AZD8931 Sequencing of the EPHA coding
Sequencing of the EPHA2 coding region of the randomly selected final cohort of 150 patients (falling into the three categories based on KS diagnosis and KSHV serology status as outlined above) resulted in a total of 57 unique variants across the 3964 base pair EPHA2 coding region, 22 of which were predicted to result in amino AZD8931 (AA) changes. While the majority (35) of these variants are recorded in the NCBI database of single nucleotide polymorphisms (dbSNP), 22 novel variants were identified in our cohort. The total variation across the coding region of EPHA2 is represented schematically in Fig. 1. Exons 3, 5, 11 and 17 contain the highest number of variants uniformly found across all patients compared to the reference sequence. Taking the size of each exon into account, exon 11 can be identified as harbouring the highest average rate of variation per bp (0.0062 variants/bp), followed by exons 17 (0.0033 variants/bp), 12 (0.0030 variants/bp) and 5 (0.0023 variants/bp). Moreover, KS+ patients (group 1) appeared to have an increased number of variations in the Pkinase-Tyr domain (exons 12–15) when compared to KS− patients (groups 2 and 3) (Fig. 1). Aggregate variation across EPHA2 was associated with increased risk of KS. Specifically, 37 (74%) KS cases (group 1) had one or more SNV versus only 15 (30%) of KS−/KSHV+ controls (group 2) (OR = 6.6 (95% CI: 2.8, 15.9), p = 2.2 × 10-5, Supplementary Table S1). Missense variants, rather than synonymous variants or variants in the UTR, appeared to be the primary drivers of this association (OR = 4.9 (95% CI: 2.0, 11.7), p = 0.0004). When aggregate variation was considered within each of the functional domains of EPHA2, it was observed that having one or more SNV in the Pkinase-Tyr domain or SAM domain was associated with increased risk of KS (OR = 4.9 (95% CI: 1.9, 12.4), p = 0.001 and OR = 13.8 (95% CI: 1.7, 111.6), p = 0.014, respectively). After removing SNVs based on LD and with MAF < 3% (Table 3), 8 individual variants were assessed statistically for association with KS development by comparing occurrence in KS+/KSHV+ (group 1) patients versus KS−/KSHV+ (group 2) patients. The variant at mRNA positions 2254 is predicted to result in a novel, non-conservative AA change from Leucine to Proline at AA position 700 and is in LD with another SNV at mRNA position 2257 predicted to result in an AA substitution from Aspartate to Alanine at the next AA position, 701, in the conserved Pkinase-Tyr domain. These heterozygous variants were found to co-occur in 8 (16%) KS+ patients (group 1) (with an additional group 1 patient showing only the variant at mRNA position 2257) and not to occur at all (0%) in KS− patients in group 2 (OR undefined, adj. p = 0.04, Table 3). Additionally, several other variants occurring in the Pkinase-Tyr domain (spanning mRNA positions 1990–2766), were found to be overrepresented in the KS+ (group 1) patients (Table 3). Within the SAM domain, a heterozygous G > T variant at mRNA position 2990, predicted to result in a substitution of Asparagine for Lysine at AA position 945, was found to be significantly more frequent among KS+/KSHV+ (group 1) patients (18%) compared to KS−/KSHV+ (group 2) patients (0%) (OR undefined, adj. p = 0.02, Table 3). Both the variant at mRNA position 2254 and at position 2990 were found to be ‘probably damaging’ when assessed for functional impact using the PolyPhen-2 prediction tool. Aggregate tests did not find statistically significant associations between EPHA2 SNVs and KSHV status. However, there was a trend indicating that having one or more missense SNV was associated with increased risk of KSHV infection. Specifically, 40 (40%) KSHV cases had one or more missense SNV compared to only 12 (24%) KSHV negative controls (OR = 2.1 (95% CI: 0.98, 4.5), p = 0.06, Supplementary Table S2). To test individual variants for association with KSHV infection, we compared the occurrence of 4 SNVs in KSHV+ groups to KSHV− patients which we identified after removing SNVs based on LD and with MAF < 3% (Table 4). We discovered a novel heterozygous C > T variant in the conserved Pkinase-Tyr domain at mRNA position 2727, which was found to be statistically significant (OR = 6.4 (95% CI: 1.4, 28.4), adj. p = 0.03), occurring in 21 (21%) KSHV+ patients (14 in group 1, and 7 in group 2) and in only 2 (4%) KSHV− (group 3) patients (Table 4). This variant is predicted to result in a non-conservative substitution of a Cysteine for Arginine at AA position 858 which is predicted to be “probably damaging” using the PolyPhen-2 prediction tool. The SNV at mRNA position 2727 was also found to be overrepresented in KS+/KSHV+ patients (14 patients (28%) in group 1 compared to KS−/KSHV+ patients (7 patients (14%) in group 2) but this was not statistically significant (Table 3). The presence of this variant in KS−/KSHV+ (group 2) patients, while to a lesser extent than in KS+/KSHV+ (group 1) patients, indicated that the occurrence of this variant in KS+/KSHV+ (group 1) patients may be a consequence of increased susceptibility to KSHV infection and thereby an indirect association with KS development.