In Vivo Assessment of Retinal Phenotypes in Axenfeld-Rieger Syndrome.

Purpose: Axenfeld-Rieger syndrome (ARS) is characterized by ocular anomalies including posterior embryotoxon, iridocorneal adhesions, corectopia/iris hypoplasia, and developmental glaucoma. Although anterior segment defects and glaucoma contribute to decreased visual acuity, the role of potential posterior segment abnormalities has not been explored. We used high-resolution retinal imaging to test the hypothesis that individuals with ARS have posterior segment pathology. Methods: Three individuals with FOXC1-ARS and 10 with PITX2-ARS completed slit-lamp and fundus photography, optical coherence tomography (OCT), OCT angiography, and adaptive optics scanning light ophthalmoscopy (AOSLO). Quantitative metrics were compared to previously published values for individuals with normal vision. Results: All individuals demonstrated typical anterior segment phenotypes. Average ganglion cell and inner plexiform layer thickness was lower in PITX2-ARS, consistent with the glaucoma history in this group. A novel phenotype of foveal hypoplasia was noted in 40% of individuals with PITX2-ARS (but none with FOXC1-ARS). Moreover, the depth and volume of the foveal pit were significantly lower in PITX2-ARS compared to normal controls, even excluding individuals with foveal hypoplasia. Analysis of known foveal hypoplasia genes failed to identify an alternative explanation. Foveal cone density was decreased in one individual with foveal hypoplasia and normal in six without foveal hypoplasia. Two individuals (one from each group) demonstrated non-foveal retinal irregularities with regions of photoreceptor anomalies on OCT and AOSLO. Conclusions: These findings implicate PITX2 in the development of the posterior segment, particularly the fovea, in humans. The identified posterior segment phenotypes may contribute to visual acuity deficits in individuals with PITX2-ARS.

Complex balanced intrachromosomal rearrangement involving PITX2 identified as a cause of Axenfeld-Rieger Syndrome.

Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Alternative Genetic Diagnoses in Axenfeld-Rieger Syndrome Spectrum.

Axenfeld-Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities. PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld-Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy-Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1-ARS. Anterior segment anomalies are not currently associated with USP9X, yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13, BCOR, and an X chromosome deletion encompassing HCCS and AMELX (linked with ocular and dental anomalies, correspondingly) were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions.

CUX1-related neurodevelopmental disorder: deep insights into phenotype-genotype spectrum and underlying pathology.

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux1+/- mouse model. Through international collaboration, we assembled the phenotypic and molecular information for 34 individuals (23 unpublished individuals). We analyze brain CUX1 expression and susceptibility to epilepsy in Cux1+/- mice. We describe 34 individuals, from which 30 were unrelated, with 26 different null and four missense variants. The leading symptoms were mild to moderate delayed speech and motor development and borderline to moderate intellectual disability. Additional symptoms were muscular hypotonia, seizures, joint laxity, and abnormalities of the forehead. In Cux1+/- mice, we found delayed growth, histologically normal brains, and increased susceptibility to seizures. In Cux1+/- brains, the expression of Cux1 transcripts was half of WT animals. Expression of CUX1 proteins was reduced, although in early postnatal animals significantly more than in adults. In summary, disease-causing CUX1 variants result in a non-syndromic phenotype of developmental delay and intellectual disability. In some individuals, this phenotype ameliorates with age, resulting in a clinical catch-up and normal IQ in adulthood. The post-transcriptional balance of CUX1 expression in the heterozygous brain at late developmental stages appears important for this favorable clinical course.

Distinct Roles of Histone Lysine Demethylases and Methyltransferases in Developmental Eye Disease.

Histone lysine methyltransferase and demethylase enzymes play a central role in chromatin organization and gene expression through the dynamic regulation of histone lysine methylation. Consistent with this, genes encoding for histone lysine methyltransferases (KMTs) and demethylases (KDMs) are involved in complex human syndromes, termed congenital regulopathies. In this report, we present several lines of evidence for the involvement of these genes in developmental ocular phenotypes, suggesting that individuals with structural eye defects, especially when accompanied by craniofacial, neurodevelopmental and growth abnormalities, should be examined for possible variants in these genes. We identified nine heterozygous damaging genetic variants in KMT2D (5) and four other histone lysine methyltransferases/demethylases (KMT2C, SETD1A/KMT2F, KDM6A and KDM5C) in unrelated families affected with developmental eye disease, such as Peters anomaly, sclerocornea, Axenfeld-Rieger spectrum, microphthalmia and coloboma. Two families were clinically diagnosed with Axenfeld-Rieger syndrome and two were diagnosed with Peters plus-like syndrome; others received no specific diagnosis prior to genetic testing. All nine alleles were novel and five of them occurred de novo; five variants resulted in premature truncation, three were missense changes and one was an in-frame deletion/insertion; and seven variants were categorized as pathogenic or likely pathogenic and two were variants of uncertain significance. This study expands the phenotypic spectra associated with KMT and KDM factors and highlights the importance of genetic testing for correct clinical diagnosis.

Congenital aniridia beyond black eyes: From phenotype and novel genetic mechanisms to innovative therapeutic approaches.

Congenital PAX6-aniridia, initially characterized by the absence of the iris, has progressively been shown to be associated with other developmental ocular abnormalities and systemic features making congenital aniridia a complex syndromic disorder rather than a simple isolated disease of the iris. Moreover, foveal hypoplasia is now recognized as a more frequent feature than complete iris hypoplasia and a major visual prognosis determinant, reversing the classical clinical picture of this disease. Conversely, iris malformation is also a feature of various anterior segment dysgenesis disorders caused by PAX6-related developmental genes, adding a level of genetic complexity for accurate molecular diagnosis of aniridia. Therefore, the clinical recognition and differential genetic diagnosis of PAX6-related aniridia has been revealed to be much more challenging than initially thought, and still remains under-investigated. Here, we update specific clinical features of aniridia, with emphasis on their genotype correlations, as well as provide new knowledge regarding the PAX6 gene and its mutational spectrum, and highlight the beneficial utility of clinically implementing targeted Next-Generation Sequencing combined with Whole-Genome Sequencing to increase the genetic diagnostic yield of aniridia. We also present new molecular mechanisms underlying aniridia and aniridia-like phenotypes. Finally, we discuss the appropriate medical and surgical management of aniridic eyes, as well as innovative therapeutic options. Altogether, these combined clinical-genetic approaches will help to accelerate time to diagnosis, provide better determination of the disease prognosis and management, and confirm eligibility for future clinical trials or genetic-specific therapies.

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.

ARHGAP35 is a novel factor disrupted in human developmental eye phenotypes.

ARHGAP35 has known roles in cell migration, invasion and division, neuronal morphogenesis, and gene/mRNA regulation; prior studies indicate a role in cancer in humans and in the developing eyes, neural tissue, and renal structures in mice. We identified damaging variants in ARHGAP35 in five individuals from four families affected with anophthalmia, microphthalmia, coloboma and/or anterior segment dysgenesis disorders, together with variable non-ocular phenotypes in some families including renal, neurological, or cardiac anomalies. Three variants affected the extreme C-terminus of the protein, with two resulting in a frameshift and C-terminal extension and the other a missense change in the Rho-GAP domain; the fourth (nonsense) variant affected the middle of the gene and is the only allele predicted to undergo nonsense-mediated decay. This study implicates ARHGAP35 in human developmental eye phenotypes. C-terminal clustering of the identified alleles indicates a possible common mechanism for ocular disease but requires further studies.

Axenfeld-Rieger syndrome: more than meets the eye.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is characterised by typical anterior segment anomalies, with or without systemic features. The discovery of causative genes identified ARS subtypes with distinct phenotypes, but our understanding is incomplete, complicated by the rarity of the condition. METHODS: Genetic and phenotypic characterisation of the largest reported ARS cohort through comprehensive genetic and clinical data analyses. RESULTS: 128 individuals with causative variants in PITX2 or FOXC1, including 81 new cases, were investigated. Ocular anomalies showed significant overlap but with broader variability and earlier onset of glaucoma for FOXC1-related ARS. Systemic anomalies were seen in all individuals with PITX2-related ARS and the majority of those with FOXC1-related ARS. PITX2-related ARS demonstrated typical umbilical anomalies and dental microdontia/hypodontia/oligodontia, along with a novel high rate of Meckel diverticulum. FOXC1-related ARS exhibited characteristic hearing loss and congenital heart defects as well as previously unrecognised phenotypes of dental enamel hypoplasia and/or crowding, a range of skeletal and joint anomalies, hypotonia/early delay and feeding disorders with structural oesophageal anomalies in some. Brain imaging revealed highly penetrant white matter hyperintensities, colpocephaly/ventriculomegaly and frequent arachnoid cysts. The expanded phenotype of FOXC1-related ARS identified here was found to fully overlap features of De Hauwere syndrome. The results were used to generate gene-specific management plans for the two types of ARS. CONCLUSION: Since clinical features of ARS vary significantly based on the affected gene, it is critical that families are provided with a gene-specific diagnosis, PITX2-related ARS or FOXC1-related ARS. De Hauwere syndrome is proposed to be a FOXC1opathy.

TCEAL1 loss-of-function results in an X-linked dominant neurodevelopmental syndrome and drives the neurological disease trait in Xq22.2 deletions.

An Xq22.2 region upstream of PLP1 has been proposed to underly a neurological disease trait when deleted in 46,XX females. Deletion mapping revealed that heterozygous deletions encompassing the smallest region of overlap (SRO) spanning six Xq22.2 genes (BEX3, RAB40A, TCEAL4, TCEAL3, TCEAL1, and MORF4L2) associate with an early-onset neurological disease trait (EONDT) consisting of hypotonia, intellectual disability, neurobehavioral abnormalities, and dysmorphic facial features. None of the genes within the SRO have been associated with monogenic disease in OMIM. Through local and international collaborations facilitated by GeneMatcher and Matchmaker Exchange, we have identified and herein report seven de novo variants involving TCEAL1 in seven unrelated families: three hemizygous truncating alleles; one hemizygous missense allele; one heterozygous TCEAL1 full gene deletion; one heterozygous contiguous deletion of TCEAL1, TCEAL3, and TCEAL4; and one heterozygous frameshift variant allele. Variants were identified through exome or genome sequencing with trio analysis or through chromosomal microarray. Comparison with previously reported Xq22 deletions encompassing TCEAL1 identified a more-defined syndrome consisting of hypotonia, abnormal gait, developmental delay/intellectual disability especially affecting expressive language, autistic-like behavior, and mildly dysmorphic facial features. Additional features include strabismus, refractive errors, variable nystagmus, gastroesophageal reflux, constipation, dysmotility, recurrent infections, seizures, and structural brain anomalies. An additional maternally inherited hemizygous missense allele of uncertain significance was identified in a male with hypertonia and spasticity without syndromic features. These data provide evidence that TCEAL1 loss of function causes a neurological rare disease trait involving significant neurological impairment with features overlapping the EONDT phenotype in females with the Xq22 deletion.

CRISPR-Cas9-mediated functional dissection of the foxc1 genomic region in zebrafish identifies critical conserved cis-regulatory elements.

FOXC1 encodes a forkhead-domain transcription factor associated with several ocular disorders. Correct FOXC1 dosage is critical to normal development, yet the mechanisms controlling its expression remain unknown. Together with FOXQ1 and FOXF2, FOXC1 is part of a cluster of FOX genes conserved in vertebrates. CRISPR-Cas9-mediated dissection of genomic sequences surrounding two zebrafish orthologs of FOXC1 was performed. This included five zebrafish-human conserved regions, three downstream of foxc1a and two remotely upstream of foxf2a/foxc1a or foxf2b/foxc1b clusters, as well as two intergenic regions between foxc1a/b and foxf2a/b lacking sequence conservation but positionally corresponding to the area encompassing a previously reported glaucoma-associated SNP in humans. Removal of downstream sequences altered foxc1a expression; moreover, zebrafish carrying deletions of two or three downstream elements demonstrated abnormal phenotypes including enlargement of the anterior chamber of the eye reminiscent of human congenital glaucoma. Deletions of distant upstream conserved elements influenced the expression of foxf2a/b or foxq1a/b but not foxc1a/b within each cluster. Removal of either intergenic sequence reduced foxc1a or foxc1b expression during late development, suggesting a role in transcriptional regulation despite the lack of conservation at the nucleotide level. Further studies of the identified regions in human patients may explain additional individuals with developmental ocular disorders.

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.

SOX11 variants cause a neurodevelopmental disorder with infrequent ocular malformations and hypogonadotropic hypogonadism and with distinct DNA methylation profile.

PURPOSE: This study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants. METHODS: Individuals with protein altering variants in SOX11 were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of SOX11 in developing human brain was defined using RNAscope. RESULTS: We reported 38 new patients with SOX11 variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of SOX11 syndrome. A distinctive pattern of blood DNA methylation was identified in SOX11 syndrome, separating SOX11 syndrome from other BAFopathies. CONCLUSION: SOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.

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.

WDR37 syndrome: identification of a distinct new cluster of disease-associated variants and functional analyses of mutant proteins.

Missense variants located in the N-terminal region of WDR37 were recently identified to cause a multisystemic syndrome affecting neurological, ocular, gastrointestinal, genitourinary, and cardiac development. WDR37 encodes a WD40 repeat-containing protein of unknown function. We identified three novel WDR37 variants, two likely pathogenic de novo alleles and one inherited variant of uncertain significance, in individuals with phenotypes overlapping those previously reported but clustering in a different region of the protein. The novel alleles are C-terminal to the prior variants and located either within the second WD40 motif (c.659A>G p.(Asp220Gly)) or in a disordered protein region connecting the second and third WD40 motifs (c.778G>A p.(Asp260Asn) and c.770C>A p.(Pro257His)). The three novel mutants showed normal cellular localization but lower expression levels in comparison to wild-type WDR37. To investigate the normal interactions of WDR37, we performed co-immunoprecipitation and yeast two-hybrid assays. This revealed the ability of WDR37 to form homodimers and to strongly bind PACS1 and PACS2 phosphofurin acidic cluster sorting proteins; immunocytochemistry confirmed colocalization of WDR37 with PACS1 and PACS2 in human cells. Next, we analyzed previously reported and novel mutants for their ability to dimerize with wild-type WDR37 and bind PACS proteins. Interaction with wild-type WDR37 was not affected for any variant; however, one novel mutant, p.(Asp220Gly), lost its ability to bind PACS1 and PACS2. In summary, this study presents a novel region of WDR37 involved in human disease, identifies PACS1 and PACS2 as major binding partners of WDR37 and provides insight into the functional effects of various WDR37 variants.

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.

Dominant variants in PRR12 result in unilateral or bilateral complex microphthalmia.

Complex microphthalmia is characterized by small eyes with additional abnormalities that may include anterior segment dysgenesis. While many genes are known, a genetic cause is identified in only 4-30% of microphthalmia, with the lowest rate in unilateral cases. We identified four novel pathogenic loss-of-function alleles in PRR12 in families affected by complex microphthalmia and/or Peters anomaly, including two de novo, the first dominantly transmitted allele, as well as the first splicing variant. The ocular phenotypes were isolated with no additional systemic features observed in two unrelated families. Remarkably, ocular phenotypes were asymmetric in all individuals and unilateral (with structurally normal contralateral eye) in three. There are only three previously reported PRR12 variants identified in probands with intellectual disability, neuropsychiatric disorders, and iris anomalies. While some overlap with previously reported cases is seen, nonsyndromic developmental ocular anomalies are a novel phenotype for this gene. Additional phenotypic expansions included short stature and normal development/cognition, each noted in two individuals in this cohort, as well as absence of neuropsychiatric disorders in all. This study identifies new associations for PRR12 disruption in humans and presents a genetic diagnosis resulting in unilateral ocular phenotypes in a significant proportion of cases.

Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome.

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.