A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (RT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs

Distribution of raw unrounded copy number estimates of <i>FCGR3A</i>.

<p>A, Analysis was carried out using 160 samples genotyped with Sequenom. Unrounded copy number estimates are in bins of 0.1, with the count of each bin displayed on the y-axis. Blue indicates samples that were called as duplications, and red samples that were called as deletions. The red bars indicate copy number loss (CN<2); black bars indicate copy number normal (CN = 2); blue bars indicate copy number gain (CN>3). <b>B</b>, Distribution frequency of absolute copy number call for the samples genotyped with Sequenom MassARRAY and PRT-REDVR shows a higher rate of copy number normal (CN = 2) being called by Sequenom.</p

Analysis of raw quantification data (logEC50) for Sequenom MassARRAY.

<p>Correlation between raw individual copy number calls from the probes designed at 5′-, 3’-, and centre regions of the genes <i>FCGR3A</i> (A, B, and C) and <i>FCGR3B</i> (D, E, and F). Note that there is a clear seperation of 2 cluster plots indicating batch effects, as circled, due to different experimental batches. <b>A</b>, <b>3’ vs 5′; B, 3’ vs centre;</b> and <b>C, 5′ vs centre</b>; <b>D</b>, <b>5′ vs 3’; E, 3’ vs centre;</b> and <b>F, 5′ vs centre.</b> A and D, correlation between the 5- and 3- prime probes were slightly affected, probably due to the probes distance.</p

Percentage of concordant results of <i>FCGR3B</i> CN calls between the three different assays.

<p>Percentage of concordant results of <i>FCGR3B</i> CN calls between the three different assays.</p

Scatterplot showing all 99 samples matched for <i>FCGR3B</i> CNV with PRT-REDVR, Sequenom and qPCR.

<p><b>A.</b> qPCR vs Sequenom. qPCR values represent raw copy number, Sequenom values represent absolute copy numbers, <b>B.</b> Sequenom vs qPCR. Both Sequenom and qPCR raw copy number values are presented, showing a low concordance rate between the two platforms, <b>C.</b> qPCR vs PRT-REDVR. qPCR values represent raw unrounded copy number, PRT-REDVR values represent absolute copy number; and <b>D.</b> Sequenom vs PRT-REDVR. Sequenom values represent raw unrounded copy numbers, PRT-REDVR values represent absolute copy number. The broader scater plots of qPCR signal intensity show wider variability of copy number calls, suggesting more copy number calls from qPCR were discordant with the Sequenom and PRT-REDVR. D exhibits a more clustered plot indicating higher agreement of CN calls between Sequenom and PRT.</p

Analysis of raw copy number quantification data for PRT.

<p>Correlation between individual duplicated PRT copy number. Individual results from FAM-labelled (Y-axis) versus Hex-labelled (X-axis) representing internal replication of the assay, are plotted for 99 samples. Colour coded according to the integer copy number of each samples as estimated in REDVR.</p

Primer sequences and single nucleotide variation (SNV) identified for <i>FCGR3A/B</i> in Sequenom MassARRAY.

<p>Primer sequences and single nucleotide variation (SNV) identified for <i>FCGR3A/B</i> in Sequenom MassARRAY.</p

Distribution of unrounded copy numbers called by qPCR and Sequenom for <i>FCGR3B</i>.

<p>Analysis was carried out using 100 samples genotyped with both qPCR and Sequenom. Unrounded copy number estimates are in bins of 0.1, with the count of each bin displayed on the y-axis.</p

Primer sequences for REDVR assay.

<p>The primer pair 38 distinguishes <i>FCGR3A</i> from <i>FCGR3B</i> while the primer pair 234 distinguishes the HNA1a region from HNA1b.</p><p>Primer sequences for REDVR assay.</p

Primer sequences for <i>FCGR3</i> PRT assay.

<p>Primer sequences for <i>FCGR3</i> PRT assay.</p