Georgina Hall

A paradigm shift in the delivery of services for diagnosis of inherited retinal disease

Objectives Current technologies for delivering gene testing are labour-intensive and expensive. Over the last 3 years, new high-throughput DNA sequencing techniques (next generation sequencing; NGS), with the capability to analyse multiple genes or entire genomes, have been rapidly adopted into research. This study examines the possibility of incorporating NGS into a clinical UK service context. Methods The study applied NGS of 105 genes to 50 patients known to be affected by inherited forms of blindness in the setting of a UK National Health Service-accredited diagnostic molecular genetics laboratory. The study assessed the ability of an NGS protocol to identify likely disease-causing genetic variants when compared with current methodologies available through UK diagnostic laboratories. Results Conventional testing is only applicable to the minority of patients with inherited retinal disease and identifies mutations in fewer than one in four of those patients tested. By contrast, the NGS assay is directed at all patients with such disorders and identifies disease-causing mutations in 50–55%, which is a dramatic increase. This includes patients with apparently ‘sporadic’ disease, and those for whom clinical management and prognosis are altered as a consequence of defining their disease at a molecular level. Conclusions The new NGS approach delivers a step change in the diagnosis of inherited eye disease, provides precise diagnostic information and extends the possibility of targeted treatments including gene therapy. The approach represents an exemplar that illustrates the opportunity that NGS provides for broadening the availability of genetic testing. The technology will be applied to many conditions that are associated with high levels of genetic heterogeneity.

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.

Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease

Purpose To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Design Case series. Participants A total of 562 patients diagnosed with IRD. Methods We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Main Outcome Measures Diagnostic yield of genomic testing. Results Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15–45) uplift in diagnostic yield. Conclusions We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.

Early onset retinal dystrophy due to mutations in LRAT: Molecular analysis and detailed phenotypic study

PURPOSE To report novel variants and characterize the phenotype associated with the autosomal recessive retinal dystrophy caused by mutations in the lecithin retinol acyltransferase (LRAT) gene. METHODS A total of 149 patients with Lebers congenital amaurosis (LCA) or early onset retinal dystrophy were screened for mutations in LCA-associated genes using an arrayed-primer extension (APEX) genotyping microarray (Asper Ophthalmics). LRAT sequencing was subsequently performed in this 148-patient panel. Patients identified with mutations underwent further detailed phenotyping. RESULTS APEX analysis identified one patient with a previously reported homozygous LRAT mutation. Sequencing of the panel identified three additional patients with novel homozygous LRAT mutations in exon 2. All four patients had severe progressive nyctalopia, visual field constriction, and photophilia in childhood. Visual acuity ranged from 0.22 logMAR to hand motion. Funduscopy revealed severe retinal pigment epithelial atrophy and minimal retinal pigmentation. Asteroid hyalosis and macular epiretinal fibrosis were frequent. All demonstrated reduced fundus autofluorescence. Optical coherence tomography identified disrupted retinal lamination, outer-retinal debris, and an unidentifiable photoreceptor layer in two cases. Full-field electroretinograms were undetectable or showed severe rod-cone dysfunction. Photopic perimetry revealed severe visual field constriction. Dark-adapted perimetry demonstrated markedly reduced photoreceptor sensitivity. Dark-adapted spectral sensitivity measurements identified functioning rods in two of three patients. All three had severely reduced L- and M-cone sensitivity and poor color discrimination. CONCLUSIONS LRAT mutations cause a severe, early childhood onset, progressive retinal dystrophy. Phenotypic similarities to the retinal dysfunction associated with RPE-specific protein 65 kDa mutations, another visual cycle gene, suggest that LRAT deficiency may show a good response to novel therapies.

Understanding the impact of genetic testing for inherited retinal dystrophy.

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.

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.

Building the Genetic Counsellor Profession in the United Kingdom: Two Decades of Growth and Development

The United Kingdom (UK) is comprised of England, Scotland, Wales and Northern Ireland and the current population is approximately 62 million (Office for National Statistics 2012). Healthcare, including genetic services and appropriate testing, is provided to all citizens free at the point of service, via a National Health Service (NHS) funded through taxation (National Health Service Choices 2012). All UK citizens have a named general practitioner who is responsible for their overall healthcare. Individuals and families in every part of the UK have access to genetic services. These are staffed by multi-disciplinary teams comprising mainly medical geneticists and genetic counsellors; the regional NHS genetics laboratories are either integrated with or very closely aligned to the clinical centres. Within the regional genetics centres, a comprehensive service is provided that includes prenatal genetics, and general, metabolic and cancer genetics relating to both paediatric and adult care (Department of Health 2010). Specialist training exists for medical geneticists, genetic counsellors and laboratory geneticists. Many clients are referred to the genetic service by a primary care practitioner (doctor, nurse or midwife working in a primary care setting, such as a community clinic), although in some cases another specialist will refer. Medical genetics clinics were first established in 1946 (Harper et al. 2010), although there was huge expansion in the provision of genetic services during the 1980’s, when testing using recombinant DNA became feasible in the clinical setting. With this expansion, a number of specialist nurses were employed to support the work of medical geneticists, obtaining the necessary knowledge and skills informally through observing colleagues. At the end of the 1980’s, ten of these practitioners met at a scientific meeting and formed the Association of Genetic Nurses and Counsellors (initially called the Genetic Nurses and Social Workers Association). In 1992 an Education Working Group was formed by members of that association and two studies were undertaken to document the role and scope of practice of genetic nurses (Skirton et al. 1997). Building on that study, the first formal recommendations for the education required for genetic nurses or counsellors were made (Skirton et al. 1998).

Biallelic Mutations in the Autophagy Regulator DRAM2 Cause Retinal Dystrophy with Early Macular Involvement

Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs(∗)3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165(∗)] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function.

Understanding the expectations of patients with inherited retinal dystrophies

Background UK genetic ophthalmology services for patients with retinal dystrophy (RD) are variable. Little research exists to define service requirements, or expectations, of patients and their families. This study aimed to explore the views and perceived benefits of genetic ophthalmology services among members of families with RD. Methods Twenty participants with known RD mutations were recruited through UK genetic ophthalmic clinics. Semistructured qualitative interviews explored interviewees’ perceptions of the role of these services. Interviews were transcribed verbatim and analysed using inductive thematic analysis. Results Interviewees’ expectations and requirements of genetic ophthalmology services were wide-ranging and often perceived to be unmet. Participant expectations were classified in three groups: (1) Medical expectations included obtaining a diagnosis and information about disease/prognosis, genetic risks and research (2) Psychosocial expectations related to participants’ need for support in adjusting to RD (3) Practical expectations included the desire for information about welfare and support. Conclusions Expectations of RD families for clinical services are complex, encompassing a range of healthcare specialties. Services that align to these expectations will need to reach beyond the diagnostic arena and provide practical and psychosocial support. The identification of measurable outcomes will facilitate future development and evaluation of service delivery models. Many of the expectations identified here map to an existing, previously validated, outcomes framework for clinical genetic services. However, an additional outcome domain, labelled ‘Independence’ was also identified; this could either be specific to vision loss or relate generally to disability caused by genetic conditions.

Valuing the benefits of genetic testing for retinitis pigmentosa: a pilot application of the contingent valuation method

Background Technological advances present an opportunity for more people with, or at risk of, developing retinitis pigmentosa (RP) to be offered genetic testing. Valuation of these tests using current evaluative frameworks is problematic since benefits may be derived from diagnostic information rather than improvements in health. This pilot study aimed to explore if contingent valuation method (CVM) can be used to value the benefits of genetic testing for RP. Methods CVM was used to elicit willingness-to-pay (WTP) values for (1) genetic counselling and (2) genetic counselling with genetic testing. Telephone and face-to-face interviews with a purposive sample of individuals with (n=25), and without (n=27), prior experience of RP were used to explore the feasibility and validity of CVM in this context. Results Faced with a hypothetical scenario, the majority of participants stated that they would seek genetic counselling and testing in the context of RP. Between participant groups, respondents offered similar justifications for stated WTP values. Overall stated WTP was higher for genetic counselling plus testing (median=£524.00) compared with counselling alone (median=£224.50). Between-group differences in stated WTP were statistically significant; participants with prior knowledge of the condition were willing to pay more for genetic ophthalmology services. Conclusions Participants were able to attach a monetary value to the perceived potential benefit that genetic testing offered regardless of prior experience of the condition. This exploratory work represents an important step towards evaluating these services using formal cost–benefit analysis.