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Biallelic pathogenic variants in ALDH1A3 are responsible for approximately 11% of recessively inherited cases of severe developmental eye anomalies. Some individuals can display variable neurodevelopmental features, but the relationship to the ALDH1A3 variants remains unclear. Here, we describe seven unrelated families with biallelic pathogenic ALDH1A3 variants: four compound heterozygous and three homozygous. All affected individuals had bilateral anophthalmia/microphthalmia (A/M), three with additional intellectual or developmental delay, one with autism and seizures and three with facial dysmorphic features. This study confirms that individuals with biallelic pathogenic ALDH1A3 variants consistently manifest A/M, but additionally display neurodevelopmental features with significant intra- and interfamilial variability. Furthermore, we describe the first case with cataract and highlight the importance of screening ALDH1A3 variants in nonconsanguineous families with A/M., (© 2023. The Author(s).)

Peters' anomaly (PA) is a rare anterior segment dysgenesis characterized by central corneal opacity and irido-lenticulo-corneal adhesions. Several genes are involved in syndromic or isolated PA (B3GLCT, PAX6, PITX3, FOXE3, CYP1B1). Some copy number variations (CNVs) have also been occasionally reported. Despite this genetic heterogeneity, most of patients remain without genetic diagnosis. We retrieved a cohort of 95 individuals with PA and performed genotyping using a combination of comparative genomic hybridization, whole genome, exome and targeted sequencing of 119 genes associated with ocular development anomalies. Causative genetic defects involving 12 genes and CNVs were identified for 1/3 of patients. Unsurprisingly, B3GLCT and PAX6 were the most frequently implicated genes, respectively in syndromic and isolated PA. Unexpectedly, the third gene involved in our cohort was SOX2, the major gene of micro-anophthalmia. Four unrelated patients with PA (isolated or with microphthalmia) were carrying pathogenic variants in this gene that was never associated with PA before. Here we described the largest cohort of PA patients ever reported. The genetic bases of PA are still to be explored as genetic diagnosis was unavailable for 2/3 of patients. Nevertheless, we showed here for the first time the involvement of SOX2 in PA, offering new evidence for its role in corneal transparency and anterior segment development., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants., Methods: By locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6 -related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available., Results: Five out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation., Conclusion: We describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Microphthalmia, anophthalmia, and anterior segment dysgenesis are severe ocular developmental defects. There is a wide genetic heterogeneity leading to these ocular malformations. By using whole genome, exome and targeted sequencing in patients with ocular developmental anomalies, six biallelic pathogenic variants (including five novel variants) were identified in the PXDN gene in four families with microphthalmia and anterior segment dysgenesis. Only 11 different mutations (11 families) have been described in this gene to date. The phenotype of these patients is variable in severity, ranging from cataract and developmental glaucoma to complex microphthalmia. Interestingly, two unrelated patients of our series presented with an ocular phenotype including aniridia and microspherophakia. However, despite various phenotypic presentations and types of mutations, no genotype-phenotype correlation could be made. Thus, this work improves our knowledge of the recessive phenotype associated with biallelic variants in this gene and highlights the importance of screening PXDN in patients with anterior segment dysgenesis with or without microphthalmia.

Purpose: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions., Methods: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGU eye ) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants., Results: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease., Conclusion: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include β-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Eye formation is the result of coordinated induction and differentiation processes during embryogenesis. Disruption of any one of these events has the potential to cause ocular growth and structural defects, such as anophthalmia and microphthalmia (A/M). A/M can be isolated or occur with systemic anomalies, when they may form part of a recognizable syndrome. Their etiology includes genetic and environmental factors; several hundred genes involved in ocular development have been identified in humans or animal models. In humans, around 30 genes have been repeatedly implicated in A/M families, although many other genes have been described in single cases or families, and some genetic syndromes include eye anomalies occasionally as part of a wider phenotype. As a result of this broad genetic heterogeneity, with one or two notable exceptions, each gene explains only a small percentage of cases. Given the overlapping phenotypes, these genes can be most efficiently tested on panels or by whole exome/genome sequencing for the purposes of molecular diagnosis. However, despite whole exome/genome testing more than half of patients currently remain without a molecular diagnosis. The proportion of undiagnosed cases is even higher in those individuals with unilateral or milder phenotypes. Furthermore, even when a strong gene candidate is available for a patient, issues of incomplete penetrance and germinal mosaicism make diagnosis and genetic counseling challenging. In this review, we present the main genes implicated in non-syndromic human A/M phenotypes and, for practical purposes, classify them according to the most frequent or predominant phenotype each is associated with. Our intention is that this will allow clinicians to rank and prioritize their molecular analyses and interpretations according to the phenotypes of their patients.

GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.

Background: Congenital cataract displays large phenotypic (syndromic and isolated cataracts) and genetic heterogeneity. Mutations in several transcription factors involved in eye development, like PITX3, have been associated with congenital cataracts and anterior segment mesenchymal disorders., Materials and Methods: Targeted sequencing of 187 genes involved in ocular development was performed in 96 patients with mainly anophthalmia and microphthalmia. Additionally, Sanger sequencing analysis of PITX3 was performed on a second cohort of 32 index cases with congenital cataract and Peters anomaly and/or sclereocornea., Results: We described five families with four different PITX3 mutations, two of which were novel. In Family 1, the heterozygous recurrent c.640_656dup (p.Gly220Profs*95) mutation cosegregated with eye anomalies ranging from congenital cataract to Peters anomaly. In Family 2, the novel c.669del [p.(Leu225Trpfs*84)] mutation cosegregated with dominantly inherited eye anomalies ranging from posterior embryotoxon to congenital cataract in heterozygous carriers and congenital sclereocornea and cataract in a patient homozygous for this mutation. In Family 3, we identified the recurrent heterozygous c.640_656dup (p.Gly220Profs*95) mutation segregating with congenital cataract. In Family 4, the de novo c.582del [p.(Ile194Metfs*115)] mutation was identified in a patient with congenital cataract, microphthalmia, developmental delay and autism. In Family 5, the c.38G>A (p.Ser13Asn) mutation segregated dominantly in a family with Peters anomaly, which is a novel phenotype associated with the c.38G>A variant compared with the previously reported isolated congenital cataract., Conclusions: Our study unveils different phenotypes associated with known and novel mutations in PITX3, which will improve the genetic counselling of patients and their families.

Axenfeld-Rieger syndrome (ARS) is a heterogeneous clinical entity transmitted in an autosomal dominant manner. The main feature, Axenfeld-Rieger Anomaly (ARA), is a malformation of the anterior segment of the eye that can lead to glaucoma and impair vision. Extra-ocular defects have also been reported. Point mutations of FOXC1 and PITX2 are responsible for about 40% of the ARS cases. We describe the phenotype of a patient carrying a deletion encompassing the 4q25 locus containing PITX2 gene. This child presented with a congenital heart defect (Tetralogy of Fallot, TOF) and no signs of ARA. He is the first patient described with TOF and a complete deletion of PITX2 (arr[GRCh37]4q25(110843057-112077858)x1, involving PITX2, EGF, ELOVL6 and ENPEP) inherited from his ARS affected mother. In addition, to our knowledge, he is the first patient reported with no ocular phenotype associated with haploinsufficiency of PITX2. We compare the phenotype and genotype of this patient to those of five other patients carrying 4q25 deletions. Two of these patients were enrolled in the university hospital in Toulouse, while the other three were already documented in DECIPHER. This comparative study suggests both an incomplete penetrance of the ocular malformation pattern in patients carrying PITX2 deletions and a putative association between TOF and PITX2 haploinsufficiency., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2., (© 2016 Chassaing et al.; Published by Cold Spring Harbor Laboratory Press.)

Disease-causing mutations affecting either one of the transcription factor genes, PITX2 or FOXC1, have been previously identified in patients with Axenfeld-Rieger syndrome (AR). We identified a family who segregate novel mutations in both PITX2 (p.Ser233Leu) and FOXC1 (c.609delC). The most severely affected individual, who presented with an atypical phenotype of corneal opacification, lens extrusion, persistent hyperplastic primary vitreous (PHPV), and subsequent bilateral retinal detachment, inherited mutations in both genes, whereas the single heterozygous mutations caused mild AR phenotypes. This is the first report of such digenic inheritance. By analyzing cognate targets of each gene, we showed that FOXC1 and PITX2 can independently regulate their own and each other's target gene promoters and do not show synergistic action in vitro. Mutation in either gene caused reduced transcriptional activation to different extents on the FOXO1 and PLOD1 promoters, whereas both mutations in combination showed the lowest level of activation. These data show how the compensatory activity of one factor, when the other is impaired, may lessen the phenotypic impact of developmental anomalies, yet reduced activity of both transcription factors increased disease severity. This suggests an under-reported mechanism for phenotypic variability whereby single mutations cause mild AR phenotypes, whereas digenic inheritance increases phenotypic severity., (© 2011 Wiley-Liss, Inc.)

Purpose: Anterior segment ocular dysgenesis (ASOD) is a broad heterogeneous group of diseases detectable at the clinical and molecular level. In a patient with bilateral congenital ASOD including aniridia and aphakia, a complex chromosomal rearrangement, inv(2)(p22.3q12.1)t(2;16)(q12.1;q12.2), was characterized at the molecular level, to identify candidate genes implicated in ASOD., Methods: After negative sequencing of the PAX6, FOXC1, and PITX2 genes, we used fluorescence in situ hybridization (FISH) and Southern blot analysis to characterize the chromosomal breakpoints. Candidate genes were selected, and in situ tissue expression analysis was performed on human fetuses and embryos., Results: Molecular analyses showed that the 16q12.2 breakpoint in this rearrangement occurs in a 625-bp region centromeric to the IRX3 gene, which belongs to the IRXB cluster. In situ hybridization expression studies showed that during early human embryonic development, the IRX3 gene is expressed in the anterior segment of the eye. Of interest, it has been shown previously that a highly conserved noncoding region (HCNCR) is located 300 kb centromeric to the IRX3 gene and induces, in a murine transgenic assay, an expression pattern fitting that of the IRX3 gene., Conclusions: The authors propose that the 16q12.2 breakpoint of this complex translocation is causally related to the ocular anterior segment dysgenesis observed in this patient. This translocation is assumed to separate the HCNCR from the IRXB cluster genes, thus deregulating the IRXB cluster and leading to the ASOD observed by a positional effect.

The eye is a complex organ whose embryonic origin and developmental dynamics are now better known. The genetic basis of eye development is interesting with respect to both fundamental notions and medical implications. They are reviewed here in the light of the latest anatomical and molecular knowledge. Anomalies of the master control pathways and differentiation cascades are described, with reference to human cases and animal models. New knowledge of the pathophysiology of inherited defects sometimes challenges old classifications, provides new diagnostic tools, and may soon lead to novel treatments.

Objective: Several hereditary ischemic small-vessel diseases of the brain have been reported during the last decade. Some of them have ophthalmological, mainly retinal, manifestations. Herein, we report on a family affected by vascular leukoencephalopathy and variable abnormalities of the anterior chamber of the eye., Methods: After the occurrence of a small, deep infarct associated with white matter lesions in a patient with a medical history of congenital cataract and amblyopia, we conducted clinical and neuroradiological investigations in 10 of her relatives., Results: Diffuse leukoencephalopathy associated with ocular malformations of the Axenfeld-Rieger type was observed in five individuals. Familial genetic analyses led to the identification of a novel missense mutation in the COL4A1 gene, p.G720D, which cosegregates with the disease., Interpretation: Our data corroborate previous observations demonstrating the role of COL4A1 in cerebral microangiopathy and expand the phenotypic spectrum associated with mutations in this gene. We delineate a novel association between the Axenfeld-Rieger anomaly and leukoencephalopathy and stroke. Ann Neurol 2007.

Purpose: Several ocular defects have been identified as a consequence of the PAX6 gene mutations. With regard to the implication of this gene in unusual phenotypes, we report a family presenting with congenital nystagmus, foveal hypoplasia, and iris hypoplasia or atypical coloboma., Design: Observational case report., Methods: The entire transcribed region of the PAX6 gene was submitted to mutation search at the DNA and mRNA levels in five affected members of a French family in test with 82 normal subjects., Results: A novel heterozygous PAX6 gene splice mutation (IVS4 + 5G>C) was identified. The mutation is located in IVS4 within the consensus donor splice site. A mutant mRNA lacking exon 4 as the sole defect was evidenced. The resultant protein was predicted to contain a cryptic ATG initiation codon in exon 3 and a slightly altered paired-domain in an open reading frame extended by 13 amino acids., Conclusions: The association of anterior segment anomalies and foveal hypoplasia with one of the slightest alterations of the PAX6 protein described to date confirms the association of variant phenotypes with hypomorphic alleles. Mutation screening of the PAX6 gene could be useful in elucidating the origin of complex ocular malformations.

PAX6, a paired box transcriptional factor, is considered as the master control gene for morphogenesis of the eye. Human PAX6 mutations have been associated with a range of eye abnormalities, including aniridia, various anterior segment defects and foveal hypoplasia. We carried out a mutational analysis of the PAX6 gene in 54 unrelated patients with aniridia or related syndromes. A deleterious variation was evidenced in 17 sporadic cases (50%) and in 13 (72%) familial cases. Twenty-four different mutations, 17 of which are novel, were found. The spectrum of PAX6 mutations was highly homogeneous: 23 mutations (96%) leading to premature stop codons (eight nonsense and four splice site mutations, 11 insertions and deletions) and only one (4%) missense mutation. Twenty-two mutations were associated with aniridia phenotypes whereas two were associated with atypical phenotypes. These latter encompassed a missense mutation (R19P) in an individual with a microphthalmia-sclerocornea and a splice site mutation (IVS4+5G > C) in a family presenting with a congenital nystagmus. Both represented the most probably hypomorphic alleles. Aniridia cases were associated with nonsense or frameshifting mutations. A careful examination of the phenotypes did not make it possible to recognise significant differences whenever the predicted protein was deprived of one or another of its functional domains. This strongly suggested that most of the truncating mutations generated null alleles by nonsense mediated mRNA decay. Our observations support the concept of dosage effects of the PAX6 mutations as well as presenting evidence for variable expressivity.

Microphthalmia and anophthalmia (MA) are severe developmental eye anomalies, many of which are likely to have an underlying genetic cause. More than 30 genes have been described, each of which is responsible for a small percentage of these anomalies. Among these, is the FOXE3 gene, which was initially described in individuals with dominantly inherited anterior segment dysgenesis and, subsequently, associated with recessively inherited primary aphakia, sclerocornea and microphthalmia. In this work, we describe 8 individuals presenting with an MA phenotype. Among them, 7 are carrying biallelic recessive FOXE3 mutations and 2 of these have novel mutations: p.(Ala78Thr) and p.(Arg104Cys). The last of our patients is carrying in the heterozygous state the recessive p.(Arg90Leu) mutation in the FOXE3 gene. To further understand FOXE3 involvement in this wide spectrum of ocular anomalies with 2 different patterns of inheritance, we reviewed all individuals with ocular abnormalities described in the literature for which a FOXE3 mutation was identified. This review demonstrates that correlations exist between the mutation type, mode of inheritance and the phenotype severity. Furthermore, understanding the genetic basis of these conditions will contribute to overall understanding of eye development, improve the quality of care, genetic counseling and, in future, gene‐based therapies. [ABSTRACT FROM AUTHOR]