Screening for amblyopia in preverbal children with photoscreening photographs: II. Sensitivity and specificity of the MTI photoscreener.

OBJECTIVE: To determine the sensitivity and specificity of vision screening using the Medical Technology and Innovations (MTI), Inc., PhotoScreener. DESIGN: Cross-sectional study. PARTICIPANTS AND TESTING: Three hundred ninety-two children less than 4 years of age received a complete ophthalmologic examination and were photographed using the MTI PhotoScreener. One hundred three children had normal examinations, and the remaining 284 children had conditions of interest for pediatric screening: ptosis, media opacity, refractive error, or strabismus. Five children were excluded. MAIN OUTCOME MEASURES: The grading of the photographs by the manufacturer's representative was compared with the results of the ophthalmologic examinations. Sensitivity and specificity of vision screening were determined. RESULTS: The analysis of all informative photographs resulted in a sensitivity of 65% and a specificity of 87%. The sensitivity of detection for children with some forms of strabismus was high, up to 95% for esotropia of 10Delta or more. Sensitivities for the detection of ptosis, media opacity, and refractive error were poor in patients where strabismus was not also present. CONCLUSIONS: The MTI PhotoScreener may play a role in preverbal vision screening; identification of two of three children with amblyopiogenic factors before age 4 would be an exciting advance in public health. However, improvement in the ability to identify children with media opacity and refractive error is necessary. Improvements may be possible with modifications of the examination failure and photograph grading criteria.

Screening for amblyopia in preverbal children with photoscreening photographs. III. improved grading criteria for hyperopia.

OBJECTIVE: To examine the ability of the Medical Technology and Innovations (MTI), Inc., Photoscreener (Cedar Falls, IA) to detect hyperopia and to improve the photograph grading criteria to screen for amblyopiogenic levels of hyperopia. DESIGN: Cross-sectional study and reanalysis. PARTICIPANTS AND TESTING: In previous work, 392 participants received a complete ophthalmologic examination and were photographed using the MTI Photoscreener. For this study, all 209 participants with normal examination findings (65 children) or hyperopia without anisometropia (144 children) were selected. The data were reanalyzed using modified photograph grading and ophthalmologic examination failure criteria. Potential reasons for why many children with hyperopia passed photoscreening were explored. MAIN OUTCOME MEASURES: We determined whether a study participant would pass or fail screening with a given photograph grading and ophthalmologic examination failure criteria. RESULTS: Most children with hyperopia of +2.00 to +3.50 diopters (D) passed screening with the MTI instrument, in most cases because their photographs lacked bright crescents. When bright crescents in at least two of the four possible meridians were the grading guideline for screening failure and the pediatric ophthalmologists' consensus hyperopia failure criteria (> +3.50 D) were adopted, the sensitivity for hyperopia detection was 100% and the specificity was 88%. Identical results were obtained using the American Academy of Ophthalmology Preferred Practice Pattern hyperopia failure criteria (>/= +4.50 D). CONCLUSIONS: The MTI photograph grading guidelines can be simplified, and the ophthalmologic examination failure criteria for hyperopia can be improved. The presence of a bright crescent in the lower or the left pupillary margin indicate hyperopia in an amblyopiogenic range (> +3.50 D).

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C.

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Mechanisms of spectral tuning in the mouse green cone pigment.

Diversification of cone pigment spectral sensitivities during evolution is a prerequisite for the development of color vision. Previous studies have identified two naturally occurring mechanisms that produce variation among vertebrate pigments by red-shifting visual pigment absorbance: addition of hydroxyl groups to the putative chromophore binding pocket and binding of chloride to a putative extracellular loop. In this paper we describe the use of two blue-shifting mechanisms during the evolution of rodent long-wave cone pigments. The mouse green pigment belongs to the long-wave subfamily of cone pigments, but its absorption maximum is 508 nm, similar to that of the rhodopsin subfamily of visual pigments, but blue-shifted 44 nm relative to the human red pigment, its closest homologue. We show that acquisition of a hydroxyl group near the retinylidene Schiff base and loss of the chloride binding site mentioned above fully account for the observed blue shift. These data indicate that the chloride binding site is not a universal attribute of long-wave cone pigments as generally supposed, and that, depending upon location, hydroxyl groups can alter the environment of the chromophore to produce either red or blue shifts.

Saccharomyces cerevisiae PAC2 functions with CIN1, 2 and 4 in a pathway leading to normal microtubule stability.

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1 delta, cin2 delta and cin4 delta. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant recombinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2 delta, cin2 delta and cin4 delta, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of beta-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.

Individual variation in size of the human red and green visual pigment gene array.

PURPOSE: To determine the size variation of the X-chromosomal human red and green visual pigment gene array in the general population using pulsed field gel electrophoresis and Southern blotting. METHODS: Peripheral blood lymphocytes were prepared from 67 anonymous males. The cells were embedded in agarose and the genomic DNA digested with restriction enzyme Not I. The resulting DNA fragments were resolved on a contour-clamped homogeneous electric field gel, and the Not I fragment containing the red and green pigment genes was visualized by Southern blot hybridization with a human green pigment cDNA probe. RESULTS: In DNA from each male, a single hybridizing fragment was observed in Not I-digested DNA. The lengths of the fragments from different males were observed to vary in steps of approximately 39 kilobases (kb), consistent with earlier studies showing a visual pigment gene repeat unit of 39 kb and a head-to-tail tandem arrangement of the red and green visual pigment genes. In the population studied, the number of repeat units per X-chromosome had a mean of 2.9 and a standard deviation of 0.94. CONCLUSIONS: The sizes of visual pigment gene arrays observed in this study resemble those determined in earlier studies based on ratios of restriction fragments resolved by conventional gel electrophoresis and visualized by whole genome Southern blotting, but differ significantly from those determined using ratios of fragments obtained by the polymerase chain reaction.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

A new member of the frizzled family from Drosophila functions as a Wingless receptor.

Receptors for Wingless and other signalling molecules of the Wnt gene family have yet to be identified. We show here that cultured Drosophila cells transfected with a novel member of the frizzled gene family in Drosophila, Dfz2, respond to added Wingless protein by elevating the level of the Armadillo protein. Moreover, Wingless binds to Drosophila or human cells expressing Dfz2. These data demonstrate that Dfz2 functions as a Wingless receptor, and they imply, in general, that Frizzled proteins are receptors for the Wnt signalling molecules.

cDNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21: expression, chromosomal localization and analysis of a highly polymorphic (CAG)n trinucleotide repeat.

The two most consistent features of the diseases caused by trinucleotide repeat expansion-neuropsychiatric symptoms and the phenomenon of genetic anticipation-may be present in forms of dementia, hereditary ataxia, Parkinsonism, bipolar affective disorder, schizophrenia and autism. To identify candidate genes for these disorders, we have screened human brain cDNA libraries for the presence of gene fragments containing polymorphic trinucleotide repeats. Here we report the cDNA cloning of CAGR1, originally detected in a retinal cDNA library. The 2743 bp cDNA contains a 1077 bp open reading frame encoding 359 amino acids. This amino acid sequence is homologous (56% amino acid identify and 81% amino acid conservation) to the Caenorhabditis elegans cell fate-determining protein mab-21. CAGR1 is expressed in several human tissues, most prominently in the cerebellum, as a message of approximately 3.0 kb. The gene was mapped to 13q13, just telomeric to D13S220. A 5'-untranslated CAG trinucleotide repeat is highly polymorphic, with repeat length ranging from six to 31 triplets and a heterozygosity of 87-88% in 684 chromosomes from several human populations. One allele from an individual with an atypical movement disorder and bipolar affective disorder type II contains 46 triplets, 15 triplets longer than any other allele detected. Though insufficient data are available to link the long repeat to this clinical phenotype, an expansion mutation of the CAGR1 repeat can be considered a candidate for the etiology of disorders with anticipation or developmental abnormalities, and particularly any such disorders linked to chromosome 13.

A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled.

In Drosophila melanogaster, the frizzled gene plays an essential role in the development of tissue polarity as assessed by the orientation of cuticular structures. Through a combination of random cDNA sequencing, degenerate polymerase chain reaction amplification, and low stringency hybridization we have identified six novel frizzled homologues from mammals, at least 11 from zebrafish, several from chicken and sea urchin, and one from Caenorhabditis elegans. The complete deduced amino acid sequences of the mammalian and nematode homologues share with the Drosophila frizzled protein a conserved amino-terminal cysteine-rich domain and seven putative transmembrane segments. Each of the mammalian homologues is expressed in a distinctive set of tissues in the adult, and at least three are expressed during embryogenesis. As hypothesized for the Drosophila frizzled protein, the frizzled homologues are likely to act as transmembrane receptors for as yet unidentified ligands. These observations predict the existence of a family of signal transduction pathways that are homologous to the pathway that determines tissue polarity in Drosophila.

The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons.

A search for POU domain sequences expressed in the human retina has led to the identification of three closely related genes: Brn-3a, Brn-3b, and Brn-3c. The structure and expression pattern of Brn-3b was reported earlier (Xiang et al., 1993); we report here the structures and expression patterns of Brn-3a and Brn-3c. Antibodies specific for each Brn-3 protein were generated and shown to label only ganglion cells in a variety of vertebrate retinas. A complex pattern of strongly and weakly immunolabeled ganglion cells was observed in mouse, cat, and monkey retinae. In mouse and cat retinae, Brn-3a and Brn-3b proteins are found in a large fraction of ganglion cells, whereas Brn-3c is present in fewer ganglion cells. In the cat retina, anti-Brn-3a immunoreactivity was strong in the small ganglion cells (gamma cells) and weak in the remaining ganglion cells (alpha and beta cells); anti-Brn-3b immunoreactivity was present in all ganglion cells; and anti-Brn3c immunoreactivity was confined to the small ganglion cells. Immunolabeling of macaque retinae following retrograde labeling from the lateral geniculate nucleus revealed strong anti-Brn-3a immunoreactivity in a minority of retrogradely labeled P-type ganglion cells, and weak Brn-3a immunoreactivity in all of the remaining P- and M-type ganglion cells. In the same retinae, strong anti-Brn-3b immunoreactivity was seen in nearly all P-type ganglion cells and weak immunoreactivity in nearly all M-type ganglion cells. Each of the Brn-3-specific antibodies also labeled subsets of neurons in the dorsal root and trigeminal ganglia, suggesting that primary somatosensory neurons and retinal ganglion cells share genetic regulatory hierarchies. In vitro selection of an optimal DNA binding site using the Brn-3b POU domain has revealed a consensus [(A/G)CTCATTAA(T/C)] that is recognized by each of the Brn-3 POU domains and is distinct from binding sites previously described for other POU domain proteins.

Autosomal dominant retinitis pigmentosa in a large family: a clinical and molecular genetic study.

PURPOSE: To characterize the pedigree, visual function phenotype, and responsible mutation in a large family with autosomal dominant retinitis pigmentosa. METHODS: Pedigree data were obtained by personal interviews and corroborated with community records. One hundred twenty-eight members of the family were examined clinically, and a subset of 12 affected subjects was further studied with dark- and light-adapted static perimetry and electroretinography. The coding region of the rhodopsin gene was polymerase chain reaction (PCR) amplified and resolved by denaturing gradient gel electrophoresis. Genomic DNA samples from nine affected and five unaffected family members were analyzed by PCR amplification and restriction enzyme digestion. RESULTS: A 14-generation pedigree was identified in which retinitis pigmentosa (RP) was inherited in an autosomal dominant fashion. Affected individuals reported early night blindness and showed vessel attenuation and bone spicule-like pigmentary changes. In these individuals, the rod electroretinogram (ERG) was not detectable, and the cone ERG was reduced in amplitude and delayed in timing. With dark-adapted perimetry, rod function could be detected in only one young patient, and it was markedly abnormal. Light-adapted perimetry indicated that cone sensitivity could be relatively well preserved in the central field, but it was diminished in the periphery even in the most mildly affected subjects. A valine345-to-leucine mutation was identified in the rhodopsin gene and shown to cosegregate in the heterozygous condition with the disease. CONCLUSIONS: The natural history of RP in this family begins with a loss of rod function, progresses to involve the cone system, and leads eventually to a severe loss of visual function. The invariance of valine345 in all functional vertebrate visual pigments sequenced to date, and the unusually conservative nature of the valine345-to-leucine mutation suggests that the carboxy terminus of rhodopsin is involved in a highly specific interaction with one or more rod proteins.

Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa.

PURPOSE: To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein. METHODS: Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels. RESULTS: There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes. CONCLUSIONS: There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.

Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: implications for the structure and function of rhodopsin.

Ten rhodopsin mutations have been found in a screen of 282 subjects with retinitis pigmentosa (RP), 76 subjects with Leber congenital amaurosis, and 3 subjects with congenital stationary night blindness. Eight of these mutations (gly51-to-ala, val104-to-ile, gly106-to-arg, arg135-to-gly, cys140-to-ser, gly188-to-glu, val209-to-met, and his211-to-arg) produce amino acid substitutions, one (gln64-to-ter) introduces a stop codon, and one changes a guanosine in the intron 4 consensus splice donor sequence to thymidine. Cosegregation of RP with gln64-to-ter, gly106-to-arg, arg135-to-gly, cys140-to-ser, gly188-to-glu, his211-to-arg, and the splice site guanosine-to-thymidine indicates that these mutations are likely to cause retinal disease. Val104-to-ile does not cosegregate and is therefore unlikely to be related to retinal disease. The relevance of gly51-to-ala and val209-to-met remains to be determined. The finding of gln64-to-ter in a family with autosomal dominant RP is in contrast to a recent report of a recessive disease phenotype associated with the rhodopsin mutation glu249-to-ter. In the present screen, all of the mutations that cosegregate with retinal disease were found among patients with RP. The mutations described here bring to 35 the total number of amino acid substitutions identified thus far in rhodopsin that are associated with RP. The distribution of the substitutions along the polypeptide chain is significantly nonrandom: 63% of the substitutions involve those 19% of amino acids that are identical among vertebrate visual pigments sequenced to date.

A locus control region adjacent to the human red and green visual pigment genes.

Deletion of sequences 5' of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a beta-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5' of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5' region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression.

Phototransduction in retinal rods involves a G-protein-mediated signaling cascade that leads to cGMP hydrolysis and the closure of a cGMP-gated channel. This channel has recently been purified from bovine retina and molecularly cloned (Kaupp et al., 1989). We report here the cloning of cDNA and genomic DNA encoding the human rod cGMP-gated channel, based upon its homology to the bovine counterpart. The human mRNA structure differs from the bovine in containing an Alu repetitive element spliced into the 5' untranslated region. The human cGMP-gated channel gene (CNCG) is located on chromosome 4 and contains at least 10 exons. One large exon encodes the carboxy-terminal two-thirds of the protein, whereas seven small exons encode the amino-terminal one-third of the protein. Alternative splicing removes one of the small exons in a subset of transcripts in the human retina, producing an internal in-frame deletion of 36 codons. When expressed in a human embryonic kidney cell line (293S), the full-length cDNA clone, but not the differentially spliced variant, produced functional ion channels broadly similar to the native channels in vertebrate rods.

Saturation mutagenesis of the yeast his3 regulatory site: requirements for transcriptional induction and for binding by GCN4 activator protein.

Expression of the yeast his3 and other amino acid biosynthetic genes is induced during conditions of amino acid starvation. The coordination of this response is mediated by a positive regulatory protein called GCN4, which binds specifically to regulatory sites upstream of all coregulated genes and stimulates their transcription. The nucleotide sequence requirements of the his3 regulatory site were determined by analysis of numerous point mutations obtained by a novel method of cloning oligonucleotides. Almost all single base pair mutations within the nine base pair sequence ATGACTCTT significantly reduce his3 induction in vivo and GCN4 binding in vitro, whereas changes outside this region have minimal effects. One mutation, which generates a sequence that most closely resembles the consensus for 15 coregulated genes, increases both the level of induction and the affinity for GCN4 protein. The palindromic nature of the optimal sequence, ATGACTCAT, suggest that GCN4 protein binds as a dimer to adjacent half-sites that possibly overlap.