A comparison of assays for accurate copy number measurement of the low-affinity FC gamma receptor genes FCGR3A and FCGR3B

• Main Authors: Abubakar, S. (Author), Aziz, A.T.A (Author), Bakar, S.A (Author), Haridan, U.S (Author), Hollox, E.J (Author), Lee, C.K.C (Author), Machado, L.R (Author), Mokhtar, U. (Author), Mustafa, M. (Author), Peng, H.B (Author), Shueb, R.H (Author), Sim, B. (Author), Yusof, N.K.N (Author), Zaid, M. (Author)
• Format: Article
• Language: English
• Published: Public Library of Science 2015
• Subjects: allele; Article; bioassay; Biological Assay; case control study; Case-Control Studies; controlled study; copy number variation; dengue; Dengue; DNA Copy Number Variations; Fc receptor; FCGR3A gene; FCGR3A protein, human; FCGR3B gene; FCGR3B protein, human; gene; gene dosage; genetic association; genetic parameters; genetic predisposition; Genetic Predisposition to Disease; genetic variability; genetics; genotype; Genotype; glycosylphosphatidylinositol anchored protein; GPI-Linked Proteins; human; Humans; measurement accuracy; paralogue ratio test restriction enzyme digest variant ratio; procedures; quantitative analysis; real time polymerase chain reaction; Real-Time Polymerase Chain Reaction; Receptors, IgG; sequence homology
• Online Access: View Fulltext in Publisher; View in Scopus

 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 (PRT-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. © 2015 Haridan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.