Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are ‘digital PCR' and ‘next-generation sequencing' (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening marker

Quantitative Proteomic (iTRAQ) Analysis of 1st Trimester Maternal Plasma Samples in Pregnancies at Risk for Preeclampsia

A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers

Quantitative Proteomics Analysis of Maternal Plasma in Down Syndrome Pregnancies Using Isobaric Tagging Reagent (iTRAQ)

Currently no specific biomarkers exist for the screening of pregnancies at risk for down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in βHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers

Identification of screening biomarkers for chromosomal anomalies and pregnancy-related disorder using quantitative plasma proteomics

Due to the risk associated with invasive procedures, a large research effort has been expended to the development of risk free alternatives. Current results indicate that we may be approaching the long sought goal of Non-Invasive Prenatal Diagnosis (NIPD), whereby it will be possible to identify hereditary single gene disorders or a chromosomal abnormality in the growing fetus. One of the routes explored for NIPD was via the enrichment of fetal cells, specifically erythroblasts, from maternal blood. After the enrichment, the putative fetal cells were examined by FISH analysis for the presence of a chromosomal anomaly. In the large a multi-centre NIFTY study it was concluded that although promising, the sensitivity and specificity was below the required clinical application. Proteomics is defined as the analysis of whole protein component of a tissue (e.g. brain), cell (e.g. yeast) or a body fluid (blood or urine). More precisely it involves the determination of identity of the protein present in the mixture and its relative and absolute quantity. Recently it is also used to identify protein modifications (e.g. phosphorylation, glycosylation). Applications of proteomics are very wide, with the major application in clinical research being applied for the better understanding of biological processes and disease state. Examples are, to find and validate new biomarkers (diagnostic and prognostic) or to understand the pharmacodynamics and pharmacokinetics of a drug compound. A specific proteome is very dynamic and can provide lot of information on the expression pattern on normal vs. disease or control vs. treated. To study the proteome in its complexity, advanced tool are required, and in this context mass spectroscopy has emerged as a powerful technique for proteomic analysis. A typical mass spectroscopy based proteomics workflow involves the digestion of protein in-gel or in liquid. Online or off-line fractionation of the peptides is performed by liquid chromatography (LC) and followed by ionization. The peptides are converted into ions and mass analysis is done on these ions, following which the mass to charge ratio is recorded. The resulting fragment masses are used to search of large protein databases search, resulting in the identification of the peptide and protein. Plasma is an attractive entity for the proteomics studies. It contains different proteins from the various organs in high or low abundance. Many pathology or disease associated proteins are often present in plasma. Due to there low abundance, a number of different strategies have been developed to detect these in the plasma proteome, of which a few are discussed below. In traditional 2D gel electrophoresis the most preferred staining method is by Coomassie Brilliant Blue (CBB) or sliver stains. After staining intensity of the protein spot is used for relative quantitation when compare with the gel which is run in parallel. But CBB has poor detection sensitivity, where as sliver stain is not compatible with the down stream mass spectroscopy analysis. To over come this problem the proteins were label with fluorescent cyanine dye (cy2, cy3 and cy5) before the 2D separation. This method in know as 2-D differential gel electrophoresis (2D DIGE) to avoid the gel to gel variation. In same gel one can run control and experimental sample as well the internal standard. Internal standard is made by mixing equal amount of control and experimental sample and is used for the relative quantitation. After the run the gel is scanned with the special Typhoon variable mode imager and DeCyder software is used for the differential analysis. Over a decade MS has evolved as a powerful technique. This has lead to the development of shotgun proteomics, which is a useful tool as it bank ready quantification using special reagent and technique. Lately there are different techniques available for the labeling which enables the quantification of the protein like stable isotope labeling of amino acid in cell cultures (SILAC), isotopic-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantitation (iTRAQ). iTRAQ is an isobaric chemical labeling approach currently the only technique capable of multiplexing up to eight different samples for relative quantification. 8-plex chemically identical iTRAQ reagents are available, named 114, 115, 116, 117, 118, 119, and 121 which have the same overall mass. Each label is composed of a peptide reactive group (NHS ester) and an isobaric tag of 145 Da that consists of a balancer group (carbonyl) and a reporter group (based on N-methyl piperazine), between the balancer and the reporter group is a fragmentation site. The peptide reactive group attaches specifically to free primary amino groups – N-termini and ε-amino groups of lysine residues. Each sample to be analyzed is tryptic digested and labeled with the single iTRAQ label after which sample are pooled for tandem mass analysis. The peptide product ion spectra is then used for the identification of the proteins and relative quantitation is derived from the peak intensities of the 8-plex iTRAQ reporter ions detected in the 114-121 m/z region of the fragmented ion spectra. Data acquired is always compared to a reference sample, and the quantity of each peptide is expressed as a ratio relative to the reference sample. As the field of shotgun proteomics is evolving rapidly, it is likely to play a role in the detection of biomarkers. iTRAQ has been shown to permit very reliable quantitation of proteins in complex mixture such as plasma, serum or urine. As such it has been suggested to be a useful tool for the detection of biomarkers. Hence, it is likely to play a key role in this field. Selected Reaction Monitoring (SRM) is very rapidly developing tool for the MS based quantitation. It exploits the unique future of the triple quadrupole. The first and third quadrupole act as a filter to select the ion of defined m/z and the second quadrupole is a collision cell. The research carried out in this thesis does however demonstrate that it is possible to mine the maternal plasma proteome for new biomarkers. Their verification will however need to be carried out in much larger studies, and using more convenient techniques

Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ)

Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers

Determination of fetal chromosome aberrations from fetal DNA in maternal blood : has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are ‘digital PCR’ and ‘next-generation sequencing’ (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as β-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers

Detection of increased amounts of cell-free fetal DNA with short PCR amplicons

AIM: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. METHOD: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). RESULTS: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. CONCLUSIONS: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy