Sequenom, Inc. v Ariosa Diagnostics, Inc.
[2019] FCA 1011
•27 June 2019
FEDERAL COURT OF AUSTRALIA
Sequenom, Inc. v Ariosa Diagnostics, Inc. [2019] FCA 1011
File number: VID 611 of 2016 Judge: BEACH J Date of judgment: 27 June 2019 Catchwords: PATENTS – non-invasive prenatal diagnosis – detection of fetal DNA in maternal serum or plasma samples – detection of fetal abnormalities for chromosomal aneuploidies or simple mutations – detection and monitoring of pregnancy-associated conditions such as pre-eclampsia – asserted grounds of invalidity – manner of manufacture – lack of inventive step – lack of utility – lack of fair basis – lack of sufficiency – false suggestion or misrepresentation – invalidity grounds rejected – Harmony Test – construction – infringement – infringement established Legislation: Patents Act 1990 (Cth) Sch 1, ss 7(2), 7(3), 13(1), 18(1), 40(2)(a), 40(3), 138(3)(d)
Statute of Monopolies 1624, 21 Jac 1 c 3, s 6
Federal Court Rules 2011 (Cth) r 34.23
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Aktiebolaget Hässle v Alphapharm Pty Ltd (2002) 212 CLR 411
Alice Corporation Pty Ltd v CLS Bank International (2014) 573 US 208
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Apotex Pty Ltd v Les Laboratories Servier [2013] FCA 1426
Apotex Pty Ltd v Sanofi-Aventis Australia Pty Ltd (2013) 253 CLR 284
Apotex Pty Ltd v Warner-Lambert Company LLC (No 2) (2016) 122 IPR 17
Ariosa Diagnostics Inc. Sequenom, Inc. 788 F3d 1371 (3d Cir 2015)
Arrow Pharmaceuticals Ltd v Merck & Co Inc (2004) 213 ALR 182
Artcraft Urban Group Pty Ltd v Streetworx Pty Ltd (2016) 245 FCR 485
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British Acoustic Films Ld v Nettlefold Productions (1936) 53 RPC 221
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Commissioner of Patents v Emperor Sports Pty Ltd (2006) 149 FCR 386
Commissioner of Patents v Microcell (1958) 102 CLR 232
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D’Arcy v Myriad Genetics Inc (2015) 258 CLR 334
Delnorth Pty Ltd v Commissioner of Patents (2013) 100 IPR 175
DSI Australia (Holdings) Pty Ltd v Garford Pty Ltd (2013) 100 IPR 19; [2013] FCA 132
EI Dupont de Nemours & Co v Imperial Chemical Industries plc (2002) 54 IPR 304
Fawcett v Homan (1896) 13 RPC 398
Foster’s Australia Ltd v Cash’s (Australia) Pty Ltd (2013) 219 FCR 529
Gilead Sciences Pty Ltd v Idenix Pharmaceuticals LLC (2016) 117 IPR 252
GlaxoSmithKline Consumer Healthcare Investments (Ireland) (No 2) Ltd v Apotex Pty Ltd (2016) 119 IPR 1; [2016] FCA 608
Grant v Commissioner of Patents (2006) 154 FCR 62
ICI Chemicals & Polymers Ltd v Lubrizol Corp Inc (1999) 45 IPR 577
Idenix Pharmaceuticals LLC v Gilead Sciences Pty Ltd (2017) 134 IPR 1
Illumina, Inc v Premaitha Health Plc [2017] EWHC 2930 (Pat)
In re O’Farrell (1988) 853 F 2d 894
Intalite International NV v Cellular Ceilings Ltd. (No. 2) [1987] RPC 537
JMVB Enterprises Pty Ltd v Camoflag Pty Ltd (2006) 154 FCR 348
KD Kanopy Australasia Pty Ltd v Insta Image Pty Ltd (2007) 71 IPR 615
Kimberly-Clark Australia Pty Ltd v Arico Trading International Pty Ltd (2001) 207 CLR 1
Kirin-Amgen Inc v Hoechst Marion Roussel Ltd (2004) 64 IPR 444; [2004] UKHL 46
Lane Fox v Kensington and Knightsbridge Electric Lighting Co Ltd [1892] 3 Ch 424
Lockwood Security Products Pty Ltd v Doric Products Pty Ltd (2004) 217 CLR 274
Lockwood Security Products Pty Ltd v Doric Products Pty Ltd (No 2) (2007) 235 CLR 173
Mayo Collaborative Services v Prometheus Laboratories, Inc (2012) 566 US 66
Meat & Livestock Australia Limited v Cargill, Inc (2018) 354 ALR 95; (2018) 129 IPR 278; [2018] FCA 51
Meyers Taylor Pty Ltd v Vicarr Industries Ltd (1977) 137 CLR 228
Minnesota Mining & Manufacturing Co v Tyco Electronics Pty Ltd (2002) 56 IPR 248
Minnesota Mining and Manufacturing Co v Beiersdorf (Australia) Ltd (1980) 144 CLR 253
National Research Development Corporation v Commissioner of Patents (1959) 102 CLR 252
Northern Territory v Collins (2008) 235 CLR 619
NV Philips Gloeilampenfabrieken v Mirabella International Pty Ltd (1995) 183 CLR 655
Olin Corporation v Super Cartridge Co Pty Ltd (1977) 180 CLR 236
Olin Mathieson Chemical Corporation v Biorex Laboratories Ltd [1970] RPC 157
Prestige Group (Australia) Pty Ltd v Dart Industries Inc (1990) 26 FCR 197
Ranbaxy Australia Pty Ltd v Warner-Lambert Co LLC (2008) 77 IPR 449
Regeneron Pharmaceuticals Inc Genentech Inc; Bayer Pharma AG v Genentech Inc [2013] EWCA Civ 93
Regeneron Pharmaceuticals Inc v Genentech [2012] EWHC 657 (Pat)
Rehm Pty Ltd v Websters Security Systems (International) Pty Ltd (1988) 81 ALR 79
Rescare Ltd v Anaesthetic Supplies Pty Ltd (1992) 111 ALR 205; [1992] FCA 811
Research Affiliates LLC v Commissioner of Patents (2014) 227 FCR 378
Sachtler GmbH and Co KG (formerly Sachtler AG) v RE Miller Pty Ltd (2005) 221 ALR 373; [2005] FCA 788
Sanofi-Aventis Australia Pty Ltd v Apotex Pty Ltd (No 3) (2011) 196 FCR 1
Sartas No 1 Pty Ltd v Koukourou & Partners Pty Ltd (1994) 30 IPR 479; [1994] FCA 936
Sigma Pharmaceuticals (Australia) Pty Ltd v Wyeth (2011) 119 IPR 194
SNF (Australia) Pty Ltd v Ciba Speciality Chemicals Water Treatments Ltd (2011) 92 IPR 46; [2011] FCA 452
The Wellcome Foundation Ltd v VR Laboratories (Aust) Pty Ltd (1981) 148 CLR 262
Tramanco Pty Ltd v BPW Transpec Pty Ltd (2014) 105 IPR 18
Warner-Lambert Co LLC v Apotex Pty Ltd (No 2) (2018) 355 ALR 44
Winner v Morey Haigh & Associates (Australasia) Pty Ltd (1996) 33 IPR 215
WM Wrigley Jr Co v Cadbury Schweppes Pty Ltd (2005) 66 IPR 298
Dates of hearing: 27 to 31 August, 3, 10 and 11 September 2018 Date of last submissions: 25 September 2018 Registry: Victoria Division: General Division National Practice Area: Intellectual Property Sub-area: Patents and associated Statutes Category: Catchwords Number of paragraphs: 1477 Counsel for the Applicant/Cross-Respondent: Mr D Shavin QC and Ms HMJ Rofe QC Solicitor for the Applicant/Cross-Respondent: Davies Collison Cave Law Counsel for the Respondents/Cross-Claimants: Mr A Ryan SC, Mr T Cordiner QC and Mr C Smith Solicitor for the First Respondent/Cross-Claimant: Herbert Smith Freehills Solicitor for the Second Respondent/Cross-Claimant: Clayton Utz Solicitor for the Third Respondent/Cross-Claimant: Gilbert + Tobin ORDERS
VID 611 of 2016 BETWEEN: SEQUENOM, INC.
Applicant
AND: ARIOSA DIAGNOSTICS, INC.
First Respondent
SONIC HEALTHCARE LIMITED (ACN 004 196 909)
Second Respondent
CLINICAL LABORATORIES PTY LTD (ACN 006 823 089)
Third Respondent
AND BETWEEN: ARIOSA DIAGNOSTICS, INC. (and others named in the Schedule)
First Cross-Claimant
AND: SEQUENOM, INC.
Cross-Respondent
JUDGE:
BEACH J
DATE OF ORDER:
27 JUNE 2019
THE COURT ORDERS THAT:
1.Within 21 days of the date of these orders, the applicant file and serve minutes of orders and submissions (limited to 3 pages) to give effect to these reasons, on costs and for the further conduct of these proceedings.
2.Within 14 days of receipt of such minutes and submissions, the respondents file and serve responding minutes of orders and submissions (limited to 3 pages).
3.The interim confidentiality order made in Order 1 of the orders dated 9 June 2017 be extended until further order, save that nothing therein applies to the Court’s reasons for judgment.
4.Liberty to apply.
Note: Entry of orders is dealt with in Rule 39.32 of the Federal Court Rules 2011.
REASONS FOR JUDGMENT
[Redacted version]BEACH J:
The applicant (Sequenom) is the patentee of Australian Patent No 727919 entitled “Non-invasive prenatal diagnosis” (the Patent). The Patent claims a priority date of 4 March 1997. It was filed on 4 March 1998 and granted on 19 April 2001. The invention claimed in the Patent provides a method for detecting the presence of a nucleic acid of fetal origin in a maternal serum or plasma sample. The Patent relates to prenatal diagnosis by detecting fetal nucleic acids in such non-cellular components of a maternal blood sample.
Sequenom seeks monetary and declaratory relief against the respondents for infringement of claims 1, 2, 3, 5, 6, 9, 13, 14, 22, 23, 25 and 26 of the Patent (the relevant claims).
The first respondent (Ariosa) is a US company that conducts and licenses others to conduct a non-invasive prenatal diagnosis test, marketed under the name “Harmony” (the Harmony Test).
The second respondent (Sonic) is an Australian pathology company which has been licensed by Ariosa to promote and supply in Australia Harmony Test results, and which through its subsidiary Sullivan Nicolaides Pty Ltd has used the Harmony Test in Australia.
The third respondent (Clinical) is an Australian pathology company which has been licensed by Ariosa to promote and supply in Australia Harmony Test results, and which has used the Harmony Test in Australia.
The respondents have cross-claimed seeking revocation of the relevant claims of the Patent on various grounds being that:
(a)the relevant claims are not for a manner of manufacture;
(b)the claimed invention lacks an inventive step;
(c)the relevant claims are not useful;
(d)the relevant claims are not fairly based;
(e)there is a lack of sufficiency; and
(f)the claimed invention was obtained by false suggestion or misrepresentation.
For most purposes I will describe the respondents collectively as Ariosa except where I need to distinguish between them for the purposes of the infringement case.
The present trial was held on issues of liability only. In summary, Sequenom has succeeded on its infringement claims save as to its case concerning the infringement of claim 26. Ariosa has failed to establish invalidity save as to claim 26 which is invalid for lack of fair basis.
For convenience I have divided my reasons into the following sections:
(a)Glossary of some terms – [10] to [84];
(b)Common general knowledge – [85] to [193];
(c)The Patent – [194] to [307];
(d)The experts – [308] to [338];
(e)Invalidity – General – [339] to [341];
(f)Manner of Manufacture – [342] to [528];
(g)Inventive Step – [529] to [797];
(h)Utility – [798] to [826];
(i)Sufficiency – [827] to [1038];
(j)Internal fair basis – [1039] to [1076];
(k)False suggestion – [1077] to [1160];
(l)Infringement – [1161] to [1476];
(m)Conclusion – [1477].
GLOSSARY OF SOME TERMS
Before discussing the science and then the Patent, it is useful to set out a glossary of some of the key terms. This glossary has been drawn from the expert evidence with some modifications to ensure its utility to both the invalidity case and the infringement case.
5' end and 3' end: In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide sugar-ring means that there will be a 5'-end, which frequently contains a phosphate group attached to the 5' carbon of the ribose ring, and a 3'-end, which typically is unmodified from the ribose -OH substituent. In a DNA double helix, the strands run in opposite directions to permit base pairing between them, which is essential for replication or transcription of the encoded information.
Allele: A form of a gene. Usually one of two alleles, each inherited from a different parent.
Allosome: One of the sex chromosomes, X or Y.
Amniocentesis: A prenatal test involving the insertion of a needle through the mother’s abdomen into the uterus to withdraw amniotic fluid containing fetal cells shed by the fetus into the amniotic fluid.
Aneuploidy: The presence of an abnormal number of chromosomes in a cell, for example, 47 chromosomes instead of the expected and normal 46 (23 pairs).
Anucleated: A cell without a nucleus.
Array Binding Region: A section that is ultimately designed to bind to the microarray used in the detection step.
Assay Pair: For the purposes of the detection step, the Harmony Test notionally “pairs up” the target loci into assay pairs (for the Non-Polymorphic Assay) and SNP allele pairs (for the Polymorphic Assay). As depicted in the confidential Product and Process Description (PPD) which I have annexed to my reasons, [Redacted]
Autosomal chromosome: A chromosome that is not an allosome (a sex chromosome). Autosomes appear in 22 pairs whose members have the same form (but differ from other pairs in a diploid cell), whereas members of an allosome pair determine sex and may differ.
Biotinylation: The process of covalently attaching biotin to, for example, DNA. Biotinylation is rapid, specific and is unlikely to perturb the natural function of DNA due to the small size of biotin. Biotin binds to streptavidin with an extremely high affinity, fast on-rate, and high specificity and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest.
Buffy coat: The fragment of un-clotted blood after centrifugation (spinning down) that contains most of the white blood cells and platelets.
Cell free DNA (cfDNA): Non-cellular DNA (i.e. DNA that is outside a cell).
Cell free fetal DNA (cffDNA): Non-cellular DNA from a fetus.
Chorionic Villus Sampling (CVS): A prenatal procedure involving the insertion of a catheter or needle through the abdomen or cervix into the placental portion of the uterus to take a sample of the chorionic villi (containing placental cells) surrounding the sac which protects the fetus.
Cordocentesis: Also known as percutaneous umbilical blood sampling or fetal blood sampling. A prenatal procedure performed after 18 weeks involving the insertion of a needle into the umbilical cord or intra hepatic vein under ultrasound guidance to withdraw fetal blood to detect conditions associated with fetal blood circulation including isoimmunisation, haemoglobinopathy, anaemia, thrombocytopenia and coagulation-factor abnormalities.
DANSR: Digital analysis of selected regions, a process of analysing sequences from assays targeted against selected genomic regions.
Denaturation: A process where proteins or nucleic acids such as DNA or RNA lose their structure. In the case of DNA, denaturation results in the disruption of the complementary base pair bonding (i.e. A – T and C – G) and separation of the double stranded helix into two single strands.
Discriminating Region: A section that is designed to allow the Harmony Test [Redacted]
Down syndrome: An aneuploidy of chromosome 21, with three chromosome 21s present instead of the normal two, typically associated with physical growth delays, characteristic facial features and mild to moderate intellectual disability. Down syndrome also includes cases in which the extra copy of all or part of the chromosome 21 is not a separate, third chromosome but is translocated or joined to another chromosome.
Edwards syndrome: Edwards syndrome is a chromosomal abnormality caused by the presence of all, or part of, an extra chromosome 18.
Electrophoresis: A method that separates fragments of DNA based on their size which can be stained to enable visual detection.
Erythrocytes: Red blood cells.
Fetal blood sampling: See Cordocentesis.
Fetal Fraction: The proportion of DNA in a sample deriving from the fetus (as opposed to the mother).
Fibrinogen: A blood clotting agent.
FISH or fluorescence in situ hybridisation: This technique is more targeted than conventional karyotyping. It utilises DNA probes which bind to specific and selected regions of interest on particular chromosomes (e.g. chromosome 21), which are then fluorescently labelled to reveal, with the aid of a fluorescent microscope, the presence, absence, relative positioning and / or copy number of the specifically targeted DNA segments.
FORTE: The Harmony Test’s bioinformatics algorithm (Fetal-Fraction Optimized Risk of Trisomy Evaluation) used to calculate risk scores for aneuploidy and determine fetal fraction and fetal sex.
Genotype: The genes in an individual’s DNA. Now this definition is very broad. In clinical practice, it would be more usual to use the term genotype to refer to the molecular genetic characteristics of an individual at a specific locus.
Heterozygous: A diploid organism is heterozygous at a gene locus when its cells contain two different alleles of a gene. The cell or organism is called a heterozygote specifically for the allele in question, and therefore, heterozygosity refers to a specific genotype.
h-index: The h-index is an author-level metric that attempts to measure both the productivity and citation impact of the publications of a scientist or scholar. The index is based on the number of the scientist’s most cited papers and the number of citations that these have received in other publications.
Homozygous: Where an individual has two copies of the same allele.
Informative locus: A polymorphic locus where the fetus has a different pair of alleles compared to its mother.
Karyotyping: The conventional form of chromosomal analysis, which is used to detect numerical and / or structural chromosome abnormalities in a sample of cells which have first been grown in culture. The method, broadly speaking, involves the use of various dyes which selectively stains the chromosomes in the sample.
LCR: Ligase chain reaction.
Leukocytes: White blood cells.
Ligation: The joining of two DNA strands. But in the context of the ligation chain reaction, ligation is the covalent joining of two adjacent nucleotides that are perfectly hybridized to the complementary target DNA.
Ligation Product: The oligonucleotides bind to their complementary sequences in the sample. Where all the three adjacent oligonucleotides bind, they are then ligated using the enzyme ligase to produce (partly) double stranded molecules called ligation products. DNA ligase enzymes are commonly used in molecular biology to join strands of DNA together by catalysing the formation of a bond between the 3' end of one DNA strand and the 5' end of an adjacent DNA strand. In the Harmony Test, the three oligonucleotides only join to form a ligation product if they are adjacent to one another (i.e. their binding sites on the nucleic acid of interest are next to each other).
Locus (plural loci): A targeted region of the genome being usually a defined or mapped position of the genome.
Microarray: Microarrays are used to detect and quantify specifically-targeted sequences of DNA and are also known as “gene chips”. A microarray is typically a glass slide onto which single stranded DNA molecules are fixed in an orderly manner at specific locations called spots (or features). A microarray may contain thousands of spots and each spot may contain a few million copies of identical DNA molecules that uniquely correspond to sequences of interest. Researchers know the identity and position of each short fragment in the microarray.
Minor Source Fraction: In the Harmony Test, the assumption is that the “minor source” of cfDNA in the sample of maternal and fetal cfDNA is of fetal origin and that the minor source fraction is an estimation of the fetal fraction.
NASBA: Nucleic acid sequence based amplification.
NIFTY Trials: The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) was a prospective, multicentre clinical project to develop non-invasive methods of prenatal diagnosis. The initial objective was to assess the utility of fetal cells in the peripheral blood of pregnant women to detect fetal chromosome abnormalities.
NIPD: Non-invasive prenatal diagnosis.
Non-Polymorphic Assay: The set of non-polymorphic oligonucleotides introduced into the sample of cfDNA in the Harmony Test.
OLA: Oligonucleotide ligation assay.
Oligo Junction: The junction of two adjacent oligonucleotides.
Oligonucleotide (oligo): See the definition for “probe”.
PCR: The polymerase chain reaction.
Percutaneous Umbilical Blood Sampling: See Cordocentesis.
Phenotype: The set of observable characteristics of an individual usually resulting from the interaction of its genotype with the environment, although not necessarily; for example, short long bones is the phenotype associated with the FGFR3 Gly380Arg (achondroplasia, common dwarfism).
Placenta: The organ comprising cells of fetal origin connecting a developing fetus to the uterine wall. It allows nutrient uptake; provides thermo-regulation to the fetus, waste elimination and gas exchange via the mother’s circulation; protects against internal infection; and produces hormones to support pregnancy.
Plasma: The supernatant or non-cellular liquid component of blood broadly comprising fibrinogen (a coagulation factor), salts, glucose, amino acids, vitamins, urea, other proteins and fats. It is obtained after centrifugation of blood collected into a test tube typically containing anticoagulant to prevent clotting.
Polymorphic Assay: The assay in the Harmony Test that is used to determine the relative proportion or dosage of markers on the selected target chromosomes. Its purpose is to estimate the minor source fraction in the sample.
Polymorphic locus: A locus that includes a known single nucleotide polymorphism (SNP).
Polymorphism: The presence of two or more different forms of a gene or sequence in an individual or population. But in clinical practice the term is usually reserved for neutral, common variants, usually present at greater than 1 to 2% of the population.
Pre-eclampsia: A disorder of pregnancy characterised by high blood pressure and a large amount of protein in the urine. The disorder usually occurs in the third trimester of pregnancy and worsens over time.
Primer: A short nucleic acid sequence that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyse this process, DNA polymerase, can only add new nucleotides to an existing strand of DNA.
Probe: A probe, sequence specific probe or oligonucleotide probe is a fragment of single stranded DNA or RNA designed to bind to and target a particular complementary sequence of DNA or RNA in a given sample.
qPCR: Quantitative PCR.
qfPCR: Quantitative fluorescent PCR.
Read Out Cassette: For the purpose of further analysis, [Redacted]
Real time PCR (RT-PCR): This can also be known as quantitative PCR (qPCR). Real time PCR refers to a form of the reaction where the data is collected in real time (i.e. “live”) compared to a traditional or standard reaction where the data is collected at the end. The distinction is important as it means the data can be used for quantification. The method of detection may be, but does not need to be, by fluorescent probe. The use of specific fluorescent probes provides sequence-specific detection of DNA fragments with a complementary sequence to the probe.
Restriction enzyme: An enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites.
RFLP: Restriction fragment length polymorphism.
Serum: The supernatant obtained after a blood sample has been allowed to clot.
SNP: Single-Nucleotide Polymorphisms or SNPs are variations in a single nucleotide at a specific position in genomes that are present within a population. For example, 60% of individuals in a population may have a gene with the sequence CGA at a given position in the genome and the remaining 40% of the population may have the sequence CGC at the same position. There are millions of SNPs present in the human genome.
SNP Allele Pair: For the purposes of the detection step, the Harmony Test notionally “pairs up” the target loci into assay pairs (for the Non-Polymorphic Assay) and SNP allele pairs (for the Polymorphic Assay). [Redacted]
STR: Short tandem repeat.
Thrombocytes: Platelets (fragments of large cells crucial to normal blood clotting).
Triple screen test: A prenatal test that measures the amounts of three substances in a pregnant woman’s blood: alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and estriol (uE3). Similar tests using two markers are also known as serum screening tests.
Trisomy 18: See Edwards Syndrome.
Trisomy 21: See Down Syndrome.
Venepuncture: The act of obtaining access to veins (e.g. using a needle), typically from the arm, for the purpose of, for example, drawing a blood sample.
VNTR: Variable number of tandem repeats.
COMMON GENERAL KNOWLEDGE
The common general knowledge in the fields of fetal medicine and molecular genetics as at 4 March 1997 (the priority date) included the following information, which I have drawn from a statement agreed to by the parties save that I have added further detail concerning the PCR process and also further detail on Sanger sequencing which should be uncontroversial. I have also deleted some of the more basic diagrams concerning DNA.
(a) Blood
Blood cells make up approximately 45% of adult blood. These cells include oxygen carrying erythrocytes (red blood cells), immune cells called leukocytes (white blood cells) and thrombocytes (platelets). In the case of pregnant women, it was also known that there were fetal cells present in the mother’s blood. Plasma, which makes up the remaining 55% of the blood, is a straw coloured fluid which contains water, blood plasma proteins (including clotting factors), minerals and dissolved nutrients (such as glucose, amino acids, and fatty acids), and waste products (such as urea and lactic acid). Serum is the name given to plasma which has had the clotting factors removed.
Whole blood can be separated by centrifugation into three layers: (a) the upper plasma layer; (b) the “buffy coat” layer which contains the leukocytes and thrombocytes; and (c) the lower layer which contains the erythrocytes.
(b) Prenatal development
Following fertilisation of an ovum (egg) by a spermatozoon in a fallopian tube, the resulting single cell zygote travels down the fallopian tube and divides to form a blastocyst. Approximately 5 days after fertilisation, the blastocyst, which consists of trophoblast cells and embryonic cells, reaches the uterus and becomes embedded in the endometrium (lining) of the uterus. The trophoblast cells, which surround the embryonic cells, proliferate and embed further into the uterine lining, eventually forming the placenta. The blastocyst becomes fully implanted approximately 7 to 12 days after fertilisation.
The Placenta
The placenta is a composite structure made up of maternal tissues as well as those derived from the fetus. Fetal blood vessels extend to the placenta via the umbilical cord and branch into many chorionic villi, providing a large surface area for the exchange of materials between fetal and maternal blood across a layer of tissue called the placental membrane. A variety of materials, including nutrients and oxygen, are exchanged between the maternal circulatory system and the fetus via chorionic villi in the placenta and the umbilical cord. Other materials passing from the fetus or placenta into the maternal blood circulation include fetal blood cells, proteins and hormones which form the basis of the Rh disease test and the biochemical screens of maternal serum for Down syndrome discussed below. Likewise, waste materials are removed from the fetus to the maternal circulation.
Pre-eclampsia
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. The early forms of pre-eclampsia are often associated with fetal growth restriction due to placental dysfunction.
(c) The human genome
The human genome represents the complete set of inherited instructions encoded in DNA in a human cell.
The human haploid nuclear genome consists of approximately 3 billion base pairs of DNA, organised into 23 chromosomes. Each chromosome carries a set of genes. A chromosome is a DNA-protein complex. In typical diploid individuals, that is, individuals with a balanced pairing of chromosomes, this is organised into a total of 46 chromosomes. The 46 chromosomes in a normal human somatic cell are made up of 22 pairs of homologous autosomes (non-sex chromosomes) and two sex chromosomes (XX for typical females and XY for typical males). One set of 23 chromosomes (comprising 22 autosomes and 1 allosome) is maternally-inherited and one set of 23 chromosomes is paternally-inherited.
DNA is a double helix molecule comprised of complementary strands of nucleotides sometimes referred to as the “building blocks” of DNA. Each nucleotide is composed of:
(a)one of four nucleotides, which are often referred to simply as “bases”, being cytosine (C), guanine (G), adenine (A) or thymine (T);
(b)a sugar called deoxyribose; and
(c)a phosphate group.
Nucleotides on the same DNA strand are joined in tandem by covalent bonds between the 5' position of the sugar on one nucleotide and the 3' position of the sugar on the phosphate on the next nucleotide. This forms a polynucleotide chain running from the 5' (or “five prime”) end to the 3' (or “three prime”) end. The two DNA strands, which run reverse-parallel to each other, 5'-3' and 3'-5', pair with complementary bases (A to T, C to G) to form “base pairs”.
Chromosomal DNA is replicated in the human cell nucleus. For this to occur the DNA-protein complexes must be disassembled and the DNA strands temporarily separated in the region of DNA being replicated. DNA helicases catalyse enzyme-dependent separation of the complementary strands from the site of previously bound initiator proteins, allowing DNA polymerase enzyme activity to synthesise two new strands using free deoxynucleotides (dNTPs). With the incorporation of each deoxynucleotide into the growing DNA strand, a pyrophosphate (two phosphate groups linked together) is released. Each strand of the original DNA molecule acts as a template for the production of a complementary strand in order to form two copies of the original DNA molecule.
Genes are functional units of DNA in the genome that code for particular proteins and non-coding RNAs. Different versions of a gene, for example, caused by variants such as single or multiple base changes, may be referred to as “alleles”. Where an individual has two copies of the same allele, that is, the same allele at a particular locus on each chromosome within one pair, they are said to be “homozygous” with respect to that allele. Where the alleles are different, i.e. there are different alleles at a particular locus on each chromosome within one pair, the individual is said to be “heterozygous” with respect to that allele.
(d) Genetic disorders
Genetic disorders are caused by changes to the genome. They may be caused by a range of mechanisms such as deletions, duplications, rearrangements and chemical modifications affecting anything from a single base to a whole chromosome or even the whole genome. Those caused by a defect in only one gene, for example, cystic fibrosis are classed as “single-gene disorders”. Other disorders may be caused by one of a number of genes. Although the spectrum is a continuum, disorders involving larger regions such as partial or whole chromosomes, for example Down syndrome, are generally referred to as “chromosomal” whilst those involving changes at the nucleotide level are generally classed as “molecular”. Genetic disorders may be inherited or arise “de novo” in the germ cell or developing embryo.
Prior to the priority date, prenatal testing was available for some single gene disorders, such as sickle cell anaemia, thalassemia and cystic fibrosis.
The cystic fibrosis gene was identified in 1989 and by the late 1990s several hundred mutations were recognized and this number has increased since that time. Prenatal diagnosis of cystic fibrosis was well-developed by the priority date and involved extracting fetal DNA from samples obtained using amniocentesis or CVS, and then analysing the extracted fetal DNA using methods such as targeted PCR.
Single-gene disorders can be inherited in an autosomal dominant fashion, which means that the disorder is expressed even if the person is heterozygous for the mutant allele, or in an autosomal recessive fashion, meaning that the disorder will only be expressed if the person is homozygous for mutant alleles. Disorders can also be inherited in an X-linked fashion in which a male will be more likely to have the disease phenotype as they only have one copy of the X chromosome.
Rhesus Disease
Rh (Rhesus) 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 gene (RhD) that encodes it in both copies of chromosome 1 which carries the RhD gene. 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.
Rh disease can cause haemolytic disease of the newborn and fetus. This typically arises in second or subsequent pregnancies when a Rh negative mother is carrying a Rh positive fetus. In other words, the child inherits from its mother a copy of chromosome 1 in which the RhD gene is deleted or mutated and a copy of chromosome 1 from the father in which the RhD gene is present and functional. Since the child possesses one functioning copy of the RhD gene, the child produces Rh factor, and is thus referred to as Rh positive.
When an Rh negative mother carries a Rh positive fetus, the fetus expresses the Rh factor on its red blood cells. During pregnancy and birth the mother may be exposed to fetal red blood cells expressing Rh factor. The mother mounts an immune response to Rh factor, which it identifies as foreign, and thus her immune system becomes sensitised to Rh factor. A Rh negative mother sensitised to Rh factor may mount a more robust immune response destroying the red blood cells of a Rh positive fetus in subsequent pregnancies.
By the priority date it was routine to give all pregnant mothers a blood test to determine their Rh status. The general approach to treatment was to treat all mothers identified as Rh negative with anti-Rh factor antibodies (so called prophylactic anti-D), ensuring that any Rh positive fetal red blood cells are masked before an immune response can be raised against them by the mother’s immune system, hence preventing issues with subsequent Rh positive pregnancies. This was, however, an inefficient approach as it inevitably involved treating Rh negative mothers carrying Rh negative fetuses, who did not need the treatment. There was, therefore, a desire to develop a way to screen for the Rh status of the fetus noninvasively. This would allow prophylactic anti-D to be given only to the Rh negative women who needed it, that is, those carrying a Rh positive fetus.
Haemoglobinopathies
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.
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 point mutations occurring in the case of beta-thalassemia may be different between the father and mother.
Sex-linked disorders
A number of diseases are known to be caused by a defective gene on the X chromosome, e.g. haemophilia. In many cases, these diseases primarily affect male fetuses because female fetuses will have another, non-defective, copy of the X chromosome. Work was on-going at the priority date to try to find ways to identify fetuses with possible sex-linked disorders. There was, therefore, a desire to develop a way to identify the sex of the fetus quickly, accurately and as early as possible in the first trimester. Further, in cases of congenital adrenal hyperplasia, which is not an X-linked condition, treatment with dexamethasone is needed to prevent virilisation in girls. This medication can be stopped once the fetus is known to be male. In severe X-linked diseases for which the parents may wish to elect termination, having a diagnosis as early as possible in the first trimester is valuable. DNA testing for the disease in such cases, which often took a week or longer, would only be done after the sex determination and could be omitted if the fetus was known to be female. Gender determination by ultrasound only becomes reliable for establishing the sex of a fetus from 18 weeks onwards.
As at the priority date, the sex of a male fetus would be apparent if a particular molecular test involved the PCR amplification of sequences known to be from the Y chromosome. In other words, if you could detect by PCR a Y-specific fragment, it was most likely that the fetus was male. This required sequence information for the Y chromosome to be known to allow for the design of primers to result in the Y specific PCR products. Further, the loci would need to be known to be unique to the Y chromosome. As at the priority date, there were Y-specific sequences published in the literature which could be used as PCR primers.
Aneuploidies
The presence of a variation in the number of chromosomes from the usual complement, that is, 46 chromosomes is referred to as aneuploidy. The absence of a single chromosome from a usual pair is referred to as monosomy, and the presence of an additional copy of a single chromosome to a usual pair is referred to as trisomy.
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.
The most common viable autosomal trisomies are trisomies of chromosomes 21, 18 and 13. Trisomy 13 and trisomy 18 often result in miscarriage, stillbirth or, in the case of viable births, neonatal death. Trisomy 21 is not usually life-threatening but can result in significant physical and mental disability. Fetuses with aneuploidies of multiple chromosomes are unlikely to survive past the early stages of pregnancy.
The additional chromosome found in cases of trisomy may be paternally or maternally inherited. In trisomies 13, 18 and 21, the extra copy of the relevant chromosome is inherited from the mother in the majority of cases, that is, over 91% maternal in trisomy 13, around 95% maternal in trisomy 18 and around 90% maternal in trisomy 21.
(e) Molecular genetic diagnosis
Molecular genetic clinical diagnosis first became possible in the 1980s following the discovery of genetic markers, that is, known sites of variation between individuals within a population located on a particular chromosome, otherwise known as genetic polymorphisms.
The process of working out which allele (marker) a person had at a particular position or set of positions on a chromosome, that is, determining the genetic make-up of the alleles at the relevant loci on an individual’s chromosomes was called genotyping.
At the priority date the vast majority of genotyping methods depended on either detecting differences by length of genetic variation, sequence, position, conformation, or methylation. Detection methods included fluorescent labels, biotinylated probes, intercalating dyes and radiolabels.
There were numerous methods but predominant among them were restriction fragment length polymorphism (RFLP) analysis, short tandem repeats (STR), variable number of tandem repeats (VNTR), single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), allele specific oligonucleotide (ASO), amplification refractory mutation system (ARMS), oligonucleotide ligation assay (OLA), pulsed field gel electrophoresis (PFGE), southern analysis, fluorescence in situ hybridisation (FISH), Sanger sequencing, pyrosequencing and reverse blots. Some of these methods are explained in more detail below.
Genetic markers used to detect human DNA per se could be as simple as detecting any one of the many repeat element families in the human genome e.g. Alu repeats. An alternative approach would be to test for the presence of a single copy gene. A more refined method of detection would rely on demonstration of differences based on polymorphism such as RFLPs detected either by genomic Southern blotting, or by PCR. Other markers commonly used at the time included STRs, VNTRs and other genetic variants.
Markers used to quantify DNA include the markers referred to in the preceding paragraph, along with determination of single or multiple copy sequences relative to a standard.
In 1997 thousands of markers that could be used for human molecular genetic testing were known. Commonly used markers included STRs, VNTRs, RFLPs and markers of structural variants. Of these variants, those used for human molecular screening/diagnosis would detect or be closely linked to the mutations underlying the specific genetic disease. Such markers would ideally be validated to document sensitivity, specificity, positive and negative predictive values. A wider number of markers may be required for diagnostic testing compared to screening.
Restriction Fragment Length Polymorphisms
Polymorphic loci at which base changes were known to affect restriction sites were known as restriction fragment length polymorphisms or RFLPs as I have referred to.
Certain naturally occurring bacterial enzymes called restriction endonucleases (or restriction enzymes) were known to cut double stranded DNA at specific points defined by the presence of a particular DNA sequence (a restriction site). Human DNA was known to contain numerous restriction sites. It was also observed after subjecting human DNA to bacterial restriction enzymes that not all individuals possess the same set of restriction sites within their DNA. If the DNA sequence at a particular locus differed from the restriction site by a single base, the relevant restriction enzyme would not cut the DNA at that site. This meant that if a section of two people’s DNA was subjected to restriction enzyme digestion the resulting fragments of DNA would either be of the same lengths (if all of the restriction sites were the same) or different lengths (if one or more of the restriction sites was absent, for example). The different fragment profiles could be observed by size separation of the products of restriction enzyme digestion by gel electrophoresis followed by a means of visualising the DNA.
Short tandem repeats
Short tandem repeats (STRs), also known as “microsatellites”, are sections of DNA made up of repeated short sequences, typically 2 to 4 base pairs long although they can be longer. It was found that different individuals often possessed different numbers of these repeated short sequences within a particular STR locus.
An individual’s STR genotype at a particular locus could be determined by creating primers for the constant (i.e. non-repetitive) DNA sequences on either side of a given STR locus and amplifying the resulting DNA fragment by PCR. Differing numbers of repeated sequences within the STR in different individuals would result in different length fragments being amplified, and the fragment lengths could be differentiated by gel electrophoresis followed by visualisation using a DNA stain or label.
Figure 3: STRs – simplified example
The diagram above shows only one allele from each person, just to simplify the explanation. In reality, each person would have two copies of the STR locus, one on each chromosome, which could be the same or different as the allele drawn out above.
The gels produced from STR amplification could require careful analysis as the repetitive nature of STRs could lead to problems during PCR amplification and “slippage” of the DNA strands resulting in DNA fragments being produced with extra or missing copies of the short repeat units. This gave rise to extra bands in the gel known as “stutter bands” or “shadow bands” which could require careful analysis, especially when genotyping individuals with similar allele sizes, for example n base pairs and n+2 base pairs for a dinucleotide repeat marker.
Figure 4: Schematic of stutter bands on a gel
STRs were used throughout the 1990s along with RFLPs as markers for linkage analysis and gene mapping. The greater heterozygosity, that is, the number of possible alleles at a given locus, of STRs, compared with RFLPs, increased the informativity of linkage analysis and assisted in the mapping of disease-causing genes. Once a disorder had been sufficiently localised to a particular part of a chromosome, that is, a defined genetic region, it was then feasible to sequence that small region to look for a gene and mutations.
Alu Repeats
Alu repeats are short repetitive sequences of DNA that are found throughout the human genome. They are the most common repeat sequences, and are present in tens of thousands of copies on every human chromosome. Alu repeats are generally not unique to a particular individual.
Amplification of Alu repeats or inter-Alu repeats may be used to establish whether human DNA is present in a sample. Primers that could be used to amplify Alu sequences had been reported by the priority date; see, for example, Nelson DL et al, “Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources” (1989) 86(17) Proc. Nat. Acad. Sci. USA 6686-6690 (Nelson 1989).
Linkage analysis
Linkage analysis is based on the principle that the closer together one locus (e.g. a gene) is to another locus (e.g. a RFLP or STR marker) on a chromosome, the more likely it is that the gene and the marker will be passed on together to the offspring. Thus the two loci are said to be “linked”.
By analysing the pattern of markers present in the genomes of various members of a family in which at least one person was affected by a genetically inherited disorder, it was sometimes found that the affected (or carrier) relative(s) in a family had a consistently different marker pattern in a particular region of the chromosome to those who were not affected by the disorder. In that situation it could therefore be inferred that the gene causing the disorder was likely to be found close to those markers. By repeating this type of analysis with other families the conclusions could be refined, allowing the approximate location of the disorder-causing gene on the chromosome to be identified, that is, mapped.
Linkage analysis could also be used in some cases to provide a diagnosis of a genetically inherited disorder, for example if a family had a history of a genetic disorder and wished to know if another member was likely to be affected by that disorder. A good example is Huntington’s disease, the symptoms of which typically only develop when a person is in their 40s, which was diagnosed by linkage analysis in the 1980s and 1990s. Linkage analysis had to be carried out for each family separately.
The process required genotyping as many family members as possible in relation to a panel of markers such as RFLPs or STRs which were known to be linked with the gene thought to cause the disorder in question. The way in which the various markers were inherited down the family line could then be assessed to determine whether the presence of a particular pattern of markers (haplotype) was associated with individuals affected by the disorder in that particular family. In cases where this was possible, the relative in question could be genotyped in respect of those markers to ascertain whether or not they were likely to be affected by the disorder too.
Direct mutation analysis
From the late 1980s onwards people started to identify the genes which were responsible for causing genetically inherited disorders. An early example was the cystic fibrosis gene which was first identified in 1989.
As new methods and new technology developed through the 1990s, considerable progress was made in identifying the genes responsible for various disorders. Once the relevant genes were isolated their sequences could be determined and, in some cases, particular mutations could be identified as being responsible for causing the disorder in question by comparing the DNA sequences of affected and unaffected individuals at the relevant locus.
The availability of more genetic information about disorder-causing genes through the 1990s meant that this was a time of expansion in molecular genetic diagnosis, particularly in the second half of the decade and through the 2000s. Rather than having to carry out linkage analysis, with all the work on family member genotyping and the complexity which that involved, it became possible to diagnose some conditions by genotyping the individual in question at the relevant locus to determine whether a disorder-causing mutation was present. This was simplest in cases where only a single gene was responsible for the disease (known as a “single gene disease”) and where the disease was caused by a dominant allele, that is, if the individual is found to have the disease-causing gene even in only one of the relevant pair of chromosomes they can still be diagnosed with the disorder. For recessive conditions, it was common practice to check the phase, that is, check the parental origin of the mutation(s).
But some mutations proved very hard to characterise. For example, a large deletion in a gene on one copy of the relevant chromosome might not be seen if the patient's other copy of the gene was normal. In that case, the normal gene would be picked up but because the assay was qualitative, the fact that the other copy was missing could go unobserved. In 1997, direct mutation analysis was conducted on a much smaller scale compared to what is done now.
By the priority date the Human Genome Project was underway with the aim of mapping and sequencing all of the genes making up the human genome. However, the project had not been completed. It would take several more years to be finished with the full genome being published in 2003.
(f) Molecular techniques
By the priority date, there was a range of molecular techniques and technologies available. Some of these included the following:
(a)Gel electrophoresis.
(b)Polymerase chain reaction (PCR), including:
(i)Nested PCR; and
(ii)Quantitative PCR techniques.
(c)Sanger sequencing.
(d)Branched DNA methods.
(e)Ligase chain reaction.
Gel electrophoresis
Gel electrophoresis was and is used to separate molecules like DNA based on their size (molecular weight). When carried out following PCR or simple restriction enzyme digestion, the reaction products would be mixed with a suitable buffer containing a dye to increase the density of the material in the sample so that it could be seen during loading into a “well” on a gel made of agarose or polyacrylamide. An electric field would then be applied to the gel, which would cause the DNA, which is negatively charged, to migrate through the gel. The gel acts like a sieve, with smaller DNA molecules moving through it more quickly than larger DNA molecules. The gel would then be stained, typically with ethidium bromide, which binds to DNA, and then visualised with UV light. The stained molecules travel a particular distance from the starting point corresponding to the size of the molecule, so different PCR amplification products or fragments generated by restriction endonucleases will form separate bands on the gel.
PCR techniques
PCR is a standard molecular biology technique that involves the amplification of specific sequences of DNA using repeated cycles of denaturation, primer annealing and extension, resulting in theory at least in exponential accumulation of DNA fragments. The basic technique is illustrated by the diagram below:
Figure 5: PCR steps: (1) denaturation; (2) annealing; (3) elongation. Template DNA shown in green; primers shown in red; new DNA strand shown in blue
DNA primers must be designed to bind to opposite DNA strands and flank the target of interest and initiate synthesis of a new DNA strand complementary to the target sequence of each template strand. These primer pairs are commonly referred to as the forward and reverse primers.
PCR requires the following components:
(a)A DNA template which is the gene or DNA sequence to be amplified.
(b)Primers, which are short single stranded pieces of DNA (i.e. an oligonucleotide) that have been synthesised to be complementary to a section of DNA. Two primers flank the gene or DNA fragment (in opposite orientation) to be amplified.
(c)DNA polymerase, being an enzyme that recognises primers and mediates the replication of the DNA template between the two primers starting from the 5' end to the 3' end by adding a complementary base to the growing strand. The most commonly used of these enzymes was Taq DNA polymerase which was derived from the heat-resistant bacteria Thermus aquaticus. As it is heat resistant it is suitable for use in PCR, which relies on thermal cycling.
(d)Nucleotides (dNTPs or deoxynucleotide triphosphates), being single units of the bases A, T, G, and C or in other words the “building blocks” for new DNA.
In broad terms, PCR involves the following steps:
(a)The first step is denaturation where the sample DNA is heated to break the weak bonds between the nucleotides of each strand so that the DNA separates into two separate strands.
(b)The second step is annealing. PCR does not copy all of the DNA in the sample but only a very specific gene or DNA sequence targeted by the primers. Two primers are used: one for each of the separated complementary single DNA strands. In this step, the primers bind, or anneal, to their complementary sequences on the DNA strands, marking the beginning of the sequence to be copied in the following step. Annealing occurs at lower temperatures than denaturation. And in this respect a balance needs to be achieved between a higher temperature which achieves greater specificity / lower mismatches, and the risk of denaturing the primers at those higher temperatures which prevents binding.
(c)The third step is extension where the reaction tube is heated to facilitate the extension of the nucleotide chain. From the start of the regions marked by the primers, nucleotides in the solution are added to the annealed primers by DNA polymerase to create a new strand of DNA complementary to each of the single DNA template strands. After this step, two identical copies of the original target DNA sequence are made. The extension must continue past the position of the primer on the complementary strand, so that in the next PCR round that new copy serves as a template for further amplification.
(d)The above steps are then repeated about 30 to 35 times to produce a sufficient amount of DNA copies of the original DNA target sequence. Too many amplification cycles beyond this leads to a building up of PCR “artefacts”, which create extra bands and impede analysis.
Let me expand further on the question of primers. PCR relies on the design of primers that will amplify a sequence of interest. As at the priority date, a number of issues needed to be considered in primer design, which sometimes required multiple rounds of optimisation, including limited sequence information and manual primer design.
The Gene Mutation Database and Human Genome Database were used at the priority date to design primers for use in DNA amplification methods for human molecular genetic testing. At the priority date the Gene Mutation Database and Human Genome Database had large collections of DNA sequences collated from the literature. In some circumstances these were of variable quality and reliability. Therefore, it was wise to validate any chosen marker. Both of these databases were searchable for specific DNA loci and the specific sequences could be downloaded and employed for primer design.
For human molecular genetic screening / diagnosis, the Gene Mutation Database had almost 500 specific disorders listed with known mutations. However, this constituted a very small percentage of total single gene disorders. For accurate human diagnostics of the majority of single gene disorders, there would be considerably more reliance on personal communication between research and clinical groups.
As I have indicated, as at the priority date, standard practice was to run 30 to 35 cycles of amplification, that is, to the end-point of the reaction. Running more than 40 cycles was known to result in a much higher chance of amplifying something other than the target. This can happen when the primers bind to a sequence of DNA that is very similar to the target or where there is contamination, that is, when some foreign DNA gets into the sample and is targeted by the primers. The risk of contamination affecting the results of a PCR assay is particularly high when the target sequence occurs in low levels in the sample or when there is no separation of the pre- and post-PCR laboratory areas or where appropriate equipment is not used, such as filter tips. Standard PCR is a non-quantitative technique.
Nested PCR
Nested PCR is a technique used to improve the specificity of the PCR reaction, that is, so that ideally only target sequences are amplified. It is particularly useful for detecting low levels of target sequence in a given DNA sample. In this method, the DNA in the sample is first amplified using “outer” primers which bind to the target sequence and possibly other non-target sequences in the sample. The sequences or “amplicons” amplified in this first round of PCR are subjected to a further PCR reaction involving one (hemi-nested) or two different “inner” primers having sequences which bind to sequences within the target amplicon.
Quantification of genetic material
Methods of quantification of genetic material were available at the priority date and included semi quantitative methods including densitometry scanning of areas under the peak (AUP) for Southern blots or bands on a gel. All of these could have been compared to internal controls. Other methods included PCR based methods consisting of endpoint PCR assays that sometimes used fluorescent markers. These methods also employed AUP measurements.
More accurate quantitative detection was achieved by quantitative PCR that measured changes in the exponential phase of PCR, again relative to a control.
The methods described above had a defined dynamic range and the quantification was relative.
Quantitative PCR
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), fluorescent labelling of PCR products and detection with laser-induced fluorescence using capillary electrophoresis or acrylamide gels and plate capture and sandwich probe hybridisation.
Real-time quantitative PCR
One type of qPCR method that was available by the priority date was real-time quantitative PCR (“real-time qPCR”). Real-time qPCR involved a PCR reaction during which accumulation of the amplified PCR product was monitored by measuring a signal created by either fluorescent dyes or fluorescent probes in the reaction sample to generate an amplification curve. That is, real time qPCR is a development of standard (or end-stage) PCR, and uses fluorescent reporter molecules to monitor the amounts of PCR product present after each PCR cycle. This allows the generation of a growth curve and enables the quantification of DNA in the exponential phase by determining the number of amplification cycles necessary to achieve a specified fluorescence level.
Quantitative fluorescent PCR
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 quantitation compared to real-time qPCR methods, as the latter quantified the PCR products in the same vessel as that used to amplify the DNA.
Sanger sequencing
Sanger sequencing is a method of direct DNA sequencing developed by Fred Sanger in about 1977. It was originally called “DNA sequencing” but is now called Sanger sequencing to distinguish it from the new generation of sequencing technologies.
The principle behind Sanger sequencing is similar to PCR except only one primer is used. Accordingly, amplification is arithmetic not exponential. The primer is a mixture of nucleotides and di-deoxynucleotides, which act as chain terminators during amplification, such that when a di-deoxynucleotide analogue is incorporated into the growing DNA strand, it prevents further extension from occurring. It is chance whether a normal or di-deoxy analogue is incorporated into the DNA extension product. But the ratio of normal to di-deoxy analogues is calculated to allow mainly normal nucleotides to be incorporated. The result is a series of extension products of various lengths depending on when a di-deoxy analogue is incorporated. This reaction is cycled 25 times to increase the number of extension products and to ensure that the reaction mixture contains extension products terminating at every base of the original sequence. Extension products are then separated according to size by gel electrophoresis, with each product differing in size by 1 base. The order of the separated extension products, terminating in a particular di-deoxy analogue, provides the sequence of the original sample DNA, which is demonstrated in Figure 6.
Figure 6
To enable detection, each di-deoxy analogue is labelled. When performing manual sequencing, usually each analogue was radiolabelled and detected on the gel after electrophoresis by autoradiography. When using automated Sanger sequencing instruments, the analogues could be labelled with one of four fluorescent colours (one for each base), such that the sequence of a sample could be determined based on the fluorescence of each analogue.
There were limitations with Sanger sequencing including that it could only sequence up to about 600 to 800 base pair fragments. If the DNA fragment was longer it was necessary to sequence multiple overlapping sequences.
Once a DNA fragment had been sequenced it was necessary to compare it to a reference sequence to identify any variations. Once sequence variations have been identified one would either be able to identify the significance of it based on previous experience or previous publications, or one could search for that mutation in a gene specific database to review what was known about the significance of that variant. Reference sequences could be sourced from the literature, gene specific databases or standard sequences.
Branched DNA methods
Branched DNA methods involve the use of capture probes and amplification of the signal through label probes.
Ligase Chain Reaction
The ligase chain reaction (“LCR”) is depicted in the Figure below.
Figure 7: Ligase Chain Reaction
LCR is a technique that can be used in some cases to discriminate between DNA sequences differing in only a single base pair of nucleotides, such as a single base mutation.
LCR typically involves the use of two complementary pairs of single stranded DNA probes or oligonucleotides, each pair designed to bind to adjacent regions on both the sense and the antisense strands of a target DNA sequence. The junction of the two adjacent oligonucleotides (“Oligo Junction”) in each pair is usually positioned so that the nucleotide at the end of one of the oligonucleotides coincides with the point mutation. If, and only if, the nucleotide in the oligonucleotide at the Oligo Junction is complementary to the nucleotide at the same position in the target sequence can an enzyme called DNA ligase join the two oligonucleotides. The process can also be used to amplify a region without a single base mutation.
If the two adjacent oligonucleotides are able to join or ligate, the joined oligonucleotides can then themselves be used as templates for further rounds of ligation mediated amplification, which, like PCR, exponentially amplifies the paired oligonucleotides containing the target DNA sequence.
If the adjacent oligonucleotides are not able to join, because the nucleotide at the Oligo Junction is not complementary to the nucleotide at that position in the target sequence or because the target sequences are not present, amplification will not occur.
(g) Prenatal testing
Triple Screen
It was known that at around 16 weeks’ gestation in the second trimester of pregnancy, three substances, namely, alpha-fetoprotein (AFP), free beta hCG and estriol, were often present in different amounts in maternal serum in pregnancies where the fetus was affected by Down syndrome and pregnancies where it was not. This led to the development of what became known as the “triple test”. Instead of involving an invasive test, measurement of the levels of these markers could be achieved using a simple blood sample from the mother at around 16 weeks’ gestation. By considering both the risk associated with the mother’s age and the risk calculated by reference to the mother’s AFP level, a more accurate estimate of the overall likelihood that the mother was carrying a fetus affected by Down syndrome could be obtained. Determining AFP levels in maternal serum was also much cheaper than invasive testing and was not associated with a risk of miscarriage.
The triple test was the standard serum screening test for Down syndrome for a long time and was eventually offered to all pregnant women regardless of age in many countries. Although the precise figures vary, as a general rule, the triple test led to the identification, after invasive testing, of about 50% of cases of Down syndrome in the tested population. In other words, of the women taking the test who are actually carrying fetuses affected by Down syndrome about half of them will be identified by the triple test.
By the priority date work had also been carried out to find new markers which could be used at 10 to 12 weeks’ gestation as there was a desire to enable diagnosis of Down syndrome within the first trimester of pregnancy, leading to the discovery in the early 1990s that free beta hCG could be used in combination with pregnancy associated plasma protein A (PAPPA) to screen reliably for Down syndrome during the first trimester of pregnancy using maternal serum. It had also been discovered that fetuses affected by Down syndrome could be identified using ultrasound scanning in what was known as the nuchal translucency test. By the priority date researchers had started to consider whether combining the nuchal translucency test with serum markers might give even more accurate results.
Consequently, in addition to the use of maternal age to assess the risk that a pregnant woman was carrying a fetus with Down syndrome, it being known for decades that the likelihood of a woman conceiving a fetus affected by Down syndrome increases as the woman gets older, biochemical screening of maternal serum was used to identify women who were at higher risk of carrying an aneuploidy fetus and only those women were referred for invasive testing.
Cytogenetic techniques
Cytogenetic techniques were available at the priority date to analyse the number and structure of chromosomes in fetal cells, which had been extracted from the pregnant woman’s amniotic fluid, by amniocentesis, or placenta, by chorionic villus sampling (CVS). Each of these techniques carried with it a risk to the fetus.
Amniocentesis involves the collection of amniotic fluid, which contains fetal cells, using a needle which is inserted through the abdomen and uterus into the amniotic sac under ultrasound guidance. It is feasible from 15 weeks.
CVS is a technique for sampling cells that are likely to have the same karyotype as the fetus although in some cases they will differ, for example where there is a confined placental mosaicism. The sample is collected from the chorionic villus of the placenta either using a catheter inserted through the vagina or using a needle inserted through the abdomen.
Once the fetal cells had been isolated and cultured they could be analysed by cytogenetic techniques to determine the number and structure of chromosomes. These techniques included the following methods.
One method was karyotyping. The “karyotype” of an individual is the number and appearance of the chromosomes in the nucleus of the cell. Traditional karyotyping involves staining dividing (metaphase) chromosomes with a dye to allow them to be visualised under a microscope. This allowed fetuses possessing an abnormal number of chromosomes (such as an extra copy of chromosome 21 in Down syndrome) or chromosomes with abnormal structures to be diagnosed.
Another method was fluorescent in situ hybridisation (FISH). FISH uses a fluorescently labelled DNA probe which is designed to bind to portions of a gene of interest. The presence of trisomy 21 may be detected by the presence of three fluorescent spots in the fetal cell, rather than the expected two. The FISH technique hybridised fluorescent probes to specific sequences within a chromosome. Probes used in FISH were large and usually isolated sections of chromosomes, as opposed to small sequences used in other molecular work. Fluorescence microscopy could then be used to detect where on the chromosomes the fluorescent probe has bound. FISH was and is used to detect chromosome translocations, aneuploidies and chromosome identification.
Whilst these approaches were accurate and reliable, the main drawback with amniocentesis and CVS was that both procedures were invasive and were understood to increase the risk of the mother suffering a miscarriage. At the priority date the risk was believed to be around 1% for amniocentesis and 2% for CVS.
(h) Y chromosome markers & sex determination
The sex of a fetus determined by cytogenetics or detection of Y chromosome markers in kits was taken into account in the assessment of sex-linked genetic disorders. For example, if a pregnant woman was a known carrier of haemophilia and was having a male child, then the male child would have haemophilia.
As at the priority date, the sex of a male fetus would be apparent if a particular molecular test involved the PCR amplification of sequences known to be from the Y chromosome. In other words, if you could detect by PCR a Y-specific fragment, it was clear that the fetus was male. This required sequence information for the Y chromosome to be known to allow for the design of primers to result in the Y specific PCR products. Further, the loci would need to be known to be unique to the Y chromosome. As at the priority date, there were Y specific sequences published in the literature, which could be used as PCR primers.
(i) Fetal cells in maternal blood
The idea that fetal cells might be present in the mother’s blood had been first proposed in 1969. The possibility of being able to access whole fetal cells by taking a maternal blood sample was of great interest to those looking for a non-invasive way of obtaining information about the fetus because it would allow analysis of the fetal genome to be carried out without the need for invasive testing and the problems associated with it. All that would be needed was a blood sample from the mother’s arm. As a result, a substantial amount of work was carried out in this area through the 1980s and the 1990s.
The approaches and techniques that had been used for non-invasive prenatal testing using fetal cells both isolated and in a background of maternal cells before the priority date are described in a paper published in 1994 by JL Simpson and S Elias entitled “Isolating Fetal Cells in Maternal Circulation for Prenatal Diagnosis” 14(13) Prenatal Diagnosis 1229-1242.
By the priority date it was known that several different types of fetal cells were present in maternal blood during pregnancy, and that these fetal cells had the potential to be used for prenatal testing. These included:
(a)haematopoietic stem cells which, in the fetus, go on to make red and white blood cells;
(b)nucleated erythrocytes, which are a type of red blood cell which is typically only found in fetuses and very young children, not in adults whose red blood cells do not contain a nucleus or, therefore, chromosomes;
(c)lymphocytes and granulocytes which are types of white blood cell; and
(d)trophoblasts which are fetal placental cells which invade the tissue of the mother’s uterine wall causing changes in its vascular structure and formation of the placenta.
I note that in 1996, C Danae Steele and others (Steele CD et al, “Prenatal Diagnosis Using Fetal Cells Isolated From Maternal Peripheral Blood: A Review” (1996) 39(4) (December) Clinical Obstetrics & Gynecology 801-813) discussed relevant different cell types in the following terms:
TROPHOBLASTS
The possible cell types that can be isolated from maternal blood and used for prenatal diagnosis include trophoblasts, lymphocytes, and erythroblasts. Trophoblasts were the first cells to be identified in the maternal circulation because of their large size, but there are several reasons that they are not ideal for prenatal diagnosis. First, very small numbers of these cells are present in maternal blood during the first trimester, when it would be ideal to perform prenatal diagnosis. Secondly, any trophoblasts that are released into the maternal blood quickly become trapped in the lung and only rarely remain in the peripheral circulation. Thirdly, syncytiotrophoblasts [placenta] do not always share the same chromosome complement as the fetus and may be either multinucleated or mosaic. For these reasons, most investigators have concluded that trophoblasts are not the ideal cells to use for prenatal diagnosis.
LYMPHOCYTES
Fetal lymphocytes are an attractive potential source of cells for prenatal diagnosis. They express the HLA class of antigens so that if the mother and father are HLA incompatible, cells may be sorted by flow cytometry and enriched for paternal antigen expressing cells. However, this approach requires paternal HLA typing and specific antisera, and is impossible when paternal and maternal HLA antigens are shared. This approach is further limited because fetal production of lymphocytes does not begin until the second trimester, and even if lymphocytes can be isolated, it is not clear that they respond to the mitogens used to produce the metaphases needed for karyotyping. Tharapel et al flow-sorted cells for HLA differences between mother and fetus but could locate only maternal cells after metaphase had been induced.
Another potential problem is that in some cases fetal lymphocytes can persist in the maternal circulation for many years after a pregnancy…
NUCLEATED ERYTHROCYTES
Fetal nucleated erythrocytes appear to be best suited for this approach to prenatal diagnosis for several reasons. First, nucleated erythrocytes are rare in the adult circulation (except in clinical circumstances of increased hematopoesis such as pregnancy), but common in the fetus, especially in early gestation when hematopoesis occurs mainly in the yolk sac. Secondly, nucleated erythrocytes express several unique antigens such as the transferrin receptor, which make it possible to use automated cell-sorting techniques to enrich samples of maternal blood for fetal cells. Thirdly, these cells produce unique fetal hemoglobin chains such as zeta and gamma, which have the potential to be used as markers to identify these fetal cells. Lastly, erythrocytes are known to have a short life-span and are unlikely to persist from one pregnancy to the next.
Recent studies suggest that most nucleated erythrocytes that occur in first trimester maternal blood samples are maternal in origin. Enrichment of nucleated erythrocytes in general will enhance the concentration of maternal cells as well as fetal. Therefore, a unique marker for fetal nucleated erythrocytes continues to be sought.
All of these cell types were known to be present in all three trimesters of pregnancy.
From at least the late 1980s there was research being done by a number of groups on processes of recognition and enrichment of fetal cells. The aims of this research included investigating the use of fetal cells isolated from maternal blood to provide a non-invasive means of diagnosis of Down syndrome, to determine the blood type of the fetus and enable the pregnancy to be managed accordingly, to provide a way to determine the sex of the fetus (which would be useful when trying to identify fetuses with possible sex-linked disorders), and to diagnose single gene disorders.
However, isolating fetal cells was not easy because they were known to occur only rarely in maternal blood and were vastly outnumbered by maternal cells in the sample. Consequently, as well as work on new or improved methods and equipment for isolating fetal cells, research was also being carried out into methods for enriching the proportion of fetal cells in a sample to try to overcome these problems. Various techniques were tried including flow sorting, which relied, for example, on finding antibodies which bound to particular antigens on fetal cells, and magnetic sorting. These methods had some success in improving rates of fetal cell isolation during the course of the 1990s. However, by the priority date fetal cells could not reliably be identified from every maternal blood sample.
Another important issue was verifying that the cells which had been isolated were actually fetal before any analysis was carried out. Fetal cell detection had been approached using various methods. Early studies used fluorescence activated cell sorting, selecting cells identified as fetal by antibodies directed at paternal HLA type (human leukocyte antigen type). Flow cytometry was also used.
Flow cytometry is the use of light scattering and absorption spectra to measure various intrinsic and extrinsic properties of whole cells suspended in a solution. The solution is passed through a narrow tube past a series of laser beams arranged at different angles relative to the direction of flow, allowing only a single cell to pass at once. The passage of the cell interrupts the laser beams, and this is detected by an array of receivers arranged opposite to the beam generators. This allows each individual cell to be detected as a unique signal event, allowing the diagnostician to record many thousands of such events and draw conclusions about the properties of a large population of cells.
The size and internal granularity of cells can be inferred from the scattering of light from different angles. Fluorescently-labelled probes which bind to cell surface markers may also be used. Using this approach, the signals produced by flow cytometry can also be used to sort populations of cells, for example to isolate a specific sub-population of interest.
Figure 8: Illustration of the basic principles of flow cytometry
Similarly, at the priority date linkage analysis involved assumptions that the target gene of interest had been or was likely to have been inherited with the adjacent or linked genetic markers (i.e. RFLP markers) detected by the analysis. The markers were used to trace the presence of a nearby allele indirectly. And determination that a fetus was male following the detection of a Y signal involved an assumption that the Y chromosome did not come from, for example, a mosaicism in the mother or a transplanted organ.
Further and in any event, the accuracy of the Harmony Test’s methods means that it is problematic to treat the identification of informative paternally inherited (fetal) markers in the Polymorphic Assay as a mere assumption.
And indeed the identification of fetal sequences in the Harmony Test is not treated in ambiguous terms in the respondents’ own instructional and marketing material. Let me give the following examples as correctly submitted by Sequenom.
The Harmony IVD Kit Instructions (emphasis added):
(a)state that “FORTE then identifies loci that are informative for estimating fetal fraction in each sample”;
(b)explain that informative loci are defined as loci “where the material genotype is homozygous for one allele, and the fetus has inherited a different allele”; and
(c)refer to the Harmony Test’s ability to “detect” minor source cfDNA at fractions from 4% (i.e. fetal DNA).
Sonic’s presentation on the Harmony Test explains that the fetal fraction is the percentage cfDNA “from the fetus” and that “the fetal fraction MUST be measured. Accurately.” (emphasis added).
Further, Mr Andrew Sparks and Mr Eric Wang, the signatories of the PPD, in an article published in 2012 that I have referred to earlier titled “Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18” explained that:
[i]nformative polymorphic loci were defined as loci where fetal alleles differ from maternal alleles. Because DANSR exhibits allele specificities >99%, informative loci were readily identified when the fetal allele proportion of a locus was measured to be between 1-20%. A maximum likelihood estimate using the binomial distribution was employed to determine the most likely fetal fraction based upon measurements from several informative loci.
Further, the Fetal Fraction Brochure explains that the DANSR and FORTE core technologies of the Harmony Prenatal Test “give you confidence by measuring fetal fraction accurately and reproducibly; [r]eporting fetal fraction to provide a confident result; [i]ncorporating fetal fraction into results to accurately distinguish high and low probability results (even at low fetal fraction)” and defines fetal fraction as “the amount of fetal cell-free DNA circulating in the mother’s blood compared to the total cell-free DNA”. The Fetal Fraction Brochure similarly states that the Harmony Test “provides better quality control as compared to other tests by accurately quantifying fetal cfDNA”.
Further, an Ariosa brochure titled “Harmony Prenatal Test: Clear Answers to Questions that Matter” dated 2016 states that the Harmony Test incorporates “precise fetal DNA measurements”.
Further, an undated presentation on the Harmony Test titled “Considerations in Selecting NIPT: Choosing the Right Test for your Practice” confirms that the Harmony Test “[a]ccurately measures fetal DNA amount using SNPs”.
Further, an Ariosa brochure titled “The Harmony IVD Kit: Bringing the leading, global NIPT brand to your laboratory” dated 2016 explains that “[s]ingle nucleotide polymorphisms (SNP) technology is also employed to distinguish and quantify fetal fraction” (emphasis added).
In my view the Harmony Test’s approach to detecting fetal DNA falls within the scope of the plain words of the detection claims of the Patent.
Moreover, I agree with Sequenom that the respondents’ attempt to deny infringement on the basis of alleged assumptions and inferences in the Harmony Test method is artificial and at odds with the views of the experts (other than Ms Norbury), the respondents’ own published material and the purposive meaning given to the term “detect” by persons skilled in the art.
Further, if it needs to be said in terms of the integers of claim 1, it is apparent from the description in the PPD that the Harmony Test is performed on the plasma component of a blood sample taken from a pregnant female.
The Harmony IVD Kit Instructions state at p 2:
The Harmony Test is intended for use in analysis of cfDNA samples isolated from plasma from pregnant women…The Harmony Test requires cfDNA that has been isolated using a commercially available cfDNA extraction kit from approximately 4mL of plasma…
Further, the Harmony Brochure states that “Harmony™ involves testing millions of short fragments of cfDNA in maternal plasma”.
Claim 2 – …comprising amplifying the fetal nucleic acid
Ariosa accepts that there is amplification in the Harmony Test for the purposes of claim 2. Accordingly, infringement is established.
Claim 3 – …wherein the fetal nucleic acid is amplified by PCR
The confidential PPD describes the use of PCR. Page 3 of the Harmony IVD Kit Instructions refers to “[t]hermal cycling of the 96 UPCR [universal polymerase chain reaction] reactions” which Professor Fisk says is a reference to amplification via PCR. Accordingly this integer is satisfied for claim 3. Accordingly, infringement is established.
Claim 5 – …by means of a sequence specific probe
Ariosa accepts that the Harmony Test uses a probe for the purposes of claim 5. Accordingly, infringement is established.
Claim 6 – …presence of a fetal nucleic acid sequence from the Y chromosome is detected
The evidence of both Professors Fisk and Lovett is that the Harmony Test Non-Polymorphic Assay involves the detection of loci on the Y chromosome. The Non-Polymorphic Assay involves looking for sequences that are of fetal origin as the Y chromosome is not possessed by pregnant women.
In response to the question, “[d]oes the interrogation of loci on the Y chromosome in the Non-Polymorphic Assay of the Harmony Test detect the presence of a nucleic acid of foetal origin in a maternal plasma sample within the meaning of claim 1 of the Patent?”, and the question, “[d]oes the interrogation of loci on the Y chromosome in the Non-Polymorphic Assay of the Harmony Test detect the presence of a foetal nucleic acid sequence from the Y chromosome within the meaning of claim 6 of the Patent?”, Professor Fisk, Professor Lovett and Ms Norbury all answered “Yes”, with Ms Norbury “noting” the “possibility” of a false positive. A Harmony Test document by Ariosa dated 2016 records that the Harmony Test’s fetal sex microarray is 99.8% accurate.
The presence of a fetal nucleic acid from the Y chromosome is detected as part of the Harmony Test. For example, in the PPD, chromosome Y is listed as a target chromosome within the Non-Polymorphic Assay, which is always run simultaneously with the Polymorphic Assay.
It is of course an assumption of the Harmony Test that the Y chromosome comes from the father.
The Harmony IVD Kit Instructions at p 23 states:
The Harmony Test was used to determine fetal sex in 787 of the 791 specimens described above. These specimens included 395 pregnancies where clinical genetic testing outcomes indicated no male fetus was present (absence of Y chromosome), and 392 pregnancies in which a male fetus was present (presence of Y chromosome).
Accordingly, infringement is established.
Claim 9 – …presence of a fetal nucleic acid from a paternally-inherited non Y chromosome is detected
The Polymorphic Assay identifies major and minor fractions of informative loci. The minor fraction is paternally inherited fetal DNA. Professor Lovett considers it implausible that any individual Harmony Test would not detect a paternally inherited (or fetal) nucleic acid.
Accordingly, infringement is established.
Claim 13 – …for determining the sex of the fetus
Claim 13 is a method for determining the sex of the fetus. It is dependent on claim 6, which provides for the detection of the presence of a fetal nucleic acid sequence from the Y chromosome.
Now Ariosa says that the Harmony Test does not definitively “determine” fetal sex. It says that the Harmony Test does not make a determination of the likelihood that a fetus is female that is based on detecting the presence of a fetal nucleic acid sequence from the Y chromosome. Rather, it determines the likelihood of a female fetus based on the absence of the detection of a sequence from the Y chromosome as stated in an example Harmony Test Report in evidence:
Fetal Sex test quantifies the Y chromosome. A “female” result indicates absence of Y chromosome and a “male” result indicatives presence of Y chromosome…
But Sequenom says that at the priority date, in the context of NIPD, “determination of fetal sex” simply meant detecting the presence of a Y DNA sequence and covered methods having varied levels of specificity (i.e. false negative rates) and sensitivity (false positive rates). That is, the claims cover the determination of the likelihood of a particular fetal condition or characteristic, such as gender.
Further, in responding to Ms Norbury’s suggestion that Example 1 of the Patent does not determine fetal sex but rather “male status”, Professor Fisk explained that:
I consider such a distinction to be completely artificial for the purposes of considering the disclosure in the Patent. The Patent conventionally and understandably equates the presence or absence of a signal from a sequence on the Y chromosome with gender determination. The Patent does not, for example, delve into very rare discrepancies between genotype and phenotype in genetic males, such as testicular feminisation or 5-alpha reductase deficiency. In my view, Example 1 clearly determined what is understood in the Field by fetal sex, albeit with a degree of confidence of less than 100%.
Consistently with Professor Fisk’s views, the Patent states that “sex determination” may be simply carried out by detecting the presence of a Y chromosome. In this context, no mention is made of the necessity to, for example, have a control for non-Y chromosome cffDNA to have additional confidence that a fetus where a Y sequence is not detected would be female.
Further, in terms of determination, as Professor Hyett and his co-authors noted in Hyett JA et al “Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancy” (2005) 25(12) Prenatal Diagnosis 1111-1116:
Lo et al. (1997) demonstrated the presence of ffDNA in the plasma of pregnant women carrying male foetuses and that this was potentially a useful source of material for genetic prenatal diagnosis (Lo, 2000). Several groups have reported their ability to determine fetal sex by positively identifying male DNA sequences (to the SRY gene) in maternal plasma (Table 1) (Lo et al., 1997, 1998; Smid et al., 1999; Zhong et al., 2000, 2001; Al-Yatama et al., 2001; Costa et al., 2001; Honda et al., 2001, 2002; Nelson et al., 2001; Rijnders et al., 2001, 2003; Sekizawa et al., 2001; Farina et al., 2002; Hromadnikova et al., 2002; Guibert et al., 2003). Early studies using conventional PCR techniques had lower sensitivity and specificity than those more recent series that have used real-time quantitative PCR…
(Emphasis added.)
In the same article, Professor Hyett relevantly noted that the “disadvantage of this technique, which uses primers to identify the male SRY gene, is that the diagnosis of a female fetus is based on a negative test result, and therefore carries the risk of falsely being negative” (emphasis added).
Further, even if determination imports an extremely high degree of accuracy, such a standard is satisfied by the Harmony Test. The Harmony IVD Kit Instructions themselves state (at page 4) that the Harmony Test can be used to “determine fetal sex”. It is not suggested that further invasive tests are needed to “confirm” this determination or diagnosis, and the respondents concede that the Harmony Test sex option is a prenatal diagnosis.
An example Harmony Test result in evidence similarly explains that the Harmony Test’s “Fetal Sex” test “…quantifies the Y chromosome. A “female” result indicates absence of Y chromosome and a “male” result indicates presence of Y chromosome” and has an accuracy of over 99% for male or female sex.
The results report the fetus to be either a “male” or “female”. They do not, for example, state that there is a “low risk” that the fetus is male.
In the joint expert report, all of the experts agreed that the Non-Polymorphic Assay of the Harmony Test is performed for determining the sex of the fetus within the meaning of claim 13 of the Patent.
Accordingly, in my view the respondents’ assertions that the Harmony Test does not definitively “determine” fetal sex for the purposes of claim 13, but rather determines the “likely chromosomal gender” is artificial and does not place the Harmony Test method outside the scope of the claims. Infringement is established.
Claim 14 – …determining the concentration of the fetal nucleic acid sequence in the maternal serum or plasma
Claim 14 of the Patent provides:
The method according to any one of claims 6 to 12, which comprises determining the concentration of the foetal nucleic acid sequence in the maternal serum or plasma.
As discussed above, Ariosa’s argument is that the term “concentration” in claim 14 is a reference to an amount per unit volume, not a ratio or relative concentration. But I have already rejected this argument.
As to infringement, the Harmony IVD Kit Instructions use the term “concentration” to refer to relative concentration. And Ms Norbury agreed that concentration was used in a relative manner in the following extract from the Instructions:
A targeted amplification process termed Digital Analysis of Selected Regions (DANSR) is used to simultaneously amplify from each of the 96 DNA specimens UPCR products corresponding to approximately 6800 ~75bp long genomic intervals including approximately 1200 non-polymorphic loci on each of chromosomes 4, 13, 18, 21, and X; approximately 100 non-polymorphic loci on chromosome Y; and approximately 700 single nucleotide polymorphism (SNP)-containing polymorphic loci on chromosomes 1-12, in proportion to the concentrations of their cognate loci and alleles in the original DNA specimens.
The Polymorphic Assay involves the detection and quantification of SNP alleles at a large number of loci (on chromosomes 1-12). The quantity and thus concentration of each allele at an informative locus in the Polymorphic Assay is estimated by detection of the different light intensities generated at each of two different wavelengths. The allele generating the lesser signal is necessarily the paternally inherited allele of fetal origin. By calculating or very accurately “estimating” fetal fraction using the procedure outlined in the PPD, the Harmony Test includes the step of determining the concentration of fetal nucleic acid sequence in the maternal serum or plasma.
Accordingly, the Harmony Test infringes claim 14.
Claims 22, 23, 25 and 26
Claims 22, 25 and 26 each refer to “a method of performing a prenatal diagnosis” and/or “providing a diagnosis”.
As I have indicated, Ariosa says that the term “prenatal diagnosis” is characterised as a type of test which provides highly accurate and reliable results on which a clinician and a patient can base a clinical decision, that is a decision in relation to treatment or intervention. But it says that other than sex determination (which is only provided if requested by the patient), the Harmony Test is a screening test. It is a screening test which provides a risk assessment that the fetus carries certain genetic disorders and, if the patient requests it, a risk assessment of sex chromosomal aneuploidy. If a patient is assessed to be high risk by the Harmony Test, the patient will be referred for further testing using invasive techniques. No clinical decisions are made based on the results of the Harmony Test.
Now Ariosa concedes that the Harmony Test fetal sex option is a prenatal diagnosis. Accordingly, the issue which remains is whether the Harmony Test’s detection of XYY and genotyping in the Polymorphic Assay is a prenatal diagnosis. And as I have said, it is.
Further, let me make the more general point that in the context of the Patent, “diagnosis” encompasses prenatal risk assessments.
The Harmony Test accurately determines, for example, fetal sex and detects fetal chromosomal aneuploidy, being explicit examples of the method of prenatal diagnosis defined in the Patent. The Harmony Test also detects and determines paternally inherited mutations and genotypes the fetus. The Harmony Test is therefore a “method of prenatal diagnosis” within the meaning of the relevant claims. By providing a determination of fetal gender (i.e. a male or female result – not a “risk score” as suggested by Ms Norbury), a fetal fraction, and of a “high” or “low” risk of aneuploidy, the Harmony Test also “provides a diagnosis” within the meaning of the claims. As noted above, Ariosa concedes that the Harmony Test’s determination of fetal sex constitutes a “diagnosis” within the meaning of the claims.
If the Harmony Test reports that there is a high risk that the fetus has XYY aneuploidy, confirmatory invasive testing may be offered to the mother. If, on the other hand, the Harmony Test reports that there is a low risk of XYY aneuploidy (and other conditions), no further testing would typically be performed. Thus, “clinical decisions” are made on the basis of the Harmony Test’s XYY results.
The Harmony Test materials give a sensitivity of 100% and a specificity of 99.7% for sex chromosome aneuploidies.
Further, in the case of sex chromosome aneuploidies, the chance of a major handicap is small. Hence the consequence of a detection of XYY aneuploidy is not in the realm of a high risk trisomy 21, 18 or 13 result, which must be confirmed by amniocentesis or CVS before termination may be offered. Thus, the Harmony Test’s XYY detection method provides a highly accurate and reliable result which is at the diagnostic end of the spectrum, even on the narrow definition of diagnosis.
Let me deal with another matter concerning the expression “a nucleic acid”. Ariosa also contends that claims 22, 23 and 25 require a diagnosis to be made on the basis of “a” (singular) nucleic acid and that this does not occur with the Harmony Test, which provides risks assessments based on complex calculations performed using information relating to a large number of different nucleic acid sequences.
More particularly, Ariosa says that claim 22 refers to detecting the presence of “a nucleic acid of foetal origin”, and then providing a diagnosis based on “the presence and/or quantity and/or sequence of the foetal nucleic acid”. Read in context in the Patent, that process requires a diagnostic conclusion to be based on an analysis of a single nucleic acid. But it says that that does not occur with the Harmony Test, which provides risks assessments based on complex calculations performed using information relating to a large number (thousands) of different nucleic acid sequences. Further, it says that claim 23 is dependent on claim 22, and has the same requirement.
I would reject what I would describe as a technical argument.
The “nucleic acid” detected in the Harmony Test’s sex determination and XYY aneuploidy detection methods may properly be identified as fetal nucleic acid from the Y chromosome comprising a number of different discrete sequences from that nucleic acid. The “nucleic acid” detected as part of the Harmony Test’s Polymorphic Assay may properly be identified as fetal nucleic acid from a non-Y chromosome.
Further, claims 22 and 23 do not in any event require a diagnosis to be made solely on the basis of the fetal nucleic acid. Claim 25 similarly only requires the nucleic acid to be indicative of a fetal condition or characteristic.
Let me now deal with another issue concerning the integer “separating the sample into a cellular and non-cellular (plasma) fraction” (claims 22 and 23).
The PPD at [4.1] provides:
A collected blood sample firstly undergoes a centrifugation protocol. This separates the blood into three components: (1) the buffy coat; (2) erythrocytes (red blood cells); and (3) plasma (the fluid part of the blood, which contains, among other things, minerals, salts, hormones and proteins). Foetal cfDNA and Maternal cfDNA are both contained within the plasma component.
Plasma is a non-cellular fraction of the blood sample.
Accordingly, the Harmony Test infringes claims 22 and 23.
As to claim 25, Ariosa says that it refers to testing for “foetal nucleic acid indicative of a maternal or foetal condition or characteristic”, but that does not occur with the Harmony Test. Ariosa says that there is no fetal nucleic acid (or even fetal nucleic acids) that are of themselves indicative of any maternal or fetal condition or characteristic that are tested for in the Harmony Test. Ariosa says that the process used is a different one, involving a complex test which utilises data aggregated from a large number of loci on various chromosomes together with data from other sources to assess the probability as to whether the fetus is likely to have an aneuploidy. But I reject such a technical argument. Further, XYY aneuploidy and fetal sex are fetal “conditions” or “characteristics” within the meaning of claim 25 and thus the Non-Polymorphic Assay of the Harmony Test infringes claim 25. Further, the Polymorphic Assay determines the relative concentration of cffDNA in a maternal plasma sample and partially genotypes the fetus at a large number of informative polymorphic loci by detecting paternally inherited SNP mutations. The partial genotype of the fetus is also a fetal characteristic within the broad language of claim 25.
I do not need to say anything further about claim 26, save to say that the Harmony Test falls within its scope if claim 26 was otherwise valid.
Conclusions
In summary, the Harmony Test falls within the scope of each of the relevant claims as correctly submitted by Sequenom.
First, the Harmony Test is performed on plasma extracted from maternal blood samples (all relevant claims).
Second, the Harmony Test is a detection method (claims 1, 2, 3, 5, 6, 9 and 22).
Third, the Harmony Tests reports the gender of the fetus (claims 6, 13, 22, 23, 25 and 26) and the risk that the fetus is suffering from aneuploidy such as XYY (claims 22, 23, 25 and 26).
Fourth, the Harmony Test detects paternally inherited mutations and determines the partial genotype of the fetus by detecting paternally inherited mutations (claims 22, 23, 25 and 26).
Fifth, the Harmony Test is a “method of performing a prenatal diagnosis” (claims 22, 23, 25 and 26). It provides a diagnosis based on the presence, quantity and sequence of the fetal nucleic acid (claims 22 and 23) and it comprises a test for fetal nucleic acid indicative of a fetal condition or characteristic (claim 25).
Sixth, in the course of the Non-Polymorphic Assay of the Harmony Test, the presence of a fetal nucleic acid sequence from the Y chromosome is detected by means of a sequence specific probe and amplified by enrichment and PCR (claims 1, 2, 3, 5 and 6).
Seventh, the Non-Polymorphic Assay is relevantly performed for determining the sex of the fetus (claim 13).
Eighth, in the course of the Polymorphic Assay of the Harmony Test, nucleic acid of fetal origin from a paternally inherited non-Y chromosome is detected by means of a sequence specific probe and amplified by enrichment and PCR (claims 1, 2, 3, 5 and 9).
Ninth, in the course of the Polymorphic Assay of the Harmony Test, the presence of a fetal nucleic acid from a paternally inherited non-Y chromosome is detected (claim 9) and the concentration of the sequence in the maternal plasma is determined (claim 14).
(e) Infringement – legal characterisation and other matters
In my view Ariosa has infringed the relevant claims (other than claim 26) of the Patent as pleaded in the second further amended statement of claim.
Further, Sonic and Clinical by their use of the Harmony Test in Australia have each infringed the relevant claims (other than claim 26) of the Patent. Sonic and Clinical have each also infringed Sequenom’s exclusive rights by promoting and supplying Harmony Test results to medical practitioners during the send out periods. Ariosa has also authorised that conduct.
Further, the Harmony Test results constitute a “product” resulting from the use of the method of Sequenom’s claimed invention and the promotion and supply of such results infringes the exclusive rights in that claimed invention granted to Sequenom.
Now in relation to the send out periods the respondents say that the importation into Australia of patient reports generated by the performance of the Harmony Test in the USA on samples collected in Australia did not constitute an infringement of the Patent.
As explained by Mr Rubano there are two ways in which the Harmony Test may be provided, either under the “Test Send Out” (TSO) model or the Ariosa cell-free System (AcfS) model. Under the TSO model, Sonic between June 2015 and October 2015 and Clinical between December 2014 and March 2016 collected samples of blood from pregnant women in Australia, and sent those to Ariosa in San Jose, California. Ariosa performed the Harmony Test in its laboratory in San Jose and generated a report for each Harmony Test performed. An original copy of the Harmony report in PDF format was automatically saved on Ariosa’s server in San Jose. The Harmony report was made available by Ariosa via an online secure document storage and sharing system known as ShareFile provided by a third party, Citrix; this is a distributed server system in which physical or virtual servers are located at various locations around the world and the data stored on one server is replicated on all other servers in the system. A copy of the Harmony report could then be downloaded by representatives of Sonic and Clinical from the ShareFile server. Since 2 February 2016, Ariosa has operated its own file sharing system, the Harmony customer portal, and has progressively migrated its TSO customers from ShareFile to the Harmony customer portal. From 25 January 2017, Sullivan Nicolaides was migrated to this portal. Reports are made available in a similar way to the ShareFile system and the Harmony customer portal is hosted on Ariosa’s server located in San Jose. Once the Harmony report was downloaded by a clinic, it was the responsibility of the clinic to provide a copy of the report to the requesting doctors and patients.
As I say, the respondents reject Sequenom’s assertion that the performance of the Harmony Test in the USA on samples collected in Australia by Sonic and Clinical infringes the Patent.
Moreover, they reject Sequenom’s characterisation of the “product” of the Harmony Test.
Section 13(1) of the Act provides that “a patent gives the patentee the exclusive rights, during the term of the patent, to exploit the invention” and to authorise others to do so. The definition of “exploit” is found in the Dictionary in Schedule 1 of the Act, which provides:
“exploit”, in relation to an invention, includes:
(a)where the invention is a product--make, hire, sell or otherwise dispose of the product, offer to make, sell, hire or otherwise dispose of it, use or import it, or keep it for the purpose of doing any of those things; or
(b)where the invention is a method or process--use the method or process or do any act mentioned in paragraph (a) in respect of a product resulting from such use.
In Apotex Pty Ltd v Warner-Lambert Company LLC (No 2) (2016) 122 IPR 17, Nicholas J held at [296] to [298] that:
The definition of “exploit” makes no reference to the patent area. As I have said, the express territorial limitation upon the patentee’s exclusive rights is found in ss 12 and 13. In my respectful view, there is therefore no reason to read down the words of either para (a) or para (b) of the definition of “exploit” to found any territorial limitation. This is because the Act expressly provides that a patent only has effect in the patent area: see also s 70 of the Patents Act 1952 (Cth).
Paragraph (b) of the definition of “exploit” refers to the doing of an act referred to in para (a) which includes to make or import a product. The patentee’s exclusive rights are infringed (subject to available defences) if another person does any such act within the patent area. The fact that the patented method is performed outside the patent area does not avoid infringement of a method claim (including a Swiss claim) if the product imported and sold in Australia was made using the patented method because “the acts of importation and sale occur within the patent area”. The relevant act of infringement is not the use of the method outside the patent area but the exploitation (by importation and sale) in Australia of a product made using the patented method.
In my respectful opinion, contrary to the approach taken by Lindgren J, the relevant territorial limitation is reflected in the language of ss 12 and 13(3) and there is therefore no justification for importing words of territorial limitation into the definition of “exploit”. It follows that I take a somewhat different approach to the construction of the definition of “exploit” to that taken by Lindgren J in Alphapharm, though I do not think the difference has any impact on whether or not Apotex threatens to infringe the Swiss claims in this case.
This finding was considered and upheld by the Full Court in Warner-Lambert Co LLC v Apotex Pty Ltd (No 2) (2018) 355 ALR 44 at [156] to [164] and [167] to [168], having also considered Lindgren J’s approach in Alphapharm Pty Ltd v H Lundbeck A/S (2008) 76 IPR 618 at [691] to [694].
The respondents deny that the Harmony Test results or reports are properly described as “products” of the claimed methods, for the purpose of the definition of “exploit”. They say that whilst a report may have been generated as a result of the performance of the Harmony Test, that report merely records information. They say that in theory, the information in the report could have been communicated to a doctor or patient in Australia by telephone. They say that the fundamental nature of information is not confined by its method of conveyance. Consistently with this, they say that to the extent that the Harmony Test or any aspect of it falls within the scope of the claims of the Patent, the Harmony Test results or reports are not a “product” within the meaning of the definition of “exploit” in the Act.
Indeed they go so far as to say that there is nothing in the Act to suggest that the importation of a “product” can extend to the importation of information, and that there is nothing in Northern Territory v Collins (2008) 235 CLR 619 that would support such an approach.
In summary, the respondents say that the Harmony Test results or reports are not a “product” that can be imported (or kept or sold) within the meaning of the definition of “exploit” in the Act. It is instead information that could be brought within Australia by any form of communication, or by a person receiving that communication directly overseas and themselves travelling to Australia whilst retaining that information in their memory.
But in my view the respondents’ arguments lack any bounce.
The term “product” is not defined in the Act. Accordingly and in context, the term is to be given its ordinary meaning, namely as covering anything resulting from the patented method that can be commercially exploited. Such an ordinary meaning was adopted in Northern Territory v Collins, where the Court was concerned with the interpretation of the word “product” in the context of s 117 of the Act; a term occurring in several places of the same statute should usually be similarly construed.
Further, it has been said that “[i]n relation to a process, the product is the state of affairs in which an effect may be observed” (Cancer Voices Australia v Myriad Genetics Inc (2013) 99 IPR 567 per Nicholas J at [88]) and that “vendible product” should be “understood as covering every end produced or artificially created state of affairs which is of utility in practical affairs and whose significance thus is economic” (CCOM Pty Ltd v Jiejing Pty Ltd (1994) 51 FCR 260 at 291 per Spender, Gummow and Heerey JJ, referring to NRDC at 276 to 277. Now I accept that these observations were made in a different context and with different qualifications. Nevertheless they provide a relevant analogy.
Moreover, interpreting the definition of “product” broadly limits the undesirable possibility that a potential infringer could utilise a patented process overseas and then import the resulting product into Australia, and so circumvent the monopoly granted by the method patent.
In my view, the reality is that the Harmony Test is carried out for the purpose of obtaining the results. The results are clearly commercially valuable; patients pay in the order of $400 to obtain them. The Harmony Test results including being recorded in electronic or paper form are the product of a process for the purposes of the definition of “exploit”. Moreover, by supplying the Harmony Test results to clinicians in Australia, Sonic and Clinical infringed the relevant claims during the send out period.
Finally, I would note that it is not now in dispute that Ariosa authorised the relevant conduct of Sonic and Clinical that I have referred to above and that it is a joint tortfeasor with them, assuming relevant infringement is established.
CONCLUSION
Sequenom has succeeded on its infringement case save for its case on infringement concerning claim 26. The respondents have failed on their invalidity case save that they have established that claim 26 is invalid for lack of fair basis. I will direct that the parties bring in minutes of orders to reflect these reasons and for the further steps to be taken in this matter. I will also hear the parties on any question of costs.
I certify that the preceding one thousand, four hundred and seventy-seven (1477) numbered paragraphs are a true copy of the Reasons for Judgment herein of the Honourable Justice Beach. Associate:
Dated: 27 June 2019
SCHEDULE
PRODUCT AND PROCESS DESCRIPTION (Confidential)
[Redacted]
SCHEDULE OF PARTIES
VID 611 of 2016 Cross-Claimants
Second Cross-Claimant:
SONIC HEALTHCARE LIMITED (ACN 004 196 909)
Third Cross-Claimant:
CLINICAL LABORATORIES PTY LTD (ACN 006 823 089)
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