Humans May Be Easier To Clone Than Sheep And Mice Because Of A Single Genetic Difference

The genetic benefit seems subtle, say the researchers, but it is so important that it creates fundamental differences between humans and other animals in the way they regulate fetal growth and cancer susceptibility. The research is published in the Aug. 15, 2001 issue of Human Molecular Genetics.

The genetic benefit they found was this: humans and other primates possess two activated copies of a gene called insulin-like growth factor II receptor (IGF2R). Offspring receive one functional copy from each parent, as expected. However, sheep, pigs, mice and virtually all non-primate mammals receive only one functional copy of this gene because of a rare phenomenon known as genomic imprinting, in which the gene is literally stamped with markings that turn off its function.

With the second copy of the gene permanently imprinted, such animals are more prone to two major problems -- developing cancer and suffering from cloning complications like overly large offspring, immature lung development, enlarged hearts and reduced immunity to disease, say the scientists.

"This is the first concrete genetic data showing that the cloning process could be less complicated in humans than in sheep," said Keith Killian, a Duke University Medical Center molecular evolutionist and first author of the study. "Only one in 300 sheep embryos takes hold, and up to half of these embryos have large offspring syndrome, which can kill the mother and the fetus. Since humans are not imprinted at IGF2R, then fetal overgrowth would not be predicted to occur if humans were cloned."

The problems associated with cloning an imprinted animal occur when scientists manipulate the fledgling embryos in the laboratory, the Duke researchers said. While the IGF2R gene remains intact, the "epigenetic" markings -- crucial information layered on top of the gene sequence -- are inadvertently damaged and alter the way the gene functions, said Randy Jirtle, professor of radiation oncology at Duke.

Jirtle compares the IGF2R gene to computer hardware that is functioning properly, and its epigenetic markings to computer software that is damaged or defective. The computer will not run if the software does not work.

Jirtle said humans are not subject to these epigenetic alterations because, according to their research, humans are not imprinted at IGF2R. For reasons unknown, he says, the very state of being imprinted appears to make imprinted animals more susceptible to epigenetic damage.

However, many scientists have believed that up to 50 percent of people are imprinted at the IGF2R gene and are more susceptible to cancer and, potentially, cloning complications. But the Duke scientists, using the latest gene mapping technology, found no evidence that any humans possess an imprinted IGF2R. Also, decades of successfully manipulating human embryos through in vitro fertilization have not resulted in large offspring syndrome.

While it is true that the IGF2R gene is frequently mutated in human breast, colon, head and neck, liver and lung cancers, the researchers said that is not because humans are imprinted at IGF2R. The gene, like any other involved in cancer, simply becomes mutated, or defective, for a variety of reasons unrelated to imprinting. Mutating two copies of the IGF2R gene is much more difficult and statistically unlikely than mutating a single copy, which is why mice and other imprinted animals are far more susceptible to cancer than are humans, said Jirtle.

Because mice and rats comprise 90 percent of the animals used in research, scientists should take into account the rodents' genetic susceptibility to cancer when they are applying their study conclusions to humans, said Killian. Clinical development of hundreds of potential disease-treating drugs have been abandoned after rodent studies have shown them to be potential carcinogens -- studies that might have had a different outcome if rodents possessed two functional copies of IGF2R. "You could theoretically give new life to thousands of discarded compounds by retesting them in animals that, like humans, have both functional copies of IGF2R," said Killian.

To test for the presence of imprinting in humans and other mammals, the Duke team used six different single nucleotide polymorphisms, distinctive genetic markers called "SNIPs." Scientists worldwide are now using SNIPs to map the entire human genome because the older method, variable number tandem repeat (VNTR), frequently produces ambiguous results.

"VNTRs are antiquated and error prone because the enzymes used to amplify the DNA jump backward and forward," said Killian. "When you go to analyze the results, the picture you get is smeared and therefore ambiguous."

The Duke team's results definitively showed that human IGF2R is not imprinted. The gene is also not imprinted in primates or our closest non-primate relatives, including tree shrews and lemurs. In fact, the team's evolutionary research shows that all these mammals lost imprinting of the IGF2R gene approximately 70 million years ago. The modern-day absence of imprinting in all primates and their closest relatives bolsters the team's genetic data showing that no humans are imprinted at IGF2R, said Jirtle. "While there are approximately 45 imprinted genes identified in mammals, IGF2R is the only gene known to have gained imprinting at one point and later lost it during primate evolution," said Jirtle.

In contrast, IGF2R is still imprinted in all other placental mammals and marsupials included in their study -- including opossums, mice, rats, sheep, cows and pigs. The divergence of imprinting among various classes of mammals millions of years ago suggests that they embarked on separate evolutionary paths that gave rise to the unique genetic traits they possess today.

"Knowing where on the evolutionary scale IGF2R imprinting appeared and subsequently vanished will enable scientists to select animal models better suited for making accurate inferences about human clonability and cancer susceptibility," said Jirtle. "If you don't know how animals are related to each other, there is no way to accurately extrapolate the experimental results from one species to another."

The study was funded by grants from the National Institutes of Health, Department of Defense and Sumitomo Chemical Co. Ltd. Other researchers on the paper include Catherine M. Nolan of the department of zoology at University College Dublin, Dublin, Ireland; Andrew A. Wylie of the department of radiation oncology at Duke University Medical Center; and Tao Li, Thanh H. Vu and Andrew R. Hoffman of the Veterans Affairs Palo Alto Health Care System and department of medicine at Stanford University School of Medicine, Palo Alto, Calif.