Three-dimensional primary myofibroblast culture reveals morphological alterations in CPAMD8 deficiency.

PURPOSE: To describe the novel clinical phenotypes and develop a three-dimensional (3D) primary myofibroblast culture model to investigate morphological alterations associated with CPAMD8 deficiency. METHODS: We identified novel CPAMD8 variants by exome sequencing. Primary myofibroblasts from human ciliary muscle were cultured in a Matrigel-based 3D system, and CPAMD8 was knocked down by siRNA to assess effects on aggregate growth, morphology, and gene expression. RESULTS: Two novel nonsense variants of the CPAMD8 gene were identified within the pedigree. We identified megalocornea, lentis ectopia, and iris hypoplasia in patients with ASGD8, whereas glaucoma was not observed. Notable alterations in corneal biometry were observed in patients with ASGD8 for the first time. Additionally, as a preliminary observation based on the two affected individuals, reduced retinal vascular density was noted. Upon knockdown of CPAMD8 expression, three-dimensional cultured myofibroblasts demonstrated restricted proliferation, as observed under a microscope and confirmed by CCK-8 assay. Furthermore, ACTA2 expression was downregulated, and the expression of certain TGF-β receptors (TGFBR2 and TGFBR3) was altered, while TGFBR1 and SMAD2 remained unchanged, suggesting a partial rather than global disruption of TGF-β signaling. These findings are consistent with structural abnormalities of the anterior segment in ASGD8. CONCLUSIONS: ASGD8 can cause abnormalities in both the anterior and posterior eye segments. Our newly developed three-dimensional myofibroblast model offers a straightforward, efficient, and cost-effective approach for investigating genetic eye disorders.