Congenital anterior segment ocular disorders: Genotype-phenotype correlations and emerging novel mechanisms.

Development of the anterior segment of the eye requires reciprocal sequential interactions between the arising tissues, facilitated by numerous genetic factors. Disruption of any of these processes results in congenital anomalies in the affected tissue(s) leading to anterior segment disorders (ASD) including aniridia, Axenfeld-Rieger anomaly, congenital corneal opacities (Peters anomaly, cornea plana, congenital primary aphakia), and primary congenital glaucoma. Current understanding of the genetic factors involved in ASD remains incomplete, with approximately 50% overall receiving a genetic diagnosis. While some genes are strongly associated with a specific clinical diagnosis, the majority of known factors are linked with highly variable phenotypic presentations, with pathogenic variants in FOXC1, CYP1B1, and PITX2 associated with the broadest spectrum of ASD conditions. This review discusses typical clinical presentations including associated systemic features of various forms of ASD; the latest functional data and genotype-phenotype correlations related to 25 ASD factors including newly identified genes; promising novel candidates; and current and emerging treatments for these complex conditions. Recent developments of interest in the genetics of ASD include identification of phenotypic expansions for several factors, discovery of multiple modes of inheritance for some genes, and novel mechanisms including a growing number of non-coding variants and alleles affecting specific domains/residues and requiring further studies.

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 Genetic Diagnoses in Septo-Optic Dysplasia.

Septo-optic dysplasia (SOD) is a developmental phenotype characterized by midline neuroradiological anomalies, optic nerve hypoplasia, and pituitary anomalies, with a high degree of variability and additional systemic anomalies present in some cases. While disruption of several transcription factors has been identified in SOD cohorts, most cases lack a genetic diagnosis, with multifactorial risk factors being thought to play a role. Exome sequencing in a cohort of families with a clinical diagnosis of SOD identified a genetic diagnosis in 3/6 families, de novo variants in SOX2, SHH, and ARID1A, and explored variants of uncertain significance in the remaining three. The outcome of this study suggests that investigation for a genetic etiology is warranted in individuals with SOD, particularly in the presence of additional syndromic anomalies and when born to older, multigravida mothers. The identification of causative variants in SHH and ARID1A further expands the phenotypic spectra associated with these genes and reveals novel pathways to explore in septo-optic dysplasia.

Comprehensive phenotypic and functional analysis of dominant and recessive FOXE3 alleles in ocular developmental disorders.

The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.

Identification of missense MAB21L1 variants in microphthalmia and aniridia.

Microphthalmia, coloboma, and aniridia are congenital ocular phenotypes with a strong genetic component but often unknown cause. We present a likely causative novel variant in MAB21L1, c.152G>T p.(Arg51Leu), in two family members with microphthalmia and aniridia, as well as novel or rare compound heterozygous variants of uncertain significance, c.184C>T p.(Arg62Cys)/c.-68T>C, and c.658G>C p.(Gly220Arg)/c.*529A>G, in two additional probands with microphthalmia, coloboma and/or cataracts. All variants were predicted as damaging by in silico programs. In vitro studies of coding variants revealed normal subcellular localization but variable stability for the corresponding mutant proteins. In vivo complementation assays using the zebrafish mab21l2 Q48Sfs*5 loss-of-function line demonstrated that though overexpression of wild-type MAB21L1 messenger RNA (mRNA) compensated for the loss of mab21l2, none of the coding variant mRNAs produced a statistically significant rescue, with p.(Arg51Leu) showing the highest degree of functional deficiency. Dominant variants in a close homolog of MAB21L1, MAB21L2, have been associated with microphthalmia and/or coloboma and repeatedly involved the same Arg51 residue, further supporting its pathogenicity. The possible role of p.(Arg62Cys) and p.(Gly220Arg) in microphthalmia is similarly supported by the observed functional defects, with or without an additional impact from noncoding MAB21L1 variants identified in each patient. This study suggests a broader spectrum of MAB21L1-associated disease.

Genetic disruption of zebrafish mab21l1 reveals a conserved role in eye development and affected pathways.

BACKGROUND: The male-abnormal 21 like (MAB21L) genes are important in human ocular development. Homozygous loss of MAB21L1 leads to corneal dystrophy in all affected individuals along with cataracts and buphthalmos in some. The molecular function and downstream pathways of MAB21L factors are largely undefined. RESULTS: We generated the first mab21l1 zebrafish mutant carrying a putative loss-of-function allele, c.107delA p.(Lys36Argfs*7). At the final stages of embryonic development, homozygous mab21l1c.107delA fish displayed enlarged anterior chambers and corneal thinning which progressed with age. Additional studies revealed increased cell death in the mutant corneas, transformation of the cornea into a skin-like epithelium, and progressive lens degeneration with development of fibrous masses in the anterior chamber. RNA-seq of wild-type and mutant ocular transcriptomes revealed significant changes in expression of several genes, including irf1a and b, stat1, elf3, krt17, tlr9, and loxa associated with immunity and/or corneal function. Abnormal expression of lysyl oxidases have been previously linked with corneal thinning, fibrosis, and lens defects in mammals, suggesting a role for loxa misexpression in the progressive mab21l1c.107delA eye phenotype. CONCLUSIONS: Zebrafish mab21l1 is essential for normal corneal development, similar to human MAB21L1. The identified molecular changes in mab21l1c.107delA mutants provide the first clues about possible affected pathways.