Normal Tension Glaucoma

A Family-based Approach to Find Normal Tension Glaucoma Genes

Project Summary. Glaucoma often runs in families. In rare cases, a large number of family members have normal tension glaucoma that is passed down from generation to generation in an obvious pattern. We have enrolled several such families with normal tension glaucoma into our research program.

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This is a pedigree or family tree of a family affected with normal tension glaucoma. The squares represent male family members while the circles represent females. The family members with black symbols have glaucoma while those with white symbols don’t have glaucoma.

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We are studying normal tension glaucoma families to find the genes that cause their eye disease using a technique called linkage analysis. We collect DNA from family members and type each family member at tens of thousands of genetic markers that are scattered across the genome. By comparing the pattern of inheritance of the genetic markers to the pattern of inheritance of glaucoma in the family, we can map the position of the gene that causes the family’s disease to a small region of the genome.

We searched for the gene that causes glaucoma in the family shown above using a stepwise approach described below. We have published two reports describing this research that showed that TBK1 gene is a new gene that causes glaucoma in this and other families. The scientific reports about this research can be viewed here (initial report and confirmatory report).

We scanned the whole genome of the family shown above and found that a segment of chromosome 12 was always co-inherited with glaucoma. This experiment showed that the gene causing glaucoma in this family is located on a small region of chromosome 12 that spanned over 200 genes. This first step in our studies narrowed our search for a glaucoma gene from the whole genome (25,000+ genes) down to around 200.

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We tested this part of chromosome 12 for large structural abnormalities (deletions of DNA or duplications of DNA) and we detected a large 700,000 bp duplication. This duplication was found in all of the family members with glaucoma and was never seen in people with normal eyes. This second step in our studies further narrowed our search for a glaucoma gene down to only four genes! The chromosome 12 duplication in this family spanned only four genes, exportin (XPOT), TANK binding kinase 1 (TBK1), Ras associated domain family member 3 (RASSF3), and glucosamine (N-acetyl)-6-sulfatase (GNS).

We tested a group of 152 additional normal tension patients from Iowa and 252 patients from Japan for similar chromosome 12 duplications. We found three more patients with overlapping chromosome 12 duplications - each of them different. Two of the glaucoma patients with the chromosome 12 duplications are Caucasian, one patient is African American, and another patient is Asian. When the extent of the chromosome 12 duplications in each patient is compared (green and blue boxes in the figure to the right), there is a small region of 300,000 base pairs of DNA and 3 genes that are duplicated in all of these patients (shown as red boxes in the figure). This part of our research further narrowed our search down to just three candidate genes for causing glaucoma in our families (XPOT, TBK1, and RASSF3).

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When we searched the world's literature on these three candidate genes, it became clear that the TBK1 gene was the best bet of the three for causing glaucoma in our families. The evidence supporting TBK1 as the glaucoma gene in these families is listed below:

• TBK1 is already known to interact with others genes that causes low pressure glaucoma, optineurin (OPTN) and toll-like receptor 4 (TLR4). This makes TBK1 guilty by association.

• TBK1, OPTN, and TLR4 all produce proteins that participate in autophagy – a process by which cells breakdown and recycle their components. Autophagy may sometimes cause cells to self-destruct (apoptosis) and may be involved in the way the optic nerve is damaged in glaucoma

We studied the activity of TBK1 in normal eyes. We found that TBK1 is most active in the tissues of the eye that are affected by low pressure glaucoma - the retinal ganglion cell layer and its projections which make up the optic nerve. These parts of the human retina are shown in the picture to the left. We used antibodies to label TBK1 protein with a purple stain. The pattern of purple labeling shows that TBK1 is active in the right part of the eye to cause nerve damage and glaucoma.


We have strong evidence that an extra copy of the TBK1 gene causes normal tension glaucoma in some families.

• TBK1 was found to be duplicated in several families with normal tension glaucoma.
• TBK1 interacts with the only other known normal tension glaucoma gene (optineurin)
• TBK1 is active in the right tissues of the eye to cause glaucoma

However, we are conducting additional studies to gather the most definitive proof that an extra copy of the TBK1 gene leads to normal tension glaucoma. We have engineered a strain of mice that have an extra copy of the TBK1 gene - one of these "transgenic" mice is shown to the right. We are currently examining these mice to see if the extra copy of TBK1 in their genome leads to disease of the optic nerve and development of glaucoma.

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Acknowledgement: The discoveries described above were made possible by the clinical skills and thoughtful research contributions of Dr. Alan L. Robin in Baltimore and Drs. Wallace L.M. Alward, Robert F. Mullins, and Young H. Kwon at the University of Iowa.

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