Pharmacogenetic analyses of variations of measures of cardiovascular risk in Alzheimer's dementia

The burden of vascular risk factors affects several organic mechanisms such as renal function and cerebral perfusion. Though albuminuria is a marker for subclinical cardiovascular damage, it has been described that in older people with arterial hypertension both low and high glomerular filtration rates lead to increased cardiovascular morbidity and mortality1, possibly due to increased vascular burden or frailty, respectively. High cerebrovascular risk leads to earlier onset of Alzheimer's dementia (AD), particularly when the vascular burden is more intense during midlife2. In healthy older people, there is a linear negative relationship between white matter hyperintensities and late-life cognitive function that increases with age3; in contrast, combined cerebrovascular risk factors may be neuroprotective in late life for patients with AD, probably due to enhanced cerebral perfusion4.

Angiotensin-converting enzyme (ACE) inhibitors such as enalapril reduce intraglomerular pressure5 as well as the risk of incident mild cognitive impairment6. The large interindividual variability in blood pressure response to ACE inhibitors is probably related to genetic differences7, while it has been demonstrated that brain-penetrating ACE inhibitors boost genetically mediated neuroprotective effects in patients with AD8. An Alu repeat insertion/deletion (I/D) polymorphism in intron 16 is the most studied functional marker of ACE, but associations of the insertion allele with AD are not consistent in all studies9, though such associations are usually confirmed by meta-analyses10. The two functional variants of ACE with the most significant effects for higher activity of the ACE are rs1800764 and rs42915, affecting neuropsychiatric symptoms and risk of the amnestic phenotype of AD11, cognitive decline8 and urea and creatinine5 variations: rs1800764 is located at about 0.2 kb from the transcription start site in the promoter of ACE in 17q23, while rs4291 is at approximately 3.8 kb from the same site8.

An atherogenic lipid profile also increases the risk of arterial hypertension and endothelium damage12. The LDLR (low-density lipoprotein receptor) gene resides within a region linked to AD in 19p13.3, whereas rs1166957613 and rs593014 are two of the most important genetic variants of the epidermal growth factor precursor homology domain of LDLR to be associated with variability in risk of AD15. The liver X receptor β (LXR-β) isoform is also expressed in the brain, regulating cholesterol homeostasis and amyloidogenesis16, while several variants of the LXR-β gene close to APOE in chromosome 19 have been linked with variable risk of AD17. LXR-β modulation may also inhibit angiotensin II by suppressing the angiotensin (AT1) receptor18.

Metabolic changes in late life lead to weaker associations between LDL-cholesterol and coronary heart disease (CHD), whereas the opposite occurs with levels of high-density lipoprotein (HDL) cholesterol19. Moreover, high blood pressure and atherogenic mechanisms give rise to increased arterial stiffness, which alters cerebral autoregulation and leads to cognitive decline2, but these mechanisms may be prevented by therapy with anti-hypertensive and lipid-lowering drugs. While neurodegeneration may affect blood pressure variation in late life20, gender differences have been reported for the associations of cerebrovascular risk factors with cognitive and functional decline21. This prospective study was aimed to estimate the variations in one year of systolic and diastolic blood pressure in patients with AD, as well as of creatinine clearance, by pharmacogenetic analyses of the effects of ACE inhibitors and statins, while also taking into account possible impacts of these cardiovascular measures over variations of the CHD risk.

Material & Methods

Consecutive outpatients with AD according to the National Institute on Aging - Alzheimer's Association criteria22 were prospectively selected from November 2010 to May 2014 at the Behavioural Neurology Section of Hospital São Paulo, Federal University of São Paulo, São Paulo, Brazil. Patients were excluded if they had mild cognitive impairment, history of kidney transplant or any form of dialytic therapy or if they did not complete one year of follow up.

After diagnostic confirmation, all patients had at least three consultations in the follow up and were assessed for proxy reports regarding age, the onset of dementia and treatment with anti-hypertensive or lipid-lowering drugs, whereas weight, gender, arterial hypertension, creatinine clearance and CHD risk were objectively assessed. For statistics, only the first and the last evaluations were considered. Fasting serum creatinine levels, total cholesterol and high-density lipoprotein cholesterol were measured at the beginning and after one year of follow up. Creatinine clearance was estimated by the formula by Cockcroft and Gault23. Framingham projections of the 10-yr absolute CHD risk24 were estimated for all patients. Blood pressure was measured in every evaluation, while the diagnosis and treatment of arterial hypertension followed the Joint National Committee (JNC) 7 report25.

This study was approved by the Institutional Ethics Committee. All patients and their legal representatives signed the written informed consent forms before the evaluation. Blood samples (5 ml) were drawn from each patient for genotyping.

Genotyping procedures: Genotyping from venous blood DNA by real-time polymerase chain reactions using TaqMan^® SNP Genotyping Assays on the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems^®, USA) was undertaken only after clinical data were collected from all patients, following the standard protocols of the manufacturer. The insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene was determined by conventional PCR26. The presence of the I/D polymorphism or ACE genotypes or haplotypes of rs1800764 and rs4291 was correlated with anti-hypertensive treatment using one of the ACE inhibitors. Presence of LDLR genotypes or haplotypes of rs11669576 (exon 8 of the LDLR gene) and rs5930 (exon 10 of the LDLR gene), or genotypes of rs2695121 (LXR-β gene), was correlated with hypocholesterolaemic therapy with a statin.

Outcome measures: The primary outcome measure was the variation in one year of creatinine clearance, systolic and diastolic blood pressure, taking the following independent variables into account: use of one of the ACE inhibitors and the ACE I/D polymorphism or ACE genotypes or haplotypes; or therapy with a statin and genotypes of the LXR-β gene or LDLR genotypes or haplotypes. In secondary analyses, the impact of changes in one year of systolic and diastolic blood pressure or creatinine clearance over variations of the CHD risk was assessed.

Statistical analysis: Paired Student's t test was employed for variations of weight, serum creatinine, creatinine clearance, blood pressure, total cholesterol, HDL-cholesterol and the CHD risk, taking baseline and final values after one year into account. The Hardy-Weinberg equilibrium for the I/D polymorphism and genotypes of ACE, LDLR and the LXR-β gene, was estimated using the Chi-square test. A general linear model with post-hoc Hochberg's GT2 was employed for variations of creatinine clearance, systolic and diastolic blood pressure, according to ACE genotypes or haplotypes or the I/D polymorphism, and use or no use of one of the ACE inhibitors; or else, LDLR genotypes or haplotypes or genotypes of the LXR-β gene, and use or no use of a statin. The general linear model was adjusted for gender, age, length of the dementia syndrome and weight variations in one year. Simple linear regressions were employed for correlations between variations of creatinine clearance, systolic and diastolic blood pressure and variations of the CHD risk, as well as for correlations regarding variations of creatinine clearance and systolic and diastolic blood pressure levels among themselves. All analyses were performed with IBM SPSS Statistics v20.0 (SPSS Inc., Chicago, IL, USA).

Results

Overall, 217 consecutive patients were included in this study. During follow up, 14 patients (6.5%) died, eight (3.7%) abandoned the study and two patients (0.9%) were excluded due to incomplete clinical data, resulting in a final sample of 193 patients.

Table I shows demographic and clinical profile for all patients. For blood pressure control, 81 patients (42.0%) used one anti-hypertensive drug, 53 patients (27.5%) used two and 21 (10.9%) used three anti-hypertensive drugs: 124 (64.2%) used ACE inhibitors, 22 (11.4%) used angiotensin-receptor blockers, 61 (31.6%) used diuretics, 22 (11.4%) used β-blockers, 21 (10.9%) used calcium channel blockers and only 38 (19.7%) who did not have arterial hypertension used no anti-hypertensive drug. No patient used an ACE inhibitor and an angiotensin receptor blocker at the same time, but all four patients who used ezetimibe were also treated with a statin. Systolic and diastolic blood pressure levels were significantly lowered after one year, as well as total cholesterol levels, weight, creatinine clearance and the CHD risk.

Table II shows genetic results for all patients. Minor allele frequencies were 0.497 for the ACE I/D polymorphism (insertion allele), 0.497 for ACE-rs1800764 (C), 0.345 for ACE-rs4291 (T), 0.078 for LDLR-rs11669576 (A), 0.345 for LDLR-rs5930 (A) and 0.381 for rs2695121 (T, LXR-β gene), all variants in Hardy-Weinberg equilibrium.

Table III shows the effects of genotypes of rs2695121 (LXR-β gene), LDLR and ACE over variations of creatinine clearance and blood pressure in one year regardless of pharmacological treatment. The presence of T alleles of rs2695121 (LXR-β gene) or A alleles of LDLR-rs5930 was associated with lower systolic blood pressure, whereas the AA genotype of LDLR-rs5930 was associated with higher creatinine clearance after one year.

Table IV shows blood pressure and creatinine clearance variations in one year according to the use or no use of ACE inhibitors, and ACE variants. ACE inhibitors had significant systolic and diastolic blood pressure lowering effects, regardless of any ACE polymorphisms or haplotypes; however, these effects were more significant for carriers of ACE Alu DD, the C allele of ACE-rs1800764 or the AT genotype of ACE-rs4291. Carriers of ACE Alu II, the T allele of ACE-rs1800764 or the A allele of ACE-rs4291 who used ACE inhibitors were protected against decreased creatinine clearance variations.

Table V shows blood pressure and creatinine clearance variations in one year according to the use or no use of statins, and genotypes of rs2695121 (LXR-β gene), as well as LDLR variants. No significant impacts over variations in blood pressure or creatinine clearance were found regarding the use or no use of statin therapy. Statins led to lower creatinine clearance for carriers of the CT genotype of rs2695121 (LXR-β gene), while carriers of the AG genotype of LDLR-rs5930 had lower blood pressure after one year when not using statins.

Creatinine clearance variations followed weight variations and were significantly affected by these factors in the general linear model (P<0.001). In spite of specific pharmacological treatment, there were no correlations between creatinine clearance and the CHD risk, neither at the beginning nor at the end of the follow up. Similarly, creatinine clearance variations were not correlated with variations of the CHD risk, but each 1 mmHg variation in systolic blood pressure led to 0.2 per cent variation in the CHD risk (P<0.001), and each 1 mmHg variation in diastolic blood pressure led to 0.1 per cent variation in the CHD risk (P=0.003). Similarly, systolic and diastolic blood pressure levels were associated at the beginning and at the end of the follow up (P<0.001), as well as their variations (P<0.001): each 1.15 mmHg variation in systolic blood pressure led to 1.00 mmHg variation in diastolic blood pressure. In contrast, creatinine clearance variations were associated neither with systolic nor with diastolic blood pressure variations.

Discussion

The burden of vascular risk factors over primary neurodegeneration in the ageing population is likely to boost the incidence of dementia in countries such as India27 and Brazil2; therefore, it is important to assess treatment strategies for vascular risk in highly populated countries. Allele frequencies of the polymorphisms investigated in the present study were in agreement with previous studies. Overall, it was found that ACE inhibitors had significant systolic and diastolic blood pressure lowering effects for all patients, regardless of any ACE polymorphisms or haplotypes, and without significant effects over creatinine clearance variations. Impacts of statin therapy were found neither for blood pressure nor for creatinine clearance variations. The presence of T alleles of rs2695121 (LXR-β gene) or A alleles of LDLR-rs5930 led to systolic blood pressure reductions. Still, the AA genotype of LDLR-rs5930 was protective regarding creatinine clearance variations.

Systolic and diastolic blood pressure variations were associated with variations of the CHD risk. In contrast, no associations were found between creatinine clearance and blood pressure or the CHD risk, suggesting independence between pathways that mediate effects of high blood pressure over renal function and CHD.

Variants ACE-rs180076428 and ACE-rs42915 and the deletion allele of the I/D polymorphism10 in ACE have been shown to be associated with boosted serum levels of the ACE while increasing the risk of arterial hypertension29, particularly for patients with CHD and cerebrovascular disease7. In our study, blood pressure lowering effects of ACE inhibitors were more significant for carriers of ACE Alu DD, the C allele of ACE-rs1800764, or the AT genotype of ACE-rs4291. Carriers of ACE Alu II, the T allele of ACE-rs1800764, or the A allele of ACE-rs4291 who used ACE inhibitors were protected regarding creatinine clearance variations. Overall, carriers of genotypes that supposedly lead to higher serum levels of the ACE led to better blood pressure response to ACE inhibitors, whereas lower serum levels had protective effects over renal function30.

The statin therapy did not affect blood pressure or creatinine clearance variations in our study: since our patients were old people, most of them probably had accumulated diffuse atherosclerotic plaques throughout life that would not be susceptible to lipid-lowering therapy; and statin therapy being initiated in late life for most of them was possibly too late to revert the effects of atherogenesis over the endothelium. Similarly, conflicting associations have been reported between statin therapy and cognitive decline in late life31. It remains to be seen whether longer-term lipid-lowering therapy might have benefits over cardiovascular measures. A meta-analysis in patients with chronic renal failure not requiring dialysis reported that the effects of statins on stroke and renal function were uncertain, while adverse effects were incompletely understood; however, statins consistently lowered death and major cardiovascular events in these patients32. While the AA genotype of LDLR-rs5930 led to increased creatinine clearance, and the AG genotype was associated with lower blood pressure after one year for patients who did not use statins, the A allele was associated with lower risk of AD when combined with other LDLR polymorphisms in an earlier study14. Another study reported the GG genotype of LDLR-rs11669576 associated with a higher risk of AD when in combination with APOE4+ haplotypes and the CC genotype of LDLR-rs592515. Hypertriglyceridaemia has also been found to be associated with AD, regardless of any genetic variants33. It may be possible that the same LDLR genotypes that reduce cardiovascular risk tend to confer neuroprotection, suggesting intertwined mechanisms, but since the assessment of the risk of AD was not an outcome measure of this study, these findings should be studied with greater depth in other populations.

Regarding the LXR-β gene, statins led to lower creatinine clearance for carriers of the CT genotype of rs2695121, whereas the presence of T alleles of rs2695121 was associated with systolic blood pressure lowering; in an earlier study, the C allele was associated with late-onset AD when in combination with other genotypes in sibpairs16. Conflicting results have been found for the association of rs2695121 with the metabolic syndrome18, even though the T allele has also been shown to be associated with obesity in women34. The T allele of rs2695121 may be associated with reduced risk of late-onset AD by lowering systolic blood pressure, while earlier studies found that late-life increases in body mass index may be neuroprotective for patients with AD4, but these findings should be confirmed when other polymorphisms of the LXR-β gene are concurrently analyzed.

More than 80 per cent of our patients had arterial hypertension according to the JNC 7 report25, confirming the burden of this cardiovascular risk factor in older people. People with dementia tend to experience greater late-life decrease in blood pressure as a feature of neurodegeneration20. This could explain the significant disparities of blood pressure variations in our findings, though our close follow up with aggressive anti-hypertensive therapy might also have affected these results.

Limitations of this study included the fact that it was conducted in a single centre, with a short follow up and no randomization, and lacking measurements of serum levels of the ACE, as well as an objective evaluation of sarcopenia. Furthermore, it was not known whether the pharmacogenetic effects of ACE inhibitors and statins were either dose-dependent or more significant when starting treatment or at any time during therapy, because many patients were already under treatment when they were included in the study. We tried to minimize these limitations by keeping observers blinded to genetic data during the evaluations. Physical activity could also be a confounding factor for our results, but most patients were sedentary; thus, the effects of exercise for our sample, if any, were probably very small. Individual variability in drug response depends on the interactions of several complex factors, including genetic and environmental issues, and must be studied in different populations.

In conclusion, our study showed that genetic determinants of cardiovascular effects affected the individual variability of the response to ACE inhibitors and statins. ACE inhibitors, but not statins, were involved in blood pressure variations in older people, regardless of any genetic variants; and lipid metabolism alleles were involved in blood pressure and creatinine clearance variability, probably due to atherogenic mechanisms. Future studies should confirm these findings in other populations, particularly when starting therapy with statins or ACE inhibitors and hence the personalization of therapeutic decisions can be achieved.