Case-control study to identify genetic risk markers for dementia .13

Patients were recruited from the dementia outpatient clinic of Chang Gung Medical Center. All examinations were performed by Drs W.-C. Hsu, H.-C. Fung, J.-C. Lin, H.-P. Hsu, C.-M. Chen, Y.-R. Wu, K.-H. Chang and L.-S. Ro (Department of Neurology, Chang Gung Memorial Hospital) after obtaining informed consent from patients and control individuals. Patients with a previous clinical history of neurological, psychiatric, somatic, or toxic causes for dementia were excluded. Evaluation included general physical and neurological assessment, the Mini-Mental State Examination (MMSE) and the Hachinski ischemia score (HIS). Laboratory studies included complete blood cell count, biochemistry analysis, erythrocyte sedimentation rate, vitamin B₁₂ and folic acid levels, thyroid-stimulating hormone level and syphilis serological testing. Each patient underwent a brain computerized tomography scan (CT) or magnetic resonance imaging (MRI). At least 2 neurologists examined all the patients, and confirmed that they fulfilled the DSM-IV criteria for dementia. The diagnosis of AD was made by consensus, according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association for probable AD (NINCDS-ADRDA) (McKhann et al., 1984). VaD was diagnosed using the criteria of the National Institute of Neurological Disorders and Stroke and the Association Internationale pour la Recherche et l’Enseignement en Neurosciences (NINDS-AIREN) (Roman et al., 1993). All VaD patients in this study had HIS of > 7. They belonged to the multi-infarct dementia subtype of VaD. The brain CT or magnetic resonance imaging of those

apparent infarction (Moroney et al., 1997). Patients whose HIS were between 4 and 7, with dementia caused by a possible combination of VD and AD and those whose brain CT study only showed peri-ventricular white matter changes were excluded. Unrelated controls without stroke and cognitive impairment were recruited from the same community; similar ethnic origin and area of residence of the controls and the patients limit the possible confounding effect of population stratification.

Genomic DNA extraction

Genomic DNA extraction from whole blood (peripheral leukocytes) or lymphoblastoid cells is carried out using DNA Extraction Kit (Cat. No.

200600, Stratagene). Three volume of 1× solution 1 was mixed well with the blood sample and incubated on ice for 5 minutes. After centrifugation at 2,000 rpm for 10 minutes, the supernatant was removed and the pellet was resuspended in 2 ml of solution 2. Then 10 μl of pronase is added to the suspension and incubated at 60°C for several hours, or at 37°C overnight for several days. The suspension was placed on ice for 10 minutes, followed by mixing with 0.8 ml of solution 3 and stood on ice for 10 minutes. The protein precipitate was removed by centrifugation at 3,400 rpm for 15 minutes at 4°C, and the supernatant containing nucleic acid was treated with 6 μl of RNase at 37°C for 15 minutes. DNA precipitate was separated out by mixing with 2.5 ml of isopropanol, transferred to a fresh tube, and centrifuged at 14,000 rpm for 1 minute. The pellet was rinsed once with 70% ethanol and air-dried. DNA was dissolved in adequate volume of ddH₂O and the concentration was determined using the spectrophotometer.

Polymerase chain reaction (PCR) and genotyping

Genotypes for APOE ε2/ε3/ε4, ACE -240 A/T and Alu I/D, IL-1α -889

C/T, IL-1β -511 C/T, as well as HSPA5 -415 G/A, -370 C/T and -180 del/G were determined by polymerase chain reaction (PCR)-based restriction and/or electrophoresis assay (Table 1). Briefly, 100 ng of genomic DNA, 0.4 μM of each primer, 100-200 μM dNTPs, 0.8-3.0 mM MgCl2, 10 mM of Tris pH 8.3, 50 mM KCl, 0.5 U Taq polymerase were prepared in a final volume of 25 μl. Genotypes for ACE Alu I/D were determined by direct electrophoresis of PCR products. Genotypes for APOE ε2/ε3/ε4, ACE -240 A/T, IL-1α -889 C/T, IL-1β -511 C/T, and HSPA5 -415 G/A and -370 C/T were determined by electrophoresis after restriction analysis. Genotypes for HSPA5 -180 del/G were determined by analyzing PCR-amplified products in a linear polyacrylamide gel on an automated MegaBACE Analyzer (Molecular Dynamics, Division of Amersham Pharmacia Biotech). Alleles del and G were confirmed by DNA sequencing. Genotypes for KLK1-130 GN were determined as previously described (Lee-Chen et al., 2004).

PCR-amplified products were analyzed in a linear polyacrylamide gel and allele sizes were determined by comparing migration relative to molecular weight standards. In addition, aliquots of the amplified products were mixed with an equal volumn of 95% formamide buffer and subjected to single strand conformation polymorphism (SSCP) analysis using GeneGel Excel gels as recommended by the manufacture (Pharmacia Biotech).

Statistical analysis

Differences in genotype frequencies between groups were assessed by the Chi-square test. The expected genotypic frequency under random mating was computed using the algorithm described by H. Levene (Levene, 1949), while Chi-square analysis was used to test for the Hardy-Weinberg equilibrium (Yeh and Boyle, 1997). The pair-wise haplotype frequencies were computed by gene counting (Hill, 1974) and tested by Chi-square test

for significance. A P-value of statistical significance was adjusted by Bonferroni correction for each set of comparison.

For HSPA5 gene, the SNPSpD method (Nyholt, 2004) was used to generate an adjusted significance threshold for correction of multiple SNP testing. The experiment-wide significance threshold of 0.030 was required to keep the type I error rate at 5%. Measures of pairwise linkage disequilibrium (LD) between SNPs including Lewontin’s standardized disequilibrium coefficients (D’), The squared pairwise correlations (Δ²), and eigenvalues (λs) were computed with the LDMAX software-part of the GOLD Command Line Tool package (Abecasis and Cookson 2000).

PHASE version 2.1 was used to infer the HSPA5 gene haplotypes (Stephens et al., 2001). The HSPA5 pairwise haplotype frequencies were computed and Chi-square tested for significance. Odds ratios (ORs) with 95%

confidence intervals (95% CI) were calculated to test the association between genotype/allele/haplotype and disease.

II. ACE promoter -240 A/T and Alu I/D reporter functional assay