The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu).

BACKGROUND: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation. METHODS AND RESULTS: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control. CONCLUSIONS: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).

Proteomic and Molecular Assessment of the Common Saudi Variant in ACADVL Gene Through Mesenchymal Stem Cells.

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) is a coenzyme encoded by ACADVL that converts very-long-chain fatty acids into energy. This process is disrupted by c.65C > A; p.Ser22∗ mutation. To clarify mechanisms by which this mutation leads to VLCAD deficiency, we evaluated differences in molecular and cellular functions between mesenchymal stem cells with normal and mutant VLCAD. Saudi Arabia have a high incidence of this form of mutation. Stem cells with mutant VLCAD were isolated from skin of two patients. Metabolic activity and proliferation were evaluated. The Same evaluation was repeated on normal stem cells introduced with same mutation by CRISPR. Mitochondrial depiction was done by electron microscope and proteomic analysis was done on patients' cells. Metabolic activity and proliferation were significantly lower in patients' cells. Introducing the same mutation into normal stem cells resulted in the same defects. We detected mitochondrial abnormalities by electron microscopy in addition to poor wound healing and migration processes in mutant cells. Furthermore, in a proteomic analysis, we identified several upregulated or downregulated proteins related to hypoglycemia, liver disorder, and cardiac and muscle involvement. We concluded experimental assays of mutant ACADVL (c.65C > A; p.Ser22∗) contribute to severe neonatal disorders with hypoglycemia, liver disorder, and cardiac and muscle involvement.

The SORCS3 gene is mutated in brothers with infantile spasms and intellectual disability.

The sortilin-related VPS10 domain-containing receptor 3 (SORCS3) is a type-I receptor transmembrane protein and a member of the vacuolar protein sorting 10 receptor family. Proteins of this family are defined to have a vacuolar protein sorting 10 domain at the N-terminus. They play important roles as a sorting agency within the cells and transport a variety of intracellular proteins between the Golgi apparatus, endosome, lysosome, secretory granules, and plasma membrane. They are also involved in signal transduction. Clinically, they have been implicated in the pathophysiology of multiple sclerosis and Alzheimer's disease. Here, we report details on two brothers deceased at 20 months and 2 years of age, respectively, with a neurological phenotype including infantile spasms, intellectual disability, global developmental delay, microcephaly, hypotonia, spastic quadriplegia, and delayed myelination. Whole exome sequencing and autozygome analysis showed homozygous missense variant in the SORCS3 gene. The pathogenicity is supported by functional studies in the patient mesenchymal stem cells. Patients' cells showed less proliferation capability than normal cells. In addition, making the same mutation in normal cells revealed a viability defect in them. This is the first study on human subjects with a SORCS3 gene defect and supports the important role of SORCS3 in the central nervous system.