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Transferrin gene polymorphism in Alzheimer's disease and dementia with Lewy bodies in humans
Neuroscience Letters 317 (2002) 13–16 www.elsevier.com/locate/neulet
Transferrin gene polymorphism in Alzheimer’s disease and dementia with Lewy bodies in humans Rafiqul I. Hussain a, Clive G. Ballard b, James A. Edwardson b, Christopher M. Morris a,* a
MRC Building, Institute for the Health of the Elderly, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, UK b Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK Received 18 June 2001; received in revised form 16 October 2001; accepted 16 October 2001
Abstract Genetic studies in Alzheimer’s disease (AD), have indicated that the apolipoprotein E locus (APO E) is a major susceptibility factor, but that it can only account for approximately 50% of AD cases. Several other studies have attempted to identify additional genetic loci associated with disease development, often based on a candidate gene approach. As several lines of evidence indicate that oxidative stress and free radical damage occur in AD, the transferrin gene (TF) has been suggested as a candidate locus for AD since it is the major transport protein for iron, which itself is a major factor in free radical generation. Previous studies have shown elevated TF C2 allele frequencies in AD, this being specifically associated with carriers of the APO E 14 allele. We have therefore determined the influence of the common polymorphisms in TF, C1 and C2, in dementia. The frequency of the C2 allele was not significantly associated with AD. Stratification of cases according to the APO E 14 allele showed a highly significant excess of the C2 allele in AD cases without the 14 allele, contrasting with previous studies. Given the contrasting findings between reports of altered TF C2 allele frequencies, the TF locus would not appear to be a strong risk factor for AD in this population. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Transferrin; Oxidative stress; Apolipoprotein E
Alzheimer’s disease (AD) is the commonest cause of dementia in older people. Its aetiology is complex, involving both environmental and genetic causes. Familial earlyonset AD is known to be caused by mutations in the amyloid precursor protein, or Presenilin 1 or Presenilin 2 [9], though these causes of AD are relatively uncommon. The 14 allele of the apolipoprotein E gene (APO E) is a major susceptibility factor for late-onset AD, and this finding in AD has emphasized the central role played by genetic factors in dementia development and progression [3,21]. Evidence suggests that other risk factor loci exist and whilst whole genome scanning has indicated several loci associated with AD, a candidate gene approach based on genes involved in the pathophysiology of AD has also frequently been employed [11,18]. Increased production of free radicals and oxidative stress may be involved in the development of AD [1]. Increased * Corresponding author. Tel.: 144-191-273-5251; fax: 144-191272-5291. E-mail address: [email protected] (C.M. Morris).
iron levels are found in the AD brain and there is an increase in the levels of the iron storage protein, ferritin, in AD [4]. Iron is found associated with senile plaques [7,15] and also with neurofibrillary tangles in AD [6,7,15]. Immunostaining for ferritin has also shown the presence of ferritin-positive microglia associated with plaques in AD [8]. There is also evidence for increased free radical formation in the brains of AD cases compared with controls, and this may be potentiated by iron [17]. Transferrin (TF) is the major circulating glycoprotein involved in the transport of iron to cells, and is produced by the liver and also by oligodendrocytes and choroidal epithelium in the central nervous system [14]. TF binds two atoms of ferric iron per molecule with very high affinity, resulting in essentially no free ferric ions in solution when the protein is in its normal physiological excess, thereby preventing free radical production. Several allelic isoforms of TF have been identified. The commonest forms are C1 and C2 (C1, Pro 570; C2, Ser 570) found at frequencies of approximately 80 and 20%, respectively. It has been suggested that the C2 protein has reduced ironbinding capacity due to the non-conservative amino acid
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 40 3- X
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R.I. Hussain et al. / Neuroscience Letters 317 (2002) 13–16
change [25]. On the basis of this, it could be argued that if free radical production was a major pathogenic event in AD, then individuals carrying the C2 allele could be at greater risk of developing the disorder. This has been suggested by several studies which have demonstrated an increase in the C2 allele frequency in AD [16,22,23]. We have therefore analyzed a large series of neuropathologically confirmed cases of AD and controls for the C1 and C2 polymorphisms in TF to determine their effect in this population. Additionally, we have genotyped a cohort of cases of dementia with Lewy bodies (DLB) to determine if any changes associated with TF are specific to AD or involved in both DLB and AD. All cases and controls were from the North of England and were part of a large ongoing study. A total of 180 cases of AD (age range, 46–93; mean ^ SD, 77.9 ^ 8.0 years), 65 DLB (age range, 65–95; mean ^ SD, 79.2 ^ 6.5 years) and 121 non-demented control cases (age range, 65–100; mean age at death, 77.9 ^ 8.7 years) were studied. AD cases were clinically diagnosed as such according to NINCDSADRDA criteria [13] and neuropathologically verified as such. DLB cases were clinically diagnosed according to consensus criteria [12] and met operationalized criteria for a diagnosis of DLB [12]. Control cases had no history of neuropsychiatric disease, and on neuropathological examination, showed age related neuropathology only (low or absent senile plaques, absent neocortical neurofibrillary tangles). Genomic DNA was isolated from frozen brain tissue using standard proteinase K digestion followed by phenol/ chloroform extraction. PCR for the TF C1 and C2 alleles was performed using the primers: TFC2 forward, 5 0 GCTGTGCCTTGATGGTACCAGGTAA-3 0 ; and TFC2 reverse, 5 0 -GGACGCAAGCTTCCTTATCT-3 0 , described by Namekata and colleagues [16]. The reactions were performed in a final volume of 20 ml standard buffer containing 5 pmol of each primer, 0.5 U Taq polymerase (Pharmacia, UK), 200 mM each deoxy-nucleotide, and 200 ng of DNA. Reaction conditions included an initial denaturation at 948C for 1 min and 30 s, followed by 40 cycles of annealing at 528C for 1 min, extension at 728C for 1 min, and denaturation at 958C for 30 s. PCR products were digested overnight at 378C with 10 U BstEII (New England Biolabs, UK) and then run on composite 3% NuSeive GTG/ 1% standard agarose gels (Flowgen, UK). The TF C2 allele was determined by the presence of a 110 bp uncut PCR product and the C1 allele by the presence of an 89 bp band. Amplification of the APO E gene containing the allelic sites was performed by previously described methods [10,24]. Statistical analysis of allele frequencies was carried out using the Chi-square test with Yates’ correction for small sample sizes when appropriate. Logistic regression analysis was performed using the presence of the APO E 14 allele, gender and age as conditional variables when analyzing for an effect of TF genotypes. Genotype data were analyzed for an effect on age at onset of AD, and duration of illness
Table 1 Allele frequency of the TF C1 and C2 polymorphisms in AD and DLB a Age
TF frequencies C2
Control (121) AD (180) DLB (65)
77.9 ^ 8.7 77.9 ^ 8.0 79.2 ^ 6.5
C1/C1
C1/C2
0.12 0.79 (95) 0.19 (23) 0.17 0.69 (124) 0.29 (52) 0.14 0.75 (49) 0.22 (14)
C2/C2 0.02 (3) 0.02 (4) 0.03 (2)
a Numbers in parentheses indicate the number of cases studied.
where accurate data were available. Clinical data were analyzed by ANOVA with post-hoc t-tests where appropriate. Analyses were carried out using SPSS for Windows and Minitab version 10. The frequency of the TF C2 polymorphism (see Table 1) was not significantly different between the AD and control cases (17 vs. 12%; x2 ¼ 2:52, df ¼ 1, P ¼ 0:11). The frequency of the different TF genotypes was also not altered between the AD population and the control population (x2 ¼ 3:77, df ¼ 2, P ¼ 0:15). No changes were observed between DLB and control populations for either TF C2 polymorphism (14 vs. 12%; x2 ¼ 0:27, df ¼ 1, P ¼ 0:61) or TF genotypes (x2 ¼ 0:25, df ¼ 2, P ¼ 0:88). No changes were observed between AD or DLB populations for either TF C2 polymorphism (x2 ¼ 0:57, df ¼ 1, P ¼ 0:45) or TF genotypes (x2 ¼ 1:39, df ¼ 2, P ¼ 0:50). Stratification of the results for APO E 14 allele status (Table 2) revealed an elevated TF C2 allele frequency in AD cases without the e4 allele when compared with control cases without the E4 allele, which was significant after correcting for multiple testing (n ¼ 12 comparisons; x2 ¼ 11:4, df ¼ 1, P ¼ 0:0011). The TF C2 allele frequency was not elevated in the similar DLB group (x2 ¼ 0:245, df ¼ 1, P ¼ 0:621). Logistic regression analysis demonstrated a significant effect of the TF C2 polymorphism on the development of AD (Wald, 5.1; P ¼ 0:02), with the presence of APO E 14 (34.9; P , 0:00001), age (31.9; P , 0:00001) and gender (4.3; P ¼ 0:04) also displaying significant effects. The TF C2 allele, however, did not show any significant effects in DLB. The TF polymorphisms were assessed for their influence on the age at onset and duration of dementia alone, and Table 2 Stratification of TF C2 allele frequencies in AD and DLB according to presence of the APO E 14 allele a TF C2 allele frequency
Control AD DLB
APO E 14-negative
APO E 14-positive
0.11 (20) 0.22 b (25) 0.03 (2)
0.14 (9) 0.16 (32) 0.21 (15)
a Numbers in parentheses indicate the number of cases studied. b P , 0:01 (x 2 test, compared with control group).
R.I. Hussain et al. / Neuroscience Letters 317 (2002) 13–16
in the presence or absence of the 14 allele. No effect was found for these polymorphisms on age at onset or duration of disease (data not shown). Several studies in widely varied populations have shown an association of the TF C2 allele and dementia, and these findings are consistent with the hypothesis that increased iron mediated free radical damage may result from the C2 allele [16,22,23]. The current study does not demonstrate any significant association between the major TF polymorphisms alone in 180 autopsy verified AD cases and over 60 cases of DLB. In the initial study [23], isoelectric focussing was used to define TF polymorphisms in a South African population of 20 AD cases. The diagnosis of AD was made clinically and errors in the clinical ascertainment may have biased the results. Furthermore, no local control samples were used, the data being derived from previously published observations which could introduce population or age bias in the sample. Age changes in TF C2 frequencies have been suggested [2], though this has been challenged [5]. Also, the small sample used in the initial study lacks statistical power. On the basis of the allele frequency reported in the previous study, our investigation would have greater than 95% power to detect a difference at P , 0:05 and 80% power to detect P , 0:005. Based on the data generated by the current study, a cohort size of over 850 cases and controls would be required to detect any significant effect of the TF C2 alone with confidence (P ¼ 0:05 at 80% power). One large study in a cohort of clinically ascertained Japanese AD cases did, however, identify a significantly increased TF C2 allele frequency [16]. The lack of pathological confirmation in the cohort may have led to potential bias, if for instance any increase in C2 allele frequency were confined to DLB cases included in the cohort as AD. In the current study, we did not, however, find any significant alteration in the TF C2 allele frequency; therefore, this may not be the case. A Norwegian study identified an increased TF C2 allele frequency in AD [22], though as with the initial study, a relatively small sample size and a lack of pathological confirmation make it difficult to say this with sufficient confidence. It is possible that the influence of the C2 allele varies in different populations. Three studies have identified an excess of TF C2 alleles in different AD populations [16,22,23], though this study has not. If the C2 allele promotes free radical production, then it may accelerate pathological damage caused as a result of the APO E 14 allele. This has been suggested by Van Rensburg [23] where an earlier onset of disease was promoted by the presence of the C2 allele in APO E 14 carriers (though we have not detected any significant influence in this study on onset of dementia (not shown)). Similar findings were, however, shown in a Japanese study, where the C2 allele associated with 14 homozygotes [16]. Whilst the C2 allele in isolation is not powerful enough to influence AD development, it may act synergistically with the allele 14. APO E itself is suggested to accelerate the disease process [19,20]
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and a similar argument could apply to TF C2. On the basis of our results, the TF C2 allele is associated with individuals not carrying the APO E 14 allele (Table 2), which contrasts with previous findings [16,23]. Given the weak association and the opposite interaction with APO E 14, any effect of the C2 allele may be due to a closely linked gene and not the TF gene itself, or due to the presence of a type I error. A recent study has suggested that an additional risk factor locus for AD may be located at the telomeric end of the short arm of chromosome 12, and additionally, with sites on chromosomes 4, 6 and 20, but no association was found with loci on chromosome 3, where the TF gene is situated [18]. Kehoe and colleagues [11] using a large sib-pair study based on UK and US AD cases identified a locus on chromosome 3, though this was not situated with the TF locus at 3q21. Due to the possibility of an association of the C2 allele in the current study, it would be useful to determine the influence of the TF gene and surrounding markers in other AD populations and also using family based methods of analysis. [1] Beard, J.L., Connor, J.R. and Jones, B.C., Iron in the brain, Nutr. Rev., 57 (1993) 157–170. [2] Beckman, L. and Beckman, G., Decrease of transferrin C2 frequency with age, Hum. Hered., 36 (1986) 254–255. [3] Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. and Pericak-Vance, M.A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families, Science, 261 (1993) 921–923. [4] Dedman, D., Treffry, A., Candy, J.M., Taylor, G.A., Morris, C.M., Bloxham, C.A., Perry, R.H., Edwardson, J.A. and Harrison, P.M., Iron and aluminium in relation to brain ferritin in normal, Alzheimer’s disease and chronic renal dialysis patients, Biochem. J., 287 (1992) 509–514. [5] Du Chesne, A., Does the transferrin C2 frequency depend on age? Hum. Hered., 43 (1993) 63–65. [6] Good, P.F., Perl, D.P., Bierer, L.M. and Schmeidler, J., Selective accumulation of aluminium and iron in the neurofibrillary tangles of Alzheimer’s disease. A laser microprobe (LAMMA) study, Ann. Neurol., 31 (1992) 286–292. [7] Goodman, L., Alzheimer’s disease. A clinico-pathologic analysis of twenty three cases with a theory on pathogenesis, J. Nerv. Ment. Dis., 117 (1953) 97–130. [8] Grundke Iqbal, I., Fleming, J., Tung, Y.-C., Lassman, H., Iqbal, K. and Joshi, J.G., Ferritin is a component of the neuritic (senile) plaque in Alzheimer’s dementia, Acta Neuropathol., 81 (1990) 105–110. [9] Hardy, J., Amyloid, the presenilins and Alzheimer’s disease, Trends Neurosci., 20 (1997) 154–159. [10] Hixon, J.E. and Vernier, D.T., Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI, J. Lipid Res., 31 (1990) 545–548. [11] Kehoe, P., Wavrant-De Vrieze, F., Crook, R., Wu, W.S., Holmans, P., Fenton, I., Spurlock, G., Norton, N., Williams, H., Williams, N., Lovestone, S., Perez-Tur, J., Hutton, M., Chartier-Harlin, M.C., Shears, S., Roehl, K., Booth, J., Van Voorst, W., Ramic, D., Williams, J., Goate, A., Hardy, J. and Owen, M.J., A full genome scan for late onset Alzheimer’s disease, Hum. Mol. Genet., 8 (1999) 237–245. [12] McKeith, I.G., Gatasko, D., Kosaka, K., Perry, E.K., Dickson, D.W., Hansen, L.A., Salmon, D.P., Lowe, J., Mirra, S.S., Byrne, E.J., Lennox, G., Quinn, N.P., Edwardson, J.A., Ince, P.G., Bergeron, C., Burns, A., Miller, B.L., Lovestone,
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R.I. Hussain et al. / Neuroscience Letters 317 (2002) 13–16 S., Collerton, D., Jansen, E.N., Ballard, C., de Vos, R.A., Wilcock, G.K. and Jellinger, K.A., Consensus guidelines for the clinical and pathological diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB International Workshop, Neurology, 47 (1996) 1113–1124. McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlen, E.M., Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of the Department Of Health and Human Services Task Forces on Alzheimer’s disease, Neurology, 34 (1984) 939–944. Morris, C.M., Candy, J.M., Bloxham, C.A. and Edwardson, J.A., Immunocytochemical localisation of transferrin in the human brain, Acta Anat., 143 (1992) 14–18. Morris, C.M., Kerwin, J.M. and Edwardson, J.A., Non-haem iron histochemistry of the normal and Alzheimer’s disease hippocampus, Neurodegeneration, 3 (1994) 267–275. Namekata, K., Imagawa, M., Terashi, A., Ohta, S., Oyama, F. and Ihara, Y., Association of transferrin C2 allele with late onset Alzheimer’s disease, Hum. Genet., 101 (1997) 126– 129. Pappolla, M.A., Omar, R.A., Kim, K.S. and Robakis, N.K., Immunohistochemical evidence of antioxidant stress in Alzheimer’s disease, Am. J. Pathol., 140 (1992) 621–628. Pericak-Vance, M.A., Bass, M.P., Yamaoka, L.H., Gaskell, P.C., Scott, W.K., Terwedow, H.A., Menold, M.M., Conneally, P.M., Small, G.W., Vance, J.M., Saunders, A.M., Roses, A.D. and Haines, J.L., Complete genomic screen in late onset familial Alzheimer’s disease. Evidence for a new
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locus on chromosome 12, J. Am. Med. Assoc., 15 (1998) 1237–1241. Roses, A.D., Apolipoprotein E affects the rate of Alzheimer’s disease expression: b-amyloid burden is a secondary consequence dependant on Apo E genotype and duration of disease, J. Neuropathol. Exp. Neurol., 53 (1994) 429–437. Roses, A.D., Apolipoprotein E is a relevant susceptibility gene that affects rate of expression of Alzheimer’s disease, Neurobiol. Aging, 15 (1994) S165–S167. Saunders, A.M., Strittmatter, W.J., Schmechel, D., St. George Hystop, P.H., Pericak-Vance, M.A., Joo, S.H., Rosi, B.L., Gusella, J.F., Crapper-MacLachlan, D.R., Alberts, M.J., Hulette, C., Crain, B., Goldgaber, D. and Roses, A.D., Association of apolipoprotein E allele 14 with late-onset familial and sporadic Alzheimer’s disease, Neurology, 43 (1993) 1467–1472. Van Lendeghem, G.F., Sikstrom, C., Beckman, L.E., Adolfsson, R. and Beckman, L., Transferrin C2, metal binding and Alzheimer’s disease, NeuroReport, 9 (1998) 177–179. Van Rensburg, S.J., Carstens, M.E., Potocnik, F.C., Aucamp, A.K. and Taljaard, J.J., Increased frequency of the transferrin C2 subtype in Alzheimer’s disease, NeuroReport, 4 (1993) 1269–1271. Wenham, P.R., Price, W.H. and Blundell, G., Apolipoprotein E genotyping by one-stage PCR, Lancet, 337 (1991) 1158– 1159. Wong, C.I. and Saha, N., Effects of transferrin genetic phenotypes on total iron binding capacity, Acta Haematol., 75 (1989) 215–218.
Transferrin gene polymorphism in Alzheimer’s disease and dementia with Lewy bodies in humans Rafiqul I. Hussain a, Clive G. Ballard b, James A. Edwardson b, Christopher M. Morris a,* a
MRC Building, Institute for the Health of the Elderly, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, UK b Institute of Human Genetics, International Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK Received 18 June 2001; received in revised form 16 October 2001; accepted 16 October 2001
Abstract Genetic studies in Alzheimer’s disease (AD), have indicated that the apolipoprotein E locus (APO E) is a major susceptibility factor, but that it can only account for approximately 50% of AD cases. Several other studies have attempted to identify additional genetic loci associated with disease development, often based on a candidate gene approach. As several lines of evidence indicate that oxidative stress and free radical damage occur in AD, the transferrin gene (TF) has been suggested as a candidate locus for AD since it is the major transport protein for iron, which itself is a major factor in free radical generation. Previous studies have shown elevated TF C2 allele frequencies in AD, this being specifically associated with carriers of the APO E 14 allele. We have therefore determined the influence of the common polymorphisms in TF, C1 and C2, in dementia. The frequency of the C2 allele was not significantly associated with AD. Stratification of cases according to the APO E 14 allele showed a highly significant excess of the C2 allele in AD cases without the 14 allele, contrasting with previous studies. Given the contrasting findings between reports of altered TF C2 allele frequencies, the TF locus would not appear to be a strong risk factor for AD in this population. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Transferrin; Oxidative stress; Apolipoprotein E
Alzheimer’s disease (AD) is the commonest cause of dementia in older people. Its aetiology is complex, involving both environmental and genetic causes. Familial earlyonset AD is known to be caused by mutations in the amyloid precursor protein, or Presenilin 1 or Presenilin 2 [9], though these causes of AD are relatively uncommon. The 14 allele of the apolipoprotein E gene (APO E) is a major susceptibility factor for late-onset AD, and this finding in AD has emphasized the central role played by genetic factors in dementia development and progression [3,21]. Evidence suggests that other risk factor loci exist and whilst whole genome scanning has indicated several loci associated with AD, a candidate gene approach based on genes involved in the pathophysiology of AD has also frequently been employed [11,18]. Increased production of free radicals and oxidative stress may be involved in the development of AD [1]. Increased * Corresponding author. Tel.: 144-191-273-5251; fax: 144-191272-5291. E-mail address: [email protected] (C.M. Morris).
iron levels are found in the AD brain and there is an increase in the levels of the iron storage protein, ferritin, in AD [4]. Iron is found associated with senile plaques [7,15] and also with neurofibrillary tangles in AD [6,7,15]. Immunostaining for ferritin has also shown the presence of ferritin-positive microglia associated with plaques in AD [8]. There is also evidence for increased free radical formation in the brains of AD cases compared with controls, and this may be potentiated by iron [17]. Transferrin (TF) is the major circulating glycoprotein involved in the transport of iron to cells, and is produced by the liver and also by oligodendrocytes and choroidal epithelium in the central nervous system [14]. TF binds two atoms of ferric iron per molecule with very high affinity, resulting in essentially no free ferric ions in solution when the protein is in its normal physiological excess, thereby preventing free radical production. Several allelic isoforms of TF have been identified. The commonest forms are C1 and C2 (C1, Pro 570; C2, Ser 570) found at frequencies of approximately 80 and 20%, respectively. It has been suggested that the C2 protein has reduced ironbinding capacity due to the non-conservative amino acid
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 40 3- X
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R.I. Hussain et al. / Neuroscience Letters 317 (2002) 13–16
change [25]. On the basis of this, it could be argued that if free radical production was a major pathogenic event in AD, then individuals carrying the C2 allele could be at greater risk of developing the disorder. This has been suggested by several studies which have demonstrated an increase in the C2 allele frequency in AD [16,22,23]. We have therefore analyzed a large series of neuropathologically confirmed cases of AD and controls for the C1 and C2 polymorphisms in TF to determine their effect in this population. Additionally, we have genotyped a cohort of cases of dementia with Lewy bodies (DLB) to determine if any changes associated with TF are specific to AD or involved in both DLB and AD. All cases and controls were from the North of England and were part of a large ongoing study. A total of 180 cases of AD (age range, 46–93; mean ^ SD, 77.9 ^ 8.0 years), 65 DLB (age range, 65–95; mean ^ SD, 79.2 ^ 6.5 years) and 121 non-demented control cases (age range, 65–100; mean age at death, 77.9 ^ 8.7 years) were studied. AD cases were clinically diagnosed as such according to NINCDSADRDA criteria [13] and neuropathologically verified as such. DLB cases were clinically diagnosed according to consensus criteria [12] and met operationalized criteria for a diagnosis of DLB [12]. Control cases had no history of neuropsychiatric disease, and on neuropathological examination, showed age related neuropathology only (low or absent senile plaques, absent neocortical neurofibrillary tangles). Genomic DNA was isolated from frozen brain tissue using standard proteinase K digestion followed by phenol/ chloroform extraction. PCR for the TF C1 and C2 alleles was performed using the primers: TFC2 forward, 5 0 GCTGTGCCTTGATGGTACCAGGTAA-3 0 ; and TFC2 reverse, 5 0 -GGACGCAAGCTTCCTTATCT-3 0 , described by Namekata and colleagues [16]. The reactions were performed in a final volume of 20 ml standard buffer containing 5 pmol of each primer, 0.5 U Taq polymerase (Pharmacia, UK), 200 mM each deoxy-nucleotide, and 200 ng of DNA. Reaction conditions included an initial denaturation at 948C for 1 min and 30 s, followed by 40 cycles of annealing at 528C for 1 min, extension at 728C for 1 min, and denaturation at 958C for 30 s. PCR products were digested overnight at 378C with 10 U BstEII (New England Biolabs, UK) and then run on composite 3% NuSeive GTG/ 1% standard agarose gels (Flowgen, UK). The TF C2 allele was determined by the presence of a 110 bp uncut PCR product and the C1 allele by the presence of an 89 bp band. Amplification of the APO E gene containing the allelic sites was performed by previously described methods [10,24]. Statistical analysis of allele frequencies was carried out using the Chi-square test with Yates’ correction for small sample sizes when appropriate. Logistic regression analysis was performed using the presence of the APO E 14 allele, gender and age as conditional variables when analyzing for an effect of TF genotypes. Genotype data were analyzed for an effect on age at onset of AD, and duration of illness
Table 1 Allele frequency of the TF C1 and C2 polymorphisms in AD and DLB a Age
TF frequencies C2
Control (121) AD (180) DLB (65)
77.9 ^ 8.7 77.9 ^ 8.0 79.2 ^ 6.5
C1/C1
C1/C2
0.12 0.79 (95) 0.19 (23) 0.17 0.69 (124) 0.29 (52) 0.14 0.75 (49) 0.22 (14)
C2/C2 0.02 (3) 0.02 (4) 0.03 (2)
a Numbers in parentheses indicate the number of cases studied.
where accurate data were available. Clinical data were analyzed by ANOVA with post-hoc t-tests where appropriate. Analyses were carried out using SPSS for Windows and Minitab version 10. The frequency of the TF C2 polymorphism (see Table 1) was not significantly different between the AD and control cases (17 vs. 12%; x2 ¼ 2:52, df ¼ 1, P ¼ 0:11). The frequency of the different TF genotypes was also not altered between the AD population and the control population (x2 ¼ 3:77, df ¼ 2, P ¼ 0:15). No changes were observed between DLB and control populations for either TF C2 polymorphism (14 vs. 12%; x2 ¼ 0:27, df ¼ 1, P ¼ 0:61) or TF genotypes (x2 ¼ 0:25, df ¼ 2, P ¼ 0:88). No changes were observed between AD or DLB populations for either TF C2 polymorphism (x2 ¼ 0:57, df ¼ 1, P ¼ 0:45) or TF genotypes (x2 ¼ 1:39, df ¼ 2, P ¼ 0:50). Stratification of the results for APO E 14 allele status (Table 2) revealed an elevated TF C2 allele frequency in AD cases without the e4 allele when compared with control cases without the E4 allele, which was significant after correcting for multiple testing (n ¼ 12 comparisons; x2 ¼ 11:4, df ¼ 1, P ¼ 0:0011). The TF C2 allele frequency was not elevated in the similar DLB group (x2 ¼ 0:245, df ¼ 1, P ¼ 0:621). Logistic regression analysis demonstrated a significant effect of the TF C2 polymorphism on the development of AD (Wald, 5.1; P ¼ 0:02), with the presence of APO E 14 (34.9; P , 0:00001), age (31.9; P , 0:00001) and gender (4.3; P ¼ 0:04) also displaying significant effects. The TF C2 allele, however, did not show any significant effects in DLB. The TF polymorphisms were assessed for their influence on the age at onset and duration of dementia alone, and Table 2 Stratification of TF C2 allele frequencies in AD and DLB according to presence of the APO E 14 allele a TF C2 allele frequency
Control AD DLB
APO E 14-negative
APO E 14-positive
0.11 (20) 0.22 b (25) 0.03 (2)
0.14 (9) 0.16 (32) 0.21 (15)
a Numbers in parentheses indicate the number of cases studied. b P , 0:01 (x 2 test, compared with control group).
R.I. Hussain et al. / Neuroscience Letters 317 (2002) 13–16
in the presence or absence of the 14 allele. No effect was found for these polymorphisms on age at onset or duration of disease (data not shown). Several studies in widely varied populations have shown an association of the TF C2 allele and dementia, and these findings are consistent with the hypothesis that increased iron mediated free radical damage may result from the C2 allele [16,22,23]. The current study does not demonstrate any significant association between the major TF polymorphisms alone in 180 autopsy verified AD cases and over 60 cases of DLB. In the initial study [23], isoelectric focussing was used to define TF polymorphisms in a South African population of 20 AD cases. The diagnosis of AD was made clinically and errors in the clinical ascertainment may have biased the results. Furthermore, no local control samples were used, the data being derived from previously published observations which could introduce population or age bias in the sample. Age changes in TF C2 frequencies have been suggested [2], though this has been challenged [5]. Also, the small sample used in the initial study lacks statistical power. On the basis of the allele frequency reported in the previous study, our investigation would have greater than 95% power to detect a difference at P , 0:05 and 80% power to detect P , 0:005. Based on the data generated by the current study, a cohort size of over 850 cases and controls would be required to detect any significant effect of the TF C2 alone with confidence (P ¼ 0:05 at 80% power). One large study in a cohort of clinically ascertained Japanese AD cases did, however, identify a significantly increased TF C2 allele frequency [16]. The lack of pathological confirmation in the cohort may have led to potential bias, if for instance any increase in C2 allele frequency were confined to DLB cases included in the cohort as AD. In the current study, we did not, however, find any significant alteration in the TF C2 allele frequency; therefore, this may not be the case. A Norwegian study identified an increased TF C2 allele frequency in AD [22], though as with the initial study, a relatively small sample size and a lack of pathological confirmation make it difficult to say this with sufficient confidence. It is possible that the influence of the C2 allele varies in different populations. Three studies have identified an excess of TF C2 alleles in different AD populations [16,22,23], though this study has not. If the C2 allele promotes free radical production, then it may accelerate pathological damage caused as a result of the APO E 14 allele. This has been suggested by Van Rensburg [23] where an earlier onset of disease was promoted by the presence of the C2 allele in APO E 14 carriers (though we have not detected any significant influence in this study on onset of dementia (not shown)). Similar findings were, however, shown in a Japanese study, where the C2 allele associated with 14 homozygotes [16]. Whilst the C2 allele in isolation is not powerful enough to influence AD development, it may act synergistically with the allele 14. APO E itself is suggested to accelerate the disease process [19,20]
15
and a similar argument could apply to TF C2. On the basis of our results, the TF C2 allele is associated with individuals not carrying the APO E 14 allele (Table 2), which contrasts with previous findings [16,23]. Given the weak association and the opposite interaction with APO E 14, any effect of the C2 allele may be due to a closely linked gene and not the TF gene itself, or due to the presence of a type I error. A recent study has suggested that an additional risk factor locus for AD may be located at the telomeric end of the short arm of chromosome 12, and additionally, with sites on chromosomes 4, 6 and 20, but no association was found with loci on chromosome 3, where the TF gene is situated [18]. Kehoe and colleagues [11] using a large sib-pair study based on UK and US AD cases identified a locus on chromosome 3, though this was not situated with the TF locus at 3q21. Due to the possibility of an association of the C2 allele in the current study, it would be useful to determine the influence of the TF gene and surrounding markers in other AD populations and also using family based methods of analysis. [1] Beard, J.L., Connor, J.R. and Jones, B.C., Iron in the brain, Nutr. Rev., 57 (1993) 157–170. [2] Beckman, L. and Beckman, G., Decrease of transferrin C2 frequency with age, Hum. Hered., 36 (1986) 254–255. [3] Corder, E.H., Saunders, A.M., Strittmatter, W.J., Schmechel, D.E., Gaskell, P.C., Small, G.W., Roses, A.D., Haines, J.L. and Pericak-Vance, M.A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families, Science, 261 (1993) 921–923. [4] Dedman, D., Treffry, A., Candy, J.M., Taylor, G.A., Morris, C.M., Bloxham, C.A., Perry, R.H., Edwardson, J.A. and Harrison, P.M., Iron and aluminium in relation to brain ferritin in normal, Alzheimer’s disease and chronic renal dialysis patients, Biochem. J., 287 (1992) 509–514. [5] Du Chesne, A., Does the transferrin C2 frequency depend on age? Hum. Hered., 43 (1993) 63–65. [6] Good, P.F., Perl, D.P., Bierer, L.M. and Schmeidler, J., Selective accumulation of aluminium and iron in the neurofibrillary tangles of Alzheimer’s disease. A laser microprobe (LAMMA) study, Ann. Neurol., 31 (1992) 286–292. [7] Goodman, L., Alzheimer’s disease. A clinico-pathologic analysis of twenty three cases with a theory on pathogenesis, J. Nerv. Ment. Dis., 117 (1953) 97–130. [8] Grundke Iqbal, I., Fleming, J., Tung, Y.-C., Lassman, H., Iqbal, K. and Joshi, J.G., Ferritin is a component of the neuritic (senile) plaque in Alzheimer’s dementia, Acta Neuropathol., 81 (1990) 105–110. [9] Hardy, J., Amyloid, the presenilins and Alzheimer’s disease, Trends Neurosci., 20 (1997) 154–159. [10] Hixon, J.E. and Vernier, D.T., Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI, J. Lipid Res., 31 (1990) 545–548. [11] Kehoe, P., Wavrant-De Vrieze, F., Crook, R., Wu, W.S., Holmans, P., Fenton, I., Spurlock, G., Norton, N., Williams, H., Williams, N., Lovestone, S., Perez-Tur, J., Hutton, M., Chartier-Harlin, M.C., Shears, S., Roehl, K., Booth, J., Van Voorst, W., Ramic, D., Williams, J., Goate, A., Hardy, J. and Owen, M.J., A full genome scan for late onset Alzheimer’s disease, Hum. Mol. Genet., 8 (1999) 237–245. [12] McKeith, I.G., Gatasko, D., Kosaka, K., Perry, E.K., Dickson, D.W., Hansen, L.A., Salmon, D.P., Lowe, J., Mirra, S.S., Byrne, E.J., Lennox, G., Quinn, N.P., Edwardson, J.A., Ince, P.G., Bergeron, C., Burns, A., Miller, B.L., Lovestone,
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