CBS | GeneID:875 | Homo sapiens
[ ] NCBI Entrez Gene
|Gene ID||875||Official Symbol||CBS|
|Also Known As||OTTHUMP00000109416; OTTHUMP00000109418; beta-thionase; cystathionine beta-synthase; methylcysteine synthase; serine sulfhydrase|
|Summary||The protein encoded by this gene acts as a homotetramer to catalyze the conversion of homocysteine to cystathionine, the first step in the transsulfuration pathway. The encoded protein is allosterically activated by adenosyl-methionine and uses pyridoxal phosphate as a cofactor. Defects in this gene can cause cystathionine beta-synthase deficiency (CBSD), which can lead to homocystinuria. [provided by RefSeq]|
Orthologs and Paralogs
|GeneID:611071||CBS||XP_853796.1||Canis lupus familiaris|
[ ] Monoclonal and Polyclonal Antibodies
|GO:0004122||Function||cystathionine beta-synthase activity|
|GO:0005506||Function||iron ion binding|
|GO:0046872||Function||metal ion binding|
|GO:0030170||Function||pyridoxal phosphate binding|
|GO:0008652||Process||cellular amino acid biosynthetic process|
|GO:0006535||Process||cysteine biosynthetic process from serine|
|GO:0019343||Process||cysteine biosynthetic process via cystathionine|
|GO:0043506||Process||regulation of JUN kinase activity|
MicroRNA and Targets
[ ] MicroRNA Sequences and Transcript Targets from miRBase at Sanger
|RNA Target||miRNA #||mat miRNA||Mature miRNA Sequence|
Chemicals and Drugs
[ ] Comparative Toxicogenomics Database from MDI Biological Lab
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
|Chemical and Interaction|
Gene and Diseases
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
|C17orf28||CBS / C17orf28||Two-hybrid||Rual JF (2005)|
|C6orf55||CBS / C6orf55||Two-hybrid||Rual JF (2005)|
|CBS||CBS / CBS||Affinity Capture-Western||Rual JF (2005)|
|CBS||CBS / CBS||Co-crystal Structure||Janosik M (2001)|
|CBS||CBS / CBS||Co-crystal Structure||Meier M (2001)|
|CBS||CBS / CBS||Two-hybrid||Rual JF (2005)|
|FXR2||CBS / FXR2||Two-hybrid||Rual JF (2005)|
- [ ] Velez DR, et al. (2009) "Spontaneous preterm birth in African Americans is associated with infection and inflammatory response gene variants." Am J Obstet Gynecol. 200(2):209.e1-209.27. PMID:19019335
- [ ] Menon R, et al. (2009) "Racial disparity in pathophysiologic pathways of preterm birth based on genetic variants." Reprod Biol Endocrinol. 7(1):62. PMID:19527514
- [ ] Luo D, et al. (2009) "[Levels of homocysteine and polymorphisms of homocysteine metabolism-related enzymes in patients with type 2 diabetes mellitus and coronary heart disease]" Wei Sheng Yan Jiu. 38(1):39-42. PMID:19267073
- [ ] Belew MS, et al. (2009) "Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli." Protein Expr Purif. 64(2):139-145. PMID:19010420
- [ ] Sawula W, et al. (2009) "Homocysteine level and metabolism in ischemic stroke in the population of Northern Poland." Clin Biochem. 42(6):442-447. PMID:19166826
- [ ] Dutta S, et al. (2009) "Correlation between cystathionine beta synthase gene polymorphisms, plasma homocysteine and idiopathic mental retardation in Indian individuals from Kolkata." Neurosci Lett. 453(3):214-218. PMID:19429038
- [ ] Pridgeon JW, et al. (2009) "Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes." FEBS J. 276(1):118-131. PMID:19019082
- [ ] Boyles AL, et al. (2009) "Oral facial clefts and gene polymorphisms in metabolism of folate/one-carbon and vitamin A: a pathway-wide association study." Genet Epidemiol. 33(3):247-255. PMID:19048631
- [ ] Singh LR, et al. (2009) "Functional rescue of mutant human cystathionine beta-synthase by manipulation of Hsp26 and Hsp70 levels in Saccharomyces cerevisiae." J Biol Chem. 284(7):4238-4245. PMID:19074437
- [ ] Zschocke J, et al. (2009) "Molecular neonatal screening for homocystinuria in the Qatari population." Hum Mutat. 30(6):1021-1022. PMID:19370759
- [ ] Franke B, et al. (2009) "An association study of 45 folate-related genes in spina bifida: Involvement of cubilin (CUBN) and tRNA aspartic acid methyltransferase 1 (TRDMT1)." Birth Defects Res A Clin Mol Teratol. 85(3):216-226. PMID:19161160
- [ ] Hosgood HD 3rd, et al. (2008) "Pathway-based evaluation of 380 candidate genes and lung cancer susceptibility suggests the importance of the cell cycle pathway." Carcinogenesis. 29(10):1938-1943. PMID:18676680
- [ ] Yang QH, et al. (2008) "Prevalence and effects of gene-gene and gene-nutrient interactions on serum folate and serum total homocysteine concentrations in the United States: findings from the third National Health and Nutrition Examination Survey DNA Bank." Am J Clin Nutr. 88(1):232-246. PMID:18614746
- [ ] Meyer K, et al. (2008) "MALDI-TOF MS Genotyping of Polymorphisms Related to 1-Carbon Metabolism Using Common and Mass-Modified Terminators." Clin Chem. ():. PMID:18988749
- [ ] Jakubowski H, et al. (2008) "Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans." FASEB J. 22(12):4071-4076. PMID:18708589
- [ ] Belkahla R, et al. (2008) "[Effect of polymorphisms on key enzymes in homocysteine metabolism, on plasma homocysteine level and on coronary artery-disease risk in a Tunisian population]" Ann Cardiol Angeiol (Paris). 57(4):219-224. PMID:18620331
- [ ] Zhu W, et al. (2008) "[CBS gene variations and serum homocysteine level associated with congenital heart defects]" Wei Sheng Yan Jiu. 37(4):463-467. PMID:18839533
- [ ] Maitland-van der Zee AH, et al. (2008) "Interactions between the single nucleotide polymorphisms in the homocysteine pathway (MTHFR 677C>T, MTHFR 1298 A>C, and CBSins) and the efficacy of HMG-CoA reductase inhibitors in preventing cardiovascular disease in high-risk patients of hypertension: the GenHAT study." Pharmacogenet Genomics. 18(8):651-656. PMID:18622257
- [ ] Chang MH, et al. (2008) "Prevalence in the United States of Selected Candidate Gene Variants: Third National Health and Nutrition Examination Survey, 1991-1994." Am J Epidemiol. ():. PMID:18936436
- [ ] Ott N, et al. (2008) "Polymorphisms in methionine synthase (A2756G) and cystathionine beta-synthase (844ins68) and susceptibility to carcinomas of the upper gastrointestinal tract." J Cancer Res Clin Oncol. 134(3):405-410. PMID:17726616
- [ ] Summers CM, et al. (2008) "Functional polymorphisms of folate-metabolizing enzymes in relation to homocysteine concentrations in systemic lupus erythematosus." J Rheumatol. 35(11):2179-2186. PMID:18785313
- [ ] Giusti B, et al. (2008) "Genetic analysis of 56 polymorphisms in 17 genes involved in methionine metabolism in patients with abdominal aortic aneurysm." J Med Genet. 45(11):721-730. PMID:18635682
- [ ] Gibson CS, et al. (2008) "Candidate genes and cerebral palsy: a population-based study." Pediatrics. 122(5):1079-1085. PMID:18977990
- [ ] Wang SS, et al. (2008) "Polymorphisms in DNA repair and one-carbon metabolism genes and overall survival in diffuse large B-cell lymphoma and follicular lymphoma." Leukemia. ():. PMID:18830263
- [ ] Giusti B, et al. (2008) "High-throughput multiplex single-nucleotide polymorphism (SNP) analysis in genes involved in methionine metabolism." Biochem Genet. 46(7-8):406-423. PMID:18427977
- [ ] Janosik M, et al. (2008) "Birth Prevalence of Homocystinuria in Central Europe: Frequency and Pathogenicity of Mutation c.1105C>T (p.R369C) in the Cystathionine Beta-Synthase Gene." J Pediatr. ():. PMID:18950795
- [ ] Barbe L, et al. (2008) "Toward a confocal subcellular atlas of the human proteome." Mol Cell Proteomics. 7(3):499-508. PMID:18029348
- [ ] Frank N, et al. (2008) "Purification and characterization of the wild type and truncated human cystathionine beta-synthase enzymes expressed in E. coli." Arch Biochem Biophys. 470(1):64-72. PMID:18060852
- [ ] Starr JM, et al. (2008) "Oxidative stress, telomere length and biomarkers of physical aging in a cohort aged 79 years from the 1932 Scottish Mental Survey." Mech Ageing Dev. 129(12):745-751. PMID:18977241
- [ ] Skibola CF, et al. (2008) "Polymorphisms in the estrogen receptor 1 and vitamin C and matrix metalloproteinase gene families are associated with susceptibility to lymphoma." PLoS ONE. 3(7):e2816. PMID:18636124
- [ ] Semmler A, et al. (2008) "Polymorphisms of homocysteine metabolism are associated with intracranial aneurysms." Cerebrovasc Dis. 26(4):425-429. PMID:18799873
- [ ] Stevens VL, et al. (2008) "No association of single nucleotide polymorphisms in one-carbon metabolism genes with prostate cancer risk." Cancer Epidemiol Biomarkers Prev. 17(12):3612-3614. PMID:19064578
- [ ] Alessio AC, et al. (2008) "Polymorphisms in the CBS gene and homocysteine, folate and vitamin B12 levels: association with polymorphisms in the MTHFR and MTRR genes in Brazilian children." Am J Med Genet A. 146A(20):2598-2602. PMID:18792976
- [ ] Boyles AL, et al. (2008) "Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts." Am J Med Genet A. 146A(4):440-449. PMID:18203168
- [ ] Velez DR, et al. (2008) "Preterm birth in Caucasians is associated with coagulation and inflammation pathway gene variants." PLoS ONE. 3(9):e3283. PMID:18818748
- [ ] Summers CM, et al. (2008) "Influence of the cystathionine beta-synthase 844ins68 and methylenetetrahydrofolate reductase 677C>T polymorphisms on folate and homocysteine concentrations." Eur J Hum Genet. 16(8):1010-1013. PMID:18398434
- [ ] Carballal S, et al. (2008) "Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase." Biochemistry. 47(10):3194-3201. PMID:18278872
- [ ] Gupta S, et al. (2008) "Cystathionine beta-synthase p.S466L mutation causes hyperhomocysteinemia in mice." Hum Mutat. 29(8):1048-1054. PMID:18454451
- [ ] Semmler A, et al. (2008) "Polymorphisms of methionine metabolism and susceptibility to meningioma formation: laboratory investigation." J Neurosurg. 108(5):999-1004. PMID:18447718
- [ ] Lim U, et al. (2007) "Gene-nutrient interactions among determinants of folate and one-carbon metabolism on the risk of non-Hodgkin lymphoma: NCI-SEER case-control study." Blood. 109(7):3050-3059. PMID:17119116
- [ ] Lee KM, et al. (2007) "One-carbon metabolism gene polymorphisms and risk of non-Hodgkin lymphoma in Australia." Hum Genet. 122(5):525-533. PMID:17891500
- [ ] Moore LE, et al. (2007) "Polymorphisms in one-carbon metabolism and trans-sulfuration pathway genes and susceptibility to bladder cancer." Int J Cancer. 120(11):2452-2458. PMID:17311259
- [ ] Lissowska J, et al. (2007) "Genetic polymorphisms in the one-carbon metabolism pathway and breast cancer risk: a population-based case-control study and meta-analyses." Int J Cancer. 120(12):2696-2703. PMID:17311260
- [ ] Cherney MM, et al. (2007) "Ferrous human cystathionine beta-synthase loses activity during enzyme assay due to a ligand switch process." Biochemistry. 46(45):13199-13210. PMID:17956124
- [ ] Harris SE, et al. (2007) "A genetic association analysis of cognitive ability and cognitive ageing using 325 markers for 109 genes associated with oxidative stress or cognition." BMC Genet. 8():43. PMID:17601350
OBJECTIVE: The objective of the study was to study the genetic risk factors of spontaneous preterm birth (PTB) in African Americans. STUDY DESIGN: Case-control analyses were performed using maternal and fetal deoxyribonucleic acid from 279 African American birth events (82 PTB and 197 term) and 1432 single-nucleotide polymorphisms from 130 candidate genes. Single-locus association and haplotype analyses were performed. RESULTS: The most significant associations were in the maternal interleukin (IL)-15 (rs10833, allele P = 2.91 x 10(-4), genotype P = 2.00 x 10(-3)) gene and the fetal IL-2 receptor B (IL-2RB) (rs84460, allele P = 1.37 x 10(-4), genotype P = 6.29 x 10(-4)) gene. The best models for these markers were additive (rs10833, odds ratio [OR], 0.30; 95% confidence interval [CI], 0.14-0.62; P = 1.0 x 10(-3); rs84460, OR, 2.32; 95% CI, 1.47-3.67; P < 1.0 x 10(-3)). The largest number of significant associations was found in genes related to infection and inflammation. There were overall a larger number of significant associations in infants than in mothers. CONCLUSION: These results support a strong role for genes involved in infection and inflammation in the pathogenesis of PTB, particularly IL-12 and IL-12RB, and indicate that in African Americans there may be complementarity of maternal and fetal genetic risks for PTB.
ABSTRACT: OBJECTIVE: To study pathophysiologic pathways in spontaneous preterm birth and possibly the racial disparity associating with maternal and fetal genetic variations, using bioinformatics tools. METHODS: A large scale candidate gene association study was performed on 1442 SNPs in 130 genes in a case (preterm birth <36 weeks) control study (term birth >37 weeks). Both maternal and fetal DNA from Caucasians (172 cases and 198 controls) and 279 African-Americans (82 cases and 197 controls) were used. A single locus association (genotypic) analysis followed by hierarchical clustering was performed, where clustering was based on p values for significant associations within each race. Using Ingenuity Pathway Analysis (IPA) software, known pathophysiologic pathways in both races were determined. RESULTS: From all SNPs entered into the analysis, the IPA mapped genes to specific disease functions. Gene variants in Caucasians were implicated in disease functions shared with other known disorders; specifically, dermatopathy, inflammation, and hematological disorders. This may reflect abnormal cervical ripening and decidual hemorrhage. In African-Americans inflammatory pathways were the most prevalent. In Caucasians, maternal gene variants showed the most prominent role in disease functions, whereas in African Americans it was fetal variants. The IPA software was used to generate molecular interaction maps that differed between races and also between maternal and fetal genetic variants. CONCLUSION: Differences at the genetic level revealed distinct disease functions and operational pathways in African Americans and Caucasians in spontaneous preterm birth. Differences in maternal and fetal contributions in pregnancy outcome are also different between African Americans and Caucasians. These results present a set of explicit testable hypotheses regarding genetic associations with preterm birth in African Americans and Caucasians.
OBJECTIVE: To explore the significance of Hey, the gene polymorphisms of methylenetetrahydrofolate reductase (MTHFR) C677 T and cystathionine beta- synthase (CBS844) ins68 in type 2 diabetes mellitus (DM) with coronary heart disease (CHD) in China. Methods We selected 70 patients with type 2 DM and CHD, 71 type 2 diabetes patients, and 85 controls in Han nationality from northern China. Hey levels were measured by fluorescence polarization immunoassay (FPIA) and the plasma folate and vitamin B12 levels by microparticle enzyme immunoassay (MEIA). The gene polymorphisms of the MTHFR C677 T were determined by PCR- RFLP assay and the gene polymorphisms of the CBS 844ins68 were determined by PCR assay. RESULTS: The plasma Hey levels in DM with CHD group (14.8 micromol/L) were significantly higher than in DM group (11.1 micromol/L) and control group (11.2 micromol/L), (P < 0.01). The levels of plasma folate and Vitamin B12 in DM with CHD group were significantly lower than in DM group and control group, (P < 0.05). The T allelic frequency of MTHFR in DM and CHD group was significantly higher than that in DM group and controls (45% vs 26.8%, 31.2%, P < 0.01). There were no significant differences in the frequencies of CBS 844ins68 polymorphism among 3 groups (P > 0.05). Logistic-regression analysis indicated that the OR of HHcy was 4.547 (95% CI 1.97-10.496) (P < 0.01), the OR of MTHFR 677 with T (including MTHFR CT genotype and Tr genotype)was 2.369 (95% CI 1.160-4.841), (P = 0.018), and the OR of CBS 844ins68 was 0.384 (95% CI 0.033-4.423), (P = 0.443). CONCLUSION: Hyperhomocysteinemia and MTHFR with T allele might be the risk factors for DM with CHD in northern Chinese Han population.
Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence ofa protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86-91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for L-Cth production, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by L-Hcys (K(i)(L-HCYS) = 2.1 +/- 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of L-Cth toL-Ser and L-Hcys were also determined and the k(cat)/K(m)(L-CTH) of this reaction is only approximately 2-fold lower than the k(cat)/K(m)(L-SER) of the physiological, condensation reaction.
OBJECTIVES: In this study we analyzed the occurrence of ischemic brain stroke in Northern Poland in regard to risk factors. DESIGN AND METHODS: 131 ischemic stroke patients and 64 controls were studied. Analyzed risk factors included conventional risk factors, total plasma homocysteine level and polymorphisms of the main enzymes of homocysteine metabolism-methylenetetrahydrofolate reductase (polymorphisms C677T and A1298C) and cystathionine beta synthase (polymorphism T833C). RESULTS: We confirmed the occurrence of a number of conventional risk factors in ischemic stroke. We found that hyperhomocysteinemia is an independent risk factor (p=0.0001). Plasma homocysteine correlated inversely with plasma vitamin B(6). We also found a relationship between C677T polymorphism type and hyperhomocysteinemia (p=0.0266). CONCLUSIONS: The occurrence of studied polymorphisms in the population of northern Poland was higher than reported previously for similar populations. However, none of the studied genetic factors were found to be significant risk factors in ischemic brain stroke.
Deficiency in cystathionine beta synthase (CBS) enzyme sometimes leads to hyperhomocysteinemia/homocystinuria, conditions often associated with mental retardation (MR). In this investigation, association of idiopathic MR (IMR) with six CBS gene polymorphisms and fasting total plasma homocysteine (plHcy) was explored. Nuclear families with IMR probands (N=180) and control subjects (N=106) were recruited. Genomic DNA was subjected to PCR amplification and RFLP analysis. plHcy was measured by enzyme immunoassay. Data obtained was subjected to statistical analyses. Linkage disequilibrium between polymorphic sites was computed. T833C/844ins68 polymorphism revealed significant maternal transmission in IMR cases. The 31bpVNTR 21 repeat allele was significantly higher in male IMR cases as compared to sex-matched controls (P=0.004). A significant difference was also noticed in genotype frequencies of male IMR cases (P=0.005). Four other sites, G919A, C1105T, G1316A and G1330A, were not polymorphic in the studied population. While no significant contribution of any particular genotype was observed, plHcy level was significantly higher in male IMR cases as compared to sex-matched controls (P=0.0001). The data presented here is probably indicative of a higher risk of IMR in male subjects in association with two CBS polymorphisms and mild elevation in plHcy concentration.
Despite the critical importance of protein ubiquitination in the regulation of diverse cellular processes, the molecular mechanisms by which cells recognize and transmit ubiquitin signals remain poorly understood. The endosomal sorting machinery component hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) contains a ubiquitin-interacting motif (UIM), which is believed to bind ubiquitinated membrane cargo proteins and mediate their sorting to the lysosomal degradation pathway. To gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a proteomic screen for Hrs UIM-interacting ubiquitinated proteins in human brain by using an in vitro expression cloning screening approach. We have identified 48 ubiquitinated proteins that are specifically recognized by the UIM domain of Hrs. Among them, 12 are membrane proteins that are likely to be Hrs cargo proteins, and four are membrane protein-associated adaptor proteins whose ubiquitination may act as a signal to target their associated membrane cargo for Hrs-mediated endosomal sorting. Other classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, proteins associated with the cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination and metabolism, suggesting the involvement of Hrs UIM-mediated ubiquitin signaling in the regulation of multiple cellular processes. We have characterized the ubiquitination of two identified proteins, Munc18-1 and Hsc70, and their interaction with Hrs UIM, and provided functional evidence supporting a role for Hsc70 in the regulation of Hrs-mediated endosome-to-lysosome trafficking.
An increased risk of facial clefts has been observed among mothers with lower intake of folic acid or vitamin A around conception. We hypothesized that the risk of clefts may be further moderated by genes involved in metabolizing folate or vitamin A. We included 425 case-parent triads in which the child had either cleft lip with or without cleft palate (CL/P) or cleft palate only (CPO), and no other major defects. We analyzed 108 SNPs and one insertion in 29 genes involved in folate/one-carbon metabolism and 68 SNPs from 16 genes involved in vitamin A metabolism. Using the Triad Multi-Marker (TRIMM) approach we performed SNP, gene, chromosomal region, and pathway-wide association tests of child or maternal genetic effects for both CL/P and CPO. We stratified these analyses on maternal intake of folic acid or vitamin A during the periconceptional period. As expected with this high number of statistical tests, there were many associations with P-values<0.05; although there were fewer than predicted by chance alone. The strongest association in our data (between fetal FOLH1 and CPO, P=0.0008) is not in agreement with epidemiologic evidence that folic acid reduces the risk of CL/P in these data, not CPO. Despite strong evidence for genetic causes of oral facial clefts and the protective effects of maternal vitamins, we found no convincing indication that polymorphisms in these vitamin metabolism genes play an etiologic role.
Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine beta-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.
We report the results of molecular neonatal screening for homocystinuria (cystathionine beta-synthase deficiency) in neonates of Qatari origin, developed in conjunction with a novel biochemical screening approach. DNA was extracted from dried blood spots (DBS); the prevalent Qatari CBS gene mutation p.R336C (c.1006C>T) and a second mutation were tested with specific TaqMan assays. Over a period of 2 years we screened 12,603 neonates and identified six affected neonates homozygous for p.R336C. There were 225 heterozygous carriers for p.R336C. One additional child with homocystinuria detected through biochemical screening was homozygous for a mutation not previously identified in Qatar. Homocystinuria in the Qatari population has an incidence of 1:1,800, the highest in the world and even higher than previously estimated. Allele frequency of the mutation p.R336C is approximately 1%, displaying a significant deviation from Hardy Weinberg equilibrium. In conclusion, first-line molecular neonatal screening is technically feasible and may be developed as an option for presymptomatic identification of genetic disorders caused by specific mutations or a limited number of prevalent mutations. However, sensitivity for the diagnosis of disorders caused by various mutations is limited even in a homogeneous population such as Qatar.
BACKGROUND: Spina bifida is a class of neural tube defects, which are congenital malformations of the central nervous system with a prevalence of 0.5 to 12 per 1000 births globally. In this article we attempt to identify genes related to folate and its metabolic pathways that are involved in the etiology of spina bifida. METHODS: We selected 50 folate metabolism-related genes and genotyped polymorphisms in those genes. Eighty-seven polymorphisms in 45 genes passed quality controls. Associations with spina bifida were investigated in 180 patients and 190 controls. For those polymorphisms that were nominally associated with spina bifida risk, the relation with serum and red blood cell folate, vitamin B(12), and homocysteine was evaluated in controls. RESULTS: A polymorphism in CUBN was significantly associated with decreased spina bifida risk, after correction for multiple testing, and was related to increased vitamin B(12) (p = 0.039) and red blood cell folate (p = 0.001). The CUBN gene encodes the intrinsic factor-cobalamin receptor (or cubilin), a peripheral membrane protein that acts as a receptor for intrinsic factor-vitamin B(12) complexes. Vitamin B(12) is an important cofactor in the folate metabolism, and low B(12) status in mothers has been linked to neural tube defects in children. Other interesting findings include nominally significant associations with polymorphisms in TRDMT1, ALDH1L1, SARDH, and SLCA19A1 (RFC1). CONCLUSION: Our study indicates interesting new candidate genes and functional pathways for further study and confirms earlier findings. None of the genes CUBN, TRDMT1, ALDH1L1, or SARDH have been investigated previously for association with spina bifida.
Common genetic variation may play an important role in altering lung cancer risk. We conducted a pathway-based candidate gene evaluation to identify genetic variations that may be associated with lung cancer in a population-based case-control study in Xuan Wei, China (122 cases and 111 controls). A total of 1260 single-nucleotide polymorphisms (SNPs) in 380 candidate genes for lung cancer were successfully genotyped and assigned to one of 10 pathways based on gene ontology. Logistic regression was used to assess the marginal effect of each SNP on lung cancer susceptibility. The minP test was used to identify statistically significant associations at the gene level. Important pathways were identified using a test of proportions and the rank truncated product methods. The cell cycle pathway was found as the most important pathway (P = 0.044) with four genes significantly associated with lung cancer (PLA2G6 minP = 0.001, CCNA2 minP = 0.006, GSK3 beta minP = 0.007 and EGF minP = 0.013), after adjusting for multiple comparisons. Interestingly, most cell cycle genes that were associated with lung cancer in this analysis were concentrated in the AKT signaling pathway, which is essential for regulation of cell cycle progression and cellular survival, and may be important in lung cancer etiology in Xuan Wei. These results should be viewed as exploratory until they are replicated in a larger study.
BACKGROUND: Abnormalities of folate and homocysteine metabolism are associated with a number of pediatric and adult disorders. Folate intake and genetic polymorphisms encoding folate-metabolizing enzymes influence blood folate and homocysteine concentrations, but the effects and interactions of these factors have not been studied on a population-wide basis. OBJECTIVE: The objective was to assess the prevalence of these genetic polymorphisms and their relation to serum folate and homocysteine concentrations. DESIGN: DNA samples from 6793 participants in the third National Health and Nutrition Examination Survey (NHANES III) during 1991-1994 were genotyped for polymorphisms of genes coding for folate pathway enzymes 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298A-->C, methionine synthase reductase (MTRR) 66A-->G, and cystathionine-beta-synthase 844ins68. The influence of these genetic variants on serum folate and homocysteine concentrations was analyzed by age, sex, and folate intake in 3 race-ethnicity groups. RESULTS: For all race-ethnicity groups, serum folate and homocysteine concentrations were significantly related to the MTHFR 677C-->T genotype but not to the other polymorphisms. Persons with the MTHFR 677 TT genotype had a 22.1% (95% CI: 14.6%, 28.9%) lower serum folate and a 25.7% (95% CI: 18.6%, 33.2%) higher homocysteine concentration than did persons with the CC genotype. Moderate daily folic acid intake (mean: 150 microg/d; 95% CI: 138, 162) significantly reduced the difference in mean homocysteine concentrations between those with the MTHFR 677 CC and TT genotypes. We found a significant interaction between MTHFR 677C-->T and MTRR 66A-->G on serum homocysteine concentrations among non-Hispanic whites. CONCLUSIONS: The MTHFR 677C-->T polymorphism was associated with significant differences in serum folate and homocysteine concentrations in the US population before folic acid fortification. The effect of MTHFR 677C-->T on homocysteine concentrations was reduced by moderate daily folic acid intake.
BACKGROUND: Large cohort studies may provide sufficient power to disentangle the role of polymorphisms related to 1-carbon metabolism and chronic diseases, but they require fast, accurate, high-throughput genotyping techniques. MALDI-TOF mass spectrometry has been adapted to rapid fine mapping using various approaches for allele discrimination. We developed a genotyping method based on MALDI-TOF MS and compared assay performance for formats based on standard and mass-modified terminators. METHODS: The assay includes 20 polymorphisms of 14 genes involved in 1-carbon metabolism (BHMT 742G>A, CBS 844ins68 and 699C>T, CTH 1364G>T, DHFR del19, NOS3 -786T>C and 894G>T, FOLR1 1314A>G, MTHFD1 -105T>C and 1958G>A, MTHFR 677C>T and 1298A>C, MTR 2756A>G, MTRR 66A>G and 524C>T, SLC19A1 80G>A, SHMT2 1420C>T, TCN2 67A>G and 776C>G, and TYMS 1494del6). RESULTS: Missing calls were observed for 4.7% of the DNA samples, attributed to failed liquid sample handling. Highly accurate genotyping was obtained by mass-modified as well as standard ddNTPs, with an average error rate of =0.1% by analysis of sample duplicates. A semiquantitative approach enabled unambiguous identification of the CBS 844ins68. Cluster plots of the relative allele intensities showed allele-specific bias according to type of minisequencing terminator and revealed a potential structural variation in the BHMT gene. CONCLUSIONS: MALDI-TOF MS-based genotyping using either standard or mass-modified terminators allows the accurate determination of single nucleotides as well as structural genetic variants. This was demonstrated with 20 polymorphisms involved in 1-carbon metabolism.
Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.
BACKGROUND: Hyperhomocysteinemia is known as an independent-risk factor for coronary-artery disease (CAD). However, the effect of homocystein metabolic enzymes polymorphisms on CAD is still controversed. We investigated the relation between homocystein metabolic key enzymes polymorphisms, homocystenemia and coronary stenosis in a Tunisian population. METHODS: Samples were collected from 251 CAD patients documented by angiography. Genotyping were performed for C677T methylene-tetrahydrofolate reductase (MTHFR), A2756G methionine-synthase (MS) and 844ins 68 cystathionine-beta-synthase (CBS). We measured fasting plasma tHcy, folate and vitamin B12. RESULTS: There was significant increase in homocysteinemia for homozygous genotypes of C677T MTHFR (p<0.001) and A2756G MS (p=0.01), but not for 844ins68 CBS (p=0.105). Potential confounders adjusted odds-ratios for significant coronary stenosis, associated with MTHFR TT, MS GG and CBS insertion, were respectively 1.78 (p=0.041); 2.33 (p=0.036) and 0.87 (p=0.823). The effect of mutated MTHFR genotype was more pronounced on homocysteinemia (21.4+/-9.1 micromol/L; p<0.001) and coronary stenosis (OR=2.73; p=0.033) at low folatemia (< or =6.1 ng/mL). CONCLUSION: MTHFR TT and MS GG genotypes increase tHcy concentration and coronary stenosis risk, especially with low folatemia.
OBJECTIVE: To study the relationship between two common CBS gene variations, tHcy level and CHDs in a nuclear family-based study. METHODS: 234 Chinese CHDs patients and their biological parents were recruited as case groups. And another 136 normal individuals and their parents were recruited as controls. The CBS gene 844ins68 and G919A variants were analyzed by PCR and PCR-ARMS methods. The serum fasting total homocysteine (tHcy) level was detected by Fluorescence Polarization Immunoassay. RESULTS: CBS 844ins68 variant was associated with high risk of CHDs, the odds ratios (ORs) between heterozygotes (DI) versus wild homozygotes (DD) were 14.19 (95% CI: 2.21-591.52), 4.37 (95% CI: 1.24-23.47) and 4.77 (95% CI: 1.38-25.37) in mothers, fathers and offspring respectively (P < 0.05). CBS G919A was significantly associated with low risk of CHDs. The ORs of offspring between heterozygotes (GA) and mutant homozygotes (AA) versus wild homozygotes (GG) were 0.45 (95% CI: 0.23-0.87) and 0.34 (95% CI: 0.11-1.01), P < 0.05. And the parents carrying GA and AA genotypes also was lower risk of CHDs. For both of above two variants, the significant relations occurred especially in ventricular septal defect subgroup. Genotype combination analysis showed the more risk alleles (I and G) the family members carried, the higher the offspring risk of CHDs. And the serum fasting tHcy level was not significantly different among various groups and genotypes. CONCLUSION: CBS gene 844ins68 and G919A variations in nuclear families could be associated with CHDs risk of offspring, but not with serum fasting tHcy level.
BACKGROUND: High homocysteine blood concentrations predispose to coronary artery disease and statins influence homocysteine levels. AIM: To study whether genes that regulate homocysteine metabolism interact with statins to modify the risk of coronary heart disease (CHD) and other cardiovascular outcomes. METHODS: The Genetics of Hypertension Associated Treatment is an ancillary study of the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). The genotyped population in the Lipid-Lowering Trial of Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial included 9624 participants randomly assigned to pravastatin or to usual care. The efficacy of pravastatin in reducing risk of all-cause mortality and CHD was compared among genotype strata (MTHFR 677 CC, CT, and TT, MTHFR 1298 AA, AC, and CC, CBSins DD and I) by examining an interaction term in a proportional hazards model. RESULTS: No evidence existed of a pharmacogenetic effect on statins with the MTHFR 1298 A>C genotype for CHD risk. However, in persons with the CC variant for the MTHFR 677 C>T genotype, a significantly protective effect against CHD [0.71 (95% CI 0.58-0.87)] was shown, although in the CT [1.25 (95% CI 0.97-1.61)] and TT groups [0.80 (95% CI 0.50-1.28)] there were no such effects (interaction hazard ratio P=0.004). The CBSins, I+ variant was associated with a significantly reduced risk for CHD among those on statin treatment [0.58 (95% CI 0.44-0.78)] whereas the DD genotype showed no effect of statin therapy [1.01 (95% CI 0.84-1.20; P=0.002 for interaction]. For the endpoint all-cause mortality, no significant differences in efficacy were noted. CONCLUSION: Polymorphisms in genes in the homocysteine pathway (MTHFR 677 C>T and CBSins) appear to modify the efficacy of pravastatin in reducing risk of cardiovascular events.
Population-based allele frequencies and genotype prevalence are important for measuring the contribution of genetic variation to human disease susceptibility, progression, and outcomes. Population-based prevalence estimates also provide the basis for epidemiologic studies of gene-disease associations, for estimating population attributable risk, and for informing health policy and clinical and public health practice. However, such prevalence estimates for genotypes important to public health remain undetermined for the major racial and ethnic groups in the US population. DNA was collected from 7,159 participants aged 12 years or older in Phase 2 (1991-1994) of the Third National Health and Nutrition Examination Survey (NHANES III). Certain age and minority groups were oversampled in this weighted, population-based US survey. Estimates of allele frequency and genotype prevalence for 90 variants in 50 genes chosen for their potential public health significance were calculated by age, sex, and race/ethnicity among non-Hispanic whites, non-Hispanic blacks, and Mexican Americans. These nationally representative data on allele frequency and genotype prevalence provide a valuable resource for future epidemiologic studies in public health in the United States.
PURPOSE: Folate deficiency is considered to increase the risk for the development of malignant tumors such as prostate and colorectal cancer. Methionine synthase (MTR) and cystathionine ss-synthase (CBS) are enzymes that play a central role in folate metabolism, thereby affecting DNA methylation and synthesis. A single A-->G substitution at nucleotide 2756 of the MTR and a 68 bp CBS insertion polymorphism in exon 8 have been associated with decreased enzyme activity. The purpose of this study is to compare the association of the MTR A2756G polymorphism and CBS insertion polymorphism with susceptibility to carcinomas of the upper gastrointestinal tract. METHODS: Using the restriction fragment length polymorphism (RFLP)-PCR, the prevalence of MTR A2756G and CBS insertion polymorphism was determined in healthy controls (n = 257) and in patients with esophageal squamous cell carcinoma (ESCC) (n = 263), Barrett's esophagus-associated esophageal adenocarcinoma (BC) (n = 89), cardiac carcinoma (CC) (n = 144), or gastric carcinoma (GC) (n = 221) from German Caucasian subjects. RESULTS: No significant difference in MTR A2756G genotype distribution was observed between controls (A/A 66.9%, A/G 29.8%, G/G 3.3%) and patients with ESCC (A/A 61.7%, A/G 36.3%, G/G 2.1%), BC (A/A 69.2%, A/G 26.9%, G/G 3.9%), CC (A/A 51.8%, A/G 44.6%, G/G 3.6%), or GC (A/A 73.4%, A/G 20.9%, G/G 5.7%). Similarly, the CBS genotype (I: allele with 68 bp insertion; N: allele without insertion) distribution among German patients with ESCC (N/N 86.8%, I/N 13.2%), BC (N/N 90.2%, I/N 9.8%), CC (N/N 90.1%, I/N 9.9%) or GC (N/N 91.3%, I/N 8.7%) was not different from healthy controls (N/N 90.4%, I/N 9.6%). The gene allele constellation I/I was not present. CONCLUSIONS: The current study suggests that there is no association between MTR A2756G polymorphism and the CBS (844ins68) insertion polymorphism and cancer of the upper gastrointestinal tract.
OBJECTIVE: To determine if functional polymorphisms of folate/homocysteine pathway enzymes are associated with homocysteine concentrations and/or coronary artery calcification (CAC) scores in patients with systemic lupus erythematosus (SLE) and controls. METHODS: We investigated 163 SLE patients and 160 controls. Functional polymorphisms in 6 genes in the folate/homocysteine pathway were genotyped: 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, cystathionine ss-synthase (CBS) 844ins68, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, thymidylate synthase (TYMS) 1494del6, and dihydrofolate reductase (DHFR) c.86+60_78. RESULTS: Homocysteine levels were higher in African American SLE patients than Caucasian patients and African American controls. Genotype distributions were significantly different in African American and Caucasian controls for 6 of the 7 polymorphisms. Genotype distributions for each polymorphism did not differ significantly between SLE patients and controls even after stratification by race. Glomerular filtration rate was strongly negatively correlated to homocysteine levels, and was therefore adjusted for as a covariate in the models of the effects of the polymorphisms on homocysteine levels. In SLE patients none of the 7 polymorphisms was associated with homocysteine concentrations. In Caucasian controls only MTHFR 677C>T and 1298A>C showed effects on homocysteine similar to what would be expected from the literature. There were no genotypic associations with median CAC scores in SLE patients or controls with and without stratification by race. CONCLUSION: Polymorphisms in folate/homocysteine metabolizing enzymes do not predict higher homocysteine levels or CAC scores in patients with SLE.
BACKGROUND: Previous studies suggested an association between abdominal aortic aneurysm (AAA) and hyperhomocysteinaemia, a complex trait determined by genetic and environmental factors. Our hypothesis was that polymorphisms in genes directly or indirectly involved in methionine metabolism may contribute to AAA susceptibility. METHOD: We studied 56 polymorphisms in MTHFR, MTR, MTRR, CBS, MTHFD1, SLC19A1, NNMT, TCN2, AHCY, BHMT, BHMT2, FOLH1, TYMS, ENOSF1, SHMT1, PON1, PON2 genes according to their demonstrated/putative function, localisation in promoter or regulatory and coding regions and/or heterozygosity values >0.300. Polymorphisms were evaluated by using a primer extension based microarray technology in 423 AAA patients and 423 matched controls. RESULTS: All polymorphisms resulted in Hardy-Weinberg equilibrium in patients and controls. At the multiple logistic regression analysis adjusted for traditional cardiovascular risk factors (sex, age, hypertension, smoking habit, dyslipidaemia, diabetes) and chronic obstructive pulmonary disease (COPD), rs8003379 MTHFD1 (odds ratio (OR) 0.41, 95% confidence interval (CI) 0.26 to 0.65) and rs326118 MTRR (OR 0.47, 95% CI 0.29 to 0.77) polymorphisms resulted in independent susceptibility factor for AAA. CONCLUSIONS: After haplotype reconstruction, logistic regression analyses adjusted for traditional risk factors and COPD showed a significant association among AAA and AHCY, FOLH1, MTHFD1, MTR, NNMT, PON1 and TYMS haplotypes. Our findings offer new insights into the pathogenesis of AAA.
OBJECTIVE: The objective of this study was to examine whether selected genetic polymorphisms in the infant are associated with later-diagnosed cerebral palsy. METHODS: A population-based case-control study was conducted of 28 single-nucleotide polymorphisms measured in newborn screening blood spots. A total of 413 children with later-diagnosed cerebral palsy were born to white women in South Australia in 1986-1999, and there were 856 control children. Distributions of genotypic frequencies were examined in total cerebral palsy, in gestational age groups, and by types of cerebral palsy and gender. Genotyping was performed by using a TaqMan assay. RESULTS: For inducible nitric-oxide synthase, possession of the T allele was more common in all children with cerebral palsy and for heterozygotes who were born at term. For lymphotoxin alpha, homozygous variant status was associated with risk for cerebral palsy and with spastic hemiplegic or quadriplegic cerebral palsy. Among term infants, heterozygosity for the endothelial protein C receptor single-nucleotide polymorphism was more frequent in children with cerebral palsy. In preterm infants, the variant A allele of interleukin 8 and heterozygosity for the beta-2 adrenergic receptor were associated with cerebral palsy risk. Interleukin 8 heterozygote status was associated with spastic diplegia. Variants of several genes were associated with cerebral palsy in girls but not in boys. CONCLUSIONS: Two of the 28 single-nucleotide polymorphisms examined were associated with all types of spastic cerebral palsy in both gestational age groups and others with cerebral palsy in gestational age or cerebral palsy subgroups. Some of these associations support previous findings. There may be a genetic contribution to cerebral palsy risk, and additional investigation is warranted of genes and gene-environment interactions in cerebral palsy.
Hyperhomocysteinemia is a well-known independent marker factor for atherothrombotic diseases and may result from acquired and genetic influences. Several polymorphisms are suspected to be associated with hyperhomocysteinemia, but data are limited and inconsistent. High-throughput genotyping technologies, such as GenomeLab SNPStream, are now available. Moreover, an appropriate selection of SNPs to be analyzed could represent a strong resource to define the role of genetic risk factors. We developed a multiplex PCR-oligonucleotide extension approach by GenomeLab platform. We selected 72 SNPs based on their putative function and frequency in the candidate genes AHCY, BHMT, BHMT2, CBS, ENOSF1, FOLH1, MTHFD1, MTHFR, MTR, MTRR, NNMT, PON1, PON2, SLC19A1, SHMT1, TCN2, and TYMS. We were able to analyze 57 of the SNPs (79%). For MTHFR C677T and A1298C and MTR A2756G SNPs, we compared data obtained with an electronic microchip technology and found 99.2% concordance. We also performed a haplotype analysis. This approach could represent a useful tool to investigate the genotype-phenotype correlation and the association of these genes with hyperhomocysteinemia and correlated diseases.
OBJECTIVES: To estimate the frequency of the cystathionine beta-synthase deficiency caused by c.1105C>T mutation in Central Europe compared to Norway, and to examine the pathogenicity of the corresponding p.R369C mutant enzyme. STUDY DESIGN: Mutation c.1105C>T was analyzed in 600 anonymous Czech newborn blood spots. Catalytic activity and quaternary structure of the p.R369C mutant was evaluated after expression in 2 cellular systems. RESULTS: Population frequency of the c.1105C>T mutation was 0.005, predicting the birth prevalence of homocystinuria of 1:40000, which increased to 1:15500 in a model including 10 additional mutations. In Escherichia coli the p.R369C mutant misfolded, and its activity was severely reduced, and expression in Chinese hamster ovary cells enabled proper folding with activity decreased to 63% of the wild-type enzyme. This decreased activity was not due to impaired K(m) for both substrates but resulted from V(max) lowered to 55% of the normal cystathionine beta-synthase enzyme. CONCLUSIONS: The c.1105C>T (p.R369C) allele is common also in the Czech population. Although the p.R369C mutation impairs folding and decreases velocity of the enzymatic reaction, our data are congruent with rather mild clinical phenotype in homozygotes or compound heterozygotes carrying this mutation.
Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
In this paper, we describe the expression and characterization of recombinant human cystathionine beta-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the N-terminus. In our two-step purification procedure we are able to obtain a highly pure enzyme in sufficient quantities for crystallography and other physical chemical methods. We have investigated the biochemical and catalytic properties of purified full-length human CBS and of two truncation mutants lacking the C-terminal domain or both the N-terminal heme-binding and the C-terminal regulatory regions. Specifically, we have determined the pH optima of the different CBS forms and their kinetic and spectral properties. The full-length and the C-terminally truncated enzyme had a broad pH 8.5 optimum while the pH optimum of the N- and C- terminally truncated enzyme was sharp and shifted to pH 9. Furthermore, we have shown unequivocally that CBS binds one mole of heme per subunit by determining both the heme and the iron content of the enzyme. The activity of the enzyme was unaffected by the redox status of the heme iron. Finally, we show that CBS is stimulated by S-adenosyl- l-methionine but not its analogs.
Telomere shortening is a biomarker of cellular senescence and is associated with a wide range of age-related disease. Oxidative stress is also associated with physiological aging and several age-related diseases. Non-human studies suggest that variants in oxidative stress genes may contribute to both telomere shortening and biological aging. We sought to test whether oxidative stress-related gene polymorphisms contribute to variance in both telomere length and physical biomarkers of aging in humans. Telomere lengths were calculated for 190 (82 men, 108 women) participants aged 79 years and associations with 384 SNPs, from 141 oxidative stress genes, identified 9 significant SNPS, of which those from 5 genes (GSTZ1, MSRA, NDUFA3, NDUFA8, VIM) had robust associations with physical aging biomarkers, respiratory function or grip strength. Replication of associations in a sample of 318 (120 males, 198 females) participants aged 50 years confirmed significant associations for two of the five SNPs (MSRA rs4841322, p=0.008; NDUFA8 rs6822, p=0.048) on telomere length. These data indicate that oxidative stress genes may be involved in pathways that lead to both telomere shortening and physiological aging in humans. Oxidative stress may explain, at least in part, associations between telomere shortening and physiological aging.
BACKGROUND: Non-Hodgkin lymphoma (NHL) is the fifth most common cancer in the U.S. and few causes have been identified. Genetic association studies may help identify environmental risk factors and enhance our understanding of disease mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: 768 coding and haplotype tagging SNPs in 146 genes were examined using Illumina GoldenGate technology in a large population-based case-control study of NHL in the San Francisco Bay Area (1,292 cases 1,375 controls are included here). Statistical analyses were restricted to HIV- participants of white non-Hispanic origin. Genes involved in steroidogenesis, immune function, cell signaling, sunlight exposure, xenobiotic metabolism/oxidative stress, energy balance, and uptake and metabolism of cholesterol, folate and vitamin C were investigated. Sixteen SNPs in eight pathways and nine haplotypes were associated with NHL after correction for multiple testing at the adjusted q<0.10 level. Eight SNPs were tested in an independent case-control study of lymphoma in Germany (494 NHL cases and 494 matched controls). Novel associations with common variants in estrogen receptor 1 (ESR1) and in the vitamin C receptor and matrix metalloproteinase gene families were observed. Four ESR1 SNPs were associated with follicular lymphoma (FL) in the U.S. study, with rs3020314 remaining associated with reduced risk of FL after multiple testing adjustments [odds ratio (OR) = 0.42, 95% confidence interval (CI) = 0.23-0.77) and replication in the German study (OR = 0.24, 95% CI = 0.06-0.94). Several SNPs and haplotypes in the matrix metalloproteinase-3 (MMP3) and MMP9 genes and in the vitamin C receptor genes, solute carrier family 23 member 1 (SLC23A1) and SLC23A2, showed associations with NHL risk. CONCLUSIONS/SIGNIFICANCE: Our findings suggest a role for estrogen, vitamin C and matrix metalloproteinases in the pathogenesis of NHL that will require further validation.
BACKGROUND: Impaired homocysteine metabolism is associated with a number of vasculopathies including extracranial aneurysms. We analyzed the possible association of nine genetic variants of homocysteine metabolism with the occurrence of intracranial aneurysms. METHODS: Caucasian patients (n = 255) treated at two German hospitals for intracranial aneurysms and local controls (n = 348) were genotyped for the following polymorphisms: methionine synthase (MTR) c.2756A-->G, methylenetetrahydrofolate reductase (MTHFR) c.677C-->T, MTHFR c.1298A-->C, cystathionine beta-synthase (CBS) c.844_855ins68, CBS c.833T-->C, dihydrofolate reductase (DHFR) c.594 + 59del19bp, glutathione S-transferase Omega-1 (GSTO1) c.428C-->A, reduced folate carrier 1 (RFC1) c.80G-->A and transcobalamin 2 (Tc2) c.776C-->G. RESULTS: The G-allele of the missense polymorphism Tc2 c.777C-->G was found to be underrepresented in patients, suggesting that this variant may protect from the formation of cerebral aneurysms [odds ratio per two risk alleles (OR) 0.48; 95% confidence interval (CI) 0.30-0.77; p = 0.002]. We obtained borderline results for the G-allele of RFC1 c.80G-->A (OR 1.64; 95% CI 1.01-2.65; p = 0.051) and the insertion allele of DHFR c.594 + 59del19bp (OR 1.61; 95% CI 1.00-2.60; p = 0.059), which were found to be overrepresented in patients. CONCLUSION: Polymorphisms of homocysteine metabolism are possible risk factors for the formation of intracranial aneurysms.
One-carbon metabolism mediates the interconversion of folates for the synthesis of precursors used in DNA synthesis, repair, and methylation. Inadequate folate nutrition or compromised metabolism can disrupt these processes and facilitate carcinogenesis. In this study, we investigated associations of 39 candidate single nucleotide polymorphisms (SNP) in 9 one-carbon metabolism genes with risk of prostate cancer using 1,144 cases and 1,144 controls from the Cancer Prevention Study-II Nutrition Cohort. None of these SNPs were significantly associated with prostate cancer risk, either overall or in cases with advanced prostate cancer. Thus, our findings do not support the hypothesis that common genetic variation in one-carbon metabolism genes influences prostate cancer risk.
Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) and cystathionine beta-synthase (CBS) genes, involved in the intracellular metabolism of homocysteine (Hcy), can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of CBS T833C, G919A and the insertion of 68-bp (844ins68) polymorphisms and their correlation with Hcy, folate and B(12) in 220 children previously genotyped for MTHFR C677T, A1298C, and MTRR A66G. The prevalence of heterozygote children for 844ins68 was 19.5%. The T833C CBS mutation was identified in association with 844ins68 in all the carriers of the insertion. Genotyping for CBS G919A mutation showed that all the children presented the GG genotype. Analysis of Hcy, B(12) and folate, according to the combination of the different genotypes of the C677T and A1298C MTHFR, A66G MTRR, and 844ins68 CBS showed that the 677TT/1298AA/68WW genotype is associated with an increase in Hcy, when compared to the 677CC/1298AC/68WW (P = 0.033) and the 677CT/1298AA/68WW genotypes (P = 0.034). Since B(12) and folate were not different between these groups, a genetic interaction between diverse polymorphisms probably influences Hcy. Our results emphasize the role of genetic interactions in Hcy levels.
Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 microg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts.
Spontaneous preterm birth (<37 weeks gestation-PTB) occurs in approximately 12% of pregnancies in the United States, and is the largest contributor to neonatal morbidity and mortality. PTB is a complex disease, potentially induced by several etiologic factors from multiple pathophysiologic pathways. To dissect the genetic risk factors of PTB a large-scale high-throughput candidate gene association study was performed examining 1536 SNP in 130 candidate genes from hypothesized PTB pathways. Maternal and fetal DNA from 370 US Caucasian birth-events (172 cases and 198 controls) was examined. Single locus, haplotype, and multi-locus association analyses were performed separately on maternal and fetal data. For maternal data the strongest associations were found in genes in the complement-coagulation pathway related to decidual hemorrhage in PTB. In this pathway 3 of 6 genes examined had SNPs significantly associated with PTB. These include factor V (FV) that was previously associated with PTB, factor VII (FVII), and tissue plasminogen activator (tPA). The single strongest effect was observed in tPA marker rs879293 with a significant allelic (p = 2.30x10(-3)) and genotypic association (p = 2.0x10(-6)) with PTB. The odds ratio (OR) for this SNP was 2.80 [CI 1.77-4.44] for a recessive model. Given that 6 of 8 markers in tPA were statistically significant, sliding window haplotype analyses were performed and revealed an associating 4 marker haplotype in tPA (p = 6.00x10(-3)). The single strongest effect in fetal DNA was observed in the inflammatory pathway at rs17121510 in the interleukin-10 receptor antagonist (IL-10RA) gene for allele (p = 0.01) and genotype (p = 3.34x10(-4)). The OR for the IL-10RA genotypic additive model was 1.92 [CI 1.15-3.19] (p = 2.00x10(-3)). Finally, exploratory multi-locus analyses in the complement and coagulation pathway were performed and revealed a potentially significant interaction between a marker in FV (rs2187952) and FVII (rs3211719) (p<0.001). These results support a role for genes in both the coagulation and inflammation pathways, and potentially different maternal and fetal genetic risks for PTB.
A high homocysteine, low folate phenotype is a feature of many diseases. The effect of the cystathionine beta-synthase (CBS) 844ins68 polymorphism on homocysteine and folate concentrations was examined alone and in the context of the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism in a Northwestern European male population. The MTHFR 677TT genotype is known to be associated with increased homocysteine and decreased folate relative to CT heterozygotes and CC homozygotes in this and other populations. MTHFR 677TT homozygotes who were also CBS 844ins68 carriers had homocysteine and folate concentrations similar to those of individuals with the MTHFR 677CT and CC genotypes. Homocysteine levels in MTHFR 677TT subjects carrying the CBS 844ins68 allele were 24.1% lower than in non-carriers (6.66 vs 8.77 micromol/l, P=0.045), and serum folate levels were 27.7% higher (11.16 vs 8.74 nmol/l, P=0.034). These findings suggest that the CBS 844ins68 allele 'normalizes' homocysteine and folate levels in MTHFR 677TT individuals.
Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.
Missense mutations in the cystathionine beta-synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild-type (Tg-hCBS) or p.S466L (Tg-S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg-S466L mice was 142+/-55 microM compared to 16+/-13 microM for hCBS mice. Tg-S466L mice also had significantly higher levels of total free homocysteine and S-adenosylhomocysteine in liver and kidney. Only 48% of Tg-S466L mice had detectable CBS protein in the liver, whereas all the Tg-hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg-S466L animals compared to Tg-hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg-S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S-adenosylmethionine (AdoMet). However, even in Tg-S466L animals that had in vitro liver CBS activity equivalent to Tg-hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo.
OBJECT: Functionally relevant polymorphisms of methionine and folate metabolism have been shown to be associated with various human cancer entities including cerebral lymphoma and glioblastoma multiforme. The authors investigated the association of 7 functional polymorphisms of methionine metabolism with meningioma formation. METHODS: This case-controlled, monocenter association study included 290 patients of Caucasian origin undergoing surgical resection for intracranial meningioma (World Health Organization [WHO] Grade I, 190 cases; WHO Grade II, 82 cases; WHO Grade III, 18 cases) and 287 age- and sex-matched local controls. The authors analyzed the following genetic variants: dihydrofolate reductase c.594+59del19, 5,10-methylenetetrahydrofolate reductase c.677C > T and c.1298A > C, 5-methyltetrahydrofolate-homocysteine S-methyltransferase (MTR) c.2756A > G, reduced folate carrier 1 c.80G > A, cystathionine beta-synthase (CBS) c.844_855ins68 and transcobalamin 2 c.776C > G. RESULTS: The variant CBS c.844_855ins68 -- that is, the allele carrying the insertion ("ins" or "i") as opposed to the wild-type allele designated as deletion ("del" or "d") -- was significantly overrepresented in meningioma patients (dd/ id/ii: 0.81/0.18/0.01) in comparison with the controls (dd/id/ii: 0.88/0.12/0; 2 df, chi-square 8.97, p = 0.011; multiple nominal regression with age and sex as covariables). In addition, explorative analyses revealed an association of the MTR c.2756A > G variant with meningioma WHO Grade III (AA/AG/GG: patients, 1.0/0/0; controls, 0.64/0.32/0.04; 2 df, chi-square 14.44, p = 0.001). CONCLUSIONS: The results of this study suggest that genetic variants of methionine metabolism are associated with meningioma formation.
We previously reported a lower risk of non-Hodgkin lymphoma (NHL) associated with high consumption of vitamin B6 and methionine, dietary determinants of one-carbon metabolism. Evidence has linked genetic variants involved in one-carbon metabolism to NHL. We investigated 30 polymorphisms in 18 genes for their main effect on NHL among 1141 incident cases and 949 population-based controls and examined gene-nutrient interactions in a subgroup of 386 cases and 319 controls who provided detailed food-frequency information. Odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for age, sex, and race. We observed a decreased risk of NHL over-all with BHMTEx8+453A>T and increased risk with CBS Ex13+41C>T, FPGS Ex15-263T>C, and SHMT1 Ex12+138C>T and Ex12+236C>T. Furthermore, significant gene-nutrient interactions limited the protective association comparing high versus low vitamin B6 to FPGS Ex15-263T>C CC (OR = 0.22; 95% CL = 0.10-0.52), MTHFS IVS2-1411T>G TT/TG (OR = 0.54; 95% CI = 0.36-0.81), and MTR Ex26-20A>G AA (OR = 0.55; 95% CI = 0.35-0.86) genotypes, and the protective association of methionine to FTHFD Ex10-40G>TGG (OR = 0.63; 95% CI = 0.44-0.91), MTHFR Ex8-62A>C CC (OR = 0.13; 95% CI = 0.04-0.39), and MTRR Ex5+136T>CTT (OR = 0.67; 95% CI = 0.47-0.97) genotypes. Warranting replication, our finding of gene-nutrient interactions in one-carbon metabolism supports their etiologic involvement in lymphomagenesis.
Dysregulation of the one-carbon metabolic pathway, which controls nucleotide synthesis and DNA methylation, may promote lymphomagenesis. We evaluated the association between polymorphisms in one-carbon metabolism genes and risk of non-Hodgkin lymphoma (NHL) in a population-based case-control study in Australia. Cases (n = 561) and controls (n = 506) were genotyped for 14 selected single-nucleotide polymorphisms in 10 genes (CBS, FPGS, FTHFD, MTHFR, MTHFS, MTR, SHMT1, SLC19A1, TCN1, and TYMS). We also conducted a meta-analysis of all studies of Caucasian populations investigating the association between MTHFR Ex5+79C > T (a.k.a., 677C>T) and NHL risk. A global test of 13 genotypes was statistically significant for diffuse large B-cell lymphoma (DLBCL; P = 0.008), but not for follicular lymphoma (FL; P = 0.27) or all NHL (P = 0.17). The T allele at MTHFR Ex5+79 was marginally significantly associated with all NHL (OR = 1.25, 95% CI = 0.98-1.59) and DLBCL (1.36, 0.96-1.93). The T allele at TYMS Ex8+157 was associated with a reduced risk of FL (0.64, 0.46-0.91). An elevated risk of NHL was also observed among carriers of the G allele at FTHFD Ex21+31 (all NHL, 1.31, 1.02-1.69; DLBCL, 1.50, 1.05-2.14). A meta-analysis of 11 studies conducted in Caucasian populations of European origin (4,121 cases and 5,358 controls) supported an association between the MTHFR Ex5+79 T allele and increased NHL risk (additive model, P = 0.01). In conclusion, the results of this study suggest that genetic polymorphisms of one-carbon metabolism genes such as MTHFR and TYMS may influence susceptibility to NHL.
We have previously reported significant inverse associations between bladder cancer risk and dietary intake of vitamins B2, B6, B12, folate and protein in a hospital-based bladder cancer case-control study conducted in Spain (1,150 cases;1,149 controls). Because these dietary factors are involved in the one-carbon metabolism pathway, we evaluated associations between bladder cancer risk and 33 single nucleotide polymorphisms (SNPs) in 8 genes (CBS, CTH, MTHFR, MTR, MTRR, SHMT1, SLC19A1 and TYMS) and interactions with dietary variables involved in this pathway. Two SNPs in the CTH gene were significantly associated with bladder cancer risk. OR (95% CI) for heterozygous and the homozygous variants compared to homozygous wild-type individuals were: 1.37 (1.04-1.80) IVS3-66 A > C and 1.22 (1.02-1.45) IVS10-430 C > T. Because the CTH gene is important for glutathione synthesis, we examined interactions with the GSTM1 gene, which codes for glutathione S-transferase muu. Increased risk for individuals with the IVS10-430 CT or TT genotype was limited to those with the GSTM1 null genotype (p-interaction = 0.02). No other SNPs were associated with risk of bladder cancer. These findings suggest that common genetic variants in the one-carbon pathway may not play an important role in the etiology of bladder cancer. However, our results provide some evidence that variation in glutathione synthesis may contribute to risk, particularly among individuals who carry a deletion in GSTM1. Additional work is needed to comprehensively evaluate genomic variation in CTH and related genes in the trans-sulfuration pathway and bladder cancer risk.
Epidemiological evidence suggests that intake of folate and other B-vitamins and genetic variants in the one-carbon metabolism pathway could influence the risk of breast cancer. Previous studies have focused on 2 polymorphisms in the methylenetetrahydrofolate gene (MTHFR A222V and E429A); however, findings are inconclusive. In a large population-based case-control study in Poland (2,386 cases, 2,502 controls), we investigated the association between breast cancer risk and 13 polymorphisms in 6 one-carbon metabolism genes (MTHFR, MTR, MTRR, CBS, SHMT1 and SLC19A1). Data suggested an association between a nonsynonymous change in the gene coding for methionine synthase (MTR D919G) and reduced breast cancer risk: OR (95% CI) = 0.84 (0.73-0.96) and 0.85 (0.62-1.15) for heterozygous and homozygote variant genotypes, respectively, compared with common homozygotes; p-trend = 0.01, false discovery rate = 0.14. We found no significant associations between other variants and breast cancer risk, including MTHFR A222V or E429A. Meta-analyses including published studies of MTHFR A222V (8,330 cases and 10,825 controls) and E429A (6,521 cases and 8,515 controls) supported the lack of an overall association; however, studies suggested an increase in risk among premenopausal women. In conclusion, this report does not support a substantial overall association between the evaluated polymorphisms in the one-carbon metabolism pathway and breast cancer risk.
Cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 degrees C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.
BACKGROUND: Non-pathological cognitive ageing is a distressing condition affecting an increasing number of people in our 'ageing society'. Oxidative stress is hypothesised to have a major role in cellular ageing, including brain ageing. RESULTS: Associations between cognitive ageing and 325 single nucleotide polymorphisms (SNPs), located in 109 genes implicated in oxidative stress and/or cognition, were examined in a unique cohort of relatively healthy older people, on whom we have cognitive ability scores at ages 11 and 79 years (LBC1921). SNPs showing a significant positive association were then genotyped in a second cohort for whom we have cognitive ability scores at the ages of 11 and 64 years (ABC1936). An intronic SNP in the APP gene (rs2830102) was significantly associated with cognitive ageing in both LBC1921 and a combined LBC1921/ABC1936 analysis (p < 0.01), but not in ABC1936 alone. CONCLUSION: This study suggests a possible role for APP in normal cognitive ageing, in addition to its role in Alzheimer's disease.