Fifteen-year hunt uncovers gene behind pseudothalidomide syndrome

A team of scientists from Colombia, the United States and elsewhere has successfully completed a 15-year-plus search for the genetic problems behind the very rare Roberts syndrome, whose physical manifestations often include cleft lip and palate and shortened limbs that resemble those of babies whose mothers took thalidomide during pregnancy.

The discovery, which is reported in the April 10 advance online section of Nature Genetics, proves that genes behind very rare inherited diseases can now be found, offering excellent opportunities to strengthen understanding of craniofacial and limb development, health and disease beyond the rare disease itself, say the researchers.

Because of advances in technology and computer analysis, the researchers were able to find the Roberts gene, called ESCO2, by studying samples from just 15 Roberts syndrome families from Colombia, Turkey, Canada and Italy and to provide insight into its biological effect.

"For decades now, we've known that the appearance and number of chromosomes were abnormal in people with Roberts syndrome, but we hadn't been able to figure out why or how," says Ethylin Jabs, M.D., a professor in the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins. "Just within the last few years have the genetic techniques, the genomic information, and the computer analysis become powerful enough to find the genetic mutations behind a disease as rare as Roberts."

Some of the techniques they used -- such as that to make many copies of DNA from a small sample -- have been around in some form for more than a decade. But others are much more recent developments. For example, the researchers found important genetic changes in part by comparing different species' genetic sequences, most of which were published only within the last four years.

"In 1989, we were collecting samples and characterizing the chromosome problem in cells from people with Roberts syndrome," Jabs remembers. "We knew it would be really important to find the gene, but it just wasn't practical at that time."

A few years later, in 1995, two Colombian geneticists started their quest to fully understand Roberts syndrome. Without their push, the gene for Roberts might still be unknown.

Colombian Hugo Vega had noticed an unusual number of patients with Roberts syndrome in the clinic at the University of Bogotá. Fairly quickly, he tracked down seven families with Roberts syndrome in two villages outside Bogotá. Four of the families share an 18th-century ancestor, he and Miriam Gordillo, then an undergraduate, discovered.

"The families have really collaborated with us, they've worked with us quite closely to help us uncover the gene behind the syndrome," says Gordillo. "Now we have about 10 affected families from outside Bogotá, and we can offer a genetic test to families at risk of Roberts syndrome."

Vega and Gordillo, a husband-and-wife team, criss-crossed the globe to continue their work and find better funding opportunities. In Japan, Vega tied the Colombian families' syndrome to a large region of chromosome 8. In The Netherlands, a post-9/11 detour, he added to his analysis samples from Turkish and Italian families with Roberts syndrome.

In 2004, Gordillo got a student visa to work with Jabs and to study for her doctorate in human genetics at Johns Hopkins. Over the past year, Gordillo analyzed the chromosome 8 region in samples from 15 families (consisting of 18 affected members and 33 unaffected members) and tied the condition to one of 6 genes.

Then, the international team compared the human sequence of the genes to those from chimpanzee, mouse, rat, chicken and zebrafish, and to the gene sequences of the affected family members. One segment of a gene called ESCO2 that was identical in all the animals contained changes that disrupted the gene's protein-making instructions in people with the syndrome. Knocking out the equivalent gene in yeast and fruit flies led to the same chromosome problems, says Gordillo.

"Comparative genomics didn't really exist even five years ago," says Jabs. "Techniques to genetically engineer yeast, fruit flies and even mice have dramatically improved in the last 15 years. And we were also able to look at when and where the gene is expressed during human development. Without these techniques, and without the powerful computer programs, we wouldn't have been able to identify this gene and confirm its role in Roberts syndrome."

The physical similarities of people with Roberts syndrome and those whose mothers took thalidomide suggest similar underlying biology, Jabs notes. Although there's some evidence that thalidomide prevents blood vessel growth, it's not clear why. If the underlying biology is related somehow, then thalidomide might affect chromosomes and cell division like ESCO2 in Roberts syndrome, Jabs speculates.

During normal cell division, every chromosome is copied, and each of the "original" chromosomes is attached to its "new" copy. While there are attachment points along the entire chromosome, the bulk of the connection is at the centromere, a chromosome's functional hub.

The chromosomes' connection allows the cell to move them together, ensuring that the two copies are lined up together at the center of the dividing cell. Once lined up, tiny molecular "motors" attach to the centromere of each copy and pull the original and the new copy away from each another as division proceeds.

However, in cells from people with Roberts syndrome, the chromosome copies are frequently not attached to each other at their centromeres and the chromosomes don't get lined up properly. As a result, the cell doesn't divide or divides very slowly, and the new cells can end up with too many or too few chromosomes (a problem also seen in cancer cells). In Roberts syndrome, the cells tend to stop growing or die, precluding proper development of the limbs, palate and other structures.

The researchers were funded by the Japanese Ministry of Education, Culture, Sports, Science and Technology; the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologia; the Smile Train Fellowship Award to the Center for Craniofacial Development and Disorders at Johns Hopkins; the Louis H. Gross Foundation; J.S. Sutland; L. and S. Pakula; and the Netherlands Organization for Health Research and Development.

Authors on the report are Hugo Vega, of the Institute of Genetics at the National University of Colombia, Bogotá; Norio Sakai, Chengzhe Xu, Keiichi Ozono and Koji Inui of Osaka University Graduate School of Medicine; Quinten Waisfisz, Djoke van Gosliga and Hans Joenje of VU University Medical Center, Amsterdam, The Netherlands; Miriam Gordillo and Ethylin Jabs of the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins; Itaru Yanagihara and Minoru Yamada of the Osaka Medical Center for Maternal and Child Health; and Hülya Kayserili of Istanbul University. Vega and Gordillo studied at the Osaka Graduate School of Medicine, also.