Background
Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics.
Methods
We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.
Results
We detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions.
Conclusion
Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.
Lack of functional evidence hampers variant interpretation, leaving a large proportion of cases with a suspected Mendelian disorder without genetic diagnosis after genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies, and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA-sequencing (RNA-seq) in routine diagnostics. To address these issues, we implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease. We detected on average 12,500 genes per sample including around 60% disease genes - a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than one week from sample preparation to result reporting and provided a median of eight disease genes per patient for inspection. A genetic diagnosis was established for 16% of the WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.
Among 15 patients with acute thrombotic disease of pelvic veins who had been submitted to operative thrombectomy and creation of arteriovenous fistula in the groin, 12 presented with stenotic lesions 3 months later. These stenoses were submitted to percutaneous angioplasty. If angioplasty failed, percutaneous placement of a vascular stent (wall stent) was performed immediately (n = 7). Stenting in cross-over-technique proved practicable in all cases. Secondary stenotic disease in the exclusively dilated area was observed in 3/5 cases and was also treated with a wall stent. In one patient with recurrent stenoses who refused stenting, extended thrombosis occurred after occlusion of the AV-fistula. At mid-term PTA was successful in only two cases. Intimal hyperplasia was observed in only one wall stent treated patient. Percutaneous treatment of iliacal stenoses in patients with postthrombotic syndrome may be performed safely under the protective effect of the fistula. With the presented technique, patency of pelvic veins could be restored in 11/12 patients with postoperative significant venous stenoses.
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