Denise L. Walker

The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by dilation of collecting ducts and by biliary dysgenesis and is an important cause of renal- and liver-related morbidity and mortality. Genetic analysis of a rat with recessive polycystic kidney disease revealed an orthologous relationship between the rat locus and the ARPKD region in humans; a candidate gene was identified. A mutation was characterized in the rat and screening the 66 coding exons of the human ortholog (PKHD1) in 14 probands with ARPKD revealed 6 truncating and 12 missense mutations; 8 of the affected individuals were compound heterozygotes. The PKHD1 transcript, approximately 16 kb long, is expressed in adult and fetal kidney, liver and pancreas and is predicted to encode a large novel protein, fibrocystin, with multiple copies of a domain shared with plexins and transcription factors. Fibrocystin may be a receptor protein that acts in collecting-duct and biliary differentiation.

Comprehensive Molecular Diagnostics in Autosomal Dominant Polycystic Kidney Disease

Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, duplication of PKD1, and a high level of unclassified variants (UCV). Present mutation detection levels are 60 to 70%, and PKD1 and PKD2 UCV have not been systematically classified. This study analyzed the uniquely characterized Consortium for Radiologic Imaging Study of PKD (CRISP) ADPKD population by molecular analysis. A cohort of 202 probands was screened by denaturing HPLC, followed by direct sequencing using a clinical test of 121 with no definite mutation (plus controls). A subset was also screened for larger deletions, and reverse transcription-PCR was used to test abnormal splicing. Definite mutations were identified in 127 (62.9%) probands, and all UCV were assessed for their potential pathogenicity. The Grantham Matrix Score was used to score the significance of the substitution and the conservation of the residue in orthologs and defined domains. The likelihood for aberrant splicing and contextual information about the UCV within the patient (including segregation analysis) was used in combination to define a variant score. From this analysis, 44 missense plus two atypical splicing and seven small in-frame changes were defined as probably pathogenic and assigned to a mutation group. Mutations were thus defined in 180 (89.1%) probands: 153 (85.0%) PKD1 and 27 (15.0%) PKD2. The majority were unique to a single family, but recurrent mutations accounted for 30.0% of the total. A total of 190 polymorphic variants were identified in PKD1 (average of 10.1 per patient) and eight in PKD2. Although nondefinite mutation data must be treated with care in the clinical setting, this study shows the potential for molecular diagnostics in ADPKD that is likely to become increasingly important as therapies become available.

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

Studies of humoral immunity to preprohypocretin in human leukocyte antigen DQB1*0602-positive narcoleptic subjects with cataplexy

BACKGROUND Canine models for narcolepsy have mutations of the hypocretin receptor 2 gene, and preprohypocretin knockout murine lines exhibit narcoleptic-like behaviors. Human narcolepsy with cataplexy is associated with human leukocyte antigen DQB1*0602 and reduced hypocretin levels in cerebrospinal fluid, suggesting an autoimmune diathesis. We tested the hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have immunoglobulin (Ig)G reactive to human preprohypocretin and its cleavage products. METHODS Serum samples of 41 DQB1*0602-positive narcoleptic subjects with cataplexy and 55 control subjects were studied, as were 19 narcoleptic and 13 control samples of cerebrospinal fluid. We tested for IgG reactive to preprohypocretin and its major cleavage products (including hypocretin 1 and 2), using immunoprecipitation assays (IP), immunofluorescence microscopy (IF) of Chinese hamster ovarian cells expressing preprohypocretin, and Western blots. RESULTS There was no evidence for IgG reactive to preprohypocretin or its cleavage products in CSF of subjects with narcolepsy as measured by IPs, Western blots, and IF. Although the IP with CSF and the C-terminal peptide showed significant differences by two methods of comparison, the control subjects had higher counts per minute than narcoleptic subjects, which was opposite to our hypothesis. CONCLUSIONS The hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have IgG reactive to preprohypocretin or its cleavage products was not supported.

Preclinical pharmacology of bizelesin, a potent bifunctional analog of the DNA-binding antibiotic CC-1065

Bizelesin (NSC-615291), a potent, bifunctional analog of the cyclopropylpyrroloindole antitumor antibiotics CC-1065 and adozelesin, has been selected by the National Cancer Institute for evaluation as a potential chemotherapeutic agent. All three compounds bind to and alkylate DNA at the N-3 position of adenine in a sequence-selective manner. Bizelesin is unique among the analogs with bifunctional alkylating capability due to two chloromethyl moieties that are converted to the cyclopropyl alkylating species that interact with DNA. A reverse-phase high-performance liquid chromatography (HPLC) assay and an L1210 cell bioassay were developed for bizelesin and subsequently applied to stability and murine pharmacokinetics studies. Following 48 h of incubation with L1210 cells the 50% growth-inhibitory concentrations (IC50) of bizelesin, adozelesin, and CC-1065 were 2.3, 3.4, and 88.1 pM, respectively. Bizelesin was stable in organic solvents but was less stable in aqueous solutions, with the half-life values obtained in buffers at pH 4, 7, and 10 being 9.6, 2.1, and <1 h, respectively. By HPLC analysis, bizelesin degradation was associated with the appearance of two peaks, the mono- and dicyclopropyl derivatives formed by base-catalyzed intramolecular alkylation of the chloromethyl groups. Bizelesin and the dicyclopropyl derivative were equipotent in the L1210 cell bioassay. Following i.v. administration of bizelesin (15 μg/kg) to male CD2F1 mice, the plasma elimination of cytotoxic activity determined with the bioassay was described by a two compartment open model; the α-phase (t1/2α) and β-phase (t1/2β) half-lives, steady-state volume of distribution (VSS), and total body clearance (ClTB) were 3.5 min, 7.3 h, 7,641 ml/kg, and 16.3 ml min−1 kg−1, respectively. The systemic drug exposure following i. p. administration was at least 10 times lower than that resulting from i. v. infusion. Following i. v. or i. p. administration the recovery of material in urine was <0.1% of the delivered dose.

CYP2D6: novel genomic structures and alleles.

Objective CYP2D6 is a polymorphic gene. It has been observed to be deleted, to be duplicated and to undergo recombination events involving the CYP2D7 pseudogene and surrounding sequences. The objective of this study was to discover the genomic structure of CYP2D6 recombinants that interfere with clinical genotyping platforms that are available today. Methods Clinical samples containing rare homozygous CYP2D6 alleles, ambiguous readouts, and those with duplication signals and two different alleles were analyzed by long-range PCR amplification of individual genes, PCR fragment analysis, allele-specific primer extension assay, and DNA sequencing to characterize alleles and genomic structure. Results Novel alleles, genomic structures, and the DNA sequence of these structures are described. Interestingly, in 49 of 50 DNA samples that had CYP2D6 gene duplications or multiplications where two alleles were detected, the chromosome containing the duplication or multiplication had identical tandem alleles. Conclusion Several new CYP2D6 alleles and genomic structures are described which will be useful for CYP2D6 genotyping. The findings suggest that the recombination events responsible for CYP2D6 duplications and multiplications are because of mechanisms other than interchromosomal crossover during meiosis.

The Metabolism of Pyrazoloacridine (NSC 366140) by Cytochromes P450 and Flavin Monooxygenase in Human Liver Microsomes

Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase. After administration of [10b-14C]PZA to mice, six urinary metabolites were detected by high-performance liquid chromatography UV and radiochromatograms including 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. Trace concentrations of 9-desmethyl-PZA and PZA N-oxide were detected in mouse plasma. PZA N-oxide and N-demethyl-PZA were detected in urine from patients after PZA administration. PZA, 9-desmethyl-PZA, and PZA N-oxide inhibited growth of A375 human melanoma cells. IC50 values were 0.17, 0.11, and 7.0 μm, respectively, for the three molecules.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1-2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD+, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F1 mice. 6AN reached peak serum concentrations of 80-90 microM and was cleared rapidly, with T1/2alpha and T1/2beta values of 7.4 and 31.3 min, respectively. Bioavailability was 80-100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

Preclinical pharmacology of ecteinascidin 729, a marine natural product with potent antitumor activity

Abstract Ecteinascidins are marine natural products with potent antiproliferative activity under evaluation as chemotherapeutic agents by the National Cancer Institute. Ecteinascidins bind the minor groove of DNA and may form covalent adducts with DNA by binding the N-2 of guanine in a fashion similar to saframycin antibiotics. The most potent ecteinascidin is ET-729 with antitumor activity observed following administration of 3.8 and 10 μg/kg to mice bearing P388 leukemia and B16 melanoma, respectively. A reverse-phase high-performance liquid chromatography (HPLC) assay and an L1210 cell bioassay were developed for ET-729 and utilized for stability and murine pharmacokinetic studies. HPLC analysis showed that ET-729 was stable in organic solvents, mobile phase and acidic buffer (t1/2 > 100 h). Stability was diminished under neutral and basic conditions (t1/2 < 14 h). Following a 48-h incubation with L1210 cells in growth medium in the absence and presence of 2.5% murine plasma, the 50% growth inhibitory concentrations (IC50) of ET-729 were 37 and 72 pM, respectively. Following intravenous administration of ET-729 to male CD2F1 mice, the disappearance of antiproliferative activity determined by the bioassay was described by a two-compartment open model. The mean values of the elimination half-life and plasma clearance were 28 min and 39.7 ml/min per kg, respectively. Following intraperitoneal administration, peak plasma concentrations of antiproliferative activity were observed 6–15 min after injection and antiproliferative concentrations remained above 1 nM for longer than 1 h. Intraperitoneal bioavailability varied over a wide range (20–91%). Antiproliferative activity was detected in every urine sample following intravenous and intraperitoneal administration, but the total 48-h urinary recovery was less than 0.1%.

Frequency of Undetected CYP2D6 Hybrid Genes in Clinical Samples: Impact on Phenotype Prediction

Cytochrome P450 2D6 (CYP2D6) is highly polymorphic. CYP2D6-2D7 hybrid genes can be present in samples containing CYP2D6*4 and CYP2D6*10 alleles. CYP2D7-2D6 hybrid genes can be present in samples with duplication signals and in samples with homozygous genotyping results. The frequency of hybrid genes in clinical samples is unknown. We evaluated 1390 samples for undetected hybrid genes by polymerase chain reaction (PCR) amplification, PCR fragment analysis, TaqMan copy number assays, DNA sequencing, and allele-specific primer extension assay. Of 508 CYP2D6*4-containing samples, 109 (21.5%) harbored CYP2D6*68 + *4-like, whereas 9 (1.8%) harbored CYP2D6*4N + *4-like. Of 209 CYP2D6*10-containing samples, 44 (21.1%) were found to have CYP2D6*36 + *10. Of 332 homozygous samples, 4 (1.2%) harbored a single CYP2D7-2D6 hybrid, and of 341 samples with duplication signals, 25 (7.3%) harbored an undetected CYP2D7-2D6 hybrid. Phenotype before and after accurate genotyping was predicted using a method in clinical use. The presence of hybrid genes had no effect on the phenotype prediction of CYP2D6*4- and CYP2D6*10-containing samples. Four of four (100%) homozygous samples containing a CYP2D7-2D6 gene had a change in predicted phenotype, and 23 of 25 (92%) samples with a duplication signal and a CYP2D7-2D6 gene had a change in predicted phenotype. Four novel genes were identified (CYP2D6*13A1 variants 1 and 2, CYP2D6*13G1, and CYP2D6*13G2), and two novel hybrid tandem structures consisting of CYP2D6*13B + *68×2 + *4-like and CYP2D6*13A1 variant 2 + *1×N were observed.