Key developmental genes identified, linked to rare syndrome

November 01, 1999

Researchers at the Massachusetts General Hospital (MGH) have identified two genes that are crucial to a key step in embryonic development and also have shown that the human version of one of these genes may be associated with a rare immune deficiency syndrome. In their study in the October 29 issue of Cell, the team from the MGH Cardiovascular Research Center (CVRC) confirmed in mice that DNA modification enzymes produced by genes called Dnmt3a and Dnmt3b control the establishment of methylation patterns - a crucial process that controls gene function. They also find that mutations in the human Dnmt3b gene may cause a condition called ICF syndrome. In addition, both genes may be associated with some forms of cancer.

"The processes controlled by these genes are critical to how an organism develops," says En Li, PhD, of the MGH CVRC, the study's senior author. "The ability to study them directly is opening up a new field in which to investigate existing questions."

In all living creatures, the process by which a fertilized egg develops into an embryo, an infant and finally an adult is governed by instructions encoded in the DNA. The basic genetic code is transmitted by the sequence of four molecules known as bases - adenine, guanine, cytosine and thymine. In vertebrate animals and some other organisms, that code can be modified - and the message that it carries changed - by a process called methylation, the addition of a methyl group (CH3) to certain cytosine molecules. Methylation plays such an important role in regulating gene function that some scientists refer to it as "the fifth base."

In mice, soon after an egg is fertilized, the methyl groups on the genes inherited from the parents are largely removed. After the egg implants in the uterus, a wave of what is called "de novo" methylation takes place, establishing a new, embryonic pattern. As cells in the developing embryo reproduce, newly synthesized DNA continually is remethylated to maintain the original pattern established by de novo methylation. The establishment and maintenance of appropriate methylation patterns are critical to the development of an embryo and in the regulation of a number of biological processes. Scientists believe that this same sort of two-step de novo and maintenance methylation process occurs in human embryos.

The study of methylation and its role in mammalian development is a primary goal of Li and his research team. Last year the group identified and cloned mouse and human versions of two genes called Dnmt3a and Dnmt3b. Biochemical evidence strongly suggested that the proteins they coded were critical and long-sought-after enzymes for controlling de novo methylation. In the current study, the MGH team created and studied a group of mouse embryos in which either or both of the candidate genes were inactivated or knocked out.

Results of the study, led by Masaki Okano, PhD, a postdoctoral fellow in Li's laboratory, showed that the "knockout mouse" embryos did not develop normally. Although those in which Dnmt3a was knocked out survive and appear normal at birth, their future growth is slow and they die at about 4 weeks. When Dnmt3b is knocked out, the embryos do not survive, showing significant growth impairment and defects in the neural tube. Embryos in which both genes are knocked out fail to achieve some of the earliest stages of development and die.

Further analysis of genes from the various knockout mice and from embryonic stem cells in which the genes were inactivated confirmed that the defects seen in the mouse embyros were the result of a lack of de novo methylation. The different patterns of defects seen in mice with only a single gene knocked out suggest that two genes have slightly different functions - probably controlling methylation of different portions of the genome. Chromosomal analysis of embryonic stem cells in which Dnmt3b had been knocked out showed substantial demethylation of chromosome segments called minor satellite repeats. These areas, located near the centromere - the central point at which the two strands of a chromosome are attached to each other - are believed to be important in maintaining a chromosome's structural stability.

It already had been known that ICF syndrome - a genetic condition that includes defects in the immune system and characteristic facial abnormalities - was also associated with chromosomal instability and a lack of methylation in the area of the minor satellite repeats. When Li's team identified and cloned the human equivalent of Dnmt3b last year, the gene mapped to an area of chromosome 20 that other researchers had associated with ICF.

In the current study, the MGH researchers analyzed cultured cells from an ICF patient and her parents to search for mutations in Dnmt3b. Two different mutations were found in the patient's Dnmt3b genes, one of which was also present in the mother's DNA. While no mutations were seen in the father genes - usually patients with the disorder inherit a mutated gene from each parent - the researchers believe that in this particular case a spontaneous mutation occurred in the second copy of Dnmt3b. The fact that no similar mutation were found in cells from 50 health individuals suggests that the genetic changes were not normally occurring variations and supports the research team's belief that mutations that alter the activity of Dnmt3b are the cause of ICF.

While ICF is an extremely rare condition - only about 20 cases have been reported - the researchers also will be investigating the possible role of both Dnmt3 genes in other diseases, particularly cancers. "We know these genes are highly activated in certain cancers, which may be responsible for inappropriate methylation and inactivation of tumor suppressor genes. In other cancers, inactivation of these genes or their enzymes may lead to centromeric demethylation and chromosome instability," Li says. "We want to understand more about the mechanisms by which methylation regulates chromosome stability and eventually investigate whether abnormal activation of de novo methylation is involved in development of cancer."

The study was supported by grants from the National Institutes of Health, Bristol Myers-Squibb and the MGH Center for Cancer Risk Analysis. Study co-authors, in addition to Li and Okano, are Daphne Bell, PhD, and Daniel Haber, MD, of the MGH Cancer Center.
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Massachusetts General Hospital

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