heparitin-sulfate and Abnormalities--Multiple

Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.

Topics: Abnormalities, Multiple; Arrhythmias, Cardiac; Genetic Diseases, X-Linked; Gigantism; Glypicans; Heart Defects, Congenital; Hedgehog Proteins; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Intellectual Disability; Signal Transduction

Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 μmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.

Topics: Abnormalities, Multiple; Catalytic Domain; Crystallography, X-Ray; Dermatan Sulfate; Enzyme Replacement Therapy; Gene Expression; Heparitin Sulfate; Humans; Iduronidase; Infant; Male; Molecular Dynamics Simulation; Mucopolysaccharidosis I; Point Mutation; Principal Component Analysis; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Substrate Specificity

Glypicans are a family of heparan sulfate proteoglycans whose members are bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Loss-of-function mutations in GPC3, one of the six mammalian glypicans, causes the Simson-Golabi-Behmel Syndrome. This is a disorder characterized by pre- and post-natal overgrowth, a broad spectrum of visceral and skeletal abnormalities, and an increased risk for the development of embryonic tumors. GPC3-null mice also display significant overgrowth. We have recently reported that GPC3 acts as a negative regulator of Hedgehog signaling during development, and that the overgrowth caused by the lack of functional GPC3 is due, at least in part, to the hyperactivation of Hedgehog signaling. Here we discuss the rationale that led us to hypothesize that GPC3 could be a negative regulator of Hedgehog signaling, and speculate about the implications of our discovery regarding the role of GPC3 in some cancer types. We also discuss our recent results of experiments that investigated the role of the core protein, the heparan sulfate chains, and the GPI anchor in GPC3 function. Finally, we propose an explanation for the tissue-specific function of GPC3.

Topics: Abnormalities, Multiple; Animals; Body Size; Glycosylphosphatidylinositols; Glypicans; Hedgehog Proteins; Heparitin Sulfate; Mice; Neoplasms; Signal Transduction; Syndrome; Wnt Proteins

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Topics: Abnormalities, Multiple; Animals; Beckwith-Wiedemann Syndrome; Body Weight; Cell Division; Female; Genotype; Glypicans; Growth Disorders; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Insulin-Like Growth Factor II; Kidney Tubules, Collecting; Male; Mandible; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Phenotype; Proteoglycans; Syndrome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome with associated visceral and skeletal abnormalities. Alterations in the glypican-3 gene (GPC3), which is located on Xq26, have been implicated in the etiology of relatively milder cases of this disorder. Not all individuals with SGBS have demonstrated disruptions of the GPC3 locus, which raises the possibility that other loci on the X chromosome could be responsible for some cases of this syndrome. We have previously described a large family with a severe form of SGBS that is characterized by multiple anomalies, hydrops fetalis, and death within the first 8 wk of life. Using 25 simple tandem-repeat polymorphism markers spanning the X chromosome, we have localized the gene for this disorder to an approximately 6-Mb region of Xp22, with a maximum LOD score of 3.31 and with LOD scores <-2.0 for all of Xq. These results demonstrate that neither the GPC3 gene nor other genes on Xq26 are responsible for all cases of SGBS and that a second SGBS locus resides on Xp22.

Topics: Abnormalities, Multiple; Chromosome Mapping; Female; Genetic Variation; Genotype; Glypicans; Haplotypes; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Hydrops Fetalis; Lod Score; Male; Pedigree; Polymorphism, Genetic; Proteoglycans; Recombination, Genetic; Syndrome; Tandem Repeat Sequences; X Chromosome

Simpson-Golabi-Behmel syndrome (SGBS) is one of the overgrowth syndromes. Microdeletions of the glypican-3 (GPC3) gene were described by Pilia et al. (1996). Glypican-3 encodes a putative extracellular proteoglycan which is expressed in embryonic mesodermal tissues and plays an important role in embryonal growth. We report a Japanese patient with SGBS who had a single base deletion in the exon 7 of the GPC3 gene. This is the first report of a single base deletion of the GPC3 gene.

Topics: Abnormalities, Multiple; Amino Acid Sequence; Base Sequence; Exons; Glypicans; Growth Disorders; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hernia, Diaphragmatic; Humans; Infant, Newborn; Male; Proteoglycans; Radiography; Sequence Deletion; Syndrome

Perlecan, a large, multi-domain, heparan sulfate proteoglycan originally identified in basement membrane, interacts with extracellular matrix proteins, growth factors and receptors, and influences cellular signalling. Perlecan is present in a variety of basement membranes and in other extracellular matrix structures. We have disrupted the gene encoding perlecan (Hspg2) in mice. Approximately 40% of Hspg2-/- mice died at embryonic day (E) 10.5 with defective cephalic development. The remaining Hspg2-/- mice died just after birth with skeletal dysplasia characterized by micromelia with broad and bowed long bones, narrow thorax and craniofacial abnormalities. Only 6% of Hspg2-/- mice developed both exencephaly and chondrodysplasia. Hspg2-/- cartilage showed severe disorganization of the columnar structures of chondrocytes and defective endochondral ossification. Hspg2-/- cartilage matrix contained reduced and disorganized collagen fibrils and glycosaminoglycans, suggesting that perlecan has an important role in matrix structure. In Hspg2-/- cartilage, proliferation of chondrocytes was reduced and the prehypertrophic zone was diminished. The abnormal phenotypes of the Hspg2-/- skeleton are similar to those of thanatophoric dysplasia (TD) type I, which is caused by activating mutations in FGFR3 (refs 7, 8, 9), and to those of Fgfr3 gain-of-function mice. Our findings suggest that these molecules affect similar signalling pathways.

Topics: Abnormalities, Multiple; Animals; Animals, Newborn; Cartilage; Cartilage Oligomeric Matrix Protein; Cell Differentiation; Cell Division; Chondrocytes; Extracellular Matrix Proteins; Gene Deletion; Gene Expression; Glycoproteins; Growth Plate; Head; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Matrilin Proteins; Mice; Mice, Transgenic; Mutagenesis, Insertional; Protein-Tyrosine Kinases; Proteoglycans; Receptor, Fibroblast Growth Factor, Type 3; Receptors, Fibroblast Growth Factor; RNA, Messenger; Thanatophoric Dysplasia

Topics: Abnormalities, Multiple; Animals; Glypicans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Insulin-Like Growth Factor II; Mice; Mice, Knockout; Proteoglycans; Somatomedins; Syndrome

Interest in glypican-3 (GPC3), a member of the glypican-related integral membrane heparan sulfate proteoglycans (GRIPS) family, has increased with the finding that it is mutated in the Simpson-Golabi-Behmel overgrowth syndrome (Pilia et al. [1996] Nat. Genet. 12:241-247). The working model suggested that the membrane-bound protein acts locally to limit tissue and organ growth and that it may function by interacting with insulin-like growth factor 2 (IGF2) to limit its local effective level. Here we have tested two predictions of the model. In situ hybridization with the mouse gene cDNA was used to study the expression pattern during embryonic and fetal development. In agreement with predictions, the gene is expressed in precisely the organs that overgrow in its absence; and the patterns of expression of Gpc3 and those reported for Igf2 are strictly correlated.

Topics: Abnormalities, Multiple; Animals; Blotting, Northern; Ectoderm; Gene Expression Regulation, Developmental; Gigantism; Glypicans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; In Situ Hybridization; Mesoderm; Mice; Phenotype; Proteoglycans; Syndrome

Recently, we have shown that mutations in the X-linked glypican 3 (GPC3) gene cause the Simpson-Golabi-Behmel overgrowth syndrome (SGBS; ). The next centromeric gene detected is another glypican, glypican 4 (GPC4), with its 5' end 120763bp downstream of the 3' terminus of GPC3. One recovered GPC4 cDNA with an open reading frame of 1668nt encodes a putative protein containing three heparan sulfate glycosylation signals and the 14 signature cysteines of the glypican family. This protein is 94.3% identical to mouse GPC4 and 26% identical to human GPC3. In contrast to GPC3, which produces a single transcript of 2.3kb and is stringently restricted in expression to predominantly mesoderm-derived tissues, Northern analyses show that GPC4 produces two transcripts, 3.4 and 4.6kb, which are very widely expressed (though at a much higher level in fetal lung and kidney). Interestingly, of 20 SGBS patients who showed deletions in GPC3, one was also deleted for part of GPC4. Thus, GPC4 is not required for human viability, even in the absence of GPC3. This patient shows a complex phenotype, including the unusual feature of hydrocephalus; but because an uncle with SGBS is less affected, it remains unclear whether the GPC4 deletion itself contributes to the phenotype.

Topics: Abnormalities, Multiple; Amino Acid Sequence; Animals; Base Sequence; Centromere; DNA; Gene Expression Regulation, Developmental; Glypicans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Mice; Molecular Sequence Data; Physical Chromosome Mapping; Proteoglycans; RNA, Messenger; Sequence Alignment; Sequence Deletion; Sequence Homology, Amino Acid; Tissue Distribution; Transcription, Genetic; Tumor Cells, Cultured; X Chromosome

To identify the proportion and type of deletions present in the glypican 3 (GPC3) gene in a group of patients with Simpson-Golabi-Behmel syndrome (SGBS).. PCR analysis using primer pairs which amplify fragments from each of the eight exons of the GPC3 gene was carried out in a series of 18 families with SGBS (approximately half of reported cases).. Deletions were detected in only five families (one reported previously). We found deletions in all exons of the gene except exon 3.. Our results suggest that large scale deletions may be less common in SGBS than was originally thought. One patient, with an exon 4 and 5 deletion, lacked the characteristic facial dysmorphic features. This raises the possibility of involvement of GPC3 gene defects in a wider range of overgrowth disorders.

Topics: Abnormalities, Multiple; Child; Exons; Face; Genetic Linkage; Glypicans; Growth Disorders; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Male; Phenotype; Polymerase Chain Reaction; Proteoglycans; Sequence Deletion; Syndrome; X Chromosome

We present the case of a male infant, born prematurely (at 33 weeks gestation) with macrosomia, disproportionate macrocephaly, facial dysmorphism, short penis and a small umbilical defect. He had a large ASD and was ventilated from birth for respiratory distress syndrome. He died at 12 hours of age despite neonatal ITU care. Post-mortem examination showed highly lobulated kidneys with nodules of blastema and foci of hamartomatous change in the medulla. Prominence of pancreatic islet cells and expansion of hepatic portal tracts were also noted. His mother has minor cervical spine abnormalities. We discuss the differential diagnosis and the difficulty in confidently assigning a diagnosis to this patient, as considerable overlap is becoming evident between Simpson-Golabi-Behmel syndrome and Perlman syndrome.

Topics: Abnormalities, Multiple; Beckwith-Wiedemann Syndrome; Chromosomes, Human, Pair 11; Face; Female; Glypicans; Growth Disorders; Heart Septal Defects, Atrial; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Infant, Newborn; Kidney; Liver; Male; Pancreas; Pregnancy; Proteoglycans; Syndrome

Topics: Abnormalities, Multiple; Animals; Beckwith-Wiedemann Syndrome; Gigantism; Growth Disorders; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Insulin-Like Growth Factor II; Proteoglycans; Receptor, IGF Type 2; Syndrome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues in which it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action.

Topics: Abnormalities, Multiple; Amino Acid Sequence; Animals; Base Sequence; Cell Line; Chromosome Aberrations; Chromosome Disorders; Chromosome Mapping; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 16; Cloning, Molecular; DNA Primers; Female; Gene Deletion; Genetic Linkage; Glypicans; Growth Disorders; HeLa Cells; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Immunologic Techniques; Insulin-Like Growth Factor II; Male; Mice; Molecular Sequence Data; Pedigree; Protein Binding; Proteoglycans; Sequence Homology, Amino Acid; Syndrome; Translocation, Genetic; Tumor Cells, Cultured; X Chromosome

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth disorder recently shown to be caused by mutations in the heparan sulfate proteoglycan GPC3 [Pilia et al., Nat Genet; 12:241-247 1996]. We have used Southern blot analysis and polymerase chain reaction amplification of intra-exonic sequences to identify four new GPC3 mutations and further characterize three previously reported SGBS mutations. De novo GPC3 mutations were identified in 2 families. In general, the mutations were unique deletions ranging from less than 0.1 kb to more than 300 kb in length with no evidence of a mutational hot spot discerned. The lack of correlation between the phenotype of 18 affected males from these 7 families and the location and size of the GPC3 gene mutations suggest that SGBS is caused by a nonfunctional GPC3 protein.

Topics: Abnormalities, Multiple; Autoradiography; Blotting, Southern; Chromosome Deletion; DNA Probes; Genotype; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Male; Mutation; Pedigree; Phenotype; Polymerase Chain Reaction; Proteoglycans; X Chromosome

Topics: Abnormalities, Multiple; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, 4-5; Dermatoglyphics; Glycosaminoglycans; Heparitin Sulfate; Humans; Infant, Newborn; Male

This male infant was first brought to attention in the neonatal period because he presented clinical and radiological evidence of multiple bone deformities. He was readmitted at 21/2 months for hydrocephaly, hepatosplenomegaly and poor somatic and psychomotor development. In addition, coarse facies, corneal opacities and stiff joints were noticed. Bone X-ray anomalies and vacuolized lymphocytes supported the clinical presumption of lysosomal storage disorder. The diagnosis of multiple sulphatase deficiency rests on the presence of MPS and sulphatides in the urine, the finding of a mixed storage process in conjunctival biopsy and the demonstration of deficiencies in arylsulphatases A, B, C, iduronate sulphatase and heparan sulphatase in serum, leukocytes and cultured fibroblasts.

Topics: Abnormalities, Multiple; Arylsulfatases; Bone and Bones; Cerebroside-Sulfatase; Chondro-4-Sulfatase; Heparitin Sulfate; Humans; Infant; Male; Mucopolysaccharidosis II; Steryl-Sulfatase; Sulfatases

Clinical heterogeneity for Sanfilippo B syndrome (MPS III B) in the same family has never been reported previously. We describe two clinically severe cases and one clinically mild case of MPS III B in a Neapolitan sibship. We could not detect N-acetyl-alpha-D-glucosaminidase activity in the sera of either the severe or mild cases. Mucopolysacchariduria mainly due to heparan sulfate excretion was consistently high in the severely affected patients and extremely variable in the mildly affected one.

Topics: Abnormalities, Multiple; Adult; Child; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Intellectual Disability; Male; Mucopolysaccharidoses; Mucopolysaccharidosis III