Workpackage 4:

New Approaches for Prenatal and Neonatal Screening

1. establishment of a SAFE New Technology Resource Centre (NTRC), which will aid in introducing new technologies into this field of research.
2. to help familiarise the diverse scientific community present in the SAFE Network with the current state of the art and how this can be applied to the task at hand. The NTRC will also function
3. to identify relevant cutting edge-technologies, radical innovations and breakthroughs occurring in diverse and related fields as a means of incorporating key-players not yet identified within the SAFE consortium.
4. to alert the SAFE network of potentially exiting new developments, thereby helping partners to keep abreast with the current rapid pace of technological development.

D4.1 Assay for most common CAH mutations. (June 2007)
D4.2 Assay for copy number variants of CYP21 in the CAH region and determination of correlation between 17-hydroxyprogesterone and copy number of active CYP21, June 2007
D4.3 Proof of concept using a retrospective study, January 2008
D4.4 Prospective study including CYP21 genotyping assay to neonatal screening program, September 2008
D4.5 Use of MLPA/real-time QPCR to detect copy number variations on neonatal DNA from Guthrie card, February 2008
D4.6 Protocol for identification of deletion borders in microdeletion syndromes, September 2008
D4.7 Characterization and functional studies of candidate genes in areas with copy number variation, December 2008
D4.8 Delivery of aptamer sequence against monoamineoxidase, February 2007
D4.9 Delivery of aptamer sequence against 17-hydroxyprogesterone using new modified oligonucleotides, June 2007
D4.10 Delivery of aptamer sequence against human chorionic gonadotropin, December 2007
D4.11 Provision of target peptide for syncytiotrophoblast membrane/ vesicles, December 2007
D4.12 Identification of targets for pre-term labour detection, February 2008
D4.13 Aptamer sequence against syncytiotrophoblast membrane/ vesicles, June 2008 D4.14 Aptamer sequence against identified pre-term labour target, August 2008
D4.15 Development of a microfluidic device that is appropriate to isolate ffDNA, March 2007
D4.16 Pre-concentration of isolated nucleic acids on a microchip from a large volume (Integration of electrokinetic trapping and isotachophoresis), August 2007
D4.17 Size separation by capillary electrophoresis and recovery of nucleic acids smaller than 300 bp (ffDNA), December 2007
D4.18 Amplification of recovered DNA, August 2008
D4.19 Applying on or more microfluidic devices (Isolation device, pre-concentration dev., capillary electrophoresis dev.) on real samples, February 2009
D4.20 Demonstration of DNA biosensor exploiting nanoparticle signal enhancement for the detection of model analyte, June 2007
D4.21 Surface chemistry optimization using mixed metal monolayer for anchoring of DNA probes, December 2007
D4.22 Demonstration of DNA biosensor with probe oligos immobilized via reductive desorption, June 2008
D4.23 Standardisation of the methodology of fetal DNA extraction from maternal blood and to study the yield of fetal DNA at different gestational ages, August 2007
D4.24 Development of non-invasive RhD genotyping using fetal DNA in Maternal blood, February 2008
D4.25 Development of non-invasive prenatal diagnosis of ?-thalassemia, August 2008
D4.26 Report on WP4 – SELEX & aptamer selection workshop, July 2007
D4.27 Report on microfluidics workshop September 2007
D4.28 Report on annual pre-term labour workshop, October 2007
D4.29 Report on annual haemoglobinopathies workshop, December 2007
D4.30 Report on annual neonatal screening workshop, January 2008

Achivements

The main achievements of Workpackage 4 are:

(i) Microfluidics for enhanced non-invasive prenatal diagnostics;
Research via the PhD bursary focused on the development of a microfluidic device that utilises capillary electrophoresis and microfluidic switching of DNA <500bp so that this DNA is available for further analysis.

(ii) Selection of aptamers against foetal cell specific markers;
Research work continued based on the aptamer targets prioritised for selection using the automated robot. Workshops to give a practical demonstration of the robot and for in depth discussions regarding the aptamer selection process and elucidiation of structure and final application to NIPD.

(iii) Nanostructured Biosensors
This work is focused on the development of structured electrode surfaces (transducers) for specific immobilization of biological recognition elements, e.g. probe DNA molecules for detecting target DNA. The general strategy is based on the use of selfassembled monolayers (SAMs) as a matrix for embedding the probe DNA molecules. Progress has been made in further characterizing nanostructures produced by selective reductive desorption of SAMs. A new method of signal enhancing based on the use of stationary palladium and gold nanoparticles as substrates DNA probe molecules for has been developed and tested with model substances.

(iv) Neonatal screening in the post-genomic era;
Research work focused on neonatal screening for congenital heart diseases or other gene copy-number variant disorders. Additionally, the PhD bursary focused on Method development for screening for a number of known chromosomal deletions using MLPA, MAPH, and will focus on other relevant technologies with a goal of attaining a chip-based system that can be applied to a variety of deletions/duplications/insertions.
Primary model systems include detection of large deletions in syndromes such as Williams-Beuren Syndrome and detectionof chromosomal duplications which may be relevant for susceptibility to inflammatory and/or infectious diseases.

(v) New technologies for haemoglobinopathies
The objective is to hold workshops to identify new technologies for the detection of haemoglobinopathies, looking both at foetal cells and cell free fetal DNA in circulating maternal blood. Based on the outcomes of the workshop, recommendations for future research objectives with the SAFE network will be made.

(vi) Pre-term labour detection
The objective is to establish a critical mass of European researchers working in the area of pre-term labour and to set up spinout group, the European Parturition Group.

(vii) New technologies – New Technology Resource Centre
The NTRC has and will continue its activities updating the various sections and expanding its contents according to the new technologies identified throughout the project.

The various technologies demonstrated within this new technology workpackage would then be extended to other areas of application such as detection of rare cells for early cancer diagnostics and/or theranostics, isolation of circulating stem cells, diagnosis of infectious disease, detection of bio terrorism agents and harmful pathogens etc.

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