BY GEAROID TUOHY PHD
BIO-OPHTHALMOLOGY
                                   Gene hunters score significant victory in battle against AMD

After an epic journey involving 15 million base pairs of DNA, 70 genes, and five years of toil, an American research team has announced the identity of one of the first "AMD genes." The research group identified "hemicentinin-1" as the smoking gun responsible for AMD in a large family afflicted by the disorder for at least four generations.The discovery, by researchers in the Casey Eye Institute at the Oregon Health & Science University, adds to a growing body of evidence that AMD, age-related macular degeneration, represents a hereditary condition. The finding should now pave the way to a better understanding of the disorder that afflicts one-in-four people over the age of 65 years. In addition, the discovery should permit identification of pre-symptomatic individuals through a relatively simple diagnostic test. Eventually, such research findings should lead to the introduction of viable treatments for AMD. In AMD, vision loss results from the death of photoreceptor cells in the central cone-rich macula responsible for fine photopic vision.

In 1998, the same research group, which is led by Dennis Schultz, reported that it had mapped an area of DNA on human chromosome that was associated with AMD in the same large family. Having further narrowed the suspect DNA to a stretch of 15 million bases, the research group focused in on 20 candidate genes. Each gene needed to be screened for mutations after which only one DNA variation - a switch from adenine to guanine at base number 16,263 - was found to be exclusively present in affected individuals. The mutation caused an amino acid change from glutamine to arginine in the hemicentin-1 protein. Examination of this sequence in eight other species indicated that such a change would most likely affect protein function, further suggesting the role of hemicentinin-1 in AMD pathology.

The research finding represents a combination of hard work, scientific skill and good judgment in determining which genes to screen. The trick in gene hunting is to reduce the vast number of potential genes that could be causing the disorder to a manageable few. This is achieved by recording both clinical and laboratory data to make associations between genetic markers in a person's DNA and the absence or presence of the disease.

A genetic marker is the equivalent of a grid reference along a motorway. As the human genetic sequence is three billion bases long, it is important to have a road map of reference points before a researcher jumps in the car. Genetic markers represent a series of reference points allowing the researcher to know relative distances between the various markers. If marker "ABC" consistently shows up in the genetic samples from afflicted patients but not in people without a disease, then a researcher may tentatively deduce that there is a relationship between the marker ABC and the disease.

This is precisely the form of analysis employed by the research team at the Casey Eye Institute. In their search, the researchers knew that the gene was somewhere along human chromosome 1. Further refinements of this "guilt by association" approach eventually led the researchers to a stretch of DNA approximately 15 million bases in length. At that point, the researchers used their experience, scientific skill and good judgement to pick out the most likely genes in this region that might be associated with a disease like AMD.

In choosing such genes, the research team gave priority to: genes that are known to be expressed in the retina; genes that have a similarity to genes known to cause retinal disorders; and genes that express proteins potentially related to drusen formation.

With the candidates narrowed to 20 genes, the researchers began the arduous task of mutation screening, which required the sequencing of each gene and the comparison of the mutation with its "normal" copy. Although the researchers found 49 variations, they found only one that turned out to be consistently associated with individuals who had the disease. This variation, known as "A16263G", in the hemicentin-1 gene, changed the amino acid composition of the corresponding protein.

More often than not, mutations in the primary DNA sequence have little or no impact on the biology of the gene. However, on occasion, a single base change such as A16263G, may change the protein derived from that gene in a fundamental manner. The mutation found in this study caused arginine rather than glutamine to be inserted into the hemicentin-1 protein. To check how significant of a change this might be, the research team examined the equivalent stretch of DNA from eight different species; they found glutamine to be conserved in all cases. The more often an amino acid is conserved across different species, the more likely it is that the amino acid is critically important at that position for the correct functioning of the protein. In addition, the change from glutamine to arginine caused by the mutation changes both the size and electrostatic charge of the amino-acid side chain of the hemicentin-1 protein; therefore, the protein's structure and function are highly likely to be affected.

Once the mutation was identified, the research group screened 1,378 individuals for the A16263G mutation. These 1,378 individuals were either members of families with AMD or from a pool of sporadic AMD cases or control subjects. Among this group, eleven individuals with the mutation were identified, seven of whom had AMD.

Examinations of the pedigree of this large family are consistent with a dominant mode of inheritance for AMD. The A16263G mutation may cause pathology in a variety of ways; one of the next steps in the research process is to learn how this primary mutation leads to the death of photoreceptor cells.

Now that the gene hunters have identified the mutation, the challenge now turns to the "gene fixers". With a clear understanding of hemicentin-1's molecular pathology, it may be quite possible to devise a treatment approach to intervene in the biology of the disease and correct the underlying malfunction. Most likely, one of the first experiments will be to genetically engineer an animal model with the disease to contain the A16263G mutation in the hemicentin-1 gene. Such a model would allow researchers to observe the pathology and biology of disease progression with the ultimate goal of finding a treatment to correct the condition.

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