While genetic association studies so far have focused
While genetic association studies so far have focused on immune-modulatory genes [, , , , ], the Eph Receptor A2 protein (EPHA2) tyrosine kinase receptor is a promising candidate for investigation of KSHV-induced KS as it potentially acts on two levels, namely susceptibility to KSHV infection, and susceptibility to KS development.
EPHA2 has recently been identified as a host receptor utilised by KSHV for entry into and trafficking within endothelial cells [22,23]. Additionally, EPHA2 has been implicated in oncogenesis: EPHA2 is upregulated on the mRNA and protein level in a wide variety of cancer cell lines and tissues, and EPHA2 signalling has been implicated in cell transformation, tumour maintenance and progression, angiogenesis and metastasis [22,, , , , ]. Although vital in the uptake mechanism of KSHV and significantly involved in oncogenesis, little is known about the pathological consequences of sequence variants in the EPHA2 gene on KSHV infection and/or KS development. To date, polymorphisms within EPHA2 have only been identified in association with cataract pathogenesis [, , , , , ]. Therefore, this study aimed to investigate potential sequence variants in the protein coding region of the EPHA2 gene in South African HIV-infected patients. We hypothesised that if such variants exist and translate into amino Cefepime changes, particularly in known functional domains, they may predispose affected individuals to KSHV infection and/or KS oncogenesis. Here we report that aggregate variation (having ≥1 rare variant with Minor Allele Frequency (MAF) < 5%) from the reference sequence (NM_004431) across the entire EPHA2 coding region, particularly driven by variation within the functionally important and conserved protein tyrosine kinase (Pkinase-Tyr) and sterile-α-motif (SAM) domains, is associated with susceptibility to KS. Moreover, we found that three novel, non-synonymous variants in the Pkinase-Tyr and SAM domains are associated with KSHV infection (c.2727C > T) or KS development (c.2254 T > C and c.2990 G > T).
Materials and methods
Results Clinical and demographic information concerning the abovementioned participant groups (total sample size n = 150, with n = 50 per group) is summarised in Table 1. All patients were HIV-infected as determined serologically. Age did not differ significantly between the three groups: median age was 36, 39 and 40 for groups 1, 2 and 3, respectively. Although the final cohort of patients had a slight overrepresentation of males (55.3%) compared to females (44.7%), the sex ratio in the three groups was similar. Population group distribution was heavily skewed towards black Africans (93.3%) and included only a minority of mixed ancestry (5.3%) and Caucasian (1.3%) individuals which was consistent across the three patient groups. Most recent CD4 counts were recorded at the time of patient recruitment. CD4 counts did not statistically differ between patient groups, although they were lowest in group 1 (KS+/KSHV+) and highest in group 3 patients (KS−/KSHV−). All KS+ patients (group 1) received ART with an average time of 298 ± 483 days before KS diagnosis, whereas a significantly smaller number of KS− patients (groups 2 and 3) were on ART medication (p < 0.0001) at the time of recruitment. Since none of the demographic parameters presented in Table 1 significantly differed, the three patient groups were considered suitable for further candidate gene association analysis. Fifty plasma samples from patients with clinically diagnosed KS were assessed by K8.1 and ORF73 ELISAs and all (95% CI 92.9–100%) were found to be KSHV seropositive as expected (Table 2A), while 31.6% (95% CI 28.3–35.1%) of the total patient cohort without KS (706 patients) were found to be KSHV seropositive (Table 2B). KSHV status was tested only once per patient with the timing relative to diagnosis of HIV infection being highly variable.