Integrator complex subunit 15 controls mRNA splicing and is critical for eye development.

The eye and brain are composed of elaborately organized tissues, development of which is supported by spatiotemporally precise expression of a number of transcription factors and developmental regulators. Here we report the molecular and genetic characterization of Integrator complex subunit 15 (INTS15). INTS15 was identified in search for the causative gene(s) for an autosomal-dominant eye disease with variable individual manifestation found in a large pedigree. While homozygous Ints15 knockout mice are embryonic lethal, mutant mice lacking a small C-terminal region of Ints15 show ocular malformations similar to the human patients. INTS15 is highly expressed in the eye and brain during embryogenesis and stably interacts with the Integrator complex to support small nuclear RNA 3' end processing. Its knockdown resulted in missplicing of a large number of genes, probably as a secondary consequence, and substantially affected genes associated with eye and brain development. Moreover, studies using human iPS cells-derived neural progenitor cells showed that INTS15 is critical for axonal outgrowth in retinal ganglion cells. This study suggests a new link between general transcription machinery and a highly specific hereditary disease.

Biallelic CDK9 variants as a cause of a new multiple-malformation syndrome with retinal dystrophy mimicking the CHARGE syndrome.

CDK9 has been considered a candidate gene involved in the CHARGE-like syndrome in a pair of cousins. We report an 8-year-old boy with a strikingly similar phenotype including facial asymmetry, microtia with preauricular tags and bilateral hearing loss, cleft lip and palate, cardiac dysrhythmia, and undescended testes. Joint contracture, no finger flexion creases, and large halluces were the same as those of a previously reported patient with homozygous CDK9 variants. The ocular phenotype included blepharophimosis, lacrimal duct obstruction, eyelid dermoids, Duane syndrome-like abduction deficit, and congenital cataracts. Optical coherence tomography and electroretinography evaluations revealed severe retinal dystrophy had developed at an early age. Trio-based whole-exome sequencing identified compound heterozygous variants in CDK9 [p.(A288T) of maternal origin and p.(R303C) of paternal origin] in the patient. Variants' kinase activities were reduced compared with wild type. We concluded that CDK9 biallelic variants cause a CHARGE-like malformation syndrome with retinal dystrophy as a distinguishing feature.

ATYPICAL FORM OF RETINOPATHY OF PREMATURITY WITH SEVERE FIBROVASCULAR PROLIFERATION IN THE OPTIC DISK REGION.

PURPOSE: To describe severe fibrovascular proliferation that developed in the optic disk region in an atypical form of retinopathy of prematurity (ROP). METHODS: Retrospective observational case reports. RESULTS: Four patients (8 eyes) with ROP were included. Three patients were born very prematurely (24-25 weeks of gestational age; weight, 500-1,000 grams); 1 patient was born at 33 weeks of gestational age. Among all eight eyes of four patients who received prompt ROP screening and underwent laser photocoagulation, six eyes had atypical and severe fibrovascular proliferation mainly in the optic disk region; the other two eyes, including one eye with classic ROP and one eye with aggressive posterior ROP, did not have the atypical form. All eight eyes had a total to partial retinal detachment. Among the six eyes with the atypical form, early vitreous surgery with lensectomy was possible in three eyes; only late vitreous surgery with lensectomy was possible in two eyes; one eye was inoperable. Three eyes had a partial or complete reattachment, whereas three eyes had a total retinal detachment. Among the six eyes with atypical fibrovascular proliferation, only two eyes obtained light perception vision. CONCLUSION: An atypical and severe form of ROP, in which fibrovascular proliferation grew mainly from the optic disk region, needs further investigation for treatment in addition to laser photocoagulation and vitreous surgery.

Different foveal schisis patterns in each retinal layer in eyes with hereditary juvenile retinoschisis evaluated by en-face optical coherence tomography.

PURPOSE: To analyze the structures of schisis in eyes with hereditary juvenile retinoschisis using en-face optical coherence tomography (OCT) imaging. METHODS: In this retrospective observational study, we reviewed the medical records of patients with hereditary juvenile retinoschisis who underwent comprehensive ophthalmic examinations including swept-source OCT. RESULTS: OCT images were obtained from 16 eyes of nine boys (mean age ± standard deviation, 10.6 ± 4.0 years). The horizontal OCT images at the fovea showed inner nuclear layer (INL) schisis in one eye (6.3 %), ganglion cell layer (GCL) and INL schisis in 12 eyes (75.0 %), INL and outer plexiform layer (OPL) schisis in two eyes (12.5 %), and GCL, INL, and OPL schisis in one eye (6.3 %). En-face OCT images showed characteristic schisis patterns in each retinal layer, which were represented by multiple hyporeflective holes in the parafoveal region in the GCL, a spoke-like pattern in the foveal region, a reticular pattern in the parafoveal region in the INL, and multiple hyporeflective polygonal cavities with partitions in the OPL. CONCLUSIONS: Our results using en-face OCT imaging clarified different patterns of schisis formation among the GCL, INL, and OPL, which lead to further recognition of structure in hereditary juvenile retinoschisis.

SOX2 nonsense mutation in a patient clinically diagnosed with non-syndromic hypogonadotropic hypogonadism.

Hypogonadotropic hypogonadism (HH) is a genetically heterogeneous condition that occurs either as an isolated disorder or as a component of congenital malformation syndromes. SOX2 is a causative gene of syndromic HH characterized by anophthalmia, microphthalmia, or coloboma and other neurological defects such as epilepsy. To date, the causal relationship between SOX2 abnormalities and non-syndromic HH remains speculative. Here, we identified a nonsense mutation of SOX2 in a male patient clinically diagnosed with non-syndromic HH. The patient had epilepsy but no additional clinical features. Ophthalmological examination revealed no abnormalities except for decreased thickness of the retinal nerve fiber layer. Audiometry showed mild sensorineural hearing impairment of both ears. Hormonal evaluation suggested isolated gonadotropin deficiency. Next-generation sequencing-based mutation screening of 13 major causative genes for HH identified a p.Lys35∗ mutation in SOX2 and excluded pathogenic mutations in other tested genes. The p.Lys35∗ mutation appeared to encode a non-functioning SOX2 protein that lacks 283 of 317 amino acids. The SOX2 mutation was absent in the maternal DNA sample, while a paternal sample was unavailable for sequence analysis. These results expand the clinical consequences of SOX2 haploinsufficiency to include non-syndromic HH. Systematic mutation screening using a next-generation sequencer and detailed evaluation of nonspecific ocular/neurological features may help identify SOX2 mutation-positive individuals among HH patients.

Successful scleral buckling of late-onset visual decrease in eye with retinal folds.

PURPOSE: To describe the outcome of scleral buckling to treat radial retinal folds (RFs) that caused a late-onset and sudden visual decrease with impairment of the fovea. METHODS: This is an observational case report. Ophthalmic examinations were performed preoperatively and postoperatively and included measurement of the best-corrected visual acuity (BCVA), fundus examination, fluorescein angiography, focal macular electroretinography (FM-ERG), and spectral-domain optical coherence tomography (OCT). RESULTS: A patient, whose case was reported previously, had a superonasal retinal detachment with a dislocated fovea and good BCVA in her right eye and a sustained BCVA of 40/50 by age 17 years and 3 months. The BCVA decreased suddenly to 20/200 at age 17 years and 11 months. Fundus examinations showed micro-dislocation of the fovea to the RFs and narrowing of the RFs compared with the previous report. FM-ERG showed an almost extinguished response, and OCT images showed abnormalities of the outer nuclear and photoreceptor layers. Encircling and radial scleral buckling was performed. Four months postoperatively, the BCVA improved to 30/50, which corresponded to the recovery of the FM-ERG response and findings on the OCT images. One year and 3 months postoperatively, the BCVA recovered to 35/50 with repositioning of the fovea. CONCLUSIONS: Simultaneous encircling and radial scleral buckling resulted in the recovery of both retinal function and structure in the current case and is a useful surgical option to treat late-onset progressive RFs due to intensification of traction.

Abnormal traction of the vitreous detected by swept-source optical coherence tomography is related to the maculopathy associated with optic disc pits.

BACKGROUND: Maculopathy associated with optic disc pits (ODP), which sometimes causes severe visual loss, usually appears in late childhood or early adulthood. However, it has long been unclear how the disease begins to develop at these ages. We evaluated the relationship between vitreous structure and maculopathy associated with ODP. METHODS: Six patients (seven eyes) with ODP were diagnosed between July 1990 and May 2013. Fundus photographs and swept-source optical coherence tomography (SS-OCT) images were evaluated retrospectively, and the vitreous at the vitreoretinal interface was visualized by reconstructing three-dimensional SS-OCT images. Vitrectomy was performed in the eyes with maculopathy. RESULTS: Among the six patients, five had ODP in one eye each and one patient had bilateral ODP. The pits were mainly located in the temporal quadrant, and maculopathy, including retinoschisis and retinal detachment, was detected in five eyes associated only with the temporal pits. A flat retinal detachment was observed in four eyes and identified within the vascular arcade except in one eye. A posterior precortical vitreous pocket (PPVP) was observed in all eyes except in one eye without maculopathy. Reconstructing images from SS-OCT showed the vitreoretinal interface abnormalities around the optic disc and the macular area in all eyes, which was completely different from the vitreoretinal interface in the normal pediatric eye. Vitrectomy was performed in four eyes with retinal detachment to resect the abnormal vitreous traction. Posterior vitreous detachment was created in two eyes. Retinal reattachment was achieved in three eyes, and subretinal fluid receded in one eye. The visual acuity improved in all four eyes. CONCLUSIONS: Abnormal traction of the vitreous due to an abnormality of the vitreoretinal interface, which may be strengthened by the development of a PPVP, generates the maculopathy associated with ODP.

Outer retinal deformity detected by optical coherence tomography in eyes with foveal hypoplasia.

PURPOSE: To investigate the relationship between vision and foveal maturity, especially in foveal hypoplasia exhibiting severe structural immaturity. METHODS: This retrospective observational case series included 42 eyes of 23 patients (mean age, 7.0 ± 5.0 years; 9 patients with foveal hypoplasia as an isolated entity and 14 patients with aniridia). A complete ophthalmic examination included measurement of best-corrected visual acuity (BCVA) and spectral-domain optical coherence tomography (SD-OCT). The sensory retina, ganglion cell complex (GCC), and outer retinal layers, including Henle's fiber layer (HFL), were measured and analyzed. RESULTS: Using SD-OCT images, eyes were classified as having a differentiated (6 eyes), diffuse (19 eyes), or no HFL (17 eyes), based on the appearance of the HFL around the foveal region. The logMAR BCVA was significantly worse (p < 0.0001) in eyes with diffuse HFL and those with no HFL than in those with differentiated HFL. Outer retinal thickness (outer plexiform layer + HFL + outer nuclear layer) was less (p = 0.0051) in eyes with no HFL than in those with differentiated HFL. The logMAR BCVA, GCC thickness, and outer retinal thickness in eyes with foveal hypoplasia with aniridia were significantly worse (p = 0.0083), thicker (p = 0.0039), and thinner (p = 0.0001), respectively, than in eyes with foveal hypoplasia as an isolated entity. CONCLUSIONS: In eyes with foveal hypoplasia with severe structural immaturity, diffuse HFL or no HFL was associated with worse vision. There was greater foveal immaturity in eyes with foveal hypoplasia with aniridia compared with foveal hypoplasia as an isolated entity.

Two-step transplantation for primary hyperoxaluria: a winning strategy to prevent progression of systemic oxalosis in early onset renal insufficiency cases.

Several transplant strategies for PH1 have been proposed, and LT is performed to correct the metabolic defects. The patients with PH1 often suffer from ESRD and require simultaneous LKT, which leads to a long wait due to the shortage of suitable organ donors. Five patients with PH1 underwent LDLT at our institute. Three of the five patients were under dialysis before LDLT, while the other two patients were categorized as CKD stage 3. An isolated LDLT was successfully performed in all but our first case, who had complicated postoperative courses and consequently died due to sepsis after retransplantation. The renal function of the patients with CKD stage 3 was preserved after LDLT. On the other hand, our second case with ESRD underwent successful LDKT six months after LDLT, and our infant case is waiting for the subsequent KT without any post-LDLT complications after the early establishment of PD. In conclusion, a two-step transplant strategy may be needed as a life-saving option for patients with PH1 and may be possible even in small infants with systemic oxalosis. While waiting for a subsequent KT, an early resumption of PD should be considered from the perspective of the long-term requirement of RRT.

Retinal hemorrhages and damages from tractional forces associated with infantile abusive head trauma evaluated by wide-field fundus photography.

We evaluated the distribution and types of retinal hemorrhages (RHs) and other damages in eyes with abusive head trauma (AHT). This retrospective, consecutive case series of AHT and non-AHT conditions involved 54 children with AHT, 43 children with head bruises, and 49 children with blunt eye trauma, each of non-AHT supported by reliable witness accounts. RHs and other damage were evaluated using ophthalmoscopy and wide-field fundus photography. A variety of RH types and other damage were identified in the AHT group but not in the non-AHT group. RHs in AHT extended from the posterior pole to the far periphery in 77% of eyes and on/near the veins in 86% and arteries in 85%, most of which were in the far periphery. Retinoschisis, white-dot lesions, and retinal folds were seen even in the far periphery. RHs on/near the veins and arteries, retinoschisis, and retinal folds suggest a traumatic mechanism of the tractional force of the vitreous that is attached to the entire retinal surface. Identifying the distribution and arterio and venous origins of RHs is a key factor in determining the association with trauma. Thus, wide-field fundus photography is useful to record and evaluate the origin of the RHs and other retinal damage.

Modeling of Retina and Optic Nerve Ischemia-Reperfusion Injury through Hypoxia-Reoxygenation in Human Induced Pluripotent Stem Cell-Derived Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are specialized projection neurons that constitute part of the retina, and the death of RGCs causes various eye diseases, but the mechanism of RGC death is still unclear. Here, we induced cell death in human induced pluripotent stem cell (hiPSC)-derived RGC-rich retinal tissues using hypoxia-reoxygenation in vitro. Flow cytometry, immunochemistry, and Western blotting showed the apoptosis and necrosis of RGCs under hypoxia-reoxygenation, and they were rescued by an apoptosis inhibitor but not by a necrosis inhibitor. This revealed that the cell death induced in our model was mainly due to apoptosis. To our knowledge, this is the first model to reproduce ischemia-reperfusion in hiPSC-derived RGCs. Thus, the efficacy of apoptosis inhibitors and neuroprotective agents can be evaluated using this model, bringing us closer to clinical applications.

Establishment and visual analysis of CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice.

In CBA/J and C3H/HeJ mice, retinitis pigmentosa is inherited as an autosomal-recessive trait due to a mutation in Pde6b, which encodes cGMP phosphodiesterase subunit b. In these strains, the Y347X mutation in Pde6b leads to the upregulation of cGMP levels, increased Ca2+ influx induces rod death, and the outer segment and rod cells entirely disappeared by 35 days after birth. In the present study, we utilized the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9-mediated gene editing to repair the Y347X mutation in CBA/J and C3H/HeJ mice. Evaluation of the established CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice, which were confirmed to have normal retinal layers by live fundoscopic imaging and histopathological analysis, revealed improved visual acuity based on the visual cliff and light/dark latency tests. Furthermore, our analyses revealed that the visible platform test was a more effective tool for testing visual behavior in these mice. The results suggest that the established strains can serve as control groups for CBA/J and C3H/HeJ in ophthalmology studies involving retinitis pigmentosa.

A pediatric case of congenital stromal corneal dystrophy caused by the novel variant c.953del of the DCN gene.

We report a 1-year-old girl with congenital stromal corneal dystrophy confirmed by genetic analysis. The ocular phenotype included diffuse opacity over the corneal stroma bilaterally. We performed a genetic analysis to provide counseling to the parents regarding the recurrence rate. Whole exome sequencing was performed on her and her parents, and a novel de novo variant, NM_001920.5: c.953del, p.(Asn318Thrfs*10), in the DCN gene was identified in the patient.

Structure of the Retinal Margin and Presumed Mechanism of Retinal Detachment in Choroidal Coloboma.

PURPOSE: To describe the vitreoretinal structure at the margin of the choroidal coloboma in infants and older patients using swept-source (SS) OCT. DESIGN: Retrospective case series. PARTICIPANTS: Nineteen eyes of 16 patients with choroidal coloboma (7 males, 9 females; average age, 12.3 ± 7.1 years). METHODS: The patients were classified into 2 groups: infants 1 year of age or younger (3 eyes) and older patients (16 eyes). Each finding on SS OCT was documented according to previously defined histopathologic findings. MAIN OUTCOME MEASURES: Description of the SS OCT features of choroidal colobomas. RESULTS: Swept-source OCT showed that the extracolobomatous retina centrally traversed the margin to continue as the marginal intercalary membrane (MICM), whereas the outer layers of the MICM were reversed at the point (point of reversal [POR]). The expected duplication was seen in all infant eyes, but in none of the older eyes whose outer layers of the MICM were ambiguous. However, at the boundary between the layered MICM and monolayered central intercalary membrane (CICM), the POR was detectable in all patients. Further SS OCT analysis showed that the MICM schisis and CICM schisis occurred simultaneously with vitreous traction. Retinal detachments (RDs) seen in 4 eyes were connected to the only MICM schisis, and a MICM break was identified in 1 eye. Swept-source OCT showed that retinal pigment epithelial hyperplasia adhered tightly to the retina and that the glial triangle was adhered tightly to the sclera, indicating barriers to the development of RD after MICM schisis. CONCLUSIONS: Swept-source OCT first visualized the POR in infant eyes and showed that the POR was identifiable despite the atrophic changes in the outer layer of the MICM in the older eyes. Based on the POR location, we confirmed that the intercalary membranes reported in previous OCT studies were clearly differentiated between the MICM and CICM. We also showed that the presence of MICM and CICM schisis resulted from vitreous traction at the coloboma margin and that MICM breaks induced RD only if the barrier that prevented the development of RD was broken.

SEVERE RECURRENT FIBROVASCULAR PROLIFERATION AFTER COMBINED INTRAVITREAL BEVACIZUMAB INJECTION AND LASER PHOTOCOAGULATION FOR AGGRESSIVE POSTERIOR RETINOPATHY OF PREMATURITY.

PURPOSE: To describe the clinical features of severe recurrent fibrovascular proliferation after intravitreal bevacizumab injections and laser photocoagulation for aggressive posterior retinopathy of prematurity. METHODS: This retrospective, nonrandomized case series reviewed the medical and ophthalmic records in the referral hospital and our hospital. PATIENTS: Four patients (seven eyes) with aggressive posterior retinopathy of prematurity. RESULTS: The patients were referred for vitrectomy with/without lensectomy for recurrent fibrovascular proliferation with a tractional retinal detachment after combined intravitreal bevacizumab injections and laser photocoagulation. Three patients were born at 22 weeks or 23 weeks' gestational age and one patient at 29 weeks' gestational age. Preoperatively, fluorescein angiography images showed all eyes had tractional retinal detachment from regrowth of fibrovascular proliferation 3 months to 5 months after the intravitreal bevacizumab injection and abnormal retinal vasculature; four eyes had a broad ischemic retina. Postoperatively, four eyes had retinal attachment and three eyes a total retinal detachment. Neovascular glaucoma developed in five of the seven eyes during the clinical course. CONCLUSION: Severe fibrovascular proliferation may recur due to widespread retinal ischemia with capillary dropout and abnormal vasculature after failed combined intravitreal bevacizumab and laser photocoagulation therapy as the initial treatment for aggressive posterior retinopathy of prematurity. Careful follow-up is important especially after anti-vascular endothelial growth factor treatment, with recognition that severe reactivation is possible.

Early laser photocoagulation for extensive retinal avascularity in infants with incontinentia pigmenti.

PURPOSE: To describe the clinical features and treatment outcomes of severe retinopathy in eyes with incontinentia pigmenti (IP) of infants within a few months of birth. STUDY DESIGN: Retrospective clinical study. METHODS: Six eyes of three patients (6-day-old girl, 5-month-old girl, and 14-day-old boy) with IP were examined and treated under general anesthesia. Ophthalmologic examinations were performed including images from wide-angle fluorescein angiography (FA), swept-source optical coherence tomography (OCT), and OCT angiography (OCTA). RESULTS: Ophthalmoscopy showed prominent vascular tortuosity in five eyes, retinal hemorrhages in four eyes, and incomplete vascular development in two eyes. FA showed extensive avascularity including the posterior pole of the retina in all cases except one eye. Prompt and intensive laser photocoagulation stabilized the pre-proliferative severe retinopathy in five eyes; however, foveal structure and vessel anomalies were detected in three of six eyes by OCT and two of five eyes by OCTA. CONCLUSION: Severe retinopathy in the neonatal period and infancy was present not only in the periphery but also in the posterior pole including the fovea, which might be related to retinal vascular maldevelopment. It is, therefore, recommended that wide-angle fundus FA examination be performed in the early postnatal period to detect early signs of severe retinopathy in infants with IP.

CrxOS maintains the self-renewal capacity of murine embryonic stem cells.

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.

Vascular abnormalities in aggressive posterior retinopathy of prematurity detected by fluorescein angiography.

PURPOSE: To evaluate fluorescein angiography (FA) in eyes with aggressive posterior retinopathy of prematurity (AP-ROP). DESIGN: Retrospective, nonrandomized case series. PARTICIPANTS: Three patients (6 eyes) with AP-ROP. METHODS: Three patients (6 eyes) diagnosed with AP-ROP during ROP screening between July 2007 and July 2008 were included in this study. Fundus photographs and FA were obtained before and after laser and surgical treatment using a wide-field digital pediatric imaging system. MAIN OUTCOME MEASURES: Fluorescein angiography and fundus photographs. RESULTS: At the initial stage of AP-ROP, FA showed vascular abnormalities, including capillary nonperfusion throughout the vascularized retina, shunting in the vascularized retina, a circumferential demarcation line, and limited vessel development, which was difficult to identify only by ophthalmoscopy. After treatment, FA showed poorly developed retinal vessels, including 4 small major vessels without an arcade pattern, small macular vessels, an inhomogeneous capillary bed, and absence of a capillary-free zone in the fovea. CONCLUSIONS: Capillary bed loss throughout the vascularized posterior retina is characteristic of AP-ROP and may exacerbate retinopathy.