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Introduction

Fcγ receptors (FcγRs) bind specifically to the γ chain of the Fc fragment of immunoglobulin G (IgG) and are located on the surface of immune cells, such as monocytes, macrophages and natural killer cells. FcγRs are directly involved in the function of these immune cells and regulate immune responses. There are 3 subtypes of FcγRs, i.e., FcγRI, FcγRII and FcγRIII, which are highly homologous to one another. FcγRI binds to IgG with high-affinity, whereas FcγRII and FcγRIII are low-affinity receptors for IgG (1,2). Each of these receptors has multiple isoforms. Among these isoforms, FcγRIIa and FcγRIIIa are encoded by the FCGR2A and FCGR3A genes, respectively. It is also known that there are non-synonymous single-nucleotide polymorphisms (SNPs) for these genes, including rs1801274 (A>G; H131R) for FCGR2A and rs396991 (T>G; F158V) for FCGR3A (3–7). These SNPs encode for functional receptors and the receptor encoded by each variant gene has a distinct binding affinity for the Fc fragment of an antibody. It was previously reported that the affinity of FcγRIIa with the H131 variant is higher compared to that with the R131 variant and the affinity of FcγRIIIa with the V158 variant is higher compared to that with the F158 variant (4,8,9). It was also reported that these SNPs are associated with a risk for the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) (10–12), and may also be involved in individual differences in the efficacy of antibody-based drugs, such as rituximab (13–16), trastuzumab (17,18) and cetuximab (19–21). However, there is currently no consensus on these findings and available information regarding the frequency of these SNPs in the Japanese population has not been adequately investigated (7).

In addition, it was recently demonstrated that copy number variations (CNVs) may also be involved in disease susceptibility (22–24). It is known that there are CNVs at the genetic locus (1q23) where FCGR2A and FCGR3A are located (6,25–28). It was also reported that CNVs are a risk factor for the development of autoimmune diseases, such as SLE (9,26,29,30). However, compared with SNPs for these genes, the involvement of CNVs in FCGR2A and FCGR3A and the risk of development of autoimmune diseases has not been adequately investigated. Although CNVs in FCGR3A have been reported previoulsy, there is no available information regarding the frequency of CNVs in FCGR2A in the Japanese population.

The aim of this study was to investigate the prevalence of SNPs and CNVs in FCGR2A and FCGR3A in the Japanese population, in order to aid the future diagnosis and treatment of autoimmune diseases and the prediction of antibody-based drug efficacy.

Materials and methods

Subjects

A total of 113 healthy Japanese volunteers (59 males and 54 females), aged 21–41 years and residing in the city of Sapporo and neighboring areas, were enrolled in this study. The study protocol was approved by the Institutional Review Board of the National Hospital Organization Hokkaido Cancer Center and written informed consent was obtained from all participating subjects.

Genomic DNA isolation

Genomic DNA was isolated from peripheral blood anticoagulated with K2-EDTA using a Puregene® DNA Isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The quantity and quality of the extracted genomic DNA were evaluated using a NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and electrophoresis with an ethidium bromide-agarose gel, respectively.

Genotyping of SNPs

The genotypes of rs1801274 (A>G) in the FCGR2A gene and rs396991 (T>G) in the FCGR3A gene were determined by the TaqMan® SNP Genotyping assay (Life Technologies, Carlsbad, CA, USA). The assay IDs were c_9077561_20 for rs1801274 and c_25815666_10 for rs396991. The polymerase chain reaction (PCR) mixture contained 5 μl of 2X TaqMan Fast Universal PCR Master mix (Life Technologies) for rs1801274 and 5 μl of 2X TaqMan Universal PCR Master mix (Life Technologies) for rs396991. Both reactions used ~100 ng of genomic DNA and 0.25 μl of 40X TaqMan SNP Genotyping assay, with the final volume adjusted to 10 μl using sterile purified water. The cycling conditions were as follows: rs1801274 was pre-warmed at 60°C for 1 min, followed by AmpliTaq Gold activation (hot start) at 95°C for 20 sec, denaturation at 95°C for 3 sec, annealing/extension at 60°C for 30 sec, repeated for 40 cycles, followed by a post-read at 60°C for 1 min; rs396991 was pre-warmed at 60°C for 1 min, followed by uracil-DNA glycosylase activation at 50°C for 2 min, AmpliTaq Gold activation (hot start) at 95°C for 10 min, denaturation at 95°C for 15 sec, annealing/extension at 60°C for 1 min, repeated for 40 cycles and followed by a post-read at 60°C for 1 min.

Following amplification of the DNA templates using the ABI PRISM® 7500 Fast Real-Time PCR system (Life Technologies), allele determinations were performed with endpoint fluorescence intensity measurements, using the 7500 System software (Life Technologies).

Determination of CNVs

For the determination of CNVs at the FCGR2A and FCGR3A loci, the TaqMan Copy Number assay (Life Technologies) was used. The assay IDs were Hs00103511_cn for FCGR2A (exon 1-intron 1) and Hs00139300_cn for FCGR3A (exon 2). In addition, TaqMan Copy Number Reference assay RNase P (Life Technologies) was used. The PCR amplifications were performed in a reaction volume of 20 μl, containing 10 μl of 2X TaqMan Universal PCR Master mix, 1.0 μl of 20X TaqMan Copy Number assay, 1.0 μl of 20X TaqMan Copy Number Reference assay and ~20 ng of template DNA. The cycling conditions were as follows: enzyme activation (hot start) at 95°C for 20 sec, followed by denaturation at 95°C for 15 sec, annealing/extension at 60°C for 1 min and repeated for 40 cycles. The copy numbers were determined using CopyCaller™ software (Life Technologies) following amplification of the DNA templates using the ABI PRISM® 7500 Fast Real-Time PCR system and measuring fluorescence intensity at the endpoint. To obtain an accurate copy number, 4 different DNA templates were used for each sample. At least 7 samples were simultaneously analyzed to determine the copy number using CopyCaller™ software (Invitrogen Life Technologies, Carlsbad, CA, USA).

Statistical analysis

The Hardy-Weinberg equilibrium (HWE) test for assessing the rs1801274 and rs396991 genotype frequency among the subjects was conducted using PowerMarker software, version 3.0 (31) and a HWE calculator including analysis for ascertainment bias software (32). The allele and CNV frequencies were compared with previously published data from healthy individuals using the Fisher’s exact test with SPSS Statistics software, version 21 (IBM Japan, Ltd., Tokyo, Japan). A two-tailed P<0.05 was considered to indicate a statistically significant difference.

Results

Genotype determinations and comparisons of allele frequencies with published data on healthy individuals

The genotype distributions for rs1801274 of FCGR2A and rs396991 of FCGR3A obtained in this study are presented in Table I. Divergence from the HWE was not observed for either SNP.

Table I FCGR2A (rs1801274; A>G) and FCGR3A (rs396991; T>G) genotypes of the subjects included in this study.

Table I

FCGR2A (rs1801274; A>G) and FCGR3A (rs396991; T>G) genotypes of the subjects included in this study.

Gene	SNP	Genotype	No. of subjects (%)
FCGR2A	rs1801274	A/A	79 (69.9)
		A/G	33 (29.2)
		G/G	1 (0.9)
FCGR3A	rs396991	T/T	64 (56.7)
		T/G	44 (38.9)
		G/G	5 (4.4)

[i] SNP, single-nucleotide polymorphism.

Comparisons of the frequencies for each SNP allele with previously published data are shown in Table II for rs1801274 and in Table III for rs396991. The minor allele frequency (MAF) for rs1801274 obtained in this study (0.155) was significantly lower among Japanese individuals compared to that among other ethnic groups (Table II). The MAF for rs396991 (0.239) was marginally different from that for rs1801274 (0.155). These frequencies were not significantly different from those reported for other Japanese populations. However, the allele frequencies for rs396991 obtained in this study for Japanese subjects were significantly different from those observed in Ethiopians and Norwegians (Table III). In particular, the T allele frequency among Japanese was higher compared to that among Ethiopians and Norwegians.

Table II Ethnic differences in the allele frequency of FCGR2A rs1801274 (A>G) in healthy subjects.

Table II

Ethnic differences in the allele frequency of FCGR2A rs1801274 (A>G) in healthy subjects.

			Allele frequency
Study	Population	No.	A	G	P-valuea	Refs.
The present study	Japanese	113	0.845	0.155	-
Iwasaki et al	Japanese	403	0.800	0.200	0.128	(7)
	Japanese Brazilians	80	0.806	0.194	0.318
	Non-Japanese Brazilians	386	0.469	0.531	<0.0001
Breunis et al	Northern-European	100	0.540	0.460	<0.0001	(6)
Van Den Berg et al	Ethiopians	77	0.470	0.530	<0.0001	(5)
	Norwegians	86	0.420	0.580	<0.0001
Reilly et al	African American	50	0.440	0.560	<0.0001	(3)
	Caucasian American	47	0.540	0.460	<0.0001

a The Fisher’s exact test was used to determine the differences between the allele frequency in this study and previously published results.

Table III Ethnic differences in the allele frequency of FCGR3A rs396991 (T>G) in healthy subjects.

Table III

Ethnic differences in the allele frequency of FCGR3A rs396991 (T>G) in healthy subjects.

			Allele frequency
Study	Population	No.	T	G	P-valuea	Refs.
The present study	Japanese	113	0.761	0.239	-
Iwasaki et al	Japanese	403	0.754	0.246	0.824	(7)
	Japanese Brazilians	80	0.713	0.287	0.300
	Non-Japanese Brazilians	386	0.715	0.285	0.177
Breunis et al	Northern-European	100	0.710	0.290	0.232	(6)
Van Den Berg et al	Ethiopians	77	0.540	0.460	<0.0001	(5)
	Norwegians	86	0.660	0.340	0.031

a The Fisher’s exact test was used to determine the differences between the allele frequency in this study and other previously published results.

CNV determination and comparisons of CNV frequencies with published data on healthy individuals

The CNVs for FCGR2A and FCGR3A from this study are presented in Table IV. There was no CNV detectable for FCGR2A, as all subjects tested had 2 copies of this gene.

Table IV Number of copy number variations in FCGR2A and FCGR3A in this study.

Table IV

Number of copy number variations in FCGR2A and FCGR3A in this study.

		Copy number variation (%)
Gene	TaqMan® Copy Number assay ID	0 copies	1 copy	2 copies	3 copies	4 copies
FCGR2A	Hs00103511_cn	0	0	113 (100)	0	0
FCGR3A	Hs00139300_cn	0	3 (2.7)	105 (92.9)	5 (4.4)	0

However, with regard to FCGR3A, 5 subjects had 3 copies of this gene and 3 subjects had a single copy (gene deletion). The associations between CNVs in FCGR3A and the rs396991 genotypes were as follows: 3 subjects with a single copy of FCGR3A had the T/T genotype and of the 5 subjects with 3 gene copies, 1 had the T/T and 4 had the T/G genotype. Comparisons between the CNVs in FCGR3A from this study and those from previously published studies are shown in Table V. There were no significant differences between our results and those of previously published studies.

Table V Ethnic differences in the frequency of copy number variation in FCGR3A in healthy subjects.

Table V

Ethnic differences in the frequency of copy number variation in FCGR3A in healthy subjects.

			Copy number variation frequency
Study	Population	No.	1 copy	2 copies	3 copies	4 copies	P-valuea	Refs.
The present study	Japanese	113	0.027	0.929	0.044	0	-
Hollox et al	Japanese	32	0	0.875	0.094	0.031	0.301	(25)
	Chinese	32	0.031	0.813	0.156	0	0.082
	Yoruba	35	0.029	0.943	0.029	0	1
	Europeans	110	0.027	0.891	0.082	0	0.356
Breunis et al	Northern-Europeans	129	0.016	0.946	0.039	0	0.606	(26)
Zhou et al	Han Chinese	146	0.041	0.877	0.075	0	0.289	(27)

a Fisher’s exact test. The differences in the copy number distributions between this study and other studies were determined by dividing the subjects into 2 groups: group A with 2 gene copies and group B with copy numbers other than 2.

Discussion

In this study, the MAF for rs1801274 (A>G, H131R) of FCGR2A was 0.155 and only 1 subject (0.9%) had the G/G genotype. A previous study indicated that the MAF of FCGR2A among Japanese and Japanese-Brazilians was 0.200 and 0.194, respectively (7). Therefore, the SNP frequency for this minor allele appears to be between 0.15–0.2 in the Japanese population. The rs1801274 allele frequency among the Japanese subjects in this study was significantly different from those previously reported among Europeans (3–6) and Africans (3,4). Therefore, there are ethnic differences in rs1801274 SNP frequency. The MAF for rs396991 (T>G, F158V) of FCGR3A was previously reported to be 0.2–0.3 among Japanese individuals (7). We confirmed this frequency in a separate population. Therefore, the prevalence of this SNP frequency was found to be marginally higher compared with that of FCGR2A rs1801274. The MAF of rs36991 was found to be significantly lower in Japanese compared with that in Ethiopians (P<0.0001) and marginally lower compared with that observed in Norwegians (P=0.031) (4). Since rs1801274 and rs396991 are both functional SNPs, it may be predicted that the affinities of FcγIIa and FcγIIIa for antibodies may display ethnic variation.

No CNVs were detected for FCGR2A. To the best of our knowledge, this was the first study to assess CNVs of this gene in Japanese individuals. However, Breunis et al (26) also failed to detect any CNVs in FCGR2A among healthy European subjects, as well as among patients with diseases such as idiopathic thrombocytopenia, Kawasaki disease, or rheumatoid arthritis. By contrast, CNVs in FCGR3A were identified [3 subjects with a gene deletion (single copy) and 5 subjects with 3 gene copies]. This finding confirmed the previously observed presence of CNVs in FCGR3A (25). Unlike that previous small study, we did not identify any individuals with 4 gene copies; however, this may be due to the low frequency of this event. We observed a similar incidence of FCGR3A CNVs between the subjects in this study and Chinese (25,27), European (25,26) and African (25,26) subjects. Therefore, it appears that CNVs in FCGR3A are a common finding across different ethnic groups. Changes in gene dosage caused by CNVs may result in individual variations of the FcγRIIIa expression.

Three subjects with a single FCGR3A copy were found to have an rs396991 T/T genotype. However, their actual genotypes were considered to be T/- (T/del) when CNVs were taken into consideration. It was also predicted that the actual genotype of 1 subject was T/T/T and the 4 subjects genotyped as T/G were either T/T/G or T/G/G. Therefore, it is recommended to take CNVs for FCGR3A into consideration when evaluating the SNP genotype.

The TaqMan Copy Number assay to determine CNVs in FCGR2A and FCGR3A used a probe for FCGR2A that recognized the region between exon 1 and intron 1 and the probe for FCGR3A recognized a region of exon 2. To confirm our results, additional CNV analysis, using multiple probes that are able to recognize various regions of each gene should be considered. Additional analysis of the genotypes of FCGR2B, FCGR2C and FCGR3B, which are linked in tandem to FCGR2A and FCGR3A at 1q23, should also be considered in future studies.

Using commercially available TaqMan-based assays, we determined the incidence of SNPs and CNVs in FCGR2A and FCGR3A among healthy Japanese individuals. These assays confirmed data previously reported by studies that utilized more complex methods, such as multiplex ligation-dependent probe amplification (26). However, a large-scale analysis of the entire FCGR region, using a next-generation genome sequencing, may be useful for a more detailed investigation of genetic variability of FCGR in the Japanese population.

The straightforward methods used in this study to obtain information regarding the FCGR SNPs and CNVs among Japanese subjects may allow the development of diagnostic methods and treatments for autoimmune diseases, including SLE, as well as genetic biomarker research to predict the efficacy of antibody-based drugs, such as trastuzumab and rituximab.

References