Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be ...challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA‐seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease‐causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin‐fibroblast‐based transcriptome analysis for molecular diagnostics. RNA‐seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA‐based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant.
For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. ...Transcriptome analysis (RNA sequencing RNA-seq) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals’ cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual’s specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics.
We present a user-friendly web application to detect pathogenic outlier gene expression and mRNA splicing in transcriptome data. Application of this pipeline in a cohort of 67 undiagnosed individuals with neurodevelopmental disorders increased the diagnostic yield by 13%. This approach will facilitate wider implementation of RNA-seq in routine genome diagnostics.
Background
We aimed to evaluate the association between public media and trends in new presentations of acute coronary syndrome (ACS) during the first wave of the coronavirus disease 2019 (COVID‑19) ...in the Netherlands.
Methods
New ACS presentations per week in 73 hospitals during the first half of 2019 and 2020 were retrieved from the national organisation Dutch Hospital Data and incidence rates were calculated. Stratified analyses were performed by region, type of ACS and patient characteristics.
Results
After the first confirmed COVID‑19 case and during lockdown, numbers declined by up to 41% (95% confidence interval (CI): 36–47%) compared to 2019. This reduction was more pronounced for non-ST-segment elevation myocardial infarction (NSTEMI) (48%; 95% CI: 39–55%) and unstable angina (UA; 50%; 95% CI: 40–59%) than for STEMI (34%; 95% CI: 23–43%). There was no association between ACS and COVID‑19 incidence rate per region. After the steep decline, a public campaign encouraged patients not to postpone hospital visits. Numbers then increased, without a rebound effect. Trends were similar irrespective of sex, age or socio-economic status. During the outbreak, compared to coronary artery bypass graft procedures, relatively more (acute) percutaneous coronary interventions for NSTEMI and UA were performed.
Conclusion
New ACS presentations decreased by up to 41%. Lockdown measures and public campaigns, rather than COVID‑19 incidence, were associated with significant changes in new ACS presentations. Even though causality cannot be established, this emphasises the role of the public media and healthcare organisations in informing patients to prevent underdiagnoses of ACS and associated health damage.
Background and Purpose
Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure, but its effects in hypertensive diabetes are unknown.
Experimental Approach
To address ...this, TGR (mRen2)27 rats (angiotensin II‐dependent hypertension model) were made diabetic with streptozotocin over 18 weeks and treated with either vehicle, angiotensinogen siRNA, the AT1 antagonist valsartan, the ACE inhibitor captopril, valsartan + siRNA or valsartan + captopril for the final 3 weeks. Mean arterial pressure (MAP) was measured via radiotelemetry.
Key Results
MAP before treatment was 153 ± 2 mmHg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a change in glomerular filtration rate. All treatments lowered MAP and cardiac hypertrophy, and the largest drop in MAP was observed with siRNA + valsartan. Treatment with siRNA lowered circulating angiotensinogen by >99%, and the lowest circulating angiotensin II and aldosterone levels occurred in the dual treatment groups. Angiotensinogen siRNA did not affect renal angiotensinogen mRNA expression, confirming its liver‐specificity. Furthermore, only siRNA with or without valsartan lowered renal angiotensin I. All treatments lowered renal angiotensin II and the reduction was largest (>95%) in the siRNA + valsartan group. All treatments identically lowered albuminuria, whereas only siRNA with or without valsartan restored podocyte foot processes and reduced glomerulosclerosis.
Conclusion and Implications
Angiotensinogen siRNA exerts renoprotection in diabetic TGR (mRen2)27 rats and this relies, at least in part, on the suppression of renal angiotensin II formation from liver‐derived angiotensinogen. Clinical trials should now address whether this is also beneficial in human diabetic kidney disease.
Secretin-stimulated pancreatic juice (PJ), collected from the duodenum, presents a valuable biomarker source for the (earlier) detection of pancreatic cancer (PC). Here, we evaluate the feasibility ...and performance of shallow sequencing to detect copy number variations (CNVs) in cell-free DNA (cfDNA) from PJ for PC detection. First, we confirmed the feasibility of shallow sequencing in PJ (n = 4), matched plasma (n = 3) and tissue samples (n = 4, microarray). Subsequently, shallow sequencing was performed on cfDNA from PJ of 26 cases (25 sporadic PC, 1 high-grade dysplasia) and 19 controls with a hereditary or familial increased risk of PC. 40 of the 45 PJ samples met the quality criteria for cfDNA analysis. Nine individuals had an 8q24 gain (oncogene MYC; 23%; eight cases (33%) and one control (6%),
= 0.04); six had both a 2q gain (STAT1) and 5p loss (CDH10; 15%; four cases (7%) and two controls (13%),
= 0.72). The presence of an 8q24 gain differentiated the cases and controls, with a sensitivity of 33% (95% CI 16-55%) and specificity of 94% (95% CI 70-100%). The presence of either an 8q24 or 2q gain with a 5p loss was related to a sensitivity of 50% (95% CI 29-71%) and specificity of 81% (95% CI 54-96%). Shallow sequencing of PJ is feasible. The presence of an 8q24 gain in PJ shows promise as a biomarker for the detection of PC. Further research is required with a larger sample size and consecutively collected samples in high-risk individuals prior to implementation in a surveillance cohort.
Uveal melanoma (UM) has a high propensity to metastasize. Prognosis is associated with specific driver mutations and copy number variations (CNVs), but limited primary tumor tissue is available for ...molecular characterization due to eye-sparing irradiation treatment. This study aimed to assess the rise in circulating tumor DNA (ctDNA) levels in UM and evaluate its efficacy for CNV-profiling of patients with UM.
In a pilot study, we assessed ctDNA levels in the blood of patients with UM (n = 18) at various time points, including the time of diagnosis (n = 13), during fractionated stereotactic radiotherapy (fSRT) treatment (n = 6), and upon detection of metastatic disease (n = 13). Shallow whole-genome sequencing (sWGS) combined with in silico size-selection was used to identify prognostically relevant CNVs in patients with UM (n = 26) from peripheral blood retrieved at the time of diagnosis (n = 9), during fSRT (n = 5), during post-treatment follow-up (n = 4), metastasis detection (n = 6), and metastasis follow-up (n = 4).
A total of 34 patients had blood analyzed for ctDNA detection (n = 18) and/or CNV analysis (n = 26) at various time points. At the time of diagnosis, 5 of 13 patients (38%) had detectable ctDNA (median = 0 copies/mL). Upon detection of metastatic disease, ctDNA was detected in 10 of 13 patients (77%) and showed increased ctDNA levels (median = 24 copies/mL, P < 0.01). Among the six patients analyzed during fSRT, three (50%) patients had detectable ctDNA at baseline and three of six (50%) patients had undetectable levels of ctDNA. During the fSRT regimen, ctDNA levels remained unchanged (P > 0.05). The ctDNA fractions were undetectable to low in localized disease, and sWGS did not elucidate chromosome 3 status from blood samples. However, in 7 of 10 (70%) patients with metastases, the detection of chromosome 3 loss corresponded to the high metastatic-risk class.
The rise in ctDNA levels observed in patients with UM harboring metastases suggests its potential utility for CNV profiling. These findings highlight the potential of using ctDNA for metastasis detection and patient inclusion in therapeutic studies targeting metastatic UM.
Aim
The aim of this article is to present the method and results of the data quality control system and audit within the Netherlands Heart Registration (NHR) using data of patients treated with ...percutaneous coronary intervention (PCI) in the Netherlands as an example.
Methods
The NHR is a Dutch nationwide registry of all cardiac interventions, comprising data from all 71 hospitals, of which 30 are cardiac intervention or heart centres. Each year, within the NHR, data validation and verification is performed by standard quality controls and monitoring visits (audits). For the audit in 2019, a sample of 50–100 medical records of patients treated with PCI in 2016 and 2017 were reviewed in each hospital by an independent auditor. The data received by the NHR were compared with the information in the hospitals’ medical records. In total 12 patient characteristics, 5 intervention variables and 3 outcome variables were screened. The value of a variable was considered discrepant if more than 10% of the medical records reviewed regarding this variable were not consistent with the reported data received by the NHR.
Results
For all variables together, the consistency was high, 97.6%. All variables, except multivessel disease (9.3% discrepancy in the 2622 medical records reviewed), had an accuracy above 95%.
Conclusion
The results of the audit of the PCI medical records show that the overall quality of the data is high. For variables such as multivessel disease it is important to improve knowledge of the definitions and to train all those involved in the registration process.
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