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1.  Next-generation DNA sequencing-based assay for measuring allelic expression imbalance (AEI) of candidate neuropsychiatric disorder genes in human brain 
BMC Genomics  2011;12:518.
Common genetic variants that regulate gene expression are widely suspected to contribute to the etiology and phenotypic variability of complex diseases. Although high-throughput, microarray-based assays have been developed to measure differences in mRNA expression among independent samples, these assays often lack the sensitivity to detect rare mRNAs and the reproducibility to quantify small changes in mRNA expression. By contrast, PCR-based allelic expression imbalance (AEI) assays, which use a "marker" single nucleotide polymorphism (mSNP) in the mRNA to distinguish expression from pairs of genetic alleles in individual samples, have high sensitivity and accuracy, allowing differences in mRNA expression greater than 1.2-fold to be quantified with high reproducibility. In this paper, we describe the use of an efficient PCR/next-generation DNA sequencing-based assay to analyze allele-specific differences in mRNA expression for candidate neuropsychiatric disorder genes in human brain.
Using our assay, we successfully analyzed AEI for 70 candidate neuropsychiatric disorder genes in 52 independent human brain samples. Among these genes, 62/70 (89%) showed AEI ratios greater than 1 ± 0.2 in at least one sample and 8/70 (11%) showed no AEI. Arranging log2AEI ratios in increasing order from negative-to-positive values revealed highly reproducible distributions of log2AEI ratios that are distinct for each gene/marker SNP combination. Mathematical modeling suggests that these log2AEI distributions can provide important clues concerning the number, location and contributions of cis-acting regulatory variants to mRNA expression.
We have developed a highly sensitive and reproducible method for quantifying AEI of mRNA expressed in human brain. Importantly, this assay allowed quantification of differential mRNA expression for many candidate disease genes entirely missed in previously published microarray-based studies of mRNA expression in human brain. Given the ability of next-generation sequencing technology to generate large numbers of independent sequencing reads, our method should be suitable for analyzing from 100- to 200-candidate genes in 100 samples in a single experiment. We believe that this is the appropriate scale for investigating variation in mRNA expression for defined sets candidate disorder genes, allowing, for example, comprehensive coverage of genes that function within biological pathways implicated in specific disorders. The combination of AEI measurements and mathematical modeling described in this study can assist in identifying SNPs that correlate with mRNA expression. Alleles of these SNPs (individually or as sets) that accurately predict high- or low-mRNA expression should be useful as markers in genetic association studies aimed at linking candidate genes to specific neuropsychiatric disorders.
PMCID: PMC3228908  PMID: 22013986
2.  Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues 
Pharmacogenetics and genomics  2008;18(9):781-791.
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 applied a rapid and accurate AEI methodology for testing 42 genes implicated in human diseases and drug response, specifically cardiovascular and CNS diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. Substantial AEI was observed in ∼55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in SOD2 and ACE 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 ∼90% of srSNPs affects mRNA folding, and hence potentially function. Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.
PMCID: PMC2779843  PMID: 18698231
3.  Low ABCB1 Gene Expression Is an Early Event in Colorectal Carcinogenesis 
PLoS ONE  2013;8(8):e72119.
The ABCB1/MDR1 gene product ABCB1/P-glycoprotein is implicated in the development of colorectal cancer (CRC). NFKB1 encodes transcription factors regulating expression of a number of genes including ABCB1. We have previously found association between the ABCB1 C-rs3789243-T polymorphism and CRC risk and interactions between the ABCB1 C-rs3789243-T and C3435T polymorphisms and meat intake in relation to CRC risk (Andersen, BMC Cancer, 2009, 9, 407). ABCB1 and NFKB1 mRNA levels were assessed in intestinal tissue from 122 CRC cases, 101 adenoma cases (12 with severe dysplasia, 89 with mild-moderate dysplasia) and from 18 healthy individuals, together with gene polymorphisms in ABCB1 and NFKB1. ABCB1 mRNA levels were highest in the healthy individuals and significantly lower in mild/moderate and severe dysplasia tissue (P<0.05 for both), morphologically normal tissues close to the tumour (P<0.05), morphologically normal tissue at a distance from the tumour (P<0.05) and CRC tissue (P<0.001). Furthermore, ABCB1 mRNA levels were lower in adenomas and carcinomas compared to morphologically normal tissue from the same individuals (P<0.01). The ABCB1 C-rs3789243-T and NFKB1 -94ins/del homozygous variant genotypes were associated with low ABCB1 mRNA levels in morphologically normal sigmoid tissue from adenoma cases (P<0.05 for both). NFKB1 mRNA levels were lower in both tumour and normal tissue from cancer patients (P<0.001) as compared to healthy individuals but we were unable to show association between NFKB1 -94ins/del genotype and NFKB1 mRNA levels. This study suggests that low ABCB1 mRNA levels are an early event in CRC development and that the two polymorphisms affect ABCB1 mRNA levels whereas low NFKB1 mRNA levels occur later in carcinogenesis. Low ABCB1 protein levels may promote colorectal carcinogenesis through increasing intracellular exposure to carcinogenic ABCB1 substrates.
PMCID: PMC3747088  PMID: 23977225
4.  Allelic expression analysis of the osteoarthritis susceptibility gene COL11A1 in human joint tissues 
The single nucleotide polymorphism (SNP) rs2615977 is associated with osteoarthritis (OA) and is located in intron 31 of COL11A1, a strong candidate gene for this degenerative musculoskeletal disease. Furthermore, the common non-synonymous COL11A1 SNP rs1676486 is associated with another degenerative musculoskeletal disease, lumbar disc herniation (LDH). rs1676486 is a C-T transition mediating its affect on LDH susceptibility by modulating COL11A1 expression. The risk T-allele of rs1676486 leads to reduced expression of the COL11A1 transcript, a phenomenon known as allelic expression imbalance (AEI). We were keen therefore to assess whether the effect that rs1676486 has on COL11A1 expression in LDH is also observed in OA and whether the rs2615977 association to OA also marked AEI.
Using RNA from OA cartilage, we assessed whether either SNP correlated with COL11A1 AEI by 1) measuring COL11A1 expression and stratifying the data by genotype at each SNP; and 2) quantifying the mRNA transcribed from each allele of the two SNPs. We also assessed whether rs1676486 was associated with OA susceptibility using a case–control cohort of over 18,000 individuals.
We observed significant AEI at rs1676486 (p < 0.0001) with the T-allele correlating with reduced COL11A1 expression. This corresponded with observations in LDH but the SNP was not associated with OA. We did not observe AEI at rs2615977.
COL11A1 is subject to AEI in OA cartilage. AEI at rs1676486 is a risk factor for LDH, but not for OA. These two diseases therefore share a common functional phenotype, namely AEI of COL11A1, but this appears to be a disease risk only in LDH. Other functional effects on COL11A1 presumably account for the OA susceptibility that maps to this gene.
PMCID: PMC3599795  PMID: 23497244
Osteoarthritis; Lumbar disc herniation; Genetics; Susceptibility; COL11A1; Allelic expression
5.  Dopamine Transporter Gene Variant Affecting Expression in Human Brain is Associated with Bipolar Disorder 
Neuropsychopharmacology  2011;36(8):1644-1655.
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.
PMCID: PMC3138671  PMID: 21525861
dopamine transporter; bipolar disorder; allelic expression imbalance; SLC6A3; rs27072; Dopamine; Depression; Unipolar/Bipolar; Pharmacogenetics/Pharmacogenomics; Neurogenetics; Allelic expression imbalance; Dopamine transporter; SLC6A3
6.  Allelic mRNA expression of sortilin-1 (SORL1) mRNA in Alzheimer’s autopsy brain tissues 
Neuroscience letters  2008;448(1):120-124.
Polymorphisms in the gene encoding SORL1, involved in cellular trafficking of APP, have been implicated in late-onset Alzheimer’s disease, by a mechanism thought to affect mRNA expression. To search for regulatory polymorphisms, we have measured allele-specific mRNA expression of SORL1 in human autopsy tissues from the prefrontal cortex of 26 Alzheimer’s patients, and 51 controls, using two synonymous marker SNPs (rs3824968 in exon 34 (11 heterozygous AD subjects and 16 controls), and rs12364988 in exon 6 (8 heterozygous AD subjects)). Significant allelic expression imbalance (AEI), indicative of the presence of cis-acting regulatory factors, was detected in a single control subject, while allelic ratios were near unity for all other subjects. We genotyped 7 SNPs in two haplotype blocks that had previously been implicated in Alzheimer’s disease. Since each of these SNPs was heterozygous in several subjects lacking AEI, this study fails to support a regulatory role for SORL1 polymorphisms in mRNA expression.
PMCID: PMC2612539  PMID: 18938222
Alzheimer’s disease; SORL1; Allelic expression imbalance
7.  Whole transcriptome RNA-Seq allelic expression in human brain 
BMC Genomics  2013;14:571.
Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals.
We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals.
These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.
PMCID: PMC3765493  PMID: 23968248
RNA-Seq; Whole transcriptome; Allele expression; mRNA expression; Functional genetics; Regulatory polymorphism; eQTL; Read alignment; Next generation sequencing; Bioinformatics
8.  ABCB1 haplotypes are associated with P-gp activity and affect a major molecular response in chronic myeloid leukemia patients treated with a standard dose of imatinib 
Oncology Letters  2014;7(4):1313-1319.
Despite the high efficacy of imatinib mesylate (IM) treatment for chronic myeloid leukemia (CML) patients, some individuals develop resistance due to impaired bioavailability. It has been previously demonstrated that the haplotypes for ATP-binding cassette subfamily B member 1 (ABCB1)with c.1236C>T, c.3435C>T and c.2677G>T/A polymorphisms markedly affect the secondary structure of ABCB1 mRNA and its activity. These modifications may affect efflux transporter activity and response to treatment with IM. The aim of the present study was to investigate the influence of ABCB1 haplotypes on P-glycoprotein (P-gp) activity, IM plasma levels and IM response. In total, 28 chronic-phase CML patients treated with a standard dose of IM (400 mg/day) were studied. The patients were selected according to the haplotypes of ABCB1, with c.1236C>T, c.3435C>T and c.2677G>T polymorphisms, and were classified into two groups based on the presence of the mutated allele in each genotype for the three ABCB1 polymorphisms. In addition, expression of P-gp and breakpoint cluster region-abelson 1 (BCR-ABL1), ABCB1 and solute carrier family 22 member 1 (SLC22A1) mRNA were evaluated. The P-gp activity in the wild-type group was found to be higher than that in the mutated group (59.1 vs. 38.3%; P=0.001). Furthermore, the patients who did not achieve major molecular response (MMR) showed a higher rate of efflux mediated by P-gp when compared with individuals who achieved MMR (64.7 vs. 45.7%; P=0.001). All patients without MMR demonstrated effluxes of >60%. In addition, patients without MMR exhibited lower plasma concentrations of IM compared with those with MMR (0.51 vs. 1.42 μg/ml; P=0.001). Higher levels of SLC22A1 mRNA were observed in patients who achieved MMR and complete molecular response (P<0.05). In conclusion, the ABCB1 1236CT/3435CT/2677GT and 1236TT/3435TT/2677TT haplotypes are associated with reduced P-gp activity and MMR in chronic-phase CML patients treated with a standard dose of IM.
PMCID: PMC3961201  PMID: 24660038
imatinib mesylate; chronic myeloid leukemia; ABCB1
9.  Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1 
BMC Medical Genetics  2014;15:53.
An osteoarthritis (OA) susceptibility locus has been mapped to chromosome 3p21, to a region of high linkage disequilibrium encompassing twelve genes. Six of these genes are expressed in joint tissues and we therefore assessed whether any of the six were subject to cis-acting regulatory polymorphisms active in these tissues and which could therefore account for the association signal.
We measured allelic expression using pyrosequencing assays that can distinguish mRNA output from each allele of a transcript single nucleotide polymorphism. We assessed RNA extracted from the cartilage and other joint tissues of OA patients who had undergone elective joint replacement surgery. A two-tailed Mann–Whitney exact test was used to test the significance of any allelic differences.
GNL3 and SPCS1 demonstrated significant allelic expression imbalance (AEI) in OA cartilage (GNL3, mean AEI = 1.04, p = 0.0002; SPCS1, mean AEI = 1.07, p < 0.0001). Similar results were observed in other tissues. Expression of the OA-associated allele was lower than that of the non-associated allele for both genes.
cis-acting regulatory polymorphisms acting on GNL3 and SPCS1 contribute to the OA association signal at chromosome 3p21, and these genes therefore merit further investigation.
PMCID: PMC4101866  PMID: 24886551
Osteoarthritis; Genetic risk; Single nucleotide polymorphism; Allelic imbalance; Linkage disequilibrium
10.  ABCB1 haplotype and OPRM1 118A > G genotype interaction in methadone maintenance treatment pharmacogenetics 
Genetic variability in ABCB1, encoding the P-glycoprotein efflux transporter, has been linked to altered methadone maintenance treatment dose requirements. However, subsequent studies have indicated that additional environmental or genetic factors may confound ABCB1 pharmacogenetics in different methadone maintenance treatment settings. There is evidence that genetic variability in OPRM1, encoding the mu opioid receptor, and ABCB1 may interact to affect morphine response in opposite ways. This study aimed to examine whether a similar gene-gene interaction occurs for methadone in methadone maintenance treatment.
Opioid-dependent subjects (n = 119) maintained on methadone (15–300 mg/day) were genotyped for five single nucleotide polymorphisms of ABCB1 (61A > G; 1199G > A; 1236C > T; 2677G > T; 3435C > T), as well as for the OPRM1 118A > G single nucleotide polymorphism. Subjects’ methadone doses and trough plasma (R)-methadone concentrations (Ctrough) were compared between ABCB1 haplotypes (with and without controlling for OPRM1 genotype), and between OPRM1 genotypes (with and without controlling for ABCB1 haplotype).
Among wild-type OPRM1 subjects, an ABCB1 variant haplotype group (subjects with a wild-type and 61A:1199G:1236C:2677T:3435T haplotype combination, or homozygous for the 61A:1199G:1236C:2677T:3435T haplotype) had significantly lower doses (median ± standard deviation 35 ± 5 versus 180 ± 65 mg/day, P < 0.01) and Ctrough (78 ± 22 versus 177 ± 97 ng/mL, P < 0.05) than ABCB1 wild-type subjects. Among subjects with the most common ABCB1 haplotype combination (wild-type with 61A:1199G:1236T:2677T:3435T), the OPRM1 118 A/G genotype was associated with a significantly higher Ctrough than 118 A/A (250 ± 126 versus 108 ± 36 ng/mL, P = 0.016). No ABCB1 haplotype group or OPRM1 genotype was associated with dose or Ctrough without taking into account confounding genetic variability at the other locus. Therefore, two interacting pharmacogenetic determinants of methadone maintenance treatment response were identified, ie, ABCB1, where variants are associated with lower methadone requirements, and OPRM1, where the variant is associated with higher methadone requirements.
These opposing pharmacogenetic effects therefore need to be considered in combination when assessing methadone maintenance treatment pharmacogenetics.
PMCID: PMC3513228  PMID: 23226062
methadone; opiate substitution treatment; ABCB1; P-glycoprotein; OPRM1; receptors; opioid; mu
11.  Polymorphisms in the xenobiotic transporter Multidrug Resistance 1 (MDR1) and interaction with meat intake in relation to risk of colorectal cancer in a Danish prospective case-cohort study 
BMC Cancer  2009;9:407.
The xenobiotic transporters, Multidrug Resistance 1 (MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2) may restrict intestinal absorption of various carcinogens, including heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons (PAH). Cyclooxygenase-2 (COX-2) derived prostaglandins promote gastrointestinal carcinogenesis, affecting angiogenesis, apoptosis, and invasiveness.
The aim of this study was to investigate if polymorphisms in these genes were associated with risk of colorectal cancer (CRC), and to investigate possible interactions with lifestyle factors such as smoking, meat consumption, and NSAID use.
The following polymorphisms were analyzed; a synonymous MDR1 C3435T (rs1045642) in exon26, G-rs3789243-A in intron3, the functional BCRP C421A (rs2231142), the two COX-2 A-1195G (rs689466) and G-765C (rs20417) in the promoter region, and the COX-2 T8473C (rs5275) polymorphisms in the 3'-untranslated region. The polymorphisms were assessed together with lifestyle factors in a nested case-cohort study of 359 cases and a random cohort sample of 765 participants from the Danish prospective Diet, Cancer and Health study.
Carriers of the variant allele of MDR1 intron 3 polymorphism were at 1.52-fold higher risk of CRC than homozygous wild type allele carriers (Incidence rate ratio (IRR) = 1.52, 95% Confidence Interval (CI): 1.12-2.06). Carriers of the variant allele of MDR1 C3435T exon 26 had a lower risk of CRC than homozygous C-allele carriers (IRR = 0.71 (CI:0.50-1.00)). There was interaction between these MDR1 polymorphisms and intake of red and processed meat in relation to CRC risk. Homozygous MDR1 C3435T C-allele carriers were at 8% increased risk pr 25 gram meat per day (CI: 1.00-1.16) whereas variant allele carriers were not at increased risk (p for interaction = 0.02). COX-2 and BCRP polymorphisms were not associated with CRC risk. There was interaction between NSAID use and MDR1 C3435T and COX-2 T8473C (p-values for interaction 0.001 and 0.04, respectively).
Two polymorphisms in MDR1 were associated with CRC risk and there was interaction between these polymorphisms and meat intake in relation to CRC risk. Our results suggest that MDR1 polymorphisms affect the relationship between meat and CRC risk.
PMCID: PMC2797527  PMID: 19930591
12.  Increased Risk for Congenital Heart Defects in Children Carrying the ABCB1 Gene C3435T Polymorphism and Maternal Periconceptional Toxicants Exposure 
PLoS ONE  2013;8(7):e68807.
The etiology of congenital heart defect (CHD) is commonly believed to involve the interaction of multiple environmental and genetic factors. This study aimed to explore the joint effects of the ABCB1 gene C3435T polymorphism and maternal periconceptional toxicants exposure on the CHD risk in a Han Chinese population.
An age and gender matched case-control study with standardized data collection involving 201 pairs was conducted. Periconceptional toxicants exposure was obtained through a structured questionnaire. A job exposure matrix (JEM) was used for toxicants exposure assessment. Genotyping of the ABCB1 C3435T polymorphism was performed by sequencing. Logistic regression analysis was performed to assess the joint effects of the ABCB1 gene C3435T polymorphism and toxicants exposure on the risk of CHD. Placenta tissues and umbilical cords were collected to investigate the impact of C3435T polymorphism on the transcription and translation activities of ABCB1 gene.
Maternal periconceptional exposures to phthalates (adjusted OR: 1.6; 95%CI: 1.0–2.6) and alkylphenolic compounds (adjusted OR:1.8; 95%CI:1.1–3.0) were associated with a higher incidence of CHDs in general. More cases were carriers of the ABCB1 CC/CT genotypes (OR: 2.0, 95%CI: 1.1–3.5, P-value: 0.021). Children carrying the CC/CT genotype and periconceptionally exposed to phthalates and alkylphenolic compounds suffered almost 3.5-fold increased risk of having CHD than non-exposed children with TT genotype (adjusted OR: 3.5, 95%CI: 1.5–7.9, P-value: 0.003), and the OR changed to 4.4 for septal defects (adjusted OR: 4.4,95%CI:1.8–10.9,P-value:0.001). The ABCB1 mRNA expression of the TT genotype was significantly higher than that of the CC genotype (P = 0.03). Compared with TT genotype, lower P-glycoprotein expression was observed for the CC/CT genotypes.
The C3435T polymorphism in the ABCB1 gene of fetus increases the risks of CHD in a Han Chinese population when the mothers are exposed to phthalates and alkylphenolic compounds during the periconceptional period, particularly for septal defects.
PMCID: PMC3714281  PMID: 23874772
13.  Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines 
BMC Molecular Biology  2012;13:25.
It has been reported that the histone deacetylase inhibitor (iHDAc) trichostatin A (TSA) induces an increase in MDR1 gene transcription (ABCB1). This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp). It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation.
A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study.
The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5′ end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used.
The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates differentially both ABCB1 promoters, downregulating the upstream promoter that is responsible for active P-glycoprotein expression. These results suggest that iHDACs such as TSA may in fact potentiate the effects of antitumour drugs that are substrates of Pgp. Finally, we also demonstrate that TSA upregulates RUNDC3B mRNA independently of the ABCB1 promoter in use.
PMCID: PMC3441908  PMID: 22846052
14.  Ethnicity-Related Polymorphisms and Haplotypes in the Human ABCB1 Gene 
Pharmacogenomics  2007;8(1):29-39.
The human multi-drug resistance gene (MDR1, ABCB1) codes for P-glycoprotein (P-gp), an important membrane-bound efflux transporter known to confer anti-cancer drug resistance as well as affect the pharmacokinetics of many drugs and xenobiotics. A number of single nucleotide polymorphisms (SNPs) have been identified throughout the ABCB1 gene which may have an effect on P-gp expression levels and function. Haplotype as well as genotype analysis of SNPs is becoming increasingly important in identifying genetic variants underlying susceptibility to human disease. Three SNPs, 1236C>T, 2677G>T, and 3435C>T have been repeatedly shown to predict changes in the function of P-gp. The frequencies with which these polymorphisms exist in a population have also been shown to be ethnically related.
In this study, 95 individuals representative of the entire ethnic make-up of the United States were compared to 101 individuals from an Ashkenazi Jewish population. These individuals were analyzed by genomic sequencing and PCR-RFLP to calculate their genotype frequencies.
Twenty-five SNPs were located in the exons of the ABCB1 gene. All of the polymorphisms identified were in parts of the ABCB1 gene product predicted to be intracellular, and 16 appear to be novel as compared to those listed by NCBI. Frequencies of the 1236C>T and 2677G>T/A/C SNPs were similar for the American and Ashkenazi populations (64.2% and 60.4% respectively for 1236C>T – χ2 is 0.30 p≤1; 55.8% and 64.4% for 2677G>T/A/C χ2 is 1.49 p≤1), but were different for 3435C>T (24.2% for the American population and 69.3% for the Ashkenazi population χ2 is 39.927 p<0.001). The 1236T/2677T/3435T haplotype occurred in 23.6% (SE 0.013) of the Ashkenazi population.
The SNP at location 3435C>T plays a significant role in the ABCB1 gene. The haplotype and genotype analysis from these data may be used as a basis for studies on the relationship between ABCB1 genotypes and drug efficacy, drug toxicity, disease susceptibility or other phenotypes.
PMCID: PMC1876748  PMID: 17187507
P-glycoprotein; ABCB1; ethnicity; haplotypes; SNPs
15.  Allelic expression imbalance at high-density lipoprotein cholesterol locus MMAB-MVK 
Human Molecular Genetics  2010;19(10):1921-1929.
Genome-wide association studies (GWAS) have identified numerous loci associated with various complex traits for which the underlying susceptibility gene(s) remain unknown. In a GWAS for high-density lipoprotein-cholesterol (HDL-C) level, one strongly associated locus contains at least two biologically compelling candidates, methylmalonic aciduria cblB type (MMAB) and mevalonate kinase (MVK). To detect evidence of cis-acting regulation at this locus, we measured relative allelic expression of transcribed SNPs in five genes using human hepatocyte samples heterozygous for the transcribed SNP. If an HDL-C-associated SNP allele differentially regulates mRNA level in cis, samples heterozygous both for a transcribed SNP and an HDL-C-associated SNP should display allelic expression imbalance (AEI) of the transcribed SNP. We designed statistical tests to detect AEI in a comprehensive set of linkage disequilibrium (LD) scenarios between the transcribed SNP and an HDL-C-associated SNP (rs7298565) in phase unknown samples. We observed significant AEI of 22% in MMAB (P = 1.4 × 10−13, transcribed SNP rs11067231), and the allele associated with lower HDL-C level was associated with greater MMAB transcript level. The same rs7298565 allele was also associated with higher MMAB mRNA level (P = 0.0081) and higher MMAB protein level (P = 0.0020). In contrast, MVK, UBE3B, KCTD10 and ACACB did not show significant AEI (P ≥ 0.05). These data suggest MMAB is the most likely gene influencing HDL-C levels at this locus and demonstrate that measuring AEI at loci containing more than one candidate gene can prioritize genes for functional studies.
PMCID: PMC2860891  PMID: 20159775
16.  The Role of Abcb5 Alleles in Susceptibility to Haloperidol-Induced Toxicity in Mice and Humans 
PLoS Medicine  2015;12(2):e1001782.
We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.
Methods and Findings
A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.
ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.
Gary Peltz and colleagues examine the role of ABCB5 alleles in haloperidol-induced toxicity in a murine genetic model and humans treated with haloperidol.
Editors' Summary
The brain is the control center of the human body. This complex organ controls thoughts, memory, speech, and movement, it is the seat of intelligence, and it regulates the function of many organs. The brain comprises many different parts, all of which work together but all of which have their own special functions. For example, the forebrain is involved in intellectual activities such as thinking whereas the hindbrain controls the body’s vital functions and movements. Messages are passed between the various regions of the brain and to other parts of the body by specialized cells called neurons, which release and receive signal molecules known as neurotransmitters. Like all the organs in the body, blood vessels supply the brain with the oxygen, water, and nutrients it needs to function. Importantly, however, the brain is protected from infectious agents and other potentially dangerous substances circulating in the blood by the “blood-brain barrier,” a highly selective permeability barrier that is formed by the cells lining the fine blood vessels (capillaries) within the brain.
Why Was This Study Done?
Although drugs have been developed to treat various brain disorders, more active and less toxic drugs are needed to improve the treatment of many if not most of these conditions. Unfortunately, relatively little is known about how the blood-brain barrier regulates the entry of drugs into the brain or about the genetic factors that affect the brain’s susceptibility to drug-induced toxicities. It is not known, for example, why about half of patients given haloperidol—a drug used to treat psychotic disorders (conditions that affect how people think, feel, or behave)—develop tremors and other symptoms caused by alterations in the brain region that controls voluntary movements. Here, to improve our understanding of how drugs enter the brain and impact its function, the researchers investigate the genetic factors that affect haloperidol-induced toxicity by genetically analyzing several inbred mouse strains (every individual in an inbred mouse strain is genetically identical) with different susceptibilities to haloperidol-induced toxicity and by undertaking a human genetic association study (a study that looks for non-chance associations between specific traits and genetic variants).
What Did the Researchers Do and Find?
The researchers used a database of genetic variants called single nucleotide polymorphisms (SNPs) and a computational genetic mapping approach to show first that variations within the gene encoding Abcb5 affected susceptibility to haloperidol-induced toxicity (indicated by changes in the length of time taken by mice to move their paws when placed on an inclined wire-mesh screen) among inbred mouse strains. Abcb5 is an ATP-binding cassette transporter, a type of protein that moves molecules across cell membranes. The researchers next showed that Abcb5 is expressed in brain capillaries, which is the location of the blood-brain barrier. Abcb5 was also expressed in cerebellar Purkinje cells, which help to control motor (intentional) movements. They also measured the measured the effect of haloperidol and the haloperidol concentration in brain tissue sections in mice that were genetically engineered to make no Abcb5 (Abcb5 knockout mice). Finally, the researchers investigated whether specific alleles (alternative versions) of ABCB5 are associated with haloperidol-induced toxicity in people. Among a group of 85 patients treated with haloperidol for a psychotic illness, one specific ABCB5 allele was associated with haloperidol-induced toxicity during the first few days of treatment.
What Do These Findings Mean?
These findings indicate that Abcb5 is a component of the blood-brain barrier in mice and suggest that genetic variants in the gene encoding this protein underlie, at least in part, the differences in susceptibility to haloperidol-induced toxicity seen among inbred mice strains. Moreover, the human genetic association study indicates that a specific ABCB5 allele also affects the susceptibility of people to haloperidol-induced toxicity. The researchers note that other ABCB5 alleles or other genetic factors that affect haloperidol-induced toxicity in people might emerge if larger groups of patients were studied. However, based on their findings, the researchers propose a new model for the genetic mechanisms that underlie inter-individual and cell type-specific differences in susceptibility to haloperidol-induced brain toxicity. If confirmed in future studies, this model might facilitate the development of more effective and less toxic drugs to treat a range of brain disorders.
Additional Information
Please access these websites via the online version of this summary at
The US National Institute of Neurological Disorders and Stroke provides information about a wide range of brain diseases (in English and Spanish); its fact sheet “Brain Basics: Know Your Brain” is a simple introduction to the human brain; its “Blueprint Neurotherapeutics Network” was established to develop new drugs for disorders affecting the brain and other parts of the nervous system
MedlinePlus provides links to additional resources about brain diseases and their treatment (in English and Spanish)
Wikipedia provides information about haloperidol, about ATP-binding cassette transporters and about genetic association (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC4315575  PMID: 25647612
17.  Clinical Significance of ABCB1 Genotyping in Oncology 
P-glycoprotein (Pgp) is a drug efflux pump that transports natural products, including taxanes and other chemotherapeutic agents, from cells. Several frequent polymorphisms in ATP binding cassette gene B1 (ABCB1) may influence Pgp levels and drug efflux. The purpose of this review was to assess the clinical significance of ABCB1 polymorphisms in oncology.
Peer-reviewed studies were identified through a search of PubMed/MEDLINE (1990-2008) and the ASCO abstracts (2003-2008) database. Included studies described clinical trials where ABCB1 genotyping was performed in patients with cancer. Search terms included ABCB1, Pgp, docetaxel, paclitaxel, irinotecan, imatinib and anticancer agent. Studies were excluded if the manuscript was not available in English.
The influence of polymorphisms in ABCB1 2677G>T/A, 3435C>T, and 1236C>T and progression-free and overall survival in 309 patients from the Australian Ovarian Cancer Study treated with paclitaxel/carboplatin demonstrated that compared to homozygote GG carriers at 2677, women with the minor T/A alleles were significantly less likely to relapse following treatment. Other trials of ABCB1 genotyping in breast and prostate cancer patients receiving taxanes have shown inconsistent results. Pharmacokinetic studies where ABCB1 was genotyped and patients received irinotecan or imatinib have also shown inconsistent results.
A number of commercially available drugs are substrates for Pgp, and the ABCB1-variant genotypes are frequent and functionally significant, which may have future implications for drug dosing.
PMCID: PMC2894614  PMID: 19401306
18.  ABCB1/MDR1 gene polymorphisms as a prognostic factor in colorectal cancer 
To analyse the single-nucleotide polymorphisms (SNPs): ABCB11236C>T, ABCB12677G>T/A, ABCB13435C>T and haplotypes in the ABCB1/MDR1 gene, which could contribute to genetic risk of colorectal cancer (CRC). Disease association between the ABCB1/MDR1 genotype, allele, haplotype frequencies and histological features, such as TNM classification, localization of primary carcinoma, grade of malignancy, histological type of tumour, lymphoid infiltration and vessel invasion were estimated. In this study, the potential role of SNPs of the ABCB1/MDR1 gene as a prognostic marker for CRC was analysed.
Materials and methods
Tumour specimens of 95 patients with CRC were studied. Using automated sequencing or PCR-RFLP method, DNA for three common SNPs of ABCB1/MDR1 was extracted and analysed. The results of genotyping and haplotype analysis with histopathological features, grading and clinical staging of neoplasms were correlated.
A statistically significant higher frequency of T1236 allele in T1/T2 (89.7%), M0 groups (81.6%) and I/II clinical staging (82.7%) in comparison with T3/T4 (68.2%), M1 groups (47.4%) and III/IV clinical staging (65.1%) was detected. Furthermore, multivariate analysis according to Cox's proportional hazard model indicated that the T1236 allele is a good, independent prognostic factor and the presence of this allele decreases the risk of death in comparison with a group without this allele (HR = 0.26; p = 0.0424). In addition, a statistically significant higher frequency of C3435 allele and significant differences in the C3435 allele distribution in N1/N2 group (91.7% and 62.5%, respectively) than N0 group (71.2% and 44.9%, respectively) was found. Each of the eight possible haplotypes was noted in M0 or I/II group and only seven in M1 or III/IV group. Haplotype T1236-G2677-C3435 only in less advanced CRC subjects (9.6% in I/II and 9.2% in M0 group) was detected. In addition, significant differences in haplotype distributions between M0 or I/II and M1 or III/IV group were found (p = 0.01 and p = 0.05, respectively).
These results suggest association between T1236 allele and T1236-G2677-C3435 haplotype and less advanced CRC, so these genetic markers may play a role as potentially good prognostic factors. Differences in haplotype distributions and degree of clinical staging may suggest that some other potential SNPs, especially in regulatory region of ABCB1/MDR1 gene, may influence P-glycoprotein function and CRC progression.
PMCID: PMC2928441  PMID: 20533057
ABCB1 gene; MDR1 gene; Polymorphism; Haplotype analysis; Colorectal cancer
19.  An intronic PICALM polymorphism, rs588076, is associated with allelic expression of a PICALM isoform 
Although genome wide studies have associated single nucleotide polymorphisms (SNP)s near PICALM with Alzheimer’s disease (AD), the mechanism underlying this association is unclear. PICALM is involved in clathrin-mediated endocytosis and modulates Aß clearance in vitro. Comparing allelic expression provides the means to detect cis-acting regulatory polymorphisms. Thus, we evaluated whether PICALM showed allele expression imbalance (AEI) and whether this imbalance was associated with the AD-associated polymorphism, rs3851179.
We measured PICALM allelic expression in 42 human brain samples by using next-generation sequencing. Overall, PICALM demonstrated equal allelic expression with no detectable influence by rs3851179. A single sample demonstrated robust global PICALM allelic expression imbalance (AEI), i.e., each of the measured isoforms showed AEI. Moreover, the PICALM isoform lacking exons 18 and 19 (D18-19 PICALM) showed significant AEI in a subset of individuals. Sequencing these individuals and subsequent genotyping revealed that rs588076, located in PICALM intron 17, was robustly associated with this imbalance in D18-19 PICALM allelic expression (p = 9.54 x 10-5). This polymorphism has been associated previously with systolic blood pressure response to calcium channel blocking agents. To evaluate whether this polymorphism was associated with AD, we genotyped 3269 individuals and found that rs588076 was modestly associated with AD. However, when both the primary AD SNP rs3851179 was added to the logistic regression model, only rs3851179 was significantly associated with AD.
PICALM expression shows no evidence of AEI associated with rs3851179. Robust global AEI was detected in one sample, suggesting the existence of a rare SNP that strongly modulates PICALM expression. AEI was detected for the D18-19 PICALM isoform, and rs588076 was associated with this AEI pattern. Conditional on rs3851179, rs588076 was not associated with AD risk, suggesting that D18-19 PICALM is not critical in AD. In summary, this analysis of PICALM allelic expression provides novel insights into the genetics of PICALM expression and AD risk.
PMCID: PMC4150683  PMID: 25169757
PICALM; Alzheimer’s disease; Next-generation sequencing; Allelic expression imbalance; Single nucleotide polymorphism
20.  Genetic Variants in ABCB1, CYP2C19, and Cardiovascular Outcomes Following Treatment with Clopidogrel and Prasugrel 
Lancet  2010;376(9749):1312-1319.
The thienopyridine clopidogrel is one of the most commonly prescribed drugs worldwide. Both clopidogrel and the third-generation thienopyridine prasugrel are subject to efflux via P-glycoprotein (encoded by ABCB1, also known as MDR1). In vitro and clinical studies suggest that ABCB1 polymorphisms, particularly C3435T, may be associated with altered drug metabolism and efficacy.
We genotyped 2,932 patients with an acute coronary syndrome (ACS) in TRITON-TIMI 38 treated with clopidogrel or prasugrel and 321 healthy individuals in whom we measured the pharmacologic response to clopidogrel or prasugrel.
Among ACS patients treated with clopidogrel, ABCB1 C3435T genotype was significantly associated with risk for the primary endpoint of cardiovascular death, MI, or stroke (P=0.0064). TT homozygotes (804/2,932 [27%] of the population) had a 72% increased risk of the primary endpoint as compared with CT /CC individuals (52/414 [12.9%] vs. 80/1,057 [7.8%], HR 1.72, 95% CI 1.22–2.44, P=0.002). ABCB1 C3435T and CYP2C19 genotypes were significant, independent predictors of the primary endpoint, and the 47% (681/1454) of the population who were either CYP2C19 reduced-function allele carriers, ABCB1 3435 TT homozygotes, or both were at significantly increased risk of cardiovascular death, MI, or stroke (HR 1.97, 95% CI 1.38–2.82, P=0.0002). In healthy subjects, 3435 TT homozygotes had a reduction in platelet aggregation with clopidogrel that was 7.3 absolute percentage points lower (i.e., less platelet inhibition) vs. CT/CC individuals (P=0.0127). ABCB1 genotypes were not significantly associated with clinical or pharmacologic outcomes among ACS or healthy individuals treated with prasugrel.
Individuals with the ABCB1 3435 TT genotype have less platelet inhibition and are at significantly increased risk of recurrent ischemic events in the setting of clopidogrel treatment. Taking into account both ABCB1 and CYP2C19, nearly half of the population carries a genotype associated with an increased risk for major adverse cardiovascular events while on standard doses of clopidogrel.
PMCID: PMC3036672  PMID: 20801494
21.  Genetic and Non-Genetic Determinants of Raltegravir Penetration into Cerebrospinal Fluid: A Single Arm Pharmacokinetic Study 
PLoS ONE  2013;8(12):e82672.
Antiretroviral drugs vary in their central nervous system penetration, with better penetration possibly conferring neurocognitive benefit during human immunodeficiency virus (HIV) therapy. The efflux transporter gene ABCB1 is expressed in the blood-brain barrier, and an ABCB1 variant (3435C→T) has been reported to affect ABCB1 expression. The integrase inhibitor raltegravir is a substrate for ABCB1. We examined whether ABCB1 3435C→T affects raltegravir disposition into cerebrospinal fluid (CSF), and explored associations with polymorphisms in other membrane transporter genes expressed in the blood-brain barrier.
Forty healthy, HIV-negative adults of European descent (20 homozygous for ABCB1 3435 C/C, 20 homozygous for 3435 T/T, each group divided equally between males and females) were given raltegravir 400 mg twice daily for 7 days. With the final dose, plasma was collected for pharmacokinetic analysis at 9 timepoints over 12 hours, and CSF collected 4 hours post dose.
The 4-hour CSF concentration correlated more strongly with 2-hour (r2=0.76, P=1.12x10-11) than 4-hour (r2=0.47, P=6.89x10-6) single timepoint plasma concentration, and correlated strongly with partial plasma area-under-the-curve values (AUC0-4h r2=0.86, P=5.15x10-16). There was no significant association between ABCB1 3435C→T and ratios of CSF-to-plasma AUC or concentration (p>0.05 for each comparison). In exploratory analyses, CSF-to-plasma ratios were not associated with 276 polymorphisms across 16 membrane transporter genes.
Among HIV-negative adults, CSF raltegravir concentrations do not differ by ABCB1 3435C→T genotype but strongly correlate with plasma exposure.
Trial Registration NCT00729924
PMCID: PMC3859605  PMID: 24349334
22.  Gene-Wide Characterization of Common Quantitative Trait Loci for ABCB1 mRNA Expression in Normal Liver Tissues in the Chinese Population 
PLoS ONE  2012;7(9):e46295.
In order to comprehensively screen genetic variants leading to differential expression of the important human ABCB1 gene in the primary drug-metabolizing organ, ABCB1 mRNA expression levels were measured in 73 normal liver tissue samples from Chinese subjects. A set of Tag SNPs. were genotyped. In addition, imputation was performed within a 500 kb region around the ABCB1 gene using the reference panels of 1,000 Genome project and HapMap III. Bayesian regression was used to assess the strength of associations by compute Bayes Factors for imputed SNPs. Through imputation and linkage disequilibrium analysis, the imputed loci rs28373093, rs1002205, rs1029421, rs2285647, and rs10235835, may represent independent and strong association signals. rs28373093, a polymorphism 1.5 kb upstream from the ABCB1 transcription start site, has the strongest association. 2677 G>A/T and 3435C>T confer a clear gene-dosage effect on ABCB1 mRNA expression. The systematic characterization of gene-wide common quantitative trait loci associated with ABCB1 mRNA expression in normal liver tissues would provide the candidate markers to ABCB1-relevant clinical phenotypes in Chinese population.
PMCID: PMC3458811  PMID: 23050008
23.  Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos 
BMC Biology  2013;11:69.
In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water.
Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a polycyclic aromatic hydrocarbon) and galaxolide and tonalide (two polycyclic musks).
We show that zebrafish Abcb4 is a cellular toxicant transporter and provides protection of embryos against toxic chemicals dissolved in the water. Zebrafish Abcb4 thus is functionally similar to mammalian ABCB1, but differs from mammalian ABCB4, which is not involved in cellular resistance to chemicals but specifically transports phospholipids in the liver. Our data have important implications: Abcb4 could affect bioavailability - and thus toxicologic and pharmacologic potency - of chemicals to zebrafish embryos and inhibition of Abcb4 therefore causes chemosensitization, that is, enhanced sensitivity of embryos to toxicants. These aspects should be considered in (eco)toxicologic and pharmacologic chemical screens with the zebrafish embryo, a major vertebrate model.
PMCID: PMC3765700  PMID: 23773777
Abcb4; Abcb5; Chemosensitization; Efflux transporters; Environment-tissue barrier; Multixenobiotic resistance; MXR; P-glycoprotein
24.  Promoter Polymorphisms in ACE (Angiotensin I–Converting Enzyme) Associated With Clinical Outcomes in Hypertension 
Genetic variants of ACE are suspected risk factors in cardiovascular disease, but the alleles responsible for the variations remain unidentified. To search for regulatory polymorphisms, allelic angiotensin I–converting enzyme (ACE) mRNA expression was measured in 65 heart tissues, followed by genotype scanning of the ACE locus. Marked allelic expression imbalance (AEI) detected in five African-American subjects was associated with single-nucleotide polymorphisms (SNPs) (rs7213516, rs7214530, and rs4290) residing in conserved regions 2−3 kb upstream of ACE. Moreover, each of the SNPs affected transcription in reporter gene assays. SNPs rs4290 and rs7213516 were tested for associations with adverse cardiovascular outcomes in hypertensive patients with coronary disease (International Verapamil SR Trandolapril Study Genetic Substudy (INVEST-GENES), n = 1,032). Both SNPs were associated with adverse cardiovascular outcomes, largely attributable to nonfatal myocardial infarction in African Americans, showing an odds ratio of 6.16 (2.43−15.60) (P < 0.0001) for rs7213516. The high allele frequency in African Americans (16%) compared to Hispanics (4%) and Caucasians (<1%) suggests that these alleles contribute to variation between populations in cardiovascular risk and treatment outcomes.
PMCID: PMC2709290  PMID: 18946466
25.  Evidence for population variation in TSC1 and TSC2 gene expression 
BMC Medical Genetics  2011;12:29.
Tuberous sclerosis complex (TSC) is an autosomal dominant neurogenetic disorder caused by mutations in one of two genes, TSC1 or TSC2, which encode the proteins hamartin and tuberin, respectively [1-3]. Common features of TSC include intractable epilepsy, mental retardation, and autistic features. TSC is associated with specific brain lesions, including cortical tubers, subependymal nodules and subependymal giant cell astrocytomas. In addition, this disease frequently produces characteristic tumors, termed hamartomas, in the kidneys, heart, skin, retina, and lungs. Disease severity in TSC can be quite variable and is not determined by the primary mutation alone. In fact, there is often considerable variability in phenotype within single families, where all affected individuals carry the same mutation. Factors suspected to influence phenotype in TSC include the specific primary mutation, random occurrence of second-hit somatic mutations, mosaicism, "modifying genes", and environmental factors. In addition to these factors, we hypothesize that differences in mRNA expression from the non-mutated TSC allele, or possibly from the mutated allele, play a part in modifying disease severity. Common genetic variants that regulate mRNA expression have previously been shown to play important roles in human phenotypic variability, including disease susceptibility. A prediction based on this idea is that common regulatory variants that influence disease severity in TSC should be detectable in non-affected individuals.
A PCR/primer extension assay was used to measure allele specific expression of TSC1 and TSC2 mRNAs in leukocytes isolated from normal volunteers. This assay can be used to measure "allelic expression imbalance" (AEI) in individuals by making use of heterozygous "marker" single nucleotide polymorphisms (SNPs) located within their mRNA.
In this study we show for the first time that TSC1 and TSC2 genes exhibit allele-specific differences in mRNA expression in blood leukocytes isolated from normal individuals.
These results support the possibility that allele-specific variation in TSC mRNA expression contributes to the variable severity of symptoms in TSC patients.
PMCID: PMC3051885  PMID: 21345208

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