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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.

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.

Leontiou C.A.,Cyprus Institute of Neurology and Genetics | Hadjidaniel M.D.,Cyprus Institute of Neurology and Genetics | Mina P.,Cyprus Institute of Neurology and Genetics | Antoniou P.,NIPD Genetics Ltd | And 2 more authors.
PLoS ONE | Year: 2015

Introduction 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. Methods Performance was tested by combining fully methylated and fully unmethylated ë-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. Results 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. Conclusion 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. © 2015 Leontiou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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.

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