Comprehensive approach for identification of functional FCGR2C alleles resulting in protein expression as a determinant for predicting predisposition to autoimmunity

Present addresses Dorit Lehmann is nowanemployeeofEisai GmbH,Germany,VerenaBergandBirgitM. Reipert of IMCUniversity ofAppliedSciences Krems,Austria,KarinHockofDaiichi Sankyo EuropeGmbH,Austria, PadmapriyaPonnuswamyofCSLBehring,Germany,Mantas MalisauskasofMerckHealthcareKGaA, and BrianA.Crowe is currently an independent consultant in Leobendorf, Austria. Abstract The balance of activating and inhibitory signals from the low affinity Fc gamma receptors modulates immune responses triggered by IgG antibody-immune complexes. In homeostasis, this leads to antigen clearance, while in autoimmune diseases to unwanted immune response. Besides the activating receptors FcɣRIIa, FcɣRIIIa, and the inhibitory FcɣRIIb receptor, a third activating receptor, FcɣRIIc, was shown to be expressed on several immune cell types, however, only in the presence of a functional FCGR2C-ORF allele. FcɣRIIc expression is associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus or systemic sclerosis. Thus, the determination of the functional FCGR2C gene resulting in protein expression on immune cells becomes highly relevant, particularly in the context of unwanted immune responses through inadvertent FcɣRIIc activation by molecules targeting stimulation of the inhibitory receptor FcɣRIIb, currently pursued by several pharmaceutical companies. The high degree of homology within the FCGR2/3 gene cluster complicates development of an accurate method for identification of FcɣRIIc expression. Here we describe a comprehensive approach to characterize genetic status of the FCGR2C gene locus consisting of cDNA sequencing, SNaPshot genotyping and low-coverage next-generation sequencing. This might enable Mendelian randomization hypothesis testing across autoimmune diseases to personalize therapies and enhance treatment outcomes.


INTRODUCTION
Human cells of the hematopoietic lineage express a spectrum of Fc gamma receptors (FcɣRs), which bind the Fc region of immunoglobulin G (IgG) and lead to immune response initiated by the antibody- receptor signaling and localization in lipid rafts. 9 Except for FCGR-2B1 and −2B3 splice variants, exon 6, which encodes cytoplasmic domain 1, is spliced in all FCGR2 mRNAs, leading to endocytosiscompetent receptors. 10 Pivotal for FcɣRIIc, the SNP rs759550223 NR_047648.1:n.268T > C in exon 3 of FCGR2C results in a change from glutamine to a STOP codon and therefore loss of protein expression. 11 Thus, the expression of FcɣRIIc critically depends on the presence of an FCGR2C-ORF allele. 11 Previous studies have estimated FCGR2C expression prevalence between 15% -45%, 12

SNaPShot SNP genotyping
Genomic DNA samples were amplified using Accu Prime Taq

Flow cytometry
Flow cytometry analysis was performed using 100 µL of whole blood.

Low coverage whole-genome sequencing (WGS)
Quantity and quality of genomic DNA were assessed by Qubit™ 2.0

Genetic association analysis
The association between genotype and disease status was analyzed using R version 4.04. The SLE population was compared with both the healthy controls from the current study, as well as healthy control data from the study performed by Nagelkerke and colleagues. 17 Data from this study was selected because a relatively large number of healthy controls were available with high quality FCGR2C genotype results.
As SLE predominately affects females, the analysis using healthy controls from the current study was limited to female population. Since the Nagelkerke and colleagues did not report the data by gender, the comparison with this data included both males and females. For each comparison, a simple logistic regression model was fitted, relating the FCGR2C-ORF genotype to the disease status. The p-value for association was computed from the likelihood ratio test, and a 95% confidence interval for the odds ratio was computed from the profile likelihood method.

Molecular approach for the determination of the genetic status of the FCGR2C gene locus
The FCGR2C gene originated from a recent evolutionary unequal cross-over event between the 5′ part of FCGR2B and the 3′ part of the FCGR2A gene, hence, the very high homology between the 3 genes. 6,18,19 In particular, there is only one amino acid difference (encoded in exon 5) compared to FCGR2B and only one amino acid difference (encoded in exon 8) compared to FCGR2A. 10,20 Besides the 99% homology between FCGR2C and respective parts of FCGR2A and ing inconsistent SNP and protein expression findings, the method using standard, short-read, low-coverage, next-generation sequencing was established to detect CNVs, as such cases were reported previously. 4 The overall workflow for determination of the genetic status of the FCGR2C gene is outlined in Figure 1B

FCGR2C genotyping by sequencing of FCGR2C mRNA-specific PCR products
To determine the status of the rs759550223 SNP in FCGR2C gene, NR_047648.1:n.268T > C, exon 3, sequencing of FCGR2C-specific PCR products was performed ( Figure 2). To this end, a PCR was designed to

FCGR2C genotyping by SNaPshot on FCGR2C-gene specific PCR products
The cDNA sequencing approach described above identifies functional

Expression of Fcpress protein in donors carrying FCGR2C ORF allele
The genotyping methods described above identified individuals with a functional FCGR2C gene, thereby revealing the potential to express      (Table 1, Supplementary table 1). Despite relatively small number of donors analyzed so far, the fact that the FCGR2C-ORF allele was detected in 23% of all donors evaluated would suggest that the FcγRIIc receptor expression is relatively frequent (Table 1). Since SLE affects predominantly women, 24 (Table 2). Thus, the results of the initial analysis of the accrued samples suggest that the presence of a func-tional FCGR2C gene may correlate with SLE in the female population.

Distribution of FCGR2C-ORF alleles in donors of different ethnicities and health status
An additional analysis, comparing the Caucasian SLE patients from the current study (both male and female) with the sample of 919 healthy Caucasian patients performed by Nagelkerke and colleagues, 17 gave an odds ratio of 2.57 with a 95% confidence interval of (1.04, 5.60) and a pvalue of 0.04. This effect is similar to the effect estimated from our data alone, but with increased precision resulting from the larger sample size, thus reaching statistical significance. This suggests that if a larger sample size had been included in the present study, the conclusion regarding the correlation of the FcγRIIc prevalence and susceptibility to autoimmunity would have been further strengthened statistically.

DISCUSSION
The towards FCGR2B in this region. 26,27 which in turn suggested that the differences between FCGR2B and FCGR2C described in publicly available genomic databases apply exclusively to FCGR2C-STOP alleles. 27 These further SNPs also raise the question of possible additional differences within intronic regions originating from the evolutionary diver- Amplification (MLPA) has become a method of choice to analyze CNV, even within complex genomic regions such as the FCGR gene cluster, due to its simplicity in design and the availability of developed computational analysis solutions. However, MLPA comes with its own shortcomings such as its inability to provide information regarding the exact location of a duplicated sequence or its orientation, its lack of sensitivity for regions not directly encompassed by the probe sets used, and the possible occurrence of false-positive results attributable to polymorphism-induced allele dropouts. 30 Here we describe an alternative method to MLPA that does not rely on the generation of appropriate primers, avoids potential amplification bias, and precisely maps the CNV boundaries. 31

ACKNOWLEDGEMENTS
Sincere thanks are given to all blood donors enabling this study, Dr.
Irina Korschinek from Ingenetix GmbH for a productive collaboration on the development of SNaPshot genotyping within the complex

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.