Campbell, C. D. | Search bioRxiv

bioRxiv2022

Porcn is essential for growth and invagination of the mammalian optic cup

Microphthalmia, anophthalmia, and coloboma (MAC) are congenital ocular malformations causing 25% of childhood blindness. The X-linked disorder Focal Dermal Hypoplasia is frequently associated with MAC and results from mutations in Porcn, a membrane bound O-acyl transferase required for palmitoylation of Wnts to activate multiple Wnt-dependent pathways. Wnt/{beta}-catenin signaling is suppressed in the anterior neural plate for initiation of eye formation and is subsequently required during differentiation of the retinal pigment epithelium (RPE). Non-canonical Wnts are critical for early eye formation in frog and zebrafish, however, it is unclear whether this also applies to mammals. We performed ubiquitous conditional inactivation of Porcn in mouse around the eye field stage. In PorcnCKO, optic vesicles (OV) arrest in growth and fail to form an optic cup. Ventral proliferation is significantly decreased in the mutant OV, with a concomitant increase in apoptotic cell death. While pan-ocular transcription factors such as PAX6, SIX3, LHX2, PAX2 are present, indicative of maintenance of OV identity, regional expression of VSX2, MITF, OTX2 and NR2F2 is downregulated. Failure of RPE differentiation in PorcnCKO is consistent with downregulation of the Wnt/{beta}-catenin effector LEF1, starting around 2.5 days after inactivation. This suggests that Porcn inactivation affects signaling later than a potential requirement for Wnts to promote eye field formation. Altogether, our data shows a novel requirement for Porcn in regulating growth and morphogenesis of the OV, likely by controlling proliferation and survival. In FDH patients with ocular manifestations, growth deficiency during early ocular morphogenesis may be the underlying cause for microphthalmia.

developmental biology↗

bioRxiv2021

Clonal hematopoiesis detection in cancer patients using cell free DNA sequencing

In the context of cancer, clonal hematopoiesis of indeterminate potential (CHIP) is associated with the development of therapy-related myeloid neoplasms and shorter overall survival. Cell-free DNA (cfDNA) sequencing is becoming widely adopted for genomic screening of cancer patients but has not been used extensively to determine CHIP status due to a requirement for matched blood and tumor sequencing. Here we present an accurate machine learning approach to determine clonal hematopoiesis (CH) status from cfDNA sequencing alone and apply our model to 4,096 oncology clinical cfDNA samples. Using this method, we determine that 26% of patients in this cohort have evidence of CH and CH is most common in lung cancer patients. Matched RNAseq data shows signals of increased inflammation, especially neutrophil activation, within the tumor microenvironment of CH-positive patients. Additionally, CH patients showed evidence of neutrophil activation systemically, pointing to a potential mechanism of action for the worse outcomes associated with CH status. Neutrophil activation may be one of many mechanisms however, as estrogen positive breast cancer patients harboring TET2 frameshift mutations had worse outcomes but similar neutrophil levels to CH-negative patients. One Sentence SummaryWe train an accurate machine learning model to detect clonal hematopoiesis in cancer and characterize associated changes in the tumor microenvironment.

cancer biology↗