|Molecular Vision 2007;
Received 6 July 2007 | Accepted 11 September 2007 | Published 13 September 2007
MYOC gene mutations in Spanish patients with autosomal dominant primary open-angle glaucoma: a founder effect in southeast Spain
María Pilar López-Garrido,3
1Servicio de Oftalmología, Hospital Virgen de los Lirios, Polígono de Caramanxel S/N, 03804 Alcoy (Alicante), Spain; 2Servicio de Oftalmología, Hospital General Universitario de Alicante, C/. Pintor Baeza 12, 03010 Alicante, Spain; 3Área de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Avda. de Almansa 14, 02006 Albacete, Spain; 4Servicio de Oftalmología, Hospital Arnau de Vilanova, C/San Clemente 12 46012 Valencia, Spain; 5Servicio de Oftalmología, Complejo Hospitalario Universitario de Albacete (Hospital Perpetuo Socorro), C/. Seminario 4, 02006 Albacete, Spain
Correspondence to: Julio Escribano, Área de Genética, Facultad de Medicina, Avda. de Almansa 14, 02006 Albacete, Spain; Phone: +34 967 599200 ext. 2928; FAX: +34 902 204130; email: firstname.lastname@example.org
Purpose: Primary open angle glaucoma (POAG) is a genetically heterogeneous disease resulting in optic disc cupping and visual impairment. It can be inherited as either a complex or a monogenic trait. Autosomal dominant POAG is the most frequent type of monogenic glaucoma. In this study, we investigated the role of myocilin MYOC in Spanish patients with autosomal dominant POAG.
Methods: We retrospectively analyzed the MYOC gene by PCR-DNA sequencing in five Southeast Spanish families and one Colombian family of Hispanic origin affected by autosomal dominant juvenile-onset open angle glaucoma (JOAG). We also analyzed two families with adult-onset POAG (AOAG).
Results: MYOC mutations D380A and P370L segregated with the disease in the five JOAG Spanish families and the Colombian family, respectively. Neither MYOC mutations nor cytochrome P4501B1 CYP1B1 mutations were detected in the AOAG families. The disease showed an insidious onset in D380A carriers, making early diagnosis difficult. A delay in diagnosis resulted in severe visual impairment. Topical medications were effective in controlling intraocular pressure (IOP) in D380A carriers, but 72.2% of them required surgery for long-term IOP control. Conversely, only 30% of AOAG patients required surgery. Mutation P370L was associated with a severe phenotype unresponsive to medical treatment. Analysis of the four MYOC-linked polymorphic microsatellite markers in the JOAG Spanish families revealed a common disease haplotype, indicating that the D380A mutation was inherited from the same founder.
Conclusions: This is the first evidence of a founder effect for a MYOC mutation in Spanish JOAG patients. Analysis of the MYOC gene in Spanish patients with JOAG is useful to identify at-risk individuals thus help prevent visual impairment through early treatment.
Primary open-angle glaucoma (POAG; OMIM 137760) is an optic neuropathy and a leading cause of definitive blindness in the world. Most POAG cases are clinically characterized by increased aqueous outflow resistance, which leads to elevation of intraocular pressure (IOP). Elevated IOP may result in the apoptotic death of retinal ganglion cells, associated with the characteristic cupping of the optic-nerve head and with the progressive loss of visual fields. On clinical examination, the trabecular meshwork (TM) appears to be completely normal and there is no evidence of secondary glaucoma. IOP elevation requires medical treatment and often a surgical procedure to prevent the optic nerve and visual field from irreversible damage [1-3]. Hence, detection of at-risk individuals is important because an optimal treatment at early stages can improve the prognosis.
Genetically, most POAG cases follow a complex (non-Mendelian) pattern of inheritance, which manifests clinically in adulthood (>40 years). However, juvenile open-angle glaucoma (JOAG), a subtype of POAG whose onset occurs at an early age (between 10 and 35 years), affects a minority of patients referred for glaucoma evaluation (0.7%)  and typically shows an autosomal dominant inheritance , although JOAG individuals manifest anatomic findings that are indistinguishable from POAG . JOAG patients tend to be more resistant to pharmacological therapy than adult-onset POAG (AOAG) subjects and they often require surgical treatment to control IOP.
At least 23 loci linked with monogenic POAG have been mapped [6-9] of which 14 have been named from glaucoma 1A (GLC1A) to glaucoma 1N (GLC1N). Five of these loci (GLC1A, GLC1J, GLC1K, GLC1M, and GLC1N) are linked with JOAG. So far, myocilin MYOC (located at locus GLC1A) is the only gene known to be involved in JOAG. Different prevalences of MYOC mutations in JOAG families have been reported, ranging from 8% to 36% of affected pedigrees [10-12]. MYOC mutations are also involved in 3-5% of non-Mendelian POAG cases as well as in AOAG pedigrees with autosomal dominant inheritance [13-15]. The MYOC gene encodes an extracellular glycoprotein of unknown function. It is ubiquitously expressed in many human tissues although the areas where it is more highly abundant are restricted to tissues of the eye such as the iris, ciliary body, and TM [16,17]. Interestingly, most disease-causing mutations map to the olfactomedin-like domain of the protein, which is encoded by exon 3 .
Here, we report the analysis of MYOC mutations in autosomal dominant POAG in seven families from southeast Spain and in one Hispanic family from Colombia.
Fourteen individuals with JOAG, four with juvenile ocular hypertension (JOHT), and 28 unaffected individuals (including partners) of five apparently unrelated Spanish families were recruited for genetic analysis. These families lived in the following cities in the southeast Spanish province of Alicante: Alcoy (GJ1), San Vicente (GJ2), Monovar (GJ3), Elda (GJ4), and Orihuela (GJ5). In addition, two members of one Hispanic family from Colombia, affected by severe JOAG (GJ6), and seven patients with AOAG, members of two Spanish families (GA1 and GA2), were also studied. In all cases, a family history of bilateral glaucoma was documented in three to five consecutive generations. The study protocol was approved by the Ethics Committee for Human Research of the Hospital Virgen de los Lirios and followed the tenets of the Declaration of Helsinki. Informed consents were obtained from all subjects included in the study.
Criteria for diagnosing glaucoma included IOP above 21 mmHg (measured by Goldmann applanation tonometry) with a progressive cupping of the optic disc and visual field deterioration (tested with Humphrey or MEDMONT M700 perimeters), open angles on gonioscopy with no history of angle closure, and absence of any ocular disease contributing to IOP elevation. Criteria for diagnosing ocular hypertension (OHT) were an IOP above 21 mmHg in the absence of damage of both the optic disc and visual field. JOAG was diagnosed when patients were younger than 40 years while AOAG was diagnosed when the onset occurred over the age of 40 years . We retrospectively analyzed a total of 36 eyes from 18 affected patients belonging to the five Spanish JOAG families, two eyes from the proband of the Colombian family, and 20 eyes from 10 members of the two AOAG families.
Sequence variation screening
Genomic DNA was extracted from the peripheral leukocytes of all studied subjects with the Perfect gDNA Blood Mini kit (Eppendorf, Madrid, Spain), according to the manufacturer's protocol. The methods used for identifying mutations in the three exons of the MYOC gene have been previously described .
Microsatellite marker haplotype analysis
To characterize a possible founder effect, members of the five JOAG families who carried the D380A mutation were genotyped for four microsatellite markers (D1S2851, D1S1165, D1S210, and D1S2815) located within a region of 2.48 Mb surrounding the MYOC gene, as previously described .
Age of mutation
Mutation-dating was carried out employing Bayesian multipoint population genetic modeling  with the DMLE+2.2 program. We used haplotype data and a population growth rate of 0.05 for this calculation. Chromosome map distances between markers and the disease mutation were obtained from the UCSC Genome Browser.
Penetrance of the D380A MYOC mutation was estimated by cumulative incidence functions, using Kaplan-Meier analysis  where age at diagnosis was substituted by survival time.
Clinical phenotype of patients
We studied the role of MYOC gene mutations in Spanish and Hispanic patients affected by Mendelian POAG. To that end, we analyzed five Spanish families (GJ1-GJ5) and one Colombian family of Hispanic origin (GJ6) with JOAG. Additionally, we studied two Spanish families (GA1 and GA2) affected by AOAG (Figure 1). The disease segregated as an autosomal dominant trait in all families. Although the five JOAG Spanish families had ancestors from the same province (Alicante), their pedigrees could not be interconnected by genealogical investigation.
Eleven (61.1%) and seven (38.9%) JOAG and JOHT patients of the Spanish families were male and female, respectively. The onset of the disease was insidious. The age at diagnosis ranged from 10 to 38 years (mean 23.2 years), and the mean IOP before medical or surgical care was 28.9±5.8 mmHg. However, three patients (Figure 1, subjects III:3, III:2, and III:1 from families GJ2, GJ3, and GJ4, respectively) were diagnosed early with OHT during childhood or adolescence. In two of them, the diagnosis was facilitated because they complained of haloes around lights at the age of 10 and 12 years with IOPs ranging from 30 to 46 mmHg (Table 1). Although most patients had a moderate elevation of IOP, those with delayed diagnosis presented severe optic nerve deterioration (Table 1). In most JOAG patients from these families, topical medications were initially effective in reducing IOP, but filtration surgery was often required for long-term IOP control. Some patients with very high IOPs required surgical intervention soon after diagnosis to prevent loss of vision. At the time of the study, 26 eyes (72.2%) of affected individuals were operated on to control IOP. The early diagnosed patients mentioned above were referred to the Hospital General Universitario de Alicante and underwent combined trabeculotomy-trabeculectomy as primary surgery. All operations in the eyes of the remaining patients and reoperations were trabeculectomies of Cairns. The mean IOP of affected eyes at the last ocular examination was 17.3±6.21 mmHg. At the last follow up, controlled IOP was achieved in 34 eyes (94.4%). Of these, 10 eyes (27.7%) responded to topical hypotensive medication, 12 eyes (33.3%) required one operation or more without medical treatment, and IOP was controlled in 10 eyes (27.7%) after surgery with additional topical hypotensive therapy. IOP was uncontrolled in two eyes (5.5%) at the last follow up (Table 1, subjects II:1 from families GJ3 and GJ5).
The patient of Hispanic origin was diagnosed with JOAG at 28 years of age with IOPs above 36 mmHg, high optic disc excavations, and bilateral visual field alterations (Table 1). In contrast to the majority of the Spanish patients, this subject was resistant to conventional drugs from the beginning of treatment and required early filtration surgery for an adequate control of IOPs. After surgery, his IOPs returned to normal values without medical treatment.
In the AOAG families, six affected individuals (60%) were male and four (40%) were female. Glaucoma was associated with high myopia in four subjects (80%) of the GA1 family. The mean age and IOP at diagnosis in these families was 51.9 years and 27.1±1.2 mmHg, respectively. The mean IOP decreased to 17.7±1.2 mmHg with pharmacological and surgical treatment. At the last follow-up, controlled IOP was achieved in 17 eyes (85%), and only six eyes (30%) required filtration surgery.
Analysis of MYOC mutations
Mutations in the three exons of MYOC were analyzed by direct PCR sequencing in all affected members of the eight POAG families. A heterozygous A>C transversion in codon 380, resulting in the substitution of Asp for Ala, was detected in all affected subjects of the five JOAG Spanish families (Figure 2A). This mutation clearly segregated with the disease (Figure 1) and was not found among 100 (200 chromosomes) Spanish control subjects (data not shown). Seven young carriers of this mutation, who did not show elevated IOP, were identified (Figure 1A; symbols with dots). At the time of this study, the age of these carriers ranged from 2 to 16 years. Therefore, they were considered to be at high risk of developing JOAG. We ruled out the presence of mutations in the coding region of CYP1B1 in all the families (data not shown).
Most affected individuals displayed a phenotype featured by onset in the third decade of life (mean 23.2 years). A Kaplan-Meier analysis was performed to estimate the penetrance of the D380A mutation as a function of the patient's age at diagnosis (Figure 3). The probability of remaining unaffected by the disease in carriers of the D380A mutation was null at 38 years (Figure 3). Thus, the penetrance of this mutation is estimated to be complete at this age and 50% by the age of 24 years.
To determine whether there was a founder effect for this mutation or if it emerged independently in each family, we determined disease-associated haplotypes using four highly polymorphic microsatellite markers, which spanned a region of 2.48 Mb containing the MYOC gene. A common haplotype (3-4-4-A-7) was linked to the disease in three families (Figure 1; GJ2, GJ3, and GJ5) and was not detected in any of the normal subjects. One mutation carrier (IV:2 from family GJ3) had a recombinant disease haplotype (3-7-4-A-7). In the other two families (GJ1 and GJ4), the smallest shared area was restricted to the closest microsatellite D1S2815 (Figure 1). These data clearly indicate that members of families GJ2, GJ3, and GJ5 inherited the mutation from a common ancestor and provide evidence of the same ancestral origin for families GJ1 and GJ4. The five JOAG Spanish families lived closely in the southeast Spanish province of Alicante (Figure 4), further supporting the existence of a founder effect.
The age of the D380A mutation was estimated to be 3,800 years (190 generations with 20 years each generation), using the DMLE+2.2 program and microsatellite data from patients and normal subjects (Figure 5). Similar results were obtained with the BDMC21 v2.1 program (data not shown) .
Direct DNA sequence analysis of the patient II:2 from the Colombian family revealed a C>T heterozygous transition in codon 370 of the MYOC gene (Figure 2B), which predicts the amino acid substitution P370L (Figure 1; family GJ6). His daughter (Figure 1, GJ6, subject III:1), who was three months old at the time of the study, inherited this mutation (Figure 2B).
The complete coding region of both the MYOC and CYP1B1 genes were analyzed in the two AOAG families (Figure 1). Although they showed a clear autosomal dominant pattern of inheritance, no mutations were detected in them. In contrast with the five JOAG families, the mean age at diagnosis in the two AOAG families was 51.9 years and the probability of remaining unaffected as a function of the patients' age at diagnosis was null by the age of 70 years, showing the severity of MYOC associated glaucoma (Figure 4).
MYOC mutations in autosomal dominant glaucoma in Spain
The human MYOC gene has been implicated in autosomal dominant POAG in several studies. However, knowledge of the role that this gene plays in Spanish patients with monogenic glaucoma is scarce. Here, we show the association of MYOC mutations with JOAG but not with AOAG in a total of seven Spanish and one Hispanic family with autosomal dominant POAG. We also report for the first time a founder effect in familial JOAG in southeast Spain.
In the present study, the heterozygous D380A MYOC mutation segregated with the disease in all the JOAG Spanish families was analyzed. This non-conservative amino acid substitution not only affects a highly conserved residue, located in the olfactomedin-like domain of the protein, but also introduces an apolar amino acid in the place of a negatively charged amino acid, probably impairing the correct folding of the protein. This hypothesis is supported by previous studies which have shown that this mutant myocilin, transiently expressed in human cell lines, accumulates intracellularly as insoluble aggregates and reduces its proteolytic processing [24-26]. Interestingly, the D380A myocilin mutation has been previously found only in a Spanish  and a British family  with autosomal dominant JOAG, indicating that it has a restricted distribution. To date, only one other MYOC mutation (V426F) has been identified in this type of monogenic glaucoma  in Spain. However, further studies with additional families from different regions of the country are required to ascertain the degree of either allelic or locus heterogeneity in this type of glaucoma in Spanish patients. Three additional mutations of the D380 codon in MYOC have been reported (D380H, D380G, and D380N) [15,30,31]. It has been suggested that substitution of the amino acid residue D380 with any of the reported amino acid results in a similar clinical presentation of glaucoma, which is intermediate between the severe phenotype associated with the P370L mutation and the milder clinical presentation observed in carriers of the Q368Stop mutation .
We ruled out the presence of both MYOC and CYP1B1 mutations in the two AOAG families analyzed. It remains to be investigated whether sequence variations of candidate genes such as OPTN and WDR36 are involved in the disease in these families.
Genotype-phenotype correlation and clinical practice
Although reduced penetrance has been described in some families with JOAG [32,33], our data show that the mutation D380A is completely penetrant at 38 years of age. Mutation carriers with a delayed diagnosis evolved gradually toward severe glaucomatous optic neuropathy. Therefore, carriers of this mutation must be carefully followed up to detect early signs of the disease before elevated IOP causes rapid optic nerve damage . Glaucoma is usually symptomless until its later stages. However, we found two patients with IOPs in the range of 30-40 mmHg who reported haloes around lights and who were diagnosed at the age of 10 and 12 years, respectively. This could be a symptom of early developing POAG, suggesting that the young cornea is more susceptible to gradual IOP elevation than the adult one . We observed intra-familial and inter-familial phenotype variability (Table 1), which could be due to differences in the stage of the disease at diagnosis, treatment, and/or to other genetic factors. Interestingly, the mean IOP of D380A carriers at diagnosis was slightly lower than that usually observed for other reported MYOC mutations [12-15,35,36], but further studies are required to ascertain whether these differences are significant. Some carriers of this mutation were diagnosed and treated at an early age, and they did not developed any optic or visual alterations even after 20 years of follow-up, showing the usefulness of the prompt detection of carriers of this mutation.
In contrast with the mutation D380A, the other MYOC mutation identified in this study, P370L, shows a wide distribution in populations worldwide including French, German, Greek, Indian, Japanese, and North American patients . However, to the best of our knowledge, this is the first report of this mutation in Hispanic subjects. The wide distribution of this mutation could be the result of independent de novo events in different ethnic backgrounds rather than the result of a founder effect since this transition affects a CpG dinucleotide that could be a mutation hotspot . In accordance with our results, it has been reported that the P370L amino acid substitution is associated with the most severe glaucoma phenotype caused by MYOC mutations, featured by a particularly early age at onset (around 12 years) with accompanying elevated IOPs (average maximum IOP>35 mmHg) and by a tendency to be unresponsive to medications [13,38]. The mutated amino acid is placed on a predicted double turn , therefore, this amino acid substitution could also impair the secondary structure of the protein, leading to its misfolding.
Founder effect and estimation of age of the D380A mutation
Our data clearly show that the D380A mutation was inherited from a common ancestor in at least three of the five Spanish families with JOAG. The evidence for a founder effect is supported by the presence of a shared disease haplotype (3-4-3-A-7) in combination with a common geographical origin of the families involved. In accordance with our data, founder MYOC mutations have also been reported in other populations around the world including French , Quebecoise , and Australian patients [40,41].
The estimation of age indicates that this is an ancient mutation which emerged approximately 3,800 year ago. However, this calculation needs to be confirmed because the method used to this purpose is of limited accuracy since it is based on a series of assumptions including the number of chromosomes analyzed, which was small in the present study.
In conclusion, our data provide strong evidence of a founder effect for the D380A MYOC mutation in Spanish patients and show that the genetic analysis of MYOC mutations could play a key role in the management of autosomal dominant JOAG in affected families from this country. The genetic analysis of MYOC in these families could not only contribute to identify at risk persons decades before the disease manifests phenotypically but also to their genetic counseling.
We are most grateful to the patients and their families for their enthusiastic cooperation in this study. We thank Dr. Samper-Giménez, Chairman of the Servicio de Oftalmología, Hospital Virgen de los Lirios and Dr. Belmonte-Martínez, Chairman of the Servicio de Oftalmología, Hospital General Universitario de Alicante, for supporting this project. We also thank Mrs. Carmen Cifuentes and Mrs. Mercedes Iñiguez de Onzoño for technical assistance and the following nurses that contributed by extracting blood samples: Mrs. Ana Torregrosa, Mrs. Francisca Climent, Mrs. Asunción Llinares and Mrs. Maria Victoria Avargues. María-Pilar López-Garrido is recipient of a fellowship from the Consejería de Sanidad de la Junta de Comunidades de Castilla-La Mancha. This study was supported in part by research grants PI052494 and 02021-00 from the Fondo de Investigaciones Sanitarias, and Consejería de Sanidad of the Junta de Comunidades de Castilla-La Mancha, respectively. The authors have no financial or proprietary conflicts relevant to the content of this paper.
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