(i) Maternal and fetal conditions and characteristics
228 The Patent states (page 3, line 29 to page 4, line 4) that the method according to the invention may be particularly useful for sex determination. It explains that this application may be carried out by simply detecting the presence of a Y chromosome. In this context, no mention is made of controls for cffDNA or validation or optimisation required for a commercial clinical sex determination test. Rather, the Patent demonstrates that using only 10µL of plasma or serum, a detection rate of 80% for plasma and 70% for serum can be obtained and that this is high enough to be useful (page 9, line 11).
229 The Patent explains (page 4, lines 5 to 8) that "the method of the invention can be applied to the detection of any paternally-inherited sequences which are not possessed by the mother".
230 Examples of the above application are stated in the Patent to include the following.
231 First, fetal rhesus D status determination in rhesus negative mothers is given as an example. I have already touched upon this to some extent, but let me re-iterate the following.
232 As explained at page 15, lines 9 to 11 of the Patent, the rhesus blood group system is involved in haemolytic disease of the newborn, transfusion reactions and autoimmune haemolytic anaemia.
233 As I have said, the Rh factor (also known as the Rh D antigen) is a protein found on the surface of red blood cells in so-called Rh positive individuals. Rh negative individuals lack this protein. Lack of this protein in Rh negative individuals is caused by a deletion or mutations of the RhD gene that encodes it in both copies of chromosome 1. If one copy of chromosome 1 contains the RhD gene and one does not, the individual still expresses the Rh factor and is considered Rh positive. I refer to my earlier discussion in the common general knowledge section.
234 Second, haemoglobinopathies, by detecting a paternally inherited mutation in the beta-globin gene not possessed by the mother, is given as an example.
235 As I have already said, haemoglobinopathies are genetic disorders in which the haemoglobin molecules in an affected individual's red blood cells are abnormal. Well-known examples of haemoglobinopathies are sickle cell anaemia and alpha- and beta-thalassemia. Alpha-thalassemia is considered a lethal disease, often leading to fetal death in the third trimester with maternal hydrops ("mirror") syndrome also commonly present. Sickle cell and beta-thalassemia can be treated but patients suffer from many symptoms and often need life-long repeated blood transfusions. Parents being confronted with a diagnosis of these fetal diseases may elect for termination.
236 Work had been carried out before the priority date to characterise the beta-globin gene and various mutations which were shown to lead to certain blood disorders. The use of PCR for these genes to determine the risk of a fetus being affected by such diseases was well known by the priority date arising from research into fetal cells in maternal circulation.
237 The point mutations occurring in the case of beta-thalassemia may be different between the father and mother. This meant that, using PCR primers, one could distinguish between fetal DNA, which should have paternally inherited markers, and remaining maternal DNA, which would not. As explained in the Patent (page 4, lines 19 to 22):
Provided that the father and mother carry different mutations, the paternal mutation can be used as an amplification target on maternal plasma and serum, so as to assess the risk that the foetus may be affected.
238 Third, reference was made to other paternally-inherited DNA polymorphisms or mutations on a Y or non-Y chromosome.
239 The Patent explains at page 4, line 30 to page 5, line 2 that this particular application will require the prior genotyping of the father and mother using a panel of polymorphic markers and then an allele for detection will be chosen which is present in the father, but is absent in the mother. The Patent explains at page 4, lines 27 to 30 that this type of analysis can be used to ascertain the presence of fetal nucleic acid in a particular maternal or serum sample, prior to diagnostic analysis such as sex determination.
240 Genetic markers commonly known and used at the priority date included polymorphic loci at which base changes were known to affect restriction sites, that is, RFLPs and STRs or microsatellites being sections of DNA made up of repeated short sequences.
241 Let me continue with my discussion of the Patent more generally. At page 5, lines 3 to 6, the Patent explains that the "plasma or serum based non-invasive diagnosis method according to the invention can be applied to screening for Down syndrome and other chromosomal aneuploidies" and gives two possible ways in which this might be done. As I have indicated, aneuploidies in autosomal and sex chromosomes are responsible for a number of genetic conditions, due to abnormal dosage of genes, including Down syndrome (trisomy of chromosome 21), Edwards syndrome (trisomy of chromosome 18), Patau syndrome (trisomy of chromosome 13), Turner syndrome (full or partial monosomy of X), Klinefelter syndrome (XXY), XYY syndrome, XXYY syndrome and Triple X syndrome.
242 Broadly, the first method involves the quantitative detection of fetal nucleic acid to screen women with higher cffDNA levels compared to normal pregnancies and the second method involves "the quantitation of foetal DNA markers on different chromosomes" (emphasis added). In this context, the Patent refers to the recent development of quantitative PCR techniques, such as real-time quantitative PCR (real-time qPCR) described in Heid CA et al, "Real time Quantitative PCR" (1996) 6(10) Genome Research 986-994 (Heid et al 1996).
243 As I have already indicated, quantitative PCR (qPCR) is a broad term that is used to refer to PCR methods which enable the products of a conventional PCR reaction to be quantified. By the priority date, amplification of sequences that were the targets of qPCR could be detected using a number of methods including agarose gels, a form of gel electrophoresis discussed above, fluorescent labelling of PCR products and detection with laser-induced fluorescence using capillary electrophoresis or acrylamide gels and plate capture and sandwich probe hybridisation.
244 As I have already said, another qPCR method available at the priority date was quantitative fluorescent PCR (qfPCR), which was a method which required the products of the PCR reaction to be detected and quantified using a post-reaction "end point" technique, namely, electrophoresis. As a result, qfPCR methods were understood to be more prone to errors in quantification compared to real-time qPCR methods, as the latter quantified the PCR products in the same vessel as that used to amplify the DNA.
245 Heid et al 1996 was received on 3 June 1996 and published later that year. The real-time qPCR method described in Heid et al 1996 measured PCR product accumulation through a hybridisation probe called the "TaqMan Probe", which is labelled with two different fluorescent dyes. One dye is a "reporter" dye and the other is a "quenching" dye. When the TaqMan Probe is intact, the "quenching" dye "quenches" or absorbs the fluorescence emission of the reporter dye, such that a fluorescence signal is not generated. However, when the TaqMan probe is cut or cleaved during amplification of a target sequence, the reporter dye's emission is no longer quenched, resulting in a detectable increase of the reporter dye fluorescence emission spectra. In such an assay, the presence and/or quantity of a target nucleic acid sequence is indirectly detected or inferred from the emission spectra levels detected by an instrument capable of measuring fluorescence in real time generated by a fluorophore that is no longer attached to the target sequence (e.g. ABI Prism 7700 Sequence Detector) (see, for example, the Patent at page 11, lines 8 to 13).
246 The background to Heid et al 1996 explains that:
Several detection systems are used for quantitative PCR and RT-PCR analysis: (1) agarose gels, (2) fluorescent labelling of PCR products and detection with laser-induced fluorescence using capillary electrophoresis (Fasco et al. 1995; Williams et al. 1996) or acrylamide gels, and (3) plate capture and sandwich probe hybridization (Mulder et al. 1994).
247 Unlike other quantitative PCR methods, real-time qPCR does not require post PCR handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays.
248 The concentration of DNA in a real-time qPCR reaction sample is determined by calculating the threshold cycle value (CT) for a sample, which is the PCR cycle number at which the concentration of DNA in a reaction sample (typically measured by the amount of fluorescence) reaches a threshold, which is placed in the exponential phase of amplification. CT values are calculated from amplification plots, such as the plot depicted below. As a consequence of the exponential nature of PCR, one cycle represents approximately a doubling in template concentration. At the priority date, qPCR was suitable for detecting a 2-fold difference.
249 The Patent describes another application (page 5, line 27 to page 6, line 2) of the accurate quantitation of fetal nucleic acid levels in the maternal serum or plasma, namely, the molecular monitoring of placental pathologies such as pre-eclampsia, in which the concentration of fetal DNA is said to be elevated.
250 As I have indicated, pre-eclampsia is a pregnancy disorder which affects about 6% of pregnancies, and is characterised by high blood pressure and elevated protein levels in the maternal urine. Pre-eclampsia generally occurs 24 to 26 weeks after fertilisation and often increases in severity until birth. Untreated, pre-eclampsia may lead to eclampsia (convulsions), bleeding in the mother's brain and death of the mother. Especially the early forms of pre-eclampsia are often associated with fetal growth restriction due to placental dysfunction.
251 The Patent says (page 6, lines 3 to 6) that it is also anticipated that it will be possible to incorporate the nucleic acid-based diagnosis methods described in the Patent into existing prenatal screening programmes and that "[s]ex determination has successfully been performed on pregnancies from 7 to 40 weeks of gestation".
252 From page 6, line 20 to page 33, the Patent illustrates the invention with reference to five Examples, but "which do not in any way limit the scope of the invention".