Publications

Normal anatomic variation in the amount of fat within the petrous temporal bone of dogs can result in a magnetic resonance (MR) imaging pseudolesion. Focal hyperintense areas in the region of the hippocampus on T1-weighted, T1-weighted, and fluid-attenuated inversion recovery imaging sequences were noted in a dog being imaged for seizure activity. Further investigation of this region, aided by the use of cadaveric specimens, led to the identification of normal anatomic variability in the amount of fat in the substantia spongiosa of the petrous temporal bone. The presence of normal adipose tissue was confirmed histopathologically. Fat suppression MR imaging sequences can be used to differentiate whether hyperintensity ventral to the hippocampus is a result of a pathologic process, or fat in the substantia spongiosa of the petrous temporal bone.

A 10-month-old dog was presented with clinical signs of fever, lethargy, inappetence, and cardiac arrhythmias. Computed tomography scan and cerebrospinal fluid analysis supported the diagnosis of steroid-responsive meningitis-arteritis. Echocardiography, electrocardiogram, and elevated serum troponin I supported a diagnosis of myocarditis. The arrhythmias resolved during treatment of the primary neurological disease, and they were considered as secondary to the meningitis.

Genome-wide association studies have been performed extensively in the last few years, resulting in many new discoveries of genomic regions that are associated with complex traits. It is often the case that a SNP found to be associated with the condition is not the causal SNP, but a proxy to it as a result of linkage disequilibrium. For the identification of the actual causal SNP, fine-mapping follow-up is performed, either with the use of dense genotyping or by sequencing of the region. In either case, if the causal SNP is in high linkage disequilibrium with other SNPs, the fine-mapping procedure will require a very large sample size for the identification of the causal SNP. Here, we show that by leveraging genetic variability across populations, we significantly increase the localization success rate (LSR) for a causal SNP in a follow-up study that involves multiple populations as compared to a study that involves only one population. Thus, the average power for detection of the causal variant will be higher in a joint analysis than that in studies in which only one population is analyzed at a time. On the basis of this observation, we developed a framework to efficiently search for a follow-up study design: our framework searches for the best combination of populations from a pool of available populations to maximize the LSR for detection of a causal variant. This framework and its accompanying software can be used to considerably enhance the power of fine-mapping studies.

OBJECTIVES

Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11), and to assess interindividual variability in BSEP expression.

METHODS

We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method. BSEP expression was assessed by real-time mRNA analysis, western blot analysis, and immunofluorescence confocal microscopy.

RESULTS

For the most part, polymorphisms were rare and ethnic-dependent. In vitro functional studies revealed several rare variants, including 616A>G, 1674G>C, 1772A>G, and 3556G>A, to be associated with significantly impaired taurocholate transport activity while the 890A>G variant trended towards impaired function but was not statistically significant. The 3556G>A variant was associated with reduced cell surface to total protein expression compared with wild-type BSEP. Expression of BSEP by mRNA and protein analysis was determined from a bank of human liver samples. Wide interindividual variability was noted in both mRNA (19-fold) and protein (31-fold) expression levels. The common variant 1331T>C was associated with significantly reduced hepatic BSEP mRNA levels.

CONCLUSION

Accordingly, our study indicates there are functionally relevant polymorphisms in ABCB11 which may be of potential relevance in the predisposition to acquired liver disorders such as drug-induced cholestasis.

Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(-)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(-)- and (S)-(+)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(-)- or (S)-(+)-FLX (100 microM), both FLX-CoA and FLX-SG were detected by sensitive liquid chromatography-tandem mass spectrometry techniques. However, these derivatives were observed primarily from (R)-(-)-FLX incubation extracts, for which the formation rates of FLX-CoA and FLX-SG were rapid, reaching maximum concentrations of 42 and 2.8 nM, respectively, after 6 min of incubation. Incubations with (S)-(+)-FLX over 60 min displayed 8.1 and 2.7% as much FLX-CoA and FLX-SG area under the concentration versus time curves, respectively, compared with corresponding incubations with (R)-(-)-FLX. Coincubation of lauric acid (1000 microM) with (R)-(-)-FLX (10 microM) led to the complete inhibition of FLX-CoA formation and a 98% inhibition of FLX-SG formation. Reaction of authentic (R,S)-FLX-CoA (2 microM) with GSH (10 mM) in buffer (pH 7.4, 37 degrees C) showed the quantitative formation of FLX-SG after 3 h of incubation. Together, these results demonstrate the stereoselective transacylation of GSH in hepatocyte incubations containing (R)-(-)-FLX, which is consistent with bioactivation by stereoselective (R)-FLX-CoA formation.

The complexities associated with advance treatment decisions may result in decisional uncertainty and poorly informed choices, particularly among vulnerable populations such as minorities and patients with limited health literacy. Using a hypothetical scenario, we assessed uncertainty about treatment preferences among 205 chronically ill, English/Spanish-speaking older adults from a county outpatient clinic in San Francisco. Participants were read a scenrio that described a very poor health state and poor outcome of life support treatment. Participants were asked to imagine that they were in this scenario and to choose either: all life support treatments; try life support with an option of stopping; or no life support. They were then asked how certain they were about this decision. Forty-five percent of participants were uncertain about their decision. In adjusted multivariate analysis, Latinos and Asian/Pacific Islanders were more likely than Whites to be uncertain about their treatment decisions, as were participants with limited versus adequate literacy and poor versus good self-rated health. Many patients may be uncertain about advance treatment preferences. Culturally sensitive, literacy-appropriate tools are needed to address decisional uncertainty and to help patients prepare for decision making about their future health care.