Human Molecular Genetics

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Human Molecular Genetics

Pages 2221-2228

©1999 Oxford University Press

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A cellular assay distinguishes normal and mutant TIGR/myocilin protein
Introduction
Results
   Normal TIGR/myocilin is Triton soluble whereas mutant protein is not
   The difference in Triton solubility between mutant and normal protein is immediate
   Triton insolubility is a general characteristic of mutant TIGR/myocilin proteins
   Human->murine amino acid substitutions in the olfactomedin-like domain of human TIGR/myocilin yield Triton-soluble proteins
   Test of ambiguous variants
Discussion
   Comparison of assay results to prior inferences about ambiguous variants
   Relationship of glaucoma phenotype to degree of Triton insolubility
   Implications for a model of glaucoma pathogenesis
Materials And Methods
   DNA constructs
   Cell culture and transient expression of TIGR/myocilin in HEK cells
   Triton X-100 extraction
   Analysis of TIGR/myocilin by SDS-PAGE and immunoblot
Acknowledgements
References

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A cellular assay distinguishes normal and mutant TIGR/myocilin protein

Zhaohui Zhou, Douglas Vollrath⁺

Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Lane Building, Room L305, Stanford, CA 94305-5120, USA

Received June 28, 1999; Revised and Accepted August 13, 1999

Glaucoma is a blinding eye disease that affects ~70 000 000 people world-wide. Mutations in the gene TIGR/MYOC have been shown to cause the most common form of the disease, primary open angle glaucoma, in selected families. Amino acid sequence variants of the gene have been found in 2-4% of sporadic primary open angle glaucoma cases. Most variants are rare and it is often difficult to definitively distinguish between a deleterious mutation and a benign variant solely on the basis of relative frequencies in patient and control groups. The function of the TIGR/myocilin protein is unknown and an assay to functionally classify variants is lacking. We sought to develop a biochemical assay to distinguish different forms of TIGR/myocilin. We investigated the Triton X-100 detergent solubility characteristics of mutant and normal forms of the protein, expressed by transfection in cultured cells. We observed a clear difference in the behavior of the two types of TIGR/myocilin; all confirmed mutant proteins tested were substantially Triton insoluble, while normal protein and controls were completely soluble. We also tested seven ambiguous variant proteins and classified them as mutant or normal on the basis of their Triton solubility. The results in some cases validated, and in other cases contradicted, earlier classifications of these variants. To our knowledge, Triton solubility is the first example of a general difference in the properties of mutant and normal forms of TIGR/myocilin. The assay we have developed will be useful for discerning protein functional information from the location of mutations, will aid genetic counseling of individuals with TIGR/myocilin variants and may provide a clue to understanding a mechanism by which mutations in TIGR/MYOC cause glaucoma.

INTRODUCTION

Glaucoma is an eye disorder that is the second leading cause of blindness world-wide after cataract (1). The most common form of glaucoma is primary open angle glaucoma (POAG). It is characterized by degeneration and cupping of the optic nerve, a characteristic pattern of visual field loss and increased intraocular pressure (IOP) in a majority of cases. The age at diagnosis for POAG ranges from <10 years to >70 years, with a majority of cases occurring after the age of 40. A number of genes have been implicated in the pathogenesis of various forms of glaucoma (2-9). Of these genes, only mutations in the gene TIGR/MYOC have been shown to cause a POAG phenotype, accounting for 2-4% of cases (4,10-13). Most mutations in TIGR/MYOC result in dominant early onset POAG, typified by high IOP.

The function(s) of the TIGR/myocilin protein and the mechanism(s) by which mutations in the gene cause glaucoma are not known. The gene was initially named trabecular meshwork-inducible glucocorticoid response (TIGR), on the basis of a high level of induction of mRNA and protein following dexamethasone treatment of human trabecular meshwork (HTM) cells (14). Kubota et al. (15) independently identified the gene and named it myocilin (MYOC) because sequence analysis revealed similarity to Dictyostelium discoideum myosin and the protein appeared to localize to the connecting cilium of retinal photoreceptor cells. The region of myosin similarity is predicted to form a coiled-coil structure (14,15) and there is a potential N-terminal signal sequence, consistent with evidence that the protein is secreted from glucocorticoid-treated HTM cells (14). Most glaucoma-causing mutations affect residues in the C-terminal half of the protein in a putative domain that shares similarity with bullfrog olfactomedin (15,16), an extracellular matrix protein of unknown function that is an abundant constituent of the frog olfactory neuro-epithelium (17). Sequence comparisons indicate that the olfactomedin-like domain is phylogenetically conserved to nematodes (18,19).

A catalog of mutant forms of TIGR/MYOC may provide clues to the protein's normal function(s) and the mechanism(s) by which mutations cause glaucoma. Extensive screening of TIGR/MYOC in glaucomatous and normal individuals has revealed many missense variants and a handful of premature termination and frameshift mutations (4,10-13,18,20-30). Some of the missense variants have been shown to co-segregate with a POAG phenotype in families with many affected members, providing statistical evidence for a causative role of these particular mutations in glaucoma pathogenesis. However, a majority of TIGR/MYOC missense variants are present at a low frequency among glaucoma patients or control groups and have not been studied in large families. In such cases, a conclusive distinction between a disease-causing mutation and a benign variant has not been possible because of the small number of normal or affected individuals identified who harbor a particular sequence change. The inability to distinguish between deleterious and benign variants not only degrades efforts to infer protein functional information from the location of mutations, it also creates a dilemma for the genetic counseling of individuals with rare TIGR/MYOC variants because it is unclear whether such individuals are at increased risk for developing POAG. An assay is needed to differentiate normal and mutant forms of TIGR/myocilin. In this report, we describe such an assay and use it to assess some of the ambiguous rare missense variants in TIGR/MYOC that have been previously classified as probable disease-causing mutations or probable benign sequence changes.

RESULTS

To study the characteristics of normal and mutant forms of TIGR/myocilin, we introduced DNA constructs encoding epitope-tagged versions of the protein (Fig. 1) into the human embryonic kidney (HEK) cell line 293T by liposome-mediated transient transfection. In the course of these studies, we noticed that mutant forms of the protein seemed to be less soluble than normal protein when extracted from cells with the non-ionic detergent Triton X-100. Triton extraction is commonly used to partition cellular proteins and many cytoskeletal proteins are relatively insoluble in Triton (31,32). We adapted a Triton extraction protocol used to study mutant and wild-type forms of the merlin protein (33,34) to the study of TIGR/myocilin, in order to determine whether the apparent difference in Triton solubility of normal TIGR/myocilin and its mutated counterparts was general and reproducible.

Figure 1. TIGR/myocilin structure and DNA constructs. The primary amino acid structure of human TIGR/myocilin is depicted above with the location of various domains and putative functional or structural regions indicated. A cassette encoding 10 amino acids that included the FLAG epitope tag was inserted immediately after codon 35 of the TIGR/myocilin cDNA. Starting with this epitope-tagged normal TIGR/myocilin construct, site-directed mutagenesis was employed to make 21 separate variants in the olfactomedin-like domain (black vertical lines).

Normal TIGR/myocilin is Triton soluble whereas mutant protein is not

The Pro370Leu mutant of TIGR/myocilin is associated with a phenotype of especially early onset POAG (average age at diagnosis ~12 years), accompanied by high IOPs in affected individuals (average maximum IOP >35 mm Hg) (18,21,26,35). This aggressive glaucoma phenotype has been observed in six Caucasian families of northern European origin (12,18,21, 24,26) and one Japanese family (10), leading to the suggestion that Pro370Leu is a strong mutant allele of TIGR/MYOC (12,21).

We compared the Triton solubility of the Pro370Leu mutant with normal TIGR/myocilin. DNA constructs encoding FLAG epitope-tagged (36) versions of the two proteins were separately transfected into HEK cells. After 2 days, cells were extracted with a buffer containing 0.5% Triton X-100. The soluble and insoluble fractions were concentrated and equivalent amounts were analyzed by SDS-PAGE and immunoblotting.

We observed a clear difference in the behavior of normal and Pro370Leu mutant proteins; nearly all of the normal epitope-tagged TIGR/myocilin is soluble, while the Pro370Leu mutant protein remains almost completely in the insoluble pellet (Fig. 2A). Comparable results have been obtained in more than 20 replications of this experiment and similar results were obtained when the same DNA constructs were introduced into HeLa, NIH 3T3 or COS7 cells (Fig. 2B), demonstrating that the phenomenon is not cell-type specific. TIGR/myocilin endogenously expressed by HEK cells is soluble, like epitope-tagged normal protein (Fig. 2A).

Figure 2. Triton X-100 extraction of normal and Pro370Leu mutant forms of TIGR/myocilin. (A) Aliquots of 0.2 µg of DNA constructs encoding FLAG epitope-tagged versions of normal or mutant TIGR/myocilin protein were transfected into HEK cells by lipofection and a Triton extraction procedure was carried out as described in Materials and Methods. Triton-soluble (S) and -insoluble (I) proteins were detected by immunoblot with the anti-FLAG monoclonal antibody, M2. Endogenous TIGR/myocilin was detected with an affinity-purified polyclonal anti-peptide antibody directed against residues 33-47. (B) The procedure in (A) was repeated for COS7, NIH 3T3 and HeLa cells. About 5-fold longer exposure times were required for the NIH 3T3 and HeLa samples along with 1 µg of each DNA construct. The doublet of protein bands observed in (A) and (B) results from N-glycosylation; treatment with N-glycosidase eliminates the upper band (data not shown).

The difference in Triton solubility between mutant and normal protein is immediate

To assess the kinetics of insolubility, we performed Triton extraction at various times after transfection of DNA constructs encoding normal or Pro370Leu mutant TIGR/myocilin. Normal and mutant proteins were detectable by immunoblot as early as 36 and 24 h after transfection, respectively (Fig. 3A). The difference in Triton solubility of the two types of protein at the earlier times is apparent and indistinguishable from that seen later, an indication that a high protein concentration within cells is not required for mutant insolubility. In fact, when large amounts of DNA were transfected into HEK cells to intentionally overproduce Pro370Leu protein, the proportion of soluble mutant protein increased (Fig. 3B). In contrast, normal TIGR/myocilin remains almost completely soluble over a similar range of DNA concentrations (data not shown).

Figure 3. Time course and DNA titration of Triton solubility of TIGR/myocilin in HEK cells. (A) Parallel transfections were carried out in 6-well culture dishes for normal and Pro370Leu mutant FLAG-tagged DNA constructs and individual wells were extracted with Triton X-100 at 12 h intervals for 72 h. Protein concentration was measured with a DC Protein Assay kit (Bio-Rad, Hercules, CA) and equivalent amounts of protein for each time point were analyzed. Proteins were detected by immunoblot with the M2 antibody. (B) Three different amounts of the Pro370Leu mutant DNA construct were transfected in individual wells of a 6-well dish and proteins were processed after 45 h.

Triton insolubility is a general characteristic of mutant TIGR/myocilin proteins

To investigate whether Triton insolubility is a general property of glaucomatous forms of TIGR/myocilin, we made DNA constructs for 11 additional TIGR/MYOC mutants, all of which affect the olfactomedin-like domain of the protein. The 11 mutations were chosen because their causative role in POAG has been firmly established (Table 1). Constructs were separately transfected into HEK cells and Triton extraction was performed for each mutant protein after 2 days (Fig. 4). Immunoblot analysis showed that most mutant proteins behaved like Pro370Leu in that they were almost completely insoluble and two mutant proteins (Gly364Val and Asp380Ala) were substantially insoluble. When compared with the nearly complete and highly reproducible solubility of normal TIGR/myocilin, these results indicate that Triton insolubility is a characteristic of bona fide glaucomatous mutant TIGR/myocilin proteins.

Figure 4. Triton insolubility of 11 familial glaucoma mutants of TIGR/myocilin. An additional 11 TIGR/myocilin mutants, for which there is strong evidence of a causative role in glaucoma (Table 1), were tested in the Triton extraction procedure after transfection into HEK cells. The figure is a composite of four experiments. A normal control was included in each experiment. An aliquot of 0.2 µg of DNA was used for each construct and exposure times varied from 10 to ~30 s.

Table 1. Comparison of genetic and biochemical evidenc for 19 TIGR/MYOC variants

Mutation	Genetic evidencea	Reference	Insoluble protein
Pathogenic mutations
Glu323Lys	3.75 (0)	21,37	+
Gly364Val	3.5 (0.05)	11	+
GlyGln367-368Val	>3 (0.01)	29	+
Gln368Stop	Higher frequency in probands	11,22	+
Pro370Leu	8.38 (0); 3.92 (0)	21,38; 18,35	+
Asp380Ala	5.48 (0)	27	+
Lys423Glu	6.62 (0)	25,39	+
Val426Phe	3.52 (0)	21,40	+
Tyr437His	13.8 (0)	11	+
Ile477Asn	11.6 (0); 6.42 (0)	11; 28	+
Ile477Ser	6.21 (0)	18,41	+
Asn480Lys	>20 (0)	30	+
Ambiguous variants
Glu352Lys	48-year-old carrier; 6/1693 probands, 0/238 normals	12; 13	-
Thr377Met	1.3 (0.2)	11	+
Lys398Arg	20/1693 probands, 4/238 normals	13	-
Arg422His	1/716 probands, 0/91 normals	11	-
Arg422Cys	0/716 probands, 1/505 in general population	11	+
Val495Ile	Small family with Thr377Met	11	-
Ile499Phe	Family with 7 affecteds and 2 carriers	18	+

^aAvailable LOD scores are given, followed by [Theta] in parentheses.

Human->murine amino acid substitutions in the olfactomedin-like domain of human TIGR/myocilin yield Triton-soluble proteins

Murine TIGR/myocilin and its human ortholog are 81% identical (42-44), consistent with a conservation of function between the murine and human proteins. If the insolubility that we observe is specific for glaucomatous mutant TIGR/myocilin proteins and not simply the result of substitutions in the olfactomedin-like domain, then changing an amino acid residue in human TIGR/myocilin to that found in the murine protein should not affect the solubility of human TIGR/myocilin. As a test of this hypothesis, we made DNA constructs encoding two human->murine substitutions in the olfactomedin-like domain of human TIGR/myocilin. One of the substitutions (Glu409Ala) results in a change in net electrostatic charge of the protein, while the other (Ala386Ser) results in a change in polarity of a residue. Of a total of 21 probable disease-causing mutations, classified on the basis of an apparent increased frequency among glaucoma patients as compared with controls, 17 resulted in a change in charge, polarity or size of a residue and were termed `non-conservative' (13). By this definition, both human->murine substitutions are non-conservative changes.

DNA constructs encoding the two TIGR/myocilin variants were transfected into HEK cells and Triton extraction was performed. Immunoblot analysis showed that neither of the human->murine substitutions had an effect on the solubility of otherwise normal human TIGR/myocilin; both proteins were almost completely soluble (Fig. 5). These controls provide further evidence that Triton insolubility is a characteristic of mutant forms of TIGR/myocilin found in familial glaucoma and indicate that the Triton extraction procedure that we have developed can be used as a biochemical assay to distinguish normal and mutant forms of TIGR/myocilin found in POAG patients, normal controls and/or the general population.

Figure 5. Triton solubility of human->murine substitutions in the olfactomedin-like domain of TIGR/myocilin. Site-directed mutagenesis was used to replace a residue in epitope-tagged human TIGR/myocilin with the corresponding amino acid encoded by the murine gene and the Triton extraction procedure was carried out after transfection into HEK cells. Normal and Pro370Leu mutant DNA constructs were included as controls.

Test of ambiguous variants

Seven TIGR/MYOC missense variants for which strong evidence for or against a causative role in glaucoma is not available (Table 1) were tested in the Triton extraction assay. Of the seven variants, three (Thr377Met, Arg422His and Ile499Phe) were previously described as probable disease-causing mutations, while three (Lys398Arg, Arg422Cys and Val495Ile) were thought unlikely to be pathogenic mutations. A seventh variant (Glu352Lys) has been described as a probable mutation (13) or as a probable benign sequence change (11,12). Results of the Triton extraction assay are consistent with previous predictions for four of the six variants (Fig. 6). Results for two of the variants are at odds with previous predictions; Arg422Cys, a putative normal variant, behaves like a mutant, demonstrating substantial insoluble protein, while Arg422His, a putative disease-causing mutation, yields a completely soluble protein. The solubility of the Glu352Lys variant protein is indistinguishable from normal TIGR/myocilin.

Figure 6. Triton solubility of ambiguous variants of TIGR/myocilin. A total of seven TIGR/myocilin missense variants, for which strong evidence for or against a causative role in glaucoma is not available, were tested in the Triton extraction assay after transfection into HEK cells. The three variants to the left were previously described as probable mutations (see text), while the three to the right were thought to be benign sequence changes. The Glu352Lys variant has been described at different times as mutant or benign. We tested five of the ambiguous variants (Glu352Lys, Thr377Met, Lys398Arg, Arg422His and Val495Ile) in COS7 cells and obtained results similar to those shown.

DISCUSSION

More than 40 amino acid sequence variants of TIGR/myocilin have been reported (4,10-13,18,20-30). Most are missense variants identified in a small number of individuals and present at an overall population frequency well below 1% and most appear to be population specific (13). It is therefore difficult to conclusively classify many of the variants as pathogenic mutations or benign sequence changes. We have shown that substantial insolubility in Triton X-100 is an attribute of mutant forms of TIGR/myocilin expressed through transfection of HEK cells; all 12 bona fide mutant proteins tested displayed substantial Triton insolubility, while normal TIGR/myocilin and controls were soluble. These observations serve as the basis for a general and reproducible cellular assay that distinguishes between mutant and normal forms of TIGR/myocilin.

Comparison of assay results to prior inferences about ambiguous variants

Seven ambiguous variant proteins were tested in the assay and classified as mutant or normal. The Lys398Arg variant was found at a frequency of ~1% among both glaucoma probands and control subjects and presumed to be a probable benign polymorphism (11,13). Our results demonstrating Triton-soluble Lys398Arg protein confirm this conclusion. A compound variant allele of Val495Ile and Thr377Met was found in two members of a single glaucoma family (11), while Thr377Met alone was found in two other families (11,12), one of which was large enough to generate suggestive, but not conclusive, statistical support for a causative role in glaucoma (11). The Val495Ile variant was presumed to be benign (11) and our assay results substantiate this conclusion as well as the conclusion that Thr377Met is a pathogenic mutation. We also tested the compound variant and found no difference in solubility between it and the Thr377Met variant (data not shown). The Ile499Phe variant has been observed in seven affected and two unaffected members of the same family (18). One of the carriers is 6 years older than the oldest affected member. The Triton insolubility of this variant supports the conclusion that it is a glaucoma-causing mutation and suggests that the oldest carrier represents a case of reduced penetrance.

Our assay results are at odds with previous predictions for two variants at the same amino acid position in TIGR/myocilin. The Arg422His variant, previously classified as a probable disease-causing mutation (11), behaves like the normal protein and we infer that it is a benign sequence change with no causative role in glaucoma. This variant was present in only one of 716 glaucoma probands tested and not found in a smaller number (596) of controls (11). In light of the genetic heterogeneity of glaucoma and its relatively high population prevalence, we suppose that the POAG manifest by this individual arises from causes unrelated to the Arg422His variant. The Arg422Cys variant, described previously as unlikely to be a pathogenic mutation (11), behaves like a mutant protein in our assay and is probably a disease-causing mutation. The mutation was identified in a single individual from the general population whose glaucoma status was not known (11). We predict that this individual is at increased risk for developing POAG as a result of a mutation in TIGR/MYOC.

The Glu352Lys variant was previously classified as both a probable benign sequence change (11,12) and as a probable disease-causing mutation (13). Our results indicate that Glu352Lys is a benign sequence change. The variant was found in one of four affected members and one 48-year-old unaffected member of an African-American family with adult onset POAG (12). Interestingly, at least three of six other glaucoma probands reported to carry the variant are also of African origin (13,45). It is therefore likely that this variant represents a benign change that is found predominantly among Africans (12) and that it was not detected among normal controls because of the smaller number of African controls tested (13).

Together, our results for the seven ambiguous variants demonstrate the utility of the assay to differentiate between rare glaucoma-causing mutations and benign sequence changes. All of the variants tested fell within the C-terminal 200 amino acids of TIGR/myocilin, where ~90% of putative mutations have been found. It remains to be seen whether the assay is applicable to variants outside this region.

Relationship of glaucoma phenotype to degree of Triton insolubility

Of the 12 bona fide pathogenic mutations tested, 10 displayed near complete Triton insolubility. The remaining two mutations, Gly364Val and Asp380Ala, display a degree of insolubility that is clearly distinguishable from normal protein and controls, but is not as complete as that observed for the other 10 mutations (Fig. 5). Similar results were obtained for Thr377Met, Arg422Cys and Ile499Phe (Fig. 6). Available descriptions of the phenotypes of family members with POAG and a mutation associated with partial solubility reveal a later mean age at diagnosis and a lower mean maximum IOP as compared with glaucoma phenotypes for some of the mutations associated with near complete insolubility (Table 2). This trend is particularly striking in light of the fact that the phenotypic data were collected from a variety of Caucasian populations by research groups in a number of different countries, so environmental and genetic background effects may diminish phenotypic differences due to mutant alleles. On the other hand, there is substantial overlap in the range of these param-eters in the two groups and the small number of examples of each type makes the significance of these apparent differences uncertain. A clear exception to the suggested correlation between degree of solubility and severity of glaucoma phenotype is the Gln368Stop mutation, which has been associated with a significantly later onset, lower IOP phenotype and incomplete penetrance (11,22). The Gln368Stop protein is completely insoluble, but it may well represent a special case because premature termination mutations can destabilize mRNA, leading to decreased production of mutant protein (46). The milder phenotype of Gln368Stop may therefore be unrelated to the characteristics of the mutant protein.

Table 2. Glaucoma phenotypes associated with insoluble and partially soluble TIGR/MYOC variants

Mutation	Age at diagnosis (years)a	Max. IOP (mm Hg)a	No. of patients	Reference
Insoluble
Glu323Lys	19 (9-43)	43 (23-59)b	11	21
GlyGln367-368Val	28 (8-54)	38	20	29
Pro370Leu	12 (5-27)	45 (25-66)	15	21
Lys423Glu	30 (8-62)	NA (22-64)	40	39
Val426Phe	26 (16-46)	43 (32-52)	12	21
Tyr437His	20 (8-41)	44 (14-77)	27	11
Ile477Asn	21 (12-41)	40 (20-52)	13	11
Asn480Lys	35 (21-51)	36 (28-48)	6	35, family C
Gln368Stop	59 (36-77)	30 (21-56)	22	11
Partially soluble
Gly364Val	34 (22-48)	36 (15-65)	16	11
Thr377Met	37 (20-60)	31 (20-50)	15	11
Ile499Phe	31 (20-40)	31 (23-40)	7	35, family A

^aThe mean is followed by the range in parentheses.
^bCompiled for 6/11 patients.

Implications for a model of glaucoma pathogenesis

The relative Triton insolubility of mutant forms of TIGR/myocilin may provide clues to understanding the mechanism by which mutations in the gene cause a dominant POAG phenotype. The complete insolubility of Pro370Leu mutant protein at the earliest detectable time (Fig. 3A) and the appearance of soluble mutant protein at high levels of expression (Fig. 3B) are consistent with a model of saturable binding of mutant protein to one or more cellular components. The cellular components might themselves be Triton insoluble, or Triton insolubility might result from a complex of mutant protein with otherwise soluble components. Normal TIGR/myocilin can form dimers and, possibly, higher multimers (47,48; Z. Zhou and D. Vollrath, unpublished data), suggesting that mutant protein may interfere with normal protein through formation of heteromultimers. A fraction of normal TIGR/myocilin may be retained in cells along with mutant protein, resulting in decreased secretion of normal protein. This model is consistent with the observation that expression of a mutant form of TIGR/myocilin in perfused human anterior segments through use of a recombinant adenovirus results in a reduction in the amount of normal, endogenous TIGR/myocilin present in the perfusate (49). Other models of pathogenesis are also possible, such as disruption by mutant protein of the processing of normal TIGR/myocilin through binding and sequestration of another protein or cell death due to accumulation of insoluble protein complexes. Regardless of the specific model, the fact that Triton insolubility of mutant protein occurs in at least four different cell lines derived from a number of different tissues and mammalian species indicates that the insolubility is likely the result of a general cellular mechanism.

To our knowledge, the assay we have developed is the first description of a biochemical difference in the general characteristics of normal and mutant forms of TIGR/myocilin protein. The assay will be useful for classifying rare TIGR/MYOC variants. In addition, a better understanding of the Triton insolubility phenomenon, including identification of proteins that interact with mutant and normal TIGR/myocilin, may provide insight into the molecular pathology of glaucoma.

MATERIALS AND METHODS

DNA constructs

A TIGR/MYOC cDNA was amplified by PCR from a human heart cDNA library (Clontech, Palo Alto, CA) with primers flanking the coding region for TIGR/myocilin, cloned into the vector pGEM-T (Promega, Madison, WI) and verified by sequencing. The cDNA was subcloned into the eukaryotic expression vector pCDNA3 (Invitrogen, Carlsbad, CA) with NotI. One copy of the FLAG epitope (36) was inserted between codons 35 and 36 of the cDNA sequence, which is just C-terminal of a probable signal sequence cleavage site in the TIGR/myocilin protein. DNA constructs encoding TIGR/myocilin variants were generated with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). All constructs were partially sequenced to confirm that they encoded the expected sequence variant. Nucleotide changes made by site-directed mutagenesis corresponded to those described in human individuals.

Cell culture and transient expression of TIGR/myocilin in HEK cells

Cells were maintained in Dulbecco's minimum essential medium supplemented with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD). Exponentially growing cells were seeded 24 h before transfection. DNA was transfected into cells grown in 6-well plates with Lipofectamine Plus (Life Technologies) according to the manufacturer's protocol. Briefly, DNA was diluted in 100 µl of serum-free medium and 6 µl of lipofection reagent was added and incubated at room temperature for 15 min. Then, 4 µl of reagent diluted in 100 µl of serum-free medium was added and incubated for another 15 min. The DNA-lipid complexes were added to cells which had been rinsed once with serum-free medium and incubated at 37°C and 5% CO₂. After 3 h, medium with serum was added to bring the final concentration of serum to 10% and the medium was changed 1 day after transfection. For HEK and COS7 cells, 0.2 µg of DNA were used for transfection in each well of a 6-well plate. One microgram of DNA was used for HeLa and NIH 3T3 cells.

Triton X-100 extraction

Other than where noted, Triton extraction was performed 45 h after transfection. Transfected HEK cells in a 6-well plate were washed with Dulbecco's phosphate-buffered saline (PBS) twice, then incubated for 40 s with 300 µl of extraction buffer (50 mM MES, 3 mM EGTA, 5 mM MgCl₂, 0.5% Triton X-100, pH 6.4). Detergent-soluble material was precipitated in 80% acetone for 4 h at -20°C and the pellet was recovered after centrifugation (14 000 r.p.m. for 30 min in an Eppendorf microfuge). One milliliter of PBS was added to the detergent-insoluble material. It was scraped off the culture well and collected by centrifugation at 14 000 r.p.m. for 10 min in an Eppendorf microfuge. Pellets containing either detergent-soluble or detergent-insoluble material were resuspended in 50 µl of Laemmli sample buffer and boiled for 10 min prior to electrophoresis.

Analysis of TIGR/myocilin by SDS-PAGE and immunoblot

HEK cell extracts were subjected to SDS-PAGE, electrophoretically transferred to NitroBind nitrocellulose membrane (MSI, Westborough, MA), blocked with 5% non-fat dry milk in TBST blocking buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h at 23°C and incubated for 1 h in blocking buffer with either an anti-FLAG monoclonal antibody M2 (Eastman Kodak, New Haven, CT) or an affinity-purified anti-TIGR polyclonal antibody (Z. Zhou and D. Vollrath, unpublished data). Unbound antibody was removed by washing three times for 10 min with blocking buffer and goat anti-mouse or goat anti-rabbit IgG horseradish peroxidase conjugate (Promega, Madison, WI), diluted to 1:10 000 in blocking buffer, was added for 1 h. The blot was subjected to two 10 min washes in blocking buffer, followed by three 10 min washes with TBST, and then treated with a chemiluminescence substrate (Pierce, Rockford, IL) and immediately exposed to ECL Hyperfilm (Amersham, Arlington Heights, IL).

ACKNOWLEDGEMENTS

We thank Drs W. James Nelson, Julia E. Richards, David L. Rimm and Neil J. Risch for discussions and comments on the manuscript, and Virna L. Babb for DNA sequencing. This study was supported by a grant from the National Institutes of Health (EY11405). Z.Z. was supported in part by a training grant from the National Institutes of Health (HG 00044).

REFERENCES

1. Quigley, H.A. (1996) Number of people with glaucoma worldwide. Br. J. Ophthalmol., 80, 389-393. MEDLINE Abstract

2. Glaser, T., Walton, D.S. and Maas, R.L. (1992) Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nature Genet., 2, 232-239. MEDLINE Abstract

3. Semina, E.V., Reiter, R., Leysens, N.J., Alward, W.L., Small, K.W., Datson, N.A., Siegel-Bartelt, J., Bierke-Nelson, D., Bitoun, P., Zabel, B.U., Carey, J.C. and Murray, J.C. (1996) Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nature Genet., 14, 392-399. MEDLINE Abstract

4. Stone, E.M., Fingert, J.H., Alward, W.L.M., Nguyen, T.D., Polansky, J.R., Sunden, S.L.F., Nishimura, D., Clark, A.F., Nystuen, A., Nichols, B.E., Mackey, D.A., Ritch, R., Kalenak, J.W., Craven, E.R. and Sheffield, V.C. (1997) Identification of a gene that causes primary open angle glaucoma. Science, 275, 668-670. MEDLINE Abstract

5. Stoilov, I., Akarsu, A.N. and Sarfarazi, M. (1997) Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum. Mol. Genet., 6, 641-647. MEDLINE Abstract

6. Bejjani, B.A., Lewis, R.A., Tomey, K.F., Anderson, K.L., Dueker, D.K., Jabak, M., Astle, W.F., Otterud, B., Leppert, M. and Lupski, J.R. (1998) Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant cause of primary congenital glaucoma in Saudi Arabia. Am. J. Hum. Genet., 62, 325-333. MEDLINE Abstract

7. Vollrath, D., Jaramillo-Babb, V.L., Clough, M.V., McIntosh, I., Scott, K.M., Lichter, P.R. and Richards, J.E. (1998) Loss-of-function mutations in the LIM-homeodomain gene, LMX1B, in nail-patella syndrome. Hum. Mol. Genet., 7, 1091-1098. MEDLINE Abstract

8. Mears, A.J., Jordan, T., Mirzayans, F., Dubois, S., Kume, T., Parlee, M., Ritch, R., Koop, B., Kuo, W.L., Collins, C., Marshall, J., Gould, D.B., Pearce, W., Carlsson, P., Enerback, S., Morissette, J., Bhattacharya, S., Hogan, B., Raymond, V. and Walter, M.A. (1998) Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly. Am. J. Hum. Genet., 63, 1316-1328. MEDLINE Abstract

9. Nishimura, D.Y., Swiderski, R.E., Alward, W.L., Searby, C.C., Patil, S.R., Bennet, S.R., Kanis, A.B., Gastier, J.M., Stone, E.M. and Sheffield, V.C. (1998) The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. Nature Genet., 19, 140-147. MEDLINE Abstract

10. Suzuki, Y., Shirato, S., Taniguchi, F., Ohara, K., Nishimaki, K. and Ohta, S. (1997) Mutations in the TIGR gene in familial primary open-angle glaucoma in Japan. Am. J. Hum. Genet., 61, 1202-1204. MEDLINE Abstract

11. Alward, W.L.M., Fingert, J.H., Coote, M.A., Johnson, T., Lerner, S.F., Junqua, D., Durcan, F.J., McCartney, P.J., Mackey, D.A., Sheffield, V.C. and Stone, E.M. (1998) Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLCIA). N. Engl. J. Med., 338, 1022-1027. MEDLINE Abstract

12. Wiggs, J.L., Allingham, R.R., Vollrath, D., Jones, K., De La Paz, M., Kern, J., Patterson, K., Babb, V.L., Del Bono, E.A., Pericak-Vance, M.A. and Haines, J.L. (1998) Prevalence of mutations in TIGR/Myocilin in patients with adult and juvenile primary open angle glaucoma. Am. J. Hum. Genet., 63, 1549-1551. MEDLINE Abstract

13. Fingert, J.H., Heon, E., Liebmann, J.M., Yamamoto, T., Craig, J.E., Rait, J., Kawase, K., Hoh, S.T., Buys, Y.M., Dickinson, J., Hockey, R.R., Williams-Lyn, D., Trope, G., Kitazawa, Y., Ritch, R., Mackey, D.A., Alward, W.L., Sheffield, V.C. and Stone, E.M. (1999) Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum. Mol. Genet., 8, 899-905. MEDLINE Abstract

14. Polansky, J.R., Fauss, D.J., Chen, P., Chen, H., Lutjen-Drecoll, E., Johnson, D., Kurtz, R.M., Ma, Z.-D., Bloom, E. and Nguyen, T.D. (1997) Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica, 211, 126-139. MEDLINE Abstract

15. Kubota, R., Noda, S., Wang, Y., Minoshima, S., Asakawa, S., Kudoh, J., Mashima, Y., Oguchi, Y. and Shimizu, N. (1997) A novel myosin-like protein (Myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression and chromosomal mapping. Genomics, 41, 360-369. MEDLINE Abstract

16. Sarfarazi, M. (1997) Recent advances in molecular genetics of glaucomas. Hum. Mol. Genet., 6, 1667-1677. MEDLINE Abstract

17. Yokoe, H. and Anholt, R.R. (1993) Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium. Proc. Natl Acad. Sci. USA, 90, 4655-4659. MEDLINE Abstract

18. Adam, M.F., Belmouden, A., Binisti, P., Brezin, A.P., Valtot, F., Bechetoille, A., Dascotte, J.-C., Copin, B., Gomez, L., Chaventre, A., Bach, J.-F. and Garchon, H.-J. (1997) Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma. Hum. Mol. Genet., 12, 2091-2097.

19. Karavanich, C.A. and Anholt, R.R. (1998) Molecular evolution of olfactomedin. Mol. Biol. Evol., 15, 718-726. MEDLINE Abstract

20. Stoilova, D., Child, A., Brice, G., Crick, R.P., Fleck, B.W. and Sarfarazi, M. (1997) Identification of a new `TIGR' mutation in a family with juvenile-onset primary open angle glaucoma. Ophthalmic Genet., 18, 109-118. MEDLINE Abstract

21. Rozsa, F.W., Shimizu, S., Lichter, P.R., Johnson, A.T., Othman, M.I., Scott, K., Downs, C.A., Nguyen, T.D., Polansky, J. and Richards, J.E. (1998) GLC1A mutations point to regions of potential functional importance on the TIGR/MYOC protein. Mol. Vis., 4, 20. MEDLINE Abstract

22. Allingham, R.R., Wiggs, J.L., Reardon, M., Jones, K., Herndon, L., Tallett, D., Broomer, B., Karolak, L., del Bono, E.A., Haines, J.L. and Pericak-Vance, M.A. (1998) Myocilin Gln368Stop mutation in families with primary open angle glaucoma. Invest. Ophthalmol. Vis. Sci., 39, 3154.

23. Mansergh, F.C., Kenna, P.F., Ayuso, C., Kiang, A.S., Humphries, P. and Farrar, G.J. (1998) Novel mutations in the TIGR gene in early and late onset open angle glaucoma. Hum. Mutat., 11, 244-251. MEDLINE Abstract

24. Michels-Rautenstrauss, K.G., Mardin, C.Y., Budde, W.M., Liehr, T., Polansky, J., Nguyen, T., Timmerman, V., Van Broeckhoven, C., Naumann, G.O., Pfeiffer, R.A. and Rautenstrauss, B.W. (1998) Juvenile open angle glaucoma: fine mapping of the TIGR gene to 1q24.3-q25.2 and mutation analysis. Hum. Genet., 102, 103-106. MEDLINE Abstract

25. Morissette, J., Clépet, C., Moisan, S., Dubois, S., Winstall, E., Vermeeren, D., Nguyen, T.D., Polansky, J.R., Côté, G., Anctil, J.-L., Amyot, M., Plante, M., Falardeau, P. and Raymond, V. (1998) Homozygotes carrying an autosomal dominant TIGR mutation do not manifest glaucoma. Nature Genet., 19, 319-321. MEDLINE Abstract

26. Stoilova, D., Child, A., Brice, G., Desai, T., Barsoum-Homsy, M., Ozdemir, N., Chevrette, L., Adam, M.F., Garchon, H.J., Pitts Crick, R. and Sarfarazi, M. (1998) Novel TIGR/MYOC mutations in families with juvenile onset primary open angle glaucoma. J. Med. Genet., 35, 989-992. MEDLINE Abstract

27. Kennan, A.M., Mansergh, F.C., Fingert, J.H., Clark, T., Ayuso, C., Kenna, P.F., Humphries, P. and Farrar, G.J. (1998) A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma. J. Med. Genet., 35, 957-960. MEDLINE Abstract

28. Richards, J.E., Ritch, R., Lichter, P.R., Rozsa, F.W., Stringham, H.M., Caronia, R.M., Johnson, D., Abundo, G.P., Willcockson, J., Downs, C.A., Thompson, D.A., Musarella, M.A., Gupta, N., Othman, M.I., Torrez, D.M., Herman, S.B., Wong, D.J., Higashi, M. and Boehnke, M. (1998) Novel trabecular meshwork inducible glucocorticoid response mutation in an eight-generation juvenile-onset primary open-angle glaucoma pedigree. Ophthalmology, 105, 1698-1707. MEDLINE Abstract

29. Angius, A., De Gioia, E., Loi, A., Fossarello, M., Sole, G., Orzalesi, N., Grignolo, F., Cao, A. and Pirastu, M. (1998) A novel mutation in the GLC1A gene causes juvenile open-angle glaucoma in 4 families from the Italian region of Puglia. Arch. Ophthalmol., 116, 793-797. MEDLINE Abstract

30. Brezin, A.P., Adam, M.F., Belmouden, A., Lureau, M.A., Chaventre, A., Copin, B., Gomez, L., De Dinechin, S.D., Berkani, M., Valtot, F., Rouland, J.F., Dascotte, J.C., Bach, J.F. and Garchon, H.J. (1998) Founder effect in GLC1A-linked familial open-angle glaucoma in Northern France. Am. J. Med. Genet., 76, 438-445. MEDLINE Abstract

31. Blikstad, I., Nelson, W.J., Moon, R.T. and Lazarides, E. (1983) Synthesis and assembly of spectrin during avian erythropoiesis: stoichiometric assembly but unequal synthesis of alpha and beta spectrin. Cell, 32, 1081-1091. MEDLINE Abstract

32. Hinck, L., Nathke, I.S., Papkoff, J. and Nelson, W.J. (1994) Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. J. Cell Biol., 125, 1327-1340. MEDLINE Abstract

33. Gutmann, D.H., Geist, R.T., Xu, H., Kim, J.S. and Saporito-Irwin, S. (1998) Defects in neurofibromatosis 2 protein function can arise at multiple levels. Hum. Mol. Genet., 7, 335-345. MEDLINE Abstract

34. Deguen, B., Merel, P., Goutebroze, L., Giovannini, M., Reggio, H., Arpin, M. and Thomas, G. (1998) Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane. Hum. Mol. Genet., 7, 217-226. MEDLINE Abstract

35. Brezin, A.P., Bechetoille, A., Hamard, P., Valtot, F., Berkani, M., Belmouden, A., Adam, M.F., Dupont de Dinechin, S., Bach, J.F. and Garchon, H.J. (1997) Genetic heterogeneity of primary open angle glaucoma and ocular hypertension: linkage to GLC1A associated with an increased risk of severe glaucomatous optic neuropathy. J. Med. Genet., 34, 546-552. MEDLINE Abstract

36. Hopp, T.P., Prickett, K.S., Price, C., Libby, R.T., March, C.J., Cerretti, P., Urdal, D.L. and Conlon, P.J. (1988) A short marker sequence useful for recombinant protein identification and purification. Biotechnology, 6, 1205-1210.

37. Lichter, P.R., Richards, J.E., Boehnke, M., Othman, M., Cameron, B.D., Stringham, H.M., Downs, C.A., Lewis, S.B. and Boyd, B.F. (1997) Juvenile glaucoma linked to the GLC1A gene on chromosome 1q in a Panamanian family. Am. J. Ophthalmol., 123, 413-416. MEDLINE Abstract

38. Richards, J.E., Lichter, P.R., Boehnke, M., Uro, J.L., Torrez, D., Wong, D. and Johnson, A.T. (1994) Mapping of a gene for autosomal dominant juvenile-onset open-angle glaucoma to chromosome Iq. Am. J. Hum. Genet., 54, 62-70. MEDLINE Abstract

39. Morissette, J., Cote, G., Anctil, J.L., Plante, M., Amyot, M., Heon, E., Trope, G.E., Weissenbach, J. and Raymond, V. (1995) A common gene for juvenile and adult-onset primary open-angle glaucomas confined on chromosome 1q. Am. J. Hum. Genet., 56, 1431-1442. MEDLINE Abstract

40. Johnson, A.T., Richards, J.E., Boehnke, M., Stringham, H.M., Herman, S.B., Wong, D.J. and Lichter, P.R. (1996) Clinical phenotype of juvenile-onset primary open-angle glaucoma linked to chromosome 1q. Ophthalmology, 103, 808-814. MEDLINE Abstract

41. Meyer, A., Bechetoille, A., Valtot, F., Dupont de Dinechin, S., Adam, M.F., Belmouden, A., Brezin, A.P., Gomez, L., Bach, J.F. and Garchon, H.J. (1996) Age-dependent penetrance and mapping of the locus for juvenile and early-onset open-angle glaucoma on chromosome 1q (GLC1A) in a French family. Hum. Genet., 98, 567-571. MEDLINE Abstract

42. Abderrahim, H., Jaramillo-Babb, V.L., Zhou, Z. and Vollrath, D. (1998) Characterization of the murine Tigr/Myoc gene. Mamm. Genome, 9, 673-675. MEDLINE Abstract

43. Fingert, J.H., Ying, L., Swiderski, R.E., Nystuen, A.M., Arbour, N.C., Alward, W.L., Sheffield, V.C. and Stone, E.M. (1998) Characterization and comparison of the human and mouse GLC1A glaucoma genes. Genome Res., 8, 377-384. MEDLINE Abstract

44. Tomarev, S.I., Tamm, E.R. and Chang, B. (1998) Characterization of the mouse Myoc/Tigr gene. Biochem. Biophys. Res. Commun., 245, 887-893. MEDLINE Abstract

45. Buys, Y., Williams-Lyn, D., Trope, G., Flanagan, J., Affitigato, L.M., Stone, E.M. and Heon, E. (1999) The role of the myocilin gene in adult-onset glaucoma: a perspective from the greater Toronto area. Invest. Ophthalmol. Vis. Sci., 40, 406.

46. Macquat, L.E. (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA, 1, 453-465. MEDLINE Abstract

47. Nguyen, T.D., Chen, P., Huang, W.D., Chen, H., Johnson, D. and Polansky, J.R. (1998) Gene structure and properties of an olfactomedin-related glycoprotein, TIGR, cloned from glucocorticoid-induced trabecular meshwork cells. J. Biol. Chem., 273, 1-10. MEDLINE Abstract

48. Raymond, V., Moisan, S., Rodrigue, M.A., Nguyen, T.D., Polansky, J.R. and Morissette, J. (1999) Evidence for homoallelic complementation as a model to account for the unaffected status of homozygotes carrying an autosomal dominant TIGR mutation. Invest. Ophthalmol. Vis. Sci., 40, 3130.

49. Caballero, M., Gonzalez, P., Russell, P., Rowlette, L.L.S. and Borras, T. (1999) Adenoviral gene transfer of a single domain of the TIGR/MYOC protein to the human perfused anterior segment cultures. Invest. Ophthalmol. Vis. Sci., 40, 3132.

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+To whom correspondence should be addressed. Tel: +1 650 723 3290; Fax: +1 650 723 7016; Email: vollrath{at}genome.stanford.edu

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