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....
Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.
This study investigated whether a polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T) modifies responses to methotrexate (MTX) in patients undergoing bone marrow transplantation. About 10% to 12% of the population carry the MTHFR TT genotype (enzyme activity, 30% of wild type [CC]). Patients (n ؍ 220) with chronic myelogenous leukemia underwent marrow allografts and were given a short course of MTX. MTX toxicity measures included the oral mucositis index (OMI), speed of engraftment (platelet and granulocyte counts), and bilirubin. Patients with lower MTHFR activity (TT genotype) had 36% higher mean OMI during days 1 to 18 (؉5.7, P ؍ .046) and 20% higher OMI between days 6 and 12 (؉3.8, P ؍ .27). Platelet counts recovered more slowly among patients with the TT genotype compared to wild type (24% slower recovery to 10 000 platelets/L, P ؍ .23; 34% slower to 20 000/ L, P ؍ .08 Folate is essential for nucleotide synthesis. The effectiveness of MTX is largely attributable to its role as an inhibitor of dihydrofolate reductase. Its metabolites also inhibit other folate enzymes, including 5,10-methylenetetrahydrofolate reductase (MTHFR), 1,3,4 which converts 5,10-methylenetetrahydrofolate to 5,10-methyltetrahydrofolate.A common MTHFR polymorphism (C677T) results in reduced activity. 5 The variant TT genotype, associated with about 30% of wild-type (CC) activity, is present in about 10% to 12% of white and Asian populations. Heterozygotes (CT) (about 60% activity) constitute approximately 40% of the population. Variations are seen in risk of acute lymphocytic leukemia, 6 colorectal neoplasia, 7-10 neural tube defects, 11,12 and possibly cardiovascular disease, 13 largely in the presence of low folate levels. Because MTX induces a low folate state, we hypothesized that toxicity would be aggravated among patients with the TT and, possibly, the CT genotype. Study design Study design and patient populationWe undertook a study of patients undergoing marrow transplantation at the Fred Hutchinson Cancer Research Center (FHCRC) from 1992 to 1999.The following criteria applied: (1) first chronic phase of chronic myelogenous leukemia 14 ; (2) first transplantation; (3) conditioning regimen: either busulfan/cyclophosphamide 15 or cyclophosphamide/total body irradiation, as described 15,16 ; (4) age at transplantation, at least 18 years; and (5) available DNA. All patients received 4 doses of MTX (intravenously, day 1, at 15 mg/m, 2 and days 3, 6, and 11, at 10 mg/m 2 ) and cyclosporine (as previously described 15 ) for prevention of GVHD. 2 All patients gave informed consent. The study was approved by the FHCRC Institutional Review Board. Data collectionData were abstracted by a single abstractor (blinded to genotypes) on previous interferon treatment, smoking, MTX administration, and, if applicable, MTX serum levels and leucovorin administration.Data from the patient database included: (1) conditioning regimen; (2) donor/matching status; (3) demographics; (4) weight, height, and calculated ...
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