By David N. Leff

From the earliest days of in utero development, all human female embryos are lyonized - not lionized. Lyonization is the process of turning off one of the two X chromosomes with which every mammal is born. An English cytogeneticist named Mary Lyon discovered the effect, named after her, in 1961.

The reason for this seeming after-thoughtlessness on the part of Mother Nature - supplying two X's, then canceling one - is actually simple and logical: Every female inherits one X chromosome from her father, another from her mother. One of the two has to go, and it does.

"What's involved in inactivating that extra X," observed molecular biologist Timothy Bestor, "is DNA methylation." He explained that methylation "is a covalent modification of genomic DNA, specifically of its cytosine residues. Methylated genes are usually turned off, and unmethylated genes turned on. Because it increases the information content of DNA, methylation is in principle capable of doing just about anything. Over the years, every possible role has been assigned to it. But the real function has always been controversial."

Bestor is an associate professor of genetics and development at Columbia University's College of Physicians & Surgeons. In today's issue of Nature, dated Nov. 11, 1999, he tackles that DNA methylation debate on two levels - clinical and genomic. The paper, of which he is corresponding author, is titled: "Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene."

That human disorder is better known as ICF syndrome, the acronym for its unabridged name: "Immunodeficiency, centromere instability and facial anomalies." With just over 40 patients diagnosed worldwide since it surfaced 21 years ago, ICF may well be one of the rarest familial syndromes known to man - and woman.

Rare But Probably Under-Diagnosed

"It was first described simultaneously in England and Italy in 1978." Bestor told BioWorld Today. "The English group was led by Maj Hulten, who is a co-author on this Nature paper. The Italian group was at the University of Pavia, led by professor L. Tieplo, who gave the syndrome its name in 1988."

As for ICF's scanty incidence, Bestor pointed out it's "probably a severely under-diagnosed condition. It's not part of the differential diagnosis in the U.S., and only two patients in North America have been diagnosed with ICF. In Western Europe there are about 40 known cases, and elsewhere in the world none at all.

"ICF children," Bestor continued, "present in infancy with recurrent bacterial and viral infections, reflecting their severe immunodeficiency. It's usually lethal. Some of them die before the age of 2. Antibiotics and gammaglobulin are fairly effective, but nonetheless most of the patients do die before adulthood."

Bestor made the added point, "Methylation of the inactive X chromosome in females with ICF syndrome doesn't appear to have any effect on X inactivation, and the severity of ICF syndrome is very similar in male and female patients."

As the syndrome's name implies, odd facial features are hallmarks of ICF, and an aid to clinical diagnosis. "They share some similarity with the facial anomalies in Down's syndrome," Bestor pointed out.

"That is, there's some macroglossia [oversize tongue], epicanthal folds [slanting eyelids], flattening of the nasal bridge, and hypertelorism [eyes set wide apart]. These facial anomalies are very mild, and not disfiguring by any means," he added, "but highly characteristic of ICF.

"However," Bestor underlined, "the definitive diagnosis is made by observation of the cytogenetic abnormalities. Those are really obvious. This disease, even though it's rare, is famous among cytogeneticists because it has by far the most extravagant chromosomal aberrations of any genetic disorder."

To introduce this aspect, Bestor recalled, "About seven years ago I gave a seminar at the Pasteur Institute in Paris, and lunched with some French scientists interested in DNA methylation. One of them was Evani Viegas-Piquinot. She had just found - and not yet published - the observation that in this disease called ICF syndrome, there's a lack of methylation at a particular site of satellite DNA, which is normally heavily methylated. This lack of methylation," Bestor observed, "occurred precisely at the chromosomal regions that are highly unstable in this syndrome. That's the key point."

Evani is senior author of the Nature paper. She, Bestor and their co-authors analyzed the DNA of five unrelated ICF patients, and found mutations in both alleles (parental gene variants) of the gene that encodes the enzyme DNA methyltransferase (DNAMT), which is critical to DNA methylation patterns. They named the gene DNAMT3B.

A Natural For Gene Therapy

"To do these studies," Bestor recounted, "We got either DNA or blood cells from the affected families. Then we directly sequenced all of the genes from each patient and both parents, looking for changes in DNA sequence that affected changes in amino acid sequence.

"In all the affecteds we found mutations in this candidate gene. And we were able to confirm that none of the unaffected parents had mutations in both alleles. If we found a mutation that was present in both alleles of one parent, we could exclude it as causative, because clearly that particular mutation couldn't cause the disease, as that parent doesn't have it."

This study, Bestor pointed out, "identified the first human genetic syndrome caused by defective DNA methylation."

He made the final point, "ICF syndrome could be an excellent target for gene therapy. There's every indication that if you restore this enzyme to the progenitors of lymphocytes, which are key cells in the immune system, it would cure the disease. Because if you restored DNMB3 activity to ICF cells, you completely restore the methylation of the DNA classical satellites, which largely account for the disorder.

"At this point," he added, "we're conducting experiments to determine the cause of defective immune-system lymphocytes in ICF. Obviously the ICF gene-therapy aspect is very attractive to us, although," he concluded, "in its present state, with only 40-odd diagnosed patients, it's not likely to be a money maker."