WP 3: Fetal Circulatory Nucleic acids

Objectives

Non-Invasive detection of paternal alleles carried foetal DNA found in maternal plasma has been an area of intense activity, and has seen the introduction of small-scale non-invasive prenatal definition of blood group status. The approaches applied are not consistent, and at present are based on a single technological platform, using real-time PCR. The SAFE Network will unify many groups working in this area to standardise genotyping assays, using RhD genotyping as a model system. The overall objective of this will be to prepare for the large-scale implementation of RhD genotyping in all D-negative women, which represents 15-18% of all pregnancies. Further genotyping strategies for NIPD need to be developed notably b-thalassemia and cystic fibrosis. In order to provide routine NIPD, multiplexing involving the detection of further foetal alleles (e.g. SRY, SNPs, STR-bi-allelic markers) will be necessary to ensure that foetal DNA is detected in every maternal plasma samples, and quantitative analysis may reveal foetal abnormality. Maternal plasma based typing may provide a methodology to test for chromosome abnormalities in pregnancy, but will require either foetal/maternal DNA separation or enzymic manipulation (telomere depletion) to specifically degrade maternal DNA. Other nucleic acids, notably RNA can be found in maternal peripheral blood, and the suitability of this material for prenatal diagnosis will be assessed. Finally it is apparent that significant attention must be paid to developing high-throughput screening techniques if routine, large scale NIPD is to be implemented.

Description of work

Workpackages will define the division of labour to meet the key objectives outlined above.
WP3.1 Definition of standardisation of RHD genotyping and large-scale testing.
WP3.2 Definition of standardisation of SRY genotyping,
WP3.5 Identification of further foetal markers suitable for internal standards and quantitation/assessment of foetal DNA in aneuploidy and preeclampsia
WP3.8 Development of assays for disease associated genes
WP3.9 Assessment of foetally-derived RNA as a target for NIPD
WP3.10 Development of new genotyping assays (gene chip technology)
WP3.11 High-throughput instrumentation and robotics in order to scale up methods for population screening.

Deliverables

D3.3 Pre-validated RHD and SRY genotyping assays, for use in screening in workshops clinical trials.
D3.4 Identification of a series of bi-allelic markers (STRs or SNPs) suitable for use as internal controls and quantification and use in screening for aneuploidy; A series of genotyping assays for monogenic multi-allele inherited diseases.
D3.12 Definition of genotyping assays using different technical platforms - sensitivity and accuracy using defined model systems and an assessment of the performance of robotic instrumentation for the extraction of maternal plasma samples.

Milestones

M3.7 Standardisation agreed at milestone 1 implemented across SAFE laboratories and efficacy of techniques compared.
M3.13 Review of WP3 data and assessment of future strategy