A discriminative analytical method for detection of CES1A1 and CES1A2/CES1A3 genetic variants

Abstract

Human carboxylesterase 1 (hCES1), encoded by the CES1 gene, is the predominant hepatic hydrolase responsible for the metabolism of many therapeutic agents, toxins, and endogenous substances. Genetic variants of CES1 can affect hCES1 function and expression and ultimately influence clinical response to drugs serving as hCES1 substrates. The CES1 gene consists of three isoforms including the functional CES1A1 and CES1A2 genes and the nonfunctional pseudogene CES1A3. Natural variants of these isoforms exert differing impacts on hCES1 function. However, the existing CES1 genotyping methods are incapable of determining whether these variants belong to CES1A1, CES1A2, or CES1A3 because of the high similarity among these three genes, as a consequence they are unable to discriminate between heterozygotes and homozygotes. We report the development of a novel long-range PCR-based, discriminative genotyping assay capable of specifically detecting the variants among CES1A1, CES1A2, and CES1A3 genes. The comparison of the genotyping results between this novel assay and those previously reported methods highlighted the necessity of applying the discriminative genotyping assay in pharmacogenetic studies involving CES1 gene.

Figure 1

Long-range PCR of ~14kb fragments of CES1A1 and CES1A3/CES1A2 genes from the PM of methylphenidate (Figure 1A). Lane 1: λ DNA/Hind III plus marker; Lane 2 and 3: the long-range PCR products of the CES1A1 gene from the methylphenidate PM; Lane 4: the CES1A3/CES1A2 long-range PCR product from the PM. Figure 1 B shows the sequencing chromatograms of CES1A1 exon 4 of the PM and his biological father and mother indicating the PM and his father are heterozygous CES1A1 G428A (Gly143Glu) whereas the mother is wild type.

Figure 2

DNA sequencing chromatograms of a homozygote of CES1A1 Gly143Glu using the novel long-range PCR-based assay (A) and conventional direct DNA sequencing (B), and the determination of the CES1A1 variant Gly143Glu utilizing the Taqman^® assay (C). The Gly143Glu homozygote was incorrectly detected as a heterozygote by both conventional direct DNA sequencing and Taqman^® assay.