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1.  Three Different Cone Opsin Gene Array Mutational Mechanisms with Genotype–Phenotype Correlation and Functional Investigation of Cone Opsin Variants 
Human Mutation  2014;35(11):1354-1362.
Mutations in the OPN1LW (L-) and OPN1MW (M-)cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of color vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the locus control region (LCR); missense mutation (p.Cys203Arg) in an L-/M-hybrid gene; and exon 3 single-nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate-to-high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease-associated exon 3 SNP haplotypes encoding LIAVA, LVAVA, or MIAVA were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 years having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here, we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease-associated haplotypes, appears to be principally responsible for this mutational mechanism.
PMCID: PMC4285181  PMID: 25168334
opsin; blue cone monochromacy; splicing; cone dystrophy; OPN1LW; OPN1MW
2.  Retinal Structure and Function in Achromatopsia: Implications for Gene Therapy 
Ophthalmology  2013;121(1):234-245.
To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy.
Cross-sectional study.
Forty subjects with ACHM.
All subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (i) continuous inner segment ellipsoid (ISe), (ii) ISe disruption, (iii) ISe absence, (iv) presence of a hyporeflective zone (HRZ), and (v) outer retinal atrophy including retinal pigment epithelial (RPE) loss. Foveal and outer nuclear layer (ONL) thickness was measured, and presence of hypoplasia determined.
Main Outcome Measures
Photoreceptor appearance on SD-OCT imaging; foveal and ONL thickness; presence of foveal hypoplasia; retinal sensitivity and fixation stability; and association of these parameters with age and genotype.
Forty subjects with mean age of 24.9 years (range 6 to 52) were included. Disease-causing variants were found in CNGA3 (n=18), CNGB3 (n=15), GNAT2 (n=4), and PDE6C (n=1). No variants were found in 2 individuals. 22.5% of subjects had a continuous ISe layer at the fovea; 27.5% had ISe disruption; 20% had an absent ISe layer; 22.5% had a HRZ; and 7.5% had outer retinal atrophy. No significant differences in age (p=0.77), mean retinal sensitivity (p=0.21) or fixation stability (p=0.34) across the 5 SD-OCT categories were evident. No significant correlation was found between age and foveal thickness (p=0.84), or between age and foveal ONL thickness (p=0.12).
The lack of clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring their impact.
PMCID: PMC3895408  PMID: 24148654
3.  Microcephaly with or without Chorioretinopathy, Lymphoedema or Mental Retardation (MCLMR); review of phenotype associated with KIF11 mutations 
Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR) (MIM #152950) is a rare autosomal dominant condition for which a causative gene has recently been identified. Mutations in the kinesin family member 11 (KIF11) gene have now been described in sixteen families worldwide. This is a review of the condition based on the clinical features of thirty seven individuals from twenty two families. This report includes nine previously unreported families and additional information for some of those reported previously.
The condition arose de novo in 8/20 families (40%). The parental results were not available for two probands. The mutations were varied, and include missense, nonsense, frameshift and splice site and are distributed evenly throughout the KIF11 gene. In our cohort, 86% had microcephaly, 78% had an ocular abnormality consistent with the diagnosis, 46% had lymphoedema, 73% had mild-moderate learning difficulties, 8% had epilepsy and 8% had a cardiac anomaly. We identified three individuals with KIF11 mutations, but no clinical features of MCLMR demonstrating reduced penetrance. The variable expression of the phenotype and presence of mildly affected individuals indicates that the prevalence may be higher than expected, and we would therefore recommend a low threshold for genetic testing.
PMCID: PMC3938398  PMID: 24281367
Microcephaly; Chorioretinal dysplasia; Lymphoedema; KIF11; MCLMR
The transcription factor nuclear factor E2-related factor 2 (Nrf2) regulates the expression of multiple cytoprotective genes that have been shown to offer protection in response to a number of insults. The present study describes a novel strategy to increase expression of Nrf2-responsive genes in brain injured mice. Under normal conditions, the adapter protein Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and promotes its proteosomal degradation in the cytoplasm. The amino acid sequence DEETGE, located at amino acid 77–82 of Nrf2, is critical for Nrf2-Keap1 interaction, and synthetic peptides containing this sequence can be used to disrupt the complex in vitro. We observed that intracerebroventricular (i.c.v.) infusion of a peptide containing the DEETGE sequence along with the cell transduction domain of the HIV-TAT protein (TAT-DEETGE) into brain-injured mice did not increase the mRNA levels for Nrf2-driven genes. However, when a calpain cleavage sequence was introduced between the TAT sequence and the DEETGE sequence, the new peptide (TAT-CAL-DEETGE) increased the mRNA levels of these genes. Increased gene expression was not observed when the TAT-CAL-DEETGE peptide was injected into uninjured animals. Furthermore, injection of TAT-CAL-DEETGE peptides before or after brain injury reduced blood-brain barrier compromise, a prominent secondary pathology that negatively influences outcome. The present strategy to increase Nrf2-responsive gene expression can be adapted to treat other insults or diseases based on their underlying mechanism(s) of cellular damage.
PMCID: PMC4264351  PMID: 21414291
blood-brain barrier; cytoprotective genes; Keap1; Nrf2; peptide-based therapy; TBI
5.  Persistent working memory dysfunction following traumatic brain injury: evidence for a time-dependent mechanism. 
Neuroscience  2009;159(2):483-491.
The prefrontal cortex is highly vulnerable to traumatic brain injury resulting in the dysfunction of many high-level cognitive and executive functions such as planning, information processing speed, language, memory, attention, and perception. All of these processes require some degree of working memory. Interestingly, in many cases, post-injury working memory deficits can arise in the absence of overt damage to the prefrontal cortex. Recently, excess GABA-mediated inhibition of prefrontal neuronal activity has been identified as a contributor to working memory dysfunction within the first month following cortical impact injury of rats. However, it has not been examined if these working memory deficits persist, and if so, whether they remain amenable to treatment by GABA antagonism. Our findings show that working memory dysfunction, assessed using both the delay match-to-place and delayed alternation t-maze tasks, following lateral cortical impact injury persists for at least 16 weeks post-injury. These deficits were found to be no longer the direct result of excess GABA-mediated inhibition of medial prefrontal cortex neuronal activity. Golgi staining of prelimbic pyramidal neurons revealed that TBI causes a significant shortening of layer V/VI basal dendrite arbors by 4 months post-injury, as well as an increase in the density of both basal and apical spines in these neurons. These changes were not observed in animals 14 days-post-injury, a time point at which administration of GABA receptor antagonists improves working memory function. Taken together, the present findings, along with previously published reports, suggest that temporal considerations must be taken into account when designing mechanism-based therapies to improve working memory function in TBI patients.
PMCID: PMC4264540  PMID: 19167462
Golgi staining; neuronal morphology; prelimbic cortex; delayed alternation T-maze; delay match-to-place; GAD
6.  Molecular modeling indicates distinct classes of missense variants with mild and severe XLRS phenotypes 
Human Molecular Genetics  2013;22(23):4756-4767.
X-linked retinoschisis (XLRS) is a vitreo-retinal degeneration caused by mutations in the RS1 gene which encodes the protein retinoschisin (RS1), required for the structural and functional integrity of the retina. Data are presented from a group of 38 XLRS patients from Moorfields Eye Hospital (London, UK) who had one of 18 missense mutations in RS1. Patients were grouped based on mutation severity predicted by molecular modeling: mild (class I), moderate (intermediate) and severe (class II). Most patients had an electronegative scotopic bright flash electroretinogram  (ERG) (reduced b/a-wave ratio) in keeping with predominant inner retinal dysfunction. An association between the type of structural RS1 alterations and the severity of b/a-wave reduction was found in all but the oldest group of patients, significant in patients aged 15–30 years. Severe RS1 missense changes were associated with a lower ERG b/a ratio than were mild changes, suggesting that the extent of inner retinal dysfunction is influenced by the effect of the mutations on protein structure. The majority of class I mutations showed no changes involving cysteine residues. Class II mutations caused severe perturbations due to the removal or insertion of cysteine residues or due to changes in the hydrophobic core. The ERG b/a ratio in intermediate cases was abnormal but showed significant variability, possibly related to the role of proline or arginine residues. We also conducted a second study, using a completely independent cohort, to indicate a genotype–ERG phenotype correlation.
PMCID: PMC3820135  PMID: 23847049
7.  Wolfram gene (WFS1) mutation causes autosomal dominant congenital nuclear cataract in humans 
European Journal of Human Genetics  2013;21(12):1356-1360.
Congenital cataracts are an important cause of bilateral visual impairment in infants. Through genome-wide linkage analysis in a four-generation family of Irish descent, the disease-associated gene causing autosomal-dominant congenital nuclear cataract was mapped to chromosome 4p16.1. The maximum logarithm of odds (LOD) score was 2.62 at a recombination fraction θ=0, obtained for marker D4S432 physically close to the Wolfram gene (WFS1). By sequencing the coding regions and intron–exon boundaries of WFS1, we identified a DNA substitution (c.1385A-to-G) in exon 8, causing a missense mutation at codon 462 (E462G) of the Wolframin protein. This is the first report of a mutation in this gene causing an isolated nuclear congenital cataract. These findings suggest that the membrane trafficking protein Wolframin may be important for supporting the developing lens.
PMCID: PMC3831071  PMID: 23531866
nuclear cataract; linkage; WFS1 gene; heterogeneity
8.  Human Cone Visual Pigment Deletions Spare Sufficient Photoreceptors to Warrant Gene Therapy 
Human Gene Therapy  2013;24(12):993-1006.
Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5–58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.
PMCID: PMC3868405  PMID: 24067079
9.  Randomized trial to evaluate nutritional status and absorption of enteral feeding after brain death 
Catecholamines and inflammatory mediators, with elevated levels after brain death, are associated with reduced function and survival of transplanted organs. Enteral nutrition reduces tissue damage and may benefit organs.
To evaluate the effects of immunomodulating enteral nutrition in organ donors.
Prospective, randomized, open-label study.
Intensive care unit.
Thirty-six brain-dead organ donors.
Donors were randomized to receive enteral nutrition containing omega-3 polyunsaturated fatty acid, antioxidants, and glutamine or standard care (fasting). Donors received hormonal replacement therapy of corticosteroid, levothyroxine, dextrose, and insulin.
Main Outcome Measures
Gastrointestinal assimilation (measured by 13carbon-labeled uracil breath analysis), quantity of organs recovered, resting energy expenditure, urine level of urea nitrogen, and serum levels of albumin, prealbumin, interleukin 6, tumor necrosis factor-α, and C-reactive protein were evaluated.
Thirteen patients (36%) assimilated 13C-labeled uracil. Resting energy expenditure was significantly higher than predicted between 10 and 14 hours after baseline in 33 donors (P = .007). Other measures were not conclusively different between fed and fasting groups. No adverse events occurred that were related to the enteral feeding.
About 30% of donors metabolized 13C-labeled uracil, although no difference in oxidation rate was found between fasting and fed donors. Corticosteroid administration lowers plasma levels of interleukin 6 and most likely contributes to greater than predicted resting energy expenditure. Thus energy needs may not be met during fasting if hormones are given. Consequences of this possible energy deficit warrant further study.
PMCID: PMC3971639  PMID: 24311403
10.  HDAC inhibition combined with behavioral therapy enhances learning and memory following traumatic brain injury 
Neuroscience  2009;163(1):1-8.
Traumatic brain injury (TBI) induces a number of pathological events ranging from neuronal degeneration and tissue loss to impaired neuronal plasticity and neurochemical dysregulation. In rodents, exposure of brain injured animals to environmental enrichment has been shown to be an effective means of enhancing learning and memory post-injury. Recently, it has been discovered that environmental enrichment may enhance neuronal plasticity through epigenetic changes that involve enhanced histone acetylation, a property that can be mimicked by the use of histone deactylase (HDAC) inhibitors. We therefore evaluated the consequences of the HDAC inhibitor sodium butyrate on the learning and memory of brain injured mice. In contrast to a previous report using a mouse neurodegeneration model, sodium butyrate (1.2g/kg daily for four weeks) did not improve learning and memory when tested after the completion of the drug treatment paradigm. In addition, sodium butyrate administration during the reported period of neurodegeneration (days 0–5) also offered no benefit. However, when administered concurrently with training in the Morris water maze task (beginning on day 14 post-injury), sodium butyrate improved learning and memory in brain injured mice. Interestingly, when these mice were subsequently tested in an associative fear conditioning task, a continued improvement was observed. Taken together, our findings indicate that HDAC inhibition may mimic some of the cognitive improvements seen following enriched environment exposure, and that the improvement is observed when the treatment is carried out current with behavioral testing.
PMCID: PMC4217276  PMID: 19531374
traumatic brain injury; HDAC; Morris water maze; hippocampus; delay fear conditioning
11.  Understanding the impact of genetic testing for inherited retinal dystrophy 
European Journal of Human Genetics  2013;21(11):1209-1213.
The capability of genetic technologies is expanding rapidly in the field of inherited eye disease. New genetic testing approaches will deliver a step change in the ability to diagnose and extend the possibility of targeted treatments. However, evidence is lacking about the benefits of genetic testing to support service planning. Here, we report qualitative data about retinal dystrophy families' experiences of genetic testing in United Kingdom. The data were part of a wider study examining genetic eye service provision. Twenty interviewees from families in which a causative mutation had been identified by a genetic eye clinic were recruited to the study. Fourteen interviewees had chosen to have a genetic test and five had not; one was uncertain. In-depth telephone interviews were conducted allowing a thorough exploration of interviewees' views and experiences of the benefits of genetic counselling and testing. Transcripts were analysed using thematic analysis. Both affected and unaffected interviewees expressed mainly positive views about genetic testing, highlighting benefits such as diagnostic confirmation, risk information, and better preparation for the future. Negative consequences included the burden of knowledge, moral dilemmas around reproduction, and potential impact on insurance. The offer of genetic testing was often taken up, but was felt unnecessary in some cases. Interviewees in the study reported many benefits, suggesting genetic testing should be available to this patient group. The benefits and risks identified will inform future evaluation of models of service delivery. This research was part of a wider study exploring experiences of families with retinal dystrophy.
PMCID: PMC3798830  PMID: 23403902
retinal dystrophy; genetic testing; service delivery; qualitative interviews
12.  Behavioral and Histopathological Alterations Resulting from Mild Fluid Percussion Injury 
Journal of Neurotrauma  2013;30(9):702-715.
The majority of people who sustain a traumatic brain injury (TBI) have an injury that can be classified as mild (often referred to as concussion). Although head CT scans for most subjects who have sustained a mild TBI (mTBI) are negative, these persons may still suffer from neurocognitive and neurobehavioral deficits. In order to expedite pre-clinical research and develop therapies, there is a need for well-characterized animal models of mTBI that reflect the neurological, neurocognitive, and pathological changes seen in human patients. In the present study, we examined the motor, cognitive, and histopathological changes resulting from 1.0 and 1.5 atmosphere (atm) overpressure fluid percussion injury (FPI). Both 1.0 and 1.5 atm FPI injury caused transient suppression of acute neurological functions, but did not result in visible brain contusion. Animals injured with 1.0 atm FPI did not show significant motor, vestibulomotor, or learning and memory deficits. In contrast, 1.5 atm injury caused transient motor disturbances, and resulted in a significant impairment of spatial learning and short-term memory. In addition, 1.5 atm FPI caused a marked reduction in cerebral perfusion at the site of injury that lasted for several hours. Consistent with previous studies, 1.5 atm FPI did not cause visible neuronal loss in the hippocampus or in the neocortex. However, a robust inflammatory response (as indicated by enhanced GFAP and Iba1 immunoreactivity) in the corpus callosum and the thalamus was observed. Examination of fractional anisotropy color maps after diffusion tensor imaging (DTI) revealed a significant decrease of FA values in the cingulum, an area found to have increased silver impregnation, suggesting axonal injury. Increased silver impregnation was also observed in the corpus callosum, and internal and external capsules. These findings are consistent with the deficits and pathologies associated with mild TBI in humans, and support the use of mild FPI as a model to evaluate putative therapeutic options.
PMCID: PMC3941923  PMID: 23301501
axonal injury; concussion; DTI; mild traumatic brain injury
13.  Analysis of Functional Pathways Altered after Mild Traumatic Brain Injury 
Journal of Neurotrauma  2013;30(9):752-764.
Concussive injury (or mild traumatic brain injury; mTBI) can exhibit features of focal or diffuse injury patterns. We compared and contrasted the cellular and molecular responses after mild controlled cortical impact (mCCI; a focal injury) or fluid percussion injury (FPI; a diffuse injury) in rats. The rationale for this comparative analysis was to investigate the brain's response to mild diffuse versus mild focal injury to identify common molecular changes triggered by these injury modalities and to determine the functional pathways altered after injury that may provide novel targets for therapeutic intervention. Microarrays containing probes against 21,792 unique messenger RNAs (mRNAs) were used to investigate the changes in cortical mRNA expression levels at 3 and 24 h postinjury. Of the 354 mRNAs with significantly altered expression levels after mCCI, over 89% (316 mRNAs) were also contained within the mild FPI (mFPI) data set. However, mFPI initiated a more widespread molecular response, with over 2300 mRNAs differentially expressed. Bioinformatic analysis of annotated Gene Ontology molecular function and biological pathway terms showed a significant overrepresentation of genes belonging to inflammation, stress, and signaling categories in both data sets. We therefore examined changes in the protein levels of a panel of 23 cytokines and chemokines in cortical extracts using a Luminex-based bead immunoassay and detected significant increases in macrophage inflammatory protein (MIP)-1α (CCL3), GRO-KC (CXCL1), interleukin (IL)-1α, IL-1β, and IL-6. Immunohistochemical localization of MIP-1α and IL-1β showed marked increases at 3 h postinjury in the cortical vasculature and microglia, respectively, that were largely resolved by 24 h postinjury. Our findings demonstrate that both focal and diffuse mTBI trigger many shared pathobiological processes (e.g., inflammatory responses) that could be targeted for mechanism-based therapeutic interventions.
PMCID: PMC3653386  PMID: 22913729
bioinformatics; GFAP; IL-1β; MIP-1α; neuroinflammation; UCHL1
14.  Identification of a Rare Coding Variant in Complement 3 Associated with Age-related Macular Degeneration 
Nature genetics  2013;45(11):10.1038/ng.2758.
Macular degeneration is a common cause of blindness in the elderly. To identify rare coding variants associated with a large increase in risk of age-related macular degeneration (AMD), we sequenced 2,335 cases and 789 controls in 10 candidate loci (57 genes). To increase power, we augmented our control set with ancestry-matched exome sequenced controls. An analysis of coding variation in 2,268 AMD cases and 2,268 ancestry matched controls revealed two large-effect rare variants; previously described R1210C in the CFH gene (fcase = 0.51%, fcontrol = 0.02%, OR = 23.11), and newly identified K155Q in the C3 gene (fcase = 1.06%, fcontrol = 0.39%, OR = 2.68). The variants suggest decreased inhibition of C3 by Factor H, resulting in increased activation of the alternative complement pathway, as a key component of disease biology.
PMCID: PMC3812337  PMID: 24036949
15.  ABCA4 Gene Screening by Next-Generation Sequencing in a British Cohort 
We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations.
We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis.
Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site–altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals.
Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies.
PCR-enrichment–based next-generation sequencing with an amplicon tagging protocol revealed two or more disease-causing variants in 37 of 79 patients with ABCA4-associated retinal disease, who had only one variant detected in prescreening with arrayed primer extension technology.
PMCID: PMC3796939  PMID: 23982839
ABCA4; next generation sequencing; Stargardt disease
16.  Abnormal retinal development associated with FRMD7 mutations 
Human Molecular Genetics  2014;23(15):4086-4093.
Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder, often associated with FRMD7 mutations. As the appearance of the retina is reported to be normal based on conventional fundus photography, IIN is postulated to arise from abnormal cortical development. To determine whether the afferent visual system is involved in FRMD7 mutations, we performed in situ hybridization studies in human embryonic and fetal stages (35 days post-ovulation to 9 weeks post-conception). We show a dynamic retinal expression pattern of FRMD7 during development. We observe expression within the outer neuroblastic layer, then in the inner neuroblastic layer and at 9 weeks post-conception a bilaminar expression pattern. Expression was also noted within the developing optic stalk and optic disk. We identified a large cohort of IIN patients (n = 100), and performed sequence analysis which revealed 45 patients with FRMD7 mutations. Patients with FRMD7 mutations underwent detailed retinal imaging studies using ultrahigh-resolution optical coherence tomography. The tomograms were compared with a control cohort (n = 60). The foveal pit was significantly shallower in FRMD7 patients (P < 0.0001). The optic nerve head morphology was abnormal with significantly decreased optic disk area, retinal nerve fiber layer thickness, cup area and cup depth in FRMD7 patients (P < 0.0001). This study shows for the first time that abnormal afferent system development is associated with FRMD7 mutations and could be an important etiological factor in the development of nystagmus.
PMCID: PMC4082370  PMID: 24688117
17.  X-linked cone dystrophy and colour vision deficiency arising from a missense mutation in a hybrid L/M cone opsin gene 
Vision research  2013;80:41-50.
In this report, we describe a male subject who presents with a complex phenotype of myopia associated with cone dysfunction and a protan vision deficiency. Retinal imaging demonstrates extensive cone disruption, including the presence of non-waveguiding cones, an overall thinning of the retina, and an irregular mottled appearance of the hyper reflective band associated with the inner segment ellipsoid portion of the photoreceptor. Mutation screening revealed a novel p.Glu41Lys missense mutation in a hybrid L/M opsin gene. Spectral analysis shows that the mutant opsin fails to form a pigment in vitro and fails to be trafficked to the cell membrane in transfected Neuro2a cells. Extensive sequence and quantitative PCR analysis identifies this mutant gene as the only gene present in the affected subject’s L/M opsin gene array, yet the presence of protanopia indicates that the mutant opsin must retain some activity in vivo. To account for this apparent contradiction, we propose that a limited amount of functional pigment is formed within the normal cellular environment of the intact photoreceptor, and that this requires the presence of chaperone proteins that promote stability and normal folding of the mutant protein.
PMCID: PMC3594517  PMID: 23337435
Colour vision; visual pigments; dichromacy; opsin mutation; cone dysfunction; retinal imaging
18.  Evaluation of the X-Linked High-Grade Myopia Locus (MYP1) with Cone Dysfunction and Color Vision Deficiencies 
X-linked high myopia with mild cone dysfunction and color vision defects has been mapped to chromosome Xq28 (MYP1 locus). CXorf2/TEX28 is a nested, intercalated gene within the red-green opsin cone pigment gene tandem array on Xq28. The authors investigated whether TEX28 gene alterations were associated with the Xq28-linked myopia phenotype. Genomic DNA from five pedigrees (with high myopia and either protanopia or deuteranopia) that mapped to Xq28 were screened for TEX28 copy number variations (CNVs) and sequence variants.
To examine for CNVs, ultra-high resolution array-comparative genomic hybridization (array-CGH) assays were performed comparing the subject genomic DNA with control samples (two pairs from two pedigrees). Opsin or TEX28 gene-targeted quantitative real-time gene expression assays (comparative CT method) were performed to validate the array-CGH findings. All exons of TEX28, including intron/exon boundaries, were amplified and sequenced using standard techniques.
Array-CGH findings revealed predicted duplications in affected patient samples. Although only three copies of TEX28 were previously reported within the opsin array, quantitative real-time analysis of the TEX28 targeted assay of affected male or carrier female individuals in these pedigrees revealed either fewer (one) or more (four or five) copies than did related and control unaffected individuals. Sequence analysis of TEX28 did not reveal any variants associated with the disease status.
CNVs have been proposed to play a role in disease inheritance and susceptibility as they affect gene dosage. TEX28 gene CNVs appear to be associated with the MYP1 X-linked myopia phenotypes.
PMCID: PMC3934550  PMID: 19098318
19.  Assessing Retinal Structure In Complete Congenital Stationary Night Blindness and Oguchi Disease 
American journal of ophthalmology  2012;154(6):987-1001.e1.
To examine retinal structure and changes in photoreceptor intensity post-dark adaptation in patients with complete congenital stationary night blindness and Oguchi disease.
Prospective observational case series.
We recruited three patients with complete congenital stationary night blindness caused by mutations in GRM6, two brothers with Oguchi disease caused by mutations in GRK1, and one normal control. Retinal thickness was measured from optical coherence tomography (OCT) images. Integrity of the rod and cone mosaic was assessed using adaptive optics scanning light ophthalmoscopy. We imaged five of the patients following a period of dark adaptation, and examined layer reflectivity on OCT in a patient with Oguchi disease under light- and dark-adapted conditions.
Retinal thickness was reduced in the parafoveal region in patients with GRM6 mutations, as a result of decreased thickness of the inner retinal layers. All patients had normal photoreceptor density at all locations analyzed. Upon removal from dark adaptation, the intensity of the rods (but not cones) in the patients with Oguchi disease gradually and significantly increased. In one Oguchi patient, the outer segment layer contrast on OCT was fourfold higher under dark-adapted versus light-adapted conditions.
The selective thinning of the inner retinal layers in patients with GRM6 mutations suggests either reduced bipolar/ganglion cell numbers or altered synaptic structure in the inner retina. Our finding that rods, but not cones, change intensity after dark adaptation suggests that fundus changes in Oguchi disease are due to changes within the rods as opposed to changes at a different retinal locus.
PMCID: PMC3498541  PMID: 22959359
20.  Genome-wide association study of age-related macular degeneration identifies associated variants in the TNXB–FKBPL–NOTCH4 region of chromosome 6p21.3 
Human Molecular Genetics  2012;21(18):4138-4150.
Age-related macular degeneration (AMD) is a leading cause of visual loss in Western populations. Susceptibility is influenced by age, environmental and genetic factors. Known genetic risk loci do not account for all the heritability. We therefore carried out a genome-wide association study of AMD in the UK population with 893 cases of advanced AMD and 2199 controls. This showed an association with the well-established AMD risk loci ARMS2 (age-related maculopathy susceptibility 2)–HTRA1 (HtrA serine peptidase 1) (P =2.7 × 10−72), CFH (complement factor H) (P =2.3 × 10−47), C2 (complement component 2)–CFB (complement factor B) (P =5.2 × 10−9), C3 (complement component 3) (P =2.2 × 10−3) and CFI (P =3.6 × 10−3) and with more recently reported risk loci at VEGFA (P =1.2 × 10−3) and LIPC (hepatic lipase) (P =0.04). Using a replication sample of 1411 advanced AMD cases and 1431 examined controls, we confirmed a novel association between AMD and single-nucleotide polymorphisms on chromosome 6p21.3 at TNXB (tenascin XB)–FKBPL (FK506 binding protein like) [rs12153855/rs9391734; discovery P =4.3 × 10−7, replication P =3.0 × 10−4, combined P =1.3 × 10−9, odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.3–1.6] and the neighbouring gene NOTCH4 (Notch 4) (rs2071277; discovery P =3.2 × 10−8, replication P =3.8 × 10−5, combined P =2.0 × 10−11, OR = 1.3, 95% CI = 1.2–1.4). These associations remained significant in conditional analyses which included the adjacent C2–CFB locus. TNXB, FKBPL and NOTCH4 are all plausible AMD susceptibility genes, but further research will be needed to identify the causal variants and determine whether any of these genes are involved in the pathogenesis of AMD.
PMCID: PMC3428154  PMID: 22694956
21.  Variations in Opsin Coding Sequences Cause X-Linked Cone Dysfunction Syndrome with Myopia and Dichromacy 
To determine the role of variant L opsin haplotypes in seven families with Bornholm Eye Disease (BED), a cone dysfunction syndrome with dichromacy and myopia.
Analysis of the opsin genes within the L/M opsin array at Xq28 included cloning and sequencing of an exon 3-5 gene fragment, long range PCR to establish gene order, and quantitative PCR to establish gene copy number. In vitro expression of normal and variant opsins was performed to examine cellular trafficking and spectral sensitivity of pigments.
All except one of the BED families possessed L opsin genes that contained a rare exon 3 haplotype. The exception was a family with the deleterious Cys203Arg substitution. Two rare exon 3 haplotypes were found and, where determined, these variant opsin genes were in the first position in the array. In vitro expression in transfected cultured neuronal cells showed that the variant opsins formed functional pigments, which trafficked to the cell membranes. The variant opsins were, however, less stable than wild type.
It is concluded that the variant L opsin haplotypes underlie BED. The reduction in the amount of variant opsin produced in vitro compared with wild type indicates a possible disease mechanism. Alternatively, the recently identified defective splicing of exon 3 of the variant opsin transcript may be involved. Both mechanisms explain the presence of dichromacy and cone dystrophy. Abnormal pigment may also underlie the myopia that is invariably present in BED subjects.
In this study, the genetic basis for X-linked cone dystrophy with dichromacy and myopia is identified as arising from sequence variants in exon 3 of the L opsin gene.
PMCID: PMC3597186  PMID: 23322568
22.  Genetic influences on plasma CFH and CFHR1 concentrations and their role in susceptibility to age-related macular degeneration 
Human Molecular Genetics  2013;22(23):4857-4869.
It is a longstanding puzzle why non-coding variants in the complement factor H (CFH) gene are more strongly associated with age-related macular degeneration (AMD) than functional coding variants that directly influence the alternative complement pathway. The situation is complicated by tight genetic associations across the region, including the adjacent CFH-related genes CFHR3 and CFHR1, which may themselves influence the alternative complement pathway and are contained within a common deletion (CNP147) which is associated with protection against AMD. It is unclear whether this association is mediated through a protective effect of low plasma CFHR1 concentrations, high plasma CFH or both. We examined the triangular relationships of CFH/CFHR3/CFHR1 genotype, plasma CFH or CFHR1 concentrations and AMD susceptibility in combined case–control (1256 cases, 1020 controls) and cross-sectional population (n = 1004) studies and carried out genome-wide association studies of plasma CFH and CFHR1 concentrations. A non-coding CFH SNP (rs6677604) and the CNP147 deletion were strongly correlated both with each other and with plasma CFH and CFHR1 concentrations. The plasma CFH-raising rs6677604 allele and raised plasma CFH concentration were each associated with AMD protection. In contrast, the protective association of the CNP147 deletion with AMD was not mediated by low plasma CFHR1, since AMD-free controls showed increased plasma CFHR1 compared with cases, but it may be mediated by the association of CNP147 with raised plasma CFH concentration. The results are most consistent with a regulatory locus within a 32 kb region of the CFH gene, with a major effect on plasma CFH concentration and AMD susceptibility.
PMCID: PMC3820139  PMID: 23873044
23.  The Effect of Cone Opsin Mutations on Retinal Structure and the Integrity of the Photoreceptor Mosaic 
To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations.
Eleven subjects were recruited, eight of whom have been previously described. Cone and rod density was measured using images of the photoreceptor mosaic obtained from an adaptive optics scanning light ophthalmoscope (AOSLO). Total retinal thickness, inner retinal thickness, and outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness were measured using cross-sectional spectral-domain optical coherence tomography (SD-OCT) images. Molecular genetic analyses were performed to characterize the OPN1LW/OPN1MW gene array.
While disruptions in retinal lamination and cone mosaic structure were observed in all subjects, genotype-specific differences were also observed. For example, subjects with “L/M interchange” mutations resulting from intermixing of ancestral OPN1LW and OPN1MW genes had significant residual cone structure in the parafovea (∼25% of normal), despite widespread retinal disruption that included a large foveal lesion and thinning of the parafoveal inner retina. These subjects also reported a later-onset, progressive loss of visual function. In contrast, subjects with the C203R missense mutation presented with congenital blue cone monochromacy, with retinal lamination defects being restricted to the ONL+HFL and the degree of residual cone structure (8% of normal) being consistent with that expected for the S-cone submosaic.
The photoreceptor phenotype associated with OPN1LW and OPN1MW mutations is highly variable. These findings have implications for the potential restoration of visual function in subjects with opsin mutations. Our study highlights the importance of high-resolution phenotyping to characterize cellular structure in inherited retinal disease; such information will be critical for selecting patients most likely to respond to therapeutic intervention and for establishing a baseline for evaluating treatment efficacy.
Subjects with OPN1LW and OPN1MW mutations showed a spectrum of retinal phenotypes with genotype-specific differences. This has implications for restoration of visual function in these subjects and highlights high-resolution retinal imaging as a complementary tool for emerging therapeutic efforts.
PMCID: PMC3816954  PMID: 23139274
25.  Caffeic Acid Phenethyl Ester Protects Blood–Brain Barrier Integrity and Reduces Contusion Volume in Rodent Models of Traumatic Brain Injury 
Journal of Neurotrauma  2012;29(6):1209-1218.
A number of studies have established a deleterious role for inflammatory molecules and reactive oxygen species (ROS) in the pathology of traumatic brain injury (TBI). Caffeic acid phenethyl ester (CAPE) has been shown to exert both antioxidant and anti-inflammatory effects. The primary objective of the present study was to examine if CAPE could be used to reduce some of the pathological consequences of TBI using rodent models. Male Sprague-Dawley rats and C57BL/6 mice were subjected to controlled cortical impact (CCI) injury. Blood–brain barrier (BBB) integrity was assessed by examining claudin-5 expression and the extravasation of Evans blue dye. The effect of post-injury CAPE administration on neurobehavioral function was assessed using vestibulomotor, motor, and two hippocampus-dependent learning and memory tasks. We report that post-TBI administration of CAPE reduces Evans blue extravasation both in rats and mice. This improvement was associated with preservation of the levels of the tight junction protein claudin-5. CAPE treatment did not improve performance in either vestibulomotor/motor function (tested using beam balance and foot-fault tests), or in learning and memory function (tested using the Morris water maze and associative fear memory tasks). However, animals treated with CAPE were found to have significantly less cortical tissue loss than vehicle-treated controls. These findings suggest that CAPE may provide benefit in the treatment of vascular compromise following central nervous system injury.
PMCID: PMC3325550  PMID: 22150135
claudin-5; neurovascular function; reactive oxygen species; tight junction; vascular permeability

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