We re-sequenced HPV16 genome (~6 kb) implicated in cervical carcinogenesis (LCR, E2, E5, E6, E7, L1, L2) to prioritize sequence variants for functional validation as biomarkers, using CaCx cases (n=74) and asymptomatic controls (n=24). Of the nucleotide variations recorded (n=271), non-synonymous changes in L2 region were significantly higher (p=0.005) among cases (2.67%) compared to controls (1.27%). Using SIFT database, 29 non-synonymous changes (frequency=0.01-0.03) predicted as deleterious to protein functions were identified. Haplotype analysis considering 110 polymorphic variations (frequency> or =0.05) within intact viral isolates (53 CaCx cases and 21 controls) using NETWORK software, confirmed Asian-American (AA, 14.86%) and European (E, 85.14%) variants, differing at 78 positions. The E-variants portrayed thirty-six haplotypes, of which, E-12 was most prevalent within cases (38.1%; 16/42) and controls (28.57%; 6/21) harboring polymorphic variations at 10 positions, in contrast to HPV16R. Cases of the E-12 haplotype harbored 7 deleterious mutations distributed within L1 (n=1), E2 (n=1), E5 (n=1), and L2 (n=4), while none within similar controls. Thus rare deleterious variations within genes implicated in productive infection over the E-12 haplotype background of intact HPV16 isolates might be of causal relevance for CaCx development.
In a novel attempt to understand the variations in DNA sequences underlying HLA class I alleles associated with HPV16-related CaCx, we determined the alleles by reconstructing SNP-based haplotypes from resequencing of the most polymorphic exons 2 and 3 of HLA-A, HLA-B and HLA-C. We also determined the impact of SNPs and transcriptional alterations of the genes on CaCx. A high density of SNPs was identified from resequencing. HLA expression was determined by real-time PCR. We identified that even a single associated HLA allele had many underlying SNP-based haplotypes. Out of the most frequent (≥5%) HLA class I alleles, HLA-B*40:06 and HLA-B*15:02 respectively imparted significant risk towards and protection from CaCx as well as HPV16 infection. Employing median-joining networks to detect clusters of sequence-variations for specific HLA alleles, we found the protective SNP-based signature, GAATTTA, in all SNP-based haplotypes of HLA-B*15:02 allele. The signature was derived from seven SNPs within HLA-B which were newly associated with the disease. Contrarily, similarly derived risk-signature, TTGCGCC, mapped only to 52% of SNP-based haplotypes of HLA-B*40:06 allele. This indicated that all SNP-based haplotypes underlying a particular associated HLA allele might or might not have a single signature of risk/protection. HLA-A, HLA-B and HLA-C expressions were downregulated among CaCx cases compared to asymptomatic infections and HPV-negative controls. HLA-A and HLA-B were repressed in both cases harbouring episomal and integrated HPV16, whereas HLA-C in only the latter. Novel genetic variations and differential downregulation-patterns of HLA class I have a significant bearing on HPV16-related CaCx pathogenesis.
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