CONGENITAL GLAUCOMA

The genetics of primary congenital glaucoma

Richard A. Armstrong D.Phil

P
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rimary congenital glaucoma (PCG) is a rare disorder accounting for between 0.01% and 0.04% of cases of total blindness.1 Although present at birth, the condition may not be recognised until infancy or even early childhood. The impact on the visual development of the child can be considerable and early recognition followed by appropriate therapy can significantly improve future quality of life. In the majority of cases, PCG is bilateral and results from interference with the outflow of aqueous humor from the eye due to a congenital defect in the development of the anterior chamber. Male infants may be at greater risk compared with females. PCG may occur as an isolated anomaly or be associated with a number of more complex syndromes such as neurofibromatosis. Although the majority of cases occur more or less sporadically in the population, there is increasing evidence that genetics may play a role in the development of the disorder. A number of cases are now recognised as familial, and within affected families the disorder exhibits either an autosomal recessive pattern of inheritance or a polygenic inheritance pattern, ie involving several genes and the environment. A previous article in the genetic series2 described the patterns of inheritance associated with juvenile and adult-onset glaucoma, the genes that have been linked to these conditions, and considered how the effects of specific gene defects could lead to the development of glaucoma. This article concentrates exclusively on PCG and considers: 1) the clinical and pathological features of the disorder, 2) the development of the anterior chamber, 3) the differential diagnosis of PCG, 4) the genetic factors that may be associated with PCG, and 5) how the presence of abnormal genes might cause PCG.

Figure 1: Section through a human eye showing the typical features of buphthalmos. Note in particular the enlarged cornea, the deep anterior chamber, atrophy of the iris, the thin sclera, and cupping of the optic disc.
blepharospasm, and a hazy enlarged cornea. Of these symptoms, photophobia is probably the most consistently present and this symptom may persist into adolescence. However, the presence of any of these signs in an infant should arouse suspicion of PCG. A cloudy cornea begins as a slight hazy opacity but later the cornea becomes more opaque with the development of stromal swelling. Increase in the size of the cornea is usually the result of a greater tension on the relatively elastic cornea and sclera of the infant eyeball and therefore the condition is usually accompanied by a general enlargement of the eye. The diameter of the infant cornea is normally 10-11mm increasing to 12mm at one year of age. As a consequence, a diameter of greater than 12mm should be considered as an indication of possible PCG in infants. Increase in the size of the cornea is also associated with an elevation of intraocular pressure (IOP). Breaks occur in Descemets membrane beginning at the periphery of the cornea parallel with the limbus. Later these breaks may begin to affect the central regions of the cornea. The optic disc may remain normal, despite the high IOP, due to the yielding characteristics of the sclera and the general enlargement of the eyeball. Nevertheless, by the time glaucoma is diagnosed in the child, the optic nerve head is likely to be abnormal. If the condition is untreated or if the pressure is not adequately controlled, then typical optic disc changes such as disc pallor and cupping may appear. Hence, measurement of the IOP is essential in infants where PCG is suspected. Tonometry can often be accomplished in a young childs eye with a handheld instrument such as a Perkins tonometer.

Buphthalmos
The term buphthalmos refers to the typical appearance of the eye in PCG (Figure 1). Without treatment, the disease progresses slowly and the eye enlarges. In particular, the cornea becomes abnormally enlarged, thin, and bulging and is accompanied by

General features of PCG

Typical clinical manifestations of PCG include photophobia, epiphora (increased tear production),

CONGENITAL GLAUCOMA

increasing opacity. The anterior chamber becomes deeper, the pupil dilated, and the iris begins to degenerate. The sclera is thin often with a bluish tinge, the consequence of the uveal pigment showing through. Later the optic disc develops pallor, cupping, and becomes atrophic. These changes may not develop continuously in all patients but become arrested at some stage. Nevertheless, without treatment the changes can progress to complete blindness.

Differential diagnosis
The diagnosis of PCG requires the presence of buphthalmos in at least one eye together with an increase in IOP before three years of age.6 If a larger than normal eye is present, however, then there may be several possible causes.7 First, the presence of an intraocular mass such as retinoblastoma should be excluded. Second, the presence of neurofibromatosis can also result in an enlarged eye.4 Third, a diffuse enlargement of the eye may be associated with a connective tissue disorder or related to axial myopia. Fourth, if enlargement of the eye is more focal it may be attributable to staphyloma, viz., a thinned area of sclera lined by the choroid and which has a tendency to bulge under the influence of increased IOP. This condition may occur when the sclera is thinned as a consequence of high myopia or as a result of injury. Fifth, if one small eye is present, the normal sized eye may be incorrectly judged as large. Sixth, bilateral enlargement of the cornea in an otherwise healthy eye may occur in congenital megalocornea, a non-progressive enlargement of the cornea to 13mm or greater. However, in this condition there is no photophobia or corneal swelling, no break in Descemets membrane, no abnormality

detectable in the anterior chamber angle, no cupping of the optic disc, and no significant increase in IOP.

Development of the anterior chamber

PCG is believed to result from a congenital defect in the development of the angle of the anterior chamber (Figure 2). As early as week 12 during development, a wedged shaped mass of mesenchyme can be identified at the anterior chamber angle, ie, at the junction of the papillary membrane and the lateral margins of the cornea. Within this wedged shaped portion of tissue there is a row of small capillaries, which are lined with mesoderm-derived vascular endothelial cells. By the fifth month, early trabeculae are apparent separated by intervening spaces and the capillaries are fused to form a single vessel (the canal of Schlemm), which is continuous with the collector channels and scleral vessels. Subsequently, the meshwork becomes specialised into inner uveal trabeculae, numerous intermediate layers of lamellar corneoscleral trabeculae, and a more loosely organised cribriform meshwork. Perforations of the cuboidal epithelial cells, which line the inner surface of the meshwork and which allow communication between the

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Associated disorders and syndromes

PCG may occur as an isolated condition or as part of a more complex syndrome. For example, in the case of a 4-year-old male infant with trisomy 13 (three copies of chromosome 13 are present), there was severe slowing down of thought processes and a reduction in physical movement (psychomotor retardation), abnormally low muscle tone (muscular hypotonia), cerebral convulsions, bilateral PCG leading to buphthalmos, and bilateral optic nerve atrophy.3 Moreover, in another study, an identical twin presented at birth with unilateral PCG accompanied by neurofibromatosis with spinal cord involvement.4 Hence, newborns with unilateral PCG should raise a suspicion of possible neurofibromatosis. Bupthalmos can also occur together with unilateral persistent hyperplastic primary vitreous cataract, a condition which arises from the persistence of the hyaloid vessel which joins the optic nerve to the back of the lens. In addition, there are a number of congenital anomalies in which glaucoma occurs with relatively high frequency but which develop during the teenage years or in the early twenties. These include such conditions as aniridia (complete or partial loss of the iris), hemangioma of the face (Sturge-Weber syndrome), defective development of the mesoderm (mesodermal dysgenesis), neurofibromatosis, Marfans syndrome, and Marchesanis syndrome. Hence, in a 28-year-old male with trisomy affecting part of chromosome 7, and partial monosomy affecting chromosome 15 resulting from a paternal chromosomal rearrangement (translocation), there was bilateral PCG accompanying the typical manifestations of the partial trisomy.5

Anterior surface of cilary body Limbus junction between the cornea and sclera

Canal of schlemm Posterior chamber Trabeculae

Iris
Anterior chamber

Figure 2: The anterior angle showing the Canal of Schlemm and the trabeculae. Diagram courtesy of Dr Mark Dunne, Aston University.

CONGENITAL GLAUCOMA

Disorder

Locus

Gene

Location

Gene abnormalities

Pure PCG

GLC3A

P4501B1 (CYP1B1)

2p21

136bp deletion exon III, Insertion exon II Deletion 5 end of III C/T transition at codon 355 Substitution at codon 368

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GLC3B GLC3C GLC1A Syndromes with PCG _ _ _

Unknown Unknown Myocilin _ _ _

1p36 14q24.3 1q21-q23

Unknown Unknown Homozygous mutations in intron 2 Trisomy Trisomy Trisomy

13 7 15

Table 1: Genes associated with Primary Congenital Glaucoma (PCG). Abbreviations: p = p arm of chromosome, q = q arm of chromosome, bp = base pairs, 5 = 5 prime end of DNA strand, C = Cytosine, T = Thymine
anterior chamber and the meshwork, occur from 15 weeks onward. The anterior chamber itself develops between the sixth and ninth month of foetal life as a chink in the mesoderm between the iris root and the developing trabeculum. If the mesoderm does not entirely regress in this region, an impervious layer may remain bridging the angle between the iris and the cornea and which impedes access of aqueous to the trabecular meshwork. different mutations were identified within the CYP1B1 gene,9 viz., a deletion of 136 nucleotides in exon III, an insertion of an extra cytosine nucleotide in exon II, and a larger deletion affecting the one end of exon III. The effect of all three mutations was to remove certain combinations of amino acids from the resulting protein. Subsequently, several further abnormalities of the CYP1B1 gene have been identified. First, a new-born infant presented with buphthalmos and an opaque cornea and these alterations were associated with a change in one nucleotide of the DNA, viz., a novel cytosine to thymine transition at codon 355, this change resulting in a protein shortened by 188 amino acids.10 Second, extensive clinical and genetic variation has been observed in Indian patients with PCG, the predominant genetic change being a substitution of one amino acid by another at codon 368 within the CYP1B1 gene.11 Soon after the identification of the first gene linked to PCG, eight families were found that exhibited no linkage to the 2p31 region. Hence, a second locus (GLC3B) was postulated and appeared to be located on the short arm of chromosome 1 (1p36 -36.1).8 In addition, there are at least four families not linked to either of these loci suggesting at least one more gene linked to PCG. Recently, a locus (GLC3C) has been found at 14q24.3 but at the time of writing the genes associated with this and the GLC3B locus have not been definitively identified. A major gene linked to primary open angle glaucoma (POAG) is the gene coding for the protein myocilin and this gene locus (GLC1A) probably accounts for 10-33% of juvenile cases. Abnormalities of the myocilin gene have been detected in families in which different members develop either PCG or POAG. In particular, homozygous mutations in intron 2 of the myocilin gene appear to be involved both in PCG and POAG. A more detailed discussion of the myocilin gene can be found in Armstrong and Smith.2

The genetics of PCG

The major genes that have been associated with PCG to date are summarized in Table 1. The majority of familial cases of PCG exhibit an autosomal recessive pattern of inheritance,8 ie both parents carry a copy of the mutant gene but do not themselves show signs of the condition. The first genetic site or locus associated with PCG (GLC3A) was discovered on the p arm of chromosome 2 (location 2p31). Of the original families studied, a significant proportion of individuals mapped to this locus.1,8 Subsequently, the human cytochrome P4501B1 gene (CYP1B1) was mapped to the critical region and is a gene that is expressed in the trabecular meshwork.9 Originally, three

How could genes cause PCG?

There have been several theories as to how genes might be implicated in the pathogenesis of PCG. First, PCG may result from the persistence of a membrane (Barkans membrane) over the inner surface of the anterior chamber angle. This explanation is now thought to be unlikely as there is no strong evidence for the existence of

CONGENITAL GLAUCOMA

this membrane during normal development. Second, PCG could be caused by a failure of cleavage of the mesenchyme to form the iridocorneal angle. This theory can also be discounted since there is considerable doubt as to whether this cleavage actually occurs during development. Third, PCG could be caused by a failure of apoptosis (programmed cell death) during angle development. However, apoptosis has been rarely observed during normal development of the anterior chamber angle in the human eye. Fourth, the CYP1B1 gene may be involved in a process within the cornea that regulates the secretion of fluid; production of excess fluid could then result in high IOP and PCG. Fifth, PCG could be due to a failure of differentiation or a change in differential growth rates affecting cells within the anterior chamber angle. There are histological studies of PCG that describe the tissue as undifferentiated or lacking the typical appearance of trabeculae with spaces between them, especially in the outer layers. Recent genetic studies offer some support for the fifth hypothesis, ie, there is a problem in the development of the anterior chamber angle. In many of the hereditary forms of PCG, especially those with a strong pattern of inheritance, the primary defect is in the CYP1B1 gene. This gene is expressed in the tissue of the anterior chamber and mutations of the gene interfere both with the integrity of the protein and with its ability to adopt a normal shape and as a consequence to bind iron containing compounds.12 CYP1B1 is also involved in the normal development and function of the eye by metabolising essential molecules used in the signalling pathway. There is, however, a yet unknown sequence of events that may link changes in the CYP1B1 gene to the final stages of anterior chamber development. In addition, the relationship between mutations of the CYP1B1 gene and PCG remains controversial since experimental (transgenic) mice in which the gene is deactivated (knockout mice) do not develop a typical glaucoma phenotype.13 Recent studies suggest that the myocilin gene may also be involved in PCG. The exact function of the protein is currently unknown but it is a cytoskeletal protein and is probably involved in the development of microtubules within ciliated epithelial

cells. Aberrant development or transport of materials within the microtubules of cells of the trabecular meshwork could then directly lead to increased IOP.

not achieve vision better than 20/50 (Snellen). Patients with PCG will require follow up care for the rest of their lives as subsequent elevation of IOP can occur at any age.

Treatment of PCG
In most cases, PCG is managed by surgery. Medical care may be used to manage the condition prior to surgery and to control pressure changes in the eye during surgery. The surgical intervention is designed to eliminate the resistance to aqueous outflow caused by the structural abnormalities within the anterior chamber angle. Goniotomy is one procedure that can be used and involves the removal of tissue with the aid of a goniolens, thus relieving the compressive forces on the anterior part of the uvea and the trabecular network. This procedure eliminates any resistance caused by incomplete development of the inner trabecular network. Another approach is to use trabeculotomy in which the canal of Schlemm is identified by dissection and the trabecular meshwork removed by passing a probe into the canal. This procedure can be carried out if the cornea is cloudy or opaque, an advantage over gonioscopy. The success rate for both procedures is about 80%. The prognosis is far poorer in infants with elevated IOP and if an opaque cornea is present at birth, while best outcomes are often achieved if the surgery is carried out between the second and the eighth week of life. If these procedures fail, usually after several attempts, then a filtering procedure is usually carried out involving trabeculectomy. If all of these methods fail then the use of shunts or the destruction of the ciliary body have been advocated. Intensive aftercare is usually required after these procedures and often involves several further examinations under anaesthetic. Complications of the surgery may involve hyphema (bleeding into the anterior chamber), infection, lens damage, and uveitis but the risks of the anaesthetic are generally regarded as the most serious possible complication. The corneal swelling may persist for several weeks even after successful reduction of IOP. Reduction in IOP is often an important indicator of subsequent visual quality of life, substantially better vision being observed in patients whose pressures remain no higher than 19mmHg. However, nearly 50% of children will

Discussion and conclusions

Recent studies have emphasised that a significant proportion of glaucoma cases are genetic and that genetically based forms of the disease are heterogenous. At least seven major genetic loci have now been identified in glaucoma. Five of these loci, designated GLC1A to GLC1E, are associated with forms of primary open angle glaucoma and two loci, designated GLC3A and GLC3B associated with PCG. In addition, a number of loci have been implicated in the secondary glaucomas (GLC2). One of the objectives of the molecular biological study of glaucoma is to establish how the disease develops as a result of the production of aberrant gene products. Many of the genes associated with glaucoma code for proteins that are likely to be directly or indirectly involved in the development and/or function of cells within the trabecular meshwork. The identification of specific defects in these genes is likely to lead to a better understanding of the mechanisms involved in PCG and glaucoma in general and to the development of alternative therapies to surgery. The CYP1B1 gene in particular, which is linked to congenital glaucoma, and is expressed in the trabecular meshwork, codes for a member of the cytochrome P450 group of proteins. These iron-binding proteins constitute a family of enzymes involved in the processes of xenobiotic metabolism, growth, and development. The discovery of the CYP1B1 gene in PCG emphasizes the importance of abnormalities in the molecular structure of proteins expressed in cells of the trabecular network as a cause of PCG. The identification of specific genetic defects leads to the possibility of more widespread screening for PCG especially in affected families and hence, the possibility of the identification of asymptomatic carriers of the disease. Early identification of 'at risk' parents may then enable earlier detection of PCG and intervention in the infant.

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References
See www.optometry.co.uk/references