Jeffrey D. Esko, Ph.D. University of California, San Diego
Hereditary multiple exostosis (HME) is a dominant genetic disorder resulting in the formation of generally benign cartilage-capped outgrowths from various bones. Recent work from a number of laboratories suggests that the disease correlates with mutations in EXT genes involved in making a complex sugar, or polysaccharide, called heparan sulfate. Since heparan sulfate interacts with many factors involved in cell growth, this discovery may shed light on the cause of the disease, which in turn may suggest new treatments. These recent discoveries have tweaked the interest of scientists from seemingly unrelated fields.
To appreciate these recent findings, an explanation of heparan sulfate, EXT, and the genetic basis of HME is in order. When we say that HME is a dominant genetic disorder, we mean that the clinical syndrome is caused by a genetic change (mutation) in a gene that is usually passed from one parent to their afflicted child. Using genetic tricks, scientists have located the mutations associated with HME to one of three related genes, called EXT1, EXT2 and EXT3. The first two genes (EXT1 and EXT2) have been cloned, which means that we know the actual sequence of the DNA that makes up the EXT1 and EXT2 genes. The sequence of these genes acts as a blueprint for making specific proteins in cells. When geneticists talk about mutations in EXT1 and EXT2, they actually mean changes in the blueprint that cause differences in the proteins encoded by these genes. Since proteins are made of amino acids, the mutations that correlate with the exostosis actually affect the amino acid sequence of the protein.
The EXT1 and EXT2 genes are shared by humans, other mammals (e.g., mice and hamsters) and even simple organisms (e.g., flies and worms). The wide distribution of EXT's suggested that the encoded proteins are fundamentally important for cells. Indeed, EXT proteins act as important catalysts (many proteins do this). These particular catalysts, also called enzymes, make it easier to form a chemical bond between special types of sugars, called N-acetylglucosamine (GlcNAc for short, pronounced glicknack) and glucuronic acid (GlcA for short, sometimes pronounced glickA). GlcNAc and GlcA combine in an alternating pattern to form a polymer, in this case a polysaccharide called heparan sulfate (pronounced hep-ah-ran sulfate).
The first connection between heparan sulfate and the EXT genes was made by Frank Tufaro and his group at the University of British Columbia. Tufaro was studying Herpes simplex virus which he and other workers had shown to require heparan sulfate to infect human and rodent cells. Using a genetic trick, Tufaro identified a gene that was required for viral infection. When he sequenced the gene he found it to be identical to EXT1. Simultaneously, Ulf Lindahl and his group in Uppsala, Sweden purified the enzyme required to make heparan sulfate and showed that it was identical to EXT2. My group at the University of California at San Diego had previously identified mutant hamster cells unable to make heparan sulfate, and using this new information we found that all of our mutant cells contained mutations in EXT1. Many of the human mutant EXTs, when studied in rodent cells, are unable to catalyze the GlcNAc and GlcA reactions, which firmly established a connection between HME and loss of the ability to make heparan sulfate.
Individuals afflicted with HME have many different mutations in EXT1 and EXT2 (so-called etiologic mutations, since they are thought to cause the disease). If you look at the layout of the EXT1 gene, the mutations occur at several different locations, with some more common than others. Many of the mutations in the hamster cell EXT1 occur in similar places.
One idea is that the EXT proteins act like an assembly line, where station A does the GlcNAc step and station B does the GlcA step. It turns out that many of the mutations affect only one of the "substations" and not the other. From a biochemist's perspective, the mutations provide insight into the inner workings of the EXTs. The results also infer that mutations in either reaction can cause disease.
Since the EXT mutations block the ability of our cultured hamster cells to make heparan sulfate, we presume that they have the same effect in the cells found in cartilage and bone where the exostoses occur. However, this relatively simple idea has not yet been confirmed, since it is difficult to obtain the appropriate cells and tissues from patients. Working with The MHE Coalition, Jacqui Hecht a the University of Texas Health Sciences Center at Houston has recently obtained cells from surgical specimens of exostoses. Hopefully, we will soon know whether the chondrocytes make less heparan sulfate and if they have less GlnNaC and GlcA transferase activity. Since the EXT mutations should be expressed in other cells as well (e.g., in blood cells), we may be able to develop other types of diagnostic procedures based on biochemical analyses, in addition to the genetic analyses already being performed.
If heparan sulfate is made by virtually all cells in the body, why do EXT mutations only affect the cartilage and bone? We do not know the answer to this question, but the solution may lie in how heparan sulfate is thought to act in cell growth, maturation and differentiation, the process by which one cell can turn into many different types of cells. Heparan sulfate is known to interact with a large number of specialized proteins that tell cells when to divide and when to change into other types of cells. Conceivably, the decrease in heparan sulfate caused by the EXT mutations creates a block in the normal flow of information, resulting in loss of control over the cartilage-producing cells. Why this effect is so restricted to bone and cartilage is unclear and needs additional research. One approach to studying this problem is to try to make similar kinds of mutations in mice, which is underway in collaboration with Daniel Wells' laboratory at the University of Houston.
As in all research, each step forward brings more questions. As the pace of research in this area increases, new biochemical and genetic insights into the nature of the disease should emerge. Although no one can guarantee a cure for HME based on these recent findings, continued research in this area will undoubtedly bring us closer to the cause and potential control of the disease.
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