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Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients
Accepted Manuscript Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients
Yanzhi Li, Zhengsheng Zhang, Linglong Deng, Feng Bai, Yongmei Shi, Hui Yu, Chunming Xie, Yonggui Yuan, Zhijun Zhang PII: DOI: Reference:
S0022-510X(17)30399-4 doi: 10.1016/j.jns.2017.06.026 JNS 15401
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
8 October 2016 23 May 2017 15 June 2017
Please cite this article as: Yanzhi Li, Zhengsheng Zhang, Linglong Deng, Feng Bai, Yongmei Shi, Hui Yu, Chunming Xie, Yonggui Yuan, Zhijun Zhang , Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/j.jns.2017.06.026
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ACCEPTED MANUSCRIPT Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients
Yanzhi Li,·Zhengsheng Zhang,·Linglong Deng,·Feng Bai,·Yongmei Shi,·Hui Yu,·Chunming
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Xie,·Yonggui Yuan,·Zhijun Zhang
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Corresponding author: Zhijun Zhang
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Yanzhi Li
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Geriatric Department of Affiliated ZhongDa Hospital, Medical School of Southeast University, Nanjing, Jiangsu 210009, China
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Zhengsheng Zhang, Feng Bai,·Yongmei Shi, Chunming Xie·
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Neurologic Department of Affiliated ZhongDa Hospital, Neuropsychiatric Institute and Medical School of Southeast University, Nanjing, Jiangsu 210009, China Linglong Deng, Hui Yu
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School of Clinical Medicine, Southeast University, Nanjing 210009, China. Yonggui Yuan
Psychiatric Department of Affiliated ZhongDa Hospital, Medical School of Southeast University, Nanjing, Jiangsu 210009, China Zhijun Zhang
(☒ )
Neurologic Department of Affiliated ZhongDa Hospital, Neuropsychiatric Institute and Medical School of Southeast University, No 87 DingJiaQiao Rd. Nanjing, China
ACCEPTED MANUSCRIPT 210009 Fax: +86 25 8326 2503; E-mail address: [email protected].
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The number of text pages of the whole manuscript is 18 pages, and the number of
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figures and tables respectively are 1 and 2.
ACCEPTED MANUSCRIPT Abstract Angiotensin-converting enzyme (ACE) gene has been implicated in amnestic mild cognitive impairment (aMCI). Most human genetic studies have focused on ACE insertion (I)/deletion (D) polymorphism and yielded conflicting results. In this work,
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we evaluated the relationships between cognitive function, serum ACE level, brain
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white matter (WM) integrity, and ACE I/D polymorphism in 48 patients with aMCI
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and 36 well matched control subjects from south China. In aMCI patients, D allele frequency was higher (D/I ratio= 0.51:0.49) than that of the control subjects (D/I
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ratio= 0.43:0.57); however, the difference was not statistically significant (p > 0.05).
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The D carriers in aMCI subjects performed significantly poorer on auditory–verbal learning test (AVLT) -delayed recall than the I homozygous group (p= 0.035). These
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carriers had higher serum ACE level than the I homozygous carriers of aMCI (p=
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0.037). In the aMCI group, D carriers showed significantly lower fractional anisotropy (FA) values in the left middle frontal gyrus, left anterior cingulate, right gyrus parahippocampalis, right inferior parietal lobule, and bilateral anterior central
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gyrus than the I homozygotes carriers. However, no significant difference was observed in FA values between I homozygotes and D carriers in the control subjects. The ACE D allele in aMCI patients may increase the risk of cognitive impairment. A high serum ACE level possibly plays an important role in the incidence of aMCI. Keywords: mild cognitive impairment·angiotensin converting enzyme gene·cognitive function·magnetic resonance imaging·diffusion tensor imaging
ACCEPTED MANUSCRIPT Highlights: A higher frequency of D allele was detected in aMCI patients. The D carriers had significantly higher serum ACE levels than I homozygotes (p<0.05) in aMCI.
aMCI D carriers showed significantly lower FA values in some memory related brain regions.
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ACCEPTED MANUSCRIPT Background Alzheimer’s disease (AD) is the most common cause of dementia in the older adult subjects and accounts for 50% to 60% of all cases. Most patients with dementia live in developing countries, and the forecasted rates of increase in China, India, and other
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South Asian countries are threefold higher than the ~100% increase estimated in 2040
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[1,2]. Mild cognitive impairment (MCI) has been recognized as a transitional phase
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between normal health and AD. Recent studies have shown that individuals with MCI tend to progress to AD at an annual rate of 10%–15% [3]. Amnestic MCI (aMCI) is
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characterized by selective memory deficits associated or not with other cognitive
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dysfunctions and represents early AD; 76% of MCI cases progressing to AD are aMCI patients [4]. This impairment has the highest probability of developing into
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dementia and even more in the presence of biomarkers (e.g., medial temporal lobe
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atrophy) [5].
AD is a heterogeneous disorder with both familial (about 1% of cases) and sporadic forms. Only the apolipoprotein E (APOE) ε4 allele has been reported as a genetic risk
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factor for sporadic cases. The continually updated meta-analysis functions of the Internet-based Alzgene database (www.alzgene.org; Gene ID = 125), which is applied to the dataset criteria to assess growing epidemiologic evidence from genetic associations, ranks angiotensin-converting enzyme (ACE) gene variation as the second most significant genetic susceptibility locus for AD following APOE ε4 [6]. A common Alu insertion (I)/deletion (D) polymorphism (rs1799752) exists in intron 16 of ACE gene. Research has shown that homozygosity for the D and I alleles are
ACCEPTED MANUSCRIPT associated with the highest and lowest plasma ACE, respectively, and that the heterozygotes have intermediate values [7]. However, the relationship between ACE gene polymorphism and AD remains contentious. Several works have reported a strong association between the ACE I allele and AD [8,9] and have suggested that the
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D allele has a significant function in the progression to AD in aging and MCI [10,11].
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Results of various diagnostic criteria for AD, MCI and normal control, racial
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differences, or other factors contributing to the development of AD are inconsistent [6,9].
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Diffusion tensor imaging (DTI) is a relatively new magnetic resonance imaging
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(MRI) technique and is more sensitive to white matter (WM) alterations before the microstructural change develops into macrostructural loss of WM measurable by
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voxel-based analyses (VBA). Several works have reported that changes in WM
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microstructure assessed with DTI can be a more sensitive parameter than gray matter data for detecting mild structural changes occurring at the early stages of various neurodegenerative diseases [12,13]. In addition, DTI has revealed early and
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progressive loss of integrity of WM that is temporally antecedent to overt clinical evaluation and cerebrospinal fluid biomarkers in AD and MCI [14,15]. The true genetic susceptibility of ACE I/D gene polymorphism on AD pathogenesis remains ambiguous because of inconsistent outcomes. In this study, the possible association of ACE I/D polymorphism with aMCI in South China was analyzed by ACE genotype and DTI analysis by the VBA method. The patients and controls were from Jiangsu province in South China, which limits the ethnic variation in the
ACCEPTED MANUSCRIPT frequency of ACE gene I/D polymorphism and enzyme level in producing false positive outcomes, thereby resulting in a case-control study.
Methods Participants
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We have been recruiting 115 aMCI patients and 93 control subjects, from 1480
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aging in the Outpatient Department of Neurology. Eighty-one cases were excluded as
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no MRI study was performed on them, while another 43 cases were excluded due to a standardized neuropsychological evaluation was not performed on them. Therefore, a
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total of 84 people from the Han ethnic group were enrolled in this study. They were
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from the Outpatient Department of Neurology, and 48 of them have aMCI (18 women and 30 men; mean age±S.D.= 72.04±4.42). Among them, 36 were designated as
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normal controls (19 women and 17 men; mean age±S.D. 71.64±3.72). The patients
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were matched according to sex and age, and all patients gave their informed consent to participate in this study, which was approved by the Institutional Ethical Review Board of the Clinic Medical College of Southeast University, Nanjing, China. The
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criteria for aMCI were as follows: (1) subjective memory impairment corroborated by subject and an informant, (2) weak objective memory performance documented by delayed recall in the auditory–verbal learning test (AVLT), (3) a clinical dementia rating of 0.5, with at least 0.5 in the memory domain, (4) normal general cognitive functioning as evaluated by a score on the mini- mental state exam (MMSE) greater than or equal to 24, (5) no or minimal impairment in 14 daily living activities, and (6) absence of dementia. Participants were excluded from the present study if they have a
ACCEPTED MANUSCRIPT history of known stroke (modified Hachinski score>4), taking ACE inhibitors within the last 3 months, alcoholism, head injury, Parkinson’s disease, epilepsy, major depression, other neurological or psychiatric illnesses, major medical illness, or severe visual or hearing loss.
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All participants underwent a standardized neuropsychological evaluation, including
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the AVLT, Rey–Osterrich complex figure test, verbal fluency test (animal fluency
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task), digit span test, symbol digit modalities test, trail making tests A and B, and clock drawing test. This battery evaluated the function of episodic memory regarding
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both verbal and visual information, semantic memory, attention, psychomotor speed,
neurologists (YM Shi and
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executive function, and visuospatial skills in all subjects. Two experienced H Yu) who are experienced
in professional
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neuropsychological scale assessments administered the process, which also included
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structured interviews with subjects and their informants. Genotyping of ACE I/D and measurement of serum ACE levels ACE polymorphisms were genotyped as previously described [16]. The primer
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sequences were designed in accordance with the forward primer given as 5΄–CTG GAG ACC ACT CCC ATC CTT TCT–3΄ and reverse primer 5΄-GAT GTG GCC ATC ACA TTC GTC AGA T–3΄ to amplify 490/190 bp fragments (I/D allele) that were run on 2% agarose gel. To reduce the incidence of mistyping ID as a DD genotype, all DD genotypes were verified using a second insertion-specific amplification with a primer pair given as: 5΄–TGG GAC CAC AGC GCC CGC CAC TAC–3΄ and 5΄–TCG CCA GCC CTC CCA TGC CCA TAA–3΄. Each blood
ACCEPTED MANUSCRIPT specimen was tested in duplicate to assure reproducibility of results. Data fro m both reactions were used to establish the ACE-ID genotype. Each allele and genotype was determined. Serum ACE level was measured using ultraviolet spectrophotometry (using a
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Hitachi 7170 full- automatic biochemical analyzer) in accordance with the instructions
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on the serum ACE level kit (Zhejiang Kuake Bioscience Technology Co., Ltd.) as
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previously described [16]. This level was measured in all subjects on the same day to minimize assay variance. The intra-assay coefficient of variation was less than 6%.
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Imaging acquisition
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MRI was performed with a General Electric 1.5 Tesla scanner (General Electric Medical Systems, Milwaukee, WI, USA). All participants laid supine with their head fixed
by a belt and
foam pads to
minimize
head
movement.
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snugly
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Diffusion-weighted imaging was acquired with a single-shot echo planar imaging sequence in alignment with the anterior–posterior commissure plane. The diffusion-sensitizing gradients were applied along 25 noncollinear directions (b= 1000
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s/mm2 ), together with an acquisition without diffusion weighting (b=0). Thirty contiguous axial slices with a slice thickness of 4 mm and no gap were acquired. The acquisition parameters were as follows: repetition time=10 000 ms; echo time=81.2 ms; matrix=128×128; field of view=240 mm×240 mm; number of excitations=2. Image analysis The diffusion tensor matrix was calculated according to the Stejskal and Tanner equation. Three pairs of eigenvalues (γ1, γ2, and γ3) and eigenvectors were obtained
ACCEPTED MANUSCRIPT by the diagonalization of the tensor matrix. The principal direction at each point was given by the eigenvector that corresponded to the largest eigenvalue, and fractional anisotropy (FA) values were calculated according to a previously described method [17]. For each participant, the b=0 image was first normalized to the standard
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Montreal Neurological Institute space using statistical parameters maps (SPM12)
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(http://www.fil.ion.ucl.ac.uk/spm). Then, the transformation matrix was applied to the
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FA map to normalize this map to the standard Montreal Neurological Institute space. All images were resampled with a final voxel size of 1×1×1 mm3 . Furthermore, each
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FA map was spatially smoothed by a 6 mm full width at half- maximum Gaussian
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kernel to decrease spatial noise and compensate for the inexact nature of normalization. Following the described procedure, statistical analysis was carried out
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through a cohort analysis (two-sample t-test) between I homozygotes and D carriers in
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aMCI and NC, age and gender as covariates. Significance reached p<0.05 (p<0.005, cluster size=118 voxels, corrected by Monte Carlo simulation). Statistical analysis
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Analysis was performed using SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). A chi-squared goodness-of- fit test was used to test the distribution of genotypes and allele frequencies for deviations from the Hardy–Weinberg equilibrium. The ACE allele and genotype distributions between patients and controls were evaluated using the chi-squared test. A K–S (Lilliefors) test was used to analyze the normality, and a Levene test was used to verify the homogeneity of group variances. An independent-sample t-test was used for the normally distributed variables. The
ACCEPTED MANUSCRIPT nonparametric Mann – Whitney U test was used for asymmetrically distributed variables. Statistical significance was defined at p<0.05 from the two-tailed test.
Results Comparison of clinical characte ristics, cognitive performance, and serum ACE
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level between aMCI and control subjects
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Significant differences in years of education were not observed between the aMCI
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and normal control (p>0.05). All scores on the neuropsychological tests in the aMCI group were significantly lower than those in normal control subjects (p < 0.05). Table
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1 shows that the serum ACE level in aMCI subjects showed no significant difference
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from the levels in normal control subjects (p=0.161).
Comparison of the frequencies of allele and genotype of ACE I/D between aMCI
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and control subjects
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The ACE genotype frequencies were 20.8% (10) for DD, 60.4% (29) for ID and 18.8% (9) for II genotypes in aMCI patients, and 16.7% (6) for DD, 52.8% (19) for ID and 30.4% (11) for II genotypes in control subjects, respectively. In aMCI patients,
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a high D allele frequency (D/I ratio=0.51:0.49) compared with the control controls (D/I ratio=0.43:0.57) was detected, and the difference was not statistically significant (p > 0.05). The observed genotype distribution was in agreement with the Hardy– Weinberg equilibrium (aMCI: p=0.147; control subjects: p=0.647). Differences in neuropsychological tests and serum ACE activity between ACE genotype subgroups in aMCI and control subjects Table 2 shows that the D carriers had significantly poorer performance on
ACCEPTED MANUSCRIPT AVLT-delayed recall than I homozygous carriers in aMCI (p<0.05). However, in the control subjects, no genotype difference existed between D carriers and I homozygotes in all tests. The D carriers had significantly higher serum ACE levels than the I homozygotes (p<0.05) in aMCI but not in control subjects.
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Differences in FA values between aMCI and control subjects, and between ACE
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genotype subgroups in both groups
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In a voxel-by- voxel contrast, several regions showed significantly lower FA values in aMCI patients than in control subjects. Table 3 show that the WM areas exhibiting
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lower FA values were the right precuneus, right lingual gyrus, bilateral middle
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temporal gyrus, and left gyrus parahippocampalis.
Table 4 and Fig. 1 show that upon comparison with aMCI I homozygotes, aMCI D
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carriers showed significantly lower FA values in bilateral hippocampus, right inferior
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parietal lobule, and bilateral anterior central gyrus. No significantly higher FA values were identified in aMCI D carriers. However, no significant differences were observed in FA values between I homozygotes and D carriers in the control subjects.
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Discussion
Biological studies have suggested that ACE gene could play an important role in aMCI pathology. This study has shown a statistically significant effect of the ACE I/D gene polymorphism on the integrity of brain WM on an aMCI group in South China by a comprehensive battery of standardized neuropsychological tests A higher frequency of D allele was detected in aMCI patients (D/I ratio=0.51:0.49) than in controls (D/I ratio=0.43:0.57), and increased homozygosity was found for the
ACCEPTED MANUSCRIPT D allele (20.8%) compared with controls (16.7%) in aMCI. The serum ACE level in aMCI was higher than that in normal control, and the difference was not statistically significant. However, the D carriers had significantly higher serum ACE levels than I homozygotes (p<0.05) in aMCI. Although the biological mechanism underlying the
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association between aMCI and ACE is unclear, the DD genotype is associated with
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increased ACE levels in plasma and in some tissues [18]. Previous, reports have
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suggested that the association of DD genotype with increased ACE is consistently found in the brain or CSF in animal models, MCI, and AD patients [7,19]. Research
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has shown that ACE in the brain can modulate cerebral blood flow [20], degrade
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β-amyloid [21], and reduce the release of acetylcholine [22], thereby influencing cognitive processing. Using ACE inhibitors in antihypertensive treatment, an apparent
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improvement in the cognitive functions of patients has been demonstrated, which has
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not been observed using other antihypertensive [23]. Treatment with brain-penetrating ACE inhibitors significantly reduces ACE activity in CSF [24] and attenuates neurodegeneration and memory impairments in aging and AD [24–26].
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Several lower FA values and impaired cognitive function in AVLT-delayed recall have been found in MCI with D carriers than in I homozygotes. An anatomopathology research has revealed that the frontal and temporal cortex tend to be severely affected by WM myelin loss in AD with D homozygotes compared with those with I carriers, even in conjunction with the APOE ε4 allele [27]. Furthermore, the association with increased age and cognitive decline is significantly stronger in people with the ACE D homozygotes than in I carriers, and this interaction is particularly strong for the
ACCEPTED MANUSCRIPT decline in tests of delayed verbal memory [28]. The delayed recall task of the AVLT represented episodic memory has been used to characterize memory impairment in MCI [29]. These findings reveal that ACE has a significant function in the progression of neurodegeneration and cognitive dysfunction.
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This study is subject to several limitations. One is the relatively small number of
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subjects, i.e., small number genetic subgroups in the aMCI and NC samples, which
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limited our ability to generalize the results. Another is the cross-sectional and case– control nature of this study. Hence, our results should be confirmed by an additional
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longitudinal study. A follow-up of the current cohort is underway.
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In conclusion, we found that serum ACE levels are elevated in those with aMCI who were D carriers than the patients with aMCI who were I homozygotes. It also
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suggested that significantly lower FA values in some regions s of the brain in aMCI D
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carriers compared with I homozygotes. The D allele of ACE gene can be a risk factor for AD. However, the hypothesis requires to be confirmed by follow- up studies. Determining the clear pathogenesis of this ACE gene in AD may help to reduce the
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worldwide health burden of AD. Results provide a window for reconsidering the significant function of gene polymorphism and suggest possible strategies for future pharmacogenomic researches. Acknowledgments This research was partly supported by the Key Program for Clinical Medicine and Science and Technology, Jiangsu Provence Clinical Medical Research Center (No. BL2013025), National Natural Science Foundation of China (Nos. 81201080 and
ACCEPTED MANUSCRIPT 81271336), and National Hi-Tech Research and Development Program of China (863 Program) (No. 2008AA02Z413). Conflict of interest/disclosure The authors declare that they have no financial or other conflicts of interest in
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relation to this research and its publication.
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[29]
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1369–75.
Med. 256 (2004) 240–6.
ACCEPTED MANUSCRIPT Table 1. Comparison of cognitive performance, serum ACE level between aMCI and
Normal Controls
Measure
(N = 48)
(N = 36)
p Value
Education
15.5 (12-16)
16 (12-16)
0.286
MMSE AVLT-delayed recall
27 (26-28) 3 (2-4)
29 (27-29) 8 (7-9.75)
< .001b < .001b
CFT-delayed recall
9.5 (5.25-16)
16 (10.75-20.38)
= .002b
Category Fluency Test Trail Making Test A
10.77 ± 3.02 92.63 ± 30.91
13.28 ± 2.68 71.81 ± 27.96
< .001 = .002c
Trail Making Test B Symbol Digit Modalities Test
164.5 (122.75-215.25) 24.58 ± 8.87
Digit Span Test Clock Drawing Test
12 (11-13) 9 (8-9)
ACE Level(U/L)
122.50±48.64
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a
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aMCI
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control subjectsa
c
118 (101.25-161.50) 35.08 ± 10.71
= .004b < .001c
13 (12-14) 9 (8.25-10)
= .009b = .027b
107.64±46.36
=.161c
Values expressed as median (interquartile range) except where noted. Mann-Whitney U test. c Independent-Sample t test aMCI: amnestic mild cognitive impairment; AVLT: auditory verbal learning test; CFT: rey-osterrieth complex figure test; MMSE: mini- mental state examination; ACE: angiotensin converting enzyme.
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b
ACCEPTED MANUSCRIPT Table 2. Comparison of neuropsychological data, serum ACE level between aMCI and control subjects according to ACE genotypea aMCI
control subjects DD/ID (N = 25)
II (N = 11)
P Value
MMSE
27 (26-28)
27 (27-28)
0.413b
29 (27.5-30)
29 (27-29)
0.208b
AVLT-delayed recall
3 (2-4)
4 (2.5-6)
0.035
8 (7-10)
8 (7-8)
0.650
CFT-delayed recall Category Fluency Test Trail Making Test A
9 (5-16) 10.56 ±2.96 94.64 ±31.06
14 (6-19) 11.67 ±3.28 83.89 ±30.39
0.268b c 0.502 0.352c
16 (8.5-22) 13.2 ±2.63 73.64 ±29.52
16 (14-20.5) 13.45 ±2.91 67.64 ±24.87
0.602b c 0.312 0.561c
Trail Making Test B Symbol Digit Modalities
165 (122-217) 23.96 ±78.46
146 (110.5-214) 27.33 ±10.54
0.526 0.307c
131 (101-171) 34.12 ±9.30
114 (101-132) 37.27 ±13.63
0.264 0.424c
Test Digit Span Test
12 (11-13)
12 (11-13.5)
0.737b
13 (12-14.5)
14 (13-14)
0.134b
Clock Drawing Test ACE Level(U/L)
9 (8-9) 129.49 ±48.21
9 (8-10) 92.22 ±39.85
9 (8.5-10) 116.76 ±46.52
9 (8-10) 86.90 ±40.59
0.755b 0.075c
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b
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a
b
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II (N = 9)
P Value
Measure
DD/ID (N = 39)
0.232b 0.037c
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Values expressed as median (interquartile range) except where noted. Mann-Whitney U test c Independent-Sample t test aMCI: amnestic mild cognitive impairment; AVLT: auditory verbal learning test; CFT: rey-osterrieth complex figure test; MMSE: mini- mental state examination; ACE: angiotensin converting enzyme.
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ACCEPTED MANUSCRIPT Table 3. Regions with lower FA values in aMCI patients compared to control subjects Areas
Hemisphere
Cluster size (voxels)
Peak t-value
Primary peak location x, y, z (mm)
precuneus lingual gyrus middle temporal gyrus
R
347
4.29
17, -74, 35
R
304
3.89
18, -58, -4
L
348
4.37
-42, -26, -12
middle temporal gyrus s
R
336
4.03
48, -24, -17
gyrus parahippocampalis
L
273
4.14
-16, -42, -3
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R: right; L: left; FA, fractional anisotropy; aMCI: amnestic mild cognitive impairment. The threshold was set at a corrected threshold of Monte Carlo < 0.05, Cluster size is more than 100 voxels. aMCI subjects , n = 48; control subjects, n = 36.
ACCEPTED MANUSCRIPT Table 4. Regions with lower FA values in D carriers compared to I homozygotes in aMCI. Hemisphere
Cluster size (voxels)
Peak t-value
Primary peak location x, y, z (mm)
Hippocampus Hippocampus
L
120
3.12
-26, -33, -5
R
153
3.25
28, -12, -20
inferior parietal lobule
R
297
3.78
36, -36, 42
anterior central gyrus
L
287
3.86
-38, -11, 16
anterior central gyrus
R
186
3.43
37, -10, 36
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Areas
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R: right; L: left; FA, fractional anisotropy; aMCI: amnestic mild cognitive impairment. The threshold was set at a corrected threshold of Monte Carlo < 0.05, Cluster size is more than 100 voxels. aMCI subjects with I homozygotes, n = 9; with D carriers, n = 39.
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Fig. 1. In aMCI group, the D carriers were compared to the I homozygotes. Results of the VBA approach highlighted that FA values were lower in the D carriers compared to those in the I homozygotes in some regions, including bilateral hippocampus, right inferior parietal lobule, bilateral anterior central gyrus. Results are displayed at p<0.05, MonteCarlo-corrected, and overlaid on the MNI template. R=right side; L=left side.
Yanzhi Li, Zhengsheng Zhang, Linglong Deng, Feng Bai, Yongmei Shi, Hui Yu, Chunming Xie, Yonggui Yuan, Zhijun Zhang PII: DOI: Reference:
S0022-510X(17)30399-4 doi: 10.1016/j.jns.2017.06.026 JNS 15401
To appear in:
Journal of the Neurological Sciences
Received date: Revised date: Accepted date:
8 October 2016 23 May 2017 15 June 2017
Please cite this article as: Yanzhi Li, Zhengsheng Zhang, Linglong Deng, Feng Bai, Yongmei Shi, Hui Yu, Chunming Xie, Yonggui Yuan, Zhijun Zhang , Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2017), doi: 10.1016/j.jns.2017.06.026
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ACCEPTED MANUSCRIPT Genetic variation in angiotensin converting-enzyme affects the white matter integrity and cognitive function of amnestic mild cognitive impairment patients
Yanzhi Li,·Zhengsheng Zhang,·Linglong Deng,·Feng Bai,·Yongmei Shi,·Hui Yu,·Chunming
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Xie,·Yonggui Yuan,·Zhijun Zhang
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Corresponding author: Zhijun Zhang
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Yanzhi Li
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Geriatric Department of Affiliated ZhongDa Hospital, Medical School of Southeast University, Nanjing, Jiangsu 210009, China
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Zhengsheng Zhang, Feng Bai,·Yongmei Shi, Chunming Xie·
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Neurologic Department of Affiliated ZhongDa Hospital, Neuropsychiatric Institute and Medical School of Southeast University, Nanjing, Jiangsu 210009, China Linglong Deng, Hui Yu
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School of Clinical Medicine, Southeast University, Nanjing 210009, China. Yonggui Yuan
Psychiatric Department of Affiliated ZhongDa Hospital, Medical School of Southeast University, Nanjing, Jiangsu 210009, China Zhijun Zhang
(☒ )
Neurologic Department of Affiliated ZhongDa Hospital, Neuropsychiatric Institute and Medical School of Southeast University, No 87 DingJiaQiao Rd. Nanjing, China
ACCEPTED MANUSCRIPT 210009 Fax: +86 25 8326 2503; E-mail address: [email protected].
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The number of text pages of the whole manuscript is 18 pages, and the number of
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figures and tables respectively are 1 and 2.
ACCEPTED MANUSCRIPT Abstract Angiotensin-converting enzyme (ACE) gene has been implicated in amnestic mild cognitive impairment (aMCI). Most human genetic studies have focused on ACE insertion (I)/deletion (D) polymorphism and yielded conflicting results. In this work,
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we evaluated the relationships between cognitive function, serum ACE level, brain
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white matter (WM) integrity, and ACE I/D polymorphism in 48 patients with aMCI
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and 36 well matched control subjects from south China. In aMCI patients, D allele frequency was higher (D/I ratio= 0.51:0.49) than that of the control subjects (D/I
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ratio= 0.43:0.57); however, the difference was not statistically significant (p > 0.05).
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The D carriers in aMCI subjects performed significantly poorer on auditory–verbal learning test (AVLT) -delayed recall than the I homozygous group (p= 0.035). These
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carriers had higher serum ACE level than the I homozygous carriers of aMCI (p=
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0.037). In the aMCI group, D carriers showed significantly lower fractional anisotropy (FA) values in the left middle frontal gyrus, left anterior cingulate, right gyrus parahippocampalis, right inferior parietal lobule, and bilateral anterior central
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gyrus than the I homozygotes carriers. However, no significant difference was observed in FA values between I homozygotes and D carriers in the control subjects. The ACE D allele in aMCI patients may increase the risk of cognitive impairment. A high serum ACE level possibly plays an important role in the incidence of aMCI. Keywords: mild cognitive impairment·angiotensin converting enzyme gene·cognitive function·magnetic resonance imaging·diffusion tensor imaging
ACCEPTED MANUSCRIPT Highlights: A higher frequency of D allele was detected in aMCI patients. The D carriers had significantly higher serum ACE levels than I homozygotes (p<0.05) in aMCI.
aMCI D carriers showed significantly lower FA values in some memory related brain regions.
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ACCEPTED MANUSCRIPT Background Alzheimer’s disease (AD) is the most common cause of dementia in the older adult subjects and accounts for 50% to 60% of all cases. Most patients with dementia live in developing countries, and the forecasted rates of increase in China, India, and other
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South Asian countries are threefold higher than the ~100% increase estimated in 2040
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[1,2]. Mild cognitive impairment (MCI) has been recognized as a transitional phase
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between normal health and AD. Recent studies have shown that individuals with MCI tend to progress to AD at an annual rate of 10%–15% [3]. Amnestic MCI (aMCI) is
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characterized by selective memory deficits associated or not with other cognitive
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dysfunctions and represents early AD; 76% of MCI cases progressing to AD are aMCI patients [4]. This impairment has the highest probability of developing into
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dementia and even more in the presence of biomarkers (e.g., medial temporal lobe
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atrophy) [5].
AD is a heterogeneous disorder with both familial (about 1% of cases) and sporadic forms. Only the apolipoprotein E (APOE) ε4 allele has been reported as a genetic risk
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factor for sporadic cases. The continually updated meta-analysis functions of the Internet-based Alzgene database (www.alzgene.org; Gene ID = 125), which is applied to the dataset criteria to assess growing epidemiologic evidence from genetic associations, ranks angiotensin-converting enzyme (ACE) gene variation as the second most significant genetic susceptibility locus for AD following APOE ε4 [6]. A common Alu insertion (I)/deletion (D) polymorphism (rs1799752) exists in intron 16 of ACE gene. Research has shown that homozygosity for the D and I alleles are
ACCEPTED MANUSCRIPT associated with the highest and lowest plasma ACE, respectively, and that the heterozygotes have intermediate values [7]. However, the relationship between ACE gene polymorphism and AD remains contentious. Several works have reported a strong association between the ACE I allele and AD [8,9] and have suggested that the
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D allele has a significant function in the progression to AD in aging and MCI [10,11].
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Results of various diagnostic criteria for AD, MCI and normal control, racial
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differences, or other factors contributing to the development of AD are inconsistent [6,9].
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Diffusion tensor imaging (DTI) is a relatively new magnetic resonance imaging
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(MRI) technique and is more sensitive to white matter (WM) alterations before the microstructural change develops into macrostructural loss of WM measurable by
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voxel-based analyses (VBA). Several works have reported that changes in WM
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microstructure assessed with DTI can be a more sensitive parameter than gray matter data for detecting mild structural changes occurring at the early stages of various neurodegenerative diseases [12,13]. In addition, DTI has revealed early and
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progressive loss of integrity of WM that is temporally antecedent to overt clinical evaluation and cerebrospinal fluid biomarkers in AD and MCI [14,15]. The true genetic susceptibility of ACE I/D gene polymorphism on AD pathogenesis remains ambiguous because of inconsistent outcomes. In this study, the possible association of ACE I/D polymorphism with aMCI in South China was analyzed by ACE genotype and DTI analysis by the VBA method. The patients and controls were from Jiangsu province in South China, which limits the ethnic variation in the
ACCEPTED MANUSCRIPT frequency of ACE gene I/D polymorphism and enzyme level in producing false positive outcomes, thereby resulting in a case-control study.
Methods Participants
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We have been recruiting 115 aMCI patients and 93 control subjects, from 1480
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aging in the Outpatient Department of Neurology. Eighty-one cases were excluded as
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no MRI study was performed on them, while another 43 cases were excluded due to a standardized neuropsychological evaluation was not performed on them. Therefore, a
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total of 84 people from the Han ethnic group were enrolled in this study. They were
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from the Outpatient Department of Neurology, and 48 of them have aMCI (18 women and 30 men; mean age±S.D.= 72.04±4.42). Among them, 36 were designated as
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normal controls (19 women and 17 men; mean age±S.D. 71.64±3.72). The patients
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were matched according to sex and age, and all patients gave their informed consent to participate in this study, which was approved by the Institutional Ethical Review Board of the Clinic Medical College of Southeast University, Nanjing, China. The
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criteria for aMCI were as follows: (1) subjective memory impairment corroborated by subject and an informant, (2) weak objective memory performance documented by delayed recall in the auditory–verbal learning test (AVLT), (3) a clinical dementia rating of 0.5, with at least 0.5 in the memory domain, (4) normal general cognitive functioning as evaluated by a score on the mini- mental state exam (MMSE) greater than or equal to 24, (5) no or minimal impairment in 14 daily living activities, and (6) absence of dementia. Participants were excluded from the present study if they have a
ACCEPTED MANUSCRIPT history of known stroke (modified Hachinski score>4), taking ACE inhibitors within the last 3 months, alcoholism, head injury, Parkinson’s disease, epilepsy, major depression, other neurological or psychiatric illnesses, major medical illness, or severe visual or hearing loss.
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All participants underwent a standardized neuropsychological evaluation, including
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the AVLT, Rey–Osterrich complex figure test, verbal fluency test (animal fluency
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task), digit span test, symbol digit modalities test, trail making tests A and B, and clock drawing test. This battery evaluated the function of episodic memory regarding
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both verbal and visual information, semantic memory, attention, psychomotor speed,
neurologists (YM Shi and
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executive function, and visuospatial skills in all subjects. Two experienced H Yu) who are experienced
in professional
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neuropsychological scale assessments administered the process, which also included
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structured interviews with subjects and their informants. Genotyping of ACE I/D and measurement of serum ACE levels ACE polymorphisms were genotyped as previously described [16]. The primer
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sequences were designed in accordance with the forward primer given as 5΄–CTG GAG ACC ACT CCC ATC CTT TCT–3΄ and reverse primer 5΄-GAT GTG GCC ATC ACA TTC GTC AGA T–3΄ to amplify 490/190 bp fragments (I/D allele) that were run on 2% agarose gel. To reduce the incidence of mistyping ID as a DD genotype, all DD genotypes were verified using a second insertion-specific amplification with a primer pair given as: 5΄–TGG GAC CAC AGC GCC CGC CAC TAC–3΄ and 5΄–TCG CCA GCC CTC CCA TGC CCA TAA–3΄. Each blood
ACCEPTED MANUSCRIPT specimen was tested in duplicate to assure reproducibility of results. Data fro m both reactions were used to establish the ACE-ID genotype. Each allele and genotype was determined. Serum ACE level was measured using ultraviolet spectrophotometry (using a
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Hitachi 7170 full- automatic biochemical analyzer) in accordance with the instructions
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on the serum ACE level kit (Zhejiang Kuake Bioscience Technology Co., Ltd.) as
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previously described [16]. This level was measured in all subjects on the same day to minimize assay variance. The intra-assay coefficient of variation was less than 6%.
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Imaging acquisition
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MRI was performed with a General Electric 1.5 Tesla scanner (General Electric Medical Systems, Milwaukee, WI, USA). All participants laid supine with their head fixed
by a belt and
foam pads to
minimize
head
movement.
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snugly
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Diffusion-weighted imaging was acquired with a single-shot echo planar imaging sequence in alignment with the anterior–posterior commissure plane. The diffusion-sensitizing gradients were applied along 25 noncollinear directions (b= 1000
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s/mm2 ), together with an acquisition without diffusion weighting (b=0). Thirty contiguous axial slices with a slice thickness of 4 mm and no gap were acquired. The acquisition parameters were as follows: repetition time=10 000 ms; echo time=81.2 ms; matrix=128×128; field of view=240 mm×240 mm; number of excitations=2. Image analysis The diffusion tensor matrix was calculated according to the Stejskal and Tanner equation. Three pairs of eigenvalues (γ1, γ2, and γ3) and eigenvectors were obtained
ACCEPTED MANUSCRIPT by the diagonalization of the tensor matrix. The principal direction at each point was given by the eigenvector that corresponded to the largest eigenvalue, and fractional anisotropy (FA) values were calculated according to a previously described method [17]. For each participant, the b=0 image was first normalized to the standard
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Montreal Neurological Institute space using statistical parameters maps (SPM12)
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(http://www.fil.ion.ucl.ac.uk/spm). Then, the transformation matrix was applied to the
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FA map to normalize this map to the standard Montreal Neurological Institute space. All images were resampled with a final voxel size of 1×1×1 mm3 . Furthermore, each
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FA map was spatially smoothed by a 6 mm full width at half- maximum Gaussian
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kernel to decrease spatial noise and compensate for the inexact nature of normalization. Following the described procedure, statistical analysis was carried out
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through a cohort analysis (two-sample t-test) between I homozygotes and D carriers in
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aMCI and NC, age and gender as covariates. Significance reached p<0.05 (p<0.005, cluster size=118 voxels, corrected by Monte Carlo simulation). Statistical analysis
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Analysis was performed using SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). A chi-squared goodness-of- fit test was used to test the distribution of genotypes and allele frequencies for deviations from the Hardy–Weinberg equilibrium. The ACE allele and genotype distributions between patients and controls were evaluated using the chi-squared test. A K–S (Lilliefors) test was used to analyze the normality, and a Levene test was used to verify the homogeneity of group variances. An independent-sample t-test was used for the normally distributed variables. The
ACCEPTED MANUSCRIPT nonparametric Mann – Whitney U test was used for asymmetrically distributed variables. Statistical significance was defined at p<0.05 from the two-tailed test.
Results Comparison of clinical characte ristics, cognitive performance, and serum ACE
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level between aMCI and control subjects
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Significant differences in years of education were not observed between the aMCI
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and normal control (p>0.05). All scores on the neuropsychological tests in the aMCI group were significantly lower than those in normal control subjects (p < 0.05). Table
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1 shows that the serum ACE level in aMCI subjects showed no significant difference
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from the levels in normal control subjects (p=0.161).
Comparison of the frequencies of allele and genotype of ACE I/D between aMCI
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and control subjects
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The ACE genotype frequencies were 20.8% (10) for DD, 60.4% (29) for ID and 18.8% (9) for II genotypes in aMCI patients, and 16.7% (6) for DD, 52.8% (19) for ID and 30.4% (11) for II genotypes in control subjects, respectively. In aMCI patients,
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a high D allele frequency (D/I ratio=0.51:0.49) compared with the control controls (D/I ratio=0.43:0.57) was detected, and the difference was not statistically significant (p > 0.05). The observed genotype distribution was in agreement with the Hardy– Weinberg equilibrium (aMCI: p=0.147; control subjects: p=0.647). Differences in neuropsychological tests and serum ACE activity between ACE genotype subgroups in aMCI and control subjects Table 2 shows that the D carriers had significantly poorer performance on
ACCEPTED MANUSCRIPT AVLT-delayed recall than I homozygous carriers in aMCI (p<0.05). However, in the control subjects, no genotype difference existed between D carriers and I homozygotes in all tests. The D carriers had significantly higher serum ACE levels than the I homozygotes (p<0.05) in aMCI but not in control subjects.
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Differences in FA values between aMCI and control subjects, and between ACE
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genotype subgroups in both groups
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In a voxel-by- voxel contrast, several regions showed significantly lower FA values in aMCI patients than in control subjects. Table 3 show that the WM areas exhibiting
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lower FA values were the right precuneus, right lingual gyrus, bilateral middle
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temporal gyrus, and left gyrus parahippocampalis.
Table 4 and Fig. 1 show that upon comparison with aMCI I homozygotes, aMCI D
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carriers showed significantly lower FA values in bilateral hippocampus, right inferior
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parietal lobule, and bilateral anterior central gyrus. No significantly higher FA values were identified in aMCI D carriers. However, no significant differences were observed in FA values between I homozygotes and D carriers in the control subjects.
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Discussion
Biological studies have suggested that ACE gene could play an important role in aMCI pathology. This study has shown a statistically significant effect of the ACE I/D gene polymorphism on the integrity of brain WM on an aMCI group in South China by a comprehensive battery of standardized neuropsychological tests A higher frequency of D allele was detected in aMCI patients (D/I ratio=0.51:0.49) than in controls (D/I ratio=0.43:0.57), and increased homozygosity was found for the
ACCEPTED MANUSCRIPT D allele (20.8%) compared with controls (16.7%) in aMCI. The serum ACE level in aMCI was higher than that in normal control, and the difference was not statistically significant. However, the D carriers had significantly higher serum ACE levels than I homozygotes (p<0.05) in aMCI. Although the biological mechanism underlying the
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association between aMCI and ACE is unclear, the DD genotype is associated with
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increased ACE levels in plasma and in some tissues [18]. Previous, reports have
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suggested that the association of DD genotype with increased ACE is consistently found in the brain or CSF in animal models, MCI, and AD patients [7,19]. Research
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has shown that ACE in the brain can modulate cerebral blood flow [20], degrade
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β-amyloid [21], and reduce the release of acetylcholine [22], thereby influencing cognitive processing. Using ACE inhibitors in antihypertensive treatment, an apparent
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improvement in the cognitive functions of patients has been demonstrated, which has
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not been observed using other antihypertensive [23]. Treatment with brain-penetrating ACE inhibitors significantly reduces ACE activity in CSF [24] and attenuates neurodegeneration and memory impairments in aging and AD [24–26].
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Several lower FA values and impaired cognitive function in AVLT-delayed recall have been found in MCI with D carriers than in I homozygotes. An anatomopathology research has revealed that the frontal and temporal cortex tend to be severely affected by WM myelin loss in AD with D homozygotes compared with those with I carriers, even in conjunction with the APOE ε4 allele [27]. Furthermore, the association with increased age and cognitive decline is significantly stronger in people with the ACE D homozygotes than in I carriers, and this interaction is particularly strong for the
ACCEPTED MANUSCRIPT decline in tests of delayed verbal memory [28]. The delayed recall task of the AVLT represented episodic memory has been used to characterize memory impairment in MCI [29]. These findings reveal that ACE has a significant function in the progression of neurodegeneration and cognitive dysfunction.
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This study is subject to several limitations. One is the relatively small number of
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subjects, i.e., small number genetic subgroups in the aMCI and NC samples, which
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limited our ability to generalize the results. Another is the cross-sectional and case– control nature of this study. Hence, our results should be confirmed by an additional
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longitudinal study. A follow-up of the current cohort is underway.
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In conclusion, we found that serum ACE levels are elevated in those with aMCI who were D carriers than the patients with aMCI who were I homozygotes. It also
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suggested that significantly lower FA values in some regions s of the brain in aMCI D
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carriers compared with I homozygotes. The D allele of ACE gene can be a risk factor for AD. However, the hypothesis requires to be confirmed by follow- up studies. Determining the clear pathogenesis of this ACE gene in AD may help to reduce the
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worldwide health burden of AD. Results provide a window for reconsidering the significant function of gene polymorphism and suggest possible strategies for future pharmacogenomic researches. Acknowledgments This research was partly supported by the Key Program for Clinical Medicine and Science and Technology, Jiangsu Provence Clinical Medical Research Center (No. BL2013025), National Natural Science Foundation of China (Nos. 81201080 and
ACCEPTED MANUSCRIPT 81271336), and National Hi-Tech Research and Development Program of China (863 Program) (No. 2008AA02Z413). Conflict of interest/disclosure The authors declare that they have no financial or other conflicts of interest in
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relation to this research and its publication.
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ACCEPTED MANUSCRIPT Table 1. Comparison of cognitive performance, serum ACE level between aMCI and
Normal Controls
Measure
(N = 48)
(N = 36)
p Value
Education
15.5 (12-16)
16 (12-16)
0.286
MMSE AVLT-delayed recall
27 (26-28) 3 (2-4)
29 (27-29) 8 (7-9.75)
< .001b < .001b
CFT-delayed recall
9.5 (5.25-16)
16 (10.75-20.38)
= .002b
Category Fluency Test Trail Making Test A
10.77 ± 3.02 92.63 ± 30.91
13.28 ± 2.68 71.81 ± 27.96
< .001 = .002c
Trail Making Test B Symbol Digit Modalities Test
164.5 (122.75-215.25) 24.58 ± 8.87
Digit Span Test Clock Drawing Test
12 (11-13) 9 (8-9)
ACE Level(U/L)
122.50±48.64
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control subjectsa
c
118 (101.25-161.50) 35.08 ± 10.71
= .004b < .001c
13 (12-14) 9 (8.25-10)
= .009b = .027b
107.64±46.36
=.161c
Values expressed as median (interquartile range) except where noted. Mann-Whitney U test. c Independent-Sample t test aMCI: amnestic mild cognitive impairment; AVLT: auditory verbal learning test; CFT: rey-osterrieth complex figure test; MMSE: mini- mental state examination; ACE: angiotensin converting enzyme.
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b
ACCEPTED MANUSCRIPT Table 2. Comparison of neuropsychological data, serum ACE level between aMCI and control subjects according to ACE genotypea aMCI
control subjects DD/ID (N = 25)
II (N = 11)
P Value
MMSE
27 (26-28)
27 (27-28)
0.413b
29 (27.5-30)
29 (27-29)
0.208b
AVLT-delayed recall
3 (2-4)
4 (2.5-6)
0.035
8 (7-10)
8 (7-8)
0.650
CFT-delayed recall Category Fluency Test Trail Making Test A
9 (5-16) 10.56 ±2.96 94.64 ±31.06
14 (6-19) 11.67 ±3.28 83.89 ±30.39
0.268b c 0.502 0.352c
16 (8.5-22) 13.2 ±2.63 73.64 ±29.52
16 (14-20.5) 13.45 ±2.91 67.64 ±24.87
0.602b c 0.312 0.561c
Trail Making Test B Symbol Digit Modalities
165 (122-217) 23.96 ±78.46
146 (110.5-214) 27.33 ±10.54
0.526 0.307c
131 (101-171) 34.12 ±9.30
114 (101-132) 37.27 ±13.63
0.264 0.424c
Test Digit Span Test
12 (11-13)
12 (11-13.5)
0.737b
13 (12-14.5)
14 (13-14)
0.134b
Clock Drawing Test ACE Level(U/L)
9 (8-9) 129.49 ±48.21
9 (8-10) 92.22 ±39.85
9 (8.5-10) 116.76 ±46.52
9 (8-10) 86.90 ±40.59
0.755b 0.075c
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II (N = 9)
P Value
Measure
DD/ID (N = 39)
0.232b 0.037c
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Values expressed as median (interquartile range) except where noted. Mann-Whitney U test c Independent-Sample t test aMCI: amnestic mild cognitive impairment; AVLT: auditory verbal learning test; CFT: rey-osterrieth complex figure test; MMSE: mini- mental state examination; ACE: angiotensin converting enzyme.
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ACCEPTED MANUSCRIPT Table 3. Regions with lower FA values in aMCI patients compared to control subjects Areas
Hemisphere
Cluster size (voxels)
Peak t-value
Primary peak location x, y, z (mm)
precuneus lingual gyrus middle temporal gyrus
R
347
4.29
17, -74, 35
R
304
3.89
18, -58, -4
L
348
4.37
-42, -26, -12
middle temporal gyrus s
R
336
4.03
48, -24, -17
gyrus parahippocampalis
L
273
4.14
-16, -42, -3
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R: right; L: left; FA, fractional anisotropy; aMCI: amnestic mild cognitive impairment. The threshold was set at a corrected threshold of Monte Carlo < 0.05, Cluster size is more than 100 voxels. aMCI subjects , n = 48; control subjects, n = 36.
ACCEPTED MANUSCRIPT Table 4. Regions with lower FA values in D carriers compared to I homozygotes in aMCI. Hemisphere
Cluster size (voxels)
Peak t-value
Primary peak location x, y, z (mm)
Hippocampus Hippocampus
L
120
3.12
-26, -33, -5
R
153
3.25
28, -12, -20
inferior parietal lobule
R
297
3.78
36, -36, 42
anterior central gyrus
L
287
3.86
-38, -11, 16
anterior central gyrus
R
186
3.43
37, -10, 36
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Areas
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R: right; L: left; FA, fractional anisotropy; aMCI: amnestic mild cognitive impairment. The threshold was set at a corrected threshold of Monte Carlo < 0.05, Cluster size is more than 100 voxels. aMCI subjects with I homozygotes, n = 9; with D carriers, n = 39.
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Fig. 1. In aMCI group, the D carriers were compared to the I homozygotes. Results of the VBA approach highlighted that FA values were lower in the D carriers compared to those in the I homozygotes in some regions, including bilateral hippocampus, right inferior parietal lobule, bilateral anterior central gyrus. Results are displayed at p<0.05, MonteCarlo-corrected, and overlaid on the MNI template. R=right side; L=left side.