Mapping the Primary Open Angle Glaucoma Gene(s)

Glaucoma is one of the leading causes of blindness in the United States. With over one million people affected, understanding the mechanism of this disease is imperative. Because many individuals with glaucoma report additional family members who are affected, there appears to be a gene or genes that cause glaucoma. This proposal seeks to identify this gene(s). Our hypothesis is that the gene causing certain types of glaucoma is part of the filter that regulates the pressure inside the eye. The most likely component of the filter to do this is a protein called a “proteoglycan”. This protein has special properties that allow it to bind water and regulate the flow through the filter. In addition, if this protein is not replaced on a fairly regular basis, the old protein will, itself, plug up the filter. Thus, we hypothesize that a proteoglycan gene may be the defect in one of the most common forms of glaucoma, called primary open-angle glaucoma.

Specific Aim 1: Determine if one of the trabecular proteoglycans is a candidate ghene for primary open-angle glaucoma. Microsatellites associated with each of the trabecular proteoglycan genes will be tested for cosegregation with glaucoma in families in which glaucoma is inherited as an autosomal dominant trait. We have identified several specific proteoglycans that make up this filter. These include syndecan, versican, perlecan, decorin and biglycan. The role of these proteoglycans in glaucoma will be explored by using a technique called “linkage analysis”. This technique follows the inheritance of markers associated with these proteoglycans. If a certain marker is always inherited with glaucoma in a certain family, there is a high likelihood that the proteoglycan associated with the marker is the glaucoma gene. If these specific genes are ruled out, we will still have gained information on the location of the glaucoma gene. Essentially, this technique will map ihe glaucoma gene to a certain region on a physical structure of DNA, called a chromosome. Each cell has 46 chromosomes. Linkage analysis will be used to determine if one of these chromosomes contains the glaucoma gene. We will be able to rule out regions on each chromosome as sites for the glaucoma gene. If one of the markers of the proteoglycans is shown to be inherited with glaucoma, we will have mapped glaucoma to the site of that marker on a specific chromosome. If the marker is ruled out, then we can rule out that region on that particular chromosome. Thus, we will have gained specific information about the location of the glaucoma gene.

Specific Aim 2: Identify families from Dr. Samples Glaucoma clinic in which glaucoma clearly is inherited. Examine willing individuals from glaucoma families for the presence or absence of glaucoma and collect blood samples. Our second specific aim is to look at additional families in which glaucoma is inherited. By gathering blood samples and establishing which individuals have glaucoma, we can further our linkage analysis. These families will allow us to determine if the map site or gene found in the first family is a common one that is found in other families. If it is the common one, we will have found one of the major gene(s) causing glaucoma. If the glaucoma gene in these families maps to a different site then we will have found evidence for more than one gene that causes glaucoma.

Specific Aim 3: If the above proteoglycans are ruled out as candidate genes in these families, examine additional trabecular extracellular matrix proteins for cosegregation with glaucoma. If the genes discussed in specific aim 1 are ruled out as glaucoma gene, our third specific aim will be to look at additional genes that are likely candidates. These genes encode other proteins that make the filter described above. A similar rationale, as in specific aim 1, will be used. We will follow the glaucoma gene through our families and see if it is inherited with one of the filter proteins. Since we know the location of these filter proteins on the chromosomes, ruling in or out one of these proteins will further define our map of the glaucoma gene.

Our long term goal is to identify the genes responsible for the inherited forms of glaucoma. Glaucoma is a complex disease, thus it is likely that there is more than one gene responsible for glaucoma. Even in the one form of glaucoma, which we are concentrating our work on (primary open-angle glaucoma), there is probably more than one type. By testing many families in which primary open-angle glaucoma is inherited, we will be able to determine if more that one gene is responsible for this form of glaucoma. Ultimately, we will be able to define the glaucoma gene and the mutation(s), which cause glaucoma.

2nd Year

Primary open-angle glaucoma is one of the most common blinding diseases in the world. About 10% of people affected with glaucoma have inherited it from one of their parents. By identifying the gene that causes glaucoma in these families, we may gain a better understanding of what causes glaucoma in people who have no family history. The exact cause of glaucoma has yet to be determined but it is generally accepted that glaucoma results from impaired outflow of the aqueous humor in the eye. That is a filter that normally would allow the aqueous humor to flow is now obstructed and pressure builds up in the eye. This in turn leads to damage to the optic nerve from the increased pressure inside the eye. The filter which becomes defective is most likely the extracellular matrix of the trabecular meshwork. Our working hypothesis is that in families in which glaucoma is clearly inherited, a defective trabecular extracellular matrix protein is responsible. Moreover, we hypothesize that there will probably be more than one gene that may cause glaucoma. That is, glaucoma is a heterogeneous group of disorders, one glaucoma family may have a defect in one gene, while a second glaucoma family will have a defect in a different gene.

Specific Aim 1: Determine if one of the trabecular proteoglycans is a candidate gene for primary open-angle glaucoma. Microsatellites associated with each of the trabecular proteoglycan genes will be tested for cosegregation in families in which glaucoma is inherited as an autosomal dominant trait. To find the glaucoma gene, the first step is to map its position on one of the 23 chromosomes that are inherited from one’s parents. A new technique has recently emerged which is much quicker and more efficient for mapping genes. This technique combines the polymerase chain reaction and microsatellite markers. Microsatellite markers are specific fingerprints for specific chromosome regions. These markers can be used to determine which chromosome was inherited from each parent. If a particular chromosome or part of a chromosome is always passed down to a person with glaucoma, then the chance of that particular chromosome carrying the glaucoma gene is very high. The proteoglycan genes may very likely be involved in glaucoma. Since the chromosome location of these genes is known, the microsatellite markers associated with these proteoglycan genes can be used to determine if these regions are always inherited with the glaucoma gene. If they are not, then that specific proteoglycan is probably not involved in glaucoma. We have looked at four proteogl ycan genes and there is no evidence for linkage of these genes with glaucoma in our first family. Thus, in this family none of these four genes is likely to be the glaucoma gene. We have also looked at two other families and at this point in time we cannot rule out three of the genes, versican, decorin, and link protein in these two families.

Specific Aim 2: Identify additional families from Dr. Samples’ Glaucoma clinic in which POAG is clearly inherited. Examine willing individuals from glaucoma families for the presence or absence of clincial signs of glaucoma and collect blood samples or skin biopsies. We have identified 11 families in which glaucoma is inherited as an autosomal dominant trait. To date over 129 individuals from these families have been examined. Blood samples or skin biopsies have been obtained from each of these individuals after informed consent forms have been signed. Twenty-seven of these individuals have adult-onset glaucoma. Eight are suspects, i.e., they carry one risk factor for glaucoma such as high intraocular pressure. One of the families has juvenile glaucoma with five affected members. This is a rare form of glaucoma associated with dramatically shortened height and probably represents a variant of Weill-Marchesani syndrome. Three of these families have been used in our linkage analysis as described in specific aims 1 and 3. These are the families that we have extended out the farthest at this time. Their pedigrees are included in the appendix. In order to bring in individuals from family 1, we made two separate field trips to Bend and a field trip to Eugene. From these three field trips we were able to collect blood from 49 family members and Dr. Samples examined 51 individuals. Altogether from family 1 we have obtained blood samples from 65 individuals. Of these seven have glaucoma and five are suspects. The spouses are also asked to participate, including being examined by Dr. Samples, since the incidence of glaucoma is fairly high in the population. Interestingly, the wife of one of the family members was diagnosed by Dr. Samples as having glaucoma and she informed us that her brother, mother and maternal aunt also have glaucoma. Unfortunately, her family is in Australia, so we won’t be able to follow up on them at this time. From family 2, we have obtained 18 blood samples. Dr. Samples has examined fourteen of the individuals. Two of the great-aunts of the proband also have glaucoma. Although they live in California, we were able to obtain blood samples from them and are in the process of obtaining their medical records. In this family there are at least eight affected members and one suspect. We are continuing to follow up on this family since there are at least fifteen potentially informative individuals that we can add to the pedigree. Dr. Samples and I will be making a field trip in January or February to Salem, Oregon to examine and collect blood samples from the six daughters of individual 94050, who has glaucoma. The daughters range in age from 34 to 50 years old. Family 3, although a relatively small family, has been very compliant. We have essentially brought in all of the individuals in the Northwest area and are continuing to contact additional family members who live elsewhere in the United States. To date we have obtained blood samples from 20 individuals, five of whom have glaucoma and one, who is a suspect.

Specific Aim 3: If the above proteoglycans are ruled out as candidate genes in these families, examine additional trabecular extracellular matrix proteins for cosegregation with glaucoma. While the proteoglycans are the most likely genes to be involved in glaucoma, there are additional proteins present in the filter of the trabecular meshwork that may be involved in glaucoma. These proteins include collagens, integrins, and elastin. The same technique used in specific aim 1 has been used to determine if these proteins could potentially cause glaucoma. Because this technique is fairly rapid, instead of concentrating just on the chromosomes to which the above proteins have been assigned, we have extended our work to eventually cover all 22 of the chromosomes that are passed on by both the mother and father. Over 15 chromosomes have been analyzed using microsatellite markers to determine if the glaucoma gene is associated with any of these markers. At this time chromosome 19 appears as the most likely region for the glaucoma gene in our three families. Statistical analysis on chromosome 19 is presently being run to determine if the findings are statistically significant. This will be extended to additional families as we bring them in.