A novel missense variant expands the phenotype and genotype of PAX6-associated foveal hypoplasia accompanied by various manifestations of anterior segment dysgenesis.

BACKGROUND: According to previous reports, PAX6-associated foveal hypoplasia (FH) could usually be accompanied by various anterior segment anomalies including variable iris changes. This study aims to exhibit unusual phenotypes of a novel missense variant of PAX6 from a Chinese pedigree. METHODS: Ophthalmic examinations including slit-lamp biomicroscopy, gonioscopy, ophthalmic ultrasound, ultrasonic biomicroscopy, optical coherence tomography, wide-field fundus imaging, and visual field test were performed to evaluate the clinical manifestations. Whole-exome sequencing (WES) and bioinformatics analysis were conducted in eight members from this pedigree to identify the causative mutation. RESULTS: WES revealed a novel heterozygous substitution of PAX6 (NM_000280.5:c.157G > A, p.(Val53Met) (chr11:31823309 C > T, hg19)), which cosegregated with the phenotype of this pedigree. All the three patients (a pair of fraternal twins and their mother) exhibited bilateral FH and anterior segment dysgenesis (ASD) including microcornea, sclerocornea, obvious symmetrical corectopia, iris stromal dysplasia, goniodysgenesis, and abnormal distribution of fundus blood vessels. The girl of the fraternal twins also demonstrated bilateral temporal deviation of lenses and abnormal tissue membrane connecting anterior chamber angle and lens anterior capsule in the right eye. The mother additionally showed apparent cataract bilaterally and cupping of the optic disc in her left eye. CONCLUSION: A novel missense variant in PAX6 gene was detected in a Chinese pedigree demonstrating bilateral FH and ASD. It is really distinctive that the ASD involves almost all parts of the anterior segment, and bilateral symmetrical corectopia is the most perceptible sign. This study expands the phenotypic and genotypic spectrum of PAX6-associated ocular diseases, and facilitates the understanding of the crucial role that PAX6 plays in the development of the eye. Meanwhile, PAX6 could be considered as a candidate pathogenic gene of bilateral symmetrical corectopia.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells.

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down). Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot. DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone. Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Ophthalmological evaluation and integrated intervention in a family with congenital aniridia in Alagoas, Brazil / Avaliação oftalmológica e intervenção integrada em família com aniridia congênita em Alagoas, Brasil

Abstract Objective: We aimed to describe the clinical and phenotypic manifestations as well as the visual prognosis of a family with CA in Northeastern Brazil. Methods: This was a cross-sectional study involving 31 individuals (56 eyes) from the same family presenting CA phenotypes. The study population resided in the municipality of Água Branca, in the backlands of the state of Alagoas, Northeastern Brazil. The clinical and phenotypic variables were analyzed. For the analysis, descriptive statistics (absolute and relative frequency and measures of central tendency and dispersion) and inferential statistics (Shapiro-Wilk and Student's t tests) were used, with 95% confidence intervals and significance set at 5%. Results: Of the 31 individuals, 18 (58.1%) were male, with a mean age of 27.45 ± 17.49 years, with no difference between sexes. Of the 56 eyes evaluated, 26 and 30 were right and left eyes, respectively; 61.3% (n = 19) individuals had complete bilateral aniridia and 25.8% (n = 8) reported a total loss of light perception in both the eyes. The most prevalent ocular abnormalities were nystagmus (n = 27; 87.09%), cataract (n = 20; 64.5%), strabismus (n = 14; 45.2%), corneal changes such as opacities and/or vascularization (n = 13; 41.93%), and ectopia lentis (n = 6; 19.4%). Further, 13 individuals underwent retinal optical coherence tomography, six man and seven women aged 9-48 (mean, 30.15 ± 15.9) years. All patients presented absence of foveal depression as well as reduced macular thickness and visual acuity. Nine subjects underwent phacoemulsification. Conclusion: The study showed wide phenotypic variation among the studied individuals, with poor visual prognosis. The study highlights the need to establish comprehensive care mechanisms for families with the disease.

Resumo Objetivo: Descrever manifestações clínicas e fenotípicas e o prognóstico visual de uma família com aniridia congênita (AC). Métodos: Trata-se de estudo transversal envolvendo 31 indivíduos (56 olhos), de uma mesma família com fenótipo de AC residindo no município de Água Branca, no sertão do estado de Alagoas, região nordeste do Brasil. Foram analisadas variáveis clínicas e fenotípicas. Para a análise, foi utilizada a estatística descritiva (frequência absoluta e relativa e medidas de tendência central e de dispersão) e inferencial (testes de Shapiro-Wilk e t Student). Considerou-se o intervalo de confiança de 95% e a significância de 5%. Resultados: Dos 31 indivíduos, 18 (58,1%) eram do sexo masculino, com média de idade de 27,45±17,49, sem diferença entre os sexos. Dos 56 olhos avaliados, 26 eram olhos direitos e 30 olhos esquerdos: 61,3% (n=19) apresentavam aniridia bilateral total; 25,8% (n=8) referiam perda total de percepção da luz em ambos os olhos. As anormalidades oculares mais prevalentes foram o nistagmo (n=27; 87,09%), catarata (n=20; 64,5%), estrabismo 14 (45,2%), alterações opacidades ou vascularização corneanas (n=13; 41,93%) e ectopia lentis (n=6; 19,4%). Os 13 indivíduos submetidos à tomografia de coerência óptica (OCT) retiniana apresentavam perda da depressão foveal, redução da espessura macular e redução da acuidade visual. Nove indivíduos foram submetidos a cirurgia de facoemulsificação. Conclusão: O estudo mostrou ampla variação fenotípica entre os indivíduos estudados, com pobre prognóstico visual. O estudo destaca a necessidade de estabelecer mecanismos de cuidado integral para as famílias com a doença.

The role of the diencephalon in the guidance of thalamocortical axons in mice.

Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.

Early and late auditory information processing show opposing deviations in aniridia.

Aniridia is a congenital disorder, predominantly caused by heterozygous mutations of the PAX6 gene. While ocular defects have been extensively characterized in this population, brain-related anatomical and functional abnormalities are emerging as a prominent feature of the disorder. Individuals with aniridia frequently exhibit auditory processing deficits despite normal audiograms. While previous studies have reported hypoplasia of the anterior commissure and corpus callosum in some of these individuals, the neurophysiological basis of these impairments remains unexplored. This study provides direct assessment of neural activity related to auditory processing in aniridia. Participants were presented with tones designed to elicit an auditory steady-state response (ASSR) at 22 Hz, 40 Hz, and 84 Hz, and infrequent broadband target tones to maintain attention during electroencephalography (EEG) recording. Persons with aniridia showed increased early cortical responses (P50 AEP) in response to all tones, and increased high-frequency oscillatory entrainment (84 Hz ASSR). In contrast, this group showed a decreased cortical integration response (P300 AEP to target tones) and reduced neural entrainment to cortical beta-band stimuli (22 Hz ASSR). Collectively, our results suggest that subcortical and early cortical auditory processing is augmented in aniridia, while functional cortical integration of auditory information is deficient in this population.

The effects of electromagnetic fields on cultured human retinal pigment epithelial cells.

A great deal of evidence has confirmed that electromagnetic fields (EMFs) can affect the central nervous system. In this study, cultured neonatal human retinal pigment epithelial (hRPE) cells were exposed to pulsed EMF of 1 mT intensity and 50 Hz frequency 8 h daily for 3 days. In addition to cell proliferation and cell death assays, immunocytochemistry for RPE65, PAX6, nestin, and cytokeratin 8/18 proteins were performed. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for NES, PAX6, RPE65, and ACTA2 gene expression. Exposed hRPE cells did not demonstrate significant change in terms of cytomorphology, cell proliferation, or cell death. Protein expression of PAX6 was decreased in treated cells compared to controls and remained unchanged for RPE65, cytokeratin 8/18, and nestin. Gene expressions of NES, RPE65, and PAX6 were decreased in treated cells as compared to controls. Gene expression of ACTA2 did not significantly change. In conclusion, viability of cultivated neonatal hRPE cells did not change after short exposure to a safe dose of pulsed EMF albeit that both gene and protein expressions of retinal progenitor cell markers were reduced. Whether longer exposure durations that are being constantly produced by widely-used electronic devices may induce significant changes in these cells, needs further investigation. Bioelectromagnetics. 39:585-594, 2018. © 2018 Wiley Periodicals, Inc.

Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary in the spinal cord.

Progenitor cells are segregated into multiple domains along the dorsoventral axis of the vertebrate neural tube, and each progenitor domain generates particular types of neurons. Selective cross-repressive interactions between pairs of class I and class II transcription factors play important roles in patterning neural progenitors into domains with clear boundaries. Here, we provide evidence that the zinc-finger protein Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary through mutually repressive interactions with the class II protein Nkx2-2. The ventral limit of Sp8 expression is complementary to the dorsal limit of Nkx2-2 expression at the pMN/p3 boundary. Sp8 and Nkx2-2 exert cross-repressive interactions, and changing the expression of Sp8 and Nkx2-2 is coupled with pMN and p3 progenitor fate conversion. Sp8 exerts its neural patterning activities by acting as a transcriptional activator. The expression of a repressive form of Sp8 results in the selective inhibition of motor neuron generation and the ectopic induction of Nkx2-2 expression. Sp8 expression is positively regulated by, but not completely dependent on, Pax6. Furthermore, whereas loss of Pax6 function alone results in disruption of the pMN/p3 domain boundary only in the rostral levels of the spinal cord, loss of both Sp8 and Pax6 functions results in disruption of the pMN/p3 domain boundary along the whole rostrocaudal axis of the spinal cord. We conclude that Sp8 plays a supplementary role to Pax6 in specifying the pMN over p3 progenitor fate through cross-repressive interactions with Nkx2-2.