Search for cholesterol absorption genes narrows to two chromosome regions

Findings with lab mice may lead to novel cholesterol-lowering drugs against heart disease

Two people eat the same egg, cheese and ham muffin for breakfast, yet one absorbs significantly more cholesterol into his or her blood than the other. Why?
The answer, and all of its implications for combating heart disease, remains stubbornly hidden within our DNA. In recent genetic studies with lab mice, however, researchers at The Rockefeller University have begun to close in on the culprit genes.

“By determining the genetic basis behind the observation that some people absorb 25 percent of cholesterol from their diet, while others absorb up to 75 percent, we hope to develop new treatments to protect this latter group,” says senior co-author Jan. L. Breslow, M.D., head of The Rockefeller University’s Laboratory of Biochemical Genetics and Metabolism and former national president of the American Heart Association.

The researchers hope that the identification of genes that regulate cholesterol absorption in mice will lead them to the location of similar genes in humans – and ultimately to the development of drugs that specifically reduce cholesterol absorption and protect against coronary heart disease, the number one cause of death in the United States.

In the Dec. 10 issue of the Proceedings of the National Academy of Sciences (published online Nov. 22), the Rockefeller scientists report the use of mouse “genetic linkage mapping” technology to narrow the location of genes responsible for regulating the absorption of plant fatty molecules called “plant sterols” – markers of cholesterol absorption – to two distinct regions on chromosome 2 and 14.

While the exact location of the genes has not been deduced, the results indicate that the researchers have indeed uncovered their general vicinity: one of the putative sites has an incredibly high probability – a billion to one – of carrying the suspected genes.

“We are excited because our data analysis shows that cholesterol absorption genes are very likely hiding in chromosome 14,” says Ephraim Sehayek, M.D., first author and principal investigator of the study and a clinical scholar at Rockefeller.

“Now that we know where to look, we can use a variety of techniques to uncover their identities.”

Cholesterol: good and bad

Cholesterol is an essentially fatty molecule found in blood and in all body cells. But, when too much of it floats throughout the blood, this waxy substance can clog arteries that feed the heart and brain, ultimately leading to heart disease and stroke. Factors that bring about a rise in the level of blood cholesterol include saturated fatty acids and trans-fatty acids, common to red meats, dairy products and margarine – as well as dietary cholesterol itself, found in meat, eggs, cheese and other animal products.

But, while scientists know much about the role of cholesterol in heart disease, they poorly understand how the body absorbs cholesterol from foods.

That’s why Sehayek and colleagues set out to discover the genes that regulate cholesterol absorption in mice. But to do this, they needed to directly measure the amount of dietary cholesterol absorbed into the blood of mice – a task confounded by the presence of non-dietary cholesterol in the blood. Cholesterol comes in two forms: dietary, which comes from foods; and non-dietary, which is made by the body. Their solution was to turn to plants.

Plants possess cholesterol-like molecules called plant sterols, which people also absorb from food. But unlike cholesterol, plant sterols are not produced by the body and thus their plasma levels directly reflect dietary absorption. In addition, previous studies have identified a rare genetic condition, beta-sitosterolemia, in which people absorb too much cholesterol and plant sterols from food and consequently develop heart disease at a young age. For these and other reasons, Sehayek chose to use plasma plant sterol levels as a marker of cholesterol absorption.

Breeding mice in search of genes

Next came the tricky part: genetic linkage analysis. In this gene-hunting technique, researchers crossbreed hundreds of mice in an attempt to link an observed trait to the genes that cause it. One experiment can take years and only sometimes results in the discovery of new genes.

The first step is to mate two strains of laboratory mice possessing opposite varieties of a trait of interest. Sehayek and colleagues began by mating a strain of mice they discovered to have low plasma plant sterol levels with another strain possessing high plasma plant sterol levels. Next, they crossbred the progeny of this mating while keeping track of each newborn animal’s plasma plant sterol levels as well as their DNA make-up; for every 10 million base pair units or so of DNA, they asked if the DNA originated from the parent with high or low plant sterol levels.

By tracking the origin of the DNA in this way hundreds of times across the entire genome, the researchers were able to link plasma levels of plant sterols in mice to two distinct patches of DNA that must control these levels. In other words, they mapped the location of plant sterol genes to regions, or loci, on chromosomes 2 and 14. “Our data analysis shows a very strong signal at chromosome 14,” says Sehayek. “This means that sterol absorption genes are most definitely somewhere in this region.”

With their genes on the DNA map, the researchers now plan to apply both genetic and molecular tools to hunt down their precise location.

In the meantime, their findings already have applications for human studies. Several researchers in the Breslow laboratory, along with Rockefeller researchers Jeffrey M. Friedman, M.D., Ph.D., and Markus Stoffel, M.D., Ph.D., are involved in ongoing studies of the island population of Kosrae, located 5,400 miles off Los Angeles in Micronesia.

The Kosraeans possess to a high degree the collection of health problems known as “Syndrome X,” including obesity, diabetes, high blood pressure and high blood cholesterol, Also, most Kosreans can trace their heritage to a relatively small “founder” population. Thus, by studying the genetic inheritance patterns of this group of islanders in a similar fashion to genetic linkage studies in mice, the researchers hope to identify the genes behind Syndrome X disorders.

The newly identified plant sterol absorption regions in the mouse now will guide the search for candidate DNA loci in the Kosrae population.

“By applying our findings in mice to human studies, we may actually gain clues to our hunt for mice genes,” says Sehayek. “It’s a back and forth process between humans and mice that hopefully will result in the discovery of novel human cholesterol absorption genes.”

Other authors of this study include Elizabeth M. Duncan and Jennie G. Ono at The Rockefeller University; Deiter Lutjohann and Klaus von Bergmann at the University of Bonn, Germany; Ashok K. Batta and Gerald Salem at the Veterans Affairs Medical Center, East Orange, N.J.

Founded by John D. Rockefeller in 1901, The Rockefeller University was this nation’s first biomedical research university. Today it is internationally renowned for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. A total of 21 scientists associated with the university have received the Nobel Prize in medicine and physiology or chemistry, 16 Rockefeller scientists have received Lasker Awards, have been named MacArthur Fellows and 11 have garnered the National Medical of Science. More than a third of the current faculty are elected members of the National Academy of Sciences.