The frequency of the APOE ε4 in AD patients
A total of 383 AD patients were enrolled in this study, 125 cases (32.6%) carried APOE ε4, among whom 4 cases (1.0%) carried the APOE ε2/ε4, 96 cases (25.1%) carried the APOE ε3/ε4, and 25 cases (6.5%) carried the APOE ε4/ε4. In addition, 29 cases (7.6%) carried the APOE ε2/ε3, and the APOE ε3/ε3 was the most frequent one (59.8%) (Fig. 1).
Association of APOE ε4 with demographic variables and clinical information
In this study, 125 cases (32.6%) were the APOE ε4 carrier group, in which 76 cases (60.8%) were female and the median age was 68.00 (62.00, 74.00) years. The data showed no significant differences in demographic variables between the two groups (Table 1).
As shown in Table 2, clinical information was compared between the APOE ε4 carrier and the APOE ε4 non-carrier groups. The median disease duration was 24.00 (12.00, 48.00) months in the APOE ε4 carrier group, which was not significantly different from the non-carrier group. The APOE ε4 carrier group had a significantly higher frequency of AD-D than the APOE ε4 non-carrier group (P < 0.001).
Cognitive function was also compared between the two groups. As far as global cognitive function, the scores of MMSE (P = 0.005) and MOCA scales (P = 0.011) were all significantly decreased in the APOE ε4 carrier group compared with that in the non-carrier group. Besides, each individual cognitive domain between the two groups was also conducted. Firstly, in terms of the memory, the scores of AVLT N1-3 (P < 0.001), AVLT N4 (P < 0.001), AVLT N5 (P < 0.001) and RFT-delayed recall (P = 0.027) in the APOE ε4 carrier group were all significantly lower than those in the non-carrier group. Secondly, in terms of the language, the APOE ε4 carrier group had significantly decreased scores of VFT (P < 0.001) and BNT scales (P = 0.023) compared with the non-carrier group. As far as the attention, the APOE ε4 carrier group spent more time on the TMT-A test (P = 0.012) and the TMT-B test (P = 0.050) than the non-carrier group did. There was no significant difference in executive function and visuospatial ability between the two groups.
Table 1 Demographic variables of APOE ε4- and ε4+ groups
|
APOE ε4- group
(n=258)
|
APOE ε4+ group
(n=125)
|
P
|
Female [n (%)]
|
149 (57.75)
|
76 (60.80)
|
0.617
|
Age [years, median (quartile)]
|
66.00 (60.00, 74.00)
|
68.00 (62.00, 74.00)
|
0.150
|
Age of onset [years, median (quartile)]
|
62.00 (55.00, 70.00)
|
65.00 (58.00, 72.00)
|
0.143
|
Education level [n (%)]
|
|
|
0.109
|
Primary school and below
|
54 (20.93)
|
27 (21.60)
|
|
Middle and high school
|
126 (48.84)
|
60 (48.00)
|
|
Bachelor’s degree and above
|
78 (30.23)
|
38 (30.40)
|
|
Smoking [n (%)]
|
61 (23.64)
|
26 (21.00)
|
0.519
|
Drinking [n (%)]
|
53 (20.54)
|
24 (19.20)
|
0.792
|
BMI [median (quartile)]
|
23.89 (21.89, 26.11)
|
23.40 (21.30, 24.90)
|
0.087
|
Resting heart rate [Times/minute, median (quartile)]
|
72.00 (69.50, 78.00)
|
72.00 (69.00, 76.00)
|
0.344
|
Systolic blood pressure [mmHg, median (quartile)]
|
132.00 (122.00, 143.00)
|
133.50 (124.75, 148.50)
|
0.195
|
Diastolic blood pressure [mmHg, median (quartile)]
|
81.00 (75.00, 87.75)
|
83.00 (75.75, 90.00)
|
0.336
|
History
|
|
|
|
Hypertension [n (%)]
|
102 (39.53)
|
43 (34.40)
|
0.404
|
Hyperlipidemia [n (%)]
|
49 (18.99)
|
29 (23.20)
|
0.489
|
Myocardial infarction [n (%)]
|
3 (1.16)
|
1 (0.80)
|
0.892
|
Atrial fibrillation [n (%)]
|
2 (0.78)
|
2 (2.00)
|
0.512
|
Diabetes mellitus [n (%)]
|
49 (18.99)
|
17 (13.60)
|
0.253
|
Hyperhomocysteinemia [n (%)]
|
3 (1.16)
|
16 (12.80)
|
0.897
|
Cerebrovascular disease [n (%)]
|
37 (14.34)
|
10 (10.00)
|
0.119
|
Thyroid disease [n (%)]
|
17 (6.59)
|
4 (3.20)
|
0.382
|
Asthma [n (%)]
|
2 (0.70)
|
2 (1.60)
|
0.510
|
Insomnia [n (%)]
|
33 (12.79)
|
9 (7.20)
|
0.207
|
Sleep apnea syndrome [n (%)]
|
14 (5.42)
|
6 (4.80)
|
0.954
|
Depression [n (%)]
|
16 (6.20)
|
11 (8.80)
|
0.686
|
Other mental disorder [n (%)]
|
10 (3.88)
|
6 (4.80)
|
0.812
|
Abbreviation: APOE, apolipoprotein E; APOE ε4-, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers.; BMI: body mass index.
Table 2 Cognitive performances of the APOE ε4- and ε4+ groups
|
APOE ε4- group
|
APOE ε4+ group
|
P
|
Disease duration [years, median (quartile)]
|
24.00 (12.00, 48.00)
|
24.00 (12.00, 48.00)
|
0.730
|
Stage of disease [n (%)]
|
|
|
|
MCI
|
150 (58.14)
|
50 (40.00)
|
0.001**
|
Dementia
|
108 (41.86)
|
75 (60.00)
|
|
Cognitive function
|
|
|
|
Global cognitive function
|
|
|
|
MMSE [points, median (quartile)]
|
22.00 (15.00, 26.00)
|
19.00 (11.00, 24.50)
|
0.005**
|
MoCA (points, mean ± SD)
|
15.01 ± 7.44
|
12.93 ± 7.41
|
0.011*
|
Individual cognitive domain
|
|
|
|
Memory
|
|
|
|
AVLT N1-3 (points, mean ± SD)
|
11.46 ± 6.04
|
8.98 ± 5.67
|
<0.001**
|
AVLT N4 [points, median (quartile)]
|
1.00 (0.00, 4.00)
|
0.00 (0.00, 2.00)
|
<0.001**
|
AVLT N5 [points, median (quartile)]
|
0.00 (0.00, 4.00)
|
0.00 (0.00, 2.00)
|
<0.001**
|
RFT delayed recall [ points, median (quartile)]
|
3.00 (0.00, 10.00)
|
0.00 (0.00, 8.00)
|
0.027*
|
Language
|
|
|
|
VFT (points, mean ± SD)
|
33.05 ± 16.64
|
26.88 ± 16.06
|
<0.001**
|
BNT [points, median (quartile)]
|
23.00 (18.00, 26.00)
|
21.00 (14.00, 25.00)
|
0.023*
|
Attention
|
|
|
|
SDMT (points, mean ± SD)
|
21.09 ± 15.27
|
19.83 ± 21.41
|
0.571
|
TMT-A time (seconds, mean ± SD)
|
105.99 ± 72.95
|
128.59 ± 76.28
|
0.012*
|
SCWT-A time [seconds, median (quartile)]
|
40.00 (30.00, 54.00)
|
49.89 (27.00, 60.00)
|
0.925
|
SCWT-B time [seconds, median (quartile)]
|
53.00 (39.00, 72.50)
|
55.50 (40.43, 80.00)
|
0.620
|
Visuospatial ability
|
|
|
|
RFT imitation [ points, median (quartile)]
|
27.25 (8.75, 34.00)
|
22.00 (2.00, 33.00)
|
0.255
|
Executive function
|
|
|
|
SCWT-C time (seconds, mean ± SD)
|
107.97 ± 73.25
|
102.81 ± 61.18
|
0.554
|
TMT-B-time [seconds, median (quartile)]
|
207.00 (122.00, 240.00)
|
240.00 (161.00, 240.00)
|
0.050
|
Abbreviation: APOE, apolipoprotein E; APOE ε4-, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers; MCI: mild cognitive impairment; MMSE, Mini-Mental State Examination; MoCA, Montreal Cognitive Assessment; AVLT, Auditory Verbal Learning Test; RFT, Rey-Osterrieth Complex Figure Test; VFT, Verbal Fluency Test; BNT, Boston Naming Test; SDMT, Symbol Digit Modalities Test; TMT, Trail Making Test; SCWT, The Stroop Color and Word Test. *P < 0.05, **P < 0.01.
Association among APOE ε4, neuropathological proteins in CSF, and cognition in AD patients
Neuropathological proteins between the APOE ε4 carrier and the non-carrier groups
Aβ1-42 level in CSF from the APOE ε4 carrier group was significantly lower than that from the non-carrier group (P = 0.023) (Table 3). There were no statistical differences in the levels of P-tau (T181), P-tau (S199), P-tau (T231), P-tau (S396) and T-tau in CSF from the APOE ε4 carrier group than that from the non-carrier group. Multiple linear regression analyses showed APOE ε4 was negatively associated with Aβ1-42 level after adjusting for age, gender and disease duration [β, -1.13; 95% CI (-2.15, -0.11); P = 0.031].
Aβ1-42 level from the APOE ε4-/-, the APOE ε4+/- and APOE ε4+/+ groups were compared (Fig. 2). In comparison to the APOE ε4-/- group, Aβ1-42 level in CSF was reduced in the APOE ε4+/- group (P = 0.879) and significantly decreased in the APOEε4+/+ group (P = 0.011). In addition, compared with the APOE ε4+/- group, Aβ1-42 level was markedly declined in the APOEε4+/+ group (P = 0.009).
Table 3 Levels of neuropathological proteins of AD in CSF from APOE ε4- and APOE ε4+ groups
|
APOE ε4- group
|
APOE ε4+ group
|
P
|
Aβ1-42 [ng/ml, median (quartile)]
|
0.79 (0.44, 1.09)
|
0.63 (0.35, 1.00)
|
0.023*
|
P-tau (T181) [ng/ml, median (quartile)]
|
58.25 (31.94, 89.84)
|
72.30 (37.94, 90.47)
|
0.737
|
P-tau (S199) [ng/ml, median (quartile)]
|
6.43 (4.29, 11.34)
|
7.21 (4.11, 11.77)
|
0.932
|
P-tau (T231) [ng/ml, median (quartile)]
|
81.79 (64.74, 107.60)
|
86.10 (59.45, 112.16)
|
0.239
|
P-tau (S396) (ng/ml, mean ± SD)
|
66.38 ± 27.47
|
60.21 ± 28.17
|
0.949
|
T-tau [ng/ml, median (quartile)]
|
89.12 (72.62, 122.93)
|
94.83 (66.18, 123.83)
|
0.365
|
Abbreviation: APOE, apolipoprotein E;APOE ε4-, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers; Aβ, β amyloid protein; T-tau, total tau; P-tau, phosphorylated tau. *P < 0.05.
Correlations between Aβ1-42 level and cognition
In the aspect of overall cognitive function, Aβ1-42 level in CSF was significantly and positively correlated with the scores of MMSE (r = 0.32, P < 0.001) and MoCA (r = 0.36, P < 0.001) scales (Fig. 3).
In terms of individual cognitive domains, Aβ1-42 level was also significantly and positively correlated with the scores of AVLT N1-3 (r = 0.26, P = 0.003), AVLT N4 (r = 0.25, P = 0.006), AVLT N5 (r = 0.20, P = 0.026), RFT- delayed recall (r = 0.27, P = 0.005), VFT (r = 0.30, P < 0.001), SDWT (r = 0.34, P < 0.001) and RFT imitation (r = 0.25, P = 0.009). These results suggested that more Aβ1-42 deposition in the brain was significantly correlated with the dramatically declined overall cognitive function and multiple cognitive domains of memory, language, attention and visuospatial ability in APOE ε4 carriers. There were no marked correlations between Aβ1-42 level and executive function (Fig. 3).
Multiple linear regression analyses further illustrated that lower Aβ1-42 level was associated with worse overall cognitive function and individual cognitive domains, including memory, language and attention, which was independent of age, disease duration and education level (Supplementary Table S1).
Association among APOE ε4, neuroinflammatory factors in CSF, and cognitive function
Association between APOE ε4 and the levels of neuroinflammatory factors
Multiple linear regression analyses were conducted to explore associations between APOE ε4 and levels of neuroinflammatory factors, including TNF-α, IL-1β, IL-6, IFN-γ, NO, ·OH, sTREM2 and YKL-40 in CSF. It was found that APOE ε4 was markedly associated with elevated NO level in CSF after adjusting for age, gender and disease duration [β, 2.24; 95% CI (0.18, 4.30); P = 0.033] (Table 4).
Table 4 Association between levels of neuroinflammatory factors in CSF and APOE ε4 in AD patients
|
Unadjusted
|
Adjusted
|
|
β (95%CI)
|
P
|
β (95%CI)
|
P
|
TNF-α (pg/mL)
|
2.08 (-0.17,4.33)
|
0.070
|
2.14 (-0.22, 4.50)
|
0.075
|
IL-1β (pg/mL)
|
0.24 (-0.69, 1.16)
|
0.618
|
0.16 (-0.81, 1.14)
|
0.744
|
IL-6 (pg/mL)
|
0.02 (-0.43, 0.48)
|
0.918
|
-0.02 (-0.49, 0.44)
|
0.920
|
IFN-γ (pg/mL)
|
-0.11 (-1.99,1.77)
|
0.906
|
-0.27 (-2.26, 1.72)
|
0.787
|
NO (μmol/L)
|
2.24 (0.18, 4.30)
|
0.033*
|
2.58 (0.40, 4.77)
|
0.021*
|
·OH (U/mL)
|
-42.85(-101.50, 5.81)
|
0.151
|
-52.03(-113.04, 8.98)
|
0.094
|
STREM-2 (pg/mL)
|
-72.30(-240.23, 95.69)
|
0.395
|
-82.51(-246.54, 81.51)
|
0.321
|
YKL-40 (pg/mL)
|
5764.47
(-6400.52, 17929.44)
|
0.347
|
3564.20
(-9368.21, 16496.62)
|
0.583
|
Age, gender, disease duration and education level were adjusted. Abbreviation: APOE, apolipoprotein E; AD, Alzheimer’s disease; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; IFN-γ, interferon-γ; NO, nitric oxide; ·OH, hydroxy radical; sTREM-2, triggering receptor expressed on myeloid cells-2; YKL-40, Tyr-Lys-Leu-40. APOE ε4-, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers. *P < 0.05.
Correlations between neuroinflammatory factors and cognition
Correlations between the levels of neuroinflammatory factors in CSF and the scores of cognitive rating scales in AD patients were displayed (Fig. 3). The NO level was significantly and positively correlated with TMT-A-time (r = 0.21, P = 0.026) and TMT-B-time (r = -0.38, P < 0.01), demonstrating that higher NO level was significantly correlated with worse attention and executive function. Moreover, IL-1β level in CSF was significantly and negatively correlated with the scores of VFT (r = -0.18, P = 0.043) and RFT-imitation scales (r = -0.21, P = 0.025), while was significantly and positively correlated with the time spent on SCWT-A (r = 0.22, P = 0.017), SCWT-B (r = 0.23, P = 0.013) and SCWT-C (r = -0.29, P < 0.01) in AD patients, indicating that the elevated IL-1β level was markedly correlated with impaired language, visuospatial ability, attention and executive function. Additionally, the levels of sTREM2 and YKL-40 were all significantly and negatively correlated with worse language function (P < 0.05).
Associations between neuroinflammatory factors in CSF and cognitive function of AD were further validated by linear regression analysis and adjusted for possible confounding factors, including age, disease duration and education level (Supplementary Table S2-S5). The results showed that the higher NO level was also associated with longer time of TMT-A [β, 3.24; 95% CI (0.00, 6.47); P = 0.050] and TMT-B [β, 5.24; 95% CI (1.11, 9.37); P = 0.013] after adjusting for the above confounding factors (Supplementary Table S2). Furthermore, the higher IL-1β level was associated with longer time of SCWT-A [β, 8.10; 95% CI (2.51, 13.68); P = 0.005] and SCWT-C [β, 6.61; 95% CI (0.27, 12.94); P = 0.041], which were independent of age, disease duration and education level (Supplementary Table S3). In addition, YKL-40 level was significantly and negatively associated with the score of BNT [β, -1.30E-4; 95% CI (-2.26E-4, 3.30E-5); P = 0.011], which was independent of age, disease duration and education level (Supplementary Table S5). The predictive value of sTREM2 for VFT was not found (Supplementary Table S4).