Showing papers by "Jo Vandesompele published in 2010"
TL;DR: It is shown that miR-9, which is upregulated in breast cancer cells, directly targets CDH1, the E-cadherin-encoding messenger RNA, leading to increased cell motility and invasiveness, and a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis
Abstract: β-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a ‘miRNA sponge’ in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin. Metastases are responsible for more than 90% of cancer-related mortality. These secondary growths arise through a multistep process that begins when cancer cells within primary tumours break away from neighbouring cells and invade the basement membrane 1 . This local invasion may frequently be triggered by contextual signals that carcinoma cells receive from the nearby stroma, causing them to undergo an epithelial–mesenchymal transition (EMT) 2 . Subsequently, metastasizing cells enter the circulation either directly or through lymphatics. Size constraints in the microvasculature cause many of these cells to be arrested at distant sites, where they may extravasate and enter the foreign tissue parenchyma. There they may remain dormant or, with low efficiency, proliferate from occult micrometastases to form angiogenic, clinically detectable metastases. The absence of EMT-inducing signals in the foreign microenvironment may cause such disseminated cells to revert to an epithelial phenotype by means of a mesenchymal–epithelial transition. Critical regulators of the metastatic process include both proteins and miRNAs 3,4
1,238 citations
01 Feb 2010
TL;DR: In this article, the authors uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasissuppressing protein E-cadherin.
Abstract: β-catenin signalling, which contributes to upregulated expression of the gene encoding vascular endothelial growth factor (VEGF); this leads, in turn, to increased tumour angiogenesis. Overexpression of miR-9 in otherwise non-metastatic breast tumour cells enables these cells to form pulmonary micrometastases in mice. Conversely, inhibiting miR-9 by using a ‘miRNA sponge’ in highly malignant cells inhibits metastasis formation. Expression of miR-9 is activated by MYC and MYCN, both of which directly bind to the mir-9-3 locus. Significantly, in human cancers, miR-9 levels correlate with MYCN amplification, tumour grade and metastatic status. These findings uncover a regulatory and signalling pathway involving a metastasis-promoting miRNA that is predicted to directly target expression of the key metastasis-suppressing protein E-cadherin. Metastases are responsible for more than 90% of cancer-related mortality. These secondary growths arise through a multistep process that begins when cancer cells within primary tumours break away from neighbouring cells and invade the basement membrane 1 . This local invasion may frequently be triggered by contextual signals that carcinoma cells receive from the nearby stroma, causing them to undergo an epithelial–mesenchymal transition (EMT) 2 . Subsequently, metastasizing cells enter the circulation either directly or through lymphatics. Size constraints in the microvasculature cause many of these cells to be arrested at distant sites, where they may extravasate and enter the foreign tissue parenchyma. There they may remain dormant or, with low efficiency, proliferate from occult micrometastases to form angiogenic, clinically detectable metastases. The absence of EMT-inducing signals in the foreign microenvironment may cause such disseminated cells to revert to an epithelial phenotype by means of a mesenchymal–epithelial transition. Critical regulators of the metastatic process include both proteins and miRNAs 3,4
1,124 citations
TL;DR: The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting.
648 citations
TL;DR: This chapter provides all relevant information for a successfully implement of qPCR-based copy number analysis, and suggests an appropriate and practical way to calculate copy numbers and to objectively interpret the results.
317 citations
TL;DR: In this paper, the miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive, and the effects of activation of the microRNA clusters on global protein expression in neuroblastoma (NB) cells are examined.
269 citations
TL;DR: A meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression found Chromosome 2p gains were associated with a significantly increased ALK expression, which was also correlated with poor survival.
Abstract: Purpose Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. Experimental design The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. Results ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. Conclusions ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.
249 citations
TL;DR: This study demonstrates that the top six most stably expressed miRNAs described herein should be validated as suitable reference genes in both high-throughput and lower throughput RT-qPCR colorectal miRNA studies.
Abstract: Background
Advances in high-throughput technologies and bioinformatics have transformed gene expression profiling methodologies. The results of microarray experiments are often validated using reverse transcription quantitative PCR (RT-qPCR), which is the most sensitive and reproducible method to quantify gene expression. Appropriate normalisation of RT-qPCR data using stably expressed reference genes is critical to ensure accurate and reliable results. Mi(cro)RNA expression profiles have been shown to be more accurate in disease classification than mRNA expression profiles. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in miRNA RT-qPCR studies.
225 citations
Journal Article•
TL;DR: In-depth sequence analysis revealed extensive post-transcriptional miRNA editing in Neuroblastoma patients, and the putative tumor suppressive microRNAs, miR-542-5p and mi-628, were expressed in favorable NBs and virtually absent in unfavorable NBs.
Abstract: Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.
181 citations
TL;DR: In this paper, the authors analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads.
Abstract: Small non-coding RNAs, in particular microRNAs(miRNAs), regulate fine-tuning of gene expression and can act as oncogenes or tumor suppressor genes. Differential miRNA expression has been reported to be of functional relevance for tumor biology. Using next-generation sequencing, the unbiased and absolute quantification of the small RNA transcriptome is now feasible. Neuroblastoma(NB) is an embryonal tumor with highly variable clinical course. We analyzed the small RNA transcriptomes of five favorable and five unfavorable NBs using SOLiD next-generation sequencing, generating a total of >188 000 000 reads. MiRNA expression profiles obtained by deep sequencing correlated well with real-time PCR data. Cluster analysis differentiated between favorable and unfavorable NBs, and the miRNA transcriptomes of these two groups were significantly different. Oncogenic miRNAs of the miR17-92 cluster and the miR-181 family were overexpressed in unfavorable NBs. In contrast, the putative tumor suppressive microRNAs, miR-542-5p and miR-628, were expressed in favorable NBs and virtually absent in unfavorable NBs. In-depth sequence analysis revealed extensive post-transcriptional miRNA editing. Of 13 identified novel miRNAs, three were further analyzed, and expression could be confirmed in a cohort of 70 NBs.
170 citations
TL;DR: It is shown that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background and concludes that Dicer 1 functions as a haploinsufficient tumor suppressor.
Abstract: Human tumors are characterized by widespread reduction in microRNA (miRNA) expression, although it is unclear how such changes come about and whether they have an etiological role in the disease. Importantly, miRNA knockdown has been shown to enhance the tumorigenic potential of human lung adenocarcinoma cells. A defect in miRNA processing is one possible mechanism for global downregulation. To explore this possibility in more detail in vivo, we have manipulated Dicer1 gene dosage in a mouse model of retinoblastoma. We show that although monoallelic loss of Dicer1 does not affect normal retinal development, it dramatically accelerates tumor formation on a retinoblastoma-sensitized background. Importantly, these tumors retain one wild-type Dicer1 allele and exhibit only a partial decrease in miRNA processing. Accordingly, in silico analysis of human cancer genome data reveals frequent hemizygous, but not homozygous, deletions of DICER1. Strikingly, complete loss of Dicer1 function in mice did not accelerate retinoblastoma formation. miRNA profiling of these tumors identified members of the let-7 and miR-34 families as candidate tumor suppressors in retinoblastoma. We conclude that Dicer1 functions as a haploinsufficient tumor suppressor. This finding has implications for cancer etiology and cancer therapy.
169 citations
TL;DR: For the first time, a T-UCR expression landscape in neuroblastoma is defined and widespread T- UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis is suggested.
Abstract: Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.
TL;DR: A widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation is uncovered and further substantiate the overlapping functions of c- MYC and MYCN in the process of tumorigenesis.
Abstract: Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.
TL;DR: Gene expression-based classification using the 144-gene PAM predictor can contribute to improved treatment stratification of neuroblastoma patients.
Abstract: Purpose To evaluate the impact of a predefined gene expression– based classifier for clinical risk estimation and cytotoxic treatment decision making in neuroblastoma patients. Patients and Methods Gene expression profiles of 440 internationally collected neuroblastoma specimens were investigated by microarray analysis, 125 of which were examined prospectively. Patients were classified as either favorable or unfavorable by a 144-gene prediction analysis for microarrays (PAM) classifier established previously on a separate set of 77 patients. PAM classification results were compared with those of current prognostic markers and risk estimation strategies. Results The PAM classifier reliably distinguished patients with contrasting clinical courses (favorable [n 249] and unfavorable [n 191]; 5-year event free survival [EFS] 0.84 0.03 v 0.38 0.04; 5-year overall survival [OS] 0.98 0.01 v 0.56 0.05, respectively; both P .001). Moreover, patients with divergent outcome were robustly discriminated in both German and international cohorts and in prospectively analyzed samples (P .001 for both EFS and OS for each). In subgroups with clinical low-, intermediate-, and high-risk of death from disease, the PAM predictor significantly separated patients with divergent outcome (low-risk 5-year OS: 1.0 v 0.75 0.10, P .001; intermediaterisk: 1.0 v 0.82 0.08, P .042; and high-risk: 0.81 0.08 v 0.43 0.05, P .001). In multivariate Cox regression models based on both EFS and OS, PAM was a significant independent prognostic marker (EFS: hazard ratio [HR], 3.375; 95% CI, 2.075 to 5.492; P .001; OS: HR, 11.119, 95% CI, 2.487 to 49.701; P .001). The highest potential clinical impact of the classifier was observed in patients currently considered as non– high-risk (n 289; 5-year EFS: 0.87 0.02 v 0.44 0.07; 5-year OS: 1.0 v 0.80 0.06; both P .001).
TL;DR: It is concluded that neuroblastoma patient outcome prediction using miRNA expression is feasible and effective and studies testing miRNA‐based predictors in comparison to and in combination with mRNA and aCGH information should be initiated.
Abstract: For neuroblastoma, the most common extracranial tumour of childhood, identification of new biomarkers and potential therapeutic targets is mandatory to improve risk stratification and survival rates. MicroRNAs are deregulated in most cancers, including neuroblastoma. In this study, we analysed 430 miRNAs in 69 neuroblastomas by stem-loop RT-qPCR. Prediction of event-free survival (EFS) with support vector machines (SVM) and actual survival times with Cox regression-based models (CASPAR) were highly accurate and were independently validated. SVM-accuracy for prediction of EFS was 88.7% (95% CI: 88.5-88.8%). For CASPAR-based predictions, 5y-EFS probability was 0.19% (95% CI: 0-38%) in the CASPAR-predicted short survival group compared with 0.78% (95%CI: 64-93%) in the CASPAR-predicted long survival group. Both classifiers were validated on an independent test set yielding accuracies of 94.74% (SVM) and 5y-EFS probabilities as 0.25 (95% CI: 0.0-0.55) for short versus 1 ± 0.0 for long survival (CASPAR), respectively. Amplification of the MYCN oncogene was highly correlated with deregulation of miRNA expression. In addition, 37 miRNAs correlated with TrkA expression, a marker of excellent outcome, and 6 miRNAs further analysed in vitro were regulated upon TrkA transfection, suggesting a functional relationship. Expression of the most significant TrkA-correlated miRNA, miR-542-5p, also discriminated between local and metastatic disease and was inversely correlated with MYCN amplification and event-free survival. We conclude that neuroblastoma patient outcome prediction using miRNA expression is feasible and effective. Studies testing miRNA-based predictors in comparison to and in combination with mRNA and aCGH information should be initiated. Specific miRNAs (e.g., miR-542-5p) might be important in neuroblastoma tumour biology, and qualify as potential therapeutic targets.
TL;DR: The robustness and accuracy of the classifier enables prospective assessment of neuroblastoma patient outcome and might be an example for development and validation of robust gene expression signatures in other cancer entities.
Abstract: Purpose: Reliable prognostic stratification remains a challenge for cancer patients, especially for diseases with variable clinical course such as neuroblastoma. Although numerous studies have shown that outcome might be predicted using gene expression signatures, independent cross-platform validation is often lacking. Experimental Design: Using eight independent studies comprising 933 neuroblastoma patients, a prognostic gene expression classifier was developed, trained, tested, and validated. The classifier was established based on reanalysis of four published studies with updated clinical information, reannotation of the probe sequences, common risk definition for training cases, and a single method for gene selection (prediction analysis of microarray) and classification (correlation analysis). Results: Based on 250 training samples from four published microarray data sets, a correlation signature was built using 42 robust prognostic genes. The resulting classifier was validated on 351 patients from four independent and unpublished data sets and on 129 remaining test samples from the published studies. Patients with divergent outcome in the total cohort, as well as in the different risk groups, were accurately classified (log-rank P 5). Conclusion: The strength of this 42-gene classifier is its small number of genes and its cross-platform validity in which it outperforms other published prognostic signatures. The robustness and accuracy of the classifier enables prospective assessment of neuroblastoma patient outcome. Most importantly, this gene selection procedure might be an example for development and validation of robust gene expression signatures in other cancer entities. Clin Cancer Res; 16(5); 1532–41
TL;DR: A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of event-free survival and overall survival outcomes than 11q status by itself, improving patient stratification.
Abstract: Purpose: The purpose of this study was to further define the biology of the 11q− neuroblastoma tumor subgroup by the integration of array-based comparative genomic hybridization with microRNA (miRNA) expression profiling data to determine if improved patient stratification is possible. Experimental Design: A set of primary neuroblastoma ( n = 160), which was broadly representative of all genetic subtypes, was analyzed by array-based comparative genomic hybridization and for the expression of 430 miRNAs. A 15-miRNA expression signature previously shown to be predictive of clinical outcome was used to analyze an independent cohort of 11q− tumors ( n = 37). Results: Loss of 4p and gain of 7q occurred at a significantly higher frequency in the 11q− tumors, further defining the genetic characteristics of this subtype. The 11q− tumors could be split into two subgroups using a miRNA expression survival signature that differed significantly in clinical outcome and the overall frequency of large-scale genomic imbalances, with the poor survival subgroup having significantly more imbalances. miRNAs from the expression signature, which were upregulated in unfavorable tumors, were predicted to target downregulated genes from a published mRNA expression classifier of clinical outcome at a higher-than-expected frequency, indicating the miRNAs might contribute to the regulation of genes within the signature. Conclusion: We show that two distinct biological subtypes of neuroblastoma with loss of 11q occur, which differ in their miRNA expression profiles, frequency of segmental imbalances, and clinical outcome. A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of event-free survival and overall survival outcomes than 11q status by itself, improving patient stratification. Clin Cancer Res; 16(11); 2971–8. ©2010 AACR.
TL;DR: The findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.
Abstract: Immunological and genetic findings implicate Th17 effector cytokines in the pathogenesis of inflammatory bowel disease (IBD). Expression of Th17 pathway-associated genes is mainly studied in colonic disease. The present study assessed the mRNA expression levels of Th17 effector cytokines (IL17A, IL17F, IL21, IL22 and IL26) and genes involved in differentiation (IL6, IL1B, TGFB1, IL23A and STAT3) and recruitment of Th17 cells (CCR6 and CCL20) by quantitative real-time PCR analysis of colonic and ileal biopsies from 22 healthy control subjects, 26 patients with Crohn's disease (CD) and 12 patients with ulcerative colitis (UC). Inflammation was quantified by measuring expression of the inflammatory mediators IL8 and TNF. Evaluation of mRNA expression levels in colonic and ileal control samples revealed that TNF, TGFB1, STAT3 and CCR6 were expressed at higher levels in the ileum than in the colon. Expression of all the Th17 pathway-associated genes was increased in inflamed colonic samples. The increased expression of these genes was predominantly observed in samples from UC patients and was associated with more intense inflammation. Although increased expression of IL17A, IL17F, IL21 and IL26 was detected in inflamed ileal samples, expression of the indispensable Th17 cell differentiation factors TGFB1 and IL23A, the signaling molecule STAT3 and the Th17 recruitment factors CCR6 and CCL20 were unchanged. Our findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.
TL;DR: Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations.
Abstract: Cancer genomes display characteristic patterns of chromosomal imbalances, often with diagnostic and prognostic relevance. Therefore assays for genome-wide copy number screening and simultaneous detection of copy number alterations in specific chromosomal regions are of increasing importance in the diagnostic work-up of tumors. We tested the performance of Multiplex Amplicon Quantification, a newly developed low-cost, closed-tube and high-throughput PCR-based technique for detection of copy number alterations in regions with prognostic relevance for neuroblastoma. Comparison with array CGH and the established Multiplex Ligation-dependent Probe Amplification method on 52 neuroblastoma tumors showed that Multiplex Amplicon Quantification can reliably detect the important genomic aberrations. Multiplex Amplicon Quantification is a low-cost and high-throughput PCR-based technique that can reliably detect copy number alterations in regions with prognostic relevance for neuroblastoma.
Journal Article•
TL;DR: In this article, the authors proposed new prognostic markers, including copy number aberrations, gene expression signatures, and epigenetic markers, for improved risk estimation at the time of diagnosis and to improve the choice of risk related therapy.
Abstract: One of the main challenges in clinical cancer research remains to be accurate outcome prediction at the time of diagnosis. Although not frequent in absolute terms, neuroblastoma represents an important clinical challenge, as it is fatal in almost half of the patients despite advances in multimodal anti-cancer therapies. Four major risk stratification systems for neuroblastoma patients are currently being used in various parts of the world. Systems are based on a combination of various clinical, histopathological, and biological factors. Accordingly, different therapeutic schemes exist ranging from wait-and-see approaches to intensive multimodal therapies. Clinical experience with the currently used risk stratification systems suggests that the stratification of patients for treatment is useful, but patients with the same clinico-pathological parameters, receiving the same treatment, can have markedly different clinical courses. Therefore, the challenge remains to identify additional tumor-specific and sensitive prognostic markers for improved risk estimation at the time of diagnosis and to improve the choice of risk-related therapy. Various studies have put forward new prognostic markers, including copy number aberrations, gene expression signatures, and epigenetic markers.
TL;DR: An open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements and a modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system.
Abstract: Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions. We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data. We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.
TL;DR: A highly validated qPCR-based copy number profiling test that supersedes other approaches for copy number screening of the SHOX region in terms of reliability, accuracy, and cost efficiency is developed.
Abstract: Context: Short stature has an incidence of three in 100 in children. Reliable molecular genetic testing may be crucial in the context of beneficial disease management. Deletions spanning or surrounding the SHOX gene account for a significant proportion of patients with idiopathic short stature (ISS) and allied disorders, such as Leri-Weill dyschondrosteosis. Objective: Several shortcomings of current strategies for copy number profiling of the SHOX region prompted us to develop an improved test for molecular diagnostics of the SHOX region. Design and Results: We introduced a quantitative PCR (qPCR)-based copy number profiling test, consisting of 11 amplicons targeting clinically relevant regions, i.e. the SHOX gene and regulatory regions. To ensure an optimal sensitivity and specificity, this test was validated in 32 controls and 18 probands with previously identified copy number changes. In addition, 152 probands with SHOX-associated phenotypes were screened, revealing 10 novel copy number changes. Concl...
TL;DR: MethGraph is a web application for easy, fast and flexible visualization and accurate in silico quality evaluation of PCR-based methylation assays, and can easily be customized and exported for use in publications or presentations.
Abstract: Abnormalities in DNA methylation of CpG islands that play a role in gene regulation affect gene expression and hence play a role in disease, including cancer. Bisulfite-based DNA methylation analysis methods such as methylation-specific PCR (MSP) and bisulfite sequencing (BiSeq) are most commonly used to study gene-specific DNA methylation. Assessing specificity and visualizing the position of PCR primers in their genomic context is a laborious and tedious task, primarily due to the sequence changes induced during the bisulfite conversion. For this purpose, we developed methGraph, a web application for easy, fast and flexible visualization and accurate in silico quality evaluation of PCR-based methylation assays. The visualization process starts by submitting PCR primer sequences for specificity assessment and mapping on the genome using the BiSearch ePCR primer-search algorithm. The next step comprises the selection of relevant UCSC genome annotation tracks for display in the final graph. A custom track showing all individual CpG dinucleotides, representing their distribution in the CpG island is also provided. Finally, methGraph creates a BED file that is automatically uploaded to the UCSC genome browser, after which the resulting image files are extracted and made available for visualization and download. The generated high-quality figures can easily be customized and exported for use in publications or presentations. methGraph is available at http://mellfire.ugent.be/methgraph/.