Outcomes of Corneal Transplant in Childhood Glaucoma.

PRCIS: Childhood glaucoma produces alterations in the postnatal development and function of various ocular structures, including the cornea. Childhood glaucoma patients present lower corneal transplant survival rates. Our series shows outcomes of corneal transplant in childhood glaucoma with survival rates of 29% at 2 years. OBJECTIVE: To investigate the surgical outcome of different types of keratoplasty in eyes with childhood glaucoma. PATIENTS AND METHODS: A retrospective review was made of the medical records from 17 eyes of 15 patients who were diagnosed with childhood glaucoma and received a corneal transplantation between January 2010 and July 2020. Patient demographics, intraocular pressure, previous ocular surgery, comorbidities, corneal transplant surgery, and follow-up outcome were collected. The primary efficacy endpoint was graft survival (in months) until failure, the latter being considered as irreversible loss of corneal transparency. Secondary efficacy points were the need for an increase in topical hypotensive therapy and the need for additional surgery. RESULTS: Seventeen eyes of 15 patients were included, 11 eyes (10 patients) with primary congenital glaucoma and 6 with other types of childhood glaucoma. Corneal transplantation was performed at the mean age of 23.76 ± 14.86 years. At the time of the transplantation, the number of topical medications was 1.35 ± 1.27, intraocular pressure was 15.00 ± 8.34 mm Hg, and patients had received up to 7 glaucoma surgeries. Descemet stripping automated endothelial keratoplasty was performed in 13 eyes (76%) and penetrating keratoplasty in 4 (24%). After surgery, 7 (41%) eyes required increased topical treatment and 2 (12%) glaucoma surgery. Twelve eyes (71%) developed graft failure at 24 months, the mean time of survival being 13.88 ± 8.25 months. CONCLUSIONS: Management of corneal decompensation in childhood glaucoma poses a challenge. In this series of childhood glaucoma with corneal transplantations, the survival rate was 29% at 24 months.

Efficacy and safety of the PreserFlo implant with mitomycin C in childhood glaucoma after previous failed glaucoma surgeries.

PURPOSE: This study aims to evaluate the efficacy and safety of the PreserFlo MicroShunt (Santen, Osaka, Japan) in lowering intraocular pressure (IOP) in childhood glaucoma patients with previous failed glaucoma surgeries. METHODS: This is a prospective case review of consecutive PreserFlo procedures performed in childhood glaucoma patients after failed surgeries. Age, sex, diagnosis, and previous glaucoma surgeries, as well as visual acuity, IOP, and treatment in the preoperative visit and all follow-up visits were collected. Outcome measures included IOP reduction from baseline, mean IOP change from baseline at month 6, medication use at 6 months, complications, adverse events, and need for further procedures. RESULTS: Fourteen patients were included, 8 (57%) males and 6 (43%) females; the mean age was 27.5 ± 13.5 years. Nine patients (64%) had at least two trabeculectomies, and 6 patients (43%) had at least one trabeculectomy and a glaucoma drainage implant. The mean IOP change from baseline was 11.3 ± 4.9 mmHg at 12 months. At 12 months, 12 patients (86%) presented ≥ 20% IOP lowering from baseline, and 11 patients (79%) presented ≥ 30%. The mean medication count decreased from 3.9 ± 0.7 (baseline) to 0.7 ± 1.3 (12 months). No intraoperative complications were reported. No adverse events were noted. No secondary filtration surgery was required, although bleb needling was required in one case, 1 month after the surgery. CONCLUSIONS: PreserFlo with MMC can be used successfully to treat uncontrolled IOP in childhood glaucoma cases with previous failed surgeries. Larger studies with longer follow-up are needed to further explore the role of the device in resistant childhood glaucoma cases.

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Retinal nerve fiber layer thickness in children with primary congenital glaucoma measured by spectral domain optical coherence tomography.

PURPOSE: To evaluate retinal nerve fiber layer (RNFL) thickness using spectral domain optical coherence tomography (SD-OCT) in a population of children diagnosed with primary congenital glaucoma (PCG). METHODS: In this cross-sectional study, 59 eyes of 59 children diagnosed with PCG and 87 eyes of 87 healthy children were evaluated by SD-OCT to measure the RNFL. The global average peripapillary RNFL thickness and sectional RNFL thickness were evaluated in both groups. Differences in global average and sectional thickness were analyzed. RESULTS: Mean age in the PCG group was 9.61 ± 3.23 years; in the control group, 8.47 ± 2.99 years (P = 0.0516). There were statistically significant differences (P < 0.007) in all sectors between both groups. CONCLUSIONS: SD-OCT is a promising tool for evaluating the eyes of children diagnosed with PCG. Future research should examine the test-retest variability of SD-OCT parameters and their ability to diagnose progression in these children.

Role of FOXC2 and PITX2 rare variants associated with mild functional alterations as modifier factors in congenital glaucoma.

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG.

Preliminary Study of the Differences in Optic Nerve Head Hemoglobin Measures Between Patients With and Without Childhood Glaucoma.

PURPOSE: To evaluate the effectiveness of quantifying color changes in the optic nerve head in retinal photographs of patients with childhood glaucoma. METHODS: In this observational study, three photographs of the optic nerve head were obtained in 28 patients with childhood glaucoma and 28 age- and sex-matched healthy participants (the childhood glaucoma and control groups, respectively). The Laguna Optic Nerve Head Hemoglobin (ONhE) software (Insoft SL, Tenerife, Spain) was used to determine hemoglobin levels in the optic nerve head. The following parameters were quantified: the hemoglobin levels in the optic nerve head across the whole disc, in 24 sectors (the optic nerve head divided by two concentric rings and eight 45-degree radial sectors), and in the vertical disc diameter (sectors 8 and 20), and the estimated cup-disc ratio and Glaucoma Discriminant Function, which combines the slope of the hemoglobin amount with the mean vertical disc diameter. RESULTS: Patient ages ranged from 9 to 14 years (median: 11 years) in the childhood glaucoma group, and 7 to 13 years (median: 9 years) in the control group (P < .061). Eyes in the childhood glaucoma group showed a significantly higher cup-disc ratio compared to eyes in the control group (0.6 ± 0.2 vs 0.5 ± 0.1, respectively; P < .0001). In the childhood glaucoma group, the Glaucoma Discriminant Function was found to be significantly lower than in the control group (-6.5 ± 31.1 vs 9.4 ± 17.1, respectively; P < .0001). There were no significant differences in the hemoglobin levels in the optic nerve head across the whole disc between eyes in the childhood glaucoma and control groups (58.2% ± 10.9% vs 58.5% ± 6.7%, respectively; P = .847). The Laguna ONhE software showed good reproducibility in measuring percentages of hemoglobin levels in both groups. CONCLUSIONS: The Laguna ONhE software is useful for patients with childhood glaucoma. However, hemoglobin levels in the optic nerve head across the whole disc may have normal values. This method had good reliability and is easy to implement in routine clinical practice. [J Pediatr Ophthalmol Strabismus. 2017;54(6):387-394.].

Whole-Exome Sequencing of Congenital Glaucoma Patients Reveals Hypermorphic Variants in GPATCH3, a New Gene Involved in Ocular and Craniofacial Development.

Congenital glaucoma (CG) is a heterogeneous, inherited and severe optical neuropathy that originates from maldevelopment of the anterior segment of the eye. To identify new disease genes, we performed whole-exome sequencing of 26 unrelated CG patients. In one patient we identified two rare, recessive and hypermorphic coding variants in GPATCH3, a gene of unidentified function, and 5% of a second group of 170 unrelated CG patients carried rare variants in this gene. The recombinant GPATCH3 protein activated in vitro the proximal promoter of CXCR4, a gene involved in embryo neural crest cell migration. The GPATCH3 protein was detected in human tissues relevant to glaucoma (e.g., ciliary body). This gene was expressed in the dermis, skeletal muscles, periocular mesenchymal-like cells and corneal endothelium of early zebrafish embryos. Morpholino-mediated knockdown and transient overexpression of gpatch3 led to varying degrees of goniodysgenesis and ocular and craniofacial abnormalities, recapitulating some of the features of zebrafish embryos deficient in the glaucoma-related genes pitx2 and foxc1. In conclusion, our data suggest the existence of high genetic heterogeneity in CG and provide evidence for the role of GPATCH3 in this disease. We also show that GPATCH3 is a new gene involved in ocular and craniofacial development.

Rare FOXC1 variants in congenital glaucoma: identification of translation regulatory sequences.

Primary congenital glaucoma (PCG) is the cause of a significant proportion of inherited visual loss in children, but the underlying mechanism is poorly understood. In this study, we assessed the relationship between PCG and FOXC1 variants by Sanger sequencing the proximal promoter and transcribed sequence of FOXC1 from a cohort of 133 PCG families with no known CYP1B1 or MYOC mutations. The pathogenicity of the identified variants was evaluated by functional analyses. Ten patients (7.5%) with no family history of glaucoma carried five different rare heterozygous FOXC1 variants with both increased (rs77888940:C>G, c.-429C>G, rs730882054:c.1134_144del(CGGCGGCGCGG), p.(G380Rfs*144) and rs35717904:A>T, c.*734A>T) and decreased (rs185790394: C>T, c.-244C>T and rs79691946:C>T, p.(P297S)) transactivation, ranging from 50 to 180% of the wild-type activity. The five variants did not show monogenic segregation, and four of them were absent in a control group (n=233). To the best of our knowledge, one of these variants (p.(G380Rfs*144)) has not previously been described. One of the FOXC1 variant carriers (p.(P297S)) also coinherited a functionally altered rare PITX2 heterozygous variant (rs6533526:C>T, c.*454C>T). Bioinformatics and functional analyses provided novel information on three of these variants. c.-429C>G potentially disrupts a consensus sequence for a terminal oligopyrimidine tract, whereas c.-244C>T may alter the RNA secondary structure in the 5'-untranslated region (UTR) that affects mRNA translation. In addition, p.(G380Rfs*144) led to increased protein stability. In summary, these data reveal the presence of translation regulatory sequences in the UTRs of FOXC1 and provide evidence for a possible role of rare FOXC1 variants as modifying factors of goniodysgenesis in PCG.

The Role of hsa-miR-548l Dysregulation as a Putative Modifier Factor for Glaucoma-Associated FOXC1 Mutations.

Mutations of the FOXC1 transcription factor are involved in a variety of autosomal dominant ocular anterior segment defects, ranging from Axenfeld-Rieger malformations to isolated glaucoma in some patients. In this study we have evaluated the possible role of the c.*734A>T FOXC1 variant as a modifier factor of the activity of two FOXC1 mutations previously identified in families primarily affected by dominant glaucoma (haplotypes p.G447_G448insDG-c.*734A>T and p.I126S-c.*734A>T). Previous bioinformatic analyses indicated that the c.*734A>T variant is located in a potential target sequence for hsa-miR-548l. Co-expression of this miRNA with a reporter cDNA construct in which the wild-type 3'UTR sequence of FOXC1 was fused to the 3'-end of the firefly luciferase coding region, led to approximately 20% decreased luciferase activity compared to the controls, confirming the presence of a target sequence for hsa-miR-548l. In contrast, this miRNA did not show any effect on the luciferase activity associated with the mutant 3'UTR FOXC1 sequence, showing that it resulted in a loss-of-function of the has-miR-548l target sequence. In addition, functional evaluation of the two glaucoma-associated haplotypes revealed increased protein levels and transactivation, compared to the corresponding individual coding mutations (approximately 1.2-fold on average). These data support the role of hsa-miR-548l as a regulator of FOXC1 translation and provide evidence for the c.*734A>T variant as a modifier factor for the activity of coding glaucoma-associated FOXC1 mutations.

Hypo- and hypermorphic FOXC1 mutations in dominant glaucoma: transactivation and phenotypic variability.

Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.