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.

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Purpose To identify disease‐causing mutations in four families with aniridia spectrum of Czech origin, to describe the associated phenotypes and to perform functional assessment of mutations located in canonical splice sites. Methods After complex ophthalmic examination of all available family members, DNA extraction from blood or saliva samples was performed. All coding regions of PAX6 gene were Sanger sequenced. In two cases of mutations which could potentially affect splicing of pre‐mRNA functional reassessment was accomplished using Exontrap vector. In one family paternity testing using a standard set of markers was performed. Results In total 13 affected individuals were examined. Except for common finding in aniridia, one individual had partial aniridia in the right eye and iris hypoplasia in the left eye, had bilateral ptosis, keratopathy with marked fibrovascular pannus, anterior polar cataracts, foveal hypoplasia, hepatopathy and trombocytopenia of unclear etiology. His affected daughter showed classical aniridia with total absence of iris and foveal hypoplasia in both eyes. Another individual had mild unilateral ptosis and anterior and posterior subcapsular cataracts. Bilateral microcornea, partial aniridia, surgically removed congenital cataracts and large posterior segment coloboma were present in another patient. In four probands, four different PAX6 mutations were identified, one mutation was novel (c.1183+1G>T). Two of detected variants were proven to affect pre‐mRNA splicing by skipping of adjutant exon sequences from mature mRNA. Conclusion Loss of visual functions in anterior segment dysgenesis disorders is often very severe, therefore establishing molecular diagnosis highly impacts patient management, enabling prenatal and preimplantation diagnostics. Our study enlarges the spectrum of known PAX6 mutations and confirms that anirida spectrum may also encompass combination of congenital ptosis, severe keratopathy and anterior polar cataracts. Vector Exontrap has been shown as an effective system for the evaluation of variants in canonical splicing sites. Funding Supported by SVV 260367/2017 and GAUK 82318‐2018.

Introduction: Achromatopsia is an autosomal recessive retinal disorder with an estimated prevalence ranging from 1 in 30.000 to 50.000. The disease is caused by mutations in six different genes. The aim of the study was to perform molecular genetic analysis in 11 unrelated probands with a clinical diagnosis of achromatopsia and to describe clinical findings in those that were found to carry biallelic pathogenic mutations. Methods: All probands and their parents underwent ophthalmic examination. Mutation detection was performed using Sanger sequencing of CNGB3 exons 6, 7, 9-13, which have been found to harbour most diseasecausing mutations in patients with achromatopsia of European origin. Results: Three known pathogenic variants in CNGB3 were identified in 2 probands. Proband 1 was a compound heterozygote for the c.819_826del; p.(Arg274Valfs*13) and c.1006G>T; p.(Glu336*). Proband 2 carried the c.1148del; p.(Thr383Ilefs*13) in a homozygous state. The best corrected visual acuity in proband 1 (aged 19 years) was 0.1 in both eyes, in proband 2 (aged 8 years) 0.05 in the right eye and 0.1 in the left eye. Both individuals had nystagmus, photophobia, and absence of colour discrimination. Fundus examination appeared normal however spectral-domain optical coherence tomography revealed subtle bilaterally symmetrical structural changes in the fovea. Conclusion: Molecular genetic analysis of Czech patients with achromatopsia was performed for the first time. Identification of diseasecausing mutations in achromatopsia is important for establishing an early diagnosis, participation in clinical trials assessing gene therapies and may be also used for preimplantation genetic diagnosis.