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‘JUNK DNA’ (NON-CODING) PATENTS: THE INVENTOR’S VIEW Malcolm J. Simons (*1) MB.ChB,MD,DMedSci,FACA,FRCPA
The ‘Junk DNA’ non-coding patents have provoked unusual interest for reasons including; 1. the widespread impression that the patents’ owner Australian Biotechnology Company Genetic Technologies Limited (GTG) interprets the patent claims to cover any test involving amplification of non-coding DNA; 2. the unusual patent license commercialization strategy adopted by GTG; 3. disbelief concerning patent novelty and obviousness; 4. reactions to the scope of the patents encompassing all eukaryotic organisms (how can a company patent DNA in all species?); and to whether society should permit such perceived monopolies. There are two families of patents. The Intron Diagnostic patent (filed August 1989) describes a method for practice of my discovery of the general utility of non-coding DNA sequence variation for Linkage Disequilibrium (LD)-based surrogate gene allele/mutation typing in single individuals (without the need for pedigree information)(*2). The Genome Mapping Patent (filed July 1990) describes a method for population-based (case-control) gene fine mapping by genome-wide LD-haplotype identification in individuals with a trait, compared with individuals lacking the trait (*3). I have become aware that reactions to the patents mainly arise from imprecision in the wording of the patent claims, and from a failure to clearly distinguish my discoveries and inventions from prior art. In particular, the use in the Intron Diagnostic patent claims of the word ‘linkage’, and the absence of the terms ‘linkage disequilibrium’ and ‘haplotype’, directs attention towards more than a decade of prior art, the latter four years employing polymerase chain reaction (PCR). An interpretation that non-coding DNA sequences occurring within genes (introns) are in genetic ‘linkage’ with the genetic locus is non-sense to many skilled in genetic art. Thus, confusion and controversy is understandable. Here I summarise my discoveries and their distinction from prior art. Prior to patent filings in 1989/90, genetic science mainly involved the study of family members for identification of linked loci by chromosome transmission. Southern blotting, using cDNA probes for detection of restriction fragment length polymorphisms (RFLP), was the main diagnostic and gene discovery DNA technology. RFLP site variations were recognized to be useful for gene mapping and for mutation diagnosis by pedigree linkage analysis from the time of publications in 1978 by Kan and Dozy (1,2), and in 1980 by Botstein et al. (3) and by Little et al. (4). Some restriction enzyme sites, located up to tens of kilobases from the gene, exhibited LD with the disease gene locus, and enabled the assignment of ‘haplotypes’ (5-9). From end-1985, with the description of sequence amplification by PCR (10), RFLP analysis by Southern blotting began to be replaced by PCR. Beginning around 1987 through to the early 1990’s, the obvious application of PCR for amplification of RFLP sites to simplify linkage analysis was reported (11-14). With the advent of PCR, research focus shifted rapidly and near-exclusively to exon amplification for detection of coding mutations and alleles. Non-coding DNA was viewed as irrelevant to the main matter of studying coding sequence responsible for translated gene products. The 10th International Histocompatibility Workshop (10IHW), culminating in 1987, applied Southern blotting for RFLP study of HLA loci distributed over 3 megabases in the major histocompatibility complex (MHC - 6p21.1-3). Now that RFLP has been largely superseded as a DNA technology, this RFLP study, involving 13 loci, 12 restriction enzymes, and 107 human cell lines, is likely to remain the largest ever performed. My analysis in 1988/89 of the Workshop RFLP DNA fragment patterns revealed that RFLP sites were non-randomly ordered according to coding HLA locus alleles, and to combinations of alleles as multi-locus haplotypes (15,16). Since some 95% of genomic DNA was known to be non-coding, the great majority of the sequence variations responsible for the RFLP patterns were likely to be located in non-coding regions. I found that RFLP allele and haplotype associations were conserved between peoples of the same HLA type but of different ethnic backgrounds. Some haplotypes extended over hundreds of kilobases. The existence of extended haplotypes occurring as supratypic HLA multi-gene blocks had been known since at least 1983 (17) so, contrary to a current view, haplotype block structure in the genome is not a recent discovery. In 1989 this extent of the non-random ordering of intron and intergenic non-coding polymorphisms as sequence information reflecting coding locus allele and multi-locus haplotypic block patterns was not known. As late as 1997, when sequencing HLA introns, Blasczyk and associates wrote: “Against all expectations, this (highly polymorphic variability of introns) is not characterised by random point mutations but by a highly systematic diversity reflecting the ancestral relationship of the HLA alleles” (18). The earliest reference involving population LD mapping, for better gene localization of a disease-associated gene that had been chromosome region-assigned by linkage, was published in 1992 (19,20). The first report of LD mapping for genome-wide searches, without prior knowledge of chromosome gene location, appeared two years later, in 1994 (21), 9 years after the description of PCR. Previous science involved extensive searches for informative RFLPs. My discovery was that it was not necessary to undertake further searches. All that was required was analysis of the sequence of small (amplifiable) non-coding regions. Any non-coding region would suffice. The utility of the polymorphic information content of any amplifiable non-coding sequence to sufficiently mark coding locus alleles/mutations and haplotypes by LD in single individuals is the basis of both families of ‘Junk DNA’ patents. My discovery was that non-coding sequence variation was sufficiently structured according to coding locus allele and multi-locus haplotypes to substitute for, and to dispense with, the linkage requirement for pedigree chromosome transmission. My expectation that the non-random, allele and haplotypic structure of non-coding sequence polymorphism would be a universal characteristic of all eukaryotic organisms, rather than being unique to HLA genes, was supported by declarations to the US Patent Office from experts in blood genetics, mouse genomics, and soybean plant genetics. While non-coding DNA may be ‘junk’ with respect to protein coding, the conservation of noncoding sequences over thousands of years in humans, and over tens of millions of years between humans and other animals, and in plants, must have reflected important, then as now largely unknown, functions. I am unaware of any current DNA molecular laboratory test for disease-associated gene diagnostics, or for HLA typing, that utilizes the method of the Intron Diagnostic patent. By contrast, all uses of population-based LD / Allele association fine-mapping seem to me to be encompassed by the Genome Mapping patent. Considering patent law rules for ‘anticipation / novelty’ and for ‘obviousness’, no prior art scientific literature has been brought to my attention that warrants disclaiming the inventions. --------------------------------------------------------------------------------------------------------------------- Footnotes: (*1) Dr. Simons has no association with GTG, and derives no benefit from any aspect of the Company’s business. (*2) US Patent No: 5,192,659. Intron Sequence Analysis Method for Detection of Adjacent and Remote Locus Alleles as Haplotypes [Intron Diagnostic patent]. (*3) US Patent No: 5,851,762. Genomic Mapping Method by Direct Haplotyping using Intron Sequence Analysis [Genomic Mapping patent].
References: 1. Kan YW, Dozy AM. “Antenatal diagnosis of sickle-cell anaemia by D.N.A. analysis of amniotic-fluid cells”. Lancet 1978 Oct. 28; 2(8096): 910-12. 2. Kan YW, Dozy AM. “Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation”. PNAS 1978 Nov; 75(11): 5631-5. 3. Botstein D, White RL, Skolnick M, and Davis RW. “Construction of a genetic linkage map in man using restriction fragment length polymorphism”. Am J Hum Genet. 1980 May; 43(3): 314-31. 4. Little PFR, Annison G, Darling S, Williamson R. “Model for antenatal diagnosis of beta-thalassaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms”. Nature 1980 May 15; 285: 144-47. 5. Rees A, Stocks J, Paul H, Ohuchi Y, Galton D. “Haplotypes identified by DNA polymorphisms at the apolipoprotein A-1 and C-III loci and hyper-triglyceridaemia. A study in a Japanese population”. Hum Genet. 1986 Feb; 72(2): 168-71. 6. Antonarakis SE, Oettgen P, Chakravarti A, Halloran SL, Hudson RR, Feisee L, Karathanasis SK. “DNA polymorphism haplotypes of the human apolipoprotein APOOA1-APOC3-APOA4 gene cluster”. Hum Genet. 1988 Nov; 80(3): 265-73. 7. Estivill X, Scambler PJ, Wainwright BJ, Hawley K, Frederick P, Schwartz M, Baiget M, Kere J, Williamson R, Farrall M. “Patterns of polymorphism and linkage disequilibrium for cystic fibrosis”. Genomics 1987 Nov; 1(3): 257-63. 8. Chebloune Y, Pagnier J, Trabuchet G, Faure C, Verdier G, Labie D, Nigon V. “Structural analysis of the 5’ flanking region of the beta-globin in African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation in Africa”. PNAS 1988 Jun; 85(12): 4431-5. 9. Leitersdorf E, Chakravarti A, Hobbs HH. “Polymorphic DNA haplotypes at the LDL receptor locus”. Am J Hum Genet. 1989 Mar; 44(3): 409-21. 10. Saiki RK., Scharf S., Faloona F., Mullis KB., Horn GT., Erlich HA., Arnheim N. “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia”. Science. 1985 Dec. 20; 230(4732): 1350-4. 11. Kogan SC, Doherty M, Gitschier J. “An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences”. New Engl. J. Med. 1987 Oct. 15; 317(16): 985-90. 12. DiLella AG, Huang WM, Woo SL. “Screening for Phenylketonuria Mutations by DNA amplification with the Polymerase Chain Reaction”. Lancet. 1988 Mar. 5; I(8584): 497-9. 13. McIntosh I, Curtis A, Millan FA, Brock DJ. “Prenatal exclusion testing for Huntington disease using the polymerase chain reaction”. Am J Med Genet. 1989 Feb; 32(2): 274-6. 14. Graham JB, Kunkel GR, Tennyson GS, Lord ST, Fowlkes DM. “The Malmo polymorphism of factor IX: establishing the genotypes by rapid analysis of DNA”. Blood 1989 Jun; 73(8): 2104-7. 15. Simons MJ, Wheeler R, Cohen D, Lalouel-JM, Dupont B. “Restriction fragment length polymorphism of HLA genes: Summary of the 10th International Histocompatibility Workshop Southern Blot analysis”. In Immunobiology of HLA: Histocompatibility Testing 1987 (Ed. Dupont B). Springer-Verlag, New York. Vol.1, 959-1023. 16. Simons M.J, Erlich H.A. “RFLP - sequence interrelations at the DPA and DPB loci”. In: Immunobiology of HLA: Histocompatibility testing 1987 (Ed. Dupont B). Springer-Verlag, New York. Vol.1, 953-958. 17. McCluskey J, Kay PH, Dawkins RL, Komori KA, Christiansen FT, McCann VJ. “Association of Specific MHC Supratypes with Rheumatoid Arthritis and Insulin-Dependent Diabetes”. Disease Markers. 1983; 1: 197-212. 18. Blasczyk R, Kotsch K, Wehling J. “The nature of polymorphism of the HLA class I non-coding regions and their contribution to the diversification of HLA”. Hereditas. 1997; 127(1-2): 7-9. 19. Hastbacka J, de la Chappelle A, Kaitila I, Sistonen P, Weaver A, Lander E. “Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland”. Nature Genetics 1992; 2: 204-211. 20. Lazzeroni LC. “A chronology of fine-scale gene mapping by linkage disequilibrium”. Statistical Methods in Medical Research 2001; 10: 57-76. 21. Houwen RH, Baharloo S, Blankenship K, Raemaekers P, Juyn J, Sandkujil LA, Freimer NB. “Genome screening by searching for shared segments: mapping a gene for benign recurrent intrahepatic cholestasis”. Nat Genet. 1994 Dec.; 8(4); 380-6.
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