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Biomedical subjects

David M Hunt

Publications and source records attributed to David M Hunt.

34 records · Page 2Linked to original sources
PubMed2004

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.

Amino Acid Substitution↗
PubMed2004

Dominant cone and cone-rod dystrophies: functional analysis of mutations in retGC1 and GCAP1.

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

Calcium↗
PubMed2004

Progressive cone dystrophy associated with mutation in CNGB3.

PURPOSE: To determine the molecular basis for phenotypic variability in a three-generation consanguineous family containing a single individual with complete achromatopsia and three individuals with progressive cone dystrophy. METHODS: Four affected individuals underwent ophthalmic examination, electrophysiological assessment, color fundus photography, and psychophysical testing. Blood samples were obtained for DNA extraction and mutation screening of the cone-specific cGMP-gated (CNG) channel protein gene CNGB3 was undertaken. RESULTS: The clinical findings in one family member were consistent with a diagnosis of complete achromatopsia, with nystagmus, photophobia, and poor visual acuity from early infancy and complete color-blindness, normal fundi, and absent cone responses with normal rod responses on electroretinography (ERG). Mutation analysis revealed her to be homozygous for the common CNGB3 achromatopsia mutation, 1148delC (Thr383fs). In contrast, the three other symptomatic individuals in the family had findings consistent with progressive cone dystrophy. Their visual problems began later in childhood (ranging from 3 to 14 years of age) and there was evidence of progressive deterioration in cone function. All three had a marked tritanopic color vision defect and fundoscopy revealed bilateral macular atrophy. Electrophysiological testing of these three subjects demonstrated clear evidence of progressive deterioration of cone responses over time; rod responses were normal. All three individuals with this progressive phenotype were found to be compound heterozygotes for the 1148delC (Thr383fs) frameshift mutation and a novel Arg403Gln missense mutation in CNGB3. CONCLUSIONS: Mutations in CNGB3, which have been shown to cause achromatopsia, are now shown to be associated with autosomal recessive progressive cone dystrophy. In this study, a novel Arg403Gln mutation was identified, located in the middle of the pore domain of the cone CNG cation channel beta-subunit, which when associated with the nonsense mutation Thr383fs, resulted in progressive cone dystrophy.

Adult↗
PubMed2003

The rod opsin pigments from two marsupial species, the South American bare-tailed woolly opossum and the Australian fat-tailed dunnart.

Rod visual pigment genes have been studied in a wide range of vertebrates including a number of mammalian species. However, no marsupials have yet been examined. To correct this omission, we have studied the rod pigments in two marsupial species, the nocturnal and frugivorous bare-tailed woolly opossum, Caluromys philander, from Central and South America, and the arhythmic and insectivorous fat-tailed dunnart, Sminthopsis crassicaudata, from Australia. Phylogenetic analysis establishes that the cloned opsin sequences are orthologues of rod opsin genes from other vertebrate species. The deduced amino acid sequences show that both possess glutamate at residue 122, a feature of rod opsins, and the corresponding gene follows the typical vertebrate rod opsin pattern of five exons separated by four introns. Compared to other vertebrates, a stretch of five residues near the C-terminus is deleted in the rod opsin of both marsupials and all eutherian mammals. From microspectrophotometric measurements, the pigments in the two species show an 8 nm difference in peak absorbance; the molecular basis for this spectral shift is discussed and two candidate substitutions are identified.

Amino Acid Sequence↗
PubMed2003

Genomic organisation and alternative splicing of human RIM1, a gene implicated in autosomal dominant cone-rod dystrophy (CORD7).

A mutation has been identified in the Rab3A-interacting molecule (RIM1) gene in CORD7, an autosomal dominant cone-rod dystrophy that localises to chromosome 6q14. The G to A point mutation results in an Arg844His substitution in the C(2)A domain of the protein that segregates with disease. This mutation is absent in over 200 control chromosomes, indicating that it is not a common polymorphism, and the almost complete sequence conservation of the C(2)A domain between human and rat RIM1 is consistent with a disease role for the change. RIM1 is expressed in brain and photoreceptors of the retina where it is localised to the pre-synaptic ribbons in ribbon synapses. The RIM1 gene is composed of at least 35 exons, spans 577 kb of genomic DNA, and encodes a protein of up to 1693 residues. The transcript shows extensive alternative splicing involving exons 17, 21-26 and 28-30.

Alternative Splicing↗
PubMed2003

An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4.

PURPOSE: To describe the phenotype of an autosomal dominant macular dystrophy and identify the chromosomal locus. METHODS: Eleven members of a five-generation, nonconsanguineous British family were examined clinically and also underwent automated perimetry, electrodiagnostic testing, fundus fluorescein angiography, and fundus autofluorescence imaging. Blood samples were taken for DNA extraction and linkage analysis was performed. RESULTS: The phenotype is characterized by bull's-eye macular dystrophy first evident in the first or second decade of life. There is mild visual impairment, central scotomata, and electrophysiological testing indicates that most affected individuals have disease confined to the central retina but older subjects have more widespread rod and cone abnormalities, demonstrated by flash ERG. Genetic linkage analysis established linkage to chromosome 4 at p15.2-16.3 with a maximum lod score of 3.03 at a recombination fraction of 0.00 for marker D4S391. The locus for this autosomal dominant macular dystrophy lies between flanking markers D4S3023 and D4S3022, and overlaps the Stargardt 4 locus. CONCLUSIONS: A new locus was identified for a bull's-eye macular dystrophy on the short arm of chromosome 4.

Adolescent↗
PubMed2003

An early-onset autosomal dominant macular dystrophy (MCDR3) resembling North Carolina macular dystrophy maps to chromosome 5.

PURPOSE: To characterize the phenotype of an autosomal dominant macular dystrophy and identify the chromosomal locus. METHODS: Thirteen members of a four-generation, nonconsanguineous British family were examined clinically and also underwent automated perimetry, fundus fluorescein angiography, and fundus autofluorescence imaging. After informed consent was obtained, blood samples were taken for DNA extraction, and genetic linkage analysis was performed. RESULTS: The retinal changes have an early age of onset and are confined to the macular region. The macular abnormalities vary from mild retinal pigment epithelium (RPE) pigmentary change to atrophy. Drusen-like deposits are present to various degrees and are characteristic of the phenotype. Subretinal neovascular membrane (SRNVM) is an established complication. Genetic linkage analysis established linkage to chromosome 5, region p13.1-p15.33 with a maximum LOD score of 3.61 at a recombination fraction of 0.00 for marker D5S630. The locus for this autosomal dominant macular dystrophy lies between flanking markers D5S1981 and D5S2031. CONCLUSIONS: A novel locus has been identified for early-onset autosomal dominant macular dystrophy on chromosome 5.

Adolescent↗
PubMed2003

Expression of PRPF31 mRNA in patients with autosomal dominant retinitis pigmentosa: a molecular clue for incomplete penetrance?

PURPOSE: To investigate whether the incomplete penetrance phenotype characteristic of adRP families linked to chromosome 19q13.4 (RP11) with mutations in the PRPF31 gene is due to differentially expressed wild-type alleles in symptomatic and asymptomatic individuals. METHODS: Real-time quantitative RT-PCR was performed on RNA from lymphoblastoid cell lines derived from a large adRP family (RP856/AD5) that segregates an 11bp deletion in exon 11 of PRPF31. The mRNA levels from only the wild-type allele of PRPF31 were assayed using a probe designed across the deletion. The Mann-Whitney U test was used to compare the median mRNA copy numbers of the symptomatic with the asymptomatic carriers of the mutant PRPF31 allele. The PRPF31 protein levels from symptomatic and asymptomatic individuals were also assayed by Western blot analysis using an antibody specific to the wild-type PRPF31 protein. RESULTS: The use of cell lines was validated by the observation that cell transformation did not alter PRPF31 expression in the cell lines compared with nucleated blood cells and donor retinas. A significant difference in wild-type PRPF31 mRNA levels was observed between symptomatic and asymptomatic individuals (P < 0.001) and was supported by Western blot analysis of the PRPF31 protein. CONCLUSIONS: Partial penetrance in RP11 could be due to the coinheritance of a PRPF31 gene defect and a low-expressed wild-type allele. This study revealed a potential avenue for future therapy in that it appears the moderate overexpression of wild-type PRPF31 may prevent clinical manifestation of the disease.

Adult↗
PubMed2002

Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31.

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology.

Alternative Splicing↗
PubMed2002

The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments.

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with lambda(max) (the peak of maximal absorbance) values generally between 400 and 430 nm, and ultraviolet-sensitive (UVS), with a lambda(max)<380 nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86-->Tyr substitution is sufficient by itself to shift the lambda(max) of the goldfish pigment from a wild-type value of 360 nm to around 420 nm, and the reverse substitution of Tyr-86-Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.

Animals↗
PubMed2002

Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes.

cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.

Amino Acid Sequence↗
PubMed2002

Spectral tuning and evolution of short wave-sensitive cone pigments in cottoid fish from Lake Baikal.

The cottoid fishes of Lake Baikal in eastern Siberia provide a unique opportunity to study the evolution of visual pigments in a group of closely related species exposed to different photic environments. Members of this species flock are adapted to different depth habitats down to >1000 m, and both the rod and cone visual pigments display short wave shifts as depth increases. The blue-sensitive cone pigments of the SWS2 class cluster into two species groups with lambda(max) values of 450 and 430 nm, with the pigment in Cottus gobio, a cottoid fish native to Britain, forming a third group with a lambda(max) of 467 nm. The sequences of the SWS2 opsin gene from C. gobio and from two representatives of the 450 and 430 nm Baikal groups are presented. Approximately 6 nm of the spectral difference between C. gobio and the 450 nm Baikal group can be ascribed to the presence of a porphyropsin/rhodopin mixture in C. gobio. Subsequent analysis of amino acid substitutions by site-directed mutagenesis demonstrates that the remainder of the shift from 461 to 450 nm arises from a Thr269Ala substitution and the shift from 450 to 430 nm at least partly from Thr118Ala and Thr118Gly substitutions. The underlying adaptive significance of these substitutions in terms of spectral tuning and signal-to-noise ratio is discussed.

Adaptation, Physiological↗
PubMed2002

Guanylate cyclase activating proteins, guanylate cyclase and disease.

A range of cone and cone-rod dystrophies (CORD) have been observed in man, caused by mutations in retinal guanylate cyclase 1 (RetGC1) and guanylate cyclase activating protein 1 (GCAP 1). The CORD causing mutations in RetGC1 are located at a mutation "hot spot" within the dimerisation domain, where R838 is the key residue. Three disease causing mutations have been found in human GCAP1, resulting in cone or cone-rod degeneration. All three mutations are dominant in their effect although the mechanism by which the P50L mutation exerts its influence remains unclear although it might act due to a haplo-insufficiency, arising from increased susceptibility to protease activity and increased thermal instability. In contrast, loss of Ca2+ sensitivity appears to be the main cause of the diseased state for the Y99C and E155G mutations. The cone and cone-rod dystrophies that are caused by mutations in RetGC1 or GCAP1 arise from a perturbation of the delicate balance of Ca2+ and cGMP within the photoreceptor cells and it is this disruption that is believed to cause cell death. The diseases caused by mutations in RetGC1 and GCAP1 prominently affect cones, consistent with the higher concentrations of these proteins in cone cells.

Amino Acid Motifs↗
PubMedDate not supplied

Progressive cone and cone-rod dystrophies: phenotypes and underlying molecular genetic basis.

The cone and cone-rod dystrophies form part of a heterogeneous group of retinal disorders that are an important cause of visual impairment in children and adults. There have been considerable advances made in recent years in our understanding of the pathogenesis of these retinal dystrophies, with many of the chromosomal loci and causative genes having now been identified. Mutations in 12 genes, including GUCA1A, peripherin/RDS, ABCA4 and RPGR, have been described to date; and in many cases detailed functional assessment of the effects of the encoded mutant proteins has been undertaken. This improved knowledge of disease mechanisms has raised the possibility of future treatments for these disorders, for which there are no specific therapies available at the present time.

DNA↗