56 research outputs found
Clinical genetic diagnostics in Danish autosomal dominant polycystic kidney disease patients reveal possible founder variants
Functional megalin is expressed in renal cysts in a mouse model of adult polycystic kidney disease
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive growth of cysts and a decline of renal function. The clinical feasibility of the number of potential disease-modifying drugs is limited by systemic adverse effects. We hypothesize that megalin, a multiligand endocytic receptor expressed in the proximal tubule, may be used to facilitate drug uptake into cysts, thereby allowing for greater efficacy and fewer side effects. METHODS: The cyst expression of various tubular markers, including megalin and aquaporin 2 (AQP2), was analysed by immunohistochemistry (IHC) of kidney sections from the ADPKD mouse model (PKD1(RC/RC)) at different post-natal ages. The endocytic function of megalin in cysts was examined by IHC of kidney tissue from mice injected with the megalin ligand aprotinin. RESULTS: Cyst lining epithelial cells expressing megalin were observed at all ages; however, the proportion decreased with age. Concomitantly, an increasing proportion of cysts revealed expression of AQP2, partial expression of megalin and/or AQP2 or no expression of the examined markers. Endocytic uptake of aprotinin was evident in megalin-positive cysts, but only in those that remained connected to the renal tubular system. CONCLUSIONS: Megalin-expressing cysts were observed at all ages, but the proportion decreased with age, possibly due to a switch in tubular origin, a merging of cysts of different tubular origin and/or a change in the expression pattern of cyst lining cells. Megalin expressed in cysts was functional, suggesting that megalin-mediated endocytosis is a potential mechanism for drug targeting in ADPKD if initiated early in the disease
Mosaicism for copy number variations in the placenta is even more difficult to interpret than mosaicism for whole chromosome aneuploidy
Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders:potentials and pitfalls
Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.</p
Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls
Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance.Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing.Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively.Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders’ mode of inheritance
11.Hypoxic ventilatory depressionと思われる一症例について(第551回千葉医学会例会・第9回麻酔科例会・第18回千葉麻酔懇話会)
FISH analysis on metaphase nuclei (top panel) of cultured cells derived from peripheral blood leukocytes of the proband of family 2 by using BAC probes for 11p15.5-15.4 (RP11-11A9, 3,236,552-3,356,012, green) and 11q22.3 (RP11-179B7, 104,298,339-104,459,797, red). The green signal on both homologues is visible only at chr11p, demonstrating the presence of an in cis duplication and excluding an unbalanced translocation. FISH analysis on interphase nuclei (bottom panel) using the BACs RP11-699D10 (2.9â3.0Â Mb, red) and RP11-11A9 (green), hybridizing within the duplication. Note that single and duplicated signals can be seen on the two homologues, respectively. The red-green-green-red order of the duplicated signals indicates that the duplication is inverted. (PDF 52Â kb
Comparison of Recombinant Human Haptocorrin Expressed in Human Embryonic Kidney Cells and Native Haptocorrin
Haptocorrin (HC) is a circulating corrinoid binding protein with unclear function. In contrast to transcobalamin, the other transport protein in blood, HC is heavily glycosylated and binds a variety of cobalamin (Cbl) analogues. HC is present not only in blood but also in various secretions like milk, tears and saliva. No recombinant form of HC has been described so far. We report the expression of recombinant human HC (rhHC) in human embryonic kidney cells. We purified the protein with a yield of 6 mg (90 nmol) per litre of cell culture supernatant. The isolated rhHC behaved as native HC concerning its spectral properties and ability to recognize both Cbl and its baseless analogue cobinamide. Similar to native HC isolated from blood, rhHC bound to the asialoglycoprotein receptor only after removal of terminal sialic acid residues by treatment with neuraminidase. Interestingly, rhHC, that compared to native HC contains four excessive amino acids (…LVPR) at the C-terminus, showed subtle changes in the binding kinetics of Cbl, cobinamide and the fluorescent Cbl conjugate CBC. The recombinant protein has properties very similar to native HC and although showing slightly different ligand binding kinetics, rhHC is valuable for further biochemical and structural studies
Mouse Transcobalamin Has Features Resembling both Human Transcobalamin and Haptocorrin
In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species
Functional in vivo analysis of Arabidopsis chromosomal high mobility group proteins of the A and B type
Maximal load of the vitamin B12 transport system: a study on mice treated for four weeks with high-dose vitamin B12 or cobinamide.
Several studies suggest that the vitamin B12 (B12) transport system can be used for the cellular delivery of B12-conjugated drugs, also in long-term treatment Whether this strategy will affect the endogenous metabolism of B12 is not known. To study the effect of treatment with excess B12 or an inert derivative, we established a mouse model using implanted osmotic minipumps to deliver saline, cobinamide (Cbi) (4.25 nmol/h), or B12 (1.75 nmol/h) for 27 days (n = 7 in each group). B12 content and markers of B12 metabolism were analysed in plasma, urine, kidney, liver, and salivary glands. Both Cbi and B12 treatment saturated the transcobalamin protein in mouse plasma. Cbi decreased the content of B12 in tissues to 33-50% of the level in control animals but did not influence any of the markers examined. B12 treatment increased the tissue B12 level up to 350%. In addition, the transcript levels for methylenetetrahydrofolate reductase in kidneys and for transcobalamin and transcobalamin receptor in the salivary glands were reduced. Our study confirms the feasibility of delivering drugs through the B12 transport system but emphasises that B12 status should be monitored because there is a risk of decreasing the transport of endogenous B12. This risk may lead to B12 deficiency during prolonged treatment
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