Vitamin D receptor (
The role of vitamin D in the development and protection of neural cells has been described in a number of studies both
The study sample included 78 patients with well-ascertained late-onset AD (median age, 75 years; range. 65-92 years; males, 47.4%; females, 52.6%) and 103 healthy controls (median age, 57 years; range, 51-90 years; males, 49.5%; females, 50.5%) (
The diagnosis of AD was established based on the current diagnostic criteria for the disease considering: i) A physical examination; ii) the results of the Mini-Mental State Examination (MMSE) questionnaire and Frontal Assessment Battery (FAB); iii) imaging results of brain CT scan and MRI; and iv) biomarker levels in the cerebrospinal fluid Αβ1-42, total-tau and P-tau. Patients were recruited from the Outpatient Clinic of the Cognitive Disorder-Dementia Unit of the Second Department of Neurology at the University General Hospital ‘ATTIKON’ (Athens, Greece). Sample collection was performed from January, 2018 to February, 2019. The present study was approved by the Scientific Council and Bioethics Committee of the University General Hospital ‘ATTIKON’ (Reg. no. 2812; December 21, 2017). Written informed consent for participation in the study and the use of their genetic data was obtained from all participants under Regulation (EU) 2016/679 (General Data Protection Regulation) and according to the Helsinki Declaration (64th World Medical Association, General Assembly, 2013).
Blood samples of patients and controls were analyzed to determine the genotypes of the SNPs TaqI (rs731236), BsmI (rs1544410) and FokI (rs2228570) of the
Genotypes of BsmI (rs1544410) and FokI (rs2228570) polymorphisms were determined using the restriction fragment length polymorphism (RFLP) method. Following the initial DNA extraction, polymerase chain reaction (PCR) was performed in order to amplify the segment of the
The analysis of the Taq1 polymorphism was performed as described in a previous study by the authors on
Data from genotype results were analyzed using SNPstats software (Catalan Institute of Oncology, 2006) (
Allele C in the TaqI polymorphism was found to be associated with a 46% lower risk (OR, 0.54; 95% CI, 0.30-0.99; P=0.045) of developing AD in the dominant model of inheritance TT vs. CT + CC (
No statistically significant differences were observed between the genotype frequency of the BsmI or FokI polymorphisms in the control and AD group (
The frequencies of the possible haplotypes produced from the three polymorphisms (TaqI, BsmI and FokI) are presented in
The TaqI and BsmI polymorphisms were in high linkage disequilibrium (D'=0.9717, r=0.8467) and haplotype analysis revealed that the TA haplotype was associated with an ~8-fold greater risk of developing AD (OR, 8.27; 95% CI, 2.70-25.28; P<0.05) (
Previous research has reported the potential association of specific
The available data regarding the association of
In the SEC cohort studied in the present study, the TaqI and BsmI polymorphisms were in high linkage disequilibrium and therefore, it was deemed appropriate to analyze the data excluding the FokI polymorphism. The analysis revealed that carriers of haplotype TA (TaqI and BsmI) had an ~8-fold greater risk of developing AD (
Another interesting finding of the present study was revealed when the data were analyzed with regards to the sex of the participants. Female carriers of haplotypes TAC and TA had an ~9- and ~14-fold greater risk of developing AD, respectively in comparison to the control female subjects. In general, data from meta-analysis studies have demonstrated that the prevalence of AD is higher in females in comparison to males, and that this difference will continue for the ensuing decades, mainly due to the longer life span of females (
The present study had certain limitations which need to be stated. Two main limitations have to be reported for the present study. The first is the statistically significant difference between average ages in the control and patient group (t-test, P<0.01; only the average ages were statistically analyzed; no other patient characteristics were statistically analyzed). The control subjects were younger than the patients. The second limitation was the small study sample, which affects the statistical power and safety of the conclusions. In addition, vitamin D and estrogen levels of the study subjects were not analyzed.
The present study aimed to investigate potential
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ND conceived the study and provided the control samples and data. MSK and ML performed the sample analysis. ED obtained the ethics committee study approval, performed the literature review, and the statistical and data analyses, and was responsible for the manuscript composition under the supervision and assistance of ND, CK and KA. DAS and AT contributed to the editing of the final manuscript. KA and CK reviewed and analyzed the results of the statistical analysis. DAS, AT, SP and VP contributed to the collection of the clinical data and patient scores. SP, PM and CK also provided the patient samples. PM contributed to the design and optimization of the RFLP analytical method used in this study and in the editing of the final manuscript. All authors discussed the results and agreed on the conclusions of the study and all authors have read and approved the final manuscript. All authors confirm the authenticity of all the raw data.
The present study was approved by the Scientific Council and Bioethics Committee of the University General Hospital ‘ATTIKON’ (Reg. no. 2812; December 21, 2017). Written informed consent for participation in the study and the use of their genetic data was obtained from all participants.
Not applicable.
DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. All the other authors declare that they have no competing interests.
Demographic data of the study groups.
Parameter | Patients | Controls |
---|---|---|
Number (n) | 78 | 103 |
Age, median (years) | 75 | 57 |
Age, mean (years) | 75 | 60 |
Max value (years) | 92 | 90 |
Min value (years) | 65 | 51 |
Range (difference between highest and smallest value) | 27 | 39 |
Males, n (%) | 37 (47.4) | 51 (49.5) |
Females, n (%) | 41 (52.6) | 52 (50.5) |
DNA sequence of forward and reverse primer for BsmI and FokI PCR amplification.
Polymorphism | Primer | DNA sequence |
---|---|---|
BsmI | Forward | 5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' |
Reverse | 5'-AACCAGCGGGAAGAGGTCAAGGG-3' | |
FokI | Forward | 5'-AGCTGGCCCTGGCACTGACTCTGCTCT-3' |
Reverse | 5'-ATGGAAACACCTTGCTTCTCCTCCCTC-3' |
Detection of BsmI and FokI genotypes based on DNA fragment sizes in gel electrophoresis.
BsmI | FokI | |
---|---|---|
PCR product | 825 bp | 265 bp |
Homozygous sample | 825 bp (AA) | 265 bp (CC) |
Homozygous sample | 650 and 175 bp (GG) | 196 and 69 bp (TT) |
Heterozygous sample | 825, 650 and 175 bp (GA) | 265, 196 and 69 bp (TC) |
Frequencies of TaqI genotypes in the different inheritance models.
TaqI rs731236 association with AD (n=181) | |||||
---|---|---|---|---|---|
Model | Genotype | Controls n (%) | AD n (%) | OR (95% CI) | P-value |
Codominant | TT | 35 (34%) | 38 (48.7) | 1.00 | 0.088 |
TC | 49 (47.6) | 32 (41.0) | 0.60 (0.32-1.14) | ||
CC | 19 (18.4) | 8 (10.3) | 0.39 (0.15-1.00) | ||
Dominant | TT | 35(34) | 40 (51.3) | 1.00 | 0.045 |
TC/CC | 68(66) | 46 (51.1) | 0.54 (0.30-0.99) | ||
Recessive | TT/TC | 84 (81.5) | 70 (89.7) | 1.00 | 0.12 |
CC | 19 (18.4) | 8 (10.3) | 0.51 (0.21-1.22) | ||
Recessive (for T allele) | CC/TC | 68 (66%) | 40 (51.3%) | 1.00 | 0.045 |
TT | 35 (34%) | 38 (48.7%) | 1.85 (1.01-3.37) |
AD, Alzheimer's disease; OR, odds ratio; CI, confidence interval.
Frequencies of BsmI genotypes in the different inheritance models.
BsmI rs1544410 association with AD (n=181) | |||||
---|---|---|---|---|---|
Model | Genotype | Controls n (%) | AD n (%) | OR (95% CI) | P-value |
Codominant | GG | 33 (32%) | 30 (38.5%) | 1.00 | 0.076 |
GA | 51 (49.5%) | 26 (33.3%) | 0.56 (0.28-1.11) | ||
AA | 19 (18.4%) | 22 (28.2%) | 1.27 (0.58-2.80) | ||
Dominant | GG | 33 (32%) | 30 (38.5%) | 1.00 | 0.37 |
GA/AA | 70 (68%) | 48 (61.5%) | 0.75 (0.41-1.40) | ||
Recessive | GG/GA | 84 (81.5%) | 56 (71.8%) | 1.00 | 0.12 |
AA | 19 (18.4%) | 22 (28.2%) | 1.74 (0.86-3.50) |
AD, Alzheimer's disease; OR, odds ratio; CI, confidence interval.
Frequencies of FokI genotypes in the different inheritance models.
FokI rs2228570 association with AD (n=181) | |||||
---|---|---|---|---|---|
Model | Genotype | Controls n (%) | AD n (%) | OR (95% CI) | P-value |
Codominant | CC | 55 (53.4%) | 34 (43.6%) | 1.00 | 0.20 |
TC | 38 (36.9%) | 39 (50%) | 1.66 (0.89-3.08) | ||
TT | 10 (9.7%) | 5 (6.4%) | 0.81 (0.25-2.57) | ||
Dominant | CC | 55 (53.4%) | 34 (43.6%) | 1.00 | 0.19 |
TC/TT | 48 (46.6%) | 44 (56.4%) | 1.48 (0.82-2.68) | ||
Recessive | CC/TC | 93 (90.3%) | 73 (93.6%) | 1.00 | 0.42 |
TT | 10 (9.7%) | 5 (6.4%) | 0.64 (0.21-1.95) |
AD, Alzheimer's disease; OR, odds ratio; CI, confidence interval.
Frequencies of possible haplotypes.
Haplotype frequencies estimation (n=181) | ||||||
---|---|---|---|---|---|---|
TaqI rs731236 | BsmI rs1544410 | FokI rs2228570 | Total | Controls | AD | Cumulative frequency |
T | G | C | 0.4093 | 0.4070 | 0.4130 | 0.4093 |
C | A | C | 0.2305 | 0.2819 | 0.1567 | 0.6398 |
T | G | T | 0.1456 | 0.1511 | 0.1383 | 0.7854 |
C | A | T | 0.1365 | 0.1305 | 0.1510 | 0.9219 |
T | A | C | 0.0587 | 0.0196 | 0.1163 | 0.9806 |
T | A | T | 0.0135 | 0.0000 | 0.0248 | 0.9941 |
C | G | C | 0.0059 | 0.0099 | 0.0000 | 1.0000 |
AD, Alzheimer's disease.
Haplotype association with AD.
Haplotype association with AD (n=181) | |||||
---|---|---|---|---|---|
TaqI rs731236 | BsmI rs1544410 | FokI rs2228570 | Frequency | OR (95% CI) | P-value |
T | G | C | 0.4095 | 1.00 | - |
C | A | C | 0.2297 | 0.47 (0.23-0.96) | 0.04 |
T | G | T | 0.1453 | 0.90 (0.43-1.88) | 0.77 |
C | A | T | 0.1373 | 1.10 (0.50-2.45) | 0.81 |
T | A | C | 0.0593 | 6.19 (1.91-20.13) | 0.0028 |
T | A | T | 0.013 | - | <0.0001 |
C | G | C | 0.006 | - | - |
AD, Alzheimer's disease; OR, odds ratio; CI, confidence interval.
Haplotype analysis with covariate sex.
Haplotype interaction with the covariate sex (n=181) | |||
---|---|---|---|
Haplotype | Frequency | Female OR (95% CI) | Male OR (95% CI) |
TGC | 0.4093 | 1.00 | 1.26 (0.37-4.32) |
CAC | 0.2299 | 0.51 (0.19-1.35) | 0.51 (0.16-1.59) |
TGT | 0.1455 | 0.79 (0.25-2.49) | 1.12 (0.38-3.34) |
CAT | 0.1371 | 0.87 (0.25-3.03) | 1.56 (0.38-6.38) |
TAC | 0.0592 | 9.27 (1.86-46.28) | 4.53 (0.68-30.11) |
TAT | 0.013 | - | - |
CGC | 0.006 | - | - |
OR, odds ratio; CI, confidence interval.
TaqI and BsmI haplotypes association with AD.
Haplotype association with AD (n=181) | ||||
---|---|---|---|---|
TaqI rs731236 | BsmI rs1544410 | Frequency | OR (95% CI) | P-value |
T | G | 0.5548 | 1.00 | - |
C | A | 0.367 | 0.67 (0.43-1.07) | 0.095 |
T | A | 0.0722 | 8.27 (2.70-25.28) | <0.05 |
C | G | 0.006 | - | 1.000 |
AD, Alzheimer's disease; OR, odds ratio; CI, confidence interval.
TaqI and BsmI haplotype analysis with covariate sex.
Haplotype interaction with the covariate sex (n=181) | |||
---|---|---|---|
Haplotype | Frequency | Female OR (95% CI) | Male OR (95% CI) |
TG | 0.5548 | 1.00 | 1.31 (0.51-3.32) |
CA | 0.367 | 0.65 (0.32-1.29) | 0.86 (0.39-1.91) |
TA | 0.0722 | 13.93 (2.95-65.87) | 5.10 (0.89-29.40) |
CG | 0.006 | - | - |
OR, odds ratio; CI, confidence interval.
Author/(Refs.) | TaqI allele | ApaI allele | Tru9I allele | BsmI allele | FokI allele | Population |
---|---|---|---|---|---|---|
Gezen-Ak |
T | C | A | G | C | AD |
Lehman |
C | A | AD | |||
Kuningas |
C | A | A | General | ||
Beydun |
T | C | G | General | ||
Present study | T | A | C | AD |
AD, Alzheimer's disease.