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. 2011 Jun 15;20(12):2482-94.
doi: 10.1093/hmg/ddr123. Epub 2011 Mar 29.

Copy Number Variations on Chromosome 12q14 in Patients With Normal Tension Glaucoma

Free PMC article

Copy Number Variations on Chromosome 12q14 in Patients With Normal Tension Glaucoma

John H Fingert et al. Hum Mol Genet. .
Free PMC article


We report identification of a novel genetic locus (GLC1P) for normal tension glaucoma (NTG) on chromosome 12q14 using linkage studies of an African-American pedigree (maximum non-parametric linkage score = 19.7, max LOD score = 2.7). Subsequent comparative genomic hybridization and quantitative polymerase chain reaction (PCR) experiments identified a 780 kbp duplication within the GLC1P locus that is co-inherited with NTG in the pedigree. Real-time PCR studies showed that the genes within this duplication [TBK1 (TANK-binding kinase 1), XPOT, RASSF3 and GNS] are all expressed in the human retina. Cohorts of 478 glaucoma patients (including 152 NTG patients), 100 normal control subjects and 400 age-related macular degeneration patients were subsequently tested for copy number variation in GLC1P. Overlapping duplications were detected in 2 (1.3%) of the 152 NTG subjects, one of which had a strong family history of glaucoma. These duplications defined a 300 kbp critical region of GLC1P that spans two genes (TBK1 and XPOT). Microarray expression experiments and northern blot analysis using RNA obtained from human skin fibroblast cells showed that duplication of chromosome 12q14 results in increased TBK1 and GNS transcription. Finally, immunohistochemistry studies showed that TBK1 is expressed in the ganglion cells, nerve fiber layer and microvasculature of the human retina. Together, these data link the duplication of genes on chromosome 12q14 with familial NTG and suggest that an extra copy of the encompassed TBK1 gene is likely responsible for these cases of glaucoma. However, animal studies will be necessary to rule out a role for the other duplicated or neighboring genes.


Figure 1.
Figure 1.
NTG pedigrees. Family members with NTG are indicated with black symbols, whereas those who did not meet diagnostic criteria for glaucoma but were considered to have unknown glaucoma status because of their age are indicated with gray symbols. Those family members with unknown glaucoma status because they were unavailable for examination are indicated with gray symbols and asterisks. (A) Pedigree 441. Members of this African-American pedigree with NTG who were included in the linkage studies are indicated with the symbol ‘×'. (B) Pedigree 458. Members of this Caucasian pedigree with NTG who contributed DNA samples are indicated with the symbol ‘×'. All affected family members in both pedigrees were found to carry their family's chromosome 12q14 duplication.
Figure 2.
Figure 2.
CNV analysis of NTG patients. Members of Pedigree 441 with NTG were investigated for CNVs by analyzing SNPs that were typed as part of the linkage analysis, by genome-wide CGH studies and by focused chromosome 12q14 CGH studies. The extent of the duplication detected in Pedigree 441 by these methods is represented by a black box. Similar analyses revealed overlapping but unique duplications in two unrelated NTG patients (Fig. 1B, Pedigree 458, IV-1 and Subject 1159-1). The extent of the duplications in these subjects is also represented by black boxes. The positions of known genes encompassed by these duplications are represented by gray boxes.
Figure 3.
Figure 3.
TBK1 expression in the human retina. RNA from human retina was used as a template in RT-PCR assays designed for genes encompassed by the chromosome 12q14 duplication in Pedigree 441 (TBK1, XPOT, RASSF3, GNS) and the control gene rhodopsin (RHO). Each assay used primers located entirely within neighboring exons and spanned an intron. Production of appropriate-sized amplicons demonstrated that each gene is expressed in the human retina. Control lanes with genomic DNA produced larger amplicons due to the included intron sequence.
Figure 4.
Figure 4.
Northern blot analysis of TBK1. Expression of TBK1 in patients with the chromosome 12q14 duplication (n = 7) was compared with expression in subjects without the duplication (n = 5) by northern blot analysis of RNA collected from fibroblast cells cultured from skin biopsies. Total RNA from fibroblast cells was first probed with a TBK1 cDNA probe. The blot was then stripped and re-probed with a β-actin (ACTB) clone. Probing with the TBK1 clone produced a single band that was quantified using the IMAGE software package and normalized to expression level of ACTB. When compared with control subjects, expression of TBK1 in patients with the chromosome 12q14 duplication was elevated 1.48-fold (P = 0.015).
Figure 5.
Figure 5.
Immunohistochemistry. (A) Representative immunohistochemical labeling of TBK1 in human donor retina shows localization to the retinal ganglion cells and axon bundles (arrows), as well as to the retinal microvasculature (asterisks). (B) No primary antibody negative control. The layers of the retina are indicated by abbreviations: NFL, nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium; CC, choriocapillaris. Some minor peroxidase reactivity is present within intravascular erythrocytes in the ‘no-primary antibody' control panel.

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