Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier/Additional Provider

Transcription

1 Proposal form for the evaluation of a genetic test for NHS Service Gene Dossier/Additional Provider TEST DISEASE/CONDITION POPULATION TRIAD Submitting laboratory: 1. Disease/condition approved name and symbol as published on the OMIM database (alternative names will be listed on the UKGTN website) 2. OMIM number for disease/condition 3. Disease/condition please provide a brief description of the characteristics of the disease/condition and prognosis for affected individuals. Please provide this information in laymen s terms. 4. Disease/condition mode of inheritance 5. Gene approved name(s) and symbol as published on HUGO database (alternative names will be listed on the UKGTN website) Manchester RGC Approved: September 2012 Retinal Degeneration includes the following conditions as follows, the full list is available in appendix 1: Retinitis pigmentosa; RP Best vitelliform macular dystrophy; VMD Bestrophin; BEST1 Cone-rod dystrophy; CORD Cone-dystrophy; COD Achromatopsia; ACHM Bardet-Biedl syndrome; BBS Usher syndrome; USH Leber congenital amaurosis; LCA Stargardt disease; STGD Retinal Degeneration conditions see appendix 1. RD describes a group of clinically overlapping retinal disorders causing visual impairment and blindness. RD affects between1: 3,500-4,000 people in Europe. [1] The disease is highly heterogeneous: Autosomal dominant (20-30%), recessive (5-20%), X-linked (5-15%). Digenic and simplex cases are described. [2] Between 40-50% of RD remains unexplained. [3] Forms of inherited retinal disease overlapping phenotypicaly include Leber Congenital Amaurosis, Macular Dystrophy, Choroideremia and Achromatopsia. Other syndromes showing a degenerative retinal phenotype included in the panel test are Usher and Bardet-Biedl. X-linked recessive, autosomal dominant and recessive forms of RD and related disorders are within the scope of the molecular screen. The screen includes 105 genes (see appendix 2). This is 105 distinct genes; 118 transcripts. 6. OMIM number for gene(s) , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

2 609502, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,611810, , , , , , , , , , , , , , , , , , , , , Gene description(s) The target enrichment consists of 105 genes,1351 exons plus intron/exon boundaries (800kb) including the Lebers Congenital Amaurosis, Macular Dystrophy, Achromatopsia genes known at the time of test design plus the genes associated with the most common forms of syndromic blindness Usher and Bardet-Biedl syndromes. 7b. Number of amplicons to provide this test 7c. MolU/Cyto band that this test is assigned to 8. Mutational spectrum for which you test including details of known common mutations Not applicable in this methodology MolU band G 2012/13 GenU band H 2013/14 Point mutations and small insertions/deletions are within the scope of the screen. This assay has not been validated for the detection of exonic deletions and insertions. 9. Technical method(s) Enrichment; a custom-design Sure Select target kit (Agilent) for the ABI 5500 system, Emulsion PCR conducted on the pull down libraries and samples run in indexed batches of 10 on the sequencer. Sequence data mapped with ABI Bioscope software using hg19 human genome as a reference. Enrichment; a custom-design Sure Select target kit (Agilent) for the ABI 5500 system, Emulsion PCR conducted on the pull down libraries and samples run in indexed batches of 10 on the sequencer. Sequence data mapped with ABI Lifescope software using hg19 human genome as a reference. Variations from reference sequences called using standard practices (Lifescope software) and only those seen more than 18x considered for further analysis. All variants called at over 18x undergo a bioinformatic analysis (standard practice for all variants identified in the laboratory) by a state registered clinical scientist. 10. Validation process Please explain how this test has been validated for use in your laboratory Validation series reported in: O'Sullivan J, et al. (2012) J Med Genet. 49:322-6 Using a test cohort of 50 patients we have: 1) Assigned criteria for transcript choice. 2) Assessed minimum coverage required in order to identify all known variants. 3) Assigned quality and coverage thresholds for SNP and indel calling. 4) Assigned criteria for filtering of benign polymorphisms

3 11a. Are you providing this test already? 11b. If yes, how many reports have you produced? 5) Assessed SNP concordance between next generation sequencing and Sanger sequencing (by comparing results from the NGS assay against previous Sanger results for a limited number of genes). Coverage of the target enrichment region: an average of 1.8 GB of sequence mapped uniquely to the genome reference hg19, with 95.5% of the target covered at 30-fold or higher. Of the 1,351 exons targeted 60 only are not adequately covered by the current assay either by virtue of being the first exon of the gene (38%) or having GC content above 65% (78%). The cost of Sanger sequencing these 60 exons would be prohibitive. When no mutations are identified in an individual we do lower the stringency of interrogation down to 14x however there will always be exons with poor coverage. We clearly state the limitation of the test by quoting the coverage eg 95.1 % of the target coding region of the transcripts, as listed below, is covered to a minimum depth of 18X. For full details please contact the laboratory. Yes As of 30-May-2012 we have 95 referrals and have reported 20 cases. 11c. Number of reports mutation 8 positive 11d. Number of reports mutation 12 negative 12. For how long have you been from February.2012 providing this service? 13a. Is there specialised local Yes clinical/research expertise for this disease? 13b. If yes, please provide details Ophthalmic Genetic expertise is available through collaboration between the Regional Clinical Genetics Service, Manchester Royal and Moorfield s Eye Hospitals. 14. Are you testing for other genes/diseases/conditions closely allied to this one? Please give details Your current activity If applicable - How many tests do you currently provide annually in your laboratory? 15a. Index cases b. Family members where mutation is known Your capacity if Gene Dossier approved How many tests will you be able to provide annually in your laboratory if this gene dossier is approved and recommended for NHS funding? All genes tested for as part of our current eye genetics service are within the expanded screening panel except RPGR exon ORF15. Current Sanger services relating to eye disorders remain unchanged and are still available. In we provided 698 clinical reports for RD following analysis using conventional technologies. In we provided 138 clinical reports for family members a. Index cases 350

4 16b. Family members where 150 mutation is known Based on experience how many 600 tests will be required nationally (UK wide) per annum? Please identify the information on which this is based 17a. Index cases b. Family members where 200 mutation is known 18. National activity (England, Scotland, Wales & Northern Ireland) If your laboratory is unable to provide the full national need please could you provide information on how the national requirement may be met. For example, are you aware of any other labs (UKGTN members or otherwise) offering this test to NHS patients on a local area basis only? This question has been included in order to gauge if there could be any issues in equity of access for NHS patients. It is appreciated that some laboratories may not be able to answer this question. If this is the case please write unknown. 600 We are aware that Oxford RGS have developed a more limited gene scan.

5 EPIDEMIOLOGY 19. Estimated prevalence of condition in the general UK population RD affects between 1: 3,500-4,000 people in Europe. [1] Please identify the information on which this is based 20. Estimated gene frequency (Carrier frequency or allele frequency) It is not possible to calculate gene frequencies in this highly heterogeneous condition. Please identify the information on which this is based 21. Estimated penetrance Please identify the information on which this is based 22. Estimated prevalence of condition in the target population. The target population is the group of people that meet the minimum criteria as listed in the Testing Criteria. Penetrance is variable between X-linked and autosomal dominant forms as well as between genes. In X-linked RD the penetrance is generally high but can vary in females due to X- inactivation. In autosomal dominant RD the penetrance is high except for those mutations found in PRPF31, PAP1 and PRPF8 which are well documented in the literature to display variable penetrance. The target population is defined by the RD phenotype, e.g. only cases with a high index of suspicion (ascertained by clinical geneticists or collaborating ophthalmologists) and a high probability of carrying an RD mutation within the screening cascade are offered mutation scanning. INTENDED USE (Please use the questions in Annex A to inform your answers) 23. Please tick the relevant clinical purpose of testing Diagnosis X Yes No Treatment X Yes No Prognosis & management X Yes No Presymptomatic testing Yes X No Carrier testing for family members Yes X No Prenatal testing Yes X No

6 TEST CHARACTERISTICS 24. Analytical sensitivity and specificity This should be based on your own laboratory data for the specific test being applied for or the analytical sensitivity and specificity of the method/technique to be used in the case of a test yet to be set up. In the validation series, of 46 SNPs previously identified by Sanger sequencing 44 were identified using the NGS method giving an estimate for analytical sensitivity of 96%. All variants missed were in RPGR ORF15 for this reason this region of the genome was excluded from the NGS panel and testing is provided separately. 25. Clinical sensitivity and specificity of test in target population The clinical sensitivity of a test is the probability of a positive test result when condition is known to be present; the clinical specificity is the probability of a negative test result when disease is known to be absent. The denominator in this case is the number with the disease (for sensitivity) or the number without condition (for specificity). In our validation cohort of 50 patients (14 ADRP, 16 presumed ARRP and 20 sporadic RP). The overall detection rate of clearly pathogenic variants was 60% for AD patients, 30% for patients with recessive disease and 30% for sporadic cases. Validation series reported in: O'Sullivan J, et al. (2012) J Med Genet. 49: Unclassified variants may be counted as potential false positive results. These account for approximately 17% of variants found in the new test. The minimum clinical specificity is therefore 83%. 26. Clinical validity (positive and negative predictive value in the target population) The clinical validity of a genetic test is a measure of how well the test predicts the presence or absence of the phenotype, clinical condition or predisposition. It is measured by its positive predictive value (the probability of getting the condition given a positive test) and negative predictive value (the probability of not getting the condition given a negative test). Positive and negative predictive values- 105 gene scan: There are insufficient data to make a reliable estimate of positive and negative predictive values in the target population given uncertainties in the penetration of some of the unclassified variants detected. Locus heterogeneity dictates that a negative screen in an affected patient does not exclude a genetic mutation as the cause of the patient s symptoms. Individual mutations in the target group have been described with a high penetrance and within a family where a mutation has been firmly associated with the condition through its inheritance the negative and positive predictive values are >99%. 27. Testing pathway for tests where more than one gene is to be tested Please include your testing strategy if more than one gene will be tested and data on the expected proportions of posi tive results for each part of the process. Please illustrate this with a flow diagram. The 105 gene scan panel is a one pass work-flow no further reflex tests are envisaged. In time the panel will be versioned to improve its scope and performance. If suspected X-linked, test for RPGR ORF15 first. CLINICAL UTILITY 28. How will the test add to the management of the patient or alter clinical outcome? The following is a summary of our clinical experience of the utility of testing for Retinal Degeneration disorders using conventional technologies but is completely relevant to the extended RD panel test presented in this gene dossier. Syndromic RD The extended RD panel will detect mutations in multi-systemic forms of RD including Bardet Biedl syndrome (BBS) and Ushers syndrome. Individual testing for bardet biedl syndrome is available through the National Specialised Commissioning Team funded bardet biedl service. The panel test is not intended to replace this. BBS mutations are also a known cause of non-syndromic RP. Therefore it is essential that these genes are covered in a panel as simplex non-syndromic RP patients with BBS

7 mutations would not fall under the remit of the nationally commissioned service. Early diagnosis in children with RP and hearing loss may improve educational support and monitoring of children with dual sensory impairment (Bolz and Roux, 2011). Given the variability of BBS, some RD patients may not be clinically diagnosed, but genetic testing revealing this diagnosis has important health implications for renal screening. Sporadic / Recessive RD Recessive RD is the most common and often most severe form of retinal dystrophy. Severe forms such as Lebers Congenital Amaurosis are present at birth. Until recently, only a minority of AR genes could be tested. Many sporadic cases are assumed to be recessive if female, or when XL forms excluded in males. Testing is important for: Confirming recessive inheritance in sporadic cases as a low offspring risk reassures an affected parent. Experience so far has revealed some cases assumed recessive in fact have a dominant mutation with a high offspring risk. Offering carrier testing in consanguineous families Prenatal testing in the more severe forms such as LCA. We were describing scenarios where an affected adult is considering their reproductive options and risk to their offspring. X-linked RD X-linked RD is one of the more severe forms and accounts for around 20% of RD (Rivolta, 2002). Mutations in 2 genes (RPGR and RP2) account for perhaps 75-90% of all X-linked RD. Mutations in these genes also cause X-linked cone-rod dystrophy (CRD), cone dystrophy (COD) and early onset severe retinal dystrophy (EOSRD). In general males with disease present early and are significantly disabled by the condition during adulthood. By contrast female carriers are more mildly affected and often develop symptoms in middle life or later. Clinical carrier testing (e.g. electrodiagnostic testing) will pick up many carriers but cannot be relied upon in early adulthood, in particular during reproductive years. The implications of X-linked RD are therefore similar to other X-linked disorders: Asymptomatic young females, in particular those with experience of their fathers/brothers with disease, are often anxious to know their carrier status. Since this cannot be provided with accuracy from clinical testing, genetic carrier testing is often sought by these females. o As a measure of clinical utility we now have experience of testing using conventional technologies in 210 probands with a family history consistent with XLRD. Subsequently carrier tests were performed for 93 females and 58 (62%) were confirmed to be carriers. o Asymptomatic young females, in whom carrier status is excluded are able to be discharged from follow-up. o Females carrying a mutation may be interested in PND (Furu et al, 1993). o Given the increasing evidence of symptoms in females this test should be regarding as a pre-symptomatic test as well as a carrier test. Asymptomatic males even those who have been reassured that they are unlikely to carry a mutation are anxious to have a formal genetic confirmation that they do not carry mutations. Males without a mutation can be excluded from follow-up. o The age at which males should be tested is debatable however, families are often keen to ensure that boys are diagnosed early since this impacts upon education, training, life choices (e.g. career choices) and management. For this reason examination, or genetic testing, under the age of 5 is often requested and is considered. Symptomatic males where males with symptoms are tested there is unlikely to be an alteration in their care pathway.

8 Symptomatic females o Genotype-phenotype correlation. There is emerging evidence that females have a strong likelihood of developing symptoms and that this is under the influence of mutation type. However at present this has no proven predictive value. Autosomal dominant RD AD RD is a progressive, heterogeneous condition accounting for 15 25% of RD (Rivolta, 2002). The implications of ADRD are therefore similar to other progressive autosomal dominant and X-linked disorders. A number of forms including RP9 and RP11 show reduced expressivity /penetrance Asymptomatic individuals, in particular those with experience of affected first degree relatives with disease, are often anxious to know their genetic status. Since this cannot be provided with accuracy from clinical testing, presymptomatic testing is often sought by these individuals. o As with other AD conditions, asymptomatic individuals, in whom affected status is excluded are able to be discharged from follow-up. o Pre-symptomatic testing via clinical examination is requested in some families with later onset AD disease it is likely that this will be requested in some families and will need to be managed as for other pre-symptomatic tests (Mezer et al, 2007) Affected individuals will often be tested in order to allow screening of the wider family. There is unlikely to be an alteration in the care pathway for affected individuals. Rivolta C, Sharon D, DeAngelis MM, Dryja TP (2002) Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns. Hum Mol Genet. 11: Moore and Burton (2008) Genetic ophthalmology in focus. Bolz H, Roux A-F (2011) Clinical Utility gene card for: Ushers syndrome. Eur J Hum Genet 19 (published online) Furu T Kaarianen H, Sankila EM, Norio R (1993) Attitudes towards prenatal testing and selective abortion among patients with retinitis pigmentosa or choroideremia as well as among their relatives. Clinic Genet 43(3): Mezer E et al (2007) Attitudes regarding predictive testing for Retinitis Pigmentosa.Ophthal Genet 28:9-15 O'Sullivan J, et al. (2012) J Med Genet. 49: How will the availability of this test impact on patient and family life? The objective of the test described here is to identify one or more causative pathological mutations in a patient with symptoms of retinal degeneration. Where this is the outcome of the test a definitive diagnosis is made for the patient and a number of consequences and impacts may follow for their family: 1. Certainty about the condition allows a potential reduction in anxiety and a greater likelihood that conditions are created for adjusting to the needs of the member of the family affected. 2. More accurate prognostic information allows planning for the future (care, education). 3. Reproductive options may be considered. 4. At risk family members may consider pre-symptomatic or carrier testing to establish their status using standard molecular techniques. 30. Benefits of the test Please provide a summary of the overall benefits of this test. This test is an extension of the scope and effectiveness of the retinal mutation scans that have been in service for a number of years. For the individual patient for whom a molecular diagnosis is established: 1. Their prognosis/possible co-morbidity pattern and a management path is better defined 2. Treatment/trial options may be opened 3. Personal life choices (reproduction, education, career) are clearer.

9 31. Is there an alternative means of diagnosis or prediction that does not involve molecular diagnosis? If so (and in particular if there is a biochemical test), please state the added advantage of the molecular test. A diagnosis of some of the conditions within the scope of the 105 gene scanning panel can be established by the clinical and family history, clinical examination and investigations such as ocular electrophysiology. In a significant proportion of patients these investigations fail to establish a diagnosis and a molecular test is a faster route to a precise diagnosis and the most appropriate clinical, social care and educational pathway. A molecular diagnosis opens the option for predictive and carrier testing in the family which is not generally available through alternative technologies. 32. Please describe any specific ethical, legal or social issues with this particular test. Given the extensive genetic screening with this panel, multiple unknown variants and unexpected results may be found. Unexpected findings may include carrier status for other retinal dystrophy genes. Patients should be appropriately consented by the referring clinician and the possibility of unexpected results should be discussed. A joint clinical and laboratory Retinal Dystrophy NGS meeting is held monthly to discuss molecular findings and clinical relevance in each case. 33. The Testing Criteria must be completed where Testing Criteria are not already available. If Testing Criteria are available, do you agree with them Yes/No Yes see below If No: Please propose alternative Testing Criteria AND please explain here the reasons for the changes. 34. Savings or investment per annum in the diagnostic pathway based on national expected activity, cost of diagnostics avoided and cost of genetic test. Please show calculations. Cost benefit analysis of genetic testing in retinal dystrophy is part of the Regard study, a Fight for Sight programme grant, aiming to develop services for inherited retinal dystrophy. Currently, a full national service evaluation is planned with cost benefit analysis of genetic testing and genetic counselling services including pilot measures around patient value using willingness to pay measures. Total annual costs of conventional testing = 229,356 Total annual costs using panel approach = 234,600 Total annual investment = 5,244 There will be savings in consultant appointments as the result will be arrived at after only one test rather than sequential testing and ongoing appointments to determine a diagnosis. Furthermore it is expected that a greater number of individuals will be picked up as the test includes a number of genes not previously available. 35. List the diagnostic tests/procedures that would no longer be required with costs. Patients would no longer require individual gene tests (see current system to test on page 13). Genetic testing will be prioritised on patient need rather than scientific restrictions with limited individual gene tests. There will be savings on those procedures which would have been required to indicate individual genetic tests and on multiple sequential genetic testing. Total cost tests/procedures no longer required

10 CASE STUDY Case example 1 - diagnostic and cascade testing (see figure 2). Autosomal dominant RP: The panel is predicted to increase pick up rates is families with a strong ADRP family history from 44% to 80%. There is a misconception that in these families there is little benefit to finding the mutation as counselling is straight forward (ie it is easy to predict the risk to offspring of an affected person). However our panel test has demonstrated that this is not always the case: Woman (II1) presented with RP (and a dominant FH). There was also hearing loss in the family, however due to the ADRP the clinician had said that Usher syndrome was unlikely (and therefore testing not indicated) and that the patient most likely had two unrelated conditions (ADRP and hearing loss). Up until the NGS all laboratory testing for this family had concentrated on identifying the cause of the ADRP in the family. NGS identified two USH2A mutations (c. 2299del and p.(glu1441x)) confirming Usher syndrome and significantly changed the counselling for this family. Simplex RP: We predict that NGS will have the biggest impact in families with RP and no discernable family history because a) there are too many genes to effectively be dealt with using Sanger and b) this represents the majority of families. Up until NGS it has not been possible to give most families with simplex RP an accurate recurrence risk. The best advice is approximately 95% of simplex RP is autosomal recessive and consequently the recurrence risk is likely to be low. However we have now encountered many families where this advice would have been unhelpful for example.

11 Finally NGS is also changing clinical practice in ways that we might not have predicted for example in the following family where we identified BBS1 mutations in a patient with presumed RP clearly this patient does not have the characteristic pattern of symptoms associated with Bardet Beidl syndrome (developmental delay, polydactyly) however in the light of the NGS result the follow up regime for this family will now include ongoing screening for renal complications. 37. For the case example, if there are cost savings, please provide these below: PRE GENETIC TEST Costs and type of imaging procedures Costs and type of laboratory pathology tests (other than molecular test proposed in this gene dossier) Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost pre genetic test POST GENETIC TEST Costs and type of imaging procedures Costs and types of laboratory pathology tests (other than molecular test proposed in this gene dossier) Cost of genetic test proposed in this gene dossier Costs and type of physiological tests (e.g. ECG) Cost and type of other investigations/procedures (e.g. biopsy) Cost outpatient consultations (genetics and non genetics) Total cost post genetic test 38. Estimated savings for case example described

12 Comparative costs of conventional screening cascade and new screening system incorporating the NGS test for 400 index patients A. Conventional system (based on retinitis pigmentosa dossier accepted by UKGTN 2007) Patients number screen Cost ( ) All RD patients meeting acceptance criteria 400 (estimated UK demand) X-linked screening cascade RD and cone rod dystrophy patients with an X-linked 200 RPGR ORF15 55,200 family history and sporadic males Patients negative after RPGR ORF15 screen 144 RPGR exons ,616 Patients negative after RPGR exons 1-14 screen 52 RP2 17,940 Patients negative after RP2 screen 4 No further action Autosomal Dominant screening cascade RD patients with a clearly autosomal dominant family history 200 Panel of common mutations and exonic hotspots 96,600 Patients negative after AD screen 112 No further action TOTAL 229,356 Total cost of conventional screening cascade 229,356 B. New screening system 1. X-linked patients (RPGR ORF15 screen by conventional sequencing (50% of patients) Patients number screen Cost ( ) All RD patients meeting acceptance criteria 400 X-linked screen RD and cone rod dystrophy patients with an X-linked 200 RPGR ORF15 55,200 family history Patients negative after X-linked screen 10 No further action TOTAL 55, AD, sporadic and AR patients using 105 gene NGS screen (50% of patients) Patients number screen Cost ( ) Non X-linked RD patients meeting acceptance ,400 criteria Patients negative after NGS screen 96 No further action TOTAL 179,400 Total cost of new screening system 234,600

13 UKGTN Testing Criteria Approved name and symbol of disease/condition(s): Retinal Degeneration panel test Approved name and symbol of gene(s): a panel of 105 genes, variants of which have been shown to be causative of Retinal Degeneration conditions Patient name: Patient postcode: OMIM number(s): OMIM number(s): Date of birth: NHS number: Name of referrer: Title/Position: Lab ID: Referrals will only be accepted from one of the following: Referrer Consultant Clinical Geneticists Consultant Ophthalmologist with special interest in retinal disorders Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria The minimum criteria for acceptance is that the index case: has been diagnosed as a result of symptoms of initial rod dysfunction followed by peripheral cone dysfunction OR a characteristic retinal appearance OR characteristic ERG. X linked RP Autosomal Dominant RP Autosomal Recessive RP Sporadic Tick if this patient meets criteria Additional Information: Please note that if the diagnosis is probable/definite and there is a single gene test for that condition, the clinician may prefer to carry out the single gene test rather than the panel test. For example Retinoschisis (available from Cambridge), Stargardt (available from Oxford), Bardet Biedl (available from Great Ormond Street) and X-linked RP as single test available from Manchester If the sample does not fulfil the clinical criteria or you are not one of the specified types of referrer and you still feel that testing should be performed please contact the laboratory to discuss testing of the sample

14 Appendix 1 Conditions included in the panel Gene HGNC Phenotype Phenotype OMIM # Gene/locus OMIM # CA4 Retinitis pigmentosa 17 # * CERKL Retinitis pigmentosa 26 # * CNGA1 Retinitis pigmentosa 49 # * CNGB1 Retinitis pigmentosa 45 # * CRB1 Leber congenital amaurosis 8 # * Pigmented paravenous chorioretinal atrophy # Retinitis pigmentosa 12, autosomal recessive # CRX Cone rod retinal dystrophy 2 # * Leber congenital amaurosis 7 # EYS Retinitis pigmentosa 25 # * FSCN2 Retinitis pigmentosa 30 # * GUCA1B Retinitis pigmentosa 48 # * IDH3B Retinitis pigmentosa 46 # * IMPDH1 Leber congenital amaurosis 11 # * Retinitis pigmentosa 10 # KLHL7 Retinitis pigmentosa 42 # * MERTK Retinitis pigmentosa 38 # * NR2E3 Enhanced S cone syndrome # * Retinitis pigmentosa 37 # NRL Retinal degeneration, autosomal recessive, clumped pigment type Retinitis pigmentosa 27 # RP1 Retinitis pigmentosa 1 # * RP9 Retinitis pigmentosa 9 # * PDE6A Retinitis pigmentosa 43 # * PDE6B Night blindness, congenital stationary, autosomal # * dominant 2 Retinitis pigmentosa 40 # PRCD Retinitis pigmentosa 36 # * PROM1 Cone rod dystrophy 12 # * Macular dystrophy, retinal, 2 # Retinitis pigmentosa 41 # Stargardt disease 4 # PRPF3 Retinitis pigmentosa 18 # * PRPF31 Retinitis pigmentosa 11 # *606419

15 PRPF8 Retinitis pigmentosa 13 # * PRPH2 Choriodal dystrophy, central areolar 2 # * Foveomacular dystrophy, adult onset, with choroidal # neovascularization Macular dystrophy, patterned # Macular dystrophy, vitelliform # Retinitis pigmentosa 7 # Retinitis pigmentosa, digenic # Retinitis punctata albescens # RGR Retinitis pigmentosa 44 # * RGS9 Bradyopsia # * RHO Night blindness, congenital stationery, autosomal # * dominant 1 Retinitis pigmentosa 4, autosomal dominant or # recessive Retinitis punctata albescens # RLBP1 Bothnia retinal dystrophy # * Fundus albipunctatus # Newfoundland rod cone dystrophy # Retinitis punctata albescens # ROM1 Retinitis pigmentosa 7, digenic # * RP2 Retinitis pigmentosa 2 # * RPE65 Leber congenital amaurosis 2 # * Retinitis pigmentosa 20 # RPGR* Cone rod dystrophy 1 # * Macular degeneration, X linked atrophic # Retinitis pigmentosa 3 # Retinitis pigmentosa, X linked, and sinorespiratory # infections, with or without deafness SAG Oguchi disease 1 # * Retinitis pigmentosa 47 # SEMA4A Cone rod dystrophy 10 # * Retinitis pigmentosa 35 # TOPORS Retinitis pigmentosa 31 # * TTC8 Bardet Biedl syndrome 8 # * Retinitis pigmentosa 51 # TULP1 Leber congenital amaurosis 15 # * Retinitis pigmentosa 14 # CEP290 Bardet Biedl syndrome 14 # * Joubert syndrome 5 # Leber congenital amaurosis 10 # Meckel syndrome type 4 # Senior Loken syndrome 6 #610189

16 AIPL1 Cone rod dystrophy # * Leber congenital amaurosis 4 # Retinitis pigmentosa, juvenile # GUCY2D Cone rod dystrophy 6 # * Leber congenital amaurosis 1 # LCA5 Leber congenital amaurosis 5 # * LRAT Leber congenital amaurosis 14 # * Retinal dystrophy, early onset severe # Retinitis pigmentosa, juvenile # RD3 Leber congenital amaurosis 12 # * RDH12 Leber congenital amaurosis 13 # * SPATA7 Leber congenital amaurosis 3 # * Retinitis pigmentosa, juvenile, autosomal recessive # ADAM9 Cone rod dystrophy 9 # * CACNA2D4 Retinal cone dystrophy 4 # * KCNV2 Retinal cone dystrophy 3B # * RIMS1 Cone rod dystrophy 7 # * RPGRIP1 Cone rod dystrophy 13 # * Leber congenital amaurosis 6 # UNC119 Cone rod dystrophy C1QTNF5 Retinal degeneration, late onset, autosomal dominant # * BEST1 Best macular dystrophy # * Bestrophinopathy # Microcornea, rod cone dystrophy, cataract, and # posterior staphyloma Retinitis pigmentosa, concentric # Retinitis pigmentosa 50 # Vitelliform macular dystrophy, adult onset # Vitreoretinochoroidopathy # ABCA4 Cone rod dystrophy 3 # * Fundus flavimaculatus # Macular degeneration, age related, 2 # Retinal dystrophy, early onset severe # Retinitis pigmentosa 19 # Stargardt disease 1 # CHM Choroideremia # * ELOVL4 Macular dystrophy, autosomal dominant, chromosome # * linked Stargardt disease 3 # CNGA3 Achromatopsia 2 # * CNGB3 Achromatopsia 3 # * Macular degeneration, juvenile # GNAT2 Achromatopsia 4 # * PDE6C Cone dystrophy 4 # *600827

17 RS1 Retinoschisis # * FZD4 Exudative vitreoretinopathy # * Retinopathy of prematurity # LRP5 Exudative vitreoretinopathy 4 # * NDP Exudative vitreoretinopathy, X linked # * Norrie disease # GUCA1A Cone dystrophy 3 # * Cone rod dystrophy 14 # TIMP3 Sorsby fundus dystrophy # * EFEMP1 Doyne honeycomb degeneration of retina # * RDH5 Fundus albipunctatus # * TEAD1 Sveinsson choreoretinal atrophy # * RAX2 Cone rod dystrophy 11 # * Macular degeneration, age related, 6 # CLRN1 Retinitis pigmentosa 61 # * Usher syndrome, type 3A # ARL6 Bardet Biedl syndrome 3 # * Retinitis pigmentosa 55 # BBS1 Bardet Biedl syndrome 1 # * BBS10 Bardet Biedl syndrome 10 # * BBS12 Bardet Biedl syndrome 12 # * BBS2 Bardet Biedl syndrome 2 # * BBS4 Bardet Biedl syndrome 4 # * BBS5 Bardet Biedl syndrome 5 # * BBS7 Bardet Biedl syndrome 7 # * BBS9 Bardet Biedl syndrome 9 # * MKKS Bardet Biedl syndrome 6 # * McKusick Kaufman syndrome # TRIM32 Bardet Biedl syndrome 11 # * DFNB31 Deafness, autosomal recessive 31 # * Usher syndrome, type 2D # GPR98 Usher syndrome, type 2C # * PCDH15 Deafness, autosomal recessive 23 # * Usher syndrome, type 1D/F digenic # Usher syndrome, type 1F # USH2A Retinitis pigmentosa 39 # * Usher syndrome, type 2A # CDH23 Deafness, autosomal recessive 12 # * Usher syndrome, type 1D # Usher syndrome, type 1D/F digenic # MYO7A Deafness, autosomal dominant 11 # * Deafness, autosomal recessive 2 # Usher syndrome, type 1B # USH1C Deafness, autosomal recessive 18 # * Usher syndrome, type 1C #276904

18 USH1G Usher syndrome, type 1G # * FAM161A Retinitis pigmentosa 28 # * C2orf71 Retinitis pigmentosa 54 # * IMPG2 Maculopathy, IMPG2 related # * Retinitis pigmentosa 56 # PDE6G Retinitis pigmentosa 57 # * SNRNP200 Retinitis pigmentosa 33 # * RBP3 Autosomal Recessive Retinitis pigmentosa None given * ZNF513 Retinitis pigmentosa 58 # * CDHR1 Cone rod dystrophy 15 # * RP1L1 Occult macular dystrophy # * OTX2 Microphthalmia, syndromic 5 # * Pituitary hormone deficiency, combined, 6 # Retinal dystrophy, early onset, and pituitary # dysfunction DHDDS Retinitis pigmentosa 59 # * PITPNM3 Cone rod dystrophy 5 # * MKS1 Bardet Biedl syndrome 13 # * Meckel syndrome, type 1 # PRPF6 Retinitis pigmentosa 60 # * UNC119 Cone rod dystrophy

19 Appendix 2 - Genes included in the next generation sequencing assay HGNC Transcript HGNC Transcript HGNC Transcript CA4 NM_ RLBP1 NM_ ELOVL4 NM_ CERKL NM_ ROM1 NM_ CNGA3 NM_ CNGA1 NM_ RP2 NM_ CNGB3 NM_ CNGB1 NM_ RPE65 NM_ GNAT2 NM_ CRB1 NM_ RPGR* NM_ PDE6C NM_ CRX NM_ SAG NM_ RS1 NM_ EYS NM_ SEMA4A NM_ FZD4 NM_ FSCN2 NM_ TOPORS NM_ LRP5 NM_ GUCA1B NM_ TTC8 NM_ NDP NM_ IDH3B NM_ TULP1 NM_ GUCA1A NM_ IDH3B NM_ CEP290 NM_ TIMP3 NM_ IMPDH1 NM_ AIPL1 NM_ EFEMP1 NM_ KLHL7 NM_ GUCY2D NM_ RDH5 NM_ MERTK NM_ LCA5 NM_ TEAD1 NM_ NR2E3 NM_ LRAT NM_ RAX2 NM_ NRL NM_ RD3 NM_ CLRN1 NM_ RP1 NM_ RDH12 NM_ CLRN1 NM_ RP9 NM_ SPATA7 NM_ ARL6 NM_ PDE6A NM_ ADAM9 NM_ BBS1 NM_ PDE6B NM_ CACNA2D4 NM_ BBS10 NM_ PRCD NM_ KCNV2 NM_ BBS12 NM_ PROM1 NM_ RIMS1 NM_ BBS2 NM_ PRPF3 NM_ RIMS1 NM_ BBS4 NM_ PRPF31 NM_ RIMS1 NM_ BBS5 NM_ PRPF8 NM_ RPGRIP1 NM_ BBS7 NM_ PRPH2 NM_ UNC119 NM_ BBS7 NM_ RGR NM_ C1QTNF5 NM_ BBS9 NM_ RGS9 NM_ BEST1 NM_ MKKS NM_ RGS9 NM_ ABCA4 NM_ TRIM32 NM_ RHO NM_ CHM NM_ DFNB31 NM_ GPR98 NM_ CDH23 NM_ IMPG2 NM_ PCDH15 NM_ MYO7A NM_ PDE6G NM_ PCDH15 NM_ USH1C NM_ SNRNP200 NM_ PCDH15 NM_ USH1C NM_ RBP3 NM_ PCDH15 NM_ USH1G NM_ ZNF513 NM_ USH2A NM_ FAM161A NM_ CDHR1 NM_ RP1L1 NM_ PITPNM3 NM_ MKS1 NM_ OTX2 NM_ MKS1 NM_ PRPF6 NM_ DHDDS NM_ Notes: (1) For some genes the analysis of multiple transcripts is required. (2) * For RPGR it is not possible to obtain high quality read for exon ORF15. Consequently this analysis is offered separately on request further details are available at:

20 Appendix 3 - Test strategy Test Criteria Clearly X linked Other referral indication ORF 15 test 105 gene panel test ORF 15 mutation Report No ORF 15 mutation (an additional request is required from referring clinician) Report