Hereditary Cancer Diagnostics with I2HCP gene panel
ABSTRACT: This dataset contains targetted DNA sequencing data generated in the context of genetic diagnostics of hereditary cancer. Target enrichment was performed with the I2HCP, a custom hereditary cancer gene panel based in Agilent SureSelect and developed by the Germans Trias i Pujol Research Institute (IGTP, Badalona, Catalonia, Spain) and the Catalan Institute of Oncology (ICO, L'Hospitalet de Llobregat, Catalonia, Spain). After the enrichment, 108 samples were sequenced in Illumina HiSeq machines and 130 in MiSeq machines. Some of the samples were sequenced on both platforms. The dataset also includes MLPA validation data for all identified copy-number alterations, annotated with exon resolution and information about negative MLPA results.
All this data was generated in the context of routine diagnostics and compiled together with MPLA validation data for a genetic diagnostics oriented benchmark of germline CNV calling tools.
Project description:We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.
Project description:There is a need recognized by the National Institute of Dental & Craniofacial Research and the National Cancer Institute to advance basic, translational and clinical saliva research. The goal of the Salivaomics Knowledge Base (SKB) is to create a data management system and web resource constructed to support human salivaomics research. To maximize the utility of the SKB for retrieval,integration and analysis of data, we have developed the Saliva Ontology and SDxMart. This article reviews the informatics advances in saliva diagnostics made possible by the Saliva Ontology and SDxMart.
Project description:The presented paper scientifically discusses the progressive diagnostics of electrical drives in robots with sensor support. The AI (artificial intelligence) model proposed by the authors contains the technical conditions of fuzzy inference rule descriptions for the identification of a robot drive's technical condition and a source for the description of linguistic variables. The parameter of drive diagnostics for a robotized workplace that is proposed here is original and composed of the sum of vibration acceleration amplitudes ranging from a frequency of 6.3 Hz to 1250 Hz of a one-third-octave filter. Models of systems for the diagnostics of mechatronic objects in the robotized workplace are developed based on examples of CNC (Computer Numerical Control) machine diagnostics and mechatronic modules based on the fuzzy inference system, concluding with a solved example of the multi-criteria optimization of diagnostic systems. Algorithms for CNC machine diagnostics are implemented and intended only for research into precisely determined procedures for monitoring the lifetime of the mentioned mechatronic systems. Sensors for measuring the diagnostic parameters of CNC machines according to precisely determined measuring chains, together with schemes of hardware diagnostics for mechatronic systems are proposed.
Project description:CONTEXT: Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders. AIM: To study the utility of MLPA in diagnosis and carrier detection for DMD. MATERIALS AND METHODS: Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared. RESULTS AND CONCLUSIONS: We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.
Project description:Background:Conventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment. Methods:We first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set). Results:By minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis. Discussion:Our experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.
Project description:Molecular diagnostics are becoming increasingly important in clinical research to stratify or identify molecularly profiled patient cohorts for targeted therapies, to modify the dose of a therapeutic, and to assess early response to therapy or monitor patients. Molecular diagnostics can also be used to identify the pharmacogenetic risk of adverse drug reactions. The articles in this CCR Focus section on molecular diagnosis describe the development and use of markers to guide medical decisions regarding cancer patients. They define sources of preanalytic variability that need to be minimized, as well as the regulatory and financial challenges involved in developing diagnostics and integrating them into clinical practice. They also outline a National Cancer Institute program to assist diagnostic development. Molecular diagnostic clinical tests require rigor in their development and clinical validation, with sensitivity, specificity, and validity comparable to those required for the development of therapeutics. These diagnostics must be offered at a realistic cost that reflects both their clinical value and the costs associated with their development. When genome-sequencing technologies move into the clinic, they must be integrated with and traceable to current technology because they may identify more efficient and accurate approaches to drug development. In addition, regulators may define progressive drug approval for companion diagnostics that requires further evidence regarding efficacy and safety before full approval can be achieved. One way to accomplish this is to emphasize phase IV postmarketing, hypothesis-driven clinical trials with biological characterization that would permit an accurate definition of the association of low-prevalence gene alterations with toxicity or response in large cohorts.
Project description:PCR diagnostics are often the first line of laboratory diagnostics and are regularly designed to either differentiate between or detect all pathogen variants of a family, genus or species. The ideal PCR test detects all variants of the target pathogen, including newly discovered and emerging variants, while closely related pathogens and their variants should not be detected. This is challenging as pathogens show a high degree of genetic variation due to genetic drift, adaptation and evolution. Therefore, frequent re-evaluation of PCR diagnostics is needed to monitor its usefulness. Validation of PCR diagnostics recognizes three stages, in silico, in vitro and in vivo validation. In vitro and in vivo testing are usually costly, labour intensive and imply a risk of handling dangerous pathogens. In silico validation reduces this burden. In silico validation checks primers and probes by comparing their sequences with available nucleotide sequences. In recent years the amount of available sequences has dramatically increased by high throughput and deep sequencing projects. This makes in silico validation more informative, but also more computing intensive. To facilitate validation of PCR tests, a software tool named PCRv was developed. PCRv consists of a user friendly graphical user interface and coordinates the use of the software programs ClustalW and SSEARCH in order to perform in silico validation of PCR tests of different formats. Use of internal control sequences makes the analysis compliant to laboratory quality control systems. Finally, PCRv generates a validation report that includes an overview as well as a list of detailed results. In-house developed, published and OIE-recommended PCR tests were easily (re-) evaluated by use of PCRv. To demonstrate the power of PCRv, in silico validation of several PCR tests are shown and discussed.
Project description:BRCA1/2 screening in Hereditary Breast and Ovarian Syndrome (HBOC) is an essential step for effective patients' management. Next-Generation Sequencing (NGS) can rapidly provide high throughput and reliable information about the qualitative and quantitative status of tumor-associated genes. Straightforwardly, bioinformatics methods play a key role in molecular diagnostics pipelines. BRCA1/2 genes were evaluated with our NGS workflow, coupled with Multiplex Amplicon Quantification (MAQ) and Multiplex Ligation-dependent Probe Amplification (MLPA) assays. Variant calling was performed on Amplicon Suite, while Copy Number Variant (CNV) prediction by in house and commercial CNV tools, before confirmatory MAQ/MLPA testing. The germline profile of BRCA genes revealed a unique HBOC pattern. Although variant calling analysis pinpointed heterozygote and homozygote polymorphisms on BRCA1 and BRCA2, respectively, the CNV predicted by our script suggested two conflicting interpretations: BRCA1 duplication and/or BRCA2 deletion. Our commercial software reported a BRCA1 duplication, in contrast with variant calling results. Finally, the MAQ/MLPA assays assessed a whole BRCA2 copy loss. In silico CNV analysis is a time and cost-saving procedure to powerfully identify possible Large Rearrangements using robust and efficient NGS pipelines. Our layout shows as bioinformatics algorithms alone cannot completely and correctly identify whole BRCA1/2 deletions/duplications. In particular, the complete deletion of an entire gene, like in our case, cannot be solved without alternative strategies as MLPA/MAQ. These findings support the crucial role of bioinformatics in deciphering pitfalls within NGS data analysis.
Project description:AIMS:Amyloidosis is caused by deposition of abnormal protein fibrils, leading to damage of organ function. Hereditary amyloidosis represents a monogenic disease caused by germline mutations in 11 amyloidogenic precursor protein genes. One of the important but non-specific symptoms of amyloidosis is hypertrophic cardiomyopathy. Diagnostics of hereditary amyloidosis is complicated and the real cause can remain overlooked. We aimed to design hereditary amyloidosis gene panel and to introduce new next-generation sequencing (NGS) approach to investigate hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance. METHODS:Design of target enrichment DNA library preparation using Haloplex Custom Kit containing 11 amyloidogenic genes was followed by MiSeq Illumina sequencing and bioinformatics identification of germline variants using tool VarScan in a cohort of 40 patients. RESULTS:We present design of NGS panel for 11 genes (TTR, FGA, APOA1, APOA2, LYZ, GSN, CST3, PRNP, APP, B2M, ITM2B) connected to various forms of amyloidosis. We detected one mutation, which is responsible for hereditary amyloidosis. Some other single nucleotide variants are so far undescribed or rare variants or represent common polymorphisms in European population. CONCLUSIONS:We report one positive case of hereditary amyloidosis in a cohort of patients with hypertrophic cardiomyopathy of unknown significance and set up first panel for NGS in hereditary amyloidosis. This work may facilitate successful implementation of the NGS method by other researchers or clinicians and may improve the diagnostic process after validation.
Project description:Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by telangiectases and arteriovenous malformations (AVMs) in particular locations described in consensus clinical diagnostic criteria published in 2000. Two genes in the transforming growth factor-beta (TGF-?) signaling pathway, ENG and ACVRL1, were discovered almost two decades ago, and mutations in these genes have been reported to cause up to 85% of HHT. In our experience, approximately 96% of individuals with HHT have a mutation in these two genes, when published (Curaçao) diagnostic criteria for HHT are strictly applied. More recently, two additional genes in the same pathway, SMAD4 and GDF2, have been identified in a much smaller number of patients with a similar or overlapping phenotype to HHT. Yet families still exist with compelling evidence of a hereditary telangiectasia disorder, but no identifiable mutation in a known gene. Recent availability of whole exome and genome testing has created new opportunities to facilitate gene discovery, identify genetic modifiers to explain clinical variability, and potentially define an increased spectrum of hereditary telangiectasia disorders. An expanded approach to molecular diagnostics for inherited telangiectasia disorders that incorporates a multi-gene next generation sequencing (NGS) HHT panel is proposed.