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Affymetrix Microarrays Uncover Genetic Mutations in Embryonic Stem Cells Approved for Federally-Funded Research; Scientists Report That Majority of Cells Develop Mutations Over Time

Posted on: Sunday, 4 September 2005, 15:00 CDT

Affymetrix Inc. (Nasdaq:AFFX) announced today that a research group led by scientists at Johns Hopkins School of Medicine and the NIH used Affymetrix GeneChip(R) microarrays to discover that eight of the stem cell lines approved for federally-funded research have mutated, raising concern about the use of the remaining 14 approved cell lines in future research or therapeutic applications. The researchers used two GeneChip microarrays -- the Human the Mapping 100K Set and Mitochondrial Resequencing Array 2.0 -- to scan the stem cell genome at a level of detail never before possible. They found previously undetectable mutations in stem cells that had been grown in the laboratory for dozens of generations.

The results of their study are published in today's online issue of Nature Genetics. Senior authors include Aravinda Chakravarti, Ph.D., director of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins, and Mahendra Rao, Ph.D. head of the Stem Cell Group at the National Institute on Aging, NIH.

Mutations occur all the time as cells grow, but inside the body, mutated cells with harmful effects are often cleared out by the immune system, whereas in the laboratory culture dish -- where there is no immune system -- mutated cells can grow uncontrolled like a cancer. Over time, any cell line grown in the laboratory -- including stem cells -- can develop mutations that are compounded over many generations. The danger is that scientists may think they are studying stem cells identical to those present in the human body, but in reality the laboratory-grown cells have mutated, and any discoveries may not accurately reflect what is happening in people.

The cell lines discovered as mutant in this study are part of the limited number of existing human embryonic stem cell lines (hESC) approved by President Bush for federally-funded research in August 2001. At the time, scientists estimated that more than 60 genetically distinct stem cell lines existed, but today only 22 are available for purchase in federally-supported research.

To find the embryonic stem cell mutations, the group used three different analysis methods -- two of them microarrays -- to compare the genomes of early generation and late generation cells. The researchers looked at human nuclear DNA for mutations using the Mapping 100K Set; they used the Human Mitochondrial Resequencing Array 2.0 to look for mutations in mitochondrial DNA. By combining two different whole-genome microarray experiments with a complementary technology that focused on a handful of individual genes, the stem cell group was able to explore the genome in many different ways that together proved far more effective than any single approach.

GeneChip Mapping 100K Set

The research team used the Affymetrix Mapping 100K Set to scan over 100,000 genetic variations called SNPs (single nucleotide polymorphisms) to find that five of the nine embryonic stem cell lines they studied were missing genes or had too many copies of certain genes. These mutations are called "copy number changes." Normally, there are two copies of a gene, one from the mother and one from the father. In cell lines with copy number changes there may be only one copy or there may be more than two copies of a gene. Copy number changes can result in cancer-like cells that grow uncontrollably because they don't have the normal genetic information used to contain cell growth.

Scientists had missed these embryonic stem cell mutations before because they were unable to look at the genome at the same level of detail as with the 100K. Previously, researchers were limited to studying copy number by measuring comparative genomic hybridization over relatively large regions or by using a microscope to visually inspect the chromosomes, making it difficult to find small deletions or amplifications.

For a review of copy number studies using microarrays go to: http://www.affymetrix.com/community/wayahead/making_copies.affx.

Human Mitochondrial Resequencing Array 2.0

The stem cell group also used microarrays to compare the sequence of stem cell mitochondrial genomes -- a small subset of genes located inside human cells -- and discovered that two of the nine cell lines they studied had developed mutations in their mitochondrial DNA.

The mitochondrial genome is a second genome on top of the DNA located in the nucleus of a cell -- three billion molecules that make up all of our chromosomes and hold the blueprint for everything that makes us human. The mitochondrial genome is made up of the same C, T, G and A molecules that make up nuclear DNA, but it's much smaller, about 16,500 molecules, and it's located in a different part of the cell -- the mitochondria. Another key difference is that in nuclear DNA, there are only two copies of the DNA blueprint, whereas mitochondria have thousands of DNA copies that hold the blueprints for the things that help humans make the energy needed to breathe, walk or even blink. The mitochondria itself is the remnant of a bacteria that invaded a cell hundreds of millions of years ago; over time that bacteria lost and gained DNA, leaving behind a small genome specialized in making proteins needed to convert sugar and oxygen into energy called ATP in a process called oxidative phosphorylation.

For more in formation on mitochondria and the mitochondrial genome, visit the Wellcome Trust's review at: http://www.wellcome.ac.uk/en/genome/thegenome/hg01f014.html.

With Affymetrix' Human Mitochondrial Resequencing Array 2.0, the stem cell research group quickly analyzed all 16,500 bases -- the exact order of every A, C T and G base -- in each stem cell's mitochondria in just one experiment. They found mutations by looking for differences in the DNA sequence between early and late batches of stem cells. Five of the six mutations identified were located in genes and 1 resulted in a functional change. Up until now, scientists looking for changes in mitochondrial DNA had to manually sequence all 16,500 bases, which took weeks of labor.

"These results represent another example of how new Affymetrix products like the Mapping 100K Set and the Human Mitochondrial Resequencing Array enable scientists to make new discoveries by understanding genetic variation at a level of detail never before possible," said Greg Yap, Vice President of DNA Analysis Products at Affymetrix. "Just like with digital cameras, higher resolution provides clearer pictures. Previous tools lacked the resolution or information content to allow researchers to identify these changes."

Methylation

Using a technology complementary to Affymetrix whole-genome microarrays, the research group focused on 14 specific genes and found that all nine stem cell lines had developed chemical modifications of their DNA -- called methylation -- in at least one of three genes. One of these genes, called RASSF1A, is also methylated in many cancers, but what effect the methylation has on the stem cells is unknown.

Potential Impact of Findings

"Of note, given that genomic alterations in hESC lines tend to accrue over time in tissue culture, it does not detract from the therapeutic potential of earlier passage lines, which appear to be largely free of such measurable changes," commented the authors of the Nature Genetics publication. "Our findings, instead, mandate periodic monitoring of hESC lines for various types of genomic mutations using well-defined assays, especially for changes that are likely to affect cellular phenotype. Finally, given the evidence for clonal aberrations in later passages, it is likely that the limited repository sanctioned for federally funded research might further deplete over time."

About Affymetrix:

Affymetrix scientists invented the world's first microarray in 1989 and began selling the first commercial microarray in 1994. Since then, Affymetrix GeneChip(R) technology has become the industry standard in molecular biology research. Affymetrix technology is used by the world's top pharmaceutical, diagnostic and biotechnology companies as well as leading academic, government and not-for-profit research institutes. More than 1,200 systems have been shipped around the world and more than 3,000 peer-reviewed papers have been published using the technology. Affymetrix' patented photolithographic manufacturing process provides the most information capacity available today on an array, enabling researchers to use a whole-genome approach to analyze the relationship between genetics and health. Headquartered in Santa Clara, Calif., Affymetrix has subsidiaries in Europe and Asia in addition to manufacturing facilities in Sacramento, Calif. and Bedford, Mass. The company has about 900 employees worldwide. For more information about Affymetrix, please visit the company's website at www.Affymetrix.com

All statements in this press release that are not historical are "forward-looking statements" within the meaning of Section 21E of the Securities Exchange Act as amended, including statements regarding Affymetrix' "expectations,""beliefs,""hopes,""intentions,""strategies" or the like. Such statements are subject to risks and uncertainties that could cause actual results to differ materially for Affymetrix from those projected, including, but not limited to risks of the Company's ability to achieve and sustain higher levels of revenue, higher gross margins, reduced operating expenses, uncertainties relating to technological approaches, manufacturing, product development (including uncertainties relating to the use of the genotyping arrays and outcome of research discussed in this press release), personnel retention, uncertainties related to cost and pricing of Affymetrix products, dependence on collaborative partners, uncertainties relating to sole source suppliers, uncertainties relating to FDA and other regulatory approvals, competition, risks relating to intellectual property of others and the uncertainties of patent protection and litigation. These and other risk factors are discussed in Affymetrix' Form 10-K for the year ended December 31, 2004 and other SEC reports, including its Quarterly Reports on Form 10-Q for subsequent quarterly periods. Affymetrix expressly disclaims any obligation or undertaking to release publicly any updates or revisions to any forward-looking statements contained herein to reflect any change in Affymetrix' expectations with regard thereto or any change in events, conditions, or circumstances on which any such statements are based.

NOTE: Affymetrix, the Affymetrix logo, and GeneChip are registered trademarks owned or used by Affymetrix Inc.

Aravinda Chakravarti sits on the Scientific Advisory Board of Affymetrix.

Source: Business Wire

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