Shining a light on the FCGR Locus: Deconvoluting the challenging genomic organisation and consequences in immune oncology

Abstract

The majority of therapeutic monoclonal antibodies (mAbs) educe their functional response by interfacing with a family of cell-surface proteins called the Fc gamma receptors (FcγRs), comprising a single high-affinity and five low-affinity receptors. The low-affinity receptors are encoded by the FCGR2A, FCGR2B, FCGR2C, FCGR3A and FCGR3B genes, positioned within a highly homologous 200kb locus on chromosome 1q23-24 that is subject to numerous single nucleotide polymorphisms (SNPs) and regions of copy number variation (CNRs). Resulting from multiple recombination and segmental duplication events, the high sequence homology represents a unique challenge for the genetic analyses of this cluster as the traditional short-read technologies often fail to produce reads that can be adequately aligned or assembled. However, the Oxford Nanopore Technologies (ONT) sequencing platform introduces the exciting opportunity to accurately resolve this locus and generate highly-detailed genomic maps of the region under different copy number states.

Previously generated nanopore sequencing data of long-range (LR)-PCR products from the low- affinity FCGR genes (n=4) were analysed and led to the development of a bioinformatic pipeline to remove spurious alignments and identify single nucleotide variation. This analysis revealed that while nanopore sequenced LR-PCR products can be utilised to analyse the low-affinity FCGR genes to some extent, they are restricted by their gene-centric nature and generating reliable sequencing reads from the FCGR2C gene proved to be difficult.

To address this challenge, ONT long-read sequencing and Bionano optical mapping were performed on peripheral blood mononuclear cell (PBMC) samples from healthy donors (n=22). The project involved the optimisation of multiple amplification-independent ONT approaches; i) whole genome sequencing (WGS) ii) cas9-mediated targeting and iii) computational enrichment with adaptive sampling. The cas9 and adaptive sampling techniques resulted in a 450- and 10-fold enrichment of the FCGR locus respectively. The newly generated nanopore data was then used to provide extensive phasing, CNR breakpoint and variant characterisation. A total of 16,326 high-quality small variants, corresponding to 2,296 unique locations, were detected. This enabled the genotyping of clinically-relevant SNPs of interest, identification of extended haplotypes and successful phasing across the 200kb region. Eight polymorphic short tandem repeat (STR) regions were found that have the potential to be of functional relevance to FcγR expression. Breakpoints for CNR1, CNR2, CNR3 and CNR4 were refined to regions of 5.4kb, 14.5kb, 7.9kb and 4.7kb respectively. A novel case of CNR4 duplication was reported and CNR4, in addition to CNR3, was shown to be more prevalent than previously suggested. In conclusion, ONT presents the possibility to overcome the inherent difficulties of characterising genomic variation in the complex FCGR locus, thereby permitting a more holistic view of the region and its regulation. Ultimately this data provides a fuller understanding of the genomic variation at the FCGR locus providing the basis for further research into the regulation of these genes, with the potential to improve risk-adapted patient stratification and offer more targeted therapeutic approaches for cancer patients.

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Identifiers

ORCID for Jon Strefford: orcid.org/0000-0002-0972-2881

ORCID for Jane Gibson: orcid.org/0000-0002-0973-8285

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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