I. Christopher Lloyd

Personalized diagnosis and management of congenital cataract by next-generation sequencing.

PURPOSE To assess the utility of integrating genomic data from next-generation sequencing and phenotypic data to enhance the diagnosis of bilateral congenital cataract (CC). DESIGN Evaluation of diagnostic technology. PARTICIPANTS Thirty-six individuals diagnosed with nonsyndromic or syndromic bilateral congenital cataract were selected for investigation through a single ophthalmic genetics clinic. METHODS Participants underwent a detailed ophthalmic examination, accompanied by dysmorphology assessment where appropriate. Lenticular, ocular, and systemic phenotypes were recorded. Mutations were detected using a custom-designed target enrichment that permitted parallel analysis of 115 genes associated with CC by high-throughput, next-generation DNA sequencing (NGS). Thirty-six patients and a known positive control were tested. Suspected pathogenic variants were confirmed by bidirectional Sanger sequencing in relevant probands and other affected family members. MAIN OUTCOME MEASURES Molecular genetic results and details of clinical phenotypes were identified. RESULTS Next-generation DNA sequencing technologies are able to determine the precise genetic cause of CC in 75% of individuals, and 85% patients with nonsyndromic CC were found to have likely pathogenic mutations, all of which occurred in highly conserved domains known to be vital for normal protein function. The pick-up rate in patients with syndromic CC also was high, with 63% having potential disease-causing mutations. CONCLUSIONS This analysis demonstrates the clinical utility of this test, providing examples where it altered clinical management, directed care pathways, and enabled more accurate genetic counseling. This comprehensive screen will extend access to genetic testing and lead to improved diagnostic and management outcomes through a stratified medicine approach. Establishing more robust genotype-phenotype correlations will advance knowledge of cataract-forming mechanisms.

Molecular findings from 537 individuals with inherited retinal disease

Background Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. Methods We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). Results Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. Conclusions Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.

Ocular motor outcomes after bilateral and unilateral infantile cataracts

We wished to study how the severity and duration of early onset visual deprivation affects eye alignment and ocular stability. Thirty-three patients (aged 1 week to 12.8 years) with infantile cataracts (16 bilateral, 17 unilateral) were examined for periods up to 61 months. Twenty-three patients were considered to have cataracts, which were a major obstacle to vision (major form deprivation), 9 of whom underwent surgery within 8 weeks of birth (mean and SD=5.2+/-2.3 weeks) and 10 after 8 weeks (mean and SD=33.9+/-29.7 months). Eye alignment and fixation stability was measured using infrared recording systems and video. Visual acuity was assessed using forced-choice preferential looking techniques in the neonates and infants and with optotypes in the children. Fifteen of the 23 (65%) patients who experienced major form deprivation exhibited a nystagmus, of which 11 (73%) were manifest latent nystagmus (MLN). Nineteen of the 23 (85%) had strabismus. Of the nine patients who underwent early surgery (< or =8 weeks), two displayed a preoperative nystagmus whilst between 10 and 39 months post-operatively 8 (89%) exhibited a nystagmus. Of the group of 10 patients with minor cataracts only 2 (1 late surgery, 1 no surgery) had nystagmus and 2 strabismus. We conclude that following optimal post-operative management of infantile cataracts a sustained nystagmus--typically an MLN--is the most likely ocular motor outcome, even when the period of deprivation is as short as 3 weeks.

Combined trabeculotomy–trabeculectomy augmented with 5‐fluorouracil in paediatric glaucoma

Background:  To describe our experience of combined trabeculotomy–trabeculectomy in paediatric glaucomas with a special emphasis on the use of 5‐fluorouracil and releasable sutures.

Pinpointing clinical diagnosis through whole exome sequencing to direct patient care: a case of Senior-Loken syndrome.

In 2002, a 2-month-old male infant was a ssessed by the general paediatric and paediatric ophthalmic services for roving eye movements and abnormal responses to visual cues. No concerns were raised about the child’s general health, but visual electrophysiology showed widespread photoreceptor cell dysfunction and retinal examination showed midperipheral fi ne pigment mottling and attenuation of retinal blood vessels (appendix). The child was diagnosed with non-syndromic infantile-onset retinal dystrophy, a common cause of visual impairment that is progressive and currently untreatable. The patient and his family have had regular follow-up and educational support and the family was referred for genetic counselling. In 2006, the proband’s younger sister presented with similar symptoms shortly after birth and we diagnosed the same condition (appendix).Genetic testing in retinal dystrophies has always been challenging because of the great genetic heterogeneity associated with these conditions. More than 20 genes have been linked with infantile-onset retinal dystrophy.

Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

Abrogation of HMX1 function causes rare oculoauricular syndrome associated with congenital cataract, anterior segment dysgenesis, and retinal dystrophy.

PURPOSE To define the phenotypic manifestation, confirm the genetic basis, and delineate the pathogenic mechanisms underlying an oculoauricular syndrome (OAS). METHODS Two individuals from a consanguineous family underwent comprehensive clinical phenotyping and electrodiagnostic testing (EDT). Genome-wide microarray analysis and Sanger sequencing of the candidate gene were used to identify the likely causal variant. Protein modelling, Western blotting, and dual luciferase assays were used to assess the pathogenic effect of the variant in vitro. RESULTS Complex developmental ocular abnormalities of congenital cataract, anterior segment dysgenesis, iris coloboma, early-onset retinal dystrophy, and abnormal external ear cartilage presented in the affected family members. Genetic analyses identified a homozygous c.650A>C; p.(Gln217Pro) missense mutation within the highly conserved homeodomain of the H6 family homeobox 1 (HMX1) gene. Protein modelling predicts that the variant may have a detrimental effect on protein folding and/or stability. In vitro analyses were able to demonstrate that the mutation has no effect on protein expression but adversely alters function. CONCLUSIONS Oculoauricular syndrome is an autosomal recessive condition that has a profound effect on the development of the external ear, anterior segment, and retina, leading to significant visual loss at an early age. This study has delineated the phenotype and confirmed HMX1 as the gene causative of OAS, enabling the description of only the second family with the condition. HMX1 is a key player in ocular development, possibly in both the pathway responsible for lens and retina development, and via the gene network integral to optic fissure closure.

Bardet-Biedl Syndrome: An Atypical Phenotype in Brothers with a Proven BBS1 Mutation

Background: To report the clinical findings in two brothers presenting with a pigmentary retinopathy and post-axial polydactyly, who were found to have a mutation in the BBS1 gene, confirming a diagnosis of Bardet-Biedl syndrome (BBS). Materials and Methods: Documentation of the clinical history, electrophysiological investigations, clinical examination and ocular findings of two brothers born to non-consanguineous white parents, with careful delineation of their clinical phenotypes. Screening of the BBS 1 gene on chromosome 11q13 by PCR-amplified exon alterations followed by direct sequencing was carried out to identify pathogenic mutations. Results: Although both probands had polydactyly and the characteristic ocular signs of BBS on both ophthalmological examination and electro-retinography, neither of them had dysmorphic facial features, obesity, hypogonadism, cognitive impairment, or renal anomalies. The first proband did have mild learning difficulties, although this did not restrict him in activities of daily living. Both probands were homozygous positive for the presence of a c.1169T > G (p.Met390Arg) mutation in BBS1. Conclusion: Although neither proband fulfilled the typical criteria for BBS, this diagnosis was confirmed on mutation analysis. These cases serve to highlight the degree of clinical variability observed in BBS which may be under-diagnosed in patients with milder phenotypes.

The Use of Autozygosity Mapping and Next-Generation Sequencing in Understanding Anterior Segment Defects Caused by an Abnormal Development of the Lens

The formation of the anterior segment of the eye is an intricate process that is dependent to a large degree on the normal development of the lens. Despite intensive study of the role of well-described eye genes, many causes of lenticular and anterior segment anomalies remain elusive. The majority of genes implicated thus far act in an autosomal dominant manner. Autosomal recessive causes are less well described; their diagnosis has been hindered by technological limitations, extreme genetic heterogeneity, a lack of understanding of eye biology and the role of many genes within the genome. The opportunity for the discovery of extremely rare autosomal recessive causes of ocular abnormalities from the study of consanguineous families is large, particularly through the powerful combination of next-generation sequencing with autozygosity mapping. Having begun to overcome the genetic heterogeneity bottleneck, it is increasingly recognised that the interpretation of genetic variants and the association of novel genes with a particular phenotype remain challenging. Nonetheless, increasing understanding of the genetic and mutational basis of lens and anterior segment abnormalities will be of enormous value to our comprehension of eye disease(s). Further, it will improve our ability to accurately interpret putative disease-causing variants with the aim of providing more personalised patient care and avoiding lifelong visual loss in children.

Clinical and molecular genetic findings in autosomal dominant OPA3-related optic neuropathy

Leber hereditary optic neuropathy and autosomal dominant optic atrophy are the two most common inherited optic neuropathies. The latter has been associated with mutations in the OPA1 and OPA3 genes. To date, only six families with OPA3-associated dominant optic atrophy have been reported. In order to identify additional families, we performed Sanger sequencing of the OPA3 gene in 75 unrelated optic neuropathy patients. Affected individuals from two families were found to harbour the c.313C > G, p.(Gln105Glu) change in heterozygous state; this genetic defect has been previously reported in four dominant optic atrophy families. Intra- and interfamilial variability in age of onset and presenting symptoms was observed. Although dominant OPA3 mutations are typically associated with optic atrophy and cataracts, the former can be observed in isolation; we report a case with no lens opacities at age 38. Conversely, it is important to consider OPA3-related disease in individuals with bilateral infantile-onset cataracts and to assess optic nerve health in those whose vision fail to improve following lens surgery. The papillomacular bundle is primarily affected and vision is typically worse than 20/40. Notably, we describe one subject who retained normal acuities into the fifth decade of life. The condition can be associated with extraocular clinical features: two affected individuals in the present study had sensorineural hearing loss. The clinical heterogeneity observed in the individuals reported here (all having the same genetic defect in OPA3) suggests that the molecular pathology of the disorder is likely to be complex.