A study of alpha-adrenoceptor gene polymorphisms and Alzheimer disease

A study of alpha-adrenoceptor gene polymorphisms and Alzheimer disease
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  J Neural Transm (2001) 108: 445–450 A study of alpha-adrenoceptor gene polymorphisms andAlzheimer disease C.-J. Hong 1,2 ,   Y.-C. Wang 3 ,   T.-Y. Liu 4 , H.-C. Liu 2,5 ,  and S.-J. Tsai 1,2 1 Department of Psychiatry, Veterans General Hospital-Taipei, and 2 School of Medicine, National Yang-Ming University, Taipei, 3 Department of Psychiatry, Yu-Li Veterans Hospital, Hualien, 4 Department of Medical Research and Education, and 5 Neurological Institute, Veterans General Hospital-Taipei, Taipei, Taiwan,Republic of ChinaReceived January 24, 2000; accepted November 21, 2000 Summary.  There exists considerable evidence implicating abnormalities of the alpha ( α )-adrenergic system in the development of Alzheimer disease(AD). We propose to investigate potential correlations between the presenceor otherwise of α -adrenoceptor polymorphisms and the presence of AD. Westudied the polymorphisms of the α 1a - and the α 2a -adrenoceptor genes in 142AD patients and 98 normal controls. The result demonstrated that none of the α 2a -adrenoceptor genotypes was associated with increased susceptibility toAD. However, there was a trend that the frequency of the C allele of the α 1a -adrenoceptor was elevated and an excess of the CC genotype (90.1%) wasfound in the subjects with AD in comparison with the controls (78.6%). Thisassociation was unrelated to the apolipoprotein E genotypes. The hypothesisthat the α 1a -adrenoceptor gene may be implicated in the pathogenesis of ADmay deserve further study. Keywords:  Alzheimer disease, association study, genetic polymorphism,alpha adrenoceptor. Introduction Biochemical and pathological studies have described abnormalities forseveral neuro-transmitter systems in Alzheimer disease (AD). Whilstcholinergic dysfunction appears to be the most salient transmitter deficitfor AD, there exists compelling evidence suggesting that abnormalities of adrenergic neuro-transmission contribute to AD pathogenesis (Hertz, 1989).Adrenoceptors within neurons taken from the adrenergic projection of ADbrains show some abnormalities, for example, Kalaria et al. (1989) noted areduction in the number of α 2 -adrenoceptors in the prefrontal cortex of AD  446C.-J. Hong et al. patients. Meana et al. (1992) demonstrated a decrease in the concentration of  α 2 -adrenoceptors in the AD frontal cortex, hypothalamus and cerebellum.Neurochemical studies, moreover, have revealed a reduced noradrenalinecontent in postmortem brains from AD patients (Arai et al., 1984; Palmer etal., 1987).The CNS adrenergic system is involved in certain aspects of cognitivefunction, such as attention and memory (Randt et al., 1971). From animalstudies, lesions in locus ceruleus projections, or the pharmacological inhibitionof noradrenaline synthesis, have elicited learning disruption (Botwinick et al.,1977; Robbins and Everitt, 1982). Conversely, drugs that stimulate α 2 -adrenoceptors may improve cognitive function for young monkeys withexperimentally-induced noradrenaline depletion, or for aged monkeyswith naturally-occurring degeneration of the production of noradrenaline(Arnsten et al., 1988; Arnsten and Goldman-Rakic, 1985; Cai et al., 1993).There are two major types of α -adrenoceptors, α 1 - and α 2 -adrenoceptors,each of which reflects at least three subtypes. The α 2 -adrenoceptors arethought to be mostly inhibitory, pre-synaptic autoreceptors that play akey role in noradrenergic neuro-transmission, regulating the release of noradrenaline in the brain (Aoki et al., 1994). Recent immuno-cytochemicalstudies of α 2a -, α 2b - and α 2c -adrenoceptor subtypes from the monkey brainhave revealed that the α 2a -adrenoceptor subtype is more densely distributedin the prefrontal cortex than elsewhere in the brain, including area 46, as wellas in the locus ceruleus  (Aoki et al., 1994).The close relationship between central α -adrenergic transmission andcognitive function, viewed in light of the abnormal adrenergic functions inAD, suggest that the α -adrenoceptor genes may play a role in memoryacquisition and can be regarded as candidate genes for susceptibility to AD.We tested the hypothesis that variants of the α 1a - and α 2a -adrenoceptor genesconfer susceptibility to AD. Methods Subjects A total of 142 AD patients and 98 controls were included in this study. Some of thesepatients and controls were included in our previous study (Hong et al., 1996). All the ADpatients were recruited from the neurology clinic of the Veterans General Hospital-Taipei. AD was diagnosed consensually by a team of neurologists, according to thecriteria established by the National Institute of Neurological and CommunicativeDisorder and Stroke and the Alzheimer and Related Disorders Association for ProbableAD (McKhann et al., 1984). Each control subject was given clinical, psychological andneurological examinations to rule out any insidious cognitive deficit. Mini-Mental StateExamination (MMSE) was done in 77 controls (range 20 to 30). The sample consistedentirely of ethnic Chinese people.Genotyping of α 1a - and α 2a -adrenoceptor gene polymorphism: For α 1a - and α 2a -adrenoceptor genotyping, peripheral venous blood was withdrawn from the studysubjects, with informed consent. Genomic DNA was extracted from peripheral bloodlymphocytes according to standard procedures.The α 1a -adrenoceptor genotype was analyzed using polymerase chain reaction(PCR). PCR was initiated at 95°C for 30 seconds, 57°C for 45 seconds and 72°C for  Alpha adrenoceptor and Alzheimer disease447one minute through a total of 35 cycles. The PCR reaction mixture contained 0.2mMdNTP, 1.0mM MgCl 2 , X1 Taq reaction buffer (MBI) and 1 µ M primers of 5  -ATGCTCCAGCCAAGAGTTCA-3   and 5  -TCCAAGAAGAGCTGGCCTTC-3  . ThePCR products were digested with five units Pst I at 37°C for four hours and analyzed usinga two percent ethidium bromide-stained agarose gel. The digested products of 502bp,were type “C”, and that of 457bp and 45bp, type “T”.For α 2a -adrenoceptor promoter genotyping, PCR was initiated at 95°C for 30 seconds,56°C for 45 seconds, and 72°C for one minute through 35 cycles. The PCR reactionmixture contained 0.2mM dNTP, 1.0mM MgCl 2 , X1 Taq reaction buffer (MBI) and 1  µ Mprimers of 5  -TCACACCGGAGGTTACTTCC-3   and 5  -TCCGACGACAGCGCGAGTT-3  . The PCR products were digested with five units Hpa II at 37°C for four hoursand analyzed on a six percent polyacrylamide gel, and then stained using ethidiumbromide. The digested products of 174, 165, 116, 62 and 5bp were type “M”, and that of 121, 53, 165, 116, 62, 5bp, type “m”. All cases were also genotyped at apolipoprotein Egene (APOE). Statistical analysis Differences of continuous variables between groups were evaluated using Student’s t-test.Comparisons of the genotype distributions and the allele frequencies of adrenoceptor-gene polymorphisms were made using the Chi-square test and Fisher’s Exact test, whererequired. For the association tests of the two receptor polymorphisms, alpha valueswere considered significant when below 0.013 (0.05 / 4 comparisons; allele and genotypefrequencies for two receptors). Results The age and sex distributions of the AD patients and the control populationwere similar (  p     0.124 and  p     0.692, respectively, Table 1). When stratifiedaccording to the sex, the age difference between the two groups is still notsignificant (  p     0.273 and  p     0.330 for male and female, respectively). Thegenotypic and allelic distributions for the adrenoceptor-gene polymorphismsfor our AD patient and control populations are given in Table 1. Neither thegenotype nor allele frequencies of the α 2a -adrenoceptor differed statisticallyfor the AD group and the normal controls (Table 1). However, there is a trendthat C allele ( c 2     5.74, df   1,  p   0.020) or C/C genotype ( c 2     6.23, df  1,  p     0.016) of the α 1a -adrenoceptor gene are more frequently found in theAD group than in the control group (Table 1).We made a comparison of genotypic distribution between control subjectsand AD cases for APOE genetic polymorphism. As expected, significantassociation was found for APOE ε 4 carriers ( c 2     15.14, df   1,  p     0.001).There is no association of the APOE polymorphism with the α 1a -adrenoceptorgenotypes in AD or control groups, except that, in the AD group, there is amildly increased frequency of the α 1a  C/T genotype ( c 2     4.06, df   1,  p    0.069) in the APOE ε 4 carriers (Table 2). This finding indicated that theincreased α 1a  C/C genotype in AD cases is not due to the excess of APOE ε 4alleles in the patients. Discussion To the best of our knowledge, this is the first study of adrenoceptor-genepolymorphisms in AD. Our data indicate that neither the α 1a - nor the α 2a -  448C.-J. Hong et al. adrenoceptor-gene polymorphisms investigated play a major role in suscepti-bility to AD.Our lack of associations between AD and the adrenoceptor-genepolymorphisms may be due to the influence of several factors.Firstly, the lack of association may represent a false negative result, inlight of the low statistical power of the sample. There is only a trend that Callele of the α 1a  adrenoceptor gene is more frequently found in the AD groupthan in the control group. Replicate studies in larger samples are needed tofully resolve the possible involvement of DNA variants in the α 1a -adrenoceptor gene in AD. Table 1. The genotype and the allele distribution of α 1a - and α 2a -adrenoceptor genepolymorphisms of AD and control groupNormalAlzheimer disease  p valuesNumber (M/F)98 (52/46)142 (80/62)  c 2     0.25, df   1,  p   0.692Age (SD)73.4 (5.5)74.6 (6.9) t      1.55, df   233,  p     0.124 α 1a -adrenoceptor  Genotype (%)C/C77 (78.6)128 (90.1)  c 2     6.23, df   1,  p     0.016C/T21 (21.4)14 (9.9)T/T0 (0.0)0 (0.0)Allele (%)C175 (89.3)270 (95.1)  c 2     5.74, df   1,  p     0.020T21 (10.7)14 (4.9) α  2a -adrenoceptor  Genotype (%)M/M7 (7.1)13 (9.2)  c 2     0.77, df   2,  p     0.681M/m43 (43.9)55 (38.7)m/m48 (49.0)74 (52.1)Allele (%)M57 (29.1)81 (28.5)  c 2     0.02, df   1,  p     0.918m139 (70.9)203 (71.5) Table 2. The α 1a -adrenoceptor and apolipoprotein E gene (APOE) genotypes in AD andcontrol groups α 1a -adrenoceptorAPOETotal2/22/32/43/33/44/4ADC/C010179335128C/T10056214Total110184397142ControlC/C052637077C/T040152021Total092789098  Alpha adrenoceptor and Alzheimer disease449 Secondly, it is possible that additional variants of the α -adrenoceptorgenes may be influencing the development of AD. Before definitely excludinga role for the adrenoceptor gene in the pathogenesis of AD, further studieswith other α -adrenoceptor variants are clearly warranted.Thirdly, AD is a heterogeneous disease and this heterogeneity is reflectedby the different features presented in the clinical and biological manife-stations (Friedland et al., 1998). It seems reasonable to suggest that multiplegenes are likely to contribute to the pathogenesis of AD. The associationbetween α -adrenoceptor gene variants and AD, examined in this study, mayonly be found in a subgroup of all AD patients. For example, Russo-Neustadtand Cotman (1997) demonstrated in 1997 that abnormally low levels of cerebellar α 2 -adrenoceptors are restricted to a subgroup of AD patients thatshow no symptoms of aggression or agitation.Finally, although the α -adrenergic system may be involved in thepathogenesis of AD, the alterations to the α -adrenergic function for thesepatients may be the result of the action of, or interaction with, other genes.Post-transcriptional or -translational processes of the adrenoceptors may beinvolved in the development of AD.In addition to memory and learning, it has been suggested that the central α -adrenergic system plays a role in the regulation of sleep-wake cycle, moodand aggression. It would be of interest to investigate the genetic variations forthe α -adrenoceptor genes in the symptomatology and course of, and responseto, therapeutic drugs in AD. Acknowledgements This work was supported by Grant NHRI-GT-EX89P604L from the National HealthResearch Institute, Taiwan, ROC. The experiments in this study were performed in theMolecular Biology Laboratory, Department of Psychiatry, Veterans General Hospital-Taipei, Taipei, Taiwan, ROC. References Aoki C, Go CG, Venkatesan C, Kurose H (1994) Perikaryal and synaptic localizationof alpha 2A-adrenergic receptor-like immunoreactivity. Brain Res 650: 181–204Arai H, Kosaka K, Iizuka R (1984) Changes of biogenic amines and their metabolites inpostmortem brains from patients with Alzheimer-type dementia. J Neurochem 43:388–393Arnsten AF, Goldman-Rakic PS (1985) Alpha 2-adrenergic mechanisms in prefrontalcortex associated with cognitive decline in aged nonhuman primates. Science 230:1273–1276Arnsten AF, Cai JX, Goldman-Rakic PS (1988) The alpha-2 adrenergic agonistguanfacine improves memory in aged monkeys without sedative or hypotensive sideeffects: evidence for alpha-2 receptor subtypes. J Neurosci 8: 4287–4298Botwinick CY, Quartermain D, Freedman LS, Hallock MF (1977) Some characteristics of amnesia induced by FLA-63 an inhibitor of dopamine beta hydroxylase. PharmacolBiochem Behav 6: 487–491Cai JX, Ma YY, Xu L, Hu XT (1993) Reserpine impairs spatial working memoryperformance in monkeys: reversal by the alpha 2-adrenergic agonist clonidine. BrainRes 614: 191–196
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