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.

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.

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.

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.

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.

Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors.

1. KCNQ1-4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole-cell perforated patch technique and cell-attached patch recording. 2. Stimulation of M1 receptors by 10 microM oxotremorine-M (Oxo-M) strongly reduced (to 0-10%) currents produced by KCNQ1-4 subunits expressed individually and also those produced by KCNQ2 + KCNQ3 and KCNQ1 + KCNE1 heteromers, which are thought to generate neuronal M-currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. 3. The activity of KCNQ2 + KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell-attached pipettes was strongly and reversibly reduced by Oxo-M applied to the extra-patch membrane. 4. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2 + KCNQ3 channels, like that of native M-channels, requires a diffusible second messenger.

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.

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.

Neuronal expression of the rat M1 muscarinic acetylcholine receptor gene is regulated by elements in the first exon.

Muscarinic acetylcholine receptor genes are members of the G-protein coupled receptor superfamily. Each member of this family studied to date appears to have a distinct expression profile, however the mechanisms determining these expression patterns remain largely unknown. We have previously isolated a genomic clone containing the M1 muscarinic receptor gene and determined its gene structure [Pepitoni, Wood and Buckley (1997) J. Biol. Chem. 272, 17112-17117]. We have now identified DNA elements responsible for driving cell specific expression in transient transfection assays of immortalized cell lines. A region of the gene spanning 974 nucleotides and containing 602 nucleotides of the first exon is sufficient to drive specific expression in cell lines. Like the M4 and M2 gene promoters, the M1 promoter contains an Sp1 motif which can recruit transcription factor Sp1 and at least one other protein, although this site does not appear to be functionally important for M1 expression in our assay. We have identified a region within the first exon of the M1 gene that regulates expression in cell lines, contains several positive and negative acting elements and is able to drive expression of a heterologous promoter. A polypyrimidine/polypurine tract and a sequence conserved between M1 genes of various species act in concert to enhance M1 transcription and are able to activate a heterologous promoter. We show that DNA binding proteins interact in vitro with single-stranded DNA derived from these regions and suggest that topology of the DNA is important for regulation of M1 expression.

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.

Structure of the m1 muscarinic acetylcholine receptor gene and its promoter.

The m1 receptor is one of five muscarinic receptors that mediate the metabotropic actions of acetylcholine in the nervous system where it is expressed predominantly in the telencephalon and autonomic ganglia. RNase protection, primer extension, and 5'-rapid amplification of cDNA ends analysis of a rat cosmid clone containing the entire m1 gene demonstrated that the rat m1 gene consists of a single 657-base pairs (bp) non-coding exon separated by a 13. 5-kilobase (kb) intron from a 2.54-kb coding exon that contains the entire open reading frame. The splice acceptor for the coding exon starting at -71 bp relative to the adenine of the initiating methionine. This genomic structure is similar to that of the m4 gene (Wood, I. C., Roopra, A., Harrington, C. A., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940 and Wood, I. C., Roopra, A., and Buckley, N. J. (1996) J. Biol. Chem. 271, 14221-14225). Like the m4 gene, the m1 promoter lacks TATA and CAAT consensus motifs, and the first exon and 5'-flanking region are not gc-rich. The 5'-flanking region also contains the consensus regulatory elements Sp-1, NZF-1, AP-1, AP-2, E-box, NFkappaB, and Oct-1. Unike the m4 promoter, there is no evidence of a RE1/NRSE silencer element in the m1 promoter. Deletional analysis and transient transfection assays demonstrates that reporter constructs containing 0.9 kb of 5'-flanking sequence and the first exon are sufficient to drive cell-specific expression of reporter gene in IMR32 neuroblastoma cells while remaining silent in 3T3 fibrobasts.

Crystalline lens parameters in infancy.

Despite the importance of lens power to ocular development, few data are available regarding infant crystalline lens parameters. Lens and corneal radii of curvature were measured in the horizontal meridian using a video-based keratophakometer, and refractive error was measured by cycloplegic retinoscopy in 19 out of 27 infants ranging in age from 3 to 18 months. The median refractive error was +1.50 D, and the median corneal power was 43.5 D. Using previously reported values for axial ocular dimensions, the median anterior and posterior lens radii of curvature were 8.7 and 5.6 mm, respectively, both substantially flatter than infant schematic eye values. The median equivalent refractive index of the lens was 1.49, considerably higher than previous reported schematic values for infants or children. There was a significant reduction in hyperopia with age (r = -0.47, P = 0.043), but no age-related trends in lens or corneal radii of curvature, suggesting that calculated values for lens power and equivalent index may undergo substantial decline with age during early childhood development as axial length increases. Most of the decrease in lens power (75%) may be due to decreases in equivalent index rather than to flattening of the surface radii of curvature. Videophakometry appears to be a feasible and useful technique for documenting the role of the crystalline lens in infant ocular development.

Neural specific expression of the m4 muscarinic acetylcholine receptor gene is mediated by a RE1/NRSE-type silencing element.

Muscarinic receptor genes are members of the G-protein receptor superfamily that, with the inclusion of the odorant receptors, is believed to contain over a thousand members. Each member of this superfamily, which has been studied to date, appears to have a distinct pattern of expression, but little work has been done on the regulation of these complex expression patterns. We have recently isolated the rat m4 muscarinic receptor gene and identified a genomic 1520-nucleotide sequence that appeared capable of directing cell-specific expression (Wood, I. C., Roopra. A., Harrington, C., and Buckley, N. J. (1995) J. Biol. Chem. 270, 30933-30940). In the present study we have constructed a set of deletion promoter constructs to more closely define the DNA elements that are responsible for m4 gene expression. We have found that deletion of a RE1/NRSE silencer element between nucleotides -574 and -550, similar to that found in other neural specific genes, results in activation of reporter expression in non-m4-expressing cells. Gel mobility shift analysis has shown that a protein present in nonexpressing cells is capable of binding to this element and is probably the recently identified neural silencer, REST/NRSF. Of the constitutively active proximal promoter only a tandem Sp-1 site appears to recruit DNA binding proteins that are present in all cells tested. This represents the first report documenting the role of this silencer in regulating expression of a member of the G-protein receptor family.

Structure of the m4 cholinergic muscarinic receptor gene and its promoter.

Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the non-coding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.

Longitudinal change of refractive error in infants during the first year of life.

Using cycloplegia, the change in ametropia of 113 infants was followed at 3 month intervals over the first year of life. Scatterplots of the spherical equivalent power show that the dioptric differences exhibit a significant myopic shift of -0.38 ds between 26 and 36 weeks and -0.38 ds between 36 and 52 weeks. The spread of the dioptric differences (95% CI) does not appear to be related to the magnitude of the ametropia present and decreases with time. By 12 months of age the frequency distribution of the spherical equivalent appears to become leptokurtic as it is in the adult. On average the astigmatism was of low degree (less than 1 dioptre cylinder) and with the rule. Anisometropia was rarely seen. The results of this longitudinal study point to an optimal time for screening and perhaps prescribing for 'abnormal' refractive error between 9 and 12 months of age.

Binding and activation of the promoter for the neural cell adhesion molecule by Pax-8.

The neural cell adhesion molecule (N-CAM), is expressed in definite spatiotemporal patterns during development. To identify factors that may influence place-dependent n-cam gene expression, we have studied the binding and activation of the n-cam promoter by Pax-8, a member of the Pax family of transcription factors. Pax-8 increased n-cam promoter activity 13.4-fold in cellular co-transfection experiments, and a short segment of the promoter (-143 to -15) mediated the response. This region of the n-cam promoter produced a DNA-protein complex when incubated with either extracts from COS-7 cells transfected with the Pax-8 expression vector or a Pax-8/GST fusion protein. Pax-8 bound to the n-cam promoter through two TGCTCC motifs (designated PBS-1 and PBS-2) that resemble paired domain binding sites. Mutation of PBS-1 and PBS-2 eliminated Pax-8 activation of the n-cam promoter. Transfection of N2A neuroblastoma cells with the Pax-8 expression vector resulted in a 5-fold increase in the transcription of the endogenous n-cam gene. The combined results suggest that Pax-8 activates transcription of the n-cam gene through binding of sequences resembling paired domain binding sites in the n-cam promoter. The data raise the possibility that the n-cam promoter may be regulated by other members of the Pax gene family.

Regulation in vitro of an L-CAM enhancer by homeobox genes HoxD9 and HNF-1.

Previous studies have shown that in vitro expression of the neural cell adhesion molecule (N-CAM) can be regulated by the products of homeobox genes HoxB9, -B8, and -C6. N-CAM is a Ca(2+)-independent immunoglobulin-related CAM that plays an important role in neural development. In the present study, we investigated whether the liver cell adhesion molecule (L-CAM) a member of the Ca(2+)-dependent CAM family (cadherins) is also regulated by homeobox-containing genes. In transient cotransfection experiments of NIH 3T3 cells, we observed that both HoxD9 and liver-enriched POU-homeodomain transcription factor, HNF-1, activated chloramphenicol acetyltransferase gene reporter constructs containing the L-CAM promoter and an enhancer present in the second intron of the chicken L-CAM gene. Using electrophoretic mobility-shift assays, we found that components of cell extracts from NIH 3T3 cells transfected with HoxD9 bound to a small region of the L-CAM enhancer having a consensus sequence that is a putative binding site for HNF-1. Components of extracts from the chicken hepatoma cell line LMH that had been transfected with an HNF-1 expression vector also bound to this same site. In nuclear run-on experiments with nuclei from LMH cells that were transfected with expression vectors for HoxD9 or HNF-1, L-CAM RNA levels were increased 33-fold and 4-fold respectively. Using the same run-on procedure, it was confirmed that nuclei prepared from normal embryonic chicken liver cells expressed the RNAs for HoxD9, HNF-1, and L-CAM. Taken together with previous observations, these data raise the possibility that homeobox-containing genes will have a widespread role in the place-dependent expression of CAMs belonging both to immunoglobulin-related and to cadherin families.

Cell adhesion alters gene transcription in chicken embryo brain cells and mouse embryonal carcinoma cells.

To determine whether changes in gene expression occur in embryonic cells as a consequence of changes in cellular aggregation, chicken embryo brain (CEB) cells isolated from 8-day embryos were allowed to aggregate or prevented from aggregating by treatment with anti-neural cell adhesion molecule (N-CAM) Fab' fragments. A subtractive hybridization cloning strategy was employed to identify genes that might show different levels of expression in the two populations of cells. In addition, the transcription rates of a number of genes specifying CAMs and transcription factors were directly estimated by using nuclear run-off transcription assays. The transcription rates of several genes, including those encoding N-CAM, Ng-CAM, alpha-N-catenin, HoxA4 (Hox1.4), a fatty acid-binding protein, and a subunit of the mitochondrially encoded cytochrome-c oxidase enzyme decreased upon CEB cell aggregation. The transcription rates of several previously unidentified genes either increased or decreased upon aggregation, while the transcription of other genes remained unchanged. The transcription rate of the N-CAM gene was 3.3-fold higher in dissociated than in aggregated CEB cells. This rate of transcription also increased when the brain tissue was dissociated into single cells and the increased rate was maintained by keeping the cells dissociated in the presence of Fab' fragments of antibodies to N-CAM. Decreased transcription rates of the N-CAM gene were also observed upon aggregation of P19 cells, a mouse embryonal carcinoma cell line. Primary chicken embryo liver cells, which aggregate primarily by calcium-dependent adhesion mechanisms, did not show changes in the N-CAM gene or in the other genes whose transcription rates changed in CEB cells and P19 cells. These observations suggest that the types of genes regulated by cell aggregation include those for CAMs themselves as well as for transcription factors that may control the expression of CAMs and other molecules significant for morphogenesis.

Comparison of the techniques of videorefraction and static retinoscopy in the measurement of refractive error in infants.

Photorefraction has been suggested as a suitable method of screening for refractive error in infants. The relative performance of cycloplegic and non-cycloplegic videorefraction and cycloplegic retinoscopy was investigated on 150 infants. Under cycloplegic conditions the correlation between findings for spherical error (Rxy = 0.70) was compatible with a previous study. However, where cycloplegia was not used for videorefraction, there was poor agreement between the two techniques in the case of astigmatic error, and all types of ametropia. Interobserver repeatability was very high both for cycloplegic retinoscopy (Rxy = 0.96 spherical error, and Rxy = 0.75 astigmatic error) and for videorefraction measurements (Rxy = 0.95 horizontal meridian of photograph and Rxy = 0.85 vertical meridian). Intraobserver repeatability was also good, both for cycloplegic retinoscopy (Rxy = 0.91 spherical error and 0.82 astigmatic error) and for videorefraction with regard to spherical errors (Rxy = 0.84). Throughout the experiments videorefraction measurements of astigmatic errors proved less consistent when compared with cycloplegic retinoscopy, and to its internal reliability.