August 1, 2005 (Vol. 25, No. 14)

New Approaches and Technologies Aim to Extract Useful Information from the Genome

Scientists have been analyzing SNPs and their role in disease for several years. Mapping of the human genome revealed that these single nucleotide alterations are responsible for not only our phenotypic traits, but also how we respond to medicine and our susceptibility to disease.

These and other issues were among the key topics for discussion at Cambridge Healthtech’s “Beyond the Genome” conference, which was held in San Francisco.

“It has become evident over the past year that large structural polymorphisms are much more frequent than people previously realized,” explains Kelly Frazer, Ph.D., vp, genomics, Perlegen Sciences (

“The idea here is that if these polymorphisms are common in the human genome, they’re likely to result in phenotypic consequences. So it’s important as we do association studies to find genetic loci associated with specific phenotypic traits; we are scanning for structural polymorphisms as well as SNPs.”

In order to find out how frequent deletions of this size (around 3,000 bp) exist and whether they are in linkage with SNPs, the company conducted a deletion discovery study in 24 individuals who also had been previously used for SNP discovery.

This allowed the researchers to address the allele frequency distribution. “The minor allele frequency distribution was similar for SNPs and for these deletion polymorphisms, indicating that some deletion polymorphisms are common in humans,” states Dr. Frazer.

The group also showed that they were in strong linkage disequilibrium with SNPs, indicating they probably have a similar evolutionary history. “When we use SNPs for association studies we are also assaying these deletion polymorphisms by proxy, meaning we’re picking up the information of which allele is present, it is very important.”

Dr. Frazer says if they had found these deletions were not assayed by SNPs, “it would have meant our association studies were not assaying for these types of common human variants. If you have a deletion of 800 bp, that’s the equivalent of base pair changes between individuals as 800 SNPs. This indicates that SNPs are sufficient to assay for these other types of variants.”

Perlegen is working with an international group to develop the HapMap, which will identify and verify millions of SNPs to compile a universal guide to human genetic variation. The company is attempting to genotype more than four million SNPs in 270 people, and says it is on track to complete this project by the end of this month.

The Genetics of Disease

Genizon Biosciences (www. has a proprietary approach for studying the genetics of common diseases. Utilizing its network of 1,000 clinical investigators and 300 nurses, the company has collected samples for its genetic studies from the Quebec Founder Population. This is a unique populationdirect descendents from the original 2,600 people who settled in Quebec in the 1600s and 1700s.

“The signal-to-noise is much higher in the Quebec population than what you find in an open population,” explains William Cheliak, Ph.D., vp, business development. “For example, the two well-known breast cancer genes, BRAC1 and BRAC2, have about 1,700 variants worldwide, but in Quebec we have only 14 variants. That’s one reason why this population is so powerful for genetic research.”

Dr. Cheliak presented information on the company’s recent genetic findings related to Crohn’s disease. Patient trios (382) from the Quebec Founder Population and 160,000 polymorphic SNP markers were utilized to conduct a whole-genome association study to discover disease-related genes.

“We found 20 novel regions that are likely to contain genes that are very important in the overall pathophysiology of this disease,” states Dr. Cheliak, “in addition to confirming two regions previously associated with Crohn’s: OCTN and the gene NOD2.”

Genizon uses its proprietary software to analyze the genotype data as the basis of understanding where the disease loci or candidate regions are located. “We have built two algorithms to help us find those things and they work on the basis of what’s called haplotypes, or pattern of adjacent genotypes. One algorithm is based on a fixed window of haplotyple sharing’, the other on a variable window approach.

“They are mathematically quite different and what we like to see is a confirmation using those two different approaches to reach the candidate region.”

The company has also invested in software that builds the interactions that occur among the genes to identify disease pathways and gain an understanding of where to target new therapies and to identify predictive biomarkers.

“This technology has very high resolution so that about one third of the regions we’ve identified as implicated in Crohn’s disease contain only one or two genes, and another third contains 35 genes,” states Dr. Cheliak. The company is currently researching an additional 25 diseases.

Risk Factors

AstraZeneca Pharmaceuticals ( presented information on an on-going study involving Perlegen Sciences and an international research consortium, PROCARDIS, which has conducted whole-genome scans in families where two members have experienced a heart attack.

The study is a case-controlled (700 cases/700 controls) SNP association study into risk factors for myocardial infarction using a high density of SNP markers in a candidate region. “We wanted to pose the question of what kind of contributions genetics will make in improving drug development success,” states Neil Gibson, Ph.D., associate, principal scientist.

Dr. Gibson said a linkage study has been completed for the samples and a significant region of linkage has been discovered. Perlegen developed a SNP map through that region (genotyped 21,500 SNPs), and AstraZeneca is now conducting high-density linkage disequilibrium mapping. “This is the first disease area where we’ve been able to go in with such a rich covering of SNPs,” he states.

The company is further analyzing the initial linkage maps in various ways: haplotype analysis, estimating the true likelihood of the signal compared to the noisethis will be used to pinpoint regions of the gene to move forward and replicate. Since it is a multinational study, what is discovered in one population can, in principle, be replicated in another population.

Hybridization Probes

Scientists at the ARUP Institute for Clinical and Experimental Pathology ( have developed a technology utilizing hybridization probes to identify SNP haplotypes in one step. “Current techniques for haplotyping are very complicated,” explains Genevieve Pont-Kingon, Ph.D., senior research scientist.

Dr. Pont-Kingdon said her group was able to design a hybridization probe to cover two SNPs that are within a 100 nucletotide range. “We found if you make a deletion inside the probe compared to the template, the template sequence loops out, and then the probe binds continuously on the template.”

She adds that each combination of SNP has a specific melting temperature (reflects probe stability due to length, nucleotide composition, and homology to template) that can be recorded by fluorescence.

As an example of how this technology works, the group used three haplotypes found in the beta 2-adrenergic receptor gene and characterized by three different SNP combinations. “We were able to discover two previously undescribed haplotypes in this gene using our probe technology,” says Dr. Pont-Kingdon.

Potential applications for this new technology include pharmacogenomicsfor example, using the beta 2-adrenergic receptor gene to examine the genetic variation in this receptor as a response to beta-blockers. The technology can also directly determine haplotypes of multiple polymorphisms in a closed-tube environment, making it a potential clinical genetic test from individual samples.

Haplotype analysis with hybridization probes on high throughput instruments would enable analysis of large numbers of samples necessary for association studies and pharmacogenomic trials. “We expect that within the next few years we will be able to do haplotyping in the clinical lab,” adds Dr. Pont-Kingdon.

ParAllele BioScience ( presented information on identifying biomarkers for cancer therapeutics via analysis of germline and somatic mutations. The company’s platform is based on two proprietary technologies: Molecular Inversion Probes (MIP) and Mismatch Repair Detection (MRD).

“We are able to work with SNPs that are most relevant to the researchers because we have a highly specific technology and can multiplex to very high levels: over 20,000-plex has been demonstrated,” states Thomas Willis, Ph.D., CSO.

“By combining our technology with the whole-genome mapping products of Affymetrix, we will jointly be able to offer a broad set of genetics research products.” The company recently announced a definitive agreement to be acquired by Affymetrix.

MIP enables copy number analysislooks for all types of chromosomal re-arrangements, including small deletions and amplifications, as well as mitotic recombinations, which change allele ratio, but not overall copy number. In addition, the technology enables highly quantitative expression analysis. MRD is used for somatic mutation detection, and according to the company, has advantages over sequencing.

“This technology allows hundreds of genes to be scanned for mutations in a single reaction. Conventional sequencing allows only the analysis of a single gene segment at a time,” explains Dr. Willis.

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