Retinitis pigmentosa with iris coloboma due to miR-204 gene variant in a Chinese family.

PURPOSE: To characterize the phenotype and genotype of a Chinese family with autosomal-dominant retinitis pigmentosa (RP) accompanied by iris coloboma. METHODS: The proband, a 34-year-old male, was examined with his family by using fundus photography, optical coherence tomography (OCT), autofluorescence, and full-field electroretinography (ffERG). Genetic analyses were conducted through whole-exome sequencing (WES) to screen for variations. RESULTS: Three members of this Chinese family were shown to be bilateral iris coloboma. The male proband and his mother exhibited typical RP feature. The proband's late grandfather had been documented manifestation of iris coloboma. The mode of inheritance was confirmed to be autosomal dominance. Through linkage analysis and WES, a heterozygous variation in the miR-204 gene (n.37C>T), a noncoding RNA gene, was identified in these three members. CONCLUSIONS: In this third independent and the first Asian family, the existence of a miR-204 variant associated with RP accompanied by iris coloboma was confirmed. Our findings reinforce the significance of miR-204 as an important factor influencing visual function in the retina. When phenotypes like RP accompanied by iris coloboma in an autosomal-dominant pattern, including in Chinese patients, miR-204 aberrations should be considered.

Cell adhesion marker expression dynamics during fusion of the optic fissure.

Tissue fusion is a critical process that is repeated in multiple contexts during embryonic development and shares common attributes to processes such as wound healing and metastasis. Ocular coloboma is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause of childhood blindness. Coloboma results from fusion failure between opposing ventral retinal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, R-cadherin, ZO-1, and the EMT transcriptional activator and cadherin regulator SNAI2, in the developing chicken embryonic eye. We find that fusion pioneer cells at the edges of the fusing optic fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression of SNAI2. This highlights the unique properties of these cells and indicates that regulation of cell adhesion factors may be a critical process in optic fissure closure.

PAX2/Renal Coloboma Syndrome Expresses Extreme Intrafamilial Phenotypic Variability.

Renal coloboma syndrome (RCS) is a disease characterized by kidney and ocular anomalies (kidney hypodysplasia and coloboma). RCS is caused, in half of the cases, by mutations in the paired box 2 (PAX2) gene, a critical organogenesis transcriptional factor. We report the case of a newborn with kidney hypodysplasia in a negative parental context where mother and father were phenotypically unaffected at the initial evaluation. The maternal family presented an important history of kidney disease with undefined diagnosis. Molecular characterization identified a PAX2 variant, classified as likely pathogenic. This variant segregates with the disease, and it was also found in the newborn, explaining his severe symptoms. It is noteworthy that the mother shows the same PAX2 variant, with an apparently negative kidney phenotype, displaying the possibility of an extreme variable expressivity of the disease. This feature suggests extreme caution in segregation analysis and family counseling of PAX2 pedigrees.

Identification of HSPA8 as an interacting partner of MAB21L2 and an important factor in eye development.

BACKGROUND: Pathogenic variants in human MAB21L2 result in microphthalmia, anophthalmia, and coloboma. The exact molecular function of MAB21L2 is currently unknown. We conducted a series of yeast two-hybrid (Y2H) experiments to determine protein interactomes of normal human and zebrafish MAB21L2/mab21l2 as well as human disease-associated variant MAB21L2-p.(Arg51Gly) using human adult retina and zebrafish embryo libraries. RESULTS: These screens identified klhl31, tnpo1, TNPO2/tnpo2, KLC2/klc2, and SPTBN1/sptbn1 as co-factors of MAB21L2/mab21l2. Several factors, including hspa8 and hspa5, were found to interact with MAB21L2-p.Arg51Gly but not wild-type MAB21L2/mab21l2 in Y2H screens. Further analyses via 1-by-1 Y2H assays, co-immunoprecipitation, and mass spectrometry revealed that both normal and variant MAB21L2 interact with HSPA5 and HSPA8. In situ hybridization detected co-expression of hspa5 and hspa8 with mab21l2 during eye development in zebrafish. Examination of zebrafish mutant hspa8hi138Tg identified reduced hspa8 expression associated with severe ocular developmental defects, including small eye, coloboma, and anterior segment dysgenesis. To investigate the effects of hspa8 deficiency on the mab21l2Arg51_Phe52del allele, corresponding zebrafish double mutants were generated and found to be more severely affected than single mutant lines. CONCLUSION: This study identifies heat shock proteins as interacting partners of MAB21L2/mab21l2 and suggests a role for this interaction in vertebrate eye development.

Novel Compound Heterozygous Variations in MPDZ Gene Caused Isolated Bilateral Macular Coloboma in a Chinese Family.

Macular coloboma (MC) is a rare congenital retinochoroidal defect characterized by lesions of different sizes in the macular region. The pathological mechanism underlying congenital MC is unknown. Novel compound heterozygous variations, c.4301delA (p.Asp1434fs*3) and c.5255C>G (p.Ser1752Ter), in the multiple PDZ domain (MPDZ) proteins were identified via whole-exome analysis on the proband with isolated bilateral macular coloboma in a Chinese family. Segregation analysis revealed that each of the unaffected parents was heterozygous for one of the two variants. The results of the in silico and bioinformatics analysis were aligned with the experimental data. The knockdown of MPDZ in zebrafish caused a decrease in the ellipsoid zone, a destruction of the outer limiting membrane, and the subsequent RPE degeneration. Overall, the loss of MPDZ in zebrafish contributed to retinal development failure. These results indicate that MPDZ plays an essential role in the occurrence and maintenance of the macula, and the novel compound heterozygous variations were responsible for an autosomal recessive macular deficiency in this Chinese family.

The Vascular Pattern In Vicinity Of Chorioretinal Coloboma: An Optical Coherence Tomography Angiography Study.

BACKGROUND: Chorioretinal coloboma is a congenital anomaly which can be present in a clinical spectrum with a possibility of significant influence on visual acuity. Optical coherence tomography (OCT) has been frequently used for the study of chorioretinal coloboma. OCT angiography (OCTA), as a non-invasive method of taking high-resolution images of chorioretinal vessels, can improve our understanding of developmental aspects of this anomaly. MATERIALS AND METHODS: This observational case series evaluated patients with chorioretinal coloboma, who were referred to the eye clinic of a university-based hospital between March 2018 and October 2019. All patients underwent comprehensive ocular examination, OCT, and OCTA using AngioVue technologies from the colobomatous sites. RESULTS: This study included OCTA imaging of five patients (six eyes) with chorioretinal coloboma lesions. Large retinal vessels, which were intact in all eyes, coursed through the coloboma in four cases and around the margin of the involved area in one case. Attenuation of the microvasculature in the vicinity of coloboma with various extents from nearly normal to severe attenuation was evident in OCTA. Five eyes of four patients had disorganized superficial vessel plexus. Also corkscrew vessels were found in one eye. CONCLUSIONS: This is the first study assessing the vascular pattern in the vicinity of chorioretinal coloboma using OCTA. OCTA revealed nearly normal to severely attenuated retinal microvasculature. At the same time, intact large retinal vessels at the level of superficial vessel plexus coursed across or around the coloboma. OCTA imaging adds new insights about vascular characteristics in the vicinity of these lesions.

Orbital Cyst with Ependymal Differentiation Associated with Microphthalmia.

BackgroundOrbital cysts associated with microphthalmia are colobomatous lesions that typically present unilaterally and posterior to the globe. Case Report: A male infant had an orbital cyst associated with microphthalmia located anterior to the globe composed of a neuroglial wall, ependymal-like epithelial lining, with synaptophysin-positive cells resembling the retinal neuronal layer. Conclusion: This orbital cyst may represent a malformation of the eye rather than an encephalocele.

Identification of candidate PAX2-regulated genes implicated in human kidney development.

PAX2 is a transcription factor essential for kidney development and the main causative gene for renal coloboma syndrome (RCS). The mechanisms of PAX2 action during kidney development have been evaluated in mice but not in humans. This is a critical gap in knowledge since important differences have been reported in kidney development in the two species. In the present study, we hypothesized that key human PAX2-dependent kidney development genes are differentially expressed in nephron progenitor cells from induced pluripotent stem cells (iPSCs) in patients with RCS relative to healthy individuals. Cap analysis of gene expression revealed 189 candidate promoters and 71 candidate enhancers that were differentially activated by PAX2 in this system in three patients with RCS with PAX2 mutations. By comparing this list with the list of candidate Pax2-regulated mouse kidney development genes obtained from the Functional Annotation of the Mouse/Mammalian (FANTOM) database, we prioritized 17 genes. Furthermore, we ranked three genes-PBX1, POSTN, and ITGA9-as the top candidates based on closely aligned expression kinetics with PAX2 in the iPSC culture system and susceptibility to suppression by a Pax2 inhibitor in cultured mouse embryonic kidney explants. Identification of these genes may provide important information to clarify the pathogenesis of RCS, human kidney development, and kidney regeneration.

Cecr2 mutant mice as a model for human cat eye syndrome.

Cat eye syndrome (CES), a human genetic disorder caused by the inverted duplication of a region on chromosome 22, has been known since the late 1890s. Despite the significant impact this disorder has on affected individuals, models for CES have not been produced due to the difficulty of effectively duplicating the corresponding chromosome region in an animal model. However, the study of phenotypes associated with individual genes in this region such as CECR2 may shed light on the etiology of CES. In this study we have shown that deleterious loss of function mutations in mouse Cecr2 effectively demonstrate many of the abnormal features present in human patients with CES, including coloboma and specific skeletal, kidney and heart defects. Beyond phenotypic analyses we have demonstrated the importance of utilizing multiple genetic backgrounds to study disease models, as we see major differences in penetrance of Cecr2-related abnormal phenotype between mouse strains, reminiscent of the variability in the human syndrome. These findings suggest that Cecr2 is involved in the abnormal features of CES and that Cecr2 mice can be used as a model system to study the wide range of phenotypes present in CES.

Intravitreal ranibizumab for the management of serous maculopathy secondary to optic disc coloboma-associated choroidal neovascularisation.

We report the case of a 19-year-old patient with symptomatic unilateral serous maculopathy associated with an optic nerve coloboma. Fluorescein angiography detected a focal late leak at the temporal edge of the coloboma which was later found to correspond with an area of choroidal neovascularisation on optical coherence tomography angiography. A course of intravitreal ranibizumab achieved good clinical and structural response. This report contributes to the evidence that maculopathies associated with cavitary optic nerve anomalies may in some instances result from choroidal neovascularisation. It also highlights the importance of angiography to identify potential choroidal neovascular membranes, particularly in the absence of haemorrhages and neovascular membranes on fundus examination and conventional optical coherence tomography.

Discovery of a novel CHD7 CHARGE syndrome variant by integrated omics analyses.

Chromodomain helicase DNA-binding protein 7 (CHD7) pathogenic variants are identified in more than 90% of infants and children with CHARGE (Coloboma of the iris, retina, and/or optic disk; congenital Heart defects, choanal Atresia, Retardation of growth and development, Genital hypoplasia, and characteristic outer and inner Ear anomalies and deafness) syndrome. Approximately, 10% of cases have no known genetic cause identified. We report a male child with clinical features of CHARGE syndrome and nondiagnostic genetic testing that included chromosomal microarray, CHD7 sequencing and deletion/duplication analysis, SEMA3E sequencing, and trio exome and whole-genome sequencing (WGS). We used a comprehensive clinical assessment, genome-wide methylation analysis (GMA), reanalysis of WGS data, and CHD7 RNA studies to discover a novel variant that causes CHD7 haploinsufficiency. The 7-year-old Hispanic male proband has typical phenotypic features of CHARGE syndrome. GMA revealed a CHD7-associated epigenetic signature. Reanalysis of the WGS data with focused bioinformatic analysis of CHD7 detected a novel, de novo 15 base pair deletion in Intron 4 of CHD7 (c.2239-20_2239-6delGTCTTGGGTTTTTGT [NM_017780.3]). Using proband RNA, we confirmed that this novel deletion causes CHD7 haploinsufficiency by disrupting the canonical 3' splice site and introducing a premature stop codon. Integrated genomic, epigenomic, and transcriptome analyses discovered a novel CHD7 variant that causes CHARGE syndrome.

Bilateral severe microphthalmia in a neonate with trisomy 8 mosaicism: A new finding.

Mosaic Trisomy 8 is a rare chromosomal abnormality estimated to occur one in 30,000 newborns. The phenotype is highly variable and the severity does not appear to be correlated with the proportion of cells that contain the additional chromosome. Ocular involvement in Trisomy 8 mosaicism has previously been described to include corneal opacities, retinal dystrophy, coloboma, and unilateral microphthalmia. We report a case of severe bilateral microphthalmia in a neonate with Trisomy 8 mosaicism, a previously unrecognized ophthalmic manifestation.

Inherited intragenic PBX1 deletion: Expanding the phenotype.

PBX1 encodes the pre-B cell leukemia homeobox transcription factor, a three amino acid loop extension (TALE) homeodomain transcription factor, which forms nuclear complexes with other TALE class homeodomain proteins that ultimately regulate target genes controlling organ patterning during embryogenesis. Heterozygous de novo pathogenic variants in PBX1 resulting in haploinsufficiency are associated with congenital anomalies of the kidneys and urinary tract, most commonly renal hypoplasia, as well as anomalies involving the external ear, branchial arch, heart, and genitalia, and they cause intellectual disability and developmental delay. Affected individuals described thus far have had de novo variants. Here, we report three related individuals with an inherited pathogenic intragenic PBX1 deletion with variable clinical features typical for this syndrome.

Confirmation of FZD5 implication in a cohort of 50 patients with ocular coloboma.

Defects in optic fissure closure can lead to congenital ocular coloboma. This ocular malformation, often associated with microphthalmia, is described in various clinical forms with different inheritance patterns and genetic heterogeneity. In recent times, the identification of an increased number of genes involved in numerous cellular functions has led to a better understanding in optic fissure closure mechanisms. Nevertheless, most of these genes are also involved in wider eye growth defects such as micro-anophthalmia, questioning the mechanisms controlling both extension and severity of optic fissure closure defects. However, some genes, such as FZD5, have only been so far identified in isolated coloboma. Thus, to estimate the frequency of implication of different ocular genes, we screened a cohort of 50 patients affected by ocular coloboma by using targeted sequencing of 119 genes involved in ocular development. This analysis revealed seven heterozygous (likely) pathogenic variants in RARB, MAB21L2, RBP4, TFAP2A, and FZD5. Surprisingly, three out of the seven variants detected herein were novel disease-causing variants in FZD5 identified in three unrelated families with dominant inheritance. Although molecular diagnosis rate remains relatively low in patients with ocular coloboma (14% (7/50) in this work), these results, however, highlight the importance of genetic screening, especially of FZD5, in such patients. Indeed, in our series, FZD5 variants represent half of the genetic causes, constituting 6% (3/50) of the patients who benefited from a molecular diagnosis. Our findings support the involvement of FZD5 in ocular coloboma and provide clues for screening this gene during current diagnostic procedures.

A molecular and cellular analysis of human embryonic optic fissure closure related to the eye malformation coloboma.

Ocular coloboma is a congenital eye malformation, resulting from a failure in optic fissure closure (OFC) and causing visual impairment. There has been little study of the epithelial fusion process underlying closure in the human embryo and coloboma aetiology remains poorly understood. We performed RNAseq of cell populations isolated using laser capture microdissection to identify novel human OFC signature genes and probe the expression profile of known coloboma genes, along with a comparative murine analysis. Gene set enrichment patterns showed conservation between species. Expression of genes involved in epithelial-to-mesenchymal transition was transiently enriched in the human fissure margins during OFC at days 41-44. Electron microscopy and histological analyses showed that cells transiently delaminate at the point of closure, and produce cytoplasmic protrusions, before rearranging to form two continuous epithelial layers. Apoptosis was not observed in the human fissure margins. These analyses support a model of human OFC in which epithelial cells at the fissure margins undergo a transient epithelial-to-mesenchymal-like transition, facilitating cell rearrangement to form a complete optic cup.

Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye.

Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (~1 in 5000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggests multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein Neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicate that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased retinal pigmented epithelium (RPE) proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during OF closure.

CHARGE syndrome without colobomas: Ophthalmic findings.

To report ophthalmic findings of patients without colobomas, and with a clinical and molecular diagnosis of CHARGE Syndrome. Retrospective study of ophthalmic findings in 67 CHARGE patients-clinically confirmed diagnosis with positive CHD7 mutation-seen in the Ophthalmology department of Cincinnati Children's Hospital Medical Center between January 1, 2008 through September 25, 2018. Criteria for inclusion in this study was absence of any form of a coloboma in either eye. In our cohort, all patients had a positive CHD7 mutation, in addition to a clinical diagnosis. 19.4% (13/67) of CHARGE patients did not have a coloboma in either eye. 69.2% (9/13) had strabismus, 76.9% (10/13) had a refractive error that warranted refractive correction, 23.1% (3/13) had amblyopia, 38.5% (5/13) had nasolacrimal duct obstruction, 30.8% (4/13) had dry eye syndrome and exposure keratopathy, 15.4% (2/13) had ptosis, 15.4% (2/13) had blepharitis, 15.4% (2/13) had Cortical Visual Impairment, 7.7% (1/13) of patients had optic nerve drusen, 7.7% (1/13) had Marcus Gunn Jaw Winking, and 7.7% (1/13) with an eyelid nevus. There are numerous ophthalmic findings in individuals with CHARGE Syndrome without colobomas. No study to date has evaluated the ophthalmic findings in CHD7 positive CHARGE patients without colobomas. These findings need to be assessed and treated to ensure optimal vision in the CHARGE patient population. Absence of coloboma does not rule out a diagnosis of CHARGE syndrome, and if there is a clinical suspicion, clinical confirmation then genetic testing would be warranted.

Exome sequencing in patients with microphthalmia, anophthalmia, and coloboma (MAC) from a consanguineous population.

Next-generation sequencing strategies have resulted in mutation detection rates of 21% to 61% in small cohorts of patients with microphthalmia, anophthalmia and coloboma (MAC), but despite progress in identifying novel causative genes, many patients remain without a genetic diagnosis. We studied a cohort of 19 patients with MAC who were ascertained from a population with high rates of consanguinity. Using single nucleotide polymorphism (SNP) arrays and whole exome sequencing (WES), we identified one pathogenic variant in TENM3 in a patient with cataracts in addition to MAC. We also detected novel variants of unknown significance in genes that have previously been associated with MAC, including KIF26B, MICU1 and CDON, and identified variants in candidate genes for MAC from the Wnt signaling pathway, comprising LRP6, WNT2B and IQGAP1, but our findings do not prove causality. Plausible variants were not found for many of the cases, indicating that our current understanding of the pathogenesis of MAC, a highly heterogeneous group of ocular defects, remains incomplete.

Ocular coloboma: Genetic variants reveal a dynamic model of eye development.

Ocular coloboma is a congenital disorder of the eye where a gap exists in the inferior retina, lens, iris, or optic nerve tissue. With a prevalence of 2-19 per 100,000 live births, coloboma, and microphthalmia, an associated ocular disorder, represent up to 10% of childhood blindness. It manifests due to the failure of choroid fissure closure during eye development, and it is a part of a spectrum of ocular disorders that include microphthalmia and anophthalmia. Use of genetic approaches from classical pedigree analyses to next generation sequencing has identified more than 40 loci that are associated with the causality of ocular coloboma. As we have expanded studies to include singleton cases, hereditability has been very challenging to prove. As such, researchers over the past 20 years, have unraveled the complex interrelationship amongst these 40 genes using vertebrate model organisms. Such research has greatly increased our understanding of eye development. These genes function to regulate initial specification of the eye field, migration of retinal precursors, patterning of the retina, neural crest cell biology, and activity of head mesoderm. This review will discuss the discovery of loci using patient data, their investigations in animal models, and the recent advances stemming from animal models that shed new light in patient diagnosis.