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Reply to: “CCL3L1 and HIV/AIDS susceptibility” and “Experimental aspects of copy number variant assays at CCL3L1”

Nature Medicine volume 15, pages 1117–1120 (2009)Cite this article

He et al. reply:

We read with interest the three correspondences1,2,3 that are directly or indirectly related to our previous publication in Nature Medicine4. Because of methodological concerns with their own C-C chemokine ligand-3–like-1 (CCL3L1) gene quantitative PCR (qPCR) assay, Urban et al.1 assume that similar limitations apply to the assay that we and others have used and, by extension, infer that all of the several reported associations for CCL3L1 copy number with HIV/AIDS suceptibility (Supplementary Table 1) are spurious. We tested the validity of this assumption. Notwithstanding their statement that they “assayed for CCL3L1 copy number using a previously described method” (described in ref. 5 here), Urban et al.1 made two major modifications to our qPCR assay, which we show adversely affect assay performance. Although they used the same probe sequences, instead of using tetramethylrhodamine (TAMRA) as the quencher, they used minor groove binder (MGB), a change that raises the melting temperature of the probe 8°C higher than the optimal temperature (Supplementary Table 2). Urban et al.1 also raise the concern that our assay is confounded, because they observed that CCL3L1 gene copy number (GCN) differed with the quality and the amount of input DNA. Our published protocol specifically recommended a fixed amount of high-quality input DNA between 2 and 10 ng5, and, consequently, we used qPCR standard curves comprising twofold dilutions from 25 to 0.78 ng. By contrast, Urban et al.1 used variable amounts of input DNA (up to 100 ng) and constructed standard curves of tenfold dilutions from 100 ng to 1 pg.

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Figure 1: Comparison of the performance of the TAMRA assay5 (color-coded blue) and MGB assay1 (color-coded red) using identical human DNA samples in concurrently run experiments as described in the Supplementary Methods.
Figure 2: Chromosome phase of CCL3L1 GCN in parent-child trios and correlations among CCL3L and CCL4L genes.

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Author information

Author notes

  1. Henry M. Jackson Foundation, Wilford Hall United States Air Force Medical Center, Lackland Air Force Base, Texas, USA.

  2. Infectious Disease Service, San Antonio Military Medical Center, Brooke Army Medical Center, Fort Sam Houston, Texas, USA.

Authors and Affiliations

  1. Veterans Administration Research Center for AIDS and HIV-1 Infection, South Texas Veterans Health Care System, San Antonio, Texas, USA

    Weijing He, Hemant Kulkarni, John Castiblanco, Una Aluyen, Robert Maldonado, Andrew Carrillo, Madeline Griffin, Amanda Lipsitt, Lisa Beachy, Robert A Clark, Matthew J Dolan, Matthew J Dolan & Sunil K Ahuja

  2. Department of Medicine, University of Texas Health Science Center, San Antonio, Texas, USA

    Weijing He, Hemant Kulkarni, John Castiblanco, Una Aluyen, Robert Maldonado, Andrew Carrillo, Madeline Griffin, Amanda Lipsitt, Lisa Beachy, Robert A Clark & Sunil K Ahuja

  3. Department of Pediatrics, University of California–San Diego School of Medicine, La Jolla, California, USA

    Chisato Shimizu & Jane C Burns

  4. Department of General Pediatrics and Pediatric Infectious Diseases, Dnepropetrovsk State Medical Academy, Dnepropetrovsk, Ukraine

    Ludmila Shostakovich-Koretskaya

  5. Laboratorio de Biología Celular y Retrovirus, Hospital de Pediatría 'J.P. Garrahan', Buenos Aires, Argentina

    Andrea Mangano & Luisa Sen

  6. Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, UK

    Robert J B Nibbs

  7. AIDS Virus Research Unit, National Institute for Communicable Diseases/National Health Laboratory Services and University of the Witwatersrand, Johannesburg, South Africa

    Caroline T Tiemessen

  8. AIDS Clinical Research Unit, University of Miami, Miller School of Medicine, Miami, Florida, USA

    Hector Bolivar

  9. Departments of Pediatrics and Genome Sciences, University of Washington and Seattle Children's Hospital, Seattle, Washington, USA

    Michael J Bamshad

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  1. Weijing He
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  21. Sunil K Ahuja
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Corresponding author

Correspondence to Sunil K Ahuja.

Supplementary information

Supplementary Tables, Supplementary Methods and Supplementary Note

Supplementary Tables 1–7, Supplementary Methods and Supplementary Note (PDF 2886 kb)

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He, W., Kulkarni, H., Castiblanco, J. et al. Reply to: “CCL3L1 and HIV/AIDS susceptibility” and “Experimental aspects of copy number variant assays at CCL3L1”. Nat Med 15, 1117–1120 (2009). https://doi.org/10.1038/nm1009-1117

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