Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r ϭ 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r ϭ 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5Ј-flanking region.CYP3A contributes to the metabolism of numerous therapeutic agents and endogenous molecules. Substrates of CYP3A include benzodiazepines, hydroxymethyl glutarylCoA reductase inhibitors, dihydropyridine calcium channel blockers, human immunodeficiency virus protease inhibitors, antiepileptics, chemotherapeutics, and immunosuppressants (Guengerich, 1999). Interindividual differences in the oral bioavailability and systemic clearance of CYP3A substrates can be attributed in large part to variable expression of CYP3A in the liver (Thummel et al., 1994) and mucosal epithelium of the small intestine (DeWaziers et al., 1990;Paine et al., 1996Paine et al., , 1997. CYP3A4 is the dominant CYP3A isoform in the liver and small intestine of most white adults, whereas CYP3A7 is primarily a fetal enzyme (Kitada and Kamataki, 1994). More recently, human CYP3A43 has been identified and cloned , although its contribution to hepatic or extrahepatic CYP3A-dependent drug clearance is thought to be negligible (Westlind et al., 2001). CYP3A5 is also found in the liver and intestinal mucosa (Wrighton et al., 1990;Paine et al., 1997) and other extrahepatic tissues, including the kidney (Haehner et al., 1996), lung (Kivistö et al., 1996), and prostate gland (Yamakoshi et al., 1999). Its expression is polymorphic, with readily detectable levels in 25 to 30% and very low or undetectable levels in 70 to 75% of livers and small intestines examined (Wrighton et al., 1990;Paine et al., 1997;Tateishi et al., 1999).The genetic basis for polymorphic CYP3A5 expression was first examined by Jounä idi et al....

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

Tacrolimus is a substrate for P-glycoprotein (P-gp) and cytochrome (CYP) P4503A. P-gp is encoded by the multiple drug resistance gene MDR1 and CYP3A is the major enzyme responsible for tacrolimus metabolism. Both MDR1 and CYP3A5 genes have multiple single nucleotide polymorphisms. The objective of this study was to evaluate whether the MDR1 exon21 and exon26 polymorphisms and the CYP3A5 polymorphism are associated with tacrolimus disposition in pediatric heart transplant patients. At 3, 6 and 12 months post transplantation, a significant difference in tacrolimus blood level per dose/kg/day was found between the CYP3A5 *1/*3 (CYP3A5 expressor) vs. *3/*3 (nonexpressor) genotypes with the *1/*3 patients requiring a larger tacrolimus dose to maintain the same blood concentration. There were no significant differences in tacrolimus blood level per dose/kg/day between MDR1 exon21 G2677T and exon 26 C3435T at 3 months, but both were found to have a significant association with tacrolimus blood level per dose/kg/day at 6 and 12 months. We conclude that specific genotypes of MDR1 and CYP3A5 in pediatric heart transplant patients require larger tacrolimus doses to maintain their tacrolimus blood concentration, and that this information could be used prospectively to manage patient's immunosuppressive therapy.

Marked interindividual variability in expression of CYP3A4 influences the disposition of many endo- and xenobiotics, including the metabolism of steroids, environmental toxins and therapeutically useful drugs. The present study was designed to determine the genetic basis of CYP3A4 variability. We analysed DNA from 82 individuals with known CYP3A4 phenotype including 53 Caucasians and 21 African-American liver donors, seven individuals who were outliers in CYP3A4 metabolism and five individuals in a family of a poor nifedipine metabolizer. In addition, we analysed DNA from the eight person DNA Polymorphism Discovery Resource subset (Coriell Institute) and 89 individuals representing nine ethnic groups. Five non-synonymous mutations in the coding region of CYP3A4 were observed. CYP3A4*14 (T44C) in exon 1 resulted in an L15P change; CYP3A4*15 (G14387A) in exon 6 resulted in a R162Q substitution; CYP3A4*10 (G14422C) in exon 6 resulted in a D174H substitution; CYP3A4*16 (C15721G) in exon 7 resulted in a T185S amino acid substitution; and CYP3A4*12 (C22002T) in exon 11 resulted in a L373F change in the CYP3A4 protein. An additional six single nucleotide polymorphisms (SNPs) in the 5'-UTR, 13 SNPs in the introns and three SNPs in the 3'-UTR were observed. Extensive population differences were observed in the frequencies of various CYP3A4 alleles. None of the 28 CYP3A4 SNPs identified in CYP3A4 phenotyped persons (most individuals being heterozygous for any CYP3A4 variant) was associated with low hepatic CYP3A4 protein expression or low CYP3A4 activity in vivo.

This paper presents a robust and fully automatic filter-based approach for retinal vessel segmentation. We propose new filters based on 3D rotating frames in so-called orientation scores, which are functions on the Lie-group domain of positions and orientations [Formula: see text]. By means of a wavelet-type transform, a 2D image is lifted to a 3D orientation score, where elongated structures are disentangled into their corresponding orientation planes. In the lifted domain [Formula: see text], vessels are enhanced by means of multi-scale second-order Gaussian derivatives perpendicular to the line structures. More precisely, we use a left-invariant rotating derivative (LID) frame, and a locally adaptive derivative (LAD) frame. The LAD is adaptive to the local line structures and is found by eigensystem analysis of the left-invariant Hessian matrix (computed with the LID). After multi-scale filtering via the LID or LAD in the orientation score domain, the results are projected back to the 2D image plane giving us the enhanced vessels. Then a binary segmentation is obtained through thresholding. The proposed methods are validated on six retinal image datasets with different image types, on which competitive segmentation performances are achieved. In particular, the proposed algorithm of applying the LAD filter on orientation scores (LAD-OS) outperforms most of the state-of-the-art methods. The LAD-OS is capable of dealing with typically difficult cases like crossings, central arterial reflex, closely parallel and tiny vessels. The high computational speed of the proposed methods allows processing of large datasets in a screening setting.

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