Abstract

Introduction

Cerebrovascular risk factors play an important role in the pathogenesis of Alzheimer’s disease dementia (AD). When present in midlife, pooled cerebrovascular risk factors synergistically increase AD risk and lower the age at AD onset.¹1. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Risk factors for age at onset of dementia due to Alzheimer’s disease in a sample of patients with low mean schooling from São Paulo, Brazil. Int J Geriatr Psychiatry. 2014;29:1033-9. As a result, these patients usually present with a combination of AD and vascular neuropathological profiles.²2. Ferreira LK, Tamashiro-Duran JH, Squarzoni P, Duran FL, Alves TC, Buchpiguel CA, et al. The link between cardiovascular risk, Alzheimer’s disease, and mild cognitive impairment: support from recent functional neuroimaging studies. Rev Bras Psiquiatr. 2014;36:344-57. Conversely, we have previously shown that higher cerebrovascular risk in late life leads to cognitive and functional stabilization or even improvement for patients with AD, possibly related to enhanced cerebral perfusion,³3. de Oliveira FF, Pivi GA, Chen ES, Smith MC, Bertolucci PH. Risk factors for cognitive and functional change in one year in patients with Alzheimer's disease dementia from São Paulo, Brazil. J Neurol Sci. 2015;359:127-32. though some studies have shown that cerebrovascular risk factors may lead to faster cognitive decline.²2. Ferreira LK, Tamashiro-Duran JH, Squarzoni P, Duran FL, Alves TC, Buchpiguel CA, et al. The link between cardiovascular risk, Alzheimer’s disease, and mild cognitive impairment: support from recent functional neuroimaging studies. Rev Bras Psiquiatr. 2014;36:344-57.

The astrocyte-secreted apolipoprotein E is a cholesterol-binding lipoprotein that transports cholesterol through cell membranes⁴4. Carter CJ. Convergence of genes implicated in Alzheimer’s disease on the cerebral cholesterol shuttle: APP, cholesterol, lipoproteins, and atherosclerosis. Neurochem Int. 2007;50:12-38. and is involved in cholinergic dysfunction,⁵5. Martins IJ, Berger T, Sharman MJ, Verdile G, Fuller SJ, Martins RN. Cholesterol metabolism and transport in the pathogenesis of Alzheimer’s disease. J Neurochem. 2009;111:1275-308. atherogenesis, and amyloidogenesis,⁶6. Del Prete M, Spaccavento S, Craca A, Fiore P, Angelelli P. Neuropsychiatric symptoms and the APOE genotype in Alzheimer’s disease. Neurol Sci. 2009;30:367-73. besides being a high-affinity ligand for the low-density lipoprotein receptor (LDLR) within the central nervous system.⁷7. Retz W, Thome J, Durany N, Harsányi A, Retz-Junginger P, Kornhuber J, et al. Potential genetic markers of sporadic Alzheimer’s dementia. Psychiatr Genet. 2001;11:115-22. The apolipoprotein E gene (APOE) is a moderately penetrant gene that is neither a prerequisite nor a sufficient agent for development of AD, despite the fact that APOE-ɛ4 is the most important genetic risk factor for incidence⁸8. Bilbul M, Schipper HM. Risk Profiles of Alzheimer disease. Can J Neurol Sci. 2011;38:580-92. and earlier onset of late-onset AD,¹1. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Risk factors for age at onset of dementia due to Alzheimer’s disease in a sample of patients with low mean schooling from São Paulo, Brazil. Int J Geriatr Psychiatry. 2014;29:1033-9. while also affecting behavioral performance²2. Ferreira LK, Tamashiro-Duran JH, Squarzoni P, Duran FL, Alves TC, Buchpiguel CA, et al. The link between cardiovascular risk, Alzheimer’s disease, and mild cognitive impairment: support from recent functional neuroimaging studies. Rev Bras Psiquiatr. 2014;36:344-57. and decreasing prospective physical activity in such patients.³3. de Oliveira FF, Pivi GA, Chen ES, Smith MC, Bertolucci PH. Risk factors for cognitive and functional change in one year in patients with Alzheimer's disease dementia from São Paulo, Brazil. J Neurol Sci. 2015;359:127-32.APOE-ɛ4 carriers are also more prone to decreased risk of dementia associated with physical activity,⁹9. Rovio S, Kåreholt I, Helkala EL, Viitanen M, Winblad B, Tuomilehto J, et al. Leisure-time physical activity at midlife and the risk of dementia and Alzheimer’s disease. Lancet Neurol. 2005;4:705-11. while neuropsychiatric assessment findings may correlate with specific biomarkers.¹⁰10. Braun M, Tupper D, Kaufmann P, McCrea M, Postal K, Westerveld M, et al. Neuropsychological assessment: a valuable tool in the diagnosis and management of neurological, neurodevelopmental, medical, and psychiatric disorders. Cogn Behav Neurol. 2011;24:107-14.

Several other genetic variants involved in cerebrovascular metabolism have been associated with AD. The two functional variants of the angiotensin-converting enzyme gene (ACE) with the most significant effects for higher activity and boosted serum levels of the angiotensin-converting enzyme (ACE) are the promoter polymorphisms rs1800764 and rs4291, affecting risk and age at onset of the amnestic phenotype of AD,¹¹11. Vardy ER, Brown K, Stopford CL, Thompson JC, Richardson AM, Neary D, et al. Cognitive phenotypes in Alzheimer’s disease and genetic variants in ACE and IDE. Neurobiol Aging. 2012;33:1486.e1-2. as well as cognitive decline and incidence of early-onset hypertension.¹²12. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Brain-penetrating angiotensin-converting enzyme inhibitors and cognitive change in patients with dementia due to Alzheimer’s disease. J Alzheimers Dis. 2014;42:S321-4. The LDLR mediates increased astrocytic expression of APOE induced by amyloid-β,⁴4. Carter CJ. Convergence of genes implicated in Alzheimer’s disease on the cerebral cholesterol shuttle: APP, cholesterol, lipoproteins, and atherosclerosis. Neurochem Int. 2007;50:12-38. whereas storage and release of cholesterol depend on the expression of the low-density lipoprotein receptor gene (LDLR), which resides within a region linked to AD in 19p13.3; rs11669576⁷7. Retz W, Thome J, Durany N, Harsányi A, Retz-Junginger P, Kornhuber J, et al. Potential genetic markers of sporadic Alzheimer’s dementia. Psychiatr Genet. 2001;11:115-22. and rs5930¹³13. Gopalraj RK, Zhu H, Kelly JF, Mendiondo M, Pulliam JF, Bennett DA, et al. Genetic association of low density lipoprotein receptor and Alzheimer’s disease. Neurobiol Aging. 2005;26:1-7. are two of the most important genetic variants of the epidermal growth factor precursor homology domain of LDLR to be associated with disrupted cholesterol metabolism and variability in the risk of AD. Cholesterol governs synaptogenesis and myelin biosynthesis,¹⁴14. Warstadt NM, Dennis EL, Jahanshad N, Kohannim O, Nir TM, McMahon KL, et al. Serum cholesterol and variant in cholesterol-related gene CETP predict white matter microstructure. Neurobiol Aging. 2014;35:2504-13. while the cholesteryl ester transfer protein (CETP) is associated with reverse cholesterol transport (from tissues to the liver)⁵5. Martins IJ, Berger T, Sharman MJ, Verdile G, Fuller SJ, Martins RN. Cholesterol metabolism and transport in the pathogenesis of Alzheimer’s disease. J Neurochem. 2009;111:1275-308.; protective cholesteryl ester transfer protein gene (CETP) variants lead to lower serum CETP levels and healthier lipid profiles,¹⁵15. Sanders AE, Wang C, Katz M, Derby CA, Barzilai N, Ozelius L, et al. Association of a functional polymorphism in the cholesteryl ester transfer protein (CETP) gene with memory decline and incidence of dementia. JAMA. 2010;303:150-8. though not all studies have shown genetically mediated lifetime cognitive effects.¹⁶16. Johnson W, Harris SE, Collins P, Starr JM, Whalley LJ, Deary IJ. No association of CETP genotype with cognitive function or age-related cognitive change. Neurosci Lett. 2007;420:189-92. Nevertheless, the A allele of rs708272 (TaqIB) is associated with lower serum CETP activity and lower coronary heart disease risk,¹⁷17. Thompson A, Di Angelantonio E, Sarwar N, Erqou S, Saleheen D, Dullaart RP, et al. Association of cholesteryl ester transfer protein genotypes with CETP mass and activity, lipid levels, and coronary risk. JAMA. 2008;299:2777-88. while the G allele of rs5882 (I422V) has been associated with lower serum CETP levels and greater white matter integrity in young adults,¹⁴14. Warstadt NM, Dennis EL, Jahanshad N, Kohannim O, Nir TM, McMahon KL, et al. Serum cholesterol and variant in cholesterol-related gene CETP predict white matter microstructure. Neurobiol Aging. 2014;35:2504-13. as well as with preserved cognitive function in longevity¹⁸18. Barzilai N, Atzmon G, Derby CA, Bauman JM, Lipton RB. A genotype of exceptional longevity is associated with preservation of cognitive function. Neurology. 2006;67:2170-5. and less medial temporal lobe atrophy in APOE-ɛ4 carriers with AD.¹⁹19. Murphy EA, Roddey JC, McEvoy LK, Holland D, Hagler DJ Jr, Dale AM, et al. CETP polymorphisms associate with brain structure, atrophy rate, and Alzheimer’s disease risk in an APOE-dependent manner. Brain Imaging Behav. 2012;6:16-26. Moreover, the nuclear liver X receptor β (LXR-β) isoform is also expressed in the brain,⁴4. Carter CJ. Convergence of genes implicated in Alzheimer’s disease on the cerebral cholesterol shuttle: APP, cholesterol, lipoproteins, and atherosclerosis. Neurochem Int. 2007;50:12-38. acting as a regulator of cholesterol homeostasis, controlling amyloidogenesis, and modulating inhibition of angiotensin II, while several variants of the LXR-β gene (NR1H2) close to APOE in chromosome 19 have also been linked with variable risk of AD.²⁰20. Infante J, Rodríguez-Rodríguez E, Mateo I, Llorca J, Vázquez-Iguera JL, Berciano J, et al. Gene-gene interaction between heme oxygenase-1 and liver X receptor-β and Alzheimer’s disease risk. Neurobiol Aging. 2010;31:710-4.

We aimed to examine associations of genetic variants of APOE, ACE, LDLR, CETP, and intron 2 of NR1H2 with age at onset of late-onset AD and with neuropsychiatric symptoms, stratified by stage of dementia.

Methods

Participants

In this cross-sectional study, consecutive outpatients with late-onset AD according to U.S. National Institute on Aging-Alzheimer’s Association criteria²¹21. McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas CH, et al. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 2011;7:263-9. were recruited from the Setor de Neurologia do Comportamento, Hospital São Paulo, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil, from October 2010 to February 2013. All outpatients with this diagnosis who were followed during the study period were recruited. Late-onset AD was defined as AD with onset of dementia syndrome after age 60 years.¹1. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Risk factors for age at onset of dementia due to Alzheimer’s disease in a sample of patients with low mean schooling from São Paulo, Brazil. Int J Geriatr Psychiatry. 2014;29:1033-9. All patients underwent magnetic resonance imaging (MRI) to evaluate either medial parietal or medial, basal, or lateral temporal atrophy or, in cases of claustrophobia or pacemaker use, a computed tomography (CT) scan to exclude vascular lesions. Patients with history of stroke were not included in the study.

A detailed self-report and/or proxy report was conducted for assessment of age at AD onset, years of schooling, gender, and history of antipsychotic and antidepressant therapy. Information on age at dementia onset was obtained through a review of medical records and confirmed after an interview with the caregiver (preferably a family member), who was required to visit the patient frequently. The time of onset of subjective memory complaints or mild cognitive impairment was not taken into account; rather, only the time of dementia onset (when functional decline actually began) was considered. Use of antipsychotics or antidepressants was quantified in patients who had been receiving them for at least 3 months before the evaluation, to ensure that proper clinical response resulting from stability of drug levels in the central nervous system had been achieved, as well as because follow-up assessments at the study facility are usually conducted in 3-month intervals.

Neuropsychiatric assessment

A thorough neuropsychiatric evaluation was conducted, with particular attention to caregiver-reported Clinical Dementia Rating (CDR)²²22. Chaves ML, Camozzato AL, Godinho C, Kochhann R, Schuh A, de Almeida VL, et al. Validity of the clinical dementia rating scale for the detection and staging of dementia in Brazilian patients. Alzheimer Dis Assoc Disord. 2007;21:210-7. and the 10-item version of the Neuropsychiatric Inventory, which does not take into account night-time behavior disturbances or appetite and eating abnormalities.²³23. Camozzatto AL, Kochhann R, Simeoni C, Konrath CA, Pedro Franz A, Carvalho A, et al. Reliability of the Brazilian Portuguese version of the Neuropsychiatric Inventory (NPI) for patients with Alzheimer’s disease and their caregivers. Int Psychogeriatr. 2008;20:383-93. Effects of body mass index on age at AD onset¹1. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Risk factors for age at onset of dementia due to Alzheimer’s disease in a sample of patients with low mean schooling from São Paulo, Brazil. Int J Geriatr Psychiatry. 2014;29:1033-9. and cognitive and functional change³3. de Oliveira FF, Pivi GA, Chen ES, Smith MC, Bertolucci PH. Risk factors for cognitive and functional change in one year in patients with Alzheimer's disease dementia from São Paulo, Brazil. J Neurol Sci. 2015;359:127-32. have been previously assessed in our population, and associations of sleep duration and sleep satisfaction with neuropsychiatric symptoms and APOE haplotypes have been described elsewhere.²⁴24. de Oliveira FF, Bertolucci PH, Chen ES, Smith Mde A. Assessment of sleep satisfaction in patients with dementia due to Alzheimer’s disease. J Clin Neurosci. 2014;21:2112-7. Ratings for the final score of each item in the Neuropsychiatric Inventory took into account the frequency and severity of each patient’s behaviors, while the total score was the sum of the subscale scores. All functional and behavioral assessments were conducted on weekday mornings, by the same examiner (FFO).

Genotyping procedures

Blood samples were collected from all patients into tubes containing 0.1% ethylenediaminetetraacetic acid (EDTA). Genomic DNA was extracted from these samples using a standard salting-out procedure for determination of APOE haplotypes (polymorphisms rs7412 and rs429358), ACE genotypes and haplotypes (polymorphisms rs1800764 and rs4291), LDLR genotypes and haplotypes (polymorphisms rs11669576 and rs5930), CETP genotypes and haplotypes (polymorphisms rs5882 and rs708272), and genotypes of rs2695121 (intron 2 of the NR1H2 gene). All single nucleotide polymorphisms (SNPs) were assessed by real-time polymerase chain reactions using TaqMan^® SNP Genotyping Assays on the Applied Biosystems^® 7500 Fast Real-Time PCR System (Applied Biosystems^®, Carlsbad, USA). Standard manufacturer protocols were followed. Genotyping procedures were carried out only after clinical data had been collected from all patients, thus ensuring observer blinding to genetic data.

Outcome measures

Primarily, we sought to determine associations of genetic variants of APOE, ACE, LDLR, CETP, and intron 2 of the LXR-β gene NR1H2 with age at AD onset and neuropsychiatric symptoms, stratified by dementia stage. Secondarily, we also investigated combined associations of gender and schooling with age at AD onset, as well as associations of gender, schooling, age at dementia onset, antipsychotic therapy, and antidepressant therapy with each neuropsychiatric symptom according to CDR scores.²²22. Chaves ML, Camozzato AL, Godinho C, Kochhann R, Schuh A, de Almeida VL, et al. Validity of the clinical dementia rating scale for the detection and staging of dementia in Brazilian patients. Alzheimer Dis Assoc Disord. 2007;21:210-7. Finally, frequencies and scores for all behavioral symptoms were compared according to stage of dementia.

Statistical analysis

Associations of APOE haplotypes, ACE genotypes and haplotypes of rs1800764 and rs4291, LDLR genotypes and haplotypes of rs11669576 and rs5930, CETP genotypes and haplotypes of rs5882 and rs708272, and genotypes of rs2695121 with age at AD onset were investigated independently by way of the Mann-Whitney U test and further assessed in combination in a multiple linear regression model that included male gender and schooling as covariates. Multiple linear regression models including a copy of each minor allele, each copy of APOE-ɛ4, male gender, years of schooling, age at dementia onset, antipsychotic therapy, and antidepressant therapy were employed at each dementia stage to investigate associations of these factors with each behavioral symptom and with Neuropsychiatric Inventory total scores. Each haplotype that was represented in at least three patients was associated with each behavioral symptom and with Neuropsychiatric Inventory total scores at each dementia stage by the Mann-Whitney U test. The threshold of significance was set at p < 0.05.

Ethical considerations

This study is part of a larger research project (1067/10) approved by the Ethics Committee of Hospital São Paulo, UNIFESP, in August 2010 (CAAE 0540.0.174.000-10). All invited patients and their legal representatives agreed to participate in the study and provided written informed consent before the evaluation.

Results

At the end of the recruitment period, 217 patients had been included. Of these, 16 (7.4%) were excluded due to missing genetic information, resulting in a final sample of 201 patients.

Table 1 shows demographic and clinical results for the final sample. More than two-thirds of patients were female, 47 patients (23.4%) received antipsychotic treatment, and 85 (42.3%) were on at least one antidepressant.

Table 2 reports genotypes and haplotypes for the final sample. Overall, 108 patients (53.7%) were APOE-ɛ4 carriers, while 93 (46.3%) were APOE-ɛ4 non-carriers. All assessed genetic variants were in Hardy-Weinberg equilibrium.

Regarding independent haplotypes that could affect age at dementia onset, we found that APOE-ɛ4/ɛ4 carriers had earlier dementia onset by 4.31 years (p = 0.001), rs5882-AA/rs708272-AG carriers had later dementia onset by 1.77 year (p = 0.048), rs5882-AG/rs708272-AA carriers had earlier dementia onset by 4.55 years (p = 0.014), and rs11669576-GG/rs5930-GG carriers had later dementia onset by 2.14 years (p = 0.021). The only isolated genotype that affected age at AD onset was rs5882-AA: carriers had later dementia onset by 1.76 year (p = 0.018), but this analysis did not survive multiple correlations. According to Table 3, the only variable to affect age at AD onset in a multiple linear regression model was the APOE haplotype: each copy of APOE-ɛ4 led to earlier dementia onset by 1.728 year (p = 0.012).

Tables 4, 5, and 6 show the results of multiple linear regression analyses regarding factors that may affect each behavioral symptom and Neuropsychiatric Inventory total score for patients who were mildly impaired (CDR = 1.0), moderately impaired (CDR = 2.0), and severely impaired (CDR = 3.0), respectively. Overall, Neuropsychiatric Inventory total scores increased according to dementia severity. For mildly impaired patients (n=78), anxiety was the most frequent and highest-scored symptom, while euphoria was the least frequent and hallucinations were the lowest-scored symptom. For moderately impaired patients (n=92), apathy was the most frequent and the highest-scored symptom, while euphoria was the least frequent and hallucinations were the lowest-scored symptom. For severely impaired patients (n=31), apathy was the most frequent and highest-scored symptom, while euphoria was the least frequent and lowest-scored symptom.

Considering associations of independent haplotypes with behavioral scores in mildly impaired patients, rs1800764-CC/rs4291-AT carriers (n=10) had more hallucinations (1.40±2.6 versus 0.15±0.4, p=0.031), rs1800764-CT/rs4291-AA carriers (n=11) had more disinhibition (1.00±1.3 versus 0.39±1.3, p=0.021) and more irritability (3.00±4.1 versus 1.12±3.0, p=0.022), rs1800764-TT/rs4291-AA carriers (n=19) had more agitation (3.32±3.1 versus 1.69±3.1, p=0.017), rs11669576-AG/rs5930-AG carriers (n=5) had more apathy (7.20±3.6 versus 3.19±3.7, p=0.026), rs11669576-AG/rs5930-GG carriers (n=8) had less apathy (1.00±2.1 versus 3.73±3.8, p=0.033), rs5882-AA/rs708272-AG carriers (n=14) had lower Neuropsychiatric Inventory total scores (9.86±6.7 versus 18.66±11.3, p=0.007) and less irritability (0.57±0.8 versus 2.80±3.1, p=0.008), rs5882-AA/rs708272-GG carriers (n=13) had higher Neuropsychiatric Inventory total scores (24.38±9.4 versus 15.62±11.0, p=0.007) and more apathy (5.31±3.4 versus 3.08±3.8, p=0.041) and more dysphoria (3.38±3.8 versus 1.34±2.2, p=0.036), rs5882-AG/rs708272-GG carriers (n=12) had less anxiety (1.42±2.7 versus 4.39±4.5, p=0.025), and rs5882-GG/rs708272-AG carriers (n=5) had more delusions (3.40±5.1 versus 0.58±1.7, p=0.027).

Considering associations of independent haplotypes with behavioral scores in moderately impaired patients, rs1800764-CC/rs4291-AT carriers (n=8) had less agitation (0.37±0.7 versus 2.76±3.5, p=0.018), rs11669576-AG/rs5930-GG carriers (n=7) had less apathy (2.00±4.5 versus 5.05±4.5, p=0.049), rs11669576-GG/rs5930-AA carriers (n=8) had less anxiety (0.12±0.4 versus 2.68±3.9, p=0.033), rs11669576-GG/rs5930-AG carriers (n=42) had less irritability (3.26±3.7 versus 1.76±2.6, p=0.034), rs11669576-GG/rs5930-GG carriers (n=30) had less irritability (1.37±2.4 versus 2.97±3.4, p=0.006), rs5882-AG/rs708272-AA carriers (n=8) had more agitation (4.75±2.9 versus 2.35±3.4, p=0.008) and more hallucinations (3.00±3.8 versus 0.89±1.9, p=0.018) and more disinhibition (4.00±5.3 versus 1.08±2.5, p=0.029), and rs5882-AG/rs708272-AG carriers (n=23) had less agitation (1.39±2.9 versus 2.94±3.5, p=0.012).

Considering associations of independent haplotypes with behavioral scores in severely impaired patients, rs1800764-CT/rs4291-AA carriers (n=3) had more aberrant motor behavior (9.33±2.3 versus 3.14±4.6, p=0.033), rs1800764-CT/rs4291-AT carriers (n=11) had less disinhibition (1.27±2.9 versus 3.35±4.0, p=0.038), rs1800764-TT/rs4291-AA carriers (n=12) had more disinhibition (4.25±4.5 versus 1.58±2.8, p=0.029), rs5882-AA/rs708272-AG carriers (n=5) had more anxiety (8.00±5.7 versus 2.35±3.6, p=0.023), rs5882-AG/rs708272-AG carriers (n=11) had more irritability (6.09±4.1 versus 2.30±3.6, p=0.028), and rs5882-AG/rs708272-GG carriers (n=8) had less delusions (0.25±0.5 versus 3.00±3.9, p=0.017).

Discussion

With the exceptions of anxiety and irritability, a trend was found for all other behavioral symptoms to be intensified according to functional decline; likewise, most significant correlations were found in later dementia stages. Anxiety is usually inversely correlated with apathy,²⁵25. de Oliveira FF, Wajman JR, Bertolucci PH, Chen ES, Smith MC. Correlations among cognitive and behavioural assessments in patients with dementia due to Alzheimer’s disease. Clin Neurol Neurosurg. 2015;135:27-33. whereas overall neuropsychiatric symptoms tend to correlate better with functional independence than with cognitive decline.²⁵25. de Oliveira FF, Wajman JR, Bertolucci PH, Chen ES, Smith MC. Correlations among cognitive and behavioural assessments in patients with dementia due to Alzheimer’s disease. Clin Neurol Neurosurg. 2015;135:27-33.,²⁶26. de Oliveira FF, Bertolucci PH, Chen ES, Smith Mde A. Pharmacological modulation of cognitive and behavioral symptoms in patients with dementia due to Alzheimer’s disease. J Neurol Sci. 2014;336:103-8. Antipsychotic therapy was associated with higher Neuropsychiatric Inventory total scores for mildly impaired patients, particularly regarding disinhibition, and with more delusions and more agitation for moderately impaired patients, probably representing the need for psychotropic treatment when more behavioral symptoms are present, while agitation does not usually improve with atypical antipsychotics in the long term.²⁶26. de Oliveira FF, Bertolucci PH, Chen ES, Smith Mde A. Pharmacological modulation of cognitive and behavioral symptoms in patients with dementia due to Alzheimer’s disease. J Neurol Sci. 2014;336:103-8. Likewise, antidepressant therapy was associated with higher Neuropsychiatric Inventory total scores for mildly impaired and severely impaired patients (particularly regarding dysphoria), and with more agitation for moderately impaired patients, also representing the expected need for such therapy according to symptoms of depression.

Male gender was associated with more irritability in mildly impaired patients, more apathy in moderately impaired patients, and higher Neuropsychiatric Inventory total scores, particularly regarding delusions, in severely impaired patients. Gender differences have been reported in other studies, particularly regarding greater anxiety and dysphoria in women.⁶6. Del Prete M, Spaccavento S, Craca A, Fiore P, Angelelli P. Neuropsychiatric symptoms and the APOE genotype in Alzheimer’s disease. Neurol Sci. 2009;30:367-73.

Schooling did not correlate with dementia onset in this sample. Nevertheless, more years of education were associated with less dysphoria in moderately impaired patients and with lower Neuropsychiatric Inventory total scores in severely impaired patients, particularly regarding agitation and irritability. Though an earlier study showed that educational attainment and AD duration did not affect behavior,²⁵25. de Oliveira FF, Wajman JR, Bertolucci PH, Chen ES, Smith MC. Correlations among cognitive and behavioural assessments in patients with dementia due to Alzheimer’s disease. Clin Neurol Neurosurg. 2015;135:27-33. patients were not stratified by dementia stage, and some associations may have been missed. Education enhances cognitive reserve and may affect the way the brain copes with the burden of AD neuropathology, thus modulating behavior.

Later dementia onset was associated with fewer hallucinations, less euphoria, and less apathy for moderately impaired patients. Considering that hallucinations are strong predictors of faster functional decline,²⁶26. de Oliveira FF, Bertolucci PH, Chen ES, Smith Mde A. Pharmacological modulation of cognitive and behavioral symptoms in patients with dementia due to Alzheimer’s disease. J Neurol Sci. 2014;336:103-8. they may also be more frequent when dementia starts earlier. A high prevalence of hallucinations and extrapyramidal signs might predict the presence of Lewy bodies in patients with AD.²⁷27. Iqbal K, Flory M, Soininen H. Clinical symptoms and symptom signatures of Alzheimer’s disease subgroups. J Alzheimers Dis. 2013;37:475-81. In severely impaired patients, later dementia onset was associated with higher Neuropsychiatric Inventory total scores, particularly regarding disinhibition, but also with less apathy and less aberrant motor behavior, a complex link that could be partly due to APOE-ɛ4 non-carriers having later AD onset.

On analysis of multiple correlations, only APOE haplotypes affected the age at dementia onset in this sample, confirming the essential role of genetically mediated neurodegeneration on AD onset.¹1. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Risk factors for age at onset of dementia due to Alzheimer’s disease in a sample of patients with low mean schooling from São Paulo, Brazil. Int J Geriatr Psychiatry. 2014;29:1033-9. Furthermore, moderately impaired APOE-ɛ4 carriers had less apathy, whereas severely impaired APOE-ɛ4 carriers had more delusions and more aberrant motor behavior. Whereas some cross-sectional studies have not found associations of APOE haplotypes with specific neuropsychiatric symptoms²⁵25. de Oliveira FF, Wajman JR, Bertolucci PH, Chen ES, Smith MC. Correlations among cognitive and behavioural assessments in patients with dementia due to Alzheimer’s disease. Clin Neurol Neurosurg. 2015;135:27-33. or sleep satisfaction,²⁴24. de Oliveira FF, Bertolucci PH, Chen ES, Smith Mde A. Assessment of sleep satisfaction in patients with dementia due to Alzheimer’s disease. J Clin Neurosci. 2014;21:2112-7. other studies have shown dose-dependent associations of copies of APOE-ɛ4 with delusions,²⁸28. Scarmeas N, Brandt J, Albert M, Devanand DP, Marder K, Bell K, et al. Association between the APOE genotype and psychopathologic symptoms in Alzheimer’s disease. Neurology. 2002;58:1182-8. aberrant motor behavior,⁶6. Del Prete M, Spaccavento S, Craca A, Fiore P, Angelelli P. Neuropsychiatric symptoms and the APOE genotype in Alzheimer’s disease. Neurol Sci. 2009;30:367-73. anxiety,²⁹29. Yoo HB, Lee HW, Shin S, Park SW, Choi JS, Jung HY, et al. Butyrylcholinesterase K and apolipoprotein ɛ4 affect cortical thickness and neuropsychiatric symptoms in Alzheimer’s disease. Curr Alzheimer Res. 2014;11:137-44. and agitation.³⁰30. Craig D, Hart DJ, McKool K, McIlroy SP, Passmore AP. Apolipoprotein E e4 allele influences aggressive behaviour in Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2004;75:1327-30. Mechanisms involve atrophy of the prefrontal cortex,²⁹29. Yoo HB, Lee HW, Shin S, Park SW, Choi JS, Jung HY, et al. Butyrylcholinesterase K and apolipoprotein ɛ4 affect cortical thickness and neuropsychiatric symptoms in Alzheimer’s disease. Curr Alzheimer Res. 2014;11:137-44. temporal lobe hypoperfusion,²⁸28. Scarmeas N, Brandt J, Albert M, Devanand DP, Marder K, Bell K, et al. Association between the APOE genotype and psychopathologic symptoms in Alzheimer’s disease. Neurology. 2002;58:1182-8. cholinergic imbalance,⁶6. Del Prete M, Spaccavento S, Craca A, Fiore P, Angelelli P. Neuropsychiatric symptoms and the APOE genotype in Alzheimer’s disease. Neurol Sci. 2009;30:367-73. and tau-mediated disruption of frontal serotonergic and dopaminergic networks.³⁰30. Craig D, Hart DJ, McKool K, McIlroy SP, Passmore AP. Apolipoprotein E e4 allele influences aggressive behaviour in Alzheimer’s disease. J Neurol Neurosurg Psychiatry. 2004;75:1327-30.APOE haplotypes have also been associated with variable frequency of hallucinations.⁶6. Del Prete M, Spaccavento S, Craca A, Fiore P, Angelelli P. Neuropsychiatric symptoms and the APOE genotype in Alzheimer’s disease. Neurol Sci. 2009;30:367-73.,²⁸28. Scarmeas N, Brandt J, Albert M, Devanand DP, Marder K, Bell K, et al. Association between the APOE genotype and psychopathologic symptoms in Alzheimer’s disease. Neurology. 2002;58:1182-8. The incidence of delusions and hallucinations tends to be dissociated, considering that in AD delusions are usually more common than hallucinations, while in Lewy body dementia syndromes, hallucinations are more common than delusions.³¹31. Oliveira FF, Machado FC, Sampaio G, Marin SM, Chen ES, Smith MC, et al. Contrasts between patients with Lewy body dementia syndromes and APOE-ɛ3/ɛ3 patients with late-onset Alzheimer disease dementia. Neurologist. 2015;20:35-41. Though APOE-ɛ4 carrier status seems to predispose to late-onset depression,³3. de Oliveira FF, Pivi GA, Chen ES, Smith MC, Bertolucci PH. Risk factors for cognitive and functional change in one year in patients with Alzheimer's disease dementia from São Paulo, Brazil. J Neurol Sci. 2015;359:127-32. particularly in women,³²32. Delano-Wood L, Houston WS, Emond JA, Marchant NL, Salmon DP, Jeste DV, et al. APOE genotype predicts depression in women with Alzheimer’s disease: a retrospective study. Int J Geriatr Psychiatry. 2008;23:632-6. we did not find correlations with dysphoria in our sample. Nevertheless, one study showed that women have earlier AD onset when they are APOE-ɛ4 carriers with a history of depression,³³33. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Assessment of risk factors for earlier onset of sporadic Alzheimer’s disease dementia. Neurol India; 2014;62:625-30. confirming that gender differences might affect dementia onset.

Considering ACE genotypes, carriers of the C allele of rs1800764 had more hallucinations when mildly impaired and less dysphoria when moderately impaired, whereas carriers of the T allele of rs4291 had less dysphoria when severely impaired. Regarding represented haplotypes in independent analyses, presence of the C allele of rs1800764 was associated with less agitation, while presence of the A allele of rs4291 was associated with more agitation, more disinhibition, more irritability, and worse aberrant motor behavior. To the best of our knowledge, these are all previously unreported associations, while only the T allele of rs4291 was reported as a susceptibility factor for unipolar major depression and hypothalamic-hypophyseal-adrenocortical axis hyperactivity in an earlier study of subjects without dementia.³⁴34. Baghai TC, Binder EB, Schule C, Salyakina D, Eser D, Lucae S, et al. Polymorphisms in the angiotensin-converting enzyme gene are associated with unipolar depression, ACE activity and hypercortisolism. Mol Psychiatry. 2006;11:1003-15. Given that angiotensin-converting enzyme degrades amyloid-β³⁵35. Ning M, Yang Y, Zhang Z, Chen Z, Zhao T, Zhang D, et al. Amyloid-β-related genes SORL1 and ACE are genetically associated with risk for late-onset Alzheimer disease in the Chinese population. Alzheimer Dis Assoc Disord. 2010;24:390-6. and is overexpressed in a compensatory manner in the hippocampus, frontal cortex, and caudate nucleus of both hemispheres of patients with AD,¹²12. de Oliveira FF, Bertolucci PH, Chen ES, Smith MC. Brain-penetrating angiotensin-converting enzyme inhibitors and cognitive change in patients with dementia due to Alzheimer’s disease. J Alzheimers Dis. 2014;42:S321-4. suppression of frontally mediated behaviors could occur by boosted enzyme levels and activity. Functional studies are needed to confirm these associations.

Considering LDLR genotypes, carriers of the A allele of rs11669576 had less anxiety and more aberrant motor behavior when moderately impaired, whereas carriers of the A allele of rs5930 had less delusions when mildly impaired, less anxiety when moderately impaired, and higher Neuropsychiatric Inventory total scores when severely impaired, particularly regarding apathy and irritability. Regarding represented haplotypes in independent analyses, presence of the G allele of rs5930 was associated with less apathy in mildly and moderately impaired patients. Still, carriers of rs11669576-GG/rs5930-GG had later dementia onset. The A allele of rs11669576 has been associated with variable risk of AD, either increasing risk when in combination with copies of APOE-ɛ4⁷7. Retz W, Thome J, Durany N, Harsányi A, Retz-Junginger P, Kornhuber J, et al. Potential genetic markers of sporadic Alzheimer’s dementia. Psychiatr Genet. 2001;11:115-22. or decreasing risk - only in women - when in combination with other LDLR polymorphisms,³⁶36. Lämsä R, Helisalmi S, Herukka SK, Tapiola T, Pirttilä T, Vepsäläinen S, et al. Genetic study evaluating LDLR polymorphisms and Alzheimer’s disease. Neurobiol Aging. 2008;29:848-55. while the A allele of rs5930 was associated with lower risk of AD when combined with other LDLR polymorphisms in an earlier study.¹³13. Gopalraj RK, Zhu H, Kelly JF, Mendiondo M, Pulliam JF, Bennett DA, et al. Genetic association of low density lipoprotein receptor and Alzheimer’s disease. Neurobiol Aging. 2005;26:1-7. Although neuropsychiatric symptoms have not previously been studied in association with variants of LDLR, genotypes associated with less AD neuropathology³⁶36. Lämsä R, Helisalmi S, Herukka SK, Tapiola T, Pirttilä T, Vepsäläinen S, et al. Genetic study evaluating LDLR polymorphisms and Alzheimer’s disease. Neurobiol Aging. 2008;29:848-55. could lead to later dementia onset and milder behavioral burden in earlier stages.

Considering CETP genotypes, carriers of the G allele of rs5882 had more delusions when mildly impaired, less dysphoria when moderately impaired, and less anxiety when severely impaired, whereas carriers of the A allele of rs708272 had higher Neuropsychiatric Inventory total scores when severely impaired, particularly regarding delusions, anxiety, and aberrant motor behavior. Regarding represented haplotypes in independent analyses, mildly impaired carriers of rs5882-AA/rs708272-GG had higher Neuropsychiatric Inventory total scores, particularly regarding apathy and dysphoria, whereas moderately impaired carriers of rs5882-AG/rs708272-AA had more agitation, more hallucinations, and more disinhibition. Carriers of rs5882-AG/rs708272-AG had less agitation when moderately impaired and more irritability when severely impaired. Carriers of rs5882-AA had later dementia onset, but this finding did not survive multiple correlations; conversely, carriers of rs5882-AG/rs708272-AA had earlier dementia onset on independent analysis. Though associations of CETP genotypes with behavioral symptoms have not been reported before, the AA genotype of rs708272 was associated with increased risk of AD for APOE-ɛ4 carriers in one meta-analysis,³⁷37. Li Q, Huang P, He QC, Lin QZ, Wu J, Yin RX. Association between the CETP polymorphisms and the risk of Alzheimer’s disease, carotid atherosclerosis, longevity, and the efficacy of statin therapy. Neurobiol Aging. 2014;35:1513.e13-23. while the GG genotype of rs5882 has been associated either with slower cognitive decline and lower risk of AD,¹⁵15. Sanders AE, Wang C, Katz M, Derby CA, Barzilai N, Ozelius L, et al. Association of a functional polymorphism in the cholesteryl ester transfer protein (CETP) gene with memory decline and incidence of dementia. JAMA. 2010;303:150-8. with faster cognitive decline and higher risk of AD,³⁸38. Yu L, Shulman JM, Chibnik L, Leurgans S, Schneider JA, De Jager PL, et al. The CETP I405V polymorphism is associated with an increased risk of Alzheimer’s disease. Aging Cell. 2012;11:228-33. or with increased risk of AD only for APOE-ɛ4 non-carriers.³⁹39. Arias-Vásquez A, Isaacs A, Aulchenko YS, Hofman A, Oostra BA, Breteler M, et al. The cholesteryl ester transfer protein (CETP) gene and the risk of Alzheimer’s disease. Neurogenetics. 2007;8:189-93. The G allele of rs708272 and the G allele of rs5882 seemed to play protective roles in our sample, possibly by improving myelin biosynthesis in the brain.

Severely impaired carriers of the T allele of rs2695121 had more anxiety and more aberrant motor behavior. Among all polymorphisms of the LXR-β gene (NR1H2), the T allele of rs2695121 is the most significant for risk of AD,²⁰20. Infante J, Rodríguez-Rodríguez E, Mateo I, Llorca J, Vázquez-Iguera JL, Berciano J, et al. Gene-gene interaction between heme oxygenase-1 and liver X receptor-β and Alzheimer’s disease risk. Neurobiol Aging. 2010;31:710-4. though it had not been previously associated with behavioral symptoms. Polymorphisms in cellular cholesterol efflux-related genes may affect the neuropsychiatric profile of patients with AD, as increased cholesterol levels induce amyloidogenesis.

An important limitation of this study is that it was conducted in a single center with no randomization. We tried to mitigate this drawback by ensuring that examiners were blinded to genetic data during the evaluations. In addition, the genotype and haplotype frequencies found were similar to those reported in most previous studies, confirming that our outpatient sample was probably representative of our population as a whole. The cross-sectional design of this study precludes deeper assumptions regarding causal relations. Even though APOE haplotypes were the only genetic factors to consistently affect age at AD onset, variants involved in cerebrovascular metabolism were associated with several behavioral symptoms, leading to the conclusion that cerebrovascular risk plays an important genetically mediated role in neuropsychiatric manifestations of AD.

Acknowledgements

This work was sponsored by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (grant 1067/10) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 2015/10109-5). FFO has received research support from CAPES and FAPESP. MCS has received grants from CAPES, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and FAPESP. PHB has received grants from FAPESP.

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