Ellis-van Creveld Syndrome in Grey Alpine Cattle: Morphologic, Immunophenotypic, and Molecular Characterization

Ellis-van Creveld (EvC) syndrome is a human autosomal recessive disorder caused by a mutation in either the EVC or EVC2 gene, and presents with short limbs, polydactyly, and ectodermal and heart defects. The aim of this study was to understand the pathologic basis by which deletions in the EVC2 gene lead to chondrodysplastic dwarfism and to describe the morphologic, immunohistochemical, and molecular hallmarks of EvC syndrome in cattle. Five Grey Alpine calves, with a known mutation in the EVC2 gene, were autopsied. Immunohistochemistry was performed on bone using antibodies to collagen II, collagen X, sonic hedgehog, fibroblast growth factor 2, and Ki67. Reverse transcription polymerase chain reaction was performed to analyze EVC1 and EVC2 gene expression. Autopsy revealed long bones that were severely reduced in length, as well as genital and heart defects. Collagen II was detected in control calves in the resting, proliferative, and hypertrophic zones and in the primary and secondary spongiosa, with a loss of labeling in the resting zone of 2 dwarfs. Collagen X was expressed in hypertrophic zone in the controls but was absent in the EvC cases. In affected calves and controls, sonic hedgehog labeled hypertrophic chondrocytes and primary and secondary spongiosa similarly. FGF2 was expressed in chondrocytes of all growth plate zones in the control calves but was lost in most EvC cases. The Ki67 index was lower in cases compared with controls. EVC and EVC2 transcripts were detected. Our data suggest that EvC syndrome of Grey Alpine cattle is a disorder of chondrocyte differentiation, with accelerated differentiation and premature hypertrophy of chondrocytes, and could be a spontaneous model for the equivalent human disease.

The Challenge of Prenatal Diagnostic Work-Up of Maternally Inherited X-Linked Opitz G/BBB: Case Report and Literature Review

Background. Prenatal diagnosis of Optiz G/BBB syndrome (OS) is challenging because the characteristic clinical features, such as facial and genitourinary anomalies, may be subtle at sonography and rather unspecific. Furthermore, molecular testing of the disease gene is not routinely performed, unless a specific diagnosis is suggested. Method. Both familial and ultrasound data were used to achieve the diagnosis of X-linked OS (XLOS), which was confirmed by molecular testing of MID1 gene (Xp22.3) at birth. Results. Sequencing of MID1 gene disclosed the nucleotide change c.1285 +1 G>T, previously associated with XLOS. Conclusions. This case illustrates current challenges of the prenatal diagnostic work-up of XLOS and exemplifies how clinical investigation, including family history, and accurate US foetal investigations can lead to the correct diagnosis.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Increased levels of plasma lysosomal enzymes in patients with Lowe syndrome

Lowe syndrome is an X-linked disorder that has a complex phenotype that includes progressive renal failure and blindness. The disease is caused by mutations in an inositol polyphosphate 5-phosphatase designated OCRL. It has been shown that the OCRL protein is found on the surface of lysosomes and that a renal tubular cell line deficient in OCRL accumulated substrate phosphatidylinositol 4,5-bisphosphate. Because this lipid is required for vesicle trafficking from lysosomes, we postulate that there is a defect in lysosomal enzyme trafficking in patients with Lowe syndrome that leads to increased extracellular lysosomal enzymes and might lead to tissue damage and contribute to the pathogenesis of the disease. We have measured seven lysosomal enzymes in the plasma of 15 patients with Lowe syndrome and 15 age-matched male controls. We find a 1.6- to 2.0-fold increase in all of the enzymes measured. When the data was analyzed by quintiles of activity for all of the enzymes, we found that 95% of values in the lowest quintile come from normal subjects whereas in the highest quintile 85% of the values are from patients with Lowe syndrome. The increased enzyme levels are not attributable to renal insufficiency because there was no difference in enzyme activity in the four patients with the highest creatinine levels compared with the six patients with the lowest creatinine values.

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

The protein deficient in Lowe syndrome is a phosphatidylinositol-4,5-bisphosphate 5-phosphatase.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene. The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets over a span of 744 aa, suggesting that OCRL may be a similar enzyme. We engineered a construct of the OCRL cDNA that encodes amino acids homologous to the platelet 5-phosphatase for expression in baculovirus-infected Sf9 insect cells. This cDNA encodes aa 264-968 of the OCRL protein. The recombinant protein was found to catalyze the reactions also carried out by platelet 5-phosphatase II. Thus OCRL converts inositol 1,4,5-trisphosphate to inositol 1,4-bisphosphate, and it converts inositol 1,3,4,5-tetrakisphosphate to inositol 1,3,4-trisphosphate. Most important, the enzyme converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The relative ability of OCRL to catalyze the three reactions is different from that of 5-phosphatase II and from that of another 5-phosphatase isoenzyme from platelets, 5-phosphatase I. The recombinant OCRL protein hydrolyzes the phospholipid substrate 10- to 30-fold better than 5-phosphatase II, and 5-phosphatase I does not cleave the lipid at all. We also show that OCRL functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase in OCRL-expressing Sf9 cells. These results suggest that OCRL is mainly a lipid phosphatase that may control cellular levels of a critical metabolite, phosphatidylinositol 4,5-bisphosphate. Deficiency of this enzyme apparently causes the protean manifestations of Lowe syndrome.

Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The disease gene has been localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The function of the encoded protein remains undetermined. In this study we have characterized mutations in 12 unrelated patients to confirm the identity of the disease gene. We have also revised the coding sequence and genomic structure for the WAS gene. To analyze further the transmittance of the disease gene, we have characterized a polymorphic microsatellite at the DXS6940 locus within 30 kb of the gene and demonstrate the inheritance of the affected alleles in families with a history of WAS.

On dendrites in Down syndrome and DS murine models: a spiny way to learn

Since the discovery in the 1970s that dendritic abnormalities in cortical pyramidal neurons are the most consistent pathologic correlate of mental retardation, research has focused on how dendritic alterations are related to reduced intellectual ability. Due in part to obvious ethical problems and in part to the lack of fruitful methods to study neuronal circuitry in the human cortex, there is little data about the microanatomical contribution to mental retardation. The recent identification of the genetic bases of some mental retardation associated alterations, coupled with the technology to create transgenic animal models and the introduction of powerful sophisticated tools in the field of microanatomy, has led to a growth in the studies of the alterations of pyramidal cell morphology in these disorders. Studies of individuals with Down syndrome, the most frequent genetic disorder leading to mental retardation, allow the analysis of the relationships between cognition, genotype and brain microanatomy. In Down syndrome the crucial question is to define the mechanisms by which an excess of normal gene products, in interaction with the environment, directs and constrains neural maturation, and how this abnormal development translates into cognition and behaviour. In the present article we discuss mainly Down syndrome-associated dendritic abnormalities and plasticity and the role of animal models in these studies. We believe that through the further development of such approaches, the study of the microanatomical substrates of mental retardation will contribute significantly to our understanding of the mechanisms underlying human brain disorders associated with mental retardation. (C) 2004 Elsevier Ltd. All rights reserved.

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus ( WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultrathin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.

Cognitive-behavioural phenotype in a group of girls from 1.2 to 12 years old with the Incontinentia Pigmenti syndrome:recommendations for clinical management

Incontinentia Pigmenti (IP, OMIM#308300) is a rare X-linked genomic disorder (about 1,400 cases) that affects the neuroectodermal tissue and Central Nervous System (CNS). The objective of this study was to describe the cognitive-behavioural profile in children in order to plan a clinical intervention to improve their quality of life. A total of 14 girls (age range: from 1 year and 2 months to 12 years and 10 months) with IP and the IKBKG/NEMO gene deletion were submitted to a cognitive assessment including intelligence scales, language and visuo-spatial competence tests, learning ability tests, and a behavioural assessment. Five girls had severe to mild intellectual deficiencies and the remaining nine had a normal neurodevelopment. Four girls were of school age and two of these showed no intellectual disability, but had specific disabilities in calculation and arithmetic reasoning. This is the first description of the cognitive-behavioural profile in relation to developmental age. We stress the importance of an early assessment of learning abilities in individuals with IP without intellectual deficiencies to prevent the onset of any such deficit.

A case of bilateral perisylvian syndrome with reading disability

Bilateral Perisylvian Syndrome (BPS) often presents with epilepsy and significant behavioral impairments that can include mental retardation, dysarthria, delayed speech development, and delayed fine motor development (Graff-Radford et al., 1986 and Kuzniecky et al., 1993). While a small subset of BPS cases have been described as having relatively isolated language delays (Leventer et al., 2010), BPS is not expected in children with dyslexia. As part of a Medical University of South Carolina, IRB approved multi-site study involving retrospective and de-identified dyslexia data, we unexpectedly identified a 14.05 year old male with evidence of BPS whose father had been diagnosed with dyslexia and dysgraphia. This child had been recruited for a neuroimaging study on dyslexia from a school specializing in educating children with dyslexia. The T1-weighted MRI scan from this child demonstrated a highly unusual perisylvian sulcal/gyral patterning that is a defining feature of BPS (Fig. 1). BPS cases exhibit bilateral dysgenesis of the Sylvian fissure and surrounding gyri, which appears to occur because of a limited or absent arcuate fasciculus (Kilinc, Ekinci, Demirkol, & Agan, 2015). This BPS case also had a relatively enlarged atrium of the lateral ventricle that is consistent with the BPS anatomical presentation and reduction of parietal white matter (Graff-Radford et al., 1986, Kilinc et al., 2015 and Toldo et al., 2011).

Mutation of SLC35D3 causes metabolic syndrome by impairing dopamine signaling in striatal D1 neurons

Funding: This work was partially supported by grants from National Basic Research Program of China (2013CB530605; 2014CB942803), from National Natural Science Foundation of China 1230046; 31071252; 81101182) and from Chinese Academy of Sciences (KSCX2-EW-R-05, KJZD-EW-L08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.