Significant association of DRD2 enhancer variant rs12364283 with heroin addiction in a Pakistani population.

The dopamine D2 receptor encoded by DRD2 has been implicated in multiple psychiatric disorders, mediated at least in part by two intronic variants affecting mRNA splicing, rs1076560 and rs2283265, and a less frequent enhancer variant, rs12364283, which increases DRD2 mRNA expression. This study tests whether these functionally validated variants confer susceptibility toward heroin addiction in a Pakistani population. A total of 540 heroin addicts and 467 healthy controls were genotyped, basic allele and genotype tests were performed. Neither rs1076560 nor rs2283265 significantly associated with heroin addiction. The enhancer rs12364283 occurs more frequently in heroin-dependent cases than controls (MAF 13% vs. 7%, respectively), revealing significant association with heroin addiction (p = 3.0E-06, OR 2.1). This study identifies rs12364283 of DRD2 as a potential risk factor for heroin addiction in the Pakistani study population. This enhancer variant had been shown to increase DRD2 mRNA expression, a possible factor in increased vulnerability to heroin addiction. Further studies are needed to validate this association of rs12364283.

Testing genetic modifiers of behavior and response to atomoxetine in autism spectrum disorder with ADHD.

BACKGROUND: Frequent non-pathogenic genetic variants may act as moderators of phenotypic severity for complex disorders such as autism spectrum disorder (ASD). We previously identified polymorphisms affecting mRNA expression of candidate genes, including tryptophan hydroxylase 2 (TPH2), dopamine beta hydroxylase (DBH), and dopamine transporter (DAT, SLC6A3). METHOD: We compare genotypes and (1) clinical response to atomoxetine, (2) scores from the Autism Diagnostic Interview-Revised (ADI-R), and (3) severity of Attention Deficit Hyperactivity Disorder (ADHD) symptoms in a cohort of patients with ASD from multiple study sites. RESULTS: There was no association between CYP2D6 metabolizer status and atomoxetine response. TPH2 rs7305115 genotype was associated with ADI-R Restrictive/Repetitive Behavior score (p=0.03). DBH rs1611115 genotype was associated with ADI-R Social score (p=0.002) and Restrictive/Repetitive Behavior score (p=0.04). The DAT intron 8 5/6 repeat was associated with ADHD symptoms (ABC Hyperactivity p=0.01 and SNAP ADHD p=0.03), replicating a previous finding. CONCLUSIONS: We find associations between ASD phenotypes and regulatory variants in catecholamine biosynthesis genes. This work may help guide future genetics studies related to ASD.

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

Working memory in pregnant women: Relation to estrogen and antepartum depression.

This article is part of a Special Issue "Estradiol and cognition". Subjective changes in concentration and memory are commonly reported by women during the second or third trimesters of pregnancy, but the nature of the problem is poorly understood. We hypothesized that these self-reports might reflect difficulties in working memory (WM). It was further hypothesized that antepartum depression (depression arising during pregnancy) may play an etiological role, either on its own or due to secondary changes in endocrine function or sleep. Using WM tasks that emphasized executive control processes mediated by the prefrontal cortex (PFC) we compared pregnant women tested at 34-36 weeks of gestation (n = 28) with age- and education-matched non-pregnant controls (n = 26). All pregnant women were screened for depression. Evidence of a WM disturbance was found, and was evident only among pregnant women showing depressive symptoms. In contrast, pregnant women who were not depressed showed WM performance that equalled, or even significantly exceeded, non-pregnant controls. No significant differences were observed on control tests of other cognitive functions. Multiple regression revealed that serum estradiol concentrations, along with severity of depressive affect but not sleep disruption, significantly predicted variation in the WM scores. In agreement with studies of estradiol and WM in other contexts, higher estradiol was associated with better WM, while higher levels of depressive symptoms predicted poorer WM. We conclude that memory disturbance during gestation might not be as widespread as commonly believed, but can be seen among women experiencing antepartum depression. The high level of WM performance found in healthy, non-depressed, pregnant women is discussed from an adaptationist perspective.

Regulatory polymorphisms in human DBH affect peripheral gene expression and sympathetic activity.

RATIONALE: Dopamine ß-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine in the central nervous system and peripherally. DBH variants are associated with large changes in circulating DBH and implicated in multiple disorders; yet causal relationships and tissue-specific effects remain unresolved. OBJECTIVE: To characterize regulatory variants in DBH, effect on mRNA expression, and role in modulating sympathetic tone and disease risk. METHODS AND RESULTS: Analysis of DBH mRNA in human tissues confirmed high expression in the locus coeruleus and adrenal gland, but also in sympathetically innervated organs (liver>lung>heart). Allele-specific mRNA assays revealed pronounced allelic expression differences in the liver (2- to 11-fold) attributable to promoter rs1611115 and exon 2 rs1108580, but only small differences in locus coeruleus and adrenals. These alleles were also associated with significantly reduced mRNA expression in liver and lung. Although DBH protein is expressed in other sympathetically innervated organs, mRNA levels were too low for analysis. In mice, hepatic Dbh mRNA levels correlated with cardiovascular risk phenotypes. The minor alleles of rs1611115 and rs1108580 were associated with sympathetic phenotypes, including angina pectoris. Testing combined effects of these variants suggested protection against myocardial infarction in 3 separate clinical cohorts. CONCLUSIONS: We demonstrate profound effects of DBH variants on expression in 2 sympathetically innervated organs, liver and lung, but not in adrenals and brain. Preliminary results demonstrate an association of these variants with clinical phenotypes responsive to peripheral sympathetic tone. We hypothesize that in addition to endocrine effects via circulating DBH and norepinephrine, the variants act in sympathetically innervated target organs.

Serotonin 2A receptor gene (HTR2A) regulatory variants: possible association with severity of depression symptoms in children with autism spectrum disorder.

OBJECTIVE AND BACKGROUND: Our aim was to characterize the association of 2 functional single nucleotide polymorphisms (rs6311 and rs6314) in the serotonin 2A receptor gene (HTR2A) with severity of depression symptoms in children with autism spectrum disorder. These polymorphisms have been shown to be associated with depression symptom severity and response to selective serotonin reuptake inhibitor drugs in adults with diagnosed depressive disorder. METHODS: Parents of 104 children with autism spectrum disorder rated their children's depressive symptoms using a validated scale based on criteria from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. We compared severity of depression symptoms across the rs6311 and rs6314 genotypes, measured from the children's genomic DNA. RESULTS: Children homozygous for the G allele of rs6311 had significantly more severe depression symptoms than those with G/A or A/A genotypes (P=0.025). The effect size (partial eta-squared) was small (ηp=0.047) but was somewhat larger when we controlled for severity of generalized anxiety disorder symptoms (P=0.006, ηp=0.072). When we restricted our analyses to white participants, our results were essentially the same as for the entire sample (P=0.004, ηp=0.086). There was no significant association between rs6314 (C/C versus T carriers) and severity of depression. CONCLUSIONS: Our findings suggest that the HTR2A functional rs6311 polymorphism, which other studies have associated with differential HTR2A mRNA expression, may modulate the severity of depression symptoms in children with autism spectrum disorder. These tentative, hypothesis-generating findings need replication with larger, independent samples.

Association of dopamine gene variants, emotion dysregulation and ADHD in autism spectrum disorder.

The aim of the present study was to evaluate the association of dopaminergic gene variants with emotion dysregulation (EMD) and attention-deficit/hyperactivity disorder (ADHD) symptoms in children with autism spectrum disorder (ASD). Three dopamine transporter gene (SLC6A3/DAT1) polymorphisms (intron8 5/6 VNTR, 3'-UTR 9/10 VNTR, rs27072 in the 3'-UTR) and one dopamine D2 receptor gene (DRD2) variant (rs2283265) were selected for genotyping based on à priori evidence of regulatory activity or, in the case of DAT1 9/10 VNTR, commonly reported associations with ADHD. A sample of 110 children with ASD was assessed with a rigorously validated DSM-IV-referenced rating scale. Global EMD severity (parents' ratings) was associated with DAT1 intron8 (ηp(2)=.063) and rs2283265 (ηp(2)=.044). Findings for DAT1 intron8 were also significant for two EMD subscales, generalized anxiety (ηp(2)=.065) and depression (ηp(2)=.059), and for DRD2 rs2283265, depression (ηp(2)=.053). DRD2 rs2283265 was associated with teachers' global ratings of ADHD (ηp(2)=.052). DAT1 intron8 was associated with parent-rated hyperactivity (ηp(2)=.045) and both DAT1 9/10 VNTR (ηp(2)=.105) and DRD2 rs2283265 (ηp(2)=.069) were associated with teacher-rated inattention. These findings suggest that dopaminergic gene polymorphisms may modulate EMD and ADHD symptoms in children with ASD but require replication with larger independent samples.

Association study of the estrogen receptor gene ESR1 with postpartum depression--a pilot study.

Perinatal mood disorders, such as postpartum depression (PPD), are costly for society, with potentially serious consequences for mother and child. While multiple genes appear to play a role in PPD susceptibility, the contributions of specific genetic variations remain unclear. Previously implicated as a candidate gene, the estrogen receptor alpha gene (ESR1) is a key player in mediating hormonal differences during pregnancy and the postpartum period. This study addresses genetic factors in perinatal mood disorders, testing nine polymorphisms in ESR1. Two hundred fifty-seven postpartum women were screened for mood disorders, including 52 women with PPD and 32 without any symptoms of mood disorders. We detected a significant association for the upstream TA microsatellite repeat with Edinburgh Postnatal Depression Scale scores (p = 0.007). The same variant was also associated with the occurrence of PPD. Separately, 11 candidate functional polymorphisms in 7 additional genes were genotyped to investigate gene-gene interaction with the ESR1 TA repeat, identifying a potential interaction with the serotonin transporter. Our results support a role for ESR1 in the etiology of PPD, possibly through the modulation of serotonin signaling. Our findings for ESR1 could have broad implications for other disorders and therapies that involve estrogens.

Cholesteryl Ester Transfer Protein (CETP) polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk.

Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.

Dopamine transporter gene variant affecting expression in human brain is associated with bipolar disorder.

The gene encoding the dopamine transporter (DAT) has been implicated in CNS disorders, but the responsible polymorphisms remain uncertain. To search for regulatory polymorphisms, we measured allelic DAT mRNA expression in substantia nigra of human autopsy brain tissues, using two marker SNPs (rs6347 in exon 9 and rs27072 in the 3'-UTR). Allelic mRNA expression imbalance (AEI), an indicator of cis-acting regulatory polymorphisms, was observed in all tissues heterozygous for either of the two marker SNPs. SNP scanning of the DAT locus with AEI ratios as the phenotype, followed by in vitro molecular genetics studies, demonstrated that rs27072 C>T affects mRNA expression and translation. Expression of the minor T allele was dynamically regulated in transfected cell cultures, possibly involving microRNA interactions. Both rs6347 and rs3836790 (intron8 5/6 VNTR) also seemed to affect DAT expression, but not the commonly tested 9/10 VNTR in the 3'UTR (rs28363170). All four polymorphisms (rs6347, intron8 5/6 VNTR, rs27072 and 3'UTR 9/10 VNTR) were genotyped in clinical cohorts, representing schizophrenia, bipolar disorder, depression, and controls. Only rs27072 was significantly associated with bipolar disorder (OR = 2.1, p = 0.03). This result was replicated in a second bipolar/control population (OR = 1.65, p = 0.01), supporting a critical role for DAT regulation in bipolar disorder.

Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia.

Dopamine D2 receptor signalling is strongly implicated in the aetiology of schizophrenia. We have recently characterized the function of three DRD2 SNPs: rs12364283 in the promoter affecting total D2 mRNA expression; rs2283265 and rs1076560, respectively in introns 5 and 6, shifting mRNA splicing to two functionally distinct isoforms, the short form of D2 (D2S) and the long form (D2L). These two isoforms differentially contribute to dopamine signalling in prefrontal cortex and in striatum. We performed a case-control study to determine association of these variants and of their main haplotypes with several schizophrenia-related phenotypes. We demonstrate that the minor allele in the intronic variants is associated with reduced expression of %D2S of total mRNA in post-mortem prefrontal cortex, and with impaired working memory behavioural performance, both in patients and controls. However, the fMRI results show opposite effects in patients compared with controls: enhanced engagement of prefronto-striatal pathways in controls and reduced activity in patients. Moreover, the promoter variant is also associated with working memory activity in prefrontal cortex and striatum of patients, and less robustly with negative symptoms scores. Main haplotypes formed by the three DRD2 variants showed significant associations with these phenotypes consistent with those of the individual SNPs. Our results indicate that the three functional DRD2 variants modulate schizophrenia phenotypes possibly by modifying D2S/D2L ratios in the context of different total D2 density.

Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues.

BACKGROUND: Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms - mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here 'structural RNA SNPs' (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have used AEI to discover and characterize regulatory polymorphisms in OPRM1, TPH2, MDR1, DRD2, and VKORC1. The objective of this study was to use AEI to determine the extent of cis-regulatory factors in pharmacogenetic genes. METHODS: We applied a rapid and accurate AEI methodology for testing 42 genes implicated in cardiovascular and central nervous system diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. RESULTS: Substantial AEI was observed in approximately 55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in ACE and SOD2 mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that approximately 90% of srSNPs affect mRNA folding, and hence potentially function. CONCLUSION: Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.

Allelic mRNA expression of X-linked monoamine oxidase a (MAOA) in human brain: dissection of epigenetic and genetic factors.

A pVNTR repeat polymorphism located in the promoter region of the X-linked MAOA gene has been associated with mental disorders. To explore the effect of polymorphisms and epigenetic factors on mRNA expression, we have measured allelic expression imbalance (AEI) in female human brain tissue, employing two frequent marker single nucleotide polymorphisms (SNPs) in exon 8 (T890G) and exon 14 (C1409T) of MAOA. This approach compares one allele against the other in the same subject. AEI ratios ranged from 0.3 to 4 in prefrontal cortex, demonstrating the presence of strong cis-acting factors in mRNA expression. Analysis of CpG methylation in the MAOA promoter region revealed substantial methylation in females but not in males. MAOA methylation ratios for the three- and four-repeat pVNTR alleles of MAOA did not correlate with X-chromosome inactivation ratios, determined at the X-linked androgen receptor locus, suggesting an alternative process of dosage compensation in females. The extent of allelic MAOA methylation was highly variable and correlated with AEI (R2=0.5 and 0.7 at two CpG loci), indicating that CpG methylation regulates gene expression. Genetic factors appeared also to contribute to the AEI ratios. Genotyping of 13 MAOA polymorphisms in female subjects showed strong association with a haplotype block spanning from the pVNTR to the marker SNP. Therefore, allelic mRNA expression is affected by genetic and epigenetic events, both with the potential to modulate biogenic amine tone in the CNS.

Genetic variants of the human dipeptide transporter PEPT1.

We tested whether genetic polymorphisms affect activity of the dipeptide transporter PEPT1, which mediates bioavailability of peptidomimetic drugs. All 23 exons and adjoining intronic sections of PEPT1 (SLC15A1) were sequenced in 247 individuals of various ethnic origins (Coriell collection). Of 38 single nucleotide polymorphisms (SNPs), 21 occurred in intronic and noncoding regions and 17 in exonic coding region, of which nine were nonsynonymous. Eight nonsynonymous variants were cloned into expression vectors and functionally characterized after transient transfection into Cos7 and Chinese hamster ovary cells. None of the variants had altered transport activity for various ligands, supporting previous results, except for the new, low-frequency PEPT1-F28Y. This variant displayed significantly reduced cephalexin uptake attributable to increased K(m). Altered pH dependence of substrate transport suggested a role for F28Y in H(+)-driven translocation. Haplotype analysis revealed significant differences among ethnic populations. To search for cis-acting polymorphisms affecting transcription and mRNA processing, we measured allelic PEPT1 mRNA expression in human intestinal biopsy samples using a frequent-marker SNP in exon 17. Of 24 heterozygous samples, significant differences in allelic mRNA levels of 20 to 30% were observed in seven tissues. However, the small difference suggests that cis-acting regulatory factors have only limited effects on transporter activity. We also measured the relative formation of a splice variant (PEPT1-RF). PEPT1-RF mRNA levels ranged from 2 to 44% of total PEPT1-related mRNA, with potential consequences for drug absorption. Together with previous results, this study reveals a relatively low level of genetic variability in polymorphisms affecting both protein function and gene regulation.

Functional characterization of the peptide transporter PEPT2 in primary cultures of human upper airway epithelium.

This study characterizes the expression and function of the peptide transporter hPepT2 (SLC15A2) in differentiated primary cultures of human upper airway lung epithelia obtained from six human donors. Genotype analysis of a SNP in exon 15 of hPepT2 genotypes in six donors revealed an expected distribution of the two main variants present at similar frequency (two AA homozygotes, two BB homozygotes, and two AB heterozygotes). Real-time PCR analysis of the hPepT2 mRNA message revealed no significant differences among genotypes. hPEPT2 was expressed on the apical membrane in all donor specimens, demonstrated by cell surface biotinylation and Western analysis (104 kD). We then compared transepithelial transport of the prototypical substrate (3)H-glycylsarcosine in all donor cultures in the absence and presence of known inhibitors of hPEPT2 to ascertain the phenotype of functionally expressed hPepT2 in the upper airway epithelium. An array of inhibitors included dipeptides, beta-lactam antibiotics, bestatin, and ACE inhibitors. hPEPT2 exhibited saturable Michaelis-Menten-type kinetic parameters for GlySar, corroborating previously reported values for K(T) and J(max). Donor-to-donor variation of transport for different substrates did not correlate with hPepT2 haplotypes in this sample cohort. These findings demonstrate functional hPEPT2 transporter expression in primary cultures of human lung epithelial cells. hPEPT2-mediated transport could serve as a strategy for noninvasive systemic delivery of peptides and peptidomimetics drugs.

Genetic variants of the human H+/dipeptide transporter PEPT2: analysis of haplotype functions.

PEPT2 is a high-affinity H+/dipeptide transporter expressed in kidney, brain, lung, and mammary gland. The physiological role of PEPT2 in kidney is to reabsorb small peptides generated by luminal peptidases. PEPT2 is also a transporter for peptide-like drugs such as penicillins and cephalosporins. We have conducted a haplotype analysis of 27 single nucleotide polymorphisms located in or near exons of the human gene encoding hPEPT2 (SLC15A2), using genotyping data from 247 genomic DNA samples from the Coriell collection. Our analysis reveals that hPEPT2 has a >6-kilobase sequence block with at least 10 abundant polymorphisms in almost complete linkage disequilibrium. As a result, only two main hPEPT2 variants exist (hPEPT2*1 and *2) with several phased amino acid substitutions, present in substantial frequencies in all ethnic groups tested. When expressed in Chinese hamster ovary cells, hPEPT2*1 and *2 displayed similar Vmax values for glycyl-sarcosine (Gly-Sar), but they differed significantly in their Km values (83 +/- 16 and 233 +/- 38 microM, respectively). Moreover, hPEPT2*1 and *2 differed in their pH sensitivity for H+/Gly-Sar transport. In addition, hPEPT2*1 and *2 generated varying levels of mRNA in nine heterozygous kidney tissue samples, including one allele expressing no detectable mRNA, suggesting the presence of cis-acting polymorphisms affecting transcription or mRNA processing. The results indicate that polymorphisms in the gene encoding hPEPT2 can alter substrate transport and therefore could affect drug disposition in vivo.

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves.

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.

Pharmacogenomics of multigenic diseases: sex-specific differences in disease and treatment outcome.

Numerous genetic variations have been shown to affect disease susceptibility and drug response. Pharmacogenomics aims at improving therapy on the basis of genetic information for each individual patient. Furthermore, sex chromosomes broadly determine biological differences between males and females. Consequently, substantial sex differences exist in phenotypic manifestation of disease and treatment response. This review discusses the role of sex in coronary artery disease, schizophrenia, and depression--complex multigenic disorders with considerable sex differences in frequency and presentation. Moreover, genetic factors underlying disease and drug response appear to differ between male and female patients. This appears to result at least in part from different physiological effects exerted by sex hormones such that polymorphisms in susceptibility genes may have physiological relevance only in males or females. However, few examples have been discovered to play a role in complex multigenic diseases, and the mechanistic basis of genetic variants as sex-dependent susceptibility factors has yet to be explored. Therefore, pharmacogenomic studies must consider sex differences in an effort to optimize individual drug therapy.