A new locus (RP31) for autosomal dominant retinitis pigmentosa maps to chromosome 9p

Retinitis pigmentosa (RP) is a debilitating disease of the retina affecting ∼1.5 million people worldwide. RP shows remarkable heterogeneity both clinically and genetically, with more than 40 genetic loci implicated, 12 of which account for the autosomal dominant form (adRP) of inheritance. We have recently identified a French Canadian family that presents with early onset adRP. After exclusion of all known loci for adRP, a genome-wide search established firm linkage with a marker from the short arm of chromosome 9 (LOD score of 6.3 at recombination fraction θ=0). The linked region is flanked by markers D9S285 and D9S1874, corresponding to a genetic distance of 31 cM, in the region 9p22-p13.     

Cell migration: Rho GTPases lead the way

Rho GTPases control signal transduction pathways that link cell surface receptors to a variety of intracellular responses. They are best known as regulators of the actin cytoskeleton, but in addition they control cell polarity, gene expression, microtubule dynamics and vesicular trafficking. Through these diverse functions, Rho GTPases influence many aspects of cell behavior. This review will focus specifically on their role in cell migration.     

A new dendritic cell vaccine generated with interleukin-3 and interferon-β induces CD8+ T cell responses against NA17-A2 tumor peptide in melanoma patients

Dendritic cells derived from monocytes cultured in the presence of type I interferon were found to induce efficient T cell responses against tumor antigens in vitro. We vaccinated eight stage III or IV melanoma patients with dendritic cells generated with interferon-β and interleukin-3, activated by poly I: C, and pulsed with the tumor-specific antigen NA17.A2. This dendritic cell vaccine was well-tolerated with only minor and transient flu-like symptoms and inflammatory reactions at the injection sites. In most patients, isotopic imaging documented dendritic cells (DC) migration from the intradermal injection site to the draining lymph nodes. Finally, mixed lymphocyte-peptide culture under limiting dilution conditions followed by tetramer labeling indicated that three out of eight patients mounted a CD8 T cell response against the NA17.A2 antigenic peptide. We conclude that DC generated in type I-IFN represent an interesting alternative to DC generated in IL-4 and GM-CSF for cancer immunotherapy.     

Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty

Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.     

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy

We report mutations in the gene for topoisomerase I-binding RS protein (TOPORS) in patients with autosomal dominant retinitis pigmentosa (adRP) linked to chromosome 9p21.1 (locus RP31). A positional-cloning approach, together with the use of bioinformatics, identified TOPORS (comprising three exons and encoding a protein of 1,045 aa) as the gene responsible for adRP. Mutations that include an insertion and a deletion have been identified in two adRP-affected families--one French Canadian and one German family, respectively. Interestingly, a distinct phenotype is noted at the earlier stages of the disease, with an unusual perivascular cuff of retinal pigment epithelium atrophy, which was found surrounding the superior and inferior arcades in the retina. TOPORS is a RING domain-containing E3 ubiquitin ligase and localizes in the nucleus in speckled loci that are associated with promyelocytic leukemia bodies. The ubiquitous nature of TOPORS expression and a lack of mutant protein in patients are highly suggestive of haploinsufficiency, rather than a dominant negative effect, as the molecular mechanism of the disease and make rescue of the clinical phenotype amenable to somatic gene therapy.     

Reconstruction of human evolutionary tree using polymorphic autosomal microsatellites

Allelic frequencies of 182 tri- and tetra-autosomal microsatellites were used to examine phylogenetic relationships among 19 extant human populations. In particular, because the languages of the Basques and Hunza Burusho have been suggested to have an ancient relationship, this study sought to explore the genetic relationship between these two major language isolate populations and to compare them with other human populations. The work presented here shows that the microsatellite allelic diversity and the number of unique alleles were highest in sub-Saharan Africans. Neighbor-joining trees based on genetic distances and principal component analyses separated populations from different continents, and are consistent with an African origin for modern humans. For the first time, with biparentally transmitted markers, the microsatellite tree also shows that the San are the first branch of the human tree before the branch leading to all other Africans. In contrast to an earlier study, these results provided no evidence of a genetic relationship among the two language isolate groups. Genetic relationships, as ascertained by these microsatellites, are dictated primarily by geographic proximity rather than by remote linguistic origin, Mantel test, R(0) = 0.484, g = 3.802 (critical g value = 1.645; P = 0.05).     

Evaluation of four capillary columns for the analysis of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers in human serum for epidemiologic studies

The separation of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether (PBDE) congeners was evaluated on four capillary columns: 60 m x 0.25 mm i.d., 0.25 microm film thickness RTX-5MS and DB-XLB capillary columns, and 60 m x 0.18 mm i.d., 0.25 microm film thickness DB-XLB and DB-5MS capillary columns. Based on performance, capacity, and cost, the RTX-5MS (60 m x 0.25 mm i.d., 0.25 microm thickness) and the DB-XLB (60 m x 0.25 mm i.d., 0.25 microm film thickness) were selected for the analysis of human serum extracts by using gas chromatography/electron-capture detection. In contrast to previous studies, the oven temperature program affords the separation of congeners that are not separated by using other combinations of capillary columns, most notably PBDE-47 and PCB 180. In addition, the method enables determination of OCPs, PCBs, and PBDEs prevalent in a single extract of serum, which can lead to considerable time savings in the analysis of large number of samples collected for epidemiologic studies.