Age-related macular degeneration: a high-resolution genome scan for susceptibility loci in a population enriched for late-stage disease

Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the central region of the retina. AMD is clinically heterogeneous, leading to geographic atrophy (GA) and/or choroidal neovascularization (CNV) at advanced stages. Considerable data exists in support of a genetic predisposition for AMD. Recent linkage studies have provided evidence in favor of several AMD susceptibility loci. We have performed a high-resolution (5-cM) genome scan of 412 affected relative pairs that were enriched for late-stage disease (GA and/or CNV). Nonparametric linkage analysis was performed using two different diagnostic criteria and also by dividing the affected individuals according to GA or CNV phenotype. Our results demonstrate evidence of linkage in regions that were suggested in at least one previous study at chromosomes 1q (236-240 cM in the Marshfield genetic map), 5p (40-50 cM), and 9q (111 cM). Multipoint analysis of affected relatives with CNV provided evidence of additional susceptibility loci on chromosomes 2p (10 cM) and 22q (25 cM). A recently identified Gln5345Arg change in HEMICENTIN-1 on chromosome 1q25 was not detected in 274 affected members in the restricted group with AMD, 346 additional patients with AMD, and 237 unaffected controls. Our results consolidate the chromosomal locations of several AMD susceptibility loci and, together with previous reports, should facilitate the search for disease-associated sequence variants.     

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K(i) towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. (S)-(+)-N-3-Benzylnirvanol and (R)-(-)-N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K(i) values of < 250nM--at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.     

Active site characteristics of CYP4B1 probed with aromatic ligands.

The active site topography of rabbit CYP4B1 has been studied relative to CYP2B1 and CYP102 using a variety of aromatic probe substrates. Oxidation of the prochiral substrate cumene by CYP4B1, but not CYP2B1 or CYP102, resulted in the formation of the thermodynamically disfavored omega-hydroxy metabolite, 2-phenyl-1-propanol, with product stereoselectivity for the (S)-enantiomer. Reaction of CYP4B1, CYP2B1, and CYP102 with phenyldiazene produced spectroscopically observable sigma-complexes for each enzyme. Subsequent oxidation of the CYP2B1 and CYP102 complexes followed by LC/ESI--MS analysis yielded heme pyrrole migration patterns similar to those in previous literature reports. Upon identical treatment, no migration products were detected for CYP4B1. Intramolecular deuterium isotope effects for the benzylic hydroxylation of o-xylene-alpha-(2)H(3), p-xylene-alpha-(2)H(3), 2-(2)H(3),6-dimethylnaphthalene, and 4-(2)H(3),4'-dimethylbiphenyl were determined for CYP4B1 and CYP2B1 to further map their active site dimensions. These probes permit assessment of the ease of equilibration, within P450 active sites, of oxidizable methyl groups located between 3 and 10 A apart [Iyer et al. (1997) Biochemistry 36, 7136--7143]. Isotope effects for the CYP4B1-mediated benzylic hydroxylation of o- and p-xylenes were fully expressed (k(H)/k(D) = 9.7 and 6.8, respectively), whereas deuterium isotope effects for the naphthyl and biphenyl derivatives were both substantially masked (k(H)/k(D) approximately equal to 1). In contrast, significant suppression of the deuterium isotope effects for CYP2B1 occurred only with the biphenyl substrate. Therefore, rapid equilibration between two methyl groups more than 6 A apart is impeded within the active site of CYP4B1, whereas for CYP2B1, equilibration is facile for methyl groups distanced by more than 8 A. Collectively, all data are consistent with the conclusion that the active site of CYP4B1 is considerably restricted relative to CYP2B1.     

Mutations in a BTB-Kelch Protein, KLHL7, Cause Autosomal-Dominant Retinitis Pigmentosa

Retinitis pigmentosa (RP) refers to a genetically heterogeneous group of progressive neurodegenerative diseases that result in dysfunction and/or death of rod and cone photoreceptors in the retina. So far, 18 genes have been identified for autosomal-dominant (ad) RP. Here, we describe an adRP locus (RP42) at chromosome 7p15 through linkage analysis in a six-generation Scandinavian family and identify a disease-causing mutation, c.449G→A (p.S150N), in exon 6 of the KLHL7 gene. Mutation screening of KLHL7 in 502 retinopathy probands has revealed three different missense mutations in six independent families. KLHL7 is widely expressed, including expression in rod photoreceptors, and encodes a 75 kDa protein of the BTB-Kelch subfamily within the BTB superfamily. BTB-Kelch proteins have been implicated in ubiquitination through Cullin E3 ligases. Notably, all three putative disease-causing KLHL7 mutations are within a conserved BACK domain; homology modeling suggests that mutant amino acid side chains can potentially fill the cleft between two helices, thereby affecting the ubiquitination complexes. Mutations in an identical region of another BTB-Kelch protein, gigaxonin, have previously been associated with giant axonal neuropathy. Our studies suggest an additional role of the ubiquitin-proteasome protein-degradation pathway in maintaining neuronal health and in disease.     

Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids

The ¹³c chemical shifts for all the carbon atoms in spironolactone have been assigned. Assignments for nine additional steroids which include the C-7β isomer of spironolactone, its C-7 thiol hydrolysis product, the 7α-thioacetate derivative of testosterone and its thiol hydrolysis product are also reported.     

Bongkrekic acid and the adenosinetriphosphate requirement of malaria parasites

The avian malaria parasite Plasmodium lophurae, when removed from its host erythrocytes into an appropriate medium, develops extracellularly in vitro. This development was inhibited by bongkrekic acid at concentrations down to 2 μg/ml. Adenosine triphosphate at high concentrations partly reversed the inhibition. Bongkrekic acid also inhibited intraerythrocytic development in vitro of the human malaria P. falciparum.