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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 outcomes of hCES1 substrate drugs. CES1 gene consists of three isoforms including the functional CES1A1 and CES1A2 genes and nonfunctional pseudogene CES1A3. Natural variants of these isoforms exert differing impacts on hCES1 function. However, the existing CES1 genotyping methods are incapable of discriminating between these variants due to the high similarity among these three genes. We report the development of a novel long-range PCR-based, discriminative genotyping assay with the capacity 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 pharmacogentic studies involving CES1 gene.
Human carboxylesterase 1 (hCES1) is the major hepatic hydrolase in human, contributing to an estimated 80%– 95% of total hydrolytic activity in the liver . hCES1 is responsible for the metabolism (hydrolysis) of many medications, drugs of abuse, environmental toxins, and endogenous substances. hCES1 is encoded by the CES1 gene, which consists of three isoforms, i.e. CES1A1, CES1A2, and CES1A3 [2–4]. Both CES1A1 and CES1A2 are functional whereas CES1A3 is a non-functional pseudogene as the result of a premature stop codon located in exon 3. The CES1A3 was also termed as CES1P1 gene in a recent publication . CES1A1 and CES1A3 are inversely located on chromosome 16 q13–q22.1. CES1A2 is a variant of the CES1A3 gene with allele frequencies of 14.4%, 5.1%, and 31.3%, respectively, in Caucasians, African-Americans, and Japanese . With the exception of the promoter region and exon 1, CES1A1 and CES1A2 are identical.
Significant interindividual variability of hCES1 expression and activity has been consistently documented in the biomedical literature [4, 6]. CES1 genetic polymorphisms are increasingly viewed as one of the major factors influencing hCES1 function and expression and have recently been associated with the therapeutic outcomes of drugs whose metabolism is known to be dependent on hCES1. Two CES1 nonsynonymous variants, Gly143Glu (rs71647871) and Asp260fs (rs71647872), located in exons 4 and 6 of CES1, respectively, were recently identified in our laboratory and were observed to produce a marked impairment in hydrolytic activity [7–9]. A previously conducted normal volunteer pharmacokinetic study of methylphenidate identified a subject who carried these two mutations. This subject was determined to be a poor metabolizer (PM) of methylphenidate as evidenced by profoundly increased blood methylphenidate concentrations and significantly altered pharmacokinetic and pharmacodynamic parameters during the course of the study . The PM’s biological father and mother were determined to carry heterozygous Gly143Glu and Asp260fs, respectively . A pharmacogenetic association study suggested that attention deficit hyperactivity disorder (ADHD) patients carrying the Gly143Glu required significantly lower dose of dl-methylphenidate for symptom reduction relative to control subjects . In addition to the variants discovered in our laboratory, several other CES1 single-nucleotide polymorphisms (SNPs) have also been reported to be functionally significant in both in vitro and clinical investigations [12, 13].
It should be noted that the genetic variants within three CES1 gene isoforms (CES1A1, CES1A2, and CES1A3) may have distinct impact on overall hCES1 activity. Since CES1A3 is a pseudogene, it is believed that associated polymorphisms are unlikely to influence hCES1 expression and activity . Furthermore, the level of mRNA transcribed from CES1A2 gene is approximately 2% of that from CES1A1 gene in human liver . The majority of hepatic hCES1 is presumably the product of the CES1A1 gene [2, 4]. Therefore CES1A1 genetic variants may exhibit a greater impact on hCES1 function relative to CES1A2 variants. Thus, when targeted studies of hCES1 activity and its catalytic efficiency are undertaken, it is essential that the CES1 genotyping method employed is able to differentiate between the variants within these 3 isoforms, However, conventional genotyping methods reported to date including direct sequencing, Taqman® assay, and PCR are incapable of specifically detecting the variants (except those in the promoter and exon 1 regions) between CES1A1 and CES1A2/CES1A3 genes due to the high similarity of DNA sequences among these genes [9, 11, 14, 15]. Another limitation of these published assays is that the homozygous variants in one CES1 gene (e.g. CES1A1) could be incorrectly reported as heterozygous due to the presence of another wild type CES1 isoform (e.g. CES1A2/CES1A3). Indeed, to date these conventional genotyping methods have identified 86 subjects carrying heterozygous Gly143Glu variant [9, 11, 14]. However, heretofore, no investigators have detected or reported a homozygous individual. It is likely that a number of individuals previously genotyped and thought to be heterozygous are actually homozygous Gly143Glu carriers, yet incorrectly identified as heterozygotes due to the intrinsic limitation of these assays.
In the present study, a long-rang PCR-based discriminative genotyping assay for CES1A1 and CES1A2/CES1A3 genes was developed utilizing the genomic DNA previously collected from the methylphenidate PM and his biological parents . Two forward primers were designed based on the differences of the promoter regions between CES1A1 and CES1A2/CES1A3 genes, whereas a DNA sequence within intron 6 was chosen as the reverse primer for the PCR reactions of both genes. The sequences of primers and PCR conditions were detailed in Supplementary Table 1. The DNA fragments yielded from the long-range PCR amplification are approximately 14 kb, which span the region of exon 1 to exon 6 of both CES1A1 and CES1A2/CES1A3 genes (Figure 1A). To determine whether the variants Gly143Glu and Asp260fs are within CES1A1 or CES1A2/CES1A3 gene, the CES1A1 and CES1A2/CES1A3 long-range PCR products from the PM and his parents were used as the DNA template for the amplification of CES1 exon 4 and exon 6, where the Gly143Glu and Asp260fs are located, respectively. Additionally, the exon 3 of CES1A2/CES1A3 of the PM and his parents was amplified for DNA sequencing to determine his genotypes of CES1A2/CES1A3. The primers for exon 3, 4, 6 amplifications and the PCR conditions are listed in Supplementary Table 2. The PCR products were then subject to bidirectional direct DNA sequencing utilizing the same primers for PCR. The DNA sequencing results of exon 4 of CES1A1 and CES1A2/CES1A3 genes demonstrated that the PM and his father are the heterozygotes of Gly143Glu in CES1A1, and his mother is wild type (Figure 1B). All of these subjects carry wild type exon 4 of CES1A2/CES1A3. Thus, it is concluded that the SNP Gly143Glu is located in CES1A1 rather than CES1A2/CES1A3 gene. The exon 3 sequencing data from CES1A2/CES1A3 long-range PCR assessments indicated that the PM and his parents carry functional CES1A2 gene instead of CES1A3 pseudogene. The sequencing data from exon 6 of CES1A1 and CES1A2/CES1A3 of the PM and his mother revealed that Asp260fs is a mutation of the CES1A2 gene. Though previous in vitro functional studies have demonstrated that catalytic activity was profoundly impaired in both mutations , the Gly143Glu may have more significant influence on the enzyme activity than the Asp260fs in vivo as CES1A1 transcription efficiency is markedly higher than CES1A2.
The assay was then applied to DNA extracted from saliva samples collected from 107 children diagnosed with ADHD who were treated with dl-methylphenidate. The investigators sought the presence of the Gly143Glu variant as well as other variants within the CES1A1 exon 4 and its adjacent intronic regions. Genotyping of Asp260fs was not pursued as previous studies have found that it is an extremely rare mutation . The study population consisted of 82 males and 25 females, including 88 Caucasians, 14 African-Americans, and 5 classified as “other” according to caregiver reports of participants’ racial designation. Subjects’ age ranged from 7 to 11 years. Among these patients, one subject was determined to be a homozygote of Gly143Glu (Figure 2A) and two subjects were heterozygous while all others from this cohort were wild type. This represents the first case of homozygous Gly143Glu that has been reported to date.
In addition to the targeted Gly143Glu variant, 6 novel variants were discovered during this study including T9298C, A9324G, T9386C, C9387A, C9604T, and G9635A located in the introns 3 and 4 of CES1A1, and subsequently reported to NCBI SNP database. Among the newly identified variants, the SNP C9604T appears to be significantly more prevalent in the African-American population relative to Caucasians (32.1% vs 2.3%). The genotype frequencies of these variants in the tested population are summarized in Supplementary Table 3. No deviation from Hardy-Weinberg Equilibrium was detected for any variants among the population.
To compare the results of this long-range PCR based genotyping assay with more conventional genotyping methods previously used by other groups and our own, the subjects carrying the Gly143Glu variant and eight randomly selected wild types were re-genotyped utilizing the Taqman® and direct sequencing assays as described in our previously published study . As anticipated, the Taqman® assay was not able to differentiate the Gly143Glu homozygote from the heterozygotes, but grouped this subject together with the heterozygotes (Figure 2C). Furthermore, the direct sequencing analysis of genomic DNA incorrectly reported that the homozygous subject was a heterozygous Gly143Glu carrier due to the fact that both CES1A1 and CES1A2/CES1A3 genes were amplified when genomic DNA was utilized as the PCR template (Figure 2B). The results highlighted the limitations of conventional genotyping assays for detecting CES1 variants. However, these more conventional assays may serve as a means for initial screening of the presence of CES1 variants as these assays are relatively inexpensive and time-effective relative to our long-range PCR-based discriminative method.
In summary, the development of a discriminative CES1 genotyping assay is described capable of specifically detecting variants between CES1A1 and CES1A2/CES1A3 genes. This newly developed method revealed that the variants Gly143Glu and Asp260fs are located in CES1A1 and CES1A2 genes, respectively. The comparison of the genotyping results from this novel method versus the commonly used Taqman® and direct sequencing assays demonstrates the limitations of the latter, and the necessity of replacing the conventional CES1 genotyping assay with a more discriminative genotyping method when conducting pharmacogenomic studies involving CES1 gene.
This study was supported in part by the National Institute of Mental Health Career Development Award (K23MH083881, Tanya E. Froehlich).
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