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Related Collections Related Articles in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 December 2001, Vol. 98, No. 12, pp. 3497-3500 CORRESPONDENCE To the editor: Transcobalamin polymorphism and homocysteine Namour et al1 suggest that codon 259 of the transcobalamin (TC) gene is a major determinant of TC polymorphism in Caucasians and that heterozygous individuals have higher plasma homocysteine. Total serum homocysteine (tHcy) is increased in patients with Alzheimer disease (AD) and may be a risk factor for cognitive decline.2-5 Furthermore, TC saturation declines with age in AD.6 We therefore determined nonfasting tHcy, TC phenotype, and codon 259 polymorphism in 144 (93 female, 51 male) healthy elderly volunteers (73) and dementia patients (71) recruited to a study of tHcy and cognition (the COBALZ II project) after ethical committee approval. Phenotypes were identified by polyacrylamide gel electrophoresis (PAGE) of neuraminidase-treated radiolabeled serum samples (Figure 1) and genotypes by solid-phase minisequencing (Figure 2).7,8 Nonfasting tHcy was assayed with the Drew Scientific DS30 Hcy Analyser (Barrow in Furness, England); Vitamin B12 and folate were assayed with the Bayer ACS 180 Automated Chemiluminescence System (Newbury, England). View larger version (125K): [in this window] [in a new window]   Figure 1. TC phenotypes revealed by PAGE. Serum (25 µL) saturated with 10 µL of radioactive B12 (57Co-B12; 200mCi/mg, 1 µCi/mL [7.4 GBq/mg, 37 kBq/mL]; Amersham Pharmacia Biotech "CT2" diluted 1:5 N-saline) was incubated with 25 µL of neuraminidase (0.2 IU/mL) in 25 µL of buffer (0.05 M sodium acetate, 0.1% CaCl2, and 0.95% NaCl, pH 5.5) at 37°C for 30 minutes. Twenty-five µL was replaced with buffer (65% [wt/vol] sucrose) and bromophenol blue as an albumin- and front-marker. PAGE was performed in a 10% polyacrylamide gel (1.5 mm × 7.0 cm × 12.5 cm) with 3.5% concentrating top gel (1.5 cm high) and Tris-glycine electrode buffer at pH 8.3 and electrophoresed at 250 V. View larger version (29K): [in this window] [in a new window]   Figure 2. TC genotypes identified by solid-phase minisequencing. (A) TCII-259C homozygotes (PP); (B) TCII-259C/G heterozygotes (PR); (C) TCII-259G homozygotes (RR). Genomic DNA was amplified by PCR using the sense primer biotin-5'-GTGCGAGAGGAGATCTTGAA-3' and the antisense primer 5'-GTAGGTCTTGTGGTTCAGAA-3'. Biotinylated products were bound to streptavidine-coated microtiter plates (Wallac, Turku, Finland) and denatured with NaOH. Thermo Sequenase DNA polymerase (Thermo Sequenase Dye Terminator Kit, Amersham, Buckinghamshire, United Kingdom), fluorescent ddNTP, and the antisense detection primer 5'-CTGTTCCCAGTTCTGCCCCA-3' were added to generate an allele-specific pattern. After the minisequence reaction, the plates were washed and the extended sequence primers were released by incubation with formamide. These were separated and analyzed by capillary electrophoresis and laser-induced fluorescence in an ABI 310 genetic analyzer (Perkin-Elmer, Cambridge, England). Codon 259 polymorphism and phenotype were highly concordant (Table 1). PAGE reveals a doublet pattern in homozygotes, triplets due to overlapping bands in heterozygotes, and a quadruplet pattern in FX phenotypes (Figure 1). Alternative cleavage of the signal peptide might generate doublets rather than definitive isotypes.9 Weaker radiolabeling of TC-X in heterozygotes resulted in misclassification of some phenotypes; it perhaps reflects differential transcription.1 Codon 259 polymorphism and phenotypic distributions paralleled previous studies (PP = 32.6%, PR = 49.3%, and RR = 18.1%), (MM = 34%, MX = 43.7%, XX = 21.5%, and FX = 0.07%).1,7                                View this table: [in this window] [in a new window]   Table 1. Distribution of TC phenotypes and genotypes with their respective median and interquartile ranges for serum B12, folate, and tHcy Thirty individuals receiving B vitamins or homocysteine-disruptive medication were excluded from further analysis. Vitamin B12, folate, and tHcy did not differ among isotypes (Table 1). The apparent trend for increasing tHcy in the presence of an X variant or codon 259R allele was not significant using the Jonckheere-Terpstra test (asymptotic P value for genotype was .16 one-sided and .33 two-sided; P value for phenotypes was .08 one-sided and .17 two-sided) or using ANOVA with a Bonferroni correction. Table 2 shows the results of a generalized linear model of known tHcy determinants, together with diagnosis as an additional independent variable.                                View this table: [in this window] [in a new window]   Table 2. Results of a generalized linear model of known tHcy determinants together with diagnosis as an additional independent variable We therefore confirm that codon 259 is a major determinant of TC polymorphism in populations where M and X phenotypes predominate. The genetic basis underlying F and S phenotypes remains elusive. TC isotype is not a significant risk factor for the development of hyperhomocysteinemia in elderly Caucasians, especially when considered in relation to established risk factors such as creatinine and folate. Andrew McCaddon, Kaj Blennow, Peter Hudson, Björn Regland, and Diane Hill Correspondence: Andrew McCaddon, Gardden Road Surgery, Rhosllanerchrugog, Wrexham, North Wales, LL14 2EN, United Kingdom Acknowledgments Supported by the Wales Office of Research and Development (WS98/2/011). A.M. is an honorary research fellow of the University of Wales College of Medicine. COBALZ II investigators also include Alan Hughes, Robert Gray, Joan Barber, Alwyn Lloyd, Gareth Davies, Steve Tandy, Jenny Duguid, John H. H. Williams, and Clare Wilkinson References 1. Namour F, Olivier J-L, Abdelmouttaleb I, et al. Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood. Blood. 2001;97:1092-1098[Abstract/Free Full Text]. 2. McCaddon A, Davies G, Hudson P, Tandy S, Cattell H. Total serum homocysteine in senile dementia of Alzheimer type. Int J Ger Psych. 1998;13:235-239[CrossRef][Medline] [Order article via Infotrieve]. 3. Clarke R, Smith AD, Jobst K, Refsum H, Sutton L, Ueland PM. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol. 1998;55:1449-1455[Abstract/Free Full Text]. 4. Lehmann M, Gottfries CG, Regland B. Identification of cognitive impairment in the elderly: homocysteine is an early marker. Dement Geriatr Cogn Disord. 1999;10:12-20[CrossRef][Medline] [Order article via Infotrieve]. 5. McCaddon A, Hudson P, Davies G, Hughes A, Williams JHH, Wilkinson C. Homocysteine and cognitive decline in healthy elderly. Dement Geriatr Cogn Disord. 2001;12:309-313[CrossRef][Medline] [Order article via Infotrieve]. 6. McCaddon A, Hudson P, Abrahamsson L, Olofsson H, Regland B. Analogues, ageing and aberrant assimilation of vitamin B12 in Alzheimer's disease. Dement Geriatr Cogn Disord. 2001;12:133-137[CrossRef][Medline] [Order article via Infotrieve]. 7. Fràter-Schröder M, Hitzig WH, Butler R. Studies on transcobalamin (TC), 1: detection of TCII isoproteins in human serum. Blood. 1979;53:193-203[Free Full Text]. 8. Blennow K, Ricksten A, Prince JA, et al. No association between the alpha2-macroglobulin (A2M) deletion and Alzheimer's disease, and no change in A2M mRNA, protein, or protein expression. J Neural Transm. 2000;107:1065-1079. 9. Rothenberg SP, Quadros EV, Regec A. Transcobalamin II. In: Banerjee R, ed. Chemistry and Biochemistry of B12. New York, NY: John Wiley & Sons; 1999:441-473. Response: Transcobalamin polymorphism, homocysteine, and aging The DNA sequencing of the transcobalamin (TC) gene expressed in various cells identified 5 amino acid substitutions at codons 198, 219, 234, 259, and 376.1,2 Codon 234 and 259 variability corresponded to the replacement of proline by arginine.2 By analyzing the DNA and sera from 159 healthy Caucasians from 3 regions of northeastern France (Lorraine, Franche-Comté, and Bourgogne), we found that the codon 259 substitution was a single nucleotide polymorphism with a biallelic distribution, while no substitution was found in the other codons.3 There is therefore no doubt that codon 259 is a major genetic determinant of the TC polymorphism. We compared the TC genotype to the isoelectric point of TC isoforms and found a concordance between the amino acid substitution and the isoelectric point. Urea was used to avoid the formation of aggregates in our isoelectric focusing (IEF) analysis of TC. McCaddon et al confirm our findings on the TC codon 259 polymorphism's biallelic distribution. They compared it to a SDS-PAGE phenotyping of TC, which identifies 4 phenotypes, instead of the 2 isoforms observed in IEF. Apparently, no urea was used in their electrophoresis analysis. Urea avoids the formation of TC aggregates and induces denaturation of TC conformation, as shown by the comparison of IEF profiles of TC from HT-29 cells with and without urea.3,4 In our opinion, the role of alternative cleavage of the signal peptide in generating TC isoforms needs therefore to be confirmed, as only 2 isoforms, not 4, were found in IEF with urea. The TC codon 259 polymorphism affects apo (unsaturated) TC isoforms concentration as shown by IEF analysis of heterozygous HT-29 cells and by sera apo TC assay from homozygous and heterozygous subjects.3 This is why we thought that this polymorphism could affect the vitamin B12 cellular availability and, consequently, the homocysteine concentration. Surprisingly, the relatively lower concentration of apo TC concentration observed in the Arg/Arg homozygous subjects was not related to an increase of homocysteinemia. Indeed, a significantly higher homocysteine concentration was found in heterozygous subjects than in subjects with either homozygous genotype, while the highest apo TC concentration was observed in subjects with Pro/Pro genotypes. An explanation could be that the binding of TC to the dimeric TC receptor described by Bose et al5 is associated with a dimerization of TC and that the heterodimer of Pro/Arg isoforms has a lower affinity than Pro/Pro or Arg/Arg homodimers. This hypothesis is currently under investigation. McCaddon et al found no relation between homocysteine concentration and TC codon 259 polymorphism. They performed their study in elderly patients, but no description of the age distribution is given. Since the publication of our paper, we performed TC genotyping and homocysteine analysis in a population of 76 healthy subjects from a preventive medicine center in Lorraine, including 40 elderly subjects. The data summarized in Table 1 confirm the influence of the TC codon 259 polymorphism on homocysteine concentration. But the difference in homocysteine concentration between heterozygous and homozygous subjects was not significant in our elderly subgroup. The influence of age as a determinant of homocysteine concentration may explain the results obtained by McCaddon et al in elderly subjects. Our and McCaddon et al's data suggest therefore that investigations of TC genotype in diseases associated with mild hyperhomocysteinemia should take into consideration the age distribution. With regard to the literature, the homocysteinemia increase in the elderly is not fully understood. One should be careful not to transpose to the elderly the role of genetic and nutritional homocysteine determinants characterized in the general population.                                View this table: [in this window] [in a new window]   Table 1. Plasma homocysteine concentration according to transcobalamin codon 259 genotype in elderly and younger subjects Farès Namour and Jean-Louis Guéant Correspondence: Jean-Louis Guéant, Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, INSERM 0014, Faculté de Médecine de Nancy, France References 1. Platica O, Janeczko R, Quadros E, Regec A, Romain R, Rothenberg SP. The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I. J Biol Chem. 1991;266:7860-7863[Abstract/Free Full Text]. 2. Li N, Seetharam S, Lindemans J, Alpers DH, Arwert F, Seetharam B. Isolation and sequence analysis of variant forms of human transcobalamin II. Biochem Biophys Acta. 1993;1172:21-30[Medline] [Order article via Infotrieve]. 3. Namour F, Olivier J-L, Abdelmoutaleb I, Adjalla C, Debard R, Guéant J-L. Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood. Blood. 2001;97:1092-1098. 4. Schohn H, Guéan JL, Girr M, et al. Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line. J Biochem. 1991;280:427-430. 5. Bose S, Feix J, Seetharam S, Seetharam B. Dimerization of transcobalamin II receptor. J Biol Chem. 1996;271:11718-11725[Abstract/Free Full Text]. 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