GPC6, a Novel Member of the Glypican Gene Family, Encodes a Product Structurally Related to GPC4 and Is Colocalized withGPC5on Human Chromosome 13

Glypicans are a family of cell surface heparan sulfate proteoglycans that appear to play an important role in cellular growth control and differentiation, as is supported by the observation that mutations in GPC3 are responsible for Simpson-Golabi-Behmel syndrome (SGBS) in humans. Recently it has been shown that the GPC4 gene is tightly clustered with GPC3 on the X chromosome and that some patients with SGBS apparently have deletions affecting both genes. We report here the identification of a human cDNA encoding a novel glypican family member, glypican-6. This cDNA encodes a predicted protein of 554 amino acids and is structurally analogous to other members of the glypican gene family, but most highly related to glypican-4. A single GPC6 mRNA of 6.2 kb is detected most abundantly in the ovary, liver, and kidney, with lower levels of mRNA expression also detected in a wide range of other adult tissues. Radiation hybrid analysis mapped the GPC6 gene to human chromosome 13 very near the GPC5 gene, a member of the glypican family bearing strong similarity to GPC3.     

Glypican-6, a new member of the glypican family of cell surface heparan sulfate proteoglycans.

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, we identified a sixth member of the glypican family. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (identity of 63%). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily comprising so far glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might in part be regulated by the presence of glypican-6 on the cell surface.     

The role of glypicans in Hedgehog signaling

Glypicans (GPCs) are a family of proteoglycans that are bound to the cell surface by a glycosylphosphatidylinositol anchor. Six glypicans have been found in the mammalian genome (GPC1 to GPC6). GPCs regulate several signaling pathways, including the pathway triggered by Hedgehogs (Hhs). This regulation, which could be stimulatory or inhibitory, occurs at the signal reception level. In addition, GPCs have been shown to be involved in the formation of Hh gradients in the imaginal wing disks in Drosophila. In this review we will discuss the role of various glypicans in specific developmental events in the embryo that are regulated by Hh signaling. In addition, we will discuss the mechanism by which loss-of-function GPC3 mutations alter Hh signaling in the Simpson–Golabi–Behmel overgrowth syndrome, and the molecular basis of the GPC5-induced stimulation of Hh signaling and tumor progression in rhabdomyosarcomas.     

Boning up on glypicans—opportunities for new insights into bone biology

Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless‐integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway‐specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein–protein and protein–carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson–Golabi–Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth. Copyright © 2013 John Wiley & Sons, Ltd.     

Rational design of chimeric antigen receptor T cells against glypican 3 decouples toxicity from therapeutic efficacy

BackgroundChimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.MethodsIn selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.ResultsWhere the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non–tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.ConclusionsBy combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.     

Glypican-5 is a tumor suppressor in non-small cell lung cancer cells

Glypican-5 (GPC5) belongs to the glypican family of proteoglycans that have been implicated in a variety of physiological processes, ranging from cell proliferation to morphogenesis. However, the role of GPC5 in human cancer remains poorly understood. We report that knockdown of GPC5 in bronchial epithelial cells promoted, and forced expression of GPC5 in non-small lung cancer (NSCLC) cells suppressed, the anchorage-independent cell growth. In vivo, expression of GPC5 inhibited xenograft tumor growth of NSCLC cells. Furthermore, we found that GPC5 was expressed predominantly as a membrane protein, and its expression led to diminished phosphorylation of several oncogenic receptor tyrosine kinases, including the ERBB family members ERBB2 and ERBB3, which play critical roles in lung tumorigenesis. Collectively, our results suggest that GPC5 may act as a tumor suppressor, and reagents that activate GPC5 may be useful for treating NSCLC.     

OCI-5/GPC3, a Glypican Encoded by a Gene That Is Mutated in the Simpson-Golabi-Behmel Overgrowth Syndrome, Induces Apoptosis in a Cell Line–specific Manner

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3–induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line– specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.     

The expanding phenotypic spectrum of female <Emphasis Type="Italic">SLC9A6</Emphasis> mutation carriers: a case series and review of the literature

Christianson syndrome (OMIM 300243), caused by mutations in the X-linked SLC9A6 gene, is characterized by severe global developmental delay and intellectual disability, developmental regression, epilepsy, microcephaly and impaired ocular movements. It shares many common features with Angelman syndrome. Carrier females have been described as having learning difficulties with mild to moderate intellectual disability, behavioural issues and psychiatric illnesses. There is little literature on the carrier female phenotype of Christianson syndrome. We describe a large extended family with three affected males, four carrier females, one presumed carrier female and one obligate carrier female with a c.190G>T, p.E64X mutation known to cause a premature stop codon in SLC9A6. We characterize and expand the clinical phenotype of female SLC9A6 mutation carriers by comparing our described family with female carriers previously discussed in the literature. In particular, we highlight the neurodevelopmental and psychiatric phenotypes observed in our family and previous reports.     

De Novo Nonsense Mutations in KAT6A,a Lysine Acetyl-Transferase Gene,Cause a Syndrome Including Microcephaly and Global Developmental Delay

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129^∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024^∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.     

A 4-Mb BAC/PAC contig and complete genomic structure of the GPC5/GPC6 gene cluster on chromosome 13q32.

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that may play a role in the control of cell division and growth regulation. So far, six members (GPC1-6) of this family are known in vertebrates. We report the construction of a high-resolution 4 Mb sequence-ready BAC/PAC contig of the GPC5/GPC6 gene cluster on chromosome region 13q32. The contig indicates that, like the GPC3/GPC4 genes on Xq26, GPC5 and GPC6 are arranged in tandem array. Both GPC5 and GPC6 are very large genes, with sizes well over 1 Mb. With a size of approximately 2 Mb, GPC5 would be the second largest human gene identified to date. Comparison of the long range gene organisation on 13q and Xq, suggests that these chromosomes share several regions of homology. Mutations and deletions affecting GPC3 are associated with the Simpson-Golabi-Behmel overgrowth syndrome. Mutational analysis of GPC5 and GPC6 in 19 patients with somatic overgrowth failed to reveal pathologic mutations in either of these genes, but identified several coding region polymorphisms.     

Spectrum of NIPBL gene mutations in Polish patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a rare multi-system genetic disorder characterised by growth and developmental delay, distinctive facial dysmorphism, limb malformations and multiple organ defects. The disease is caused by mutations in genes responsible for the formation and regulation of cohesin complex. About half of the cases result from mutations in the NIPBL gene coding delangin, a protein regulating the initialisation of cohesion. To date, approximately 250 point mutations have been identified in more than 300 CdLS patients worldwide. In the present study, conducted on a group of 64 unrelated Polish CdLS patients, 25 various NIPBL sequence variants, including 22 novel point mutations, were detected. Additionally, large genomic deletions on chromosome 5p13 encompassing the NIPBL gene locus were detected in two patients with the most severe CdLS phenotype. Taken together, 42 % of patients were found to have a deleterious alteration affecting the NIPBL gene, by and large private ones (89 %). The review of the types of mutations found so far in Polish patients, their frequency and correlation with the severity of the observed phenotype shows that Polish CdLS cases do not significantly differ from other populations.     

Glypicans in growth control and cancer

The name glypican has been assigned to a family of heparan sulfate (HS) proteoglycans that are linked to the cell membrane by a glycosyl-phosphatidylinositol anchor. To date, six family members of this family have been identified in mammals (GPC1 to GPC6) and two in Drosophila. Glypicans are expressed predominantly during development, and they are thought to play a role in morphogenesis. As HS-carrying molecules, glypicans were initially considered potential regulators of heparin-binding growth factors. This has been recently confirmed by genetic interaction experiments showing that glypicans regulate wingless signaling in Drosophila. The involvement of glypicans in the in vivo regulation of other heparin-binding growth factors, such as fibroblast growth factors, remains to be determined. Interestingly and unexpectedly, a role for GPC3 in the regulation of insulin-like growth factors has been proposed. This hypothesis is based on the phenotype of patients with Simpson-Golabi-Behmel syndrome (SGBS), an overgrowth and dysmorphic syndrome in which the GPC3 gene is mutated. Thus, it is possible that glypicans regulate different kinds of growth factors in a tissue-specific manner. In addition to its involvement in SGBS, down-regulation of GPC3 has been recently associated with the progression of several types of malignant tumors, including mesotheliomas and ovarian cancer. A role for GPC1 in pancreatic cancer progression has also been proposed.     

Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

Malignant gliomas are highly lethal neoplasms with limited treatment options. We previously found that the heparan sulfate proteoglycan glypican 1 (GPC1) is universally and highly expressed in human gliomas. In this study, we investigated the biological activity of GPC1 expression in both human glioma cells and normal astrocytes in vitro. Expression of GPC1 inactivates the G₁/S checkpoint and strongly stimulates DNA replication. Constitutive expression of GPC1 causes DNA rereplication and DNA damage, suggesting a mutagenic activity for GPC1. GPC1 expression leads to a significant downregulation of the tumor suppressors pRb, Cip/Kip cyclin-dependent kinase inhibitors (CKIs), and CDH1, and upregulation of the pro-oncogenic proteins cyclin E, cyclin-dependent kinase 2 (CDK2), Skp2, and Cdt1. These GPC1-induced changes are accompanied by a significant reduction in all types of D cyclins, which is independent of serum supplementation. It is likely that GPC1 stimulates the so-called Skp2 autoinduction loop, independent of cyclin D-CDK4/6. Knockdown of Skp2, CDK2, or cyclin E, three key elements within the network modulated by GPC1, results in a reduction of the S phase and aneuploid fractions, implying a functional role for these regulators in GPC1-induced S phase entry and DNA rereplication. In addition, a significant activation of both the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways by GPC1 is seen in normal human astrocytes even in the presence of growth factor supplement. Both pathways are constitutively activated in human gliomas. The surprising magnitude and the mitogenic and mutagenic nature of the effect exerted by GPC1 on the cell cycle imply that GPC1 may play an important role in both glioma tumorigenesis and growth.     

Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case

Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems.     

Keppen-Lubinsky Syndrome Is Caused by Mutations in the Inwardly Rectifying K+ Channel Encoded by KCNJ6

Keppen-Lubinsky syndrome (KPLBS) is a rare disease mainly characterized by severe developmental delay and intellectual disability, microcephaly, large prominent eyes, a narrow nasal bridge, a tented upper lip, a high palate, an open mouth, tightly adherent skin, an aged appearance, and severe generalized lipodystrophy. We sequenced the exomes of three unrelated individuals affected by KPLBS and found de novo heterozygous mutations in KCNJ6 (GIRK2), which encodes an inwardly rectifying potassium channel and maps to the Down syndrome critical region between DIRK1A and DSCR4. In particular, two individuals shared an in-frame heterozygous deletion of three nucleotides (c.455_457del) leading to the loss of one amino acid (p.Thr152del). The third individual was heterozygous for a missense mutation (c.460G>A) which introduces an amino acid change from glycine to serine (p.Gly154Ser). In agreement with animal models, the present data suggest that these mutations severely impair the correct functioning of this potassium channel. Overall, these results establish KPLBS as a channelopathy and suggest that KCNJ6 (GIRK2) could also be a candidate gene for other lipodystrophies. We hope that these results will prompt investigations in this unexplored class of inwardly rectifying K⁺ channels.     

Glypican-6 stimulates intestinal elongation by simultaneously regulating Hedgehog and non-canonical Wnt signaling

We report here that Glypican-6 (GPC6)-null mice display at birth small intestines that are 75% shorter than those of normal littermates. Notably, we demonstrate that the role of GPC6 in intestinal elongation is mediated by both Hedgehog (Hh) and non-canonical Wnt signaling. Based on results from in vitro experiments, we had previously proposed that GPC6 stimulates Hh signaling by interacting with Hh and Patched1 (Ptc1), and facilitating/stabilizing their interaction. Here we provide strong support to this hypothesis by showing that GPC6 binds to Ptc1 in the mesenchymal layer of embryonic intestines. This study also provides experimental evidence that strongly suggests that GPC6 inhibits the activity of Wnt5a on the intestinal epithelium by binding to this growth factor, and reducing its release from the surrounding mesenchymal cells. Finally, we show that whereas the mesenchymal layer of GPC6-null intestines displays reduced cell proliferation and a thinner smooth muscle layer, epithelial cell differentiation is not altered in the mutant gut.     

Preparation, characterization and antimicrobial activity of 6-amino-6-deoxychitosan

6-Amino-6-deoxychitosans with molecular weights from 0.23 × 10⁴ to 1.41 × 10⁴ and degree of substitution from 0.85 to 0.96 were prepared via N-phthaloylation, tosylation, azidation, hydrazinolysis and reduction of azide groups. Their structures were characterized by FT-IR, ¹H NMR, ¹³C NMR, gel permeation chromatography (GPC) and elemental analysis. The antimicrobial activities of 6-amino-6-deoxychitosans against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Aspergillus niger were investigated. The results showed that 6-amino-6-deoxychitosans had a wide spectrum of effective antimicrobial activities. Compared with chitosan, 6-amino-6-deoxychitosans had much better antimicrobial activities. Their minimum inhibitory concentrations (MICs) were between 0.025% and 0.1% (w/v) in acetic/sodium acetate solution with different pH from 5.4 to 7.5. 6-Amino-6-deoxychitosans could also inhibit growth of bacteria tested in distilled water under pH 6.6-8.45. The antimicrobial mechanism was complex and the positive charge on the amino groups was not the sole factor resulting in the antimicrobial activities.     

Spectrum of <Emphasis Type="Italic">JAG1</Emphasis> gene mutations in Polish patients with Alagille syndrome

Alagille syndrome (ALGS) is an autosomal dominant disorder characterized by developmental abnormalities in several organs including the liver, heart, eyes, vertebrae, kidneys, and face. The majority (90-94 %) of ALGS cases are caused by mutations in the JAG1 (JAGGED1) gene, and in a small percent of patients (～1 %) mutations in the NOTCH2 gene have been described. Both genes are involved in the Notch signaling pathway. To date, over 440 different JAG1 gene mutations and ten NOTCH2 mutations have been identified in ALGS patients. The present study was conducted on a group of 35 Polish ALGS patients and revealed JAG1 gene mutations in 26 of them. Twenty-three different mutations were detected including 13 novel point mutations and six large deletions affecting the JAG1 gene. Review of all mutations identified to date in individuals from Poland allowed us to propose an effective diagnostic strategy based on the mutations identified in the reported patients of Polish descent. However, the distribution of mutations seen in this cohort was not substantively different than the mutation distribution in other reported populations.     

11p13 deletions can be more frequent than the PAX6 gene point mutations in Polish patients with aniridia

Aniridia is a rare, bilateral, congenital ocular disorder causing incomplete formation of the iris, usually characterized by iris aplasia/hypoplasia. It can also appear with other ocular anomalies, such as cataracts, glaucoma, corneal pannus, optic nerve hypoplasia, macular hypoplasia, or ectopia lentis. In the majority of cases, it is caused by mutation in the PAX6 gene, but it can also be caused by microdeletions that involve the 11p13 region. Twelve unrelated patients of Polish origin with a clinical diagnosis of aniridia were screened for the presence of microdeletions in the 11p13 region by means of multiplex ligation probe amplification (MLPA). Additionally, the coding regions of the PAX6 gene were sequenced in all probands. MLPA examination revealed different size deletions of the 11p13 region in five patients. In three cases, deletions encompassed the entire PAX6 gene and a few adjacent genes. In one case, a fragment of the PAX6 gene was deleted only. In the final case, the deletion did not include any PAX6 sequence. Our molecular findings provide further evidence of the existence of the distant 3′ regulatory elements in the downstream region of the PAX6 gene, which is known from other studies to influence the level of protein expression. Sequence analysis of the PAX6 gene revealed the three different point mutations in the remaining four patients with aniridia. All the detected mutations were reported earlier. Based on accomplished results, the great diversity of the molecular basis of aniridia was found. It varies from point mutations to different size deletions in the 11p13 region which encompass partly or completely the PAX6 gene or cause a position effect.