Novel procedure with improved resolution and specificity for amplification and differentiation of variants of the gene encoding carboxylesterase 1.

Carboxylesterase 1 (CES1) is implicated in the metabolism of several commonly used drugs and other xenobiotics. The gene encoding this enzyme, CES1, is duplicated in some individuals. The original gene copy is called CES1A1. The duplicated version, CES1A2, is a hybrid of CES1A1 and the CES1-related pseudogene, CES1P1. Variants of CES1A2 with a weak and a strong promoter, respectively, have been reported. In addition, there are chimeric subtypes of CES1A1 that contain a segment of CES1P1. Collectively, this represents challenges to the genotyping of CES1 that previous procedures have had difficulties in solving, frequently leading to loss of specificity and inaccurate genotyping. Here, we report a novel and specific procedure that can selectively amplify CES1A1 and CES1A2 and accurately determine their variants. This procedure may be useful for personalization of treatments with drugs metabolized by CES1.

The impact of CES1 genotypes on the pharmacokinetics of methylphenidate in healthy Danish subjects.

AIMS: This study investigated the influence of CES1 variations, including the single nucleotide polymorphism (SNP) rs71647871 (G143E) and variation in copy number, on the pharmacokinetics of a single oral dose of 10 mg methylphenidate. METHODS: CES1 genotype was obtained from 200 healthy Danish Caucasian volunteers. Based on the genotype, 44 (19 males and 25 females) were invited to participate in an open, prospective trial involving six predefined genotypes: three groups with two, three and four CES1 copies, respectively; a group of carriers of the CES1 143E allele; a group of individuals homozygous for CES1A1c (CES1VAR); and a group having three CES1 copies, in which the duplication, CES1A2, had increased transcriptional activity. Plasma concentrations of methylphenidate and its primary metabolites were determined at scheduled time points. RESULTS: Median AUC of d-methylphenidate was significantly larger in the group carrying the 143E allele (53.3 ng ml-1  h-1 , range 38.6-93.9) than in the control group (21.4 ng ml-1  h-1 , range 15.7-34.9) (P < 0.0001). Median AUC of d-methylphenidate was significantly larger in the group with four CES1 copies (34.5 ng ml-1  h-1 , range 21.3-62.8) than in the control group (P = 0.01) and the group with three CES1 copies (23.8 ng ml-1  h-1 , range 15.3-32.0, P = 0.03). There was no difference between the groups with two and three copies of CES1. CONCLUSIONS: The 143E allele resulted in an increased AUC, suggesting a significantly decreased CES1 enzyme activity. Surprisingly, this was also the case in subjects with homozygous duplication of CES1, perhaps reflecting an undiscovered mutation affecting the activity of the enzyme.

The impact of human CES1 genetic variation on enzyme activity assessed by ritalinic acid/methylphenidate ratios.

The present clinical trial investigated the impact of selected SNPs in CES1 on the metabolic activity of the enzyme. For this purpose, we used methylphenidate (MPH) as a pharmacological probe and the d-RA/d-MPH (metabolite/parent drug) ratios as a measure of enzymatic activity. This metabolic ratio (MR) was validated against the AUC ratios (AUCd -RA /AUCd -MPH ). CES1 SNPs from 120 volunteers were identified, and 12 SNPs fulfilling predefined inclusion criteria were analysed separately, comparing the effect of each genotype on the metabolic ratios. The SNP criteria were as follows: presence of Hardy-Weinberg equilibrium, a minor allele frequency ≥ 0.01 and a clearly interpretable sequencing result in at least 30% of the individuals. Each participant ingested 10 mg of racemic methylphenidate, and blood samples were drawn prior to and 3 hours after drug administration. The SNP analysis confirmed the considerable impact of rs71647871 (G143E) in exon 4 on drug metabolism. In addition, three volunteers with markedly lower median MR, indicating decreased CES1 activity, harboured the same combination of three SNPs in intron 5. The median MR for these SNPs was 8.2 for the minor allele compared to 16.4 for the major alleles (P = 0.04). Hence, one of these or the combination of these SNPs could be of clinical significance considering that the median MR of the G143E group was 5.4. The precise genetic relationship of this finding is currently unknown, as is the clinical significance.

Novel approach for CES1 genotyping: integrating single nucleotide variants and structural variation.

AIM: Development of a specific procedure for genotyping of CES1A1 (CES1) and CES1A2, a hybrid of CES1A1  and the pseudogene CES1P1. MATERIALS & METHODS: The number of CES1A1 and CES1A2  copies and that of CES1P1  were determined using real-time PCR. Long range PCRs followed by secondary PCRs allowed sequencing of single nucleotide variants in CES1A1 and CES1A2. RESULTS & CONCLUSION: A procedure consisting of two main steps was developed. Its first main step, the copy number determination, informed about presence of CES1A2 . This information enabled choice of PCR in the second main step, which selectively amplified CES1A1 and, if present, also CES1A2, for subsequent sequencing. Examination of 501 DNA samples suggested that our procedure is specific with potential for personalization of drug treatments.

Reappraisal of the genetic diversity and pharmacogenetic assessment of CES1.

The CES1 gene encodes a hydrolase that metabolizes important drugs. Variants generated by exchange of segments with CES1P1 complicate genotyping of CES1. Using a highly specific procedure we examined DNA samples from 200 Caucasians and identified 46 single nucleotide variants (SNVs) in CES1 and 21 SNVs in CES1A2, a hybrid composed of CES1 and CES1P1. Several of these SNVs were novel. The frequencies of SNVs with a potential functional impact were below 0.02 suggesting limited pharmacogenetic potential for CES1 genotyping. In silico PCR revealed that the majority of the primer pairs for amplification of CES1 or CES1A2 in three previous studies lacked specificity, which partially explains a limited overlap with our findings.

The Pharmacokinetics of Enalapril in Relation to CES1 Genotype in Healthy Danish Volunteers.

This study investigated the influence of variations in the carboxylesterase 1 gene (CES1) on the pharmacokinetics of enalapril. Forty-three healthy, Danish, Caucasian volunteers representing different pre-defined genotypes each received 10 mg of enalapril. At specified time-points, plasma concentrations of enalapril and the active metabolite enalaprilat were measured. The volunteers were divided into six different groups according to their genetic profile of CES1: group 1 (control group, n = 16) with two CES1 copies without non-synonymous SNPs in the exons; group 2 (n = 5) with four copies of CES1; group 3 (n = 6) harbouring the G143E polymorphism; group 4 (n = 2) with three CES1 copies and increased transcriptional activity of the duplication (CES1A2); group 5 (n = 4) harbouring the CES1A1c variant; and group 6 (n = 10) with three CES1 copies and the common promoter with low transcriptional activity of the duplication. The median AUC of enalaprilat in the control group was not significantly different from any of the other five groups (297 ng/ml x h in the control group versus 310, 282, 294, 344 and 306 ng/ml x h in groups 2-6, respectively). The terminal half-life of enalaprilat was significantly longer in group 6 compared with the control group (26.7 hr versus 12.7 hr, respectively). However, this was not considered clinically relevant. In conclusion, none of the selected variations of CES1 had a clinically relevant impact on the metabolism of enalapril.

Pharmacodynamic Impact of Carboxylesterase 1 Gene Variants in Patients with Congestive Heart Failure Treated with Angiotensin-Converting Enzyme Inhibitors.

BACKGROUND: Variation in the carboxylesterase 1 gene (CES1) may contribute to the efficacy of ACEIs. Accordingly, we examined the impact of CES1 variants on plasma angiotensin II (ATII)/angiotensin I (ATI) ratio in patients with congestive heart failure (CHF) that underwent ACEI dose titrations. Five of these variants have previously been associated with drug response or increased CES1 expression, i.e., CES1 copy number variation, the variant of the duplicated CES1 gene with high transcriptional activity, rs71647871, rs2244613, and rs3815583. Additionally, nine variants, representatives of CES1Var, and three other CES1 variants were examined. METHODS: Patients with CHF, and clinical indication for ACEIs were categorized according to their CES1 genotype. Differences in mean plasma ATII/ATI ratios between genotype groups after ACEI dose titration, expressed as the least square mean (LSM) with 95% confidence intervals (CIs), were assessed by analysis of variance. RESULTS: A total of 200 patients were recruited and 127 patients (63.5%) completed the study. The mean duration of the CHF drug dose titration was 6.2 (SD 3.6) months. After ACEI dose titration, there was no difference in mean plasma ATII/ATI ratios between subjects with the investigated CES1 variants, and only one previously unexplored variation (rs2302722) qualified for further assessment. In the fully adjusted analysis of effects of rs2302722 on plasma ATII/ATI ratios, the difference in mean ATII/ATI ratio between the GG genotype and the minor allele carriers (GT and TT) was not significant, with a relative difference in LSMs of 0.67 (95% CI 0.43-1.07; P = 0.10). Results of analyses that only included enalapril-treated patients remained non-significant after Bonferroni correction for multiple parallel comparisons (difference in LSM 0.60 [95% CI 0.37-0.98], P = 0.045). CONCLUSION: These findings indicate that the included single variants of CES1 do not significantly influence plasma ATII/ATI ratios in CHF patients treated with ACEIs and are unlikely to be primary determinants of ACEI efficacy.

Linkage and whole genome sequencing identify a locus on 6q25-26 for formal thought disorder and implicate MEF2A regulation.

Formal thought disorder is a major feature of schizophrenia and other psychotic disorders. It is heritable, found in healthy relatives of patients with schizophrenia and other mental disorders but knowledge of specific genetic factors is lacking. The aim of this study was to search for biologically relevant high-risk variants. Formal thought disorder was assessed in participants in the Copenhagen Schizophrenia Linkage Study (N=236), a unique high-risk family study comprised of six large pedigrees. Microsatellite linkage analysis of formal thought disorder was performed and subsequent haplotype analysis of the implicated region using phased microsatellite and SNP genotypes. Whole genome sequencing (N=3) was used in the attempt to identify causative variants in the linkage region. Linkage analysis of formal thought disorder resulted in a single peak at chromosome 6(q26-q27) centred on marker D6S1277, with a maximum LOD score of 4.0. Phasing and fine mapping of the linkage peak identified a 5.5Mb haplotype (chr6:162242322-167753547, hg18) in 31 individuals, all belonging to the same pedigree sharing the haplotype from a common ancestor. The haplotype segregated with increased total thought disorder index score (P=4.9 × 10(-5)) and qualitatively severe forms of thought disturbances. Whole genome sequencing identified a novel nucleotide deletion (chr6:164377205 AG>A, hg18) predicted to disrupt the potential binding of the transcription factor MEF2A. The MEF2A binding site is located between two genes previously reported to associate with schizophrenia, QKI (HGNC:21100) and PDE10A (HGNC:8772). The findings are consistent with MEF2A deregulation conferring risk of formal thought disorder.

Individualization of treatments with drugs metabolized by CES1: combining genetics and metabolomics.

CES1 is involved in the hydrolysis of ester group-containing xenobiotic and endobiotic compounds including several essential and commonly used drugs. The individual variation in the efficacy and tolerability of many drugs metabolized by CES1 is considerable. Hence, there is a large interest in individualizing the treatment with these drugs. The present review addresses the issue of individualized treatment with drugs metabolized by CES1. It describes the composition of the gene encoding CES1, reports variants of this gene with focus upon those with a potential effect on drug metabolism and provides an overview of the protein structure of this enzyme bringing notice to mechanisms involved in the regulation of enzyme activity. Subsequently, the review highlights drugs metabolized by CES1 and argues that individual differences in the pharmacokinetics of these drugs play an important role in determining drug response and tolerability suggesting prospects for individualized drug therapies. Our review also discusses endogenous substrates of CES1 and assesses the potential of using metabolomic profiling of blood to identify proxies for the hepatic activity of CES1 that predict the rate of drug metabolism. Finally, the combination of genetics and metabolomics to obtain an accurate prediction of the individual response to CES1-dependent drugs is discussed.

Porcine Parkin: molecular cloning of PARK2 cDNA, expression analysis, and identification of a splicing variant.

Parkin, encoded by the PARK2 gene, is an E3 ligase which functions as an integral component of the cytoplasmic ubiquitin/proteasomal protein degradation pathway. Mutations in the PARK2 gene, resulting in the loss of parkin function, leads to autosomal recessive juvenile Parkinsonism (AR-JP). This work reports the cloning and characterization of the porcine (Sus scrofa) PARK2 cDNA (SsPARK2) and splicing variants hereof. The PARK2 cDNA was amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine PARK2 cDNA codes for a protein of 461 amino acids which shows a high similarity to orangutan (91%), human (86%), and to rat (82%) parkin. A splicing variant of the porcine PARK2 with a complete deletion of exon 9 was also identified. Expression analysis by quantitative real-time RT-PCR revealed presence of PARK2 transcript in all examined organs and tissues. Differential expression was observed, with very high levels of PARK2 mRNA in cerebellum, heart, and kidney. In addition, expression analysis showed that porcine PARK2 transcripts could be detected early in embryo development in different brain regions. The porcine PARK2 orthologue was mapped to chromosome 1p24-25. Single nucleotide polymorphism (SNP) analysis revealed seven SNPs in the porcine PARK2 gene, one missense and one silent mutation in exon 7 and five SNPs in intron 7.