Molecular Vision 2004; 10:260-264 <http://www.molvis.org/molvis/v10/a33/> Received 17 October 2003 | Accepted 1 April 2004 | Published 2 April 2004

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Exclusion of 14 candidate loci for primary open angle glaucoma in Finnish families

Susanna Lemmelä,¹ Tero Ylisaukko-oja,^1,2 Eva Forsman,³ Irma Järvelä^1,4
 
 

¹Department of Medical Genetics, University of Helsinki; ²Department of Molecular Medicine, National Public Health Institute; ³Population Genetics Unit, Folkhälsan Institute of Genetics; ⁴Laboratory of Molecular Genetics, HUCH-Laboratory Diagnostics, Helsinki, Finland

Correspondence to: Irma Järvelä, MD, PhD, Laboratory of Molecular Genetics, HUCH-Laboratory Diagnostics, PO Box 140, 00290 Helsinki, Finland; Phone: +358-9-47175905; FAX: +358-9-47174001; email: irma.jarvela@hus.fi

Abstract

Purpose: The aim of the present study was to examine the genetic background of primary open angle glaucoma (POAG) in the Finnish population by analyzing previously reported candidate loci GLC1B on 2cen-q13, GLCIC on 3q21-q24, GLC1D on 8q23, GLC1F on 7q35-q36, as well as other candidate regions on chromosomes 2p14, 2q33-34, 10p12-13, 14q11, 14q21-22, 17p13, 17q25, and 19q12-14. In addition, we analysed loci for the MYOC gene on 1q23-24 and the OPTN gene on 10p14-15.

Methods: Eight Finnish families (92 family members; 27 individuals with POAG; 19 individuals with ocular hypertension or glaucoma suspicion) were genotyped using 35 microsatellite markers on the candidate loci and analyzed for linkage.

Results: No significant evidence for linkage was found in two point and multipoint linkage analyses to the tested markers in the analyzed families.

Conclusions: Our results support further genetic heterogeneity underlying POAG and encourage a search of novel genetic loci and predisposing genes in order to understand the genetic mechanisms underlying POAG.

Introduction

Glaucoma is one of the leading causes of blindness worldwide. It is a heterogeneous group of disorders that have in common a characteristic optic neuropathy with associated visual field loss. Primary open angle glaucoma (POAG) has been verifiably indicated to have a strong genetic component; still a simple Mendelian model of inheritance does not adequately explain the inheritance of this disorder. Consequently, POAG is likely to have a complex genetic etiology. Several susceptibility genes and so far unrecognized environmental factors are supposed to lie behind the glaucoma phenotype. The first identified POAG locus, GLC1A on 1q23-24 [1], includes trabecular meshwork-induced glucocorticoid response protein (TIGR) [2], also known as myocilin (MYOC). TIGR/MYOC plays an important role in the pathogenesis of autosomal dominant juvenile glaucoma with high intraocular pressure and is involved in a small but significant subset of adult onset POAG. The second glaucoma susceptibility gene, optineurin (OPTN), was identified on chromosome 10p14-15 in locus GLC1E [3,4]. Sequence alterations in this gene were found in 16.7% of hereditary forms of normal-tension glaucoma in Caucasian families. In a subsequent study, Met98Lys mutation in the OPTN gene, previously found to be associated with an increased risk of glaucoma [4], was not associated with POAG in glaucoma patients of North American origin [5]. A recent Chinese study revealed new putative mutations and polymorphisms in the OPTN gene and suggested a different mutation pattern of OPTN in Chinese individuals than in Caucasians [6]. No mutations but some polymorphisms have been identified in the OPTN gene in Japanese patients with POAG [7].

In addition to GLC1A and GLC1E, several genetic loci contributing to adult onset POAG susceptibility have been identified so far. Regions on chromosomes 2cen-q13 (GLC1B) [8], 3q21-24 (GLC1C) [9], 8q23 (GLC1D) [10], and 7q35-36 (GLC1F) [11] have been linked to hereditary glaucoma, in most cases by using a small number of large families exhibiting the autosomal dominant mode of inheritance. Additional loci on chromosomes 2p, 14q, 17q, 17p, and 19q were reported in a genome-wide scan for adult-onset POAG based on sibpair multipoint analysis [12]. Another genome-wide scan for POAG in families of African origin indicated putative linkage to chromosome 2q and 10p and yielded positive LOD scores on chromosome 14q [13]. The regions on chromosome 2 and 10 do not overlap with GLC1B and GLC1E.

The most common form of glaucoma is adult onset primary open-angle glaucoma (POAG), but there are several secondary variants, including exfoliative glaucoma (EG). One of the symptoms in EG is exfoliation on the lens capsule and anterior segment of the eye. Exfoliation is commonly diagnosed in Scandinavian countries, where its prevalence is 20-30% in over 50 year old individuals [14,15]. No loci associated with this variant have been identified so far.

We have previously excluded the coding regions of the TIGR/MYOC and OPTN genes in eight Finnish families with POAG and EG [16]. Two polymorphisms were found in the TIGR/MYOC gene and three polymorphisms in the OPTN gene. One of the identified OPTN polymorphisms was novel. In the present study, we have analyzed the same family material for linkage at four previously reported susceptibility loci for POAG (GLC1B on chromosome 2, GLC1C on chromosome 3, GLC1D on chromosome 7, GLC1F on chromosome 8), and regions with putative linkage on previous genome-scans [12,13] on chromosomes 2, 10, 14, 17, 19. In addition, we analyzed the GLC1A and GLC1E loci in order to exclude intronic and promoter regions of the TIGR/MYOC and OPTN genes.

Methods

Family material

A total of eight families were included in this study, three of them having POAG as the only diagnosis and five had both POAG and EG or ES (exfoliative glaucoma and exfoliative syndrome, Table 1). In all families, the inheritance of the disease resembled that of the autosomal dominant trait. Altogether, 92 family members from these families were included in the genotyping. Of these, 27 were classified as POAG patients with narrow diagnostic classification (liability class 1, LC1), 19 family members had POAG-like features or ocular hypertension and were included as affected in a broad phenotypic category (liability class 2, LC2), and 46 were considered as unaffected. EG was diagnosed in three patients. These individuals were considered to be unaffected. The clinical characteristics and pedigree structures of the families under study have been described earlier [16]. The study was approved by the ethical committee of the Helsinki University Hospital.

Genotyping

DNA was extracted from 10 ml of peripheral blood using a genomic DNA purification kit (PureGene®, Gentrasystems, Minneapolis, MI) according to the manufacturer's instructions. PCR reactions were performed in 15 μl reaction volumes containing 20 ng of genomic DNA, 6 pmol of both primers, 0.2 mM of dNTP, 1.5-3.0 mM MgCl₂, 10 mM TrisHCl, 50 mM KCl, 0.1% Triton X-100, and 0.23 U Dynazyme polymerase enzyme (Finnzymes Oy, Espoo, Finland). The reactions were performed using an MJ Research thermocycler and the hotstart procedure, that is, polymerase enzyme was added only after the first denaturation step of 5 min at 95 °C. Polymerase chain reactions were carried out in 35 cycles as follows; 30 s at a temperature specific for each primer (50-60 °C), 30 s at 72 °C, and 30 s at 95 °C. An elongation step of 5 min at 72 °C terminated the reaction after the last annealing.

Markers were selected from the Marshfield Medical Research Foundation map. The primer sequences were from the Genome Database. RepeatMasker was used for planning intragenic markers for the TIGR/MYOC and OPTN genes. The primers for these markers were designed using the Primer3 program (version 0.2) and the primer sequences are available from the authors. Forward primers were labelled at the 5'- end with 6-FAM, TET, NED, VIC, PET, or HEX fluorescent dye. PCR-products were pooled and electrophoresed on an ABI 377 or ABI3730 DNA sequencer (Applera Corporation, Norwalk, CT). Two independent individuals assigned the genotypes using the Genotyper 2.0 or Genemapper 3.0 softwares (Applera Corporation). Genotype errors were checked using the PEDCHECK (version 1.1) computer program [17].

Statistical analysis

Two point linkage analyses were performed using the MLINK program of the LINKAGE package [18,19]. Allele frequencies were derived from the data by the DOWNFREQ 2.1-program [20]. An affected-only approach was taken in all analyses due to late onset of this disease and lack of reliable penetrance ratios. The analyses were made under the assumption of an autosomal dominant mode of inheritance with a low phenocopy rate (0.01, 0.75, 0.75) and a rare disease allele frequency (0.0001). The LOD scores were generated under both homogeneity and heterogeneity. Tests for heterogeneity and calculations of proportion of linked families (α) were carried out using the HOMOG 3.35 program [21]. We employed the non-parametric option of SIMWALK2.83 for multipoint analyses since it is capable of utilizing the complete information of the complex pedigrees in the current data set. We considered the statistics B of SIMWALK, which is based on the maximum number of alleles descending from any one-founder allele [22].

Results

Ninety-two family members of eight Finnish families were analyzed by pairwise linkage analysis using 35 microsatellite markers on 14 loci. All markers were tested using the affected-only autosomal dominant model. The results of the two point LOD score analyses are shown in Table 2. No evidence for linkage was obtained at any of the 14 candidate loci including GLC1A, GLC1B, GLC1C, GLCID, GLC1E, GLC1F, 2q, 2p, 10p, 14q, 14p, 17q, 17p, and 19q. The slightly interesting region in the analyses was on chromosome 8 with markers D8S257 and D8S1471, which showed maximum LOD scores of 0.27 (LC1, α=1.00, θ=0.08) and 1.24 (LC2, α=0.83, θ=0.10), respectively. These two markers were located some 12 cM apart. The surrounding markers did not give any evidence of linkage. Non-parametric multipoint analysis was performed for markers on chromosome 8 under the broad phenotypic category. Statistic B of Simwalk 2 showed the highest evidence for linkage at marker D8S592 (-log₁₀(P)=0.560). The best markers at MYOC and OPTN loci were D1S2815 (Z_max=0.62, LC2, α=1.00, θ=0.24) and D10S1721 (Z_max=0.60, LC2, α=1.00, θ=0.18), respectively.

Discussion

Although a genetic component for glaucoma is well established, only two predisposing genes are known to date. Mutations in the TIGR/MYOC (myocilin) gene have been found, mainly in juvenile open angle glaucoma, but also in elderly populations with POAG [2], whereas mutations and/or polymorphisms of the optineurin gene (OPTN) have been shown to associate with POAG and normal tension glaucoma [4-7]. We have previously excluded the coding regions of these genes in the eight Finnish families [16].

In the present study, we have examined the same eight families [16] for a total of 14 chromosomal loci. These included the TIGR/MYOC (GLC1A) and OPTN (GLC1E) loci to exclude also the intronic regions of these genes in our families. None of these regions showed statistically significant evidence for linkage. Two markers, D8S257 and D8S1471 on chromosome 8 at locus GLC1D provided slightly positive pairwise LOD scores. These markers were among the four best markers in original genome scan by Triftan and colleagues [10]. However, the non-parametric multipoint analysis at this locus did not yield increased linkage. Consequently, raised pairwise LOD scores were overestimates, probably due to recombinations revealed in multipoint analysis but not shown in pairwise linkage analyses. These negative results together with earlier exclusion of TIGR/MYOC and OPTN genes in these families suggests that different, still unknown, susceptibility genes cause glaucoma in the families in current data set.

The Finnish population is one of the best-studied genetic isolates. Finland is characterized by enrichment of several inherited diseases and their mutations that are common in this population due to the founder effect and genetic drift [23,24]. Families selected to this study originated from fishing villages around the Tammisaari region on the south coast of Finland. This region was first inhabited some 10,000 years ago and is called the early settlement area [23]. Swedish migrants arrived in the twelfth century. At present, these areas belong to the Swedish speaking areas of Finland and are subisolates, which are nearest to the European gene pool. Focusing on a small subisolate should provide both genetic and environmental homogeneity, and it is probable that these families have inherited the same predisposing alleles identical-by-decent (IBD).

In conclusion, our results support further genetic heterogeneity underlying POAG. Based on this study and our earlier study of OPTN and TIGR/MYOC, the known genes and loci are not as important in the etiology of glaucoma as originally thought, and do not play a role in the etiology of POAG in our family material. A search for novel genes and novel mutations responsible for glaucoma is important in order to understand the genetic component of the disease. This study encourages the genome-wide screen to reveal the susceptibility loci and predisposing genes in this material.

Acknowledgements

We are grateful for the families who participated in the study. Financial support of Finska Läkaresällskapet, Glaucoma LUX Foundation, and the Sigrid Jusélius Foundation, is acknowledged.

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