A novel polymorphism of the brain-derived neurotrophic factor (BDNF ...

2 downloads 0 Views 120KB Size Report
Keywords: Alzheimer's disease; brain-derived neurotrophic factor (BDNF); late-onset; association study; polymorphism; apolipoprotein E; sporadic. Several lines ...
Molecular Psychiatry (2001) 6, 83–86  2001 Macmillan Publishers Ltd All rights reserved 1359-4184/01 $15.00 www.nature.com/mp

ORIGINAL RESEARCH ARTICLE

A novel polymorphism of the brain-derived neurotrophic factor (BDNF) gene associated with late-onset Alzheimer’s disease H Kunugi1, A Ueki2, M Otsuka2, K Isse3, H Hirasawa4, N Kato5, T Nabika6, S Kobayashi7 and S Nanko1 1

Department of Psychiatry, Teikyo University School of Medicine, Tokyo, Japan; 2Department of Neurology, Jichi Medical School, Omiya Medical Center, Omiya City, Japan; 3Department of Psychiatry, Tokyo Metropolitan Toshima Hospital, Tokyo, Japan; 4Department of Psychiatry, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan; 5Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan; 6Department of Laboratory Medicine, Shimane Medical University, Shimane, Japan; 7Department of Internal Medicine, Shimane Medical University, Shimane, Japan

Keywords: Alzheimer’s disease; brain-derived neurotrophic factor (BDNF); late-onset; association study; polymorphism; apolipoprotein E; sporadic Several lines of evidence have suggested altered functions of the brain-derived neurotrophic factor (BDNF) in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD). In the search for polymorphisms in the 5⬘-flanking and 5⬘-noncoding regions of the BDNF gene, we found a novel nucleotide substitution (C270T) in the noncoding region. We performed an association study between this polymorphism and AD in a Japanese sample of 170 patients with sporadic AD (51 early-onset and 119 late-onset) and 498 controls. The frequency of individuals who carried the mutated type (T270) was significantly more common in patients with late-onset AD than in controls (P = 0.00004, odds ratio: 3.8, 95% CI 1.9–7.4). However, there was no significant difference in the genotype distribution between the patients with early-onset AD and the controls, although this might be due to the small sample size of the early-onset group. Our results suggest that the C270T polymorphism of the BDNF gene or other unknown polymorphisms, which are in linkage disequilibrium, give susceptibility to late-onset AD. We obtained no evidence for the possible interactions between the BDNF and apolipoprotein E (APOE) genes, suggesting that the possible effect of the BDNF gene on the development of late-onset AD might be independent of the APOE genotype. Molecular Psychiatry (2001) 6, 83–86.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by loss and atrophy of basal forebrain cholinergic neurons and the limbic structures.1 Mutations of the genes encoding presenilin-1,2 presenilin-2,3 and amyloid precursor protein4 cause familial AD, and the ⑀4 allele of the apolipoprotein E (APOE) gene gives susceptibility to familial and sporadic AD.5 However, these genetic markers cannot explain the overall genetic susceptibility, and additional genes may be involved in the development of AD. Since neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),

and neurotrophin-3 (NT-3) promote the development, regeneration, survival and maintenance of function of neurons,6,7 polymorphisms of genes encoding these proteins may confer susceptibility to neurodegenerative changes as in AD. For the BDNF protein, reduced mRNA expression was observed in post-mortem hippocampus and temporal cortex of patients with AD.8 Immunohistochemical and Western blotting studies revealed a selective decline of the BDNF/TrkB neurotrophic signaling pathway in the frontal cortex and hippocampus in AD.9 Lower BDNF levels in the entorhinal cortex were also reported in AD.10 BDNF has been suggested to have therapeutic effects on AD and other neurodegenerative diseases.11,12 These several lines of evidence make the BDNF gene an important candidate gene for AD. We searched for polymorphisms in the 5⬘-flanking and 5⬘-noncoding regions of the BDNF gene reported by Shintani et al.13 We detected a novel polymorphism of single nucleotide substitution (C270T) in the 5⬘-noncoding region by single strand conformational polymorphism (SSCP) analysis and direct sequencing. This polymorphism could be detected by polymerase chain reaction (PCR) amplification and digestion by a restriction enzyme of AvaII, Cfr13I, Eco47I, Eco0109I, HinfI, PpuMI, Sau96I, or SinI. Differential band patterns observed in polyacrylamide gel electrophoresis of PCR products digested by HinfI are shown in Figure 1. The genotype distributions for patients with AD and controls are shown in Table 1. To ensure any conclusion, we employed three control groups (see Methods). The genotype distribution for any of the patient or control groups was not significantly deviated from the Hardy–Weinberg equilibrium. There was only one individual who was homozygous for the mutated type (T270): a male patient who developed AD at the age of 75 years. The frequencies of heterozygotes were similar in the three control groups (3–5%), irrespective of their differential distributions in age and sex. Thus the effect of age and sex on the genotype distribution was considered to be minimal, which was the rationale

BDNF and Alzheimer’s disease H Kunugi et al

84

Figure 1 Band patterns of the HinfI restriction site (C270T) of the brain-derived neurotrophic factor (BDNF) gene. Lane M: molecular weight marker; lane I: 223-bp PCR product amplified by the primers BDNFPR8F and BDNFPR8R; lane II: digested PCR products of homozygote for the wild-type (C270); lane III: heterozygote; lane IV: homozygote for the mutated type (T270).

whereby we combined the three control groups in the following statistical analysis. The frequency of individuals who carried the T270 allele was significantly increased for the patients with late-onset AD than for the age- and sex-matched controls (control A) (␹2 = 7.4, df = 1, P = 0.007, odds ratio: 3.2, 95% CI 1.3–7.7). When the three control groups were combined, the difference in the frequency of individuals carrying the T270 allele between patients and controls was highly significant, and the confidential interval of the odds ratio became narrower (␹2 = 16.8, df = 1, P = 0.00004, odds ratio: 3.8, 95% CI 1.9–7.4). However, the frequency of individuals carrying the T270 allele in the patients with early-onset AD was not significantly different from that in control A (␹2 = 0.1, df = 1, P = 0.79, odds ratio: 1.2, 95% CI 0.3–4.7) or that in the total controls (␹2 = 0.5, df = 1, P = 0.50, odds ratio: 1.6, 95% CI 0.4–5.6).

When allelewise comparisons were performed, the frequency of the T270 allele was significantly increased for the patients with late-onset AD (7.6%), compared to that in control A (2.5%; ␹2 = 8.1, df = 1, P = 0.005, odds ratio: 3.2, 95% CI 1.4–7.6) and that in the total controls (2.1%; ␹2 = 18.7, df = 1, P = 0.00002, odds ratio: 3.8, 95% CI 2.0–7.2). However, the frequency of the T270 allele in the patients with early-onset AD (2.9%) was not significantly different from that in control A (␹2 = 0.1, df = 1, P = 0.79, odds ratio: 1.2, 95% CI 0.3–4.6) or that in the total controls ( ␹2 = 0.3, df = 1, P = 0.58, odds ratio: 1.4, 95% CI 0.4–4.8). In order to examine the possible interaction between the BDNF and APOE genes, we genotyped the patients with AD with respect to the APOE gene. Among the 119 patients with late-onset AD, 58 patients carried the ⑀4 allele of the APOE gene (APOE⑀4+ group) and the remaining 61 did not (APOE⑀4− group). There was no significant difference in the frequency of individuals carrying the mutated type (T270) of the BDNF gene between the two groups (Table 1; ␹2 = 0.8, df = 1, P = 0.37). We found a single nucleotide substitution polymorphism in the 5⬘-noncoding region of the BDNF gene (C270T). Our results suggest that this polymorphism is associated with susceptibility to late-onset AD. Supposing that the 270T allele itself has a risk-increasing effect, it may result from alteration in the translation efficacy. Another possibility is that other as yet unknown polymorphisms, which are in linkage disequilibrium with the C270T, give susceptibility to lateonset AD. The obtained odds ratio was 3.8, which was comparable to that of the APOE gene in our Japanese sample, since 49% of patients with late-onset AD and 19% of controls carried the ⑀4 allele of the APOE gene (odds ratio 4.2). However, the population attributable risk of carrying the T270 allele was not very large (for lateonset AD: 10.5%, 95% CI 3.5–17.0%) as the allele fre-

Table 1 Genotype distributions for the C270T polymorphism of the brain-derived neurotrophic factor (BDNF) gene among patients with Alzheimer’s disease and controls Mean age (SD)

n

Genotype distribution (%) C/C

Alzheimer’s disease Total Early onset (⬍65 yrs) APOE⑀4+ APOE⑀4− Late onset (ⱖ65 yrs) APOE⑀4+ APOE⑀4− Controls Total Control A Control B Control C

Molecular Psychiatry

C/T

T/T

74 64 65 64 78 78 78

(9) (6) (6) (6) (6) (6) (6)

170 51 24 27 119 58 61

150 48 23 25 102 48 54

(88.2%) (94.1%) (95.8%) (92.6%) (85.7%) (82.8%) (88.5%)

19 3 1 2 16 9 7

(11.2%) (5.9%) (4.2%) (7.4%) (13.4%) (15.5%) (11.5%)

1 0 0 0 1 1 0

(0.6%) (0.0%) (0.0%) (0.0%) (0.8%) (1.7%) (0.0%)

55 77 32 57

(21) (5) (14) (8)

498 162 170 166

477 154 164 159

(95.8%) (95.1%) (96.5%) (95.8%)

21 (4.2%) 8 (4.9%) 6 (3.5%) 7 (4.2%)

0 0 0 0

(0.0%) (0.0%) (0.0%) (0.0%)

BDNF and Alzheimer’s disease H Kunugi et al

quency for the mutated type (T270) was relatively low (7.6% for the late-onset AD and 2.1% for the total controls). Although there was no significant association between the C270T polymorphism and early-onset AD, this might be attributable to the small number of subjects. When a power analysis was performed, the sample size of the early-onset group (n = 51) and the total controls (n = 498) had a power of approximately 60% to detect a significant association (P ⬍ 0.05) if there is an effect of carrying the T270 allele on the development of early-onset AD similar to that observed for the late-onset AD. Since there was no significant difference in the frequency of individuals carrying the mutated type (T270) of the BDNF gene between the APOE⑀4+ and the APOE⑀4− groups, the possible effect of T270 on susceptibility to late-onset AD might be independent of the APOE genotype. The observed association between the BDNF gene and late-onset AD in the current study is feasible since several lines of evidence have suggested altered functions of BDNF in AD.8–10 If our results are replicated in other samples, it will provide new insight into the genetic risk of AD and possible therapeutic effects of BDNF protein for the illness.

Methods Subjects To search for polymorphisms, genomic DNA from 50 patients with AD were examined. An observed polymorphism was examined for an association analysis in a sample of 170 patients with AD and three control groups. The patients were diagnosed by neurologists as meeting the NINCDS-ADRDA criteria14 for ‘probable AD’. Fifty-one patients (23 men and 28 women) were of early-onset (⬍65 years) and 119 (36 and 83) lateTable 2

onset. Mean ages for the early- and late-onset groups were 64 (SD 6) and 78 (SD 6) years, respectively. All these patients were biologically unrelated Japanese who had no family history of AD. To ensure any conclusion, we employed three control groups. The first control group (control A) consisted of 162 medical patients matched for age (mean: 77, SD 5) and sex ratio (50 men and 112 women) with the late-onset AD patients. The second control group (control B; 83 men and 87 women; mean age 32 (SD 14) years) was recruited from hospital staffs who were not assessed for neurological symptoms, although they showed good social functioning. The other control group (control C; 68 men and 99 women; mean age 57 (SD 8)) consisted of medical patients who were screened by magnetic resonance images (MRIs) of the brain and had no apparent neurodegenerative change. All the control subjects were unrelated Japanese. Informed consent for the participation of the study was obtained from close relatives for the patient group and from the controls. This study was approved by institutional ethical committees.

85

SSCP analysis and genotyping Venous blood was drawn and genomic DNA was extracted according to standard procedures. We searched for polymorphisms for the 5⬘-flanking and 5⬘noncoding regions of the BDNF gene reported by Shintani et al.13 Polymerase chain reaction (PCR) amplifications and single strand conformational polymorphism (SSCP) analyses were performed by using eight sets of primers (Table 2) which encompassed a 1.7-kb fragment from nucleotide number −1405 to +330 (GenBank accession X60202). The SSCP analysis was performed on at least two differential conditions for each target sequence. A nucleotide substitution was determined by using ABI prism 373 autosequencer (Perkin Elmer, Japan).

Oligonucleotide primer sets used for SSCP analysis

Nucleotide number of PCR producta

Forward primer

−1472 苲 −1229

BDNFPR1F

−1281 苲 −1075

BDNFPR2F

−1126 苲 −904

BDNFPR3F

−946 苲 −764

BDNFPR4F

−421 苲 −208

BDNFPR5F

−255 苲 −10

BDNFPR6F

−52苲173

BDNFPR7F

108苲330

BDNFPR8F

5⬘-CGAATCACCTACCCC CACTCT-3⬘ 5⬘-TTTTGATTCTGGTAAT TCGTG-3⬘ 5⬘-CGGCGAACTAGCATG AAATCT-3⬘ 5⬘-TAAGCGAAGGGAACG TGAAAA-3⬘ 5⬘-GAGACCTCGGGGCGT GCGATT-3⬘ 5⬘-GACTCGCAGGGCGAT GTCGCGG-3⬘ 5⬘-GACCAATCGAAGCTC AACCGA-3⬘ 5⬘-CAGAGGAGCCAGCCC GGTGCG-3⬘

Reverse primer

BDNFPR1F BDNFPR2F BDNFPR3F BDNFPR4F BDNFPR5F BDNFPR6F BDNFPR7F BDNFPR8F

5⬘-CCTCCGCTGCCTCTG AAATAG-3⬘ 5⬘-AAGCTCCATTTGATCT CGGCA-3⬘ 5⬘-AAAACTCCCACACTC TATTAT-3⬘ 5⬘-GACCTCGCTAAAGCC ACACGC-3⬘ 5⬘-CTGTCCTCACCTCCT CCCCTA-3⬘ 5⬘-GGGTCAGACATTATTT AGCTC-3⬘ 5⬘-GCGGTGGGTGTCTCA TTAAAG-3⬘ 5⬘-CTCCTGCACCAAGCC CCATTC-3⬘

a

The nucleotide numbering was according to Shintani et al.13 Molecular Psychiatry

BDNF and Alzheimer’s disease H Kunugi et al

86

Genotyping for a detected polymorphism was done by PCR with primers of BDNFPR8F and BDNFPR8R (Table 2) and digestion by the restriction enzyme HinfI, followed by 5% polyacrylamide gel electrophoresis with ethidium bromide staining (Figure 1). To examine the possible interaction between the BDNF and APOE genes, we genotyped the patients with AD as to the APOE gene, according to the methods of Wenham et al.15 Statistical analysis The presence of Hardy–Weinberg equilibrium for the genotypic distributions was examined by using the ␹2 test for goodness of fit. The difference in the genotype distribution between patients and controls was examined by using the ␹2 test for independence. We further examined the possible interaction between the BDNF and APOE genes; we compared genotype distribution of the BDNF gene between AD patients with the ⑀4 allele of the APOE gene and those without. All Pvalues reported are two-tailed. Acknowledgments This work was supported by the Pharmacopsychiatry Research Foundation. We thank Ms N Okuyama for her assistance in the laboratory.

References 1 Terry RD. Neuropathological changes in Alzheimer disease. Prog Brain Res 1994; 101: 383–390. 2 Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M et al. Cloning of a gene bearing mis-sense mutations in earlyonset familial Alzheimer’s disease. Nature 1995; 375: 754–760. 3 Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH et al. Candidate gene for the chromosome 1 familial Alzheimer’s disease locus. Science 1995; 269: 973–977.

Molecular Psychiatry

4 Goate A, Chartier-Harlin M-C, Mullan M, Brown J, Crawford F, Fidani L et al. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature 1991; 349: 704–706. 5 Saunders AM, Strittmatter WJ, Schmechel D, St. George-Hyslop PH, Pericak-Vance MA, Joo SH et al. Association of apolipoprotein E allele ⑀4 with late-onset familial and sporadic Alzheimer’s disease. Neurology 1993; 43: 1467–1472. 6 Maisonpierre PC, Belluscio L, Friedman B, Alderson RF, Wiegand SJ, Furth ME et al. NT3, BDNF and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 1990; 5: 501–509. 7 Maisonpierre PC, Le Beau MM, Espinosa III R, Ip NY, Belluscio L, de la Monte SM et al. Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations. Genomics 1991; 10: 558–568. 8 Connor B, Young D, Yan Q, Faull RL, Synek B, Dragunow M. Brainderived neurotrophic factor is reduced in Alzheimer’s disease. Brain Res Mol Brain Res 1997; 49: 71–81. 9 Ferrer I, Marin C, Rey MJ, Ribalta T, Goutan E, Blanco R et al. BDNF and full-length and truncated TrkB expression in Alzheimer disease. Implications in therapeutic strategies. J Neuropathol Exp Neurol 1999; 58: 729–739. 10 Narisawa-Saito M, Wakabayashi K, Tsuji S, Takahashi H, Nawa H. Regional specificity of alterations in NGF, BDNF and NT-3 levels in Alzheimer’s disease. Neuroreport 1996; 7: 2925–2928. 11 Knusel B, Gao H. Neurotrophins and Alzheimer’s disease: beyond the cholinergic neurons. Life Sci 1996; 58: 2019–2027. 12 Lindsay RM. Neurotrophic growth factors and neurodegenerative diseases: therapeutic potential of the neurotrophins and ciliary neurotrophic factor. Neurobiol Aging 1994; 15: 249–251. 13 Shintani A, Ono Y, Kaisho Y, Igarashi K. Characterization of the 5⬘-flanking region of the human brain-derived neurotrophic factor gene. Biochem Biophys Res Commun 1992; 182: 325–332. 14 McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer’s disease. Neurology 1984; 34: 939–944. 15 Wenham PR, Price WH, Blundell GB. Apolipoprotein E genotyping by one-stage PCR. Lancet 1991; 337: 1158–1159.

Correspondence: H Kunugi, Department of Psychiatry, Teikyo University School of Medicine 11–1, Kaga 2 Chome, Itabashi-ku, Tokyo, 173–8605, Japan. E-mail: hkunugi얀med.teikyo-u.ac.jp Received 1 May 2000; revised 3 July 2000; accepted 6 July 2000