High throughput genetic analysis of limited numbers of cells

Prenatal diagnosis mainly utilises Invasive sampling of fetal tissue by amniocentesisnor chorionic villus sampling. It is most frequently performed in relation to Downnsyndrome, whose risk increases with maternal age. However, prenatal diagnosis isnincreasingly being used in relation to other genetic diseases for which tests arenbecoming increasingly available, especially in the context of an indicative familynhistory. Although widespread, accurate and reliable, these techniques have a 0.5-n2% procedurally related risk of miscarriage and therefore are not offered to low risknpregnancies. More recently, several non-invasive prenatal screening strategies tondetect indirect indicators of disease have been implemented in order to better targetnthese invasive techniques. These screens detect the majority of Down syndromenaffected fetuses, however they have a small but significant (5-10%) false positivenrate. Therefore a rapid, non-invasive, accurate and low cost alternative method ofnprenatal screening, or ideally, diagnosis is of great potential value. Such a methodnwould allow wider use of screening in lower risk groups, thereby extending the usefulnrange of genetic diagnosis.n Fetal cells have repeatedly been shown to be present in the cervical sump ofnpregnant women. These cells can be collected by routine pap smears and couldnprovide a non-invasive source of fetal cells. This thesis describes the evaluation,ndevelopment and application of technologies to enrich, isolate and analyse fetal cellsnpresent in minimally invasive pap smears for prenatal genetic screening.n A variety of techniques currently exist for prenatal detection of aneuploidy. However,nthe literature is significantly lacking in comparison between these methods. MultiplexnFluorescent Polymerase Chain Reaction (MFPCR) of short tandem repeatnsequences was evaluated on a model case of tetrasomy 18p detected prenatally.nComparisons were made to conventional techniques of non-fluorescent PCR,nkaryotyping and FISH. Tetrasomy 18p is an ideal model system, as it requiresnaccurate determination of copy number for diagnosis. MFPCR was the onlyntechnique to accurately confirm the presence of four copies of chromosome 18pnmaterial. Techniques for removal of PCR contaminants from PCR product were alsonevaluated. One technique, ammonium acetate/ethanol precipitation was clearlynsuperior facilitating application of MFPCR product analysis to high throughputncapillary electrophoresis systems.nn Fetal cell detection techniques and subsequent enrichment of fetal cells fromnuntreated pap smear samples are described. Four highly specific anti-fetalnantibodies were evaluated by double positive immunohistochemistry with subsequentnconfirmation of cell origin by MFPCR DMA fingerprinting. A minimum specificity ofn54% was achieved using antibodies LK26 and anti-placental lactogen for detection ofnfetal cells in untreated pap smears. The presence of fetal cells in pap smears wasnconfirmed in 29 of 32 samples having an average frequency of 0.35%. A fetal cellnenrichment strategy using gradient centrifugation and magnetic acfivated cell sortingnwas then developed to enhance the frequency of fetal cells in the final sample. Fetalncell origin and an average frequency of 37% in the enriched sample was confirmednby MFPCR DMA fingerprinting with efficient isolation from 90% (n=10) of samples.n The application of multiplex single nucleotide polymorphism (SNP) genotyping to lowncopy number samples and single cells was then performed. SNP genotypes have anwide range of potential applications including predictive medicine, forensicnidentification and pharmacogenomics. SNP analysis of amniotic fluid was performednwithout the need for DNA extraction, significantly increasing the repertoire of prenatalngenetic diagnoses available to pregnant women. SNP genotyping of single cells wasndemonstrated using single amniotic fluid and buccal cells. SNP analysis of singlenfetal cells isolated from 'minimally invasive' pap smears is also described providingnboth a SNP genotype and the opportunity to confirm that the fetus is consistent withnbeing the progeny of the mother.n The technologies described in this thesis provide 'proof of principle' that fetal cellsncan be reliably enriched and isolated from minimally invasive pap smears. Thesencells have been shown to be amenable to molecular genetic analysis by MFPCR andnSNP genotyping for detection of major forms of aneuploidy and single gene defects.nValidation of these preliminary results through a clinical trial would provide thennecessary data for application of fetal cell analysis from pap smears as a routinenprenatal genetic screening program. Analysis of fetal cells from pap smears wouldnbe a significant advancement on current prenatal genetic screening programs sincenscreening would be performed much earlier in gestation as well as being more rapid,nsafer and significantly less expensive.n