Fledgling Melbourne biotech Haplomics Technologies, established in 2003, has lodged provisional patents on two powerful new technologies for diagnosing genetic disorders, and identifying genes involved in inherited genetic diseases.

The new techniques are the invention of GTG’s co-founder, and inventor of its controversial ‘junk’ DNA patents, New Zealand-born immunogeneticist Dr. Malcolm Simons. Simons quit GTG in 2000 after a rift with co-founder and current CEO Dr. Mervyn Jacobson.

Simons believes Haplomics’ new technologies represent a new paradigm in gene discovery, and will be a powerful tool for disentangling the contributions of genetic variation, variation in the cell’s RNA-based operating system, and environmental factors, to genetic diseases, genomic disorders like cancer and autoimmune diseases, as well as individual susceptibility to infection.

Crucially, Simons’ new techniques would allow geneticists to conduct gene discovery research, and diagnose genetic diseases, without being dependent on GTG’s marker technology.

Hunting Haploids
Today’s genetic tests, and gene-hunting research, are performed on mixed, diploid DNA—it has been almost impossible to distinguish the separate, haploid contributions of the male and female parents to individual’s genetic makeup, or to discern how they interact.

At this week’s 14th International HLA and Immunogenetics Workshop and Conference at the University of Melbourne, Simons described how he has implemented his ideas on a ‘haplotyping’ chip—a DNA microarray that will allow researchers to identify all sequence variation in corresponding, multi-gene segments of an individual’s paired chromosomes.

Haplomics’ HiSNP-typing chip will identify single nucleotide polymorphisms (SNPs) in gene exons that result in amino-acid substitutions in the encoded protein, and SNPs that result in so-called synonymous substitutions without altering the amino acid sequence of the eventual protein.

The haploid segments, spanning multiple genes on corresponding segments of paired chromosomes, will garner complete information about DNA sequence variation across multiple, linked genes.

It will enable identification of the separate contributions of the two haplotypes.

Tip of the Iceberg
Simons said DNA microarray technology had advanced to the stage where a single chip can carry hundreds of thousands of DNA probes. But until now, microarrays have been used to detect SNPs scattered across chromosomes, and separated by some thousands of nucleotides.

These represent SNPs identified by previous research. Simons said researchers have now realised that these widely spaced SNPs represent just the tip of the iceberg, and do not fully account for haplotype differences.

This technique, which Simons terms ‘interval SNPing’, fails to recognise the wealth of variation in the long intervals between selected SNPs. Interval SNPing, more commonly known as ‘resequencing’, was the preferred technology for the multi-million dollar international HapMap Project.

“In a number of multi-gene complexes, such as the HLA (human leucocyte antigen) complex, which spans 4 million nucleotides on chromosome 6, as many as six or eight SNPs may occur within an interval of only 25 nucleotides,” Simons said.

The interval SNPing approach is tantamount to striding across a  chromosome in seven-league boots—it misses an enormous number of SNPs and other forms of DNA-sequence variation in the intervening tracts of chromosome.

These unrecognised SNPs may contribute to genetic disorders, or disease susceptibility. Simons said his ‘overlapping SNP’ technique would blanket chromosome segments with huge numbers of overlapping probes to capture all sequence variation.

Focus on Immunity
Initially, Haplomics is focusing on the HLA complex, headquarters of the human immune system, and the 150,000 nucleotide killer-cell immunoglobulin-like receptor (KIR) complex on chromosome 19, which is at the core of innate immunity, and also modulates the adaptive immune response.

The KIR gene complex is involved in the outcome of tissue and organ transplants—immunogeneticists have recently identified the KIR complex as the source of a mysterious mechanism that allows some transplant patients to accept bone marrow or organ grafts from non-matching donors.

KIR genes have also been implicated in susceptibility to autoimmune disorders like insulin-dependent diabetes, multiple sclerosis and rheumatoid arthritis.

Simons used proprietary Oligo-Select software to identify 34,000-odd probes required to detect all variation in the major genes involved in tissue-matching.

Haplomics’ first chip, which is being delivered this week, detects variation at only nine of the more than 220 genes in the HLA complex

While the number of probes theoretically required to detect all variation in just nine HLA genes is potentially enormous, over the millennia, natural selection has winnowed the number of HLA gene combinations to a number that can be easily accommodated  on a single chip.

Test of Time
Simons said certain combinations of HLA alleles have stood the test of time, and been welded by natural selection into integrated, functional units that tend to escape recombination, and are inherited en bloc as haplotypes extending up to tens of kilobases.

He expects the patterns of SNP combinations within these extended haplotype ‘blocks’ to be more revealing than those obtained with interval SNPing. He also expects synonymous substitutions in the DNA code, that do not alter amino acid sequence of the encoded protein, will be informative in assigning haplotypes.

Linked genes in the same block of chromosome are described as being in cis phase, but Simons’ technique opens up the possibility of detecting trans interactions—’cross-talk’ between alleles at the same locus on the paired chromosomes, and epistatic interactions between different loci on complementary chromosomes.

“Being able to distinguish the two separate haplotypes is the Holy Grail of gene discovery,” Simons said. “It is recognised as being a far more powerful tool for identifying genes involved in disease, drug response and other traits than techniques based on single SNPs alone.”

IP Protection
Given that Haplomics’ new techniques have the potential to revolutionise research and genetic testing, GTG and its rivals—including Applera—may be keenly interested in the infant biotech’s new IP.

GTG would have an interest in protecting its IP position against any rival gene testing technology that does not rely on Simons’ original invention, which detects disease-causing alleles of known genes by their association with markers in non-coding DNA—both in introns and inter-genic ‘junk’ DNA. The DNA marker and the disease-causing allele are ‘linked’ as extended haplotypes.

The GTG gene-search, another Simons invention, allows geneticists to identify anonymous genes through linkage disequilibrium. Linkage disequilibrium describes a tendency of a DNA marker and an anonymous gene to be co-inherited at a frequency much greater than would be predicted from the distance between them on the chromosome.

Simons can lay claim to being the first researcher in the world to realise that long-distance associations between markers in ‘junk DNA’ and particular diseases could be used to locate and identify genes involved in relatively rare inherited disorders, where no multi-generation, extended pedigrees are available.

The GTG patent for this discovery describes how the technique can be applied to map disease genes, by identifying highly conserved haplotypes shared by affected, unrelated individuals—even individuals from different cultural or ethnic groups.

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