DNA Samples
Informed written consent was obtained from subjects prior to the study according to research protocols approved by the Ethics Committee of Tsukuba University. Schizophrenic patients (N = 102) were examined; this group consisted of 61 men (mean age, 46.2 ± 12.0 years; mean age at onset of schizophrenia, 25.2 ± 7.4 years) and 41 women (mean age, 47.5 ± 15.5 years; mean age at onset of schizophrenia, 27.0 ± 10.6 years) who matched the DSM-IV criteria for schizophrenia [13]. Patients were further divided into subtypes (22 paranoid, 38 disorganized, 4 catatonic, 29 residual, and 9 undifferentiated), and classified as to longitudinal course specifiers (64 episodic, 31 continuous, 5 single episode, and 2 other or unspecified). Furthermore, 42 patients were categorized with prominent negative symptoms, and 46 patients had a family history of psychoses in first- or second-degree relatives.
The control group consisted of 100 unrelated healthy volunteers (30 men, mean age 32.1 ± 11.4 years and 70 women, mean age 43.1 ± 12.0 years) who were hospital employees living in the same city as the patients. Each volunteer was interviewed by 2 psychiatrists in order to rule out subjects with a family history of mental illness. All patients and controls were of Japanese origin.
Genomic DNA samples were prepared from whole blood collected in disodium ethylenediamine tetra-acetic acid (EDTA; 3 mg/L) according to the sodium iodide method (DNA Extractor WB Kit, Wako Pure Chemical Industries, Osaka, Japan).
Polymerase Chain Reaction Conditions
A set of polymerase chain reaction (PCR) primers that spanned the (CTG)n repeat region in exon 1 was used to produce DNA fragments. The primers used were NOT-LR-F (forward): 5'-CCCTGCCTGAAGAGGGACAG-3' and NOT-LR-R (reverse): 5'-TCTGGGTCTGACCACTGAGAC-3'. These primers were designed using information from a previous report [4] and the GenBank sequence (accession number: U89335). The 5'-terminus of NOT-LR-F was labeled with indodicarbocyanine fluorescent dye (Amersham Pharmacia Biotech, Uppsala, Sweden) for fluorescence-based, single-strand conformational polymorphism (SSCP) analysis.
The PCR mixture contained the following: 0.5 ng genomic DNA, 0.25 μM of each primer, 0.2 mM of each deoxynucleotide triphosphate, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.5 U of Taq DNA polymerase (HotStarTaq, QIAGEN, Hilden, Germany) in a final volume of 25 μl.
The amplification reaction was performed as follows: an initial denaturation at 95°C for 15 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 30 seconds, with a final extension step of 72°C for 10 minutes (GeneAmp 9600, PE Applied Biosystems, Foster City, CA).
PCR products were visualized by ethidium bromide staining under UV light after electrophoresis on 2% agarose gels.
SSCP Analysis
A DNA sequencer (ALF express, Amersham Pharmacia Biotech) was used to perform fluorescence-based SSCP analysis. PCR products were mixed with loading buffer containing 99.5% deionized formamide and 0.5% blue dextran. The solution was denatured at 97°C for 5 minutes, then immediately cooled on ice. Single-stranded PCR products were analyzed by electrophoresis on 7% polyacrylamide gels (49:1, acrylamide:bisacrylamide ratio) containing 7 M urea in 0.5X Tris-borate-EDTA buffer at 50°C. The data were analyzed using the software package Fragment Manager (Amersham Pharmacia Biotech).
PCR Product Sequencing
PCR products showing altered band patterns by SSCP analysis were purified by centrifugation to recover the DNA (Microcon tube, Millipore, Bedford, MA). DNA sequences of the PCR products were directly determined using a Genetic Analyzer (ABI PRISM TM 310, PE Applied Biosystems) after termination-dideoxy-cycle sequencing (Sequencing Reaction Kit-FS, PE Applied Biosystems) with the forward primer (NOT-LR-F).
Statistical Analysis
Deviation of genotype distribution as derived from the Hardy-Weinberg equilibrium equation was calculated using the chi-square test for goodness of fit. Association analyses were performed using Fisher's exact probability test (2-sided) using InStat-2.01 (GraphPad Software, San Diego, CA). Simulations using the Monte Carlo method [14] were performed using ARLEQUIN (version 2.00, University of Geneva) software.
