A comprehensive mutation analysis of RP2 and RPGR in a North American cohort of families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP. Mutations in an alternatively spliced RPGR exon, ORF15, have recently been shown to account for 60% of XLRP in a European cohort of 47 families. We have performed, in a North American cohort of 234 families with RP, a comprehensive screen of the RP2 and RPGR (including ORF15) genes and their 5' upstream regions. Of these families, 91 (39%) show definitive X-linked inheritance, an additional 88 (38%) reveal a pattern consistent with X-linked disease, and the remaining 55 (23%) are simplex male patients with RP who had an early onset and/or severe disease. In agreement with the previous studies, we show that mutations in the RP2 gene and in the original 19 RPGR exons are detected in <10% and approximately 20% of XLRP probands, respectively. Our studies have revealed RPGR-ORF15 mutations in an additional 30% of 91 well-documented families with X-linked recessive inheritance and in 22% of the total 234 probands analyzed. We suggest that mutations in an as-yet-uncharacterized RPGR exon(s), intronic changes, or another gene in the region might be responsible for the disease in the remainder of this North American cohort. We also discuss the implications of our studies for genetic diagnosis, genotype-phenotype correlations, and gene-based therapy.     

The human secretin gene: fine structure in 11p15.5 and sequence variation in patients with autism

Secretin is a peptide hormone involved in digestion that has been studied as a potential therapeutic agent in patients with autism. We characterized the human secretin locus to determine whether mutations in this gene might play a role in a fraction of autism patients. While the secretin gene (SCT) was not found to be mutated in the majority of autistic patients, rare heterozygous sequence variants were identified in three patients. We also investigated length variation in a variable number of tandem repeats (VNTR) immediately upstream of SCT and found no significant differences in length between patients with autism and normal controls. SCT is located on 11p15.5, adjacent to DRD4 and HRAS. This region has been reported to be associated with both autism and attention deficit hyperactivity disorder (ADHD). Although imprinting is a characteristic of some genes in the vicinity, we could find no evidence for methylation of SCT in lymphoblast cells from patients or control individuals.     

Generation of H2O2 in Brain Mitochondria

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.     

Structure of the dnaA region of the endosymbiont, Buchnera aphidicola, of aphid Schizaphis graminum

Buchnera aphidicola is an intracellular prokaryote (endosymbiont)that lives in the body cavity of the aphid. Phylogenetic studiesindicated that it is closely related to Escherichia coli andmembers of Enterobacteria. The gene order of the region containingthe dnaA gene is well conserved in many bacteria. Seven genesof the endosymbiont of the aphid Schizaphis graminum, gyrB,dnaN, dnaA, rpmH, rnpA, yidD, and 60K, were found to be homologousin sequence and relative location to those of E. coli. We havefurther sequenced the region downstream of the 60K gene to elucidatethe boundary of the conserved region, and found that one moregene, thdF , is conserved. The comparison of gene organizationsof the dnaA region of the related bacteria supported the closephylogenetic relationship of B. aphidicola to E. coli. In addition,we have identified groES and groEL genesnext to the thdF gene.GroEL protein was reported to be expressed at an elevated levelin the endosymbionts of aphids, and is considered to play animportant role in their association with the aphid host. Comparisonof the structure of the groE operon with that of the endosymbiontof the aphid Acyrthosiphon pisum revealed the conservation ofa sequence resembling the E. coli consensus heat shock promoter,and this sequence may be responsible for the high expressionof the groEL gene in aphid endosymbionts.     

Generation of hydrogen peroxide on oxidation of NADH by hepatic plasma membranes

The oxidation of NADH by mouse liver plasma membranes was shown to be accompanied by the formation of H₂O₂. The rate of H₂O₂ formation was less than one-tenth the rate of oxygen uptake and much slower than the rate of reduction of artificial electron acceptors. The optimum pH for this reaction was 7.0 and theK _m value for NADH was found to be 3×10^–6 M. The H₂O₂-generating system of plasma membranes was inhibited by quinacrine and azide, thus distinguishing it from similar activities in endoplasmic reticulum and mitochondria. Both NADH and NADPH served as substrates for plasma membrane H₂O₂ generation. Superoxide dismutase and adriamycin inhibited the reaction. Vanadate, known to stimulate the oxidation of NADH by plasma membranes, did not increase the formation of H₂O₂. In view of the growing evidence that H₂O₂ can be involved in metabolic control, the formation of H₂O₂ by a plasma membrane NAD(P)H oxidase system may be pertinent to control sites at the plasma membrane.