By David N. Leff

Back around 1965, at Temple University Hospital in Philadelphia, a pediatric endocrinologist named Angelo DiGeorge described the syndrome that now bears his name.

"He identified this syndrome," observed molecular geneticist and pediatric cardiologist Antonio Baldini, at Baylor College of Medicine in Houston, "as a combination of an immune system problem and hypocalcemia. DiGeorge also noted facial abnormalities as a clinical entity. At that time," Baldini pointed out, "people didn't know there was such a thing as T-cell immunity, which is defective in these patients. They didn't know the difference between B-cell immunity and T-cell immunity, which had not been discovered yet.

"The DiGeorge syndrome," Baldini continued, "is usually referred to as very severely affecting patients with cardiovascular defects, absent thymus and parathyroid glands, plus craniofacial anomalies. And this syndrome is caused in 99 percent of the cases by deletion of genes on human chromosome 22 that include Tbx1. The deletion," he added, "is estimated to be present in one of every 4,000 infants born alive."

Baldini is senior author of a paper in the current issue of Nature, dated March 1, 2001, titled: "Tbx1 haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice."

"If we eliminate one copy of this Tbx1 gene from chromosome 22," Baldini told BioWorld Today, "we get aortic heart defects, which affect the connection of the heart to the systemic circulation. The heart itself is normal, but its connection with the circulation is abnormal. If we eliminate both copies of the gene, the phenotype becomes much more severe, and is called truncus arteriosus. That means instead of having two great arteries coming out of the heart, these mutant DiGeorge mice have only one artery.

"The 'T' of Tbx1," he explained, "stands for 'T-box' - a DNA-binding domain of the gene-encoded protein. It's a particular amino acid sequence that is conserved through evolution, from fruit flies down to us. And this part of the protein is designed to bind other genes to DNA, and control their expression. That is, Tbx1 is a master sequence. The T," he added, "is more of a historical heritage. T was a classic mouse mutant that had a tail defect, which is why it's called T."

Taking Big Bites Out Of Chromosomes

Baldini pointed out, "The human defect is a chromosomal deletion, missing one piece of chromosome 22. This occurs because of some problem in humans. In mice we never see that. So," he recounted, "we designed the same chromosome deletion in the mouse, using a technology called chromosome engineering. This allowed us to single out a specific piece of DNA from the mouse chromosome by inserting special DNA sequences that are recognized by particular enzymes. Then we expressed a particular viral enzyme, which cut out that piece, and rejoined the gap ends.

"You can do this with anything you like," Baldini said. "We did it from 1.2 million base pairs down to 200 kb. The technology," he observed, "was invented by Allen Bradley, a co-author of the Nature paper." (Bradley now directs the Wellcome Trust's Sanger Genome Center in Cambridge, UK - a major participant in the International Consortium of the Human Genome Project.)

"We also reinserted some of that cut-out material," Baldini related, "which cured the mouse phenotype by transgenic rescue. We didn't know exactly what genes were contained in those pieces of DNA. What helped us to define exactly what genes were there was the genome sequence deposited in the Human Genome Project database. Chromosome 22 was the first human chromosome to be sequenced. And that particular portion of the mouse genome was available for a couple of years, too. (See BioWorld Today, Dec. 13, 1999, p. 1.)

"If we remove one copy of this Tbx-1 gene," he continued, "then we have a set of abnormalities that concern the development of a particular embryonic blood vessel called the fourth pharyngeal arch artery. Its defective development - in humans, mice and many other organisms - results in aortic defects.

"We didn't know that Tbx-1 was the DiGeorge syndrome's master gene. So its discovery as the main player for development of this particular embryonic heart structure was very relevant to that syndrome. The gene had been isolated before but nobody knew what it was doing. We were looking for the gene responsible for the abnormalities seen in a mouse model of the DiGeorge syndrome. We had done that mutant mouse line a couple of years ago. It carried a large chromosome 22 deletion presenting with heart defects. So this Nature paper describes how we went from that mouse, in which we eliminated 15 genes from the chromosome, down to identification of the Tbx1 gene, which proved responsible for that phenotype."

Then Baldini and his co-authors selected 100 human patients with particular clinical presentations similar to the DiGeorge syndrome, but without the typical deletion. "We hypothesized," he recounted, "that some of these cases may have a mutation in Tbx1. So we sequenced that gene in all 100 patients to see if there were any abnormalities, but we haven't found any. We probably need to look in many more patients to draw a conclusion. So far it's still up in the air."

Step Toward Therapy: A Mouse Of Prevention

Baldini sees no apparent clinical applications from his findings so far. "We are hoping to devise in a mouse a way to prevent these abnormalities, which is the closest you can get to therapy. We want to do that after we understand how Tbx1 works. Then we can try to neutralize the negative effect of the loss of these genes by providing either a target of this gene or something else - we don't know yet.

"The strategy we have used," he observed, "allowed us to test large pieces of genomic DNA at a time. So for one extensive deletion we could test the function of many genes. And if we find an interesting effect, we can identify the gene responsible - analyze genomes a big chunk at a time."

Baylor is not patenting his work, Baldini noted. "The use of this mouse will be reserved for search of genes involved in behavior and psychiatric disorders. There will be a license on this. We are ready," he concluded, "to discuss applications with research scientists, and pharmacological applications when applicable." n