PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted ...gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.
To uncover the genetic events leading to transformation of pediatric low-grade glioma (PLGG) to secondary high-grade glioma (sHGG).
We retrospectively identified patients with sHGG from a ...population-based cohort of 886 patients with PLGG with long clinical follow-up. Exome sequencing and array CGH were performed on available samples followed by detailed genetic analysis of the entire sHGG cohort. Clinical and outcome data of genetically distinct subgroups were obtained.
sHGG was observed in 2.9% of PLGGs (26 of 886 patients). Patients with sHGG had a high frequency of nonsilent somatic mutations compared with patients with primary pediatric high-grade glioma (HGG; median, 25 mutations per exome; P = .0042). Alterations in chromatin-modifying genes and telomere-maintenance pathways were commonly observed, whereas no sHGG harbored the BRAF-KIAA1549 fusion. The most recurrent alterations were BRAF V600E and CDKN2A deletion in 39% and 57% of sHGGs, respectively. Importantly, all BRAF V600E and 80% of CDKN2A alterations could be traced back to their PLGG counterparts. BRAF V600E distinguished sHGG from primary HGG (P = .0023), whereas BRAF and CDKN2A alterations were less commonly observed in PLGG that did not transform (P < .001 and P < .001 respectively). PLGGs with BRAF mutations had longer latency to transformation than wild-type PLGG (median, 6.65 years range, 3.5 to 20.3 years v 1.59 years range, 0.32 to 15.9 years, respectively; P = .0389). Furthermore, 5-year overall survival was 75% ± 15% and 29% ± 12% for children with BRAF mutant and wild-type tumors, respectively (P = .024).
BRAF V600E mutations and CDKN2A deletions constitute a clinically distinct subtype of sHGG. The prolonged course to transformation for BRAF V600E PLGGs provides an opportunity for surgical interventions, surveillance, and targeted therapies to mitigate the outcome of sHGG.
The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508. It has been predicted that Orkambi® could treat ...patients with rarer mutations of similar “theratype”; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function—similar to ΔF508‐CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient‐derived tissue. A CRISPR/Cas9‐edited bronchial epithelial cell line bearing this mutation enabled studies showing that an “amplifier” compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this “amplifier” effect was recapitulated in patient‐derived nasal cultures—providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF‐causing mutation. We propose that this multi‐disciplinary approach, including creation of CRISPR/Cas9‐edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.
Synopsis
Potential strategies for improving function in a rare CF‐causing mutation based on a CRISPR/Cas9‐edited bronchial cell line and patient‐derived nasal cultures.
Molecular dynamic simulations predicted the consequences of the rare mutation c.3700 A>G on CFTR protein (ΔI1234_R1239‐CFTR) structure.
Misprocessing and altered function of ΔI1234_R1239‐CFTR can be partially ameliorated by small molecule modulators of ΔF508‐CFTR.
A CRISPR/Cas9‐edited HBE cell line recapitulates the endogenous expression of ΔI1234_R1239‐CFTR and response to ΔF508‐CFTR modulators.
A novel small molecule amplifier (PTI‐CH) improves the effect of the corrector (VX‐809) and potentiator (VX‐770) on ΔI1234_R1239‐CFTR in CRISPR/Cas9‐engineered and patient‐specific tissues.
Potential strategies for improving function in a rare CF‐causing mutation based on a CRISPR/Cas9‐edited bronchial cell line and patient‐derived nasal cultures.
Osteosarcomas are copy number alteration (CNA)-rich malignant bone tumors. Using microarrays, fluorescence in situ hybridization, and quantitative PCR, we characterize a focal region of chr3q13.31 ...(osteo3q13.31) harboring CNAs in 80% of osteosarcomas. As such, osteo3q13.31 is the most altered region in osteosarcoma and contests the view that CNAs in osteosarcoma are nonrecurrent. Most (67%) osteo3q13.31 CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs LOC285194 and BC040587 and, in some cases, a tumor suppressor gene that encodes the limbic system-associated membrane protein (LSAMP). Ubiquitous changes occur in these genes in osteosarcoma, usually involving loss of expression. Underscoring their functional significance, expression of these genes is correlated with the presence of osteo3q13.31 CNAs. Focal osteo3q13.31 CNAs and LOH are also common in cell lines from other cancers, identifying osteo3q13.31 as a generalized candidate region for tumor suppressor genes. Osteo3q13.31 genes may function as a unit, given significant correlation in their expression despite the great genetic distances between them. In support of this notion, depleting either LSAMP or LOC285194 promoted proliferation of normal osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1. Moreover, genetic deletions of LOC285194 or BC040587 were also associated with poor survival of osteosarcoma patients. Our findings identify osteo3q13.31 as a novel region of cooperatively acting tumor suppressor genes.
The prioritization and identification of disease-causing mutations is one of the most significant challenges in medical genomics. Currently available methods address this problem for non-synonymous ...single nucleotide variants (SNVs) and variation in promoters/enhancers; however, recent research has implicated synonymous (silent) exonic mutations in a number of disorders.
We have curated 33 such variants from literature and developed the Silent Variant Analyzer (SilVA), a machine-learning approach to separate these from among a large set of rare polymorphisms. We evaluate SilVA's performance on in silico 'infection' experiments, in which we implant known disease-causing mutations into a human genome, and show that for 15 of 33 disorders, we rank the implanted mutation among the top five most deleterious ones. Furthermore, we apply the SilVA method to two additional datasets: synonymous variants associated with Meckel syndrome, and a collection of silent variants clinically observed and stratified by a molecular diagnostics laboratory, and show that SilVA is able to accurately predict the harmfulness of silent variants in these datasets.
SilVA is open source and is freely available from the project website: http://compbio.cs.toronto.edu/silva
silva-snv@cs.toronto.edu
Supplementary data are available at Bioinformatics online.
ABSTRACT
We have developed PhenoTips: open source software for collecting and analyzing phenotypic information for patients with genetic disorders. Our software combines an easy‐to‐use interface, ...compatible with any device that runs a Web browser, with a standardized database back end. The PhenoTips’ user interface closely mirrors clinician workflows so as to facilitate the recording of observations made during the patient encounter. Collected data include demographics, medical history, family history, physical and laboratory measurements, physical findings, and additional notes. Phenotypic information is represented using the Human Phenotype Ontology; however, the complexity of the ontology is hidden behind a user interface, which combines simple selection of common phenotypes with error‐tolerant, predictive search of the entire ontology. PhenoTips supports accurate diagnosis by analyzing the entered data, then suggesting additional clinical investigations and providing Online Mendelian Inheritance in Man (OMIM) links to likely disorders. By collecting, classifying, and analyzing phenotypic information during the patient encounter, PhenoTips allows for streamlining of clinic workflow, efficient data entry, improved diagnosis, standardization of collected patient phenotypes, and sharing of anonymized patient phenotype data for the study of rare disorders. Our source code and a demo version of PhenoTips are available at http://phenotips.org.
PhenoTips is an open source software package for collecting and analyzing phenotypic information for patients with genetic disorders. Our tool closely mirrors clinician workflows, facilitating the recording of observations during the patient encounter. Its easy‐to‐use interface, compatible with any device that runs a Web browser, is coupled with a standardized database where phenotypic information is represented using the Human Phenotype Ontology. In addition to data collection, PhenoTips analyzes a wide range of measurements, and provides exam and diagnosis assistance
Medulloblastoma is the prototype of treatment success in modern pediatric neuro-oncology. Unfortunately, 20% to 30% of tumors recur despite maximal resection and multimodal therapy. Multiple biologic ...prognostic markers have been investigated to predict recurrences, but controversy remains regarding their clinical utility. Because p53 immunopositivity is an adverse prognostic marker in pediatric medulloblastoma and TP53 mutations are associated with chemotherapy and radiation therapy resistance, we aimed to determine the extent and role of TP53 mutations in pediatric medulloblastoma treatment failure.
One hundred eight of 111 consecutive patients diagnosed with medulloblastoma in our institution from 1995 to 2007 were included. Median follow-up time was 5.3 years in survivors. All samples were immunostained for p53 and erbB-2. Histologic grade and immunostaining were scored by two blinded reviewers. For 49 patients, frozen material was available for TP53 sequencing. The main outcome measures were overall and progression-free survival.
Sixteen percent of sequenced medulloblastomas harbored a TP53 mutation. As a screening test, p53 immunohistochemistry was 100% sensitive and 83% specific for a TP53 mutation. Strikingly, all mutated tumors recurred early, and 5-year survival for average-risk patients was 0% for TP53-mutated medulloblastoma compared with 74% +/- 8% for wild-type medulloblastoma (P < .0001). Furthermore, 75% of recurrences in average-risk patients were associated with TP53 mutations. On multivariate analysis, TP53 mutation status was the strongest adverse prognostic factor (hazard ratio = 10.4, P = .003).
Lack of long-term survival in TP53-mutated medulloblastomas highlights the role of TP53 mutations in medulloblastoma resistance to conventional therapies and the need for alternative treatments, and prospective validation of these findings is needed.
Background and Introduction: Delivering care through telemedicine directly into the patient's home is increasingly feasible, valuable, and beneficial. However, qualitative data on how patients' and ...physicians' perceive these virtual house calls are lacking. We conducted a qualitative analysis of perceptions of these visits for Parkinson's disease to (1) determine how patients and physicians perceive virtual visits and (2) identify components contributing to positive and negative perceptions.
Qualitative survey data were collected from patients and physicians during a 12-month randomized controlled trial of virtual house calls for Parkinson's disease. Data from 149 cases were analyzed using case-based qualitative content analysis and quantitative sentiment analysis techniques.
Positive and negative perceptions of virtual visits were driven by three themes: (1) personal benefits of the virtual visit, (2) perceived quality of care, and (3) perceived quality of interpersonal engagement. In general, participants who identified greater personal benefit, high quality of care, and good interpersonal engagement perceived visits positively. Technical problems with the software were commonly mentioned. The sentiment analysis for patients was strongly favorable (+2.5) and moderately favorable for physicians (+0.8). Physician scores were lowest (-0.3) for the ability to perform a detailed motor examination remotely.
Patients and providers generally view telemedicine favorably, but individual experiences are dependent on technical issues.
Satisfaction with and effectiveness of remote care will likely increase as common technical problems are resolved.
...all known genetic predispositions will be available and, depending on the data sharing policy, accessible to a wide range of researchers and, possibly, the public at large--this, at a time when we ...are still seeking to understand the social, clinical, and personal implications of genetic information. More research and policy analysis on the issues associated with data release is clearly needed, including an analysis of the actual harms and benefits resulting from publicly accessible data; the implications for family members and relevant communities; the appropriate balance between public access and individual privacy interests; and considerations regarding compensation for research-related injury resulting from participation in personal genome research.
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