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. 2012;7(11):e50204.
doi: 10.1371/journal.pone.0050204. Epub 2012 Nov 30.

Characterization of LEDGF/p75 Genetic Variants and Association With HIV-1 Disease Progression

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Free PMC article

Characterization of LEDGF/p75 Genetic Variants and Association With HIV-1 Disease Progression

Peter Messiaen et al. PLoS One. .
Free PMC article

Abstract

Background: As Lens epithelium-derived growth factor (LEDGF/p75) is an important co-factor involved in HIV-1 integration, the LEDGF/p75-IN interaction is a promising target for the new class of allosteric HIV integrase inhibitors (LEDGINs). Few data are available on the genetic variability of LEDGF/p75 and the influence on HIV disease in vivo. This study evaluated the relation between LEDGF/p75 genetic variation, mRNA expression and HIV-1 disease progression in order to guide future clinical use of LEDGINs.

Methods: Samples were derived from a therapy-naïve cohort at Ghent University Hospital and a Spanish long-term-non-progressor cohort. High-resolution melting curve analysis and Sanger sequencing were used to identify all single nucleotide polymorphisms (SNPs) in the coding region, flanking intronic regions and full 3'UTR of LEDGF/p75. In addition, two intronic tagSNPs were screened based on previous indication of influencing HIV disease. LEDGF/p75 mRNA was quantified in patient peripheral blood mononuclear cells (PBMC) using RT-qPCR.

Results: 325 samples were investigated from patients of Caucasian (n = 291) and African (n = 34) origin, including Elite (n = 49) and Viremic controllers (n = 62). 21 SNPs were identified, comprising five in the coding region and 16 in the non-coding regions and 3'UTR. The variants in the coding region were infrequent and had no major impact on protein structure according to SIFT and PolyPhen score. One intronic SNP (rs2737828) was significantly under-represented in Caucasian patients (P<0.0001) compared to healthy controls (HapMap). Two SNPs showed a non-significant trend towards association with slower disease progression but not with LEDGF/p75 expression. The observed variation in LEDGF/p75 expression was not correlated with disease progression.

Conclusions: LEDGF/p75 is a highly conserved protein. Two non-coding polymorphisms were identified indicating a correlation with disease outcome, but further research is needed to clarify phenotypic impact. The conserved coding region and the observed variation in LEDGF/p75 expression are important characteristics for clinical use of LEDGINs.

Conflict of interest statement

Competing Interests: All authors have declared that no competing interests exist. Prof Dr Bruno Verhasselt serves as a PLOS ONE Academic editor. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. LEDGF/p75 protein domains, gene structure and primate alignment.
LEDGF/p75 functional protein domains (green) with the integrase binding domain (IBD, indicated in red) and the PWWP domain, which regulates chromatin association. LEDGF/p75 spliced RNA with different exons (light and dark purple) is linked to the protein domains. The third line represents the PSIP1 gene region with the coding regions (purple) and non coding regions (introns are grey, 5′UTR and 3′UTR are respectively orange and yellow). The location of the discussed SNPs in this work is indicated (red arrows: SNPs detected after screening; blue arrows: 2 tagSNPs in African cohort). The second panel shows the LEDGF/p75 protein alignment of humans and four primate species based on the reference sequences. The integrase binding domain (red box) is indicated, showing no variation in the four primates. PWWP = proline-tryptophan-tryptophan-proline domain; NLS = nuclear localization signal; AT = adenine-thymine rich DNA binding region; IBD = integrase binding domain; UTR = untranslated region.
Figure 2
Figure 2. Phenotypic impact of observed genetic variants in the PSIP1 coding region.
Box-plots showing the association of individual observed SNPs in the coding region with CD4 decline (top), average log viral load (middle) and LEDGF/p75 mRNA expression (bottom).The data are combined for Africans and Caucasians. SNPs not in accordance with Hardy-Weinberg law (rs35678110) were excluded. In case of insufficient data to create a boxplot (limited amount of data points) a bar representing the mean of the values is shown.
Figure 3
Figure 3. Phenotypic impact of three genetic variants in the PSIP1 non-coding region.
(A) Differential CD4 decline for patients carrying wild-type or variant alleles of rs2737828 and rs2737835 (Caucasian) and rs16933270 (African), based on the mean CD4 slope and a similar starting point. P values to estimate significance of difference are indicated. (B) Differential expression of LEDGF/p75 mRNA and HRP2 mRNA in PBMCs from patients carrying wild-type or variant alleles of rs2737828 and rs2737835 (Caucasian) and rs16933270 (African). Horizontal bars indicate the mean expression levels.
Figure 4
Figure 4. Biological variability and correlation of LEDGF/p75 mRNA with HRP2 mRNA expression.
(A) Scatter-plot showing LEDGF/p75 mRNA and HRP2 expression in identical patient samples (n = 68). Pearson r-values and p-values are indicated. (B) Scatter-plot showing the biological variation of LEDGF/p75 mRNA expression in two samples from 24 patients at different time points. t1 = time point 1; t2 = time point 2.

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Grant support

This work was partly supported by the Flemish Agency for Innovation by Science and Technology (CellCoVir - IWT file nr 60813). Linos Vandekerckhove is supported by the National Fund for Scientific Research – Belgium as Principal Investigator. The Spanish RIS cohort and HIV BioBank are integrated in the Spanish AIDS Research Network supported by Instituto de Salud Carlos III (Grant RD06/0006/0035) and Fundación para la Investigación y Prevención del SIDA en España (FIPSE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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