muscle weakness from birth, which improved over time. Other shared features include history of polyhydramnios, hypotonia, scoliosis, and high-arched palate. Distal arthrogryposis and nemaline rods, findings reported in the first patient with TNNT3-related congenital myopathy, were not observed in the patient reported here.ConclusionsThis report provides further evidence for the association of biallelic TNNT3 variants with severe recessive congenital myopathy with or without nemaline rods and distal arthrogryposis. TNNT3 sequencing and copy number analysis should be incorporated into the workup of congenital myopathies.

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Section: Discussionmentioning

confidence: 99%

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy

et al. 2021

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Rowlands

Taylor

Rice

et al. 2021

Preprint

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BackgroundRNA-sequencing of patient biosamples is a promising approach to delineate the impact of genomic variants on splicing, but variable gene expression between tissues complicates selection of appropriate tissues. Relative expression level is often used as a metric to predict RNA-sequencing utility. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA-sequencing, overcoming some issues with using expression values alone.ResultsWe derive a novel metric, Minimum Required Sequencing Depth (MRSD), for all genes across three human biosamples (whole blood, lymphoblastoid cell lines (LCLs) and skeletal muscle). MRSD estimates the depth of sequencing required from RNA-sequencing to achieve user-specified sequencing coverage of a gene, transcript or group of genes of interest. MRSD predicts levels of splice junction coverage with high precision (90.1-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that LCLs are the optimum source of RNA, of the three investigated biosamples, for 69.3% of gene panels. Our approach demonstrates that up to 59.4% of variants of uncertain significance in ClinVar predicted to impact splicing could be functionally assayed by RNA-sequencing in at least one of the investigated biosamples.ConclusionsWe demonstrate the power of MRSD as a metric to inform choice of appropriate biosamples for the functional assessment of splicing aberrations. We apply MRSD in the context of Mendelian genetic disorders and illustrate its benefits over expression-based approaches. We anticipate that the integration of MRSD into clinical pipelines will improve variant interpretation and, ultimately, diagnostic yield.

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“…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.…”

Section: Aberrant Gene Expression Levelsmentioning

confidence: 97%

“…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.…”

Section: Studies Utilising Rna-seq In Genetic Diagnosticsmentioning

confidence: 99%

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

2021

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Purpose of Review Whole exome sequencing (WES) and whole-genome sequencing (WGS) are frontline approaches for the genetic diagnosis of rare diseases. However, WES/WGS fails in up to 75% of cases. Transcriptomics via RNA-sequencing (RNA-Seq) is a novel approach that aims to increase the diagnostic yield in rare diseases. Recent Findings Recent publications focus on the success of RNA-Seq for increasing diagnosis rates in WES/WGS-negative patients in up to 36% of cases, across a range of different diseases, sample sizes, and tissue types. Summary RNA-Seq is beneficial for aiding prioritisation of causative variants currently not detected or often overlooked by WES/WGS alone. An improvement in diagnostic yields has been demonstrated using multiple source tissues, with muscle and fibroblasts being the most representative, but the more accessible blood still demonstrating diagnostic success, particularly in neuromuscular disorders. The introduction of RNA-Seq to the genetic diagnosis toolbox promises to be a useful complementary tool to WES/WGS for improving genetic diagnosis in patients with rare disease.

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

Cited by 111 publications

References 54 publications

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy

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

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

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