Secondary (additional) findings from the 100,000 Genomes Project: Disease manifestation, health care outcomes, and costs of disclosure.

PURPOSE: The UK 100,000 Genomes Project offered participants screening for additional findings (AFs) in genes associated with familial hypercholesterolemia (FH) or hereditary cancer syndromes including breast/ovarian cancer (HBOC), Lynch, familial adenomatous polyposis, MYH-associated polyposis, multiple endocrine neoplasia (MEN), and von Hippel-Lindau. Here, we report disclosure processes, manifestation of AF-related disease, outcomes, and costs. METHODS: An observational study in an area representing one-fifth of England. RESULTS: Data were collected from 89 adult AF recipients. At disclosure, among 57 recipients of a cancer-predisposition-associated AF and 32 recipients of an FH-associated AF, 35% and 88%, respectively, had personal and/or family history evidence of AF-related disease. During post-disclosure investigations, 4 cancer-AF recipients had evidence of disease, including 1 medullary thyroid cancer. Six women with an HBOC AF, 3 women with a Lynch syndrome AF, and 2 individuals with a MEN AF elected for risk-reducing surgery. New hyperlipidemia diagnoses were made in 6 FH-AF recipients and treatment (re-)initiated for 7 with prior hyperlipidemia. Generating and disclosing AFs in this region cost £1.4m; £8680 per clinically significant AF. CONCLUSION: Generation and disclosure of AFs identifies individuals with and without personal or familial evidence of disease and prompts appropriate clinical interventions. Results can inform policy toward secondary findings.

Intraretinal Microvascular Abnormalities and Venous Beading Have Different Genetic Profiles in Caucasian Patients with Non-Proliferative Diabetic Retinopathy.

Diabetic Retinopathy (DR) is a leading cause of preventable visual impairment in the working age population. Despite the increasing prevalence of DR, there remain gaps in our understanding of its pathophysiology. This is a prospective case-control study comparing the genetic profiles of patients with no DR vs. non-proliferative DR (NPDR) focusing on intraretinal microvascular abnormalities (IRMA) and venous beading (VB) in Caucasians. A total of 596 participants were recruited to the study; 199 with moderate/severe NPDR and 397 with diabetes for at least 5 years without DR. Sixty-four patients were excluded due to technical issues. In total, 532 were analysed; 181 and 351 were in the NPDR group and no DR group, respectively. Those with severe IRMA and VB had distinctly different genetic profiles from each other and from the no DR group, which further supports the theory that these two features of DR might have different etiologies. This also suggests that IRMA and VB are independent risk factors for the development of PDR and may have different pathophysiologies. If these findings are confirmed in larger studies, this could pave the way for personalised treatment options for those more at risk of developing different features of NPDR.

Phenotypic and Genetic Characteristics in a Cohort of Patients with Usher Genes.

Background: This study aimed to compare phenotype−genotype correlation in patients with Usher syndrome (USH) to those with autosomal recessive retinitis pigmentosa (NS-ARRP) caused by genes associated with Usher syndrome. Methods: Case notes of patients with USH or NS-ARRP and a molecularly confirmed diagnosis in genes associated with Usher syndrome were reviewed. Phenotypic information, including the age of ocular symptoms, hearing impairment, visual acuity, Goldmann visual fields, fundus autofluorescence (FAF) imaging and spectral domain optical coherence tomography (OCT) imaging, was reviewed. The patients were divided into three genotype groups based on variant severity for genotype-phenotype correlations. Results: 39 patients with Usher syndrome and 33 patients with NS-ARRP and a molecular diagnosis in an Usher syndrome-related gene were identified. In the 39 patients diagnosed with Usher syndrome, a molecular diagnosis was confirmed as follows: USH2A (28), MYO7A (4), CDH23 (2), USH1C (2), GPR98/VLGR1 (2) and PCDH15 (1). All 33 patients with NS-ARRP had variants in USH2A. Further analysis was performed on the patients with USH2A variants. USH2A patients with syndromic features had an earlier mean age of symptom onset (17.9 vs. 31.7 years, p < 0.001), had more advanced changes on FAF imaging (p = 0.040) and were more likely to have cystoid macular oedema (p = 0.021) when compared to USH2A patients presenting with non-syndromic NS-ARRP. Self-reported late-onset hearing loss was identified in 33.3% of patients with NS-ARRP. Having a syndromic phenotype was associated with more severe USH2A variants (p < 0.001). Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort. Conclusions: Patients with Usher syndrome, whatever the associated gene in this cohort, tended to have an earlier onset of retinal disease (other than GPR98/VLGR1) when compared to patients presenting with NS-ARRP. Analysis of genetic variants in USH2A, the commonest gene in our cohort, showed that patients with a more severe genotype were more likely to be diagnosed with USH compared to NS-ARRP. USH2A patients with syndromic features have an earlier onset of symptoms and more severe features on FAF and OCT imaging. However, a third of patients diagnosed with NS-ARRP developed later onset hearing loss. Eighteen novel variants in genes associated with Usher syndrome were identified in this cohort, thus expanding the genetic spectrum of known pathogenic variants. An accurate molecular diagnosis is important for diagnosis and prognosis and has become particularly relevant with the advent of potential therapies for Usher-related gene

Whole genome sequencing in a Knobloch syndrome family confirms the molecular diagnosis.

BACKGROUND: To establish the molecular diagnosis in two brothers presenting with the ocular features of Knobloch Syndrome using whole genome sequencing (WGS). METHODS: Clinical examination and ophthalmological phenotyping were completed under general anaesthesia. DNA samples were tested on a targeted retinal dystrophy next-generation sequencing panel. Subsequently, WGS was performed to identify additional variants. RESULTS: Clinical examination confirmed the diagnosis of Knobloch Syndrome. Targeted sequencing identified a novel heterozygous frameshift pathogenic variant in COL18A1, c.2864dupC; p.(Gly956ArgfsX20), inherited from their mother. A second paternally inherited heterozygous missense variant was identified in both brothers, c.5014 G > A; p.(Asp1672Asn), which was initially considered to have too high frequency to be pathogenic (MAF 8.8%). This led to an in-depth analysis of the COL18A1 locus using WGS data, which confirmed that Asp1672Asn is a likely pathogenic hypomorphic allele. CONCLUSION: To date, all confirmed genetic diagnoses of Knobloch syndrome are attributable to variants in COL18A1. The family described here has a heterozygous novel loss of function variant. Detailed analysis of WGS data combined with family segregation studies concluded that although Asp1672Asn has a high population frequency, it is the most likely second pathogenic variant in our family. This supports the hypothesis that this is a hypomorphic allele, which, in combination with a loss of function pathogenic variant, leads to Knobloch syndrome.To our knowledge, this is the first time that WGS has been used to confirm a molecular diagnosis of Knobloch syndrome in this way and has provided further insight into the molecular mechanisms in this rare disorder.

An Overview of the Genetics of ABCA4 Retinopathies, an Evolving Story.

Stargardt disease (STGD1) and ABCA4 retinopathies (ABCA4R) are caused by pathogenic variants in the ABCA4 gene inherited in an autosomal recessive manner. The gene encodes an importer flippase protein that prevents the build-up of vitamin A derivatives that are toxic to the RPE. Diagnosing ABCA4R is complex due to its phenotypic variability and the presence of other inherited retinal dystrophy phenocopies. ABCA4 is a large gene, comprising 50 exons; to date > 2000 variants have been described. These include missense, nonsense, splicing, structural, and deep intronic variants. Missense variants account for the majority of variants in ABCA4. However, in a significant proportion of patients with an ABCA4R phenotype, a second variant in ABCA4 is not identified. This could be due to the presence of yet unknown variants, or hypomorphic alleles being incorrectly classified as benign, or the possibility that the disease is caused by a variant in another gene. This underlines the importance of accurate genetic testing. The pathogenicity of novel variants can be predicted using in silico programs, but these rely on databases that are not ethnically diverse, thus highlighting the need for studies in differing populations. Functional studies in vitro are useful towards assessing protein function but do not directly measure the flippase activity. Obtaining an accurate molecular diagnosis is becoming increasingly more important as targeted therapeutic options become available; these include pharmacological, gene-based, and cell replacement-based therapies. The aim of this review is to provide an update on the current status of genotyping in ABCA4 and the status of the therapeutic approaches being investigated.

Targeted next generation sequencing and family survey enable correct genetic diagnosis in CRX associated macular dystrophy - a case report.

BACKGROUND: We present 3 members of a family with macular dystrophy, originally diagnosed as Stargardt disease, with a significantly variable age at onset, caused by a heterozygous mutation in CRX. CASE PRESENTATION: A 43-year-old female with bull's eye maculopathy, whose sister was diagnosed with Stargardt disease previously at another centre, was found to have a single ABCA4 variant. Further examination of the family revealed that the asymptomatic father was also affected, indicating a dominant pattern of inheritance. In addition, the ABCA4 variant was not identified in the sister originally diagnosed with Stargardt disease. Next generation sequencing identified a heterozygous c.121C > T, p.R41W missense mutation in CRX in all 3 affected members. CONCLUSIONS: We describe a common phenotype, but with variable age at onset, with autosomal dominant inheritance and reduced penetrance in a family found to have a pathogenic sequence variant in CRX. This illustrates the importance of panel based molecular genetic testing accompanied by family studies to establish a definitive diagnosis.

"Genetic and clinical findings in an ethnically diverse retinitis pigmentosa cohort associated with pathogenic variants in EYS".

BACKGROUND AND OBJECTIVES: The EYS gene is an important cause of autosomal recessive retinitis pigmentosa (arRP). The objective of this study is to report on novel pathogenic variants in EYS and the range of associated phenotypes. SUBJECTS AND METHODS: This retrospective case series at a tertiary referral centre for inherited retinal diseases describes patients with an IRD and at least two variants in the EYS gene. Phenotyping included multimodal retinal imaging; genotyping molecular genetic analysis using targeted next generation sequencing. Sanger sequencing verification and analysis of novel variants using in silico approaches to determine their predicted pathogenicity. RESULTS: Eight male and four female patients were included. Age at onset ranged from 11 to 62 years with variable symptom presentation; ten patients showed classical features of retinitis pigmentosa, albeit with great variation in disease severity and extent. Two patients had atypical phenotypes: one with localised inferior sector pigmentation and a mild RP phenotype with changes predominantly at the posterior pole. Eighteen variants in EYS were identified, located across the gene: six were novel. Eight variants were missense, two altered splicing, one was a whole exon duplication and the remainder were predicted to result in premature truncation of the protein. CONCLUSION: The marked variability in severity and age of onset in most patients in this ethnically diverse cohort adds to growing evidence that that mild phenotypes are associated with EYS variants. Similarly, the two atypical cases add to the growing diversity of EYS disease as do the six novel pathogenic variants described.

The role of multimodal imaging and vision function testing in ABCA4-related retinopathies and their relevance to future therapeutic interventions.

The aim of this review article is to describe the specific features of Stargardt disease and ABCA4 retinopathies (ABCA4R) using multimodal imaging and functional testing and to highlight their relevance to potential therapeutic interventions. Standardised measures of tissue loss, tissue function and rate of change over time using formal structured deep phenotyping in Stargardt disease and ABCA4R are key in diagnosis, and prognosis as well as when selecting cohorts for therapeutic intervention. In addition, a meticulous documentation of natural history will be invaluable in the future to compare treated with untreated retinas. Despite the familiarity with the term Stargardt disease, this eponymous classification alone is unhelpful when evaluating ABCA4R, as the ABCA4 gene is associated with a number of phenotypes, and a range of severity. Multimodal imaging, psychophysical and electrophysiologic measurements are necessary in diagnosing and characterising these differing retinopathies. A wide range of retinal dystrophy phenotypes are seen in association with ABCA4 mutations. In this article, these will be referred to as ABCA4R. These different phenotypes and the existence of phenocopies present a significant challenge to the clinician. Careful phenotypic characterisation coupled with the genotype enables the clinician to provide an accurate diagnosis, associated inheritance pattern and information regarding prognosis and management. This is particularly relevant now for recruiting to therapeutic trials, and in the future when therapies become available. The importance of accurate genotype-phenotype correlation studies cannot be overemphasised. This approach together with segregation studies can be vital in the identification of causal mutations when variants in more than one gene are being considered as possible. In this article, we give an overview of the current imaging, psychophysical and electrophysiological investigations, as well as current therapeutic research trials for retinopathies associated with the ABCA4 gene.

Genetic and Clinical Findings in an Ethnically Diverse Cohort with Retinitis Pigmentosa Associated with Pathogenic Variants in CERKL.

Autosomal recessive retinitis pigmentosa is caused by mutations in over 40 genes, one of which is the ceramide kinase-like gene (CERKL). We present a case series of six patients from six unrelated families diagnosed with inherited retinal dystrophies (IRD) and with two variants in CERKL recruited from a multi-ethnic British population. A retrospective review of clinical data in these patients was performed and included colour fundus photography, fundus autofluorescence (AF) imaging, spectral domain-optical coherence tomography (SD-OCT), visual fields and electroretinogram (ERG) assessment where available. Three female and three male patients were included. Age at onset ranged from 7 years old to 45 years, with three presenting in their 20s and two presenting in their 40s. All but one had central visual loss as one of their main presenting symptoms. Four patients had features of retinitis pigmentosa with significant variation in severity and extent of disease, and two patients had no pigment deposition with only macular involvement clinically. Seven variants in CERKL were identified, of which three are novel. The inherited retinopathies associated with the CERKL gene vary in age at presentation and in degree of severity, but generally are characterised by a central visual impairment early on.

Novel Pathogenic Sequence Variants in NR2E3 and Clinical Findings in Three Patients.

A retrospective review of the clinical records of patients seen at the Oxford Eye Hospital identified as having NR2E3 mutations was performed. The data included symptoms, best-corrected visual acuity, multimodal retinal imaging, visual fields and electrophysiology testing. Three participants were identified with biallelic NR2E3 pathogenic sequence variants detected using a targeted NGS gene panel, two of which were novel. Participant I was a Nepalese male aged 68 years, and participants II and III were white Caucasian females aged 69 and 10 years old, respectively. All three had childhood onset nyctalopia, a progressive decrease in central vision, and visual field loss. Patients I and III had photopsia, patient II had photosensitivity and patient III also had photophobia. Visual acuities in patients I and II were preserved even into the seventh decade, with the worst visual acuity measured at 6/36. Visual field constriction was severe in participant I, less so in II, and fields were full to bright targets targets in participant III. Electrophysiology testing in all three demonstrated loss of rod function. The three patients share some of the typical distinctive features of NR2E3 retinopathies, as well as a novel clinical observation of foveal ellipsoid thickening.

Association of Clinical and Genetic Heterogeneity With BEST1 Sequence Variations.

Importance: Detailed phenotypic information on the spectrum of fundus abnormalities and clinical variability of all phenotypes associated with sequence variations in BEST1 is limited. Objective: To report a detailed phenotypic and genetic analysis of a patient cohort with sequence variations in BEST1. Design, Setting, and Participants: This retrospective case series took place at the Oxford Eye Hospital in Oxford, UK. Thirty-six patients from a single center with disease-causing sequence variations in BEST1 from 25 different families were analyzed. Data were collected from November 2017 to June 2018, and analysis began April 2018. Main Outcomes and Measures: Results of ocular phenotyping and genetic testing using targeted next-generation sequencing to identify BEST1 sequence variations. Results: Thirty-six patients from 25 families with disease-causing sequence variations in BEST1 were included. Of 36 patients, 20 (55.6%) were female. Three distinct clinical phenotypes were identified: autosomal recessive bestrophinopathy (ARB), best vitelliform macular dystrophy (BVMD), and adult-onset vitelliform macular dystrophy. The ARB phenotype group comprised 18 patients from 9 families with age in years at symptom onset ranging from less than 10 to 40s. All patients showed a common phenotype of fundus autofluorescence abnormalities, and spectral-domain optical coherence tomography features were similar in all patients with schitic and cystoid changes. A phenotype of a beaten metallic retinal appearance extending from the mid periphery to the far periphery was identified in 8 patients. Four patients from 1 family with ARB were previously reported to have autosomal recessive retinitis pigmentosa but were reclassified as having ARB as part of this study. The BVMD phenotype group comprised 16 patients from 14 families with age at symptom onset ranging from less than 10 to 70s. Fundus features were localized to the macula and consistent with the stage of BVMD. In the adult-onset vitelliform macular dystrophy phenotype group, the age in years at symptom onset varied from 50s to 70s in 2 patients from 2 families. Fundus features included small vitelliform lesions. Where available, electro-oculogram results demonstrated a reduced or absent light rise in all patients with ARB and BVMD. Genetic testing identified 22 variants in BEST1. Conclusions and Relevance: These findings support the notion that ARB, BVMD, and adult-onset vitelliform macular dystrophy are clinically distinct and recognizable phenotypes and suggest that the association of autosomal recessive retinitis pigmentosa with sequence variations in BEST1 should be rereviewed.

Novel homozygous splicing mutations in ARL2BP cause autosomal recessive retinitis pigmentosa.

Purpose: Mutations in ARL2BP, encoding ADP-ribosylation factor-like 2 binding protein, have recently been implicated as a cause of autosomal recessive retinitis pigmentosa (arRP), with three homozygous variants identified to date. In this study, we performed next-generation sequencing to reveal additional arRP cases associated with ARL2BP variants. Methods: Whole-genome sequencing (WGS) or whole-exome sequencing (WES) was performed in 1,051 unrelated individuals recruited for the UK Inherited Retinal Disease Consortium and NIHR-BioResource Rare Diseases research studies. Sanger sequencing was used to validate the next-generation sequencing data, and reverse transcriptase (RT)-PCR analysis was performed on RNA extracted from blood from affected individuals to test for altered splicing of ARL2BP. Detailed phenotyping was performed, including clinical evaluation, electroretinography, fundus photography, fundus autofluorescence imaging, and spectral-domain optical coherence tomography. Results: Homozygous variants in ARL2BP (NM_012106.3) were identified in two unrelated individuals with RP. The variants, c.207+1G>A and c.390+5G>A, at conserved splice donor sites for intron 3 and intron 5, respectively, were predicted to alter the pre-mRNA splicing of ARL2BP. RT-PCR spanning the affected introns revealed that both variants caused abnormal splicing of ARL2BP in samples from affected individuals. Conclusions: This study identified two homozygous variants in ARL2BP as a rare cause of arRP. Further studies are required to define the underlying disease mechanism causing retinal degeneration as a result of mutations in ARL2BP and any phenotype-genotype correlation associated with residual levels of the wild-type transcript.

Diabetic macular oedema: under-represented in the genetic analysis of diabetic retinopathy.

Diabetic retinopathy, a complication of both type 1 and type 2 diabetes, is a complex disease and is one of the leading causes of blindness in adults worldwide. It can be divided into distinct subclasses, one of which is diabetic macular oedema. Diabetic macular oedema can occur at any time in diabetic retinopathy and is the most common cause of vision loss in patients with type 2 diabetes. The purpose of this review is to summarize the large number of genetic association studies that have been performed in cohorts of patients with type 2 diabetes and published in English-language journals up to February 2017. Many of these studies have produced positive associations with gene polymorphisms and diabetic retinopathy. However, this review highlights that within this large body of work, studies specifically addressing a genetic association with diabetic macular oedema, although present, are vastly under-represented. We also highlight that many of the studies have small patient numbers and that meta-analyses often inappropriately combine patient data sets. We conclude that there will continue to be conflicting results and no meaningful findings will be achieved if the historical approach of combining all diabetic retinopathy disease states within patient cohorts continues in future studies. This review also identifies several genes that would be interesting to analyse in large, well-defined cohorts of patients with diabetic macular oedema in future candidate gene association studies.

Unravelling the genetics of inherited retinal dystrophies: Past, present and future.

The identification of the genes underlying monogenic diseases has been of interest to clinicians and scientists for many years. Using inherited retinal dystrophies as an example of monogenic disease we describe the history of molecular genetic techniques that have been pivotal in the discovery of disease causing genes. The methods that were developed in the 1970's and 80's are still in use today but have been refined and improved. These techniques enabled the concept of the Human Genome Project to be envisaged and ultimately realised. When the successful conclusion of the project was announced in 2003 many new tools and, as importantly, many collaborations had been developed that facilitated a rapid identification of disease genes. In the post-human genome project era advances in computing power and the clever use of the properties of DNA replication has allowed the development of next-generation sequencing technologies. These methods have revolutionised the identification of disease genes because for the first time there is no need to define the position of the gene in the genome. The use of next generation sequencing in a diagnostic setting has allowed many more patients with an inherited retinal dystrophy to obtain a molecular diagnosis for their disease. The identification of novel genes that have a role in the development or maintenance of retinal function is opening up avenues of research which will lead to the development of new pharmacological and gene therapy approaches. Neither of which can be used unless the defective gene and protein is known. The continued development of sequencing technologies also holds great promise for the advent of truly personalised medicine.

Characterization of CDH3-Related Congenital Hypotrichosis With Juvenile Macular Dystrophy.

IMPORTANCE: Congenital hypotrichosis with juvenile macular dystrophy (HJMD) is a rare disorder presenting in childhood and adolescence with central visual disturbance and sparse scalp hair. Reported retinal imaging is lacking, and whether the condition is progressive remains unclear. OBJECTIVE: To investigate a series of patients with HJMD due to biallelic mutations in CDH3 and thereby characterize the disorder. DESIGN, SETTING, AND PARTICIPANTS: Ten patients from 10 families underwent detailed clinical assessment, including serial retinal imaging and electrophysiologic evaluation, at Moorfields Eye Hospital, St James's University Hospital, and Calderdale Royal Infirmary. Patients ranged in age from 3 to 17 years at onset and 5 to 57 years at last assessment. The molecular genetic investigation included bidirectional Sanger sequencing of all exons and intron-exon boundaries of CDH3 and whole-exome sequencing in 2 patients. The study was conducted from June 5, 2013, to January 15, 2016, with final follow-up completed on December 15, 2015. MAIN OUTCOMES AND MEASURES: Results of clinical assessment and molecular genetic testing. RESULTS: All 10 patients (7 male and 3 female) presented with central visual disturbance in childhood and had lifelong sparse scalp hair with normal facial hair. Fundus examination revealed chorioretinal atrophy of the posterior pole contiguous with the disc in all but 1 patient that was associated with marked loss of autofluorescence on fundus autofluorescence imaging. Optical coherence tomography (OCT) demonstrated variable degrees of atrophy of the outer retina, retinal pigment epithelium, and choroid, with outer retinal tubulations frequently observed. One patient had mild disruption of the inner segment ellipsoid band on OCT and additional mild digit abnormalities. Electrophysiologic evaluation in 5 patients demonstrated macular dysfunction with additional mild, generalized retinal dysfunction in 2 patients. Eight patients had more than 1 evaluation; of these, 5 patients showed deterioration of visual acuity over time, 1 patient remained stable, and 2 patients had severe visual loss at presentation that precluded assessment of visual deterioration. The area of atrophy did not progress with time, but retinal thickness decreased on OCT. Electrophysiologic evaluation in 1 patient found deterioration of macular function after 13 years of follow-up, but the mild, generalized photoreceptor dysfunction remained stable. Biallelic mutations were identified in all patients, including 6 novel mutations. CONCLUSIONS AND RELEVANCE: These results suggest that CDH3-related disease is characterized by a childhood-onset, progressive chorioretinal atrophy confined to the posterior pole. The disease is readily distinguished from other juvenile macular dystrophies by the universally thin and sparse scalp hair. Patients may have additional limb abnormalities.

Hypotrichosis and juvenile macular dystrophy caused by CDH3 mutation: A candidate disease for retinal gene therapy.

Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder that causes childhood visual impairment. HJMD is caused by mutations in CDH3 which encodes cadherin-3, a protein expressed in retinal pigment epithelium (RPE) cells that may have a key role in intercellular adhesion. We present a case of HJMD and analyse its phenotypic and molecular characteristics to assess the potential for retinal gene therapy as a means of preventing severe visual loss in this condition. Longitudinal in vivo imaging of the retina showed the relative anatomical preservation of the macula, which suggested the presence of a therapeutic window for gene augmentation therapy to preserve visual acuity. The coding sequence of CDH3 fits within the packaging limit of recombinant adeno-associated virus vectors that have been shown to be safe in clinical trials and can efficiently target RPE cells. This report expands the number of reported cases of HJMD and highlights the phenotypic characteristics to consider when selecting candidates for retinal gene therapy.

Identification of rod- and cone-specific expression signatures to identify candidate genes for retinal disease.

Recent advances in technology have greatly increased our ability to identify genetic variants in individuals with retinal disease. However, determining which are likely to be pathogenic remains a challenging task. Using a transgenic coneless (cl) mouse model, together with rodless (rd/rd) and rodless/coneless (rd/rd cl) mice, we have characterised patterns of gene expression in the rod and cone photoreceptors at a genome-wide level. We examined the expression of >27,000 genes in the mice lacking rods, cones or both and compared them with wild type animals. We identified a list of 418 genes with highly significant changes in expression in one or more of the transgenic strains. Pathway analysis confirmed that expected Gene Ontology terms such as phototransduction were over-represented amongst these genes. However, many of these genes have no previously known function in the retina. Gene set enrichment analysis further demonstrated that the mouse orthologues of known human retinal disease genes were significantly enriched amongst those genes with decreased expression. Comparison of our data to human disease loci with no known causal genetic changes has highlighted genes with significant changes in expression making these strong candidates for further screening. These data add to the current literature through the utilisation of the specific cl and rd/rd cl models. Moreover, this study identifies genes that appear to be implicated in photoreceptor function thereby providing a valuable filter for variants identified by high-throughput sequencing in individuals with retinal disease.

Detailed phenotypic and genotypic characterization of bietti crystalline dystrophy.

OBJECTIVE: To provide a detailed phenotype/genotype characterization of Bietti crystalline dystrophy (BCD). DESIGN: Observational case series. PARTICIPANTS: Twenty patients from 17 families recruited from a multiethnic British population. METHODS: Patients underwent color fundus photography, near-infrared (NIR) imaging, fundus autofluorescence (FAF) imaging, spectral domain optical coherence tomography (SD-OCT), and electroretinogram (ERG) assessment. The gene CYP4V2 was sequenced. MAIN OUTCOME MEASURES: Clinical, imaging, electrophysiologic, and molecular genetics findings. RESULTS: Patients ranged in age from 19 to 72 years (median, 40 years), with a visual acuity of 6/5 to perception of light (median, 6/12). There was wide intrafamilial and interfamilial variability in clinical severity. The FAF imaging showed well-defined areas of retinal pigment epithelium (RPE) loss that corresponded on SD-OCT to well-demarcated areas of outer retinal atrophy. Retinal crystals were not evident on FAF imaging and were best visualized with NIR imaging. Spectral domain OCT showed them to be principally located on or in the RPE/Bruch's membrane complex. Disappearance of the crystals, revealed by serial recording, was associated with severe disruption and thinning of the RPE/Bruch's membrane complex. Cases with extensive RPE degeneration (N = 5) had ERGs consistent with generalized rod and cone dysfunction, but those with more focal RPE atrophy showed amplitude reduction without delay (N = 3), consistent with restricted loss of function, or that was normal (N = 2). Likely disease-causing variants were identified in 34 chromosomes from 17 families. Seven were novel, including p.Met66Arg, found in all 11 patients from 8 families of South Asian descent. This mutation appears to be associated with earlier onset (median age, 30 years) compared with other substitutions (median age, 41 years). Deletions of exon 7 were associated with more severe disease. CONCLUSIONS: The phenotype is highly variable. Several novel variants are reported, including a highly prevalent substitution in patients of South Asian descent that is associated with earlier-onset disease. Autofluorescence showed sharply demarcated areas of RPE loss that coincided with abrupt edges of outer retinal atrophy on SD-OCT; crystals were generally situated on or in the RPE/Bruch's complex but could disappear over time with associated RPE disruption. These results support a role for the RPE in disease pathogenesis.

Expression of coiled-coil protein 1, a novel gene downstream of FGF2, in the developing brain.

Fibroblast growth factor 2 (FGF2) plays an important role in cortical development. However, the genes downstream of FGF2 that mediate its effect are largely unknown. We have performed a microarray screening of genes regulated by FGF2 using primary cortical neuron culture derived from embryonic day 14.5 (E14.5) mouse forebrains. In this study, we have analysed a previously uncharacterised gene encoding a 180-amino acid protein, hereby named 'coiled-coil protein 1 (ccp1)', that showed a modest up-regulation upon FGF2 stimulation. Northern blots and RT-PCR showed specific expression of ccp1 in multiple tissues including adult and embryonic brains. In situ hybridizations revealed that ccp1 was expressed in the cortical plate between Reelin and Tbr1-positive layers in the dorsal cortex at E15.5. Furthermore, the expression pattern of ccp1 at E13.5-E14.5 reflected some of the aspects of tangential migration of cortical progenitors during the early phase. We observed that the expressed ccp1 protein was localised to endo/lysosomal compartment in the cell body as well as to vesicles present in the processes of primary cortical neurons and oligodendrocyte cell line.