Advanced diagnostic genetic testing in inherited retinal disease: experience from a single tertiary referral centre in the UK National Health Service.

In 2013, as part of our genetic investigation of patients with inherited retinal disease, we utilized multigene panel testing of 105 genes known to cause retinal disease in our patient cohorts. This test was performed in a UK National Health Service (NHS) accredited laboratory. The results of all multigene panel tests requested between 1.4.13 and 31.8.14 were retrospectively reviewed. All patients had been previously seen at Moorfields Eye Hospital, London, UK and diagnosed with an inherited retinal dystrophy after clinical examination and detailed retinal imaging. The results were categorized into three groups: (i) Testing helped establish a certain molecular diagnosis in 45 out of 115 (39%). Variants in USH2A (n = 6) and RP1 (n = 4) were most common. (ii) Definitive conclusions could not be drawn from molecular testing alone in 13 out of 115 (11%) as either insufficient pathogenic variants were discovered or those identified were not consistent with the phenotype. (iii) Testing did not identify any pathogenic variants responsible for the phenotype in 57 out of 115 (50%). Multigene panel testing performed in an NHS setting has enabled a molecular diagnosis to be confidently made in 40% of cases. Novel variants accounted for 38% of all identified variants. Detailed retinal phenotyping helped the interpretation of specific variants. Additional care needs to be taken when assessing polymorphisms in genes that have been infrequently associated with disease, as historical techniques were not as rigorous as contemporary ones. Future iterations of sequencing are likely to offer higher sensitivity, testing a broader range of genes, more rapidly and at a reduced cost.

A new locus for autosomal dominant retinitis pigmentosa on chromosome 7p.

Autosomal dominant retinitis pigmentosa (adRP) is known to result from mutations in two different retinal genes--rhodopsin and peripherin--while a third locus has been implicated by linkage data. However, families have been reported in which all three known loci have been excluded. We report linkage of adRP in one such family to two microsatellite markers on chromosome 7p. D7S435 has previously been localized to 7p13-15.1; D7S460, previously only localized to chromosome 7, maps to within 2 cM of D7S435 with a lod score of 12.15. Two point linkage analysis between these markers and adRP gave lod scores of 5.65 (theta = 0) and 4.19 (theta = 0.046) for D7S460 and D7S435, respectively. Multipoint analysis gave a maximum lod score of 8.22. These data strongly suggest a new adRP locus on chromosome 7p.

PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans.

PAX6 is widely expressed in the central nervous system. Heterozygous PAX6 mutations in human aniridia cause defects that would seem to be confined to the eye. Magnetic resonance imaging (MRI) and smell testing reveal the absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of aniridia cases, which shows that PAX6 haploinsuffiency causes more widespread human neuro developmental anomalies.

ADVIRC is caused by distinct mutations in BEST1 that alter pre-mRNA splicing.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T-->C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G-->A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G-->C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.

A novel connexin50 mutation associated with congenital nuclear pulverulent cataracts.

PURPOSE: To screen for mutations of connexin50 (Cx50)/GJA8 in a panel of patients with inherited cataract and to determine the cellular and functional consequences of the identified mutation. METHODS: All patients in the study underwent a full clinical examination and leucocyte DNA was extracted from venous blood. The GJA8 gene was sequenced directly. Connexin function and cellular trafficking were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: Screening of the GJA8 gene identified a 139 G to A transition that resulted in the replacement of aspartic acid by asparagine (D47N) in the coding region of Cx50. This change co-segregated with cataract among affected members of a family with autosomal dominant nuclear pulverulent cataracts. While pairs of Xenopus oocytes injected with wild type Cx50 RNA formed functional gap junction channels, pairs of oocytes injected with Cx50D47N showed no detectable intercellular conductance. Co-expression of Cx50D47N did not inhibit gap junctional conductance of wild type Cx50. In transiently transfected HeLa cells, wild type Cx50 localised to appositional membranes and within the perinuclear region, but Cx50D47N showed no immunostaining at appositional membranes with immunoreactivity confined to the cytoplasm. Incubation of HeLa cells transfected with Cx50D47N at 27 degrees C resulted in formation of gap junctional plaques. CONCLUSIONS: The pulverulent cataracts present in members of this family are associated with a novel GJA8 mutation, Cx50D47N, that acts as a loss-of-function mutation. The consequent decrease in lens intercellular communication and changes associated with intracellular retention of the mutant connexin may contribute to cataract formation.

Lens biology: development and human cataractogenesis.

Cataract, or opacification of the lens of the eye, is the commonest cause of visual impairment world-wide. It is only treatable at present by surgical removal. Recent advances in our understanding of the genetics of human cataract, in particular the inherited congenital form, together with the development of an array of animal models have provided valuable new insights into normal vertebrate lens biology and the mechanisms that underlie cataract formation. In this article, we review the current state of research in these areas and discuss thinking regarding the relationship between the phenotypes observed and the underlying genotype in inherited cataract.

A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract.

PURPOSE: To identify the gene responsible for autosomal dominant lamellar pulverulent cataract in a four-generation British family and characterise the functional and cellular consequences of the mutation. METHODS: Linkage analysis was used to identify the disease locus. The GJA8 gene was sequenced directly. Functional behaviour and cellular trafficking of connexins were examined by expression in Xenopus oocytes and HeLa cells. RESULTS: A 262C>A transition that resulted in the replacement of proline by glutamine (P88Q) in the coding region of connexin50 (Cx50) was identified. hCx50P88Q did not induce intercellular conductance and significantly inhibited gap junctional activity of co-expressed wild type hCx50 RNA in paired Xenopus oocytes. In transfected cells, immunoreactive hCx50P88Q was confined to the cytoplasm but showed a temperature sensitive localisation at gap junctional plaques. CONCLUSIONS: The pulverulent cataract described in this family is associated with a novel GJA8 mutation and has a different clinical phenotype from previously described GJA8 mutants. The cataract likely results from lack of gap junction function. The lack of function was associated with improper targeting to the plasma membrane, most probably due to protein misfolding.

The pattern of retinal ganglion cell dysfunction in Leber hereditary optic neuropathy.

Leber inherited optic neuropathy (LHON) is characterized by subacute bilateral loss of central vision due to dysfunction and loss of retinal ganglion cells (RGCs). Comprehensive visual electrophysiological investigations (including pattern reversal visual evoked potentials, pattern electroretinography and the photopic negative response) performed on 13 patients with acute and chronic LHON indicate early impairment of RGC cell body function and severe axonal dysfunction. Temporal, spatial and chromatic psychophysical tests performed on 7 patients with acute LHON and 4 patients with chronic LHON suggest severe involvement or loss of the midget, parasol and bistratified RGCs associated with all three principal visual pathways.

Age related macular degeneration and sun exposure, iris colour, and skin sensitivity to sunlight.

BACKGROUND/AIM: It has been suggested that sun exposure may be a risk factor for age related macular degeneration (AMD) and that skin sensitivity to sunlight and iris colour could be confounding factors. The aim was to investigate this further in the white population. METHODS: 446 cases with end stage AMD were compared with 283 spouse controls. Data on sun exposure, places of residence, iris colour, subjective assessment of change in iris colour, hair colour at age 20, and skin sensitivity were obtained using a questionnaire. Iris colour was graded clinically by comparison with standard photographs. AMD was graded using stereoscopic colour fundus photographs as well as clinical examination and was defined as the presence of geographic atrophy or choroidal neovascularisation. All variables were included in a multiple logistic regression model including age, sex, and smoking. RESULTS: There was no association between AMD and sun exposure or related factors except for the suggestion of an association between sunburn prone skin type and geographic atrophy which reached borderline significance. CONCLUSIONS: No significant association between AMD and sun exposure, iris colour, change in iris colour, or hair colour was demonstrated.

Smoking and age related macular degeneration: the number of pack years of cigarette smoking is a major determinant of risk for both geographic atrophy and choroidal neovascularisation.

BACKGROUND/AIMS: There is evidence that smoking is a risk factor for age related macular degeneration (AMD). However, not all studies have demonstrated this association and several key questions about the role of smoking in AMD have still to be determined. The aim of this study was to further investigate this relation for both choroidal neovascularisation (CNV) and geographic atrophy (GA). METHODS: To investigate the relation between smoking and the risk of developing age related macular degeneration (AMD) in white people, 435 cases with end stage AMD were compared with 280 controls. All subjects had graded stereoscopic colour fundus photography and AMD was defined as the presence of GA or CNV. Smoking history was assessed using multiple parameters in a detailed questionnaire. RESULTS: Comparison of current and former smokers with non-smokers was consistent with smoking being a risk factor for AMD but did not reach statistical significance. There was a strong association between AMD and pack years of cigarette smoking (p = 0.002), the odds ratio increasing with the amount smoked; for subjects with more than 40 pack years of smoking the odds ratio was 2.75 (95% CI 1.22 to 6.20) compared with non-smokers. Both types of AMD showed a similar relation; smoking more than 40 pack years of cigarettes was associated with an odds ratio of 3.43 (95% CI 1.28 to 9.20) for GA and 2.49 (95% CI 1.06 to 5.82) for CNV. Stopping smoking was associated with reduced odds of AMD and the risk in those who had not smoked for over 20 years was comparable to non-smokers. The risk profile was similar for males and females. Passive smoking exposure was associated with an increased risk of AMD (OR 1.87; 95% CI 1.03 to 3.40) in non-smokers. CONCLUSIONS: The authors have demonstrated a strong association between the risk of both GA and CNV and pack years of cigarette smoking. This provides support for a causal relation between smoking and AMD. They also show an increased risk for AMD in non-smokers exposed to passive smoking. Stopping smoking appears to reduce the risk of developing AMD.

X-linked retinoschisis: clinical phenotype and RS1 genotype in 86 UK patients.

BACKGROUND: Inactivating mutations of the gene RS1 lead to X-linked retinoschisis, a progressive retinal dystrophy characterised by schisis within the inner layers of the neuroretina. The mutation spectrum is large and the phenotype variable. AIM: To determine whether there is a correlation between mutation type and disease severity. METHODS: We identified the causative mutation in 86 affected patients and examined each of these patients in detail. Different categories of mutation were compared for each phenotypic characteristic. RESULTS: We found a reduction in visual acuity with increasing age and worsening macular pathology in patients over 30 years old (p < or = 0.001), but there was no correlation between mutation type and severity of disease. Furthermore, we found a wide variation in phenotype even within families. CONCLUSIONS: Identifying the causative mutation in patients with X-linked retinoschisis is helpful in confirming diagnosis and in counselling of family members but cannot be used to predict prognosis for an individual patient.

A detailed study of the phenotype of an autosomal dominant cone-rod dystrophy (CORD7) associated with mutation in the gene for RIM1.

AIM: To characterise the phenotype of an autosomal dominant cone-rod dystrophy (CORD7) associated with the Arg844His mutation in RIM1. METHODS: Eight members of a four generation, non-consanguineous British family were examined clinically and underwent electrophysiological testing, automated dark adapted perimetry, dark adaptometry, colour vision assessment, colour fundus photography, fundus fluorescein angiography (FFA), and fundus autofluorescence (AF) imaging. RESULTS: The majority of affected individuals described a progressive deterioration of central vision, night vision, and peripheral visual field usually between the third and fourth decades. The visual acuity ranged from 6/6 to 3/60. Colour vision testing showed mild to moderate dyschromatopsia in the majority of individuals. Fundus changes comprised a range of macular appearances varying from mild retinal pigment epithelial (RPE) disturbance to extensive atrophy and pigmentation. In some individuals retinal vessels were attenuated and in two subjects peripheral areas of retinal atrophy were present. An absent or severely reduced PERG was detected in all subjects, indicative of marked macular dysfunction. Full field ERG showed abnormal rod and cone responses. AF imaging revealed decreased macular AF centrally surrounded by a ring of increased AF in the majority of individuals. "Bull's eye" lesions were present in two individuals, comprising of a ring of decreased perifoveal AF bordered peripherally and centrally by increased AF. Photopic sensitivity testing demonstrated elevated central visual field thresholds with additional superior greater than inferior peripheral field loss. There were rod and cone sensitivity reductions in the central and peripheral visual fields, with the inferior retina being more affected than the superior. CONCLUSIONS: The detailed phenotype is described of the autosomal dominant cone-rod dystrophy, CORD7, which is associated with a point mutation in RIM1, a gene encoding a photoreceptor synaptic protein. The pattern of disease progression and long term visual outcome facilitates improved genetic counselling and advice on prognosis. Such phenotypic data will be invaluable in the event of future therapy.

Posterior polar cataract is the predominant consequence of a recurrent mutation in the PITX3 gene.

BACKGROUND: The authors recently identified three large genetically unrelated families with an identical 17 base pair duplication mutation in exon 4 of the PITX3 gene. Here, they report the detailed clinical phenotype. METHODS: Affected and unaffected individuals in the three families with autosomal dominant posterior polar cataract underwent full clinical examination and donated blood samples for DNA extraction and molecular genetic studies. RESULTS: In all three families, an identical 17 base pair duplication mutation in PITX3 was identified which co-segregated with disease status in the family. All affected individuals had bilateral progressive posterior polar cataracts. In one family, posterior polar cataract was the only clinical abnormality but in the other two families, one of 10 affected individuals and four of 11 affected individuals also had anterior segment mesenchymal dysgenesis (ASMD). CONCLUSION: Mutations in the PITX3 gene in humans result in posterior polar cataract and variable ASMD. The gene encodes a transcription factor which has a key role in lens and anterior segment development. The mechanism by which the mutant protein gives rise to such a regional pattern of lens opacity remains to be elucidated.

A detailed phenotypic study of "cone dystrophy with supernormal rod ERG".

AIMS: To characterise the detailed phenotype of "cone dystrophy with supernormal rod ERG" in a case series of 10 patients. METHODS: 10 affected patients were examined clinically and underwent colour fundus photography, with nine undergoing detailed electrophysiological testing. Five patients were assessed further with fundus autofluorescence (AF) imaging, automated photopic and dark adapted perimetry, and dark adaptometry. Detailed colour vision assessment was performed in six subjects. Blood samples were taken from four patients for DNA extraction and mutation screening of NR2E3 was undertaken. RESULTS: The onset of symptoms was in the first and second decades of life. Subjects presented with reduced central vision and marked photophobia. All individuals were myopic and colour vision testing revealed severely reduced colour discrimination predominantly along the red-green axes; tritan colour vision was relatively well preserved. Nyctalopia is a later feature of the disorder. Funduscopy and AF imaging revealed a range of macular appearances. There was electrophysiological evidence of marked macular dysfunction, reduced and delayed cone responses, and supernormal and delayed rod responses. Photopic and dark adapted perimetry revealed central scotomata with widespread peripheral sensitivity loss. No disease causing sequence variants in NR2E3 were identified. CONCLUSIONS: The largest case series to date has been described of the clinical, psychophysical and electrophysiological characteristics of this unusual cone dystrophy with supernormal rod responses. Electrophysiological data were consistent with a post-phototransduction, but pre-inner nuclear layer, site of dysfunction. While the definitive diagnosis can only be made with electrophysiological testing, several characteristics that may increase suspicion of this diagnosis are presented.

The genetics of strabismus.

Strabismus (misalignment of the eyes; also known as "squint") comprises a common heterogeneous group of disorders characterised by a constant or intermittent ocular deviation often associated with amblyopia (uniocular failure of normal visual development) and reduced or absent binocular vision. The associated poor cosmetic appearance may also interfere with social and psychological development. Extensive twin and family studies suggest a significant genetic component to the aetiology of strabismus. The complexity of the molecular basis of strabismus is now beginning to be elucidated with the identification of genetic loci and disease causing genes. Currently greater insights have been gained into the incomitant subtype (differing magnitude of ocular misalignment according to direction of gaze), whereas less is known about the pathogenesis of the more common childhood concomitant strabismus. It is hoped that a greater understanding of the molecular genetics of these disorders will lead to improved knowledge of disease mechanisms and ultimately to more effective treatment. The aim of this paper is to review current knowledge of the molecular genetics of both incomitant and concomitant strabismus.

The genetics of inherited macular dystrophies.

The inherited macular dystrophies comprise a heterogeneous group of disorders characterised by central visual loss and atrophy of the macula and underlying retinal pigment epithelium (RPE). The different forms of macular degeneration encompass a wide range of clinical, psychophysical and histological findings. The complexity of the molecular basis of monogenic macular disease is now beginning to be elucidated with the identification of many of the disease-causing genes. Age related macular degeneration (ARMD), the leading cause of blind registration in the developed world, may also have a significant genetic component to its aetiology. Genes implicated in monogenic macular dystrophies are good candidate susceptibility genes for ARMD, although to date, with the possible exception of ABCA4, none of these genes have been shown to confer increased risk of ARMD. The aim of this paper is to review current knowledge relating to the monogenic macular dystrophies, with discussion of currently mapped genes, chromosomal loci and genotype-phenotype relationships. Inherited systemic disorders with a macular dystrophy component will not be discussed.

The genetics of childhood cataract.

Human congenital cataract has a diverse aetiology. In the proportion of cases where the cause is genetic, the disease shows wide phenotypic and genetic heterogeneity. Over the past few years, much research has been devoted to mapping the genes that underlie the disorder. This has been helped by the extensive array of naturally occurring and genetically engineered mouse cataract models and the abundance of human candidate genes. Most progress to date has been in the identification of genetic mutations causing autosomal dominant congenital cataract where eight genes have been implicated in cataractogenesis. Overall there is good correlation between the genetic mutations so far identified and the resulting lens phenotype but it is clear that mutations at more that one locus may give rise to similar forms of cataract. The identification of genes causing inherited forms of cataract will improve our understanding of the mechanisms underlying cataractogenesis in childhood and provide further insights into normal lens development and physiology. Perhaps more importantly, it is likely that some of the genes causing early onset cataract will be implicated in age related cataract which remains the commonest cause of blindness in the world.

Identification of the gene for Nance-Horan syndrome (NHS).

BACKGROUND: The disease intervals for Nance-Horan syndrome (NHS [MIM 302350]) and X linked congenital cataract (CXN) overlap on Xp22. OBJECTIVE: To identify the gene or genes responsible for these diseases. METHODS: Families with NHS were ascertained. The refined locus for CXN was used to focus the search for candidate genes, which were screened by polymerase chain reaction and direct sequencing of potential exons and intron-exon splice sites. Genomic structures and homologies were determined using bioinformatics. Expression studies were undertaken using specific exonic primers to amplify human fetal cDNA and mouse RNA. RESULTS: A novel gene NHS, with no known function, was identified as causative for NHS. Protein truncating mutations were detected in all three NHS pedigrees, but no mutation was identified in a CXN family, raising the possibility that NHS and CXN may not be allelic. The NHS gene forms a new gene family with a closely related novel gene NHS-Like1 (NHSL1). NHS and NHSL1 lie in paralogous duplicated chromosomal intervals on Xp22 and 6q24, and NHSL1 is more broadly expressed than NHS in human fetal tissues. CONCLUSIONS: This study reports the independent identification of the gene causative for Nance-Horan syndrome and extends the number of mutations identified.

A locus for isolated cataract on human Xp.

PURPOSE: To genetically map the gene causing isolated X linked cataract in a large European pedigree. METHODS: Using the patient registers at Birmingham Women's Hospital, UK, we identified and examined 23 members of a four generation family with nuclear cataract. Four of six affected males also had complex congenital heart disease. Pedigree data were collated and leucocyte DNA extracted from venous blood. Linkage analysis by PCR based microsatellite marker genotyping was used to identify the disease locus and mutations within candidate genes screened by direct sequencing. RESULTS: The disease locus was genetically refined to chromosome Xp22, within a 3 cM linkage interval flanked by markers DXS9902 and DXS999 (Zmax=3.64 at theta=0 for marker DXS8036). CONCLUSIONS: This is the first report of a locus for isolated inherited cataract on the X chromosome. The disease interval lies within the Nance-Horan locus suggesting allelic heterogeneity. The apparent association with congenital cardiac anomalies suggests a possible new oculocardiac syndrome.

Mapping of a novel locus for achromatopsia (ACHM4) to 1p and identification of a germline mutation in the alpha subunit of cone transducin (GNAT2).

OBJECTIVE: To determine the molecular basis for achromatopsia using autozygosity mapping and positional candidate gene analysis. DESIGN AND METHODS: A large consanguineous Pakistani family containing six subjects with autosomal recessive complete achromatopsia was ascertained. After excluding linkage to the two known achromatopsia genes (CNGA3 and CNGB3), a genome wide linkage screen was undertaken. RESULTS: Significant linkage was detected to a 12 cM autozygous segment between markers D1S485 and D1S2881 on chromosome 1p13. Direct sequence analysis of the candidate gene GNAT2 located within this interval identified a frameshift mutation in exon 7 (c842_843insTCAG; M280fsX291) that segregated with the disease. CONCLUSIONS: The GNAT2 gene codes for cone alpha-transducin, the G protein that couples the cone pigments to cGMP-phosphodiesterase in phototransduction. Although cone alpha-transducin has a fundamental role in cone phototransduction, mutations in GNAT2 have not been described previously. Since mutations in the CNGA3 gene may cause a variety of retinal dystrophies (complete and incomplete achromatopsia and progressive cone dystrophy), GNAT2 mutations may also prove to be implicated in other forms of retinal dystrophy with cone dysfunction.