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    • "As such there have been a number of association studies focused on CCL3L1 copy number variation and HIV-1 susceptibility [7,18,19]. However, the reported associations are under dispute and the accuracy of CCL3L1 measurement is a major factor in the debate [20-23]. "
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    ABSTRACT: Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion.
    Full-text · Article · Aug 2011 · BMC Genomics
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    • "Without a gold-standard it is not possible to reliably assess which assay is better, but any misclassifications could lead to incorrect associations. Additionally, there are several other issues such as dye chemistry, reaction specificity/conditions and DNA concentrations that might also affect the assays [19]. Even with exact copy numbers, while there may not be differences at the sequence level, there may be differences at the expression levels and therefore may confound the overall association at the protein level rather than the nucleotide sequence level. "
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    ABSTRACT: Copy number variations (CNVs) of the gene CC chemokine ligand 3-like1 (CCL3L1) have been implicated in HIV-1 susceptibility, but the association has been inconsistent. CCL3L1 shares homology with a cluster of genes localized to chromosome 17q12, namely CCL3, CCL3L2, and, CCL3L3. These genes are involved in host defense and inflammatory processes. Several CNV assays have been developed for the CCL3L1 gene. Through pairwise and multiple alignments of these genes, we have shown that the homology between these genes ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions, with CCL3L1 and CCL3L3 being identical. By use of MEGA 4 and BioEdit, we aligned sense primers, anti-sense primers, and probes used in several previously described assays against pre-multiple alignments of all four chemokine genes. Each set of probes and primers aligned and matched with overlapping sequences in at least two of the four genes, indicating that previously utilized RT-PCR based CNV assays are not specific for only CCL3L1. The four available assays measured median copies of 2 and 3-4 in European and African American, respectively. The concordance between the assays ranged from 0.44-0.83 suggesting individual discordant calls and inconsistencies with the assays from the expected gene coverage from the known sequence. This indicates that some of the inconsistencies in the association studies could be due to assays that provide heterogenous results. Sequence information to determine CNV of the three genes separately would allow to test whether their association with the pathogenesis of a human disease or phenotype is affected by an individual gene or by a combination of these genes.
    Full-text · Article · Mar 2010 · BMC Research Notes
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    ABSTRACT: The number of copies of the gene encoding a ligand for an HIV co-receptor have been found to influence the susceptibility to HIV infection and AIDS progression. New studies dispute this conclusion. The studies are contested by the authors of the original findings, and highlight the inherent difficulties in accurately measuring gene copy numbers (pages 1110–1112, 1112–1115, 1115–1117 and 1117–1120).
    Full-text · Article · Oct 2009 · Nature medicine
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