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Sept 15, 2003

GTG's Inventions Concerning 'Junk' DNA

I was interested to read of the patent claims of Genetic Technologies Ltd. (GTG), as discussed in the interview with Mervyn Jacobson ("Playing by Aussie Rules," Aug. 2003 Bio·IT World, page 24). Two areas concern me: First, GTG's claim that its principals made important discoveries concerning the nature of "junk" DNA that were not appreciated at the time (1989) by the scientific community. Second, the patents held by GTG arising out of these discoveries.

According to GTG's Web site, co-founders Jacobson and Malcolm Simons in 1989 "resolved to prove the non-coding ('junk' DNA) region of the human HLA gene complex on chromosome 6 is in reality not 'junk', but in fact a valuable and highly ordered reservoir of useful genetic information, largely overlooked by the rest of the world. The commercial mission then evolved that GeneType would seek exclusive ownership over access to this important genetic information and ultimately, to exploit it globally for profit." (Gene Type was ultimately drawn into GTG.)

There are several important issues here: first, the characterisation of non-coding DNA as "a valuable and highly ordered reservoir of useful genetic information" and second, that this was "largely overlooked by the rest of the world." (The specific reference to the HLA gene complex is not especially significant, given the subsequent generalisation of GTG's claims.)


The Value of Junk DNA 
The first claim is based upon the understanding, laid out in several GTG patents (see below) that polymorphism(s) in non-coding DNA, provided it is linked to a gene, can be used to predict the identity of a functional variant within the gene.

Was this a new area for science? No; precisely this idea had been explicitly demonstrated in 1978 by Kan and Dozy (references 1, 2), who identified the human sickle-cell mutation by analysis of a linked polymorphic marker (restriction fragment length polymorphism, RFLP) located outside the beta-globin gene. Their Lancet publication was a landmark in human molecular genetics.

In 1980, my colleagues and I published a paper (3) using linked polymorphisms located within the introns of the adjacent fetal globin genes to diagnose beta thalassemia. We wrote then: "The use of linked polymorphisms should, therefore, be applicable to antenatal diagnosis both of beta-thalassaemia and of any other single-gene defect for which there is a DNA probe specific for a sequence linked to the affected locus." Our paper explicitly raised the necessity of identifying haplotypes of polymorphic markers before diagnostic conclusions could be reached, and discussed these in the context of linkage disequilibrium.

That same year, Phillips et al. (4) published a paper using the polymorphisms described in those previous papers to diagnose sickle-cell anemia. They wrote: "Studies of other Black families and individuals provide evidence for linkage disequilibrium in the [globin] gene complex involving the four [RFLPs] ... which span 33 kilobases." Note the use of the words "linkage disequilibrium" to describe the non-random association of multiple variants along a chromosome region.

Was Kan and Dozy's 1978 discovery subsequently lost to science? Clearly not: In 1980, Botstein and colleagues (5) put forward their influential proposal to construct a human genome linkage map using polymorphisms, building precisely upon these findings. Further, a search of PubMed for "RFLP" from 1978 to 1988 yields 2,317 references, of which 171 concern prenatal diagnosis, suggesting that this type of genetic variation was widely studied.

It is unclear to me why, in 1989, it was necessary to prove the idea that linked polymorphisms could be used to analyse functional variation: The fundamental principles and practice had been widely published, and these could be simply applied to any gene, including the HLA complex. Importantly, the concepts of haplotypes, linkage disequilibrium, and linkage had all been identified as directly relevant to the DNA-based analyses then available.

GTG's contention that its principals had discovered something that was "largely overlooked" is not supported by the scientific literature. The comment that non-genic DNA is "a valuable and highly ordered reservoir of useful genetic information" is simply a restatement of what was first demonstrated in 1978 and applied widely. In this strict sense, such DNA can never be truly "junk" by virtue of its linkage to genes and must always be of potential utility.


GTG's Patents 
GTG has three patents relevant here: U.S. Patent #5,192,659 (3.9.1993) is entitled "Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes." The later patent #5,612,179 (3.18.1997) has the same title. U.S. Patent 5,851,762 (12.22.1998) is entitled "Genomic mapping method by direct haplotyping using intron sequence analysis."

The 1993 patent is focused mainly upon human HLA genes, and states: "The present invention is based on the finding that intron sequences contain genetic variations that are characteristic of adjacent and remote alleles on the same chromosome. In particular, DNA sequences that include a sufficient number of intron sequence nucleotides can be used for direct determination of haplotype." The 1997 patent extends the earlier patent to cover all genes.

The 1998 patent is a more complex patent because it extends the utility of methodology of the '93/'97 patents to creating haplotype maps of the human (or other organism) and using these for association analyses and disease gene mapping. The methodology of association analysis of various sorts had been previously widely employed and discussed (see, for example, 6-8) and there appears to be no claimed novelty in the patent in this respect.

My reading of the three patents is that if the 1993 patent falls, then they all fall, since the methodology underpins all three.

In purely scientific terms, the material supporting the 1993 patent is inadequate in recognising the extent of scientific knowledge from the late 1970s and early 1980s. The papers I have discussed (particularly references 3 and 4) are prior examples of constructing haplotypes of intron variations linked to a mutational variant and using these as a diagnostic tool. Therefore the view that these are novel claims is open to challenge. A detailed search of the literature post-1980 produces numerous additional examples.

In short, the information at the heart of the three patents was widely appreciated and published more than a decade prior to the patenting by GTG in 1993 of the idea of using non-coding variations to infer the state of linked variations. These earlier publications used both intronic and extragenic variations, clearly stated that the approaches were based upon linkage disequilibrium and analysing haplotypes, and explicitly contained comments that the approach was generalisable to any gene.

PETER LITTLE 
School of Biotechnology & Biomolecular Sciences,
University of New South Wales
Sydney, Australia
p.little@unsw.edu.au

References 
1. Kan, Y.W., Dozy, A.M. "Antenatal diagnosis of sickle-cell anaemia by D.N.A. analysis of amniotic-fluid cells." Lancet 2, 910-2; 1978.

2. Kan, Y.W., Dozy, A.M. "Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation." PNAS 75, 5631-5; 1978.

3. Little, P.F., Annison, G., Darling, S., Williamson, R., Camba, L., Modell, B. "Model for antenatal diagnosis of beta-thalassaemia and other monogenic disorders by molecular analysis of linked DNA polymorphisms." Nature 285, 144-7; 1980.

4. Phillips, J.A., et al. "Prenatal diagnosis of sickle cell anemia by restriction and endonuclease analysis: HindIII polymorphisms in gamma-globin genes extend test applicability." PNAS 77, 2853-6; 1980.

5. Botstein, D., White, R.L., Skolnick, M., Davis, R.W. "Construction of a genetic linkage map in man using restriction fragment length polymorphisms." Am J Hum Genet. 32, 314-31; 1980.

6. Hastbacka, J., de la Chapelle, A., Kaitila, I., Sistonen, P., Weaver, A., Lander, E. "Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland." Nat Genet. 2, 204-11; 1992.

7. Feder, J.N., Gnirke, A., et al. "A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis." Nat Genet. 13, 399-408; 1996.

8. Risch, N., Merikangas, K. "The future of genetic studies of complex human diseases." Science 273, 1516-7; 1996.



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