Distribution of WDR36 DNA Sequence Variants in Patients with Primary Open-Angle Glaucoma

Abstract

purpose. To determine the distribution of WDR36 sequence variants in a cohort of patients with primary open-angle glaucoma (POAG) in the United States.

methods. All of the 23 coding exons and flanking introns of the WDR36 gene were sequenced in 118 probands from families with at least two members affected by POAG, 6 probands from juvenile-onset POAG families, and 108 control individuals.

results. Thirty-two WDR36 sequence variants were found in this population of patients with POAG. Nonsynonymous single-nucleotide polymorphisms (SNPs), including those previously described as “disease-causing” and “disease susceptibility,” were found in 17% of POAG patients and 4% of control subjects. Although the distribution of WDR36 variants in the pedigrees did not show consistent segregation with the disease, the WDR36 sequence variants were found more frequently in patients with more severe disease.

conclusions. The results of this study suggest that abnormalities in WDR36 alone are not sufficient to cause POAG. The association of WDR36 sequence variants with more severe disease in affected individuals suggests that defects in the WDR36 gene can contribute to POAG and that WDR36 may be a glaucoma modifier gene.

Primary open angle glaucoma (POAG) is a complex inherited disease that is likely to result from defects in multiple susceptibility genes as well as environmental factors. The disease causes a characteristic degeneration of the optic nerve that is frequently associated with elevated intraocular pressure (high-tension glaucoma) but can occur even if the intraocular pressure is low or normal (low-tension glaucoma). 

Two genes have been identified as factors that contribute to POAG, defects in the myocilin gene (GLC1A) primarily causes elevated pressure ^{1 2} and the optineurin gene (GLC1E), appears to contribute to disease in familial low-tension glaucoma. ^{3 4} In addition to these genes, five other glaucoma gene loci (GLC1B, GLC1C, GLC1D, GLC1F, GLC1G) have been identified using large affected pedigrees and Mendelian linkage approaches (Samples JR, et al. IOVS 2004;44:ARVO E-Abstract 4622). ^{5 6 7 8 9 10} Sib pair-based whole genome analyses of typical late-onset families have also identified chromosomal regions likely to contain POAG susceptibility genes, including 14q, ¹¹ 15q, ¹² 2q, ¹³ and 10p. ¹³  

GLC1G was initially defined as a 2-Mb region by linkage studies using POAG pedigrees (Samples JR, et al. IOVS 2004;44:ARVO E-Abstract 4622). ^{9 10} The WDR36 gene is located within the critical genetic interval defined by these linkage studies, and recently Monemi et al., ⁹ characterized WDR36 as the third POAG gene by identifying WDR36 DNA sequence variants in patients with high- and low-tension glaucoma. A total of 24 sequence variants were identified: Four were defined as disease-causing (5% of 130 POAG families, and 0% of 200 control subjects), 3 as disease-predisposing (11% of POAG families and 2% of control subjects), and 17 as polymorphisms with equal distribution between POAG cases and control subjects. 

WDR36 is a member of the WD40 repeat protein family and may be involved in T-cell activation. ¹⁴ Recently, T-cell-mediated responses have been hypothesized to participate in glaucoma associated optic nerve degeneration. ¹⁵ The purpose of this study is to determine the distribution of WDR36 DNA sequence variants in a well-studied cohort of patients with POAG in the United States. 

Methods

Patients

This research adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients and family members after explanation of the nature and possible consequences of the study. The research was approved by the institutional review boards of the Massachusetts Eye and Ear Infirmary and Duke University School of Medicine. 

POAG family probands were defined as age of diagnosis greater than 35 years, intraocular pressure greater than 22 mm Hg in both eyes without medications or 19 mm Hg on two or more medications, glaucomatous optic nerve damage in both eyes, and visual field loss in at least one eye. Glaucomatous optic nerve damage was defined as cup-to-disc ratio higher than 0.7 or focal loss of the nerve fiber layer (notch) associated with a specific visual field defect. Visual fields were performed using automated perimetry (mainly Humphrey) and were scored with a modified six-stage system adapted from that published by Quigley. ^{12 16} Level-I patients are the most severely affected and met all criteria including elevated IOP, bilateral optic nerve damage, and visual field loss in at least one eye. Level-II patients were less severely affected and satisfied any two of these three criteria. Control subjects were age matched and were collected from the same geographic regions as the probands. Control subjects had no evidence of glaucoma and no family history of glaucoma. 

For this study 118 probands from families with at least two members affected by POAG (a level-I proband with all other affected family members either level I or level II) were screened for WDR36 sequence variants. We also included six probands from juvenile-onset POAG (JOAG) families (defined as POAG except with age of onset before 35 years). 

PCR Amplification and DNA Sequencing

Genomic DNA from peripheral blood was prepared from all individuals by using standard techniques (Gentra, Minneapolis, MN). All the 23 coding exons and flanking introns of the WDR36 gene (GenBank accession no. NM_139281/ http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) were sequenced using nested PCR strategies for amplification. Amplification conditions for PCR were (per 25 μL PCR reaction): 20 mM Tris-HCl (pH 8.4); 50 mM KCl; 1.5 mM MgCl₂; 200 μM each of dATP, dCTP, dGTP, and dTTP; 100 ng forward PCR primer; 100 ng reverse PCR primer; 30 ng genomic DNA; and 0.5 U Taq DNA polymerase (Platinum Taq; Invitrogen-Life Technologies, Rockville, MD). PCR products were amplified using a “touchdown” strategy whereby the annealing temperature is lowered incrementally over the course of the reaction. Initial thermocycler conditions were as follows: 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. After two cycles at an annealing temperature of 65°C, the temperature was lowered to 63°C for two cycles, then to 61°C for two cycles, 59°C for two cycles, 57°C for two cycles, and finally 55°C for 30 additional cycles (40 cycles total). 

Oligonucleotides for amplification and sequencing were selected using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi/ provided in the public domain by the Massachusetts Institute of Technology, Cambridge, MA) and were located at least 40 bases from each splice site. Primer sequences used to amplify each exon are available on request. 

Amplified genomic DNA from an affected proband or control was directly sequenced using sequencing chemistries (BigDye ver. 3.1; Applied Biosystems, Inc. [ABI], Foster City, CA) and an automated sequencer (model 3100 or 3730; ABI). In some cases, pools of amplified genomic DNA from two individuals were initially sequenced and pools displaying DNA sequence variants were resequenced individually. Sequences from pooled samples were analyzed (Sequencher Software; Gene Codes Corp., Ann Arbor, MI). 

Results

Twelve nonsynonymous single-nucleotide polymorphisms (SNPs) were identified in the population of patients used for this study. Complete results were available for 118 POAG probands, 6 JOAG probands, and 108 control subjects. With the exception of the common variant I264V, only heterozygous changes were found. Mutations that were previously reported as disease-causing ⁹ were found in 3% of POAG probands (4/118) and 2% of control subjects (2/108). DNA sequence variants reported to be “potential disease-susceptibility mutations” were found in 2% of POAG probands (3/118) and 2% of control subjects (2/108). One previously reported nonsynonymous SNP (I264V) is a common sequence variant and was found evenly distributed between POAG probands and control subjects (Table 1) . 

Six nonsynonymous SNPs were found in this population that were not previously reported. Five of these six sequence variants are evolutionarily conserved (Table 2)and were found in 8 of 118 POAG probands and 1 of 108 control subjects. The C470Y variant was found in one juvenile POAG proband and was not found in control subjects. This was the only nonsynonymous SNP found in the six JOAG probands. When taken together, nonsynonymous SNPs (excluding the common I264V variant) were found in 20 of 118 POAG probands (17%) compared with 5 of 108 control subjects (4%). 

Seven synonymous SNPs, 4 of which were novel, and 13 intronic changes were found, including 9 that had not been previously described (Table 1) . Specific associations between POAG and these sequence changes were not found, and none of the intronic changes or synonymous SNPs was expected to affect splice sites. 

The distribution of WDR36 variants in the pedigrees did not show consistent segregation with the disease. In the 16 pedigrees with nonsynonymous SNPs (excluding the I264V common variant), a WDR36 sequence variant was found in only 25 of 44 affected individuals, and in 4 of 16 unaffected family members. However, among the affected individuals with WDR36 variants, we found that individuals with glaucoma and a WDR36 variant had more severe disease (level I) than those affected individuals without a WDR36 variant (Table 3 ; P < 0.001, χ², P = 0.0019, the Fisher exact test). Of interest, two of the pedigrees have known mutations in myocilin (Thr377Met, pedigree 27; Gln368Stop, pedigree 125) that are consistently present in every affected individual (both level-I and -II affected individuals). However, the level-I individuals in these two pedigrees had a WDR36 variant in addition to the myocilin mutation (Fig. 1) . 

Level-I patients have evidence of (1) elevated intraocular pressure, (2) optic nerve changes consistent with glaucoma, and (3) visual field changes that are consistent with the abnormalities in the optic nerve. Level-II patients have two of these three criteria. In this study, most of the level-II patients met the intraocular pressure but failed to meet either the visual field or optic nerve requirements for level-I status (15/18). Of the level-II patients without WDR36 variants, the majority (11/13) did not meet the required optic nerve or visual field criteria, whereas of the 5 level-II patients that had WDR36 variants, 4 did not meet the optic nerve or visual field criteria, and 1 failed to meet the intraocular pressure criterion (Table 4) . Other parameters, including age of diagnosis, did not appear to correlate significantly with the presence of WDR36 variants (age of diagnosis is 54.85 years with WDR36 defects and 58.65 years without WDR36 defects, P = 0.12, two-tailed test). Age of onset cannot be practically measured in a glaucoma population, because of the lack of symptoms early in the course of the disease that alert the patient to a visual problem. 

Discussion

In this patient population we found nonsynonymous WDR36 DNA sequence variants in 17% of POAG probands compared with 5% of control subjects (excluding the I264V common variant) indicating that abnormalities in WDR36 could contribute to POAG in up to 17% of this patient population. 

However, in the affected pedigrees that carried a WDR36 sequence variant, we found that the sequence variants did not consistently segregate with the occurrence of the disease, arguing that abnormalities in WDR36 alone are not sufficient to cause POAG. We did find, however, that affected individuals who carried a WDR36 sequence variant were more likely to have more severe disease than were affected patients who did not carry a variant. These results suggest that while defects in the WDR36 gene may contribute to the glaucomatous disease process, WDR36 most likely acts as a glaucoma modifier gene. 

Genes that modify the action or expression of a dominant gene are emerging as important determinants of the phenotypic variation observed in genetic disorders. In mouse studies, the segregating background genes can modify the age of onset, rate of progression, or severity of disease expression. ¹⁷ It is becoming increasingly apparent that human complex disorders arise because of multiple genetic interactions (epistasis) and gene environment interactions. ¹⁸ Epistatic interactions caused by modifier genes may make the disease more severe or less severe. The identification of modifier genes will help define the molecular pathways responsible for the disease as well as provide new information that may lead to the development of biomarkers for the disease as well as novel therapeutics. 

Although the function of the WDR36 protein and its role in glaucoma is not known, there is some evidence to suggest that WDR36 may participate in the activation of T cells in response to IL-2. ¹⁴ Previous studies have suggested that some patients with glaucoma may have an alteration of cellular immunity that is IL-2 dependent. ¹⁹ Recently, other studies have suggested that T-cell responses may influence optic nerve degeneration in glaucoma in humans ¹⁵ and in a mouse glaucoma model. ²⁰ In our study most of the level-II patients who did not have WDR36 gene defects met the affected criterion for intraocular pressure, but were scored level II because of incomplete evidence of optic nerve disease or visual field defects, which could support the hypothesis that WDR36 may contribute to increased susceptibility of optic nerve degeneration in the setting of elevated intraocular pressure. It is interesting to speculate that WDR36 may contribute to glaucoma by modifying optic nerve degeneration; however, further work defining the role of the protein in POAG is necessary before this conclusion can be reached. 

POAG is a common disorder with a complex inheritance that is likely to result from contributions of multiple genes and possibly environmental conditions. Genetic contributions to this disease may influence intraocular pressure, optic nerve degeneration or both. Mendelian autosomal dominant and recessive forms of glaucoma are caused by single gene defects that are associated with extreme phenotypes: either highly elevated intraocular pressure or severe optic nerve degeneration. Most patients with POAG do not have extreme phenotypes, and the underlying genetic etiologies are not thought to result from single gene defects, but from contributions of multiple genetic factors that independently cause moderate alterations in intraocular pressure and optic nerve disease but collectively cause more severe disease. Genes that contribute to POAG may not cause clinical evidence of the disease unless they are coupled with other genes or environmental factors. If disease features are dependent on the combined effects of multiple factors then the identification and characterization of any one disease-predisposing factor can be difficult when using traditional linkage approaches. In this study, we provide evidence to suggest that WDR36 may influence disease severity and may contribute to the disease process. The identification of glaucoma susceptibility and glaucoma modifying genes are important steps toward the complete molecular definition of POAG. 

 Supported by National Eye Institute Grants R01 EY15586 (JLW), P30 EY14104, R01 EY15543 (RRA), R01 EY13315 (MAH), and the Goldberg Foundation.

 Submitted for publication November 17, 2005; revised January 24, 2006; accepted April 24, 2006.

 Disclosure: M.A. Hauser, None; R.R. Allingham, None; K. Linkroum, None; J. Wang, None; K. LaRocque-Abramson, None; D. Figueiredo, None; C. Santiago-Turla, None; E.A. del Bono, None; J.L. Haines, None; M.A. Pericak-Vance, None; J.L. Wiggs, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

WDR36 Sequence Variants in POAG Probands and Control Subjects

	Protein Domain	POAG	Control
Nonsynonymous
L25P* (t/c)	None	3/118	2/108
D33E (c/g)	None	1/118	1/108
H212P* (at/cc)	WD40	2/118	0/108
I264V, † (a/g)	WD40	43/118	50/108
A353S (g/t)	WD40	1/118	0/108
I361V (a/g)	WD40	1/118	0/108
A449T, ‡ (g/a)	WD40	1/118	0/108
C470Y (g/a)	WD40	1/6 (JOAG)	0/108
R529Q, ‡ (g/a)	WD40	1/118	0/108
I604V (a/g)	WD40	3/118	0/108
D658G, ‡ (a/g)	WD40	2/118	2/108
M671V, † (a/g)	WD40	4/118	0/108
Synonymous
T100T (c/g)	None	1/118	0/81
G134G, † (c/t)	WD40	3/118	ND
Y141Y (t/c)	WD40	9/118	ND
Q197Q (g/a)	WD40	2/118	0/79
L661L (t/c)	WD40	1/118	0/83
V714V, † (c/g)	None	28/118	ND
V727V, † (a/t)	Utp21	50/118	ND
Intronic
IVS3 − 113 (g/a)	—	16/118	ND
IVS5 + 30, § (t/c)	—	23/118	49/79
IVS5 − 25 (c/t)	—	1/118	ND
IVS8 − 8 (c/g)	—	2/118	0/77
IVS12 + 90, § (c/t)	—	23/118	55/77
IVS14 + 89, § (c/a)	—	19/118	43/74
IVS16 − 28 (a/g)	—	16/118	28/72
IVS18 + 35 (t/g)	—	1/118	ND
IVS21 + 60, § (g/c)	—	5/118	ND
IVS21 − 75 (g/a)	—	1/118	ND
IVS21 − 23 (a/g)	—	1/118	ND
IVS22 − 8 (t/g)	—	6/118	ND
3′UTR + 93 (a/c)	—	4/118	ND

 ND, not determined. Nomenclature based on GenBank accession no. NM_139281.

*  Previously designated as disease-susceptibile.

†  Previously designated as amino acid polymorphism.

‡  Previously designated as disease-causing.

§  Previously identified intronic polymorphism.

Table 2.

 

Amino Acid Alignment and Evolutionary Conservation of Six Novel Nonsynonymous SNPs

	D33E	H212P	A353S	I361V	C470Y	I604V
Human	VPLDTLK	AILHPST	TNGADNA	RIWIFDG	KLSCSTW	DFSISVL
Chimp	VPLDTLK	AILHPST	TNGADNA	RIWIFDG	KLSCSTW	DFSISVL
Rat	– – –GG– –	TILHPST	TNGADNA	RIWIFDG	KLSCSTW	DFSITVL
Mouse	– – –GV– –	TILHPST	TNGADNA	RIWIFDG	KLSCSTW	DFSIAVL
Dog	– – –GF– –	AILHPST	TNGADNA	RIWIFDG	KLSCSTW	DFSICVL

Table 3.

 

Distribution of WDR36 Sequence Variants in Affected and Unaffected Members of POAG Pedigrees

Sequence Variant	Pedigree	Level I Affected with Variant/Total	Level II Affected with Variant/Total	Unaffected with Variant/Total
Leu25Pro	27	2/2	1/2	0/0
Leu25Pro	94	3/4	0/0	0/0
Leu25Pro	603	1/1	1/1	0/0
Asp33Glu	5010	1/1	1/4	1/5
His212Pro	5015	1/1	0/2	0/0
His212Pro	5445	1/1	1/2	0/1
Ala353Ser	552	1/2	0/1	1/4
Ile361Val	5079	2/2	0/0	1/3
Ala449Thr	5063	1/1	1/2	0/0
Arg529Gln	572	1/2	0/0	1/2
Ile604Val	5083	1/1	0/1	0/0
Ile604Val	5135	1/2	0/0	1/1
Ile604Val	5474	1/2	0/0	0/0
Asp658Gly	125	1/1	0/1	0/0
Asp658Gly	5086	1/1	0/1	0/0
Met671Val	5472	1/1	0/1	0/0
		20/26 (77%)*	5/18 (28%)	4/16

*   P < 0.001, χ²; P = 0.0019, Fisher exact test.

Figure 1.

 

Distribution of WDR36 sequence variants in selected POAG pedigrees. The affected status of the individual (either level I or level II) is shown immediately below the pedigree. The WDR36 sequence variant is shown below the pedigree in bold and the myocilin sequence variant in italic Myocilin sequence variants were found only in pedigrees 27 and 125.

Table 4.

 

Clinical Features of Level II Affected Individuals

Pedigree-Individual	Level II Clinical Feature	WDR36 Variant
27-1063	Normal visual field	Leu25Pro
27-1235	Normal visual field	—
603-3526	Normal visual field	Leu25Pro
5010-103	C/D ratio < 0.7 OU	—
5010-107	C/D ratio < 0.7 OU	—
5010-110	C/D ratio < 0.7 OU	Asp33Glu
5010-112	Normal visual field	—
5015-101	C/D ratio < 0.7 OU	—
5015-9000	C/D ratio < 0.7 OU	—
5445-1	IOP < 21	—
5445-102	IOP < 21	His212Pro
552-3125	Normal visual field	—
5063-102	C/D ratio < 0.7 OU	—
5063-104	Normal visual field	Ala449Thr
5083-100	C/D ratio < 0.7 OU	—
125-2013	Normal visual field	—
5086-105	IOP < 21	—
5472-101	C/D ratio < 0.7 OU	—

 The clinical feature that prevented a designation of level-I affected status is listed. C/D, cup-to-disc.