Exploring the relationship between polymorphic (TG/CA)n repeats Biochemically, this feature of CA-repeats may affect the movement of RNA polymerases, thus modulat- ing gene expression levels.6 It has also been shown that intronic CA sequences constitute novel and widespread regulatory elements of alternative spli- cing,7 which, in turn, adds another layer of com- plexity in the human genome.8 The abundance of intronic CA-repeats and gene expression has been found to be inversely corre- lated for some highly expressed housekeeping genes, indicating a putative global mechanism of gene expression.9 This observation raises the ques- tion of whether CA-repeats in intronic regions are functional as a gene regulation mechanism for other human genes as well. In other words, does this feature represent a unique class of gene regu- lators for the whole genome? Interestingly, the relationship between the length polymorphisms of CA-repeats located in the ﬁrst introns or intron 1 regions (I1Rs) and gene expression has recently been reported in several cancer- or disease-related genes, including the genes encoding the epidermal growth factor receptor (EGFR),10 hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2)11 and interferon-gamma (IFNG).12 These studies indi- cated a correlation between gene expression level and the polymorphic status of CA-repeats in the I1Rs. While gene expression could be regulated by various genetic or non-genetic factors — such as the presence of cis- or trans- regulatory elements,13 – 15 copy number variants (CNVs)16 and DNA methylation,17 — a genome-wide search for genes with similar structure to these genes could potentially provide evidence that this particular feature (I1 repeats) represents a class of universal gene regulation mechanism. Structurally, CA- repeats can be divided into cat- egories by length, which may reﬂect their biologi- cal properties.18 For example, short repeats (6 n , 12 units) have a very low propensity for poly- morphisms, while long repeats (n 12 units) are more likely to be polymorphic and functional.19,20 More than 90 per cent of the CA-repeats of n 12 units were found to display length polymorphisms and may act as cis-regulators of transcription.21 In general, CA- repeats of n 12 units in highly RESEARCH expressed housekeeping genes show a down- regulatory effect on transcription, which suggests an inverse correlation with the proportion of repeats in introns.9 Therefore, we sought to explore the relationship between gene expression and rela- tively long repeats (n 12 units) located within the I1Rs. In particular, using a public database of small repeats and the RefSeq-supported22 genes, we searched for genes with I1 (TG/CA)n repeats in the proximity of their neighbouring exons in the human genome. Since genes within the same bio- logical pathways are potentially under similar levels of evolutionary pressure, our next goal was to evaluate if there were any enriched or over- represented known pathways and biological pro- cesses in the genes with this particular feature. Known functional annotations, such as those main- tained at the Gene Ontology (GO)23 and Kyoto Encyclopaedia of Genes and Genomes (KEGG)24 databases, were searched for the classiﬁcation of the genes. Statistically signiﬁcant pathways and biologi- cal processes were found to be enriched in the genes with I1 (TG/CA)n repeats. Genotyping of the I1 repeats showed that the majority of these repeats were polymorphic in a panel of human lymphoblastoid cell lines (LCLs) derived from apparently healthy Caucasian individuals of north- ern and western European ancestry from the International HapMap Project7,25 (CEU samples; ie Caucasians from Utah, USA). We then tried to evaluate experimentally the relationship between mRNA expression and the polymorphisms of the I1 repeats in these LCLs. Materials and methods Dataset (TG/CA)n (where n 6) repeats in introns were identiﬁed in the human genome using Satellog (http://satellog.bcgsc.ca),26 which is a database for the identiﬁcation and prioritisation of pure repeat unit microsatellite repeats. Satellog runs on v. 34 of the human genome (version hg16, July 2003) and provides the ability to identify repeats based on user-speciﬁed characteristics, such as repeat unit, period, length and genomic coordinates. To collect # HENRY STEWART PUBLICATIONS 1479–7364. HUMAN GENOMICS. VOL 3. NO 3. 236–245 APRIL 2009 237