Apolipoprotein ɛ 4 Allele and Dental Occlusion Deficiency as Risk Factors for Alzheimer’s Disease

Abstract

Compromised dentition has been suggested to pose a significant risk factor for dementia. It was mainly investigated through insufficient tooth number, disregarding contact between opposing teeth (dental occlusion). The ɛ4 allele of apolipoprotein (APOE4) is the primary genetic marker for the late onset of Alzheimer’s disease (AD). However, APOE4 and dental occlusion have not yet been investigated as possible associated risk factors for AD. The study was aimed to examine the impact of dental status and different APOE gene variants on AD occurrence. Secondly, sociodemographic variables were investigated as factors potentially associated with AD. The case-control study included two groups: 116 patients with AD (according to the NINDS-ADRDA criteria) and 63 controls (Mini-Mental State Examination scores ≥24). The analysis of APOE gene polymorphism was conducted through PCR reaction. Dental examination included recording of number of teeth, presence of fixed or removable dentures, and number of functional tooth units (FTU). Regression analysis was used to investigate the joint effect of the clinical and genetic variables on AD. Results showed that patients with AD were more often carriers of ɛ3/ɛ4 genotype and ɛ4 allele, had lower number of teeth and FTU, and were less likely to be married, live in home, and had less chronic diseases, compared to the controls. Regression analysis showed that presence of APOE4 allele and the number of total FTU remained associated with AD, even when adjusted for age, sex, and level of education. In conclusion, deficient dental occlusion and presence of APOE4 may independently increase risk for AD.

Keywords

Alzheimer’s disease apolipoproteins E dental occlusion tooth loss

INTRODUCTION

The etiology of Alzheimer’s disease (AD) is widely believed to be a result of the combination of genetic and environmental risk factors [1, 2]. The ɛ4 allele of the apolipoprotein gene (APOE4) has proven to be a major genetic risk factor for the late onset of AD [1 , 3–5]. Several studies suggest that poor oral health can pose a significant risk factor for dementia [6 –8]. In studies that examined non-genetic risk factors for AD, tooth loss was marked as significant [9, 10]. Several mechanisms have been suggested to possibly underlie this association. The literature suggests that compromised dentition affects dietary quality and nutrient intake, including reliance on mashed food, too many calories, ingestion of high-fat food and fewer fruits and vegetables [11 –13]. Tooth loss has also been related to structural brain differences such as significantly lower total brain volume and gray matter volume in individuals with complete or partial tooth loss [10]. Despite the evidence-based arguments that masticatory dysfunction may show both short- and long-term effects on the brain and thus influence brain activity and cognitive performance [14, 15], most of these studies have considered number of teeth as a criterion, disregarding contacts between opposing teeth (dental occlusion). A study conducted in an animal model concluded that occlusal disharmony caused by cutoff of maxillary molar cusps produced psychological stress, inducing an increase in hippocampal amyloid-β, known for its role in neurodegeneration and dementia [15]. The results of a clinical study suggest that unbalanced occlusion may lead to left-right unbalance in the activity of the locus coeruleus, causing a difference in hemispheric excitability and finally to an impairment of cognitive performance [14].

So far, APOE4 and dental occlusion have not yet been investigated as possible associated risk factors for AD. The authors hypothesized that the APOE4 variant, number of natural teeth, and occlusal contacts were associated with AD. Thus, the aim of the study was to examine the association of different APOE genetic variants, number of natural teeth, and number of occluding tooth units with AD occurrence. Secondly, sociodemographic variables: sex, age, education, marital status, chronic diseases, and residence, were investigated as factors potentially associated with AD.

MATERIAL AND METHODS

Subjects

Two groups of subjects aged over 65 years were recruited for the study: AD patients and controls without dementia. Patients with a stable form of AD diagnosis have been recruited consecutively at the Institute of Neurology, University Hospital Centre and the Gerontology Centre. Clinical diagnosis of probable AD had been established based on the NINDS-ADRDA criteria [16]. The control subjects without dementia (Mini-Mental State Examination (MMSE) scores ≥24) were consecutively recruited at the Institute of Geriatric Medicine, University Hospital Centre and the Gerontology Centre. Exclusion criteria for both groups included significant change in dental status in the last five years.

Sociodemographic data such as sex, age, education, marital status, chronic diseases, and residence of patients were also collected. Written consents were obtained from subjects’ relatives. The study was approved by the Ethical Committee of the School of Dental Medicine, (decision number 36/18) and conducted from November 2015 till December 2017.

Genetic analysis

Genomic DNA was isolated from buccal swabs using a commercial kit for isolation (PureLink TM Genomic DNA Mini Kit, Invitrogen), according to the manufacturer’s recommendations. Isolated DNA was stored at –20°C until further analysis. A polymerase chain reaction (PCR) was performed using 200 ng of genomic DNA as template in 25 ml reaction mixture containing 20 pmol of PCR primers APOE-A (5^′-TCCAAGGAGCTGCAGGCGGCGCA-3^′) and APOE-B (5^′-ACAGAATTCGCCCCGGCCTGGTACACTGCCA-3^′), 1U of Taq DNA polymerase (Gibco BRL, Gaithersburg, MD, USA), 1.0 mM MgCl2 75 mM Tris-HCL (pH 9.0), 20 mM (NH₄)2SO₄ and 10% dimethyl sulphide. PCR amplification consisted of 35 cycles of 30 s at 95°C, 45 s at 63°C, and 45 s at 72°C. PCR products (270 bp) were digested using 5 U of HhaI (Thermo Fisher Scientific, Inc.). After digestion, products were separated on a 10% polyacrylamide gel and visualized on a UV transilluminator after ethidium bromide staining. The fragment lengths were as follows: for ɛ2 allele: 91 bp, 83 bp, and 38 bp; for ɛ3 allele: 91 bp, 48 bp, 38 bp, and 35 bp; for ɛ4 allele: 72 bp, 48 bp, 38 bp, and 35 bp.

Dental examination

Dental examination included recordings of number of teeth, presence of fixed or removable dentures (in edentulous and partial edentulous patients), and number of functional teeth units (FTU). As for the number of teeth, natural teeth and fixed dentures were noted without difference. The following groups were made based on number of present teeth: 0 (edentulous), 1–9, 10–19, and 20 or more teeth [17]. For calculation of FTU, pairs of opposing posterior natural teeth and artificial teeth on implant-supported, fixed, and/or removable prostheses were counted. Two opposing premolars were defined as one FTU and two opposing molars were defined as two FTU, with a maximum of 12 FTU constituting a complete dentition (third molars were excluded). Two categories were included: total number of FTU and number of FTU with natural or fixed prosthetic teeth [18].

Statistical analysis

The differences between the groups were evaluated using Pearson chi-square and Mann-Whitney tests. Factors with p < 0.05 on bivariate analysis were included in the logistic regression analysis and were adjusted for sex, age and education level. All analyses were performed with the SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA). The level of significance was set at 0.05.

RESULTS

In total, 179 patients participated in the study; 116 with AD and 63 control subjects. Sociodemographic data are shown in Table 1. AD patients were less often married, residents in their own home, and had fewer other chronic diseases (p = 0.033, p = 0.001, p = 0.021, respectively) (Table 1).

Table 1

Sociodemographic data in patients with AD (N = 116) and controls (N = 63)

General characteristics	AD group N (%)	Control group N (%)	p
Sex
Male	39 (33.6)	28 (44.4)	0.196
Female	77 (66.4)	35 (55.6)
Age (y)
65–74	61 (52.6)	27 (42.9)	0.295
75–84	45 (38.8)	30 (47.6)
>85	10 (8.6)	6 (9.5)
Marital status
Married	62 (53.4)	47 (74.6)	0.033
Divorced	9 (7.8)	4 (6.3)
Widow	42 (36.2)	12 (19.0)
Single	3 (2.6)	0 (0)
Education
Elementary school	55 (47.4)	22 (34.9)	0.189
High school	33 (28.4)	24 (38.1)
Bachelor degree	9 (7.8)	9 (14.3)
University	19 (16.4)	8 (12.7)
Residence
Home	43 (37.1)	40 (63.5)	0.001
Residential home	73 (62.9)	23 (36.5)
Chronic diseases
None	34 (29.3)	7 (11.1)	0.021
1–2	62 (53.4)	41 (65.1)
≥3	20 (17.2)	15 (23.8)

AD, Alzheimer’s disease.

The majority of subjects in both groups had at least one ɛ3 allele (Table 2). Genotype ɛ3/ɛ4 and presence of allele ɛ4 were more often recorded in patients with AD (p = 0.002, p = 0.004, respectively).

Table 2

APOE4 genotype and allele distribution in patients with AD (N = 116) and controls (N = 63)

APOE4 genotypes and alleles	AD group N (%)	Control group N (%)	p
Genotype
E3/E3	52 (44.8)	47 (74.6)	0.002
E3/E4	61 (52.6)	15 (23.8)
E2/E4	1 (0.9)	0 (0.0)
E2/E3	2 (1.7)	1 (1.6)
Alleles
E2	3 (1.3)	1 (0.8)	0.004
E3	167 (72.0)	110 (87.3)
E4	62 (26.7)	15 (11.9)

AD, Alzheimer’s disease.

Number of natural teeth and FTU are shown in Table 3. The number of teeth and mean value of total FTU were significantly higher in the control subjects (p = 0.043, p < 0.001, respectively).

Table 3

Number of natural teeth and FTU of participants with AD (N = 116) and controls (N = 63)

	AD group	Control group	p
Teeth number (N (%))
0	40 (34.5)	20 (31.7)	0.043
1–9	38 (32.8)	14 (22.2)
10–19	18 (15.5)	21 (33.3)
≥20	20 (17.2)	8 (12.7)
FTU (Mean (±SD))
Total FTU	3.88 (±4.82)	9.10 (±4.57)	0.000
Natural or fixed FTU	1.36 (±2.51)	1.48 (±2.85)	0.950

AD, Alzheimer’s disease; FTU, functional tooth units.

The results of the logistic regression analysis are presented in Table 4. Number of total FTU and presence of APOE4 allele remained significant as independent risk factors for AD even when adjusted for age, sex, and level of education.

Table 4

Factors entered into the multivariate models (p < 0.05 on bivariate analysis) for AD

Parameters	B	Exp (B)	95% CI	p
Marital status (married = 0; other = 1)	0.812	2.252	0.982–5.162	0.055
	^*0.816	^*2.261	^*0.959–5.330	^*0.062
Number of teeth (≤9 = 0;>9 = 1)	–0.566	0.568	0.260–1.242	0.156
	^*–0.609	^*0.544	^*0.240–1.231	^*0.144
Total FTU	0.196	1.217	1.123–1.318	<0.001
	^*0.215	^*1.240	^*1.138–1.353	^* <0.001
Residence (home = 0; other = 1)	0.596	1.815	0.801–4.113	0.153
	^*0.618	^*1.855	^*0.798–4.314	^*0.151
APOE4 allele (No = 0; Yes = 1)	–1.372	0.254	0.110–0.586	0.001
	^*–1.254	^*0.285	^*0.121–0.672	^* 0.004
Other diseases (No = 0; Yes = 1)	–1.018	0.361	0.128–1.021	0.555
	^*–0.902	^*0.406	^*0.138–1.190	^*0.100

^*Adjusted for age, sex, and level of education.

DISCUSSION

The connection between AD and oral health has been confirmed in both directions: patients’ AD diagnosis has repercussions on oral health and more evidence is found about the involvement of oral health factors in AD etiology and pathogenesis [7, 19]. The results of the current study have shown that patients with AD are more often APOE4 carriers, with less teeth and less occlusal contacts, single, residents in residential homes, and with less chronic diseases. However, only APOE4 allele and number of total FTU were independent risk factors for AD, regardless of patient’s sex, age, and level of education.

Statistically higher incidence of APOE4 allele in patients with AD confirms the well-known role of APOE4 as a genetic predictor of AD [5, 20]. Carriers of at least one APOE4 allele have higher chances of more pronounced symptoms, compared to non-carriers [5, 21]. Also, some researchers found difficulties in everyday functioning in APOE4 carriers and lower cognitive performance even when AD was not developed [22, 23]. Studies which investigated brain tissue in APOE4 carriers found some structural changes such as regional cortical thinning, amyloid augmentation, molecular alteration as a consequence of non-specific neurodegenerative changes, decrease of hippocampal volume, and lower functional connectivity of the hippocampus with medial frontal, parietal, and lateral temporal cortical regions [24, 25].

In most literature data, number of teeth is used as a parameter for dental status, and numerous studies showed significantly lower number of teeth in patients with dementia which is in accordance with our study. Having fewer than 11 teeth was considered a significant risk factor for AD development [26, 27]. Also, some studies indicated that healthy subjects with more teeth showed better results in cognitive tests [28, 29]. Complete or partial tooth loss has also been related to structural brain differences such as significantly lower total brain volume and gray matter volume [10].

However, number of teeth alone cannot fully represent oral functioning since teeth can be configured in an unfavorable way, without occlusal contacts. It has been suggested previously that the number of FTU better represents dental status because teeth disposition notably influences the masticatory function [11 , 30]. According to our results, total number of FTU presents an independent risk factor for AD, but on contrary, this was not the case with number of teeth or natural/fixed FTU. Although masticatory function with removable dentures has been considered affected by many factors, including denture retention, stability and patient’s adaptation, current findings show that the existence of posterior occlusal contacts, regardless of type, are crucial regarding appearance of AD. Finally, these results suggest that FTU might represent a better parameter than number of teeth when investigating possible associations between dental factors and AD.

Studies exploring both dental and genetic factors as predictors of AD are scarce. It was shown that coexistence of at least one APOE4 allele and less than eight teeth brought greater risk of mild memory impairment, but either risk factor alone did not [31]. In a longitudinal study on aging and AD, association was found between low number of teeth and the prevalence and incidence of dementia, but only in participants without APOE4, suggesting that APOE4 was a strong risk factor for AD that significantly modified the association between tooth number and dementia [7]. On the contrary, the results of the current study showed that both APOE4 and FTU play a role as independent risk factors for AD development. Evidence from previous animal and clinical studies [4, 15] suggests that accumulation of amyloid-β mediates both association of APOE4 and FTU with cognitive function impairment. Another mechanism suggested to be involved in association between dental status and AD is mediated via periodontitis-related immune mediators such as circulating cytokines (TNF-α and IL-6) produced as a response to periodontal pathogens [6, 7]. It has been proposed that elevated cytokine levels present an overlap in the inflammatory mechanism of AD and periodontal disease [32]. Other researchers hypothesized that individuals who have inherent susceptibility traits have inadequate neutralization of invading agents, which can result in chronic inflammatory process, the loss of cytoarchitectural integrity in brain and periodontal tissues and the loss of function [33]. Thus, the current observation might be the result of co-linearity, as AD might not directly be associated with the loss of tooth contact, but mediated through periodontal inflammation. Future studies should elucidate whether neurological factors related to reduced tooth contact or inflammation caused by periodontal pathogens underlies the association between dental status and AD.

Patients with AD in this study were less likely to be married, live in their own home, and have other chronic diseases, compared to the healthy controls. Accordingly, the elderly without partners had higher chances to develop AD, compared to the ones who were married [34]. In addition, AD patients living in institutions had more cognitive impairment in comparison to those living in home [35]. It can be assumed that emotional factors of the elderly without partners or living in residential home can lead to social problems and isolation and may act as a potential trigger for some diseases, including dementia. Interestingly, another study showed better dental status in the elderly with partners but no difference in their cognitive function, which gives different insight into this relationship [28]. The majority of the investigations found somatic comorbidities to be associated with lower cognitive performance in late-onset AD [35]. On the other hand, it has been observed that patients with AD may under-report symptoms other than cognitive impairment, although their comorbidity is similar to patients without dementia [35].

The main limitations are the relatively small sample size and known shortcomings of case control study design. Case control studies are retrospective and cannot confirm the nature of the obtained relationship. However, they are convenient for disease studies with a long latency between the exposure and the manifestation, which is the case with AD, and are convenient for risk factors that have not been tested, which is the case for some risk factors related to dental status and AD.

Conclusion

Patients with AD are more often APOE4 carriers, have lower number of teeth, and lower number of FTU. They are more often single, live in residential home more frequently, and have less chronic diseases. Presence of APOE4 allele and number of total FTU have been recognized as risk factors for AD.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/19-1283r1).

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