Aberrant gene expression yet undiminished retinal ganglion cell genesis in iPSC-derived models of optic nerve hypoplasia.

BACKGROUND: Optic nerve hypoplasia (ONH), the leading congenital cause of permanent blindness, is characterized by a retinal ganglion cell (RGC) deficit at birth. Multifactorial developmental events are hypothesized to underlie ONH and its frequently associated neurologic and endocrine abnormalities; however, environmental influences are unclear and genetic underpinnings are unexplored. This work investigates the genetic contribution to ONH RGC production and gene expression using patient induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs). MATERIALS AND METHODS: iPSCs produced from ONH patients and controls were differentiated to ROs. RGC genesis was assessed using immunofluorescence and flow cytometry. Flow-sorted BRN3+ cells were collected for RNA extraction for RNA-Sequencing. Differential gene expression was assessed using DESeq2 and edgeR. PANTHER was employed to identify statistically over-represented ontologies among the differentially expressed genes (DEGs). DEGs of high interest to ONH were distinguished by assessing function, mutational constraint, and prior identification in ONH, autism and neurodevelopmental disorder (NDD) studies. RESULTS: RGC genesis and survival were similar in ONH and control ROs. Differential expression of 70 genes was identified in both DESeq2 and edgeR analyses, representing a ~ 4-fold higher percentage of DEGs than in randomized study participants. DEGs showed trends towards over-representation of validated NDD genes and ONH exome variant genes. Among the DEGs, RAPGEF4 and DMD had the greatest number of disease-relevant features. CONCLUSIONS: ONH genetic background was not associated with impaired RGC genesis but was associated with DEGs exhibiting disease contribution potential. This constitutes some of the first evidence of a genetic contribution to ONH.

Whole genome sequencing unveils genetic heterogeneity in optic nerve hypoplasia.

Optic nerve hypoplasia (ONH) is a congenital malformation with a reduced number of retinal ganglion cell axons in a thin optic nerve. It is a common cause of visual impairment in children and ONH is associated with neurodevelopmental disorders, pituitary hormone deficiencies, and brain malformations. In most cases, the aetiology is unknown, but both environmental factors and genetic causes have been described. This study aimed to identify genetic variants underlying ONH in a well-characterised cohort of individuals with ONH. We performed array comparative genomic hybridization and whole genome sequencing in 29 individuals with ONH. Rare variants were verified by Sanger sequencing and inheritance was assessed in parental samples. We identified 11 rare single nucleotide variants (SNVs) in ten individuals, including a homozygous variant in KIF7 (previously associated with Joubert syndrome), a heterozygous de novo variant in COL4A1 (previously described in an individual with porencephaly), and a homozygous variant in COL4A2. In addition, one individual harboured a heterozygous variant in OPA1 and a heterozygous variant in COL4A1, both were inherited and assessed as variants of unknown clinical significance. Finally, a heterozygous deletion of 341 kb involving exons 7-18 of SOX5 (associated with Lamb-Schaffer syndrome) was identified in one individual. The overall diagnostic yield of pathogenic or likely pathogenic variants in individuals with ONH using whole genome sequencing was 4/29 (14%). Our results show that there is a genetic heterogeneity in ONH and indicate that genetic causes of ONH are not rare. We conclude that genetic testing is valuable in a substantial proportion of the individuals with ONH, especially in cases with non-isolated ONH.

Optic nerve hypoplasia in miniature poodle dogs: A preliminary genetic and candidate gene association study.

OBJECTIVE: To conduct a genetic and candidate gene association study with samples from phenotype-ascertained dogs to identify putative disease-associated gene/mutation for optic nerve hypoplasia (ONH) in the miniature poodle. ANIMALS STUDIED: A total of 43 miniature poodles from the United States and Europe, nine affected bilaterally with ONH, were included in the study. Pedigree information was recorded. PROCEDURES: A pedigree including all animals studied was assembled. Twenty-one genes typically expressed in ganglion cells or that are associated with ocular malformations and have a critical function in eye and neural retina development were selected. Exons and exon-intron boundaries of eight genes were sequenced in four ONH cases and four controls. Furthermore, cases and controls were genotyped with the Illumina CanineHD BeadChip to obtain genotypes for 13 additional candidate genes for haplotype association. RESULTS: The assembled pedigree connected all ONH-affected dogs to a possible common founder. Identified variants and haplotypes of the tested candidate genes did not segregate with the phenotype using Identity by Descent approach assuming autosomal recessive inheritance with variable but yet unknown penetrance. CONCLUSIONS: Pedigree analysis did not reveal the inheritance pattern. There is no evidence of association of the evaluated candidate genes with ONH; therefore, the screened candidate genes can provisionally be ruled out as causally associated with the disease.

Mutations in LAMB2 Are Associated With Albuminuria and Optic Nerve Hypoplasia With Hypopituitarism.

CONTEXT: Mutations in LAMB2, encoding the basement membrane protein, laminin ß2, are associated with an autosomal recessive disorder characterized by congenital nephrotic syndrome, ocular abnormalities, and neurodevelopmental delay (Pierson syndrome). CASE DESCRIPTION: This report describes a 12-year-old boy with short stature, visual impairment, and developmental delay who presented with macroscopic hematuria and albuminuria. He had isolated growth hormone deficiency, optic nerve hypoplasia, and a small anterior pituitary with corpus callosum dysgenesis on his cranial magnetic resonance imaging, thereby supporting a diagnosis of optic nerve hypoplasia syndrome. Renal histopathology revealed focal segmental glomerulosclerosis. Using next-generation sequencing on a targeted gene panel for steroid-resistant nephrotic syndrome, compound heterozygous missense mutations were identified in LAMB2 (c.737G>A p.Arg246Gln, c.3982G>C p.Gly1328Arg). Immunohistochemical analysis revealed reduced glomerular laminin ß2 expression compared to control kidney and a thin basement membrane on electron microscopy. Laminin ß2 is expressed during pituitary development and Lamb2-/- mice exhibit stunted growth, abnormal neural retinae, and here we show, abnormal parenchyma of the anterior pituitary gland. CONCLUSION: We propose that patients with genetically undefined optic nerve hypoplasia syndrome should be screened for albuminuria and, if present, screened for mutations in LAMB2.

Atonal homolog 7 (ATOH7) loss-of-function mutations in predominant bilateral optic nerve hypoplasia.

Optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality caused by underdevelopment of retinal ganglion cells (RGCs). Despite being a rare disease, ONH is the most common optic disk anomaly in ophthalmological practice. So far, mutations in several genes have been identified as causative; however, many cases of ONH remain without a molecular explanation. The early transcription factor atonal basic-helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitor cells and has a crucial role in RGC development. Previous studies have identified several mutations in the ATOH7 locus in cases of eye developmental diseases such as non-syndromic congenital retinal non-attachment and persistent hyperplasia of the primary vitreous. Here we present two siblings with a phenotype predominated by bilateral ONH, with additional features of foveal hypoplasia and distinct vascular abnormalities, where whole-exome sequencing identified two compound heterozygous missense mutations affecting a conserved amino acid residue within the bHLH domain of ATOH7 (NM_145178.3:c.175G>A; p.(Ala59Thr) and c.176C>T; p.(Ala59Val)). ATOH7 expression constructs with patient single nucleotide variants were cloned for functional characterization. Protein analyses revealed decreased protein amounts and significantly enhanced degradation in the presence of E47, a putative bHLH dimerization partner. Protein interaction assays revealed decreased heterodimerization and DNA-binding of ATOH7 variants, resulting in total loss of transcriptional activation of luciferase reporter gene expression. These findings strongly support pathogenicity of the two ATOH7 mutations, one of which is novel. Additionally, this report highlights the possible impact of altered ATOH7 dimerization on protein stability and function.

Non-Cell Autonomous Roles for CASK in Optic Nerve Hypoplasia.

Purpose: Heterozygous mutations in the essential X-linked gene CASK associate with optic nerve hypoplasia (ONH) and other retinal disorders in girls. CASK+/- heterozygous knockout mice with mosaic CASK expression exhibit ONH with a loss of retinal ganglion cells (RGCs) but no changes in retinal morphology. It remains unclear if CASK deficiency selectively affects RGCs or also affects other retinal cells. Furthermore, it is not known if CASK expression in RGCs is critical for optic nerve (ON) development and maintenance. Methods: The visual behavior of CASK+/- mice was assessed and electroretinography (ERG) was performed. Using a mouse line with a floxed CASK gene that expresses approximately 40% CASK globally in all cells (hypomorph) under hemizygous and homozygous conditions, we investigated effects of CASK reduction on the retina and ON. CASK then was completely deleted from RGCs to examine its cell-autonomous role. Finally, for the first time to our knowledge, we describe a hemizygous CASK missense mutation in a boy with ONH. Results: CASK+/- heterozygous mutant mice display reduced visual contrast sensitivity, but ERG is indistinguishable from wildtype. CASK hypomorph mice exhibit ONH, but deletion of CASK from RGCs in this background does not exacerbate the condition. The boy with ONH harbors a missense mutation (p.Pro673Leu) that destabilizes CASK and weakens the crucial CASK-neurexin interaction. Conclusions: Our results demonstrate that mosaic or global reduction in CASK expression and/or function disproportionately affects RGCs. CASK expression in RGCs does not appear critical for cell survival, indicating a noncell autonomous role for CASK in the development of ON.

Homozygous stop mutation in AHR causes autosomal recessive foveal hypoplasia and infantile nystagmus.

Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing, we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. AHR is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. Further, it has been shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in Ahr-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus. We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.