Pedigree of the family with primary open-angle glaucoma (POAG) that maps to the GLC1G locus. Arrow indicates the proband. Circles indicate female members; slashes, dead; squares, male members; solid symbols, members diagnosed as having POAG; numbers in diamonds, the numbers of asymptomatic individuals in the sibship. The descendants of individuals collected after the initial report of linkage are enclosed in boxes. The microsatellite markers are listed in the top left corner. The microsatellite marker alleles are written below each person’s identification number. The disease haplotype is boxed.
Kramer PL, Samples JR, Monemi S, Sykes R, Sarfarazi M, Wirtz MK. The Role of the WDR36 Gene on Chromosome 5q22.1 in a Large Family With Primary Open-Angle Glaucoma Mapped to This Region. Arch Ophthalmol. 2006;124(9):1328–1331. doi:10.1001/archopht.124.9.1328
JANEY L.WIGGSMD, PhDAuthor Affiliations: Departments of Neurology (Dr Kramer), Molecular and Medical Genetics (Drs Kramer and Wirtz), and Ophthalmology, Casey Eye Institute (Drs Samples and Wirtz and Ms Sykes), Oregon Health and Sciences University, Portland; and the Molecular Ophthalmic Genetics Laboratory and the Department of Surgery, University of Connecticut Health Center, Farmington (Drs Monemi and Sarfarazi).
To determine whether mutations in the WD40-repeat 36 (WDR36) gene are responsible for primary open-angle glaucoma (POAG) that maps to the GLC1G locus in a family with 16 affected family members.
Ninety-two family members underwent clinical evaluation for POAG on the basis of intraocular pressures, cupping of discs, and visual fields after informed consent was obtained. All 23 exons of WDR36 were sequenced in DNA from 5 affected and 2 unaffected family members.
Sixteen family members showed evidence of POAG. A number of sequence variations were identified in family members; most of the variations were previously described single-nucleotide polymorphisms also present in the general population. The 3 new sequence changes were all intronic; 2 were found in only 1 of the family members undergoing screening.
Several polymorphisms, including known single-nucleotide polymorphisms, were identified; however, none of these were consistent with disease-causing mutations. A mutation in a noncoding region of WDR36 may be responsible for POAG in this family, or another gene in this region may be the actual cause of glaucoma in this family.
The finding that the WDR36 gene is probably not the responsible gene in this family further documents the genetic heterogeneity of POAG.
Adult-onset primary open-angle glaucoma (POAG) is the most common form of glaucoma and a leading cause of blindness in adults older than 40 years. Family history is a significant risk factor for this condition. Eight genetic loci have so far been identified for POAG, including regions on chromosomes 1q (GLC1A),1 2q (GLC1B),2 3q (GLC1C),3 8q (GLC1D),4 10p (GLC1E),5 7q (GLC1F),6 5q (GLC1G),7,8 and 15q (GLC1I).9 Of these, disease-causing mutations have subsequently been identified in the myocilin (GLC1A),10 optineurin (GLC1E),11 and WD40-repeat 36 (GLC1G)12 genes.
We initially reported linkage of the GLC1G locus to a 6.6-megabase (Mb) region on chromosome 5q in a large kindred with POAG.7,8 Based on this information, along with data from 7 additional families with POAG that were consistent with linkage to an overlapping 35-Mb region, Monemi et al12 reduced the critical region to approximately 2 Mb and subsequently identified several disease-causing mutations in the WD40-repeat 36 (WDR36) gene. None of these mutations, however, was identified in the family with POAG. These results suggest that there is a second POAG locus on chromosome 5q, in close proximity to the WDR36 gene.
This study was approved by the Oregon Health Sciences University institutional review board. After obtaining informed consent, we examined and obtained blood samples from 92 family members of a multigenerational family with POAG in Oregon that emigrated from the Netherlands. In our initial report of linkage to chromosome 5q22.1,7,8 which was the basis for narrowing the candidate region in the study by Monemi et al,12 we identified 13 individuals with definite POAG. Subsequently, we expanded the family and identified 3 additional affected individuals. These individuals appear in boxes in the pedigree illustrated in the Figure. The current pedigree thus includes 16 living affected family members (9 men and 7 women).
Family members were examined by gonioscopy with a 4-mirror lens (Carl Zeiss Inc, Thornwood, NY) and graded according to the Becker-Schaffer grading system, with grade 4 indicating that the iridocorneal angle is at least 40°. We measured visual fields using automatic static threshold perimetry with a Humphrey field analyzer (Carl Zeiss Ophthalmic Systems, Dublin, Calif) using the 30-2 test point pattern. We used the glaucoma hemifield test to determine whether the field was glaucomatous or normal.13 Criteria for the diagnosis of POAG were as previously described.3 Briefly, a glaucomatous visual field with a vertical cup-disc ratio of 0.7 or more was the strictest criterion, which 10 of the affected family members met. One individual (FIV:11), dead at the time of the study, had previously received a diagnosis of POAG from his ophthalmologist and started latanoprost (Xalatan) therapy followed by laser trabeculectomy in the right eye. Because of the prior diagnosis and treatment, he was considered to be affected with POAG for the purposes of our study. The remaining 5 affected family members had cup-disc ratios greater than 0.7. All affected individuals are being treated with intraocular pressure–lowering medication. All individuals with intraocular pressure of less than 20 mm Hg and a vertical optic cup-disc ratio of 0.3 or less were considered unaffected. There was no evidence of pigment dispersion syndrome in this family because increased pigmentation in the trabecular meshwork was not observed in any affected individual. The mean ± SD age at diagnosis was 63.7 ± 12.6 years and ranged from 38 to 79 years.
We isolated DNA as previously described3 or by using a purification kit and microsatellite markers (Invitrogen, Carlsbad, Calif). For mutation screening, primers were designed to flank the intron-exon boundaries of the genes selected for screening. We performed polymerase chain reaction amplification using the genomic DNA of affected individuals. Direct sequencing was conducted with a commercially available terminator cycle sequencing kit (ABI-Big Dye; Applied Biosystems Inc, Foster City, Calif) and run on a genetic analyzer and DNA sequencer (ABI-3100; Applied Biosystems Inc).
After a genome-wide linkage scan and fine mapping of potential linkage regions, we identified linkage in this family to a 6.6-Mb region on chromosome 5q, flanked by microsatellite markers D5S1721 and D5S2051, as previously reported.7,8 Haplotypes for these markers in the original and expanded branches of this family are presented in the Figure. Fifteen of the 16 affected individuals carry the haplotype 2-3-1 at the internal markers D5S485, D5S2084, and D5S475, respectively. One affected individual (FIV:14) does not appear to carry the purported disease haplotype and may be a phenocopy. Clinical features of this individual are not distinct from those of other affected family members (Table 1). Two-point logarithm of the odds (LOD) scores are 2.71 and 2.56 at D5S2084 and D5S475 (θ = 0) with all of the affected family members included in the analysis. The linked alleles are very common and often homozygous; thus, the LOD score is lower than might be expected with this family structure.
As a result of the identification of mutations recently proposed in WDR36, all 23 exons of this gene were sequenced in a subset of 7 family members (5 affected and 2 unaffected individuals). The 2 unaffected individuals were 58 and 67 years old at their last examination by one of us (J.R.S.). Results are shown in Table 2. None of the 4 predicted or the 3 potential disease-causing mutations identified by Monemi et al12 occurred in any of these 7 individuals. A number of amino acid and intronic polymorphisms, most of which were identified by Monemi et al12 in their sample of 130 families with POAG, also occurred in this large family.
Several previously reported polymorphisms were identified in this family. However, none of these met the accepted criteria for disease-causing status because they were also present in the general population. The 3 novel polymorphisms did not segregate with the disease in the family and therefore were also ruled out as causing POAG. Also, the novel and known single-nucleotide polymorphisms were not found in a donor or an acceptor splice site, and thus it is unlikely that they would alter splicing of the WDR36 gene. Mutations in the promoter of WDR36 may be responsible for POAG in this family. On the other hand, it may be that the occurrence of glaucoma in this family is the result of mutations in another gene in the region. Alternatively, the WDR36 gene may not be the defective gene in the GLC1G locus. In addition to the other 6 genes in the 2-Mb region of linkage identified by Monemi et al,12 work is under way to sequence genes upstream in the adjacent 4.6-Mb region that encompasses the critical linkage region for this family.
Correspondence: Mary K. Wirtz, PhD, Department of Ophthalmology, Casey Eye Institute, Oregon Health and Science University, 3375 SW Terwilliger Blvd, Portland, OR 97239-4197 (email@example.com).
Submitted for Publication: November 9, 2005; final revision received February 1, 2006; accepted February 3, 2006.
Financial Disclosure: None reported.
Funding/Support: This study was supported by grants R01 EY009947 (Dr Sarfazi), RO1 EY11650-07, 5P30EY010572-099003, and M01 RR000334 from the National Institutes of Health; by the American Health Assistance Foundation; and by an unrestricted grant from Research to Prevent Blindness.