A de novo frameshift in HNRNPK causing a Kabuki-like syndrome with nodular heterotopia.

Kabuki syndrome is a heterogeneous condition characterized by distinctive facial features, intellectual disability, growth retardation, skeletal abnormalities and a range of organ malformations. Although at least two major causative genes have been identified, these do not explain all cases. Here we describe a patient with a complex Kabuki-like syndrome that included nodular heterotopia, in whom testing for several single-gene disorders had proved negative. Exome sequencing uncovered a de novo c.931_932insTT variant in HNRNPK (heterogeneous nuclear ribonucleoprotein K). Although this variant was identified in March 2012, its clinical relevance could only be confirmed following the August 2015 publication of two cases with HNRNPK mutations and an overlapping phenotype that included intellectual disability, distinctive facial dysmorphism and skeletal/connective tissue abnormalities. Whilst we had attempted (unsuccessfully) to identify additional cases through existing collaborators, the two published cases were 'matched' using GeneMatcher, a web-based tool for connecting researchers and clinicians working on identical genes. Our report therefore exemplifies the importance of such online tools in clinical genetics research and the benefits of periodically reviewing cases with variants of unproven significance. Our study also suggests that loss of function variants in HNRNPK should be considered as a molecular basis for patients with Kabuki-like syndrome.

Is lymphatic endoglin expression a risk marker for breast cancer metastasis? Results of a pilot study.

BACKGROUND: Studies have identified endoglin as a biological marker that is overexpressed on the microvessels of certain solid cancers (breast, colorectal cancer, and head and neck squamous cell cancers). There is, at present, no immunohistochemical marker that can discriminate between lymph node-negative and or lymph node-positive breast cancer tissue. METHODS: The expression of endoglin was quantified by immunohistochemistry and assessment of microvessel density in 53 surgical specimens. These were comprised of breast tumor tissue that had not spread to the regional lymph nodes (lymph node-negative breast tumor tissue: 20 specimens), breast tumor tissue had spread to regional lymph nodes (lymph node-positive breast tumor tissue: 21 specimens), and normal breast tissue as a control (12 specimens). RESULTS: Significant difference was observed between the expression of endoglin on microvessels of lymph node-negative and lymph node-positive breast cancer tissue (p<0.05). This significant difference was shown to be due to endoglin expression on lymphatic vessels (p<0.02), rather than on blood vessels (p>0.05). CONCLUSIONS: These findings are the first to suggest that endoglin expression on breast tumor lymphatic vessels may have diagnostic potential as a discriminator between lymph node-negative and lymph node-positive breast cancer. Further studies would be required to confirm this.

LYVE-1, the lymphatic system and tumor lymphangiogenesis.

Previous research into hyaluronan (HA) has focused on the role of this abundant tissue glycosaminoglycan in promoting cell migration through interactions with its transmembrane receptor CD44 on inflammatory leukocytes and tumor cells. The recent discovery of a new HA receptor, LYVE-1 (lymphatic vessel endothelial HA receptor), expressed predominantly in lymphatic vessels, highlights another aspect of HA biology: its continuous transit through the lymphatic system and its potential involvement in lymph node homing by CD44+ leukocytes and tumor cells. The functional role of LYVE-1 in lymphatic vessels and its application as a marker to study tumor lymphangiogenesis are important areas of investigation.

Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999) J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.

Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action.

Monocyte/macrophages play important roles in regulating tissue growth and angiogenesis through the controlled release of heparin-binding growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin binding epidermal growth factor. The action of these potent growth mediators is known to be regulated by adsorption to heparan sulfate proteoglycans (HSPGs) on the surface and within the extracellular matrix of other neighboring cells, which respectively promote or restrict interactions with their signal-transducing receptors on target cells. Here we report on the nature of HSPGs inducibly expressed on the surface of macrophages that confer these cells with the capacity to regulate endogenous growth factor activity. We reveal that activated human macrophages express only a single major 48-kDa cell surface HSPG, syndecan-2 (fibroglycan) as the result of de novo RNA and protein synthesis. In addition, we demonstrate this macrophage HSPG selectively binds the macrophage-derived growth factors FGF-2, vascular endothelial growth factor and heparin binding EGF and can present FGF-2 in a form that transactivates receptor-bearing BaF32 cells. These results define a novel and unique proteoglycan profile for macrophages and imply a key role for syndecan-2 in the delivery of sequestered growth factors by inflammatory macrophages for productive binding to their appropriate target cells in vivo.

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Isolation of multiple dimeric forms of phosphoribulokinase from an alga and a higher plant.

Dimeric phosphoribulokinase from either spinach (Spinacia oleracea) leaf or from the green alga, Scenedesmus obliquus can be separated into three distinct forms by hydrophobic interaction chromatography. Variation of the redox conditions prior to and during chromatography resulted in specific forms of phosphoribulokinase being eluted. It is suggested that three dimeric forms of phosphoribulokinase differ in the extent of disulfide bond formation between Cys-16 and Cys-55 in each of the two subunits. Phosphoribulokinase-3, isolated under the most oxidising conditions and exhibiting unusual kinetics, has properties consistent with those expected of an oxidised form of the enzyme in which Cys-16 and Cys-55 are completely oxidised to form a disulfide bond in each subunit. Phosphoribulokinase-1 is the completely reduced form predominating following incubation of extracts with dithiothreitol. Phosphoribulokinase-2, the intermediate species in which only one subunit possesses the disulfide, predominates only when extracts, previously reduced by high concentrations of 2-mercaptoethanol, are allowed to stand overnight in the presence of air prior to chromatography.

Properties of two high-molecular-mass forms of glyceraldehyde-3-phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity.

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-Mr glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G.R., Ortwerth, B.J. & Koeppe, O.J. (1970) J. Biol. Chem. 245, 4193-4198]. The other, a glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-Mr glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach. Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-Mr glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2). Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.

Multiple forms of O-methyltransferase involved in the microbial conversion of abietic acid into methyl abietate by Mycobacterium sp.

Six out of seven tested strains of mycobacteria transformed abietic acid to methyl abietate in shake culture. The conversion carried out by Mycobacterium sp. MB 3683 was induced by the substrate and stimulated by methionine. Fractionation of the cell extract of Mycobacterium sp. MB 3683 on DEAE cellulose, Ultrogel AcA 44 and MONO Q resulted in the separation of three distinct methyltransferase activities which could also esterify palmitic acid. The separated forms of the methyltransferase exhibited different activities towards these two substrates.