Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

Construction and Identification of a Novel Mice Model of Microphthalmia.

Purpose: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia. Methods: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice. Results: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation. Conclusions: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease. Translational Relevance: This study developed a novel animal model to facilitate clinical research on microphthalmia.

Comprehensive genetic analysis uncovers the mutational spectrum of MFRP and its genotype-phenotype correlation in a large cohort of Chinese microphthalmia patients.

PURPOSE: Microphthalmia is a severe congenital ocular disease featured by abnormal ocular development. The aim of this study was to detail the genetic and clinical characteristics of a large cohort of Chinese patients with microphthalmia related to MFRP variants, focusing on uncovering genotype-phenotype correlations. METHODS: Fifty microphthalmia patients from 44 unrelated Chinese families were recruited. Whole-exome sequencing (WES) was conducted to analyze the coding regions and adjacent intronic regions of MFRP. Axial lengths (AL) were measured for all probands and available family members. Protein structures of mutations with high frequency in our cohort were predicted. The genotype-phenotype correlations were explored by statistical analysis. RESULTS: Sixteen MFRP variants were detected in 17 families, accounting for 38.64 % of all microphthalmia families. There were 9 novel mutations (c.427+1G>C, c.428-2A>C, c.561_575del:p.A188_E192del, c.836G>A:p.C279Y, c.1010_1021del:p.H337_E340del:p.Y479*, c.1516_1517del:p.S506Pfs*66, c.1561T>G:p.C521G, c.1616G>A:p.R539H, and c.1735C>T:p.P579S) and six previously reported variants in MFRP, with p.E496K and p.H337_E340del being highly frequent, found in eight (47.06 %) and two families (11.76 %), respectively. Seven variants (43.75 %) were located in the C-terminal cysteine-rich frizzled-related domain (CRD) (7/16, 43.75 %). Protein prediction implicated p.E496K and p.H337_E340del mutations might lead to a destabilization of the MFRP protein. The average AL of all 42 eyes was 16.02 ± 1.05 mm, and 78.36 % of eyes with AL < 16 mm harbored p.E496K variant. Twenty-six eyes with variant variant had shorter AL than that of the other 16 eyes without this variant (p = 0.006), highlighting a novel genotype-phenotype correlation. CONCLUSIONS: In this largest cohort of Chinese patients with microphthalmia, the 9 novel variants, high frequency of p.E496W, and mutation hotspots in CRD reveals unique insights into the MFRP mutation spectrum among Chinese patients, indicating ethnic variability. A new genotype-phenotype correlation that p.E496K variant associated with a shorter AL is unveiled. Our findings enhance the current knowledge of MFRP-associated microphthalmia and provide valuable information for prenatal diagnosis as well as future therapy.

Disruption of common ocular developmental pathways in patient-derived optic vesicle models of microphthalmia.

Genetic perturbations influencing early eye development can result in microphthalmia, anophthalmia, and coloboma (MAC). Over 100 genes are associated with MAC, but little is known about common disease mechanisms. In this study, we generated induced pluripotent stem cell (iPSC)-derived optic vesicles (OVs) from two unrelated microphthalmia patients and healthy controls. At day 20, 35, and 50, microphthalmia patient OV diameters were significantly smaller, recapitulating the "small eye" phenotype. RNA sequencing (RNA-seq) analysis revealed upregulation of apoptosis-initiating and extracellular matrix (ECM) genes at day 20 and 35. Western blot and immunohistochemistry revealed increased expression of lumican, nidogen, and collagen type IV, suggesting ECM overproduction. Increased apoptosis was observed in microphthalmia OVs with reduced phospho-histone 3 (pH3+) cells confirming decreased cell proliferation at day 35. Pharmacological inhibition of caspase-8 activity with Z-IETD-FMK decreased apoptosis in one patient model, highlighting a potential therapeutic approach. These data reveal shared pathophysiological mechanisms contributing to a microphthalmia phenotype.

A founder variant expands the phenotype of WNT7B-related PDAC syndrome.

Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia, and Cardiac defects (PDAC) syndrome is a genetically heterogeneous multiple congenital malformation syndrome. Although pathogenic variants in RARB and STRA6 are established causes of PDAC, many PDAC cases remain unsolved at the molecular level. Recently, we proposed biallelic WNT7B variants as a novel etiology based on several families with typical features of PDAC syndrome albeit with variable expressivity. Here, we report three patients from two families that share a novel founder variant in WNT7B (c.739C > T; Arg247Trp). The phenotypic expression of this variant ranges from typical PDAC features to isolated genitourinary anomalies. Similar to previously reported PDAC-associated WNT7B variants, this variant was found to significantly impair WNT7B signaling activity further corroborating its proposed pathogenicity. This report adds further evidence to WNT7B-related PDAC and expands its variable expressivity.

Pathogenic variants of MFRP and PRSS56 genes are major causes of nanophthalmos in Japanese patients.

BACKGROUND: Nanophthalmos (NNO) is a rare condition with significantly shorter axial length than normal. Several genes are known to cause NNO, among them the MFRP and PRSS56 genes have been reported to cause majority of NNOs. The purpose of this study was to determine the genetic basis of Japanese patients with NNO. MATERIALS AND METHODS: We studied seven patients with NNO. Whole exome sequencing (WES) and Sanger sequencing were performed to determine the variants causing the NNO. We also reviewed the medical charts of the patients to determine the phenotype of these seven patients. RESULTS: WES revealed that four patients from three families carried homozygous frameshift variants of the PRSS56 gene (c.1066dupC). Two novel variants of the MFRP gene were detected in the other two patients: one proband had a homozygous missense variant (c.1486 G>A) and the other had a compound heterozygous variant (c.1486 G>A and c.662_663insT). The axial length of the eight eyes with the PRSS56 variant was 15.69 ± 0.48 mm (mean ± SD) and that for the 4 eyes with the MFRP variant was 15.63 ± 0.69 mm. Three of the six cases with the PRSS56 or MFRP variant had the uveal effusion syndrome. CONCLUSIONS: NNOs in Japanese patients are caused by variants of the PRSS56 and MFRP genes as in other ethnic populations. In addition, two new variants of the MFRP gene were found in our cohort. The phenotypes and anomalies in Japanese patients with NNO were similar to those reported for other ethnic populations.

Progressive Cone-Rod Dystrophy and RPE Dysfunction in Mitfmi/+ Mice.

Mutations in the mouse microphthalmia-associated transcription factor (Mitf) gene affect retinal pigment epithelium (RPE) differentiation and development and can lead to hypopigmentation, microphthalmia, deafness, and blindness. For instance, an association has been established between loss-of-function mutations in the mouse Mitf gene and a variety of human retinal diseases, including Waardenburg type 2 and Tietz syndromes. Although there is evidence showing that mice with the homozygous Mitfmi mutation manifest microphthalmia and osteopetrosis, there are limited or no data on the effects of the heterozygous condition in the eye. Mitf mice can therefore be regarded as an important model system for the study of human disease. Thus, we characterized Mitfmi/+ mice at 1, 3, 12, and 18 months old in comparison with age-matched wild-type mice. The light- and dark-adapted electroretinogram (ERG) recordings showed progressive cone-rod dystrophy in Mitfmi/+ mice. The RPE response was reduced in the mutant in all age groups studied. Progressive loss of pigmentation was found in Mitfmi/+ mice. Histological retinal sections revealed evidence of retinal degeneration in Mitfmi/+ mice at older ages. For the first time, we report a mouse model of progressive cone-rod dystrophy and RPE dysfunction with a mutation in the Mitf gene.

Phenotypic consequences of a nanophthalmos-associated TMEM98 variant in human and mouse.

Nanophthalmos is characterised by shorter posterior and anterior segments of the eye, with a predisposition towards high hyperopia and primary angle-closure glaucoma. Variants in TMEM98 have been associated with autosomal dominant nanophthalmos in multiple kindreds, but definitive evidence for causation has been limited. Here we used CRISPR/Cas9 mutagenesis to recreate the human nanophthalmos-associated TMEM98 p.(Ala193Pro) variant in mice. The p.(Ala193Pro) variant was associated with ocular phenotypes in both mice and humans, with dominant inheritance in humans and recessive inheritance in mice. Unlike their human counterparts, p.(Ala193Pro) homozygous mutant mice had normal axial length, normal intraocular pressure, and structurally normal scleral collagen. However, in both homozygous mice and heterozygous humans, the p.(Ala193Pro) variant was associated with discrete white spots throughout the retinal fundus, with corresponding retinal folds on histology. This direct comparison of a TMEM98 variant in mouse and human suggests that certain nanophthalmos-associated phenotypes are not only a consequence of a smaller eye, but that TMEM98 may itself play a primary role in retinal and scleral structure and integrity.

ALDH1A3-related congenital microphthalmia-8 due to a novel frameshift variant.

Microphthalmia (MCOP) is a group of rare developmental malformations of eye with often reduced size of the eyeball, leading to blindness. Affecting about 1 in 7000 live births, MCOP can occur due to either environmental or genetic factors. Isolated microphthalmia-8 (MCOP8) has been proved to be caused by autosomal recessive mutations of the ALDH1A3 gene (MIM*600463) encoding aldehyde dehydrogenase 1 family, member A3. Herein, we report an 8-year-old boy with vision problems since birth from a first-cousin consanguineous parents. The main symptoms of the patient included severe bilateral microphthalmia, cyst in the left eye and blindness. The child developed behavioral disorders at the age of 7. It should be noted that there is no family history of the disease. To identify the genetic factor underlying the pathogenesis in this case Whole Exome Sequencing (WES) was performed and followed by Sanger sequencing. A novel pathogenic variant, c.1441delA (p.M482Cfs*8), in the ALDH1A3 gene was detected by WES in the proband. Further prenatal diagnosis is highly suggested to the family for the future pregnancies.

SOX2 pathogenic variants with normal eyes: Expanding the phenotypic spectrum.

SOX2 pathogenic variants, though rare, constitute the most commonly known genetic cause of clinical anophthalmia and microphthalmia. However, patients without major ocular malformation, but with multi-system developmental disorders, have been reported, suggesting that the range of clinical phenotypes is broader than previously appreciated. We detail two patients with bilateral structurally normal eyes along with 11 other previously published patients. Our findings suggest that there is no obvious phenotypic or genotypic pattern that may help set apart patients with normal eyes. Our patients provide further evidence for broadening the phenotypic spectrum of SOX2 mutations and re-appraising the designation of SOX2 disorder as an anophthalmia/microphthalmia syndrome. We emphasize the importance of considering SOX2 pathogenic variants in the differential diagnoses of individuals with normal eyes, who may have varying combinations of features such as developmental delay, urogenital abnormalities, gastro-intestinal anomalies, pituitary dysfunction, midline structural anomalies, and complex movement disorders, seizures or other neurological issues.

Microphthalmia, aphakia and agenesis of the optic chiasm and tract in a wild fox squirrel (Sciurusniger).

We describe gross and histopathological features of multiple ocular and neuro-ophthalmic abnormalities in a fox squirrel (Sciurus niger). Ophthalmic findings included severe bilateral microphthalmos, with the right eye more affected than the left. Histopathology confirmed severe microphthalmia, aphakia, disorganized retinal tissue and small optic nerves, as well as agenesis of the optic chiasm and optic tract. This combination of neuro-ophthalmic abnormalities has not been previously described in wild animals.

Disruption of fetal eye development caused by insulin-induced maternal hypoglycemia in rats.

We previously revealed that insulin-induced severe and long-lasting maternal hypoglycemia in rats caused anophthalmia and microphthalmia in fetuses; however, it remained unclear whether hypoglycemia-induced eye anomalies were developmental retardation or disruption, and when and how they developed. Hence, we induced hypoglycemia in pregnant Sprague-Dawley rats by injecting insulin from Days 6 to 11 of pregnancy and performed periodical histopathological examination of fetal eyes from embryonic days (E)10 to 20. On E10, optic vesicle had developed normally both in the control and insulin-treated group; however, on E11, optic cup (OC) had developed in the control group but not in the insulin-treated group. On E12, neural retina (NR), retinal pigmented epithelium (RPE), lens, and presumptive cornea had been observed in the control group. In contrast, lens pit and OC with remaining space between RPE and NR had developed in the insulin-treated group. From E13 to E15, developmental disruption characterized by defects, hypoplasia, and degeneration in the retina, lens, and cornea was observed in the insulin-treated group, resulting in anophthalmia or microphthalmia on E20. Moreover, the expression of MITF and chx10, which are essential for early eye development by expressing in the presumptive retina and lens and regulating each other's expression level, was ectopic and suppressed on E11. In conclusion, insulin-induced maternal hypoglycemia caused developmental disruption, but not simple developmental retardation of fetal eyes, and its trigger might be a failure of presumptive retina and lens to interact on E11.

Long-read genome sequencing resolves a complex 13q structural variant associated with syndromic anophthalmia.

Microphthalmia, anophthalmia, and coloboma (MAC) are a heterogeneous spectrum of anomalous eye development and degeneration with genetic and environmental etiologies. Structural and copy number variants of chromosome 13 have been implicated in MAC; however, the specific loci involved in disease pathogenesis have not been well-defined. Herein we report a newborn with syndromic degenerative anophthalmia and a complex de novo rearrangement of chromosome 13q. Long-read genome sequencing improved the resolution and clinical interpretation of a duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) and terminal deletion. Sequence features at the breakpoint junctions suggested microhomology-mediated break-induced replication (MMBIR) of the maternal chromosome as the origin. Comparing this rearrangement to previously reported copy number alterations in 13q, we refine a putative dosage-sensitive critical region for MAC that might provide new insights into its molecular etiology.

Review of 37 patients with SOX2 pathogenic variants collected by the Anophthalmia/Microphthalmia Clinical Registry and DNA research study.

SOX2 variants and deletions are a common cause of anophthalmia and microphthalmia (A/M). This article presents data from a cohort of patients with SOX2 variants, some of whom have been followed for 20+ years. Medical records from patients enrolled in the A/M Research Registry and carrying SOX2 variants were reviewed. Thirty-seven patients were identified, ranging in age from infant to 30 years old. Eye anomalies were bilateral in 30 patients (81.1%), unilateral in 5 (13.5%), and absent in 2 (5.4%). Intellectual disability was present in all with data available and ranged from mild to profound. Seizures were noted in 18 of 27 (66.6%) patients, usually with abnormal brain MRIs (10/15, 66.7%). Growth issues were reported in 14 of 21 patients (66.7%) and 14 of 19 (73.7%) had gonadotropin deficiency. Genitourinary anomalies were seen in 15 of 19 (78.9%) male patients and 5 of 15 (33.3%) female patients. Patients with SOX2 nucleotide variants, whole gene deletions or translocations are typically affected with bilateral or unilateral microphthalmia and anophthalmia. Other associated features include intellectual disability, seizures, brain anomalies, growth hormone deficiency, gonadotropin deficiency, and genitourinary anomalies. Recommendations for newly diagnosed patients with SOX2 variants include eye exams, MRI of the brain and orbits, endocrine and neurology examinations. Since the clinical spectrum associated with SOX2 alleles has expanded beyond the originally reported phenotypes, we propose a broader term, SOX2-associated disorder, for this condition.

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.

Glaucoma Syndromes: Insights into Glaucoma Genetics and Pathogenesis from Monogenic Syndromic Disorders.

Monogenic syndromic disorders frequently feature ocular manifestations, one of which is glaucoma. In many cases, glaucoma in children may go undetected, especially in those that have other severe systemic conditions that affect other parts of the eye and the body. Similarly, glaucoma may be the first presenting sign of a systemic syndrome. Awareness of syndromes associated with glaucoma is thus critical both for medical geneticists and ophthalmologists. In this review, we highlight six categories of disorders that feature glaucoma and other ocular or systemic manifestations: anterior segment dysgenesis syndromes, aniridia, metabolic disorders, collagen/vascular disorders, immunogenetic disorders, and nanophthalmos. The genetics, ocular and systemic features, and current and future treatment strategies are discussed. Findings from rare diseases also uncover important genes and pathways that may be involved in more common forms of glaucoma, and potential novel therapeutic strategies to target these pathways.

R-Ras Deficiency in Pericytes Causes Frequent Microphthalmia and Perturbs Retinal Vascular Development.

PURPOSE: The retinal vasculature is heavily invested by pericytes. Small GTPase R-Ras is highly expressed in endothelial cells and pericytes, suggesting importance of this Ras homolog for the regulation of the blood vessel wall. We investigated the specific contribution of pericyte-expressed R-Ras to the development of the retinal vasculature. METHODS: The effect of R-Ras deficiency in pericytes was analyzed in pericyte-targeted conditional Rras knockout mice at birth and during the capillary plexus formation in the neonatal retina. RESULTS: The offspring of these mice frequently exhibited unilateral microphthalmia. Analyses of the developing retinal vasculature in the eyes without microphthalmia revealed excessive endothelial cell proliferation, sprouting, and branching of the capillary plexus in these animals. These vessels were structurally defective with diminished pericyte coverage and basement membrane formation. Furthermore, these vessels showed reduced VE-cadherin staining and significantly elevated plasma leakage indicating the breakdown of the blood-retinal barrier. This defect was associated with considerable macrophage infiltration in the retina. CONCLUSIONS: The normal retinal vascular development is dependent on R-Ras expression in pericytes, and the absence of it leads to unattenuated angiogenesis and significantly weakens the blood-retinal barrier. Our findings underscore the importance of R-Ras for pericyte function during the normal eye development.

Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA): An Unconventional Mitochondrial Disorder.

Mitochondrial disorders, although heterogeneous, are traditionally described as conditions characterized by encephalomyopathy, hypotonia, and progressive postnatal organ failure. Here, we provide a systematic review of Linear Skin Defects with Multiple Congenital Anomalies (LSDMCA), a rare, unconventional mitochondrial disorder which presents as a developmental disease; its main clinical features include microphthalmia with different degrees of severity, linear skin lesions, and central nervous system malformations. The molecular basis of this disorder has been elusive for several years. Mutations were eventually identified in three X-linked genes, i.e., HCCS, COX7B, and NDUFB11, which are all endowed with defined roles in the mitochondrial respiratory chain. A peculiar feature of this condition is its inheritance pattern: X-linked dominant male-lethal. Only female or XX male individuals can be observed, implying that nullisomy for these genes is incompatible with normal embryonic development in mammals. All three genes undergo X-inactivation that, according to our hypothesis, may contribute to the extreme variable expressivity observed in this condition. We propose that mitochondrial dysfunction should be considered as an underlying cause in developmental disorders. Moreover, LSDMCA should be taken into consideration by clinicians when dealing with patients with microphthalmia with or without associated skin phenotypes.

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.