RNA Sequencing as a Diagnostic Tool

Ketkar, Shamika; Burrage, Lindsay C.; Lee, Brendan

doi:10.1001/jama.2022.22843

Cited by 11 publications

(9 citation statements)

References 5 publications

(15 reference statements)

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“…When implementing RNA-seq as an adjunct to DNA sequencing in rare disease clinical diagnostics, many factors should be considered in addition to analytical validity, including clinical validity, clinical utility, cost and turnaround time (TAT) (25). For example, we noted that the operation of the modified polyA+ method is simpler than that of the ribo-minus method, with a shorter processing time per sample by an average of 0.5 hours.…”

Section: Discussionmentioning

confidence: 99%

Systematic evaluation of the two main blood-based RNA-seq approaches for Mendelian disease diagnosis

Yang

Chen

et al. 2023

Preprint

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Background: As an adjunct to diagnostic exome sequencing and whole genome sequencing, RNA sequencing (RNAseq) has been demonstrated to improve diagnostic yield for Mendelian diseases. However, systematic evaluation of the associated experimental and computational processes and the establishment of robust and efficient practices for RNA diagnostics implemented in the clinic to analyse readily accessible whole blood samples are still required. Methods: We simulated clinical conditions in which each patients sample is tested only once, and we evaluated the two typical experimental protocols (polyA selection and rRNA depletion) by comparing the expression profiles, aberrant splicing events and monoallelic expression (MAE) identified from 11 patients in clinical settings with different bioinformatics software. Results: We demonstrated that a higher proportion of unique reads from polyA selection than rRNA depletion were mapped to exons or exon_intron junction regions (84.54% vs. 40.14%), resulting in more detectable OMIM genes (TPM > 1) in the blood (65.29% vs. 59.79%); thus, the rRNA depletion method requires a median of 258 more valid reads per gene to achieve the same level of gene quantification. Moreover, although the transcriptome profiling of protein-coding genes in the two methods is highly correlated, polyA-selection offers more sensitive detection of MAE variants and aberrant splicing under common filtering conditions in combination with DROP. Conclusions: A combination of polyA+ and DROP is recommended when implementing blood-based RNA-seq for the diagnosis of Mendelian diseases in clinical practice, and filtering criteria for aberrant expression, aberrant splicing and MAE variants are suggested for reference.

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Section: Discussionmentioning

confidence: 99%

Systematic evaluation of the two main blood-based RNA-seq approaches for Mendelian disease diagnosis

Yang

Chen

et al. 2023

Preprint

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“…15 Among genetic diagnostic tools, RNA-seq is relatively low cost and high throughput, and is increasingly being used as an adjunct to exome sequencing. 25 The clinical use of RNA-seq to evaluate variants with uncertain pathological significance has been demonstrated in Mendelian disorders. 12 In patients with undiagnosed genetic diseases, RNA-seq has improved diagnostic yield by 7.5% to 36% through evidencing the pathogenicity of variants previously misinterpreted or missed by exome or genome sequencing.…”

Section: Discussionmentioning

confidence: 99%

“…Among genetic diagnostic tools, RNA‐seq is relatively low cost and high throughput, and is increasingly being used as an adjunct to exome sequencing 25 . The clinical use of RNA‐seq to evaluate variants with uncertain pathological significance has been demonstrated in Mendelian disorders 12 .…”

Section: Discussionmentioning

confidence: 99%

Alu Retrotransposition Event in SPAST Gene as a Novel Cause of Hereditary Spastic Paraplegia

et al. 2023

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ObjectivesTo diagnose the molecular cause of hereditary spastic paraplegia (HSP) observed in a four‐generation family with autosomal dominant inheritance.MethodsMultiplex ligation‐dependent probe amplification (MLPA), whole‐exome sequencing (WES), and RNA sequencing (RNA‐seq) of peripheral blood leukocytes were performed. Reverse transcription polymerase chain reaction (RT‐PCR) and Sanger sequencing were used to characterize target regions of SPAST.ResultsA 121‐bp AluYb9 insertion with a 30‐bp poly‐A tail flanked by 15‐bp direct repeats on both sides was identified in the edge of intron 16 in SPAST that segregated with the disease phenotype.ConclusionsWe identified an intronic AluYb9 insertion inducing splicing alteration in SPAST causing pure HSP phenotype that was not detected by routine WES analysis. Our findings suggest RNA‐seq is a recommended implementation for undiagnosed cases by first‐line diagnostic approaches. © 2023 International Parkinson and Movement Disorder Society.

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“…However, the transcriptome is specific to tissues/organs, and some genes cannot be detected in clinically accessible tissues such as blood, skin fibroblast cells, or simple tissue samples. Therefore, only variants with splicing damage potential in a particular group of genes can be validated using RT‐PCR or RNA‐seq (Ketkar et al., 2023). Minigenes can be used to investigate splice errors in vitro but may not reflect splicing events in vivo.…”

Section: Discussionmentioning

confidence: 99%

Prenatal diagnosis and preimplantation genetics testing of 3M syndrome in a Chinese family with novel biallelic variants of CUL7

Wang,

He,

Wang

et al. 2023

Molec Gen & Gen Med

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Background3M syndrome is a rare autosomal recessive developmental disorder characterized by pre and postnatal growth deficiency, dysmorphic facial features, and normal intelligence. 3M syndrome should be suspected in a proband with a combination of characteristic or recognizable dysmorphic features. The diagnosis of 3M syndrome could be confirmed by identifying biallelic variants in CUL7, OBSL1, or CCDC8.MethodsWhole‐exome sequencing (WES) was performed to identify genetic causes. Reverse‐transcription polymerase chain reaction (RT‐PCR) was performed to detect aberrant splicing events. Haplotypes were constructed using multiplex PCR and sequencing. Variants of the parental haplotype and target likely pathogenic variants were detected by PCR and Sanger sequencing from the embryos. Copy number variant (CNV) detection was performed by next‐generation sequencing.ResultsWe present the case of a nonconsanguineous Chinese couple with one abnormal pregnancy, where the fetus showed 3M phenotypes of shortened long bones. WES identified two novel heterozygous mutations in CUL7: NM_014780.5:c.354del (p.Gln119ArgfsTer52) and NM_014780.5:c.1373‐15G>A. RT‐PCR from RNA of the mother's peripheral blood leucocytes showed that c.1373‐15G>A caused the insertion of a 13‐bp extra intron sequence and encoded the mutant p.Leu459ProfsTer25. Both variants were classified as likely pathogenic according to ACMG/AMP guidelines and Clinical Genome Resource specifications. During genetic counseling, the options of prenatal diagnosis through chorionic villus sampling or amniocentesis, adoption, sperm donation, and electing not to reproduce, as well as preimplantation genetic testing for monogenic disorders (PGT‐M), were discussed. The couple hopes to conceive a child of their own and refused to accept the 25% risk during the next pregnancy and opted for PGT‐M. They finally successfully delivered a healthy baby through PGT‐M.ConclusionThis study expanded the mutation spectrum of CUL7, detected the aberrant splicing event of CUL7 via RT‐PCR, constructed the haplotype for PGT‐M, and demonstrated the successful delivery of a healthy baby using PGT‐M.

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RNA Sequencing as a Diagnostic Tool

Abstract: This JAMA Insights discusses RNA sequencing, which allows detection of qualitative and quantitative changes in RNA expression across the genome in clinical samples, and how it may improve molecular diagnostic rates achieved by diagnostic exome sequencing and whole-genome sequencing alone.

Cited by 11 publications

References 5 publications

Systematic evaluation of the two main blood-based RNA-seq approaches for Mendelian disease diagnosis

Systematic evaluation of the two main blood-based RNA-seq approaches for Mendelian disease diagnosis

Alu Retrotransposition Event in SPAST Gene as a Novel Cause of Hereditary Spastic Paraplegia

Prenatal diagnosis and preimplantation genetics testing of 3M syndrome in a Chinese family with novel biallelic variants of CUL7

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