Molecular Vision 2006; 12:360-366 <http://www.molvis.org/molvis/v12/a41/> Received 20 October 2005 | Accepted 12 April 2006 | Published 17 April 2006

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Presymptomatic genetic diagnosis for consulters from a large Chinese family with juvenile open angle glaucoma

Yuhong Chen,¹ Deke Jiang,² Bo Wan,² Long Yu,² Xinghuai Sun¹
 
 

¹State Key Laboratory of Genetic Engineering, Department of Ophthalmology and Vision Science, Eye and ENT Hospital and ²State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China

Correspondence to: Xinghuai Sun, Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Fenyang Road 83, 200031, Shanghai, China; Phone: +86-21-64371591; FAX: +86-21-64377151; email: xhsun@shmu.edu.cn

Abstract

Purpose: To provide genetic counseling and presymptomatic diagnosis to family members by investigating the genetic cause of a large primary juvenile open angle glaucoma (JOAG) pedigree with an autosomal dominant pattern.

Methods: Ocular examinations were performed on all members of the pedigree in order to determine their disease status. Subjects were labeled as affected individuals, unaffected individuals, and suspects. Genomic DNA was extracted from the peripheral blood of the family members. Three exons of MYOC, 12 coding exons of OPTN, and two coding exons of CYP1B1 were amplified by polymerase chain reaction (PCR). Direct DNA sequencing was used to identify mutations in MYOC, OPTN, and CYP1B1. Presymptomatic diagnoses were made for the consulters based on the results of both clinical examination and genetic analysis.

Results: One heterozygous mutation in the MYOC gene was identified in all patients of the pedigree. It was a cytosine to thymine transition at nucleotide 1,109, which corresponded to an amino acid residue change from proline to leucine at codon 370. The Pro370Leu mutation correlated with a severe JOAG phenotype as previously reported. Two adolescents, who were labeled as suspects, were detected to carry the same mutation and thus had a high risk of developing glaucoma. Close follow-up at regular intervals was recommended. In addition, no pathogenic mutations of OPTN and CYP1B1 were detected.

Conclusions: The Pro370Leu mutation of the MYOC gene contributed to JOAG in this pedigree. Early onset, rapid development, poor response to medicine and high penetrance are characteristics of this mutation. Genetic analysis is valuable for providing genetic counseling and presymptomatic diagnosis to members in typical autosomal inheritance pedigrees with JOAG.

Introduction

Glaucoma is a complex, heterogeneous eye disease that affects nearly 67 million people worldwide with about 6.7 million suffering from bilateral blindness as a result [1]. A cross-sectional population study showed that glaucoma is the major cause of blindness among adult Chinese, and that primary open-angle glaucoma (POAG) is the predominant form of the disease [2]. POAG is characterized by open anterior chamber angle, atrophy of the optic nerve, loss of the peripheral visual field, and high intraocular pressure in the majority of cases. POAG varies in its age of onset and has been classified into juvenile onset: juvenile primary open angle glaucoma (JOAG) and late onset: chronic primary open angle glaucoma (COAG).

Family history is the most important risk factor for POAG in different ethnic groups, including the Chinese population [3]. First-degree relatives of individuals affected with POAG carry up to ten times increased risk of developing POAG when compared to the general population [4]. Fortunately, unlike most other genetic diseases, identification of individuals at an early or preclinical stage of POAG provides an opportunity for better treatment of the disease. Most patients can save their vision with existing treatments. Thus, presymptomatic diagnosis is not only efficient but also crucial for suspects in large families suffering from glaucoma.

MYOC (myocilin)/TIGR (trabecular meshwork inducible glucocorticoid response) was the first identified POAG gene, which was designated as GLC1A [5]. Typically, the gene co-segregates with JOAG pedigrees in an autosomal dominant inheritance pattern [6]. OPTN (optineurin) was mapped to the GLC1E locus on 10p14 [7]. Sequence alterations in this gene are often found in COAG and normal tension glaucoma (NTG) [8], but were also recently discovered in JOAG [9]. CYP1B1 (cytochrome P-4501B1) is the major gene for primary congenital glaucoma (PCG) [10]. Sequence alterations in CYP1B1 have been detected in POAG families as well, acting as a modifier of MYOC [11]. CYP1B1 mutations might pose a significant risk for early-onset POAG [12]. Each of these three genes contributes in part to POAG, among which MYOC has been extensively studied.

The incidence of mutation in MYOC was reported to be from 8% [13] to 36% [14] in JOAG patients, while only 3-5% [15] in sporadic POAG patients. Genetic analysis of glaucoma families has been studied by predictive testing of MYOC mutations [16-24]. DNA testing for those mutations with high penetrance and severe phenotype is of high predictive value. It was also reported that predictive glaucoma testing in appropriate circumstances was acceptable (96%) to patients and their families [25]. In this report we conducted DNA testing and provided presymptomatic diagnoses of glaucoma for fourth and fifth generations requesting genetic counseling from a large JOAG pedigree in China. After careful ophthalmologic examination of each member of the family, the status of glaucoma of each individual was determined. The genes MYOC, OPTN, and CYP1B1 were screened by sequencing in order to investigate the genetic cause of the JOAG pedigree. Individuals received medical counseling based on both genetic and clinical results.

Methods

Clinical examination and diagnosis of juvenile primary open angle glaucoma

Clinic examinations including visual acuity, slit lamp biomicroscopy, applanation tonometry, gonioscopy, funduscopy, and perimetry were performed. All subjects were divided into three groups: (1) affected individuals, (2) unaffected individuals, and (3) suspects. POAG is defined by a normal appearing anterior chamber angle along with two of the following criteria: elevation of intraocular pressure (IOP >21 mm Hg), characteristic visual field defects, and glaucomatous optic nerve head changes (cup-disc ratio >0.6 or notches). Subjects were subclassified as JOAG if it occurred before 35 years of age. Subjects meeting only one of these criteria were defined as suspects. Individuals without any manifestation were defined as unaffected. Patients were diagnosed and treated at the Eye and ENT hospital, Fudan University, China. All participants gave informed consent after receiving explanation of the nature and possible consequences of the study.

Genetic analysis

Genomic DNA was purified from 200 μl blood samples using the QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany). The coding sequences of MYOC (GenBank AF001620), OPTN (GenBank AF420371), and CYP1B1 (GenBank U56438) were amplified by polymerase chain reaction (PCR) [26-28]. Amplifications of exons were carried out in a 25 μl reaction system containing 50 ng of genomic DNA mixed with 10X buffer, 50 pmol primers, 2.5 mmol/l dNTP (deoxyribonucleoside triphosphate), and 1.25 U Taq polymerase (Takara, Tokyo, Japan). PCR conditions were as follows: an initial denaturation at 94 °C for 2.5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at a temperature specific for each primer for 45 s, and extension at 72 °C for one min. A final extension at 72 °C for five min completed the reaction. The reaction was performed with a GeneAmp PCR system 9600 (Applied Biosystems, Foster City, CA). Direct sequencing with forward and reverse amplification primers was performed on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) according to the BigDye Terminator version 3.1 protocol.

Three glaucoma associated genes, MYOC, OPTN, and CYP1B1, were screened in each surviving individual except III:6 whose blood sample was not available. Presymptomatic genetic diagnoses were determined for consulters (family members who seek information and instruction about their disease status) according to the phenotype of JOAG, the pattern of inheritance, their clinical status, and genetic analysis results of their family. Then follow-up plans were established for consulters.

Results

Phenotypes of the patients

The family studied comprised five generations that exhibited an autosomal dominant pattern of inheritance (Figure 1). Nine patients in total were identified with JOAG (Table 1). The information for II:1, II:3, and II:5 were not integrated because of eyeball atrophy or cornea opacity. Their diagnoses were based on medical histories.

Onset ages of all patients were under 35 years of age. The earliest onset age was 14 years old. Exact ages of the disease onset were not known for II:1, II:3, and II:5 because of relatively advanced glaucoma at the time of first presentation. Most of the patients had typical glaucoma changes in optic disk and visual field (Figure 2), with all showing more damage to the optic nerve head in the right eye than in the left.

All affected family members showed notable increase in intraocular pressure (IOP) greater than 30 mm Hg. The IOP of these patients could not be controlled with available antiglaucoma medication. Subjects II:1, II:3, and II:5 were unable to acquire proper medical therapy. However, all remaining patients underwent antiglaucoma surgeries, and subjects III:7 and IV:5 had repeated operations due to failure of their first procedures.

Clinical examination of the consulters

There were altogether ten consulters with no visible optic head damage and visual field defects (Table 2) and among which five subjects (IV:6, IV:7, IV:11, IV:12, and V:1) were labeled as suspects because of maximum IOP higher than 21 mm Hg. All suspects were younger than 25 years old. No evidence for glaucoma was found in the other five young consulters.

Mutation screen of MYOC, OPTN, and CYP1B1

Direct sequence analysis revealed a heterozygous change at nucleotide 1,109 of MYOC, from cytosine to thymine (C1109T). This base pair change results in the substitution of the amino acid proline with leucine at codon 370 (Pro370Leu). Results were confirmed by sequencing of the reverse strand. The mutation was detected in all affected members. Among the ten consulters, two adolescents (IV:11, IV:12), who were labeled as suspects, also carried the same mutation (Pro370Leu) in MYOC while the other consulters lacked the mutation (Table 2). The Pro370Leu mutation was not detected in 120 unrelated controls.

Polymorphisms in MYOC (Gly12Arg, Arg76Lys), OPTN (Thr34Thr, Met98Lys), and CYP1B1 (-13 from ATG, Arg48Gly, Ala119Ser, Val243Val, Val432Leu, and Asp449Asp) were also found, but none showed cosegregration with the disease. Furthermore, no obvious variability was observed in clinical phenotype among MYOC mutants who carried OPTN or CYP1B1 variants compared to those who did not (Figure 1).

Presymptomatic diagnosis for consulters

Diagnoses of glaucoma for consulters were revised after the Pro370Leu mutation in MYOC was identified as the disease-causing mutation in this family. The two adolescents (IV:11, IV:12) carrying the Pro370Leu mutation were defined as preclinical status and have a high risk of developing glaucoma in the next few years. However, the other suspects (IV:6, IV:7, and V:1) without the mutation are at a much lower risk for JOAG. In order to give mutation carriers proper medication at an early stage of glaucoma, follow-up plans were established. The two adolescents were asked to come for applanation tomometry and funduscopy every month and perimetry every six months.

Discussion

The MYOC gene consists of three exons separated by two introns and encodes a protein of 504 amino acids. Myocilin is a secreted 55-57 kDa glycoprotein that forms dimers and multimers [29] and has several characteristic structural motifs including a myosin-like domain, a leucine zipper region, and an olfactomedin domain [30]. More than 70 mutations in MYOC have been found, and 90% of previously reported mutations occur within exon 3 [31]. The mutations in MYOC are found in 3.86% of Caucasian patients with POAG (including normal tension glaucoma) or ocular hypertension, 3.30% of patients of African descendants (including African Americans and black residents in Africa), and 4.44% of Asian patients [31].

Characteristics of many mutations through large glaucoma pedigrees have been documented [16-20]. Among all the mutations in MYOC, Pro370Leu appears to be one of the most severe and the most common gene defect causing JOAG. The present study reveals that the Pro370Leu mutation of MYOC mainly contributed to the early onset glaucoma in the pedigree studied. Median onset age of affected individuals with Pro370Leu was 22.6 years (range: 14-31 years) in this family. All patients had surgery owing to intraocular pressure from 30-54 mm Hg (average 41 mm Hg). Our results are consistent with previous studies [13,14,32-35] that demonstrated the Pro370Leu mutation results in typical JOAG phenotypes: (1) development at an early age, (2) high peaking of IOP, and (3) poor response to medications. Furthermore, Pro370Leu covers a wide range of population groups [36]. It has been identified in families in North America [13,14,37], France [32], Japan [33], Greece [11], Germany [34], and India [35] (Table 3).

The penetrance of MYOC is age-related and varies for different mutations [14]. In our study's pedigree high penetrance of Pro370Leu was presented. At the age of 20 years or older, nine of nine (100%) carriers were affected by glaucoma; at age of 15 years or older, nine of 10 (90%) carriers were affected; and at age 10 years or older, nine of 11 (82%) carriers were affected. Thus, the two suspects (IV:11 and IV:12), who were found to carry the Pro370Leu mutation, were at a high risk of developing JOAG. It is important to closely monitor the two young boys in order to detect any early signs of the development of POAG and ensure proper medication at the early stages of the disease. For example, if the IOP rises gradually by a fluctuating shape or if any optic nerve head change appears with nerve fiber layer becoming thinner before visual field defects appear, then medication should be prescribed immediately. In this family all patients showed signs of relatively advanced glaucoma except subject III:13, who was diagnosed and treated at an early stage. This is the best example of early intervention for glaucoma. Thus, monitoring of these two young boys with early diagnosis and proper medical treatment would improve their prognoses. However, the risk of individuals not carrying the MYOC mutation in this family was no more than that of the general population in China (1.76%) [38], and the frequency of their clinical examinations could be reduced.

Ethnic difference in the frequency and types of MYOC mutations among patients with POAG has been examined by case-control mutation studies in various populations. Although the mutation pattern shows some differences in Chinese sporadic patients, frequency of disease-causing mutations in MYOC is similar to that of other ethnic groups, and no common MYOC mutation that causes POAG was found [39,40]. At present, screening tests in whole populations for MYOC defects are not feasible due to the low prevalence of MYOC-associated glaucoma. However, predictive DNA testing for people at high risk of developing glaucoma may be beneficial, especially in early onset type of glaucoma pedigrees. Thus early glaucoma therapy can then be provided to these patients before occurrence of any vision loss, perhaps even before any symptoms are observed.

Acknowledgements

We are grateful for the family who participated in this study. We acknowledge financial support of the National Natural Science Foundation of China, grant number (30024001).

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