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Scientists Capture Endocrine Tumor Gene

By Sharon Ricks

On the Front Page...
Sunita Agarwal stepped into Rm. 9C216 in Bldg. 10 at 7 a.m. on Jan. 21 ready to hunt. It didn't matter that the morning was still dark or that the temperature outside was near freezing. The visiting associate was anxious to search for the gene that plagues families with tumors of the parathyroid, the pituitary and the islet cells in the pancreas. She had no idea that the day would bring an end to 3 years of exploration for NIDDK's Metabolic Diseases Branch nor that its accomplishments would be published in the Apr. 18 issue of Science. She was just eager to get started.

A member of Dr. Stephen Marx's genetics and endocrinology section, Agarwal was part of a dedicated team of 20 NIH scientists from NIDDK, NHGRI, NCI, NLM and three scientists from the University of Oklahoma who came together to pursue the gene which, when mutated, causes multiple endocrine neoplasia, type 1 (MEN1). "It's been a synergistic and positive collaboration," explains Dr. Allen Spiegel, NIDDK scientific director and chief of the branch. Members of the NIDDK/NICHD Interinstitute Endocrine Training Program, the Clinical Center radiology department, the NCI Surgery Branch and members of 65 MEN1 families also contributed.

The chase began in the 1970's, when Marx, then an NIDDK clinical associate, began to characterize the clinical features of families with MEN1 and to take care of patients. "People weren't finding genes at that time, but we were developing methods for diagnosis and treatment," he says. By 1988, Marx had begun to analyze genetic linkage in DNA samples, looking for the molecular basis for the neoplasia. The same year, a Swedish research team tracked the location of the gene to the long arm of chromosome 11, and Marx later confirmed their work. They suggested it was a tumor suppressor, a gene that acts like a set of brakes on abnormal cell growth, explains Spiegel. In MEN1, all brakes are lost and cells can grow out of control, forming multiple endocrine tumors that can cause hyperparathyroidism, pituitary tumors, severe ulcers and pancreatic cancer.

The gene team included: (in front row, from l) S. Agarwal (DDK), Z. Zhuang (NCI), S. Chandrasekharappa (NHGRI), S. Olufemi (NHGRI), S. Guru (NHGRI); (in second row, from l) M. Kester (DDK), M. Boguski (NCBI/NLM), Y. Kim (DDK), S. Marx (DDK), J. Crabtree (NHGRI); (in third row, from l) M. Emmert-Buck (NCI), L. Debelenko (NCI), J. Weisemann (NCBI/NLM), P. Manickam (NHGRI); (fourth row, from l) A. Spiegel (DDK), I. Lubensky (NCI), C. Heppner (DDK), L. Burns (DDK), F. Collins (NHGRI). Not shown are L. Liotta (NCI), Y. Wang and B. Roe (University of Oklahoma) and Q. Dong (formerly of DDK, now University of Sydney, Australia).

Even with this information, scientists both in Sweden and the U.S. found the gene elusive until NHGRI director Dr. Francis Collins and his colleague, Dr. Settara C. Chandrasekharappa, joined the effort in 1994. They introduced positional cloning to assemble the nearly 3 million base pairs of continuous DNA from a small part of chromosome 11 surrounding the MEN1 gene. "This systematic and powerful process of positional cloning was instrumental in finding the gene," says Chandrasekharappa, the paper's lead author.

The pace escalated as NCI's Drs. Lance Liotta and Michael Emmert-Buck applied a technique they had developed in 1996 to study tumor tissue for loss of a segment of the normal copy of chromosome 11, which helped narrow the candidate region. Collins and Chandrasekharappa cloned and partially sequenced the region, identifying more than 18 new genetic markers, and created a physical map. Oklahoma University scientists used this map to sequence the critical genomic region, and with help from NLM bioinformatics experts, Drs. Mark Boguski and Jane Weisemann, many candidate genes were identified. Next, NIDDK scientists analyzed DNA from individuals with MEN1 from 15 different families.

That's what Agarwal, a postdoc working with NIDDK biologist Mary Beth Kester, was doing early on Jan. 21. They used a sophisticated mutation detection technique called dideoxy fingerprinting introduced to the section by Lee Burns, a molecular biologist there. Kester and Agarwal examined two gel samples from MEN1 patients which showed band shifts that were different from normal human samples.

"We kind of knew it was the gene," says Agarwal. "Mary Beth said, 'This is it,' but others were not convinced." By 10 a.m. Friday, the disbelief dissipated. "We got the sequences of the gene alterations, and Kester saw a stop mutation and a four-base pair deletion, and we knew we had two protein truncating mutations. It was the first candidate gene in which we had seen any mutations only in patients and not in normals." They looked for and found different mutations in other patient samples. A mother, her son and nephew all with MEN1 had the same mutation. "By Sunday, we were all excited," says Agarwal. "Everyone wanted to see the data, and everyone was trying to find Collins so he could tell others to stop working on other genes."

The MEN1 gene has 10 exons and 610 amino acids. Boguski at NLM has been over it, he says, up, down and sideways, and he declares it to be unlike anything else that's ever been seen. "This gene could indicate the presence of an entirely new pathway for the control of cell growth," says Collins. Since the gene's protein product, menin, is expressed throughout the body, researchers suspect it may contribute to other more common, non-hereditary endocrine tumors and unexpected cancers. Menin will also serve as a target for drugs designed to prevent or treat benign and malignant endocrine tumors. Already peptides have been made to immunize rabbits, and researchers are screening the antibodies. Mouse models of the disease are being made. A screening test to allow doctors to eliminate repeated testing in patients who have normal MEN1 genes is now feasible and will need to be prospectively evaluated.

The paper is dedicated to the memory of Dr. Gerald D. Aurbach, former chief of NIDDK's Metabolic Diseases Branch. "It was his work that brought MEN1 patients here in the first place," says Marx. "He recognized the importance this gene could have." Aurbach died in November 1991.

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