Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

Abstract

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

Allelic recombination between distinct genomic locations generates copy number diversity in human β-defensins

“…S1), such that completely null (zero-copy) haplotypes could be created by simple allelic recombination between such chromosomes. By contrast, in typing Ͼ1,500 unrelated individuals for ␤-defensin copy number (18)(19)(20)24), we have observed only a single example of an individual with 1 copy, suggesting that zero-copy haplotypes are infrequent and may be very rare. However, a zero-copy haplotype is most likely to be found in combination with a 2-copy haplotype and thus will escape unambiguous detection by diploid copy number measurement alone; we do not yet have sufficiently precise data on frequencies of haplotypes with different copy numbers at each site to establish whether the observed frequency of 1-copy individuals represents a significant departure from the predictions of a neutral model.…”

“…We determined copy numbers of the variable ␤-defensin CNV in Centre d'Etude du Polymorphisme Humain (CEPH) family members using the paralogue ratio test (PRT), combined with analysis of variant ratios at microsatellites EPEV-1 (18) and EPEV-3, and the multiallelic length polymorphism including the indel rs5889219. PRT is a development of the multiplex comparative PCR approach that uses a single primer pair to amplify both test and reference loci, leading to more accurate and robust copy number determination (24). Ratios of products from multiallelic length polymorphisms could be used to confirm copy number measurements; for example, amplification of 3 variants with yields in the ratio 2:2:1 strongly suggests a copy number of 5 (see, for example, the rs5889219 profile of 133306 in Fig.…”

“…Copy number of the ␤-defensin repeat unit in CEPH family members was determined by combining different and complementary methods of analysis. A PRT used comparative PCR between the heat-shock protein pseudogene HSPDP3 and an unlinked reference locus on chromosome 5 as described (24) to produce an estimate of copy number per diploid genome. We used analysis of 3 multiallelic loci within each repeat, both to confirm the copy numbers determined by PRT and to distinguish repeat units in segregation analysis.…”

“…In the more frequent copy number range, low ␤-defensin copy number has been reported to be associated with Crohn's disease of the colon (23) and high copy number with predisposition to psoriasis (20). This common variation may be involved in predisposition to other inflammatory disorders, but progress in these association studies has been hampered by the difficulty of measuring copy number accurately for large numbers of samples (24,25).…”

Evolution of the rapidly mutating human salivary agglutinin gene ( DMBT1 ) and population subsistence strategy

“…1B) separately. To this end, we designed several paralog ratio tests (PRTs), a form of quantitative PCR that is particularly robust in accurately calling CNVs (27). These PRTs were used to estimate copy number at each CNV in 270 individuals from HapMap phase 1.…”