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Biomedical subjects

David M Hunt

Publications and source records attributed to David M Hunt.

At least 19 recordsLinked to original sources
PubMed2006

Mutations in the gene KCNV2 encoding a voltage-gated potassium channel subunit cause "cone dystrophy with supernormal rod electroretinogram" in humans.

"Cone dystrophy with supernormal rod electroretinogram (ERG)" is an autosomal recessive disorder that causes lifelong visual loss combined with a supernormal ERG response to a bright flash of light. We have linked the disorder to a 0.98-cM (1.5-Mb) region on chromosome 9p24, flanked by rs1112534 and rs1074449, using homozygosity mapping in one large consanguineous pedigree. Analysis of one gene within this region, KCNV2, showed a homozygous nonsense mutation. Mutations were also found in 17 alleles of 10 other unrelated families with the same disorder. In situ hybridization demonstrated KCNV2 expression in human rod and cone photoreceptors. The precise function of KCNV2 in human photoreceptors remains to be determined, although this work suggests that mutations might perturb or abrogate I(KX), the potassium current within vertebrate photoreceptor inner segments, which has been shown to set their resting potential and voltage response.

Chromosomes, Human, Pair 9↗
PubMed2006

Spectral tuning of the long wavelength-sensitive cone pigment in four Australian marsupials.

The molecular basis for the spectral tuning of longwave-sensitive (LWS) visual pigments in mammals have been described in a wide range of placental species, including the primates. However, little is known about the molecular mechanisms in marsupial LWS pigments. Here, we have studied and compared the LWS opsins in four Australian marsupials: two diprotodonts and two polyprotodonts. Phylogenetic analysis establishes that all LWS marsupial sequences form a distinct clade from the placental mammals that is subdivided into diprotodont and polyprotodont groups. Amino acid sequences reveal that substitutions at sites 277 and 285 are largely responsible for the spectral shifts in marsupial LWS pigments and species comparison indicates that the ancestral gene most likely encoded Tyr277 and Ala180. Amino acid substitutions are discussed in the context of spectral shifts in marsupial LWS and in relation to the mechanisms in primate pigments.

Amino Acid Sequence↗
PubMed2006

Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations.

Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.

Amino Acid Sequence↗
PubMed2006

Evolution of the cichlid visual palette through ontogenetic subfunctionalization of the opsin gene arrays.

The evolution of cone opsin genes is characterized by a dynamic process of gene birth and death through gene duplication and loss. However, the forces governing the retention and death of opsin genes are poorly understood. African cichlid fishes have a range of ecologies, differing in habitat and foraging style, which make them ideal for examining the selective forces acting on the opsin gene family. In this work, we present data on the riverine cichlid, Oreochromis niloticus, which is an ancestral outgroup to the cichlid adaptive radiations in the Great African lakes. We identify 7 cone opsin genes with several instances of gene duplication. We also characterize the spectral sensitivities of these genes through reconstitution of visual pigments. Peak absorbances demonstrate that each tilapia cone opsin gene codes for a spectrally distinct visual pigment: SWS1 (360 nm), SWS2b (423 nm), SWS2a (456 nm), Rh2b (472 nm), Rh2a beta (518 nm), Rh2a alpha (528 nm), and LWS (561 nm). Furthermore, quantitative reverse transcription polymerase chain reaction at 3 ontogenetic time points demonstrates that although only 4 genes (SWS2a, Rh2a alpha and beta, and LWS) are expressed in adults, mRNAs for the other genes are all expressed during ontogeny. Therefore, subfunctionalization through differential ontogenetic expression may be a key mechanism for preservation of opsin genes. The distinct peak absorbances of these preserved opsin genes provide a palette from which selection creates the diverse visual sensitivities found among the cichlid species of the lacustrine adaptive radiations.

Animals↗
PubMed2006

A study of the nuclear trafficking of the splicing factor protein PRPF31 linked to autosomal dominant retinitis pigmentosa (ADRP).

In this study the mechanism of nuclear importation of the splicing factor PRPF31 is examined and the impact of two disease-linked mutations, A194E and A216P, assessed. Using pull-down assays with GST-tagged importin proteins, we demonstrate that His-tagged PRPF31 interacts with importin beta1 for translocation to the nucleus, with no requirement for importin alpha1. The A194E and A216P mutations have no affect on this interaction. Fluorescence recovery after photobleaching (FRAP) was used to estimate the rate of movement of EGFP-tagged PRPF31 into the nuclei of live cells. The kinetics indicated a two-component recovery process; a fast component with tau approximately 6 s and a slow component with tau approximately 80 s. The mutations affected neither component. We conclude that the two mutations have no negative effect on interaction with the nuclear importation machinery. Reduced mutant protein solubility resulting in an insufficiency of splicing activity in cells with a very high metabolic demand remains the most likely explanation for the disease pathology in ADRP patients.

Active Transport, Cell Nucleus↗
PubMed2005

Mix and match color vision: tuning spectral sensitivity by differential opsin gene expression in Lake Malawi cichlids.

Cichlid fish of the East African Rift Lakes are renowned for their diversity and offer a unique opportunity to study adaptive changes in the visual system in rapidly evolving species flocks. Since color plays a significant role in mate choice, differences in visual sensitivities could greatly influence and even drive speciation of cichlids. Lake Malawi cichlids inhabiting rock and sand habitats have significantly different cone spectral sensitivities. By combining microspectrophotometry (MSP) of isolated cones, sequencing of opsin genes, and spectral analysis of recombinant pigments, we have established the cone complements of four species of Malawi cichlids. MSP demonstrated that each of these species predominately expresses three cone pigments, although these differ between species to give three spectrally different cone complements. In addition, rare populations of spectrally distinct cones were found. In total, seven spectral classes were identified. This was confirmed by opsin gene sequencing, expression, and in vitro reconstitution. The genes represent the four major classes of cone opsin genes that diverged early in vertebrate evolution. All four species possess a long-wave-sensitive (LWS), three spectrally distinct green-sensitive (RH2), a blue-sensitive (SWS2A), a violet-sensitive (SWS2B), and an ultraviolet-sensitive (SWS1) opsin. However, African cichlids determine their spectral sensitivity by differential expression of primarily only three of the seven available cone opsin genes. Phylogenetic analysis suggests that all percomorph fish have similar potential.

Animals↗
PubMed2005

Isolation and characterization of murine Cds (CDP-diacylglycerol synthase) 1 and 2.

Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2005

Cone topography and spectral sensitivity in two potentially trichromatic marsupials, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus).

The potential for trichromacy in mammals, thought to be unique to primates, was recently discovered in two Australian marsupials. Whether the presence of three cone types, sensitive to short- (SWS), medium- (MWS) and long- (LWS) wavelengths, occurs across all marsupials remains unknown. Here, we have investigated the presence, distribution and spectral sensitivity of cone types in two further species, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus). Immunohistochemistry revealed that SWS cones in the quokka are concentrated in dorso-temporal retina, while in the quenda, two peaks were identified in naso-ventral and dorso-temporal retina. In both species, MWS/LWS cone spatial distributions matched those of retinal ganglion cells. Microspectrophotometry (MSP) confirmed that MWS and LWS cones are spectrally distinct, with mean wavelengths of maximum absorbance at 502 and 538 nm in the quokka, and at 509 and 551 nm, in the quenda. Although small SWS cone outer segments precluded MSP measurements, molecular analysis identified substitutions at key sites, accounting for a spectral shift from ultraviolet in the quenda to violet in the quokka. The presence of three cone types, along with previous findings in the fat-tailed dunnart and honey possum, suggests that three spectrally distinct cone types are a feature spanning the marsupials.

Animals↗
PubMed2005

A review of sleeve fractures of the patella in children.

Patella fractures are rare in children and the sleeve fracture is a particular form of fracture that only occurs in children where it is the most common patella fracture. It is caused by rapid muscle contraction. Diagnosis may be difficult both clinically and on the X-ray which may look normal if there is no bony fragment. Patella alta is the best sign and ultrasound is very helpful. Awareness of the existence of the injury is all important. This fracture differs from straightforward avulsion because of the "sleeve" of periosteum which is pulled off the patella and will continue to form bone if not treated thus enlarging or even duplicating the patella. Treatment involves prompt reduction and, usually, internal fixation of the disrupted patella tendon.

Child↗
PubMed2005

X-linked cone dysfunction syndrome with myopia and protanopia.

PURPOSE: To perform a detailed clinical, psychophysical, and molecular assessment of members of 4 families with an unusual X-linked cone dysfunction syndrome associated with myopia. PARTICIPANTS: Affected and unaffected members of 4 British nonconsanguineous families. METHODS: Subjects underwent both detailed clinical examination and psychophysical testing. After informed consent was obtained, blood samples were taken for DNA extraction, and molecular genetic analysis was performed. The strategy for molecular analysis was to amplify the coding regions of the long and middle wavelength-sensitive cone opsin genes and the upstream locus control region by polymerase chain reaction and to examine these fragments for mutations by sequencing of DNA. RESULTS: The phenotype was almost identical in all 4 families, consisting of moderate to high myopia, astigmatism, moderately reduced acuity, and normal fundi. Electroretinography showed abnormal cone but normal rod responses. Psychophysical testing showed a selective impairment of long cones in combination with well-preserved middle cone and short cone function. There was no evidence to suggest that the phenotype was progressive. Molecular analysis of the X-linked opsin gene array in the 4 families indicated that affected males have inherited the same X-chromosome from their mother. In 2 families, a long/middle hybrid gene was detected. In a third family, the commonly described deleterious Cys203Arg amino acid substitution was identified in both the long and middle opsin genes. In the fourth family, the only abnormality was absence of a middle opsin exon 2; the cause of the protanopia in this family is uncertain. CONCLUSIONS: The X-linked cone dysfunction syndrome associated with myopia and dichromacy described here has many similarities to Bornholm eye disease, a condition previously mapped to Xq28. Except for the Cys203Arg substitution in one family, no alterations in the opsin gene array were identified that could underlie the cone dysfunction. It is therefore possible that the cone dysfunction may have a genetic origin different from that of the dichromacy.

Adolescent↗
PubMed2005

Mutation in the gene GUCA1A, encoding guanylate cyclase-activating protein 1, causes cone, cone-rod, and macular dystrophy.

PURPOSE: To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 4-generation family and to examine the phenotype and the degree of intrafamilial variability. DESIGN: Prospective case series. PARTICIPANTS: Six affected individuals from a nonconsanguineous British family. METHODS: Detailed ophthalmologic examination, color fundus photography, autofluorescence imaging, and electrophysiologic assessment were performed. Blood samples were taken for DNA extraction, and mutation screening of GUCA1A, the gene encoding guanylate cyclase-activating protein 1 (GCAP1), was undertaken. RESULTS: All affected subjects complained of mild photophobia and reduced central and color vision. Onset was between the third and fifth decade, with subsequent gradual deterioration of visual acuity and color vision. Visual acuity ranged between 6/9 and counting fingers. Color vision was either absent or markedly reduced along all 3 color axes. A range of macular appearances was seen, varying from mild retinal pigment epithelial disturbance to extensive atrophy. Electrophysiologic testing revealed a range of electrophysiologic abnormalities: isolated cone electroretinography abnormalities, reduced cone and rod responses (with cone loss greater than rod), and isolated macular dysfunction. The 4 coding exons of GUCA1A were screened for mutations in affected and unaffected family members. A single transition, A319G, causing a nonconservative missense substitution, Tyr99Cys, segregated uniquely in all affected subjects. CONCLUSIONS: The Tyr99Cys GUCA1A mutation has been previously shown to cause autosomal dominant progressive cone dystrophy. This is the first report of this mutation also causing both cone-rod dystrophy and isolated macular dysfunction. The phenotypic variation described here exemplifies the intrafamilial heterogeneity of retinal dysfunction that can be observed in persons harboring the same mutation and chromosomal segment.

Adult↗
PubMed2005

Cone-rod dystrophy, intrafamilial variability, and incomplete penetrance associated with the R172W mutation in the peripherin/RDS gene.

PURPOSE: To determine the underlying molecular genetic basis of a retinal dystrophy identified in a 5-generation family, and to examine the phenotype and degree of intrafamilial variability. DESIGN: Family genetic study. PARTICIPANTS: Nine affected individuals from a nonconsanguineous British family. METHODS: Ophthalmologic examination, color vision testing, fundus photography, autofluorescence imaging, and electrophysiological assessment were performed. The clinical notes of 2 additional deceased affected family members were also reviewed. Blood samples were taken for DNA extraction, with linkage analysis being performed, and subsequent mutation screening of the peripherin/RDS gene. RESULTS: Linkage analysis established a disease interval on chromosome 6p, which harbored the retinal candidate gene, peripherin/RDS. The 3 coding exons of the peripherin/RDS gene were subsequently screened for mutations in affected and unaffected family members. A nonconservative missense substitution, Arg172Trp (R172W), segregated uniquely in all affected subjects. The majority of subjects carrying the R172W peripherin/RDS mutation complained of reduced central vision starting in the second or third decade, with subsequent gradual deterioration of visual acuity and color vision. Three affected individuals complained of nyctalopia. A range of macular appearances were seen, varying from a typical granular appearance to extensive macular atrophy. Autofluorescence imaging in the majority of individuals identified a highly characteristic speckled macular appearance. All affected subjects had abnormal pattern electroretinograms (ERGs) consistent with macular dysfunction and 4 subjects showed additional full-field ERG abnormalities, providing evidence of generalized retinal dysfunction. There was marked variation in the clinical phenotype in those individuals who carried the R172W peripherin/RDS mutation, ranging from severe cone-rod dystrophy to asymptomatic individuals with normal retinal function. CONCLUSIONS: The Arg172Trp (R172W) peripherin/RDS mutation has been previously reported to cause a fully penetrant progressive macular dystrophy with high intrafamilial and interfamilial consistency of phenotype. This is the first report describing marked intrafamilial variation associated with this mutation, including nonpenetrance. These findings are clinically important in relation to advice on prognosis and accurate genetic counseling.

Adolescent↗
PubMed2005

Colour vision and speciation in Lake Victoria cichlids of the genus Pundamilia.

Lake Victoria cichlids are one of the most speciose groups of vertebrates. Selection on coloration is likely playing an important role in their rapid speciation. To test the hypothesis that sensory biases could explain species differences in mating preferences and nuptial coloration, we studied seven populations of four closely related species of the genus Pundamilia that differ in visual environment and male nuptial colour. Microspectrophotometry determined that the wavelength of maximum absorption (lambdamax) of the rod pigment and three cone pigments were similar in all four species. Only the long wavelength sensitive (LWS) pigment varied among species, with 3-4 nm shifts in lambdamax that correlated with differences in the LWS opsin sequence. These subtle shifts in lambdamax coincided with large shifts in male body colour, with red species having longer LWS pigments than blue species. Furthermore, we observed within and between species a correlation between water transparency and the proportion of red/red vs. red/green double cones. Individuals from turbid water had more red/red double cones than individuals from clear water. The variation in LWS lambdamax and in the proportion of red/red double cones could lead to differences in perceived brightness that may explain the evolution of variation in male coloration. However, other factors, such as chromophore shifts and higher order neural processing, should also be investigated to fully understand the physiological basis of differential responses to male mating hues in cichlid fish.

Africa, Eastern↗
PubMed2005

Adaptations to an extreme environment: retinal organisation and spectral properties of photoreceptors in Antarctic notothenioid fish.

The Notothenioid suborder of teleosts comprises a number of species that live below the sea ice of the Antarctic. The presence of 'antifreeze' glycoproteins in these fish as an adaptation to freezing temperature has been well documented but little is known about the adaptations of the visual system of these fish to a light environment in which both the quantity and spectral composition of downwelling sunlight has been reduced by passage through ice and snow. In this study, we show that the red/long-wave sensitive (LWS) opsin gene is not present in these fish but a UV-sensitive short-wave sensitive (SWS1) pigment is expressed along with blue-sensitive (SWS2) and green/middle-wave sensitive (Rh2) pigments. The identity and spectral location of maximal absorbance of the SWS1 and Rh2 pigments was confirmed by in vitro expression of the recombinant opsins followed by regeneration with 11-cis retinal. Only the SWS2 pigment showed interspecific variations in peak absorbance. Expression of the Rh2 opsin is localised to double cone receptors in both the central and peripheral retina, whereas SWS2 opsin expression is present only in the peripheral retina. SWS1 cones could not be identified by either microspectrophotometry or in situ hybridisation, presumably reflecting their low number and/or uneven distribution across the retina. A study of photoreceptor organisation in the retina of two species, the shallower dwelling Trematomus hansoni and the deeper dwelling Dissostichus mawsoni, identified a square mosaic in the former, and a row mosaic in the latter species; the row mosaic in Dissostichus mawsoni with less tightly packed cone photoreceptors allows for a higher rod photoreceptor density.

Adaptation, Biological↗
PubMed2004

Purification, characterisation and intracellular localisation of aryl hydrocarbon interacting protein-like 1 (AIPL1) and effects of mutations associated with inherited retinal dystrophies.

Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA), a major cause of childhood blindness, yet the cellular function of the encoded protein has yet to be fully elucidated. In order to investigate the biochemistry of AIPL1, we have developed a system for the expression of the recombinant protein in bacteria and its subsequent purification. The secondary structure and thermostability of wild-type and mutant proteins have been examined by circular dichroism (CD) spectroscopy. Some of the variants, notably W278X and P376S, had markedly different secondary structure compositions, indicating that the proteins had not folded properly, whilst W278X and T114I were particularly thermolabile. When eukaryotic cells were transfected with the AIPL1 expression constructs, we show by immunofluorescence microscopy that wild-type protein is distributed throughout the nucleus and cytoplasm. Several of the mutants give similar results. With two of the disease-associated variants (W278X and A336Delta2), however, the protein remains in the cytoplasm in aggresome-like particles. These particles were shown to be ubiquitinated, indicating that the mutant protein had been tagged for proteosomal degradation. On this basis, we can conclude that wild-type protein is expressed in a soluble and folded manner, and that some of the disease-associated mutant proteins are nonfunctional because they are insoluble and are degraded by the cell. Other mutations appear to have a more localised effect on secondary structure, which does not result in insolubility or affect protein targeting, but reduces the stability of the protein at human body temperature.

Adaptor Proteins, Signal Transducing↗
PubMed2004

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment.

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.

Amino Acid Sequence↗