Cell-Type Specificity of Mosaic Chromosome 1q Gain Resolved by snRNA-seq in a Case of Epilepsy With Hyaline Protoplasmic Astrocytopathy.

Objectives: Mosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathologic finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. Methods: We present the case of a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA sequencing (snRNA-seq) of brain tissue from the second resection. Results: snRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. Discussion: snRNA-seq may be used to infer the cell-type specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

Elastase 3B mutation links to familial pancreatitis with diabetes and pancreatic adenocarcinoma.

While improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis sparked us to reexamine a large kindred originally reported over 50 years ago with an autosomal dominant inheritance pattern of chronic pancreatitis, diabetes and pancreatic adenocarcinoma. Whole exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease Elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines and in CRISPR-Cas9 engineered mice indicate that this mutation causes translational upregulation of CELA3B, which upon secretion and activation by trypsin leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatitic proteases have been previously linked to hereditary pancreatitis, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease.

Mutations in Cx30 that are linked to skin disease and non-syndromic hearing loss exhibit several distinct cellular pathologies.

Connexin 30 (Cx30), a member of the large gap-junction protein family, plays a role in the homeostasis of the epidermis and inner ear through gap junctional intercellular communication (GJIC). Here, we investigate the underlying mechanisms of four autosomal dominant Cx30 gene mutations that are linked to hearing loss and/or various skin diseases. First, the T5M mutant linked to non-syndromic hearing loss formed functional gap junction channels and hemichannels, similar to wild-type Cx30. The loss-of-function V37E mutant associated with Clouston syndrome or keratitis-ichthyosis-deafness syndrome was retained in the endoplasmic reticulum and significantly induced apoptosis. The G59R mutant linked to the Vohwinkel and Bart-Pumphrey syndromes was retained primarily in the Golgi apparatus and exhibited loss of gap junction channel and hemichannel function but did not cause cell death. Lastly, the A88V mutant, which is linked to the development of Clouston syndrome, also significantly induced apoptosis but through an endoplasmic-reticulum-independent mechanism. Collectively, we discovered that four unique Cx30 mutants might cause disease through different mechanisms that also likely include their selective trans-dominant effects on coexpressed connexins, highlighting the overall complexity of connexin-linked diseases and the importance of GJIC in disease prevention.

Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing.

Pannexin1 (Panx1), a channel-forming glycoprotein is expressed in neonatal but not in aged mouse skin. Histological staining of Panx1 knockout (KO) mouse skin revealed a reduction in epidermal and dermal thickness and an increase in hypodermal adipose tissue. Following dorsal skin punch biopsies, mutant mice exhibited a significant delay in wound healing. Scratch wound and proliferation assays revealed that cultured keratinocytes from KO mice were more migratory, whereas dermal fibroblasts were more proliferative compared with controls. In addition, collagen gels populated with fibroblasts from KO mice exhibited significantly reduced contraction, comparable to WT fibroblasts treated with the Panx1 blocker, probenecid. KO fibroblasts did not increase α-smooth muscle actin expression in response to TGF-ß, as is the case for differentiating WT myofibroblasts during wound contraction. We conclude that Panx1 controls cellular properties of keratinocytes and dermal fibroblasts during early stages of skin development and modulates wound repair upon injury.

Loss of pannexin 1 attenuates melanoma progression by reversion to a melanocytic phenotype.

Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and ß-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.