RESUMEN
Heterozygous missense mutations in several genes in the WNT5A signaling pathway cause autosomal dominant Robinow syndrome 1 (DRS1). Our objective was to clarify the functional impact of a missense mutation in WNT5A on the skeleton, one of the main affected tissues in RS. We delivered avian replication competent retroviruses (RCAS) containing human wild-type WNT5A (wtWNT5A), WNT5AC83S variant or GFP/AlkPO4 control genes to the chicken embryo limb. Strikingly, WNT5AC83S consistently caused a delay in ossification and bones were more than 50% shorter and 200% wider than controls. In contrast, bone dimensions in wtWNT5A limbs were slightly affected (20% shorter, 25% wider) but ossification occurred on schedule. The dysmorphology of bones was established during cartilage differentiation. Instead of stereotypical stacking of chondrocytes, the WNT5AC83S-infected cartilage was composed of randomly oriented chondrocytes and that had diffuse, rather than concentrated Prickle staining, both signs of disrupted planar cell polarity (PCP) mechanisms. Biochemical assays revealed that C83S variant was able to activate the Jun N-terminal kinase-PCP pathway similar to wtWNT5A; however, the activity of the variant ligand was influenced by receptor availability. Unexpectedly, the C83S change caused a reduction in the amount of protein being synthesized and secreted, compared to wtWNT5A. Thus, in the chicken and human, RS phenotypes are produced from the C83S mutation, even though the variant protein is less abundant than wtWNT5A. We conclude the variant protein has dominant-negative effects on chondrogenesis leading to limb abnormalities.
Asunto(s)
Condrocitos/citología , Condrogénesis , Anomalías Craneofaciales/metabolismo , Enanismo/metabolismo , Extremidades/embriología , Deformidades Congénitas de las Extremidades/metabolismo , Anomalías Urogenitales/metabolismo , Proteína Wnt-5a/genética , Animales , Animales Modificados Genéticamente , Cartílago/metabolismo , Polaridad Celular/fisiología , Embrión de Pollo , Pollos , Condrogénesis/genética , Anomalías Craneofaciales/genética , Modelos Animales de Enfermedad , Enanismo/genética , Células HEK293 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Deformidades Congénitas de las Extremidades/genética , Mutación Missense , Fenotipo , Anomalías Urogenitales/genética , Vía de Señalización Wnt , Proteína Wnt-5a/metabolismoRESUMEN
OBJECTIVE: To evaluate the long-term structural and microvascular retinal effects of internal limiting membrane peeling for full-thickness macular hole (FTMH) using en face adaptive optics optical coherence tomography (AO-OCT), conventional OCT, and OCT angiography (OCTA). DESIGN: Interventional case series. PARTICIPANTS: Patients with FTMH treated with vitrectomy, internal limiting membrane peeling, and gas tamponade. METHODS: Eleven eyes with FTMH that had at least 12 months of postoperative follow-up were enrolled in the study. En face AO-OCT was used to image the superficial retina in the peeled and nonpeeled areas. En face structural OCT was performed to image the inner retinal dimples (IRDs), macular thickness, and retinal nerve fibre layer (RNFL). En face OCTA was used to examine the integrity of the peripapillary nerve fibre layer (NFL) plexus. RESULTS: AO-OCT showed RFNL wrapping around the IRDs, and no obvious peripapillary NFL plexus dropout was seen with OCTA. Scattered hyper-reflective dots were observed on the surface of the peeled retina in all patients imaged with AO-OCT. No significant differences were found in IRD number (91.5 ± 24.4 versus 77.2 ± 14.7; P = 2.07), IRD proportionate area (8.36 ± 3.34 versus 7.53 ± 2.60; P = 0.159), or macular thickness between the 6- and 12-month (or greater) postoperative visits. CONCLUSION: IRDs do not to progress beyond 6 months postoperatively, and no obvious damage to RFNL and peripapillary NFL plexus was detected. Hyper-reflective dots on the surface of the retina suggestive of possible Müller cell reactive gliosis were identified with AO-OCT.