Exploring the relationship between polymorphic (TG/CA)n repeats randomly selected for sequencing validation. The sequencing results were consistent with the geno- typing data (data not shown). Evaluation of the relationship between repeat length polymorphism and gene expression The relationship between genotype and gene expression level was evaluated in the remaining eight genes whose repeat genotypes were consistent with the HWE (Table 4). Real-time PCR was per- formed to measure their gene expression levels (Table S4 in the supplementary material). When grouping the samples based on repeat length geno- types, among the eight genes we tested, no statisti- cally signiﬁcant associations were observed using combinations of genotypes, including ‘samples with allele 1’ versus ‘other samples’, ‘sum of alleles in genotype A’ versus ‘sum of alleles in genotype B’ and ‘low category’ (with relatively low numbers of repeats ‘1/1’) versus ‘other genotypes’. The two most signiﬁcant relationships were INSR (r ¼ – 0.23; p ¼ 0.088) and CLMN (r ¼ – 0.19; p ¼ 0.15) for the ‘low category’ (genotype 1/1) versus ‘other geno- types’ comparison. Discussion Understanding gene regulation is critical to under- standing the underlying mechanisms of health-related phenotypes, such as the risks of Table 4. Polymorphic I1 repeats in the unrelated CEU samples Chromosome Gene INSR NRG2 PARD3 CD38 CACNA1E AHR CLMN VLDLR aRelative repeat length. 19 10 5 4 1 7 9 14 Repeat lengtha 1–8 1–6 1–5 1–5 1–8 1–4 1–6 1–11 RESEARCH common diseases. Except for a few classes of elements, such as the promoter regions,34 the puta- tive role of most non-coding elements (eg simple tandem repeats) in gene regulation, however, has not been comprehensively investigated. In this work, we explored the relationship between gene expression and a class of dinucleotide repeats in the intron 1 regions — that is, I1 (TG/CA)n repeats. We focused on relatively long repeats because they are more likely to be polymorphic,21 and therefore potentially more likely to have a role in gene regulation. We further limited our analysis set of repeats to the ﬂanking regions of 2.5 kb windows (Figure 1). This allowed us to focus on the relationship between gene expression and neighbouring repeats, which also were the targets of a couple of previous studies of EGFR,10 HSD11B211 and IFNG.12 Overall, approximately 2 per cent of known human genes have this particular feature based on our criteria. The 330 genes con- taining long I1 repeats were generally distributed evenly across the human genome, although chromosome 11 was found to be under- represented. This under-representation could be due to the arbitrary cut-offs (ie the 2.5 kb windows and n 12) we chose to use in the ﬁnal analysis set. We conﬁrmed that relatively long (TG/CA)n repeats in the I1Rs are universal across the human genome and exist in hundreds of human genes. Furthermore, a couple of biological pro- cesses were found to be enriched among these 330 genes using different annotation databases. For example, the GO and PANTHER biological pro- cesses related to metal ion transport, as well as the KEGG and PANTHER pathways related to calcium signalling, were found to be enriched, indicating that these repeats may play a role in the function or regulation of certain signalling pathways. Interestingly, the PANTHER biological processes BP00040: mRNA transcription and BP00044: mRNA transcription regulation were enriched among these genes, hinting at a potential relationship between I1 repeats and transcriptional gene expression. By genotyping the I1 repeats of ten highly vari- able genes expressed in the unrelated HapMap # HENRY STEWART PUBLICATIONS 1479–7364. HUMAN GENOMICS. VOL 3. NO 3. 236–245 APRIL 2009 243