Developing long read sequencing and its application and understanding the role of ERAP1 in cervical carcinoma.

Abstract

Long read sequencing of components of the antigen processing and presentation (APP) pathway, specifically endoplasmic reticulum aminopeptidase 1 (ERAP1), could provide a new tool for identifying those women that are at high risk of poor cervical cancer prognosis. ERAP1 edits the peptide repertoire presented to cytotoxic T lymphocytes (CTLs) through N-terminal trimming of peptide precursors to the optimal length for stable MHC I binding prior to presentation. Single nucleotide polymorphisms (SNPs) in ERAP1 exist in multiple combinations that form distinct haplotypes that when expressed as allotypes, alter ERAP1 trimming function. Individual ERAP1 SNPs have been associated with increased cervical cancer risk in GWAS studies, however a cause and effect relationship between ERAP1 allotypes and cervical cancer progression has not been established. In this study, the identification of ERAP1 allotypes from HeLa and 293T cells using long read sequencing by MinION enabled the establishment of a methodological pipeline, including optimisation of each step of the protocol, identification of the limitations of this technology and the development of a robust bioinformatics analysis pipeline. Subsequently, ERAP1 allotypes from a total of 81 patients at varying stages of cervical carcinoma were identified using long read sequencing. In this cohort, a total of 14 ERAP1 allotypes were identified and those were found in 28 distinct ERAP1 allotype combinations affecting the enzyme’s trimming function given that both chromosomal copies are codominantly expressed. Functional analysis showed that the majority of patients with a high CD8+/TIL number had ERAP1 allotypes with efficient trimming ability and better overall prognosis. ERAP1 allotype identification and CD8+/TILs could be a useful tool for identifying those women at high risk of poor prognosis so that women can receive early treatment.

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Identifiers

ORCID for Edward James: orcid.org/0000-0001-8638-7928

ORCID for Tim Elliott: orcid.org/0000-0003-1097-0222

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