Cosmc-dependent mucin-type O-linked glycosylation is essential for podocyte function

Mucin-type O-linked glycosylation, a posttranslational modification affecting the stability and biophysical characteristics of proteins, requires C1GalT1 (T synthase) and its obligate, X-linked chaperone Cosmc. Hypomorphic C1GalT1 mutations cause renal failure via not yet established mechanisms. We hypothesize that impaired Cosmc-dependent O-glycosylation in podocytes is sufficient to cause disease. Podocyte-specific Cosmc knockout mice were generated and phenotyped to test this hypothesis. Female heterozygous mice displaying mosaic inactivation of Cosmc in podocytes due to random X-linked inactivation were also examined. Mice with podocyte-specific Cosmc deletion develop profound albuminuria, foot process effacement, glomerular sclerosis, progressive renal failure, and impaired survival. Glomerular transcriptome analysis reveals early changes in cell adhesion, extracellular matrix organization, and chemokine-mediated signaling pathways, coupled with podocyte loss. Expression of the O-glycoprotein podoplanin was lost, while Tn antigen, representing immature O-glycans, was most abundantly found on podocalyxin. In contrast to hemizygous male and homozygous female animals, heterozygous female mosaic animals developed only mild albuminuria, focal foot process effacement, and nonprogressive kidney disease. Ultrastructurally, Cosmc-deficient podocytes formed Tn antigen-positive foot processes interdigitating with those of normal podocytes but not with other Cosmc-deficient cells. This suggests a cell nonautonomous mechanism for mucin-type O-glycoproteins in maintaining podocyte function. In summary, our findings demonstrated an essential and likely cell nonautonomous role for mucin-type O-glycosylation for podocyte function.

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

Nix confers heritable sex-conversion in Aedes aegypti and myo-sex is needed for male flight

A dominant and hemizygous male-determining locus (M locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix is a male-determining factor (M factor) in the M locus and its transient expression in females (m/m) results in partial masculinization. Here, we show that the Nix transgene alone was sufficient to convert females into fertile males, which continued to produce sex-converted progeny in subsequent generations. However, assisted mating with wild-type females was necessary, as the converted m/m males could not fly. Knockout of myo-sex, a myosin heavy chain gene and the only other protein-coding gene reported in the M locus, rendered wild-type males flightless. Thus, Nix alone converts female Ae. aegypti to fertile males and myo-sex is required for male flight. Only female Ae. aegypti mosquitoes bite and transmit disease-causing viruses. Nix-mediated female-to-male conversion is 100% penetrant and stable over many generations, indicating great potential for mosquito control.

PLS3 Mutations in X-Linked Osteoporosis: Clinical and Bone Characteristics of Two Novel Mutations

Background and Objectives: Plastin 3 (PLS3) mutations are associated with an X-linked osteoporosis. Here we describe two new families with novel mutations, including one with a whole gene PLS3 deletion, and review the literature on 9 previously reported cases. Results: Hemizygous male carriers presented with multiple peripheral bone fractures, low bone mineral density (BMD), and vertebral compression fractures. Heterozygous female carriers did not have a history of fragility fractures, although 1 individual presented with low BMD. Apart from greyish-tinged sclera, no other extraskeletal features of osteogenesis imperfecta were identified. Histomorphometry from a transiliac bone biopsy in one of our index patients demonstrated significantly low trabecular bone volume with increased bone turnover. Bisphosphonate treatment was associated with a reduction in the fracture rate and increased bone density. Conclusion: Hemizygous mutations in PLS3 may cause a monogenic form of X-linked osteoporosis presenting in childhood with a nonspecific phenotype. No characteristic ocular, dental, or joint abnormalities are defined. When genetic testing is undertaken to investigate for primary causes of bone fragility, we suggest PLS3 be included in order not to miss this diagnosis.

An unusual case of hereditary nephrogenic diabetes insipidus (HNDI) affecting mother and daughter

Hereditary nephrogenic diabetes iInsipidus (HNDI) is an uncommon disorder due to a resistance to anti-diuretic hormone leading to a reduced urinary concentrating ability. The X-linked form is fully expressed in hemizygous male patients, but diabetes insipidus may also present in heterozygous females where it must be distinguished from autosomal and other secondary causes. We report a mother and daughter in the same family with HNDI due to a heterozygous deletion in exon 1 of the

Splenectomy Favours Development of Extramedullary Hematopoiesis in the GATA-1low Model of Myelofibrosis.

Mice with megakaryocytic-targeted ablation of GATA-1 expression (GATA-1low mice) develop while age a syndrome similar to idiopathic myelofibrosis. These mice present with life-long thrombocytopenia and extensive proliferation of differentiation-impaired megakaryocytes. Although GATA-1 affects erythroid cell production, GATA-1low mice are not anemic because of increased erythropoiesis in the spleen. At 8–10-month, GATA-1low mice develop anemia, osteosclerosis, marrow and spleen fibrosis and neoangiogenesis. From 15-month, extensive extramedullary hematopoiesis in the liver occurs. Myelofibrosis is developed by hemizygous male and heterozygous females alike. In order to evaluate the role of the spleen in the development of extramedullary hematopoiesis, young (6-month) wild-type littermates (WT) and GATA-1low mice (both hemizygous males and heterozygous females) were splenectomized. Three- and 9–12 months later, the myelofibrotic trait expressed by the treated animals was compared with that of un-manipulated controls. Although all the mice survived surgery, a dramatic difference in mortality was observed between hemizygous males, which died of severe anemia within 2-weeks, and heterozygous females (no death recorded). Heterozygous GATA-1low females developed a modest anemia 9–12-months after surgery (Htc, 40.1±2.7% vs. 48.2±5.5 in splenectomized GATA-1low and WT littermates, respectively, p<0.01), comparable to that presented by the age-matched untreated mutants. Platelet counts increased 2-to-3-fold in both WT and GATA-1low mice, but remained significantly lower than normal in the latter. There was an increase in the number of CD16+ NK cells in the blood of splenectomized GATA-1low mice (287±61 vs. 61±19 CD16+ cells/mL in splenectomized GATA-1low and WT littermates, respectively), but no other abnormality in blood cell counts was observed. At 3 months post-splenectomy, the number of bone marrow cells in GATA-1low mice increased up to normal [from 4.0(±1.2)x106 to 8.0(±1.3)x106 cells/femur in untreated and splenectomized mutants, respectively]. Histologic evaluation of bone sections showed no overt change in the extent of marrow fibrosis compared to age-matched untouched mutants, while the extent of trabecular bone was reduced. No fibrosis developed in WT mice at any time point after splenectomy, nor were changes in total marrow cellularity observed. There was no sign of extramedullary hematopoiesis at 3 month post-splenectomy in either WT or GATA-1low mice, while at the later time point, mutant mice showed a prominent involvement of the liver with high numbers of megakaryocytes, isolated or in clusters, and maturing cells of all hematopoietic lineages, that in extreme cases diffusely infiltrated the parenchyma. Extramedullary hematopoiesis was not found in either the lung or the kidney. In conclusions, splenectomy affected the extent but not the timing of the development of extramedullary hematopoiesis in GATA-1low mice suggesting that the number of stem cells present in the marrow of old splenectomized animals was sufficient to colonize the liver. The observation that splenectomy per se does not result in extramedullary hematopoiesis support the hypothesis that it is not fibrosis of the spleen, but a second hit within the stem cell compartment, that is possibly responsible for the development of extramedullary hematopoiesis in this mouse model of myelofibrosis.

Sox3 Is Required for Gonadal Function, but Not Sex Determination, in Males and Females

ABSTRACT Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (−/−) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (−/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.