The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals

BACKGROUND: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. RESULTS: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. CONCLUSION: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species

Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase

AbstractCytochrome P450 2A6 (CYP2A6) is a polymorphic enzyme responsible for the oxidation of certain precarcinogens and drugs and is the major nicotine C-oxidase. The role of CYP2A6 for nicotine elimination was emphasised recently by the finding that smokers carrying defective CYP2A6 alleles consumed fewer cigarettes [Pianezza et al. (1998) Nature 393, 750]. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe reaction for the CYP2A6 enzyme. The present study describes an allele-specific PCR genotyping method that identifies the major defective CYP2A6 allele and accurately predicts the phenotype. An allele frequency of 1–3% was observed in Finnish, Spanish, and Swedish populations, much lower than described previously

Genetic polymorphism of human drug metabolising enzymes : structural and functional studies

There is a pronounced interindividual variability in the levels and activity of many drug metabolising enzymes, which might cause differences in the sensitivity to and the toxicity of many clinically used drugs as well as environmental compounds such as nicotine and precarcinogens. In the present investigation the molecular genetic basis for some of the differences in the CYP2A6, CYP2D6, CYP2E1 and GSTM1 enzymes have been elucidated. Cytochrome P450 2D6 (CYP2D6) exhibits a marked interindividual and interethnic variability, with many Asian and black African populations have a generally reduced CYP2D6 activity compared to Caucasians. In Chinese, who are known to having reduced capacity for metabolism of CYP2D6 substrates, we have identified and characterised the CYP2D6*10 allele. This allele was shown to carry two missense mutations, P34S and S486T, and heterologous expression in COS-1 cells revealed that the P34S substitution yields an unstable enzyme that IS rapidly degraded. Indeed CYP2D6*10 was found to be the most common CYP2D6 allele among Chinese. Among black African populations, it has been shown that the presence of the CYP2D6*17 allele correlates well to reduced in vivo activity. The three missense mutations found in this allele, T107I, R296C and S486T, were introduced in all eight possible combinations into a CYP2D6 cDNA and were expressed in yeast. This revealed that a combination of the T107I and R296C substitutions yielded an enzyme with a 5-fold higher apparent Km for the substrates, whereas no effect was seen when the amino acid substitutions were introduced separately. In a Saudi Arabian population we found a low frequency of these reduced activity alleles and inactive alleles but a Egli frequency of the CYP2D6 gene duplication which is consistent with earlier phenotyping studies in this population. The expression of ethanol-inducible P450 2E1 (CYP2E1) is also known to exhibit striking interindividual variability. In order to elucidate possible genetic causes for this variation, we screened all exons for mutations using genomic DNA from almost 200 unrelated individuals. Two novel alleles were found, yielding enzymes with the amino add substitutions R76H and V3891, respectively. These alleles were however very rare, implicating a selective pressure for active CYP2E1 enzyme, most likely because of an important endogenous function. Sequencing and characterisation of the 5' flanking region of the CYP2E1 gene revealed however the presence of a polymorphic repeat region, with an allele frequency of 23 % in a Chinese population, but only 1 % in Swedes. This polymorphism might be of importance for interindividual differences in the regulation of the CYP2E1 gene. Cytochrome P450 2A6 (CYP2A6) is the major human nicotine C-oxidase. In fact, data has been presented where a correlation was seen between the number of defective CYP2A6*2 and CYP2A6*3 alleles in an individual and the risk of becoming a smoker as well as the number of cigarettes being smoked. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe drug for the CYP2A6 enzyme. Improved genotyping methods were developed which could correctly predict the CYP2A6 phenotype. We could not, however, detect any CYP2A6*3 alleles in contrast to the 2-28 % previously reported and propose that a previously unknown common gene conversion event in the 3' flanking region of the CYP2A6 gene is the reason for this discrepancy. Furthermore, we found that the CYP2A6 gene deletion was due to an unequal cross-over event between the CYP2A6 gene and the related CYP2A7 gene. A PCR-based genotyping method was developed which showed a frequency of the deletion allele to be 15 % in a Chinese population, but only 1 % in Europeans. Finally, the molecular mechanism behind the ultrarapid glutathione S-transferase M1 (GSTM1) activity found in certain individuals was studied. Southern blotting and PCR analysis revealed a novel GSTM1 allele with a duplication of the active GSTM1 gene, which most likely occurred through an unequal cross-over event. A quantitative PCR method was developed which accurately quantified the number of GSTM1 genes. In summary, several novel cytochrome P450 and GSTM1 alleles have been identified and characterised. These are of putative importance for explanation of impaired drug metabolism and possibly for smoking behaviour and genetically determined sensitivity to carcinogens. These findings form a basis for future molecular epidemiological studies and for use in patients as a tool for individualised drug therapy resulting in less side effects and better efficacy

Didaktiska fördjupningskurser för universitetslärare inom och utom lärarutbildningen.

2010 föreslog Lärarutbildningsnämnden (LUN) ett projekt kallat Didaktiska fördjupningskurser för universitetslärare inom och utom lärarutbildningen, vilket skulle utföras inom ramen för PIL:s högskolepedagogiska utvecklingsverksamhet. Bakgrunden till projektet ligger i Göteborgs universitets vision om kompletta akademiska miljöer. Lärarutbildningen, som är en mycket decentraliserad utbildning, har dock mycket begränsade möjligheter att skapa en sådan miljö. Den fakultetsgemensamma forskarskolan Centrum för utbildningsvetenskap och lärarforskning (CUL) är en del av en långsiktig strategi för att skapa en komplett miljö. Vidare har LUN konstaterat att didaktiska fördjupningskurser för lärarutbildare kan vara ett annat sätt att hantera ovanstående problematik genom att skapa en gemensam högskoledidaktisk grund för lärarutbildarna på GU. Projektförslaget hade som inriktning att utveckla två högskoledidaktiska fördjupningskurser om 7,5 högskolepoäng (hp) vardera. Den första kursen skulle vara av mer allmän högskoledidaktisk karaktär och den andra kursen skulle vara riktad mot de av GU:s lärare som medverkar inom lärarprogrammen. En arbetsgrupp tillsattes för att genomföra projektet under hösten 2011. Resultatet av detta arbete är förslag till tre kursplaner (se bilaga 1, 2 och 3) om 5 hp vardera inom fältet högskolepedagogik. Dessa kurser syftar till att bygga på, fördjupa och utveckla de kunskaper och färdigheter som lärare vid GU tillägnat sig inom ramen för befintliga kurser i grundläggande högskolepedagogik som ges av PIL (HPE100-serien). Den första kursen – Allmän högskoledidaktik, 5 hp – fokuserar på grundläggande didaktiska frågor med fokus på begreppet användbarhet. Den andra kursen – Ämnesdidaktik för lärarutbildare, 5 hp – fokuserar på de interna och externa aspekter av samverkan som karaktäriserar lärarutbildningen. Den tredje kursen - Fördjupningsarbete i högskoledidaktik, 5 hp – syftar till att kursdeltagaren inom ramen för ett självständigt arbete ska utforska lärarutbildningens dubbla konstnärliga/vetenskapliga grund i relation till det egna ämnes-/kunskapsfältet. Kursernas övergripande mål är att stärka en formell och reell didaktisk kompetensutveckling av GU:s lärarkår och förväntas därigenom bidra till en ökad utbildningskvalitet inom Göteborgs universitet i allmänhet och för lärarprogrammen i synnerhet

Late onset hyperornithinemia-hyperammonemia-homocitrullinuria syndrome - how web searching by the family solved unexplained unconsciousness: a case report

Abstract Background Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a rare inherited urea cycle disorder, can remain undiagnosed for decades and suddenly turn into an acute life-threatening state. Adult presentation of hyperornithinemia-hyperammonemia-homocitrullinuria syndrome has rarely been described, but is potentially underdiagnosed in the emergency room. In the case of acute hyperammonemia, prompt diagnosis is essential to minimize the risk of brain damage and death. Case presentation We present the diagnostics, clinical course, and treatment of a 48-year-old Caucasian man presenting with unexplained unconsciousness in the emergency room. A web search by a family member led to the suspicion of urea cycle disorder. Subsequent analysis of plasma ammonia and amino acids in plasma and urine demonstrated a pattern typical for hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. The diagnosis was confirmed by genetic analysis which revealed two heterozygous mutations in the SLC25A15 gene. The cause of the hyperammonemia crisis was acute upper gastrointestinal hemorrhage, leading to protein overload and subsequent cerebral edema. Continuous renal replacement therapy, scavenger treatment, and tightly controlled nutrition were useful in preventing hyperammonemia and recurrence of cerebral edema. Conclusions The case emphasizes the importance of taking rare metabolic genetic disorders into consideration in patients with prolonged unexplained unconsciousness

Isolated 46,XY gonadal dysgenesis in two sisters caused by a Xp21.2 interstitial duplication containing the DAX1 gene.

Context: Testis development is a tightly regulated process that requires an efficient and coordinated spatiotemporal action of many factors, and it has been shown that several genes involved in gonadal development exert a dosage effect. Chromosomal imbalances have been reported in several patients presenting with gonadal dysgenesis as part of severe dysmorphic phenotypes. Results: We screened for submicroscopic DNA copy number variations in two sisters with an apparent normal 46, XY karyotype and female external genitalia due to gonadal dysgenesis, and in which mutations in known candidate genes had been excluded. By high-resolution tiling bacterial artificial chromosome array comparative genome hybridization, a submicroscopic duplication at Xp21.2 containing DAX1 ( NR0B1) was identified. Using fluorescence in situ hybridization, multiple ligation probe amplification, and PCR, the rearrangement was further characterized. This revealed a 637-kb tandem duplication that in addition to DAX1 includes the four MA-GEB genes, the hypothetical gene CXorf21, GK, and part of the MAP3K7IP3 gene. Sequencing and analysis of the breakpoint boundaries and duplication junction suggest that the duplication originated through a coupled homologous and nonhomologous recombination process. Conclusions: This represents the first duplication on Xp21.2 identified in patients with isolated gonadal dysgenesis because all previously described XY subjects with Xp21 duplications presented with gonadal dysgenesis as part of a more complex phenotype, including mental retardation and/or malformations. Thus, our data support DAX1 as a dosage sensitive gene responsible for gonadal dysgenesis and highlight the importance of considering DAX1 locus duplications in the evaluation of all cases of 46, XY gonadal dysgenesis