Bharuthram Avani, Paximadis Maria, Picton Anabela C P, Tiemessen Caroline T
Centre for HIV and STIs, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa; Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Centre for HIV and STIs, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa; Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Infect Genet Evol. 2014 Jul;25:28-35. doi: 10.1016/j.meegid.2014.03.028. Epub 2014 Apr 12.
The controversy surrounding the findings that copy number variation, of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. This study evaluated the standard method of quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course.
围绕CCL3编码基因的拷贝数变异影响HIV-1感染和疾病进展这一发现的争议，部分归因于用于拷贝数评估的方法所获得的可变结果。与CCL3一样，编码CC趋化因子CCL4的基因，也是CCR5受体的天然配体，被发现以群体特异性的多拷贝数形式存在，并已显示出对HIV-1具有保护作用。本研究评估了用于CCL4L基因拷贝数测定的定量实时PCR（qPCR）和液滴数字PCR（ddPCR）的标准方法。CCL4编码基因包括CCL4，每个二倍体基因组（pdg）中有两个拷贝，以及非等位基因CCL4L基因，由CCL4L1和CCL4L2组成，它们在pdg中均以多拷贝形式存在。使用qPCR和ddPCR在来自南非黑人（n = 23）和白种人（n = 32）人群组的一组未感染HIV-1的个体中测定CCL4L、CCL4L1和CCL4L2的拷贝数。与qPCR（r = 0.87，p < 0.0001）相比，观察到ddPCR产生的CCL4L拷贝数与CCL4L1和CCL4L2拷贝数之和之间具有更强的相关性（r = 0.99，p < 0.0001）。实时qPCR在较高拷贝数时表现出更大的不准确性，这与我们的黑人个体队列特别相关，与白种人（0 - 4）相比，黑人个体的CCL4L拷贝数范围更高（3 - 6），且群体中位数更高（分别为4和2）。黑人人群中CCL4L1（黑人：2，0 - 4，白种人：0，0 - 2）和CCL4L2（黑人：2，1 - 5，白种人：2，0 - 3）的中位数和范围也更高。对于评估CCL4基因拷贝数变异，液滴数字PCR被证明是一种远比qPCR优越的方法，其准确性对于研究可变基因拷贝数对宿主感染表型结果和疾病进程的贡献至关重要。
