TaqMan-based real-time polymerase chain reaction for detection of FUT2 copy number variations: Identification of novel Alu-mediated deletion

Abstract

BACKGROUND: The human FUT2 locus, which encodes a secretor-type α(1,2)fucosyltransferase, is known to be highly polymorphic. In addition to many single-nucleotide polymorphisms, three recombination alleles with a deletion of complete or partial FUT2 coding region have been reported. STUDY DESIGN AND METHODS: To detect copy number variations (CNVs) of the FUT2 gene including three recombinant alleles by a high-throughput system, we developed a triplex TaqMan real-time polymerase chain reaction (PCR) method. The relative number of copies of two regions of the FUT2 gene, the 5′ flanking (FUT2-5′) and FUT2-promoter (Prom) regions, were determined by comparing the number of threshold cycles (Ct) to those of the albumin gene (ALB) as the internal control (ÎCt). RESULTS: The mean 2-δδCt values (FUT2-5′/ALB or Prom/ALB) obtained from 237 samples with known FUT2 copy numbers clearly differentiated two nonoverlapping intervals that corresponded to the one-copy-number samples ranging from 0.42 to 0.59 and two-copy-number samples ranging from 0.81 to 1.19; no FUT2-5′ signal for recombination alleles was detected in homozygotes. Using this assay, we detected an individual in a Chinese population with a loss of one copy of the FUT2-5′ region resulting from a novel Alu-mediated FUT2 deletion (approx. 4 kb). CONCLUSIONS: The TaqMan real-time PCR method was able to detect the number of copies of FUT2 and distinguish different kinds of known CNVs. This system is robust, fast, and suitable for high-throughput analysis. © 2010 American Association of Blood Banks.