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 Submitting laboratory: Sheffield RGC 1. Disorder/condition approved name (please provide UK spelling if different from US) and symbol as published on the OMIM database (alternative names will be listed on the UKGTN website). If NGS panel test, please provide a name. If this submission is for a panel test please complete the Excel spread sheet, Appendix 1, available for download from the UKGTN website, and list all of the conditions grouped by sub panels if applicable. Neurogenetic Motor Disorders and/or Dementia 152 Gene Panel 2. OMIM number for disorder/condition If a panel test see 1 above. If a number of subpanels exist with different clinical entry points e.g. cancer panel test but different subpanels for different types of cancer (breast cancer, colon, phaeochromocytoma), then please list the sub panels here providing name of each sub panel. See Appendix 1 3a. Disorder/condition to help commissioners to understand the impact of this condition please provide, in laymen s terms (e.g tubes in the kidney (renal tubule) or low sugar in the blood (hypoglycaemia), a brief (2-5 sentences/no more than 50 words) description of how the disorder(s) affect individuals and prognosis. Hereditary Motor Disorders: Movement disorders are neurological conditions that affect the ability to control movement and may cause involuntary movements. There can be excess movement or a lack of movement and the speed, quality and fluency of the movement can all be affected. Hereditary Spastic Paraplegia (HSP): HSP is a weakness and stiffness of the leg muscles, which gradually gets worse. It is caused by the deterioration of the long spinal nerves that control voluntary movement. The muscles in the legs do not receive relax or contract messages leading to spasticity (stiff and rigid muscles) and weakness. Amyotrophic Lateral Sclerosis (ALS) with or without Frontotemporal Dementia (FTD): ALS, or Motor Neurone Disease (MND), is a progressive disease of the motor neurons, or nerves, of the brain and spinal cord with up to 15% also developing FTD. Messages gradually stop reaching muscles, leading to weakness and wasting, and affecting how a person walks, talks, eats, drinks and/or breathes. The clinical sensitivity of this test is estimated to be approximately 10% for sporadic cases, since it is believed that the majority of ALS cases are non-familial. Dementia: Dementia is a common disorder, particularly Alzheimer type dementia and generally involves memory loss and difficulties with thinking, problem-solving or language. The most common familial forms are early onset. Dementia is caused when the brain is damaged by diseases, such as with Alzheimer disease, or a series of strokes. Spinal Muscular Atrophy (SMA): Spinal muscular atrophies (SMAs) are hereditary disorders characterised by degeneration of motor neurons in the spinal cord and brainstem. Classification is by distribution of weakness (distal, proximal, bulbar) and mode of inheritance. Age of onset is variable and many forms are progressive, overlapping clinically with HSP and ALS.

2 3b. Disorder/condition if required please expand on the description of the disorder provided in answer to Q3a. Hereditary Spastic Paraplegia (HSP): HSP refers to a group of inherited disorders that cause weakness and stiffness of the leg muscles, which gradually gets worse over time. A person with HSP is likely to have inherited an abnormal gene from one or both parents that causes the long nerves in their spine, which control voluntary movement, to deteriorate. This means that muscles in the lower body do not receive the correct messages telling them to relax or contract and leads to spasticity (abnormally stiff and rigid muscles) and weakness in the leg muscles, which progressively gets worse. Most forms of HSP mainly affect the lower body the arms and hands are usually unaffected. On average, the symptoms of HSP start in mid-adulthood, although it can start as late as age 70 or in young children. Amyotrophic Lateral Sclerosis (ALS) with or without Frontotemporal Dementia (FTD): ALS, also known as Motor Neurone Disease (MND) is a progressive disease that attacks the motor neurones, or nerves, in the brain and spinal cord. This means messages gradually stop reaching muscles, which leads to weakness and wasting. This can affect how a person walks, talks, eats, drinks and breathes. However, not all symptoms necessarily happen to everyone and it is unlikely they will all develop at the same time, or in any specific order. Up to 15% of ALS patients also develop FTD, the second most common cause of dementia. It is characterised by degeneration of the frontal and temporal lobes of the brain resulting in progressive changes in personality, behaviour and language. Additionally, up to 15% of people diagnosed with FTD go on to develop ALS. Although there is currently no cure for MND, symptoms can be managed to help patients achieve the best possible quality of life. Dementia: Dementia is a general term for a decline in mental ability which is severe enough to interfere with daily life and generally involves memory loss and difficulties with thinking, problem-solving or language. Alzheimer disease (AD) is the most common type of dementia, accounting for 60-80% of cases, with vascular dementia, which occurs after a stroke, being the second most common type. Symptoms can vary widely, but at least two of the following core mental functions must have significant impairment in order to be considered as dementia: (1) memory, (2) communication and language, (3) ability to focus and pay attention, (4) reasoning and judgement, (5) visual perception. Many dementias are progressive. The most common familial forms are early onset but later-onset forms are also beginning to be shown to have a genetic basis. Spinal Muscular Atrophy (SMA): The spinal muscular atrophies (SMAs) are a clinically and genetically heterogeneous group of neuromuscular disorders caused by degeneration of anterior horn cells. SMA may affect a person s ability to crawl and walk, to move their arms, hands, head and neck, as well as their breathing and swallowing, and hence is often fatal. Different types of SMA can vary greatly in severity and the effects vary between individuals, even within members of the same family. The most common cause of SMA is homozygous deletion of exon 7 within the SMN1 gene, which causes infantile-onset autosomal recessive distal SMA, but there are many other types which are classified by the distribution of weakness and the mode of inheritance. 4. Disorder/condition mode of inheritance If this submission is for a panel test, please complete the mode of inheritance for each condition in the table in appendix 1. See Appendix 1 5. Gene approved name(s) and symbol as published on HGNC database (alternative names will be listed on the UKGTN website) If this submission is for a panel test please complete the Excel spread sheet, Appendix 1, available for download from the UKGTN website, and list all of the genes grouped by sub panels if applicable. See Appendix 1

3 6a. OMIM number(s) for gene(s) If a panel test see 5. above See Appendix 1 6b. HGNC number(s) for gene(s) If a panel test see 5. above See Appendix 1 7a. Gene description(s) If this submission is for a panel test, please provide total number of genes and if there are subpanels, please also list the number genes per sub panel. The whole panel consists of 152 genes subdivided into 4 subpanels for HSP (88 genes), FALS with or without FTD (42 genes), Dementia (27 genes) and SMA (29 genes). Please note that there is some clinical overlap between groups and some genes are associated with more than one phenotype. 7b. Number of amplicons to provide this test (molecular) or type of test (cytogenetic) (n/a for panel tests) n/a 7c. GenU band that this test is assigned to for index case testing. For NGS panel tests if there are sub panels, please provide GenU per subpanel. HSP 88 gene subpanel: Band H FALS with or without FTD 42 gene subpanel: Band G Dementia 27 gene subpanel: Band G SMA 29 gene subpanel: Band G 8. Mutational spectrum for which you test including details of known common mutations (n/a for panel tests) n/a 9a. Technical method(s) please describe the test. Testing Workflow Library Preparation SureSelect Shearing of genomic DNA using the Covaris E220 sonicator. End repair, A tailing and ligation of adaptors using SureSelectXT library system (Agilent Technologies). Enrichment by SureSelect target enrichment (Agilent Technologies) using custom in-house designed probes. Samples have barcode tags added following target enrichment. Sequencing on the Illumina HiSeq using the HiSeq Rapid SBS Kit v2 performing 2 x 108 bp paired end reads. Data Analysis Based on the open source Best Practices workflow by the Broad Institute (for additional information, see BWA alignment of reads to human genome build hg19. Generation of depth of coverage reports and checked using Alamut v 2.6 (Rev0) (Interactive Biosoftware). A minimum threshold of 30-fold read depth is set for exonic sequences and intronic sequences up to and including 5 bp from exon. A minimum threshold of 18-fold read depth is set for intronic sequences from 6 bp to 25 bp from exon together with a visual check of all reads.

4 Identification of variants using HaplotypeCaller. Annotation from dbsnp (currently dbsnp138 but updated with new releases) Variants filtered against in-house polymorphism lists and Best Practice Guidelines for the evaluation of pathogenicity and the reporting of sequence variants in clinical molecular genetics (Association for Clinical Genetic Science). Post analysis Confirmation of all clinically significant sequence variants by Sanger sequencing. Filling of clinically appropriate gaps with low depth of coverage by Sanger sequencing. Creation of a diagnostic report combining clonal and Sanger sequence data. 9b. For panel tests, please specify the strategy for dealing with gaps in coverage. Any clinically relevant gaps with a low (<30x) depth of coverage will be filled by Sanger sequencing if appropriate. 9c. Does the test include MLPA? (For panel tests, please provide this information in appendix 1) MLPA kits from MRC-Holland are available and in use in the laboratory as follows: P165-C1 HSP ATL1 and SPAST genes. P213-B1 Hereditary spastic paraplegias-2 REEP1 and SPG7 genes P306-B1 SPG11 SPG11 gene N.B: this does not include MLPA for SMN1 as this is an exclusion criterion for this test. 9d. If NGS is used, does the lab adhere to the Association of Clinical Genetic Science Best Practice Guidelines for NGS? Yes 10. Is the assay to be provided by the lab or is it to be outsourced to another provider? If to be outsourced, please provide the name of the laboratory and a copy of their ISO certificate or their CPA number. To be provided by the laboratory. 11. Validation process Please explain how this test has been validated for use in your laboratory, including calculations of the sensitivity and specificity for the types of mutations reported to cause the clinical phenotype. Note that the preferred threshold for validation and verification is 95% sensitivity (with 95% Confidence Intervals). Your internal validation documentation can be submitted as an appendix (and will be included in the published Gene Dossier available on the website). The validation information should include data on establishing minimum read depth and horizontal coverage for the regions of interest, reproducibility of the pipeline, accuracy of variant calling, filtering of common variants and artefacts. If this submission is for a panel test, please provide a summary of evidence of instrument and pipeline validation and complete the tables below. For panel tests: Sensitivity >95% (95% CI) Read depth minimum cut off: 30-fold within coding exons and +/- 5bp into exons; 18-fold +/-25 to +/-6 of coding exons. Previously tested NGS test concordant results Number of patient samples Unique variants (total) SNV Del (1bp to 21bp) and Ins (1bp to bp) CNV n/a n/a n/a Currently CNVs are not part of the routine detection pipeline. The table below includes data from the 2015 EQA scheme. NGS False negative

5 Genomic DNA samples from 8 patients that have been tested using the previous version of the HSP (4 samples) and FALS (4 samples) panels were re-tested using the new panel design. Known variants NGS test concordant results NGS False negative Reference sample details Patient samples previously tested for HSP (4 samples) and FALS (4 samples). SNV Indel (1bp to 11 bp) CNV n/a n/a n/a Unique variants (total) Two samples were tested through the EQA schemes run jointly by EMQN and UKNEQAS for Molecular Genetics. Known variants NGS test concordant results NGS False negative Reference sample details EMQN 2014 NGS EQA scheme (Part 1) and EMQN 2015 NGS (v Germline) EQA scheme Unique variants (total) SNV Indel (1bp to X bp) CNV n/a n/a n/a Specificity >95% (95% CI) Variant confirmed NGS by other method False positive Number of patient samples with a variant detected by NGS Unique variants (total) (0.72%)* SNV Indel (1bp to 67 bp) CNV n/a * all were indicated likely to be false positives from low quality score. 12a. Are you providing this test already? No. We are providing NGS testing for motor disorders based on our previous UKGTN submission for 96 genes associated with Neurogenetic disorders, submitted in January 2014 (#236). We also still offer Sanger sequencing for the SPAST, ATL1 and REEP1 genes associated with HSP and SOD1 and TARDBP associated with FALS, if required. 12b. If yes, how many reports have you produced? HSP Panel Sanger Based Tests FALS Panel 698 (SPAST, ATL1 and REEP1 genes) 445 Sanger Based Tests 132 (SOD1 and TARDBP genes) 90 NGS Based Tests NGS Based Tests

6 12c. Number of reports with a pathogenic (or likely pathogenic) mutation identified? HSP Panel FALS Panel Sanger Based Tests NGS Based Tests 109 (16%) 231 (52%) Sanger Based Tests NGS Based Tests 8 (6%) 22 (24%) 12d. Please provide the time period in which these reports have been produced and whether in a research or a full clinical diagnostic setting. Data from Sanger sequencing services is from 2011 to NGS services from 2014 to Jan All reports have been produced in a full clinical diagnostic setting. 13a. Is there specialised local clinical/research expertise for this disorder? Yes 13b. If yes, please provide details Professor Dame Pamela Shaw, based at the Sheffield Institute of Translational Neuroscience (SITraN) is an internationally recognised specialist in hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis (ALS). She and Dr Christopher McDermott run a motor neurone disorders clinic in which they see referrals with ALS from around the UK. They also have an active research programme on various aspects of ALS and HSP and have published widely on both subjects. 14. If using this form as an Additional Provider application, please explain why you wish to provide this test as it is already available from another provider. n/a

7 EPIDEMIOLOGY 15. Estimated prevalence and/or incidence of conditions in the general UK population For panel tests, please provide estimates for the conditions grouped by phenotypes being tested. Prevalence is total number of persons with the condition(s) in a defined population at a specific time (i.e. new and existing cases). e.g. CF prevalence approx. 12 per 100,000 with UK population of approx. 63 million the prevalence of affected individuals in the UK is Incidence is total number of newly identified cases in a year in a defined population. e.g. CF incidence 1/2650 live births in a UK population with 724,000 live births in a year = 273 new cases a year. Please identify the information on which this is based. Hereditary Spastic Paraplegia: The prevalence of HSP is estimated at 3-10 per 100,000 in Europe (source: Salinas et al. Hereditary spastic paraplegia: clinical features and pathogenetic mechanisms. Lancet Neurol Dec; 7(12): ). It has not been possible to ascertain the incidence for HSP in the UK population. Familial Amyotrophic Lateral Sclerosis with or without Frontotemporal Dementia: The prevalence of ALS, combining both sporadic and familial cases, is approximately 4.5 per 100,000 with an incidence of approximately 2 per 100,000 (source: Hoppitt T. et al. A Systematic Review of the Incidence and Prevalence of Long-Term Neurological Conditions in the UK. Neuroepidemiology. 2011; 36(1):19 28). Dementia: The prevalence of dementia in the UK is estimated at approximately 1265 per 100,000. Of these cases, approximately 4.7% are early-onset (i.e. under the age of 65) and the prevalence is increasing (Source: Alzheimer s Society, Dementia 2014: Opportunity for change). The incidence is estimated at approximately 323 per 100,000 (Source: Matthews FE et al. A two decade dementia incidence comparison from the Cognitive Function and Ageing Studies I and II. Nature Communications. 2016; 7: article number 11398). Spinal Muscular Atrophy (SMA): The prevalence of SMA is approximately 20 per 100,000 live births with an incidence of 2.6/100,000 (source: Orphanet Reports Prevalence and incidence of rare diseases 2015). However, the sensitivity for SMN1 deletion and sequence analysis for the detection of SMA types I III is probably almost 100% in a clinically well-defined patient group. The proportion of non 5q-SMA is <2% in SMA I+II, <10% in SMA III, and >90% for SMA IV patients (source: Rudnik-Schöneborn et al. Clinical Utility Gene Card Update (2015) European Journal of Human Genetics 23, doi: /ejhg ). 16. Estimated gene frequency (Carrier frequency or allele frequency) Please identify the information on which this is based. n/a for panel tests. n/a 17. Estimated penetrance of the condition. Please identify the information on which this is based. n/a for panel tests n/a 18. Estimated prevalence of conditions in the population of people that will be tested. n/a for panel tests. n/a INTENDED USE (Please use the questions in Annex A to inform your answers) 19. Please tick either yes or no for each clinical purpose listed. Panel Tests: a panel test would not be used for pre symptomatic testing, carrier testing and pre natal testing as the familial mutation would already be known in this case and the full panel would not be required. Yes No Treatment Yes No Prognosis & management Yes No Presymptomatic testing (n/a for Panel Tests) Yes No Carrier testing for family members (n/a for Panel Tests) Yes No Prenatal testing (n/a for Panel Tests) Yes No

8 TEST CHARACTERISTICS 20. Analytical sensitivity and specificity The analytical sensitivity of a test is the proportion of positive results correctly identified by the test (true positive/true positive + false negative). The analytical specificity of a test is the proportion of negative results correctly identified by the test (true negative/true negative + false positive). This should be based on your own laboratory data for (a) the specific test being applied for or (b) the analytical sensitivity and specificity of the method/technique to be used in the case of a test yet to be set up. Please specify any types of mutations reported to cause the clinical phenotype that cannot be detected by the test. Note that the preferred threshold is 95% sensitivity (with 95% Confidence Intervals). Validation data for the Hiseq analysis pipeline shows that both the test sensitivity and test specificity is 95% with confidence intervals of 95%. There is not currently a formal pipeline for the investigation of CNVs although it is hoped analysis will be introduced shortly. The NGS panels will not detect dosage changes, rearrangements or large triplet (or 6bp) repeat expansions such as those seen in SBMA and the C9orf72 repeat expansion. We have included testing strategies for each subpanel in question 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 disorder is known to be absent. The denominator in this case is the number with the disorder (for sensitivity) or the number without condition (for specificity). Please provide the best estimate. UKGTN will request actual data after one year service. For a panel test, the expected percentage diagnostic yield for the test in the target population can be presented as an alternative to clinical sensitivity and specificity? Hereditary Spastic Paraplegia: The diagnostic yield for the previous version of the panel test was ~50%. It is expected that this will be increased to approximately 60-65% with this version. The clinical specificity is close to 100%. Familial Amyotrophic Lateral Sclerosis with or without Frontotemporal Dementia: The clinical sensitivity of this test is estimated to be approximately 10% for sporadic cases, since it is believed that the majority of ALS cases are non-familial. In cases where there is a clear dominant family history, the clinical sensitivity will be higher at 40%. The clinical specificity is close to 100%. Dementia: The clinical sensitivity of this test is estimated to be greater than 60%. The clinical specificity is close to 100%. Spinal Muscular Atrophy (SMA): The clinical sensitivity of this test is estimated to be approximately 30%. The clinical specificity is close to 100%. 22. 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). Not currently requested for panel tests n/a

9 23. Testing pathway for tests where more than one gene is to be tested sequentially Please include your testing strategy if more than one gene will be tested and data on the expected proportions of positive results for each part of the process. Please illustrate this with a flow diagram. This will be added to the published Testing Criteria. The Clinician should choose the testing strategy as detailed below: HEREDITARY SPASTIC PARAPLEGIA: HSP 88 gene Panel Negative: 35-40% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 60-65% Positive # FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS WITH OR WITHOUT FRONTOTEMPORAL DEMENTIA: C9orf72 Expansion testing Negative: Familial: 50-60% Sporadic: 92% FALS 42 gene Panel Negative: Familial: 30-36% Sporadic: 83% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: Familial: 40-50% Sporadic: 8% Positive: Familial: 20-24% Sporadic: 9% Positive # DEMENTIA: Frontotemporal Dementia 2% Dementia 98% C9orf72 Expansion testing Negative: 1.8% Dementia 27 gene Panel Negative: 40% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 0.2% Positive: 59% Positive #

10 SPINAL MUSCULAR ATROPHY (SMA): SMN1 deletion testing Negative: 2% to 90% SMA 29 gene Panel Negative: 1.4% to 63% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 10% to 98% depending on severity Positive: 0.6% to 27% Positive # * Negative and where a single heterozygous pathogenic mutation is identified in a recessive gene, following discussion with clinician. # Exact incidence of large deletions within each panel of genes is currently unknown

11 CLINICAL UTILITY 24. How will the test change the management of the patient and/or alter clinical outcome? Please summarise in 2-3 sentences no more than 50 words. Hereditary Spastic Paraplegia: A positive result will provide prognostic information and inform family members of their risk. Drugs to reduce clonus and muscle tightness in clinically diagnosed individuals include benzodiazepines, baclofen, tizanidine, dantrolene, and botox injections. Neuroimaging and other biochemical testing will no longer be necessary. Familial Amyotrophic Lateral Sclerosis with or without Frontotemporal Dementia: A positive result will improve prognostication enabling long-term care plans. Identification of a causative mutation also facilitates genetic counselling and accurate assessment of risks for offspring and other family members, and may also provide information with respect to the likelihood of developing symptoms of FTD. Dementia: A positive result will improve prognostication enabling long-term care plans. Identification of a causative mutation also facilitates genetic counselling and accurate assessment of risks for offspring and other family members. Spinal Muscular Atrophy (SMA): A positive result will reduce the diagnostic odyssey that can be difficult for severely affected patients and inform family members of their risk. It may remove the need for EMG and / or muscle biopsy. 25. Please provide full description on likely impact on management of patient and describe associated benefits for family members. If there are any cost savings AFTER the diagnosis, please detail them here. Hereditary Spastic Paraplegia: Although no cures or specific drug treatments exist for HSP, a positive test will enable a diagnosis to be made. This will provide prognostic information to the patient and will help inform regarding risk to other family members, since the disease is so heterogeneous. Drugs to reduce clonus and muscle tightness in clinically diagnosed individuals include benzodiazepines, baclofen, tizanidine, dantrolene, and botox injections. A positive test will remove the need for expensive neuroimaging and other biochemical testing often performed in such patients and their family members. Familial Amyotrophic Lateral Sclerosis with or without Frontotemporal Dementia: Although no cures or specific drug treatments exist for ALS, the benefits of a positive test result include improved prognostication enabling long-term care plans to be initiated. Close family can anticipate long term needs of the affected individual and hence plan for the future. Identification of a causative mutation also facilitates genetic counselling and accurate assessment of risks for offspring and other family members, and may also provide information with respect to the likelihood of developing symptoms of FTD. Feedback from patient groups suggests that families affected by the consequences of the disorder would find this helpful. Dementia: The test will greatly improve the speed and accuracy of diagnosis. It will reduce the number of sequential genetic tests that are required and the associated cost. An early confirmed diagnosis will allow initiation of treatment early in the disease course which will allow better management of symptoms. A positive test result can also improve prognostication enabling long-term care plans to be initiated. Close family can anticipate long term needs of the affected individual and hence plan for the future. Identification of a causative mutation also facilitates genetic counselling and accurate assessment of risks for offspring and other family members. Spinal Muscular Atrophy (SMA): A positive test will enable a diagnosis to be made. This will provide prognostic information to the patient and will help inform regarding risk to other family members, since the disease is so heterogeneous.

12 26a. If this test was not available, what would be the consequences for patients and family members? Please describe in not more than 50 of words. There would be a continued need for other costly or invasive means of diagnosis, such as neuroimaging, biopsy and lumbar puncture. It might not be possible to clarify prognosis for the patient or to offer prenatal diagnosis; additionally, risks to other family members, including offspring, could not be clarified. 26b. The consequences for patients and family members if this test was not available if required please expand on the response provided in question 26a. n/a 27. 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. Hereditary Spastic Paraplegia: No alternative means of diagnosis is available. Clinical diagnosis is generally based on phenotype, and can include neuroimaging. Familial Amyotrophic Lateral Sclerosis with or without Frontotemporal Dementia: The diagnosis of ALS or FTD is essentially made on clinical grounds, with support from imaging and neurophysiological tests. The added advantage of molecular testing is in defining patients with complex family histories and variable clinical presentations as familial rather than sporadic. Dementia: There is no alternative means of accurate diagnosis. CADASIL can be diagnosed by MRI and skin biopsy. However the skin biopsy may be negative and is a difficult invasive procedure and CADASIL diagnosis by MRI is not specific Spinal Muscular Atrophy (SMA): No alternative means of diagnosis is available. Clinical diagnosis is generally based on phenotype, and can include neuroimaging, EMG and muscle biopsy. 28. Please list any genes where the main phenotype associated with that gene is unrelated to the phenotype being tested by the panel. For example, lung cancer susceptibility when testing for congenital cataract because ERCC6 gene (primarily associated with lung cancer) is included in a panel test for congenital cataract. n/a 29. If testing highlights a condition that is very different from that being tested for, please outline your strategy for dealing with this situation. The only circumstance in which this is likely to arise would be if any variants of potential clinical significance were found in genes associated with overlapping phenotypes e.g. ALS2 mutations may be associated with both ALS and HSP. If this should happen, after detailed evaluation of the variant identified, the findings would be discussed in some detail with the referring clinician and the importance of referral to Clinical Genetics (if not already involved) would be emphasised. Since there are clinical overlaps between the four phenotypic groups covered by this panel test, it is anticipated that the possibility of identifying a mutation in a gene that could potentially result in manifestation of additional phenotypic features would have been discussed with the patient prior to testing. Both the HSP and SMA subpanels include the HEXA gene associated with Tay-Sachs disease, and therefore there is the potential to identify heterozygote carriers of this disorder. This is of most relevance in an Ashkenazi Jewish individual, where the carrier rate is approximately 1 in 27, whereas the carrier rate outside this ethnic group is relatively low (approximately 1 in 300 in the European and American populations). However, if the situation arose, after detailed evaluation of the variant identified, the findings would be discussed in some detail with the referring clinician and the importance of referral to Clinical Genetics (if not already involved) would be emphasised.

13 30. If a panel test, is this replacing an existing panel/multi gene test and/or other tests currently carried out by your lab e.g. Noonan Spectrum Disorders 12 Gene Panel replaced multigene Sanger test for KRAS, RAF1, PTPN11 and SOS1? If so, please provide details below. These panel tests will replace the Sanger sequencing services currently available in the Sheffield laboratory as follows: HSP genes - SPAST gene sequencing, ATL1 gene sequencing, REEP1 gene sequencing ALS genes SOD1 gene sequencing, TARDBP gene sequencing, FUS gene sequencing They will also supersede the two smaller subpanels of HSP and FALS genes associated with the Neurogenetic Disorders 96 Gene Panel. 31. Please describe any specific ethical, legal or social issues with this particular test. The purpose of the panel test is confirming the diagnosis of neurogenetic disorders, often with no treatment currently available. This would subsequently lead to the development of specific Sanger sequencing tests to be utilised for diagnostic, prenatal or presymptomatic testing in relatives. There are ethical issues relating to the principles of identifying individuals not currently affected, but at risk of developing untreatable conditions. 32. REAL LIFE CASE STUDY Please provide a case study that illustrates the benefits of this test A sample was received from a 15 year old girl with a family history of complex HSP with agenesis of the corpus callosum. She and her brother were both affected, as was a male cousin. There was parental consanguinity and hence the clinical geneticist was querying whether the HSP was due to the SPG11 gene, which is recessive and associated with thin corpus callosum. The family had been known to the Clinical Genetics department for some years but they had never been able to access SPG11 testing in the UK. The testing was available in a German laboratory but the cost was very high at over 2000 euros so the clinician had hesitated to send a sample to that laboratory. The panel test performed did not identify a mutation in the SPG11 gene but did detect a homozygous insertion deletion mutation in the DDHD2 gene, which also underlies a recessive phenotype. Subsequent family studies confirmed that the girl s parents were both heterozygous carriers of this mutation and that her affected brother and cousin were also homozygous. This confirmed a diagnosis of HSP type 54. If the panel test had not been available the sample would probably have been sent to Germany for SPG11 testing, at a cost in excess of that of the panel test, and the diagnosis would not have been made.

14 UKGTN Testing Criteria Test name: Hereditary Spastic Paraplegia 88 Gene Panel Approved name and symbol of disorder/condition(s): See Appendix 1 Approved name and symbol of gene(s): See Appendix 1 OMIM number(s): OMIM number(s): Patient name: Patient postcode: 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 Geneticist Consultant Adult Neurologist Consultant Paediatric Neurologist Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Hereditary Spastic Paraplegias: progressive bilateral lower-extremity weakness and increased muscle tone and other causes excluded AND Evidence of inheritance ( Autosomal dominant, Autosomal recessive or X- linked) or, if isolated case, must be clinically entirely typical AND Mutation will affect treatment/prognostic advice/genetic risk assessment/testing in family Tick if this patient meets criteria Additional Information: Please see testing strategy below For panel tests: At risk family members where familial mutation is known do not require a full panel test but should be offered analysis of the known mutation. 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.

15 Testing Strategy: HEREDITARY SPASTIC PARAPLEGIA HSP 88 gene Panel Negative: 35-40% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 60-65% Positive # * Negative and where a single heterozygous pathogenic mutation is identified in a recessive gene, following discussion with clinician. # Exact incidence of large deletions within each panel of genes is currently unknown

16 UKGTN Testing Criteria Test name: Familial Amyotrophic Lateral Sclerosis (FALS) with or without Frontotemporal Dementia 42 gene panel Approved name and symbol of disorder/condition(s): See Appendix 1 Approved name and symbol of gene(s): See Appendix 1 Patient name: Patient postcode: Name of referrer: Title/Position: Referrals will only be accepted from one of the following: Referrer Consultant Clinical Geneticist Consultant Adult Neurologist Consultant Paediatric Neurologist Date of birth: NHS number: Lab ID: OMIM number(s): OMIM number(s): Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria The presence of: Evidence of lower motor neuron (LMN) degeneration by clinical, electrophysiologic, or neuropathologic examination AND Evidence of upper motor neuron (UMN) degeneration by clinical examination AND Progressive spread of symptoms or signs within a region or to other regions, as determined by history or examination TOGETHER WITH The absence of: Electrophysiologic and pathologic evidence of other disease processes that might explain the signs of LMN and/or UMN degeneration AND Neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiologic signs. AND Relentless progression of symptoms & signs during follow-up period AND Additional family history with affected first degree relative or two second degree relatives with the same or a related presentation AND Exclusion of C9orf72 expansion mutation Tick if this patient meets criteria Additional Information: Please see testing strategy below For panel tests: At risk family members where familial mutation is known do not require a full panel test but should be offered analysis of the known mutation. 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.

17 Testing Strategy: FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS WITH OR WITHOUT FRONTOTEMPORAL DEMENTIA C9orf72 Expansion testing Negative: Familial: 50-60% Sporadic: 92% FALS 42 gene Panel Negative: Familial: 30-36% Sporadic: 83% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: Familial: 40-50% Sporadic: 8% Positive: Familial: 20-24% Sporadic: 9% Positive # * Negative and where a single heterozygous pathogenic mutation is identified in a recessive gene, following discussion with clinician. # Exact incidence of large deletions within each panel of genes is currently unknown

18 UKGTN Testing Criteria Test name: Spinal Muscular Atrophy 29 Gene Panel Approved name and symbol of disorder/condition(s): See Appendix 1 Approved name and symbol of gene(s): See Appendix 1 Patient name: Patient postcode: Name of referrer: Title/Position: Referrals will only be accepted from one of the following: Referrer Consultant Clinical Geneticist Consultant Adult Neurologist Consultant Paediatric Neurologist Date of birth: OMIM number(s): OMIM number(s): NHS number: Lab ID: Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Tick if this patient meets criteria The presence of: Evidence of lower motor neuron (LMN) degeneration by clinical, electrophysiologic, or neuropathologic examination AND Progressive spread of symptoms or signs within a region or to other regions, as determined by history or examination TOGETHER WITH The absence of: Electrophysiologic and pathologic evidence of other disease processes that might explain the signs AND Evidence of upper motor neuron (UMN) degeneration by clinical examination AND Neuroimaging evidence of other disease processes that might explain the observed clinical and electrophysiologic signs. AND evidence of progression of symptoms & signs during follow-up period AND Exclusion of SMN1 deletion Additional Information: Please see testing strategy below For panel tests: At risk family members where familial mutation is known do not require a full panel test but should be offered analysis of the known mutation. 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.

19 Testing Strategy: SPINAL MUSCULAR ATROPHY SMN1 deletion testing Negative: 2% to 90% SMA 29 gene Panel Negative: 1.4% to 63% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 10% to 98% depending on severity Positive: 0.6% to 27% Positive # * Negative and where a single heterozygous pathogenic mutation is identified in a recessive gene, following discussion with clinician. # Exact incidence of large deletions within each panel of genes is currently unknown

20 Test name: Dementia 27 Gene Panel UKGTN Testing Criteria Approved name and symbol of disorder/condition(s): See Appendix 1 Approved name and symbol of gene(s): See Appendix 1 OMIM number(s): OMIM number(s): Patient name: Patient postcode: 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 Geneticist Consultant Adult Neurologist Consultant Psychiatrist Tick if this refers to you. Minimum criteria required for testing to be appropriate as stated in the Gene Dossier: Criteria Early onset (<55 yrs) cognitive decline where acquired causes (e.g. stroke, tumour) have been excluded. Tick if this patient meets criteria Additional Information: Please see testing strategy below For panel tests: At risk family members where familial mutation is known do not require a full panel test but should be offered analysis of the known mutation. 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.

21 Testing Strategy: DEMENTIA Frontotemporal Dementia Dementia 2% 98% C9orf72 Expansion testing Negative: 1.8% Dementia 27 gene Panel Negative: 40% *see below MLPA Dosage analysis where available Negative # not confirmed Positive: 0.2% Positive: 59% Positive # * Negative and where a single heterozygous pathogenic mutation is identified in a recessive gene, following discussion with clinician. # Exact incidence of large deletions within each panel of genes is currently unknown

22 IS IT A REASONABLE COST TO THE PUBLIC? 36. Based on experience what will be the national (UK wide) expected activity for requesting this test, per annum, for: Index cases: 630 (400 HSP / 80 FALS +/- FTD / 50 Dementia / 100 SMA) Family members where mutation is known: 190 (120 HSP / 25 FALS +/- FTD / 15 Dementia / 30 SMA) If a NGS panel test, it is recognised that the full panel will not be used to test family members where the familial mutation is known. Please provide expected number of tests to inform completion of Q If your laboratory does not have capacity to provide the full national need please suggest 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. If you are unable to answer this question please write unknown. The technology / equipment currently available in the laboratory provides the capacity to cope with over 4,000 tests a year, with further investment underway. There would therefore be no issues in dealing with 630 tests a year. 38. In order to establish the potential costs/savings that could be realised in the diagnostic care pathway, please list the tests/procedures that are no longer required to make a diagnosis for index cases where index cases would have the molecular genetic test proposed in this gene dossier at an earlier stage in the pathway. It is the tests/procedures that would be stopped for patients that are eligible for the gene test. This information will be used to calculate the overall investment / savings required in Q39 Example: The introduction of a 95 gene panel for syndromic and non syndromic hearing loss would allow those patients who are recognised early enough in their pathway to diagnosis to be offered the genetic test instead of having sequential gene tests for individual genes already available and repeated ECGs, ERGs & renal ultrasounds as part of the diagnostic pathway although these may still be required as part of management after diagnosis. Subpanels for HSP, FALS and Dementia are cost neutral. Type of test Cost ( ) Imaging procedures N/A Laboratory pathology tests SMA Not currently available (other than molecular/cyto genetic test proposed in this Gene Dossier) Physiological tests (e.g. ECG) EMG 200 (SMA) Other investigations/procedures (e.g. biopsy) Muscle Biopsy 292 (SMA) Associated inpatient stays in the diagnostic pathway N/A Total cost of tests/procedures to be stopped 492 (SMA) (please write n/a if the genetic test does not replace any other tests procedures in the diagnostic care pathway) If any of the tests/procedures listed above would be carried out on individuals after having the genetic test because the genetic test did not pick up a pathogenic mutation (i.e. negatives), please indicate the costs for these tests to continue to diagnosis. 492 for SMA subpanel only. For example a panel test replaces single gene tests that have been included above, but after the panel test an individual that tests negative would not need to have these single gene tests, because the genes were on the NGS panel.

23 39. Please complete the Excel spread sheet available to download from the UKGTN website to calculate the estimated investment or savings, based on the expected annual activity of index & family cases (Q36 above) and using the information provided in Q38. Please submit this separately. Costs only apply to SMA subpanel: Number of index cases expected annually 100 Number of family member tests expected annually 30 Cost to provide index case test 975 Cost to provide family member test 105 Costs associated with tests/procedures for index cases if the genetic test in this Gene Dossier was not available 492 Costs associated with tests/procedures for index cases that test negative for the genetic test in this Gene Dossier 492 Total annual costs for diagnostic tests prior to introduction of the genetic test submitted for evaluation in this Gene 49,200 Dossier Total annual costs to provide genetic test 97,500 Additional savings or investment for 100% pick up rate for index cases 48,300 Percentage of index cases expected not to find a pathogenic mutation (negatives) 70% Number of index cases estimated to not find a pathogenic mutation (negatives) 70 Costs or savings to provide additional tests for index cases that test negative 34,440 Total savings / investment prior to application of marginal reduction if applicable 82,740 If a panel test and there are genes on the panel test that are already available on either other panel tests or single 10% gene tests please estimate/suggest a marginal percentage reduction of the investment/savings. If you feel this is NOT applicable please leave this as 0%. Marginal percentage reduction if applicable applied to the savings/investment 8,274 TOTAL INVESTMENT for tests for INDEX CASES 74,466 Total costs for family members 3,150 If family testing is already available for any of the genes on this panel across the Network, please estimate the associated funding for these tests. 525 TOTAL INVESTMENT for tests for FAMILY MEMBERS 2,625 ADDITIONAL INVESTMENT FOR ALL ACTIVITY EXPECTED PER ANNUM 77, Please indicate the healthcare outcomes that apply to this test after diagnosis. It is recognised that all tests recommended by the UKGTN for NHS service improve clinical management and, if a familial mutation is found, allows for prenatal testing and therefore these are not included in the list below. Healthcare outcomes 1. Alerts significant clinical co-morbidities Yes 2. Reduces mortality/saves lives Yes 3. Avoids irreversible harm Yes 4. Avoids diagnostic procedures/tests (some of which may be Yes invasive) and/or multiple hospital appointments 5. Avoids incorrect management (e.g. medication or treatment) Yes that could be harmful 6. Confirms targeted therapy/management Yes 7. Earlier diagnosis allowing commencement of treatment earlier Yes with associated improved prognosis 8. Enables access to educational and social support Yes 9. At risk family members that test negative for a familial mutation can be discharged from follow up Yes 10. At risk family members that test positive for a familial mutation Yes have appropriate follow up Does this apply to this test?

24 Appendix 1 Genes in panel test and associated conditions. Rows that are highlighted in yellow indicate that the gene was being fully analysed in the context of a single separate UKGTN test at time of submission. OMIM standard name of condition (please provide the conditions that the test is for which may NOT necssarily be the condition that is linked to the gene on OMIM) OMIM symbol of condition OMIM number of condition Mode of inheritance HGNC standard name of gene HGNC symbol of the gene HGNC numb er of the gene OMIM number of the gene Evidence of association between gene and condition Pubmed ID: % of horizontal coverage of gene MLPA Hereditary Spastic Paraplegia 88 gene panel Adrenoleukodystrophy ALD XLR Ataxia, spastic, 5, autosomal recessive SPAX AR Spastic paraplegia 9A, autosomal dominant SPG9A AD Spastic paralysis, infantile onset ascending IAHSP AR Spastic paraplegia 63 SPG AR Pettigrew syndrome PGS XLR Spastic paraplegia 47, autosomal recessive SPG AR Spastic paraplegia 51, autosomal recessive SPG AR Spastic paraplegia 50, autosomal recessive SPG AR Spastic paraplegia 52, autosomal recessive SPG AR ATP binding cassette subfamily D member 1 ABCD AFG3-like AAA ATPase 2 AFG3L aldehyde dehydrogenase 18 family member A1 ALDH18A ALS2, alsin Rho guanine nucleotide exchange factor ALS , No , No , No , No adenosine monophosphate deaminase 2 AMPD No adaptor related protein complex 1 sigma 2 subunit AP1S No adaptor-related protein complex 4, beta 1 subunit AP4B adaptor-related protein complex 4, epsilon 1 subunit AP4E adaptor-related protein complex 4, mu 1 subunit AP4M , No , No , No adaptor-related protein complex 4, sigma 1 subunit AP4S No