Northern Chinese Han populations with sporadic Alzheimer’s disease and the role of urokinase-type plasminogen activator gene promoter polymorphisms****☆
Di Han1, Yan Wang2, Hongyun Li1,Jianping Jia3

1Department of Emergency Neurology, Medical School Hospital of Qingdao University, Qingdao  266003, Shandong Province, China
2Department of Neurology, Medical School Hospital of Qingdao University, Qingdao  266003, Shandong Province, China
3Department of Neurology, Xuanwu Hospital of the Capital Medical University, Beijing  100053, China

Di Han☆, Doctor, Department of Emergency Neurology, Medical School Hospital of Qingdao University, Qingdao 266003, Shandong Province, China

Corresponding author: Di Han, Department of Emergency Neurology, Medical School Hospital of Qingdao University, Qingdao 266003, Shandong Province, China
drhan6@hotmail.com

Supported by: the National Key Technology R&D Program in the Eleventh Five-year Plan Period, No. 2006BAI02B01*; the National Basic Research 973 Program, No. 2006CB500700*; the Beijing Natural Science Foundation, No. 7071004*; Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality

Abstract
BACKGROUND: Polymorphisms of urokinase-type plasminogen activator gene (PLAU) have re-cently been reported to be associated with sporadic Alzheimer’ disease (SAD). However, most studies have focused on the exon region of this gene, and there is no report on the association between promoter polymorphisms of the PLAU and SAD.
OBJECTIVE: To determine whether SAD is associated with promoter polymorphisms of PLAU in Northern Han Chinese.
DESIGN, TIME AND SETTING: A case-control study was performed at Neurology Laboratory of Xuanwu Hospital of the Capital Medical University from September 2006 to July 2008.
PARTICIPANTS: A total of 397 participants living in Beijing were assigned to SAD [n = 196, in-cluding 103 males and 93 females, mean age of (64 ± 7) years] and control [n = 201, including 108 males and 93 females, mean age of (68 ± 6) years] groups. The patients were diagnosed and met the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorder Association criteria for possible Alzheimer’s disease. Controls received clinical, mental, and neurological examinations to rule out cognitive deficiencies. All controls had Mini-Mental Status Examination scores > 27.
METHODS: Genotypes of PLAU and apolipoprotein-E were examined in 196 patients with SAD and 201 age- and sex-matched controls from the same community using polymerase chain reac-tion-restriction fragment length polymorphism method. SPSS 11.5 software was used for data analysis, distribution of allele and genotypic frequency were calculated, and Hardy-Weinberg was also performed in this study.
MAIN OUTCOME MEASURES: The main outcome measures were allele and genotype frequency differences in the promoter region of the PLAU gene between SAD and control subjects.
RESULTS: In Chinese Han populations, the two polymorphisms in PLAU promoter were -25 C/T (rs2227579) and 43 G/T (rs2227580). Detection of these promoter polymorphisms revealed sig-nificant differences in allele and genotype frequency for -25 C/T and 43 G/T when 196 SAD patients were compared with 201 controls (P ≤ 0.05). Logistic analyses indicated that, compared with C/T and T/T genotypes, the -25 C/C genotype resulted in a 1.5-fold risk for developing SAD (adjusted odds ratio = 1.510, 95% confidence interval: 0.198-2.281, P = 0.010), while the 43G/G genotype resulted in a 1.3-fold risk for SAD (adjusted odds ratio = 1.300, 95% confidence interval: 0.178-2.051, P = 0.030).
CONCLUSION: The present study provided evidence that promoter polymorphisms of PLAU are associated with development of SAD in Northern Han Chinese.
Key Words: PLAU gene; promoter; Alzheimer’s disease; polymorphism

INTRODUCTION
  
Alzheimer’s disease (AD) is the most common form of dementia in the elderly[1], clinically characterized by progressive-onset memory loss and cognitive decline, as well as neuropathological evidence of amyloid plaques and neurofibrillary tangles in the brains of affected individuals[2-3]. Genetic factors, such as mutations in presenilin-1, presenilin-2, and amyloid precursor protein genes, are thought to be associated with familial AD[4-5]. These mutations affect amyloid precursor protein metabolism such that more amyloid beta peptide (Aβ) is produced. To date, the only unequivocal genetic risk factor for sporadic AD (SAD) is apolipoprotein E gene (APOE)[6-10]. However, 50% of SAD cases do not carry the APOE ε4 allele, indicating that other risk factors may exist[11].
Although SAD pathogenesis remains obscure, it is generally accepted that cerebral accumulation of Aβ plays an important role. The amount of neurotoxic Aβ in the brain is determined by Aβ production through amyloid precursor protein processing and Aβ degradation[12-13]. Plasma A?42 levels and SAD have been linked to the same region on chromosome 10q[14]. The plasminogen activator urinary (PLAU) gene is comprised of 11 exons[15-16] that encode urokinase-type plasminogen activator (uPA), which converts plasminogen to plasmin. Aβ aggregates induce PLAU expression, resulting in increased plasmin, which degrades both aggregated and non-aggregated forms of Aβ. Previous studies[17-21] have demonstrated a significant association between PLAU and SAD. Because of the involvement of uPA in Aβ catabolism, the present study analyzed the association between PLAU promoter polymorphisms and SAD in the Northern Chinese Han population.

SUBJECTS AND METHODS

Design
A case-control study of gene polymorphism.
Time and setting
The experiment was performed at the Neurology Laboratory of Xuanwu Hospital of the Capital Medical University from September 2006 to July 2008.
Subjects
The SAD group consisted of 196 SAD patients [103 males and 93 females, mean age of (64 ± 6.8) years] living in Beijing, who were inpatients and outpatients of Beijing Xuanwu Hospital. No patients reported a family history of SAD.
Inclusion criteria: All SAD patients were diagnosed and met the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorder Association (NINCDS/ADRDA) criteria for probable AD[22]. Criteria for clinical diagnosis of probable AD include the following: dementia established by clinical examination, documented by the Mini-Mental Test, and confirmed by neuropsychological tests; deficits in two or more areas of cognition; progressive worsening of memory and other cognitive functions; no disturbance of consciousness; onset age of 40–90 years, most often after age 65; and absence of systemic disorders or other brain diseases that could account for progressive deficits in memory and cognition.
Exclusion criteria: Patients with evidence of vascular and “mixed” dementia.
The control group consisted of 201 healthy controls [108 males and 93 females, mean age of (68 ± 6.5) years] from the same community. No controls reported a family history of SAD.
Inclusion criteria: Controls were healthy check-out staff from Beijing Xuanwu Hospital. They received clinical, mental, and neurological examinations to rule out cognitive deficiencies, anxiety, depression, and other mental or physical disease. Mini-Mental Test scores were > 27.
All subjects were representative of northern Chinese Han populations. According to Administrative Regulations for Medical Institutions[23], informed consent was obtained from the participants, and the study protocol was approved by the Institute Ethical Committee. Following stratification by the APOE ε4 allele, the subjects were divided into APOE ε4 carrier and APOE ε4 non-carrier subgroups.
Methods
Drugs and instruments
The primary drugs and instruments are as follows:

DNA extraction
A total of 5 mL venous blood was collected from each subject, 1 mmol/L EDTA was added, and the tube was centrifuged at 3 000 r/min for 10 minutes to separate plasma and blood cells. Twice the volume of red blood cell lysis buffer was added to the blood cells, mixed, and incubated for 20 minutes, followed by centrifugation at  3 000 r/min for 10 minutes to remove supernatant. This process was repeated three times, and each time the supernatant was discarded. A total of 3 mL white blood cell lysis buffer, 200 μL SDS, and 70 μL protease K was added to the mixture and incubated at 55 °C overnight. Saturated sodium chloride (1 mL) was added and mixed by oscillation, the sample was centrifuged at 3 000 r/min for 15 minutes, and the supernatant was aspirated. Following addition of twice the volume of anhydrous ethanol and gentle shaking, DNA flocculation was observed, and then the DNA was removed. Following addition of 1 mL 75% ethanol to wash the DNA, the ethanol was removed and the DNA was dried at 55 °C for 2–3 hours. The DNA was dissolved in 500 μL 1× Tris and EDTA, incubated at 55 °C overnight, dissolved, and subsequently stored at –80 °C.
Sequencing of the PLAU promoter
The promoter sequence of the PLAU gene used for analysis was selected from the complete genomic contig sequence obtained from GenBank (National Institutes of Health genetic sequence database, Gene ID: 5328). The DNA fragment of the PLAU promoter was amplified using primers designed by Premier 5.0 (Primers Biosoft International, USA) (http://www.primerbiosoft.com/) (forward: 5’-TTC AAT AGG AAG CAC CAA CAG-3’; reverse: 5’-TGG GCA GCA TCA GTC AAA G-3’). Primer sets covered a 1297-bp sequence from –1 207 to +90, relative to the translation initiation codon (ATG). The PLAU promoter was selectively screened in 10 randomly selected controls and 10 SAD patients using direct sequencing. Primer synthesis and sequencing of polymerase chain reaction (PCR) products were performed by Shanghai Sangon of China and the Gene Center of North China.
Polymorphism genotyping
Each 25 μL amplification reaction contained 9.8 μL double-distilled H2O, 0.5 μL (20 μmol) from each primer, 0.6 μL (2.5 mmol) dNTPs, 0.1 μL LATaq DNA polymerase (TaKaRa, Shiga, Japan), 12.5 μL PCR buffer (2 ×), and 1.0 μL DNA template. Following initial denaturation at  94 °C for 5 minutes, the reaction mixture was subjected to 30 cycles of denaturation at 94 °C for 40 seconds, annealing at 57 °C for 30 seconds, extension at 72 °C for 1.5 minutes, followed by a final extension at 72 °C for 6 minutes. Polymorphisms were analyzed by restriction enzyme digestion of PCR products amplified from genomic DNA. To separate cleavage fragments, the primers were redesigned (forward: 5’- TCG CAG CAC AGT GCG GAG AC -3’; reverse: 5’-GCA TCA GTC AAA GCA AGA GCG-3’). After standard PCR for 30 cycles, the PCR products (516 bp) were digested with restriction endonuclease MspI and SmII respectively. PCR fragments and the DNA marker were separated on a 2.5% agarose gel and visualized with an ultraviolet transilluminator following ethidium bromide staining. APOE genotyping was performed on all subjects using previously described methods[24]. DNA was amplified utilizing a PCR thermal cycler along with the following primers – forward: 5’-AGA CGC GGG CAC GGC TGT CCA AGG A-3’ and reverse: 5’-CCC TCG CGG GCC CCG GCC TGG TAC AC-3’. The PCR products were digested with HhaI and the fragments were separated by electrophoresis on a 20% polyacrylamide non-denaturing gel for 8 hours. Following electrophoresis, the gel was stained with ethidium bromide for 30 minutes and DNA fragments were visualized by UV illumination.
Main outcome measures
The main outcome measures were allele and genotype frequency differences of the PLAU gene promoter between SAD patients and controls.
Design, enforcement, and evaluation
This study was designed by Jianping Jia, and implementation of the study was performed by Di Han. Study evaluation was performed by Yan Wang.
Statistical analysis
Statistical analysis of genotype distributions and allele frequencies was performed by chi-square test (SPSS Version 11.5, SPSS, Chicago, IL, USA). Hardy-Weinberg equilibrium (HWE) was tested using the HWE program, as previously described[25]. Linkage disequilibrium was analyzed using a previously described program[26-27], and D’ and r2 were calculated online (http://analysis.bio-x.cn/myAnalysis.php). The strength of association between alleles or genotypes and SAD was evaluated using the Odds Ratio (OR) presented with 95% confidence intervals (CI). A P value < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of the experimental animals
A total of 196 SAD patients and 201 healthy controls were included in the final analysis
Comparison of baseline data between SAD group and control group
The comparison of baseline data between the SAD and control groups is shown in Table 1. There were no significant differences in gender, sex, education level, or other baseline data between the two groups (P > 0.05).
SNP detection and HWE testing
The 1 297-bp fragment of the proximal PLAU promoter contained two polymorphisms -25C/T (rs2227579, Figure 1) and 43G/T (rs2227580, Figure 2). All genotype distributions from these polymorphisms were in Hardy-Weinberg equilibrium in the SAD (-25 C/T: P = 0.202, 43 G/T: P = 0.505) and control (-25 C/T: P = 0.847, 43 G/T: P = 0.439) groups.
Genotype distribution and allele frequency
There was no HWE deviation in polymorphisms from the SAD cases and controls. The -25 C/T genotype and C allele were more frequent in SAD than in controls (genotype: P = 0.015; allele: P = 0.004). In terms of 43 G/T, the GG genotype and G allele were more frequent in patients than in controls (genotype: P = 0.021; allele:  P = 0.006) (Tables 2 and 3).

Association between PLAU promoter polymorphisms and SAD
As expected, the APOE ε4 allele was more prevalent in SAD than in controls (P < 0.004). The subjects were then assigned to two subgroups according to APOE ε4 status. Following stratification by the APOE ε4 allele, distributions of -25 C/T in the APOE ε4 non-carrier subgroup exhibited differences between SAD and controls (genotype: P = 0.038; allele: P = 0.031). However, in the APOE ε4 carrier subgroup, there was no significant difference (genotype: P = 0.169; allele: P = 0.063). In the APOE ε4 carrier subgroup, the 43 G/T differences (genotype: P = 0.039; allele: P = 0.029), but not in subjects without the APOE ε4 allele (genotype: P = 0.156; allele: P = 0.254). After adjusting for age, gender, and APOE ε4 status through the use of logistical analyses, there was a 1.5-fold increased risk for the -25 C/C genotype to develop SAD compared with the C/T and T/T genotypes (adjusted OR = 1.510, 95% CI: 0.198–2.281, P = 0.010). Compared with the G/T and T/T genotypes, there was a 1.3-fold increased risk to develop SAD for the 43G/G genotype (adjusted OR = 1.300, 95% CI: 0.178–2.051, P = 0.030). At these loci, linkage disequilibrium between alleles was analyzed, demonstrating no linkage disequilibrium between -25 C/T and 43 G/T (D’ = 0.031, r2 = 0.000).

DISCUSSION

Because PLAU is located in a linkage region of SAD on Chromosome 10q, it is a reasonable functional and positional candidate gene. The protein product uPA is functionally involved in Aβ degradation by plasmin activation. An association between PLAU polymorphism and SAD has only been demonstrated in the exon region. Finckh et al[18] genotyped the PLAU C/T polymorphism (rs2227564) in 347 Caucasian patients with SAD and 291 Caucasian control subjects. Results demonstrated that SAD was associated with the C/C genotype, with an odds ratio of 1.89. These results suggested that PLAU is a susceptibility gene for SAD, the C allele is a recessive risk factor, and the T allele confers protection. Additional studies have confirmed the association between PLAU and SAD[17-21].

In recent years, substantial evidence has confirmed that promoter variants in certain genes may be relevant to pathogenesis of particular diseases by altering transcriptional activity[28-30]. Because the PLAU gene participates in Aβ degradation, regulatory variation may play a role in SAD genetic susceptibility. However, there are no reports regarding the possible relationship between the PLAU promoter and SAD.
The present study analyzed core sequences of the proximal PLAU promoter to elucidate the genetic contribution of PLAU to SAD development. In the Chinese Han population, -25 C/T and 43 G/T polymorphisms were detected. With regard to the C/C genotype and C allele, a greater number of SAD patients, in particular those lacking the APOE ε4 allele, exhibited -25 C/T, compared with controls. However, there was no difference in APOE ε4 carriers. These results suggest that the -25C/T polymorphism is associated with SAD and is independent of APOE ε4 allele. In addition, the G allele and G/G genotype of 43 G/T were more frequent in the SAD group than in controls, in particular in patients carrying APOE ε4 allele, but not in patients lacking the APOE ε4 allele. The findings indicate that 43 G/T is associated with SAD and could interact synergistically with the APOE ε4 allele. The results are consistent with previous studies[17-21] showing an association between PLAU and SAD.
The present results demonstrated that -25 C/T and 43 G/T polymorphisms were significantly associated with SAD susceptibility, suggesting that these specific polymorphisms were susceptibility factors for SAD. These two polymorphisms could affect transcriptional regulation of PLAU, thereby altering uPA expression and further contributing to SAD development. However, the complex mechanisms relevant to this process await further exploration with functional assays. To the best of our knowledge, this is the first study aimed at identifying the association between promoter region polymorphisms of the PLAU gene and SAD development in the northern Chinese Han population.

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 (Edited by Li L/Ji H/Song LP)