A duplication on chromosome 16q12 affecting the IRXB gene cluster is associated with autosomal dominant cone dystrophy with early tritanopic color vision defect.

Cone dystrophies are a rare subgroup of inherited retinal dystrophies and hallmarked by color vision defects, low or decreasing visual acuity and central vision loss, nystagmus and photophobia. Applying genome-wide linkage analysis and array comparative genome hybridization, we identified a locus for autosomal dominant cone dystrophy on chromosome 16q12 in four independent multigeneration families. The locus is defined by duplications of variable size with a smallest region of overlap of 608 kb affecting the IRXB gene cluster and encompasses the genes IRX5 and IRX6. IRX5 and IRX6 belong to the Iroquois (Iro) protein family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissue in vertebrates, including the eye and the retina. All patients presented with a unique progressive cone dystrophy phenotype hallmarked by early tritanopic color vision defects. We propose that the disease underlies a misregulation of the IRXB gene cluster on chromosome 16q12 and demonstrate that overexpression of Irx5a and Irx6a, the two orthologous genes in zebrafish, results in visual impairment in 5-day-old zebrafish larvae.

Nitrite Reductase Activity in Engineered Azurin Variants.

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

Large deletions of the KCNV2 gene are common in patients with cone dystrophy with supernormal rod response.

Cone dystrophy with supernormal rod response (CDSRR) is considered to be a very rare autosomal recessive retinal disorder. CDSRR is associated with mutations in KCNV2, a gene that encodes a modulatory subunit (Kv8.2) of a voltage-gated potassium channel. In this study, we found that KCNV2 mutations are present in a substantial fraction (2.2-4.3%) of a sample of 367 independent patients with a variety of initial clinical diagnoses of cone malfunction, indicating that CDSRR is underdiagnosed and more common than previously thought. In total, we identified 20 different KCNV2 mutations; 15 of them are novel. A new finding of this study is the substantial proportion of large deletions at the KCNV2 locus that accounts for 15.5% of the mutant alleles in our sample. We determined the breakpoints and size of all five different deletions, which ranged between 10.9 and 236.8 kb. Two deletions encompass the entire KCNV2 gene and one also includes the adjacent VLDLR gene. Furthermore, we investigated N-terminal amino acid substitution mutations for its effect on interaction with Kv2.1 using yeast two-hybrid technology. We found that these mutations dramatically reduce or abolish this interaction suggesting a lack of assembly of heteromeric Kv channels as one underlying pathomechanism of CDSRR.

Fetal MRI for prediction of neonatal mortality following preterm premature rupture of the fetal membranes.

BACKGROUND: Lung MRI volumetrics may be valuable for fetal assessment following early preterm premature rupture of the foetal membranes (pPROM). OBJECTIVE: To evaluate the predictive value of MRI lung volumetrics after pPROM. MATERIALS AND METHODS: Retrospective cohort study of 40 fetuses after pPROM in a large, tertiary, perinatal referral center. Fetuses underwent MRI lung volumetrics. Estimated lung volume was expressed as percentage of expected lung volume (our own normal references). Primary outcome was neonatal mortality due to respiratory distress before discharge from hospital. RESULTS: Gestational age range was 16-27 weeks. Estimated-to-expected lung volume was 73% in non-survivors and 102% in survivors (P < 0.05). There were no survivors with a lung volume less than 60% of expected. By logistic regression, mortality could be predicted with a sensitivity of 80%, specificity of 86% and accuracy of 85%. CONCLUSION: Fetal MR lung volumetrics may be useful for predicting mortality due to respiratory distress in children with early gestational pPROM.

Interaction of thiazolidinediones (glitazones) with the ATP-binding cassette transporters P-glycoprotein and breast cancer resistance protein.

AIMS: Thiazolidinediones (glitazones) are frequently prescribed antidiabetic drugs commonly used in combination drug regimens. To evaluate the risk of drug-drug interactions, we therefore aimed to systematically investigate the inhibitory and inductive effects of all glitazones on 2 of the most relevant drug transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in vitro. METHODS: The inhibition of P-gp and BCRP was assessed by fluorometric assays quantifying the increase in the intracellular concentration of fluorescent P-gp or BCRP substrates caused by their combination with the glitazones. The induction of mRNA expression was quantified by real-time RT-PCR after the treatment of HuH-7 cells with the respective compounds for 4 days. RESULTS: Rosiglitazone and troglitazone significantly inhibited P-gp and BCRP function and induced mRNA expression of BCRP but not of P-gp. Pioglitazone, which exhibited very low solubility, could only be tested up to 0.5 micromol/l and did not provoke an effect in any of the assays. CONCLUSIONS: After comparison of the in vitro data and published clinical studies, it seems unlikely that the inhibition of BCRP and P-gp by rosiglitazone plays a role in the clinical situation. In contrast, BCRP induction by rosiglitazone might be of relevance in vivo, but has to be verified in dedicated clinical studies.

In vitro and ex vivo evidence for modulation of P-glycoprotein activity by progestins.

The well known gender-related differences in drug action may partly be explained by changes in activity and expression of drug metabolising enzymes, but also by modulation of active drug transport systems (e.g. P-glycoprotein, Pgp) by sexual steroids, which is yet not well investigated. Because many women are using hormones (e.g. as oral contraceptives) we investigated the influence of different synthetic progestins on Pgp activity. Pgp inhibition of progesterone, medroxyprogesterone, chlormadinone, cyproterone, levonorgestrel, norethisterone, desogestrel, and norgestimate was measured in vitro in two Pgp over-expressing cell lines (L-MDR1, P388/dx cells) and the corresponding parental cell lines by means of calcein assay, and ex vivo in human peripheral blood mononuclear cells (PBMCs) by rhodamine123 efflux. For most progestins tested, concentrations needed to double baseline fluorescence (f2) in L-MDR1 cells were similar to that of the potent Pgp inhibitor quinidine, whereas levonorgestrel and norethisterone did not reach f2. The results in P388/dx cells essentially confirmed our findings in L-MDR1 cells. Additionally, Pgp inhibitory activity of all progestins tested was also shown ex vivo in PBMCs. The potent Pgp inhibition by several synthetic progestins in vitro and ex vivo suggests that such an interaction might be clinically relevant despite generally low plasma concentrations of progestins. The results may be of particular importance for Pgp substrates, such as protease inhibitors and chemotherapeutic agents, for which intracellular concentrations are critical.

Rhodopsin F45L Allele Does Not Cause Autosomal Dominant Retinitis Pigmentosa in a Large Caucasian Family.

PURPOSE: To ascertain the potential pathogenicity of a retinitis pigmentosa (RP)-causing RHO F45L allele in a family affected by congenital achromatopsia (ACHM). METHODS: Case series/observational study that included two patients with ACHM and 24 extended family members. Molecular genetic analysis was performed to identify RHO F45L carrier status in the family and a control population. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Comprehensive clinical testing included acuity, color vision, and dilated fundus examination. Electroretinography was used to assess rod and cone function. RESULTS: Five carriers of the RHO F45L allele alone (24-80 years) and three carriers in combination with a heterozygous CNGA3 mutant allele (10-64 years) were all free of the classic symptoms and signs of RP. In heterozygous carriers of both mutations, SD-OCT showed normal retinal thickness and intact outer retinal layers; rod and cone densities were within normal limits on AOSLO. The phenotype in two individuals affected with ACHM and harboring the RHO F45L allele was indistinguishable from that previously reported for ACHM. CONCLUSIONS: The RHO F45L allele is not pathogenic in this large family; hence, the two ACHM patients would unlikely develop RP in the future. TRANSLATIONAL RELEVANCE: The combined approach of comprehensive molecular analysis of individual genomes and noninvasive cellular resolution retinal imaging enhances the current repertoire of clinical diagnostic tools, giving a substantial impetus to personalized medicine.

Cone-rod dystrophy associated with amelogenesis imperfecta in a child with neurofibromatosis type 1.

PURPOSE: To report a case of a 9-year-old child with neurofibromatosis type 1 (NF1) and Jalili syndrome, the latter denoting a rare combination of cone-rod dystrophy and amelogenesis imperfecta. METHODS: Detailed ophthalmological and electrophysiological examinations were carried out and blood samples were taken from the patient and her father for molecular genetic analysis by direct DNA sequencing of the NF1 and the ancient conserved domain protein 4 (CNNM4) gene. RESULTS: The diagnosis of neurofibromatosis type 1 (NF1) could be confirmed clinically and genetically. Furthermore, cone-rod dystrophy and amelogenesis imperfecta could be observed as typical features of a rare condition, acknowledged as Jalili syndrome. The diagnosis was assured on the basis of clinical examinations and molecular genetic analysis of the CNNM4 gene, which was previously shown to cause Jalili syndrome. CONCLUSION: Our case shows a unique combination of NF1 and Jalili syndrome. The random association of two diseases is unusual and deserves attention. This case highlights the importance not only of detailed clinical examination, but also of molecular genetic analysis, which together provide a precise diagnosis.

Factors determining sensitivity or resistance of tumor cell lines towards artesunate.

Clinical oncology is still challenged by the development of drug resistance of tumors that result in poor prognosis for patients. There is an urgent necessity to understand the molecular mechanisms of resistance and to develop novel therapy strategies. Artesunate (ART) is an anti-malarial drug, which also exerts profound cytotoxic activity towards cancer cells. We first applied a gene-hunting approach using cluster and COMPARE analyses of microarray-based transcriptome-wide mRNA expression profiles. Among the genes identified by this approach were genes from diverse functional groups such as structural constituents of ribosomes (RPL6, RPL7, RPS12, RPS15A), kinases (CABC1, CCT2, RPL41), transcriptional and translational regulators (SFRS2, TUFM, ZBTB4), signal transducers (FLNA), control of cell growth and proliferation (RPS6), angiogenesis promoting factors (ITGB1), and others (SLC25A19, NCKAP1, BST1, DBH, FZD7, NACA, MTHFD2). Furthermore, we applied a candidate gene approach and tested the role of resistance mechanisms towards established anti-cancer drugs for ART resistance. By using transfected or knockout cell models we found that the tumor suppressor p16(INK4A) and the anti-oxidant protein, catalase, conferred resistance towards ART, while the oncogene HPV-E6 conferred sensitivity towards ART. The tumor suppressor p53 and its downstream protein, p21, as well as the anti-oxidant manganese-dependent superoxide dismutase did not affect cellular response to ART. In conclusion, our pharmacogenomic approach revealed that response of tumor cells towards ART is multi-factorial and is determined by gene expression associated with either ART sensitivity or resistance. At least some of the functional groups of genes (e.g. angiogenesis promoting factors, cell growth and proliferation-associated genes signal transducers and kinases) are also implicated in clinical responsiveness of tumors towards chemotherapy. It merits further investigation, whether ART is responsive in clinically refractory tumors and whether the genes identified in the present study also determine clinical responsiveness towards ART.

Modulation of human BCRP (ABCG2) activity by anti-HIV drugs.

OBJECTIVES: The safety and effectiveness of highly active antiretroviral therapy (HAART) is challenged by viral resistance to antiretrovirals and the frequent occurrence of drug interactions which may limit the access of these drugs to the target sites. In particular, drug distribution and elimination may be modified by active efflux transporters. While P-glycoprotein is well evaluated in this regard, the interaction of antiretrovirals with the ABC transporter BCRP (ABCG2) is far from being elucidated. The aim of this study was therefore to investigate the influence of all important anti-HIV drugs on BCRP activity in vitro in one assay to allow unrestricted comparison of the results. METHODS: BCRP inhibition was assessed by an increase in pheophorbide A accumulation in MDCKII-BCRP cells and compared with the corresponding parental cell line MDCKII lacking human BCRP. RESULTS: According to the IC(50) estimation, the rank order for BCRP inhibition was lopinavir > nelfinavir > delavirdine > efavirenz > saquinavir > atazanavir > amprenavir > abacavir. Whereas nevirapine and zidovudine exerted weak inhibition, the inhibitory potency for ritonavir and tipranavir could not be estimated due to their low solubility and all other tested compounds (indinavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir and zalcitabine) were devoid of an effect. CONCLUSIONS: Taken together, our study demonstrates significant inhibition of BCRP by many anti-HIV drugs. These results suggest that inhibition of BCRP might contribute to drug-drug interactions observed during HAART in vivo and possibly also the superior effectiveness of combination antiretroviral therapy.

Extracts and kavalactones of Piper methysticum G. Forst (kava-kava) inhibit P-glycoprotein in vitro.

Root extracts from kava-kava (Piper methysticum G. Forst) are clinically used for the treatment of anxiety and restlessness. Due to reported cases of liver toxicity, kava-kava extracts were withdrawn from the market in several countries in 2002. Because the efflux transporter P-glycoprotein (P-gp) is involved in the absorption, distribution, and excretion of many drugs and often participates in drug-drug interactions, we studied the effect of a crude kava extract and the main kavalactones kavain, dihydrokavain, methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin on the P-gp-mediated efflux of calcein-acetoxymethylester in the P-gp-overexpressing cell line P388/dx and the corresponding cell line P388. The crude extract and the kavalactones showed a moderate to potent inhibitory activity with f2) (concentration needed to double baseline fluorescence) values of 170 microg/ml and 17 to 90 microM, respectively. The f2 value of yangonin could not be determined due to its higher lipophilicity. In conclusion, our results for the first time demonstrate P-gp-inhibitory activity of kava-kava and its components in vitro.