Glaucoma Association of Genetic Variants in the TMCO1 with Clinical Parameters Related to Glaucoma and Characterization of the in the Eye

Shiwani Sharma,1,9 Kathryn P. Burdon,1,9 Glyn Chidlow,2 Sonja Klebe,1,3 April Crawford,1 David P. Dimasi,1 Alpana Dave,1 Sarah Martin,1 Shahrbanou Javadiyan,1 John P. M. Wood,2 Robert Casson,2 Patrick Danoy,4 Kim Griggs,3 Alex W. Hewitt,5 John Landers,1 Paul Mitchell,6 David A. Mackey,5,7,8 and Jamie E. Craig1

PURPOSE. Glaucoma is the leading cause of irreversible RESULTS. The data suggest that individuals homozygous for the blindness worldwide. Primary open angle glaucoma (POAG) rs4656461 risk allele (GG) are 4 to 5 years younger at diagnosis is the most common subtype. We recently reported association than noncarriers of this allele. Our data demonstrate expres- of genetic variants at chromosomal loci, 1q24 and 9p21, with sion of the TMCO1 protein in most tissues in the human eye, POAG. In this study, we determined association of the most including the trabecular meshwork and retina. However, the subcellular localization differs from that reported in other significantly associated single nucleotide polymorphism (SNP) studies. We demonstrate that the endogenous protein localizes rs4656461, at 1q24 near the TMCO1 gene, with the clinical to the cytoplasm and nucleus in vivo and ex vivo. In the parameters related to glaucoma risk and diagnosis, and nucleus, the protein localizes to the nucleoli. determined ocular expression and subcellular localization of the human TMCO1 protein to understand the mechanism of its CONCLUSIONS. This study shows a relationship between genetic variation in and around TMCO1 with age at diagnosis of POAG involvement in POAG. and provides clues to the potential cellular function/s of this METHODS. Association of SNP rs4656461 with five clinical gene. (Invest Ophthalmol Vis Sci. 2012;53:4917–4925) DOI: parameters was assessed in 1420 POAG cases using linear 10.1167/iovs.11-9047 regression. The TMCO1 gene was screened for mutations in 95 cases with a strong family history and advanced disease. Ocular laucoma refers to a group of neurodegenerative ocular expression and subcellular localization of the TMCO1 protein Gdiseases united by a clinically characteristic optic neurop- were determined by immunolabeling and as GFP-fusion. athy. Open-angle glaucoma (OAG) is common, with a prevalence of approximately 3% in people older than 50 years.1 The disease has long been known to have a genetic From the Departments of 1Ophthalmology and 3Pathology, component. First-degree relatives of affected patients exhibit a Flinders University, Flinders Medical Centre, Adelaide, Australia; 9-fold increased relative risk of developing primary OAG 2South Australian Institute of Ophthalmology, Hanson Institute & (POAG) compared with the general population.2 The Glauco- 4 The University of Adelaide, Adelaide, Australia; The University of ma Inheritance Study in Tasmania (GIST) revealed that a Queensland Diamantina Institute, Princess Alexandra Hospital, 5 positive family history is found in approximately 60% of cases Brisbane, Australia; Centre for Eye Research Australia, University 3 of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, of POAG when family members are examined, and the disease Australia; 6Centre for Vision Research, Department of Ophthalmol- is more severe in people with a family history of glaucoma, ogy and Westmead Millennium Institute, University of Sydney, compared with those with ‘‘sporadic’’ glaucoma.4 Mutations in Westmead, Australia; 7Lions Eye Institute, University of Western the myocilin gene (MYOC) are the most common reported Australia, Centre for Ophthalmology and Visual Science, Perth, genetic cause of POAG and account for approximately 3% to Australia; and 8Discipline of Medicine, University of Tasmania, 5% of cases.5 Mutations in optineurin (OPTN) and WDR36 Hobart, Australia. have also been implicated.5 Recent genome-wide 9 These authors contributed equally to the work presented here association studies have identified common variants at several and should therefore be regarded as equivalent authors. new genetic loci are associated with POAG, including CAV1 Supported by National Health and Medical Research Council and CAV2 on 7q31; CDKN2B-AS1, CDKN2A, and (NHMRC) of Australia, Glaucoma Australia, and Ophthalmic Re- 6,7 search Institute of Australia. Support was also provided by the CDKN2B on 9p21; and TMCO1 on 1q24. The mechanism of NHMRC Career Development Award (KPB) and the Practitioner disease association is not yet clear for any of these genetic Fellowship (JEC). associations and very little is known about the normal function Submitted for publication November 9, 2011; revised March 27 of TMCO1 and its role in the eye. and May 27, 2012; accepted June 12, 2012. The TMCO1 gene encodes the Transmembrane and coiled- Disclosure: S. Sharma,None;K.P. Burdon,None;G. coil domains-1 protein which belongs to the DUF841 Chidlow,None;S. Klebe,None;A. Crawford,None;D.P. Dimasi, superfamily of unknown function.8 Thegeneislocated None; A. Dave,None;S. Martin,None;S. Javadiyan,None;J.P.M. approximately 6 Mb upstream of the known POAG gene Wood,None;R. Casson,None;P. Danoy,None;K. Griggs,None; MYOC on . The protein sequence is highly A.W. Hewitt,None;J. Landers,None;P. Mitchell,None;D.A. 9 Mackey,None;J.E. Craig,None conserved across mammalian species. The gene is expressed 9 Corresponding author: Shiwani Sharma, Department of Oph- in a variety of human adult and fetal tissues at varying levels, thalmology, Flinders University, GPO Box 2100, Adelaide 5001, including ocular tissues.6 Immunohistochemistry revealed Australia; shiwani.sharma@flinders.edu.au. cytoplasmic labeling in the rat retinal ganglion cell layer.6

Investigative Ophthalmology & Visual Science, July 2012, Vol. 53, No. 8 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. 4917

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The green fluorescent protein (GFP)-fusion of TMCO1 has provided written informed consent. Approval was obtained from the been reported to localize to endoplasmic reticulum and Golgi Human Research Ethics Committees (HRECs) of Southern Adelaide apparatus in COS7 cells,10 and to the mitochondria in porcine Health Service/Flinders University, University of Tasmania, and The PK-15 cells.8 These reports suggest that the subcellular Royal Victorian Eye and Ear Hospital. All participants were screened for localization of the protein may differ depending on cell type. mutations in the MYOC gene by direct sequencing of exon 3 on an ABI A homozygous frame-shift mutation in the TMCO1 gene has PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) been shown to cause a rare recessive syndrome in the Amish, with BigDye Terminators (Applied Biosystems) according to standard consisting of craniofacial dysmorphism, skeletal anomalies, and protocols. Primer sequences are available on request. mental retardation, now known as ‘‘TMCO1 defect syn- drome.’’9 There are no reports of glaucoma in this family. Genotyping and Association Analysis Our recent study failed to find any coding mutations in POAG patients carrying two risk haplotypes at the TMCO1 locus, Genotyping of the rs4656461 SNP was conducted as previously indicating that coding mutations in TMCO1 are unlikely to reported.6 Briefly, samples originally included in the discovery phase of account for the association observed with POAG.6 the genome-wide association scan were genotyped on Omni1 SNP The most associated single nucleotide polymorphism (SNP) arrays (Illumina Inc., San Diego, CA), whereas those included in the at the 1q24 locus (rs4656461) is just 30 to TMCO1 and the replication phase were typed with iPlex Gold chemistry on an Autoflex second ranked SNP rs7518099 is within intron 2 of this gene and Mass Spectrometer (Sequenom Inc., San Diego, CA). Each clinical trait is in almost complete linkage disequilibrium with rs4656461. In was assessed for association with SNP rs4656461 (the glaucoma- addition, the highest ranked imputed SNP, rs7524755 is within associated SNP tagging the at-risk haplotype as reported previously6) the 30 UTR of TMCO1.6 All the associated SNPs are within the using linear regression under a dominant genetic model. This model same linkage disequilibrium block as TMCO1 with no other was chosen to maintain statistical power because this SNP is relatively transcripts in the block. The common haplotype across the rare (minor allele frequency approximately 12%) and only 24 gene, which is efficiently tagged by the most associated SNP, homozygous patients were observed in our cohort. SNP genotypes increases the risk of POAG. This association was discovered in a were coded as carriers of the POAG risk allele (GA or GG genotype) cohort of POAG patients with severe blinding disease, and noncarriers (AA genotype), and analyzed in the model as a replicated in a second similar cohort and another cohort with categorical variable. The assumptions of linear regression were met. A less-severe glaucoma.6 From these data, however, it is as yet P value of 0.01 was required to account for the multiple testing of five unclear whether TMCO1 genetic variation can be used to traits (highest recorded IOP, age at diagnosis, mean deviation, cup:disc pinpoint a distinct subtype of POAG or if TMCO1 mutations ratio, and central corneal thickness). Significantly associated traits were might contribute to some cases of familial OAG. then assessed under multivariate analysis including each of the other In the present study, we further investigated the TMCO1 traits as well as sex in the model. Analyses were conducted including gene for association with clinical traits relevant to glaucoma and excluding patients with known MYOC mutations. All analyses were risk and diagnosis, and screened for mutations in patients with conducted using IBM-SPSS Statistics V19 (IBM Corp., Armonk, NY). a strong family history of POAG. In addition, we investigated the detailed expression pattern of TMCO1 in human eye and Sequencing of the TMCO1 Gene determined the subcellular localization of the protein in relevant cell lines and in vivo in the eye. Ninety-five participants from the ANZRAG and GIST with advanced POAG (defined as best-corrected visual acuity worse than 6/60 due to POAG, or a reliable 24-2 Visual Field with a mean deviation of worse than 22 db or at least 2 of 4 central fixation squares affected with a METHODS Pattern SD of less than 0.5%) and a strong family history of POAG were Patient Recruitment and Data Collection selected for re-sequencing of the protein coding regions of the TMCO1 gene. Family history was defined as three or more relatives diagnosed Participants were drawn from the Australian & New Zealand Registry of with glaucoma. This was self-reported in ANZRAG but determined by Advanced Glaucoma (ANZRAG), the GIST, the Blue Mountains Eye ophthalmic examination of extended families in GIST. Each exon and Study (BMES), and patients attending the eye clinic at Flinders Medical flanking intron of the TMCO1 gene was sequenced as previously Centre (Adelaide, Australia). All participants were included in the study described.6 Sequences were compared with the TMCO1 reference reporting the association of TMCO1 with POAG and the cohorts are sequence (GenBank Accession NM_019,026) using Sequencher described in more detail in that report.6 This cohort was also examined V4.10.1 (GeneCodes Corporation, Ann Arbor, MI). for genotype-phenotype relationships at another POAG locus on 11 chromosome 9p21 in an independent study. POAG was defined by Subcellular Localization concordant findings of typical glaucomatous visual field defects on the Humphrey 24-2 test (Humphrey 30-2 test was used in BMES), with A GFP-TMCO1 fusion construct was generated by cloning the open- corresponding optic disc rim thinning, including an enlarged cup-disc reading frame of the protein in pEGFP-C1 (Clontech Laboratories, Inc., ratio (‡ 0.7), or cup-disc ratio asymmetry (‡0.2) between the two Mountain View, CA). For this, TMCO1 cDNA was amplified from the eyes. Clinical exclusion criteria were pseudoexfoliation or pigmentary human retina as previously described6 and cloned in-frame with GFP at glaucoma; angle closure or mixed mechanism glaucoma; secondary EcoRI and XbaI sites in the vector. In-frame cloning was confirmed by glaucoma due to aphakia, rubella, rubeosis, or inflammation; infantile sequencing. Expression of the fusion protein was demonstrated in glaucoma; and glaucoma in the presence of a known associated transiently transfected HEK293A cells by Western blotting as previous- syndrome. Available clinical data were extracted from each study, and ly described,12 except that protein extraction was performed in included sex, age at diagnosis, highest recorded IOP, visual field mean radioimmunoprecipitation assay buffer (10 mM HEPES, pH7.5, 150 mM deviation, vertical cup:disc ratio, central corneal thickness and NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM genotype at SNP rs4656461. Where full clinical records were available, EDTA, Protease Inhibitor Cocktail [Roche Diagnostics Australia Pty Ltd., age at diagnosis was defined as the first occasion on which the patient Sydney, NSW, Australia], 57 lM phenylmethylsulfoxylfluoride, 2 mM demonstrated definitive glaucomatous visual field loss, as defined in the sodium orthovanadate, 10 mM sodium pyrophosphate, and 20 mM inclusion criteria. When this information was not available, the sodium fluoride). For subcellular localization, 3 3 105 SH-SY5Y human chronological age of the patient at the initiation of treatment for neuroblastoma cells were seeded onto glass coverslips in six-well tissue glaucoma (eye drops, laser, or surgery) was taken. All participants culture plates. The cells were cultured in a 1:1 mixture of Dulbecco’s

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modified Eagle’s medium and Ham’s F12 medium (GIBCO, Invitrogen TABLE 1. Clinical Characteristics of the Cohort Australia Pty Ltd., Mulgrave, Victoria, Australia) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, in a humidified Clinical Trait n Mean 6 SD or % (n) atmosphere at 378C and 5% CO . On the following day, the cells were 2 Highest recorded IOP, mm Hg 1275 26.5 6 10.0 transfected with either GFP-TMCO1 fusion construct or empty vector Age at diagnosis, y 1141 62.4 6 14.0 using Lipofectamine 2000 (Invitrogen Australia Pty Ltd.) as per the Mean deviation, dB 658 17.8 6 9.4 manufacturer’s protocol. Approximately 48 hours after transfection, Cup:disc ratio 1146 0.87 6 0.13 the cells were incubated with 1 lM BODIPY-TR-Ceramide (Molecular Central corneal thickness, lm 693 518 6 41 Probes, Invitrogen Australia Pty Ltd.) for 2 hours to label the Golgi Sex, % male 1389 46.6% (647) apparatus or with 0.25 lM MitoTracker Red CM-H2XRos (Molecular Probes) for 30 minutes to label mitochondria. After incubation, the cells were washed several times with PBS, fixed in 4% paraformalde- were hybridized with the rabbit anti-TMCO1 primary antibody (1:1000; hyde/PBS, and mounted on microscope slides in ProLong Gold antifade Sigma) followed by hybridization with the Alexa Flour 488 conjugated reagent with 40,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). anti-rabbit IgG secondary antibody (1:1000; Molecular Probes). For Confocal microscopy was performed on a Leica TCS SP5 inverted double labeling, the cells were hybridized with the anti-TMCO1 Spectral confocal microscope equipped with LAS AF software (Leica antibody and mouse antinucleolin monoclonal antibody (1:500; Microsystems Pty Ltd., North Ryde, Australia). Abcam). The anti-TMCO1 antibody was detected with Alexa Flour 488 conjugated anti-rabbit IgG secondary antibody and antinucleolin Immunolabeling with Alexa Fluor 594 conjugated anti-mouse IgG secondary antibody (1:500; Molecular Probes). The single- and double-labeled cells were For immunohistochemical labeling of the TMCO1 protein in human mounted, and confocal microscopy performed as described previously. eye, the eye tissue was obtained from deceased donors through the Eye Bank of South Australia, following the guidelines of the Southern Adelaide Health Service/Flinders University HREC. The tissue was RESULTS obtained within 12 hours of donor death and average donor age was 70 years. The tissue from donors without a history of ocular disease, such Characterization of the Glaucoma Phenotype as keratoconus, pterygium, refractive surgery, inflammation, tumor, Associated with TMCO1 Genetic Variants and glaucoma, was obtained. Specificity of the rabbit anti-human TMCO1 antibody (Sigma-Aldrich, Pty Ltd., Castle Hill, NSW, Australia) Genotype information for SNP rs4656461 and clinical charac- used for immunolabeling was demonstrated in human optic nerve by terization of the disease were available for 1420 individuals. Western blotting as previously described.13 For immunolabeling, the Descriptive statistics of each measured clinical trait are given in eye tissue was fixed in buffered-formalin and embedded in paraffin; 4- Table 1. For some individuals, the data for some of the clinical lm-thick paraffin-embedded sections of the human eye were immuno- traits was not available. Each clinical trait was assessed for labeled for the TMCO1 protein as previously described14 except for the association with SNP rs4656461 (Table 2). Age at diagnosis was following variations. After hybridization with the rabbit anti-human significantly decreased in carriers of the POAG risk allele (G) at TMCO1 primary antibody (1:1000), the sections were hybridized with this SNP (P ¼ 0.004). Vertical cup:disc ratio was increased (P ¼ the NovoLink Polymer complex reagent (Leica Microsystems, Ban- 0.017); however, this result did not survive Bonferroni nockburn, IL) and visualized by Chromogen substrate coloration correction for the five traits assessed and the effect size (Dako, Glostrup, Denmark). Sections were counterstained with estimate was small. Exclusion of 20 patients carrying hematoxylin and mounted in DePeX (Merck KGaA, Darmstadt, pathogenic mutations in the MYOC gene did not significantly Germany). Light microscopy was performed on an Olympus BX41 alter the results. microscope attached with a digital DP20/DP70 camera using CellSens To explore the relationship between SNP rs4656461 and Standard Photography software (Olympus Corporation, Tokyo, Japan). age at diagnosis further, multiple linear regression was conducted, including additional clinical covariates in the Double immunofluorescent labeling in human eye sections was model. Carriers of POAG risk alleles were diagnosed more performed as previously described.15 Visualization of nucleolin, coilin, than 3 years earlier than patients with no risk alleles at this and SC35 was achieved using a three-step procedure (primary locus ( 0.024, Table 3). Highest recorded IOP also antibody, biotinylated secondary antibody, streptavidin-conjugated P ¼ contributed significantly to the model with a higher IOP also AlexaFluor 594), whereas TMCO1 was labeled by a two-step procedure leading to a slightly younger age at diagnosis (0.26 years, P (primary antibody, secondary antibody conjugated to AlexaFluor 488). ¼ 0.0002). Exclusion of patients with MYOC mutations improved In brief, tissue sections were deparaffinized. Next, antigen retrieval was the significance of the observed association with age at achieved by microwaving the sections in 10 mM citrate buffer (pH 6.0). diagnosis. The B coefficient increased to 3.54 (P 0.011), Tissue sections were then blocked in PBS containing 3% normal horse ¼ indicating a slightly stronger relationship when these generally serum and subsequently incubated overnight at room temperature in young age at diagnosis patients were removed. In the the appropriate combination of primary antibodies. On the following multivariate analysis, the association with IOP was still day, sections were incubated with biotinylated anti-mouse secondary significant but slightly attenuated with the exclusion of MYOC antibody (1:250) for the three-step procedure plus the anti-rabbit secondary antibody conjugated to AlexaFluor 488 (1:250; Molecular Probes) for the two-step procedure for 30 minutes, followed by TABLE 2. Association of SNP rs4656461 with Each Clinical Trait under streptavidin-conjugated AlexaFluor 594 (1:500; Molecular Probes) for 1 a Dominant Model hour. Sections were then mounted using antifade mounting medium Clinical Trait B SE P Value and examined under a confocal fluorescence microscope. The following antibodies were used: rabbit anti-human TMCO1 Highest recorded IOP 0.07 0.60 0.908 (ARP49429; Aviva Systems Biology, San Diego, CA), mouse anti-human Age at diagnosis 2.56 0.88 0.004 nucleolin (ab13541; Abcam, Cambridge, MA), mouse anti-human coilin Mean deviation 0.58 0.77 0.465 (ab11822; Abcam), and mouse anti-SC35 (ab11826; Abcam). Cup:disc ratio 0.02 0.008 0.017 For immunolabeling of endogenous TMCO1 in SH-SY5Y cells, 3 3 Central corneal thickness 1.43 3.29 0.665 105 cells were seeded onto glass coverslips in six-well plates. Three days later, labeling was performed as previously described.12 The cells P < 0.01 is considered significant and is indicated in bold.

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TABLE 3. Multiple Regression for Age at Diagnosis pattern of the GFP-TMCO1 fusion was seen in SRA 01/04 lens epithelial cells (data not shown). Clinical Trait B SE P Value For determining expression and in vivo localization of TMCO1 in the human eye, immunolabeling was performed in Highest recorded IOP 0.26 0.07 0.0002 human ocular sections with the anti-TMCO1 antibody. Speci- Mean deviation 0.02 0.09 0.841 Cup:disc ratio 8.55 7.16 0.233 ficity of the antibody was demonstrated by detection of the expected approximately 21-kDa protein band in human optic Central corneal thickness 0.03 0.02 0.078 Sex 1.78 1.36 0.193 nerve by Western blotting (Fig. 2A). In human ocular sections, positive immunolabeling was observed in the iridocorneal rs4656461 G allele carriers 3.13 1.39 0.024 angle, trabecular meshwork, ciliary body, and retina (Fig. 2B). R2 ¼ 0.068. P < 0.01 is considered significant. In the trabecular meshwork, positive immunolabeling was primarily seen as 1 to 2 dots in the nucleus along with carriers (B ¼0.243, P ¼ 0.001). The mean age at diagnosis by cytoplasmic labeling (Figs. 2B-b, 2B-c). A similar pattern of genotype of rs4656461 is shown in Table 4. Consistent with nuclear labeling and cytoplasmic labeling was observed in cells the multivariate model, the mean age at diagnosis was in the ciliary body (Figs. 2B-d, 2B-e). In the retina, dot-like nuclear and positive cytoplasmic labeling was observed in the decreased 2 to 3 years for each POAG risk allele in the total photoreceptor, bipolar, and ganglion cell layers (Figs. 2B-f, 2B- cohort. This is also evident when MYOC carriers are excluded. g). Cytoplasmic labeling was more prominent in the photore- No significant association of rs4656461 with age at diagnosis ceptor outer segments and ganglion cells than in bipolar cells. was observed in the MYOC mutation carriers (ANOVA P ¼ These data suggest that the TMCO1 protein is expressed in 0.971). The borderline association with cup:disc ratio was not most ocular tissues and corroborate the previous finding of significant after inclusion of IOP, age at diagnosis, central TMCO1 transcript in these tissues.6 Cytoplasmic labeling corneal thickness (CCT), and sex in the model (P ¼ 0.066, data correlates with that previously reported by us in rat retinal not shown). ganglion cells.6 Nuclear and cytoplasmic labeling of the protein Of 320 carriers of the rs4546461 risk allele (G), 60% (n ¼ in the eye indicates that TMCO1 is likely a multifunctional 195) reported a family history of glaucoma. By comparison, protein with functions both in the cytoplasm and nucleus. only 51% of noncarriers (331 of 643) reported a family history. However, as all nuclear are produced in the cytoplasm This demonstrates an enrichment for family history of and then translocated to the nucleus, it is possible that the glaucoma among TMCO1 risk allele carriers (odds ratio ¼ cytoplasmic labeling of TMCO1 represents its production 1.47, 95% confidence interval [1.12–1.93], P ¼ 0.007). To rather than function in this compartment. identify heritable rare variants that may contribute to the POAG Next, we investigated the identity of the discrete subnuclear phenotype, 95 patients with a strong family history of the regions immunopositive for TMCO1 in the cells in ocular disease were selected for resequencing of the gene. Although tissues. We hypothesized that these subnuclear regions several previously reported SNPs, present in the general represent nucleoli, Cajal body, or nuclear speckles. Nucleolin population, were observed, no novel pathogenic mutations is a protein that frequently localizes to the nucleoli and is were detected in the protein coding regions of the gene in this therefore used as a nucleolar marker16; however, it is also subset of individuals. known to localize to the perinucleolar compartment.17 The coilin protein marks the Cajal body and SC35 the speckles in the nucleus.18 To determine whether TMCO1 localizes to the Subcellular Localization of TMCO1 and Expression nucleolus, Cajal body, or speckles in the cells in ocular tissues, in Mammalian Eye we individually assessed its colocalization with nucleolin, coilin, and SC35 proteins. Double labeling with the anti- To explore the subcellular localization of the TMCO1 protein TMCO1 and antinucleolin antibodies revealed colocalization of in cell lines relevant to the retina, GFP-TMCO1 fusion was TMCO1 and nucleolin in the nucleus to discrete subcellular transiently expressed in SH-SY5Y human neuroblastoma cells. regions in the cells in human ocular sections. Representative Before that, expression of the fusion protein by the fusion colocalization in the retinal ganglion cells can be seen in Figure construct in HEK293A cells was demonstrated by Western 3. The two proteins were also observed in the cytoplasm in blotting (Fig. 1A). In SH-SY5Y cells, the fusion protein was ocular cells where they colocalized with each other. However, found uniformly distributed in the cytoplasm, or concentrated double labeling with anti-TMCO1 and anticoilin antibodies and around the nucleus, or aggregated on one side of the nucleus with anti-TMCO1 and anti-SC35 antibodies revealed that (Fig. 1B and data not shown). However, labeling of these cells TMCO1 did not colocalize with coilin or SC35 in the nucleus with the Golgi apparatus or mitochondrial marker did not or cytoplasm (Fig. 3). These data indicate that subnuclear show any colocalization of the protein with either organelle TMCO1 in the cells in ocular tissues localizes to nucleoli. (Fig. 1B, top and middle panels). In GFP-expressing control Finally, we investigated whether endogenous TMCO1 in SH- cells, the protein distributed throughout the cell and, as SY5Y cells mimics in vivo localization in the human eye. expected, did not colocalize with the Golgi apparatus or Immunolabeling with the anti-TMCO1 antibody revealed mitochondria (Fig. 1B, bottom panels). A similar localization distribution of the protein in the cytoplasm and nucleus in a

TABLE 4. Mean Age (Years) at Diagnosis by rs4656461 Genotype

All Patients MYOC Carriers Non-MYOC Carriers

Genotype n Mean Age SD n Mean Age SD n Mean Age SD

AA 769 63.3 14.1 11 46.3 19.4 758 63.5 13.9 AG 351 60.9 13.7 5 48.6 14.8 346 61.0 13.6 GG 21 58.7 14.4 2 47.0 8.5 19 59.9 14.5 The G allele at this SNP is the risk allele for glaucoma. Analysis on MYOC mutation carriers and noncarriers is also shown.

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FIGURE 1. (A) Fusion protein encoded by the GFP-TMCO1 construct. Cell lysates from HEK 293A cells transiently transfected with the fusion construct (GFP-TMCO1) or pEGFP-C1 vector (GFP) were analyzed by Western blotting with the anti-GFP antibody. The band of approximately 50 kDa seen in the TMCO1-GFP lane corresponds with the expected size of the fusion protein and that of approximately 28 kDa in the GFP lane corresponds with the size of GFP. The greater than 28-kDa protein band seen in the TMCO1-GFP lane may represent fusion protein degradation. Molecular sizes of the protein standards are indicated in kilodaltons. (B) GFP-TMCO1 fusion localization in SH-SY5Y human neuroblastoma cells. Cells transiently transfected with the fusion construct (GFP-TMCO1) or pEGFP-C1 vector (GFP) (green channel) were either stained with BODIPY- TR-Ceramide for labeling the Golgi apparatus (Golgi) or MitoTracker Red CM-H2XRos for labeling mitochondria (Mito) (red channel), nuclei counterstained with DAPI (blue channel), and visualized by confocal microscopy. GFP-TMCO1 can be seen in the cytoplasm (green). However, in overlayed images from the three channels (Merge) it neither colocalizes with the Golgi apparatus nor mitochondria. In cells expressing GFP as a control, the protein distributed in the nucleus and cytoplasm.

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FIGURE 2. (A) Endogenous TMCO1 protein in human optic nerve. Protein lysate from optic nerve tissue were analyzed by Western blotting with the rabbit anti-TMCO1 antibody. The approximately 21-kDa band corresponds with the expected size of the TMCO1 protein. Molecular sizes of the protein standards are indicated in kilodaltons. (B) Immunolabeling of TMCO1 in the human eye. Paraffin sections of human eye were immunolabeled with the anti-TMCO1 antibody and imaged by light microscopy. Positive immunolabeling can be seen as red signal in the (a) iridocorneal angle, (b, c) trabecular meshwork, (d, e) ciliary body, and (f, g) retina. Nuclei are stained blue. Pigment in panels a, c, and e is marked by the letter ‘‘p.’’ Immunolabeling of TMCO1 as an intense spot in the nucleus can be seen in cells in the trabecular meshwork (c), ciliary body (e), and retina (g). Cytoplasmic immunolabeling is also visible in these tissues. Images are, a at 310; b, d, f at 340; and c, e, g at 3600, original magnification. The presented results are representative of experiments on eyes from four independent donors.

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FIGURE 3. Immunolabeling of TMCO1 with nucleolin, coilin, and SC35 in human retina. Confocal microscopy was performed on sections immunolabeled with anti-TMCO1 (green) antibody together with either antinucleolin, anticoilin, or anti-SC35 antibodies (red). Nuclei (blue)were stained with DAPI. Images from the three color channels overlayed (merge) for determining colocalization. Top row, TMCO1 localized to discrete regions in the nucleus colocalizes with nucleolin (highlighted by arrows). Middle row, TMCO1 does not colocalize with coilin (green and red separated in merged image). Bottom row, TMCO1 does not colocalize with SC35 (green and red distinct in merged image). Scale bar: 5 lm.

punctate fashion in these cells (Fig. 4, bottom left panel). The gene is not well elucidated and it is not yet known how this signal was stronger in the nucleus compared with the gene contributes to glaucoma. This study has shown that cytoplasm. In addition, in a small proportion of cells, instead POAG patients carrying the glaucoma risk alleles at SNP of being evenly distributed in the nucleus, the protein was rs4656461 tend to be diagnosed with the disease several years concentrated to discrete subnuclear regions as seen in vivo in earlier than patients without these alleles. However, this study the eye (Fig. 4, top left panel). To determine if these TMCO1- was not able to show association of rs4656461 with IOP, positive subnuclear regions represent nucleoli, we assessed cup:disc ratio, mean deviation, or central corneal thickness. colocalization of TMCO1 with nucleolin in SH-SY5Y cells. Thus, apart from a slightly earlier onset, TMCO1-related Double labeling with the anti-TMCO1 and antinucleolin glaucoma does not appear to be a clinically distinct subtype antibodies revealed TMCO1 localization as described previous- of POAG. These SNPs may be severity factors for POAG, which ly, and localization of nucleolin to discrete subnuclear regions could influence the age at onset of disease. This idea is (Fig. 4, second column). In cells with subnuclear localization of consistent with our previous work showing an increased odds TMCO1, the protein showed complete colocalization with ratio for association in patients with blinding POAG compared nucleolin in some cells (Fig. 4, top row), as observed in vivo, with patients with less severe POAG.6 In patients with a family and exhibited incomplete colocalization in other cells (Fig. 4, history of advanced glaucoma, there may be a tendency for middle row). In cells where TMCO1 was evenly distributed in increased monitoring and thus earlier diagnosis (pseudo- the nucleus, the protein did not colocalize with nucleolin (Fig. anticipation). This phenomenon may contribute to the current 4, bottom row). Double labeling with anti-TMCO1 and findings, given the observation of an increased family history in anticoilin and with anti-TMCO1 and anti-SC35 antibodies risk allele carriers. This is a limitation of our approach; showed that TMCO1 does not colocalize with coilin or SC35 however, the same association with age of diagnosis was not 11 in SH-SY5Y cells (data not shown). From these data, TMCO1 observed at the CDKN2B-AS1 gene in this same cohort. Thus, appears to shuttle between the cytoplasm and nucleus and we believe pseudo-anticipation does not have a major affect on transiently localize to the nucleolus or the perinucleolar the current findings. compartment. MYOC glaucoma is also well known to have a younger age of onset, but within this small group of patients, the TMCO1 gene does not appear to be modifying this risk, although the DISCUSSION power of this analysis is minimal in this cohort with a limited number of participants carrying both MYOC mutations and Genetic variation in and around the TMCO1 gene at 1q24 has TMCO1 risk alleles. The inclusion of patients with MYOC been shown to be associated with POAG.6 The function of this mutations in the current analysis also does not account for the

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FIGURE 4. Immunolabeling of TMCO1 and nucleolin in human SH-SY5Y neuroblastoma cells. Confocal microscopy was performed on cells immunolabeled with the anti-TMCO1 (green) and antinucleolin (red) antibodies. Nuclei (blue) were stained with DAPI. Images from the three color channels overlayed (merge) for determining colocalization. Top row, TMCO1 localized to discrete regions in the nucleus colocalizes with nucleolin (yellow in merged image). Middle row, TMCO1, although localized to discrete regions in the nucleus, does not completely colocalize with nucleolin (green and red somewhat separated in merged image). Bottom row, TMCO1 evenly distributed in the nucleus does not colocalize with nucleolin (green and red distinct in merged image). Images were taken with a 363 objective. Brightness and contrast of images in the bottom row were enhanced for improving visibility of the cytoplasmic signal.

younger age of diagnosis observed in TMCO1 risk allele in-depth and functional analysis of the locus is required to carriers. The methods used for the collection of age at determine this. diagnosis data do have some limitations. Where complete As an initial step toward functional analysis of the TMCO1 clinical records were not available to confirm the age of gene, we determined subcellular localization of the encoded definitive glaucomatous visual loss, self-reported age at protein and protein expression in the human eye. Expression treatment initiation was used as a surrogate. However, it is of the protein in all the ocular tissues is consistent with its unlikely that any bias introduced by this limitation would be reported ubiquitous expression.9 Localization of the GFP- distributed differently across genotypes. Nevertheless, replica- TMCO1 fusion in cultured mammalian cells in this study tion of the observed association in independent POAG cohorts contradicted the previous reports. Iwamuro et al.10 reported is required to confirm the current findings. localization of the human TMCO1-GFP fusion to the endoplas- By re-sequencing of 95 patients with a strong family history mic reticulum and Golgi apparatus in COS-7 cells but did not of POAG, coding variants do not appear responsible for the determine colocalization with the organelle-specific markers. disease phenotype in these families deemed likely to have Zhang et al.,8 however, reported localization of the GFP fusion highly penetrant dominant mutations. This is not surprising of porcine TMCO1 to mitochondria in PK-15 porcine kidney given that the amino acid sequence of this protein is cells and colocalization with the mitochondrial marker. In this completely conserved among all mammals investigated to study, we observed neither colocalization of human GFP- date.9 This implies a crucial role for this ubiquitously expressed TMCO1 with the endoplasmic reticulum and Golgi apparatus, protein. The absence of coding variants in POAG patients in nor mitochondria in cultured human neuroblastoma and lens this study suggests genetic variation in regulatory elements epithelial cells. In addition, the fusion protein did not may be responsible for the observed association signal. Further colocalize with the Golgi apparatus or distribute to the

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endoplasmic reticulum in COS-7 cells under our experimental 4. Wu J, Hewitt AW, Green CM, et al. Disease severity of familial conditions (data not shown), which further contradicts glaucoma compared with sporadic glaucoma. Arch Ophthal- findings in these cells by Iwamuro et al.10 Furthermore, we mol. 2006;124:950–954. observed cytoplasmic and nuclear localization as inclusions of 5. Allingham RR, Liu Y, Rhee DJ. The genetics of primary open endogenous TMCO1 in the human ocular tissues. In SH-SY5Y angle glaucoma: a review. Exp Eye Res. 2009;88:837–844. neuroblastoma cells also, endogenous TMCO1 was found in 6. Burdon KP, Macgregor S, Hewitt AW, et al. Genome-wide the cytoplasm and nucleus and in some cells as nuclear association study identifies susceptibility loci for open angle inclusions. Together, our data suggest that GFP-TMCO1 fusion glaucoma at TMCO1 and CDKN2B-AS1. Nat Genet. 2011;43: localizes differently from endogenous TMCO1. This may be 574–578. because TMCO1 protein is smaller than GFP (approximately 21 7. Thorleifsson G, Walters GB, Hewitt AW, et al. Common vs. 28 kDa); thus, the latter leads to aberrant localization of the variants near CAV1 and CAV2 are associated with primary former or due to overexpression of the fusion protein. Grossly open-angle glaucoma. Nat Genet. 2010;42:906–909. different localization patterns of the GFP fusion and endoge- 8. Zhang Z, Mo D, Cong P, et al. Molecular cloning, expression nous protein seen in this study highlight that ectopically patterns and subcellular localization of porcine TMCO1 gene. expressed GFP fusions may not always reflect biological Mol Biol Rep. 2010;37:1611–1618. localization of the protein of interest. 9. Xin B, Puffenberger EG, Turben S, Tan H, Zhou A, Wang H. The functional significance of the cytoplasmic localization Homozygous frameshift mutation in TMCO1 causes a syn- of endogenous TMCO1 is as yet unclear. However, the drome with craniofacial dysmorphism, skeletal anomalies, and conspicuous subnuclear localization in vivo in the human mental retardation. Proc Natl Acad Sci U S A. 2010;107:258– eye and ex vivo in some SH-SY5Y cells, and colocalization with 263. nucleolin is very interesting. Nucleolin, the major component 10. Iwamuro S, Saeki M, Kato S. Multi-ubiquitination of a nascent of the nucleolus, also localizes to the perinucleolar compart- membrane protein produced in a rabbit reticulocyte lysate. J 17,19 ment, nucleoplasm, and the cell surface. The nucleolus is Biochem. 1999;126:48–53. conventionally involved in ribosome biogenesis but also has 11. Burdon KP, Crawford A, Casson RJ, et al. Glaucoma risk alleles nonconventional roles, including regulation of tumor suppres- at CDKN2B-AS1 are associated with lower intraocular pres- sor and oncogene activities, nuclear export, and control of sure, normal-tension glaucoma, and advanced glaucoma 20 aging. The perinucleolar compartment is associated with [published online ahead of print April 20, 2012]. Ophthal- 17 malignancy. Interestingly, the CDKN2B gene and the mology. doi:10.1016/j.ophtha.2012.02.004 antisense RNA gene CDKN2B-AS1 recently associated with 12. Sharma S, Ang SL, Shaw M, et al. Nance-Horan syndrome POAG by us and others are involved in tumor suppression and 6,21 protein, NHS, associates with epithelial cell junctions. Hum cell-cycle regulation. From the present data, it is not Mol Genet. 2006;15:1972–1983. unreasonable to speculate that TMCO1 may also play a role 13. Burdon KP, Sharma S, Hewitt AW, et al. Genetic analysis of the in tumor suppression and/or cell-cycle regulation. Further- clusterin gene in pseudoexfoliation syndrome. Mol Vis. 2008; more, involvement of the nucleolus in controlling aging and 14:1727–1736. association of the risk allele of SNP rs4656461 near the TMCO1 gene with earlier age at diagnosis of glaucoma raise the 14. Sharma S, Chataway T, Burdon KP, et al. Identification of possibility of a role of this gene in aging. In our view, this gene LOXL1 protein and Apolipoprotein E as components of surgically isolated pseudoexfoliation material by direct mass is likely involved in cell cycle regulation in glaucoma. In spectrometry. Exp Eye Res. 2009;89:479–485. conclusion, the present study provides clues to the potential cellular function(s) of TMCO1 and shows a relationship 15. Chidlow G, Ebneter A, Wood JP, Casson RJ. The optic nerve between genetic variation in and around this gene with age head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure at diagnosis of POAG. It also suggests that SH-SY5Y cells are a in a rat model of glaucoma. Acta Neuropathol. 2011;121:737– useful model for investigating function. Further TMCO1 751. longitudinal studies will investigate whether TMCO1 genotyp- ing can predict severity or progression of glaucoma. 16. Ginisty H, Sicard H, Roger B, Bouvet P. Structure and functions of nucleolin. J Cell Sci. 1999;112:761–772. 17. Pollock C, Huang S. The perinucleolar compartment. Cold References Spring Harb Perspect Biol. 2010;2:a000679. 1. Mitchell P, Smith W, Attebo K, Healey PR. Prevalence of open- 18. Sleeman JE. Dynamics of the mammalian nucleus: can angle glaucoma in Australia. The Blue Mountains Eye Study. microscopic movements help us to understand our genes? Ophthalmology. 1996;103:1661–1669. Philos Transact A Math Phys Eng Sci. 2004;362:2775–2793. 2. Wolfs RC, Klaver CC, Ramrattan RS, van Duijn CM, Hofman A, 19. Mongelard F, Bouvet P. Nucleolin: a multiFACeTed protein. de Jong PT. Genetic risk of primary open-angle glaucoma. Trends Cell Biol. 2007;17:80–86. Population-based familial aggregation study. Arch Ophthalmol. 20.OlsonMO,HingoraniK,SzebeniA.Conventionaland 1998;116:1640–1645. nonconventional roles of the nucleolus. Int Rev Cytol. 2002; 3. Green CM, Kearns LS, Wu J, et al. How significant is a family 219:199–266. history of glaucoma? Experience from the Glaucoma Inheri- 21. Ramdas WD, van Koolwijk LM, Lemij HG, et al. Common tance Study in Tasmania. Clin Experiment Ophthalmol. 2007; genetic variants associated with open-angle glaucoma. Hum 35:793–799. Mol Genet. 2011;20:2464–2471.

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