Impact of genetic variations and transcriptional alterations of HLA class I genes on cervical cancer pathogenesis Damayanti Das Ghosh 1 , Indranil Mukhopadhyay 1 , Amrapali Bhattacharya 2 , Rahul Roy Chowdhury 3 , Nidhu Ranjan Mandal 3 , Sudipta Roy 4 and Sharmila Sengupta 2 1 Human Genetics Unit, Indian Statistical Institute, Kolkata, India 2 Cancer Genomics and Epigenomics, National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, Kalyani, West Bengal, India 3 Department of Gynecology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India 4 Department of Pathology, Sri Aurobindo Seva Kendra, Kolkata, West Bengal, India 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. Cervical carcinoma (CaCx) is the second common cancer among Indian women with annual global incidence and mortality of 122,844 and 67,477, respectively (Globocan 2012. http://www-dep.iarc.fr/). Persistent high-risk human papillo- mavirus (HPV)-infection is the primary etiological factor for CaCx. HPV infection is spontaneously cleared by majority of infected individuals. Innate and adaptive immune responses block viral infection and eliminate infected cells. However, escape from exposure to the host immune-system is an important mechanism in viral persistence. 1 The class I Human Leukocyte Antigen (HLA), in the Major Histocompatibility Complex (MHC) of the short arm of chromosome 6 (6p21.3), is polygenic including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G. HLA class I molecules, expressed on the surface of nucleated cells, are glycoproteins of 45 kDa having three extracellular domains (a 1 , a 2 and a 3 ), a transmembrane domain and a cytoplasmic anchor-segment. The peptide-binding cleft, formed by the a 1 and a 2 domains, presents endogenous antigens of altered self-cells triggering cytotoxic T-lymphocyte (CTL) response. Different genetic variations at the peptide-binding cleft might influence antigen-presentation altering CaCx-risk. HLA-A, HLA-B and HLA-C, exhibit multiple codominant alleles formed by combinations of sequence-motifs generated by mutation, gene-conversion and recombination. Adding to the Key words: cervical cancer, HPV16, HLA class I alleles, SNP, differential expression Abbreviations: HPV: human papillomavirus; HLA: human leuko- cyte antigen; MHC: major histocompatibility complex; SNP: single nuteotide polymorphism; CaCx: cervical cancer; SBT: sequence based typing; NK cell: natural killer cell; PCR: polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; FDR: false discover rate; SSO: sequence specific oligo; GWAS: genome wide association study; IHWG: international histocompat- ibility working group; LD: linkage disequilibrium Additional Supporting Information may be found in the online version of this article. Author’s contribution: DDG and SS conceived and designed the experiments. DDG and AB performed experiments. DDG and IM analyzed the data. RRC, SR, NRM and IM contributed reagents/ materials/analysis tools. DDG and SS wrote the article. Grant sponsor: Department of Biotechnology, Government of India; Grant number: BT/PR8014/Med/14/1220/2006; Grant sponsor: Indian Statistical Institute, Kolkata (Intramural Grant). DOI: 10.1002/ijc.30681 History: Received 28 Aug 2016; Accepted 23 Feb 2017; Online 7 Mar 2017 Correspondence to: Sharmila Sengupta, National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S, Kalyani 741251, West Bengal, India, E-mail: ssg1@nibmg.ac. in, sharmilasg@gmail.com, sharmilasg2003@yahoo.com Cancer Genetics and Epigenetics Int. J. Cancer: 140, 2498–2508 (2017) V C 2017 UICC International Journal of Cancer IJC