SUMO1 gene implicated in cleft lip and palate defects

Researchers supported by the National Institutes of Health report in the current issue of the journal Science that a much-studied gene called SUMO1, when under expressed, can cause cleft lip and palate, one of the world's most common birth defects.

With several genes already implicated in causing cleft lip and palate, the authors note their addition to the list comes with a unique biological twist. The SUMO1 gene encodes a small protein that is attached to the protein products of at least three previously discovered "clefting" genes during facial development, in essence linking them into or near a shared regulatory pathway and now hotspot for clefting.

"The big challenge for research on cleft lip and palate is to move from studying individual genes to defining individual protein networks," said Dr. Richard Maas, a scientist at Brigham and Women's Hospital and Harvard University Medical School in Cambridge, Mass. and senior author on the paper. His research is supported by NIH's National Institute of Dental and Craniofacial Research (NIDCR) and the National Institute of General Medical Sciences (NIGMS).

"By protein network, I mean a nexus of proteins that interact in a highly regulated way," he continued. "It's at this dynamic, real-time level that science will begin to see the big picture and tease out more of the needed insights to understand and hopefully eventually prevent cleft lip and palate in newborns. What's exciting about SUMO1 is it allows us for the first time to begin to connect at least some of the dots and hopefully lock into a highly informative protein network that feeds into additional protein networks to form the palate, or roof of the mouth."

According to Maas, their discovery also offers a prime example of the power of genomic research, the comparative study of individual or sets of related genes among species, from yeast to human. The discovery also highlights the utility of comprehensive gene databases, DNA libraries, and other publicly accessible genomic resources to accelerate the pace of modern science.

Maas said the work that led to this weeks's Science paper began several months ago when a clinician sent a blood sample from a five-year-old patient who had been born with a cleft lip and palate but no other obvious abnormalities. The sample arrived as part of an international program in which Maas's lab participates, called the Developmental Genome Project, or DGAP.

Launched in the late 1990s, the NIGMS-supported project relies on clinicians to send to DGAP-affiliated laboratories DNA samples from consenting patients with birth defects that appear to be caused by chromosome rearrangement, particularly so-called "balanced translocations." A balanced translocation means that during the normal cell cycle, two chromosomes stick together, break, and form again incorrectly with parts of each chromosome switching places.

"DGAP builds on the hypothesis that the translocation splits a gene involved in the developmental process, renders it non functional, and causes a visible birth defect," said Dr. Fowan Alkuraya, a post-doctoral fellow in Maas's laboratory and co-lead author on the study. "In theory, the translocation will lead us to a biologically informative gene. The challenge is to prove that theory and reality are one and the same."

As the first step in the process, Alkuraya and colleagues found that the split gene in the patient's DNA sample encoded SUMO1, a small protein that is known to attach to the back of newly formed proteins to modify their function. "This was intriguing news because SUMO1 often attaches to, or tags, proteins to undergo a biochemical process called sumoylation, which influences their behavior," said Maas. "At least three of the previously identified clefting genes are known to be sumoylated and, if SUMO1 turned out to be involved in clefting, it might lead us to a relevant protein network."

To determine whether SUMO1 was indeed a clefting gene, the Maas lab turned to their experimental model of choice, the mouse. After establishing that SUMO1 is expressed in the region of the developing mouse where the palate forms, the scientists asked the next logical question: What happens if SUMO1 is expressed at abnormally low levels as the palate forms?

The scientists turned to a research consortium called BayGenomics that employs so-called "knockout," or gene inactivation, technology to for the systematic study of the individual genes with the mouse genome to decipher their possible functions. The consortium, supported by NIH's National Heart, Lung, and Blood Institute (NHLBI), has assembled a repository of embryonic stem cells for research purposes in which each available line has a different gene knocked out, or inactivated.

The Maas lab ordered the stem cell line in which SUMO1 had been partially inactivated, implanted them into female mice, and waited. The result: Four of 46 newborn mice had clefts of the palate or face. "That's about the incidence that we see in human families with a history of cleft lip and palate," said Dr. Irfan Saadi, a co-lead author on the study and post-doctoral fellow in the Maas lab. "So we weren't put off by the low incidence at all. It's what we would have expected."

In additional work, the scientists found that when SUMO1 and the sumoylated clefting gene Eya1 were both inactivated, clefting increased to 36 percent of newborn mouse pups, an indication that their proteins interact during palate development and a point that additional experiments further confirmed.

"Ten years ago, this work might have taken our laboratory years to perform," said Maas. "But with the genomic resources that are now readily available, we can get answers in a matter of weeks or months and, just as importantly, we spend a greater proportion of our time thinking through the biology rather than worrying why an assay isn't working."

With more tools and data to sift through, Maas noted that the long held distinctions between syndromic and non-syndromic cleft lip and palate have begun to blur. Traditionally, "syndromic" means babies are born with cleft lip and/or palate, in addition to other birth defects. "Non-syndromic" refers newborns who have cleft lip and/or palate only.

"Clefting reflects the combined actions of multiple gene products, rarely only one gene and its protein," said Maas. "That's why it's likely that what we now call non-syndromic has a very heterogenous mixture of manifestations, too. It's just that the other manifestations are so subtle or not immediately obvious that we don't recognize them. Through our work and that of our colleagues, we can begin to better define these conditions."