Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the ...contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.
Lysosomes orchestrate degradation and recycling of exogenous and endogenous material thus controlling cellular homeostasis. Little is known how this organelle changes during cancer. Here we ...investigate the intracellular landscape of lysosomes in a cellular model of bladder cancer. Employing standardized cell culture on micropatterns we identify a phenotype of peripheral lysosome positioning prevailing in bladder cancer cell lines but not normal urothelium. We show that lysosome positioning is controlled by phosphatidylinositol-3-phosphate (PtdIns3P) levels on endomembranes which recruit FYVE-domain containing proteins for lysosomal dispersion. We identify transcription factor EB (TFEB) as an upstream regulator of PtdIns3P production by VPS34 that is activated in aggressive bladder cancer cells with peripheral lysosomes. This conceptually clarifies the dual role of TFEB as regulator of endosomal maturation and autophagy, two distinct processes controlled by PtdIns3P. Altogether, our findings uncover peripheral lysosome positioning, resulting from PtdIns3P production downstream of TFEB activation, as a potential biomarker for bladder cancer.
Abstract
N
6
-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t
6
A) is a universal modification essential for translational accuracy and efficiency. The t
6
A pathway uses two ...sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in
YRDC
cause an extremely severe form of GAMOS whereas mutations in
GON7
, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose ...symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.
N
-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t
A) is a universal modification essential for translational accuracy and efficiency. The t
A pathway uses two sequentially acting ...enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.
Abstract Objective To monitor and quantify uteroplacental perfusion in rat pregnancies by Doppler ultrasound (DUS) and contrast-enhanced ultrasound (CEUS). Methods Fourteen rats were randomized in ...two groups (the CEUS group and the control group). On days 8, 11, 14, 17, 19 and 20 of gestation, we used DUS to measure the resistance index (RI), pulsatility index and blood velocity in the uterine, arcuate and umbilical arteries in both groups. On days 14, 17 and 20, one group was also examined by CEUS. Quantitative perfusion parameters were calculated in 4 compartments (mesometrial triangle, placenta, umbilical cord and fetus) and compared. Results The DUS measurement showed that the RI of the uterine and arcuate arteries decreased ( p < 0.01) from day 14 to day 17, while velocity increased each of these arteries ( p < 0.01 and p < 0.05, respectively). Quantification of uteroplacental perfusion by CEUS in bolus mode revealed that blood volume and local blood flow increased from day 14 to day 20 in the mesometrial triangle ( p < 0.01) and the placenta ( p < 0.05). In the CEUS destruction-replenishment mode, the perfusion parameters showed trends similar to those observed in bolus mode. No microbubbles were detected in the umbilical vein or fetal compartments. The weights of pups in the two groups did not differ significantly. Conclusions CEUS estimates of placental perfusion complement the data provided by DUS.
Background Orthostatic hypotension (OH) is highly prevalent in the elderly, and this population can be exposed to serious complications, including falls and cognitive disorders, as well as overall ...mortality. However, the pathophysiology of OH is still poorly understood, and innovative methods of cerebral blood flow (CBF) assessment have been required to accurately investigate cerebrovascular reactivity in OH. Objectives We want to compare brain tissue pulsatility (BTP) changes during an orthostatic challenge in elderly patients over 80 with and without OH. Materials and Methods Forty-two subjects aged 80 and over were recruited from the geriatric unit of the Hospital of Tours, France, and were divided into two groups according to the result of an orthostatic challenge. The noninclusion criteria were any general unstable medical condition incompatible with orthostatic challenge, having no temporal acoustic window, severe cognitive impairment (Mini Mental Status Examination <15), history of stroke, and legal guardianship. We used the novel and highly sensitive ultrasound technique of tissue pulsatility imaging to measure BTP changes in elderly patients with (n = 22) and without OH (n = 17) during an orthostatic challenge. Results We found that the mean brain tissue pulsatility related to global intracranial pulsatility, but not maximum brain tissue pulsatility related to large arteries pulsatility, decreased significantly in OH patients, with a delay compared with the immediate drop in peripheral blood pressure. Conclusion Global pulsatile CBF changes and small vessels pulsatility, rather than changes in only large arteries, may be key mechanisms in OH to account for the links between OH and cerebrovascular disorders.
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