KvSNP: accurately predicting the effect of genetic variants in voltage-gated potassium channels.

MOTIVATION: Non-synonymous single nucleotide polymorphisms (nsSNPs) in voltage-gated potassium (Kv) channels cause diseases with potentially fatal consequences in seemingly healthy individuals. Identifying disease-causing genetic variation will aid presymptomatic diagnosis and treatment of such disorders. NsSNP-effect predictors are hypothesized to perform best when developed for specific gene families. We, thus, created KvSNP: a method that assigns a disease-causing probability to Kv-channel nsSNPs. RESULTS: KvSNP outperforms popular non gene-family-specific methods (SNPs&GO, SIFT and Polyphen) in predicting the disease potential of Kv-channel variants, according to all tested metrics (accuracy, Matthews correlation coefficient and area under receiver operator characteristic curve). Most significantly, it increases the separation of the median predicted disease probabilities between benign and disease-causing SNPs by 26% on the next-best competitor. KvSNP has ranked 172 uncharacterized Kv-channel nsSNPs by disease-causing probability. AVAILABILITY AND IMPLEMENTATION: KvSNP, a WEKA implementation is available at www.bioinformatics.leeds.ac.uk/KvDB/KvSNP.html. CONTACT: d.r.westhead@leeds.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptional repression by neuron-restrictive silencer factor is mediated via the Sin3-histone deacetylase complex.

A large number of neuron-specific genes characterized to date are under the control of negative transcriptional regulation. Many promoter regions of neuron-specific genes possess the repressor element repressor element 1/neuron-restrictive silencing element (RE1/NRSE). Its cognate binding protein, REST/NRSF, is an essential transcription factor; its null mutations result in embryonic lethality, and its dominant negative mutants produce aberrant expression of neuron-specific genes. REST/NRSF acts as a regulator of neuron-specific gene expression in both nonneuronal tissue and developing neurons. Here, we shown that heterologous expression of REST/NRSF in Saccharomyces cerevisiae is able to repress transcription from yeast promoters engineered to contain RE1/NRSEs. Moreover, we have taken advantage of this observation to show that this repression requires both yeast Sin3p and Rpd3p and that REST/NRSF physically interacts with the product of the yeast SIN3 gene in vivo. Furthermore, we show that REST/NRSF binds mammalian SIN3A and HDAC-2 and requires histone deacetylase activity to repress neuronal gene transcription in both nonneuronal and neuronal cell lines. We show that REST/NRSF binding to RE1/NRSE is accompanied by a decrease in the acetylation of histones around RE1/NRSE and that this decrease requires the N-terminal Sin3p binding domain of REST/NRSF. Taken together, these data suggest that REST/NRSF represses neuronal gene transcription by recruiting the SIN3/HDAC complex.

Two types of K(+) channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell.

The potassium M current was originally identified in sympathetic ganglion cells, and analogous currents have been reported in some central neurons and also in some neural cell lines. It has recently been suggested that the M channel in sympathetic neurons comprises a heteromultimer of KCNQ2 and KCNQ3 (Wang et al., 1998) but it is unclear whether all other M-like currents are generated by these channels. Here we report that the M-like current previously described in NG108-15 mouse neuroblastoma x rat glioma cells has two components, "fast" and "slow", that may be differentiated kinetically and pharmacologically. We provide evidence from PCR analysis and expression studies to indicate that these two components are mediated by two distinct molecular species of K(+) channel: the fast component resembles that in sympathetic ganglia and is probably carried by KCNQ2/3 channels, whereas the slow component appears to be carried by merg1a channels. Thus, the channels generating M-like currents in different cells may be heterogeneous in molecular composition.

Dominant-negative subunits reveal potassium channel families that contribute to M-like potassium currents.

M-currents are K+ currents generated by members of the KCNQ family of K+ channels (Wang et al., 1998). However, in some cells, M-like currents may be contaminated by members of other K+ channel gene families, such as the erg family (Meves et al., 1999; Selyanko et al., 1999). In the present experiments, we have used the acute expression of pore-defective mutants of KCNQ3 (DN-KCNQ3) and Merg1a (DN-Merg1a) as dominant negatives to separate the contributions of these two families to M-like currents in NG108-15 neuroblastoma hybrid cells and rat sympathetic neurons. Two kinetically and pharmacologically separable components of M-like current could be recorded from NG108-15 cells that were individually suppressed by DN-Merg1a and DN-KCNQ3, respectively. In contrast, only DN-KCNQ3, and not DN-Merg1a, reduced currents recorded from sympathetic neurons. Pharmacological tests suggested that the residual current in DN-KCNQ3-treated sympathetic neurons was carried by residual KCNQ channels. Ineffectiveness of DN-Merg1a in sympathetic neurons was not caused by lack of expression, as judged by confocal microscopy of Flag-tagged DN-Merg1a. These results accord with previous inferences regarding the roles of erg and KCNQ channels in generating M-like currents. This experimental approach should therefore be useful in delineating the contributions of members of these two gene families to K+ currents in other cells.

Differential tetraethylammonium sensitivity of KCNQ1-4 potassium channels.

In Shaker-group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1-4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, > 30; KCNQ4, 3.0; KCNQ2 + KCNQ3, 3.8; and KCNQ2 + KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.

Spatial tuning of static and dynamic local stereopsis.

The range of spatial tuning for channels that process static and dynamic disparities was investigated in the central visual field by measuring stereoscopic thresholds as a function of the difference in size of spatially filtered bar-like patterns presented to the two eyes. Spatial tuning functions were revealed by an elevation of stereothreshold as the difference between the widths of bar patterns increased. Functions tuned to low spatial frequencies (0.075-2 c/deg) were classified as transient since their stereosensitivity was greater for dynamic (1 Hz) than static disparities. Functions tuned to high spatial frequencies (2.4-19 c/deg) were classified as sustained since their stereosensitivity was equal for dynamic and static disparities. When equal width patterns were presented to the two eyes, stereothreshold increased with spatial periods greater than 0.4 deg according to a constant 6 deg phase disparity. This size-disparity correlation suggests that large disparities are processed by spatial filters tuned to disparities proportional to their receptive field dimensions.

Evaluation of the validity and reliability of A-scan ultrasound biometry with a single use disposable cover.

BACKGROUND: The UK Medical Devices Agency has suggested that ophthalmic practitioners should, where practicable and not compromising clinical outcome, restrict corneal contact devices to single patient use to minimise a remote theoretical risk of transmission of new variant Creutzfeldt-Jakob disease (vCJD). This study reports on a modified technique of ultrasound A-scan biometry that complies with the MDA recommendations. METHODS: The right eyes of 37 consecutive hospital patients had a series of biometry readings taken with a Humphrey 820 A-scan instrument with a plane wave transducer use d conventionally and with the addition of a disposable latex cover. RESULTS: Intrasessional repeatability of axial length measurements was similar for conventional readings--mean difference 0.027 mm, 95% confidence intervals (CI) +/- 0.44 mm and those taken with a disposable cover (0.028 mm, CI +/- 0.38). Intersessional repeatability was equivalent with (0.002 mm, CI +.- 0.51) and without a cover (0.03 mm, CI +/- 0.51). Readings with a cover were not significantly different from those without (paired t test; p >0.05), but tended to be greater (mean difference 0.085 mm, CI +/- 0.60). CONCLUSIONS: These findings suggest that corneal contact biometry with a disposable cover is a viable and theoretically safer alternative to the conventional technique.

Contrast threshold of random dot stereograms in anisometropic amblyopia: A clinical investigation.

The contrast thresholds of random dot stereograms were obtained for both anisometropic amblyopic and normal patients. The elevation of the contrast threshold found in both amblyopic and non-amblyopic eyes of the anisometropes reflects the relative strength of the binocular inhibition mechanism present. These findings were related to the standard clinical tests used in this investigation and other recent psychophysical and physiological investigations.

A longitudinal study of the biometric and refractive changes in full-term infants during the first year of life.

Changes in ocular axial dimensions and refraction were followed longitudinally, using ultrasonography and retinoscopy, during the first year of life (mean ages 4-53 weeks) of a group of 20 full-term infants (10 male, 10 female). Using a mixed-model regression analysis, axial length changes as a function of time were found to be best described by a quadratic expression (AL=17.190+0.128x-0.0013x(2), where AL is the axial length in mm and x is the age in weeks), while anterior chamber depth changed linearly (ACD=2.619+0.018x, where ACD is the anterior chamber depth in mm): lens thickness was essentially constant. Spherical equivalent refraction through most of the first year showed a steady reduction in hypermetropia (SER=2.982-0.032x, where SER is the spherical equivalent refraction in dioptres): astigmatism also tended to diminish. Mean hyperopic refractive errors through the year were negatively correlated with corresponding axial lengths (SER=12.583-0.541AL), but some individual subjects showed marked departures from this pattern. These results are discussed in relation to concepts of emmetropization.

Repression and activation of muscarinic receptor genes.

The specific cellular response to muscarinic receptor activation is dependent upon appropriate expression of each of the five muscarinic receptor genes by individual cells. Here we summarise recent work describing some of the genomic regulatory elements and transcriptional mechanisms that control expression of the M1 and M4 genes.

Multiple chromatin modifications important for gene expression changes in cardiac hypertrophy.

Cardiac hypertrophy is an increase in the size of cardiac myocytes to generate increased muscle mass, usually driven by increased workload for the heart. Although important during postnatal development and an adaptive response to physical exercise, excessive hypertrophy can result in heart failure. One characteristic of hypertrophy is the re-expression of genes that are normally only expressed during foetal heart development. Although the involvement of these changes in gene expression in hypertrophy has been known for some years, the mechanisms involved in this re-expression are only now being elucidated and the transcription factor REST (repressor element 1-silencing transcription factor) has been identified as an important repressor of hypertrophic gene expression.

A review of autorefractors.

The overall performance of autorefractors in this study as defined by reliability, validity and rejection rate statistics, is similar to other hospital based studies but lower than in an optometric clinic. The performance of individual autorefractors varies according to the interaction between the underlying optical/detector mechanism and the media and pathological changes of a given eye. The autorefraction data proves a useful complement to the refractive data gained by conventional techniques, notwithstanding the poorer performance of the autorefractors in the presence of senescent pathology.

Stereopsis with spatially-degraded images.

Experiments are described in which stereo-thresholds were determined under conditions of monocular and binocular dioptric blur, or when spatially-filtered, computer-generated images were used. The results show that, in general, monocular image degradation in combination with an undegraded image in the other eye produces a worse stereo-performance than does the corresponding binocular combination of degraded images. High-pass spatial-frequency filtering, where image frequencies greater than or equal to 4 c/deg are retained, provides better stereo-acuity than low-pass filtering, where only frequencies less than or equal to 4 c/deg are present.

Behavioral control of visual field screening using a microcomputer.

A computer algorithm is described which automatically controls the presentation of visual field targets and analyzes the subject's responses. Data relating to the procedure's reliability and validity are reported along with experience using it with normal subjects on an IBM PC compatible. Suggestions are made for the further development of the microcomputer approach to visual field investigations.

Computer-assisted optic nerve head assessment.

An IBM-compatible microcomputer-based system has been developed for the assessment of pallor area size, shape and degree of pallor in the optic nerve head. The system allows the instantaneous capture of optic nerve head images, and software analysis of these images produces measurements of the pallor area in about 2 min. The various methods of image analysis are presented. These are enhancement, thresholding, and digital filtering for edge detection. The results of their application to the images of the optic nerve head are shown. The results of the system's analysis on a particular optic nerve head are also shown. The potential uses and difficulties of such a system are discussed.

Inter- and intra-image variability in computer-assisted optic nerve head assessment.

A computer-assisted system for optic nerve head analysis was used to measure one eye from each of 15 subjects. These comprised three groups of five, being glaucomatous, ocular hypertensive, and normal subjects. Each eye was imaged with the system 10 times. The 150 images produced were analysed in a randomized masked fashion to test the inter-image variability of the measurements. Linear pallor: disc ratios had mean coefficients of variation of 6.6% (normal), 4.8% (ocular hypertensive) and 2.6% (glaucomatous). This trend of reducing coefficient of variation from normal to ocular hypertensive to glaucomatous was reflected in other parameters also. Intra-image variability was assessed by analysing one image from each subject 10 times, in a randomized masked fashion. The mean coefficients of variation of the linear pallor: disc ratios were 6.3% (normal), 3.6% (ocular hypertensive) and 3.2% (glaucomatous). Fisher's F-test showed that no parameters had significantly lower intra-image rather than inter-image variance (at the 5% level). Variability is apparently due to operator variations rather than the image variations.

Measurement of Friedmann Visual Field Analyzer tests in primary open-angle glaucoma.

A method is described to allow an IBM PC/AT compatible microcomputer program to compute a score from the visual field data collected from a standard Friedmann Visual Field Analyzer (FVFA) Mk II testing procedure. The method could, in principle, be applied to any visual field data which provide quantitative information on the sensitivity of different retinal locations. The score takes into account the depth of any defects, their position in the visual field, and the clustering of defects into scotomata. Visual fields in 119 normal eyes, 82 ocular hypertensive (OH) eyes, and 75 glaucomatous eyes were assessed to provide the data necessary to implement this method. Analyzing the frequency of missed points against point luminance demonstrates that the most effective cut-off from which to regard points as defective is a miss at 0.8 log units or more above the working threshold. A statistical method of analyzing the distribution of defects following Bayesian principles is described and shows that points in the superior nasal, superior arcuate, and inferior arcuate areas provide the most information for detecting glaucoma. The program, written in Turbo Pascal v5.5, incorporates these findings in producing the score.

Computerized Arden grating technique for the measurement of the contrast sensitivity function.

We developed a computerized contrast sensitivity function (CSF) measurement technique using a computer graphics board to reproduce Arden grating type stimuli. The reliability of the procedure was examined by evaluating the standard deviation of repeated test differences. The validity of the procedure was tested by comparison with both a manual method of adjustment technique and with Regan low contrast letter charts. The procedure has been performed at a number of exposure speeds to account for the effect of reaction time and attention of the results. The optimum exposure time for the procedure has been found to be greater than or equal to 22 s. As a result of the benefits of automation the test is quick, produces test-retest correlation coefficients for the five spatial frequencies tested which are similar to those for the method of adjustment procedure (0.76 to 0.91 for the computerized test, 0.66 to 0.88 for the signal generator measurements), and produces values which correspond closely with the method of adjustment measurement. It has the advantages of utilizing hardware already available in many computer systems, having a constant mean luminance across the screen, and having a constant and optimized presentation time.

Inter-observer variability in a computer-assisted optic nerve head assessment system.

A computer-assisted optic nerve head analysis system was used to measure parameters of the optic nerve head from one eye from each of 15 subjects. The subjects comprised three groups of five, being glaucomatous, ocular hypertensive, and normal. The 15 images produced were analysed in a randomized masked fashion by ten observers of varying levels of experience to test the inter-observer variability of the measurements. An analysis of variance demonstrated significant differences in the measurements made by the different observers. Fisher's F-test showed that all the parameters assessing the pallor area of the optic nerve head had significantly lower inter-image variance (reported previously) compared with inter-observer variance (at the 5% level). Variability in taking measurements of the pallor area of the optic nerve head is mainly due to observer variations rather than the image variations.