Molecular Vision 2003; 9:217-222 <http://www.molvis.org/molvis/v9/a32/> Received 19 November 2002 | Accepted 29 April 2003 | Published 30 May 2003

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The role of TIGR and OPTN in Finnish glaucoma families: a clinical and molecular genetic study

Eva Forsman,¹ Susanna Lemmelä,² Teppo Varilo,³ Paula Kristo,² Henrik Forsius,¹ Eeva-Marja Sankila,⁴ Irma Järvelä^2,5
 
(The first two authors contributed equally to this publication)
 
 

¹Population Genetics Unit, Folkhälsan Institute of Genetics, Helsinki, Finland; Departments of ²Medical Genetics and ⁴Ophthalmology, University of Helsinki, Helsinki, Finland; ³Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland; ⁵HUCH-Laboratory Diagnostics, Helsinki University Hospital, Helsinki, Finland

Correspondence to: Irma Järvelä, MD, PhD, Laboratory of Molecular Genetics, HUCH-Laboratory Diagnostics, PL 140, Haartmaninkatu 2, 00290 Helsinki, Finland; Phone: 358-9-4717 5905; FAX: 358-9-4717 4001; email: irma.jarvela@hus.fi

Abstract

Purpose: The aim of the present study was to analyze the role of the two primary open angle glaucoma (POAG) genes, trabecular meshwork-induced glucocorticoid response (TIGR/MYOC) and optineurin (OPTN), in Finnish glaucoma families originating from southern coast of Finland.

Methods: In total, 136 patients were examined to determine their ophthalmological status. Genealogical studies were performed using church records. Direct PCR-sequencing of the coding regions of the TIGR and OPTN genes was performed in 11 subjects.

Results: Inheritance resembling autosomal dominant mode was detected in eight families with open-angle glaucoma. Glaucoma was diagnosed in 53 subjects, of them 44 had POAG, 7 had exfoliative glaucoma (EG), and 2 had other types of glaucoma. Of the first degree relatives, 22 out of 79 (28%) were glaucoma suspects. No mutations in these families were identified. Instead, two polymorphisms in the TIGR gene and three polymorphisms in the OPTN gene, in which one was novel, were found in three phenotypes: POAG, exfoliative glaucoma, and exfoliation syndrome.

Conclusions: Our results give evidence that novel, unidentified genes will underlie POAG and exfoliation syndrome in the Finnish population.

Introduction

A positive family history was found as an important risk factor for primary open angle glaucoma (POAG) in population based studies [1,2]. Siblings' risk to have POAG was nearly ten times higher compared with the controls in the Rotterdam Study [3]. A genetic component was first confirmed by linking familial open angle glaucoma to chromosome 1q21-31 [4]. The first glaucoma gene was mapped in juvenile onset POAG (J-POAG) [5] and called trabecular meshwork induced glucocorticoid response protein (TIGR) or myocilin (MYOC). It's mutation, GlnStop368, also has been detected in adult-onset glaucoma in different glaucoma populations [6-8]. Recently, the second gene, optineurin (OPTN), was identified at chromosome 10p14 [9]. Sequence alterations in this gene were found in 16.7% of hereditary forms of normal-tension glaucoma families. Altogether six different gene loci [10] that are connected with adult onset POAG have been identified so far. Recent molecular genetic studies support the hypothesis that glaucoma is caused by several susceptibility genes and so far unrecognised environmental factors.

The prevalence and genetics of exfoliation syndrome (ES) have been widely studied in different populations [11-14]. A variety of models for inheritance have been suggested depending on the study material. No loci associated with exfoliation syndrome have so far been identified. Tarkkanen [15] has studied several families where open-angle glaucoma without exfoliation (POAG), with exfoliation (Exfoliative Glaucoma; EG), and ES without glaucoma, were present in same pedigrees. The frequency of exfoliation was 7% among relatives of POAG patients over 60 years of age [16].

So far no molecular genetic studies have been performed in POAG and EG in the Finnish population. In the present study we have analysed the role of the two known genes, TIGR and OPTN, in Finnish families originating from the Southern Coast of Finland both with POAG and with EG by direct PCR-sequencing to find out their mutation frequency in different populations.

Methods

Patient material

The eight unrelated families with POAG and EG were collected from Tammisaari region (Figure 1). The inclusion criteria for the study were at least three known glaucoma patients in different generations including the probands who were followed up in a non-referral office by one of the authors (EF). The status of optic disc and visual fields of all glaucoma patients were reevaluated. When the patient was not available for an examination, the ophthalmologist who was responsible for glaucoma care was contacted to confirm the diagnosis. The exact glaucoma diagnosis of deceased individuals was checked in hospital records. Other family members without known glaucoma history were invited to a complete ophthalmological examination, with gonioscopy, visual fields (Humphrey Field Analyzer program 24-2) and stereo disc photos. When the glaucoma hemifield test showed a result (borderline, outside normal limits, or unreliable), visual fields were re-tested twice to confirm the findings.

The diagnostic criteria for glaucoma were two of the three characters: (1) elevated IOP>22 mmHG, (2) presence of glaucomatous changes in the optic nerve head, and (3) glaucomatous visual field defect. This criteria also covered glaucoma diagnosis in hospital records or other ophthalmologists' records. The following definitions were used for grading the newly examined subjects: The disc was graded as glaucomatous when the cup/disc (C/D) ratio was 0.6 or greater or when there was localised thinning of the rim (notching) or asymmetry of more than 0.2 in the C/D ratio between the eyes. The visual field was graded as glaucomatous when a cluster of three or more nonedge points were depressed on the pattern deviation plot at a p<5% level and at least one of them was depressed at a p<1% level [17]. Persons were classified as glaucoma suspects if only one of the diagnostic criteria was fulfilled or if a disc haemorrhage was detected.

The patient was also classified as a glaucoma patient when siblings or children of the patient had confirmed the diagnosis and reported the patient's regular use of eye drops. The study was approved by the ethical committee of Helsinki University Hospital. All participants gave an informed consent.

Genealogical studies

The genealogical study was performed in accordance with published criteria [18]. The names, dates, and places of birth of the patients' parents were used to trace ancestors back to the middle of the 1800s from local church and civil registers. Microfilm and microfiche copies of the church records in the Finnish National Archives were used for all the earlier periods.

PCR-sequencing

DNA was extracted from 10 ml of peripheral blood using a genomic DNA purification kit (PureGene®, Gentrasystems, Minneapolis, MI, USA) according to the manufacturer's instructions. The coding regions as well as flanking splice sites of the OPTN and TIGR genes were analysed. Each exon (4-16) of OPTN was amplified by PCR with primers shown in Table 1. The three exons of TIGR were amplified in six different PCR-reactions listed in Table 2. PCR amplifications were performed in a 50 μl volume containing 100-150 ng genomic DNA, 10 pmol of each primer, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl, 50 mM KCl and 0.1% Triton X-100, 125 μM nucleotide mix (dNTP) and 1U Dynazyme polymerase-enzyme (Finnzymes Oy, Espoo, Finland).

Amplification was performed in a DNA Thermal Cycler (MJ Research, Waltham, MA). Polymerase chain reaction conditions were as follows: 10 or 3 min at 95 °C followed by 35-40 cycles of denaturation step: 40 s at 95 °C, annealing step:40 s at temperature specific for each primer (50-65 °C), Elongation step: 1 min at 68 or 72 °C and final extension for 5 min at 72 °C terminated the reaction after final annealing. Sequencing was performed using cycle sequencing with Big Dye Terminator kit (version 3) supplied by Applied Biosystems (ABI, Foster City, CA, USA), and reactions were run on an ABI 3100 capillary sequencer according to the manufacturer's instructions. Mutations were first checked manually and verified by BLAST-program. All possible mutations were verified by new PCR and new sequence analysis from both strands.

Results

Altogether eight families were identified (Figure 2), five of them having POAG as the only diagnosis and three had both POAG and EG. In all families the disease was inherited, resembling autosomal dominant mode of inheritance. No common ancestors were found for these families in genealogical studies extending to the beginning of the eighteenth century.

Clinical characteristics of 136 family members in these eight families were collected (Table 3). Glaucoma was observed in a total of 53 cases; 44 of them had POAG, 7 had EG, and 2 had other types of glaucoma. The male-to-female ratio was 19:25 in POAG patients and 1:6 in EG patients. The male-to-male transmission was found in families 1 and 6 (Figure 2), but in family 6 both parents had glaucoma. One of the POAG patients had juvenile type diagnosis at the age of 21 years, while the age at diagnosis was between 40 and 81 years in the other 43 patients. The intraocular pressure (IOP) in POAG patients was at the time of diagnosis less than 22 mmHg in 8/44 (18%), between 23 and 30 mmHg in 22/44 (50%) and more than 31 mmHg in 7/44 (16%), in seven patients the IOP is unknown. Of the 79 other family members examined, none had glaucoma, but 22 (28%) were glaucoma suspects. The cause of suspicion was IOP>22 mmHg in 7/22 (31.8%), asymmetry between the discs in 8/22 (36.4%), suspicious disc in 6/22 (27.3%), disc haemorrhage in 1/22 (4.5%). According to the family history, the mothers in the first generation of four families (Figure 2) were blind because of glaucoma, but the diagnosis could not be confirmed.

A total of 11 subjects (8 with POAG, 2 with EG, and 1 with ES), representing different families and phenotypes (Figure 2), were chosen for sequencing of the coding regions of the TIGR and OPTN genes. No disease-causing mutations were identified in the TIGR gene in these families. Instead, two polymorphisms (Figure 2), Tyr347Tyr in exon 3 and Arg76Lys in exon 1, were found. Tyr347Tyr was found in one mother with EG and her son with POAG. All individuals were heterozygous for this polymorphism. Arg76Lys was present in 1/11 patient from POAG families.

Optineurin (OPTN) was sequenced for mutations in 13 exons (except the first three uncoding exons) and the flanking splice sites. No mutations were identified. Instead, two known polymorphisms (Figure 2), Thr34Thr and Glu163Glu [9] and one novel 553-5C located in intron 6, were found. Thr34Thr was detected in 6 of 11 (54.5%) of patients, from which 4 had POAG, 1 had EG, and 1 had ES. Glu163Glu was identified in one patient with EG. 553-5C was identified in 7 of 11 (63.6%) patients, 5 of which had POAG, one EG, and one ES. This polymorphism does not change splice sites or make any splice site-like formation.

Discussion

The autosomal dominant mode of inheritance was apparent in the eight pedigrees. In this study, 28% of the first degree relatives to the glaucoma patients were classified as glaucoma suspects. Their age at examination was lower than the age at diagnosis of the glaucoma patients. Their possible risk to develop glaucoma will be doubled every decade [19] in addition to their genetic risk [3]. For this group of patients the identification of the glaucoma gene/genes would be especially beneficial providing a probability with a DNA test to identify a possible glaucoma patient earlier and thus to lower a risk for a possible visual handicap in the future.

Two genes, TIGR/MYOC and OPTN, are known to be associated with POAG. In TIGR, more than 50 mutations have been identified, the majority of them being associated with juvenile onset glaucoma (JOAG). However, mutations in TIGR have also been found in elderly population with POAG [5]. Recently, optineurin gene (OPTN) was identified at chromosome 10 (GLC1E locus) to underlie both POAG and normal tension glaucoma [9]. In this study we have analyzed the role of these two genes in eight Finnish families representing three different phenotypes: POAG, EG, and ES.

No causative mutations were identified in the OPTN and TIGR genes in a total of 11 subjects. Instead, three polymorphisms in OPTN and two polymorphisms in TIGR gene were found. Two of them in OPTN, Thr34Thr and Glu163Glu, have been previously reported [9]. In addition, one novel polymorphism, 553-5C in intron 6, was identified. This polymorphism lies 5 nt from the 5' end of exon 7 and was identified both in five POAG, one EG, and one ES patient. Further studies are needed to clarify the role of 553-5C polymorphism as a risk allele for these disorders.

The polymorphism in POAG Tyr347Tyr of the TIGR gene has been reported at least in two studies [6,20]. This polymorphism is located in the third exon on the region coding for the olfactomedin-like domain (codons 324-502) [5,21,22]. This area shows a strong sequence homology to a protein found in the neuro-olfactory epithelium of the nose, called olfactomedin [23,24]. The vast majority of TIGR mutations associated with POAG lie within this region suggesting that this area plays an important, though as yet unknown, part in the pathogenesis of POAG. Interestingly, we found Tyr347Tyr in a mother with EG and her son with normal tension glaucoma (NTG; Figure 2). Since the son is only 61 years old, it is still possible he will develop exfoliation at his later age.

The second polymorphism, Arg76Lys in the TIGR gene, was identified in one POAG patient. This polymorphism has not been classified as a causative mutation because of its similar frequencies in control and primary open-angle glaucoma populations [7,25]. In addition Arg76Lys has been observed in at least four studies [26-29]. This polymorphism lies outside of olfactomedin-like region and also outside the leucine zipper region (codons 117-166) which has been implicated as another potential site for TIGR dimerisation and oligomerisation. Perhaps this sequence variation has not a critical effect on individuals phenotype because it lies on a less important region of the MYOC/TIGR gene.

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 only in this population due to founder effect and genetic drift [30,31]. Families selected to this study originated from fishing villages around the Tammisaari region on the south coast of Finland (th is also called the early settlement area) [30]. This region was first inhabitated some 10,000 years ago. A migration wave of the south genetically "founded" the current population some 2,000 years ago. Swedish migrants arrived in the twelfth century. These areas belong to the Swedish speaking areas of Finland and are subisolates nearest to European. Consequently, it could be assumed that the gene pool would be similar to that of Europeans in general.

POAG is a common disease most probably caused by many different susceptibility genes with a high prevalence also in Finland. In these kind of studies negative findings are useful since they show important data about the mutations of the novel genes in different populations. Our results provide evidence that the role of OPTN in POAG might be smaller than originally thought [9], at least in this population, and novel genes should be searched that are responsible for glaucoma. It is also noteworthy that several phenotypes, POAG, ES, and EG, were included in our families. Polymorphism Tyr347Tyr in the TIGR gene and Thr34Thr and Glu163Glu in the OPTN gene have previously been reported with POAG [6,9,20]. In this study, these variants were also present in three patients with exfoliation. The novel polymorphism 553-5C was detected in all phenotypes. A separate gene for ES has not yet been found. Thus we do not know if exfoliative glaucoma is caused by one or more genes [32]. Further studies both with families and sporadic glaucoma patients are needed to detect susceptibility genes for these different phenotypes in the future.

Acknowledgements

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

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