Molecular and computational characterization of ABCB11 and ABCG5 variants in Tunisian patients with neonatal/infantile low-GGT intrahepatic cholestasis: Genetic diagnosis and genotype-phenotype correlation assessment.

Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.

Use of Traditional and Proteomic Methods in the Assessment of a Preclinical Model of Preeclampsia.

Recent studies have demonstrated downregulation of breast cancer resistance protein (BCRP/ABCG2) in placenta obtained from women with preeclampsia (PE). BCRP is highly expressed in placenta and plays an important role in preventing xenobiotics from entering the fetal compartment. Although PE is often therapeutically managed with drugs that are substrates of BCRP, there are limited studies on the impact of PE on fetal drug exposure. Due to ethical concerns, use of preclinical models is an important approach. Thus, by using proteomic and traditional methods, we characterized transporter changes in an immunologic rat model of PE to determine its utility and predictive value for future drug disposition studies. PE was induced by daily administration of low-dose endotoxin (0.01-0.04 mg/kg) to rats on gestational days (GD) 13-16, urine was collected, and rats were sacrificed on GD17 or GD18. PE rats shared similar phenotype to PE patients, including proteinuria, and increased levels of tumor necrosis factor α and interleukin 6. Transcript and protein levels of Bcrp were significantly downregulated in placenta of PE rats on GD18. multidrug resistance 1a, multidrug resistance 1b, and organic anion transporting polypeptide 2B1 mRNA were also decreased in PE. Proteomics revealed activation of various hallmarks of PE including immune activation, oxidative stress, endoplasmic reticulum stress and apoptosis. Overall, our results demonstrated that the immunologic PE rat model exhibits numerous similarities to human PE along with dysregulation of placental transporters. Therefore, this model may be useful in examining the impact of PE on the maternal and fetal disposition of BCRP substrates. SIGNIFICANCE STATEMENT: Fully characterizing preclinical models of disease is necessary to determine their validity to human conditions. Combining traditional and proteomic methods of model characterization, we identified numerous phenotypic similarities between our model of preeclampsia and human disease. The alignment with human pathophysiological changes allows for more confident use of this preclinical model.

Preclinical assessment of drug-drug interaction of fb-PMT, a novel anti-cancer thyrointegrin αvß3 antagonist.

The objective of the current study was to identify potential drug-drug interactions (DDIs) with the drug candidate fb-PMT, a novel anticancer thyrointegrin αvß3 antagonist. This was accomplished by using several in vitro assays to study interactions of fb-PMT with both cytochrome P450 (CYP) enzymes and drug transporters, two common mechanisms leading to adverse drug effects. In vitro experiments showed that fb-PMT exhibited weak reversible inhibition of CYP2C19 and CYP3A4. In addition, fb-PMT did not show time-dependent inhibition with any of the seven CYP isoforms tested, including 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. Human liver microsomal incubations demonstrated that fb-PMT is stable. Potential transporter-mediated DDIs with fb-PMT were assessed with two ATP binding cassette (ABC) family transporters (P-glycoprotein and breast cancer resistance protein) using Caco2 cells and seven solute carrier family (SLC) transporters (organic cation transporter OCT2, organic anion transporters OAT1 and OAT3, organic anion transporter peptides OATP1B1 and OATP1B3, and the multidrug and toxic extrusion proteins MATE1 and MATE2-K using transfected HEK293 cells). Fb-PMT was not a substrate for any of the nine transporters tested in this study, nor did it inhibit the activity of seven of the transporters tested. However, fb-PMT inhibited the uptake of rosuvastatin by both OATP1B1 and OATP1B3 with half-maximal inhibitory concentrations greater than 3 and less than 10 µM. In summary, data suggest that the systemic administration of fb-PMT is unlikely to lead to DDIs through CYP enzymes or ABC and SLC transporters in humans.

Waste or die: The price to pay to stay alive.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell.

Using single molecule Molecular Inversion Probes as a cost-effective, high-throughput sequencing approach to target all genes and loci associated with macular diseases.

Macular degenerations (MDs) are a subgroup of retinal disorders characterized by central vision loss. Knowledge is still lacking on the extent of genetic and nongenetic factors influencing inherited MD (iMD) and age-related MD (AMD) expression. Single molecule Molecular Inversion Probes (smMIPs) have proven effective in sequencing the ABCA4 gene in patients with Stargardt disease to identify associated coding and noncoding variation, however many MD patients still remain genetically unexplained. We hypothesized that the missing heritability of MDs may be revealed by smMIPs-based sequencing of all MD-associated genes and risk factors. Using 17,394 smMIPs, we sequenced the coding regions of 105 iMD and AMD-associated genes and noncoding or regulatory loci, known pseudo-exons, and the mitochondrial genome in two test cohorts that were previously screened for variants in ABCA4. Following detailed sequencing analysis of 110 probands, a diagnostic yield of 38% was observed. This established an ''MD-smMIPs panel," enabling a genotype-first approach in a high-throughput and cost-effective manner, whilst achieving uniform and high coverage across targets. Further analysis will identify known and novel variants in MD-associated genes to offer an accurate clinical diagnosis to patients. Furthermore, this will reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.

Analysis of Multiple Drug Resistance Mechanism in Different Types of Soft Tissue Sarcomas: Assessment of the Expression of ABC-Transporters, MVP, YB-1, and Analysis of Their Correlation with Chemosensitivity of Cancer Cells.

Chemotherapy of soft tissue sarcomas (STS) is restricted by low chemosensitivity and multiple drug resistance (MDR). The purpose of our study was the analysis of MDR mechanism in different types of STS. We assessed the expression of ABC-transporters, MVP, YB-1, and analyzed their correlation with chemosensitivity of cancer cells. STS specimens were obtained from 70 patients without metastatic disease (2018-2020). Expression level of MDR-associated genes was estimated by qRT-PCR and cytofluorimetry. Mutations in ABC-transporter genes were captured by exome sequencing. Chemosensitivity (SI) of STS to doxorubicin (Dox), ifosfamide (Ifo), gemcitabine (Gem), and docetaxel (Doc) was analyzed in vitro. We found strong correlation in ABCB1, ABCC1, and ABCG2 expression. We demonstrated strong negative correlations in ABCB1 and ABCG2 expression with SI (Doc) and SI (Doc + Gem), and positive correlation of MVP expression with SI (Doc) and SI (Doc + Gem) in undifferentiated pleomorphic sarcoma. Pgp expression was shown in 5 out of 44 STS samples with prevalence of synovial sarcoma relapses and it is strongly correlated with SI (Gem). Mutations in MDR-associated genes were rarely found. Overall, STS demonstrated high heterogeneity in chemosensitivity that makes reasonable in vitro chemosensitivity testing to improve personalized STS therapy, and classic ABC-transporters are not obviously involved in MDR appearance.

Assessment of AAV Dual Vector Safety in theAbca4-/- Mouse Model of Stargardt Disease.

Purpose: Adeno-associated viral (AAV) gene therapy treatment for Stargardt disease currently requires a dual vector approach owing to the size of the ATP-binding cassette transporter family member gene (ABCA4). The nature of the dual vector system creates the potential for adverse events. Here we have investigated an overlapping adeno-associated viral ABCA4 dual vector system for signs of toxicity in Abca4-/- mice as a prelude to dual vector first in human clinical trials. Methods: Abca4-/- mice received a subretinal injection of a 1:1 5':3' dual vector mix; 5' vector only; 3 ' vector only; a GFP reporter vector; or diluent only (sham). All vectors were adeno-associated virus-8 Y733F. Mice were subsequently assessed for signs of toxicity as measured by loss in retinal structure by optical coherence tomography and retinal function by electroretinography up to 6 months after injection. Results: Subretinal delivery of the dual vector system and its comprising parts induced no structural or functional changes relative to paired uninjected eyes beyond those observed in the sham control cohort. Histologic changes were limited to the superior retina where the injection was performed. Electroretinography analysis confirmed the dual vector system inferred no functional changes beyond those observed in the sham control cohort. Conclusions: An optimized overlapping dual vector system for the treatment of Stargardt disease shows no additional signs of toxicity beyond those observed from a sham injection. Translational Relevance: This presentation of safety of a dual vector system for the treatment of Stargardt disease encourages its future use in clinical trial.

ABCA7 Genotype Moderates the Effect of Aerobic Exercise Intervention on Generalization of Prior Learning in Healthy Older African Americans.

African Americans are at elevated risk for age-related cognitive decline, with double the prevalence of Alzheimer's disease (AD) compared to Caucasians Americans. Various behavioral, biological, and lifestyle factors may underlie this health disparity, but little is known about the relative importance and interactions among these different risk factors in African Americans. While the neuroprotective effects of aerobic exercise on biomarkers are well established, few studies have examined the differential benefits of exercise based on genetic risk for AD. Furthermore, evidence is limited regarding the potential moderating effects of ABCA7, a gene known to confer significantly greater AD risk in African Americans. In a case-control matched sample of 56 healthy older African Americans, we investigated the effect of an aerobic exercise intervention on a hippocampus-related assessment of generalization following rule learning, in individuals who were carriers of the ABCA7 rs3764650 non-risk (TT) or high-risk (GG) genotype. Following the exercise-intervention, the non-risk group made significantly fewer generalization errors, while there was no improvement in generalization for the high-risk group. For the controls, no changes in generalization scores were observed regardless of genotype status. Our results indicate that the ongoing adverse effects of ABCA7 high-risk genotype may diminish the benefits associated with aerobic exercise. As such, the potential disease-modifying effects of aerobic exercise on AD-related neuropathology may be limited to carriers of the ABCA7 rs3764650 non-risk genotype.

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease.

PURPOSE: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

Assessment of HBEC-5i endothelial cell line cultivated in astrocyte conditioned medium as a human blood-brain barrier model for ABC drug transport studies.

Endothelial cells are main components of the Blood-Brain Barrier (BBB) and form a tight monolayer that regulates the passage of molecules, with the ATP-Binding Cassette (ABC) transporters efflux pumps. We have developed a human in vitro model of HBEC-5i endothelial cells cultivated alone or with human astrocytes conditioned medium on insert. HBEC-5i cells showed a tight monolayer within 14 days, expressing ZO-1 and claudin 5, a low apparent permeability to small molecules, with a TEER stability during five days. The P-gp, BCRP, MRPs transporters were well expressed and functional. Accumulation and efflux ratio measurement with different ABC transporters substrates (Rhodamine 123, BCECF AM, Hoechst 33342) and inhibitors (verapamil, Ko143, probenecid and cyclosporin A) were conducted. At barrier level, the functionality of ABC transporters was three-fold enhanced in astrocyte conditioned medium. We validated our model by the transport of pharmacological substrates: caffeine, rivaroxaban, and methotrexate. The rivaroxaban and methotrexate were released with an efflux ratio >3 and were decreased by more than half with inhibitors. HBEC-5i model could be used as relevant tool in preclinical studies for assessing the permeability of therapeutic molecules to cross human BBB.

Cofactor Regeneration Using Permeabilized Escherichia coli Expressing NAD(P)+-Dependent Glycerol-3-Phosphate Dehydrogenase.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes NAD(P)+-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles.

The expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the "housekeeping" genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and improved normalization of genome-wide expression-profiling data.

In Vitro Assessment of the Effect of Antiepileptic Drugs on Expression and Function of ABC Transporters and Their Interactions with ABCC2.

ABC transporters have a significant role in drug disposition and response and various studies have implicated their involvement in epilepsy pharmacoresistance. Since genetic studies till now are inconclusive, we thought of investigating the role of xenobiotics as transcriptional modulators of ABC transporters. Here, we investigated the effect of six antiepileptic drugs (AEDs) viz. phenytoin, carbamazepine, valproate, lamotrigine, topiramate and levetiracetam, on the expression and function of ABCB1, ABCC1, ABCC2 and ABCG2 in Caco2 and HepG2 cell lines through real time PCR, western blot and functional activity assays. Further, the interaction of AEDs with maximally induced ABCC2 was studied. Carbamazepine caused a significant induction in expression of ABCB1 and ABCC2 in HepG2 and Caco2 cells, both at the transcript and protein level, together with increased functional activity. Valproate caused a significant increase in the expression and functional activity of ABCB1 in HepG2 only. No significant effect of phenytoin, lamotrigine, topiramate and levetiracetam on the transporters under study was observed in either of the cell lines. We demonstrated the interaction of carbamazepine and valproate with ABCC2 with ATPase and 5,6-carboxyfluorescein inhibition assays. Thus, altered functionality of ABCB1 and ABCC2 can affect the disposition and bioavailability of administered drugs, interfering with AED therapy.

Rv1458c: a new diagnostic marker for identification of Mycobacterium tuberculosis complex in a novel duplex PCR assay.

PURPOSE: We explored the efficiency of Rv1458c, the gene encoding a putative ABC drug transporter specific for the Mycobacterium tuberculosis complex (MTBC), as a diagnostic marker. METHODOLOGY: A 190 bp region of Rv1458c and a 300 bp region of hsp65 were targeted in a novel duplex PCR assay and the results were compared with those for PCR restriction analysis(PRA) using the restriction enzymes NruI and BamHI. Species identification of a subset of the isolates (n=50) was confirmed by sequencing. Clinical isolates of M. tuberculosis (n=426) obtained from clinically suspected patients of pulmonary tuberculosis and mycobacterial (n=13) and non-mycobacterial (n=8) reference strains were included in the study. RESULTS: The duplex PCR assay correctly identified 320/426 isolates as MTBC and 106/426 isolates as non-tuberculous mycobacteria(NTM). The test was 100 % specific and sensitive when compared with NruI/BamHI PCR restriction analysis and highlighted the use of Rv1458c as a diagnostic marker for MTBC. CONCLUSION: The duplex PCR assay could be developed for use as a screening test to identify MTBC in clinical specimens in peripheral laboratories with limited resources.

Pre-Hepatectomy Assessment of Bile Transporter Expression by Gadoxetic Acid-Enhanced MRI in a Rat Model of Liver Cirrhosis.

BACKGROUND: Gadoxetic acid is a liver-specific intravenous T1 magnetic resonance (MR) contrast agent that is excreted via the hepatobiliary system. We hypothesize that hepatocyte expressions of bile transporters (OATP1 and MRP2) correlate with dynamic profile of Gadoxetic acid enhanced (GE)-MR imaging (MRI). METHODS: Two groups of rats, control (n = 6) and cirrhosis (n = 12), received gadoxetic acid enhanced MRI followed by 70% hepatectomy. The change in MR signal intensity from the baseline before the contrast injection (ΔSI) was analyzed every minute for 30 min. Dynamic signal intensity retention ratio (DSR) was defined as the mean ΔSI of the third 10-minmin period divided by the first 10-minmin period. Real-time PCR was utilized to quantify mRNA expressions. RESULTS: Compared to the control, cirrhosis group demonstrated lower mRNA levels of OATP1 (0.038 ± 0.020 vs. 0.232 ± 0.0979; p = 0.004), MRP2 (0.201 ± 0.084 vs. 0.7567 ± 0.254; p = 0.002), and OATP1/MRP2 mRNA ratio (0.193 ± 0.065 vs. 0.342 ± 0.206; p = 0.032). DSR was higher in the cirrhosis group (0.678 ± 0.554 vs -0.125 ± 0.839; p = 0.033). In the cirrhosis group, there was an inverse correlation between the ratios of OATP1/MRP2 mRNA and DSR (R = -0.709, p = 0.01). CONCLUSION: Bile transporters OATP1/MRP2 mRNA expression ratio in rat liver tissue decreased with DMN-induced liver injury. The expressions of bile transporters correlated with GE-MRI DSR. The GE-MRI DSR has potential utility in qualifying OATP1/MRP2 mRNA expression.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

BACKGROUND: Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. METHODS: The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. RESULTS: SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable. CONCLUSION: Inhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Drug interaction profile of the HIV integrase inhibitor cabotegravir: assessment from in vitro studies and a clinical investigation with midazolam.

1. Cabotegravir (CAB; GSK1265744) is a potent HIV integrase inhibitor in clinical development as an oral lead-in tablet and long-acting injectable for the treatment and prevention of HIV infection. 2. This work investigated if CAB was a substrate for efflux transporters, the potential for CAB to interact with drug-metabolizing enzymes and transporters to cause clinical drug interactions, and the effect of CAB on the pharmacokinetics of midazolam, a CYP3A4 probe substrate, in humans. 3. CAB is a substrate for Pgp and BCRP; however, its high intrinsic membrane permeability limits the impact of these transporters on its intestinal absorption. 4. At clinically relevant concentrations, CAB did not inhibit or induce any of the CYP or UGT enzymes evaluated in vitro and had no effect on the clinical pharmacokinetics of midazolam. 5. CAB is an inhibitor of OAT1 (IC50 0.81 µM) and OAT3 (IC50 0.41 µM) but did not or only weakly inhibited Pgp, BCRP, MRP2, MRP4, MATE1, MATE2-K, OATP1B1, OATP1B3, OCT1, OCT2 or BSEP. 6. Based on regulatory guidelines and quantitative extrapolations, CAB has a low propensity to cause clinically significant drug interactions, except for coadministration with OAT1 or OAT3 substrates.

Mutations in ABCA7 in a Belgian cohort of Alzheimer's disease patients: a targeted resequencing study.

BACKGROUND: ABCA7 was identified as a risk gene for Alzheimer's disease in genome-wide association studies (GWAS). It was one of the genes most strongly associated with risk of Alzheimer's disease in a Belgian cohort. Using targeted resequencing, we investigated ABCA7 in this cohort with the aim to directly detect rare and common variations in this gene associated with Alzheimer's disease pathogenesis. METHODS: We did massive parallel resequencing of ABCA7 after HaloPlex target enrichment of the exons, introns, and regulatory regions in 772 unrelated patients with Alzheimer's disease (mean age at onset 74·6 years [SD 8·9]) recruited at two memory clinics in Flanders, Belgium, and 757 geographically matched community-dwelling controls (mean age at inclusion 73·9 years [8·0]). After bioinformatic processing, common variants were analysed with conditional logistic regression and rare variant association analysis was done in Variant Association Tools. To explore an observed founder effect, additional unrelated patients with Alzheimer's disease (n=183, mean age at onset 78·8 years [SD 6·0]) and control individuals (n=265, mean age at inclusion 56·9 years [10·8]) from the same cohort who had not been included in massive parallel resequencing because of insufficient biosamples were screened for the ABCA7 frameshift mutation Glu709fs with Sanger sequencing. The effect of loss-of-function mutations on ABCA7 expression was investigated with quantitative real-time PCR in post-mortem brains of patients (n=3) and control individuals (n=4); nonsense mediated mRNA decay was investigated in lymphoblast cell lines from three predicted loss-of-function mutation carriers from the cohort of 772 patients with Alzheimer's disease. FINDINGS: An intronic low-frequency variant rs78117248 (minor allele frequency 3·8% in 58 patients with Alzheimer's disease and in controls 1·8% in 28 controls) showed strongest association with Alzheimer's disease (odds ratio 2·07, 95% CI 1·31-3·27; p=0·0016), and remained significant after conditioning for the GWAS top single nucleotide polymorphisms rs3764650, rs4147929, and rs3752246 (2·00, 1·22-3·26; p=0·006). We identified an increased frequency of predicted loss-of-function mutations in the patients compared with the controls (relative risk 4·03, 95% CI 1·75-9·29; p=0·0002). One frameshift mutation (Glu709fs) showed a founder effect in the study population, and was found to segregate with disease in a family with autosomal dominant inheritance of Alzheimer's disease. Expression of ABCA7 was reduced in the two carriers of loss-of-function mutations found only in patients with Alzheimer's disease (Glu709fs and Trp1214*) compared with four non-carrier controls (relative expression 0·45, 95% CI 0·25-0·84; p=0·002) and in lymphoblast cell lines from three carriers of Glu709fs compared with those from two non-carrier controls. INTERPRETATION: We propose that a low-frequency variant can explain the association between ABCA7 and Alzheimer's disease, and the evidence of loss-of-function mutations in this risk gene suggests that partial loss-of-function of ABCA7 could be a potential pathogenetic mechanism of Alzheimer's disease. FUNDING: Belgian Science Policy Office Interuniversity Attraction Poles program P7/16, Alzheimer Research Foundation, King Baudouin Foundation AB Fund, Methusalem Excellence Program initiative of the Flemish Government, Flanders Impulse Program on Networks for Dementia Research, Research Foundation Flanders, Agency for Innovation by Science and Technology Flanders, University of Antwerp Research Fund, and European Union's Seventh Framework Programme for Research, Technological development and Demonstration (AgedBrainSYSBIO).

The Use of Transporter Probe Drug Cocktails for the Assessment of Transporter-Based Drug-Drug Interactions in a Clinical Setting-Proposal of a Four Component Transporter Cocktail.

Probe drug cocktails are used clinically to assess the potential for drug-drug interactions (DDIs), and in particular, DDIs resulting from coadministration of substrates and inhibitors of cytochrome P450 enzymes. However, a probe drug cocktail has not been identified to assess DDIs involving inhibition of drug transporters. We propose a cocktail consisting of the following substrates to explore the potential for DDIs caused by inhibition of key transporters: digoxin (P-glycoprotein, P-gp), rosuvastatin (breast cancer resistance protein, BCRP; organic anion transporting polypeptides, OATP), metformin (organic cation transporter, OCT; multidrug and toxin extrusion transporters, MATE), and furosemide (organic anion transporter, OAT). Furosemide was evaluated in vitro, and is a substrate of OAT1 and OAT3, with Km values of 38.9 and 21.5 µM, respectively. Furosemide was also identified as a substrate of BCRP, OATP1B1, and OATP1B3. Furosemide inhibited BCRP (50% inhibition of drug transport: 170 µM), but did not inhibit OATP1B1, OATP1B3, OCT2, MATE1, and MATE2-K at concentrations below 300 µM, and P-gp at concentrations below 2000 µM. Conservative approaches for the estimation of the likelihood of in vivo DDIs indicate a remote chance of in vivo transporter inhibition by these probe drugs when administered at low single oral doses. This four component probe drug cocktail is therefore proposed for clinical evaluation.