Keywords

17.1 Introduction

Glaucoma comprises several conditions that affect the optic nerve leading to structural changes characterized by loss of retinal nerve fiber layer and optic disc cupping with corresponding visual field defects. The pathophysiology of glaucoma involves many combined mechanisms and metabolic pathways, but the main one includes the increase of intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor production at the ciliary body and its outflow through the trabecular meshwork (conventional outflow) and ciliary muscle fibers (uveoscleral outflow). In general terms, the glaucomas are classified according to their etiology, anterior chamber anatomy, and age of onset. In respect to etiology, glaucoma can be divided into primary (with no identified cause) and secondary to an ocular or systemic condition. Glaucomas can also be classified according to the anatomic characteristic of the anterior chamber angle (open angle or closed angle), and finally depending on the age of onset (adult or pediatric) [1,2,3].

In 2013, an international consortium of glaucoma specialists named Childhood Glaucoma Research Network proposed a classification of pediatric glaucomas based on clinical aspects and in the context of clinical and ocular features in which the diagnosis was made. Therefore, both primary congenital glaucoma (PCG) and juvenile open-angle glaucoma are classified as primary childhood glaucomas, since none is associated with acquired ocular anomalies, systemic disease, or syndromes [4]. Among the pediatric glaucomas, PCG is the most prevalent, with an incidence varying between 1:1250 and 1:30,000, depending on the population that is investigated. In general, the more inbred the population, the higher the incidence [5].

17.2 Epidemiology

In Brazil, there is no robust study indicating the prevalence/incidence of PCG. Available data comes from studies performed at University Hospitals. One of these studies evaluated 72 children with pediatric glaucoma, showing 61.5% with PCG and 38.5% with secondary congenital glaucoma [6].

Other studies have evaluated the causes of visual impairment in children at low vision services in Brazilian public hospitals. It is important to emphasize that Brazil is a country with a continental dimension, with the North region being less developed than the South region. These socioeconomic characteristics lead to different frequencies in causes of visual impairment in children: infections tend to be the leading cause in the North, whereas retinopathy of prematurity is more frequent in the South. In a study that evaluated children from the rural area, the main causes of low vision were congenital cataract and toxoplasmosis (14.0%) followed by congenital glaucoma [7]. Another study, performed at the University of São Paulo, the most developed state in Brazil, involving 3210 cases, also showed macular toxoplasmosis as the first cause of visual impairment in children with low vision (20.7%), followed by retinal dystrophies (12.2%), retinopathy of prematurity (11.8%), ocular malformations (11.6%), and congenital glaucoma (10.8%) [8].

A recent update about the causes of childhood blindness worldwide situates glaucoma as the third leading cause in the Americas. Interestingly, glaucoma does not appear as one of the three main causes of blindness in the other regions of the world (Africa, Eastern Mediterranean, Europe, Southeast Asia, and Western Pacific) [9].

17.3 Mechanisms and Clinical Features

The presumed mechanism in PCG development is related to a dysfunction in the outflow system, particularly at the conventional outflow. Trabecular meshwork and Schlemm canal are both structures derived from the neural crest and mesodermal lineage. During the embryonic period, these tissues do not complete their maturation, which results in decreased outflow and IOP rise. The effect of increased IOP in ocular structures go beyond glaucomatous optic nerve damage, including ocular globe enlargement (buphthalmos), increase in corneal diameter (megalocornea), and breaks in the corneal endothelium (Haab striae). These ocular alterations lead to a classical clinical triad of GCP symptoms including epiphora, blepharospasm, and photophobia [10, 11].

The Childhood Glaucoma Research Network has introduced a classification guide to PCG diagnosis defined as the presence of two or more of the following criteria: IOP greater than 21 mmHg, optic disc cupping, corneal abnormalities related to PCG, ocular enlargement evaluated by axial length or progressive myopia, and visual field defect consistent with glaucoma [4]. The treatment of PCG aims at reducing IOP and controlling amblyopia. In the vast majority of cases, IOP control requires initially an angular surgical procedure in order to improve aqueous humor outflow. PCG is mostly bilateral and asymmetrical with no sex preference in familial cases, but with higher male prevalence in sporadic cases [3, 10].

17.4 Genetic Aspects

The majority of PCG cases are sporadic and families have been reported with an autosomal recessive inheritance pattern and variable penetrance. Since the observation of inheritance patterns and the advances of molecular biology tools, several families have been evaluated, leading to the identification of genes associated with PCG. Genetic heterogeneity has been observed in PCG, what means that the same clinical phenotype results from variants in different loci/genes or that different patients, with the same genetic disease, present with different alterations in the same gene [3, 11]. Five loci have been identified (GLC3A–GLC3E) and, among these loci, variants in three genes have been associated with PCG [12].

Sarfarazi et al. identified the first gene, cytochrome P4501B1 (CYP1B1), located on chromosome 2p21 linked to GLC3A locus in Pakistani families [13, 14]. CYP1B1 belongs to the cytochrome P450 family of membrane-bound oxidase enzymes and codes for P4501B1, a monooxygenase probably involved in the metabolism of a variety of substrates, including steroids and retinoids. Different from other P450 proteins, CYP1B1 is highly expressed outside the liver, particularly in tissues responsible for IOP homeostasis: trabecular meshwork and ciliary body [15, 16].

The mechanism through which disease-associated variants cause PCG is not completely understood. It is suggested that the enzyme codified by the gene would participate in metabolic pathways involved in the development of the anterior chamber, particularly, in the formation of the trabecular meshwork, via degradation of certain metabolites, as well as in the clearance of reactive oxygen species. Hence, variants in the CYP1B1 gene could compromise the development and differentiation of this tissue, leading to IOP elevation and consequent optic nerve damage [17, 18].

The CYP1B1 gene consists of three exons, one non-coding, and two introns [19]. More than 150 variants associated with PCG have been described, according to the “The Human Gene Mutation Database” (HGMD) [20]. The distribution of mutations can vary worldwide, from 14 to 30% in North American and European populations, from 15 to 20% in Chinese and Japanese populations and from 90 to 100% in Saudi Arabians and Slovakian Gypsies [3, 21,22,23,24,25,26]. The type of disease-associated variants can also be more frequent in certain populations. For example, E387K seems to be a founder variant in Slovakian Gypsies, G61E is a founder mutation in the Middle Eastern population and R390H is common among Asian populations [27].

Following the identification of GLC3A locus and its corresponding CYP1B1 gene, two other loci, GLC3B and GLC3C, located on 1p36 and 14q24, respectively, were reported, but no PCG-associated variants have been identified [28, 29].

The GLC3D locus is also located on 14q24 and encompasses the latent transforming growth factor beta-binding protein 2 (LTBP2 gene). LTBP2 gene was identified through linkage analysis in Pakistani and Iranian PCG families presenting with autosomal recessive inheritance pattern. This gene encodes an extracellular matrix protein expressed in tissues with high concentration of elastic fibers with putative function in elastin microfibril assembly and cell adhesion. Its expression in ocular tissues such as the trabecular meshwork and ciliary body, as well as its role in anterior chamber development, make disease-causing variants in LTBP2 gene a reasonable cause of PCG. Unlike the worldwide distribution of CYP1B1 gene variants, LTBP2 alterations have been reported in few populational groups [11, 30].

The most recently identified locus is GLC3E, which contains the tunica interna endothelial cell kinase (TEK) gene. This gene was not found in a family-based linkage study, but in transgenic mice that harbored deletions in TEK gene or in both major angiopoietin ligands. These transgenic mice had a developmental loss of Schlemm’s canal, resulting in IOP rise and ganglion cell loss compatible with a PCG phenotype. These findings led to a candidate gene approach involving 189 unrelated PCG patients, of whom ten presented heterozygous disease-causing variants in the human TEK gene [31].

17.4.1 CYP1B1 Gene Screening in Brazilian PCG Patients

The first study that described the analysis of the CYP1B1 gene in PCG Brazilian patients was a collaboration between two Brazilian Universities from the state of São Paulo and the group directed by Dr. Mansoor Sarfarazi [32]. Fifty-two patients were evaluated through single-strand conformation polymorphism and Sanger sequencing: 51.9% presented positive family history, consanguinity was reported by 26.9 and 84.6% had bilateral PCG. Fifty percent of the patients showed disease-associated variants. The majority of them were present in familial versus sporadic cases (55.6 versus 41.7%) and in bilateral versus unilateral disease (55.8 versus 12.5%). Homozygosity was reported in 57.7% of the cases, heterozygosity in 15.4%, and compound heterozygosity in 26.9%. Eleven different mutations have been identified, four of them described for the first time (g.3860C>T, g.4340delG, g.8165C>G and g.8214_8215delAG). The 4340delG variant was present in 46.0% (12/26) of PCG cases positive for CYP1B1 alterations (nine homozygotes, two compound heterozygotes, and one heterozygote), associated with a severe phenotype, coursing with early onset (91.7% of the cases in the first month), worse clinical prognosis (all bilateral cases, high IOP in 11/12 cases), and limited response to surgical treatment. The most frequent haplotype observed among Brazilian patients was 5′-CCGGTA-3′, which was associated with at least seven mutations and probably with 4340delG.

In a report from Hollander et al., a deeper genotype–phenotype correlation for CYP1B1 variants was performed. The trabeculectomy specimens from patients harboring variants were analyzed showing different extent of goniodysgenesis dependent on the genotype. One of the patients was a compound heterozygote for 4340delG and C209R. This patient showed severe goniodysgenesis, with agenesis of the Schelemm’s canal [33].

In a joint study of Brazilian and American families, three variants in the CYP1B1 gene have been identified in two Brazilian families (g.8037_8046dupTCATGCCACC in homozygosity, g.8182delG, and p.Glu387Lys in compound heterozygosity) by Sanger sequencing. In both pedigrees, the disease presented with corneal edema, early onset and high IOP. The same variants were reported in one of the American families (g.8037_8046dupTCATGCCACC and p.Glu387Lys), who also showed the p.268delSNF variant. Patients who harbor these variants shared a common haplotype, indicating a common founder between these two populations [34].

Della Paolera et al. conducted another study with 30 patients from the state of São Paulo [35]. PCG was bilateral in 66.7% of the cases and unilateral in 33.3%. All patients underwent a surgical procedure before the age of 3 months and all cases were sporadic, with no consanguineous marriage being reported. Thirty percent of the patients (9/30) presented CYP1B1 variants, detected by Sanger sequencing and ten different mutations were described, two of them for the first time (4523delC and L378Q). Four of the patients presented variants in compound heterozygosis, two in homozygosis and in three patients only one mutant allele was identified. Prognosis was worse in patients who harbored alterations in the CYP1B1 gene: mean IOP at diagnosis was higher, more surgical procedures were necessary for IOP control (the risk of patients positive for CYP1B1 alterations to undergo more than one surgical procedure was nine times greater than the negative ones), and all patients had bilateral glaucoma. Different from the first study conducted in the population from São Paulo state, the 4340delG variant was present in only two (6.7%) patients in heterozygosis. Two patients presented one of the new variants, 4635delT, in homozygosis. Both patients had severe bilateral disease, with two to three surgeries in each eye to control IOP and important visual function impairment. The 4523delC and L378Q alterations were present in compound heterozygosity in three members from the same family, all with high IOP at diagnosis, difficult surgical control and poor visual function.

Few years ago, a study involving Indian and Brazilian GCP patients evaluated 301 and 150 patients, respectively [36]. This study encompassed Brazilian patients from two previous studies as well as 68 new cases [32, 35]. A frequency of approximately 44.0% disease-associated variants in the CYP1B1 gene has been reported in both populations. Despite the similar frequency, variants in homozygosis were more frequent in the Indian cohort (24.2% versus 16.7%) while compound heterozygosis was more frequent in the Brazilian cohort (12.7% versus 6.0%), which is probably due to the higher rate of consanguineous marriages among Indians. Both populations exhibited significant allelic heterogeneity. Thirty-nine variants were reported in Indian patients, while 17 in Brazilian patients. Most of these variants were population specific. Thirty-three were present only in Indian patients, while 11 were reported only in Brazilian patients. Six variants were shared between both groups (g.8037_8046dup10, g.8214_8215delAG, p.R368H, p.P437L, p.A443G, and p.S476P). The most prevalent alterations were R368H in India and 4340delG in Brazil. The R368H was observed in only three Brazilian patients (in homozygosity and compound heterozygosity) and the 4340delG was observed only in the Brazilian cohort. Regarding haplotype distribution, as observed in previous studies, the 5′-CCGGTA-3′ was a risk haplotype, associated with most variants.

In the group of Brazilian patients, 44.0% of the patients showed CYP1B1 disease-associated variants. Although not statistically significant, age of onset was lower in the group positive for CYP1B1 alterations. This group also showed higher frequency of family history and consanguinity. When all Brazilian PCG samples were evaluated (52 from the first study, 30 from the second study, and 68 from this study) no association was observed between alterations in the CYP1B1 gene, IOP, and corneal diameter. In this report, the number of surgeries and number of affected eyes were not evaluated in relation to CYP1B1 changes.

Another example of a patient from the Southeast Brazil is a 2-month-old male infant with bilateral PCG who was screened for variants in the CYP1B1 gene. Glaucoma was diagnosed when he was less than 1 month old. The patient presented IOP of 26 mmHg in the right eye and 28 mmHg in the left eye, axial length of 21.49 mm in the right eye and 22.20 mm in the left eye, as well as buphthalmos, megalocornea, and corneal edema. The child has been submitted to four surgeries to control IOP. The CYP1B1 gene screening showed the presence of two different variants (compound heterozygosity): p.E387K inherited from the father and p.R444Ter, inherited from the mother. As far as we know the R444Ter variant is being described for the first time in Brazil. The parents had no glaucoma or family history of glaucoma and no consanguinity (Fig. 17.1, data not published).

Fig. 17.1
figure 1

Primary congenital glaucoma patient harboring variants in the CYP1B1 gene. (a) Photograph of the patient at diagnosis showing corneal edema and buphthalmos. (b) Left eye after surgeries for IOP reduction. (c) Right eye after surgeries for IOP reduction. (d) Chromatogram showing the p.E387K variant (GAA-AAA) in heterozygosis. (e) Chromatogram showing the p.R444Ter variant (CGA-TGA) in heterozygosis. Photographs are courtesy of Dr. Christiane Rolim de Moura from Federal University of São Paulo

Full size image

Recently, a study was conducted by Coêlho and collaborators who evaluated 17 PCG patients from an ethnically diverse population from the Northeast Brazil through next-generation sequencing [37]. Most of the patients had bilateral glaucoma (88.2%), the age at diagnosis ranged from 0 to 9 years and in 52.9% of the patients at least two surgical procedures were required. The late diagnosis reflects the poor health care quality in the Northeast compared to the Southeast region of Brazil. Disease-associated variants were present in 23.5% of the patients, three compound heterozygotes and one homozygote, and five different variants were reported, two of which were described for the first time in Brazilian patients (p.G61E and p.Y81N). No genotype–phenotype correlation was observed.

All variants that have been reported in Brazilian PCG patients are depicted in Table 17.1.

Table 17.1 Distribution of CYP1B1 mutations associated with PCG observed in Brazilian cohorts
Full size table

As previously reported, the Brazilian population is highly admixed and heterogeneous. It is the result of several immigration events accompanied by the miscegenation of three major ancestral roots: Amerindians, Europeans, and Africans. Genetic composition varies from region to region, but it has been shown that the urban population is more uniform than previously thought. For autosomal markers, the proportion of European, African, and Amerindian ancestries was estimated between 70 and 77%, 13 and 19%, and 9 and 10%, respectively [38,39,40]. Accordingly, the study by Rolim et al. evaluated ancestry markers in PCG patients from the State of Minas Gerais and reported that the proportion of Europeans, Africans, and Amerindians ranged from 74 to 83%, 11 to 18%, and 4 to 9%, respectively. The authors demonstrated that African ancestry was more frequent in PCG cases than in controls (although with no statistical significance) and that it was associated with a higher number of surgeries to control IOP, suggesting that it might act as risk factor for the disease when in high proportion [41].

17.5 Summary

The studies evaluating the participation of the CYP1B1 gene as causative for PCG in Brazil have shown a frequency of disease-associated variants ranging from 23.5 to 50.0%. This important contribution strongly suggests that this gene is worth being tested in Brazilian PCG patients. Most of the patients present compound heterozygosity in their genotype, reinforcing the admixture profile of the Brazilian population.

The most frequent disease-associated variants in Brazil are g.4340delG, followed by g.8037_8046dupTCATGCCACC, and g.7901_7913delGAGTGCAGGCAGA. It is important to notice that only one study was performed in the Northeast region, with only 17 PCG patients included. This was enough to identify two new variants in Brazil, which emphasizes the need for additional studies in all regions of Brazil, in order to obtain a more realistic representation of PCG in this population.

Twenty years after the identification of CPYP1B1 gene, two variants remain exclusively identified in Brazil: g.4523delC and L378Q. The other variants are shared with several population groups, but four were reported in only one other country: South Korea (p.Q19Ter), Mexico (g.4635delT), and India (g.8214_8215delAG and p.S476P). It would be interesting to evaluate if these four disease-associated variants are originated from a common founder or if they are de novo events.

Brazil can contribute in the understanding of the genetic basis of PCG by searching for new genes using family-based approach, as well as investigating the genes recently associated with PCG, LTBP2, and TEK. The latter seems to be more promising, since their disease-associated variants appear to be more spread in different populations than LTBP2 variants. Finally, it is important that more collaborative studies are made to better reveal the genetic basis of PCG and to establish genotype–phenotype correlations applicable in precision medicine. For example, if TEK alterations are associated with the absence of Schlemm’s canal, the primary angle surgery might not be the ideal surgical treatment option.