Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Cardiac microvascular endothelial cells express a functional Ca 2+-sensing receptor

The mechanism whereby extracellular Ca2+ exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca2+-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd 3+, La3+ and neomycin, elicited a heterogeneous intracellular Ca2+ signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP3) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na +/Ca2+ exchanger upon substitution of extracellular Na+ unmasked the Ca2+ signal triggered by an increase in extracellular Ca2+ levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca2+ response to the CaSR agonist La3+. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca2+ from intracellular InsP3-sensitive stores.Facultad de Ciencias Exacta

Genotype–phenotype correlations in individuals with pathogenic RERE variants

Heterozygous variants in the arginine-glutamic acid dipeptide repeats gene (RERE) have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). Here, we report nine individuals with NEDBEH who carry partial deletions or deleterious sequence variants in RERE. These variants were found to be de novo in all cases in which parental samples were available. An analysis of data from individuals with NEDBEH suggests that point mutations affecting the Atrophin-1 domain of RERE are associated with an increased risk of structural eye defects, congenital heart defects, renal anomalies, and sensorineural hearing loss when compared with loss-of-function variants that are likely to lead to haploinsufficiency. A high percentage of RERE pathogenic variants affect a histidine-rich region in the Atrophin-1 domain. We have also identified a recurrent two-amino-acid duplication in this region that is associated with the development of a CHARGE syndrome-like phenotype. We conclude that mutations affecting RERE result in a spectrum of clinical phenotypes. Genotype–phenotype correlations exist and can be used to guide medical decision making. Consideration should also be given to screening for RERE variants in individuals who fulfill diagnostic criteria for CHARGE syndrome but do not carry pathogenic variants in CHD7

Genotype–phenotype correlations in individuals with pathogenic RERE variants

Heterozygous variants in the arginine‐glutamic acid dipeptide repeats gene (RERE) have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). Here, we report nine individuals with NEDBEH who carry partial deletions or deleterious sequence variants in RERE. These variants were found to be de novo in all cases in which parental samples were available. An analysis of data from individuals with NEDBEH suggests that point mutations affecting the Atrophin‐1 domain of RERE are associated with an increased risk of structural eye defects, congenital heart defects, renal anomalies, and sensorineural hearing loss when compared with loss‐of‐function variants that are likely to lead to haploinsufficiency. A high percentage of RERE pathogenic variants affect a histidine‐rich region in the Atrophin‐1 domain. We have also identified a recurrent two‐amino‐acid duplication in this region that is associated with the development of a CHARGE syndrome‐like phenotype. We conclude that mutations affecting RERE result in a spectrum of clinical phenotypes. Genotype–phenotype correlations exist and can be used to guide medical decision making. Consideration should also be given to screening for RERE variants in individuals who fulfill diagnostic criteria for CHARGE syndrome but do not carry pathogenic variants in CHD7.We describe nine unrelated individuals who carry partial deletions or putatively deleterious sequence variants in RERE. An analysis of clinical and molecular data from individuals with mutations affecting RERE suggests the existence of novel genotype‐phenotype correlations and demonstrates that a high percentage of RERE pathogenic variants affect a histidine‐rich region in the Atrophin‐1 domain. We have also identified a recurrent two‐amino‐acid duplication in this region that is associated with the development of a CHARGE syndrome‐like phenotype.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143789/1/humu23400_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143789/2/humu23400.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143789/3/humu23400-sup-0001-SuppMat.pd

Investigation of Developmental Disorders: Genetic Discovery and Functional Validation

The genetic basis of Mendelian developmental disorders continues to be a significant gap in knowledge. Genetic discovery prompts and improves diagnostic yield, compelling the human genetics community to identify etiologies for all disorders to end great uncertainty accompanying a lack of genetic diagnosis. Disease presentations lacking clinical stratification emphasize the need for molecular diagnosis. CHARGE syndrome, a monogenic disorder due to variants in CHD7, shares great phenotypic overlap with other developmental disorders. The number of individuals with features of CHARGE that do not harbor CHD7 variants was indicative that variants in other genes may hold underlying etiology. Exome sequencing was applied to a cohort of individuals that exhibited clinical CHARGE features but lacked a molecular diagnosis. Analysis revealed that some individuals had pathogenic variants in genes previously associated with other Mendelian disorders. A notable fraction of the cohort did not have a pathogenic variant uncovered by exome sequencing, emphasizing investigation of novel genetic mechanisms may be required to solve remaining diagnostic odysseys. Rare disease also presents unique challenges for genetic discovery as few individuals globally will share the same clinical features. This is evidenced by the discovery of CIT in autosomal recessive primary microcephaly that is notable for a severe reduction in head circumference as well as brain growth. Human genetics findings impinge on mitotic machinery as an underlying determinant of brain size, and CIT suited as well as expanded this biological description for its novel role in cytokinesis. Individuals with loss-of-function truncating variants in CIT exhibit severe microlissencephaly, and post-mortem histological analysis demonstrates significant cytoarchitecture disorganization and the hallmark feature of cytokinesis failure – binucleation. CIT loss-of-function missense variants causing a reduction in kinase activity without altering expression are also a genetic basis for more moderate primary microcephaly, highlighting an unmet need to understand the pathogenic mechanisms leading to clear CIT phenotypic-genotypic correlations. Using human models of neurodevelopment, neuroepithelial architecture changes are uncovered for affected cerebral organoids with homozygous CIT frameshift and kinase missense variants. Time-lapse imaging identified a delay in late mitotic progression of organoids with CIT frameshift variants but fails to identify significant delays upon loss of kinase activity alone. This finding indicated a contributing mechanism to the spectrum of clinical severity associated with differing CIT variant types. Defects to cytokinetic kinetics in the absence of CIT kinase activity are not obvious, but a moderate amount of mitotic failure is observed and possibly related to acute changes in the phosphoproteome. The biological insight conferred from these genetic discovery and functional validation studies in human models will continue to empower our understanding of health and development, holding promise for a foreseeable future where all causes and underlying pathology of Mendelian disease are uncovered.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/174598/1/moccia_1.pd

Mutations in Citron Kinase Cause Recessive Microlissencephaly with Multinucleated Neurons

Primary microcephaly is a neurodevelopmental disorder that is caused by a reduction in brain size as a result of defects in the proliferation of neural progenitor cells during development. Mutations in genes encoding proteins that localize to the mitotic spindle and centrosomes have been implicated in the pathogenicity of primary microcephaly. In contrast, the contractile ring and midbody required for cytokinesis, the final stage of mitosis, have not previously been implicated by human genetics in the molecular mechanisms of this phenotype. Citron kinase (CIT) is a multi-domain protein that localizes to the cleavage furrow and midbody of mitotic cells, where it is required for the completion of cytokinesis. Rodent models of Cit deficiency highlighted the role of this gene in neurogenesis and microcephaly over a decade ago. Here, we identify recessively inherited pathogenic variants in CIT as the genetic basis of severe microcephaly and neonatal death. We present postmortem data showing that CIT is critical to building a normally sized human brain. Consistent with cytokinesis defects attributed to CIT, multinucleated neurons were observed throughout the cerebral cortex and cerebellum of an affected proband, expanding our understanding of mechanisms attributed to primary microcephaly

Ca2+ signaling in injured in situ endothelium of rat aorta

The inner wall of excised rat aorta was scraped by a microelectrode and Ca2+ signals were investigated by fluorescence microscopy in endothelial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i), followed by a long-lasting decay phase due to Ca2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P2X and P2Y receptors agonists and antagonists, such as suramin, alpha,beta-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La3+ and Ni2+, but not to Gd3+. Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area. These results suggest that endothelium scraping: (i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+