suggest that if they can learn the makeup of the tip link, they’ll be that much closer to understanding how the gate mechanism operates.

“This research identifies protocadherin-15 to be one of the proteins associated with the tip link, thus finally answering a question that has been baffling researchers for years,” says James F. Battey, Jr., M.D., Ph.D., director of the NIDCD. “Thanks to the collaborative effort among these researchers, we are now at the closest point we have ever been to understanding the mechanism by which the ear converts mechanical energy — or energy of motion — into a form of energy that the brain can recognize as sound.”

NIDCD’s Zubair M. Ahmed, Ph.D., and Thomas B. Friedman, Ph.D., together with the University of Sussex’s Richard Goodyear, Ph.D., and Guy P. Richardson, Ph.D., and others used several lines of evidence to identify a protein that Drs. Goodyear and Richardson had earlier found to comprise tip links in the inner ears of young chicks. The protein is referred to as the “tip-link antigen” (TLA) because it induces the production of special antibodies, which bind to the protein at the stereocilia tips.

Using mass spectrometry, a laboratory technique that breaks down a substance into its individual components, the researchers analyzed the makeup of the TLA and found two peptide sequences that match up to key segments of the protein protocadherin-15 in humans, mice, and chickens, suggesting that the two proteins are evolutionarily comparable. Additional experiments using western blot analysis, a technique that identifies individual proteins in a substance by separating them from one another by mass and testing how they react to certain antibodies, demonstrated that the antibody that recognizes protocadherin-15 in mice also binds to the TLA.

The team also analyzed the amino acid sequences of protocadherin-15 and discovered four distinct forms — three of which are present in various developmental stages of the mouse inner ear. The researchers refer to the three alternative forms found in the inner ear as CD1, CD2, and CD3 because the sequential variations occur in the protein’s “cytoplasmic domain” — a stretch of amino acids anchored inside the stereocilium. (The fourth form, referred to as SI, is likely to be secreted.) With the help of two imaging techniques that use antibodies to label a targeted protein, the team found that the distribution of protocadherin-15 along the stereocilium varies by form, with the CD3 form stationed only at the tips of the stereocilia in mature hair cells, while the CD1 form is found along the lengths of the stereocilia in mature cells, but not at the tips. In contrast, the CD2 form is expressed along the lengths of stereocilia during hair cell development, but is not present in mature hair cells.

Finally, the team found that a chemical known to break tip links — called BAPTA — had no effect on the CD1 and CD2 forms of protocadherin-15 but destroyed the CD3 form. Likewise, just as tip links are known to reappear roughly four hours after the chemical is removed, the CD3 form returned within four to 24 hours upon removal of the chemical.

Based on these findings, the researchers conclude that, not only is protocadherin-15 now identified as the tip-link antigen, but it is distributed in a specific way in relation to the tip-link complex. They propose that the CD3 form of protocadherin-15, located at the tip of the shorter stereocilium, may link directly or indirectly to the CD1 form on the adjacent, taller stereocilium. This scenario could help explain how tip links that are broken in real-life situations, such as from excessive exposure to loud noise, could cause temporary hearing loss until the link re-establishes itself and hearing is restored.

In future studies, the scientists plan to delve more deeply into the role that protocadherin-15 plays in the tip-link complex and whether it interacts with other components in the formation of the tip link. They also hope to determine how tip links can be stimulated to re-form, once broken.

The work was supported by the NIDCD and The Wellcome Trust, London, UK. Other researchers on the project represent the NIH’s National Human Genome Research Institute, Bethesda, MD; University of Cambridge, UK; Brigham Young University, Provo, UT; the National Centre of Excellence in Molecular Biology, Lahore, Pakistan; and the University of Kentucky, Lexington.

NIDCD supports and conducts research and research training on the normal and disordered processes of hearing, balance, smell, taste, voice, speech and language and provides health information, based upon scientific discovery, to the public. For more information about NIDCD programs, see the Web site at www.nidcd.nih.gov.

The National Institutes of Health (NIH) — The Nation's Medical Research Agency — includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.

Scientists Identify an Inherited Gene That Strongly Affects Risk for the Most Common Form of Melanoma

Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, have identified a link between inherited and acquired genetic factors that dramatically increase the chance of developing a very common type of melanoma. This finding appears in an online version of Science* on June 29, 2006, and was a collaborative effort led by scientists at NCI and the University of California San Francisco. Also involved in the study were researchers at the University of Pennsylvania, Philadelphia, and Bufalini Hospital in Cesena, Italy**.

“Knowing who is at greater risk for melanoma due to heredity, and understanding the pathways leading to cancer, are important steps in addressing a disease which is expected to be diagnosed in over 62,000 Americans in 2006,” said National Institutes of Health Director Elias A. Zerhouni, M.D. People with fair skin are generally at increased risk of developing melanoma. Differences in skin color, or pigmentation, are due largely to the melanocortin-1 receptor (MC1R) gene. Everyone has two copies of MC1R; one inherited from the mother and one from the father, and either can be the standard form or a variant. Some variant forms of MC1R are responsible for traits such as fair skin, freckling, and red hair. But MC1R may do much more than influence pigmentation.

“We previously observed that subjects who inherit one or two variant forms of the MC1R gene had a modest increase in risk of developing melanoma, even if they have darker pigmentation,” said Maria Teresa Landi, M.D., Ph.D., lead study investigator at NCI. “We have now discovered that MC1R dramatically predisposes individuals with no excessive sun exposure and variable pigmentation to developing a particular type of melanoma.”

Melanomas, which are tumors that arise from cells which produce skin pigment, can occur on all parts of the body where these cells are present. Caucasians have a much higher chance than other populations of developing these tumors on skin areas that are exposed to the sun. Sun exposure has many effects on skin, including causing chronic sun damage, with wrinkling on areas subject to high exposure over a lifetime. Sun exposure may also lead to mutations in cancer-causing genes, such as BRAF, which are frequent in melanoma.

According to Boris Bastian, M.D., University of California, San Francisco, “The relationship between BRAF mutations in melanoma and sun exposure is complex and intriguing. On the one hand, sun exposure appears necessary for development of BRAF mutations; melanomas on areas such as the soles of feet and palms of hands, which have low exposure, have low mutation frequencies compared to the approximately 60 percent mutation frequency in sun-induced melanomas on skin without chronic sun damage. On the other hand, melanomas developing in older subjects with sufficient accumulated sun exposure to produce chronic damage also exhibit lower BRAF mutation frequencies.”

Because melanomas on skin areas with few signs of chronic sun-induced damage occur in younger people and exhibit frequent mutations in BRAF, the researchers hypothesized that there were inherited genetic factor(s) that predispose to the development of these melanomas with BRAF mutations. An interesting candidate for this genetic risk factor was the MC1R gene.

To determine if there was an association between inherited variant forms of MC1R and the development of BRAF-mutant melanoma, the researchers studied the skin surrounding the melanomas in 85 patients from the Bufalini Hospital of Cesena, Italy, and 112 patients from the Department of Dermatology at the University of California, San Francisco, and identified subjects with no or little signs of chronic sun damage. They then sequenced MC1R genes in normal cells and BRAF in tumor cells and found that BRAF mutations were more frequent in non-chronic sun-induced melanoma cases with hereditary genetic variant forms of MC1R.

Landi MT, Bauer J, Pfeiffer RM, Elder DE, Hulley B, Minghetti P, Calista D, Kanetsky PA, Pinkel D, Bastian BC. MCIR Germline Variants Confer Risk for BRAF-Mutant Melanoma. Science, online edition, June 29, 2006.

** Departments of Dermatology and Pathology and Comprehensive Cancer Center, University of California, San Francisco; Department of Dermatology, Eberhard Karls University, Tübingen, Germany; Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pa.; Department of Dermatology, M. Bufalini Hospital, Cesena, Italy; Department of Laboratory Medicine, University of California, San Francisco; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pa.

By categorizing patients into two groups, those with no variant forms of MC1R versus those who had at least one variant, the scientists found that BRAF mutations were six to 13 times more frequent in those with at least one MC1R variant form. Looking more closely, the investigators found that the risk for melanoma with BRAF mutations rose with increasing number of MC1R variant forms. Comparing data from melanoma patients and healthy controls, the risk for melanomas with BRAF mutations increased from seven times for individuals with one MC1R variant form, to 17 times for those with two variant forms, when compared with individuals with the standard MC1R.

The study results show that normal variations in the MC1R gene in Caucasians have a very specific effect on melanoma susceptibility. Additional inherited factors that affect susceptibility may also be present, but they have yet to be discovered. “The mechanism by which variant forms of the MC1R gene facilitate development of melanomas with BRAF mutations is currently unknown,” said Landi. “One possibility is that people with MC1R variant forms and variable pigmentation generate more reactive chemicals in their cells as a result of the ultraviolet exposure in sunlight. These reactive chemicals can induce mutations, like those in the BRAF gene, which may lead to cancer.”

Clinical trials for melanoma using pharmaceutical drugs directed against the BRAF gene are ongoing. Knowledge of predisposing factors in the development of BRAF mutations, such as MC1R, might aid prevention and therapeutic strategies in the future.

For more information about cancer, please visit the NCI Web site at http://www.cancer.gov, or call NCI’s Cancer Information Service at 1-800-4-CANCER (1-800-422-6237).

The National Institutes of Health (NIH) — The Nation's Medical Research Agency — includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.