RESEARCH at 958C, 20 seconds at 568C and 40 seconds at 728C. PCR products were separated in the ABI 3700 DNA sequencer (Applied Biosystems, Foster City, CA, USA), and the genotypes were read using GeneMapperw software v4.0 (Applied Biosystems). The Hardy – Weinberg equilibrium (HWE) was tested for the frequencies of each polymorphism. The relative lengths of the repeats were used in further analyses. We set the location of the ﬁrst peak to appear in the GeneMapperw software as 1. The following peaks were read based on the distance from the ﬁrst peak. Three random homozygous samples of two genes (the genes encoding the insulin receptor [INSR] and calmin [CLMN]) were sequenced to validate the genotyping. Real-time PCR We quantiﬁed the transcriptional expression levels of two genes, INSR and CLMN, by real-time PCR in the 59 unrelated CEU samples. For each cell line, 3 mg of total RNA was used and was reversely transcribed into cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The MxPro – Mx3000P QPCR System (Stratagene, La Jolla, CA, USA) was used to quantify the expression levels of each gene. The reactions were performed in a 96-well plate, in a ﬁnal volume of 25 ml containing iQTM SYBRw Green Supermix (Bio-Rad), 500 nM of forward and reverse primers, respectively, and cDNA tem- plate corresponding to 4.5 ng of total RNA. The thermal cycling conditions were 10 minutes at 958C, followed by 40 cycles of 30 seconds at 958C, 1 minute at 558C and 30 seconds at 728C, com- pleting with one cycle of 1 minute at 958C, 30 seconds at 558C and 30 seconds at 958C. A house- keeping gene, the gene encoding FK-506-binding protein 1A (FKBP1A), was included as the internal control. Primer sequences are listed in Table 1. Association of the repeat genotypes with gene expression Samples were grouped into classes based on their relative repeat genotypes. For example, the samples can be grouped into either ‘low category’ (with Zhang et al. relatively low numbers of repeats ‘1/1’) or ‘high category’ (with other genotypes such as ‘2/5’ or ‘1/7’). Other combinations, such as ‘samples with allele 1’ versus ‘other samples’ and ‘sum of alleles in genotype A’ versus ‘sum of alleles in genotype B’, were also tested. Gene expression levels from real- time PCR were then evaluated for correlation with the repeat genotypes in the 59 unrelated CEU samples. Linear regression was performed using the stat::lm function in the R Statistical Package.33 Results Identifying genes with I1 (TG/CA)n repeats A total of 5,424 (TG/CA)n (where n 6) repeats in all introns were identiﬁed in the human genome (version hg16) using Satellog.26 By mapping the repeats to the human genome, a total of 1,137 repeats were found to be located in the I1Rs (Table S1 in the supplementary material), corre- sponding to 839 unique human genes. We grouped these genes according to whether they contained short (six to 11 units) repeats (704 repeats and 536 genes) or long ( 12 units) repeats (433 repeats and 388 genes). We further limited our analysis dataset to the simple repeats within the 2.5 kb windows either downstream of exon 1 or upstream of exon 2, as well as to I1Rs larger than 2.5 kb (Figure 1). In total, our ﬁnal analysis set comprised 371 long (TG/CA)n repeats (n 12) (corresponding to 330 genes) in either the 50 (205 repeats and 187 genes) or 30 (194 repeats and 187 genes) 2.5 kb windows (Table 2 and Table S2 in the supplementary material). Among these, 44 genes had long repeats in both the 50 and 30 windows. Chromosomal distribution of the genes containing I1 (TG/CA)n repeats The chromosomal distribution of the 330 genes containing long I1 (TG/CA)n repeats was com- pared against the distribution of the human genome (version hg16). Figure 2 shows the chro- mosomal distribution of these genes. At FDR , 5 per cent, chromosome 11 was found to be under- represented relative to the whole genome (eight genes; p ¼ 8.3 1024; FDR ¼ 0.02). No other 240 # HENRY STEWART PUBLICATIONS 1479–7364. HUMAN GENOMICS. VOL 3. NO. 3. 236–245 APRIL 2009