Disruption of common ocular developmental pathways in patient-derived optic vesicle models of microphthalmia.

Genetic perturbations influencing early eye development can result in microphthalmia, anophthalmia, and coloboma (MAC). Over 100 genes are associated with MAC, but little is known about common disease mechanisms. In this study, we generated induced pluripotent stem cell (iPSC)-derived optic vesicles (OVs) from two unrelated microphthalmia patients and healthy controls. At day 20, 35, and 50, microphthalmia patient OV diameters were significantly smaller, recapitulating the "small eye" phenotype. RNA sequencing (RNA-seq) analysis revealed upregulation of apoptosis-initiating and extracellular matrix (ECM) genes at day 20 and 35. Western blot and immunohistochemistry revealed increased expression of lumican, nidogen, and collagen type IV, suggesting ECM overproduction. Increased apoptosis was observed in microphthalmia OVs with reduced phospho-histone 3 (pH3+) cells confirming decreased cell proliferation at day 35. Pharmacological inhibition of caspase-8 activity with Z-IETD-FMK decreased apoptosis in one patient model, highlighting a potential therapeutic approach. These data reveal shared pathophysiological mechanisms contributing to a microphthalmia phenotype.

Generation of the induced pluripotent stem cell line IOCVi001-A from a patient with the MFRP-related retinitis pigmentosa-nanophthalmos syndrome.

Retinitis pigmentosa (RP) is the most common retinal degeneration in humans and is characterized by the progressive degeneration of rods and cones and retinal pigment epithelium. We generated the IOCVi001-A induced pluripotent stem cell (iPSC) line from dermal fibroblast of a patient with a homozygous c.498_499insC (p.(Asn167Glnfs⁎34) variant in the Membrane-type frizzled related protein (MFRP) gene, a genetic defect causing a syndrome characterized by RP and small eye size (nanophthalmos). IOCVi001-A displayed normal stemness, expressed pluripotent stem cell markers and displayed a normal karyotype. This iPSC line can be used for in vitro disease modeling for complex forms of RP.

Emerging roles of the MiT/TFE factors in cancer.

The microphthalmia/transcription factor E (MiT/TFE) transcription factors (TFs; TFEB, TFE3, MITF, and TFEC) play a central role in cellular catabolism and quality control and are subject to extensive layers of regulation that influence their localization, stability, and activity. Recent studies have highlighted a broader role for these TFs in driving diverse stress-adaptation pathways, which manifest in a context- and tissue-dependent manner. Several human cancers upregulate the MiT/TFE factors to survive extreme fluctuations in nutrients, energy, and pharmacological challenges. Emerging data suggest that reduced activity of the MiT/TFE factors can also promote tumorigenesis. Here, we outline recent findings relating to novel mechanisms of regulation and activity of MiT/TFE proteins across some of the most aggressive human cancers.

The Regulation of MiTF/TFE Transcription Factors Across Model Organisms: from Brain Physiology to Implication for Neurodegeneration.

The microphthalmia/transcription factor E (MiTF/TFE) transcription factors are responsible for the regulation of various key processes for the maintenance of brain function, including autophagy-lysosomal pathway, lipid catabolism, and mitochondrial homeostasis. Among them, autophagy is one of the most relevant pathways in this frame; it is evolutionary conserved and crucial for cellular homeostasis. The dysregulation of MiTF/TFE proteins was shown to be involved in the development and progression of neurodegenerative diseases. Thus, the characterization of their function is key in the understanding of the etiology of these diseases, with the potential to develop novel therapeutics targeted to MiTF/TFE proteins and to the autophagic process. The fact that these proteins are evolutionary conserved suggests that their function and dysfunction can be investigated in model organisms with a simpler nervous system than the mammalian one. Building not only on studies in mammalian models but also in complementary model organisms, in this review we discuss (1) the mechanistic regulation of MiTF/TFE transcription factors; (2) their roles in different regions of the central nervous system, in different cell types, and their involvement in the development of neurodegenerative diseases, including lysosomal storage disorders; (3) the overlap and the compensation that occur among the different members of the family; (4) the importance of the evolutionary conservation of these protein and the process they regulate, which allows their study in different model organisms; and (5) their possible role as therapeutic targets in neurodegeneration.

Disruption of fetal eye development caused by insulin-induced maternal hypoglycemia in rats.

We previously revealed that insulin-induced severe and long-lasting maternal hypoglycemia in rats caused anophthalmia and microphthalmia in fetuses; however, it remained unclear whether hypoglycemia-induced eye anomalies were developmental retardation or disruption, and when and how they developed. Hence, we induced hypoglycemia in pregnant Sprague-Dawley rats by injecting insulin from Days 6 to 11 of pregnancy and performed periodical histopathological examination of fetal eyes from embryonic days (E)10 to 20. On E10, optic vesicle had developed normally both in the control and insulin-treated group; however, on E11, optic cup (OC) had developed in the control group but not in the insulin-treated group. On E12, neural retina (NR), retinal pigmented epithelium (RPE), lens, and presumptive cornea had been observed in the control group. In contrast, lens pit and OC with remaining space between RPE and NR had developed in the insulin-treated group. From E13 to E15, developmental disruption characterized by defects, hypoplasia, and degeneration in the retina, lens, and cornea was observed in the insulin-treated group, resulting in anophthalmia or microphthalmia on E20. Moreover, the expression of MITF and chx10, which are essential for early eye development by expressing in the presumptive retina and lens and regulating each other's expression level, was ectopic and suppressed on E11. In conclusion, insulin-induced maternal hypoglycemia caused developmental disruption, but not simple developmental retardation of fetal eyes, and its trigger might be a failure of presumptive retina and lens to interact on E11.

R-Ras Deficiency in Pericytes Causes Frequent Microphthalmia and Perturbs Retinal Vascular Development.

PURPOSE: The retinal vasculature is heavily invested by pericytes. Small GTPase R-Ras is highly expressed in endothelial cells and pericytes, suggesting importance of this Ras homolog for the regulation of the blood vessel wall. We investigated the specific contribution of pericyte-expressed R-Ras to the development of the retinal vasculature. METHODS: The effect of R-Ras deficiency in pericytes was analyzed in pericyte-targeted conditional Rras knockout mice at birth and during the capillary plexus formation in the neonatal retina. RESULTS: The offspring of these mice frequently exhibited unilateral microphthalmia. Analyses of the developing retinal vasculature in the eyes without microphthalmia revealed excessive endothelial cell proliferation, sprouting, and branching of the capillary plexus in these animals. These vessels were structurally defective with diminished pericyte coverage and basement membrane formation. Furthermore, these vessels showed reduced VE-cadherin staining and significantly elevated plasma leakage indicating the breakdown of the blood-retinal barrier. This defect was associated with considerable macrophage infiltration in the retina. CONCLUSIONS: The normal retinal vascular development is dependent on R-Ras expression in pericytes, and the absence of it leads to unattenuated angiogenesis and significantly weakens the blood-retinal barrier. Our findings underscore the importance of R-Ras for pericyte function during the normal eye development.

Genotype-phenotype spectrum in isolated and syndromic nanophthalmos.

PURPOSE: To (i) describe a series of patients with isolated or syndromic nanophthalmos with the underlying genetic causes, including novel pathogenic variants and their functional characterization and (ii) to study the association of retinal dystrophy in patients with MFRP variants, based on a detailed literature review of genotype-phenotype correlations. METHODS: Patients with nanophthalmos and available family members received a comprehensive ophthalmological examination. Genetic analysis was based on whole-exome sequencing and variant calling in core genes including MFRP, BEST1, TMEM98, PRSS56, CRB1, GJA1, C1QTNF5, MYRF and FAM111A. A minigene assay was performed for functional characterization of a splice site variant. RESULTS: Seven patients, aged between three and 65 years, from five unrelated families were included. Novel pathogenic variants in MFRP (c.497C>T, c.899-3C>A, c.1180G>A), and PRSS56 (c.1202C>A), and a recurrent de novo variant in FAM111A (c.1706G>A) in a patient with Kenny-Caffey syndrome type 2, were identified. In addition, we report co-inheritance of MFRP-related nanophthalmos and ADAR-related Aicardi-Goutières syndrome. CONCLUSION: Nanophthalmos is a genetically heterogeneous condition, and the severity of ocular manifestations appears not to correlate with variants in a specific gene. However, retinal dystrophy is only observed in patients harbouring pathogenic MFRP variants. Furthermore, heterozygous carriers of MFRP and PRSS56 should be screened for the presence of high hyperopia. Identifying nanophthalmos as an isolated condition or as part of a syndrome has implications for counselling and can accelerate the interdisciplinary care of patients.

Two sisters with microphthalmia and anterior segment dysgenesis secondary to a PAX6 pathogenic variant with clinically healthy parents: a case of gonadal mosaicism?

PURPOSE: Genetic analysis of two siblings with complex microphthalmia, with clinically healthy parents. STUDY DESIGN: Clinical and experimental. METHODS: The patients underwent a detailed ophthalmic evaluation, including visual acuity, fundus examination, gonioscopy, ultrasound examination, and optical coherence tomography. Lensectomy with anterior vitrectomy was conducted in both patients. Additionally, in patient p1, electroencephalography analysis was performed. Genetic analysis was carried out using array comparative genomic hybridization (aCGH) and whole exome sequencing (WES). Bidirectional Sanger sequencing was conducted for validation and segregation analysis of the identified variant in the family. RESULTS: The aCGH results were normal. The heterozygous PAX6 variant c.52G>C (p.Gly18Arg) was identified in the proband (p1) through WES analysis. Sanger sequencing of exon 5 of PAX6 confirmed the presence of the variant in the other affected sibling (patient p2) but did not allow for identification of the variant in the parents' DNA isolated from leukocytes and buccal cells. CONCLUSIONS: The description of the variant in PAX6 in two siblings with clinically healthy parents who are negative for the mutation in DNA from leukocytes and buccal cells represents the possibility of parental gonadal mosaicism. Detection of germ cell mosaicism in the parents is essential to provide genetic counseling to the family regarding the risk of reoccurrence. Furthermore, we also report a pathogenic variant in PAX6 that to our knowledge has not so far been reported in patients with partial aniridia and therefore broadens the spectrum of the variants associated with aniridia.

Neogenin-loss in neural crest cells results in persistent hyperplastic primary vitreous formation.

Neogenin is a transmembrane receptor critical for multiple cellular processes, including neurogenesis, astrogliogenesis, endochondral bone formation, and iron homeostasis. Here we present evidence that loss of neogenin contributes to pathogenesis of persistent hyperplastic primary vitreous (PHPV) formation, a genetic disorder accounting for ~ 5% of blindness in the USA. Selective loss of neogenin in neural crest cells (as observed in Wnt1-Cre; Neof/f mice), but not neural stem cells (as observed in GFAP-Cre and Nestin-Cre; Neof/f mice), resulted in a dysregulation of neural crest cell migration or delamination, exhibiting features of PHPV-like pathology (e.g. elevated retrolental mass), unclosed retinal fissure, and microphthalmia. These results demonstrate an unrecognized function of neogenin in preventing PHPV pathogenesis, implicating neogenin regulation of neural crest cell delamination/migration and retinal fissure formation as potential underlying mechanisms of PHPV.

The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development.

Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans-yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3'UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3'UTR. Further, we demonstrate that Sox2 3'UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24-Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

A Novel Mutation p.S93R in CRYBB1 Associated with Dominant Congenital Cataract and Microphthalmia.

Purpose: To identify the pathogenetic mutations in a four-generation Chinese family with dominant congenital cataracts and microphthalmia.Methods: A four-generation Chinese family with dominant congenital cataracts were recruited. Genomic DNAs were collected from their peripheral blood leukocytes and subjected to whole exome sequencing. The genetic mutations were identified by bioinformatic analyses and verified by Sanger sequencing.Results: Whole exome sequencing revealed a c.279C>G point mutation in the CRYBB1 gene which was further verified by Sanger sequencing. The nucleotide replacement results in a novel mutation p.S93R in a conserved residue of ßB1 crystallin which is predicted to disrupt normal ßB1 structure and function.Conclusions: We identified a novel missense mutation p.S93R in CRYBB1 in a Chinese family with autosomal dominant congenital cataracts and microphthalmia. This serine residue is extremely conserved evolutionarily in more than 50 ßγ-crystallins of many species. These data will be very helpful to further understand the structural and functional features of crystallins.

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

PURPOSE: To investigate the role of fibroblast growth factors (FGFs) in the production of neural retina (NR) and retinal pigmented epithelium (RPE) in a human pluripotent stem cell model of early retinal development. METHODS: Human induced pluripotent stem cell (hiPSC) lines from an individual with microphthalmia caused by a functional null mutation (R200Q) in visual system homeobox 2 (VSX2), a transcription factor involved in early NR progenitor cell (NRPC) production, and a normal sibling were differentiated along the retinal and forebrain lineages using an established protocol. Quantitative and global gene expression analyses (microarray and RNAseq) were used to investigate endogenous FGF expression profiles in these cultures over time. Based on these results, mutant and control hiPSC cultures were treated exogenously with selected FGFs and subjected to gene and protein expression analyses to determine their effects on RPE and NR production. RESULTS: We found that FGF9 and FGF19 were selectively increased in early hiPSC-derived optic vesicles (OVs) when compared to isogenic cultures of hiPSC-derived forebrain neurospheres. Furthermore, these same FGFs were downregulated over time in (R200Q)VSX2 hiPSC-OVs relative to sibling control hiPSC-OVs. Interestingly, long-term supplementation with FGF9, but not FGF19, partially rescued the mutant retinal phenotype of the (R200Q)VSX2 hiPSC-OV model. However, antagonizing FGF9 in wild-type control hiPSCs did not alter OV development. CONCLUSIONS: Our results show that FGF9 acts in concert with VSX2 to promote NR differentiation in hiPSC-OVs and has potential to be used to manipulate early retinogenesis and mitigate ocular defects caused by functional loss of VSX2 activity. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Mutation in the mouse histone gene Hist2h3c1 leads to degeneration of the lens vesicle and severe microphthalmia.

During an ENU (N-ethyl-N-nitrosourea) mutagenesis screen, we observed a dominant small-eye mutant mouse with viable homozygotes. A corresponding mutant line was established and referred to as Aey69 (abnormality of the eye #69). Comprehensive phenotyping of the homozygous Aey69 mutants in the German Mouse Clinic revealed only a subset of statistically significant alterations between wild types and homozygous mutants. The mutation causes microphthalmia without a lens but with retinal hyperproliferation. Linkage was demonstrated to mouse chromosome 3 between the markers D3Mit188 and D3Mit11. Sequencing revealed a 358 A-> C mutation (Ile120Leu) in the Hist2h3c1 gene and a 71 T-> C (Val24Ala) mutation in the Gja8 gene. Detailed analysis of eye development in the homozygous mutant mice documented a perturbed lens development starting from the lens vesicle stage including decreasing expression of crystallins as well as of lens-specific transcription factors like PITX3 and FOXE3. In contrast, we observed an early expression of retinal progenitor cells characterized by several markers including BRN3 (retinal ganglion cells) and OTX2 (cone photoreceptors). The changes in the retina at the early embryonic stages of E11.5-E15.5 happen in parallel with apoptotic processes in the lens at the respective stages. The excessive retinal hyperproliferation is characterized by an increased level of Ki67. The hyperproliferation, however, does not disrupt the differentiation and appearance of the principal retinal cell types at postnatal stages, even if the overgrowing retina covers finally the entire bulbus of the eye. Morpholino-mediated knock-down of the hist2h3ca1 gene in zebrafish leads to a specific perturbation of lens development. When injected into zebrafish zygotes, only the mutant mouse mRNA leads to severe malformations, ranging from cyclopia to severe microphthalmia. The wild-type Hist2h3c1 mRNA can rescue the morpholino-induced defects corroborating its specific function in lens development. Based upon these data, it is concluded that the ocular function of the Hist2h3c1 gene (encoding a canonical H3.2 variant) is conserved throughout evolution. Moreover, the data highlight also the importance of Hist2h3c1 in the coordinated formation of lens and retina during eye development.

Ocular phenotypic consequences of a single copy deletion of the Yap1 gene (Yap1 +/-) in mice.

Purpose: To identify the effects of a single copy deletion of Yap1 (Yap1 +/-) in the mouse eye, the ocular phenotypic consequences of Yap1 +/- were determined in detail. Methods: Complete ophthalmic examinations, as well as corneal esthesiometry, the phenol red thread test, intraocular pressure, and Fourier-domain optical coherence tomography were performed on Yap1 +/- and age-matched wild-type (WT) mice between eyelid opening (2 weeks after birth) and adulthood (2 months and 1 year after birth). Following euthanasia, enucleated eyes were characterized histologically. Results: Microphthalmia with small palpebral fissures, corneal fibrosis, and reduced corneal sensation were common findings in the Yap1 +/- mice. Generalized corneal fibrosis precluded clinical examination of the posterior structures. Histologically, thinning and keratinization of the corneal epithelium were observed in the Yap1 +/- mice in comparison with the WT mice. Distorted collagen fiber arrangement and hypercellularity of keratocytes were observed in the stroma. Descemet's membrane was extremely thin and lacked an endothelial layer in the Yap1 +/- mice. The iris was adherent to the posterior cornea along most of its surface creating a distorted contour. Most of the Yap1 +/- eyes were microphakic with swollen fibers and bladder cells. The retinas of the Yap1 +/- mice were normal at 2 weeks and 2 months of age, but the presence of retinal abnormalities, including retinoschisis and detachment, was markedly increased in the Yap1 +/- mice at 1 year of age. Conclusions: The results show that the heterozygous deletion of the Yap1 gene in mice leads to complex ocular abnormalities, including microphthalmia, corneal fibrosis, anterior segment dysgenesis, and cataract.

SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.

Partial Rescue of Ocular Pigment Cells and Structure by Inducible Ectopic Expression of Mitf-M in MITF-Deficient Mice.

Purpose: Complete deficiency of microphthalmia transcription factor (MITF) in Mitfmi-vga9/mi-vga9 mice is associated with microphthalmia, retinal dysplasia, and albinism. We investigated the ability of dopachrome tautomerase (DCT) promoter-mediated inducible ectopic expression of Mitf-M to rescue these phenotypic abnormalities. Methods: A new mouse line was created with doxycycline-inducible ectopic Mitf-M expression on an Mitf-deficient Mitfmi-vga9 background (DMV mouse). Adult DMV mice were phenotypically characterized and tissues were collected for histology, immunohistochemistry, and evaluation of Mitf, pigmentary genes, and retinal pigment epithelium (RPE) gene expression. Results: Ectopic Mitf-M expression was specifically induced in the eyes, but was not detected in the skin of DMV mice. Inducible expression of Mitf-M partially rescued the microphthalmia, RPE structure, and pigmentation as well as a subset of the choroidal and iris melanocytes but not cutaneous melanocytes. RPE function and vision were not restored in the DMV mice. Conclusions: Ectopic expression of Mitf-M during development of Mitf-deficient mice is capable of partially rescuing ocular and retinal structures and uveal melanocytes. These findings provide novel information about the roles of Mitf isoforms in the development of mouse eyes.

Mutation of frizzled8a delays neural retinal cell differentiation and results in microphthalmia in zebrafish.

Eye formation in vertebrates involves highly coordinated processes, and the differentiation of various eye tissues is regulated by conserved transcription factors and signalling pathways. Mutations in key genes of the regulatory hierarchy lead to congenital disorders and ocular diseases. The Wnt signalling pathway plays a key role in different aspects of eye development, and several Wnt receptors of the Frizzled family are required for eye specification and differentiation. However, their precise function in these processes remains elusive. Here we show that mutation of the frizzled8a gene in zebrafish leads to microphthalmia. The differentiation of retinal layers is delayed, and retinal progenitor cells in microphthalmic embryos fail to normally exit the cell cycle to enter into the post-mitotic state. They exhibit delayed differentiation associated with enhanced apoptosis, which results in abnormal lamination of retinal layers, reduction in the number of retinal cells, and small eye phenotype. These findings suggest that Frizzled8a plays a specific role in regulating cell cycle progression during the differentiation of retinal progenitor cells.

Relative anterior microphthalmos in oculodentodigital dysplasia.

Here, we report a patient with oculodentodigital dysplasia (ODDD) caused by the c. 413G>A, p.Gly138Asp mutation in the gap junction protein alpha-1 gene. The patient suffered from characteristic dysmorphic features of ODDD. Ophthalmological investigation disclosed microcornea and a shallow anterior chamber, as expected. Surprisingly, the patient had a normal axial length and moderate myopia on both eyes. To the best of our knowledge, this is the first report on ODDD associated with relative anterior microphthalmos and myopia.

SMARCA4 inactivating mutations cause concomitant Coffin-Siris syndrome, microphthalmia and small-cell carcinoma of the ovary hypercalcaemic type.

SMARCA4 chromatin remodelling factor is mutated in 11% of Coffin-Siris syndrome (CSS) patients and in almost all small-cell carcinoma of the ovary hypercalcaemic type (SCCOHT) tumours. Missense mutations with gain-of-function or dominant-negative effects are associated with CSS, whereas inactivating mutations, leading to loss of SMARCA4 expression, have been exclusively found in SCCOHT. We applied whole-exome sequencing to study a 15-year-old patient with mild CSS who concomitantly developed SCCOHT at age 13 years. Interestingly, our patient also showed congenital microphthalmia, which has never previously been reported in CSS patients. We detected a de novo germline heterozygous nonsense mutation in exon 19 of SMARCA4 (c.2935C > T;p.Arg979*), and a somatic frameshift mutation in exon 6 (c.1236_1236delC;p.Gln413Argfs*88), causing complete loss of SMARCA4 immunostaining in the tumour. The immunohistochemical findings are supported by the observation that the c.2935C > T mutant transcript was detected by reverse transcription polymerase chain reaction at a much lower level than the wild-type allele in whole blood and the lymphoblastoid cell line of the proband, confirming nonsense-mediated mRNA decay. Accordingly, immunoblotting demonstrated that there was approximately half the amount of SMARCA4 protein in the proband's cells as in controls. This study suggests that SMARCA4 constitutional mutations associated with CSS are not necessarily non-truncating, and that haploinsufficiency may explain milder CSS phenotypes, as previously reported for haploinsufficient ARID1B. In addition, our case supports the dual role of chromatin remodellers in developmental disorders and cancer, as well as the involvement of SMARCA4 in microphthalmia, confirming previous findings in mouse models and the DECIPHER database. Finally, we speculate that mild CSS might be under-recognized in a proportion of SCCOHT patients harbouring SMARCA4 mutations. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.