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A Risky Factor in Heart Disease
    For all the controversy over heart disease and diets, there is no question that there are genetic components to the equation that cannot be ignored. This past year, scientists at Berkeley Lab identified a new apolipoprotein that appears to play a significant role in controlling triglyceride levels in the blood. Triglycerides are one of the two major blood fats, along with cholesterol, that are important risk factors in heart disease development.

Dr. Eddy Rubin, a geneticist who heads the Genome Sciences Department in Berkeley Lab's Life Sciences Division, and Len Pennacchio, a member of Rubin's research group, led the team that found the new gene, which they named apoAV. They used transgenic mice to identify and test the gene, then compared their findings to clinical studies on humans to determine its influence on triglyceride levels.

"By comparing the sequence of the genomes of humans and mice we have found a genetic jewel that had been missed when the sequence of the human genome alone was analyzed," says Rubin. "ApoAV appears to have a major role in lipid metabolism in both humans and mice."

"By comparing the sequence of the genomes of humans and mice, we found a genetic jewel that had been missed."

Heart disease remains the leading cause of death in the United States. The majority of these deaths are the result of atherosclerosis-the hardening of arteries through the buildup of plaque deposited primarily by plasma lipids such as low density lipoprotein (LDL), also known as "bad cholesterol," and triglycerides. Many Americans have sought to reduce atherosclerosis through careful diet, but dietary effects can vary widely among individuals depending upon their genetic and metabolic profiles.

It is well established that a cluster of apolipoprotein genes along human chromosome 11-known as the apoAI/CIII/AIV region-has a major influence on plasma lipid profiles and, consequently, atherosclerosis susceptibility. It is also well known that mutations in DNA sequences within this region can contribute to severely elevated triglyceride levels. Since the DNA for the apoAI/CIII/AIV region has now been fully sequenced as a result of the Human Genome Project, Rubin, Pennacchio, and the other members of the team searched for additional genes in the region by looking for any sequences that had been conserved through evolution in both humans and mice.

  Eddy Rubin, head of Berkeley Lab's Genome Sciences Department (right) and Len Pennacchio, a member of Rubin's research group, led the team that found a new gene--apoAV--which plays a significant role in controlling blood triglyceride levels, an important risk factor in heart disease development.

"The approach we took was based on the concept that if a segment of the genome has been conserved over the 60 million years since humans and mice are believed to have diverged, then the sequence within the segment probably encodes an important biological function," explains Rubin. "Accordingly, in scanning the genomes of mice and humans we focused our attention specifically on those sequences shared by humans and mice."

ApoAV was introduced into a strain of mice through standard transgenic technology and engineered to overexpress itself. Other mice were engineered to lack apoAV. Comparing the two groups revealed a dramatic contrast.

Says Pennacchio, "Mice expressing human apoAV showed a 300 percent decrease in plasma triglyceride concentrations, while the knockout mice lacking apoAV showed a 400 percent increase."

Having identified apoAV and determined its influence on triglyceride concentrations in mice, the researchers next examined the relationship between DNA-sequence polymorphisms in the apoAV gene and plasma lipid levels in humans.

"To serve as genetic markers for association studies, we identified single nucleotide polymorphisms (SNPs) across and surrounding the human apoAV gene," says Rubin. "Four markers with relatively high minor allele frequencies were obtained and scored in approximately 500 random unrelated Caucasian individuals (mostly males) who had been phenotyped for numerous lipid parameters before and after consumption of high- and low-fat diets."

A human/mouse sequence comparison plot reveals apoAV adjacent to the gene ZNF259. The sequence conservation curve is colored to indicate where human/mouse sequence alignments achieve greater than 50% similarity (translated exons are red, untranslated exons green, noncoding conserved sequences are blue). ApoAV was identified through this cross-sequence analysis. The gene modulates triglyceride levels in both humans and mice.  

A second human association study using a different experimental design was also performed, this time on some 400 individuals drawn from the extreme ends of a clinical study encompassing several thousand subjects. In both studies, a strong link was found between plasma triglyceride levels and SNPs across the apoAV gene.

Says Rubin, "The two human studies indicated that a common sequence variation in the vicinity of apoAV is inherited in more than 10 percent of the population and appears to result in a 20- to 30-percent increase in an individual's blood triglyceride levels."

Rubin and Pennacchio believe it may be possible to use apoAV polymorphisms as prognostic indicators for hyper-triglyceridemia susceptibility and that apoAV modulation could be a potential strategy to reduce this cardiovascular disease risk factor. The next step, they say, is to identify DNA sequence variations that lead to lower apoAV protein concentrations in the blood.

Working with Rubin and Pennacchio on this study were Dr. Ronald Krauss, a physician with Berkeley Lab's Life Sciences Division recognized as a leading authority on genetic risk factors in heart disease, plus Michael Olivier and David Cox at Stanford University's Human Genome Center, Jaroslav Hubacek and Jonathan Cohen of the University of Texas Southwestern Medical Center, and Jean-Charles Fruchart of the University of Lille in France.

-- Lynn Yarris

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