ABSTRACT: Objective: to compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofavir (TDF) or zidovidine (AZT). There were significant divergence between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processin, with some convergence at 18 months. Compared to HIV negative controls the ABC group, but not AZT or TDF, showed enrichment of genes controlling adherence junction at six months and 184 months (adjusted p <0.05), and cell matrix adhesion (adjusted p<3.4E-0.5) at 6 months. Tight junction (p=0.03), gap junction (p<0.05) and leukocyte transendothelial migration (p=0.03) were over-expressed in ABC compared to TDF at 6 m. Enrichment pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group compared with the other treatments, There was little difference between AZT and TDF. Conclusion: After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are taking place in other tissues including the coronary vasculature, they may contribute to the observed increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens. 31 HIV treatment naïve patients were randomised to receive either zidovidine (AZT)/lamivudine or tenofavir (TDF)/emtricitabine in fixed dose preparations. Both groups also received efavirenz. Patients were tested before treatment (AZT n=15, TDF n=16) and at 6 (AZT n=9, TDF n=12) and 18-24 (AZT n= 8, TDF n=9) months after starting therapy. Tests included extensive biochemical tests, tests of body fat distribution and subcutaneous fat biopsies. 15 HIV negative patients were similarly tested. Patients and controls were matched for age, ethnicity, gender, weight, BMI, pretreatment CD4 count, viral load, and family history of cardiovascular disease and diabetes. In a second part of the study patients on their initial antiretroviral regimen containihg abacavir (ABC)/lamivudine plus efavirenz for 6 months (n=9) and 184 months (n=10) were similarly tested. the ABC-treated group were matched in all the listed characteristice with the other two groups. None of the patients had ever experienced an AIDS-defining disease or developed lipodystrophy over the study period. Fat biopsies were snap frozen until assayed. Microarray methods: RNA was extracted for microarray and array data generated using a Qiagen RNeasy Lipid Tissue Mini Kit. Total RNA was converted into labeled cDNA using the WT-Ovation Pico RNA Amplification System and samples were hybridized to an Agilent Whole Human Genome Microarray 4x44K (AMADID 014850, Agilent Technologies). The Agilent arrays were scanned with the Agilent Technologies DNA Microarray Scanner G2505C with default settings. Scanned images were extracted and initial quality control performed in Agilent Feature Extraction (AFE) software version 10.7.3.1. Additional quality assessment was performed using the R arrayQualityMetrics package. As no major experimental problems could be detected, all microarrays were included in subsequent analysis steps. The dataset was read into R and processed using the Agi4x44PreProcess package with options to use the AFE Processed Signal as foreground signal and BG Used signal as background adjusted signal. The data were then normalized by the quantile method using the limma function normalize Between Arrays. Probeset filtering was performed using the Agi4x44PreProcess filter method using AFE provided flags to identify features with quantification errors of the signal, reducing the 45,015 features on the Agilent Whole Human Genome Microarray 4x44K array by 38%, or 17,264 features. Residual technical batch effects were corrected using the ComBat method implemented in the SVA R package.Since patients were not randomised to the ABC treatment all samples were treated as unrelated. Gene expression differences between treatment arms were assessed using the linear modeling features implemented in the limma package in R. Multiple testing across contrasts was performed using the limma global method with Benjamini-Hochberg approach for control of false discovery rate (FDR). Gene set enrichment analysis of GO terms and KEGG pathways was performed using the conditional hypergeometric test implemented in the GOstats R package. Statistical significance of gene set enrichment was performed using the Benjamini-Hochberg correction method implemented in the EMA R package .
