Serotoninergic Polymorphisms (5-HTTLPR and 5-HT2A): Association Studies with Psychosis in Alzheimer Disease

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1 GENETIC TESTING Volume 7, Number 4, 2003 Mary Ann Liebert, Inc. Serotoninergic Polymorphisms (5-HTTLPR and 5-HT2A): Association Studies with Psychosis in Alzheimer Disease A. ROCCHI, D. MICHELI, R. CERAVOLO, M.L. MANCA, G. TOGNONI, G. SICILIANO, and L. MURRI ABSTRACT Patients with Alzheimer disease (AD) often exhibit psychotic symptoms associated with cognitive impairment. A few association studies have been carried out to determine if the serotonin transporter and receptor genes are potential risk factors for AD and/or associated psychopathology. The aim of this study was to investigate the association of a serotonin transporter gene-linked polymorphic region (5-HTTLPR) and the 5-HT2A receptor T102C polymorphism with the risk of developing dementia and/or psychotic symptoms in a group of sporadic AD patients from Italy. No significant differences in the distribution of allele and genotype frequencies of 5-HTTLPR and 5-HT2A T102C were found between patient and control groups. However, a significant association between the C102/C102 5-HT2A genotype and psychotic symptoms (p, 0.001) was observed. Our data strongly confirm results from previous studies suggesting that the C102 allele of the 5-HT2A receptor is associated with the occurrence of psychotic symptoms in AD. On the contrary, the serotonin transporter does not appear to be a major susceptibility factor in the pathophysiology of the disease. INTRODUCTION ALZHEIMER DISEASE (AD) is often complicated by psychiatric symptoms, which occur in one-third of patients at an early stage of the disease (Burns et al., 1990a,b; Ballard et al., 1999; Paulsen et al., 2000). The possible hypotheses for psycho-pathology in AD involve selective loss of different neuronal populations, alterations of specific neurotransmitter systems, and genetic risk factors (Forstl et al., 1994; Holmes et al., Blusztajn and Berse, 2000). Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter involved in the control of mood and a variety of traits and behaviors. Serotonin dysfunction has been implicated in the pathogenesis of many psychiatric diseases such as depression, obsessive-compulsive disorder, schizophrenia, and in the psychotic symptoms of patients diagnosed as having AD (Meltzer et al., 1998; Cross, 1990). Postmortem and biopsy studies of brains from patients with AD showed a decrease in the levels of 5-HT, of 5-HT receptors, and of the 5-HT transporter (5- HTT) sites (Bowen et al., 1983; Cheng et al., 1991; Tejani-Butt et al., 1995). In the 59 promoter region of the 5-HTT gene (chromosomal location 17q11.1 q12), there is an insertion/deletion polymorphism of one or more units of a base pair repeat, known as 5-HTTLPR (Lesch et al., 1994), which is the site of origin of a number of different alleles. The most common alleles are named long (L) and short (S), and they are characterized by a differential rate of transcription, with the long allele being more efficient (Collier et al., 1996; Heils et al., 1996). Recently, an allelic association between the 5-HTTLPR polymorphism and late-onset AD has been reported, but other authors did not confirm these data (Li et al., 1997; Oliveira et al., 1998; Hu et al., 2000; Zill et al., 2000). In the human 5-HT2A receptor gene, localized on chromosome 13q14 q21 (Sparkes et al., 1991), a single-nucleotide polymorphism (SNP) derived from a silent mutation is present at position 102 (T102C). Recent observations (Holmes et al., 1998; Nacmias et al., 2001) indicate that this and other common genetic polymorphisms in the 5-HT2A and 5-HT2C receptor genes may be risk factors for psychotic symptoms in the course of AD. Several studies investigated the association between apolipoprotein E (ApoE) genotype and psychotic symptoms in AD, but only inconclusive results were reported (Ramachandran et al., 1996; Harwood et al., 1999). In light of these considerations, we analyzed the distribution of the polymorphisms in the 5-HTT gene promoter region, 5- HT2A receptor, and ApoE genes in Italian patients with spo- Department of Neuroscience Neurological Clinics, University of Pisa, Pisa, Italy. 309

2 310 ROCCHI ET AL. TABLE 1. GENOTYPE DISTRIBUTION AND ALLELIC FREQUENCIES OF THE 5-HT2A T102C POLYMORPHISM IN AD AND CONTROL GROUPS Allele frequencies Groups n CC (%) TC (%) TT (%) C (%) T (%) AD (27.0) 64 (47.0) 35 (26.0) 136 (50.37) 134 (49.63) Controls (24.4) 40 (44.4) 28 (31.1) 84 (47.0)0 96 (53.0)0 radic AD, to obtain more information about their possible roles as AD risk factors and/or genetic determinants of predisposition to the development of AD-associated psychopathology. This is the first association study about 5-HTT and AD performed in an Italian population sample. The 5-HT2A receptor polymorphism has already been analyzed in Italy (Nacmias et al., 2001), but independently from the serotonin transporter. Our study will make possible a more comprehensive view of the involvement of the serotonin transmitter system in the pathophysiology of AD. Patients MATERIALS AND METHODS A total of 135 consecutive AD patients (90 females and 45 males, mean age at onset and SD: years) presenting to the University of Pisa, Alzheimer Disease Centre, Department of Neurosciences, were recruited. The diagnosis of probable AD was made according to DSM-IV and NINCDS-ADRDA criteria (McKhann et al., 1984). Cognitive assessment was made by means of a Mini-Mental State Examination (MMSE) and a complete neuropsychological battery, including reasoning, language, short- and long-term memory, and sustained and divided attention tasks. The Clinical Dementia Rating Scale and Instrumental Activities of Daily Living Scale were also performed to assess the functional ability of each AD patient. Behavioral assessment was carried on by means of the Neuropsychiatric Inventory (NPI), which is a care-giver-based instrument that evaluates 10 troublesome behaviors in patients with dementia. The 10 domains assessed by using the NPI are delusions, hallucinations, agitation, depression, anxiety, euphoria, apathy, disinhibition, irritability, and abnormal motor output. The cognitive evaluation and NPI were administered at the same time. Each patient or, when requested, his legal guardian gave an informed written consent to carry on the study. As controls, we studied a group of 90 age- and sex-matched non-demented individuals (mean age and SD: years). All control subjects were evaluated to exclude the concomitant finding of other neurological or psychiatric disorders. DNA analysis Genomic DNA was extracted from peripheral blood lymphocytes, according to standard procedures (Maniatis et al., 1982). 5-HT2A T102C and 5-HTTLPR polymorphisms were typed by a method involving polymerase chain reaction (PCR) amplification. The amplification of 5-HT2A T102C was followed by Msp I digestion (Holmes et al., 1998). PCR conditions for typing 5-HTTLPR were performed according to Collier and co-workers (Collier et al., 1996). This protocol is engineered to discriminate only between the two most common alleles (L or S). The various genotypes were detected on a 3.5% metaphor-agarose gel after electrophoresis. The ApoE genotypes were detected using the oligonucleotide primers 59-TCGGCCGCAGGGCGCTGATGG-3 9 and 59- TCGCGGGCCCCGGCCTGGTA-3 9. PCR amplification was performed by using 25 ng of genomic DNA, 0.1 mm of each primer and 1.5 mm MgCl 2 in a final volume of 20 ml. The sample was subjected to one cycle of 2 min at 95 C, then 30 cycles of 30 sec duration at 90 C, 30 sec at 67 C, 30 sec at 72 C, and finally 72 C for 7 min. The PCR product was digested using the enzyme Hha I. The frequency of ApoE alleles was compared with epidemiological data from the literature (Mayeux et al., 1998). Statistical analysis Comparison of genotype distributions and allele frequencies between patients and controls were evaluated by x 2 statistics. In all tests, a significance level of p, 0.05 was required. TABLE 2. GENOTYPE DISTRIBUTION AND ALLELIC FREQUENCIES OF THE 5-HT2A T102C POLYMORPHISM IN AD CASES WITH PSYCHOSIS AND WITHOUT PSYCHOSIS Allele frequencies Groups n CC (%) TC (%) TT (%) C (%) T (%) AD with (44.8) 24 (41.4) 8 (13.8) 77 (66.0) 41 (34.0) psychosis AD without (13.0) 40 (51.9) 27 (35.1) 59 (39.0) 93 (61.0) pychosis Total (27.0) 64 (47.0) 35 (26.0) 136 (50.37) 134 (49.63)

3 SEROTONINERGIC POLYMORPHISMS IN AD 311 TABLE 3. GENOTYPE DISTRIBUTION AND ALLELE FREQUENCIES OF THE 5-HT2A T102C POLYMORPHISM IN AD PATIENTS WITH AND WITHOUT PSYCHOTIC SYMPTOMS AFTER STRATIFICATION ACCORDING TO THE PRESENCE OR ABSENCE OF APOE «4 ALLELE AD with psychosis AD without psychosis ApoE«41 ApoE«42 ApoE«41 ApoE«42 n 5 26 n 5 32 n 5 26 n 5 51 TT (%) 3 (11.5) 5 (15.6) 10 (38.5) 16 (31.4) TC (%) 11 (42.3) 13 (40.6) 12 (46.2) 29 (56.8) CC (%) 12 (46.2) 14 (43.8) 4 (15.4) 6 (11.8) Alleles T (%) 17 (32.7) 23 (35.9) 32 (61.5) 61 (59.8) C (%) 35 (67.3) 41 (64.1) 20 (38.5) 41 (40.2) RESULTS Clinical features of patients On the basis of the NPI assessment, AD patients were divided into two subgroups: patients with psychotic symptoms (n 5 58, 43%) and without psychotic symptoms (n 5 77, 57%). In more detail, all psychotic patients had delusions and half had hallucinations. The most common delusions were of burglary and infidelity, whereas visual hallucinations were more prevalent than auditory ones. Trends for lower MMSE (p ), greater agitation (p ), and anxiety (p ), with lower aberrant motor behavior (p ) were present in the psychotic group with respect to the nonpsychotic group. No significant difference was observed between the two subgroups (psychotic vs. nonpsychotic patients) in terms of duration of the disease ( years, vs years). Also, no difference was reported between psychotic and nonpsychotic AD patients regarding severity of the dementia and functional ability in daily living, as assessed by Clinical Dementia Rating and Instrumental Activity of Daily Living Scale. At the time of genetic testing, AD psychotic patients were taking the following neuroleptic drugs: 22 subjects, olanzapine; 18, risperidone; 5, clozapine. Thirteen patients were not in therapy because their behavioral disorder was newly diagnosed at the time of the examination. Of the AD patients, 125 out of all 135 included in the study were taking for cognitive pharmacological support, cholinesterase inhibitors at optimal doses (54, rivastigmine; 10, galantamine; 61, donepezil). In the clinical follow-up at 2 years, as expected, AD psychotic patients exhibited an accelerated cognitive decline, when compared to AD non-psychotic patients. 5-HT2A T102C polymorphism Table 1 shows the genotype distribution and allele frequencies of the 5-HT2A T102C polymorphism in cases and controls. The genotype and allele distributions of sporadic AD cases did not deviate from the Hardy-Weinberg equilibrium and were comparable to controls. On the contrary, when we considered the presence or not of psychotic symptoms, the allele and genotype frequencies differed significantly between AD patients with and without psychiatric features (x , p, 0.001). For AD subjects with psychosis, 44.8% were homozygous for the C102 allele, compared to 13% of AD without psychosis (Table 2), indicating a segregation of the CC genotype with psychotic symptoms, whereas the TT genotype was associated with absence of psychosis (35.1% vs. 13.8%). In terms of allelic frequency, the C allele was overrepresented in the AD group with psychosis (66%); conversely, in patients without psychosis, there was a higher frequency of the T allele (61%). A stratification of the 5-HT2A data based on the presence or absence of the ApoE «4 allele (patients with one or more «4 alleles were considered as «41 and the others as «42) did not show any association between ApoE genotypes and the presence of psychotic symptoms, or between ApoE and 5-HT2A genotypes (Table 3). TABLE 4. GENOTYPE DISTRIBUTION AND ALLELE FREQUENCIES OF THE 5-HTTLPR POLYMORPHISM IN AD PATIENTS, WITH AND WITHOUT PSYCHOTIC SYMPTOMS, AND CONTROLS Genotype Allele frequencies Group n SS (%) LS (%) LL (%) S (%) L (%) AD cases (23.0) 54 (40.0) 50 (37.0) 116 (43.0) 154 (57.0) AD with (18.9) 28 (48.3) 19 (32.8) 50 (43.0) 66 (57.0) psychosis AD without (26.0) 26 (33.8) 31 (40.2) 66 (43.0) 88 (57.0) psychosis Controls (23.3) 38 (42.2) 31 (34.5) 80 (44.0) 100 (56.0)

4 312 ROCCHI ET AL. TABLE 5. GENOTYPE DISTRIBUTION AND ALLELE FREQUENCIES OF THE 5-HTTLPR POLYMORPHISM IN AD PATIENTS WITH AND WITHOUT PSYCHOTIC SYMPTOMS, AFTER STRATIFICATION OF PATIENTS INTO APOE«41 AND APOE«42 GROUPS AD with psychosis AD without psychosis ApoE«41 ApoE«42 ApoE«41 ApoE«42 n 5 26 n 5 32 n 5 26 n 5 51 LL (%) 9 (34.6) 9 (28.1) 10 (38.5) 21 (41.2) LS (%) 12 (46.2) 18 (56.3) 8 (30.8) 18 (35.3) SS (%) 5 (19.2) 5 (15.6) 8 (30.8) 12 (23.5) Alleles f (L) 58% 56% 54% 59% f (S) 42% 44% 46% 41% 5-HTTLPR polymorphism Table 4 shows the genotype distribution and allele frequencies of the 5-HTTLPR polymorphism in cases and controls. The genotype and allele distributions of sporadic AD cases did not deviate from the Hardy-Weinberg equilibrium and were comparable to controls. Table 4 also displays the genotype and allele frequencies of 5-HTTLPR in the two subgroups of AD patients with and without psychosis: No correlation was present between psychotic symptoms and 5-HTTLPR. To assess a possible association between 5-HTT and ApoE polymorphism, patients were grouped according to the presence (ApoE «41) or absence (ApoE «42) of the «4 allele. No differences could be detected between carriers and noncarriers of the «4 allele in the frequency distribution of alleles and genotypes of the 5-HTT polymorphism, both in psychotic and nonpsychotic patients (Table 5). ApoE polymorphism Allelic and genotype frequency distributions of the ApoE polymorphism in our sample are in Hardy-Weinberg equilibrium, and, as shown in Table 6, confirm the results obtained in the numerous studies about the association between AD and ApoE: There is a highly significant difference in the genotype and allele frequencies of the ApoE «4 polymorphism between patients and controls (x , p, 0.001). DISCUSSION The T102C polymorphism in the 5-HT2A gene has been previously considered a susceptibility factor for the development of schizophrenia, bipolar affective disorders, and eating disorders (Williams et al., 1996; Collier et al., 1997; Sorbi et al., 1998). Moreover, a significant loss of 5-HT2A receptors was reported in postmortem and in vivo studies on AD patients with behavioral alterations (Cross et al., 1986; Blin et al., 1993). Results from our study strongly confirm that genetic variation at the 5-HT2A gene locus is associated with the development of psychotic symptoms in AD and, with regard to this, the C102 allele might play a significant role in the clinical course of the disease. The finding of such a positive association points to this genotype as a risk factor for the occurrence of psychosis in AD. Because the T102C polymorphism does not cause a change in the 5-HT2A amino acid sequence, our results may be the consequence of the existence of a functional polymorphism elsewhere in the gene, which could be in linkage disequilibrium with the T102C variation. Actually, as reported in a paper by Arranz et al. (1998a), the T102C polymorphism has been shown to be in linkage disequilibrium with the G/A promoter polymorphism of the same gene, which would affect the expression levels of the receptor protein. Association studies of the serotonin transporter gene in AD and related psychopathology have yielded conflicting results (Kunugi et al., 2000; Zill et al., 2000; Sukonick et al., 2001; Tsai, 2001). Our data confirm that there was no association between 5-HTTLPR and psychotic symptoms in AD, suggesting that the genetic variation at the 5-HTT locus does not constitute a susceptibility factor for either AD risk or the development of psychotic symptoms in AD patients. Our data fail to detect any synergistic effect between ApoE and polymorphisms of the serotoninergic system on the risk for AD. This is because the genotypic distributions of the 5- HTTLPR and 5-HT2A polymorphisms are not significantly different between carriers and noncarriers of the ApoE «4 allele, both in the AD and control groups. However, in accordance with previous studies, in our population sample, the «4 allele TABLE 6. GENOTYPE DISTRIBUTION AND ALLELIC FREQUENCIES OF THE APOE POLYMORPHISM IN AD PATIENTS AND CONTROLS AD Controls n n 5 90 E2E2 0 0 E2E3 2 4 E2E4 1 0 E3E E3E E4E Sum Alleles (%) E2 1.1% 2.3% E3 75.6% 94.3% E4 23.3% 3.4%

5 SEROTONINERGIC POLYMORPHISMS IN AD 313 was found to be the major determinant of susceptibility to sporadic late-onset AD. Several studies analyzed the association between psychotic symptoms and the presence of the ApoE «4 allele (Forsell et al., 1998; Levy et al., 1999). Earlier work reported an increase of the «4 frequency in psychotic patients (Ramachandran et al., 1996); however, later work did not find a positive association (Harwood et al., 1999). In the present study, we failed to detect any association between a particular genotype in 5-HT2A or 5-HTT and the presence or absence of the «4 allele in patients stratified according to a clinical diagnosis of psychosis. Therefore, we have to conclude that ApoE is not a key factor in the development of psychotic symptoms during the course of AD. In conclusion, these rather common polymorphisms of genes of the serotoninergic system are not involved in the basic mechanisms of AD pathogenesis, but 5-HT2A seems to act like a genetic risk factor for the development of psychotic symptoms in those with AD. This datum can be significant in light of recent studies on schizophrenic patients (Arranz et al., 1998a,b) showing an association between the T102C and two other polymorphisms in the 5-HT2A gene and response to clozapine, an atypical antipsychotic drug with affinity for serotonin receptor binding sites. The C102 homozygous genotype was more frequent in patients who were clozapine-resistant than in patients who were responsive. In summary, this study shows that in patients with sporadic AD, psychotic symptoms are strongly associated with the C102 allele of the 5-HT2A gene and supports the usefulness of genetic screening in the search for determinant factors of noncognitive symptomatology in AD. 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