Copy number variation (CNV) represents a significant proportion of human genomic variation and it is increasingly becoming clear that CNV is involved in phenotypic variation and disease (Itsara et al, 2009). We are interested in understanding the relationship between copy number variation at loci critical to development, growth and disease. Our current region of interest focuses on a genomic domain spanning genes important for pluripotency, energy utilisation and the immune response. Our analysis of the current genome build reveals part of this region to exist mainly as an ancient (pre-simian) tandem duplication, with corresponding gene duplications. SNP genotyping across this interval in a case control study demonstrated association with rheumatoid arthritis and also revealed hints for CNV (Lorentzen et al, 2007). Due to its complexity and limited LD with surrounding DNA the region is not detected using current genome wide SNP technologies, and therefore targetted CNV analysis is required. Assays for CNV within the tandem duplication were developed based on an adaption to the paralogous ratio test (PRT)(Armour et al, 2007). In these assays the relative number of copies between the tandem repeats is assessed using oligonucleotide primers that amplify simultaneously from both repeats. During this analysis we discovered that either repeat can be duplicated or deleted, thereby representing a complex CNV. From these investigations we have developed an accurate copy number counting assay and demonstrated a significant association between rheumatoid arthritis and copy number of this tandem duplication. This region is of substantial interest as, in addition to the association with RA, it has been implicated in a number of disorders through the data from the rat genome database including QTLs related to insulin resistance and heart disease. Mouse models determine that homozygous nulls for this gene are lethal whereas heterozygous null are related to insulin resistance and increased body mass. Furthermore, changes in protein levels of one of the genes within the CNV in humans has been linked to cancer, insulin resistance and Alzheimer's disease. Given the wide range of disease associations and the role pluripotency, energy transfer and immune response plays in growth and development it is likely that changes in copy number at this region will have an impact on various underlying biochemical measures. Therefore it would be prudent, in addition to traditional case control studies that we are planning, to examine the role of copy number in relation to various endophenotypes. For this the highly characterised ALSPAC cohort is ideal.

For this purpose we have designed and assessed a number of assays based on the paralog ratio test (PRT). These assays allow the assessment of copy number between the repeats, and thereby can indicate which of the two repeats are changing in copy number. The PRT is relatively unique among copy number counting methods in that it requires very little DNA to produce accurate counting. It would therefore be feasible to rapidly genotype the entire ALSPAC cohort with multiple assays using 100 ng DNA, including repeats.

The results of these assays would then be analysed in relation to various biochemical and physiological measures listed below.