SRPK3 Is Essential for Cognitive and Ocular Development in Humans and Zebrafish, Explaining X-Linked Intellectual Disability.

OBJECTIVE: Intellectual disability is often the outcome of neurodevelopmental disorders and is characterized by significant impairments in intellectual and adaptive functioning. X-linked intellectual disability (XLID) is a subset of these disorders caused by genetic defects on the X chromosome, affecting about 2 out of 1,000 males. In syndromic form, it leads to a broad range of cognitive, behavioral, ocular, and physical disabilities. METHODS: Employing exome or genome sequencing, here we identified 4 missense variants (c.475C > G; p.H159D, c.1373C > A; p.T458N, and c.1585G > A; p.E529K, c.953C > T; p.S318L) and a putative truncating variant (c.1413_1414del; p.Y471*) in the SRPK3 gene in 9 XLID patients from 5 unrelated families. To validate SRPK3 as a novel XLID gene, we established a knockout (KO) model of the SRPK3 orthologue in zebrafish. RESULTS: The 8 patients ascertained postnatally shared common clinical features including intellectual disability, agenesis of the corpus callosum, abnormal eye movement, and ataxia. A ninth case, ascertained prenatally, had a complex structural brain phenotype. Together, these data indicate a pathological role of SRPK3 in neurodevelopmental disorders. In post-fertilization day 5 larvae (free swimming stage), KO zebrafish exhibited severe deficits in eye movement and swim bladder inflation, mimicking uncontrolled ocular movement and physical clumsiness observed in human patients. In adult KO zebrafish, cerebellar agenesis and behavioral abnormalities were observed, recapitulating human phenotypes of cerebellar atrophy and intellectual disability. INTERPRETATION: Overall, these results suggest a crucial role of SRPK3 in the pathogenesis of syndromic X-linked intellectual disability and provide new insights into brain development, cognitive and ocular dysfunction in both humans and zebrafish. ANN NEUROL 2024.

Eye tracking: empirical foundations for a minimal reporting guideline.

In this paper, we present a review of how the various aspects of any study using an eye tracker (such as the instrument, methodology, environment, participant, etc.) affect the quality of the recorded eye-tracking data and the obtained eye-movement and gaze measures. We take this review to represent the empirical foundation for reporting guidelines of any study involving an eye tracker. We compare this empirical foundation to five existing reporting guidelines and to a database of 207 published eye-tracking studies. We find that reporting guidelines vary substantially and do not match with actual reporting practices. We end by deriving a minimal, flexible reporting guideline based on empirical research (Section "An empirically based minimal reporting guideline").

SLC38A8 mutations result in arrested retinal development with loss of cone photoreceptor specialization.

Foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis is an autosomal recessive disorder arising from SLC38A8 mutations. SLC38A8 is a putative glutamine transporter with strong expression within the photoreceptor layer in the retina. Previous studies have been limited due to lack of quantitative data on retinal development and nystagmus characteristics. In this multi-centre study, a custom-targeted next generation sequencing (NGS) gene panel was used to identify SLC38A8 mutations from a cohort of 511 nystagmus patients. We report 16 novel SLC38A8 mutations. The sixth transmembrane domain is most frequently disrupted by missense SLC38A8 mutations. Ninety percent of our cases were initially misdiagnosed as PAX6-related phenotype or ocular albinism prior to NGS. We characterized the retinal development in vivo in patients with SLC38A8 mutations using high-resolution optical coherence tomography. All patients had severe grades of arrested retinal development with lack of a foveal pit and no cone photoreceptor outer segment lengthening. Loss of foveal specialization features such as outer segment lengthening implies reduced foveal cone density, which contributes to reduced visual acuity. Unlike other disorders (such as albinism or PAX6 mutations) which exhibit a spectrum of foveal hypoplasia, SLC38A8 mutations have arrest of retinal development at an earlier stage resulting in a more under-developed retina and severe phenotype.

Genotypic and Phenotypic Spectrum of Foveal Hypoplasia: A Multicenter Study.

PURPOSE: To characterize the genotypic and phenotypic spectrum of foveal hypoplasia (FH). DESIGN: Multicenter, observational study. PARTICIPANTS: A total of 907 patients with a confirmed molecular diagnosis of albinism, PAX6, SLC38A8, FRMD7, AHR, or achromatopsia from 12 centers in 9 countries (n = 523) or extracted from publicly available datasets from previously reported literature (n = 384). METHODS: Individuals with a confirmed molecular diagnosis and availability of foveal OCT scans were identified from 12 centers or from the literature between January 2011 and March 2021. A genetic diagnosis was confirmed by sequence analysis. Grading of FH was derived from OCT scans. MAIN OUTCOME MEASURES: Grade of FH, presence or absence of photoreceptor specialization (PRS+ vs. PRS-), molecular diagnosis, and visual acuity (VA). RESULTS: The most common genetic etiology for typical FH in our cohort was albinism (67.5%), followed by PAX6 (21.8%), SLC38A8 (6.8%), and FRMD7 (3.5%) variants. AHR variants were rare (0.4%). Atypical FH was seen in 67.4% of achromatopsia cases. Atypical FH in achromatopsia had significantly worse VA than typical FH (P < 0.0001). There was a significant difference in the spectrum of FH grades based on the molecular diagnosis (chi-square = 60.4, P < 0.0001). All SLC38A8 cases were PRS- (P = 0.003), whereas all FRMD7 cases were PRS+ (P < 0.0001). Analysis of albinism subtypes revealed a significant difference in the grade of FH (chi-square = 31.4, P < 0.0001) and VA (P = 0.0003) between oculocutaneous albinism (OCA) compared with ocular albinism (OA) and Hermansky-Pudlak syndrome (HPS). Ocular albinism and HPS demonstrated higher grades of FH and worse VA than OCA. There was a significant difference (P < 0.0001) in VA between FRMD7 variants compared with other diagnoses associated with FH. CONCLUSIONS: We characterized the phenotypic and genotypic spectrum of FH. Atypical FH is associated with a worse prognosis than all other forms of FH. In typical FH, our data suggest that arrested retinal development occurs earlier in SLC38A8, OA, HPS, and AHR variants and later in FRMD7 variants. The defined time period of foveal developmental arrest for OCA and PAX6 variants seems to demonstrate more variability. Our findings provide mechanistic insight into disorders associated with FH and have significant prognostic and diagnostic value.

Optic Nerve Head and Retinal Abnormalities Associated with Congenital Fibrosis of the Extraocular Muscles.

Congenital fibrosis of the extraocular muscles (CFEOM) is a congenital cranial dysinnervation disorder caused by developmental abnormalities affecting cranial nerves/nuclei innervating the extraocular muscles. Autosomal dominant CFEOM arises from heterozygous missense mutations of KIF21A or TUBB3. Although spatiotemporal expression studies have shown KIF21A and TUBB3 expression in developing retinal ganglion cells, it is unclear whether dysinnervation extends beyond the oculomotor system. We aimed to investigate whether dysinnervation extends to the visual system by performing high-resolution optical coherence tomography (OCT) scans characterizing retinal ganglion cells within the optic nerve head and retina. Sixteen patients with CFEOM were screened for mutations in KIF21A, TUBB3, and TUBB2B. Six patients had apparent optic nerve hypoplasia. OCT showed neuro-retinal rim loss. Disc diameter, rim width, rim area, and peripapillary nerve fiber layer thickness were significantly reduced in CFEOM patients compared to controls (p < 0.005). Situs inversus of retinal vessels was seen in five patients. Our study provides evidence of structural optic nerve and retinal changes in CFEOM. We show for the first time that there are widespread retinal changes beyond the retinal ganglion cells in patients with CFEOM. This study shows that the phenotype in CFEOM extends beyond the motor nerves.

Can Structural Grading of Foveal Hypoplasia Predict Future Vision in Infantile Nystagmus?: A Longitudinal Study.

PURPOSE: To evaluate structural grading and quantitative segmentation of foveal hypoplasia using handheld OCT, versus preferential looking (PL), as predictors of future vision in preverbal children with infantile nystagmus. DESIGN: Longitudinal cohort study. PARTICIPANTS: Forty-two patients with infantile nystagmus (19 with albinism, 17 with idiopathic infantile nystagmus, and 6 with achromatopsia) were examined. METHODS: Spectral-domain handheld OCT was performed in preverbal children up to 36 months of age. Foveal tomograms were graded using our 6-point grading system for foveal hypoplasia and were segmented for quantitative analysis: photoreceptor length, outer segment (OS) length, and foveal developmental index (FDI; a ratio of inner layers versus total foveal thickness). Patients were followed up until they could perform chart visual acuity (VA) testing. Data were analyzed using linear mixed regression models. Visual acuity predicted by foveal grading was compared with prediction by PL, the current gold standard for visual assessment in infants and young children. MAIN OUTCOME MEASURES: Grade of foveal hypoplasia, quantitative parameters (photoreceptor length, OS length, FDI), and PL VA were obtained in preverbal children for comparison with future chart VA outcomes. RESULTS: We imaged 81 eyes from 42 patients with infantile nystagmus of mean age 19.8 months (range, 0.9-33.4 months; standard deviation [SD], 9.4 months) at the first handheld OCT scan. Mean follow-up was 44.1 months (range, 18.4-63.2 months; SD, 12.0 months). Structural grading was the strongest predictor of future VA (grading: r = 0.80, F = 67.49, P < 0.0001) compared with quantitative measures (FDI: r = 0.74, F = 28.81, P < 0.001; OS length: r = 0.65; F = 7.94, P < 0.008; photoreceptor length: r = 0.65; F = 7.94, P < 0.008). Preferential looking was inferior to VA prediction by foveal grading (PL: r = 0.42, F = 3.12, P < 0.03). CONCLUSIONS: Handheld OCT can predict future VA in infantile nystagmus. Structural grading is a better predictor of future VA than quantitative segmentation and PL testing. Predicting future vision may avert parental anxiety and may optimize childhood development.

Homozygous stop mutation in AHR causes autosomal recessive foveal hypoplasia and infantile nystagmus.

Herein we present a consanguineous family with three children affected by foveal hypoplasia with infantile nystagmus, following an autosomal recessive mode of inheritance. The patients showed normal electroretinography responses, no signs of albinism, and no anterior segment or brain abnormalities. Upon whole exome sequencing, we identified a homozygous mutation (c.1861C>T;p.Q621*) in the aryl hydrocarbon receptor (AHR) gene that perfectly co-segregated with the disease in the larger family. AHR is a ligand-activated transcription factor that has been intensively studied in xenobiotic-induced toxicity. Further, it has been shown to play a physiological role under normal cellular conditions, such as in immunity, inflammatory response and neurogenesis. Notably, knockout of the Ahr gene in mouse impairs optic nerve myelin sheath formation and results in oculomotor deficits sharing many features with our patients: the eye movement disorder in Ahr-/- mice appears early in development and presents as conjugate horizontal pendular nystagmus. We therefore propose AHR to be a novel disease gene for a new, recessively inherited disorder in humans, characterized by infantile nystagmus and foveal hypoplasia.

Abnormal retinal development associated with FRMD7 mutations.

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder, often associated with FRMD7 mutations. As the appearance of the retina is reported to be normal based on conventional fundus photography, IIN is postulated to arise from abnormal cortical development. To determine whether the afferent visual system is involved in FRMD7 mutations, we performed in situ hybridization studies in human embryonic and fetal stages (35 days post-ovulation to 9 weeks post-conception). We show a dynamic retinal expression pattern of FRMD7 during development. We observe expression within the outer neuroblastic layer, then in the inner neuroblastic layer and at 9 weeks post-conception a bilaminar expression pattern. Expression was also noted within the developing optic stalk and optic disk. We identified a large cohort of IIN patients (n = 100), and performed sequence analysis which revealed 45 patients with FRMD7 mutations. Patients with FRMD7 mutations underwent detailed retinal imaging studies using ultrahigh-resolution optical coherence tomography. The tomograms were compared with a control cohort (n = 60). The foveal pit was significantly shallower in FRMD7 patients (P < 0.0001). The optic nerve head morphology was abnormal with significantly decreased optic disk area, retinal nerve fiber layer thickness, cup area and cup depth in FRMD7 patients (P < 0.0001). This study shows for the first time that abnormal afferent system development is associated with FRMD7 mutations and could be an important etiological factor in the development of nystagmus.

A novel interaction between FRMD7 and CASK: evidence for a causal role in idiopathic infantile nystagmus.

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder of eye movement that can be caused by mutations in the FRMD7 gene that encodes a FERM domain protein. FRMD7 is expressed in the brain and knock-down studies suggest it plays a role in neurite extension through modulation of the actin cytoskeleton, yet little is known about its precise molecular function and the effects of IIN mutations. Here, we studied four IIN-associated missense mutants and found them to have diverse effects on FRMD7 expression and cytoplasmic localization. The C271Y mutant accumulates in the nucleus, possibly due to disruption of a nuclear export sequence located downstream of the FERM-adjacent domain. While overexpression of wild-type FRMD7 promotes neurite outgrowth, mutants reduce this effect to differing degrees and the nuclear localizing C271Y mutant acts in a dominant-negative manner to inhibit neurite formation. To gain insight into FRMD7 molecular function, we used an IP-MS approach and identified the multi-domain plasma membrane scaffolding protein, CASK, as a FRMD7 interactor. Importantly, CASK promotes FRMD7 co-localization at the plasma membrane, where it enhances CASK-induced neurite length, whereas IIN-associated FRMD7 mutations impair all of these features. Mutations in CASK cause X-linked mental retardation. Patients with C-terminal CASK mutations also present with nystagmus and, strikingly, we show that these mutations specifically disrupt interaction with FRMD7. Together, our data strongly support a model whereby CASK recruits FRMD7 to the plasma membrane to promote neurite outgrowth during development of the oculomotor neural network and that defects in this interaction result in nystagmus.

Gender- and Ethnicity-Related Differences in Optic Nerve Head Topography in Healthy Indian and Caucasian Participants.

We investigated the effect of ethnicity and gender on optic nerve head morphology in healthy subjects using spectral-domain optical coherence tomography (SD-OCT). Thirty-five Indian (i.e. Indian subcontinent) females, 34 Caucasian females, 32 Indian males, and 32 Caucasian males were examined using SD-OCT (Copernicus, Optopol Technology). Disc and rim areas were larger in Caucasian males compared with females but smaller in Indians males compared with females. Indian participants had significantly larger cup areas and volumes without significant differences in retinal nerve fibre layer (RNFL) thicknesses between groups. Gender and ethnicity differences should be considered in assessment of patients.

A novel portable flip-phone based visual behaviour assay for zebrafish.

The optokinetic reflex (OKR) serves as a vital index for visual system development in early life, commonly observed within the first six months post-birth in humans. Zebrafish larvae offer a robust and convenient model for OKR studies due to their rapid development and manageable size. Existing OKR assays often involve cumbersome setups and offer limited portability. In this study, we present an innovative OKR assay that leverages the flexible screen of the Samsung Galaxy Z Flip to optimize setup and portability. We conducted paired slow-phase velocity measurements in 5-day post-fertilization (dpf) zebrafish larvae (n = 15), using both the novel flip-phone-based assay and a traditional liquid-crystal display (LCD) arena. Utilizing Bland-Altman plots, we assessed the agreement between the two methods. Both assays were efficacious in eliciting OKR, with eye movement analysis indicating high tracking precision in the flip-phone-based assay. No statistically significant difference was observed in slow-phase velocities between the two assays (p = 0.40). Our findings underscore the feasibility and non-inferiority of the flip-phone-based approach, offering streamlined assembly, enhanced portability, and the potential for cost-effective alternatives. This study contributes to the evolution of OKR assay methodologies, aligning them with emerging research paradigms.

ERG Responses in Albinism, Idiopathic Infantile Nystagmus, and Controls.

Purpose: Our primary aim was to compare adult full-field ERG (ffERG) responses in albinism, idiopathic infantile nystagmus (IIN), and controls. A secondary aim was to investigate the effect of within-subject changes in nystagmus eye movements on ffERG responses. Methods: Dilated Ganzfeld flash ffERG responses were recorded using DTL electrodes under conditions of dark (standard and dim flash) and light adaptation in 68 participants with albinism, 43 with IIN, and 24 controls. For the primary aim, the effect of group and age on ffERG responses was investigated. For the secondary aim, null region characteristics were determined using eye movements recorded prior to ffERG recordings. ffERG responses were recorded near and away from the null regions of 18 participants also measuring the success rate of recordings. Results: For the primary aim, age-adjusted photopic a- and b-wave amplitudes were consistently smaller in IIN compared with controls (P < 0.0001), with responses in both groups decreasing with age. In contrast, photopic a-wave amplitudes increased with age in albinism (P = 0.0035). For the secondary aim, more intense nystagmus significantly reduced the success rate of measurable responses. Within-subject changes in nystagmus intensity generated small, borderline significant differences in photopic b-wave peak times and a-and b-wave amplitudes under scotopic conditions with standard flash. Conclusions: Age-adjusted photopic ffERG responses are significantly reduced in IIN adding to the growing body of evidence of retinal abnormalities in IIN. Differences between photopic responses in albinism and controls depend on age. Success at obtaining ffERG responses could be improved by recording responses at the null region.

Single cell dual-omic atlas of the human developing retina.

The development of the retina is under tight temporal and spatial control. To gain insights into the molecular basis of this process, we generate a single-nuclei dual-omic atlas of the human developing retina with approximately 220,000 nuclei from 14 human embryos and fetuses aged between 8 and 23-weeks post-conception with matched macular and peripheral tissues. This atlas captures all major cell classes in the retina, along with a large proportion of progenitors and cell-type-specific precursors. Cell trajectory analysis reveals a transition from continuous progression in early progenitors to a hierarchical development during the later stages of cell type specification. Both known and unrecorded candidate transcription factors, along with gene regulatory networks that drive the transitions of various cell fates, are identified. Comparisons between the macular and peripheral retinae indicate a largely consistent yet distinct developmental pattern. This atlas offers unparalleled resolution into the transcriptional and chromatin accessibility landscapes during development, providing an invaluable resource for deeper insights into retinal development and associated diseases.

Potential of handheld optical coherence tomography to determine cause of infantile nystagmus in children by using foveal morphology.

OBJECTIVE: To investigate the feasibility of handheld (HH) ultra-high-resolution spectral-domain optical coherence tomography (SD-OCT) in young children with nystagmus, to determine its sensitivity and specificity in classifying foveal abnormalities, and to investigate its potential to determine the cause of infantile nystagmus with the use of foveal morphology. DESIGN: Prospective, case-control study. PARTICIPANTS AND CONTROLS: A total of 50 patients with nystagmus and 50 healthy control subjects (mean age, 3.2 years; range, 0-8 years). METHODS: Each patient was scanned using HH SD-OCT (Bioptigen Inc., Research Triangle Park, NC) without sedation, and foveal morphology was classified into 1 of 4 categories: (1) typical foveal hypoplasia (predicting clinical diagnosis of albinism, PAX6 mutations, or isolated foveal hypoplasia); (2) atypical foveal hypoplasia (predicting achromatopsia); (3) other foveal changes (corresponding to retinal dystrophies); and (4) normal fovea (predicting idiopathic or manifest latent nystagmus). An independent interpretation of the HH SD-OCT scans by masked examiners was performed, and the sensitivity and specificity of the predicted diagnosis were calculated. MAIN OUTCOME MEASURES: The success rate of image acquisition and sensitivity and specificity of the HH SD-OCT in classifying foveal abnormalities. RESULTS: In 94% of examinations, HH SD-OCT was successful. Twenty-three patients had typical foveal hypoplasia (category 1). Of these patients, 21 were diagnosed with albinism and 2 were diagnosed with PAX6 mutations. Five patients were classified as atypical (category 2) and diagnosed with achromatopsia. Six patients had other abnormal foveal morphology (category 3) and were diagnosed with retinal dystrophy. Sixteen patients had normal foveal morphology (category 4). Of these patients, 12 were diagnosed with idiopathic nystagmus and 4 were diagnosed with manifest latent nystagmus. Sensitivities of HH SD-OCT for classifying typical or atypical foveal hypoplasia, other abnormal foveal morphology, and normal morphology were 92.8%, 86.7%, 41.1%, and 88.4%, respectively, with specificities of 91.4%, 94.8%, 97.7% and 95.1%, respectively. CONCLUSIONS: We demonstrate excellent feasibility of HH SD-OCT in the diagnosis of conditions associated with infantile nystagmus. The HH SD-OCT classification of foveal abnormalities was highly sensitive and specific. This classification was used to determine the underlying cause of infantile nystagmus. Handheld SD-OCT in early childhood can facilitate focused investigations and earlier diagnosis. This is important in an era when potentially time-sensitive treatment, such as gene therapy, is imminent.

Abnormal foveal morphology in carriers of oculocutaneous albinism.

BACKGROUND/AIMS: To investigate the foveal morphology in carriers of oculocutaneous albinism (OCA) using spectral domain optical coherence tomography (SD-OCT). A cross-sectional, observational study. METHODS: Handheld SD-OCT (Envisu C2300) was used to acquire horizontal scans through the centre of the fovea in biological parents of patients with OCA (n=28; mean age±SD=40.43±8.07 years) and age-matched and ethnicity-matched controls (n=28; mean age±SD=38.04±10.27 years). Sequence analysis was performed for variants in known genes associated with OCA. Best-corrected visual acuity (BCVA), presence of foveal hypoplasia and grade, foveal, parafoveal and perifoveal thickness measurements of total retinal layers (TRL), inner retinal layers (IRL) and outer retinal layers (ORL) thickness were measured. RESULTS: Foveal hypoplasia was identified in 32.14% of OCA carriers; grade 1 in all cases. OCA carriers demonstrated significant thicker TRL thickness (median difference: 13.46 µm, p=0.009) and IRL thickness (mean difference: 8.98 µm, p<0.001) at the central fovea compared with controls. BCVA of carriers was between -0.16 and 0.18 logMAR (mean: 0.0 logMAR). No significant differences in BCVA was noted between OCA carriers or controls (p=0.83). In the OCA carriers, we identified previously reported pathogenic variants in TYR, OCA2 and SLC45A2, novel OCA2 variants (n=3) and heterozygosity of the pathogenic TYR haplotype. CONCLUSION: We have, for the first time, identified foveal abnormalities in OCA carriers. This provides clinical value, particularly in cases where limited phenotype data are available. Our findings raise the possibility that previously reported mild cases of foveal hypoplasia or isolated foveal hypoplasia could correspond to OCA carrier status.