Vitreoretinopathy in Asymptomatic Children With CTNNB1 Syndrome.

Importance: Previous studies have identified familial exudative vitreoretinonpathy (FEVR) in patients with CTNNB1 syndrome based on severe congenital ocular phenotypes. However, ophthalmoscopy may not be sufficient to detect vision-threatening vitreoretinopathy in all patients. Objective: To report a consecutive retrospective case series of 11 patients with CTNNB1 variants who had previously unremarkable ophthalmoscopic examination results and to describe their detailed ophthalmic phenotypes. Design, Setting, and Participants: This retrospective case series was conducted at the Children's Hospital of Philadelphia from October 2022 to November 2023 among patients with identified variants in CTNNB1 and previously documented normal results in office retinal examinations. These consecutive patients subsequently underwent an examination under anesthesia with fluorescein angiography. Detailed genotype information was analyzed for all patients, and each variant was mapped on the CTNNB1 gene to observe any associations with severity of vitreoretinopathy. Main Outcomes and Measures: Number of patients with vitreoretinopathy and number requiring treatment for vitreoretinopathy. Results: The mean (SD) age at the time of CTNNB1 syndrome diagnosis was 2 (1) years, and the mean (SD) age at examination was 6 (3) years for the 11 total patients. A total of 9 patients had a diagnosis of strabismus, and 5 patients had undergone strabismus surgery. FEVR was present in 5 of 11 patients and in 9 eyes. The presence of disease requiring treatment was identified in 6 eyes, including 1 retinal detachment. Detailed genotype analysis of the patients found no clearly delineated high-risk loci in CTNNB1 in association with high severity of FEVR. Conclusions and Relevance: In this case series study, nearly all patients with CTNNB1 syndrome required ophthalmic care for refractive error and strabismus, and a subset also required treatment for FEVR. These findings support consideration of ultra-widefield fluorescein angiography among individuals with CTNNB1 syndrome when feasible, including the use of sedation if such an assessment is not possible in the office setting.

Anti-TRPM1 autoantibody-positive unilateral melanoma associated retinopathy (MAR) triggered by immunotherapy recapitulates functional and structural details of TRPM1-associated congenital stationary night blindness.

Purpose: To describe the retinal phenotype of an unusual case of anti-TRPM1 autoantibody-positive unilateral melanoma-associated retinopathy (MAR) triggered by nivolumab therapy and compare with the phenotype of TRPM1-associated Congenital Stationary Night Blindness (TRPM1-CSNB). Observations: Unilateral MAR was diagnosed 3 months after starting nivolumab therapy for consolidation of a successfully treated melanoma. Retinal autoantibodies against TRPM1 were identified. ffERG, microperimetry and static chromatic perimetry confirmed unilateral ON-Bipolar Cell (ON-BPC) dysfunction and central rod sensitivity losses in the left eye; the contralateral eye was normal. There was borderline ganglion cell (GCL) and inner nuclear layer (INL) thinning, but a significantly thinner inner plexiform layer (IPL) in the affected compared to the unaffected eye. Longitudinal reflectivity profiles (LRPs) demonstrated an abnormal inner plexiform layer (IPL) lamination in the involved eye. Nearly identical changes were documented in two cases of TRMP1-cCSNB and in a case of anti-TRPM1 autoantibody-negative MAR. The functional changes partially recovered with discontinuation of the medication without added immunosuppression. Conclusions and Importance: Comparisons between the affected and unaffected eye in this unilateral MAR case revealed inner retinal abnormalities and abnormal lamination of the IPL associated with the classical retina-wide ON-BPC dysfunction, and localized central rod-mediated sensitivity losses. A nearly identical structural phenotype in two cases of cCSNB and a case of anti-TRPM1 autoantibody-negative MAR supports a specific structural-functional phenotype for these conditions with ON-BPC dysfunction.

Characterising the refractive error in paediatric patients with congenital stationary night blindness: a multicentre study.

BACKGROUND/AAIMS: Congenital stationary night blindness (CSNB) is an inherited retinal disease that is often associated with high myopia and can be caused by pathological variants in multiple genes, most commonly CACNA1F, NYX and TRPM1. High myopia is associated with retinal degeneration and increased risk for retinal detachment. Slowing the progression of myopia in patients with CSNB would likely be beneficial in reducing risk, but before interventions can be considered, it is important to understand the natural history of myopic progression. METHODS: This multicentre, retrospective study explored CSNB caused by variants in CACNA1F, NYX or TRPM1 in patients who had at least 6 measurements of their spherical equivalent of refraction (SER) before the age of 18. A mixed-effect model was used to predict progression of SER overtime and differences between genotypes were evaluated. RESULTS: 78 individuals were included in this study. All genotypes showed a significant myopic predicted SER at birth (-3.076D, -5.511D and -5.386D) for CACNA1F, NYX and TRPM1 respectively. Additionally, significant progression of myopia per year (-0.254D, -0.257D and -0.326D) was observed for all three genotypes CACNA1F, NYX and TRPM1, respectively. CONCLUSIONS: Patients with CSNB tend to be myopic from an early age and progress to become more myopic with age. Patients may benefit from long-term myopia slowing treatment in the future and further studies are indicated. Additionally, CSNB should be considered in the differential diagnosis for early-onset myopia.

Co-Occurrence of Pallister-Killian Syndrome and Burkitt Lymphoma in a Patient with Near-Normal Neurocognitive Development.

Background: Pallister-Killian syndrome (PKS) is typically recognized by its features that include developmental delay, seizures, sparse temporal hair, and facial dysmorphisms. PKS is most frequently caused by mosaic supernumerary isochromosome 12p. Case Presentation: Here, we report a patient with PKS who was subsequently diagnosed with Burkitt lymphoma. Following the successful treatment of lymphoma, this patient demonstrated very mild intellectual disability despite the diagnosis of PKS, which is usually associated with severe developmental delay. Discussion: This is the first reported patient with PKS and a hematologic malignancy. Although there is no significant reported association of tetrasomy 12p with cancer, the co-occurrence of two rare findings in this patient suggests a potential relationship. The localization of AICDA, a gene for which overexpression has been implicated in promoting t(8;14) noted in our patient's lymphoma, raises a potential mechanism of pathogenesis. In addition, this case indicates that children with PKS can demonstrate near-normal cognitive development.

Monoallelic intragenic POU3F2 variants lead to neurodevelopmental delay and hyperphagic obesity, confirming the gene's candidacy in 6q16.1 deletions.

While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.

Ocular Biomarkers of Riboflavin Transporter Deficiency.

BACKGROUND: To describe the clinical presentation with a focus on ocular manifestations and response to riboflavin supplementation of 3 patients with riboflavin transporter deficiency (RTD) caused by mutations in SLC52A2 ( SLC52A2- RTD). METHODS: This is a retrospective review of records of 3 children (aged 18, n = 2 and age = 8, n = 1) with SLC52A2- RTD. Patients underwent comprehensive ophthalmic evaluations including color vision testing, pattern visual-evoked potentials (pVEPs, 1 patient) and spectral domain optical coherence tomography (SD-OCT) imaging. Patients received riboflavin supplements from the time of the molecular diagnosis of RTD. RESULTS: Two unrelated 18-year-old patients with SLC52A2- RTD had a symptomatic onset with sensorineural hearing loss and auditory neuropathy/dys-synchrony since age 3 and 11, respectively. On examination 7 years after symptomatic onset, they showed subnormal visual acuities (20/30 and 20/60, both eyes, respectively), preserved color vision, and a thin but measurable retinal ganglion cell layer (GCL) and nerve fiber (RNFL). The inner and outer nuclear layers were normal. The asymptomatic SLC52A2- positive brother of one of these patients started riboflavin supplementation right after the molecular diagnosis and had normal vision and SD-OCTs 7 years later. Onset of riboflavin supplementation in one of the 2 symptomatic cases resulted in acute improvement of the pattern visual-evoked potential and vision. CONCLUSIONS: Retinal ganglion cells and their axons are uniquely susceptible to RTD compared with other highly energy-dependent retinal neurons, such as photoreceptors, raising the possibility for alternative mechanisms of disease or protection. Riboflavin supplementation results in acute functional improvement of vision and long-term preservation of GCL and RNFL if initiated early.

Fleck-like lesions in CEP290-associated leber congenital amaurosis: a case series.

PURPOSE: To provide a detailed ophthalmic phenotype of a small cohort of patients with Leber Congenital Amaurosis (LCA) caused by mutations in CEP290 (CEP290-LCA) with a focus on elucidating the origin of yellow-white lesions observed in 30% of patients with this condition. METHODS: This is a retrospective review of records of five patients with CEP290-LCA. Patients had comprehensive ophthalmic evaluations. Visual function was assessed with full-field electroretinograms (ffERGs) and full-field sensitivity testing (FST). Multimodal imaging was performed with spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF) with short- (SW) and near-infrared (NIR) excitation wavelengths. RESULTS: All patients showed relative structural preservation of the foveal and near midperipheral retina separated by a pericentral area of photoreceptor loss. Yellow-white, fleck-like lesions in an annular distribution around the near midperiphery co-localized with hyperreflective lesions on SD-OCT. The lesions located between the inner segment ellipsoid signal and the apical retinal pigment epithelium (RPE). The inner retina was normal. Longitudinal observations in one of the patients indicates the abnormalities may represent an intermediate stage in the degenerative process between the near normal appearing retina previously documented in young CEP290-LCA patients and the pigmentary retinopathy observed along the same region in older individuals. CONCLUSIONS: We speculate that fleck-like lesions in CEP290-LCA correspond to malformed, rudimentary or degenerated, including shed, photoreceptor outer segments. The topography and possible origin of the abnormalities may inform the planning of evolving genetic therapies for this disease.

Severe Familial Exudative Vitreoretinopathy, Congenital Hearing Loss, and Developmental Delay in a Child With Biallelic Variants in FZD4.

Importance: Familial exudative vitreoretinopathy (FEVR) is a nonsyndromic autosomal dominant retinal disorder commonly caused by variants in the FZD4 gene. This study investigates the potential role beyond ocular abnormalities for FZD4 gene variants in patients with FEVR. Objective: To evaluate the role of FZD4 in symptoms beyond those associated with FEVR through a patient with biallelic variants in FZD4. Design, Setting, and Participants: This case series included the DNA testing and phenotyping of 1 patient proband and her parents, combined with signaling assays, to determine the association of patient-derived compound heterozygous variants on FZD4 signaling and biologic function. Main Outcomes and Measures: FZD4 genes were tested using next-generation sequencing and Sanger sequencing. Cell-based assays measured the effect of the variants on FZD4 signaling. Results: The proband presented with absent red reflexes from complete tractional retinal detachments diagnosed at 3 days of age and failed the newborn screening hearing test. Auditory brainstem response at 6 months of age showed bilateral mild to moderate high-frequency sensorineural hearing loss. The patient manifested developmental delays in speech and walking. Intravenous fluorescein angiography (IVFA) of the patient's parents detected stage 1 FEVR. Genetic testing revealed 2 FZD4 variants in the patient, each variant found in 1 parent. Signaling assays confirmed that the presence of both variants was associated with significantly worse signaling activity compared with the heterozygous state. Conclusions and Relevance: Results of this case series suggest that extraocular syndromic FEVR was associated with FZD4 variants. The decrease in FZD4 signaling owing to the biallelic nature of the disease resulted in hearing deficits, developmental delays, and a more severe retinal phenotype.

RP1-associated recessive retinitis pigmentosa caused by paternal uniparental disomy.

BACKGROUND: We report on a patient with a juvenile-onset inherited retinal degeneration (IRD) associated with homozygous RP1 mutations inherited by uniparental disomy (UPD). MATERIAL AND METHODS: A 6-year-old healthy girl failed school vision screening and was diagnosed with a bull's eye maculopathy. She underwent complete ophthalmic examination, full-field electroretinograms (ERG), kinetic fields, full-field sensitivity testing (FST), and retinal imaging with spectral domain optical coherence tomography (SD-OCT) and near-infrared (NIR) and short wavelength (SW) fundus autofluorescence (FAF). RESULTS: Visual acuities were relatively preserved (20/30+). There was subtle foveal depigmentation but an otherwise normal fundus examination. SD-OCT revealed a relatively preserved fovea with thinning of the photoreceptor outer nuclear layer with increasing distance from the foveal center coinciding with marked attenuation of the NIR and less marked loss of the SW-FAF signal. ERGs were non-detectable. Kinetic visual fields were generally full to large (V-4e) target but constricted to ~10°of eccentricity to I-4e stimuli. Dark-adapted thresholds by FST were rod-mediated and elevated by ~2 log units. Homozygous pathogenic mutations in RP1 (c.1720_1721del; p.Ser574Asnfs*8) were identified. Family member testing revealed father and siblings to be unaffected carriers; the mother carried wild-type alleles. Further testing suggested UPD of chromosome 8. CONCLUSION: This report adds support to UPD as a mechanism of inheritance in IRDs and stresses the importance of familial testing for genetic diagnosis and counseling. Consistent with earlier descriptions of autosomal recessive RP1-IRDs our patient showed an early rod and cone photoreceptor degeneration.

Molecular Diagnostic Outcomes from 700 Cases: What Can We Learn from a Retrospective Analysis of Clinical Exome Sequencing?

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.

NMNAT1-ASSOCIATED CONE-ROD DYSTROPHY: EVIDENCE FOR A SPECTRUM OF FOVEAL MALDEVELOPMENT.

PURPOSE: To describe in detail the phenotype of two siblings with biallelic NMNAT1 mutations. METHODS: A 4-year-old male patient (P1) and his 7-year-old sister (P2), product of a nonconsanguineous union of Egyptian ancestry, underwent a comprehensive ophthalmic examination, retinal imaging with spectral domain optical coherence tomography and near infrared (NIR) fundus autofluorescence (FAF), and full-field electroretinograms (ERG). RESULTS: Patients had blurred vision and nystagmus at ∼3 years of age. P2 was hyperopic (+6D). Visual acuity in P1 was 20/100 at age 3 and remained at ∼20/125 at age 4; P2 visual acuity was 20/70 at age 4 and declined to ∼20/200 at age 7. ERGs recorded in P1 showed relatively large rod-mediated responses but nearly undetectable cone signals. There was foveal/parafoveal depigmentation. Spectral domain optical coherence tomography showed hypoplastic foveas, a thin outer nuclear layer centrally but normal thickness beyond the vascular arcades. At the foveal center, cone outer segments were absent and the outer nuclear layer was further hyporreflective. The inner retina was mostly within normal limits. There was central depigmentation on near infrared fundus autofluorescence. Biallelic mutations were identified in NMNAT1: One was previously reported (c.769 G>A; pGlu257Lys), and the other one (c.245T>C; pVal82Ala) was novel. CONCLUSION: NMNAT1 mutations cause a consistent phenotype characterized by early-onset, progressive, cone>rod retinawide dysfunction and predominantly central abnormalities ranging from a hypoplastic to an atrophic fovea, supporting a critical role for NMNAT1 in central retinal development and maintenance. Relatively preserved inner retina and detectable photoreceptors may become therapeutic targets.

Pathogenic NR2F1 variants cause a developmental ocular phenotype recapitulated in a mutant mouse model.

Pathogenic NR2F1 variants cause a rare autosomal dominant neurodevelopmental disorder referred to as the Bosch-Boonstra-Schaaf Optic Atrophy Syndrome. Although visual loss is a prominent feature seen in affected individuals, the molecular and cellular mechanisms contributing to visual impairment are still poorly characterized. We conducted a deep phenotyping study on a cohort of 22 individuals carrying pathogenic NR2F1 variants to document the neurodevelopmental and ophthalmological manifestations, in particular the structural and functional changes within the retina and the optic nerve, which have not been detailed previously. The visual impairment became apparent in early childhood with small and/or tilted hypoplastic optic nerves observed in 10 cases. High-resolution optical coherence tomography imaging confirmed significant loss of retinal ganglion cells with thinning of the ganglion cell layer, consistent with electrophysiological evidence of retinal ganglion cells dysfunction. Interestingly, for those individuals with available longitudinal ophthalmological data, there was no significant deterioration in visual function during the period of follow-up. Diffusion tensor imaging tractography studies showed defective connections and disorganization of the extracortical visual pathways. To further investigate how pathogenic NR2F1 variants impact on retinal and optic nerve development, we took advantage of an Nr2f1 mutant mouse disease model. Abnormal retinogenesis in early stages of development was observed in Nr2f1 mutant mice with decreased retinal ganglion cell density and disruption of retinal ganglion cell axonal guidance from the neural retina into the optic stalk, accounting for the development of optic nerve hypoplasia. The mutant mice showed significantly reduced visual acuity based on electrophysiological parameters with marked conduction delay and decreased amplitude of the recordings in the superficial layers of the visual cortex. The clinical observations in our study cohort, supported by the mouse data, suggest an early neurodevelopmental origin for the retinal and optic nerve head defects caused by NR2F1 pathogenic variants, resulting in congenital vision loss that seems to be non-progressive. We propose NR2F1 as a major gene that orchestrates early retinal and optic nerve head development, playing a key role in the maturation of the visual system.

Expansion of the Genotypic and Phenotypic Spectrum of WASF1-Related Neurodevelopmental Disorder.

In humans, de novo truncating variants in WASF1 (Wiskott-Aldrich syndrome protein family member 1) have been linked to presentations of moderate-to-profound intellectual disability (ID), autistic features, and epilepsy. Apart from one case series, there is limited information on the phenotypic spectrum and genetic landscape of WASF1-related neurodevelopmental disorder (NDD). In this report, we describe detailed clinical characteristics of six individuals with WASF1-related NDD. We demonstrate a broader spectrum of neurodevelopmental impairment including more mildly affected individuals. Further, we report new variant types, including a copy number variant (CNV), resulting in the partial deletion of WASF1 in monozygotic twins, and three missense variants, two of which alter the same residue, p.W161. This report adds further evidence that de novo variants in WASF1 cause an autosomal dominant NDD.

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

A Virtual Reality Orientation and Mobility Test for Inherited Retinal Degenerations: Testing a Proof-of-Concept After Gene Therapy.

PURPOSE: To test the ability of a virtual reality (VR) orientation and mobility (O&M) protocol to serve a measure of functional vision for patients with inherited retinal degenerations (IRDs). METHODS: A VR-O&M protocol designed using a commercially available VR hardware was tested in normally sighted control subjects (n=7; ages 10-35yo; Average 22.5yo) and patients with RPE65-associated Leber Congenital Amaurosis (n=3; ages 7-18yo; Average 12.7yo), in two of them before and after gene therapy. Patients underwent perimetry and full-field sensitivity testing. VR-O&M parameters correlated with the visual dysfunction. RESULTS: Visual acuities in RPE65 patients were on average worse than 20/200, dark-adapted sensitivity losses >5 log units, and fields constricted between 20° and 40°. Before treatment, patients required ~1000-fold brighter environment to navigate, had at least x4 more collisions, and were slower both to orient and navigate compared to control subjects. Improvements in cone- (by 1-2 L.u.) and rod-mediated (by >4 L.u.) sensitivities post-treatment led to fewer collisions (at least by half) at ~100-fold dimmer luminances, and to x4 times faster navigation times. CONCLUSION: This study provides proof-of-concept data in support for the use of VR-O&M systems to quantify the impact that the visual dysfunction and improvement of vision following treatments has on functional vision in IRDs. The VR-O&M was useful in potentially challenging scenarios such as in pediatric patients with severe IRDs. TRANSLATIONAL RELEVANCE: A VR-O&M test will provide much needed flexibility, both in its deployment as well as in the possibility to test various attributes of vision that may be impacted by gene therapy in the setting of translational studies. PRECIS: This study provides proof-of-concept data in support for the use of a virtual reality orientation and mobility test to quantify the impact of the disease and of treatments thereof on functional vision in inherited retinal degenerations.

Palmoplantar keratoderma with deafness phenotypic variability in a patient with an inherited GJB2 frameshift variant and novel missense variant.

BACKGROUND: Variants in the GJB2 gene encoding the gap junction protein connexin-26 (Cx26) can cause autosomal recessive nonsyndromic hearing loss or a variety of phenotypically variable autosomal dominant disorders that effect skin and hearing, such as palmoplantar keratoderma (PPK) with deafness and keratitis-ichthyosis-deafness (KID) syndrome. Here, we report a patient with chronic mucocutaneous candidiasis, hyperkeratosis with resorption of the finger tips, profound bilateral sensorineural hearing loss, and normal hair and ocular examination. Exome analysis identified a novel missense variant in GJB2 (NM_004004.5:c.101T>A, p.Met34Lys) that was inherited from a mosaic unaffected parent in the setting of a well-reported GJB2 loss of function variant (NM_004004.5:c.35delG, p.Gly12Valfs*2) on the other allele. METHOD: Rat epidermal keratinocytes were transfected with cDNA encoding wildtype Cx26 and/or the Met34Lys mutant of Cx26. Fixed cells were immunolabeled in order to assess the subcellular location of the Cx26 mutant and cell images were captured. RESULTS: Expression in rat epidermal keratinocytes revealed that the Met34Lys mutant was retained in the endoplasmic reticulum, unlike wildtype Cx26, and failed to reach the plasma membrane to form gap junctions. Additionally, the Met34Lys mutant acted dominantly to wildtype Cx26, restricting its delivery to the cell surface. CONCLUSION: Overall, we show the p.Met34Lys variant is a novel dominant acting variant causing PPK with deafness. The presence of a loss a function variant on the other allele creates a more severe clinical phenotype, with some features reminiscent of KID syndrome.

ENHANCED S-CONE SYNDROME: VISUAL FUNCTION, CROSS-SECTIONAL IMAGING, AND CELLULAR STRUCTURE WITH ADAPTIVE OPTICS OPHTHALMOSCOPY.

PURPOSE: To describe in detail the phenotype of a patient with enhanced S-cone syndrome. METHODS: We describe a 13-year-old boy who presented with blurred vision, vitreous cells, cystoid macular edema refractory to steroid treatment, and a negative uveitic workup. The patient underwent a complete ophthalmic examination, full-field electroretinograms (ffERG), automatic static perimetry and multimodal imaging with spectral domain optical coherence tomography, and adaptive optics scanning laser ophthalmoscopy (AOSLO). RESULTS: Spectral domain optical coherence tomography demonstrated cystoid macular edema and a hyperthick, delaminated midperipheral retina. Fluorescein angiography did not demonstrate macular leakage. Rod-mediated ffERGs were undetectable, and there was a supernormal response to short-wavelength stimuli compared with photopically matched longer wavelengths of light consistent with enhanced S-cone syndrome. Gene screening was positive for compound heterozygous mutations NR2E3: a known (c.119-2 A>C) and a novel (c.119-1G>A) mutation. By perimetry, sensitivities were normal or above normal for short-wavelength stimuli; there was no detectable rod-mediated vision. AOSLO demonstrated higher than normal cone densities in the perifoveal retina and evidence for smaller outer segment cone diameters. CONCLUSION: Evidence for supernumerary cones (at least twice the normal complement) by AOSLO and spectral domain optical coherence tomography was associated with supernormal S-cone sensitivities and electroretinogram responses confirming previous in vivo findings in postmortem human specimens. Smaller than normal cones in enhanced S-cone syndrome may represent "hybrid" photoreceptors analogous to the rd7/rd7 murine model of the disease.

EP300-related Rubinstein-Taybi syndrome: Highlighted rare phenotypic findings and a genotype-phenotype meta-analysis of 74 patients.

Pathogenic variants in the homologous and highly conserved genes-CREBBP and EP300-are causal for Rubinstein-Taybi syndrome (RSTS). CREBBP and EP300 encode histone acetyltransferases (HAT) that act as transcriptional co-activators, and their haploinsufficiency causes the pathology characteristic of RSTS by interfering with global transcriptional regulation. Though generally a well-characterized syndrome, there is a clear phenotypic spectrum; rare associations have emerged with increasing diagnosis that is critical for comprehensive understanding of this rare syndrome. We present 12 unreported patients with RSTS found to have EP300 variants discovered through gene sequencing and chromosomal microarray. Our cohort highlights rare phenotypic features associated with EP300 variants, including imperforate anus, retained fetal finger pads, and spina bifida occulta. Our findings support the previously noted prevalence of pregnancy-related hypertension/preeclampsia seen with this disease. We additionally performed a meta-analysis on our newly reported 12 patients and 62 of the 90 previously reported patients. We demonstrated no statistically significant correlation between phenotype severity (within the domains of intellectual disability and major organ involvement, as defined in our Methods section) and variant location and type; this is in contrast to the conclusions of some smaller studies and highlights the importance of large patient cohorts in characterization of this rare disease.

HACE1 deficiency leads to structural and functional neurodevelopmental defects.

OBJECTIVE: We aim to characterize the causality and molecular and functional underpinnings of HACE1 deficiency in a mouse model of a recessive neurodevelopmental syndrome called spastic paraplegia and psychomotor retardation with or without seizures (SPPRS). METHODS: By exome sequencing, we identified 2 novel homozygous truncating mutations in HACE1 in 3 patients from 2 families, p.Q209* and p.R332*. Furthermore, we performed detailed molecular and phenotypic analyses of Hace1 knock-out (KO) mice and SPPRS patient fibroblasts. RESULTS: We show that Hace1 KO mice display many clinical features of SPPRS including enlarged ventricles, hypoplastic corpus callosum, as well as locomotion and learning deficiencies. Mechanistically, loss of HACE1 results in altered levels and activity of the small guanosine triphosphate (GTP)ase, RAC1. In addition, HACE1 deficiency results in reduction in synaptic puncta number and long-term potentiation in the hippocampus. Similarly, in SPPRS patient-derived fibroblasts, carrying a disruptive HACE1 mutation resembling loss of HACE1 in KO mice, we observed marked upregulation of the total and active, GTP-bound, form of RAC1, along with an induction of RAC1-regulated downstream pathways. CONCLUSIONS: Our results provide a first animal model to dissect this complex human disease syndrome, establishing the first causal proof that a HACE1 deficiency results in decreased synapse number and structural and behavioral neuropathologic features that resemble SPPRS patients.

Automated Clinical Exome Reanalysis Reveals Novel Diagnoses.

Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel gene-disease and variant-disease associations are expected to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories.