Nonallelic Homologous Recombination of the FCGR2/3 Locus Results in Copy Number Variation and Novel Chimeric FCGR2 Genes with Aberrant Functional Expression

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ORIGINAL ARTICLE Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression

SQ Nagelkerke1, CE Tacke2, WB Breunis2, J Geissler1, JWR Sins2, B Appelhof3, TK van den Berg1, M de Boer1 and TW Kuijpers1,2

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped 44000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Genes and Immunity (2015) 16, 422–429; doi:10.1038/gene.2015.25; published online 2 July 2015

INTRODUCTION the different FCGR2 genes, as well as the nomenclature used in the Immunoglobulin G (IgG) binds via its Fc domain to IgG receptors present study). Because the FCGR2C gene shares the last exon, (Fc-gamma receptors, FcγRs), and ligation and crosslinking of the which contains an immunoreceptor tyrosine-based activation FcγRs can induce a variety of effector functions in the cell motif, with FCGR2A, the FcγRIIc is also an activating receptor. expressing them. In humans, there are various activating and one However, FcγRIIc is only expressed in some individuals, as a 3 single inhibitory FcγR (FcγRIIb), with differential expression on consequence of the common p.Gln57Stop polymorphism, or as a various leukocyte subsets.1 Human FcγRs can be distinguished result of splice-site mutations,15 rendering FCGR2C a pseudogene into a high-affinity FcγR (FcγRI, encoded by the FCGR1A gene at unless the FCGR2C-ORF allele is present. Furthermore, the chromosome 1q21) and five low-affinity FcγRs (the different segmental duplication created two different but highly homo- isoforms of FcγRII and FcγRIII, all of which are encoded by the logous FCGR3 genes, FCGR3A (CD16a) and FCGR3B (CD16b), which FCGR2/3 locus at chromosome 1q23.3). The various FCGR2 and differ slightly in their extracellular domains but totally in the way FCGR3 genes show multiple genetic variations including function- they attach to the cell membrane and in expression pattern.1,2 ally relevant single-nucleotide polymorphisms (SNPs) and copy We have previously shown that the CNV at this locus occurs in number variations (CNVs).2 These SNPs and CNVs have been all genes except for the flanking FCGR2A and FCGR2B genes.16 associated with various autoimmune3–9 and infectious Since then, different methods have shown that CNV involves diseases,10,11 and with efficacy of immunotherapy,12,13 and it is reasonably well-defined regions of the locus,6,7,17 and it was likely that other disease associations will be identified in the suggested that CNV at the FCGR2/3 locus results from nonallelic future. However, the genetic analysis of this locus is complicated, homologous recombination (NAHR) of the two paralogous as it shows a very high degree of homology between the genes. repeats.17 The FCGR2/3 locus contains three FCGR2 and two FCGR3 genes Having routinely tested various large cohorts of healthy in two paralogous repeats of 82 kb. An unequal crossover event individuals and patients (including 44000 samples in total) with between the activating FCGR2A (CD32a) and the inhibitory FCGR2B our multiplex-ligation-dependent probe amplification (MLPA) (CD32b), the two genes that flank the region, has led to a assay, we could further define the copy number variable regions segmental duplication in which FCGR2C (CD32c) was formed,14 (CNRs) present at this locus, now describing four distinct CNRs. with the resulting FCGR2C gene being highly homologous to Furthermore, we saw that the NAHR break points for some of the FCGR2B in the first six exons and highly homologous to FCGR2A in CNRs are within genes, suggesting the presence of chimeric the last two exons (see Supplementary Figure 1 for an overview of genes at the locus. Indeed, we discovered that rearrangements

1Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 3Department of Genome Analysis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Correspondence: SQ Nagelkerke, Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam 1066 CX, The Netherlands. E-mail: [email protected] or [email protected] Received 30 March 2015; revised 17 May 2015; accepted 29 May 2015; published online 2 July 2015 NAHR of the FCGR2/3 locus results in chimeric FCGR2 genes SQ Nagelkerke et al 423 of the FCGR2/3 locus can result in the fusion of a part of either Figure 1c, and CNR numbering reflects the prevalence in the study FCGR2A or FCGR2B to other parts of the locus (although their population, as described below. copy number is never increased or decreased). The chimeric CNR1 includes the genes of FCGR2C, HSPA7 and FCGR3B. The nature of these genes has major consequences for expression break point is somewhere within the 30-kb intergenic DNA and function. between FCGR3B and FCGR2B. CNR2 includes exon8 and the 3ʹ-untranslated region (3ʹ-UTR) of FCGR2A, HSPA6, FCGR3A and the proximal part of FCGR2C, RESULTS including exon7 but not exon8 or the 3ʹ-UTR of FCGR2C. Four distinct patterns of CNV exist at the FCGR2/3 locus CNR3 includes the complete genes of FCGR3A, FCGR2C and Our MLPA assay detected CNV in genomic DNA at probe-binding HSPA7. The break point responsible for this CNR lies in the sites, as tested in 4357 individuals of different ethnic background. intergenic regions between FCGR3A/B and HSPA6/7. Among the different probes, seven ‘pairs’ of probes were designed CNR4 includes exons of FCGR2C after exon3, HSPA7, FCGR3B and on paralogous sequence variants (PSVs) that distinguish the two the first exons of FCGR2B, at least including exon3. 82-kb repeat units in the original segmental duplication at this locus (Figure1a and b). Four of these pairs represent unambiguous Recombination and chimeric genes of FCGR2A and FCGR2C: Exon8 (that is, not polymorphic in either of the two repeats) PSVs, and and the 3ʹ-UTR of FCGR2A and FCGR2C contain unambiguous PSVs three pairs detect polymorphic sequences in FCGR2C, being Our MLPA analysis showed that the break point for CNR2 appears nonpolymorphic in their counterpart in FCGR2A and FCGR2B. It has to lie within genes. Therefore, in case of a deletion, it creates an previously been shown that no unambiguous PSVs exist in a FCGR2A/2C chimeric gene consisting of the first seven exons of 7 24.5-kb block including the FCGR2C gene. FCGR2A and exon8 and the 3ʹ-UTR of FCGR2C (Figure 2a), whereas We observed that, when CNV was present in a sample, it always a duplication gives rise to an FCGR2C/2A chimeric gene (Figure 3a). included a complete and adjacent series of seven probes To explore and confirm this possibility, further evidence was representing one of the two probes in a pair, and both when obtained using various sequencing approaches. Although FCGR2A there was a duplication or a deletion. This suggests that CNV at and FCGR2C are highly homologous in exon8 and the 3ʹ-UTR, one the FCGR2/3 locus always involves an 82-kb region, compatible difference in exon8 and up to 13 potential differences in the with NAHR of the 82-kb repeat units, with the break points of the 3ʹ-UTR (Supplementary Figure 1) have been found before by NAHR events in between two of the paralogous pairs. We were sequencing of fosmid clones in a small group of 8 individuals.7 able to distinguish distinct patterns in these CNV regions, as To confirm the difference in exon8 as an unambiguous PSV, we previously suggested as so-called CNRs.6 We report on these CNRs specifically amplified leukocyte cDNA of FCGR2A and FCGR2C with in greater detail now and show that CNR1 variations can actually specific forward primers and a common reverse primer that result from different break points, and describe a novel fourth anneals to both genes, in a large selection of individuals with no variant, CNR4. The extent of the CNRs we identified is shown in deletion or duplication of CNR2. This demonstrated the PSV in

a Two paralogous repeats of 82 kb with >98% sequence homology

proximal repeat distal repeat * * * exon7 ORF intron3 exon8 exon5 exon1 Stop intron3 exon8 exon5 exon1 MLPA exon7 exon1 exon1 probes FCGR2A/C FCGR2A HSPA6 FCGR3A FCGR3A FCGR2C FCGR2C FCGR2C HSPA7 FCGR3B FCGR3B FCGR2B/C FCGR2C FCGR2B/C

b 5’ 3’ Chromosome 1q23.3 FCGR2A HSPA6 FCGR3A FCGR2C HSPA7 FCGR3B FCGR2B

c CNR1 (0,1,2,3,4,5) CNR2 CNV (1,2,3,4,5) Regions CNR3 (CNRs) (1,2,3) CNR4 (1,2)

Figure 1. Four distinct CNV regions exist at the FCGR2/3 locus. (a) Seven pairs of MLPA probes detect paralogous sequence variants on homologous positions in the locus. Colors indicate which probes form a pair, arrows indicate the approximate binding position on chromosome 1. Underlined probes always bind to the position that is indicated by the arrow, but in addition can bind to a polymorphic variant in the other 82-kb repeat. Probes indicated with an asterisk are speciﬁc for the polymorphic variant that only occurs in the repeat indicated by the arrow, and thus are speciﬁc, but do not bind to the other polymorphic variants at this site. (b) the FCGR2/3 locus at 1q23.3, with the genes and orientation of the genes. Note that gene sizes are enlarged to increase the readability. (c) Black lines indicate the extent of CNV for the different CNRs that we have found at this locus, with the dashed lines referring to the approximate break points on the chromosome that lead to these CNRs. Observed copy number indicates which copy numbers of that CNR have been observed in the present study.

FCGR2A HSPA6 FCGR3A FCGR2C HSPA7 FCGR3B FCGR2B

A A

B B NAHR Breakpoint

2. deletion allele of CNR2 FCGR2A/2C HSPA7 FCGR3B FCGR2B

B A

Crossover in intron7 fuses the distal part of FCGR2C, with differences in exon8 and the 3’UTR, to the proximal part of FCGR2A, deleting the distal part of the wild-type FCGR2A

FCGR2A FCGR2A/C wild-type common reverse primer

Heterozygous FCGR2A/C FCGR2A/2C chimera common reverse primer

Heterozygous FCGR2A FCGR2A/2C chimera specific reverse primer

Heterozygous FCGR2C FCGR2A/2C chimera specific reverse primer

FCGR2A c.818

Neutrophils Monocytes Neutrophil ROS production 20000 20000 0.5 p<0.0001 p<0.05 p<0.001 15000 15000 0.4 MFI MFI 0.3 10000 10000 0.2

5000 AT10 5000 AT10 0.1

0 0 Max slope IgG/PMA 0.0

FCGR2A FCGR2A FCGR2A chimera chimera chimera wild-type FCGR2A/2C wild-type FCGR2A/2C wild-type FCGR2A/2C

Figure 2. FCGR2A/2C chimeric genes result in a lower protein expression and lower IgG-induced respiratory burst than wild-type FCGR2A genes. (a) Schematic overview of deletion of CNR2 creating an FCGR2A/2C chimeric gene. (b) Upper two sequences: sequencing analysis of specifically amplified FCGR2A cDNA with an FCGR2A/2C common reverse primer confirms the heterozygous presence of c.818C (encoding p.273 P), in FCGR2A/2C chimeric donors. Lower two sequences: sequencing analysis of FCGR2A cDNA in heterozygous chimeric donors with reverse primers specificforFCGR2A and FCGR2C reveals that the wild-type allele contains the common c.818 T (encoding p.273 L), whereas the chimeric allele contains c.818C. Data are representative of five heterozygous FCGR2A/2C chimeric donors. (c) Median fluorescence intensities of monoclonal antibody AT10, detecting FcγRII, corrected for isotype control, on human neutrophils and monocytes of genotyped donors. FCGR2A/2C chimeric donors n = 5, and 4 of these donors were measured twice on separate days with similar results, average is shown. Wild- type individuals n = 140. (d) IgG-induced respiratory burst by neutrophils in response to 17.5 mg ml − 1 IgG, compared with the response to 100 ng ml − 1. Phorbol 12-Myristate 13-Acetate (PMA). FCGR2 A/2C chimeric donors n = 5, and 4 of these donors were measured twice on separate days with similar results, average is shown. Wild-type individuals n = 29.

exon8 to be unambiguous: a leucine at the 14th amino acid CNR2 deletion leads to FCGR2 A/2C chimeric genes with decreased position within the exon of FCGR2A (p.273Leu), versus a proline FcγRIIa expression and function at the 14th amino acid position in exon8 of FCGR2C (p.280Pro; Five healthy individuals with a heterozygous deletion of CNR2 Figure 2b, Supplementary Table 2a and b). We discovered another, were available for the isolation of mRNA from leukocytes. When newly described unambiguous PSV in the 3ʹ-UTR of the genes: a sequencing the PCR products from cDNAs of these donors nucleotide insertion (T) at 100 bp downstream of the termination amplified with an FCGR2A-specific forward primer and an codon in all FCGR2C alleles, but in none of the FCGR2A alleles FCGR2A/C-common reverse primer, we found heterozygosity at (Supplementary Table 2a and b). the PSV sites in FCGR2A in all five donors (Figure 2b and

1.Misaligned homologous sequences in NAHR

FCGR2A HSPA6 FCGR3A FCGR2C HSPA7 FCGR3B FCGR2B

A A

B B

2. duplication allele of CNR2 NAHR Breakpoint

FCGR2A HSPA6 FCGR3A FCGR2C/2A HSPA6 FCGR3A FCGR2C HSPA7 FCGR3B FCGR2B

B A

Crossover in intron7 fuses the distal part of FCGR2A, with differences in exon8 and the 3’UTR, to the proximal part of FCGR2C, deleting the distal part of the wild-type FCGR2C

NK Cells 1200 p<0.0001 Donor #1 1000 cDNA FCGR2C 800 p<0.01

MFI p<0.0001

600 400 2B6 200 Donor #3 0 cDNA FCGR2C -Stop classical classical -ORF (1x) -ORF (1x) FCGR2C FCGR2C c.169 FCGR2C c.839 FCGR2C FCGR2C/2A Figure 3. FCGR2C/2A chimeric genes of the classical FCGR2C-ORF haplotype have a higher protein expression than normal classical FCGR2C- ORF haplotypes. (a) Schematic overview of a duplication of CNR2 creating an FCGR2C/2A chimeric gene. (b) Example of individual 454 reads of FCGR2C cDNA from two donors that have one FCGR2C/2A gene and one copy of a classical FCGR2C-ORF, showing that transcripts with an ORF in exon3 derive from an FCGR2C/2A chimeric gene in donor #1, whereas the transcripts with an ORF in exon3 derive from a wild-type FCGR2C in donor #3. (c) Median ﬂuorescence intensities of monoclonal antibody 2B6, corrected for isotype control, on human NK cells of genotyped donors. FCGR2C-stop: donors that cannot express FcγRIIc, n = 101. Classical FCGR2C-ORF: donors with one copy of FCGR2C-ORF in a wild-type FCGR2C gene, n = 20, including donor #3 (represented by an open circle). Classical FCGR2C/2A-ORF: donors #1 and #2, with a classical FCGR2C- ORF haplotype in a chimeric FCGR2C/2A gene. Donor #1 was measured twice on separate days, donor #2 was measured three times on separate days, average is shown. To ensure a fair comparison, individuals with a deletion of CNR1, who express FcγRIIb on NK cells15 (n = 15), as well as individuals with 2 copies of FCGR2C-ORF (n = 7), who have a higher expression of FcγRIIc,16 were excluded from this analysis. Individuals with the nonclassical FCGR2C-ORF allele that is not expressed15 (n = 8) were grouped with FCGR2C-stop individuals.

Supplementary Table 2c). Using specific reverse primers on the (Supplementary Table 3a), but not in the three donors that also ins/del T in the 3ʹ-UTR with a specific FCGR2A forward primer we had one FCGR2C-ORF allele (Supplementary Table 3b). As the also amplified the cDNA from chimeric and normal genes sequencing was done on cDNA, it could reflect the increased specifically (Figure 2b and Supplementary Table 2c). To determine stability of the FCGR2C-ORF mRNA2 resulting in a bias for this if these chimeric FCGR2A/2C genes were functionally different allele. Therefore we continued with 454 next-generation sequen- from wild-type FCGR2A genes, we tested expression levels of cing of the cDNA of these donors, and in this way we could FcγRIIa on blood cells of these donors, and found a significantly confirm the presence of both wild-type and chimeric alleles in lower expression on neutrophils (Figure 2c). On monocytes, the these donors in individual reads (Supplementary Table 3c). difference was less clear, with all donors with chimeric FCGR2A/2C Furthermore, the individual reads of the 454 sequencing revealed genes within the normal range, although the difference was still that in donor #1 and #2 the FCGR2C-ORF haplotype was in the significant (Figure 2c). Furthermore, the IgG-induced NADPH- chimeric allele, whereas in donor #3 it was in one of the non- oxidase activity in neutrophils from these donors was significantly chimeric alleles (Figure 3b and Supplementary Table 3c). As the decreased compared with the donors with wild-type FCGR2A FCGR2C/2A chimeric genes containing an FCGR2C-ORF can be genes (Figure 2d). expressed, we tested the expression levels of FcγRIIc on NK cells, using monoclonal antibody 2B6. Although the monoclonal anti- CNR2 duplication leads to FCGR2C/2A chimeric genes and body 2B6 detects both FcγRIIc and FcγRIIb, it can be used for the increased FcγRIIc expression levels if the chimeric gene is of the specific detection of FcγRIIc on NK cells, because these cells classical FCGR2C-ORF haplotype normally do not express FcγRIIb. Only in individuals with a Eight healthy individuals with a heterozygous duplication of CNR2 deletion of CNR1, FcγRIIb is present on NK cells.7,15 Therefore, were available for the isolation of mRNA from leukocytes. Sanger individuals with a deletion of CNR1 were excluded from this sequencing of cDNAs amplified with an FCGR2C-specific forward analysis, to ensure detection of FcγRIIc only. We found that primer and an FCGR2A/C-common reverse primer revealed the expression of FcγRIIc was significantly higher in donor #1 and #2 heterozygosity at the PSV sites (as expected from the MLPA than in individuals with an FCGR2C-ORF in a wild-type FCGR2C results) in the donors that only had FCGR2C-Stop genes gene (Figure 3c).

1.misaligned homologous sequences in NAHR

FCGR2A HSPA6 FCGR3A FCGR2C HSPA7 FCGR3B FCGR2B

A A

B B

NAHR Breakpoint

2. deletion allele of CNR4 FCGR2A HSPA6 FCGR3A FCGR2C/2B

B A

Crossover fuses the proximal part of FCGR2C, which in most cases contains a Stop codon in exon 3, to the distal part of FCGR2B, deleting the proximal part of the wild-type FCGR2B

Expression FcRIIb on B cells 25000 p<0.0001 20000 FCGR2B

wild-type MFI 15000 10000 2B6 Heterozygous 5000 FCGR2B-Stop 0

-Stop FCGR2B c.169 FCGR2B wild-type FCGR2B (heterozygous) Figure 4. Deletion of CNR4 leads to the novel FCGR2B-Stop allele with decreased expression of FcγRIIb. (a) Schematic overview of NAHR of CNR4 creating an FCGR2B-Stop allele. Upper panel: two misaligned chromosomes with the distal 82-kb repeat unit of chromosome A aligned to the proximal 82-kb repeat unit of chromosome B. Black lines indicate crossover break points that result in NAHR. The homology between the 82-kb repeat units is reflected by shading. Lower panel: deletion of CNR4 creates an FCGR2B-Stop allele. (b) Sequencing analysis of specifically amplified genomic FCGR2B confirms the presence of the stop codon in heterozygous FCGR2B-Stop donors, data are representative of all three heterozygous FCGR2B-Stop donors. (c) Median fluorescence intensities of monoclonal antibody 2B6, corrected for isotype control, on circulating B cells in genotyped individuals. In this case, monoclonal antibody 2B6 detects only FcγRIIb, because FcγRIIc is not expressed on B cells.2 FCGR2B wild-type donors n = 144, heterozygous FCGR2B-Stop donors n = 3 (including 1 vasculitis and 1 SLE patient, who were clinically stable at the time of testing. Neither of them was on systemic anti-inflammatory medication).

FCGR2B/C-ORF probe, with a concomitant increase of the probe Table 1. Prevalence of CNR copy numbers in the study population detecting the FCGR2C-stop sequence. We reasoned that in these FCGR2C CNR1 CNR2 CNR3 CNR4 cases, the stop codon at position 57 in must have been introduced into the FCGR2B gene, which is normally not 0 copies 10 (0.2%) 0 0 0 polymorphic at this position, by a deletion of a novel CNR, that 1 copy 373 (8.6%) 43 (1.0%) 3 (0.1%) 5 (0.1%) is, CNR4 (Figure 4a). This deletion thus would create an FCGR2B- 2 copies 3432 (78.7%) 4132 (94.8%) 4333 (99.5%) 4352 (99.9%) Stop allele. Sequence analysis of FCGR2B genomic DNA specifically 3 copies 497 (11.4%) 178 (4.1%) 21 (0.5%) NDa fi fi a ampli ed by long-range PCR con rmed the stop codon in one of 4 copies 44 (1.0%) 3 (0.1%) 0 ND the FCGR2B alleles (Figure 4b). We found this novel FCGR2B-Stop 5 copies 1 (0.0%) 1 (0.0%) 0 NDa allele to be very rare, as it occurred only in five out of 4357 Abbreviations: CNR, copy number variable region; ND, not determined. individuals tested, including two unrelated healthy individuals, a Prevalence of all the different CNRs in the study population (n = 4357). pediatric patient and his healthy father, and one patient with Number of individuals is shown for each amount of copies, followed by systemic lupus erythematosus (SLE). All individuals identified thus percentage of the study population in brackets. Some individuals have far were of European descent, and were heterozygous for the deletions/duplication in more than one CNR (not shown in the table). a FCGR2C deletion. The pediatric patient, his father and the SLE patient were Because of multiple polymorphic sites in , no unambiguous PSVs γ exist in a 24.5-kb block including the FCGR2C gene and the intergenic available for further testing. Surface expression of Fc RIIb on region proximal to FCGR2C.7 Therefore, distinguishing CNR1 from CNR4 is circulating B cells revealed that expression levels were indeed only possible in the case of deletions. It will never be possible to lower than in control donors with two normal FCGR2B alleles distinguish between CNR1 and CNR4 in the case of duplications, and all (Figure 4c). duplications of this type were classified as CNR1. Prevalence of CNRs at the locus, inheritance and detectability by whole-exome sequencing Deletion of CNR4 results in FCGR2B-Stop alleles and causes a Table 1 shows the prevalence of the CNRs in the study population. lowered FcγRIIb expression in heterozygotes CNR1 is the most prevalent, both in the case of deletions and We also identified for the first time, several individuals that had a duplications, and is the only CNR for which we found individuals heterozygous deletion of FCGR3B, HSPA7 and exon8 of FCGR2C with homozygous deletions. Rare individuals with five copies of that was accompanied by a decrease of probe binding of the the same CNR were found for both CNR1 and CNR2, indicating

Genes and Immunity (2015) 422 – 429 © 2015 Macmillan Publishers Limited NAHR of the FCGR2/3 locus results in chimeric FCGR2 genes SQ Nagelkerke et al 427 that it is possible that one chromosome contains two duplications. individuals with a duplication showed an increase in the c.798+1A Analysis of parent–offspring trios revealed that CNR duplications variant that only occurs in FCGR2C, except for those with an and deletions showed normal Mendelian inheritance FCGR2C-ORF haplotype. These data suggest that the break point is (Supplementary Figure 2), also in the cases with more than one between exon7 and exon8. duplication on the same chromosome. Furthermore, this analysis CNR2 was identified as the cause of two novel chimeric genes at revealed that a deletion of one CNR and a duplication of another this locus. The chimeric FCGR2A/2C gene may have a large impact CNR on the same chromosome also occurred in rare cases on the functioning of the immune system. This chimeric gene (Supplementary Figure 2). leads to decreased expression levels of the major activating IgG Finally, we wanted to test whether the chimeric genes at the receptor on phagocytes, FcγRIIa, resulting in markedly reduced locus can also be detected by next-generation sequencing activity in response to IgG. Differences in the 3ʹ-UTR can techniques such as whole-exome or whole-genome sequencing. theoretically explain that the wild-type FCGR2A 3ʹ-UTR ensures Because of the high sequence homology of the locus, it is better stability of the mRNA, as indicated by the fact that when impossible to correctly map all reads obtained with next- the FCGR2A 3ʹ-UTR is introduced into FCGR2C, the expression of an generation sequencing techniques. We performed MLPA analysis open-reading frame (ORF) FcγRIIc increases (Figure 3c). Although on 10 subjects for which whole-exome sequence data the chimeric FCGR2A/2C gene greatly influences reactivity to IgG was available to compare the results. This showed that the in vitro, heterozygosity of this variant occurs in healthy individuals. FCGR2A/2C chimeric gene could not be reliably detected by Any association with a low copy number of FCGR3A, as for whole-exome sequencing, because this indicated heterozygosity instance reported recently for SLE and rheumatoid arthritis,20 may at the leucine/proline difference at p.273 in FCGR2A in all 10 have derived from a deletion of CNR2 and the presence of a samples, whereas only 1 of these samples actually carried a concomitant chimeric FCGR2A/2C gene. In that sense, low copy heterozygous FCGR2A/2C chimeric gene, which has a proline at number of FCGR3A may function as a tagging variant for chimeric p.273 (Supplementary Table 4). On the other hand, in cases such FCGR2A/2C genes. Detailed analyses by MLPA, that can distinguish as the FCGR2C-ORF genotype, whole-exome sequencing failed to CNR2 from CNR3 as the cause for low copy number of FCGR3A, will identify the three individuals that were genotyped by MLPA as help to determine if the actual association is a low copy number of FCGR2C-ORF (Supplementary Table 4), because only FCGR2C-Stop FCGR3A itself or the chimeric FCGR2A/2C gene. We suppose that sequences were mapped to FCGR2C, whereas FCGR2C-ORF the impact of this chimeric gene will be much stronger in sequences were wrongly mapped to FCGR2B. Together, these individuals homozygous for this deletion (~1 in 20 000 individuals data indicate that whole-exome sequencing is currently unable to of European descent). These individuals will have a complete lack adequately detect FCGR2 chimeric genes. of FCGR3A, and a lowered expression of FCGR2A. Thus, homozygosity for this FCGR2A/2C chimera implies that all immunoreceptor tyrosine-based activation motif-dependent activating DISCUSSION low-affinity FcγRs are severely affected, presumably resulting in Having investigated the extent of CNV at the FCGR2/3 locus in reduced uptake of IgG-opsonized pathogens and a phenotype of 44000 individuals, we provide the most comprehensive descrip- severe and recurrent infections. tion of CNRs at this locus, including the characterization of FCGR2C/2A chimeric genes are more prevalent than the chimeric genes that have a significant impact on phenotypic and FCGR2A/2C chimeric genes, but can only have functional relevance functional alterations directly relevant to human immunity. if they are of the FCGR2C-ORF variant, which is the minority of The formation of these chimeric genes is the result of NAHR of cases (26% of the individuals identified with an FCGR2C/2A gene a segmental duplication, which often leads to genomic instability had an FCGR2C-ORF, but the actual prevalence will be less, and disease.18 Our study extends the previous assumptions because not all of these individuals will have the FCGR2C-ORF in regarding CNRs at the FCGR2/3 locus. Following our earlier the chimeric gene). We showed that these chimeric genes have a findings,3,16 the so-called CNR1, 2 and 3 were suggested to higher protein expression than normal, which suggests that these explain the duplications and deletions at this locus,6 but the break individuals may be even more prone to autoimmune diseases points were not defined. We now offer a more detailed CNR model or auto-inflammation associated with the FCGR2C-ORF variant. of CNR1, 2, 3 and 4 that explains the functionally relevant CNV and The determination whether an FCGR2C/2A chimeric gene contains chimeric genes at the locus. an ORF is complicated, and as these alleles are rare, we suggest to CNR1 includes the genes of FCGR2C, HSPA7 and FCGR3B. consider these cases as normal classical FCGR2C-ORF alleles in Although it has been previously argued that the break points for genetic association studies in large cohorts. CNR1 may differ slightly between individuals,17 it is impossible to CNR4 includes exons of FCGR2C after exon3, HSPA7, FCGR3B and exactly define a break point for this CNR, as FCGR2C can be the first exons of FCGR2B, including exon3. As mentioned above, sequence identical to FCGR2B in the first six exons,19 as well as in in the case of duplication alleles, the different break points of the intergenic region proximal to the FCGR2B and FCGR2C genes.7 CNR1 and CNR4 cannot be distinguished as a result of the However, in most deletion alleles there is a deletion of the polymorphic nature of the FCGR2C gene. However, as these sites FCGR2C-specific p.57Ter sequence (data not shown), which shows are not polymorphic in FCGR2B, the break point can be accurately that in most cases FCGR2C is included in CNR1. defined in deletion alleles. CNR2 includes FCGR2A, HSPA6, FCGR3A and the proximal part of The CNR4-related chimeric gene product derived from a FCGR2C. In addition, it includes the distal part of FCGR2A, and we deletion was designated FCGR2B-Stop in analogy to the show that this involves not only the 3ʹ-UTR as previously most common FCGR2C-Stop variant which is a nonexpressed suggested,6 but also exon8. Exon8 and the 3ʹ-UTR of FCGR2C are pseudogene. Deletion of CNR4 thus silences the FCGR2B gene. not included, and the NAHR crossover break point for this CNR is Apparently, heterozygosity for this variant does not necessarily in between exon7 and exon8, creating chimeric genes. Because cause disease, as we have found this allele in three healthy adults. exon7 is polymorphic in FCGR2C, it could be argued that the break Its identification in two patients with vasculitis and SLE may point may even be proximal to exon7. However, there is only one suggest that this allelic variant of FCGR2B predisposes to PSV in exon7 of FCGR2A and FCGR2C, c.777+1G in FCGR2A, which autoimmunity, but its prevalence is low. Knockout mice is polymorphic in FCGR2C7 (c.798+1A4G). This PSV was detected completely deficient for FcγRIIb are viable, but prone to severe, by our MLPA probes, and our data revealed that none of the early onset autoimmunity, at least when on a C57BL/6 individuals with a deletion showed a decrease in probe binding to background.21,22 We estimate homozygosity for the FCGR2B-Stop the variant c.777+1G in FCGR2A (data not shown). Conversely, all variant to occur in ~ 1:1 000 000 individuals of European descent.

© 2015 Macmillan Publishers Limited Genes and Immunity (2015) 422 – 429 NAHR of the FCGR2/3 locus results in chimeric FCGR2 genes SQ Nagelkerke et al 428 Unexplained cases of early onset severe autoimmunity may Isolation of RNA actually be the consequence of homozygosity at this allele. The mRNA was isolated from 10 × 106 purified peripheral blood mononuclear FCGR2B-Stop variant will not readily be identified by next- cells by use of the QIAamp RNA blood mini kit (Qiagen), according to the generation whole-exome or whole-genome sequencing, because manufacturer’s instructions. Subsequently, first-strand cDNA was synthe- FCGR2B sized with the Superscript III first-strand synthesis system for RT-PCR (Life of the complete overlap of the -stop sequence with the μ FCGR2C FCGR2B Technologies, Darmstadt, Germany). In short, RNA was primed with 2.5 M common -stop sequence, which causes all the - oligo(dT) for 5 min at 65 °C. Reverse transcription was performed with FCGR2C − 1 stop reads to be allocated to . This notion is supported by 10 U μl Superscript III in the presence of 5 mM MgCl2,20mM Tris-HCl, our finding that all FCGR2C-ORF sequences in individuals 50 mM KCl (pH 8.4, reverse transcriptase buffer), 0.5 mM 2′-deoxynucleoside − genotyped to be positive for the FCGR2C-ORF haplotype were 5′-triphosphate and 2 U μl 1 RNaseOUT (without DTT) for 50 min at 50 °C. invariably allocated to FCGR2B (Supplementary Table 4). If an Thereafter, Superscript III was inactivated by incubation for 5 min at 85 °C, FCGR2B-Stop is suspected, direct staining of FcγRIIb on B cells and followed by chilling on ice. Immediately thereafter, 2 U RNase H were fi fi FCGR2B added, and the mixture was incubated at 37 °C for 20 min. Subsequently, con rmation by gene-speci c sequencing by long-range cDNA was stored at − 20 °C until further use. PCR, as described above, would suffice. In conclusion, we present a comprehensive model that explains FCGR2A/2C and FCGR2C/2A chimeric genes all the CNV at the FCGR2/3 locus, together with a characterization of novel chimeric genes that show greatly altered expression cDNA from genotyped donors was used for PCR reactions. An overview of primer sequences is given in Supplementary Table 1. Specific forward levels and function which may lead to extreme disease primers for FCGR2A (FCGR2A-1) and FCGR2C (FCGR2C) were used in phenotypes when homozygously present. The fact that these combination with a common reverse primer for FCGR2A and FCGR2C chimeric genes have never been described before probably is the (FCGR2A/C). Another specific forward primer for FCGR2A (FCGR2A-2) was result of the extremely high level of genetic homology between used in combination with specific reverse primers for FCGR2A (FCGR2A-no the different parts of the locus. Our study is crucial for insT) or FCGR2C (FCGR2C-insT). The PCR conditions were 95 °C for 2 min, FCGR followed by 40 cycles of 95 °C for 20 s, 62 °C for 1 min and 72 °C for 1 min, understanding the consequences of the CNV of genes and fi FCGR2 followed by an elongation at 72 °C for 2 min. PCR products were puri ed occurrence of different chimeras, which will help accurate by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Amersham, genotyping and correct interpretation of sequence data at this UK) and sequenced with BigDye terminator cycle sequencing (v 1.1; highly relevant immune reactivity locus. Applied Biosystems, Paisley, UK) with different sequence primers, including both the sense primers that were used for the PCR reaction, as well as two nested sequence primers (FCGR2A/C-seq1 and FCGR2A/C-seq2). MATERIALS AND METHODS Subject groups 454 sequencing The study population consisted of cohorts of healthy individuals and cDNA from six individuals that had an FCGR2C/2A chimeric gene as various patient cohorts that were genotyped for FCGR2/3 CNV and SNPs for determined by MLPA was amplified with the FCGR2C forward primer and the purpose of genetic association studies. These studies will be reported common FCGR2A/C reverse primer mentioned in Supplementary Table 1. elsewhere (Tsang-a-Sjoe, Nagelkerke, Kuijpers, Voskuijl et al., submitted, The PCR amplified full-length FCGR2C transcripts were processed in the and Nagelkerke, Tacke, Kuijpers et al., submitted). In total, the study 454FLX+ workflow. 454 Lib-L libraries were prepared by ligating the GS FLX population consisted of 4357 individuals. The cohorts are described in Titanium Rapid Library MID adaptors, to the PCR fragments on a SPRI detail in the Supplementary Materials and Methods. Workstation (Beckman Coulter, Woerden, The Netherlands). Seven libraries The study was approved by the Medical Ethics Committee of the with different MIDs were equimolar pooled and further processed for Academic Medical Center and Sanquin in Amsterdam, Netherlands, and emulsion-PCR, using the GS FLX Titanium MV emPCR Kit (Lib-L). The was performed in accordance with the Declaration of Helsinki principles. enriched DNA beads were subsequently loaded in one lane of the 4 Written informed consent was obtained from all participating donors and region-divided picotiterplate, and sequenced on the 454FLX+ platform using the GS FLX Titanium Sequencing kit XL+, with flow pattern B, using patients. the GS FLX+, GS FLX and GS Junior System Software Package v2.9 (Roche, Basel, Switzerland). After Image Processing the Signal Processing was MLPA started for Long Amplicons #1. Average readlengths were 1150 bp and Genomic DNA was isolated from whole blood with the QIAamp Blood Mini more than 2000 filter-passed reads were obtained per sample. kit (Qiagen, Hilden, Germany), or from saliva with the Oragene DNA self- collection kit (DNA Genotek, Kanata, ON, Canada), according to the FCGR2B-Stop sequencing manufacturer’s instructions. fi fi FCGR FCGR2A FCGR2B FCGR2C A gene-speci c long-range PCR was performed to amplify only CNV and SNPs in the low-af nity genes , , , FCGR2B genomic DNA without FCGR2C amplification. In brief, FCGR3A FCGR3B FCGR fi and were routinely determined with an -speci c a 15-kb fragment was amplified with the nonspecific FCGR2B/C sense MLPA assay (MRC-Holland, Amsterdam, The Netherlands). The MLPA assay primer (Supplementary Table 1), annealing in the promoter region and an was performed according to the manufacturer’s protocol, essentially as FCGR2B-specific antisense primer (FCGR2B). The PCR was performed 3,16 described previously. Fourteen of the probes included in the FCGR with the Primestar GXL TaKaRa polymerase (Westburg, Leusden, The MLPA were used in determining the extent of CNRs at the FCGR2/3 locus: Netherlands), conditions were 30 cycles of 98 °C for 10 s and 68 °C for these included eight gene-specific probes to determine the CNV of the 15 min. PCR products were purified with ExoSAP-IT kit (Affymetrix, Santa genes, as shown in Figure 1. The six other probes that were used for Clara, CA, USA) according to the manufacturer’s instructions and detecting CNRs detected polymorphic sequences in some of the genes. sequenced with BigDye terminator cycle sequencing (v 1.1; Applied These were probe specific for the stop codon in exon3 of the FCGR2C gene, Biosystems) with the sequence primer FCGR2B/C-seq. rs759550223 c.169C4T (p.Gln57*; FCGR2C-Stop probe in Figure 1) and a nonspecific FCGR2B/C probe to detect the ORF in exon3 (FCGR2B/C ORF Whole-exome sequencing probe in Figure 1). Furthermore, a probe specific for the intron3 variant FCGR2C FCGR2C Ten individuals were tested by MLPA and whole-exome sequencing to c.392-20G in (rs530707246, intron3 probe in Figure 1), and compare the two genotyping methods. These individuals were patients fi − FCGR2C, a nonspeci c probe detecting the c.392 20C variant in as well as with a pontocerebellar hypoplasia phenotype, and were not suspected to − FCGR2B FCGR2B/C c.392 20C in intron3 of ( intron3 probe in Figure 1). have genetic alterations at the FCGR2/3 locus. Whole-exome sequencing Finally, a specific probe was included for the splice-site mutation at the was performed on genomic DNA isolated from EDTA-blood. Library was border of exon7/intron7 in FCGR2C (rs76277413, c.798 +1A variant, FCGR2C prepared with the Agilent Human All Exon 50 Mb kit (Agilent Technologies, exon7 probe in Figure 1) and a nonspecific probe detecting c.798+1G in Santa Clara, CA, USA). Sequencing was done on a SOLiD5500 xl (Applied FCGR2C, as well as c.777+1G in FCGR2A (FCGR2A/C exon7 probe in Biosystems) or Illumina HiSeq 2000 instrument (Illumina, San Diego, Figure 1). CA, USA). Reads were aligned to the human genome reference19 with

Genes and Immunity (2015) 422 – 429 © 2015 Macmillan Publishers Limited NAHR of the FCGR2/3 locus results in chimeric FCGR2 genes SQ Nagelkerke et al 429 BWA-MEM23 and variants were called using GATK’s version 2.7–4 3 Breunis WB, van Mirre E, Bruin M, Geissler J, de Boer M, Peters M et al. Copy UnifiedGenotyper (Broad Institute, Cambridge, MA, USA).24 Ingenuity number variation of the activating FCGR2C gene predisposes to idiopathic Variant Analysis (Qiagen) and Integrative Genomics Viewer version 2.3.40 thrombocytopenic purpura. Blood 2008; 111:1029–1038. (Broad Institute, Cambridge, MA, USA) enabled variant analysis. 4 Fanciulli M, Norsworthy PJ, Petretto E, Dong R, Harper L, Kamesh L et al. FCGR3B copy number variation is associated with susceptibility to systemic, but not fi Nat Genet – Flow cytometry organ-speci c, autoimmunity. 2007; 39: 721 723. 5 Khor CC, Davila S, Breunis WB, Lee YC, Shimizu C, Wright VJ et al. Genome-wide The following monoclonal antibodies were used in flow cytometry: association study identifies FCGR2A as a susceptibility locus for Kawasaki disease. anti-CD3 clone SK7, Pe-Cy7 labeled, anti-CD14 clone M5E2, Pe-Cy7 Nat Genet 2011; 43: 1241–1246. labeled, anti-CD19 clone HIB19, APC labeled and anti-CD56 clone B159, et al. APC labeled (BD Pharmingen, San Diego, CA, USA), anti-FcγRII clone AT10, 6 Niederer HA, Willcocks LC, Rayner TF, Yang W, Lau YL, Williams TN FITC-labeled (AbD Serotec, Oxford, UK), and anti-FcγRIIb/c clone 2B6, Alexa Copy number, linkage disequilibrium and disease association in the FCGR locus. Hum Mol Genet – Fluor 488 labeled (a generous gift from MacroGenics, Rockville, MD, USA). 2010; 19: 3282 3294. et al. Whole-blood leukocytes were isolated by lysis of red blood cells by an 7 Mueller M, Barros P, Witherden AS, Roberts AL, Zhang Z, Schaschl H Genomic isotonic ammonium-chloride buffer. 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Genetic polymorphism studies in periodontitis and − 1 or 100 ng ml phorbol 12-myristate 13-acetate. Results were compared Fcgamma receptors. J Periodontal Res 2012; 47: 273–285. after correction for the values from unstimulated and PMA-stimulated 11 Adu B, Dodoo D, Adukpo S, Hedley PL, Arthur FK, Gerds TA et al. Fc gamma samples. For each experiment, the maximum slope of H2O2 production was receptor IIIB (FcgammaRIIIB) polymorphisms are associated with clinical malaria in determined by calculating the maximum value of the derivative of the ghanaian children. PLoS One 2012; 7: e46197. H2O2 concentration over time. Measurements were performed every 30 s. fi 12 Mellor JD, Brown MP, Irving HR, Zalcberg JR, Dobrovic A. A critical review of the The maximum slope was based on ve sequential time points. role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. J Hematol Oncol 2013; 6:1. Statistical analysis 13 Tamura K, Shimizu C, Hojo T, Akashi-Tanaka S, Kinoshita T, Yonemori K et al. In case of multiple expression values per donor, the mean of these values FcgammaR2A and 3A polymorphisms predict clinical outcome of trastuzumab in was taken for the statistical analyses. Expressions between groups were both neoadjuvant and metastatic settings in patients with HER2-positive compared using Mann–Whitney tests (two groups) or a Kruskal–Wallis test breast cancer. Ann Oncol 2011; 22: 1302–1307. with post hoc Mann–Whitney tests (42 groups) using GraphPad Prism 14 Warmerdam PA, Nabben NM, van de Graaf SA, van de Winkel JG, Capel PJ. The software (GraphPad Software, La Jolla, CA, USA). Two-sided tests were used human low affinity immunoglobulin G Fc receptor IIC gene is a result of an and a P-value of o0.05 after Bonferroni correction was considered unequal crossover event. J Biol Chem 1993; 268: 7346–7349. significant, uncorrected P-values are shown in Figures 2–4. 15 van der Heijden J, Breunis WB, Geissler J, de Boer M, van den Berg TK, Kuijpers TW. Phenotypic variation in IgG receptors by nonclassical FCGR2C alleles. J Immunol 2012; 188: 1318–1324. CONFLICT OF INTEREST 16 Breunis WB, van Mirre E, Geissler J, Laddach N, Wolbink G, van der Schoot E et al. The authors declare no conflict of interest. Copy number variation at the FCGR locus includes FCGR3A, FCGR2C and FCGR3B but not FCGR2A and FCGR2B. Hum Mutat 2009; 30: E640–E650. 17 Machado LR, Hardwick RJ, Bowdrey J, Bogle H, Knowles TJ, Sironi M et al. ACKNOWLEDGEMENTS Evolutionary history of copy-number-variable locus for the low-affinity Fcgamma receptor: mutation rate, autoimmune disease, and the legacy of helminth infec- We thank Dr Marc Bijl, Mr Michel Tsang-a-Sjoe, Professor Dr Alexandre Voskuijl, tion. Am J Hum Genet 2012; 90: 973–985. Dr Bart Biemond, Mrs Lisa Vogelpoel and Professor Dr Martin Hibberd for help in 18 Emanuel BS, Shaikh TH. Segmental duplications: an 'expanding' role in genomic sample collection, and Professor Dr Frank Baas for help with data analysis. This work Nat Rev Genet – was supported by a grant from the Landsteiner Foundation for Blood transfusion instability and disease. 2001; 2: 791 800. Research (LSBR 0916). CET has received a grant of the Ter Meulen Fund, Royal 19 Su K, Wu J, Edberg JC, McKenzie SE, Kimberly RP. Genomic organization of classical fi Genes Immun – Netherlands Academy of Arts and Sciences (TMF2012/227). human low-af nity Fcgamma receptor genes. 2002; 3:S51 S56. 20 Chen JY, Wang CM, Chang SW, Cheng CH, Wu YJ, Lin JC et al. Association of FCGR3A and FCGR3B copy number variations with systemic lupus erythematosus and rheu- AUTHOR CONTRIBUTIONS matoid arthritis in Taiwanese patients. 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