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Key heart gene also regulates immune system

Variants in the cysteine-rich with EGF-like domains 1 (CRELD1) gene are associated with nonsyndromic atrioventricular septal defect, and mice lacking the gene die before birth, making CRELD1 a critical player in embryonic heart development. In adulthood, CRELD1 expression is ubiquitous, yet murine embryonic lethality poses challenges to determining the gene’s function outside of heart development. As recently reported in Nature Immunology (https://doi.org/10.1038/s41590-020-00811-2), Bonaguro and colleagues make use of the extreme variance in CRELD1 expression in large human cohorts to find that CRELD1 modulates immune system homeostasis. The researchers examined whole-blood RNA-sequencing data in 95 individuals. By binning the top and bottom 10th percentiles, they generated a highly expressing CRELD1 group (CRELD1^HI) and a lowly expressing CRELD1 group (CRELD1^LO). When the researchers assessed immune-cell populations in blood between the groups, they found that CRELD1^LO donors had lower numbers of naive CD4⁺ and CD8⁺ T cells and more effector memory cells. The team then assessed the relationship between low CRELD1 expression and low numbers of naive CD4⁺ T cells by generating viable conditional CRELD1 knockout mice. Young mice lacking CRELD1 were healthy, with T-cell numbers comparable to those in control mice. However, CRELD1-deficient mice older than 11 months had significantly lower T-cell numbers than controls. The researchers also saw markers of immunosenescence in the knockout mice, but not the controls. Together the results indicate that CRELD1 knockout mice mirror the phenotypic features seen in CRELD1^LO individuals. RNA sequencing and gene set enrichment analysis of naive CD4⁺ T cells further showed differences in T-cell homeostasis, activation, and apoptosis-associated programs between CRELD1 knockout and control mice, as well as downregulation of Wnt signaling. When the researchers activated CD4⁺ T cells by T-cell-receptor stimulation, naive CD4⁺ T cells from knockout mice initially hyperproliferated but were subject to increased apoptosis, leading to reduced overall proliferation and T-cell loss compared with cells from control mice. The researchers then correlated the results back to humans. Differential gene expression analysis across 285 human CD4⁺ T-cell transcriptomes revealed a similar enrichment signature between the CRELD1-deficient mice and low-CRELD1-expressing individuals, and principal component analysis between CRELD1^LO and CRELD1^HI individuals revealed clear phenotypic differences. The authors conclude that together the data indicate a role for CRELD1 in regulating immune system homeostasis. —V. L. Dengler, News Editor

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Variant in Cl⁻/H⁺ exchanger may underlie early-onset neurodegenerative disorder

Neurons depend on the clearance of intracellular aggregates for normal function, a process that in turn is dependent on ion homeostasis and transport across endosomal and lysosomal membranes. The Cl⁻/H⁺ exchanger CLC6, encoded by CLCN6, functions mainly in late endosomes of the nervous system. In mice, lack of CLCN6 leads to mild lysosomal storage disruption; however, the impact of CLCN6 variants in human disease is not well established. In a recently published study in the American Journal of Human Genetics (https://doi.org/10.1016/j.ajhg.2020.11.004), Polovitskaya and colleagues report that a missense variant in CLCN6 is associated with a severe, early-onset neurodegenerative disorder. The researchers identified a de novo change, c.1658A>G (p.Tyr553Cys), in CLCN6 in three unrelated individuals with unclassified and extreme neurodegeneration. Partial sequence alignment revealed that the affected residue is highly conserved, while in silico predictive tools classified the variant as damaging/deleterious. All three individuals presented with developmental delay with early-onset regression, generalized hypotonia, and respiratory insufficiency requiring intervention. Magnetic resonance imaging additionally uncovered bilateral diffusion restriction in cerebral peduncles, dorsal brainstem, and/or dorsal midbrain. To determine the impact of the p.Tyr553Cys variant on ion transport, the researchers performed whole-cell patch-clamp analysis on Chinese hamster ovary cells transfected with wild-type or variant expression constructs. Currents in variant-transfected cells activated more slowly upon depolarization than wild-type transfected cells and reached higher steady-state amplitudes. In addition, currents in variant-transfected cells were insensitive to acidic extracellular pH, suggesting that transport activity would not decrease at the low pH of late endosomes, as in wild-type. When the researchers transfected wild-type CLC6 into HeLa cells, they saw that CLC6 localized to small cytoplasmic vesicles at late endosomes and lysosomes. However, CLC6-Tyr553Cys transfection resulted in obviously large vesicles (more than 2 µm). Live-cell imaging revealed that vesicle enlargement likely occurred via fusion events. In contrast, the team did not detect a difference in vesicle size or intracellular pH between control and patient-derived fibroblasts. The authors conclude that the CLCN6 missense variant is associated with a severe clinical phenotype that allows clinical diagnosis. Overall, the work establishes CLCN6 as a neurological disorder–associated gene. —V. L. Dengler, News Editor

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