Christopher Bruce | University of Leicester (original) (raw)
Papers by Christopher Bruce
British Journal of Pharmacology, 1997
We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels... more We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist. Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al. In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentration...
Gut, 2010
Introduction Neonatal cholestasis is the presenting clinical feature of serious and potentially l... more Introduction Neonatal cholestasis is the presenting clinical feature of serious and potentially life limiting liver diseases such as progressive familial intrahepatic cholestasis (PFIC), arthrogryposis-renal-cholestasis (ARC) syndrome and Niemann Pick C (NPC) disease. A single rapid molecular test to confirm the diagnosis would reduce the delay from molecular genetic investigation at multiple diagnostic centres thus facilitating optimal clinical management and counselling. We have designed a resequencing microarray (BRUM1) capable of simultaneously sequencing multiple genes associated with neonatal cholestasis. Aim To assess the utility of BRUM1 as a first-line molecular investigation for patients with neonatal cholestasis in whom an inherited causes is suspected. Method DNA from 95 infants with neonatal cholestasis in whom an inherited cause was suspected was amplified by PCR and hybridised to BRUM1 (validated against reference sequencing with 98.9% agreement (CI 0.97 to >0.99))...
F1000Research, 2013
Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be ... more Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS) is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR) has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with 'lownormal' gamma glutamyl transpeptidase (gGT) cholestasis without known molecular diagnoses. Methods: 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of "low gGT cholestasis" such as ARC syndrome and bile acid biosynthesis disorders were excluded. Results: Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%.
Nature Genetics, 2010
A r t i c l e s Generation of distinct apical and basolateral plasma membrane domains is essentia... more A r t i c l e s Generation of distinct apical and basolateral plasma membrane domains is essential for various epithelial functions, including lumen formation 1-3. Cohesive epithelial layers act as physical ion-selective barriers. This barrier function is highly dependent on the integrity of apical junctional complexes (AJCs), comprising tight junctions and adherens junctions, that link adjacent epithelial cells 4,5. Generation of AJCs is one of the steps in a complex mechanism of cell polarization that is dependent on multiple regulatory pathways 6. Better insight into these pathways is essential for understanding the development and physiology of the many organs that rely on intact cell polarity for their function. Investigation of the molecular pathology of human diseases can shed new light on the intracellular pathways in mammalian cells, and many common disorders are associated with defects in apical-basolateral polarity 7-10. ARC (MIM208085) is a severe autosomal recessive multisystem disorder that affects the development and function of several organs, particularly the liver and kidneys. Germline mutations in VPS33B are found in approximately 75% of individuals with ARC 11. VPS33B encodes VPS33B, the homolog of yeast class C vacuolar protein sorting (vps) protein Vps33p, from the Sec1-Munc18 family of proteins regulating SNARE-mediated membrane fusion. Class C vps proteins participate in at least two multiprotein complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS), which interact with yeast orthologs of Rab5 and Rab7 GTPases and are crucial for several steps in vesicular trafficking pathways 12-15. Recent studies suggested that mammalian homologs of the yeast constituents of HOPS can be localized to early and late endosomes and lysosomes and regulate a number of intracellular processes 16-18. Whereas the Vps33a homolog of yeast Vps33p forms part of the mammalian HOPS complex, the pathway involving Vps33b remains unknown, although interaction with other class C vps protein homologs has been proposed 16,19,20. To elucidate the molecular basis of ARC and gain insights into the role of VPS33B
Nanomedicine, 2010
Aims: In cancer therapy, research has focused on the development of nanocarriers that can aid dia... more Aims: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization. Materials & methods: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications. The intracellular distribution after internalization has been analyzed by ultrasensitive SR-XFM and genotoxicity evaluated using γ-H2AX DNA damage foci phosphorylation assay. Results: We demonstrate the unprecedented capability of SR-XFM for extremely sensitive nanoimaging and intracellular elemental distribution analysis of noble metal nanoparticles in cells. Furthermore, we show that, depending o...
Molecular Genetics and Metabolism, 2010
Journal of Inherited Metabolic Disease, 2011
Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolon... more Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were >85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C > T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A > G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A > G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.
Journal of Biological Chemistry, 2006
Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cell... more Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.
Human Mutation, 2012
Arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisys... more Arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B
Human Molecular Genetics, 2009
Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility ... more Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease. In the current study, we investigated a combined panel of 98 SNPs, including 70 tag SNPs, across an extended 800 kb region of the TSHR to refine association in a cohort of 768 GD subjects and 768 matched controls. In total, 28 SNPs revealed association with GD (P < 0.05), with strongest SNP associations at rs179247 (x 2 = 32.45,
Dalton Transactions, 2010
The MDA-MB-231 (human breast carcinoma) cell line was maintained in continuous logarithmic cultur... more The MDA-MB-231 (human breast carcinoma) cell line was maintained in continuous logarithmic culture in Dulbecco's Modified Eagle's Medium (DMEM) (+ 4.5 g/l glucose, Gibco BRL TM , Invitrogen Corporation, GB) supplemented with 10% FBS (Invitrogen), 2 mM l-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 10 mM Hepes buffer (Sigma) and antibiotics (Antibiotics Antimycotic 100× diluted to 1×, Sigma). Cells from confluent monolayers were removed from flasks by 1% trypsin (Trypsin-EDTA 10× diluted to 1× using PBS, Sigma). Cell viability was determined by the trypan blue dye exclusion test. Extended incubation cellular toxicity 10,000 cells were seeded in 100 µL complete medium per well of 96-multiwell flatbottom microtiter plates (Corning Costar) and incubated for 24 hrs at 37 °C, 5 % CO 2 for cells to adhere. Cells were then treated with 100 µL compound of concentrations between 6.5 and 200 µM followed by further incubation of 1-6 days. At the end of each incubation period cell viability was established by the MTT assay and IC 50 value calculated. Volume cellular toxicity 10,000 cells were seeded in 100 µl of complete medium in each well of 96-multiwell flatbottom microtiter plates (Corning Costar) and incubated at 37 °C, 5 % CO 2 for 24 hours
Cancer Genetics and Cytogenetics, 2003
We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a p... more We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a patient (tumor cell sample designated as MA214) with bilateral Wilms tumor (WT). We have now physically refined the breakpoints and identified putative gene targets within this region. The translocation breakpoints are contained within a 2.5-Mbp region on 5q21 containing four candidate genes and a 1.3-Mbp region on 6q21 that contains three candidate genes. To explore the role of this region in WT genesis, we have performed loss of heterozygosity (LOH) analysis with markers flanking the translocation breakpoints in tumor from MA214 and a panel of sporadic WT. Alleles were retained for all informative markers used in the MA214 tumor. In sporadic tumors LOH was found in 6 of 63 (9.5%) and 5 of 62 (8%) informative cases for flanking markers D6S301 and D6S1592 on 6q21. LOH was found in 3 of 58 (5.2%) and 2 of 54 (3.6%) for flanking markers D5S495 and D5S409 on 5q21. These preliminary data suggest LOH at the t(5;6)(q21;q21) region is unlikely to be a mechanism for tumor development in MA214, but may be important for a subgroup of sporadic WT.
British Journal of Pharmacology, 1997
We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels... more We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist. Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al. In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentration...
Gut, 2010
Introduction Neonatal cholestasis is the presenting clinical feature of serious and potentially l... more Introduction Neonatal cholestasis is the presenting clinical feature of serious and potentially life limiting liver diseases such as progressive familial intrahepatic cholestasis (PFIC), arthrogryposis-renal-cholestasis (ARC) syndrome and Niemann Pick C (NPC) disease. A single rapid molecular test to confirm the diagnosis would reduce the delay from molecular genetic investigation at multiple diagnostic centres thus facilitating optimal clinical management and counselling. We have designed a resequencing microarray (BRUM1) capable of simultaneously sequencing multiple genes associated with neonatal cholestasis. Aim To assess the utility of BRUM1 as a first-line molecular investigation for patients with neonatal cholestasis in whom an inherited causes is suspected. Method DNA from 95 infants with neonatal cholestasis in whom an inherited cause was suspected was amplified by PCR and hybridised to BRUM1 (validated against reference sequencing with 98.9% agreement (CI 0.97 to >0.99))...
F1000Research, 2013
Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be ... more Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS) is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR) has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with 'lownormal' gamma glutamyl transpeptidase (gGT) cholestasis without known molecular diagnoses. Methods: 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of "low gGT cholestasis" such as ARC syndrome and bile acid biosynthesis disorders were excluded. Results: Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%.
Nature Genetics, 2010
A r t i c l e s Generation of distinct apical and basolateral plasma membrane domains is essentia... more A r t i c l e s Generation of distinct apical and basolateral plasma membrane domains is essential for various epithelial functions, including lumen formation 1-3. Cohesive epithelial layers act as physical ion-selective barriers. This barrier function is highly dependent on the integrity of apical junctional complexes (AJCs), comprising tight junctions and adherens junctions, that link adjacent epithelial cells 4,5. Generation of AJCs is one of the steps in a complex mechanism of cell polarization that is dependent on multiple regulatory pathways 6. Better insight into these pathways is essential for understanding the development and physiology of the many organs that rely on intact cell polarity for their function. Investigation of the molecular pathology of human diseases can shed new light on the intracellular pathways in mammalian cells, and many common disorders are associated with defects in apical-basolateral polarity 7-10. ARC (MIM208085) is a severe autosomal recessive multisystem disorder that affects the development and function of several organs, particularly the liver and kidneys. Germline mutations in VPS33B are found in approximately 75% of individuals with ARC 11. VPS33B encodes VPS33B, the homolog of yeast class C vacuolar protein sorting (vps) protein Vps33p, from the Sec1-Munc18 family of proteins regulating SNARE-mediated membrane fusion. Class C vps proteins participate in at least two multiprotein complexes, class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS), which interact with yeast orthologs of Rab5 and Rab7 GTPases and are crucial for several steps in vesicular trafficking pathways 12-15. Recent studies suggested that mammalian homologs of the yeast constituents of HOPS can be localized to early and late endosomes and lysosomes and regulate a number of intracellular processes 16-18. Whereas the Vps33a homolog of yeast Vps33p forms part of the mammalian HOPS complex, the pathway involving Vps33b remains unknown, although interaction with other class C vps protein homologs has been proposed 16,19,20. To elucidate the molecular basis of ARC and gain insights into the role of VPS33B
Nanomedicine, 2010
Aims: In cancer therapy, research has focused on the development of nanocarriers that can aid dia... more Aims: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization. Materials & methods: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications. The intracellular distribution after internalization has been analyzed by ultrasensitive SR-XFM and genotoxicity evaluated using γ-H2AX DNA damage foci phosphorylation assay. Results: We demonstrate the unprecedented capability of SR-XFM for extremely sensitive nanoimaging and intracellular elemental distribution analysis of noble metal nanoparticles in cells. Furthermore, we show that, depending o...
Molecular Genetics and Metabolism, 2010
Journal of Inherited Metabolic Disease, 2011
Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolon... more Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.
Journal of Biological Chemistry, 2006
Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cell... more Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.
Human Mutation, 2012
Arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisys... more Arthrogryposis-renal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B
Human Molecular Genetics, 2009
Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility ... more Graves' disease (GD) is a common autoimmune disease (AID) that shares many of its susceptibility loci with other AIDs. The thyroid stimulating hormone receptor (TSHR) represents the primary autoantigen in GD, in which autoantibodies bind to the receptor and mimic its ligand, thyroid stimulating hormone, causing the characteristic clinical phenotype. Although early studies investigating the TSHR and GD proved inconclusive, more recently we provided convincing evidence for association of the TSHR region with disease. In the current study, we investigated a combined panel of 98 SNPs, including 70 tag SNPs, across an extended 800 kb region of the TSHR to refine association in a cohort of 768 GD subjects and 768 matched controls. In total, 28 SNPs revealed association with GD (P < 0.05), with strongest SNP associations at rs179247 (x 2 = 32.45,
Dalton Transactions, 2010
The MDA-MB-231 (human breast carcinoma) cell line was maintained in continuous logarithmic cultur... more The MDA-MB-231 (human breast carcinoma) cell line was maintained in continuous logarithmic culture in Dulbecco's Modified Eagle's Medium (DMEM) (+ 4.5 g/l glucose, Gibco BRL TM , Invitrogen Corporation, GB) supplemented with 10% FBS (Invitrogen), 2 mM l-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 10 mM Hepes buffer (Sigma) and antibiotics (Antibiotics Antimycotic 100× diluted to 1×, Sigma). Cells from confluent monolayers were removed from flasks by 1% trypsin (Trypsin-EDTA 10× diluted to 1× using PBS, Sigma). Cell viability was determined by the trypan blue dye exclusion test. Extended incubation cellular toxicity 10,000 cells were seeded in 100 µL complete medium per well of 96-multiwell flatbottom microtiter plates (Corning Costar) and incubated for 24 hrs at 37 °C, 5 % CO 2 for cells to adhere. Cells were then treated with 100 µL compound of concentrations between 6.5 and 200 µM followed by further incubation of 1-6 days. At the end of each incubation period cell viability was established by the MTT assay and IC 50 value calculated. Volume cellular toxicity 10,000 cells were seeded in 100 µl of complete medium in each well of 96-multiwell flatbottom microtiter plates (Corning Costar) and incubated at 37 °C, 5 % CO 2 for 24 hours
Cancer Genetics and Cytogenetics, 2003
We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a p... more We have previously described the physical localization of a constitutional t(5;6)(q21;q21) in a patient (tumor cell sample designated as MA214) with bilateral Wilms tumor (WT). We have now physically refined the breakpoints and identified putative gene targets within this region. The translocation breakpoints are contained within a 2.5-Mbp region on 5q21 containing four candidate genes and a 1.3-Mbp region on 6q21 that contains three candidate genes. To explore the role of this region in WT genesis, we have performed loss of heterozygosity (LOH) analysis with markers flanking the translocation breakpoints in tumor from MA214 and a panel of sporadic WT. Alleles were retained for all informative markers used in the MA214 tumor. In sporadic tumors LOH was found in 6 of 63 (9.5%) and 5 of 62 (8%) informative cases for flanking markers D6S301 and D6S1592 on 6q21. LOH was found in 3 of 58 (5.2%) and 2 of 54 (3.6%) for flanking markers D5S495 and D5S409 on 5q21. These preliminary data suggest LOH at the t(5;6)(q21;q21) region is unlikely to be a mechanism for tumor development in MA214, but may be important for a subgroup of sporadic WT.