Agency: European Commission | Branch: FP7 | Program: CP | Phase: ICT-2011.3.2 | Award Amount: 10.96M | Year: 2012
This project will develop a family of In-Vitro Diagnostic Systems that will transfer for the first time novel Non Invasive Prenatal Diagnostic (NIPD) methods in tube to a LabonaChip format. This family will consist of three research systems chosen to cover all prenatal diagnosis models:\n- ANGELAB1. This system will use a LabonaChip to extract fetal DNA from the mothers plasma based on differences in methylated pattern between fetus and mother and captured by specific magnetophoresis, plus qPCR for monogenic diseases with a known mutation for: Spinal Muscular Atrophy, Cystic Fibrosis (CF), and X-linked disorders. It will be used only for high risk population.\n- ANGELAB2. It will use a LabonaChip to carry out immunoprecipitation (MeDIP) for fetal DNA extraction from mothers blood and real time quantitative Polymerase Chain Reaction (real time qPCR) for Aneuploidies of chromosomes 13, 18, 21, X and Y. It will be used for population screening purposes.\n- ANGELAB3. This system will use the DNA sample provided from ANGELAB1 or ANGELAB2 and it will carry out Digital PCR on a LabonaChip to detect: CF (unknown multiple mutations), b-thalassemia and achondroplasia. I will be used for population screening purposes.\nAll systems can manage 8 samples at a time. They can be modularly assembled to themselves, offering a system adaptable to different throughput needs.\nThe consortium will also develop and integrate a LabonaChip Pilot Production Line (LPPL):\n- LPPL: This pilot line setup will provide a sustainable and economic LabonChip manufacturing. Materials, processes, and their life cycle will be considered. The environmental assessment will be carried out following ISO 14044. Quality control tools will be integrated along the entire production chain of LabonaChips.\nThis project will end with the implementation of these three diagnostic systems as pilot routines in two Hospitals through a technical validation of 700 pregnancies.
Papageorgiou E.A.,The Cyprus Institute of Neurology and Genetics |
Papageorgiou E.A.,NIPD Genetics Ltd |
Patsalis P.C.,The Cyprus Institute of Neurology and Genetics
Genome Medicine | Year: 2012
Non-invasive prenatal diagnosis (NIPD) has substantial medical importance as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Several approaches have been demonstrated as being proof-of concept for NIPD of chromosomal aneuploidies. These approaches include cell-based and cell-free detection methods, involving the investigation of fetal cells in the maternal circulation, formaldehyde treatment of maternal plasma, DNA methylation studies using sodium bisulfite or restriction enzymes, protein-based studies, identification of fetal-specific mRNAs and digital polymerase chain reaction (PCR) approaches, and recently next-generation sequencing and methylated DNA immunoprecipitation real-time quantitative PCR-based approaches. Although all these NIPD methods have both advantages and limitations, some are moving closer to clinical implementation. Biotechnology companies dedicated to the development of NIPD tests such as the sequencing- or methylation-based approaches are finalizing large clinical trials. It is expected that these new technologies will facilitate safer, more sensitive and accurate prenatal diagnostic tests in the near future. In this review, we highlight the most recent advances in methods for NIPD of aneuploidies, and we discuss their future implications in clinical practice. © 2012 BioMed Central Ltd.
Kypri E.,University of Texas at Austin |
Kypri E.,NIPD Genetics Ltd. |
Falkenstein K.,University of Texas at Austin |
De Lozanne A.,University of Texas at Austin
Traffic | Year: 2013
While loss of the protein Lyst causes abnormal lysosomes in patients with Chediak-Higashi syndrome, the contribution of Lyst to lysosome biology is not known. Previously we found that the Dictyostelium ortholog of Lyst, LvsB, is a cytosolic protein that associates with lysosomes and post-lysosomes to prevent their inappropriate fusion. Here we provide three lines of evidence that indicate that LvsB contributes to lysosome function by antagonizing the function of DdRab14, a protein that promotes homotypic fusion among lysosomes. (1) Instead of restricting DdRab14 to lysosomes, cells that lack LvsB expand DdRab14 localization to include post-lysosomes. (2) Expression of activated DdRab14 phenocopies the loss of LvsB, causing inappropriate heterotypic fusion between lysosomes and post-lysosomes and their subsequent enlargement. (3) Conversely, expression of inactivated DdRab14 suppresses the phenotype of LvsB null cells and restores their lysosomal size and segregation from post-lysosomes. Our data suggest a scenario where LvsB binds to late lysosomes and promotes the inactivation of DdRab14. This inactivation allows the lysosomes to mature into post-lysosomes for eventual secretion. We propose that human Lyst may function similarly to regulate Rab-dependent fusion of lysosomal compartments. © 2013 John Wiley & Sons A/S.
Patsalis P.C.,Cyprus Institute of Neurology and Genetics |
Tsaliki E.,NIPD Genetics Ltd |
Koumbaris G.,NIPD Genetics Ltd |
Koumbaris G.,Cyprus Institute of Neurology and Genetics |
And 4 more authors.
Expert Opinion on Biological Therapy | Year: 2012
Introduction: Non-invasive prenatal diagnosis (NIPD) of Down syndrome is rapidly evolving. Currently, two applications for NIPD of Down syndrome have been developed with potential and have displayed positive results; the NIPD using next-generation sequencing technologies and the NIPD using the methylated DNA immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (qPCR). Areas covered: The MeDIP real-time qPCR approach is based on the identification of differentially methylated regions (DMRs) and their use for discriminating normal from Down syndrome cases. DMRs were identified using high-resolution oligo-arrays. A subgroup of DMRs was selected for further investigation. Through the design of a discriminant equation which combines the results obtained from different DMRs, normal and abnormal cases are correctly classified indicating 100% sensitivity and specificity. Expert opinion: Previous studies have also identified DMRs between non-pregnant female blood and placental DNA. However, these methods have been associated with a number of limitations including the low sensitivity and/or specificity of the assays, the limited number of identified DMRs or methylation sensitive sites and SNPs located on DMRs. These limitations have been overawed by the development of the MeDIP real-time qPCR-based methodology. © 2012 Informa UK, Ltd.
Kyriakou S.,The Cyprus Institute of Neurology and Genetics |
Kypri E.,NIPD Genetics Ltd |
Spyrou C.,NIPD Genetics Ltd |
Tsaliki E.,NIPD Genetics Ltd |
And 4 more authors.
Prenatal Diagnosis | Year: 2013
Objective: The goal of this study is to evaluate the amount of free fetal DNA (ffDNA), total DNA, and 'fetal fraction' found in maternal plasma and whether these influence the enrichment ratios of differentially methylated regions (DMRs) and the correct classification of trisomy 21 using the methylated DNA immunoprecipitation-quantitative polymerase chain reaction (MeDIP-qPCR)-based noninvasive prenatal diagnostic methodology applied in peripheral blood. Methods: Absolute quantification of ffDNA using DYS14 and total DNA using β-globin was applied in 83 maternal plasma samples. The quantification values for all 83 samples were correlated with the enrichment ratios of all seven DMRs and D-values that were obtained from the diagnostic formula of MeDIP-qPCR method. Results: Our analysis concluded that trisomy 21 samples had significantly higher ffDNA and total DNA levels compared with those of normal samples. Enrichment ratios of the majority of DMRs studied exhibited no association with ffDNA, total DNA, and 'fetal fraction', and only a small portion of DMRs exhibited moderate association. Correlation studies of ffDNA, total DNA, and fetal fraction with the diagnostic D-value showed weak to no association but without affecting the classification of trisomy 21. Conclusion: Overall, the variability of ffDNA and total DNA among maternal samples does not affect the correct trisomy 21 classification using MeDIP-qPCR methodology applied in peripheral blood. © 2013 John Wiley & Sons, Ltd.
Agency: European Commission | Branch: H2020 | Program: ERC-POC | Phase: ERC-PoC-2015 | Award Amount: 150.00K | Year: 2016
Scientific and medical evidence indicate that non-invasive prenatal testing, known as non-invasive prenatal testing (NIPT), is a safer alternative to invasive tests that might put the pregnancy at risk. Modern NIPT examine traces of fetal DNA in the maternal bloodstream to determine whether the fetus is at risk of chromosomal abnormalities such as, but not limited to, Down syndrome (trisomy 21), Patau syndrome (trisomy 13) and Edwards syndrome (trisomy 18). In this ERC Proof of Concept Grant (mR-NIPD), we anticipate to correlate already discovered DNA biomarkers of the ERC NIPD (funded ERC) with biomarkers in mRNA transcripts. The method, directly related to the currently funded ERC, will use m6A-specific methylated mRNA immunoprecipitation combined with Next Generation Sequencing (MeRIP-Seq) on fetal and maternal mRNA samples. As a result, we aim to increase the number of fetal specific biomarkers and provide a novel, cost-effective non-invasive prenatal test that will be accessible to all pregnant women independent from social and economic status
PubMed | Childhood Hospital Materno Infantil, Ultrasound and Fetal Medicine Center, Medical University of Bialystok, University of Szeged and 6 more.
Type: Journal Article | Journal: Clinical chemistry | Year: 2016
There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT).We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing.Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction.The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.
PubMed | NIPD Genetics Ltd and The Cyprus Institute of Neurology & Genetics
Type: Comparative Study | Journal: PloS one | Year: 2015
Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.Performance was tested by combining fully methylated and fully unmethylated -DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.
NIPD Genetics Ltd | Date: 2016-05-20
The invention provides methods for non-invasive prenatal testing that allow for detecting risk of chromosomal and subchromosomal abnormalities, including but not limited to aneuploidies, microdeletions and microduplications, insertions, translocations, inversions and small-size mutations including point mutations and mutational signatures. The methods of the invention utilize a pool of TArget Capture Sequences (TACS) to enrich for sequences of interest in a mixed sample containing both maternal and fetal DNA, followed by massive parallel sequencing and statistical analysis of the enriched population to thereby detect the risk of a genetic abnormality in the fetal DNA. Kits for carrying out the methods of the invention are also provided.
Nipd Genetics Ltd | Date: 2011-01-26
The invention provides methods and compositions for noninvasive prenatal diagnosis of fetal aneuploidies. A large panel of differentially methylated regions (DMRs) have been identified. Certain of these DMRs are hypomethylated in adult female blood DNA and hypermethylated in fetal DNA, whereas others are hypermethylated in adult female blood DNA and hypomethylated in fetal DNA. Moreover, DMRs that are hypomethylated in adult female blood DNA and hypermethylated in fetal DNA have been shown to accurately predict a fetal aneuploidy in fetal DNA present in a maternal blood sample during pregnancy. In the methods of the invention, hypermethylated DNA is physically separated from hypomethylated DNA, preferably by methylated DNA immunoprecipitation.