|Molecular Vision 2006;
Received 30 June 2005 | Accepted 14 February 2006 | Published 14 February 2006
A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q
Chi Pui Pang,1
Bao Jian Fan,1 Oscar Canlas,2
Dan Yi Wang,1 Stéphane Dubois,3 Pancy Oi Sin Tam,1
Dennis Shun Chiu Lam,1 Vincent Raymond,3 Robert
1Department of Ophthalmology & Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China; 2Jose B. Lingad Memorial Regional Hospital, San Fernando, Philippines; 3Laboratory of Ocular Genetics and Genomics, Molecular Endocrinology and Oncology Research Center, Laval University Hospital Research Centre, Québec City, Québec, Canada; 4Department of Ophthalmology, New York Eye and Ear Infirmary, New York City, NY; 5Department of Ophthalmology, New York Medical College, Valhalla, NY
Correspondence to: Prof. Chi Pui Pang, Department of Ophthalmology &
Visual Sciences, The Chinese University of Hong Kong, Hong Kong Eye
Hospital, 147K Argyle Street, Kowloon, Hong Kong; Phone: +852 27623169;
FAX: +852 27159490; email: email@example.com
Dr. Dr. Fan is now at the Department of Biochemistry, The University of Hong Kong, Hong Kong, China
Purpose: To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG) and to screen the novel glaucoma gene WD repeat domain 36 (WDR36).
Methods: Complete ophthalmic examination and genomic DNA were obtained from 27 family members, in which nine were confirmed JOAG patients. Myocilin (MYOC), optineurin (OPTN), and WDR36 were screened for mutations by polymerase chain reaction and direct sequencing. Genome-wide scanning was carried out using the ABI PRISM Linkage Mapping Set MD-10. Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on chromosome 5q were also analyzed. The significance of LOD scores was tested with simulation analyses using FASTLINK. Haplotypes were constructed using Simwalk2.
Results: MYOC or OPTN mutations were excluded in all family members. A maximum LOD score value of 4.82 at θ=0.00 was obtained for the marker D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at θ=0.00. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32 within a region of 36 Mb flanked by D5S2051 and D5S2090. Screening of the novel WDR36 glaucoma-associated gene, which lies centromeric to the disease interval, revealed no mutations within any of the 23 coding exons or splicing junctions.
Conclusions: Our results provided the mapping of a novel locus for JOAG at 5q and excluded coding or splicing junctions mutations within the WDR36 gene.
Glaucoma is a leading cause of vision impairment and blindness in both developed and developing countries. Primary open angle glaucoma (POAG) occurs most frequently, accounting for more than 50% of all cases of the disease . Juvenile-onset primary open angle glaucoma (JOAG) is a subset of POAG that appears earlier in life and is often inherited in an autosomal dominant manner .
POAG is a genetically heterogeneous disorder, but the molecular basis of most cases remains unknown. Seven chromosomal loci have been linked with POAG: 1q23 (GLC1A) , 2cen-q13 (GLC1B) , 3q21-q24 (GLC1C) , 8q23 (GLC1D) , 10p15-p14 (GLC1E) , 7q35-q36 (GLC1F) , and 5q22.1 (GLC1G) . A genome-wide scan involving an initial pedigree set of 113 affected sib-pairs and a second pedigree set of 69 affected sib-pairs has reported additional association to regions 2p14, 14q11, 14q21-q22, 17p13, 17q25, and 19q12-q14 . Another genome-wide scan on 146 POAG families of African descent suggested possible linkage to 2q33.3-q37.3 and 10p12-p13 . A recent genome-wide scan revealed putative glaucoma loci on 9q22 and 20p12 in 25 families with juvenile-onset POAG . Among these loci, only three of them (1q21-q31, 9q22, and 20p12) contributed to JOAG while the others exclusively accounted for adult-onset POAG [3,12]. However, none of these loci showed linkage with POAG in a study of eight Finnish POAG families .
To date, only three genes have been identified from these loci. The first gene identified from GLC1A is myocilin (MYOC; OMIM 601652) . In Caucasians, about 2-4% of POAG cases are due to MYOC mutations [15-17], although it can be as high as 36% in JOAG families . In our previous studies, the prevalence of MYOC mutations was about 1.1-1.5% in Chinese POAG patients [19,20]. The second gene for POAG was identified from GLC1E as optineurin (OPTN; OMIM 602432) . Mutations in OPTN were initially found in 16.7% of families with hereditary and adult-onset POAG and 12% of sporadic patients with POAG, the majority of them with intraocular pressure (IOP) of less than 22 mm Hg . Two subsequent studies on Caucasian POAG patients reported no glaucoma-causing mutations in OPTN. One study involved 801 patients of variable age onset  and the other had 86 adult-onset patients . A study of 148 Japanese patients with normal tension glaucoma and 165 with POAG also found no specific glaucoma-causing mutations in OPTN . We found OPTN mutations to account for 1.6% of sporadic POAG in Chinese patients . The third gene for POAG was recently characterized as the WD repeat domain 36 (WDR36; GenBank NM_139281) gene at GLC1G . WDR36 is a novel gene with 23 exons and encodes for a 951 amino acid protein with several WD40 repeats. In the original study reporting WDR36 as the GLC1G gene, four mutations were found associated with more than 5% of all sporadic cases of POAG.
Besides the 17 aforementioned genetic loci, additional loci are expected to be involved in the etiology of this group of eye disorders. In this study, we present the chromosomal mapping of a novel locus to the 5q region in one JOAG family.
As previously reported a large family was recruited from the Ibanez region of the Philippines . The study protocol was approved by the Ethics Committee for Human Research, the Chinese University of Hong Kong. In accordance with the tenets of the Declaration of Helsinki, informed consent was obtained from all living family members after explanation of the nature and possible consequences of the study. This five-generation family had a total of 95 members, in which 22 were affected with JOAG. Complete ophthalmic examinations were given to 27 family members, in which nine were confirmed JOAG patients. Peripheral venous whole blood from these subjects was collected for genomic DNA extraction. The other family members did not agree to participate in this study. Their clinical information was obtained through previous medical records. The incompleteness of the pedigree structure might pose a founder-effect that may increase the possibility of false-positive linkage. JOAG was defined as meeting all of the following criteria: exclusion of secondary causes (e.g., trauma, uveitis, or steroid-induced glaucoma), anterior chamber angle open (grade III or IV gonioscopy), an IOP greater than or equal to 22 mm Hg in both eyes by applanation tonometry, characteristic optic disc damage or typical visual field loss by Humphrey automated perimeter with the Glaucoma Hemifield Test, and diagnosis before age 35. Visual acuity was determined using Snellen eye chart. For the affected subjects, age at diagnosis ranged from 12 to 33 years (mean±SD: 19±4.2 years), the highest IOP from 24 to 44 mm Hg (mean±SD: 32±6.3 mm Hg), and cup to disc ratio from 0.7 to 0.9 (mean±SD: 0.8±0.04).
Genotyping was carried out using the ABI PRISM Linkage Mapping Set MD-10 (Applied Biosystems, Foster City, CA) comprising 382 microsatellite markers with an average spacing of 10 cM. Polymerase chain reactions (PCRs) were performed following the manufacturer's protocols in a Perkin-Elmer 9700 thermocycler (Applied Biosystems). The PCR products were subsequently pooled and separated on a 5% denaturing polyacrylamide gel in an ABI 377 DNA sequencer (Applied Biosystems). The Genescan and Genotyper software packages (Applied Biosystems) were used to call genotypes. The GenoPedigree and GenBase software packages (Applied Biosystems) were used to draw pedigree and to export data for linkage analysis. Family relationships were confirmed by observation of Mendelian inheritance of genotypes of microsatellite makers from all panels of the ABI Linkage Mapping Set MD-10 using PedCheck program , which also provided significant error-checking for the genotyping. For fine mapping, an additional 45 microsatellite markers flanking the promising region on chromosome 5 were analyzed in a similar fashion.
Two-point linkage analyses were performed with the MLINK and ILINK programs from the FASTLINK version 4.1P software package [28,29]. Regions containing markers that yielded LOD scores >3.0 were further analyzed by multipoint analyses using the LINKMAP program. According to Sarfarazi et al. , the JOAG gene frequency was set as 0.0001. An autosomal dominant mode of inheritance was used with one liability class with penetrance values 0, 1, 1, respectively, since no skipped generations were observed in this family. The significance of LOD scores was tested with simulation analyses using FASTLINK version 4.1P. Haplotypes for markers from the fine mapping regions were constructed using Simwalk2 version 2.83 .
The MYOC and OPTN genes were screened for sequence alterations by PCR and direct sequencing as previously reported [19,25]. In addition, the MYOC promoter polymorphism, MYOC.mt1 (-1000C>G), was investigated by restriction endonuclease assays as previously reported . The WDR36 gene was screened as follows. Primers used to obtain the initial amplicons are given in Table 1. Initial PCRs were performed on an Applied Biosystems Gene Amp PCR System 9700 (96 wells, Applied Biosystems) in a total volume of 50 μl containing 100 ng of genomic DNA, 10 pmol of each primer, 200 μM dNTPs, 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, and 1 U of platinum Taq DNA polymerase (Invitrogen, Burlington ON). Cycling conditions were as follows: the first denaturation step of 5 min at 95 °C, 35 cycles of denaturation (95 °C for 30 s), annealing (56 °C for 30 s), elongation (72 °C for 30 s), and a final single elongation step of 7 min. PCR products were diluted in 5 volumes of PB buffer (Qiagen, Mississauga ON), transferred on a Whattman GF/C filter plate, washed twice with 80% ethanol/20 mM Tris (pH 7.5), and eluted in 50 μl of water. Samples were quantified by the PicoGreen reagent protocol. A second PCR was performed using the sequencing primers as described in Table 1 on an Applied Biosystems Gene Amp PCR System 9700 (96 wells) or 9700 Viper (384 wells) machines to incorporate the sequencing dyes (Big Dyes, version 3.1 from ABI), using a protocol of 25 cycles of denaturation (95 °C for 10 s) and annealing (55 °C for 5 s), followed by one last step of elongation (59 °C for 2 min). PCR products were purified by the ABI ethanol-EDTA precipitation protocol, collected using a Beckman-Coulter Allegra 6R centrifuge (Beckman-Coulter, Fullerton, CA), and resuspended in a 50% HiDi-formamide solution. Samples were then run on an Applied Biosystems Prism 3730xl DNA Analyzer automated sequencer. Sequence data were analyzed using the Staden preGap4 and Gap4 programs. Each amplicon was sequenced on one strand. As no variations or discrepancies were observed compared to the wild-type sequence, the complementary strand was not sequenced.
As previously reported , the five generations of vertical inheritance of the JOAG phenotype displayed a direct male-to-male transmission with similar numbers of affected males and females. It was consistent with an autosomal dominant pattern of inheritance. Examination of the Mendelian inheritance of genotypes of all microsatellite markers showed that most markers in one male subject in the third generation had a Mendelian inheritance inconsistent with those in the other family members, indicating he was not a genetically related member of this family. Thus he and his son, together with his spouse, were excluded from our linkage analysis. To increase the statistical power, 23 informative meioses were used in our linkage analysis (Figure 1). Among them, there were a total of eight JOAG patients. For the purpose of linkage analysis, only JOAG patients were considered affected. The other family members were considered as normal or unaffected. Because the minimum and median ages at diagnosis were, respectively, 15 years (V:12) and 20 years old, this pedigree was considered a juvenile-onset, but not an adult-onset, open-angle glaucoma family.
Mutation screening of MYOC and OPTN
Since MYOC and OPTN are associated with autosomal dominant open angle glaucoma [14,21], we screened both genes for sequence alterations in all 27 family members whose blood samples were available. Three polymorphisms (R76K, -83G>A, and -1000G>C) in MYOC and four polymorphisms (T34T, M98K, R545Q, and IVS7+24G>A) in OPTN were found in this family. However, none of them segregated with JOAG and were thus not associated with glaucoma in the family .
After exclusion of MYOC and OPTN as disease-causing genes in our family, a genome-wide search was carried out using the ABI MD-10 marker set. Microsatellite markers from this set are spaced on a 10 cM average grid on all 22 autosomes. Following a first scan, markers located in three regions on chromosomes 2, 5, and 7 demonstrated two-point LOD scores >1.0 (Table 2). Among these three regions, one marker at D5S436, on the long arm of chromosome 5 band q32, gave a LOD score value of 2.41 at θ=0.00, showing a suggestive linkage to JOAG. Two adjacent markers at loci D5S410 and D5S471 gave LOD score values of 1.48 and 1.05, respectively. This potential chromosome region on 5q23-q33 between D5S471 and D5S410 was thus further evaluated by fine mapping. The other two regions on chromosomes 2 and 7 that gave two-point LOD scores >1.0 (Table 2) were not further pursued in this study.
In the flanking region of D5S471 and D5S410, 45 additional microsatellite markers were genotyped. Linkage analysis of most of these markers gave positive LOD score values (Table 3). A maximum two-point LOD score of 2.41 at θ=0.00 was obtained for D5S436. Markers D5S1505, D5S2098, and D5S402 also had LOD score values over 2.0. Multipoint analysis of these markers did not increase the LOD score values. When only affected meioses were used in two-point linkage analysis, a maximum LOD score of 1.81 at θ=0.00 were obtained for D5S2065, D5S1384, D5S2098, D5S2011, and D5S638. When the affected status of the unaffected family members was considered as normal for those with age greater than or equal to 35 years or as unknown for those with age <35 years in two-point linkage analysis, a maximum LOD score of 4.82 at θ=0.00 were obtained for D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at θ=0.00. We used FASTLINK to generate 10,000 unlinked replicates with allele frequencies of D5S2011 and then computed the best LOD score for each replicate. No replicates exceeded the true LOD score of 4.82 and gave an empirical p<0.0001.
Haplotype analysis and recombination mapping
A total of 12 microsatellite markers within the flanking region of D5S471 and D5S410 were used to construct the haplotypes (Figure 1). At the time of study, inspection of the haplotype transmission data identified a common disease haplotype that has been clearly inherited by all seven affected subjects (II:3, III:2, III:6, III:8, III:12, IV:10, and IV:11) in this family, by the founder patient (I:1), and by three other at-risk subjects at 7 (IV:1), 3 (IV:3), and 13 (IV:8) years of age. These three phenotypically normal subjects carry the disease haplotype, but were still too young to show any sign of glaucoma and may develop glaucoma at some time in future.
Further inspection of the haplotypes in this pedigree revealed two critical recombination events in two affected individuals (IV:10 and IV:11) that limited the location of the JOAG locus telomeric to D5S2051 and centromeric to D5S2090, within a region of about 36 Mb (Figure 1, Figure 2). This region was located on 5q22.1-q32.
Screening the WDR36 gene at GLC1G for mutations
Recently, WDR36 was characterized as the third glaucoma gene . The gene mapped at the GLC1G locus on chromosome 5q22.1. Even though WDR36 localized centromerically to the present disease interval at chromosome 5q22.1-q32 between positions 111,037,156 bp and 147,210,429 bp using the Human Genome Sequence (Build 35.1), the discrepancy that currently exists between the genetic and physical maps may still position WDR36 within our disease region, thus making the gene a good candidate for the disorder in our family. We therefore screened for mutations in all 23 coding exons and splicing junctions of the WDR36 gene in three affected patients carrying the disease haplotype (subjects II:3, III:6, and III:8). Two asymptomatic persons, spouse III:3 and subject III:4, who did not harbor the disease haplotype, were used as controls. No sequence variations were detected in any of the three affected nor the two control persons in the 23 exons and splicing junctions. All sequences obtained were wild-type. Our data thus excluded coding sequences and splicing junctions of the WDR36 gene as sites for potential mutations.
Candidate gene search
Within the 5q22.1-q32 region between D5S2051 and D5S2090, a total of 311 genes have been annotated in the NCBI Map Viewer (Build 35.1). Such a large number of genes pose technical difficulties for candidate gene search by mutation analysis. It will be helpful to further confine the interval by recruiting more family members to identify additional recombinational events.
To date at least 15 genetic loci have been linked to POAG. Among those, there is evidence that JOAG can be caused by multiple independent genes located at heterogeneous loci . The mapping of the first locus for JOAG was followed by the localization of several loci predicted to be involved in this severe type of glaucoma . Two additional loci have been recently described . In this study, we provide evidence for a novel locus that should harbor a gene responsible for JOAG, excluding MYOC, OPTN, and WDR36. We detected significant linkage with DNA markers at 5q23-q33. Fine mapping with additional markers within this region supported the linkage. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32. Monemi and colleagues  reported a probable adult-onset POAG locus on 5q33-q35, a region of approximately 36 cM between D5S2497 and D5S498. They termed this locus GLC1G and claimed that it had been narrowed down to an interval of <5 cM by using additional small families, from which one potential candidate gene was proposed . More recently, the same group further refined their mapping interval, and new data resulting from discrepancies between the genetic and physical maps positioned several families previously mapped to the 5q33-q35 region more centromeric to chromosome 5q22.1. Saturation mapping of these linked families was subsequently performed. Meanwhile, Samples et al.  mapped a new POAG locus to a 3.8 Mb region between D5S2084 and D5S492 on chromosome 5 in a large Oregon family. By combining data obtained from Samples et al. , Monemi et al.  finally identified the third POAG gene as WDR36 at GLC1G on 5q22.1.
The JOAG locus identified in our study was very close to but did not overlap with the GLC1G minimal interval between D5S1466 and D5S2051 (Figure 2). However, discrepancy between the genetic and physical maps might still position the WDR36 gene within our disease interval. We therefore screened WDR36 for mutations and did not find any sequence variations in the coding exons or splicing junctions of the WDR36 gene. Although we cannot rule out other variations outside these regions (within the introns or the promoter sequence), our data do suggest the presence of a novel JOAG gene on 5q. In addition, our haplotype analysis and recombination mapping excluded the interval between D5S2084 and D5S492 from being the disease-causing locus in our pedigree. This whole region had been reported by Samples et al. . Since the 5q22.1-q32 region covers a large distance of about 36 Mb, recruitment of more family members and further mapping should help to refine this region. We conclude that a novel genetic locus on 5q22.1-q32 has been identified for juvenile-onset POAG.
We are grateful to the family who participated in this study. This study was supported in part by a block grant of the Chinese University of Hong Kong, a direct grant (2040997) from the Medical Panel, the Chinese University of Hong Kong, and in part from the Canadian Institutes of Health Research, grant number MOP-64219. The authors thank Shanghai Genecore Biotechnologies for technical assistance in some of the genotyping work. VR is a Fonds de la Recherche en Santé du Québec National Investigator.
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